U.S. patent number 6,943,240 [Application Number 09/954,987] was granted by the patent office on 2005-09-13 for nucleic acids for high throughput screening of cpg-based immuno-agonist/antagonist.
This patent grant is currently assigned to Coley Pharmaceuticals GmbH. Invention is credited to Stefan Bauer, Grayson Lipford, Hermann Wagner.
United States Patent |
6,943,240 |
Bauer , et al. |
September 13, 2005 |
Nucleic acids for high throughput screening of CpG-based
immuno-agonist/antagonist
Abstract
The invention pertains to murine TLR9 and related TLR9s which
include murine-specific amino acids, as well as nucleic acids which
encode those polypeptides. The present invention also includes
fragments and biologically functional variants of the murine TLR9.
The invention further relates to methods of using such murine and
non-murine TLR9 nucleic acids and polypeptides, especially in
methods for screening for agonists and antagonists of
immunostimulatory CpG nucleic acids. Also included are murine TLR9
inhibitors which inhibit murine TLR9 activity by inhibiting the
expression or function of murine TLR9. In a further aspect the
present invention pertains to murine TLR7 and murine TLR8, as well
as related TLR7 and TLR8 molecules which include murine-specific
amino acids, as well as nucleic acids which encode those
polypeptides. The present invention also includes fragments and
biologically functional variants of the murine TLR7 and TLR8.
Methods are included for screening for ligands of TLR7 and TLR8, as
well as for inhibitors and agonists and antagonists of signaling
mediated by TLR7 and TLR8.
Inventors: |
Bauer; Stefan (Munich,
DE), Lipford; Grayson (Dusseldorf, DE),
Wagner; Hermann (Eching, DE) |
Assignee: |
Coley Pharmaceuticals GmbH
(Langenfeld, DE)
|
Family
ID: |
27499675 |
Appl.
No.: |
09/954,987 |
Filed: |
September 17, 2001 |
Current U.S.
Class: |
536/23.1;
435/320.1; 435/325 |
Current CPC
Class: |
A61P
37/00 (20180101); C07K 14/705 (20130101) |
Current International
Class: |
C07K
14/435 (20060101); C07K 14/705 (20060101); C07H
021/00 () |
Field of
Search: |
;536/23.1
;435/320.1,325 |
References Cited
[Referenced By]
U.S. Patent Documents
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|
Primary Examiner: Marschel; Ardin H.
Assistant Examiner: Smith; Carolyn L.
Attorney, Agent or Firm: Wolf, Greenfield & Sacks,
P.C.
Parent Case Text
RELATED APPLICATIONS
This invention claims benefit of U.S. Provisional Application No.
60/233,035, filed Sep. 15, 2000; U.S. Provisional Application No.
60/263,657, filed Jan. 23, 2001; U.S. Provisional Application No.
60/291,726, filed May 17, 2001 abn, and U.S. Provisional
Application No. 60/300,210, filed Jun. 22, 2001.
Claims
What is claimed is:
1. An isolated nucleic acid molecule which encodes a Toll-like
receptor 9 (TLR9) polypeptide HTLR9-CXXCm, said polypeptide
comprising an amino acid sequence of SEQ ID NO:6 except for
substitution of amino acids 269-274 of SEQ ID NO:6 with amino acids
269-274 of SEQ ID NO:3.
2. An expression vector comprising the isolated nucleic acid
molecule of claim 1 operably linked to a promoter.
3. A host cell comprising the expression vector of claim 2.
4. The host cell of claim 3, further comprising at least one
expression vector selected from the group consisting of: (a) an
expression vector comprising a nucleic acid molecule which encodes
an isolated TLR7 polypeptide operably linked to a promoter, and (b)
an expression vector comprising a nucleic acid molecule which
encodes an isolated TLR8 polypeptide operably linked to a
promoter.
5. The host cell of claim 3, further comprising a reporter gene
construct comprising a reporter gene operatively linked to a
promoter sensitive to NF-.kappa.B.
6. The host cell of claim 3, wherein the host cell is a 293
fibroblast cell.
7. The host cell of claim 5, wherein the host cell is a 293
fibroblast cell.
Description
FIELD OF THE INVENTION
The invention pertains to signal transduction by immunostimulatory
nucleic acids.
BACKGROUND OF THE INVENTION
Bacterial DNA is a potent immunomodulatory substance. Yamamoto S et
al., Microbiol Immunol 36:983-997 (1992). It has been hypothesized
to be a pathogen-derived ligand recognized by an unidentified
pathogen recognition receptor that initiates a host of innate and
adaptive immune responses. Wagner H, Adv Immunol 73:329-368 (1999).
CpG motif-containing oligodeoxynucleotides (CpG ODN) can mimic the
biology of bacterial DNA. Krieg A M et al., Nature 374:546-549
(1995). CpG ODN and DNA vectors have recently been shown to be of
clinical value due to immunostimulatory, hematopoietic and adjuvant
qualities.
The adaptive immune system appeared approximately 450 million years
ago when a transposon that carried the forerunners of the
recombinase activating genes, RAG-1 and RAG-2, was inserted into
the germ line of early jawed vertebrates. Agarwal A. et al., Nature
394:744 (1998). The ability to mount an adaptive immune response
allowed organisms to remember the pathogens that they had already
encountered, and natural selection made the adaptive immune
response a virtually universal characteristic of vertebrates.
However, this did not lead to discarding the previous form of host
defense, the innate immune system. Indeed, this earlier form of
host defense has been coopted to serve a second function,
stimulating and orienting the primary adaptive immune response by
controlling the expression of costimulatory molecules.
It had been surmised for a decade that cells of the innate immune
system bear receptors for conserved molecular patterns associated
with microbial pathogens. According to this model, when the protein
antigens derived from pathogens are processed and presented as
peptides that serve as the stimulus for specific T cell receptors,
pattern recognition receptors (PRRs) on the antigen-presenting
cells also induce the synthesis of costimulatory molecules,
cytokines, and chemokines. These activated antigen-presenting cells
serve to attract and activate the antigen-specific T cells that are
essential to all adaptive immune responses. Janeway C A J, Cold
Spring Harbor Symp Quant Biol 54:1 (1989); Fearon D T et al.,
Science 272:50 (1996); and Medzhitov R et al., Cell 91:295 (1997).
It was known that the substances that can induce costimulation
include bacterial lipopolysaccharide (LPS), synthetic
double-stranded RNA, glycans, and mannans. Furthermore,
experimental evidence indicated that the processed antigen ligand
for the T cell had to be on the same cell as the costimulatory
molecule. This is obviously of crucial importance for maintaining
self-tolerance; bystander presentation of costimulatory molecules
would mean that tolerance would be lost whenever an infection
occurred.
To validate this model, it was necessary to identify receptors for
microbial patterns that, upon binding pathogen ligands, initiate
signaling cascades leading to the production of costimulatory
molecules and cytokines. Molecules such as mannose binding protein
(MBP) do not qualify for this role, because they activate
proteolytic cascades or promote phagocytosis but are not known to
induce costimulation. The break-through came with the
identification of a human homologue of Drosophila Toll initially
cloned as a cDNA and later named hTLR4 (for human Toll-like
receptor). Medzhitov R et al., Nature 388:394 (1997); Rock F L et
al., Proc Natl Acad Sci USA 95:588 (1998); Chaudhary P M et al.,
Blood 91:4020-4027 (1998).
Toll-like receptors (TLRs) are a family of germline-encoded
transmembrane proteins that facilitate pathogen recognition and
activation of the innate immune system. Hoffmann J A et al.,
Science 284, 1313-1318 (1999); Rock F L et al., Proc Natl Acad Sci
USA 95:588-593 (1998). TLRs engage conserved pathogen-derived
ligands and subsequently activate the TLR/IL-1R signal transduction
pathway to induce a variety of effector genes. Medzhitov R et al.,
Mol Cell 2:253-258 (1998); Muzio M et al., J Exp Med 187:2097-2101
(1998).
So far, ten different mammalian TLRs have been described. Rock F L
et al., Proc Natl Acad Sci USA 95:588-593 (1998); Chaudhary P M et
al., Blood 91:4020-4027 (1998); Takeuchi O et al., Gene 231:59-65
(1999); Aderem A. et al., Nature 406:782-7 (2000). So far, genetic
data suggest that the TLRs have unique functions and are not
redundant. Ligands for and the function of most of these TLRs,
aside from TLR2 and TLR4, remain to be elucidated.
It turns out that an LPS-binding and signaling receptor complex is
assembled when hTLR4 interacts with LPS bound to CD14, a peripheral
membrane protein held to the cell surface by a
glycosyl-phosphoinositol tail. The presence of LPS binding protein
(LBP) further increases signaling. The hTLR4 protein has a
leucine-rich repeat sequence in its extracellular domain that
interacts with CD14 complexed with LPS. TLR4 then transduces the
LPS signal across the membrane because destructive mutation of this
gene lead to an LPS-unresponsive state in mice, which are also
deficient in the clearance of Gram-negative bacteria. Poltorak A et
al., Science 282:2085 (1998); Qureshi S T et al., J Exp Med
189:615-625 (1999); Eden C S et al., J Immunol 140:180 (1988). It
has since become apparent that humans, like flies, have numerous
Toll-like receptors (TLRs).
TLR4 and other TLRs have a cytoplasmic Toll/IL-1 receptor (TIR)
homology domain. This domain communicates with a similar domain on
an adapter protein (MyD88) that interacts with TLR4 by means of a
like:like interaction of TIR domains. The next interaction is
between the adapter and a kinase, through their respective "death
domains." The kinase in turn interacts with tumor necrosis factor
(TNF) receptor-associated factor-6 (TRAF6). Medzhitov R et al., Mol
Cell 2:253 (1998); Kopp E B et al., Curr Opin Immunol 11:15 (1999).
After TRAF6, two sequential kinase activation steps lead to
phosphorylation of the inhibitory protein I.kappa.B and its
dissociation from NF-.kappa.B. The first kinase is a
mitogen-activated kinase kinase kinase (MAPKKK) known as NIK, for
NF-.kappa.B-inducing kinase. The target of this kinase is another
kinase made up of two chains, called I.kappa.B kinase .alpha.
(IKK.alpha.) and I.kappa.B kinase .beta. (IKK.beta.), that together
form a heterodimer of IKK.alpha.:IKK.beta., which phosphorylates
I.kappa.B. NF-.kappa.B translocates to the nucleus to activate
genes with .kappa.B binding sites in their promoters and enhancers
such as the genes encoding interleukin-1.beta. (IL-1.beta.), IL-6,
IL-8, the p40 protein of IL-12, and the costimulatory molecules
CD80 and CD86.
The types of cells that respond to CpG DNA-B cells, dendritic cells
(DCs) and macrophages--are also stimulated by other
pathogen-derived pattern-recognition factors, such as LPS. In
general, the PRRs of the innate immune system are situated on the
cell surface, where they are probably best able to detect microbes.
Although cell-surface proteins that bind DNA are well described,
and have been proposed to mediate immune activation by CpG motif
(Liang H et al., J Clin Invest 98:1119-1129 (1998)), this binding
is sequence-independent and does not bring about cell activation.
Krieg A M et al., Nature 374:546-549 (1995); Yamamoto T et al.,
Microbiol Immunol 38:831-836 (1994); Hacker H et al., EMBO J.
17:6230-6240 (1998). Because CpG ODNs that have been immobilized to
prevent cell uptake are nonstimulatory (Krieg A M et al., Nature
374:546-549 (1995); Manzel L et al., Antisense Nucleic Acid Drug
Dev 9:459-464 (1999)), it appears that CpG ODN probably work by
binding to an intracellular receptor. In support of this
hypothesis, drugs such as chloroquine, which interfere with the
endosomal acidification/processing of ODNs, specifically block the
immune stimulatory effects of CpG DNA. Hacker H et al., EMBO J.
17:6230-6240 (1998); Macfarlane D E et al., J Immunol 160:1122-1131
(1998); Yi A K et al., J Immunol 160:4755-4761 (1998). It has been
proposed that an endosomal step is required for the CpG-induced
signal transduction pathways. Hacker H et al., EMBO J. 17:6230-6240
(1998); Yi A K et al., J Immunol 160:4755-4761 (1998). How the
information contained in unmethylated CpG-motifs of bacterial DNA
trigger changes in gene expression has not previously been
discovered.
Since the receptor for bacterial DNA has been unknown, development
of screening for optimal CpG motifs through direct binding analysis
has been limited. An additional complication appears to be
species-specific selectivity for CpG sequence, i.e., an optimal
sequence for one species may not be optimal for another.
SUMMARY OF THE INVENTION
Nucleic acids encoding three Toll-like receptors, Toll-like
receptor 7 (TLR7), TLR8, and TLR9 of the mouse have now been
identified, isolated, cloned and sequenced by the inventors. The
invention in general provides isolated nucleic acid molecules
encoding TLRs and isolated fragments of those nucleic acid
molecules; isolated TLR polypeptides and isolated fragments of
those polypeptides; expression vectors containing the foregoing
nucleic acid molecules; host cells having the foregoing expression
vectors; fusion proteins including the TLR polypeptides and
fragments thereof; and screening methods useful for identifying,
comparing, and optimizing agents which interact with these TLRs,
particularly agents that alter the expression of and signaling
associated with these TLR molecules. In preferred embodiments the
screening methods are high throughput screening methods.
The invention in some aspects arises from the surprising discovery
that TLR9 is involved in immunostimulatory nucleic acid
(ISNA)-induced immunostimulation. The invention also stems in part
from the surprising discovery that TLR9 transduces immune
activating signals in response to ISNA in a manner that is both
sequence-specific and species-specific.
In a first aspect the invention provides isolated nucleic acid
molecules which encode full-length murine TLR9. According to this
aspect of the invention, isolated nucleic acid molecules are
provided which are selected from the group consisting of (a)
nucleic acid molecules which hybridize under stringent conditions
to a nucleic acid molecule having a nucleotide sequence set forth
as SEQ ID NO:1, and which code for a murine TLR9 having an amino
acid sequence set forth as SEQ ID NO:3; (b) nucleic acid molecules
that differ from the nucleic acid molecules of (a) in codon
sequence due to degeneracy of the genetic code; and (c) complements
of (a) or (b). In a certain embodiment, the isolated nucleic acid
molecule codes for SEQ ID NO:3, where SEQ ID NO:3 represents the
deduced amino acid sequence of full-length murine TLR9. In some
embodiments the isolated nucleic acid molecule comprises the
nucleotide sequence of SEQ ID NO:1 or SEQ ID NO:2, where these
correspond to full-length cDNA and the open reading frame for
murine TLR9, respectively.
The term "stringent conditions" as used herein refers to combined
conditions based on parameters including salt, temperature, organic
solvents, and optionally other factors with which the paractioner
skilled in the art is familiar. Nucleic acid hybridization
parameters may be found in references which compile such methods,
e.g., Molecular Cloning: A Laboratory Manual, J. Sambrook, et al.,
eds., Second Edition, Cold Spring Harbor Laboratory Press, Cold
Spring Harbor, N.Y., 1989, or Current Protocols in Molecular
Biology, F. M. Ausubel, et al., eds., John Wiley & Sons, Inc.,
New York. More specifically, stringent conditions, as used herein,
refers, for example, to hybridization at 65.degree. C. in
hybridization buffer (3.5.times.SSC, 0.02% Ficoll, 0.02% polyvinyl
pyrrolidone, 0.02% bovine serum albumin, 2.5 mM NaH.sub.2 PO.sub.4
(pH7), 0.5% SDS, 2 mM EDTA). SSC is 0.15M sodium chloride/0.15M
sodium citrate, pH7; SDS is sodium dodecyl sulfate; and EDTA is
ethylenediaminetetraacetic acid. After hybridization, the membrane
upon which the DNA is transferred is washed with 2.times.SSC at
room temperature and then with 0.1-0.5.times. SSC/0.1.times.SDS at
temperatures up to 68.degree. C. There are other conditions,
reagents, and so forth which can be used, which result in a similar
degree of stringency. The skilled artisan will be familiar with
such conditions, and thus they are not given here. It will be
understood, however, that the skilled artisan will be able to
manipulate the conditions in a manner to permit the clear
identification of alleles of murine TLR nucleic acids of the
invention. The skilled artisan also is familiar with the
methodology for screening cells and libraries for expression of
such molecules which then are routinely isolated, followed by
isolation of the pertinent nucleic acid molecule and
sequencing.
The invention in a second aspect provides isolated TLR9
polypeptides or fragments thereof. The isolated TLR9 polypeptides
or fragments thereof include at least one amino acid of a murine
TLR9 selected from the group consisting of amino acids 2, 3, 4, 6,
7, 18, 19, 22, 38,44, 55, 58, 61, 62, 63, 65, 67, 71, 80, 84, 87,
88, 91, 101, 106, 109, 117, 122, 123, 134, 136, 140, 143, 146, 147,
157, 160, 161, 167, 168, 171, 185, 186, 188, 189, 191, 199, 213,
217, 220, 227, 231, 236, 245, 266, 269, 270, 271, 272, 273, 274,
278, 281, 285, 297, 298, 301, 305, 308, 311, 322, 323, 325, 326,
328, 332, 335, 346, 348, 353, 355, 358, 361, 362, 365, 367, 370,
372, 380, 381, 382, 386, 389, 392, 394, 397, 409, 412, 413, 415,
416, 419, 430, 432, 434, 435, 438, 439, 443, 444, 446, 447, 448,
450, 451, 452, 454, 455, 459, 460, 463, 465, 466, 468, 469, 470,
472, 473, 474, 475, 478, 488, 489, 494, 495, 498, 503, 508, 510,
523, 531, 539, 540, 543, 547, 549, 561, 563, 565, 576, 577, 579,
580, 587, 590, 591, 594, 595, 597, 599, 601, 603, 610, 611, 613,
616, 619, 632, 633, 640, 643, 645, 648, 650, 657, 658, 660, 667,
670, 672, 675, 679, 689, 697, 700, 703, 705, 706, 711, 715, 716,
718, 720, 723, 724, 726, 729, 731, 735, 737, 743, 749, 750, 751,
752, 754, 755, 759, 760, 772, 774, 780, 781, 786, 787, 788, 800,
814, 821, 829, 831, 832, 835, 844, 857, 858, 859, 862, 864, 865,
866, 879, 893, 894, 898, 902, 910, 917, and 927 of SEQ ID NO:3,
wherein the TLR9 polypeptide or fragment thereof has an amino acid
sequence which is identical to a human TLR9 polypeptide or fragment
thereof except for the at least one amino acid of murine TLR9. The
TLR9 polypeptide or fragment thereof in certain embodiments
according to this aspect of the invention further includes at least
one amino acid of murine TLR9 selected from the group consisting of
amino acids 949, 972, 975, 976, 994, 997, 1000, 1003, 1004, 1010,
1011, 1018, 1023, and 1027 of SEQ ID NO:3. Thus specifically
excluded from this aspect of the invention are TLR9 fragments
restricted to the C-terminal 95 amino acids and fragments
thereof.
In certain embodiments the TLR9 polypeptide and fragments thereof
according to this aspect of the invention exclude those TLR9
polypeptides and fragments thereof which differ from human TLR9 and
fragments thereof only by one or more conservative amino acid
substitutions at particular sites noted above. As is well known in
the art, a "conservative amino acid substitution" refers to an
amino acid substitution which generally does not alter the relative
charge or size characteristics of the polypeptide in which the
amino acid substitution is made. Conservative substitutions of
amino acids typically include substitutions made amongst amino
acids within the following groups: methionine (M), isoleucine (I),
leucine (L), valine (V); phenylalanine (F), tyrosine (Y),
tryptophan (W); lysine (K), arginine (R), histidine (H); alanine
(A), glycine (G); serine (S), threonine (T); glutamine (O),
asparagine (N); and glutamic acid (E), aspartic acid (D).
According to this and other aspects of the invention, with
reference to TLR "polypeptides and fragments thereof," "fragments
thereof" refers to polypeptide fragments having stretches of
contiguous amino acid residues that are at least about 8 amino
acids long. Generally the fragments are at least about 10 amino
acids long; more generally at least 12 amino acids long; often at
least about 14 amino acids long; more often at least about 16 amino
acids long; typically at least 18 amino acids long; more typically
at least 20 amino acids long; usually at least 22 amino acids long;
and more usually at least 24 amino acids long. Certain preferred
embodiments include larger fragments that are, for example, at
least about 30 amino acids long, at least about 40 amino acids
long, at least about 50 amino acids long, at least about 100 amino
acids long, at least about 200 amino acids long, and so on, up to
and including fragments that are a single amino acid shorter than
full-length TLR polypeptide.
In certain embodiments, the human TLR9 has an amino acid sequence
set forth as SEQ ID NO:6.
In preferred embodiments, the isolated TLR9 polypeptides or
fragments thereof include an amino acid sequence selected from the
group consisting of SEQ ID NO:3 and fragments of SEQ ID NO:3. In
some embodiments according to this aspect of the invention, the
isolated TLR9 polypeptides or fragments thereof include
combinations of the foregoing human and murine TLR9
polypeptides.
In certain preferred embodiments the isolated TLR9 polypeptide or
fragment thereof is an extracytoplasmic domain (also referred to
herein as extracellular domain) of TLR9, or a portion thereof. As
described in greater detail further herein, TLR7, TLR8, and TLR9
have certain structural and functional domains. Structural domains
of these TLRs include but are not limited to an extracytoplasmic
domain, a transmembrane domain, and a cytoplasmic domain. The
extracytoplasmic domain extends into the lumen of
endosomal/lysosomal vesicles. The cytoplasmic domain includes a
Toll/interleukin-1 receptor-like domain (also referred to as
Toll/IL-1R domain, TIR homology domain, or TIR domain). In murine
TLR9 the extracytoplasmic, transmembrane, and cytoplasmic domains
correspond to amino acids 1 to about 819, about 820 to about 837,
and about 838 to about 1032, respectively.
As mentioned above, it has been discovered according to the
invention that TLR9 is involved in immune activation induced by
certain nucleic acid molecules referred to in the art as
immunostimulatory nucleic acids (ISNAs), including CpG nucleic
acids. It is believed by the inventors that binding of ISNA to TLR9
leads to signal transduction involving the TIR domain of TLR9. Thus
in certain embodiments according to this aspect of the invention,
the isolated TLR9 polypeptide or fragment thereof selectively binds
to an ISNA, including an ISNA that is a CpG nucleic acid.
Also included according to this aspect of the invention are
isolated TLR9 polypeptides or fragments thereof which are portions
of the extracytoplasmic domain believed by the inventors to
interact with immunostimulatory nucleic acids such as CpG nucleic
acids. In certain embodiments such portions include an MBD motif
set forth as any one of SEQ ID NOs: 126, 127, 210, and 211. In
certain embodiments portions of the extracytoplasmic domain
believed by the inventors to interact with immunostimulatory
nucleic acids include a CXXC motif set forth as any one of SEQ ID
NOs: 196, 197, and 198.
According to a third aspect of the invention, isolated nucleic acid
molecules are provided which encode the foregoing isolated TLR9
polypeptides or fragments thereof. The isolated nucleic acid
molecules according to this aspect of the invention specifically
exclude certain expressed sequence tags (ESTs) identified by the
following GenBank accession numbers: AA162495, AA197442, AA273731,
AA794083, AA915125, AA968074, AI428529, AI451215, AI463056,
AI893951, AV142833, AV326033, AV353853, AW048117, AW048548,
AW215685, AW549817, BB179985, BB215203, BB283380, BB285606,
BB312895, BB497196, BB622397, BF016670, BF150116, BF161011,
BF300296, BF385702, BF539367, BF784415, BG863184, BG922959,
BG967012, BG974917, BI105291, BI153921, BI651868, BI653892, and
W76964.
In a fourth aspect the invention provides isolated nucleic acid
molecules which encode full-length murine TLR7. According to this
aspect of the invention, isolated nucleic acid molecules are
provided which are selected from the group consisting of (a)
nucleic acid molecules which hybridize under stringent conditions
to a nucleic acid molecule having a nucleotide sequence set forth
as SEQ ID NO:173, and which code for a murine TLR7 having an amino
acid sequence set forth as SEQ ID NO:175; (b) nucleic acid
molecules that differ from the nucleic acid molecules of (a) in
codon sequence due to degeneracy of the genetic code; and (c)
complements of (a) or (b). In a certain embodiment, the isolated
nucleic acid molecule codes for SEQ ID NO:175, where SEQ ID NO:175
represents the deduced amino acid sequence of full-length murine
TLR7. In some embodiments the isolated nucleic acid molecule
comprises the nucleotide sequence of SEQ ID NO:173 or SEQ ID
NO:174, where these correspond to full-length cDNA and the open
reading frame for murine TLR7, respectively.
The invention in a fifth aspect provides isolated TLR7 polypeptides
or fragments thereof. The isolated TLR7 polypeptides or fragments
thereof include at least one amino acid of a murine TLR7 selected
from the group consisting of amino acids 4, 8, 15, 16, 18, 21, 23,
24, 25, 27, 37, 39, 40, 41, 42, 44, 45, 61, 79, 83, 86, 89, 92, 96,
103, 109, 111, 113, 119, 121, 127, 128, 131, 145, 148, 151, 164,
172, 176, 190, 202, 203, 204, 205, 222, 225, 226, 228, 236, 238,
243, 250, 253, 266, 268, 271, 274, 282, 283, 287, 288, 308, 313,
314, 315, 325, 328, 331, 332, 341, 343, 344, 347, 351, 357, 360,
361, 362, 363, 364, 365, 366, 370, 371, 377, 378, 387, 388, 389,
392, 397, 398, 413, 415, 416, 419, 421, 422, 425, 437, 438, 440,
446, 449, 453, 454, 455, 456, 462, 470, 482, 486, 487, 488, 490,
491, 493, 494, 503, 505, 509, 511, 529, 531, 539, 540, 543, 559,
567, 568, 574, 583, 595, 597, 598, 600, 611, 613, 620, 624, 638,
645, 646, 651, 652, 655, 660, 664, 665, 668, 669, 672, 692, 694,
695, 698, 701, 704, 714, 720, 724, 727, 728, 733, 738, 745, 748,
755, 762, 777, 780, 789, 803, 846, 850, 851, 860, 864, 868, 873,
875, 884, 886, 888, 889, 890, 902, 903, 911, 960, 967, 970, 980,
996, 1010, 1018, 1035, and 1045 of SEQ ID NO:175, wherein the TLR7
polypeptide or fragment thereof has an amino acid sequence which is
identical to a human TLR7 polypeptide or fragment thereof except
for the at least one amino acid of murine TLR7.
In certain embodiments the TLR7 polypeptide and fragments thereof
according to this aspect of the invention exclude those TLR7
polypeptides and fragments thereof which differ from human TLR7 and
fragments thereof only by one or more conservative amino acid
substitutions at particular sites noted above.
In certain embodiments, the human TLR7 has an amino acid sequence
set forth as SEQ ID NO:170.
In preferred embodiments, the isolated TLR7 polypeptides or
fragments thereof include an amino acid sequence selected from the
group consisting of SEQ ID NO:175 and fragments of SEQ ID NO:175.
In some embodiments according to this aspect of the invention, the
isolated TLR7 polypeptides or fragments thereof include
combinations of the foregoing human and murine TLR7
polypeptides.
In certain preferred embodiments the isolated TLR7 polypeptide or
fragment thereof is an extracytoplasmic domain of TLR7, or a
portion thereof. In certain embodiments according to this aspect of
the invention, the isolated TLR7 polypeptide or fragment thereof
selectively binds to an ISNA, including an ISNA that is a CpG
nucleic acid. Also included according to this aspect of the
invention are isolated TLR7 polypeptides or fragments thereof which
are portions of the extracytoplasmic domain believed by the
inventors to interact with immunostimulatory nucleic acids such as
CpG nucleic acids. In certain embodiments such portions include an
MBD motif set forth as any one of SEQ ID NOs: 203, 204, 212, and
213. In certain embodiments portions of the extracytoplasmic domain
believed by the inventors to interact with immunostimulatory
nucleic acids include a CXXC motif set forth as any one of SEQ ID
NOs: 196, 199, and 200.
According to a sixth aspect of the invention, isolated nucleic acid
molecules are provided which encode the foregoing isolated TLR7
polypeptides or fragments thereof. The isolated nucleic acid
molecules according to this aspect of the invention specifically
exclude certain ESTs identified by the following GenBank accession
numbers: AA176010, AA210352, AA241310, AA266000, AA266744,
AA276879, AA288480, AA871870, AI119722, AI449297, AI466859,
AI604175, AV322307, BB033376, BB116163, BB210788, BB464985,
BB466708, BB636153, BF101884, BF124798, BF143871, BG067922,
BG080980, BG082140, BG871070, BG964747, BG976560, BI150306,
BI411471, and C87987.
In a seventh aspect the invention provides isolated nucleic acid
molecules which encode full-length murine TLR8. According to this
aspect of the invention, isolated nucleic acid molecules are
provided which are selected from the group consisting of (a)
nucleic acid molecules which hybridize under stringent conditions
to a nucleic acid molecule having a nucleotide sequence set forth
as SEQ ID NO:190, and which code for a murine TLR8 having an amino
acid sequence set forth as SEQ ID NO:192; (b) nucleic acid
molecules that differ from the nucleic acid molecules of (a) in
codon sequence due to degeneracy of the genetic code; and (c)
complements of (a) or (b). In a certain embodiment, the isolated
nucleic acid molecule codes for SEQ ID NO:192, where SEQ ID NO:192
represents the deduced amino acid sequence of full-length murine
TLR8. In some embodiments the isolated nucleic acid molecule
comprises the nucleotide sequence of SEQ ID NO:190 or SEQ ID
NO:191, where these correspond to full-length cDNA and the open
reading frame for murine TLR8, respectively.
The invention in an eighth aspect provides isolated TLR8
polypeptides or fragments thereof. The isolated TLR8 polypeptides
or fragments thereof include at least one amino acid of a murine
TLR8 selected from the group consisting of amino acids 5, 6, 9, 10,
14, 15, 18, 21, 22, 23, 24, 25, 26, 27, 28, 30, 39, 40, 41, 43, 44,
50, 51, 53, 55, 61, 67, 68, 74, 80, 85, 93, 98, 99, 100, 104, 105,
106, 107, 110, 114, 117, 119, 121, 124, 125, 134, 135, 138, 145,
155, 156, 157, 160, 161, 162, 163, 164, 166, 169, 170, 174, 180,
182, 183, 186, 187, 191, 193, 194, 196, 197, 199, 200, 207, 209,
210, 227, 228, 230, 231, 233, 234, 241, 256, 263, 266, 267, 268,
269, 272, 274, 275, 276, 280, 285, 296, 298, 299, 300, 303, 305,
306, 307, 310, 312, 320, 330, 333, 335, 343, 344, 345, 346, 347,
349, 351, 356, 362, 365, 366, 375, 378, 379, 380, 381, 383, 384,
386, 387, 392, 402, 403, 408, 414, 416, 417, 422, 426, 427, 428,
429, 430, 431, 433, 437, 438, 439, 440, 441, 444, 445, 449, 456,
461, 463, 471, 483, 486, 489, 490, 494, 495, 496, 505, 507, 509,
512, 513, 519, 520, 523, 537, 538, 539, 541, 542, 543, 545, 554,
556, 560, 567, 569, 574, 575, 578, 586, 592, 593, 594, 595, 597,
599, 602, 613, 617, 618, 620, 621, 623, 628, 630, 633, 639, 641,
643, 644, 648, 655, 658, 661, 663, 664, 666, 668, 677, 680, 682,
687, 688, 690, 692, 695, 696, 697, 700, 702, 703, 706, 714, 715,
726, 727, 728, 730, 736, 738, 739, 741, 746, 748, 751, 752, 754,
757, 764, 766, 772, 776, 778, 781, 784, 785, 788, 791, 795, 796,
801, 802, 806, 809, 817, 820, 821, 825, 828, 829, 831, 839, 852,
853, 855, 858, 863, 864, 900, 903, 911, 918, 934, 977, 997, 1003,
1008, 1010, 1022, 1023, 1024, 1026, and 1030 of SEQ ID NO:192,
wherein the TLR8 polypeptide or fragment thereof has an amino acid
sequence which is identical to a human TLR8 polypeptide or fragment
thereof except for the at least one amino acid of murine TLR8.
In certain embodiments the TLR8 polypeptide and fragments thereof
according to this aspect of the invention exclude those TLR8
polypeptides and fragments thereof which differ from human TLR8 and
fragments thereof only by one or more conservative amino acid
substitutions at particular sites noted above.
In certain embodiments, the human TLR8 has an amino acid sequence
set forth as SEQ ID NO:184.
In preferred embodiments, the isolated TLR8 polypeptides or
fragments thereof include an amino acid sequence selected from the
group consisting of SEQ ID NO:192 and fragments of SEQ ID NO:192.
In some embodiments according to this aspect of the invention, the
isolated TLR8 polypeptides or fragments thereof include
combinations of the foregoing human and murine TLR8
polypeptides.
In certain preferred embodiments the isolated TLR8 polypeptide or
fragment thereof is an extracytoplasmic domain of TLR8, or a
portion thereof. In certain embodiments according to this aspect of
the invention, the isolated TLR8 polypeptide or fragment thereof
selectively binds to an ISNA, including an ISNA that is a CpG
nucleic acid. Also included according to this aspect of the
invention are isolated TLR8 polypeptides or fragments thereof which
are portions of the extracytoplasmic domain believed by the
inventors to interact with immunostimulatory nucleic acids such as
CpG nucleic acids. In certain embodiments such portions include an
MBD motif set forth as any one of SEQ ID NOs: 205, 206, 214, and
215. In certain embodiments portions of the extracytoplasmic domain
believed by the inventors to interact with immunostimulatory
nucleic acids include a CXXC motif set forth as any one of SEQ ID
NOs: 196, 201, and 202.
According to a ninth aspect of the invention, isolated nucleic acid
molecules are provided which encode the foregoing isolated TLR8
polypeptides or fragments thereof. The isolated nucleic acid
molecules according to this aspect of the invention specifically
exclude certain ESTs identified by the following GenBank accession
numbers: AA116795, AA268605, AA920337, AI529457, AI849892,
AV097766, AV117427, AV164719, AV169968, AW551677, BB143750,
BB214171, BB243478, BB244318, BB254686, BB256660, BB258368,
BB278984, BB291470, BB292008, BB364655, BB373674, BB428800,
BB439876, BB444812, BB445724, BB465766, BB470182, BB535086,
BB573907, BB573981, BB607650, BF135656, BF722808, BG299237,
BG918020, BG919592, and W39977.
In a further aspect, the invention provides TLR expression vectors
comprising the foregoing isolated nucleic acid molecules operably
linked to a promoter. Thus in certain embodiments pertaining to
TLR9, the expression vector includes an isolated nucleic acid
molecule according to the first aspect or the third aspect of the
invention, operably linked to a promoter. In other embodiments,
relating to TLR7, the expression vector includes an isolated
nucleic acid molecule according to the fourth aspect or the sixth
aspect of the invention, operably linked to a promoter. In yet
other embodiments, relating to TLR8, the expression vector includes
an isolated nucleic acid molecule according to the seventh aspect
or the ninth aspect of the invention, operably linked to a
promoter.
The expression vectors according to this aspect of the invention
are designed and constructed so that when they are introduced into
a cell, under proper conditions they direct expression of the gene
product encoded by the incorporated isolated nucleic acid molecule.
For example, the promoter can be constitutively active or it can be
inducible or repressible upon interaction with a suitable inducer
or repressor compound.
According to another aspect, host cells are provided that include a
TLR expression vector of the invention. While any suitable method
can be used, an expression vector typically is introduced into a
cell by transfection or transformation. The host cells transformed
or transfected with the TLR expression vectors are in some
embodiments co-transformed or co-transfected with another
expression vector useful for the expression of another polypeptide.
Alternatively, a host cell can be tranformed or transfected with an
expression vector capable of directing expression of a TLR
polypeptide or fragment thereof of the invention and (i) at least
one additional TLR polypeptide or fragment thereof, or (ii) at
least one non-TLR polypeptide or fragment thereof. In certain
preferred embodiments, the host cell includes separate expression
vectors for any combination of TLR7, TLR8, and TLR9. In some
embodiments, a co-transformed or co-transfected expression vector
may be useful for detection or regulation of TLR expression or
TLR-related signaling. Specifically, in certain preferred
embodiments the host cell includes an expression vector providing a
reporter construct capable of interacting with a TIR domain.
In another aspect, the invention provides agents which selectively
bind the isolated TLR polypeptides and fragments thereof of the
invention. In certain embodiments the agent does not bind a human
TLR polypeptide or fragment thereof, wherein the human TLR is
selected from human TLR7, TLR8, and TLR9. In certain embodiments
the agent is a polypeptide, preferably one selected from the group
consisting of monoclonal antibodies, polyclonal antibodies, Fab
antibody fragments, F(ab').sub.2 antibody fragments, Fv antibody
fragments, antibody fragments including a CDR3 region, and fusion
proteins and other polypeptides including any such antibodies or
antibody fragments.
Also provided are agents which selectively bind the foregoing
isolated nucleic acid molecules, preferably antisense nucleic acid
molecules which selectively bind to any of the foregoing isolated
nucleic acid molecules encoding a TLR polypeptide or fragment
thereof. In some embodiments the agent is an isolated nucleic acid
molecule which hybridizes under stringent conditions to an isolated
nucleic acid moleucle provided according to any of the first,
third, fourth, fifth, sixth, and eighth aspects of the invention.
In certain preferred embodiments the agent is an isolated nucleic
acid molecule having a nucleotide sequence which is complementary
to an isolated nucleic acid moleucle provided according to any of
the first, third, fourth, fifth, sixth, and eighth aspects of the
invention.
In still other aspects of the invention, methods for inhibiting TLR
expression and TLR signaling in a cell are provided. The methods
include contacting the cell with an amount of an agent effective to
inhibit TLR expression and TLR signaling in the cell, wherein the
TLR is selected from the group consisting of TLR7, TLR8, and TLR9.
In some embodiments the agent brought into contact with the cell is
selected from the group consisting of monoclonal antibodies,
polyclonal antibodies, Fab antibody fragments, F(ab').sub.2
antibody fragments, Fv antibody fragments, antibody fragments
including a CDR3 region, and fusion proteins and other polypeptides
that include any such antibodies or antibody fragments. In some
embodiments the cell is contacted with an antisense nucleic acid
specific for the TLR, in an amount effective to inhibit TLR
expression in the cell. In some embodiments the cell is contacted
with an agent such as a cytokine or small molecule, in an amount
effective to inhibit TLR expression in the cell.
In yet another aspect the invention provides a method for
identifying nucleic acid molecules which interact with a TLR
polypeptide or a fragment thereof. The method involves contacting a
TLR polypeptide selected from the group consisting of TLR7, TLR8,
TLR9, and nucleic acid-binding fragments thereof with a test
nucleic acid molecule; and measuring an interaction of the test
nucleic acid molecule with the TLR polypeptide or fragment thereof.
Nucleic acid-binding fragments of TLRs preferably include the
extracytoplasmic domain or subportions thereof, such as those which
include at least an MBD motif, a CXXC motif, or both an MBD motif
and a CXXC motif.
In this and other aspects of the invention involving methods of use
of TLR polypeptides and fragments thereof, in some embodiments the
TLR polypeptide or fragment thereof is TLR7. Likewise in this and
other aspects of the invention involving methods of use of TLR
polypeptides and fragments thereof, in some embodiments the TLR
polypeptide or fragment thereof is TLR8. Also in this and other
aspects of the invention involving methods of use of TLR
polypeptides and fragments thereof, in some embodiments the TLR
polypeptide or fragment thereof is TLR9.
In this and other aspects of the invention involving methods of use
of TLR polypeptides and fragments thereof, in some embodiments the
TLR polypeptide or fragment thereof is expressed in a cell. The
cell expressing the TLR polypeptide or fragment thereof may
naturally express the TLR polypeptide or fragment thereof, or it
may be a host cell as provided by other aspects of the instant
invention.
In this and other aspects of the invention involving methods of use
of TLR polypeptides and fragments thereof, in some embodiments the
TLR polypeptide or fragment thereof is an isolated TLR polypeptide
or fragment thereof. In certain preferred embodiments the isolated
TLR polypeptide or fragment thereof is immobilized on a solid
support, for example a multiwell plate, a slide, a BIAcore chip, a
bead, a column, and the like. The immobilization can be
accomplished by any chemical or physical method suitable for the
purpose of the assay to be performed according to the method of the
invention.
In certain embodiments the TLR polypeptide or fragment thereof is
fused with an Fc fragment of an antibody. The Fc fragment portion
of such a fusion molecule may be useful, for example, for attaching
the TLR polypeptide or fragment thereof to a substrate, or for
providing a target for detecting the presence of the TLR
polypeptide or fragment thereof. The Fc fragment can be selected
from any suitable vertebrate species and will typically, but not
necessarily, be derived from an antibody belonging to the IgG class
of antibodies. For example, the Fc can be a human or a murine
Fc.gamma.. In certain embodiments the TLR polypeptide or fragment
thereof is fused with an Fc fragment of an antibody with a specific
cleavage site at or near the junction between the TLR polypeptide
or fragment thereof and the Fc fragment. In one preferred
embodiment the cleavage site is a thrombin protease recognition
site. In a preferred embodiment the TLR polypeptide or fragment
thereof fused with the Fc fragment includes a TLR extracytoplasmic
domain.
In certain embodiments the interaction involving the TLR polyeptide
or fragment thereof and the test nucleic acid molecule is binding
between the TLR polypeptide or fragment thereof and the test
nucleic acid molecule.
In certain embodiments according to this aspect of the invention,
the measuring is accomplished by a method selected from the group
consisting of enzyme-linked immunosorbent assay (ELISA),
biomolecular interaction assay (BIA), electromobility shift assay
(EMSA), radioimmunoassay (RIA), polyacrylamide gel electrophoresis
(PAGE), and Western blotting.
In certain embodiments the measuring is accomplished by a method
comprising measuring a response mediated by a TLR signal
transduction pathway. For example, the response mediated by a TLR
signal transduction pathway can be selected from the group
consisting of induction of a gene under control of NF-.kappa.B
promoter and secretion of a cytokine. In certain preferred
embodiments the gene under control of NF-.kappa.B promoter is
selected from the group consisting of IL-8, IL-12 p40,
NF-.kappa.B-luc, IL-12 p40-luc, and TNF-luc. In certain preferred
embodiments the secreted cytokine is selected from the group
consisting of IL-8, TNF-.alpha., and IL-12 p40.
In another embodiment the method according to this aspect of the
invention can be used to determine if the test nucleic acid
molecule is an immunostimulatory nucleic acid. The method involves
the additional steps of comparing (a) the response mediated by a
TLR signal transduction pathway as measured in the presence of the
test nucleic acid molecule with (b) a response mediated by a TLR
signal transduction pathway as measured in the absence of the test
nucleic acid molecule; and determining the test nucleic acid
molecule is an immunostimulatory nucleic acid when (a) exceeds
(b).
In yet another embodiment the method according to this aspect of
the invention can be used to determine if the response to the test
nucleic acid molecule is stronger or weaker than a response to a
reference nucleic acid molecule. The method involves the additional
steps of comparing the response to a reference response when the
TLR polypeptide is independently contacted with a reference nucleic
acid molecule; and determining if the response is stronger or
weaker than the reference response. In this embodiment the test
nucleic acid molecule and the reference nucleic acid molecule are
not able to compete or interact. For example, the reference
response can be a parallel control or a historical control.
In another embodiment the method involves the additional steps of
comparing the response to a reference response when the TLR
polypeptide is concurrently contacted with a reference nucleic acid
molecule; and determining if the response is stronger or weaker
than the reference response. In this embodiment the test nucleic
acid molecule and the reference nucleic acid molecule are
potentially able to compete or interact since they are both
present, for example, in a single reaction.
In another aspect the invention provides a screening method for
identifying an immunostimulatory nucleic acid. The method according
to this aspect involves contacting a functional TLR selected from
the group consisting of TLR7, TLR8, and TLR9 with a test nucleic
acid molecule; detecting presence or absence of a response mediated
by a TLR signal transduction pathway in the presence of the test
nucleic acid molecule arising as a result of an interaction between
the functional TLR and the test nucleic acid molecule; and
determining the test nucleic acid molecule is an ISNA when the
presence of a response mediated by the TLR signal transduction
pathway is detected. A functional TLR refers to a TLR polypeptide
or fragment thereof that can bind with a ligand and as a
consequence of the binding engage at least one step or additional
polypeptide in a TLR signal transduction pathway.
In one embodiment the method according to this aspect of the
invention includes the further step of comparing (a) the response
mediated by the TLR signal transduction pathway arising as a result
of an interaction between the functional TLR and the test nucleic
acid molecule with (b) a response arising as a result of an
interaction between the functional TLR and a reference ISNA. In
this and other screening assays of the instant invention, in
preferred embodiments the screening method is performed on a
plurality of test nucleic acids. In certain preferred embodiments
the response mediated by the TLR signal transduction pathway is
measured quantitatively, and the response mediated by the TLR
signal transduction pathway associated with each of the plurality
of test nucleic acid molecules is compared with a response arising
as a result of an interaction between the functional TLR and a
reference ISNA.
In certain preferred embodiments a subset of the plurality of test
nucleic acid molecules is selected based on the ability of the
subset to produce a specific response mediated by the TLR signal
transduction pathway. For example, the specific response can be
induction of a specific cytokine or panel of cytokines, e.g., Th1
cytokines, or, alternatively, inhibition of a specific cytokine or
panel of cytokines, e.g., Th2 cytokines. The specific response can
be induction, or, alternatively, inhibition of a specific class or
subclass of antibody or panel of classes or subclasses of
antibodies, e.g., Th1-associated antibodies or Th2-associated
antibodies. The specific response in some embodiments can be
activation or inhibition of certain types of immune cells, e.g., B
cells, dendritic cells (DCs), and natural killer (NK) cells. In
some embodiments the specific response can be induction or
inhibition of proliferation of certain types of immune cells, e.g.,
B cells, T cells, NK cells, dendritic cells, monocytes/macrophages.
The subset of the plurality of test nucleic acids is therefore
selected on the basis of the common association between the test
nucleic acids of the subset and the particular type of response
mediated by the TLR signal transduction pathway. The particular
type of response mediated by the TLR signal transduction pathway is
typically, but not necessarily, an immune cell response.
In certain embodiments the response mediated by a TLR signal
transduction pathway is selected from the group consisting of
induction of a gene under control of NF-.kappa.B promoter and
secretion of a cytokine. In certain preferred embodiments the gene
under control of NF-.kappa.B promoter is selected from the group
consisting of IL-8, IL-12 p40, NF-.kappa.B-luc, IL-12 p40-luc, and
TNF-luc. In certain preferred embodiments the cytokine is selected
from the group consisting of IL-8, TNF-.alpha., and IL-12 p40.
In certain preferred embodiments the reference ISNA is a CpG
nucleic acid.
In certain preferred embodiments the test nucleic acid molecule is
a CpG nucleic acid.
According to this and other aspects of the invention involving
functional TLR in a screening assay, in some embodiments the
functional TLR is expressed in a cell. In some embodiments the
functional TLR is naturally expressed by the cell. In certain
preferred embodiments the cell is an isolated mammalian cell that
naturally expresses the functional TLR. Whether the cell expresses
the TLR naturally or the cell expresses the TLR because an
expression vector having an isolated nucleic acid molecule encoding
the TLR operatively linked to a promoter has been introduced into
the cell, in some embodiments the cell further includes an
expression vector comprising an isolated nucleic acid which encodes
a reporter construct selected from the group consisting of IL-8,
IL-12 p40, NF-.kappa.B-luc, IL-12 p40-luc, and TNF-luc, operatively
linked to a promoter.
Also according to this and other aspects of the invention involving
functional TLR in a screening assay, in certain embodiments the
functional TLR is part of a cell-free system.
Also according to this and other aspects of the invention involving
functional TLR in a screening assay, in certain embodiments the
functional TLR is part of a complex with another TLR. In certain
preferred embodiments the complex is a complex of TLR9 and TL R7.
In certain preferred embodiments the complex is a complex of TLR9
and TLR8.
Also according to this and other aspects of the invention involving
functional TLR in a screening assay, in certain embodiments the
functional TLR is part of a complex with a non-TLR protein selected
from the group consisting of MyD88, IRAK, TRAF6, I.kappa.B,
NF-.kappa.B, and functional homologues and derivatives thereof.
Further according to this and and other aspects of the invention
involving functional TLR in a screening assay, in certain
embodiments the response mediated by a TLR signal transduction
pathway is selected from the group consisting of induction of a
gene under control of NF-.kappa.B promoter and secretion of a
cytokine.
Also according to this and and other aspects of the invention
involving functional TLR in a screening assay, in certain
embodiments the gene under control of NF-.kappa.B promoter is
selected from the group consisting of IL-8, IL-12 p40,
NF-.kappa.B-luc, IL-12 p40-luc, and TNF-luc.
Also according to this and and other aspects of the invention
involving functional TLR in a screening assay, in certain
embodiments wherein the cytokine is selected from the group
consisting of IL-8, TNF-.alpha., and IL-12 p40.
In a further aspect, the invention provides a screening method for
comparing TLR signaling activity of a test compound with an ISNA.
The method entails contacting a functional TLR selected from the
group consisting of TLR7, TLR8, and TLR9 with a reference ISNA and
detecting a reference response mediated by a TLR signal
transduction pathway; contacting a functional TLR selected from the
group consisting of TLR7, TLR8, and TLR9 with a test compound and
detecting a test response mediated by a TLR signal transduction
pathway; and comparing the test response with the reference
response to compare the TLR signaling activity of the test compound
with the ISNA.
In certain embodiments according to this aspect of the invention,
the reference ISNA is a CpG nucleic acid.
In certain embodiments according to this aspect of the invention,
the test compound is a polypeptide. In certain embodiments the test
compound is part of a combinatorial library of compounds.
In certain embodiments the functional TLR is contacted with the
reference ISNA and the test compound independently. Accordingly, in
certain embodiments the screening method is a method for
identifying an ISNA mimic, and the test compound is determined to
be an ISNA mimic when the test response is similar to the reference
response obtained with the reference ISNA. A test response is
similar to the reference response when the test and reference
responses are qualitatively alike, even if not quantitatively
alike. Thus, for example, the test and reference responses are
considered alike when both responses include induction of a
Th1-like immune response. The test response can be quantitatively
less than, about the same as, or greater than the reference
response.
In certain other embodiments the functional TLR is contacted with
the reference ISNA and the test compound concurrently to produce a
test-reference response mediated by a TLR signal transduction
pathway, wherein the test-reference response may be compared to the
reference response. In certain preferred embodiments the screening
method is a method for identifying an ISNA agonist, wherein the
test compound is an ISNA agonist when the test-reference response
is greater than the reference response. In certain preferred
embodiments the screening method is a method for identifying an
ISNA antagonist, wherein the test compound is an ISNA antagonist
when the test-reference response is less than the reference
response.
In a further aspect the invention provides a screening method for
identifying species specificity of an ISNA. The method according to
this aspect of the invention involves contacting a functional TLR
selected from the group consisting of TLR7, TLR8, and TLR9 of a
first species with a test ISNA; contacting a functional TLR
selected from the group consisting of TLR7, TLR8, and TLR9 of a
second species with the test ISNA; measuring a response mediated by
a TLR signal transduction pathway associated with the contacting
the functional TLR of the first species with the test ISNA;
measuring a response mediated by the TLR signal transduction
pathway associated with the contacting the functional TLR of the
second species with the test ISNA; and comparing (a) the response
mediated by a TLR signal transduction pathway associated with the
contacting the functional TLR of the first species with the test
ISNA with (b) the response mediated by the TLR signal transduction
pathway associated with the contacting the functional TLR of the
second species with the test ISNA. In preferred embodiments the TLR
of the first species corresponds to the TLR of the second species,
e.g., the TLR of the first species is human TLR9 and the TLR of the
second species is murine TLR9. In certain embodiments the
functional TLR may be expressed in a cell, part of cell-free
system, or part of a complex with another TLR or with a non-TLR
protein, as previously described.
In yet another aspect the invention provides a method for
identifying lead compounds for a pharmacological agent useful in
the treatment of disease associated with TLR9 signaling activity.
The method according to this aspect of the invention involves
providing a cell comprising a TLR9 polypeptide or fragment thereof
as provided in the second aspect of the invention; contacting the
cell with a candidate pharmacological agent under conditions which,
in the absence of the candidate pharmacological agent, cause a
first amount of TLR9 signaling activity; and determining a second
amount of TLR9 signaling activity as a measure of the effect of the
pharmacological agent on the TLR9 signaling activity, wherein a
second amount of TLR9 signaling activity which is less than the
first amount indicates that the candidate pharmacological agent is
a lead compound for a pharmacological agent which reduces TLR9
signaling activity and wherein a second amount of TLR9 signaling
activity which is greater than the first amount indicates that the
candidate pharmacological agent is a lead compound for a
pharmacological agent which increases TLR9 signaling activity.
These and other aspects of the invention are described in greater
detail below.
BRIEF DESCRIPTION OF THE FIGURES
FIG. 1 is two paired bar graphs showing (A) the induction of
NF-.kappa.B and (B) the amount of IL-8 produced by 293 fibroblast
cells transfected with human TLR9 in response to exposure to
various stimuli, including CpG-ODN, GpC-ODN, LPS, and medium.
FIG. 2 is a bar graph showing the induction of NF-.kappa.B produced
by 293 fibroblast cells transfected with murine TLR9 in response to
exposure to various stimuli, including CpG-ODN, methylated CpG-ODN
(Me-CpG-ODN), GpC-ODN, LPS, and medium.
FIG. 3 is a series of gel images depicting the results of reverse
transcriptase-polymerase chain reaction (RT-PCR) assays for murine
TLR9 (mTLR9), human TLR9 (hTLR9), and glyceraldehyde-3-phosphate
dehydrogenase (GAPDH) in untransfected control 293 cells, 293 cells
transfected with mTLR9 (293-mTLR9), and 293 cells transfected with
hTLR9 (293-hTLR9).
FIG. 4 is a graph showing the degree of induction of
NF-.kappa.B-luc by various stimuli in stably transfected 293-hTLR9
cells.
FIG. 5 is a graph showing the degree of induction of
NF-.kappa.B-luc by various stimuli in stably transfected 293-mTLR9
cells.
FIG. 6 is an image of a Coomassie-stained polyacrylamide gel
depicting the presence of soluble hTLR9 in the supernatants of
yeast cells transfected with hTLR9, either induced (lane 1) or not
induced (lane 2).
FIG. 7 is a graph showing proliferation of human B cells in
response to various stimuli, including Escherichia coli (E. coli)
DNA, DNase-digested E. coli DNA, CpG-ODN, GpC-ODN, and LPS.
FIG. 8 is two paired bar graphs showing induction of (top) IL-8 and
(bottom) TNF in plasmacytoid dendritic cells (CD123+DC) and
monocyte-derived dendritic cells (MDDC) in response to various
stimuli, including E. coli DNA, DNase-digested E. coli DNA,
CpG-ODN, GpC-ODN, and LPS.
FIG. 9 is a series of images of stained gels showing results of
semi-quantitative RT-PCR comparing relative levels of human TLR9,
TLR2, and TLR4 mRNA expression in human peripheral blood cells:
MDDC (lane 1), purified CD 14+monocytes (lane 2), B cells (lane 3),
CD123+DC (lane 4), CD4+T cells (lane 5), and CD8+T cells (lane 6).
GAPDH a control for equalizing amounts of cDNA.
FIG. 10 is a pair of graphs showing amounts of IL-12 induced in (A)
human peripheral blood mononuclear cells (PBMC) and (B) murine
splenocytes in response to shown concentrations of various ODN;
including ODN 2006 (filled circles; SEQ ID NO: 112), 2006-GC (open
circles; SEQ ID NO:118), 1668 (filled triangles; SEQ ID NO:84), and
1668-GC (open triangles; SEQ ID NO:85).
FIG. 11 is a quartet or graphs depicting responsiveness of 293
cells transfected with hTLR9 (left panels) or mTLR9 (right panels)
upon stimulation with shown concentrations of various ODN,
including ODN 2006 (filled circles; SEQ ID NO: 112), 2006-GC (open
circles; SEQ ID NO:118), 1668 (filled triangles; SEQ ID NO:84), and
1668-GC (open triangles; SEQ ID NO:85). Responses are shown in
terms of induction of NF-.kappa.B-luc (upper panels) and IL-8
(lower panels).
FIG. 12 is a bar graph depicting the dose-response of 293-hTLR9
cells to E. coli DNA (black bars) and to DNase-digested E. coli DNA
(gray bars).
FIG. 13 is a pair of graphs showing the responsiveness of (A)
293-hTLR9 and (B) 293-mTLR9 cells to shown concentrations of
phosphodiester versions of ODN 2006 (filled circles; SEQ ID NO:
112), 2006-GC (open circles; SEQ iD NO:118), 1668 (filled
triangles; SEQ ID NO:84), and 1668-GC (open triangles; SEQ ID
NO:85).
FIG. 14 is a pair of graphs snowing the responsiveness of 293-hTLR9
and 293-mTLR9 cells to shown concentrations of ODN 5002 (filled
circles SEQ ID NO:132) and ODN 5007 (open circles; SEQ ID
NO:98).
FIG. 15 is a bar graph showing the response of 293 cells
transfected with mTLR9 to CpG-ODN 1668 (SEQ ID NO:84) is inhibited
in a dose-dependent manner by co-exposure to non-CpG-ODN PZ2 (SEQ
ID NO:43).
FIG. 16 is a bar graph showing the response of 293-hTLR9 cells to
CpG-ODN (black bars) or to TNF (gray bars) in the presence of shown
amounts of blocking non-CpG-ODN.
FIG. 17 is a bar graph showing blockade of response of 293-hTLR9
cells to CpG-ODN, but not to IL-1 or TNF, in the presence of
Bafilomycin A (gray bars). Control treatment with dimethyl
sulfoxide (DMSO) is shown in black bars.
FIG. 18 is a graph showing the effect of varying concentrations of
dominant negative human MyD88 on the induction of NF-.kappa.B in
293-hTLR9 cells stimulated with CpG-ODN (open circles), TNF-.alpha.
(filled circles), or control (filled triangles).
FIG. 19 is a series of three Western blot images showing the
response of various polyclonal antibodies to purified hTLR9-FLAG
and mTLR9-FLAG: upper panel, anti-human and anti-mouse
intracellular; middle, anti-mouse extracellular; and lower,
anti-human extracellular. Arrows indicate position of TLR9 in each
blot.
FIG. 20 is a bar graph depicting the responsiveness of native form
hTLR9 and hTLR9 variant form hTLR9-CXXCm to various stimuli at
different concentrations. ODN are as follows: ODN 1668 (SEQ ID
NO:84) and ODN 2006 (SEQ ID NO:112).
FIG. 21 is a bar graph depicting the responsiveness of native form
mTLR9 and mTLR9 variant form mTLR9-CXXCm to various stimuli at
different concentrations. ODN are as follows: ODN 1668 (SEQ ID
NO:84) and ODN 2006 (SEQ ID NO:112).
FIG. 22 is a bar graph showing the responsiveness of native form
mTLR9, mTLR9 variant form mTLR9-Phmut, and mTLR9 variant form
mTLR9-MBDmut to various stimuli at different concentrations. ODN
are as follows: ODN 1668 (SEQ ID NO:84) and ODN 2006 (SEQ ID NO:
112).
FIG. 23 is a bar graph showing the responsiveness or native form
hTLR9, hTLR9 variant form hTLR9-PHmut, and hTLR9 variant form
hTLR9-MBDmut to various stimuli at different concentrations. ODN
are as follows: ODN 1668 (SEQ ID NO:84) and ODN 2006 (SEQ ID
NO:112).
FIG. 24 is a bar graph showing the responsiveness of native form
mTLR9 and mTLR9 variant form mTLR9-TIRh to various stimuli at
different concentrations. ODN are as follows: ODN 1668 (SEQ ID
NO:84) and ODN 2006 (SEQ ID NO:112).
FIG. 25 is a bar graph showing the responsiveness of native form
hTLR9 and hTLR9 variant form hTLR9-TIRm to various stimuli at
different concentrations. ODN are as follows: ODN 1668 (SEQ ID
NO:84) and ODN 2006 (SEQ ID NO:112).
FIG. 26 is a series of linear maps representing various features of
human TLR7, TLR8, and TLR9 polypeptides.
FIG. 27 is an image of a silver stained polyacrylamide gel and
schematic representation of a fusion protein in which the
extracellular domain of human TLR9 (hTLR9) is fused to a human IgG1
Fc domain (hIgG-Fc) with a thrombin protease recognition site
interposed. From left to right, the gel was loaded with (1)
supernatant of transfectants; (2) lysates of transfectants, treated
with thrombin; (3) untreated lysates of transfectants; (4)
molecular weight markers; (5) supernatant of mock transfectants;
(6) lysates of mock transfectants, treated with thrombin; and (7)
untreated lysates of mock transfectants. Soluble hTLR9 and Fc are
the products released from intact hTLR9-IgG-Fc following thrombin
treatment. Molecular weights are indicated along the right side of
the silver stain gel image.
BRIEF DESCRIPTION OF SELECTED SEQUENCES
SEQ ID NO:1 is the nucleotide sequence encoding a cDNA for murine
TLR9.
SEQ ID NO:2 is the nucleotide sequence encoding the coding region
of murine TLR9.
SEQ ID NO:3 is the amino acid sequence of a murine TLR9 encoded by
SEQ ID NO:1.
SEQ ID NO:173 is the nucleotide sequence encoding a cDNA for murine
TLR7.
SEQ ID NO:174 is the nucleotide sequence encoding the coding region
of murine TLR7.
SEQ ID NO:175 is the amino acid sequence of a murine TLR7 encoded
by SEQ ID NO:173.
SEQ ID NO:190 is the nucleotide sequence encoding a cDNA for murine
TLR8.
SEQ ID NO:191 is the nucleotide sequence encoding the coding region
of murine TLR8.
SEQ ID NO:192 is the amino acid sequence of a murine TLR8 encoded
by SEQ ID NO:190.
DETAILED DESCRIPTION OF THE INVENTION
The present invention in one aspect involves the identification of
cDNAs encoding mouse TLR9, referred to herein as murine TLR9 and,
equivalently, mTLR9. The nucleotide sequence of the cDNA for murine
TLR9 is presented as SEQ ID NO:1, the coding region of the cDNA for
murine TLR9 is presented as SEQ ID NO:2, and the amino acid
sequence of the murine TLR9 is presented as SEQ ID NO:3. The
closely related human TLR9 (equivalently, hTLR9) was deposited in
GenBank under accession numbers AF245704 and NM.sub.-- 017742.
The nucleotide sequence of the cDNA for murine TLR9 presented as
SEQ ID NO:1 is 3200 nucleotides long and includes the open reading
frame (ORF, bases 40-3135) presented as SEQ ID NO:2 which spans
3096 nucleotides (excluding the stop codon). The amino acid
sequence of the murine TLR9 presented as SEQ ID NO:3 is 1032 amino
acids (aa) long, and it is believed to include an extracellular
domain (aa 1-819), a transmembrane domain (aa 820-837), and an
intracellular domain (aa 838-1032).
The amino acid sequence of human TLR9 (SEQ ID NO:6) and the amino
acid sequence of the murine TLR9 (SEQ ID NO:3) are thus both 1032
amino acids long. Comparison of the aligned amino acid sequences
for the murine and the human TLR9 molecules reveals a single base
insertion at aa 435 of the murine TLR9 and a single base deletion
at aa 860 of the human TLR9. (See Table 4 below.)
Whereas much of the polypeptide presented herein is identical to
human TLR9, murine TLR9 has several single amino acid differences.
These differences in amino acids are specifically amino acids 2, 3,
4, 6, 7, 18, 19, 22, 38, 44, 55, 58, 61, 62, 63, 65, 67, 71, 80,
84, 87, 88, 91, 101, 106, 109, 117, 122, 123, 134, 136, 140, 143,
146, 147, 157, 160, 161, 167, 168, 171, 185, 186, 188, 189, 191,
199, 213, 217, 220, 227, 231, 236, 245, 266, 269, 270, 271, 272,
273, 274, 278, 281, 285, 297, 298, 301, 305, 308, 311, 322, 323,
325, 326, 328, 332, 335, 346, 348, 353, 355, 358, 361, 362, 365,
367, 370, 372, 380, 381, 382, 386, 389, 392, 394, 397, 409, 412,
413, 415, 416, 419, 430, 432, 434, 435, 438, 439, 443, 444, 446,
447, 448, 450, 451, 452, 454, 455, 459, 460, 463, 465, 466, 468,
469, 470, 472, 473, 474, 475, 478, 488, 489, 494, 495, 498, 503,
508, 510, 523, 531, 539, 540, 543, 547, 549, 561, 563, 565, 576,
577, 579, 580, 587, 590, 591, 594, 595, 597, 599, 601, 603, 610,
611, 613, 616, 619, 632, 633, 640, 643, 645, 648, 650, 657, 658,
660, 667, 670, 672, 675, 679, 689, 697, 700, 703, 705, 706, 711,
715, 716, 718, 720, 723, 724, 726, 729, 731, 735, 737, 743, 749,
750, 751, 752, 754, 755, 759, 760, 772, 774, 780, 781, 786, 787,
788, 800, 814, 821, 829, 831, 832, 835, 844, 857, 858, 859, 862,
864, 865, 866, 879, 893, 894, 898, 902, 910, 917, 927, 949, 972,
975, 976, 994, 997, 1000, 1003, 1004, 1010, 1011, 1018, 1023, and
1027 of SEQ ID NO:3
In some forms the mouse protein mTLR9 contains a signal sequence at
the N-terminus (amino acids 1-26) which allows transport to the
endoplasmic reticulum and subsequently to the cell surface or
intracellular compartments. A transmembrane region (amino acids
820-837) anchors the protein to the cell membrane. The cytoplasmic
tail contains a Toll/IL-1 receptor (TIR) homology domain which is
believed to function in signaling upon ligand binding.
Leucine-rich-repeats (LRR) can be found in the extracellular region
(a common feature of TLRs) and may be involved in ligand binding or
dimerization of the molecule.
Both mouse and human TLR9 have an N-terminal extension of
approximately 180 amino acids compared to other TLRs. An insertion
also occurs at amino acids 253-268, which is not found in TLRs 1-6
but is present in human TLR7 and human TLR8. (See FIG. 26.) This
insert has two CXXC motifs which participate in forming a CXXC
domain. The CXXC domain resembles a zinc finger motif and is found
in DNA-binding proteins and in certain specific CpG binding
proteins, e.g., methyl-CpG binding protein-1 (MBD-1). Fujita N et
al., Mol Cell Biol 20:5107-5118 (2000). Both human and mouse TLR9
CXXC domains occur at aa 253-268:
CXXC motif: GNCXXCXXXXXXCXXC SEQ ID NO:196 Human TLR9:
GNCRRCDHAPNPCMEC SEQ ID NO:197 Murine TLR9: GNCRRCDHAPNPCMIC SEQ ID
NO:198
An additional motif involved in CpG binding is the MBD motif, also
found in MBD-1, listed below as SEQ ID NO:125. Fujita, N et al.,
Mol Cell Biol 20:5107-18 (2000); Ohki I et al., EMBO J.
18:6653-6661 (1999). Amino acids 524-554 of hTLR9 and aa 525-555 of
mTLR9 correspond to the MBD motif of MBD-1 as shown:
MBD motif: SEQ ID NO:125 MBD-1
R-XXXXXXX-R-X-D-X-Y-XXXXXXXXX-R-S-XXXXXX-Y SEQ ID NO:126 hTLR9
Q-XXXXXXX-K-X-D-X-Y-XXXXXXXXX-R-L-XXXXXX-Y SEQ ID NO:127 mTLR9
Q-XXXXXXX-K-X-D-X-Y-XXXXXXXXX-Q-L-XXXXXX-Y SEQ ID NO:210 hTLR9
Q-VLDLSRN-K-L-D-L-Y-HEHSFTELP-R-L-EALDLS-Y SEQ ID NO:211 mTLR9
Q-VLDLSHN-K-L-D-L-Y-HWKSFSELP-Q-L-QALDLS-Y
Although the signaling functions of MBD-1 and TLR9 are quite
different, the core D-X-Y is involved in CpG binding and is
conserved. The C-terminal octamer S-XXXXXX-Y of the MBD motif may
not be involved in binding and the S is not conserved by TLR9. The
other mismatches are highly conserved or moderately conserved;
example R to K or R to Q. These changes could explain MBD-1 as a
methyl-CpG binder and TLR9 as a non-methyl-CpG binder. The
differences between mouse and human TLR9 may explain inter-species
differences in CpG-motif sequence selectivity. See FIG. 14 for
inter-species sequence selectivity.
As discussed in Example 11 below and shown in FIGS. 22 and 23, the
D-X-Y core of this MBD motif occurs as D-L-Y in both mTLR9 (aa
535-537) and hTLR9 (aa 534-536). Substitution of A for D and A for
Y in the D-X-Y core, resulting in A-L-A in place of D-L-Y, destroys
receptor activity for mTLR9 and hTLR9 alike.
The invention involves in one aspect murine TLR9 nucleic acids and
polypeptides, as well as therapeutics relating thereto. The
invention also embraces isolated functionally equivalent variants,
useful analogs and fragments of the foregoing nucleic acids and
polypeptides; complements of the foregoing nucleic acids; and
molecules which selectively bind the foregoing nucleic acids and
polypeptides.
The murine TLR9 nucleic acids and polypeptides of the invention are
isolated. As used herein with respect to nucleic acids, the term
"isolated" means: (i) amplified in vitro by, for example,
polymerase chain reaction (PCR); (ii) recombinantly produced by
cloning; (iii) purified, as by cleavage and gel separation; or (iv)
synthesized by, for example, chemical synthesis. An isolated
nucleic acid is one which is readily manipulable by recombinant DNA
techniques well known in the art. Thus, a nucleotide sequence
contained in a vector in which 5' and 3' restriction sites are
known or for which PCR primer sequences have been disclosed is
considered isolated, but a nucleic acid sequence existing in its
native state in its natural host is not. An isolated nucleic acid
may be substantially purified, but need not be. For example, a
nucleic acid that is isolated within a cloning or expression vector
is not pure in that it may comprise only a tiny percentage of the
material in the cell in which it resides. Such a nucleic acid is
isolated, however, as the term is used herein because it is readily
manipulable by standard techniques known to those of ordinary skill
in the art. An isolated nucleic acid as used herein is not a
naturally occurring chromosome.
As used herein with respect to polypeptides, "isolated" means
separated from its native environment and present in sufficient
quantity to permit its identification or use. Isolated, when
referring to a protein or polypeptide, means, for example: (i)
selectively produced by expression cloning or (ii) purified as by
chromatography or electrophoresis. Isolated proteins or
polypeptides may be, but need not be, substantially pure. The term
"substantially pure" means that the proteins or polypeptides are
essentially free of other substances with which they may be found
in nature or in vivo systems to an extent practical and appropriate
for their intended use. Substantially pure polypeptides may be
produced by techniques well known in the art. Because an isolated
protein may be admixed with a pharmaceutically acceptable carrier
in a pharmaceutical preparation, the protein may comprise only a
small percentage by weight of the preparation. The protein is
nonetheless isolated in that it has been separated from the
substances with which it may be associated in living systems, i.e.,
isolated from other proteins.
As used herein a murine TLR9 nucleic acid or murine TLR9
polypeptide also embraces homologues and alleles of murine TLR9. In
general homologues and alleles typically will share at least 40%
nucleotide identity and/or at least 50% amino acid identity to the
sequences of specified nucleic acids and polypeptides,
respectively. Thus homologues and alleles of murine TLR9 typically
will share at least 40% nucleotide identity and/or at least 50%
amino acid identity to the sequences of murine TLR9 nucleic acids
and TLR9 polypeptides, respectively. In some instances homologues
and alleles will share at least 50% nucleotide identity and/or at
least 65% amino acid identity and in still other instances will
share at least 60% nucleotide identity and/or at least 75% amino
acid identity. Preferably the homologues and alleles will share at
least 80% nucleotide identity and/or at least 90% amino acid
identity, and more preferably will share at least 90% nucleotide
identity and/or at least 95% amino acid identity. Most preferably
the homologues and alleles will share at least 95% nucleotide
identity and/or at least 99% amino acid identity. The homology can
be calculated using various publicly available software tools
developed by the National Center for Biotechnology Information
(NCBI, Bethesda, Md.) that can be obtained through the internet.
Exemplary tools include the BLAST system available from the NCBI,
used with default settings. Pairwise and ClustalW alignments
(BLOSUM30matrix setting) as well as Kyte-Doolittle hydropathic
analysis can be obtained, for example, using the MacVector sequence
analysis software (Oxford Molecular Group). Watson-Crick
complements of the foregoing nucleic acids also are embraced by the
invention.
As used herein a murine TLR9 nucleic acid or murine TLR9
polypeptide also embraces homologues and alleles of murine TLR9. In
general homologues and alleles typically will share at least 40%
nucleotide identity and/or at least 50% amino acid identity to the
sequences of specified nucleic acids and polypeptides,
respectively. Thus homologues and alleles of murine TLR9 typically
will share at least 40% nucleotide identity and/or at least 50%
amino acid identity to the sequences of murine TLR9 nucleic acids
and TLR9 polypeptides, respectively. In some instances homologues
and alleles will share at least 50% nucleotide identity and/or at
least 65% amino acid identity and in still other instances will
share at least 60% nucleotide identity and/or at least 75% amino
acid identity. Preferably the homologues and alleles will share at
least 80% nucleotide identity and/or at least 90% amino acid
identity, and more preferably will share at least 90% nucleotide
identity and/or at least 95% amino acid identity. Most preferably
the homologues and alleles will share at least 95% nucleotide
identity and/or at least 99% amino acid identity. The homology can
be calculated using various publicly available software tools
developed by the National Center for Biotechnology Information
(NCBI, Bethesda, Md.) that can be obtained through the internet
(ftp:/ncbi.nlm.nih.gov/pub/). Exemplary tools include the BLAST
system available from the NCBI at http://www.ncbi.nlm.nih.gov, used
with default settings. Pairwise and ClustalW alignments (BLOSUM30
matrix setting) as well as Kyte-Doolittle hydropathic analysis can
be obtained, for example, using the MacVector sequence analysis
software (Oxford Molecular Group). Watson-Crick complements of the
foregoing nucleic acids also are embraced by the invention.
Alleles of the murine TLR9 nucleic acids of the invention can be
identified by conventional techniques. For example, alleles of
murine TLR9 can be isolated by hybridizing a probe which includes
at least a fragment of SEQ ID NO:1 or SEQ ID NO:2 under stringent
conditions with a cDNA library and selecting positive clones. Thus,
an aspect of the invention is those nucleic acid sequences which
code for murine TLR9 polypeptides and which hybridize to a nucleic
acid molecule consisting of SEQ ID NO:1 or SEQ ID NO:2 under
stringent conditions.
In screening for murine TLR nucleic acids, a Southern blot may be
performed using the foregoing stringent conditions, together with a
radioactive probe. After washing the membrane to which the DNA is
finally transferred, the membrane can be placed against X-ray film
to detect the radioactive signal. Corresponding non-radioactive
methods are also well known in the art and can be used to similar
effect.
The murine TLR nucleic acids of the invention also include
degenerate nucleic acids which include alternative codons to those
present in the native materials. For example, serine residues are
encoded by the codons AGC, AGT, and TCA, TCC, TCG and TCT. Each of
the six codons is equivalent for the purposes of encoding a serine
residue. Thus, it will be apparent to one of ordinary skill in the
art that any of the serine-encoding nucleotide triplets may be
employed to direct the protein synthesis apparatus, in vitro or in
vivo, to incorporate a serine residue into an elongating murine TLR
polypeptide. Similarly, nucleotide sequence triplets which encode
other amino acid residues include, but are not limited to: CCA,
CCC, CCG and CCT (proline codons); CGA, CGC, CGG, CGT, AGA and AGG
(arginine codons); ACA, ACC, ACG and ACT (threonine codons); AAC
and AAT (asparagine codons); and ATA, ATC and ATT (isoleucine
codons). As is well known by those of ordinary skill in the art,
other specific amino acid residues may be encoded similarly by
multiple nucleotide sequences. Thus, the invention embraces
degenerate nucleic acids that differ from the biologically isolated
nucleic acids in codon sequence due to the degeneracy of the
genetic code. The above-noted codon degeneracy notwithstanding, it
is well appreciated by those skilled in the art that there are
certain codon usage preferences in specific host organisms, such
that in practice it may be preferred to select or to avoid certain
degenerate codons in a particular host.
The invention also provides modified nucleic acid molecules which
include additions, substitutions and deletions of one or more
nucleotides. The modified nucleic acid molecules according to this
aspect of the invention exclude fully native human TLR9 nucleic
acid molecules (GenBank Accession No. AF245704 (SEQ ID NO:4) or
GenBank Accession No. NM.sub.-- 017442 (SEQ ID NO:5)). In preferred
embodiments, these modified nucleic acid molecules and/or the
polypeptides they encode retain at least one activity or function
of the unmodified nucleic acid molecule and/or the polypeptides,
such as signaling activity, etc. In certain embodiments, the
modified nucleic acid molecules encode modified polypeptides,
preferably polypeptides having conservative amino acid
substitutions as are described elsewhere herein. The modified
nucleic acid molecules are structurally related to the unmodified
nucleic acid molecules and in preferred embodiments are
sufficiently structurally related to the unmodified nucleic acid
molecules so that the modified and unmodified nucleic acid
molecules hybridize under stringent conditions known to one of
skill in the art.
For example, modified nucleic acid molecules which encode
polypeptides having single amino acid changes can be prepared. Each
of these nucleic acid molecules can have one, two or three
nucleotide substitutions exclusive of nucleotide changes
corresponding to the degeneracy of the genetic code as described
herein. Likewise, modified nucleic acid molecules which encode
polypeptides having two amino acid changes can be prepared which
have, e.g., 2-6 nucleotide changes. Numerous modified nucleic acid
molecules like these will be readily envisioned by one of skill in
the art, including for example, substitutions of nucleotides in
codons encoding amino acids 2 and 3, 2 and 4, 2 and 5, 2 and 6, and
so on. In the foregoing example, each combination of two amino
acids is included in the set of modified nucleic acid molecules, as
well as all nucleotide substitutions which code for the amino acid
substitutions. Additional nucleic acid molecules that encode
polypeptides having additional substitutions (i.e., 3 or more),
additions or deletions (e.g., by introduction of a stop codon or a
splice site(s)) also can be prepared and are embraced by the
invention as readily envisioned by one of ordinary skill in the
art. Any of the foregoing nucleic acids or polypeptides can be
tested by routine experimentation for retention of structural
relation or activity to the nucleic acids and/or polypeptides
disclosed herein.
The invention also provides isolated fragments of SEQ ID NO:1 and
SEQ ID NO:2. The fragments can be used as probes in Southern blot
assays to identify such nucleic acids, or they can be used in
amplification assays such as those employing PCR. Smaller fragments
are those comprising 12, 13, 14, 15, 16, 17, 18, 20, 22, 25, 30,
40, 50, or 75 nucleotides, and every integer therebetween, and are
useful, e.g., as primers for nucleic acid amplification procedures.
As known to those skilled in the art, larger probes such as 200,
250, 300, 400 or more nucleotides are preferred for certain uses
such as Southern blots, while smaller fragments will be preferred
for uses such as PCR. Fragments also can be used to produce fusion
proteins for generating antibodies or determining binding of the
polypeptide fragments. Likewise, fragments can be employed to
produce non-fused fragments of the murine TLR9 polypeptides,
useful, for example, in the preparation of antibodies, in
immunoassays, and the like. The foregoing nucleic acid fragments
further can be used as antisense molecules to inhibit the
expression of murine TLR9 nucleic acids and polypeptides,
particularly for therapeutic purposes as described in greater
detail below.
The invention also includes functionally equivalent variants of the
murine TLR9, which include variant nucleic acids and polypeptides
which retain one or more of the functional properties of the murine
TLR9. Preferably such variants include the murine-specific
N-terminal domain (e.g., amino acids 1-819 or amino acids 1-837 of
SEQ ID NO:3). For example, variants include a fusion protein which
includes the extracellular and transmembrane domains of the murine
TLR9 (i.e., amino acids 1-837) which retains the ability to
interact with extracellular molecules in a manner similar to intact
murine TLR9. Alternative variants include, for example, a fusion
protein which includes the cytoplasmic domain of murine TLR9 (i.e.,
amino acids 838-1032) which retains the ability to interact with
intracellular molecules in a manner similar to intact murine TLR9.
Still other functionally equivalent variants include truncations,
deletions, point mutations, or additions of amino acids to the
sequence of SEQ ID NO:3 which retain functions of SEQ ID NO:3. For
example, the FLAG peptide sequence (DYKDDDDK) can be added at the
N-terminal end, or green fluorescent protein (GFP) can be added at
the C-terminal end. All such addition variant polypeptides are
preferably made by translation of modified nucleic acids based on
SEQ ID NO:1 or SEQ ID NO:2 with corresponding appropriate coding
nucleic acid sequence appended thereto with maintenance of the
proper reading frame.
Functionally equivalent variants also include a murine TLR9 which
has had a portion (e.g., of the N-terminus) removed or replaced by
a similar domain from another TLR (e.g., a "domain-swapping"
variant). Examples of such domain-swapping variants include at
least two types: those involving swapping a TLR9 domain from one
species with a TLR9 domain from another species, and those
involving swapping a TLR domain from TLR9 with a TLR domain from
another TLR. In certain preferred embodiments the swapping involves
corresponding domains between the different TLR molecules. It is
believed that certain such domain-swapping variants are not
functionally equivalent in a literal sense, insofar as they can
function in a manner superior to either or both intact parent TLR
molecules from which a particular domain-swapping variant derives.
For example, the TLR/IL-1R signaling mediated by human TLR9 could
be limited, not by the capacity of its extracellular domain to
interact with CpG ODN, but rather by the capacity of its
cytoplasmic domain to engage the TLR/IL-1R signaling pathway. In
such a circumstance, it could be advantageous to substitute a more
potent cytoplasmic domain from a TLR9 from another species. Such a
domain-swapping variant, e.g., chimeric hTLR9/mTLR9, could be used
in screening assays for CpG immuno-agonist/antagonists to increase
the sensitivity of the assay, without changing the species
specificity.
Other functionally equivalent variants will be known to one of
ordinary skill in the art, as will be methods for preparing such
variants. The activity of a functionally equivalent variant can be
determined using the methods provided herein, and in references
that have described assays using other TLRs and TLRs of other
species. Such variants are useful, inter alia, for evaluating
bioavailability of drugs, in assays for identification of compounds
which bind to and/or regulate the signaling function of the murine
TLR9, and for determining the portions of the murine TLR9 which are
required for signaling activity.
Variants which are non-functional also can be prepared as described
above. Such variants are useful, for example, as negative controls
in experiments testing TLR9 signaling activity. Examples of
non-functional variants include those incorporating a mutation of
proline at aa 915 to histidine (P915H) which renders both mTLR9 and
hTLR9 nonfunctional with respect to signaling. Futher examples of
non-functional variants include those incorporating a mutation of
the D-X-Y core of the MBD motif to A-L-A, as discussed above, to
render both mTLR9 and hTLR9 nonfunctional with respect to CpG DNA
binding.
A murine TLR9 nucleic acid, in one embodiment, is operably linked
to a gene expression sequence which can direct the expression of
the murine TLR9 nucleic acid within a eukaryotic or prokaryotic
cell. A "gene expression sequence" is any regulatory nucleotide
sequence, such as a promoter sequence or promoter-enhancer
combination, which facilitates the efficient transcription and
translation of the nucleic acid to which it is operably linked.
With respect to murine TLR9 nucleic acid, the "gene expression
sequence" is any regulatory nucleotide sequence, such as a promoter
sequence or promoter-enhancer combination, which facilitates the
efficient transcription and translation of the murine TLR9 nucleic
acid to which it is operably linked. The gene expression sequence
may, for example, be a mammalian or viral promoter, such as a
constitutive or inducible promoter. Constitutive mammalian
promoters include, but are not limited to, the promoters for the
following genes: hypoxanthine phosphoribosyl transferase (HPRT),
adenosine deaminase, pyruvate kinase, .beta.-actin promoter, and
other constitutive promoters. Exemplary viral promoters which
function constitutively in eukaryotic cells include, for example,
promoters from the simian virus (e.g., SV40), papilloma virus,
adenovirus, human immunodeficiency virus (HIV), Rous sarcoma virus
(RSV), cytomegalovirus (CMV), the long terminal repeats (LTR) of
Moloney murine leukemia virus and other retroviruses, and the
thymidine kinase (TK) promoter of herpes simplex virus. Other
constitutive promoters are known to those of ordinary skill in the
art. The promoters useful as gene expression sequences of the
invention also include inducible promoters. Inducible promoters are
expressed in the presence of an inducing agent. For example, the
metallothionein (MT) promoter is induced to promote transcription
and translation in the presence of certain metal ions. Other
inducible promoters are known to those of ordinary skill in the
art.
In general, the gene expression sequence shall include, as
necessary, 5' non-transcribing and 5' non-translating sequences
involved with the initiation of transcription and translation,
respectively, such as a TATA box, capping sequence, CAAT sequence,
and the like. Especially, such 5' non-transcribing sequences will
include a promoter region which includes a promoter sequence for
transcriptional control of the operably joined murine TLR9 nucleic
acid. The gene expression sequences optionally include enhancer
sequences or upstream activator sequences as desired.
Generally a nucleic acid coding sequence and a gene expression
sequence are said to be "operably linked" when they are covalently
linked in such a way as to place the transcription and/or
translation of the nucleic acid coding sequence under the influence
or control of the gene expression sequence. Thus the murine TLR9
nucleic acid sequence and the gene expression sequence are said to
be "operably linked" when they are covalently linked in such a way
as to place the transcription and/or translation of the murine TLR9
coding sequence under the influence or control of the gene
expression sequence. If it is desired that the murine TLR9 sequence
be translated into a functional protein, two DNA sequences are said
to be operably linked if induction of a promoter in the 5' gene
expression sequence results in the transcription of the murine TLR9
sequence and if the nature of the linkage between the two DNA
sequences does not (1) result in the introduction of a frame-shift
mutation, (2) interfere with the ability of the promoter region to
direct the transcription of the murine TLR9 sequence, or (3)
interfere with the ability of the corresponding RNA transcript to
be translated into a protein. Thus, a gene expression sequence
would be operably linked to a murine TLR9 nucleic acid sequence if
the gene expression sequence were capable of effecting
transcription of that murine TLR9 nucleic acid sequence such that
the resulting transcript might be translated into the desired
protein or polypeptide.
The murine TLR9 nucleic acid molecules and the murine TLR9
polypeptides (including the murine TLR9 inhibitors described below)
of the invention can be delivered to the eukaryotic or prokaryotic
cell alone or in association with a vector. In its broadest sense,
a "vector" is any vehicle capable of facilitating: (1) delivery of
a nucleic acid or polypeptide to a target cell, (2) uptake of a
nucleic acid or polypeptide by a target cell, or (3) expression of
a nucleic acid molecule or polypeptide in a target cell. In this
particular setting, a "vector" is any vehicle capable of
facilitating: (1) delivery of a murine TLR9 nucleic acid or
polypeptide to a target cell, (2) uptake of a murine TLR9 nucleic
acid or polypeptide by a target cell, or (3) expression of a murine
TLR9 nucleic acid molecule or polypeptide in a target cell.
Preferably, the vectors transport the murine TLR9 nucleic acid or
polypeptide into the target cell with reduced degradation relative
to the extent of degradation that would result in the absence of
the vector. Optionally, a "targeting ligand" can be attached to the
vector to selectively deliver the vector to a cell which expresses
on its surface the cognate receptor (e.g., a receptor, an antigen
recognized by an antibody) for the targeting ligand. In this
manner, the vector (containing a murine TLR9 nucleic acid or a
murine TLR9 polypeptide) can be selectively delivered to a specific
cell. In general, the vectors useful in the invention are divided
into two classes: biological vectors and chemical/physical vectors.
Biological vectors are more useful for delivery/uptake of murine
TLR9 nucleic acids to/by a target cell. Chemical/physical vectors
are more useful for delivery/uptake of murine TLR9 nucleic acids or
murine TLR9 proteins to/by a target cell.
Biological vectors include, but are not limited to, plasmids,
phagemids, viruses, other vehicles derived from viral or bacterial
sources that have been manipulated by the insertion or
incorporation of the nucleic acid sequences of the invention, and
free nucleic acid fragments which can be linked to the nucleic acid
sequences of the invention. Viral vectors are a preferred type of
biological vector and include, but are not limited to, nucleic acid
sequences from the following viruses: retroviruses, such as Moloney
murine leukemia virus; Harvey murine sarcoma virus; murine mammary
tumor virus; Rous sarcoma virus; adenovirus; adeno-associated
virus; SV40-type viruses; polyoma viruses; poxyiruses; Epstein-Barr
viruses; papilloma viruses; herpes virus; vaccinia virus; and polio
virus. One can readily employ other vectors not named but known in
the art.
Preferred viral vectors are based on non-cytopathic eukaryotic
viruses in which non-essential genes have been replaced with the
gene of interest. Non-cytopathic viruses include retroviruses, the
life cycle of which involves reverse transcription of genomic viral
RNA into DNA with subsequent proviral integration into host
cellular DNA. In general, the retroviruses are
replication-deficient (i.e., capable of directing synthesis of the
desired proteins, but incapable of manufacturing an infectious
particle). Such genetically altered retroviral expression vectors
have general utility for the high-efficiency transduction of genes
in vivo. Standard protocols for producing replication-deficient
retroviruses (including the steps of incorporation of exogenous
genetic material into a plasmid, transfection of a packaging cell
line with plasmid, production of recombinant retroviruses by the
packaging cell line, collection of viral particles from tissue
culture media, and infection of the target cells with viral
particles) are provided in Kriegler, M., "Gene Transfer and
Expression, A Laboratory Manual," W.H. Freeman Co., New York (1990)
and Murray, E. J., ed., "Methods in Molecular Biology," vol. 7,
Humana Press, Inc., Clifton, N.J. (1991).
Another preferred virus for certain applications is the
adeno-associated virus (AAV), a double-stranded DNA virus. The AAV
can be engineered to be replication-deficient and is capable of
infecting a wide range of cell types and species. It further has
advantages, such as heat and lipid solvent stability; high
transduction frequencies in cells of diverse lineages; and lack of
superinfection inhibition thus allowing multiple series of
transductions. Reportedly, the AAV can integrate into human
cellular DNA in a site-specific manner, thereby minimizing the
possibility of insertional mutagenesis and variability of inserted
gene expression. In addition, wild-type AAV infections have been
followed in tissue culture for greater than 100 passages in the
absence of selective pressure, implying that the AAV genomic
integration is a relatively stable event. The AAV can also function
in an extrachromosomal fashion.
Expression vectors containing all the necessary elements for
expression are commercially available and known to those skilled in
the art. See, e.g., Molecular Cloning: A Laboratory Manual, J.
Sambrook, et al., eds., Second Edition, Cold Spring Harbor
Laboratory Press, Cold Spring Harbor, N.Y., 1989. Cells are
genetically engineered by the introduction into the cells of
heterologous DNA (RNA) encoding a murine TLR9 polypeptide or
fragment or variant thereof. That heterologous DNA (RNA) is placed
under operable control of transcriptional elements to permit the
expression of the heterologous DNA in the host cell.
Preferred systems for mRNA expression in mammalian cells are those
such as pRc/CMV (available from Invitrogen, Carlsbad, Calif.) that
contain a selectable marker such as a gene that confers G418
resistance (which facilitates the selection of stably transfected
cell lines) and the human CMV enhancer-promoter sequences.
Additionally, suitable for expression in primate or canine cell
lines is the pCEP4 vector (Invitrogen), which contains an Epstein
Barr virus (EBV) origin of replication, facilitating the
maintenance of plasmid as a multicopy extrachromosomal element.
Another expression vector is the pEF-BOS plasmid containing the
promoter of polypeptide Elongation Factor 1.alpha., which
stimulates efficiently transcription in vitro. The plasmid is
described by Mishizuma and Nagata (Nucleic Acids Res 18:5322
(1990)), and its use in transfection experiments is disclosed by,
for example, Demoulin (Mol Cell Biol 16:4710-4716 (1996)). Still
another preferred expression vector is an adenovirus, described by
Stratford-Perricaudet, which is defective for E1 and E3 proteins (J
Clin Invest 90:626-630 (1992)).
In addition to the biological vectors, chemical/physical vectors
may be used to deliver a nucleic acid or polypeptide to a target
cell and facilitate uptake thereby. As used herein, a
"chemical/physical vector" refers to a natural or synthetic
molecule, other than those derived from bacteriological or viral
sources, capable of delivering an isolated nucleic acid or
polypeptide to a cell. As used herein with respect to a murine TLR9
nucleic acid or polypeptide, a "chemical/physical vector" refers to
a natural or synthetic molecule, other than those derived from
bacteriological or viral sources, capable of delivering the
isolated murine TLR9 nucleic acid or polypeptide to a cell.
A preferred chemical/physical vector of the invention is a
colloidal dispersion system. Colloidal dispersion systems include
lipid-based systems including oil-in-water emulsions, micelles,
mixed micelles, and liposomes. A preferred colloidal system of the
invention is a liposome. Liposomes are artificial membrane vesicles
which are useful as a delivery vector in vivo or in vitro. It has
been shown that large unilamellar vesicles (LUV), which range in
size from 0.2-4.0 .mu.m can encapsulate large macromolecules. RNA,
DNA, and intact virions can be encapsulated within the aqueous
interior and be delivered to cells in a biologically active form
(Fraley et al., Trends Biochem Sci 6:77 (1981)). In order for a
liposome to be an efficient nucleic acid transfer vector, one or
more of the following characteristics should be present: (1)
encapsulation of the nucleic acid of interest at high efficiency
with retention of biological activity; (2) preferential and
substantial binding to a target cell in comparison to non-target
cells; (3) delivery of the aqueous contents of the vesicle to the
target cell cytoplasm at high efficiency; and (4) accurate and
effective expression of genetic information.
Liposomes may be targeted to a particular tissue by coupling the
liposome to a specific ligand such as a monoclonal antibody, sugar,
glycolipid, or protein. Ligands which may be useful for targeting a
liposome to a particular cell will depend on the particular cell or
tissue type. Additionally when the vector encapsulates a nucleic
acid, the vector may be coupled to a nuclear targeting peptide,
which will direct the murine TLR9 nucleic acid to the nucleus of
the host cell.
Liposomes are commercially available from Gibco BRL, for example,
as LIPOFECTIN.TM. and LIPOFECTACE.TM., which are formed of cationic
lipids such as N-[1-(2,3 dioleyloxy)-propyl]-N,N,
N-trimethylammonium chloride (DOTMA) and dimethyl
dioctadecylammonium bromide (DDAB). Methods for making liposomes
are well known in the art and have been described in many
publications.
Other exemplary compositions that can be used to facilitate uptake
by a target cell of nucleic acids in general, and nucleic acids
encoding the murine TLR9 in particular, include calcium phosphate
and other chemical mediators of intracellular transport,
microinjection compositions, electroporation and homologous
recombination compositions (e.g., for integrating a murine TLR9
nucleic acid into a preselected location within a target cell
chromosome).
The invention also embraces so-called expression kits, which allow
the artisan to prepare a desired expression vector or vectors. Such
expression kits include at least separate portions of the
previously discussed coding sequences. Other components may be
added, as desired, as long as the previously mentioned sequences,
which are required, are included.
It will also be recognized that the invention embraces the use of
the murine TLR9 cDNA sequences in expression vectors to transfect
host cells and cell lines, be these prokaryotic (e.g., E. coli), or
eukaryotic (e.g., 293 fibroblast cells (ATCC, CRL-1573), MonoMac-6,
THP-1, U927, CHO cells, COS cells, yeast expression systems and
recombinant baculovirus expression in insect cells). Especially
useful are mammalian cells such as human, pig, goat, primate,
rodent, guinea pig, etc. They may be of a wide variety of tissue
types, and include primary cells and cell lines. The expression
vectors require that the pertinent sequence, i.e., those nucleic
acids described supra, be operably linked to a promoter.
The invention also provides isolated murine TLR9 polypeptides which
include the amino acid sequences of SEQ ID NO:3 and fragments
thereof, encoded by the murine TLR9 nucleic acids described above.
Murine TLR9 polypeptides also embrace alleles, functionally
equivalent variants and analogs (those non-allelic polypeptides
which vary in amino acid sequence from the disclosed murine TLR9
polypeptides by 1, 2, 3, 4, 5, or more amino acids) provided that
such polypeptides retain TLR9 activity. Non-functional variants
also are embraced by the invention; these are useful as antagonists
of TLR9 signaling function, as negative controls in assays, and the
like. Such alleles, variants, analogs and fragments are useful, for
example, alone or as fusion proteins for a variety of purposes
including as a component of assays.
Fragments of a polypeptide preferably are those fragments which
retain a distinct functional capability of the intact polypeptide,
in particular as a receptor of various molecules. Accordingly,
fragments of a TLR9 polypeptide preferably are those fragments
which retain a distinct functional capability of the TLR9
polypeptide, in particular as a receptor of various molecules. Of
particular interest are fragments that bind to ISNAs, including,
for example, fragments that bind CpG nucleic acids. Other
functional capabilities which can be retained in a fragment of a
polypeptide include signal transduction (e.g., TLR/IL-1R signaling
by murine TLR9), interaction with antibodies and interaction with
other polypeptides (such as would be found in a protein complex).
Those skilled in the art are well versed in methods that can be
applied for selecting fragments which retain a functional
capability of the murine TLR9. Confirmation of the functional
capability of the fragment can be carried out by synthesis of the
fragment and testing of the capability according to standard
methods. For example, to test the signaling activity of a murine
TLR9 fragment, one inserts or expresses the fragment in a cell in
which signaling can be measured. Such methods, which are standard
in the art, are described further herein.
The invention embraces variants of the murine TLR9 polypeptides
described above. As used herein, a "variant" of a polypeptide is a
polypeptide which contains one or more modifications to the primary
amino acid sequence of a polypeptide. Accordingly, a "variant" of a
murine TLR9 polypeptide is a polypeptide which contains one or more
modifications to the primary amino acid sequence of a murine TLR9
polypeptide. Modifications which create a murine TLR9 variant can
be made to a murine TLR9 polypeptide for a variety of reasons,
including 1) to reduce or eliminate an activity of a murine TLR9
polypeptide, such as signaling; 2) to enhance a property of a
murine TLR9 polypeptide, such as signaling, binding affinity for
nucleic acid ligand or other ligand molecule, protein stability in
an expression system, or the stability of protein-protein binding;
3) to provide a novel activity or property to a murine TLR9
polypeptide, such as addition of an antigenic epitope or addition
of a detectable moiety, e.g., luciferase, FLAG peptide, GFP; 4) to
establish that an amino acid substitution does or does not affect
molecular signaling activity; or 5) reduce immunogenicity of a
murine TLR9 polypeptide. Modifications to a murine TLR9 polypeptide
are typically made to the nucleic acid which encodes the murine
TLR9 polypeptide, and can include deletions, point mutations,
truncations, amino acid substitutions and additions of amino acids
or non-amino acid moieties. Alternatively, modifications can be
made directly to the polypeptide, such as by cleavage, addition of
a linker molecule, addition of a detectable moiety (for example,
biotin, fluorophore, radioisotope, enzyme, or peptide), addition of
a fatty acid, and the like.
Modifications also embrace fusion proteins comprising all or part
of the murine TLR9 amino acid sequence. One of skill in the art
will be familiar with methods for predicting the effect on protein
conformation of a change in protein sequence, and can thus "design"
a variant murine TLR9 according to known methods. One example of
such a method is described by Dahiyat and Mayo in Science 278:82-87
(1997), whereby proteins can be designed de novo. The method can be
applied to a known protein to vary a only a portion of the
polypeptide sequence. By applying the computational methods of
Dahiyat and Mayo, specific variants of a murine TLR9 polypeptide
can be proposed and tested to determine whether the variant retains
a desired conformation.
Variants include murine TLR9 polypeptides which are modified
specifically to alter a feature of the polypeptide unrelated to its
physiological activity. For example, cysteine residues can be
substituted or deleted to prevent unwanted disulfide linkages.
Similarly, certain amino acids can be changed to enhance expression
of a murine TLR9 polypeptide by eliminating proteolysis by
proteases in an expression system (e.g., dibasic amino acid
residues in yeast expression systems in which KEX2 protease
activity is present).
Mutations of a nucleic acid which encode a murine TLR9 polypeptide
preferably preserve the amino acid reading frame of the coding
sequence, and preferably do not create regions in the nucleic acid
which are likely to hybridize to form secondary structures, such as
hairpins or loops, which can be deleterious to expression of the
variant polypeptide.
Mutations can be made by selecting an amino acid substitution, or
by random mutagenesis of a selected site in a nucleic acid which
encodes the polypeptide. Variant polypeptides are then expressed
and tested for one or more activities to determine which mutation
provides a variant polypeptide with a desired property. Further
mutations can be made to variants (or to non-variant murine TLR9
polypeptides) which are silent as to the amino acid sequence of the
polypeptide, but which provide preferred codons for translation in
a particular host. The preferred codons for translation of a
nucleic acid in, e.g., E. coli, are well known to those of ordinary
skill in the art. Still other mutations can be made to the
noncoding sequences of a murine TLR9 gene or cDNA clone to enhance
expression of the polypeptide.
The activity of variants of murine TLR9 polypeptides can be tested
by cloning the gene encoding the variant murine TLR9 polypeptide
into a prokaryotic or eukaryotic (e.g., mammalian) expression
vector, introducing the vector into an appropriate host cell,
expressing the variant murine TLR9 polypeptide, and testing for a
functional capability of the murine TLR9 polypeptides as disclosed
herein. For example, the variant murine TLR9 polypeptide can be
tested for ability to provide signaling, as set forth below in the
examples. Preparation of other variant polypeptides may favor
testing of other activities, as will be known to one of ordinary
skill in the art.
The skilled artisan will also realize that conservative amino acid
substitutions may be made in murine TLR9 polypeptides to provide
functionally equivalent variants of the foregoing polypeptides,
i.e., variants which retain the functional capabilities of the
murine TLR9 polypeptides. As used herein, a "conservative amino
acid substitution" refers to an amino acid substitution which does
not alter the relative charge or size characteristics of the
polypeptide in which the amino acid substitution is made. Variants
can be prepared according to methods for altering polypeptide
sequence known to one of ordinary skill in the art such as are
found in references which compile such methods, e.g., Molecular
Cloning: A Laboratory Manual, J. Sambrook, et al., eds., Second
Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor,
N.Y., 1989, or Current Protocols in Molecular Biology, F. M.
Ausubel, et al., eds., John Wiley & Sons, Inc., New York.
Exemplary functionally equivalent variants of the murine TLR9
polypeptides include conservative amino acid substitutions of SEQ
ID NO:3. Conservative substitutions of amino acids include
substitutions made amongst amino acids within the following groups:
(a) M, I, L, V; (b) F, Y, W; (c) K, R, H; (d) A, G; (e) S, T; (f)
Q, N; and (g) E, D.
Conservative amino acid substitutions in the amino acid sequence of
murine TLR9 polypeptide to produce functionally equivalent variants
of murine TLR9 typically are made by alteration of the nucleic acid
sequence encoding murine TLR9 polypeptides (e.g., SEQ ID NO:1 and
SEQ ID NO:2). Such substitutions can be made by a variety of
methods known to one of ordinary skill in the art. For example,
amino acid substitutions may be made by PCR-directed mutation,
site-directed mutagenesis according to the method of Kunkel
(Kunkel, Proc Natl Acad Sci USA 82:488-492 (1985)), or by chemical
synthesis of a gene encoding a murine TLR9 polypeptide. The
activity of functionally equivalent fragments of murine TLR9
polypeptides can be tested by cloning the gene encoding the altered
murine TLR9 polypeptide into a bacterial or mammalian expression
vector, introducing the vector into an appropriate host cell,
expressing the altered murine TLR9 polypeptide, and testing for the
ability of the murine TLR9 polypeptide to mediate a signaling
event. Peptides which are chemically synthesized can be tested
directly for function.
A variety of methodologies well known to the skilled practitioner
can be utilized to obtain isolated murine TLR9 polypeptide
molecules. The polypeptide may be purified from cells which
naturally produce the polypeptide by chromatographic means or
immunological recognition. Alternatively, an expression vector may
be introduced into cells to cause production of the polypeptide. In
another method, mRNA transcripts may be microinjected or otherwise
introduced into cells to cause production of the encoded
polypeptide. Translation of mRNA in cell-free extracts such as the
reticulocyte lysate system also may be used to produce polypeptide.
Those skilled in the art also can readily follow known methods for
isolating murine TLR9 polypeptides. These include, but are not
limited to, immunochromatography, HPLC, size-exclusion
chromatography, ion-exchange chromatography and immune-affinity
chromatography.
The invention as described herein has a number of uses, some of
which are described elsewhere herein. For example, the invention
permits isolation of the murine TLR9 polypeptide molecules by,
e.g., expression of a recombinant nucleic acid to produce large
quantities of polypeptide which may be isolated using standard
protocols. As another example, the isolation of the murine TLR9
gene makes it possible for murine TLR9 to be used in methods for
assaying molecular interactions involving TLR9.
As discussed further in the Examples below, it has been discovered
according to one aspect of the invention that responsiveness to
ISNA can be reconstituted in ISNA-unresponsive cells by introducing
into ISNA-unresponsive cells an expression vector that directs the
expression of murine TLR9 (and certain homologues and variants
thereof). Cells so reconstituted also exhibit responses to
substances other than phosphorothioate ISNA, e.g., E. coli DNA,
phosphodiester CpG-ODN, and even methylated CpG-ODN.
Also as discussed further in the Examples below, it has been
discovered according to certain aspects of the instant invention
that TLR9 not only confers upon cells the ability to signal in
response to binding ISNA, but also confers both sequence
specificity and species specificity to such signaling responses.
Thus murine TLR9 signaling in response to CpG-ODN 1668, reportedly
an optimal murine ISNA, was found to be significantly stronger than
the corresponding murine TLR9 signaling response to CpG-ODN 2006,
reportedly an optimal human ISNA. The converse was also found to be
true, i.e., human TLR9 signaling in response to CpG-ODN 2006 was
found to be significantly stronger than the corresponding human
TLR9 signaling response to CpG-ODN 1668. Furthermore, it has been
discovered according to the instant invention that certain types of
cells preferentially express TLR9. For example, TLR9 is strongly
expressed in B cells and plasmacytoid dendritic cells (CD123+DC),
but only weakly by T cells, monocyte-derived dendritic cells
(MDDC), and CD14+monocytes. In contrast, TLR2 and TLR4 are strongly
expressed by MDDC and CD14+monocytes, but relatively weakly by B
cells, CD123+DC, and T cells.
The invention also embraces agents which bind selectively to the
murine TLR9 nucleic acid molecules or polypeptides as well as
agents which bind to variants and fragments of the polypeptides and
nucleic acids as described herein. The agents include polypeptides
which bind to murine TLR9, and antisense nucleic acids, both of
which are described in greater detail below. The agents can inhibit
or increase murine TLR9-mediated signaling activity (antagonists
and agonists, respectively).
Some of the agents are inhibitors. A murine TLR9 inhibitor is an
agent that inhibits murine TLR9-mediated signaling across a cell
membrane.
As used herein "TLR9 signaling" refers to an ability of a TLR9
polypeptide to activate the TLR/IL-1R (TIR) signaling pathway, also
referred to herein as the TLR signal transduction pathway. Without
meaning to be held to any particular theory, it is believed that
the TLR/IL-1R signaling pathway involves signaling via the
molecules myeloid differentiation marker 88 (MyD88) and tumor
necrosis factor (TNF) receptor-associated factor 6 (TRAF6), leading
to activation of kinases of the I.kappa.B kinase complex and the
c-jun NH.sub.2 -terminal kinases (e.g., JNK 1/2). Hacker H et al.,
J Exp Med 192:595-600 (2000). A molecule which inhibits TLR9
activity (an antagonist) is one which inhibits TLR9-mediated
activation of the TLR/IL-1R signaling pathway, and a molecule which
increases TLR9 signaling (an agonist) is one which increases
TLR9-mediated activation of the TLR/IL-1R signaling pathway.
Changes in TLR9 activity can be measured by assays such as those
disclosed herein, including expression of genes under control of
.kappa.B-sensitive promoters and enhancers. Such naturally
occurring genes include the genes encoding IL-1.beta., IL-6, IL-8,
the p40 subunit of interleukin 12 (IL-12p40), and the costimulatory
molecules CD80 and CD86. Other genes can be placed under the
control of such regulatory elements (see below) and thus serve to
report the level of TLR9 signaling. Additional nucleotide sequence
can be added to SEQ ID NO:1 or SEQ ID NO:2, preferably to the 5' or
the 3' end of SEQ ID NO:2, to yield a nucleotide sequence encoding
a chimeric polypeptide that includes a detectable or reporter
moiety, e.g., FLAG, luciferase (luc), green fluorescent protein
(GFP) and others known by those skilled in the art. These are
discussed in greater detail in the Examples below.
In one embodiment the murine TLR9 inhibitor is an antisense
oligonucleotide that selectively binds to a murine TLR9 nucleic
acid molecule, to reduce the expression of murine TLR9 (or TLR9 of
another species) in a cell. This is desirable in virtually any
medical condition wherein a reduction of TLR9 signaling activity is
desirable.
As used herein, the term "antisense oligonucleotide" or "antisense"
describes an oligonucleotide that is an oligoribonucleotide,
oligodeoxyribonucleotide, modified oligoribonucleotide, or modified
oligodeoxyribonucleotide which hybridizes under physiological
conditions to DNA comprising a particular gene or to an mRNA
transcript of that gene and, thereby, inhibits the transcription of
that gene and/or the translation of that mRNA. The antisense
molecules are designed so as to interfere with transcription or
translation of a target gene upon hybridization with the target
gene or transcript. Those skilled in the art will recognize that
the exact length of the antisense oligonucleotide and its degree of
complementarity with its target will depend upon the specific
target selected, including the sequence of the target and the
particular bases which comprise that sequence. It is preferred that
the antisense oligonucleotide be constructed and arranged so as to
bind selectively with the target under physiological conditions,
i.e., to hybridize substantially more to the target sequence than
to any other sequence in the target cell under physiological
conditions.
Based upon SEQ ID NO:1 and SEQ ID NO:2, or upon allelic or
homologous genomic and/or cDNA sequences, one of skill in the art
can easily choose and synthesize any of a number of appropriate
antisense molecules for use in accordance with the present
invention. In order to be sufficiently selective and potent for
inhibition, such antisense oligonucleotides should comprise at
least 10 and, more preferably, at least 15 consecutive bases which
are complementary to the target, although in certain cases modified
oligonucleotides as short as 7 bases in length have been used
successfully as antisense oligonucleotides. Wagner R W et al., Nat
Biotechnol 14:840-844 (1996). Most preferably, the antisense
oligonucleotides comprise a complementary sequence of 20-30 bases.
Although oligonucleotides may be chosen which are antisense to any
region of the gene or mRNA transcripts, in preferred embodiments
the antisense oligonucleotides correspond to N-terminal or 5'
upstream sites such as translation initiation, transcription
initiation or promoter sites. In addition, 3'-untranslated regions
may be targeted. Targeting to mRNA splicing sites has also been
used in the art but may be less preferred if alternative mRNA
splicing occurs. In addition, the antisense is targeted,
preferably, to sites in which mRNA secondary structure is not
expected (see, e.g., Sainio et al., Cell Mol Neurobiol
14(5):439-457 (1994)) and at which polypeptides are not expected to
bind. Thus, the present invention also provides for antisense
oligonucleotides which are complementary to allelic or homologous
cDNAs and genomic DNAs corresponding to murine TLR9 nucleic acid
containing SEQ ID NO:1 or SEQ ID NO:2.
In one set of embodiments, the antisense oligonucleotides of the
invention may be composed of "natural" deoxyribonucleotides,
ribonucleotides, or any combination thereof. That is, the 5' end of
one native nucleotide and the 3' end of another native nucleotide
may be covalently linked, as in natural systems, via a
phosphodiester internucleoside linkage. These oligonucleotides may
be prepared by art-recognized methods which may be carried out
manually or by an automated synthesizer. They also may be produced
recombinantly by vectors.
In preferred embodiments, however, the antisense oligonucleotides
of the invention also may include "modified" oligonucleotides. That
is, the oligonucleotides may be modified in a number of ways which
do not prevent them from hybridizing to their target but which
enhance their stability or targeting or which otherwise enhance
their therapeutic effectiveness.
The term "modified oligonucleotide" as used herein describes an
oligonucleotide in which (1) at least two of its nucleotides are
covalently linked via a synthetic internucleoside linkage (i.e., a
linkage other than a phosphodiester linkage between the 5' end of
one nucleotide and the 3' end of another nucleotide) and/or (2) a
chemical group not normally associated with nucleic acids has been
covalently attached to the oligonucleotide. Preferred synthetic
internucleoside linkages are phosphorothioates, alkylphosphonates,
phosphorodithioates, phosphate esters, alkylphosphonothioates,
phosphoramidates, carbamates, carbonates, phosphate triesters,
acetamidates, carboxymethyl esters and peptides.
The term "modified oligonucleotide" also encompasses
oligonucleotides with a covalently modified base and/or sugar. For
example, modified oligonucleotides include oligonucleotides having
backbone sugars which are covalently attached to low molecular
weight organic groups other than a hydroxyl group at the 3'
position and other than a phosphate group at the 5' position. Thus
modified oligonucleotides may include a 2'-O-alkylated ribose
group. In addition, modified oligonucleotides may include sugars
such as arabinose instead of ribose. The present invention, thus,
contemplates pharmaceutical preparations containing modified
antisense molecules that are complementary to and hybridizable
with, under physiological conditions, nucleic acids encoding murine
TLR9 polypeptides, together with pharmaceutically acceptable
carriers.
Agents which bind murine TLR9 also include binding peptides and
other molecules which bind to the murine TLR9 polypeptide and
complexes containing the murine TLR9 polypeptide. When the binding
molecules are inhibitors, the molecules bind to and inhibit the
activity of murine TLR9. When the binding molecules are activators,
the molecules bind to and increase the activity of murine TLR9. To
determine whether a murine TLR9 binding agent binds to murine TLR9
any known binding assay may be employed. For example, the binding
agent may be immobilized on a surface and then contacted with a
labeled murine TLR9 polypeptide. The amount of murine TLR9 which
interacts with the murine TLR9 binding agent or the amount which
does not bind to the murine TLR9 binding agent may then be
quantitated to determine whether the murine TLR9 binding agent
binds to murine TLR9.
The murine TLR9 binding agents include molecules of numerous size
and type that bind selectively or preferentially to murine TLR9
polypeptides, and complexes of both murine TLR9 polypeptides and
their binding partners. These molecules may be derived from a
variety of sources. For example, murine TLR9 binding agents can be
provided by screening degenerate peptide libraries which can be
readily prepared in solution, in immobilized form or as phage
display libraries. Combinatorial libraries also can be synthesized
of peptides containing one or more amino acids. Libraries further
can be synthesized of peptoids and non-peptide synthetic
moieties.
Phage display can be particularly effective in identifying binding
peptides useful according to the invention. Briefly, one prepares a
phage library (using, e.g., m13, fd, or lambda phage), displaying
inserts from 4 to about 80 amino acid residues using conventional
procedures. The inserts may represent, for example, a completely
degenerate or biased array. One then can select phage-bearing
inserts which bind to the murine TLR9 polypeptide. This process can
be repeated through several cycles of reselection of phage that
bind to the murine TLR9 polypeptide. Repeated rounds lead to
enrichment of phage bearing particular sequences. DNA sequence
analysis can be conducted to identify the sequences of the
expressed polypeptides. The minimal linear portion of the sequence
that binds to the murine TLR9 polypeptide can be determined. One
can repeat the procedure using a biased library containing inserts
containing part or all of the minimal linear portion plus one or
more additional degenerate residues upstream or downstream thereof.
Yeast two-hybrid screening methods also may be used to identify
polypeptides that bind to the murine TLR9 polypeptides. Thus, the
murine TLR9 polypeptides of the invention, or a fragment thereof,
can be used to screen peptide libraries, including phage display
libraries, to identify and select peptide binding partners of the
murine TLR9 polypeptides of the invention. Such molecules can be
used, as described, for screening assays, for purification
protocols, for interfering directly with the functioning of murine
TLR9 and for other purposes that will be apparent to those of
ordinary skill in the art.
The invention also embraces agents which bind selectively to
certain regulatory sequences associated with the murine TLR9
nucleic acid molecules described herein. The agents include
polypeptides which bind to transcription and translation regulatory
sequences of murine TLR9, and antisense nucleic acids, both of
which are described in greater detail below. The agents can inhibit
or increase murine TLR9 expression, as well as signaling activity
(antagonists and agonists, respectively). Agents which bind
selectively to regulatory sequences associated with the murine TLR9
nucleic acid molecules can be identified using methods familiar to
those of skill in the art. For example, a promoter region including
at least 100, 200, 300, 400, 500, or more nucleotides upstream (5')
of the coding region of murine TLR9 can be identified by isolating,
from appropriate genomic DNA, such nucleotide sequences using the
sequences of SEQ ID NO:1 or SEQ ID NO:2 as primers or as probes,
and then inserting the promoter region DNA into an appropriate
expression vector so as to control the expression of TLR9 or some
other reporter gene, introducing the TLR9 promoter vector into an
appropriate host cell, and screening for TLR9 or reporter
expression by those cells following their incubation in the
presence and absence of various test agents. A reporter gene other
than TLR9 can include, for example, an enzyme, a cytokine, a cell
surface antigen, luciferase, chloramphenicol acetyl transferase
(CAT), etc. An agent that inhibits expression of TLR9 or the
reporter under the control of the TLR9 promoter is classified as a
TLR9 expression inhibitor. Conversely, an agent that augments
expression of TLR9 or reporter under the control of the TLR9
promoter is classified as a TLR9 expression enhancer. It was
discovered according to the instant invention, for example, that
the cytokine IL-4 inhibits the expression of TLR9. In this manner
it is possible to identify agents that can be administered in
conjunction with ISNA, for example by local administration, to
enhance response to the ISNA. Such an enhancing effect might be
desirable, for example, in the setting of immunization or
vaccination. Conversely, it it is possible to identify agents that
can be administered in conjunction with a ISNA, for example by
local administration, to inhibit response to the ISNA. Such an
inhibiting response might be desirable, for example, in the setting
of gene replacement therapy.
Therefore the invention generally provides efficient methods of
identifying pharmacological agents or lead compounds for agents
useful in the treatment of conditions associated with TLR9 activity
and the compounds and agents so identified. Generally, the
screening methods involve assaying for compounds which inhibit or
enhance signaling through murine TLR9. Such methods are adaptable
to automated, high throughput screening of compounds. Examples of
such high throughput screening methods are described in U.S. Pat.
Nos. 6,103,479; 6,051,380; 6,051,373; 5,998,152; 5,876,946;
5,708,158; 5,443,791; 5,429,921; and 5,143,854.
A variety of assays for pharmacological agents are provided,
including labeled in vitro protein binding assays, signaling assays
using detectable molecules, etc. For example, protein binding
screens are used to rapidly examine the binding of candidate
pharmacological agents to a murine TLR9. The candidate
pharmacological agents can be derived from, for example,
combinatorial peptide or nucleic acid libraries. Convenient
reagents for such assays are known in the art. An exemplary
cell-based assay of signaling involves contacting a cell having a
murine TLR9 with a candidate pharmacological agent under conditions
whereby the induction of a detectable molecule can occur. Specific
conditions are well known in the art and are described, for
example, in Hacker H et al., J Exp Med 192:595-600 (2000), and
references cited therein. A reduced degree of induction of the
detectable molecule in the presence of the candidate
pharmacological agent indicates that the candidate pharmacological
agent reduces the signaling activity of murine TLR9. An increased
degree of induction of the detectable molecule in the presence of
the candidate pharmacological agent indicates that the candidate
pharmacological agent increases the signaling activity of murine
TLR9.
Murine TLR9 used in the methods of the invention can be added to an
assay mixture as an isolated polypeptide (where binding of a
candidate pharmaceutical agent is to be measured) or as a cell or
other membrane-encapsulated space which includes a murine TLR9
polypeptide. In the latter assay configuration, the cell or other
membrane-encapsulated space can contain the murine TLR9 as a
polypeptide or as a nucleic acid (e.g., a cell transfected with an
expression vector containing a murine TLR9). In the assays
described herein, the murine TLR9 polypeptide can be produced
recombinantly, isolated from biological extracts, or synthesized in
vitro. Murine TLR9 polypeptides encompass chimeric proteins
comprising a fusion of a murine TLR9 polypeptide with another
polypeptide, e.g., a polypeptide capable of providing or enhancing
protein-protein binding, enhancing signaling capability,
facilitating detection, or enhancing stability of the murine TLR9
polypeptide under assay conditions. A polypeptide fused to a murine
TLR9 polypeptide or fragment thereof may also provide means of
readily detecting the fusion protein, e.g., by immunological
recognition or by fluorescent labeling.
The assay mixture also comprises a candidate pharmacological agent.
Typically, a plurality of assay mixtures are run in parallel with
different agent concentrations to obtain a different response to
the various concentrations. Typically, one of these concentrations
serves as a negative control, i.e., at zero concentration of agent
or at a concentration of agent below the limits of assay detection.
Candidate pharmaceutical agents encompass numerous chemical
classes, although typically they are organic compounds. Preferably,
the candidate pharmacological agents are small organic compounds,
i.e., those having a molecular weight of more than 50 yet less than
about 2500. Polymeric candidate agents can have higher molecular
weights, e.g., oligonucleotides in the range of about 2500 to about
12,500. Candidate agents comprise functional chemical groups
necessary for structural interactions with polypeptides, and may
include at least an amine, carbonyl, hydroxyl or carboxyl group,
preferably at least two of the functional chemical groups and more
preferably at least three of the functional chemical groups. The
candidate agents can comprise cyclic carbon or heterocyclic
structure and/or aromatic or polyaromatic structures substituted
with one or more of the above-identified functional groups.
Candidate agents also can be biomolecules such as nucleic acids,
peptides, saccharides, fatty acids, sterols, isoprenoids, purines,
pyrimidines, derivatives or structural analogs of the above, or
combinations thereof and the like. Where the agent is a nucleic
acid, the agent typically is a DNA or RNA molecule, although
modified nucleic acids having non-natural bonds or subunits are
also contemplated.
Candidate agents are obtained from a wide variety of sources,
including libraries of natural, synthetic, or semisynthetic
compounds, or any combination thereof. For example, numerous means
are available for random and directed synthesis of a wide variety
of organic compounds and biomolecules, including expression of
randomized oligonucleotides, synthetic organic combinatorial
libraries, phage display libraries of random peptides, and the
like. Alternatively, libraries of natural compounds in the form of
bacterial, fungal, plant and animal extracts are available or
readily produced. Additionally, natural and synthetically produced
libraries and compounds can be readily modified through
conventional chemical, physical, and biochemical means. Further,
known pharmacological agents may be subjected to directed or random
chemical modifications such as acylation, alkylation,
esterification, amidification, etc., to produce structural analogs
of the agents.
Therefore, a source of candidate agents are libraries of molecules
based on known TLR9 ligands, e.g., CpG oligonucleotides shown
herein to interact with TLR9, in which the structure of the ligand
is changed at one or more positions of the molecule to contain more
or fewer chemical moieties or different chemical moieties. The
structural changes made to the molecules in creating the libraries
of analog inhibitors can be directed, random, or a combination of
both directed and random substitutions and/or additions. One of
ordinary skill in the art in the preparation of combinatorial
libraries can readily prepare such libraries based on existing TLR9
ligands.
A variety of other reagents also can be included in the mixture.
These include reagents such as salts, buffers, neutral proteins
(e.g., albumin), detergents, etc. which may be used to facilitate
optimal protein-protein and/or protein-nucleic acid binding. Such a
reagent may also reduce non-specific or background interactions of
the reaction components. Other reagents that improve the efficiency
of the assay such as protease inhibitors, nuclease inhibitors,
antimicrobial agents, and the like may also be used.
The mixture of the foregoing assay materials is incubated under
conditions whereby, but for the presence of the candidate
pharmacological agent, the murine TLR9 mediates TLR/IL-1R
signaling. For determining the binding of a candidate
pharmaceutical agent to a murine TLR9, the mixture is incubated
under conditions which permit binding. The order of addition of
components, incubation temperature, time of incubation, and other
parameters of the assay may be readily determined. Such
experimentation merely involves optimization of the assay
parameters, not the fundamental composition of the assay.
Incubation temperatures typically are between 4.degree. C. and
40.degree. C. Incubation times preferably are minimized to
facilitate rapid, high throughput screening, and typically are
between 1 minute and 10 hours.
After incubation, the level of signaling or the level of specific
binding between the murine TLR9 polypeptide and the candidate
pharmaceutical agent is detected by any convenient method available
to the user. For cell-free binding type assays, a separation step
is often used to separate bound from unbound components. The
separation step may be accomplished in a variety of ways. For
example, separation can be accomplished in solution, or,
conveniently, at least one of the components is immobilized on a
solid substrate, from which the unbound components may be easily
separated. The solid substrate can be made of a wide variety of
materials and in a wide variety of shapes, e.g., microtiter plate,
microbead, dipstick, resin particle, etc. The substrate preferably
is chosen to maximize signal-to-noise ratios, primarily to minimize
background binding, as well as for ease of separation and cost.
Separation may be effected for example, by removing a bead or
dipstick from a reservoir, emptying or diluting a reservoir such as
a microtiter plate well, rinsing a bead, particle, chromatographic
column or filter with a wash solution or solvent. The separation
step preferably includes multiple rinses or washes. For example,
when the solid substrate is a microtiter plate, the wells may be
washed several times with a washing solution, which typically
includes those components of the incubation mixture that do not
participate in specific bindings such as salts, buffer, detergent,
non-specific protein, etc. Where the solid substrate is a magnetic
bead, the beads may be washed one or more times with a washing
solution and isolated using a magnet.
Detection may be effected in any convenient way for cell-based
assays such as measurement of an induced polypeptide within, on the
surface of, or secreted by the cell. Examples of detection methods
useful in such cell-based assays include fluorescence-activated
cell sorting (FACS) analysis, bioluminescence, fluorescence,
enzyme-linked immunosorbent assay (ELISA), reverse
transcriptase-polymerase chain reaction (RT-PCR), and the like.
A variety of methods may be used to detect the label, depending on
the nature of the label and other assay components. For example,
the label may be detected while bound to the solid substrate or
subsequent to separation from the solid substrate. Labels may be
directly detected through optical or electron density, radioactive
emissions, nonradiative energy transfers, etc., or indirectly
detected with antibody conjugates, streptavidin-biotin conjugates,
etc. Methods for detecting the labels are well known in the
art.
The murine TLR9 binding agent may also be an antibody or a
functionally active antibody fragment. Antibodies are well known to
those of ordinary skill in the science of immunology. As used
herein, the term "antibody" means not only intact antibody
molecules but also fragments of antibody molecules retaining
specific target binding ability. Such fragments are also well known
in the art and are regularly employed both in vitro and in vivo. In
particular, as used herein, the term "antibody" means not only
intact immunoglobulin molecules but also the well-known active
fragments F(ab').sub.2 and Fab. F(ab').sub.2 and Fab fragments
which lack the Fc fragment of intact antibody clear more rapidly
from the circulation and may have less non-specific tissue binding
than an intact antibody (Wahl R L et al., J Nucl Med 24:316-325
(1983)).
Monoclonal antibodies may be made by any of the methods known in
the art utilizing murine TLR9, or a fragment thereof, as an
immunogen. Alternatively the antibody may be a polyclonal antibody
specific for murine TLR9 which inhibits murine TLR9 activity. The
preparation and use of polyclonal antibodies are also known to one
of ordinary skill in the art.
Significantly, as is well known in the art, only a small portion of
an antibody molecule, the paratope, is involved in the binding of
the antibody to its epitope (see, in general, Clark, W. R. (1986)
The Experimental Foundations of Modern Immunology, Wiley &
Sons, Inc., New York; Roitt, I. (1991) Essential Immunology, 7th
Ed., Blackwell Scientific Publications, Oxford). The pFc' and Fc
regions, for example, are effectors of the complement cascade but
are not involved in antigen binding. An antibody from which the
pFc' region has been enzymatically cleaved, or which has been
produced without the pFc' region, designated an F(ab').sub.2
fragment, retains both of the antigen binding sites of an intact
antibody. Similarly, an antibody from which the Fc region has been
enzymatically cleaved, or which has been produced without the Fc
region, designated an Fab fragment, retains one of the antigen
binding sites of an intact antibody molecule. Proceeding further,
Fab fragments consist of a covalently bound antibody light chain
and a portion of the antibody heavy chain denoted Fd. The Fd
fragments are the major determinant of antibody specificity (a
single Fd fragment may be associated with up to ten different light
chains without altering antibody specificity) and Fd fragments
retain epitope-binding ability in isolation.
Within the antigen-binding portion of an antibody, as is well-known
in the art, there are complementarity determining regions (CDRs),
which directly interact with the epitope of the antigen, and
framework regions (FRs), which maintain the tertiary structure of
the paratope (see, in general, Clark, 1986; Roitt, 1991). In both
the heavy chain Fd fragment and the light chain of IgG
immunoglobulins, there are four framework regions (FR1 through FR4)
separated respectively by three complementarity determining regions
(CDR1 through CDR3). The CDRs, and in particular the CDR3 regions,
and more particularly the heavy chain CDR3, are largely responsible
for antibody specificity.
The sequences of the antigen-binding Fab' portion of the
anti-murine TLR9 monoclonal antibodies identified as being useful
according to the invention in the assays provided above, as well as
the relevant FR and CDR regions, can be determined using amino acid
sequencing methods that are routine in the art. It is well
established that non-CDR regions of a mammalian antibody may be
replaced with corresponding regions of non-specific or
hetero-specific antibodies while retaining the epitope specificity
of the original antibody. This technique is useful for the
development and use of "humanized" antibodies in which non-human
CDRs are covalently joined to human FR and/or Fc/pFc' regions to
produce a functional antibody. Techniques to humanize antibodies
are particularly useful when non-human animal (e.g., murine)
antibodies which inhibit murine TLR9 activity are identified. These
non-human animal antibodies can be humanized for use in the
treatment of a human subject in the methods according to the
invention. Examples of methods for humanizing a murine antibody are
provided in U.S. Pat. Nos. 4,816,567, 5,225,539, 5,585,089,
5,693,762 and 5,859,205. Other antibodies, including fragments of
intact antibodies with antigen-binding ability, are often referred
to as "chimeric" antibodies.
Thus, as will be apparent to one of ordinary skill in the art, the
present invention also provides for F(ab').sub.2 and Fab fragments
of an anti-murine TLR9 monoclonal antibody; chimeric antibodies in
which the Fc and/or FR and/or CDR1 and/or CDR2 and/or light chain
CDR3 regions of an anti-murine TLR9 antibody have been replaced by
homologous human or non-human sequences; chimeric F(ab').sub.2
fragment antibodies in which the FR and/or CDR1 and/or CDR2 and/or
light chain CDR3 regions of an anti-murine TLR9 antibody have been
replaced by homologous human or non-human sequences; and chimeric
Fab fragment antibodies in which the FR and/or CDR1 and/or CDR2
and/or light chain CDR3 regions have been replaced by homologous
human or non-human sequences.
According to the invention murine TLR9 inhibitors also include
"dominant negative" polypeptides derived from SEQ ID NO:3. A
dominant negative polypeptide is an inactive variant of a
polypeptide, which, by interacting with the cellular machinery,
displaces an active polypeptide from its interaction with the
cellular machinery or competes with the active polypeptide, thereby
reducing the effect of the active polypeptide. For example, a
dominant negative receptor which binds a ligand but does not
transmit a signal in response to binding of the ligand can reduce
the biological effect of expression of the receptor. As shown in
the Examples below, TLR9 polypeptides which incorporate the
substitution of histidine for proline at aa 915 (P915H mutation)
are functionally inactive and are dominant negative with respect to
the native TLR9 polypeptide.
The end result of the expression of a dominant negative murine TLR9
polypeptide of the invention in a cell is a reduction in TLR9
activity such as signaling through the TIR pathway. One of ordinary
skill in the art can assess the potential for a dominant negative
variant of a murine TLR9 polypeptide and, using standard
mutagenesis techniques, create one or more dominant negative
variant polypeptides. For example, given the teachings contained
herein of a murine TLR9 polypeptide, one of ordinary skill in the
art can modify the sequence of the murine TLR9 polypeptide by
site-specific mutagenesis, scanning mutagenesis, partial gene
deletion or truncation, and the like. See, e.g., U.S. Pat. No.
5,580,723 and Molecular Cloning: A Laboratory Manual, J. Sambrook,
et al., eds., Second Edition, Cold Spring Harbor Laboratory Press,
Cold Spring Harbor, N.Y., 1989. The skilled artisan then can test
the population of mutagenized polypeptides for diminution in murine
TLR9 activity and/or for retention of such an activity. Other
similar methods for creating and testing dominant negative variants
of a murine TLR9 polypeptide will be apparent to one of ordinary
skill in the art.
Each of the compositions according to this aspect of the invention
is useful for a variety of therapeutic and non-therapeutic
purposes. For example, the murine TLR9 nucleic acids of the
invention are useful as oligonucleotide probes. Such
oligonucleotide probes can be used herein to identify genomic or
cDNA library clones possessing an identical or substantially
similar nucleic acid sequence. A suitable oligonucleotide or set of
oligonucleotides, which is capable of hybridizing under stringent
hybridization conditions to the desired sequence, a variant or
fragment thereof, or an anti-sense complement of such an
oligonucleotide or set of oligonucleotides, can be synthesized by
means well known in the art (see, for example, Synthesis and
Application of DNA and RNA, S. A. Narang, ed., 1987, Academic
Press, San Diego, Calif.) and employed as a probe to identify and
isolate the desired sequence, variant or fragment thereof by
techniques known in the art. Techniques of nucleic acid
hybridization and clone identification are disclosed by Sambrook,
et al., Molecular Cloning: A Laboratory Manual, J. Sambrook, et
al., eds., Second Edition, Cold Spring Harbor Laboratory Press,
Cold Spring Harbor, N.Y., 1989.
To facilitate the detection of a desired nucleic acid sequence, or
variant or fragment thereof, whether for cloning purposes or for
the mere detection of the presence of the sequence, the
above-described probes may be labeled with a detectable group. Such
a detectable group may be any material having a detectable physical
or chemical property. Such materials have been well developed in
the field of nucleic acid hybridization and, in general, many
labels useful in such methods can be applied to the present
invention. Particularly useful are radioactive labels. Any
radioactive label may be employed which provides for an adequate
signal and has a sufficient half-life. If single stranded, the
oligonucleotide may be radioactively labeled using kinase
reactions. Alternatively, oligonucleotides are also useful as
nucleic acid hybridization probes when labeled with a
non-radioactive marker such as biotin, an enzyme or a fluorescent
group. See, for example, Leary J J et al., Proc Natl Acad Sci USA
80:4045 (1983); Renz M et al., Nucleic Acids Res 12:3435 (1984);
and Renz M, EMBO J 6:817 (1983).
Additionally, complements of the murine TLR9 nucleic acids can be
useful as antisense oligonucleotides, e.g., by delivering the
antisense oligonucleotide to an animal to induce a murine TLR9
"knockout" phenotype. The administration of antisense RNA probes to
block gene expression is discussed in Lichtenstein C, Nature
333:801-802 (1988).
Alternatively, the murine TLR9 nucleic acid of the invention can be
used to prepare a non-human transgenic animal. A "transgenic
animal" is an animal having cells that contain DNA which has been
artificially inserted into a cell, which DNA becomes part of the
genome of the animal which develops from that cell. Preferred
transgenic animals are primates, mice, rats, cows, pigs, horses,
goats, sheep, dogs and cats. Animals suitable for transgenic
experiments can be obtained from standard commercial sources such
as Charles River (Wilmington, Mass.), Taconic (Germantown, N.Y.),
Harlan (Indianapolis, Ind.), etc. Transgenic animals having a
particular property associated with a particular disease can be
used to study the effects of a variety of drugs and treatment
methods on the disease, and thus serve as genetic models for the
study of a number of human diseases. The invention, therefore,
contemplates the use of murine TLR9 knockout and transgenic animals
as models for the study of disorders involving TLR9-mediated
signaling. A variety of methods known to one of ordinary skill in
the art are available for the production of transgenic animals
associated with this invention.
Inactivation or replacement of the endogenous TLR9 gene can be
achieved by a homologous recombination system using embryonic stem
cells. The resultant transgenic non-human mammals having a
TLR9.sup.-/- knockout phenotype may be made transgenic for the
murine TLR9 and used as a model for screening compounds as
modulators (agonists or antagonists/inhibitors) of the murine TLR9.
In this manner, such therapeutic drugs can be identified.
Additionally, a normal or mutant version of murine TLR9 can be
inserted into the germ line to produce transgenic animals which
constitutively or inducibly express the normal or mutant form of
murine TLR9. These animals are useful in studies to define the role
and function of murine TLR9 in cells.
Generally, doses of active compounds would be from about 0.01 mg/kg
per day to 1000 mg/kg per day. It is expected that doses ranging
from 50-500 mg/kg will be suitable and in one or several
administrations per day. Lower doses will result from other forms
of administration, such as intravenous administration. In the event
that a response in a subject is insufficient at the initial doses
applied, higher doses (or effectively higher doses by a different,
more localized delivery route) may be employed to the extent that
patient tolerance permits. Multiple doses per day are contemplated
to achieve appropriate systemic levels of compound, although fewer
doses typically will be given when compounds are prepared as slow
release or sustained release medications.
The antagonists, agonists, nucleic acids, and polypeptides of
murine TLR9 useful according to the invention may be combined,
optionally, with a pharmaceutically acceptable carrier. Thus the
invention also provides pharmaceutical compositions and a method
for preparing the pharmaceutical compositions which contain
compositions of this aspect of the invention. The pharmaceutical
compositions include any one or combination of the antagonists,
agonists, nucleic acids and polypeptides of murine TLR9 useful
according to the invention and, optionally, a pharmaceutically
acceptable carrier. Each pharmaceutical composition is prepared by
selecting an antagonist, agonist, nucleic acid or polypeptide of
murine TLR9 useful according to the invention, as well as any
combination thereof, and, optionally, combining it with a
pharmaceutically acceptable carrier.
The term "pharmaceutically acceptable carrier" as used herein means
one or more compatible solid or liquid fillers, diluents or
encapsulating substances which are suitable for administration into
a human. The term "carrier" denotes an organic or inorganic
ingredient, natural or synthetic, with which the active ingredient
is combined to facilitate the application. The components of the
pharmaceutical compositions also are capable of being co-mingled
with the molecules of the present invention, and with each other,
in a manner such that there is no interaction which would
substantially impair the desired pharmaceutical efficacy.
The pharmaceutical compositions may contain suitable buffering
agents, including, without limitation: acetic acid in a salt;
citric acid in a salt; and phosphoric acid in a salt.
The pharmaceutical compositions also may contain, optionally,
suitable preservatives, such as benzalkonium chloride,
chlorobutanol, parabens, and thimerosal.
When administered, the pharmaceutical preparations of the invention
are applied in pharmaceutically acceptable amounts and in
pharmaceutically acceptable compositions. Such preparations may
routinely contain salts, buffering agents, preservatives,
compatible carriers, and optionally other therapeutic agents. When
used in medicine, the salts should be pharmaceutically acceptable,
but non-pharmaceutically acceptable salts may conveniently be used
to prepare pharmaceutically acceptable salts thereof and are not
excluded from the scope of the invention. Such pharmacologically
and pharmaceutically acceptable salts include, but are not limited
to, those prepared from the following acids: hydrochloric,
hydrobromic, sulfuric, nitric, phosphoric, maleic, acetic,
salicylic, citric, formic, malonic, succinic, and the like. Also,
pharmaceutically acceptable salts can be prepared as alkaline metal
or alkaline earth salts, such as sodium, potassium or calcium
salts.
A variety of administration routes are available. The particular
mode selected will depend, of course, upon the particular compound
selected, the severity of the condition being treated, and the
dosage required for therapeutic efficacy. The methods of the
invention, generally speaking, may be practiced using any mode of
administration that is medically acceptable, meaning any mode that
produces effective levels of the active compounds without causing
clinically unacceptable adverse effects. Such modes of
administration include oral, rectal, topical, nasal, intradermal,
or parenteral routes. The term "parenteral" includes, without
limitation, subcutaneous, transdermal, intravenous, intra-arterial,
intrathecal, intramuscular, intraperitoneal, mucosal (apart from
gastrointestinal mucosa), pulmonary, intralesional, and
infusion.
The pharmaceutical compositions may conveniently be presented in
unit dosage form and may be prepared by any of the methods
well-known in the art of pharmacy. All methods include the step of
bringing the active agent into association with a carrier which
constitutes one or more accessory ingredients. In general, the
compositions are prepared by uniformly and intimately bringing the
active compound into association with a liquid carrier, a finely
divided solid carrier, or both, and then, if necessary, shaping the
product.
Compositions suitable for oral administration may be presented as
discrete units, such as capsules, tablets, lozenges, each
containing a predetermined amount of the active compound. Other
compositions include suspensions in aqueous liquids or non-aqueous
liquids such as a syrup, elixir or an emulsion.
Compositions suitable for parenteral administration conveniently
comprise a sterile aqueous preparation of the antagonists,
agonists, nucleic acids, or polypeptides of murine TLR9, which is
preferably isotonic with the blood of the recipient. This aqueous
preparation may be formulated according to known methods using
suitable dispersing or wetting agents and suspending agents. The
sterile injectable preparation also may be a sterile injectable
solution or suspension in a non-toxic parenterally acceptable
diluent or solvent, for example, as a solution in 1,3-butane diol.
Among the acceptable vehicles and solvents that may be employed are
water, Ringer's solution, and isotonic sodium chloride solution. In
addition, sterile, fixed oils are conventionally employed as a
solvent or suspending medium. For this purpose any bland fixed oil
may be employed including synthetic mono- or di-glycerides. In
addition, fatty acids such as oleic acid may be used in the
preparation of injectables. Carrier formulation suitable for oral,
subcutaneous, intravenous, intrathecal, intramuscular, etc.
administrations can be found in Remington's Pharmaceutical
Sciences, Mack Publishing Co., Easton, Pa.
Other delivery systems can include time-release, delayed release or
sustained release delivery systems such as the biological/chemical
vectors is discussed above. Such systems can avoid repeated
administrations of the active compound, increasing convenience to
the subject and the physician. Many types of release delivery
systems are available and known to those of ordinary skill in the
art. Use of a long-term sustained release implant may be desirable.
Long-term release, are used herein, means that the implant is
constructed and arranged to delivery therapeutic levels of the
active ingredient for at least 30 days, and preferably 60 days.
Long-term sustained release implants are well-known to those of
ordinary skill in the art and include some of the release systems
described above.
In another aspect the invention involves the identification of
cDNAs encoding mouse TLR7 and mouse TLR8, referred to herein as
murine TLR7 and murine TLR8 and, equivalently, mTLR7 and mTLR8,
respectively. The nucleotide sequence of the cDNA for murine TLR7
is presented as SEQ ID NO:173, the coding region of the cDNA for
murine TLR7 is presented as SEQ ID NO:174, and the amino acid
sequence of the murine TLR7 is presented as SEQ ID NO:175. The
closely related human TLR7 (equivalently, hTLR7) was previously
deposited in GenBank under accession numbers AF245702 and AF240467.
The nucleotide sequence of the cDNA for murine TLR7 presented as
SEQ ID NO:173 is 3357 nucleotides long and includes the ORF
spanning bases 117-3266, presented as SEQ ID NO:174, which spans
3150 nucleotides (excluding the stop codon). The amino acid
sequence of the murine TLR7 presented as SEQ ID NO:175 is 1050
amino acids long.
The nucleotide sequence of the cDNA for murine TLR8 is presented as
SEQ ID NO:190, the coding region of the cDNA for murine TLR8 is
presented as SEQ ID NO:191, and the amino acid sequence of the
murine TLR8 is presented as SEQ ID NO:192. The closely related
human TLR8 (equivalently, hTLR8) was previously deposited in
GenBank under accession numbers AF245703 and AF246971.
Like both human and murine TLR9, human TLR7 and human TLR8 each
contains one CXXC motif and one MBD motif. The hTLR7 CXXC motif
contains amino acids 258-273, and the hTLR8 CXXC motif contains
amino acids 255-270.
CXXC motif: GNCXXCXXXXXXCXXC SEQ ID NO:196 hTLR9: GNCRRCDHAPNPCMEC
SEQ ID NO:197 mTLR9: GNCRRCDHAPNPCMIC SEQ ID NO:198 hTLR7:
GNCPRCYNAPFPCAPC SEQ ID NO:199 mTLR7: GNCPRCYNVPYPCTPC SEQ ID
NO:200 hTLR8: GNCPRCFNAPFPCVPC SEQ ID NO:201 mTLR8:
GNCPRCYNAPFPCTPC SEQ ID NO:202
Also like human and murine TLR9, human TLR7 and TLR8 also have a
single MBD motif. The the hTLR7 MBD motif spans amino acids
545-575, and the hTLR8 MBD motif amino acids spans 533-563.
MBD motif SEQ ID NO:125 MBD-1
R-XXXXXXX-R-X-D-X-Y-XXXXXXXXX-R-S-XXXXXX-Y SEQ ID NO:126 hTLR9
Q-XXXXXXX-K-X-D-X-Y-XXXXXXXXX-R-L-XXXXXX-Y SEQ ID NO:127 mTLR9
Q-XXXXXXX-K-X-D-X-Y-XXXXXXXXX-Q-L-XXXXXX-Y SEQ ID NO:203 hTLR7
R-XXXXXXX-R-X-D-X-L-XXXXXXXXX-K-L-XXXXXX-S SEQ ID NO:204 mTLR7
R-XXXXXXX-R-X-D-X-L-XXXXXXXXX-S-L-XXXXXX-S SEQ ID NO:205 hTLR8
K-XXXXXXX-R-X-D-X-D-XXXXXXXXX-D-L-XXXXXX-Y SEQ ID NO:206 mTLR8
K-XXXXXXX-R-X-D-X-D-XXXXXXXXX-D-L-XXXXXX-H SEQ ID NO:212 hTLR7
R-YLDFSNN-R-L-D-L-L-HSTAFEELH-K-L-EVLDIS-S SEQ ID NO:213 mTLR7
R-YLDFSNN-R-L-D-L-L-YSTAFEELQ-S-L-EVLDLS-S SEQ ID NO:214 hTLR8
K-YLDLTNN-R-L-D-F-D-NASALTELS-D-L-EVLDLS-Y SEQ ID NO:215 mTLR8
K-YLDLTNN-R-L-D-F-D-DNNAFSDLH-D-L-EVLDLS-H
The core D-X-Y in the MBD motif is involved in CpG binding of the
MBD-1 protein and is conserved in TLR9 but only partially conserved
in TLR8 and TLR7 (Y to D or L). The other mismatches are highly or
moderately conserved; example R to K, Q, or D. These changes could
explain MBD-1 as a methyl-CpG binder and TLR9 as a binder for
CpG-DNA. The modification in the core sequence (D-X-Y) in hTLR7
(D-X-L) and TLR8 (D-X-D) is likely a structural basis for the
recognition of different nucleic acid motifs. Combined with the
presence of a CXXC domain TLR7 and TLR8 appear certainly to be
nucleic acid binding receptors relevant to the innate immune system
and thus clinical value.
The invention involves in one aspect murine TLR7 and murine TLR8
nucleic acids and polypeptides, as well as therapeutics relating
thereto. The invention also embraces isolated functionally
equivalent variants, useful analogs and fragments of the foregoing
murine TLR7 and murine TLR8 nucleic acids and polypeptides;
complements of the foregoing murine TLR7 and murine TLR8 nucleic
acids; and molecules which selectively bind the foregoing murine
TLR7 and murine TLR8 nucleic acids and polypeptides.
The murine TLR7 and murine TLR8 nucleic acids and polypeptides of
the invention are isolated. The term "isolated," with respect to
murine TLR7 and murine TLR8 nucleic acids and polypepetides, has
the same meaning as used elsewhere herein.
As used herein a murine TLR7 nucleic acid refers to an isolated
nucleic acid molecule which codes for a murine TLR7 polypeptide.
Such nucleic acid molecules code for murine TLR7 polypeptides which
include the sequence of SEQ ID NO:175 and fragments thereof. The
nucleic acid molecules include the nucleotide sequences of SEQ ID
NO:173, SEQ ID NO:174, and nucleotide sequences which differ from
the sequences of SEQ ID NO:173 and SEQ ID NO:174 in codon sequence
due to the degeneracy of the genetic code.
Also as used herein a murine TLR8 nucleic acid refers to an
isolated nucleic acid molecule which codes for a murine TLR8
polypeptide. Such nucleic acid molecules code for murine TLR8
polypeptides which include the sequences of SEQ ID NO:193, and
fragments thereof. The nucleic acid molecules include the
nucleotide sequences of SEQ ID NO:190, SEQ ID NO:191, and
nucleotide sequences which differ from the sequences of SEQ ID
NO:190 and SEQ ID NO:191 in codon sequence due to the degeneracy of
the genetic code.
The murine TLR7 and murine TLR8 nucleic acids of the invention also
include alleles as well as fragments of the foregoing nucleic
acids. Such fragments can be used, for example, as probes in
hybridization assays and as primers in a polymerase chain reaction.
Preferred murine TLR7 nucleic acids include the nucleic acid
sequence of SEQ ID NO:173 and SEQ ID NO:174. Preferred murine TLR8
nucleic acids include the nucleic acid sequence of SEQ ID NO:190
and SEQ ID NO:191. Complements of the foregoing nucleic acids also
are embraced by the invention.
As used herein a murine TLR7 nucleic acid or murine TLR7
polypeptide also embraces homologues and alleles of murine TLR7.
Likewise, as used herein a murine TLR8 nucleic acid or murine TLR8
polypeptide also embraces homologues and alleles of murine TLR8.
Homologues and alleles of murine TLR7 and murine TLR8 comply with
the degrees of nucleotide and amino acid identity as previously set
forth herein in reference to homologues and alleles of murine
TLR9.
Alleles of the murine TLR7 and murine TLR8 nucleic acids of the
invention can be identified by conventional techniques. For
example, alleles of murine TLR7 can be isolated by hybridizing a
probe which includes at least a fragment of SEQ ID NO:173 or SEQ ID
NO:174 under stringent conditions with a cDNA library and selecting
positive clones. Thus, an aspect of the invention is those nucleic
acid sequences which code for murine TLR7 polypeptides and which
hybridize to a nucleic acid molecule consisting of SEQ ID NO:173 or
SEQ ID NO:174 under stringent conditions. Likewise, an aspect of
the invention is those nucleic acid sequences which code for murine
TLR8 polypeptides and which hybridize to a nucleic acid molecule
consisting of SEQ ID NO:190 or SEQ ID NO:191 under stringent
conditions. Stringent conditions in this context has the same
meaning as described elsewhere herein, including the use of a
suitable hybridization buffer and a temperature of about 65.degree.
C.
In screening for murine TLR7 or murine TLR8 nucleic acids, a
Southern blot may be performed using the stringent conditions
previously described herein, together with a radioactive probe.
After washing the membrane to which the DNA is finally transferred,
the membrane can be placed against X-ray film to detect the
radioactive signal. Corresponding non-radioactive methods are also
well known in the art and can be used to similar effect.
The murine TLR7 and murine TLR8 nucleic acids of the invention also
include degenerate nucleic acids which include alternative codons
to those present in the native materials, as previously described
herein.
The invention also provides modified nucleic acid molecules which
include additions, substitutions and deletions of one or more
nucleotides. The modified nucleic acid molecules according to this
aspect of the invention exclude fully native human TLR7 (SEQ ID
NO:168, SEQ ID NO:169, GenBank Accession No. AF245702, and GenBank
Accession No. AF240467) and fully native human TLR8 nucleic acid
molecules (SEQ ID NO:182, SEQ ID NO:183, GenBank Accession No.
AF245703, and GenBank Accession No.AF246971). In preferred
embodiments, these modified nucleic acid molecules and/or the
polypeptides they encode retain at least one activity or function
of the unmodified nucleic acid molecule and/or the polypeptides,
such as signaling activity, etc. In certain embodiments, the
modified nucleic acid molecules encode modified polypeptides,
preferably polypeptides having conservative amino acid
substitutions as are described elsewhere herein. The modified
nucleic acid molecules are structurally related to the unmodified
nucleic acid molecules and in preferred embodiments are
sufficiently structurally related to the unmodified nucleic acid
molecules so that the modified and unmodified nucleic acid
molecules hybridize under stringent conditions known to one of
skill in the art.
The invention also provides isolated fragments of nucleotide
sequences for murine TLR7 (SEQ ID NO:173 and SEQ ID NO:174) and for
murine TLR8 (SEQ ID NO:190 and SEQ ID NO:191). The fragments can be
used as probes in Southern blot assays to identify such nucleic
acids, or can be used in amplification assays such as those
employing PCR. Smaller fragments are those comprising 12, 13, 14,
15, 16, 17, 18, 20, 22, 25, 30, 40, 50, or 75 nucleotides, and
every integer therebetween, and are useful, e.g., as primers for
nucleic acid amplification procedures. As known to those skilled in
the art, larger probes such as 200, 250, 300, 400 or more
nucleotides are preferred for certain uses such as Southern blots,
while smaller fragments will be preferred for uses such as PCR.
Fragments also can be used to produce fusion proteins for
generating antibodies or determining binding of the polypeptide
fragments. Likewise, fragments can be employed to produce non-fused
fragments of the murine TLR7 and murine TLR8 polypeptides, useful,
for example, in the preparation of antibodies, in immunoassays, and
the like. The foregoing nucleic acid fragments further can be used
as antisense molecules to inhibit the expression of murine TLR7 and
murine TLR8 nucleic acids and polypeptides.
The invention also includes functionally equivalent variants of the
murine TLR7 and murine TLR8, which include variant nucleic acids
and polypeptides which retain one or more of the functional
properties of the murine TLR7 and murine TLR8. Preferably such
variants include the murine-specific N-terminal domain.
Functionally equivalent variants also include a murine TLR7 or
murine TLR8 which has had a portion (e.g., of the N-terminus)
removed or replaced by a similar domain from another TLR (e.g., a
"domain-swapping" variant). Examples of such domain-swapping
variants include those involving swapping a TLR7 domain from
another species and swapping a TLR domain from another TLR.
Other functionally equivalent variants will be known to one of
ordinary skill in the art, as will be methods for preparing such
variants. The activity of a functionally equivalent variant can be
determined using the methods provided herein, and in references
that have described assays using other TLRs and TLRs of other
species. Such variants are useful, inter alia, for evaluating
bioavailability of drugs, in assays for identification of compounds
which bind to and/or regulate the signaling function of the murine
TLR7 and murine TLR8, and for determining the portions of the
murine TLR7 and murine TLR8 which are required for signaling
activity.
Variants which are non-functional also can be prepared as described
above. Such variants are useful, for example, as negative controls
in experiments testing TLR7 and TLR8 signaling activity. Examples
of non-functional variants include those incorporating a truncation
or mutation of amino acids deemed critical to ligand binding or
signaling activity.
In certain embodiments a murine TLR7 or murine TLR8 nucleic acid is
operably linked to a gene expression sequence which can direct the
expression of the murine TLR7 or murine TLR8 nucleic acid within a
eukaryotic or prokaryotic cell. The terms "gene expression
sequence" and "operably linked" are as previously described
herein.
The murine TLR7 and murine TLR8 nucleic acid molecules and the
murine TLR7 and murine TLR8 polypeptides of the invention can be
delivered to a eukaryotic or prokaryotic cell alone or in
association with a vector. As applied to murine TLR7 and murine
TLR8 nucleic acid molecules, a "vector" is any vehicle capable of
facilitating: (1) delivery of a murine TLR7 or murine TLR8 nucleic
acid or polypeptide to a target cell, (2) uptake of a murine TLR7
or murine TLR8 nucleic acid or polypeptide by a target cell, or (3)
expression of a murine TLR7 or murine TLR8 nucleic acid molecule or
polypeptide in a target cell.
In addition to the biological vectors, chemical/physical vectors
may be used to deliver a murine TLR7 or murine TLR8 nucleic acid or
polypeptide to a target cell and facilitate uptake thereby. As used
herein with respect to a murine TLR7 or murine TLR8 nucleic acid or
polypeptide, a "chemical/physical vector" refers to a natural or
synthetic molecule, other than those derived from bacteriological
or viral sources, capable of delivering the isolated murine TLR7 or
murine TLR8 nucleic acid or polypeptide to a cell.
Other exemplary compositions that can be used to facilitate uptake
by a target cell of the murine TLR7 or murine TLR8 nucleic acids
include calcium phosphate and other chemical mediators of
intracellular transport, microinjection compositions,
electroporation and homologous recombination compositions (e.g.,
for integrating a murine TLR7 or murine TLR8 nucleic acid into a
preselected location within a target cell chromosome).
It will also be recognized that the invention embraces the use of
the murine TLR7 and murine TLR8 cDNA sequences in expression
vectors to transfect host cells and cell lines, be these
prokaryotic (e.g., E. coli), or eukaryotic (e.g., 293 fibroblast
cells (ATCC, CRL-1573), MonoMac-6, THP-1, U927, CHO cells, COS
cells, yeast expression systems and recombinant baculovirus
expression in insect cells). Especially useful are mammalian cells
such as human, pig, goat, primate, rodent, guinea pig, etc. They
may be of a wide variety of tissue types, and include primary cells
and cell lines. The expression vectors require that the pertinent
sequence, i.e., those nucleic acids described supra, be operably
linked to a promoter.
The invention also provides isolated murine TLR7 and isolated
murine TLR8 polypeptides which include the amino acid sequences of
SEQ ID NO:175, SEQ ID NO:192, and fragments thereof, encoded by the
murine TLR7 and murine TLR8 nucleic acids described above. Murine
TLR7 and murine TLR8 polypeptides also embrace alleles,
functionally equivalent variants and analogs (those non-allelic
polypeptides which vary in amino acid sequence from the disclosed
murine TLR7 and murine TLR8 polypeptides by 1, 2, 3, 4, 5, or more
amino acids) provided that such polypeptides retain murine TLR7 or
murine TLR8 activity. Non-functional variants also are embraced by
the invention; these are useful as antagonists of TLR7 and TLR8
signaling function, as negative controls in assays, and the like.
Such alleles, variants, analogs and fragments are useful, for
example, alone or as fusion proteins for a variety of purposes
including as a component of assays.
The invention also embraces variants of the murine TLR7 and murine
TLR8 polypeptides described above. Modifications which create a
murine TLR7 variant or murine TLR8 variant can be made to a murine
TLR7 or murine TLR8 polypeptide for a variety of reasons, including
1) to reduce or eliminate an activity of a murine TLR7 or murine
TLR8 polypeptide, such as signaling; 2) to enhance a property of a
murine TLR7 or murine TLR8 polypeptide, such as signaling, binding
affinity for nucleic acid ligand or other ligand molecule, protein
stability in an expression system, or the stability of
protein-protein binding; 3) to provide a novel activity or property
to a murine TLR7 or murine TLR8 polypeptide, such as addition of an
antigenic epitope or addition of a detectable moiety, e.g.,
luciferase, FLAG peptide, GFP; 4) to establish that an amino acid
substitution does or does not affect molecular signaling activity;
or 5) reduce immunogenicity. Modifications to a murine TLR7 or
murine TLR8 polypeptide are typically made to the nucleic acid
which encodes the murine TLR7 or murine TLR8 polypeptide, and can
include deletions, point mutations, truncations, amino acid
substitutions and additions of amino acids or non-amino acid
moieties. Alternatively, modifications can be made directly to the
polypeptide, such as by cleavage, addition of a linker molecule,
addition of a detectable moiety (for example, biotin, fluorophore,
radioisotope, enzyme, or peptide), addition of a fatty acid, and
the like.
Modifications also embrace fusion proteins comprising all or part
of the murine TLR7 or murine TLR8 amino acid sequence.
Variants include murine TLR7 and murine TLR8 polypeptides which are
modified specifically to alter a feature of each polypeptide
unrelated to its physiological activity. For example, cysteine
residues can be substituted or deleted to prevent unwanted
disulfide linkages. Similarly, certain amino acids can be changed
to enhance expression of a murine TLR7 or murine TLR8 polypeptide
by eliminating proteolysis by proteases in an expression system
(e.g., dibasic amino acid residues in yeast expression systems in
which KEX2 protease activity is present).
Mutations of a nucleic acid which encode a murine TLR7 or murine
TLR8 polypeptide preferably preserve the amino acid reading frame
of the coding sequence, and preferably do not create regions in the
nucleic acid which are likely to hybridize to form secondary
structures, such as hairpins or loops, which can be deleterious to
expression of the variant polypeptide. Methods of making mutations
of murine TLR7 or murine TLR8 are as described elsewhere herein
with reference to making mutations of murine TLR9.
The activity of variants of murine TLR7 and murine TLR8
polypeptides can be tested by cloning the gene encoding the variant
murine TLR7 or murine TLR8 polypeptide into a prokaryotic or
eukaryotic (e.g., mammalian) expression vector, introducing the
vector into an appropriate host cell, expressing the variant murine
TLR7 or murine TLR8 polypeptide, and testing for a functional
capability of the murine TLR7 or murine TLR8 polypeptides as
disclosed herein.
The skilled artisan will also realize that conservative amino acid
substitutions may be made in murine TLR7 and murine TLR8
polypeptides to provide functionally equivalent variants of the
foregoing polypeptides, i.e., variants which retain the functional
capabilities of the murine TLR7 and murine TLR8 polypeptides.
A variety of methodologies well known to the skilled practitioner
can be utilized to obtain isolated murine TLR7 and murine TLR8
polypeptide molecules, as previously described in reference to
murine TLR9 polypeptides.
The invention as described herein has a number of uses, some of
which are described elsewhere herein. For example, the invention
permits isolation of the murine TLR7 and the murine TLR8
polypeptide molecules by, e.g., expression of a recombinant nucleic
acid to produce large quantities of polypeptide which may be
isolated using standard protocols. As another example, the
isolation of the murine TLR7 gene makes it possible for murine TLR7
to be used in methods for assaying molecular interactions involving
TLR7.
The invention also embraces agents which bind selectively to the
murine TLR7 or murine TLR8 nucleic acid molecules or polypeptides
as well as agents which bind to variants and fragments of the
polypeptides and nucleic acids as described herein. The agents
include polypeptides which bind to murine TLR7 or murine TLR8, and
antisense nucleic acids, both of which are described in greater
detail below. Some agents can inhibit or increase murine
TLR7-mediated signaling activity (antagonists and agonists,
respectively), and some can inhibit or increase murine
TLR8-mediated signaling activity.
In one embodiment the murine TLR7 inhibitor is an antisense
oligonucleotide that selectively binds to a murine TLR7 nucleic
acid molecule, to reduce the expression of murine TLR7 (or TLR7 of
another species) in a cell. This is desirable in virtually any
medical condition wherein a reduction of TLR7 signaling activity is
desirable. Based upon SEQ ID NO:173 and SEQ ID NO:174, or upon
allelic or homologous genomic and/or cDNA sequences, one of skill
in the art can easily choose and synthesize any of a number of
appropriate antisense molecules for use in accordance with the
present invention.
In one embodiment the murine TLR8 inhibitor is an antisense
oligonucleotide that selectively binds to a murine TLR8 nucleic
acid molecule, to reduce the expression of murine TLR8 (or TLR8 of
another species) in a cell. This is desirable in virtually any
medical condition wherein a reduction of TLR8 signaling activity is
desirable. Based upon SEQ ID NO:190 and SEQ ID NO:191, or upon
allelic or homologous genomic and/or cDNA sequences, one of skill
in the art can easily choose and synthesize any of a number of
appropriate antisense molecules for use in accordance with the
present invention.
Anti sense oligonucleotides for murine TLR7 or murine TLR8 can
include "natural" and "modified" oligonucleotides as previously
described herein.
Agents which bind murine TLR7 or murine TLR8 also include binding
peptides and other molecules which bind to the murine TLR7 or
murine TLR8 polypeptide and complexes containing the murine TLR7 or
murine TLR8 polypeptide, respectively. When the binding molecules
are inhibitors, the molecules bind to and inhibit the activity of
murine TLR7 or murine TLR8. When the binding molecules are
activators, the molecules bind to and increase the activity of
murine TLR7 or murine TLR8. To determine whether a murine TLR7 or
murine TLR8 binding agent binds to murine TLR7 or murine TLR8, any
known binding assay may be employed. For example, the binding agent
may be immobilized on a surface and then contacted with a labeled
murine TLR7 or murine TLR8 polypeptide. The amount of murine TLR7
or murine TLR8 which interacts with the murine TLR7 or murine TLR8
binding agent, or the amount which does not bind to the murine TLR7
or murine TLR8 binding agent, may then be quantitated to determine
whether the murine TLR7 or murine TLR8 binding agent binds to
murine TLR7 or murine TLR8.
The murine TLR7 or murine TLR8 binding agents include molecules of
numerous size and type that bind selectively or preferentially to
murine TLR7 or murine TLR8 polypeptides, and to complexes involving
murine TLR7 or murine TLR8 polypeptides and their binding partners.
These molecules may be derived from a variety of sources. For
example, murine TLR7 or murine TLR8 binding agents can be provided
by screening degenerate peptide libraries which can be readily
prepared in solution, in immobilized form or as phage display
libraries. Combinatorial libraries also can be synthesized of
peptides containing one or more amino acids. Libraries further can
be synthesized of peptoids and non-peptide synthetic moieties.
Exemplary methods useful for identifying murine TLR7 and murine
TLR8 binding peptides are analogous to those described herein with
reference to methods for identifying murine TLR9 binding peptides
murine, and thus are not repeated here.
Therefore the invention generally provides efficient methods of
identifying pharmacological agents or lead compounds for agents
useful in the treatment of conditions associated with TLR7 and TLR8
activity, and the compounds and agents so identified. Generally,
the screening methods involve assaying for compounds which inhibit
or enhance the expression of or signaling through murine TLR7 or
murine TLR8. Such methods are adaptable to automated, high
throughput screening of compounds.
A variety of assays for pharmacological agents are provided,
including labeled in vitro protein binding assays, signaling assays
using detectable molecules, etc. For example, protein binding
screens are used to rapidly examine the binding of candidate
pharmacological agents to a murine TLR7 or murine TLR8. The
candidate pharmacological agents can be derived from, for example,
combinatorial peptide or nucleic acid libraries. Convenient
reagents for such assays are known in the art. An exemplary
cell-based assay of signaling involves contacting a cell having a
murine TLR7 or murine TLR8 with a candidate pharmacological agent
under conditions whereby the induction of a detectable molecule can
occur. A reduced degree of induction of the detectable molecule in
the presence of the candidate pharmacological agent indicates that
the candidate pharmacological agent reduces the signaling activity
of murine TLR7 or murine TLR8. An increased degree of induction of
the detectable molecule in the presence of the candidate
pharmacological agent indicates that the candidate pharmacological
agent increases the signaling activity of murine TLR7 or murine
TLR8.
Murine TLR7 and murine TLR8 used in the methods of the invention
can be added to an assay mixture as an isolated polypeptide (where
binding of a candidate pharmaceutical agent is to be measured) or
as a cell or other membrane-encapsulated space which includes a
murine TLR7 or murine TLR8 polypeptide. In the latter assay
configuration, the cell or other membrane-encapsulated space can
contain the murine TLR7 or murine TLR8 as a polypeptide or as a
nucleic acid (e.g., a cell transfected with an expression vector
containing a nucleic acid molecule encoding murine TLR7). In the
assays described herein, the murine TLR7 or murine TLR8 polypeptide
can be produced recombinantly, isolated from biological extracts,
or synthesized in vitro. Murine TLR7 or murine TLR8 polypeptides
encompass chimeric proteins comprising a fusion of a murine TLR7 or
murine TLR8 polypeptide with another polypeptide, e.g., a
polypeptide capable of providing or enhancing protein-protein
binding, enhancing signaling capability, facilitating detection, or
enhancing stability of the murine TLR7 or murine TLR8 polypeptide
under assay conditions. A polypeptide fused to a murine TLR7 or
murine TLR8 polypeptide or fragment thereof may also provide means
of readily detecting the fusion protein, e.g., by immunological
recognition or by fluorescent labeling.
The assay mixture also comprises a candidate pharmacological agent,
as previously described in reference to murine TLR9. Candidate
pharmacologic agents are obtained from a wide variety of sources,
including libraries of natural, synthetic, or semisynthetic
compounds, or any combination thereof. Presently, natural ligands
of murine TLR7 and murine TLR8 are unknown, but they appear not to
include CpG-ODN.
A variety of other reagents also can be included in the assay
mixture. These include reagents such as salts, buffers, neutral
proteins (e.g., albumin), detergents, etc. which may be used to
facilitate optimal protein-protein and/or protein-nucleic acid
binding. Such a reagent may also reduce non-specific or background
interactions of the reaction components. Other reagents that
improve the efficiency of the assay such as protease inhibitors,
nuclease inhibitors, antimicrobial agents, and the like may also be
used.
The mixture of the foregoing assay materials is incubated under
conditions whereby, but for the presence of the candidate
pharmacological agent, the murine TLR7 or murine TLR8 mediates
TLR7-mediated or TLR8-mediated signaling, preferably TLR/IL-1R
signaling. For determining the binding of a candidate
pharmaceutical agent to a murine TLR7 or murine TLR8, the mixture
is incubated under conditions which permit binding. The order of
addition of components, incubation temperature, time of incubation,
and other parameters of the assay may be readily determined. Such
experimentation merely involves optimization of the assay
parameters, not the fundamental composition of the assay.
Incubation temperatures typically are between 4.degree. C. and
40.degree. C. Incubation times preferably are minimized to
facilitate rapid, high throughput screening, and typically are
between 1 minute and 10 hours.
After incubation, the level of signaling or the level of specific
binding between the murine TLR7 or murine TLR8 polypeptide and the
candidate pharmaceutical agent is detected by any convenient method
available to the user, as described elsewhere herein.
The murine TLR7 or murine TLR8 binding agent may also be an
antibody or a functionally active antibody fragment. Antibodies,
including monoclonal antibodies and antibody fragments, are well
known to those of ordinary skill in the science of immunology and
are as described elsewhere herein. Monoclonal antibodies may be
made by any of the methods known in the art utilizing murine TLR7
or murine TLR8, or a fragment thereof, as an immunogen.
Alternatively the antibody may be a polyclonal antibody specific
for murine TLR7 or murine TLR8 which inhibits murine TLR7 or murine
TLR8 activity. The preparation and use of polyclonal antibodies are
also known to one of ordinary skill in the art.
The sequences of the antigen-binding Fab' portion of the
anti-murine TLR7 or anti-murine TLR8 monoclonal antibodies
identified as being useful according to the invention in the assays
provided above, as well as the relevant FR and CDR regions, can be
determined using amino acid sequencing methods that are routine in
the art. Such sequence information can be used to generate
humanized and chimeric antibodies, as well as various fusion
proteins and binding fragments, as described elsewhere herein.
Thus, as will be apparent to one of ordinary skill in the art, the
present invention also provides for F(ab').sub.2 and Fab fragments
of an anti-murine TLR7 or anti-murine TLR8 monoclonal antibody;
chimeric antibodies in which the Fc and/or FR and/or CDR1 and/or
CDR2 and/or light chain CDR3 regions of an anti-murine TLR7 or
anti-murine TLR8 antibody have been replaced by homologous human or
non-human sequences; chimeric F(ab').sub.2 fragment antibodies in
which the FR and/or CDR1 and/or CDR2 and/or light chain CDR3
regions of an anti-murine TLR7 or anti-murine TLR8 antibody have
been replaced by homologous human or non-human sequences; and
chimeric Fab fragment antibodies in which the FR and/or CDR1 and/or
CDR2 and/or light chain CDR3 regions have been replaced by
homologous human or non-human sequences.
According to the invention murine TLR7 and murine TLR8 inhibitors
also include "dominant negative" polypeptides derived from SEQ ID
NO:175 or SEQ ID NO:192, respectively. The end result of the
expression of a dominant negative murine TLR7 or dominant negative
murine TLR8 polypeptide of the invention in a cell is a reduction
in TLR7 or murine TLR8 activity such as signaling through the TIR
pathway. One of ordinary skill in the art can assess the potential
for a dominant negative variant of a murine TLR7 or dominant
negative murine TLR8 polypeptide and, using standard mutagenesis
techniques, create one or more dominant negative variant
polypeptides.
Each of the compositions according to this aspect of the invention
is useful for a variety of therapeutic and non-therapeutic
purposes. For example, the murine TLR7 and murine TLR8 nucleic
acids of the invention are useful as oligonucleotide probes. Such
oligonucleotide probes can be used herein to identify genomic or
cDNA library clones possessing an identical or substantially
similar nucleic acid sequence. Methods of hybridization, synthesis
of probes, and detection are generally as described elsewhere
herein.
Additionally, complements of the murine TLR7 and murine TLR8
nucleic acids can be useful as antisense oligonucleotides, e.g., by
delivering the antisense oligonucleotide to an animal to induce a
murine TLR7 or murine TLR8 "knockout" phenotype.
Alternatively, the murine TLR7 and murine TLR8 nucleic acids of the
invention can be used to prepare a non-human transgenic animal. The
invention, therefore, contemplates the use of murine TLR7 and
murine TLR8 knockout and transgenic animals as models for the study
of disorders involving TLR7- and murine TLR8-mediated signaling. A
variety of methods known to one of ordinary skill in the art are
available for the production of transgenic animals associated with
this invention.
Inactivation or replacement of the endogenous TLR7 or TLR8 gene can
be achieved by a homologous recombination system using embryonic
stem cells. The resultant transgenic non-human mammals having a
TLR7.sup.-/- or TLR8.sup.-/- knockout phenotype may be made
transgenic for the murine TLR7 or murine TLR8 and used as a model
for screening compounds as modulators (agonists or
antagonists/inhibitors) of the murine TLR7 or murine TLR8. In this
manner, such therapeutic drugs can be identified.
Additionally, a normal or mutant version of murine TLR7 or murine
TLR8 can be inserted into the germ line to produce transgenic
animals which constitutively or inducibly express the normal or
mutant form of murine TLR7 or murine TLR8. These animals are useful
in studies to define the role and function of murine TLR7 or murine
TLR8 in cells.
The antagonists, agonists, nucleic acids, and polypeptides of
murine TLR7 and murine TLR8 useful according to the invention may
be combined, optionally, with a pharmaceutically acceptable
carrier. Thus the invention also provides pharmaceutical
compositions and a method for preparing the pharmaceutical
compositions which contain compositions of this aspect of the
invention. The pharmaceutical compositions include one or any
combination of the antagonists, agonists, nucleic acids and
polypeptides of murine TLR7 and murine TLR8 useful according to the
invention and, optionally, a pharmaceutically acceptable carrier.
Each pharmaceutical composition is prepared by selecting an
antagonist, agonist, nucleic acid or polypeptide of murine TLR7 and
murine TLR8 useful according to the invention, as well as any
combination thereof, and, optionally, combining it with a
pharmaceutically acceptable carrier.
A variety of administration routes are available, as described
previously herein. The particular mode selected will depend, of
course, upon the particular compound selected, the severity of the
condition being treated, and the dosage required for therapeutic
efficacy.
Likewise, a variety of formulations are contemplated, including, by
analogy those discussed above in reference to murine TLR9, unit
dose solids, liquids, extended release formulations, etc.
Screening Assays
In another aspect the invention provides methods for screening
candidate compounds that act as ISNA mimics, agonists or
antagonists in ISNA-induced immunomodulation via TLR7, TLR8, and
TLR9. Preferably the screening method can be adapted to accommodate
high throughput screening assays, as can be achieved, for example,
through the use of multiwell arrays of samples in conjunction with
robotic or automated array handling devices.
Immunostimulatory nucleic acids include but are not limited to CpG
nucleic acids.
A "CpG nucleic acid" or a "CpG immunostimulatory nucleic acid" as
used herein is a nucleic acid containing at least one unmethylated
CpG dinucleotide (cytosine-guanine dinucleotide sequence, i.e. "CpG
DNA" or DNA containing a 5' cytosine followed by 3' guanine and
linked by a phosphate bond) and activates a component of the immune
system. The entire CpG nucleic acid can be unmethylated or portions
may be unmethylated but at least the C of the 5' CG 3' must be
unmethylated.
In one embodiment a CpG nucleic acid is represented by at least the
formula:
wherein X.sub.1 and X.sub.2 are nucleotides, N is any nucleotide,
and N.sub.1 and N.sub.2 are nucleic acid sequences composed of from
about 0-25 N's each. In some embodiments X.sub.1 is adenine,
guanine, or thymine and/or X.sub.2 is cytosine, adenine, or
thymine. In other embodiments X.sub.1 is cytosine and/or X.sub.2 is
guanine.
In other embodiments the CpG nucleic acid is represented by at
least the formula:
wherein X.sub.1, X.sub.2, X.sub.3, and X.sub.4 are nucleotides; N
is any nucleotide; and N.sub.1 and N.sub.2 are nucleic acid
sequences composed of from about 0-25 N's each. In some
embodiments, X.sub.1 X.sub.2 are nucleotides selected from the
group consisting of: GpT, GpG, GpA, ApA, ApT, ApG, CpT, CpA, CpG,
TpA, TpT, and TpG; and X.sub.3 X.sub.4 are nucleotides selected
from the group consisting of: TpT, CpT, ApT, TpG, ApG, CpG, TpC,
ApC, CpC, TpA, ApA, and CpA. In some embodiments, X.sub.1 X.sub.2
are GpA or GpT and X.sub.3 X.sub.4 are TpT. In other embodiments
X.sub.1 or X.sub.2 or both are purines and X.sub.3 or X.sub.4 or
both are pyrimidines or X.sub.1 X.sub.2 are GpA and X.sub.3 or
X.sub.4 or both are pyrimidines.
In another embodiment the CpG nucleic acid is represented by at
least the formula:
wherein X.sub.1, X.sub.2, X.sub.3, and X.sub.4 are nucleotides; N
is any nucleotide; and N.sub.1 and N.sub.2 are nucleic acid
sequences composed of from about 0-25 N's each. In some
embodiments, X.sub.1 X.sub.2 are nucleotides selected from the
group consisting of: GpT, GpG, GpA, ApA, ApT, ApG, CpT, CpA, CpG,
TpA, TpT, and TpG; and X.sub.3 X.sub.4 are nucleotides selected
from the group consisting of: TpT, CpT, ApT, TpG, ApG, CpG, TpC,
ApC, CpC, TpA, ApA, and CpA. In some embodiments, X.sub.1 X.sub.2
are GpA or GpT and X.sub.3 X.sub.4 are TpT. In other embodiments
X.sub.1 or X.sub.2 or both are purines and X.sub.3 or X.sub.4 or
both are pyrimidines or X.sub.1 X.sub.2 are GpA and X.sub.3 or
X.sub.4 or both are pyrimidines.
Examples of CpG nucleic acids according to the invention include
but are not limited to those listed in Table 1, such as SEQ ID
NOs:21-29, 31-42, 44, 46-50, 52-62, 64-75, 77-88, 90-117,
119-124.
TABLE 1 Exemplary CpG nucleic acids AACGTTCT SEQ ID NO:21
AAGCGAAAATGAAATTGACT SEQ ID NO:22 ACCATGGACGAACTGTTTCCCCTC SEQ ID
NO:23 ACCATGGACGACCTGTTTCCCCTC SEQ ID NO:24
ACCATGGACGAGCTGTTTCCCCTC SEQ ID NO:25 ACCATGGACGATCTGTTTCCCCTC SEQ
ID NO:26 ACCATGGACGGTCTGTTTCCCCTC SEQ ID NO:27
ACCATGGACGTACTGTTTCCCCTC SEQ ID NO:28 ACCATGGACGTTCTGTTTCCCCTC SEQ
ID NO:29 AGATTTCTAGGAATTCAATC SEQ ID NO:30 AGCGGGGGCGAGCGGGGGCG SEQ
ID NO:31 AGCTATGACGTTCCAAGG SEQ ID NO:32 ATCGACTCTCGAGCGTTCTC SEQ
ID NO:33 ATGACGTTCCTGACGTT SEQ ID NO:34 ATGGAAGGTCCAACGTTCTC SEQ ID
NO:35 ATGGAAGGTCCAGCGTTCTC SEQ ID NO:36 ATGGACTCTCCAGCGTTCTC SEQ ID
NO:37 ATGGAGGCTCCATCGTTCTC SEQ ID NO:38 CAACGTT SEQ ID NO:39
CACGTTGAGGGGCAT SEQ ID NO:40 CAGGCATAACGGTTCCGTAG SEQ ID NO:41
CCAACGTT SEQ ID NO:42 CTCCTAGTGGGGGTGTCCTAT SEQ ID NO:43
CTGATTTCCCCGAAATGATG SEQ ID NO:44 CTGCTGAGACTGGAG SEQ ID NO:45
GAGAACGATGGACCTTCCAT SEQ ID NO:46 GAGAACGCTCCAGCACTGAT SEQ ID NO:47
GAGAACGCTCGACCTTCCAT SEQ ID NO:48 GAGAACGCTCGACCTTCGAT SEQ ID NO:49
GAGAACGCTGGACCTTCCAT SEQ ID NO:50 GAGCAAGCTGGACCTTCCAT SEQ ID NO:51
GATTGCCTGACGTCAGAGAG SEQ ID NO:52 GCATGACGTTGAGCT SEQ ID NO:53
GCGGCGGGCGGCGCGCGCCC SEQ ID NO:54 GCGTGCGTTGTCGTTGTCGTT SEQ ID
NO:55 GCTAGACGTTAGCGT SEQ ID NO:56 GCTAGACGTTAGTGT SEQ ID NO:57
GCTAGATGTTAGCGT SEQ ID NO:58 GCTTGATGACTCAGCCGGAA SEQ ID NO:59
GGAATGACGTTCCCTGTG SEQ ID NO:60 GGGGTCAACGTTGACGGGG SEQ ID NO:61
GGGGTCAGTCTTGACGGGG SEQ ID NO:62 GTATTTCCCAGAAAAGGAAC SEQ ID NO:63
GTCCATTTCCCGTAAATCTT SEQ ID NO:64 GTCGCT SEQ ID NO:65 GTCGTT SEQ ID
NO:66 TACCGCGTGCGACCCTCT SEQ ID NO:67 TATGCATATTCCTGTAAGTG SEQ ID
NO:68 TCAACGTC SEQ ID NO:69 TCAACGTT SEQ ID NO:70 TCAAGCTT SEQ ID
NO:71 TCAGCGCT SEQ ID NO:72 TCAGCGTGCGCC SEQ ID NO:73 TCATCGAT SEQ
ID NO:74 TCCACGACGTTTTCGACGTT SEQ ID NO:75 TCCAGGACTTCTCTCAGGTT SEQ
ID NO:76 TCCATAACGTTCCTGATGCT SEQ ID NO:77 TCCATAGCGTTCCTAGCGTT SEQ
ID NO:78 TCCATCACGTGCCTGATGCT SEQ ID NO:79 TCCATGACGGTCCTGATGCT SEQ
ID NO:80 TCCATGACGTCCCTGATGCT SEQ ID NO:81 TCCATGACGTGCCTGATGCT SEQ
ID NO:82 TCCATGACGTTCCTGACGTT SEQ ID NO:83 TCCATGACGTTCCTGATGCT SEQ
ID NO:84 TCCATGAGCTTCCTGATGCT SEQ ID NO:85 TCCATGCCGGTCCTGATGCT SEQ
ID NO:86 TCCATGCGTGCGTGCGTTTT SEQ ID NO:87 TCCATGCGTTGCCTTGCGTT SEQ
ID NO:88 TCCATGCTGGTCCTGATGCT SEQ ID NO:89 TCCATGGCGGTCCTGATGCT SEQ
ID NO:90 TCCATGTCGATCCTGATGCT SEQ ID NO:91 TCCATGTCGCTCCTGATGCT SEQ
ID NO:92 TCCATGTCGGTCCTGATGCT SEQ ID NO:93 TCCATGTCGGTCCTGCTGAT SEQ
ID NO:94 TCCATGTCGTCCCTGATGCT SEQ ID NO:95 TCCATGTCGTTCCTGATGCT SEQ
ID NO:96 TCCATGTCGTTCCTGTCGTT SEQ ID NO:97 TCCATGTCGTTTTTGTCGTT SEQ
ID NO:98 TCCTGACGTTCCTGACGTT SEQ ID NO:99 TCCTGTCGTTCCTGTCGTT SEQ
ID NO:100 TCCTGTCGTTCCTTGTCGTT SEQ ID NO:101 TCCTGTCGTTTTTTGTCGTT
SEQ ID NO:102 TCCTTGTCGTTCCTGTCGTT SEQ ID NO:103
TCGATCGGGGCGGGGCGAGC SEQ ID NO:104 TCGTCGCTGTCTCCGCTTCTT SEQ ID
NO:105 TCGTCGCTGTCTCCGCTTCTTCTTGCC SEQ ID NO:106
TCGTCGCTGTCTGCCCTTCTT SEQ ID NO:107 TCGTCGCTGTTGTCGTTTCTT SEQ ID
NO:108 TCGTCGTCGTCGTT SEQ ID NO:109 TCGTCGTTGTCGTTGTCGTT SEQ ID
NO:110 TCGTCGTTGTCGTTTTGTCGTT SEQ ID NO:111
TCGTCGTTTTGTCGTTTTGTCGTT SEQ ID NO:112 TCTCCCAGCGCGCGCCAT SEQ ID
NO:113 TCTCCCAGCGGGCGCAT SEQ ID NO:114 TCTCCCAGCGTGCGCCAT SEQ ID
NO:115 TCTTCGAA SEQ ID NO:116 TGCAGATTGCGCAATCTGCA SEQ ID NO:117
TGCTGCTTTTGTGCTTTTGTGCTT SEQ ID NO:118 TGTCGCT SEQ ID NO:119
TGTCGTT SEQ ID NO:120 TGTCGTTGTCGTT SEQ ID NO:121
TGTCGTTGTCGTTGTCGTT SEQ ID NO:122 TGTCGTTGTCGTTGTCGTTGTCGTT SEQ ID
NO:123 TGTCGTTTGTCGTTTGTCGTT SEQ ID NO:124
Other ISNAs include but are not limited to T-rich nucleic acids,
poly G nucleic acids, and nucleic acids having phosphate modified
backbones, such as phosphorothioate backbones.
A "T rich nucleic acid" or "T rich immunostimulatory nucleic acid"
is a nucleic acid which includes at least one poly T sequence
and/or which has a nucleotide composition of greater than 25% T
nucleotide residues and which activates a component of the immune
system. A nucleic acid having a poly-T sequence includes at least
four Ts in a row, such as 5'TTTT3'. Preferably the T rich nucleic
acid includes more than one poly T sequence. In preferred
embodiments the T rich nucleic acid may have 2, 3, 4, etc poly T
sequences. One of the most highly immunostimulatory T rich
oligonucleotides discovered according to the invention is a nucleic
acid composed entirely of T nucleotide residues. Other T rich
nucleic acids have a nucleotide composition of greater than 25% T
nucleotide residues, but do not necessarily include a poly T
sequence. In these T rich nucleic acids the T nucleotide resides
may be separated from one another by other types of nucleotide
residues, i.e., G, C, and A. In some embodiments the T rich nucleic
acids have a nucleotide composition of greater than 30%, 40%, 50%,
60%, 70%, 80%, 90%, and 99%, T nucleotide residues and every
integer % in between. Preferably the T rich nucleic acids have at
least one poly T sequence and a nucleotide composition of greater
than 25% T nucleotide residues.
In one embodiment the T rich nucleic acid is represented by at
least the formula:
wherein X.sub.1, X.sub.2, X.sub.3, and X.sub.4 are nucleotides. In
one embodiment X.sub.1 X.sub.2 is TT and/or X.sub.3 X.sub.4 is TT.
In another embodiment X.sub.1 X.sub.2 are any one of the following
nucleotides TA, TG, TC, AT, AA, AG, AC, CT, CC, CA, CG, GT, GG, GA,
and GC; and X.sub.3 X.sub.4 are any one of the following
nucleotides TA, TG, TC, AT, AA, AG, AC, CT, CC, CA, CG, GT, GG, GA,
and GC.
In some embodiments it is preferred that the T-rich nucleic acid
does not contain poly C(CCCC), poly A (AAAA), poly G (GGGG), CpG
motifs, or multiple GGs. In other embodiments the T-rich nucleic
acid includes these motifs. Thus in some embodiments of the
invention the T rich nucleic acids include CpG dinucleotides and in
other embodiments the T rich nucleic acids are free of CpG
dinucleotides. The CpG dinucleotides may be methylated or
unmethylated.
Poly G containing nucleic acids are also immunostimulatory. A
variety of references, including Pisetsky and Reich, 1993 Mol.
Biol. Reports, 18:217-221; Krieger and Herz, 1994, Ann. Rev.
Biochem., 63:601-637; Macaya et al., 1993, PNAS, 90:3745-3749;
Wyatt et al., 1994, PNAS, 91:1356-1360; Rando and Hogan, 1998, In
Applied Antisense Oligonucleotide Technology, ed. Krieg and Stein,
p. 335-352; and Kimura et al., 1994, J. Biochem. 116, 991-994 also
describe the immunostimulatory properties of poly G nucleic
acids.
Poly G nucleic acids preferably are nucleic acids having the
following formulas:
wherein X.sub.1, X.sub.2, X.sub.3, and X.sub.4 are nucleotides. In
preferred embodiments at least one of X.sub.3 and X.sub.4 are a G.
In other embodiments both of X.sub.3 and X.sub.4 are a G. In yet
other embodiments the preferred formula is 5' GGGNGGG 3', or 5'
GGGNGGGNGGG 3' wherein N represents between 0 and 20 nucleotides.
In other embodiments the Poly G nucleic acid is free of
unmethylated CG dinucleotides. In other embodiments the poly G
nucleic acid includes at least one unmethylated CG
dinucleotide.
Nucleic acids having modified backbones, such as phosphorothioate
backbones, also fall within the class of immunostimulatory nucleic
acids. U.S. Pat. Nos. 5,723,335 and 5,663,153 issued to Hutcherson,
et al. and related PCT publication WO95/26204 describe immune
stimulation using phosphorothioate oligonucleotide analogues. These
patents describe the ability of the phosphorothioate backbone to
stimulate an immune response in a non-sequence specific manner.
The ISNAs may be double-stranded or single-stranded. Generally,
double-stranded molecules may be more stable in vivo, while
single-stranded molecules may have increased activity. The terms
"nucleic acid" and "oligonucleotide" refer to multiple nucleotides
(i.e. molecules comprising a sugar (e.g. ribose or deoxyribose)
linked to a phosphate group and to an exchangeable organic base,
which is either a substituted pyrimidine (e.g. cytosine (C),
thymine (T) or uracil (U)) or a substituted purine (e.g., adenine
(A) or guanine (G)) or a modified base. As used herein, the terms
refer to oligoribonucleotides as well as oligodeoxyribonucleotides.
The terms shall also include polynucleosides (i.e., a
polynucleotide minus the phosphate) and any other organic
base-containing polymer. The terms "nucleic acid" and
"oligonucleotide" also encompass nucleic acids or oligonucleotides
with a covalently modified base and/or sugar. For example, they
include nucleic acids having backbone sugars which are covalently
attached to low molecular weight organic groups other than a
hydroxyl group at the 3' position and other than a phosphate group
at the 5' position. Thus modified nucleic acids may include a
2'-O-alkylated ribose group. In addition, modified nucleic acids
may include sugars such as arabinose instead of ribose. Thus the
nucleic acids may be heterogeneous in backbone composition thereby
containing any possible combination of polymer units linked
together such as peptide-nucleic acids (which have amino acid
backbone with nucleic acid bases). In some embodiments the nucleic
acids are homogeneous in backbone composition.
The substituted purines and pyrimidines of the ISNAs include
standard purines and pyrimidines such as cytosine as well as base
analogs such as C-5 propyne substituted bases. Wagner R W et al.,
Nat Biotechnol 14:840-844 (1996). Purines and pyrimidines include
but are not limited to adenine, cytosine, guanine, thymine,
5-methylcytosine, 2-aminopurine, 2-amino-6-chloropurine,
2,6-diaminopurine, hypoxanthine, and other naturally and
non-naturally occurring nucleobases, substituted and unsubstituted
aromatic moieties.
The ISNA is a linked polymer of bases or nucleotides. As used
herein with respect to linked units of a nucleic acid, "linked" or
"linkage" means two entities are bound to one another by any
physicochemical means. Any linkage known to those of ordinary skill
in the art, covalent or non-covalent, is embraced. Such linkages
are well known to those of ordinary skill in the art. Natural
linkages, which are those ordinarily found in nature connecting the
individual units of a nucleic acid, are most common. The individual
units of a nucleic acid may be linked, however, by synthetic or
modified linkages.
Whenever a nucleic acid is represented by a sequence of letters it
will be understood that the nucleotides are in 5' to 3' order from
left to right and that "A" denotes adenine, "C" denotes cytosine,
"G" denotes guanine, "T" denotes thymidine, and "U" denotes uracil
unless otherwise noted.
Immunostimulatory nucleic acid molecules useful according to the
invention can be obtained from natural nucleic acid sources (e.g.,
genomic nuclear or mitochondrial DNA or cDNA), or are synthetic
(e.g., produced by oligonucleotide synthesis). Nucleic acids
isolated from existing nucleic acid sources are referred to herein
as native, natural, or isolated nucleic acids. The nucleic acids
useful according to the invention may be isolated from any source,
including eukaryotic sources, prokaryotic sources, nuclear DNA,
mitochondrial DNA, etc. Thus, the term nucleic acid encompasses
both synthetic and isolated nucleic acids.
The term "isolated" as used herein with reference to an ISNA means
substantially free of or separated from components which it is
normally associated with in nature, e.g., nucleic acids, proteins,
lipids, carbohydrates or in vivo systems to an extent practical and
appropriate for its intended use. In particular, the nucleic acids
are sufficiently pure and are sufficiently free from other
biological constituents of host cells so as to be useful in, for
example, producing pharmaceutical preparations. Because an isolated
nucleic acid of the invention may be admixed with a
pharmaceutically-acceptable carrier in a pharmaceutical
preparation, the nucleic acid may comprise only a small percentage
by weight of the preparation. The nucleic acid is nonetheless
substantially pure in that it has been substantially separated from
the substances with which it may be associated in living
systems.
The ISNAs can be produced on a large scale in plasmids, (see
Molecular Cloning: A Laboratory Manual, J. Sambrook, et al., eds.,
Second Edition, Cold Spring Harbor Laboratory Press, Cold Spring
Harbor, N.Y., 1989) and separated into smaller pieces or
administered whole. After being administered to a subject the
plasmid can be degraded into oligonucleotides. One skilled in the
art can purify viral, bacterial, eukaryotic, etc. nucleic acids
using standard techniques, such as those employing restriction
enzymes, exonucleases or endonucleases.
For use in the instant invention, the ISNAs can be synthesized de
novo using any of a number of procedures well known in the art. For
example, the .beta.-cyanoethyl phosphoramidite method (Beaucage S L
and Caruthers M H, Tetrahedron Let 22:1859 (1981)); nucleoside
H-phosphonate method (Garegg et al., Tetrahedron Let 27:4051-4054
(1986); Froehler et al., Nucl Acid Res 14:5399-5407 (1986); Garegg
et al., Tetrahedron Let 27:4055-4058 (1986); Gaffney et al.,
Tetrahedron Let 29:2619-2622 (1988)). These chemistries can be
performed by a variety of automated oligonucleotide synthesizers
available in the market.
ISNAs having modified backbones, such as phosphorothioate
backbones, also fall within the class of immunostimulatory nucleic
acids. U.S. Pat. Nos. 5,723,335 and 5,663,153 issued to Hutcherson,
et al. and related PCT publication WO95/26204 describe immune
stimulation using phosphorothioate oligonucleotide analogues. These
patents describe the ability of the phosphorothioate backbone to
stimulate an immune response in a non-sequence specific manner.
The ISNA may be any size of at least 6 nucleotides but in some
embodiments are in the range of between 6 and 100 or in some
embodiments between 8 and 35 nucleotides in size. Immunostimulatory
nucleic acids can be produced on a large scale in plasmids. These
may be administered in plasmid form or alternatively they can be
degraded into oligonucleotides before administration.
"Palindromic sequence" shall mean an inverted repeat (i.e., a
sequence such as ABCDEE'D'C'B'A' in which A and A', B and B', etc.,
are bases capable of forming the usual Watson-Crick base pairs and
which includes at least 6 nucleotides in the palindrome. In vivo,
such sequences may form double-stranded structures. In one
embodiment the nucleic acid contains a palindromic sequence. In
some embodiments when the nucleic acid is a CpG nucleic acid, a
palindromic sequence used in this context refers to a palindrome in
which the CpG is part of the palindrome, and optionally is the
center of the palindrome. In another embodiment the nucleic acid is
free of a palindrome. A nucleic acid that is free of a palindrome
does not have any regions of 6 nucleotides or greater in length
which are palindromic. A nucleic acid that is free of a palindrome
can include a region of less than 6 nucleotides which are
palindromic.
A "stabilized ISNA" shall mean a nucleic acid molecule that is
relatively resistant to in vivo degradation (e.g. via an exo- or
endo-nuclease). Stabilization can be a function of length or
secondary structure. Nucleic acids that are tens to hundreds of kbs
long are relatively resistant to in vivo degradation. For shorter
nucleic acids, secondary structure can stabilize and increase their
effect. For example, if the 3' end of an oligonucleotide has
self-complementarity to an upstream region, so that it can fold
back and form a sort of stem loop structure, then the
oligonucleotide becomes stabilized and therefore exhibits more
activity.
Some stabilized ISNAs of the instant invention have a modified
backbone. It has been demonstrated that modification of the
oligonucleotide backbone provides enhanced activity of the ISNAs
when administered in vivo. Nucleic acids, including at least two
phosphorothioate linkages at the 5' end of the oligonucleotide and
multiple phosphorothioate linkages at the 3' end, preferably 5, may
provide maximal activity and protect the oligonucleotide from
degradation by intracellular exo- and endo-nucleases. Other
modified oligonucleotides include phosphodiester modified
oligonucleotide, combinations of phosphodiester and
phosphorothioate oligonucleotide, methylphosphonate,
methylphosphorothioate, phosphorodithioate, and combinations
thereof. Each of these combinations and their particular effects on
immune cells is discussed in more detail in U.S. Pat. Nos.
6,194,388 and 6,207,646, the entire contents of which is hereby
incorporated by reference. It is believed that these modified
oligonucleotides may show more stimulatory activity due to enhanced
nuclease resistance, increased cellular uptake, increased protein
binding, and/or altered intracellular localization. Both
phosphorothioate and phosphodiester nucleic acids are active in
immune cells.
Other stabilized ISNAs include: nonionic DNA analogs, such as
alkyl- and aryl-phosphates (in which the charged phosphonate oxygen
is replaced by an alkyl or aryl group), phosphodiester and
alkylphosphotriesters, in which the charged oxygen moiety is
alkylated. Oligonucleotides which contain diol, such as
tetraethyleneglycol or hexaethyleneglycol, at either or both
termini have also been shown to be substantially resistant to
nuclease degradation.
For use in vivo, ISNAs are preferably relatively resistant to
degradation (e.g., via endo- and exo-nucleases). Secondary
structures, such as stem loops, can stabilize nucleic acids against
degradation. Alternatively, nucleic acid stabilization can be
accomplished via phosphate backbone modifications. One type of
stabilized nucleic acid has at least a partial phosphorothioate
modified backbone. Phosphorothioates may be synthesized using
automated techniques employing either phosphoramidate or
H-phosphonate chemistries. Aryl- and alkyl-phosphonates can be
made, e.g., as described in U.S. Pat. No. 4,469,863; and
alkylphosphotriesters (in which the charged oxygen moiety is
alkylated as described in U.S. Pat. No. 5,023,243 and European
Patent No. 092,574) can be prepared by automated solid phase
synthesis using commercially available reagents. Methods for making
other DNA backbone modifications and substitutions have been
described. Uhlmann E and Peyman A, Chem Rev 90:544 (1990);
Goodchild J, Bioconjugate Chem 1:165 (1990).
Other sources of immunostimulatory nucleic acids useful according
to the invention include standard viral and bacterial vectors, many
of which are commercially available. In its broadest sense, a
"vector" is any nucleic acid material which is ordinarily used to
deliver and facilitate the transfer of nucleic acids to cells. The
vector as used herein may be an empty vector or a vector carrying a
gene which can be expressed. In the case when the vector is
carrying a gene the vector generally transports the gene to the
target cells with reduced degradation relative to the extent of
degradation that would result in the absence of the vector. In this
case the vector optionally includes gene expression sequences to
enhance expression of the gene in target cells such as immune
cells, but it is not required that the gene be expressed in the
cell.
A basis for certain of the screening assays is the presence of a
functional TLR 7, TLR8, or TLR9 in a cell. The functional TLR in
some instances is naturally expressed by the cell. In other
instances, expression of the functional TLR can involve
introduction or reconstitution of a species-specific TLR9 into a
cell or cell line that otherwise lacks the TLR or lacks
responsiveness to ISNA, resulting in a cell or cell line capable of
activating the TLR/IL-1R signaling pathway in response to contact
with an ISNA. Examples of cell lines lacking TLR9 or ISNA
responsiveness include, but are not limited to, 293 fibroblasts
(ATCC CRL-1573), MonoMac-6, THP-1, U937, CHO, and any TLR9
knock-out. The introduction of the species-specific TLR into the
cell or cell line is preferably accomplished by transient or stable
transfection of the cell or cell line with a TLR-encoding nucleic
acid sequence operatively linked to a gene expression sequence (as
described above).
The species-specific TLR, including TLR7, TLR8, and TLR9, is not
limited to a murine TLR, but rather can include a TLR derived from
murine or non-murine sources. Examples of non-murine sources
include, but are not limited to, human, bovine, canine, feline,
ovine, porcine, and equine. Other species include chicken and fish,
e.g., aquaculture species.
The species-specific TLR, including TLR7, TLR8, and TLR9, also is
not limited to native TLR polypeptides. In certain embodiments the
TLR can be, e.g., a chimeric TLR in which the extracellular domain
and the cytoplasmic domains are derived from TLR polypeptides from
different species. Such chimeric TLR polypeptides, as described
above, can include, for example, a human TLR extracellular domain
and a murine TLR cytoplasmic domain, each domain derived from the
corresponding TLR7, TLR8, or TLR9 of each species. In alternative
embodiments, such chimeric TLR polypeptides can include chimeras
created with different TLR splice variants or allotypes. Other
chimeric TLR polypeptides useful for the purposes of screening ISNA
mimics, agonists and antagonists can include chimeric polypeptides
created with a TLR of a first type, e.g., TLR9, and another TLR,
e.g., TLR7 or TLR8, of the same or another species as the TLR of
the first type. Also contemplated are chimeric polypeptides which
incorporate sequences derived from more than two polypeptides,
e.g., an extracellular domain, a transmembrane domain, and a
cytoplasmic domain all derived from different polypeptide sources,
provided at least one such domain derives from a TLR7, TLR8, or
TLR9 polypeptide. As a further example, also contemplated are
constructs such as include an extracellular domain of one TLR9, an
intracellular domain of another TLR9, and a non-TLR reporter such
as luciferase, GFP, etc. Those of skill in the art will recognize
how to design and generate DNA sequences coding for such chimeric
TLR polypeptides.
The screening assays can have any of a number of possible readout
systems based upon either TLR/IL-1R signaling pathway or other
assays useful for assaying response to ISNAs. It has been reported
that immune cell activation by CpG immunostimulatory sequences is
dependent in some way on endosomal processing. It is not yet known
whether TLR9 is directly involved in this endosomal pathway, or if
there is some intermediary between TLR9 and the endosome.
In preferred embodiments, the readout for the screening assay is
based on the use of native genes or, alternatively, cotransfected
or otherwise co-introduced reporter gene constructs which are
responsive to the TLR/IL-1R signal transduction pathway involving
MyD88, TRAF6, p38, and/or ERK. Hacker H et al., EMBO J.
18:6973-6982 (1999). These pathways activate kinases including
.kappa.B kinase complex and c-Jun N-terminal kinases. Thus reporter
genes and reporter gene constructs particularly useful for the
assays can include a reporter gene operatively linked to a promoter
sensitive to NF-.kappa.B. Examples of such promoters include,
without limitation, those for NF-.kappa.B, IL-1.beta., IL-6, IL-8,
IL-12 p40, CD80, CD86, and TNF-.alpha.. The reporter gene
operatively linked to the TLR7-, TLR8-, or TLR9-sensitive promoter
can include, without limitation, an enzyme (e.g., luciferase,
alkaline phosphatase, .beta.-galactosidase, chloramphenicol
acetyltransferase (CAT), etc.), a bioluminescence marker (e.g.,
green-fluorescent protein (GFP, U.S. Pat. No. 5,491,084), etc.), a
surface-expressed molecule (e.g., CD25), and a secreted molecule
(e.g., IL-8, IL-12 p40, TNF-.alpha.). In preferred embodiments the
reporter is selected from IL-8, TNF-.alpha., NF-.kappa.B-luciferase
(NF-.kappa.B-luc; Hacker H et al., EMBO J. 18:6973-6982 (1999)),
IL-12 p40-luc (Murphy T L et al., Mol Cell Biol 15:5258-5267
(1995)), and TNF-luc (Hacker H et al., EMBO J. 18:6973-6982
(1999)). In assays relying on enzyme activity readout, substrate
can be supplied as part of the assay, and detection can involve
measurement of chemiluminescence, fluorescence, color development,
incorporation of radioactive label, drug resistance, or other
marker of enzyme activity. For assays relying on surface expression
of a molecule, detection can be accomplished using FACS analysis or
functional assays. Secreted molecules can be assayed using
enzyme-linked immunosorbent assay (ELISA) or bioassays. Many such
readout systems are well known in the art and are commercially
available.
In another aspect the invention provides a screening method for
identifying an immunostimulatory nucleic acid molecule (ISNA). The
method entails contacting a functional TLR selected from the group
consisting of TLR7, TLR8, and TLR9 with a test nucleic acid
molecule; detecting the presence or absence of a response mediated
by a TLR signal transduction pathway in the presence of the test
nucleic acid molecule arising as a result of an interaction between
the functional TLR and the test nucleic acid molecule; and
determining the test nucleic acid molecule is an ISNA when the
presence of a response mediated by the TLR signal transduction
pathway is detected. "Functional TLR" and a "cell expressing
functional TLR" are as described elsewhere herein. A response
mediated by a TLR signal transduction pathway includes induction of
a gene under control of a promoter responsive to the TLR/IL-1R
signaling pathway, including but not limited to promoters
responsive to NF-.kappa.B. The biological response thus can
include, e.g., secretion of IL-8 and luciferase activity in a cell
transfected with NF-.kappa.B-luc, IL-12 p40-luc, or TNF-luc. A test
nucleic acid molecule can include a DNA, RNA, or modified nucleic
acid molecule as described herein. In some embodiments the test
nucleic acid molecule is a CpG nucleic acid.
Preferably, the test nucleic acid molecule is a sequence variant of
a reference ISNA, containing at least one alternative base, at
least one alternative internucleotide backbone linkage, or at least
one alternative sugar moiety as compared to the particular
reference ISNA. In a preferred embodiment the test nucleic acid
molecule is a member of a library of such test nucleic acid
molecules.
According to one embodiment of this method, comparison can be made
to a reference ISNA. The reference ISNA may be any ISNA, including
a CpG nucleic acid. In preferred embodiments the screening method
is performed using a plurality of test nucleic acids. Preferably
comparison of test and reference responses is based on comparison
of quantitative measurements of responses in each instance.
The method can be used to select a subset of test nucleic acid
molecules based on their ability to induce a similar specific
response mediated by the TLR signal transduction pathway. For
instance, the method can be used to classify test CpG nucleic acids
as predominantly B-cell activating CpG nucleic acids, or as
predominantly IFN-.alpha. inducing CpG nucleic acids. Other new
classes of ISNAs may be identified and characterized using the
method.
Application of this method permits the identification of ISNAs,
delineation of sequence specificity of a given TLR, and also
optimization of ISNA sequences. Identification of ISNAs involves
screening candidate ISNAs as above and selecting any ISNA that
induces a response as defined. Delineation of sequence specificity
involves screening candidate ISNAs as above with reference to a
particular TLR9, selecting any ISNAs that induce a response as
defined, and categorizing ISNAs that do and do not induce a
response on the basis of their sequence. Optimization of ISNA
sequences involves an iterative application of the method as
described, further including the steps of selecting the best
sequence at any given stage or round in the screening and
substituting it as a benchmark or reference in a subsequent round
of screening. This latter process can further include selection of
parameters to modify in choosing and generating candidate ISNAs to
screen.
In another aspect the invention provides screening method for
identifying species specificity of an ISNA. The method involves
contacting a functional TLR selected from the group consisting of
TLR7, TLR8, and TLR9 of a first species with a test ISNA;
contacting a functional TLR selected from the group consisting of
TLR7, TLR8, and TLR9 of a second species with the test ISNA;
measuring a response mediated by a TLR signal transduction pathway
associated with the contacting the functional TLR of the first
species with the test ISNA; measuring a response mediated by the
TLR signal transduction pathway associated with the contacting the
functional TLR of the second species with the test ISNA; and
comparing (a) the response mediated by a TLR signal transduction
pathway associated with the contacting the functional TLR of the
first species with the test ISNA with (b) the response mediated by
the TLR signal transduction pathway associated with the contacting
the functional TLR of the second species with the test ISNA. The
functional TLR may be expressed by a cell or it may be part of a
cell-free system. The functional TLR may be part of a complex, with
either another TLR or with another protein, e.g., MyD88, IRAK,
TRAF6, I.kappa.B, NF-.kappa.B, or functional homologues and
derivatives thereof. Thus for example a given ODN can be tested
against a panel of 293 fibroblast cells transfected with TLR7,
TLR8, or TLR9 from various species and optionally cotransfected
with a reporter construct (e.g., NF-.kappa.B-luc) sensitive to
TLR/IL-1R activation pathways. Thus in another aspect, the
invention provides a method for screening species selectivity with
respect to a given nucleic acid sequence.
As mentioned above, the invention in one aspect provides a
screening method for comparing TLR signaling activity or a test
compound against corresponding TLR signaling activity of a
reference ISNA. The methods generally involve contacting a
functional TLR selected from the group consisting of TLR7, TLR8,
and TLR9 with a reference ISNA and detecting a reference response
mediated by a TLR signal transduction pathway; contacting a
functional TLR selected from the group consisting of TLR7, TLR8,
and TLR9 with a test compound and detecting a test response
mediated by a TLR signal transduction pathway; and comparing the
test response with the reference response to compare the TLR
signaling activity of the test compound with the ISNA. Assays in
which the test compound and the reference ISNA contact the TLR
independently may be used to identify test compounds that are ISNA
mimics. Assays in which the test compound and the reference ISNA
contact the TLR concurrently may be used to identify test compounds
that are ISNA agonists and ISNA antagonists.
An ISNA mimic as used herein is a compound which causes a response
mediated by a TLR signal transduction pathway. As used herein the
term "response mediated by a TLR signal transduction pathway"
refers to a response which is characteristic of an ISNA-TLR
interaction. As demonstrated herein responses which are
characteristic of ISNA-TLR interactions include the induction of a
gene under control of an ISNA-specific promoter such as a
NF-.kappa.B promoter, increases in Th1 cytokine levels, etc. The
gene under the control of the NF-.kappa.B promoter may be a gene
which naturally includes an NF-.kappa.B promoter or it may be a
gene in a construct in which an NF-.kappa.B promoter has been
inserted. Genes which naturally include the NF-.kappa.B promoter
include but are not limited to IL-8, IL-12 p40, NF-.kappa.B-luc,
IL-12 p40-luc, and TNF-luc. Increases in Th1 cytokine levels is
another measure characteristic of an ISNA-TLR interaction.
Increases in Th1 cytokine levels may result from increased
production or increased stability or increased secretion of the Th1
cytokines in response to the ISNA-TLR interaction. Th1 cytokines
include but are not limited to IL-2, IFN-.gamma., and IL-12. Other
responses which are characteristic of an ISNA-TLR interaction
include but are not limited to a reduction in Th2 cytokine levels.
Th2 cytokines include but are not limited to IL-4, IL-5, and
IL-10.
The response which is characteristic of an ISNA-TLR interaction may
be a direct response or an indirect response. A direct response is
a response that arises directly as a result of the ISNA-TLR
interaction. An indirect response is a response which involves the
modulation of other parameters prior to its occurrence.
An ISNA agonist as used herein is a compound which causes an
enhanced response to an ISNA mediated by a TLR signal transduction
pathway. Thus an ISNA agonist as used herein is a compound which
causes an increase in at least one aspect of an immune response
that is ordinarily induced by the reference ISNA. For example, an
immune response that is ordinarily induced by an ISNA can
specifically include TLR7-, TLR8-, or TLR9-mediated signal
transduction in response to immunostimulatory CpG nucleic acid. An
ISNA agonist will in some embodiments compete with ISNA for binding
to TLR7, TLR8, or TLR9. In other embodiments an ISNA agonist will
bind to a site on TLR7, TLR8, or TLR9 that is distinct from the
site for binding ISNA. In yet other embodiments an ISNA agonist
will act via another molecule or pathway distinct from TLR7, TLR8,
or TLR9.
An ISNA antagonist as used herein is a compound which causes a
decreased response to an ISNA mediated by a TLR signal transduction
pathway. Thus an ISNA antagonist as used herein is a compound which
causes a decrease in at least one aspect of an immune response that
is ordinarily induced by the reference ISNA. For example, an immune
response that is ordinarily induced by an ISNA can specifically
include TLR7-, TLR8-, or TLR9-mediated signal transduction in
response to immunostimulatory CpG nucleic acid. An ISNA antagonist
will in some embodiments compete with ISNA for binding to TLR7,
TLR8, or TLR9. In other embodiments an ISNA antagonist will bind to
a site on TLR7, TLR8, or TLR9 that is distinct from the site for
binding ISNA. In yet other embodiments an ISNA antagonist will act
via another molecule or pathway distinct from TLR7, TLR8, or
TLR9.
The screening methods for comparing TLR signaling activity of a
test compound with signaling activity of an ISNA involve contacting
at least one test compound with a functional TLR selected from
TLR7, TLR8, and TLR9 under conditions which, in the absence of a
test compound, permit a reference ISNA to induce at least one
aspect of an immune response. The functional TLR may be expressed
by a cell or it may be part of a cell-free system. A cell
expressing a functional TLR is a cell that either naturally
expresses the TLR, or is a cell into which has been introduced a
TLR expression vector, or is a cell manipulated to express TLR in a
manner that allows the TLR to be expressed by the cell and to
transduce a signal under conditions which normally permit signal
transduction by the signal transducing portion of the TLR. The TLR
can be a native TLR or it can be a fragment or variant thereof, as
described above. According to these methods, the test compound is
contacted with a functional TLR or TLR-expressing cell before,
after, or simultaneously with contacting a reference ISNA with the
functional TLR or TLR-expressing cell. A response of the functional
TLR or TLR-expressing cell is measured and compared with the
corresponding response that results or would result under the same
conditions in the absence of the test compound. Where it is
appropriate, the response in the absence of the test compound can
be determined as a concurrent or historical control. Examples of
such responses include, without limitation, a response mediated
through the TLR signal transduction pathway, secretion of a
cytokine, cell proliferation, and cell activation. In a preferred
embodiment, the measurement of a response involves the detection of
IL-8 secretion (e.g., by ELISA). In another preferred embodiment
the measurement of the response involves the detection of
luciferase activity (e.g., NF-.kappa.B-luc, IL-12 p40-luc, or
TNF-luc).
Examples of reference ISNAs include, without limitation, those
listed in Table 1 (above). In some preferred embodiments the
reference ISNA is a CpG nucleic acid.
Test compounds can include but are not limited to peptide nucleic
acids (PNAs), antibodies, polypeptides, carbohydrates, lipids,
hormones, and small molecules. Test compounds can further include
variants of a reference ISNA incorporating any one or combination
of the substitutions described above. Test compounds can be
generated as members of a combinatorial library of compounds.
In preferred embodiments, the methods for screening test compounds,
test nucleic acid molecules, test ISNAs, and candidate
pharmacological agents can be performed on a large scale and with
high throughput by incorporating, e.g., an array-based assay system
and at least one automated or semi-automated step. For example, the
assays can be set up using multiple-well plates in which cells are
dispensed in individual wells and reagents are added in a
systematic manner using a multiwell delivery device suited to the
geometry of the multiwell plate. Manual and robotic multiwell
delivery devices suitable for use in a high throughput screening
assay are well known by those skilled in the art. Each well or
array element can be mapped in a one-to-one manner to a particular
test condition, such as the test compound. Readouts can also be
performed in this multiwell array, preferably using a multiwell
plate reader device or the like. Examples of such devices are well
known in the art and are available through commercial sources.
Sample and reagent handling can be automated to further enhance the
throughput capacity of the screening assay, such that dozens,
hundreds, thousands, or even millions of parallel assays can be
performed in a day or in a week. Fully robotic systems are known in
the art for applications such as generation and analysis of
combinatorial libraries of synthetic compounds. See, for example,
U.S. Pat. Nos. 5,443,791 and 5,708,158.
The invention will be more fully understood by reference to the
following examples. These examples, however, are merely intended to
illustrate the embodiments of the invention and are not to be
construed to limit the scope of the invention.
EXAMPLES
Example 1
Method of Cloning the Mouse TLR9
Alignment of human TLR9 protein sequence with mouse EST database
using tfasta yielded 7 hits with mouse EST sequences aa197442,
ai451215, aa162495, aw048117, ai463056, aw048548, and aa273731. Two
primers were designed that bind to aa197442 EST sequence for use in
a RACE-PCR to amplify 5' and 3' ends of the mouse TLR9 cDNA. The
library used for the RACE PCR was a mouse spleen marathon-ready
cDNA commercially available from Clonetech. A 5' fragment with a
length of 1800 bp obtained by this method was cloned into Promega
pGEM-T Easy vector. After sequencing of the 5' end, additional
primers were designed for amplification of the complete mouse TLR9
cDNA. The primer for the 5' end was obtained from the sequence of
the 5' RACE product whereas the primer for the 3' end was selected
from the mouse EST sequence aa273731.
Three independent PCR reactions were set up using a murine
macrophage RAW264.7 (ATCC TIB-71) cDNA as a template, and the
resulting amplification products were cloned into the pGEM-T Easy
vector. The inserts were fully sequenced, translated into protein
and aligned to the human protein sequence. One out of three clones
was error-free based on alignment comparison (clone mtlr932e.pep).
The cDNA sequence for mTLR9 is SEQ ID NO:1, is presented in Table
2. The ATG start codon occurs at base 40, and a TAG termination
codon occurs at base 3136. SEQ ID NO:2 (Table 3), corresponding to
bases 40-3135 of SEQ ID NO:1, is the coding region for the
polypeptide of SEQ ID NO:3.
TABLE 2 cDNA Sequence for Murine TLR9 (5' to 3'; SEQ ID NO:1)
tgtcagaggg agcctcggga gaatcctcca tctcccaaca tggttctccg tcgaaggact
60 ctgcacccct tgtccctcct ggtacaggct gcagtgctgg ctgagactct
ggccctgggt 120 accctgcctg ccttcctacc ctgtgagctg aagcctcatg
gcctggtgga ctgcaattgg 180 ctgttcctga agtctgtacc ccgtttctct
gcggcagcat cctgctccaa catcacccgc 240 ctctccttga tctccaaccg
tatccaccac ctgcacaact ccgacttcgt ccacctgtcc 300 aacctgcggc
agctgaacct caagtggaac tgtccaccca ctggccttag ccccctgcac 360
ttctcttgcc acatgaccat tgagcccaga accttcctgg ctatgcgtac actggaggag
420 ctgaacctga gctataatgg tatcaccact gtgccccgac tgcccagctc
cctggtgaat 480 ctgagcctga gccacaccaa catcctggtt ctagatgcta
acagcctcgc cggcctatac 540 agcctgcgcg ttctcttcat ggacgggaac
tgctactaca agaacccctg cacaggagcg 600 gtgaaggtga ccccaggcgc
cctcctgggc ctgagcaatc tcacccatct gtctctgaag 660 tataacaacc
tcacaaaggt gccccgccaa ctgcccccca gcctggagta cctcctggtg 720
tcctataacc tcattgtcaa gctggggcct gaagacctgg ccaatctgac ctcccttcga
780 gtacttgatg tgggtgggaa ttgccgtcgc tgcgaccatg cccccaatcc
ctgtatagaa 840 tgtggccaaa agtccctcca cctgcaccct gagaccttcc
atcacctgag ccatctggaa 900 ggcctggtgc tgaaggacag ctctctccat
acactgaact cttcctggtt ccaaggtctg 960 gtcaacctct cggtgctgga
cctaagcgag aactttctct atgaaagcat caaccacacc 1020 aatgcctttc
agaacctaac ccgcctgcgc aagctcaacc tgtccttcaa ttaccgcaag 1080
aaggtatcct ttgcccgcct ccacctggca agttccttca agaacctggt gtcactgcag
1140 gagctgaaca tgaacggcat cttcttccgc tcgctcaaca agtacacgct
cagatggctg 1200 gccgatctgc ccaaactcca cactctgcat cttcaaatga
acttcatcaa ccaggcacag 1260 ctcagcatct ttggtacctt ccgagccctt
cgctttgtgg acttgtcaga caatcgcatc 1320 agtgggcctt caacgctgtc
agaagccacc cctgaagagg cagatgatgc agagcaggag 1380 gagctgttgt
ctgcggatcc tcacccagct ccactgagca cccctgcttc taagaacttc 1440
atggacaggt gtaagaactt caagttcacc atggacctgt ctcggaacaa cctggtgact
1500 atcaagccag agatgtttgt caatctctca cgcctccagt gtcttagcct
gagccacaac 1560 tccattgcac aggctgtcaa tggctctcag ttcctgccgc
tgactaatct gcaggtgctg 1620 gacctgtccc ataacaaact ggacttgtac
cactggaaat cgttcagtga gctaccacag 1680 ttgcaggccc tggacctgag
ctacaacagc cagcccttta gcatgaaggg tataggccac 1740 aatttcagtt
ttgtggccca tctgtccatg ctacacagcc ttagcctggc acacaatgac 1800
attcataccc gtgtgtcctc acatctcaac agcaactcag tgaggtttct tgacttcagc
1860 ggcaacggta tgggccgcat gtgggatgag gggggccttt atctccattt
cttccaaggc 1920 ctgagtggcc tgctgaagct ggacctgtct caaaataacc
tgcatatcct ccggccccag 1980 aaccttgaca acctccccaa gagcctgaag
ctgctgagcc tccgagacaa ctacctatct 2040 ttctttaact ggaccagtct
gtccttcctg cccaacctgg aagtcctaga cctggcaggc 2100 aaccagctaa
aggccctgac caatggcacc ctgcctaatg gcaccctcct ccagaaactg 2160
gatgtcagca gcaacagtat cgtctctgtg gtcccagcct tcttcgctct ggcggtcgag
2220 ctgaaagagg tcaacctcag ccacaacatt ctcaagacgg tggatcgctc
ctggtttggg 2280 cccattgtga tgaacctgac agttctagac gtgagaagca
accctctgca ctgtgcctgt 2340 ggggcagcct tcgtagactt actgttggag
gtgcagacca aggtgcctgg cctggctaat 2400 ggtgtgaagt gtggcagccc
cggccagctg cagggccgta gcatcttcgc acaggacctg 2460 cggctgtgcc
tggatgaggt cctctcttgg gactgctttg gcctttcact cttggctgtg 2520
gccgtgggca tggtggtgcc tatactgcac catctctgcg gctgggacgt ctggtactgt
2580 tttcatctgt gcctggcatg gctacctttg ctggcccgca gccgacgcag
cgcccaagct 2640 ctcccctatg atgccttcgt ggtgttcgat aaggcacaga
gcgcagttgc ggactgggtg 2700 tataacgagc tgcgggtgcg gctggaggag
cggcgcggtc gccgagccct acgcttgtgt 2760 ctggaggacc gagattggct
gcctggccag acgctcttcg agaacctctg ggcttccatc 2820 tatgggagcc
gcaagactct atttgtgctg gcccacacgg accgcgtcag tggcctcctg 2880
cgcaccagct tcctgctggc tcagcagcgc ctgttggaag accgcaagga cgtggtggtg
2940 ttggtgatcc tgcgtccgga tgcccaccgc tcccgctatg tgcgactgcg
ccagcgtctc 3000 tgccgccaga gtgtgctctt ctggccccag cagcccaacg
ggcagggggg cttctgggcc 3060 cagctgagta cagccctgac tagggacaac
cgccacttct ataaccagaa cttctgccgg 3120 ggacctacag cagaatagct
cagagcaaca gctggaaaca gctgcatctt catgcctggt 3180 tcccgagttg
ctctgcctgc 3200
TABLE 3 Coding region for murine TLR9 (SEQ ID NO:2) atggttctcc
gtcgaaggac tctgcacccc ttgtccctcc tggtacaggc tgcagtgctg 60
gctgagactc tggccctggg taccctgcct gccttcctac cctgtgagct gaagcctcat
120 ggcctggtgg actgcaattg gctgttcctg aagtctgtac cccgtttctc
tgcggcagca 180 tcctgctcca acatcacccg cctctccttg atctccaacc
gtatccacca cctgcacaac 240 tccgacttcg tccacctgtc caacctgcgg
cagctgaacc tcaagtggaa ctgtccaccc 300 actggcctta gccccctgca
cttctcttgc cacatgacca ttgagcccag aaccttcctg 360 gctatgcgta
cactggagga gctgaacctg agctataatg gtatcaccac tgtgccccga 420
ctgcccagct ccctggtgaa tctgagcctg agccacacca acatcctggt tctagatgct
480 aacagcctcg ccggcctata cagcctgcgc gttctcttca tggacgggaa
ctgctactac 540 aagaacccct gcacaggagc ggtgaaggtg accccaggcg
ccctcctggg cctgagcaat 600 ctcacccatc tgtctctgaa gtataacaac
ctcacaaagg tgccccgcca actgcccccc 660 agcctggagt acctcctggt
gtcctataac ctcattgtca agctggggcc tgaagacctg 720 gccaatctga
cctcccttcg agtacttgat gtgggtggga attgccgtcg ctgcgaccat 780
gcccccaatc cctgtataga atgtggccaa aagtccctcc acctgcaccc tgagaccttc
840 catcacctga gccatctgga aggcctggtg ctgaaggaca gctctctcca
tacactgaac 900 tcttcctggt tccaaggtct ggtcaacctc tcggtgctgg
acctaagcga gaactttctc 960 tatgaaagca tcaaccacac caatgccttt
cagaacctaa cccgcctgcg caagctcaac 1020 ctgtccttca attaccgcaa
gaaggtatcc tttgcccgcc tccacctggc aagttccttc 1080 aagaacctgg
tgtcactgca ggagctgaac atgaacggca tcttcttccg ctcgctcaac 1140
aagtacacgc tcagatggct ggccgatctg cccaaactcc acactctgca tcttcaaatg
1200 aacttcatca accaggcaca gctcagcatc tttggtacct tccgagccct
tcgctttgtg 1260 gacttgtcag acaatcgcat cagtgggcct tcaacgctgt
cagaagccac ccctgaagag 1320 gcagatgatg cagagcagga ggagctgttg
tctgcggatc ctcacccagc tccactgagc 1380 acccctgctt ctaagaactt
catggacagg tgtaagaact tcaagttcac catggacctg 1440 tctcggaaca
acctggtgac tatcaagcca gagatgtttg tcaatctctc acgcctccag 1500
tgtcttagcc tgagccacaa ctccattgca caggctgtca atggctctca gttcctgccg
1560 ctgactaatc tgcaggtgct ggacctgtcc cataacaaac tggacttgta
ccactggaaa 1620 tcgttcagtg agctaccaca gttgcaggcc ctggacctga
gctacaacag ccagcccttt 1680 agcatgaagg gtataggcca caatttcagt
tttgtggccc atctgtccat gctacacagc 1740 cttagcctgg cacacaatga
cattcatacc cgtgtgtcct cacatctcaa cagcaactca 1800 gtgaggtttc
ttgacttcag cggcaacggt atgggccgca tgtgggatga ggggggcctt 1860
tatctccatt tcttccaagg cctgagtggc ctgctgaagc tggacctgtc tcaaaataac
1920 ctgcatatcc tccggcccca gaaccttgac aacctcccca agagcctgaa
gctgctgagc 1980 ctccgagaca actacctatc tttctttaac tggaccagtc
tgtccttcct gcccaacctg 2040 gaagtcctag acctggcagg caaccagcta
aaggccctga ccaatggcac cctgcctaat 2100 ggcaccctcc tccagaaact
ggatgtcagc agcaacagta tcgtctctgt ggtcccagcc 2160 ttcttcgctc
tggcggtcga gctgaaagag gtcaacctca gccacaacat tctcaagacg 2220
gtggatcgct cctggtttgg gcccattgtg atgaacctga cagttctaga cgtgagaagc
2280 aaccctctgc actgtgcctg tggggcagcc ttcgtagact tactgttgga
ggtgcagacc 2340 aaggtgcctg gcctggctaa tggtgtgaag tgtggcagcc
ccggccagct gcagggccgt 2400 agcatcttcg cacaggacct gcggctgtgc
ctggatgagg tcctctcttg ggactgcttt 2460 ggcctttcac tcttggctgt
ggccgtgggc atggtggtgc ctatactgca ccatctctgc 2520 ggctgggacg
tctggtactg ttttcatctg tgcctggcat ggctaccttt gctggcccgc 2580
agccgacgca gcgcccaagc tctcccctat gatgccttcg tggtgttcga taaggcacag
2640 agcgcagttg cggactgggt gtataacgag ctgcgggtgc ggctggagga
gcggcgcggt 2700 cgccgagccc tacgcttgtg tctggaggac cgagattggc
tgcctggcca gacgctcttc 2760 gagaacctct gggcttccat ctatgggagc
cgcaagactc tatttgtgct ggcccacacg 2820 gaccgcgtca gtggcctcct
gcgcaccagc ttcctgctgg ctcagcagcg cctgttggaa 2880 gaccgcaagg
acgtggtggt gttggtgatc ctgcgtccgg atgcccaccg ctcccgctat 2940
gtgcgactgc gccagcgtct ctgccgccag agtgtgctct tctggcccca gcagcccaac
3000 gggcaggggg gcttctgggc ccagctgagt acagccctga ctagggacaa
ccgccacttc 3060 tataaccaga acttctgccg gggacctaca gcagaa 3096
The deduced amino acid sequence for murine TLR9 (SEQ ID NO:3),
comprising 1032 amino acid residues, is shown in Table 4 below in
the aligned sequence comparison as mtlr932e.pep. The deduced amino
acid sequence for human TLR9 (SEQ ID NO:6), comprising 1032 amino
acid residues, is shown in Table 4 below in the aligned sequence
comparison as htlr9.pro.
TABLE 4 Amino Acid Sequence of Murine and Human TLR9 . : . : . : .
: . : . : 60 ht1r9.pro
MGFCRSAHLPLSLLVQAIMLAMTLALGTLPAFLPCELQPHGLVNCNWLFLKSVPHFSMAA 60
mt1r932e.pep
MVLRRRTLHPLSLLVQAAVLAETLALGTLPAFLPCELKPHGLVDCNWLFLKSVPRFSAAA 60 . :
. : . : . : . : . : 120 ht1r9.pro
PRGNVTSLSLSSNRIHHLHDSDFAHLPSLRHLNLKWNCPPVGLSPMHFPCHMTIEPSTFL 120
mt1r932e.pep
SCSNITRLSLISNRIHHLHNSDFVHLSNLRQLNLKWNCPPTGLSPLHFSCHMTIEPRTFL 120 .
: . : . : . : . : . : 180 ht1r9.pro
AVPTLEELNLSYNNIMTVPALPKSLISLSLSHTNILMLDSASLAGLHALRFLFMDGNCYY 180
mt1r932e.pep
AMRTLEELNLSYNGITTVPRLPSSLVNLSLSHTNILVLDANSLAGLYSLRVLFMDGNCYY 180 .
: . : . : . : . : . : 240 ht1r9.pro
KNPCRQALEVAPGALLGLGNLTHLSLKYNNLTVVPRNLPSSLEYLLLSYNRIVKLAPEDL 240
mt1r932e.pep
KNPCTGAVKVTPGALLGLSNLTHLSLKYNNLTKVPRQLPPSLEYLLVSYNLIVKLGPEDL 240 .
: . : . : . : . : . : 300 ht1r9.pro
ANLTALRVLDVGGNCRRCDHAPNPCMECPRHFPQLHPDTFSHLSRLEGLVLKDSSLSWLN 300
mt1r932e.pep
ANTLTSLRVLDVGGNCRRCDHAPNPCIECGQKSLHLHPETFHHLSHLEGLVLKDSSLHTLN 300 .
: . : . : . : . : . : 360 aa197442.pep LNLSFNYRKKVSFARLHLASSF 22
ht1r9.pro
ASWFRGLGNLRVLDLSENFLYKCITKTKAFQGLTQLRKLNLSFNYQKRVSFAHLSLAPSF 360
mt1r932e.pep
SSWFQGLVNLSVLDLSENFLYESINETNAFQNLTRLRKNLSFNYRKKVSFARLHLASSF 360 . :
. : . : . : . : . : 420 mousepep1 C 1 aa197442.pep
KNLVSLQELNMNGIFFRLLNKYTLRWLADLPKLHTLHLQMNFINQAQLSIFGTFRALRFV 82
ht1r9.pro
GSLVALKELDMHGIFFRSLDETTLRPLARLPMLQTLRLQMNFINQAQLGIFRAFPGLRYV 420
mt1r932e.pep
KNLVSLQELNMNGIFFRSLNKYTLRWLADLPKLHTLHLQMNFINAQAQLSIFGTFRALRFV 420 .
: . : . : . : . : . : 480 mousepep1 DLSDNRISGPSTLSEA 17 humanpep1
PAPVDTPSSEDFRPNC 16 aa197442.pep
DLSDNRISGPSTLSEATPEEADDAEQEELLSADPHPAPLSTPASKNFMDRCKNFKFNMDL 142
ht1r9.pro
DLSDNRISGASELT-ATMGEADGGEKVWLQPGDLAPAPVTPSSEDFRPNCSTLNFTLDL 479
mt1r932e.pep
DLSDNRIGSGPSTLSEATPEEDDAEQEELLSADPHPAPLSTPASKNFMDRCKNFKFTMDL 480 .
: . : . : . : . : . : 540 aa197442.pep
SRNNLVTITAEMFVNLSRLQCLSLSHNSIAQAVNGS 178 ht1r9.pro
SRNNLVTVQPEMFAQLSHLQCLRLSHNCISQAVNGSQFLPLTGLQVLDLSRNKLDLYHEH 539
mt1r932e.pep
SPRNNLVTIKPEMFVNLSRLQCLSLSHNSIAQAVNGSQFLPLTNLQVLDLSHNKLDLYHWK 540 .
: . : . : . : . : . : 600 aa162495.pep
YNSQPFSMKGIGHNFSFVTHLSMLQSLSLAHNDIHTRVSSHLNSNS 46 ht1r9.pro
SFTELPRLEALDLSYNSQPFGMQGVGHNFSFVAHLRTLRHLSLAHNNIHSQVSQQLCSTS 599
mt1r932e.pep
SFSELPQLQALDLSYNSQPFSMKGIGHNFVAHLSMLHSLSLAHNDIHTRVSSHLNSNS 600 . :
. : . : . : . : . : 660 aa162495.pep
VRFLDFSGNGMGRMWDEGGLYLHFFQGLSGVLKLDLSQNNLHILRPQNLDNLPKSLKLLS 106
ht1r9.pro
LRALDFSGNALGHMWAEGDLYLHFFQGLSGLIWLDLSQNRLHTLLPQTLRNLPKSLQVLR 659
mt1r932e.pep
VRFLDFSGNGMGRMWDEGGLYLHFFQGLSGLLKLDLSQNNLHILRPQNLDNLPKSLKLLS 660 .
: . : . : . : . : . : 720 aa162495.pep
LRDNYLSFFNWTSLSFLPNLEVLDLAGNQLKALTNGTLPNGTLLQKLDVSSNSIVS 162
ht1r9.pro
LRDNYLAFFKWWSLHFLPKLEVLDLAGNRLKALTNGSLPAGTRLRRLDVSCNSISFVAPG 719
mt1r932e.pep
LRDNYLSFFNWTSLSFLPNLEVLDLAGNQLKALTNGTLPNGTLLQKLDVSSNSIVSVVPA 720 .
: . : . : . : . : . : 780 ai451215.pep
PIVMNLTVLDVRSNPLHCACGAAFVDLLLEVQT 33 ht1r9.pro
FFSKAKELRELNLSANALKTVDHSWFGPLASALQILDVSANPLHCACGAAFMDFLLEVQA 779
mt1r932e.pep
FFALAVELKEVLSHNILKTVDRSWFGPIVMLTVLDVRSNPLHCACGAAFVDLLLEVQT 780 . :
. : . : . : . : . : 840 ai451215.pep
KVPGLANGVKCGSPGQLQGRSIFAQDLRLCLDEVLSWDCFGLSLLAVAVGMVVPILHHLC 93
ht1r9.pro
AVPGLPSRVKCGSPGQLQGLSIFAQDLRLCLDEALSWDCFALSLLAVALGLGVPMLHHLC 839
mt1r932e.pep
KVPGLANGVKCGSPGQLQGRSIFAQDLRLCLDEVLSWDCFGLSLLAVAVGMVPILHHLC 840 . :
. : . : . : . : . : 900 ai451215.pep
GWDVWYCFHLCLAWLPLLAR-SRRSAQTLPYDAFVVFDKAQSAVADWVYNELRVRLEERR 152
ht1r9.pro
GWDLWYCFHLCLAWLPWRGRQSGRDEDALPYDAFVVFDKTQSAVADWVYNELRGQLEECR 899
mt1r932e.pep
GWDVWYCFHLCLAWLPLLAR-SRRSAQALPYDAFVVFDKAQSAVADWVYNELRVRLEERR 899 .
: . : . : . : . : . : 960 aa273731.pep AHTDRVSGLLRTSFLLAQQRLL 22
ai463056.pep EDRDWLPGQTLFENLWASIYGSRKTLFVLAHTDRVSGLLRTSFLLAQQRLL 51
ai451215.pep GR 154 ht1r9.pro
GRWALRLCLEERDWLPGKTLFENLWASVYGSRKTLFVLAHTDRVSGLLRASFLLAQQRLL 959
mt1r932e.pep
GRRLRLCLEDRDWLPGQTLFENLWASIYGSRKTLFVLAHTDRVSGLLRTSFLLAQQRLL 959 . :
. : . : . : . : . : 1020 humanpep2 H 1 mousepep2 H 1 aa273731.pep
EDRKDVVVLVILRPDAXPSRYVRLRQRLCRQSVLFWPQRPNGQGGFWAQLSTALTRDNRH 82
ai463056.pep
EDRKDVVVLVILRPDAHRSRYVRLRQRLCRQSVLFWPQQPNGQGGFWAQLSTALTRDNRH 111
ht1r9.pro
EDRKDVVVLVILSPDGRRSRYVRLRQRLCRQSVLLWPHQPSGQRSFWAQLGMALTRDNHH 1019
mt1r932e.pep
EDRKDVVVLVILRPDAHRSRYVRLRQRLCRQSVLFWPQQPNGQGGFWAQLSTALTRDNRH 1019 .
: . : . : . : . : . : 1080 humanpep2 FYNRNFCQGPTAE 14 mousepep2
FYNQNFCRGPTAE 14 aa273731.pep FYNQNFCRGPTAE 95 ai463056.pep
FYNQNFCRGPTA 123 ht1r9.pro FYNRNFCQGPTAE 1032 mt1r932e.pep
FYNQNFCRGPTAE 1032
The following SEQ ID NOs correspond to the sequences as shown in
Table 4: htlr9.pro: SEQ ID NO:6; mtlr932e.pep: SEQ ID NO:3;
aa197442.pep: SEQ ID NO:8; mousepep1: SEQ ID NO:17; humanpep1: SEQ
ID NO:19; aa162495.pep: SEQ ID NO:14; ai451215.pep: SEQ ID NO:16;
aa273731.pep: SEQ ID NO:10; ai463056.pep: SEQ ID NO:12; humanpep2:
SEQ ID NO:20; and mousepep2: SEQ ID NO:18.
Example 2
Reconstitution of TLR9 Signaling in 293 Fibroblasts
The cloned mouse TLR9 cDNA (see above) and human TLR9 cDNA (gift
from B. Beutler, Howard Hughes Medical Institute, Dallas, Tex.) in
pT-Adv vector (from Clonetech) were cloned into the expression
vector pcDNA3.1(-) from Invitrogen using the EcoRI site. Utilizing
a "gain of function" assay it was possible to reconstitute human
TLR9 (hTLR9) and murine TLR9 (mTLR9) signaling in CpG DNA
non-responsive human 293 fibroblasts (ATCC, CRL-1573). The
expression vectors mentioned above were transfected into 293
fibroblast cells using the calcium phosphate method.
Since NF-.kappa.B activation is central to the IL-1/TLR signal
transduction pathway (Medzhitov R et al., Mol Cell 2:253-258
(1998); Muzio M et al., J Exp Med 187:2097-2101 (1998)), cells were
transfected with hTLR9 or co-transfected with hTLR9 and a
NF-.kappa.B-driven luciferase reporter construct. Human fibroblast
293 cells were transiently transfected with (FIG. 1A) hTLR9 and a
six-times NF-.kappa.B-luciferase reporter plasmid (NF-.kappa.B-luc,
kindly provided by Patrick Baeuerle, Munich, Germany) or (FIG. 1B)
with hTLR9 alone. After stimulus with CpG-ODN (2006, 2 .mu.M,
TCGTCGTTTTGTCGTTTTGTCGTT, SEQ ID NO:112), GpC-ODN (2006-GC, 2
.mu.M, TGCTGCTTTTGTGCTTTTGTGCTT, SEQ ID NO:118), LPS (100 ng/ml) or
media, NF-.kappa.B activation by luciferase readout (8 h, FIG. 1A)
or IL-8 production by ELISA (48 h, FIG. 1B) were monitored. Results
are representative of three independent experiments. FIG. 1 shows
that cells expressing hTLR9 responded to CpG-DNA but not to
LPS.
FIG. 2 demonstrates the same principle for the transfection of
mTLR9. Human fibroblast 293 cells were transiently transfected with
mTLR9 and the NF-.kappa.B-luc construct (FIG. 2). Similar data was
obtained for IL-8 production (not shown). Thus expression of TLR9
(human or mouse) in 293 cells results in a gain of function for
CpG-DNA stimulation similar to hTLR4 reconstitution of LPS
responses.
To generate stable clones expressing human TLR9, murine TLR9, or
either TLR9 with the NF-.kappa.B-luc reporter plasmid, 293 cells
were transfected in 10 cm plates (2.times.10.sup.6 cells/plate)
with 16 .mu.g of DNA and selected with 0.7 mg/ml G418 (PAA
Laboratories GmbH, Colbe, Germany). Clones were tested for TLR9
expression by RT-PCR, for example as shown in FIG. 3. The clones
were also screened for IL-8 production or NF-.kappa.B-luciferase
activity after stimulation with ODN. Four different types of clones
were generated.
293-hTLR9-luc: expressing human TLR9 and 6-fold
NF-.kappa.B-luciferase reporter 293-mTLR9-luc: expressing murine
TLR9 and 6-fold NF-.kappa.B-luciferase reporter 293-hTLR9:
expressing human TLR9 293-mTLR9: expressing murine TLR9
FIG. 4 demonstrates the responsiveness of a stable 293-hTLR9-luc
clone after stimulation with CpG-ODN (2006, 2 .mu.M), GpC-ODN
(2006-GC, 2 .mu.M), Me-CpG-ODN (2006 methylated, 2 .mu.M;
TZGTZGTTTTGTZGTTTTGTZGTT, Z=5-methylcytidine, SEQ ID NO:128), LPS
(100 ng/ml) or media, as measured by monitoring NF-.kappa.B
activation. Similar results were obtained utilizing IL-8 production
with the stable clone 293-hTLR9. 293-mTLR9-luc were also stimulated
with CpG-ODN (1668, 2 .mu.M; TCCATGACGTTCCTGATGCT, SEQ ID NO:84),
GpC-ODN (1668-GC, 2 .mu.M; TCCATGAGCTTCCTGATGCT, SEQ ID NO:85),
Me-CpG-ODN (1668 methylated, 2 .mu.M; TCCATGAZGTTCCTGATGCT,
Z=5-methylcytidine, SEQ ID NO:207), LPS (100 ng/ml) or media, as
measured by monitoring NF-.kappa.B activation (FIG. 5). Similar
results were obtained utilizing IL-8 production with the stable
clone 293-mTLR9. Results are representative of at least two
independent experiments. These results demonstrate that CpG-DNA
non-responsive cell lines can be stably genetically complemented
with TLR9 to become responsive to CpG DNA in a motif-specific
manner. These cells can be used for screening of optimal ligands
for innate immune responses driven by TLR9 in multiple species.
Example 3
Expression of Soluble Recombinant Human TLR9 in Yeast Cells (Pichia
pastoris)
Human TLR9 cDNA coding for amino acids 1 to 811 was amplified by
PCR using the primers 5'-ATAGAATTCAATAATGGGTTTCTGCCGCAGCGCCCT-3'
(SEQ ID NO:194) and 5'-ATATCTAGATCCAGGCAGAGGCGCAGGTC-3' (SEQ ID
NO:195), digested with EcoRI and XbaI, cloned into the yeast
expression vector pPICZB (Invitrogen, Groningen, Netherlands) and
transfected into yeast cells (Pichia pastoris). Clones were
selected with the antibiotic zeozin and protein production of
soluble human TLR9 was induced with methanol (see FIG. 6: SDS-PAGE,
Coomassie stained, arrow marks hTLR9; lane 1: supernatant of
culture induced with methanol; lane 2: supernatant of culture not
induced). Thus TLR9 protein can be isolated from transfectants and
further utilized for protein studies and vaccination purposes.
Example 4
hTLR9 Expression Correlates with CpG-DNA Responsiveness
Bacterial DNA has been described as a mitogen for both murine and
human B cells. Although LPS is also mitogenic for murine B cells,
it is generally accepted that LPS is not a mitogen for human B
cells. FIG. 7 demonstrates that human B cells proliferate after
stimulation with E. coli DNA or a CpG-ODN but not Dnase-digested E.
coli DNA or a control GpC-ODN. Purified human B cells were
stimulated with 50 .mu.g/ml E. coli DNA, a comparable amount of
DNase I-digested E. coli DNA, 2 .mu.M CpG-ODN (2006), 2 .mu.M
GpC-ODN (2006-GC) or 100 ng/ml LPS. B cell proliferation was
monitored at day two by .sup.3 H-thymidine uptake. These data
demonstrate that it was DNA within the E. coli DNA preparation that
was mitogenic and that a CpG-motif within the ODN was required.
Human dendritic cells (DC) have been claimed to be responsive to
CpG-DNA. While analyzing human dendritic cell responses to CpG-DNA,
we noted that plasmacytoid DC (CD123+DC) produced IFN-.alpha., TNF,
GM-CSF, and IL-8 upon exposure to CpG-DNA but not to LPS (FIG. 8
and unpublished data). The converse was true for stimulation of
monocyte-derived dendritic cells (MDDC) (FIG. 8 and unpublished
data). Purified CD123+DC or MDDC were stimulated with 50 .mu.g/ml
E. coli DNA, a comparable amount of DNase I-digested E. coli DNA, 2
.mu.M CpG-ODN (2006), 2 .mu.M GpC-ODN (2006-GC) or 100 ng/ml LPS
(FIG. 8). IL-8 and TNF concentration was determined by
enzyme-linked immunosorbent assay (ELISA). The CD123+DC response
was DNA- and CpG-motif restricted. Monocyte-derived dendritic cells
(MDDC) however demonstrated the converse response pattern, a
response to LPS but not CpG-DNA. Due to this segregated response we
analyzed TLR expression.
We have shown that CpG-DNA utilizes the Toll/IL-1R (TIR) signal
transduction pathway implying the need for a TIR domain in the
CpG-DNA signaling receptor. Hacker H et al., J Exp Med 192:595-600
(2000). It was further demonstrated that TLR9-deficient mice are
non-responsive to CpG-ODN. Hemmi H et al., Nature 408:740-5. By
semi-quantitative RT-PCR both B cells and CD123+DC yielded positive
signals for hTLR9 while MDDC, monocytes and T cells were weak to
negative (FIG. 9). The cDNAs were prepared from monocyte-derived
dendritic cells (MDDC), lane 1; purified CD14+monocytes, lane 2; B
cells, lane 3; CD123+DC, lane 4; CD4+T cells, lane 5; and CD8+T
cells, lane 6. cDNA amounts were normalized based on GAPDH amount
determined by TAG-MAN PCR (Perkin-Elmer). RT-PCR was performed for
30 cycles on normalized cDNA diluted 1:5 for human TLR2, 4 and 9,
while GAPDH was diluted 1:125. We also tested for hTLR2 and hTLR4
expression. MDDC and monocytes were positive while B cells, T cells
and CD123+DC were weak to negative (FIG. 9). Weak signals delivered
by PCR could be explained by contaminating cells, however a strong
positive signal implies expression. These data demonstrated a clear
correlation between hTLR9 mRNA expression and B cell or CD123+DC
responsiveness to CpG-DNA (FIGS. 7 and 8). A correlation could also
be shown for hTLR2 and hTLR4 expression and MDDC responsiveness to
LPS (FIG. 8). This data demonstrates that hTLR9 is a relevant
receptor for CpG-DNA responses and that its expression determines
responsiveness. If TLR9 expression could be modulated, agonism or
antagonism of CpG-DNA responses could be achieved.
Example 5
Species Specificity of TLR9 Signaling
By iterative examination of the flanking sequences surrounding CG
dinucleotides, CpG-motifs have been identified. Paradoxically, or
by twist of nature, the human optimal CpG-motif, GTCGTT (SEQ ID
NO:66), is different from the murine motif, GACGTT (SEQ ID NO:129).
Human peripheral blood mononuclear cells (PBMC) (FIG. 10A) and
murine splenocytes (FIG. 10B) were stimulated with ODN 2006 (filled
circle, TCGTCGTTTTGTCGTTTTGTCGTT, SEQ ID NO:112), ODN 2006-GC (open
circle), ODN 1668 (filled triangle, TCCATGACGTTCCTGATGCT, SEQ ID
NO:84) or ODN 1668-GC (open triangle, TCCATGAGCTTCCTGATGCT, SEQ ID
NO:85) at indicated concentrations and IL-12 production was
monitored after 8 hours. FIG. 10A shows that titration of the
optimal human ODN, 2006, on PBMC induces IL-12 production. The
optimal murine sequence, 1668, however was much less effective in
eliciting IL-12 from PBMC. The two control GpC-ODNs were
essentially negative. The converse was true for murine splenocytes
(FIG. 10B), in that the murine sequence induced optimal IL-12 while
the human sequence was much less effective. It should also be noted
that the K.sub.ac (concentration of half-maximal activation) of
murine splenocytes for 1668 was greater than human PBMC for 2006
(compare FIG. 10A to FIG. 10B).
Because stable TLR9 transfectants mirrored primary cell
responsiveness to CpG-DNA (FIGS. 4 and 5), it was hypothesized that
stable transfectants could potentially discern species-specific
CpG-motifs through TLR9 receptors. Therefore 293-hTLR9-luc
(expressing human TLR9 and 6-fold NF-.kappa.B-luc reporter),
293-mTLR9-luc (expressing murine TLR9 and 6-fold NF-.kappa.B-luc
reporter), 293-hTLR9 (expressing human TLR9) and 293-mTLR9
(expressing murine TLR9) clones were tested for CpG-DNA motif
responsiveness. FIG. 11 shows titration curves for 2006 or 1668 and
their controls versus either hTLR9 or mTLR9 cells. Depicted are
both NF-.kappa.B-driven luciferase and IL-8 production as readout.
In both 293 hTLR9-luc and 293-mTLR9-luc cells stimulation with
CpG-DNA resulted in NF-.kappa.B activation, as determined by
measurement of the induced expression of firefly luciferase under
the control of a minimal promotor containing six tandem
NF-.kappa.B-binding sites. After lysis of the cells luciferase can
be detected photometrically based on an enzymatic reaction by
luciferase which creates photons. IL-8 production was monitored
using enzyme-linked immunosorbent assay (ELISA). FIG. 11 depicts
clones stimulated with ODN 2006 (filled circle), ODN 2006-GC (open
circle), ODN 1668 (filled triangle) or ODN 1668-GC (open triangle)
at indicated concentrations and NF-.kappa.B activation or IL-8
production were measured after 10 and 48 hours, respectively.
Results shown in FIG. 11 are representative of three independent
experiments. Strikingly, CpG-motif sequence specificity was
conferred in a species-specific manner by TLR9. Additionally, the
half-maximal concentration for either 2006 or 1668 appears nearly
the same as those determined on primary cells (compare FIG. 10 and
FIG. 11). These data demonstrate that TLR9 is the CpG-DNA receptor
and that exquisite specificity to CpG-DNA sequence is conferred by
TLR9.
Example 6
Use of Stable TLR9 Clones to Test Responsiveness to Substances
Other Than Phosphorothioate ODN
As described in the foregoing Examples, the stable TLR9 clones were
initially screened for fidelity of phosphorothioate CpG-ODN
reactivity. The 293-hTLR9 cells demonstrated reactivity to CpG-DNA
and not LPS in a CpG-motif dependent manner (FIGS. 4 and 5). In the
present example the stable TLR9 transfectants were tested for
responsiveness to additional DNAs. NF-.kappa.B activation was
monitored after stimulation with E. coli DNA (black bars) or E.
coli DNA digested with DNAse I (gray bars) in 293-hTLR9-luc cells.
FIG. 12 demonstrates an E. coli DNA dose-dependent induction of
NF-.kappa.B-driven luciferase expression to a level comparable to
phosphorothioate CpG-ODN (FIG. 11). Activity was destroyed by DNase
I digestion, indicating specificity of response to DNA and not
contaminant bacterial products. The stable TLR9 transfectants can
be used to screen the activity of DNAs from various species or
vector DNAs intended for immune system stimulation. In particular,
TLR9 transfectants can be used to screen and compare the
immunostimulatory activity of DNAs from various species of
pathogens, DNA constructs, DNAs intended for use as vaccines, gene
replacement therapeutics, and nucleic acid vectors.
293-hTLR9-luc cells also were stimulated with the phosphodiester
variants of ODN 2006 (filled circle), ODN 2006-GC (open circle),
ODN 1668 (filled triangle) or ODN 1668-GC (open triangle) at
indicated concentrations, and NF-.kappa.B activation was monitored
after 12 hours (FIG. 13A). Likewise, 293-mTLR9-luc cells were
stimulated with the phosphodiester variants of ODN 2006 (filled
circle), ODN 2006-GC (open circle), ODN 1668 (filled triangle) or
ODN 1668-GC (open triangle) at indicated concentrations, and
NF-.kappa.B activation was monitored after 12 hours (FIG. 13B).
These assays show that the stable TLR9 transfectants responded to
DNAs other than phosphorothioate-modified ODN. These data
demonstrate the utility of stable TLR transfectants for screening
for agonists of the TLR9 receptor.
Example 7
TLR9 Determines CPG-ODN Activity
Although 2006 and 1668 are discussed in terms of CpG-motif
differences, they are very different in several aspects (see Table
5 for comparison). The lengths are different, 24 versus 20
nucleotides, and 2006 has four CG dinucleotides compared to one in
1668. Additional differences are the CG position relative to the 5'
and 3' ends and also 5' sequence differences. In order to determine
if motif specificity is a quality of the motif and not the global
sequence environment, for this experiment several sequences were
produced holding these variales constant. As a starting point, the
1668 sequence was modified by converting the central C to T and the
distal TG to CG, thereby creating a second CG in the resulting
sequence 5000 (SEQ ID NO:130, Table 5). Then point nucleotide
changes were made, progressing toward a 2006-like sequence, 5007
(SEQ ID NO:98). The ODN 5002 (SEQ ID NO:132) is most like 1668 with
the exception that C's at positions 12 and 19 have been converted
to T's. The last 16 nucleotides of ODN 5007 are the same as the
last 15 nucleotides of 2006 with the exception of an additional T.
The ODN concentration of half-maximal activation (K.sub.ac) was
determined by producing ODN titration curves using either
293-hTLR9-luc or 293-mTLR9-luc cells and NF-.kappa.B-driven
luciferase expression as a readout. Example curves are given in
FIG. 14. Stable transfectants 293-hTLR9-luc and 293-mTLR9-luc were
stimulated with ODN 5002 (filled circle) or ODN 5007 (open circle)
at indicated concentrations and NF-.kappa.B activation was
monitored after 12 hours. Results shown in FIG. 14 are
representative of three independent experiments. Values for
K.sub.ac for multiple ODN are given in Table 5. Similar results
were obtained for those ODN tested with 293-hTLR9 and 293-mTLR9
cells utilizing IL-8 as readout.
TABLE 5 CpG-DNA sequence specificity of human and murine TLR9
signaling activity 293- 293- SEQ CpG- hTLR9 mTLR9 ID DNA Sequence
K.sub.ac (nM) K.sub.ac (nM) NO: 1668 TCCATGACGTTCCTGATGCT
>10,000 70 84 1668- TCCATGAGCTTCCTGATGCT >10,000 >10,000
85 GC 2006 TCGTCGTTTTGTCGTTTTGTCGTT 400 >10,000 112 2006-
TGCTGCTTTTGTGCTTTTGTGCTT >10,000 >10,000 118 GC 5000
TCCATGACGTTCTTGACGCT 10,000 82 130 5001 TCCATGACGTTCTTGACGTT 7,000
55 131 5002 TCCATGACGTTCTTGATGTT 7,000 30 132 5003
TCCATGACGTTTTTGATGTT 10,000 30 133 5004 TCCATGTCGTTCTTGATGTT 5,000
400 134 5005 TCCATGTCGTTTTTGATGTT 3,000 2,000 135 5006
TCCATGTCGTTTTTGTTGTT 3,000 650 136 5007 TCCATGTCGTTTTTGTCGTT 700
1,000 98 5002 TCCATGACGTTCTTGATGTT ND 30 132 5008
TCCATGACGTTATTGATGTT ND 40 137 5009 TCCATGACGTCCTTGATGTT ND
>10,000 138 5010 TCCATGACGTCATTGATGTT ND >10,000 139
In previous unpublished work by the inventors, it had been noted
that a CA substitution converting the mouse CpG-motif from GACGTTC
to GACGTCA was deleterious. To extend our examination of the motif,
three more ODN were created to dissect this effect (5008-5010, SEQ
ID NOs:137-139, Table 5).
The activity displayed by the 293-hTLR9-luc clone increased with
progressive nucleotide substitutions converting the mouse sequence
toward the human sequence (Table 5, sequences 5000-5007). The
converse was true for the 293-mTLR9-luc clone, which showed highest
activity for the mouse sequences. The originally hypothesized
CpG-motif was purine-purine-CG-pyrimidine-pyrimidine. Most notable
to motif definition as determined by TLR9 genetic complementation
was the non-conservative pyrimidine for purine change A to T
immediately 5' of the CG (Table 5). These changes improved
293-hTLR9-luc responsiveness but diminished 293-mTLR9-luc
responsiveness. These results support the notion that the preferred
mouse motif contains ACG while the preferred human sequence
contains TCG. The conservative pyrimidine for pyrimidine change T
to C in the mouse motif, ACGTT versus ACGTC (5002 versus 5009),
completely destroyed 293-mTLR9 responsiveness. Although not a
complete iterative analysis of the CpG-motif, the data refine our
understanding of the motif. More importantly these data strongly
support direct CpG-motif engagement by TLR9.
Example 8
Antagonist Definition
It has been demonstrated that DNA uptake and endosomal maturation
are required for signal initiation by CpG-DNA. It has been
hypothesized that in order for DNA to enter the endosomal/lysosomal
compartment a non-CpG dependent uptake receptor may be required.
293 cells were transiently transfected with mTLR9 treated with
either medium only or 1.0 .mu.M CpG-ODN 1668 (FIG. 15).
Additionally the 1668-treated TLR9 transfectants were
simultaneously exposed to various doses of a non-CpG ODN (PZ2;
5'-CTCCTAGTGGGGGTGTCCTAT-3', SEQ ID NO:43). IL-8 production was
monitored after 48 h by ELISA. FIG. 15 shows that PZ2, in a
dose-dependent manner, was able to antagonize the activation of
TLR9-transfected cells stimulated with a CpG ODN.
FIG. 16 demonstrates that the stable TLR9 transfectants,
293-hTLR9-luc cells, are sensitive to non-CpG-ODN blockade.
293-hTLR9-luc cells were incubated with CpG-ODN (0.5 .mu.M) (black
bars) or TNF-.alpha. (10 ng/ml) (gray bars) and increasing
concentrations of a blocking ODN (5'-HHHHHHHHHHHHHHWGGGGG-3', SEQ
ID NO:140; H=A, T, C; W=A, T) as indicated. NF-.kappa.B activation
was monitored after 12 hours and is presented as percent yields.
Thus both mTLR9 and hTLR9 activity can be blocked by
non-stimulatory ODN. The blockade is specific to blocking ODN since
the TNF-driven NF-.kappa.B signal was not diminished. Antagonism of
CpG-DNA responses could thus be defined in stable TLR9 cells and
therefore high throughput screening can be done for TLR9
antagonist.
Bafilomycin A poisons the proton pump needed for H.sup.+ transport
into endosomes, which is required for endosomal maturation. FIG. 17
shows that blockade of endosomal maturation in 293-hTLR9-luc cells
fully blocks CpG-ODN induction of NF-.kappa.B. 293-hTLR9-luc cells
were preincubated with 10 nM Bafilomycin A (gray bars) or
dimethylsulfoxide (DMSO) control (black bars) for 30 min and
stimulated with CpG-ODN (2006, 0.5 .mu.M), IL-1 (10 ng/ml) or
TNF-.alpha. (10 ng/ml) as indicated. NF-.kappa.B activation was
monitored after 12 hours and is presented as percent yields. The
blockade was specific to CpG-DNA generated signal because both IL-1
and TNF induction of NF-.kappa.B was unaffected. These data
demonstrate that 293 cells stably complemented with hTLR9 behave in
a manner similar to primary CpG-DNA responsive cells, in that
cellular uptake and endosomal maturation are required for induction
of signal by CpG-DNA. Thus the stable transfectants can be used as
indicator for TLR9 drug antagonist.
CpG-DNA signaling appears to occur via a Toll/IL-1R-like pathway.
It was shown in the mouse that CpG-DNA signaling is dependent on
MyD88, IRAK and TRAF6. Hacker H et al., J Exp Med 192:595-600
(2000). Hemmi et al. demonstrated that mTLR9-deficient mice lack
activation of IRAK upon CpG-ODN stimulation. Hemmi H et al., Nature
408:740-5 (2000). FIG. 18 shows that CpG-DNA signaling via human
TLR9 was MyD88 dependent. hTLR9 (293-hTLR9) was co-transfected with
a six-times NF-.kappa.B luciferase reporter plasmid and increasing
concentrations of the dominant negative human MyD88 expression
vector. Cells were not stimulated (filled circles), stimulated with
CpG-ODN (2006, 2 .mu.M) (open circles) or TNF-.alpha. (10 ng/ml)
(filled triangles) and NF-.kappa.B activation was monitored after
12 hours. Results are representative of at least two independent
experiments. FIG. 18 demonstrates that dominant negative MyD88
blocks NF-.kappa.B induction in 293-hTLR9 cells following CpG-DNA
stimulation. The blockade of MyD88 did not affect NF-.kappa.B
induction via TNF induced signal transduction. In general these
data confirm the central role of MyD88 to TLR signaling and
specifically the role of MyD88 in CpG-DNA initiation of signal.
Thus human cells transfected with TLR9 can be used as indicators to
find molecules to antagonize CpG-DNA via genetic mechanisms.
Example 9
Antibody Production
Peptides for human and mouse TLR9 were designed for coupling to a
carrier protein and injected into rabbits to obtain anti-peptide
polyclonal antisera. Mouse peptide 1 (mousepep1, see Table 4) can
be found in EST aa197442 and peptide 2 (mousepep2, see Table 4) in
EST aa273731 and ai463056. Human peptide 1 (humanpep1, see Table 4)
and peptide 2 (humanpep2, see Table 4) were taken from the
published human sequence.
Three rabbit antisera were generated by this method:
anti-mousepep1, specific for the extracellular domain of murine
TLR9; anti-humanpep1, specific for the extracellular domain of
hTLR9; and antisera against a combination of mousepep2 and
humanpep2, specific for the cytoplasmic domain of both murine and
human TLR9. Immunoprecipitates with anti-FLAG antibody were
electrophoresed by PAGE and, using standard Western blotting
techniques, transferred to membrane and probed with the various
antisera. FIG. 19 shows the response to hTLR9-FLAG and mTLR9-FLAG.
The TLR9 in these blots are indicated with arrows, while the lower
molecular weight bands represent anti-FLAG antibody.
Example 10
Mutation Adjacent to the CXXC-Domain (hTLR9-CXXCm, mTLR9-CXXCh)
The CXXC motif resembles a zinc finger motif and is found in
DNA-binding proteins and in certain specific CpG binding proteins,
e.g. methyl-CpG binding protein-1 (MBD-1). Fujita N et al., Mol
Cell Biol 20:5107-5118 (2000). Human and murine TLR9 contain two
CXXC motifs. The CXXC domain is highly conserved between human and
murine TLR9 but followed by 6 amino acids (aa) which differ quite
substantially in polarity and size. By the use of a site-specific
mutagenesis kit (Stratagene, La Jolla, Calif., USA) these six amino
acid residues (human: PRHFPQ 269-274); mouse: GQKSLH 269-274) were
interchanged between human and murine TLR9. These mutations were
generated by the use of the primers
5'-CTGCATGGAGTGCGGCCAAAAGTCCCTCCACCTACATCCCGATAC-3' (SEQ ID NO:141)
and 5'-GTATCGGGATGTAGGTGGAGGGACTTTTGGCCGCACTCCATGCAG-3' (SEQ ID
NO:142) for human TLR9 and the primers
5'-CTGTATAGAATGTCCTCGTCACTTCCCCCAGCTGCACCCTGAGAC-3' (SEQ ID NO:143)
and 5'-GTCTCAGGGTGCAGCTGGGGGAAGTGACGAGGACATTCTATACAG-3' (SEQ ID
NO:144) for murine TLR9 according to the manufacturer's
protocol.
SEQ ID NO:145 CXXC motif: CXXCXXXXXXCXXC SEQ ID NO:146 Wildtype
hTLR9: CRRCDHAPNPCMECPRHFPQ aa 255-274 SEQ ID NO:147 hTLR9-CXXCm:
CRRCDHAPNPCMECGQKSLH aa 255-274 SEQ ID NO:148 Wildtype mTLR9:
CRRCDHAPNPCMICGQKSLH aa 255-274 SEQ ID NO:149 mTLR9-CXXCh:
CRRCDHAPNPCMICPRHFPQ aa 255-274
For the stimulation of the hTLR9 variant hTLR9-CXXCm, 293 cells
were transiently transfected with hTLR9 or hTLR9-CXXCm and
stimulated after 16 hours with ODN 2006 and ODN 1668 at
concentrations indicated (FIG. 20). 48 hours after stimulation
supernatant was harvested and IL-8 production was measured by
ELISA. The data show that hTLR9 can be improved by converting the
human CXXC domain to the murine CXXC domain. For the stimulation of
the mTLR9 variant mTLR9-CXXCh, 293 cells were transiently
transfected with mTLR9 or mTLR9-CXXCh and stimulated after 16 hours
with ODN 2006 and ODN 1668 at concentrations indicated (FIG. 21).
48 hours after stimulation supernatant was harvested and IL-8
production was measured by ELISA. It appears that the human CXXC
domain may diminish mTLR9-CXXCh activity relative to the wild type
mTLR9.
Example 11
Mutation in the MBD Motif (hTLR9-MBDmut, mTLR9-MBDmut)
The MBD motif is a domain recently described for CpG binding in the
protein MBD-1. Fujita N et al., Mol Cell Biol 20:5107-5118 (2000);
Ohki I et al., EMBO J. 18:6653-6661 (1999). Human and murine TLR9
contain this motif at position 524-554 and 525-555,
respectively.
MBD-1 SEQ ID NO:125 R-XXXXXXX-R-X-D-X-Y-XXXXXXXXX-R-S-XXXXXX-Y
hTLR9 SEQ ID NO:126 Q-XXXXXXX-K-X-D-X-Y-XXXXXXXXX-R-L-XXXXXX-Y
mTLR9 SEQ ID NO:127 Q-XXXXXXX-K-X-D-X-Y-XXXXXXXXX-Q-L-XXXXXX-Y
The core of this domain consists of D-L-Y in human TLR9 (aa
534-536) and mouse TLR9 (aa 535-537). Through site-specific
mutagenesis D534 and Y536 in human TLR9, and D535 and Y537 in
murine TLR9, were mutated to alanines creating the sequence A-L-A
for human (aa 534-536) and murine TLR9 (aa 535-537). These
mutations were generated by the use of the primers
5'-CACAATAAGCTGGCCCTCGCCCACGAGCACTC-3' (SEQ ID NO:150) and
5'-GAGTGCTCGTGGGCGAGGGCCAGCTTATTGTG-3' (SEQ ID NO:151) for human
TLR9 and the primers 5'-CATAACAAACTGGCCTTGGCCCACTGGAAATC-3' (SEQ ID
NO:152) and 5'-GATTTCCAGTGGGCCAAGGCCAGTTTGTTATG-3' (SEQ ID NO:153)
for murine TLR9 according to the manufacturer's protocol.
For the stimulation of mTLR9 variant, mTLR9-MBDmut, 293 cells were
transiently transfected with mTLR9 or mTLR9-MBD-mut and stimulated
after 16 hours with ODN 2006 and ODN 1668 at concentrations
indicated (FIG. 22). 48 hours after stimulation supernatant was
harvested and IL-8 production was measured by ELISA. For the
stimulation of hTLR9 variant, hTLR9-MBDmut, 293 cells were
transiently transfected with hTLR9 or hTLR9-MBD-mut and stimulated
after 16 hours with ODN 2006 and ODN 1668 at concentrations
indicated (FIG. 23). 48 hours after stimulation supernatant was
harvested and IL-8 production was measured by ELISA. The disruption
of the putative CpG binding domain DXY in TLR9 destroyed receptor
activity. These data demonstrate that the MBD motif is most likely
involved in CpG-DNA binding and can be thus be manipulated to
better understand CpG-DNA binding and efficacy.
Example 12
Proline to Histidine Mutation in the TIR-domain (hTLR9-PHmut,
mTLR9-PHmut)
Toll-like receptors have a cytoplasmic Toll/IL-1 receptor (TIR)
homology domain which initiates signaling after binding of the
adapter molecule MyD88. Medzhitov R et al., Mol Cell 2:253-8
(1998); Kopp E B et al., Curr Opin Immunol 11:15-8 (1999). Reports
by others have shown that a single-point mutation in the signaling
TIR-domain in murine TLR4 (Pro712 to His) or human TLR2 (Pro681 to
His) abolishes host immune response to lipopolysaccharide or
gram-positive bacteria, respectively. Poltorak A et al., Science
282:2085-8 (1998); Underhill D M et al., Nature 401:811-5 (1999).
Through site-specific mutagenesis the equivalent Proline at
position 915 of human and murine TLR9 were mutated to Histidine
(Pro915 to His). These mutations were generated by the use of the
primers 5'-GCGACTGGCTGCATGGCAAAACCCTCTTTG-3' (SEQ ID NO:154) and
5'-CAAAGAGGGTTTTGCCATGCAGCCAGTCGC-3' (SEQ ID NO:155) for human TLR9
and the primers 5'-CGAGATTGGCTGCATGGCCAGACGCTCTTC-3' (SEQ ID
NO:156) and 5'-GAAGAGCGTCTGGCCATGCAGCCAATCTCG-3' (SEQ ID NO:157)
for murine TLR9 according to the manufacturer's protocol.
For the stimulation of mTLR9 variant, mTLR9-PHmut, 293 cells were
transiently transfected with mTLR9 or mTLR9-PHmut and stimulated
after 16 hours with ODN 2006 and ODN 1668 at concentrations
indicated (FIG. 22). 48 hours after stimulation supernatant was
harvested and IL-8 production was measured by ELISA. For the
stimulation of hTLR9 variant, hTLR9-PHmut, 293 cells were
transiently transfected with hTLR9 or hTLR9-PHmut and stimulated
after 16 hours with ODN 2006 and ODN 1668 at concentrations
indicated (FIG. 23). 48 hours after stimulation supernatant was
harvested and IL-8 production was measured by ELISA. These data
demonstrate that TLR9 activity can be destroyed by the Pro to His
mutation. This mutation has the potential to be used as a dominant
negative to block TLR9 activity thus a genetic variant could
compete for ligand or signaling partners and disrupt signaling.
Example 13
Exchange of the TIR-Domain Between Murine and Human TLR9
(hTLR9-TIRm, mTLR9-TIRh)
Toll-like receptors have a cytoplasmic Toll/IL-1 receptor (TIR)
homology domain that initiates signaling after binding of the
adapter molecule MyD88. Medzhitov R et al., Mol Cell 2:253-8
(1998); Kopp E B et al., Curr Opin Immunol 11:15-8 (1999). This is
also true for TLR9. To generate molecules consisting of human
extracellular TLR9 and murine TIR domain (hTLR9-TIRm) or murine
extracellular TLR9 and human TIR domain (mTLR9-TIRh), the following
approach was chosen. Through site-specific mutagenesis a ClaI
restriction site was introduced in human and murine TLR9. For human
TLR9 the DNA sequence 5'-GGCCTCAGCATCTTT-3' (3026-3040, SEQ ID
NO:158) was mutated to 5'-GGCCTATCGATTTTT-3' (SEQ ID NO:159),
introducing a ClaI site (underlined in the sequence) but leaving
the amino acid sequence (GLSIF, aa 798-802) unchanged. For murine
TLR9 the DNA sequence 5'-GGCCGTAGCATCTTC-3' (2434-2447, SEQ ID
NO:160) was mutated to 5'-GGCCTATCGATTTTT-3' (SEQ ID NO:161),
introducing a ClaI site and creating the amino acid sequence
(GLSIF, aa 799-803) which differs in one position (aa 800) from the
wildtype murine TLR9 sequence (GRSIF, aa 799-803) but is identical
to the human sequence.
hTLR9-TIRm. The primers used for human TLR9 were
5'-CAGCTCCAGGGCCTATCGATTTTTGCACAGGACC-3' (SEQ ID NO:162) and
5'-GGTCCTGTGCAAAAATCGATAGGCCCTGGAGCTG-3' (SEQ ID NO:163). For
creating an expression vector containing the extracellular portion
of human TLR9 connected to the murine TIR domain, the human
expression vector was cut with ClaI and limiting amounts of EcoRI
and the fragment coding for the murine TIR domain generated by a
ClaI and EcoRI digestion of murine TLR9 expression vector was
ligated in the vector fragment containing the extracellular portion
of hTLR9. Transfection into E.coli yielded the expression vector
hTLR9-TIRm (human extracellular TLR9-murine TIR-domain).
mTLR9-TIRh. The primers used for murine TLR9 were
5'-CAGCTGCAGGGCCTATCGATTTTCGCACAGGACC-3' (SEQ ID NO:164) and
5'-GGTCCTGTGCGAAAATCGATAGGCCCTGCAGCTG-3' (SEQ ID NO:165). For
creating an expression vector containing the extracellular portion
of murine TLR9 connected to the human TIR domain, the murine
expression vector was cut with ClaI and limiting amounts of EcoRI
and the fragment coding for the human TIR domain generated by a
ClaI and EcoRI digestion of human TLR9 expression vector was
ligated in the vector fragment containing the extracellular portion
of mTLR9. Transfection into E. coli yielded the expression vector
mTLR9-TIRh (murine extracellular TLR9-human TIR-domain).
For the stimulation of the mTLR9 variant, mTLR9-TIRh, 293 cells
were transiently transfected with mTLR9 or mTLR9-TIRh and
stimulated after 16 hours with ODN 2006 and ODN 1668 at
concentrations indicated (FIG. 24). 48 hours after stimulation
supernatant was harvested and IL-8 production was measured by
ELISA. For the stimulation of the hTLR9 variant, hTLR9-TIRm, 293
cells were transiently transfected with hTLR9 or hTLR9-TIRm and
stimulated after 16 hours with ODN 2006 and ODN 1668 at
concentrations indicated (FIG. 25). 48 hours after stimulation
supernatant was harvested and IL-8 production was measured by
ELISA. Replacement of the murine TLR9-TIR domain with human does
not significantly affect mTLR9 activity. Replacement of the human
TLR9-TIR with murine however appears to have a negative effect on
hTLR9. These data demonstrate that manipulations could be made to
influence TLR9 activities.
Example 14
TLR9-fusion Protein with Green-fluorescent-protein (hTLR9-GFP,
mTLR9-GFP)
Human and murine TLR9 were individually cloned into the vector
pEGFP-N1 (Clontech, Palo Alto, Calif., USA) to create expression
vectors encoding human and murine fusion proteins consisting of an
N-terminal TLR9 protein fused to C-terminal green-fluorescent
protein (GFP). These constructs can be used to trace TLR9
localization and expression. Such detections can be used for
staining in FACS analysis, confocal microscopy and Western blot, or
for purification of polypeptides and subsequent antibody
production.
Example 15
TLR9-fusion Protein with FLAG-peptide (hTLR9-FLAG, mTLR9-FLAG)
Human and murine TLR9 were individually cloned into the vector
pFLAG-CMV-1 (Sigma, St. Louis, Mo., USA) to create expression
vectors encoding human and murine fusion proteins consisting of an
N-terminal leader peptide (preprotrypsin, which is cleaved
intracellularly during processing of the protein), FLAG-peptide
(DYKDDDDK) and TLR9 protein which does not contain its own signal
peptide. These constructs can be used to trace TLR9 localization
and expression, e.g., using anti-FLAG antibodies. Such detections
can be used for staining in FACS analysis, confocal microscopy and
Western blot, or for purification of polypeptides and subsequent
antibody production.
Example 16
Method of Cloning Human TLR7
Two accession numbers in the GenBank database, AF245702 and
AF240467, describe the DNA sequence for human TLR7. To create an
expression vector for human TLR7, human TLR7 cDNA was amplified
from a cDNA made from human peripheral mononuclear blood cells
(PBMC) using the primers 5'-CACCTCTCATGCTCTGCTCTCTTC-3' (SEQ ID
NO:166) and 5'-GCTAGACCGTTTCCTTGAACACCTG-3' (SEQ ID NO:167). The
fragment was cloned into pGEM-T Easy vector (Promega), cut with the
restriction enzyme NotI and ligated into a NotI-digested pcDNA3.1
expression vector (Invitrogen). The insert was fully sequenced and
translated into protein. The cDNA sequence for hTLR7 is SEQ ID
NO:168, is presented in Table 6. The open reading frame starts at
base 124, ends at base 3273, and codes for a protein of 1049 amino
acids. SEQ ID NO:169 (Table 7), corresponding to bases 124-3273 of
SEQ ID NO:168 (Table 6), is the coding region for the polypeptide
of SEQ ID NO:170 (Table 8).
The protein sequence of the cloned hTLR7 cDNA matches the sequence
described under the GenBank accession number AF240467. The sequence
deposited under GenBank accession number AF245702 contains two
amino acid changes at position 725 (L to H) and 738 (L to P).
TABLE 6 cDNA Sequence for Human TLR7 (5' to 3'; SEQ ID NO:168)
agctggctag cgtttaaacg ggccctctag actcgagcgg ccgcgaattc actagtgatt
60 cacctctcat gctctgctct cttcaaccag acctctacat tccattttgg
aagaagacta 120 aaaatggtgt ttccaatgtg gacactgaag agacaaattc
ttatcctttt taacataatc 180 ctaatttcca aactccttgg ggctagatgg
tttcctaaaa ctctgccctg tgatgtcact 240 ctggatgttc caaagaacca
tgtgatcgtg gactgcacag acaagcattt gacagaaatt 300 cctggaggta
ttcccacgaa caccacgaac ctcaccctca ccattaacca cataccagac 360
atctccccag cgtcctttca cagactggac catctggtag agatcgattt cagatgcaac
420 tgtgtaccta ttccactggg gtcaaaaaac aacatgtgca tcaagaggct
gcagattaaa 480 cccagaagct ttagtggact cacttattta aaatcccttt
acctggatgg aaaccagcta 540 ctagagatac cgcagggcct cccgcctagc
ttacagcttc tcagccttga ggccaacaac 600 atcttttcca tcagaaaaga
gaatctaaca gaactggcca acatagaaat actctacctg 660 ggccaaaact
gttattatcg aaatccttgt tatgtttcat attcaataga gaaagatgcc 720
ttcctaaact tgacaaagtt aaaagtgctc tccctgaaag ataacaatgt cacagccgtc
780 cctactgttt tgccatctac tttaacagaa ctatatctct acaacaacat
gattgcaaaa 840 atccaagaag atgattttaa taacctcaac caattacaaa
ttcttgacct aagtggaaat 900 tgccctcgtt gttataatgc cccatttcct
tgtgcgccgt gtaaaaataa ttctccccta 960 cagatccctg taaatgcttt
tgatgcgctg acagaattaa aagttttacg tctacacagt 1020 aactctcttc
agcatgtgcc cccaagatgg tttaagaaca tcaacaaact ccaggaactg 1080
gatctgtccc aaaacttctt ggccaaagaa attggggatg ctaaatttct gcattttctc
1140 cccagcctca tccaattgga tctgtctttc aattttgaac ttcaggtcta
tcgtgcatct 1200 atgaatctat cacaagcatt ttcttcactg aaaagcctga
aaattctgcg gatcagagga 1260 tatgtcttta aagagttgaa aagctttaac
ctctcgccat tacataatct tcaaaatctt 1320 gaagttcttg atcttggcac
taactttata aaaattgcta acctcagcat gtttaaacaa 1380 tttaaaagac
tgaaagtcat agatctttca gtgaataaaa tatcaccttc aggagattca 1440
agtgaagttg gcttctgctc aaatgccaga acttctgtag aaagttatga accccaggtc
1500 ctggaacaat tacattattt cagatatgat aagtatgcaa ggagttgcag
attcaaaaac 1560 aaagaggctt ctttcatgtc tgttaatgaa agctgctaca
agtatgggca gaccttggat 1620 ctaagtaaaa atagtatatt ttttgtcaag
tcctctgatt ttcagcatct ttctttcctc 1680 aaatgcctga atctgtcagg
aaatctcatt agccaaaactc ttaatggcag tgaattccaa 1740 cctttagcag
agctgagata tttggacttc tccaacaacc ggcttgattt actccattca 1800
acagcatttg aagagcttca caaactggaa gttctggata taagcagtaa tagccattat
1860 tttcaatcag aaggaattac tcatatgcta aactttacca agaacctaaa
ggttctgcag 1920 aaactgatga tgaacgacaa tgacatctct tcctccacca
gcaggaccat ggagagtgag 1980 tctcttagaa ctctggaatt cagaggaaat
cacttagatg ttttatggag agaaggtgat 2040 aacagatact tacaattatt
caagaatctg ctaaaattag aggaattaga catctctaaa 2100 aattccctaa
gtttcttgcc ttctggagtt tttgatggta tgcctccaaa tctaaagaat 2160
ctctctttgg ccaaaaatgg gctcaaatct ttcagttgga agaaactcca gtgtctaaag
2220 aacctggaaa ctttggacct cagccacaac caactgacca ctgtccctga
gagattatcc 2280 aactgttcca gaagcctcaa gaatctgatt cttaagaata
atcaaatcag gagtctgacg 2340 aagtattttc tacaagatgc cttccagttg
cgatatctgg atctcagctc aaataaaatc 2400 cagatgatcc aaaagaccag
cttcccagaa aatgtcctca acaatctgaa gatgttgctt 2460 ttgcatcata
atcggtttct gtgcacctgt gatgctgtgt ggtttgtctg gtgggttaac 2520
catacggagg tgactattcc ttacctggcc acagatgtga cttgtgtggg gccaggagca
2580 cacaagggcc aaagtgtgat ctccctggat ctgtacacct gtgagttaga
tctgactaac 2640 ctgattctgt tctcactttc catatctgta tctctctttc
tcatggtgat gatgacagca 2700 agtcacctct atttctggga tgtgtggtat
atttaccatt tctgtaaggc caagataaag 2760 gggtatcagc gtctaatatc
accagactgt tgctatgatg cttttattgt gtatgacact 2820 aaagacccag
ctgtgaccga gtgggttttg gctgagctgg tggccaaact ggaagaccca 2880
agagagaaac attttaattt atgtctcgag gaaagggact ggttaccagg gcagccagtt
2940 ctggaaaacc tttcccagag catacagctt agcaaaaaga cagtgtttgt
gatgacagac 3000 aagtatgcaa agactgaaaa ttttaagata gcattttact
tgtcccatca gaggctcatg 3060 gatgaaaaag ttgatgtgat tatcttgata
tttcttgaga agccttttca gaagtccaag 3120 ttcctccagc tccggaaaag
gctctgtggg agttctgtcc ttgagtggcc aacaaacccg 3180 caagctcacc
catacttctg gcagtgtcta aagaacgccc tggccacaga caatcatgtg 3240
gcctatagtc aggtgttcaa ggaaacggtc tagaatcgaa ttcccgcggc cgccactgtg
3300 ctggatatct gcagaattcc accacactgg actagtggat ccgagctcgg
taccaagctt 3360 aagtttaaac cgc 3373
TABLE 7 Coding Region for Human TLR7 (5' to 3'; SEQ ID NO:169)
atggtgtttc caatgtggac actgaagaga caaattctta tcctttttaa cataatccta
60 atttccaaac tccttggggc tagatggttt cctaaaactc tgccctgtga
tgtcactctg 120 gatgttccaa agaaccatgt gatcgtggac tgcacagaca
agcatttgac agaaattcct 180 ggaggtattc ccacgaacac cacgaacctc
accctcacca ttaaccacat accagacatc 240 tccccagcgt cctttcacag
actggaccat ctggtagaga tcgatttcag atgcaactgt 300 gtacctattc
cactggggtc aaaaaacaac atgtgcatca agaggctgca gattaaaccc 360
agaagcttta gtggactcac ttatttaaaa tccctttacc tggatggaaa ccagctacta
420 gagataccgc agggcctccc gcctagctta cagcttctca gccttgaggc
caacaacatc 480 ttttccatca gaaaagagaa tctaacagaa ctggccaaca
tagaaatact ctacctgggc 540 caaaactgtt attatcgaaa tccttgttat
gtttcatatt caatagagaa agatgccttc 600 ctaaacttga caaagttaaa
agtgctctcc ctgaaagata acaatgtcac agccgtccct 660 actgttttgc
catctacttt aacagaacta tatctctaca acaacatgat tgcaaaaatc 720
caagaagatg attttaataa cctcaaccaa ttacaaattc ttgacctaag tggaaattgc
780 cctcgttgtt ataatgcccc atttccttgt gcgccgtgta aaaataattc
tcccctacag 840 atccctgtaa atgcttttga tgcgctgaca gaattaaaag
ttttacgtct acacagtaac 900 tctcttcagc atgtgccccc aagatggttt
aagaacatca acaaactcca ggaactggat 960 ctgtcccaaa acttcttggc
caaagaaatt ggggatgcta aatttctgca ttttctcccc 1020 agcctcatcc
aattggatct gtctttcaat tttgaacttc aggtctatcg tgcatctatg 1080
aatctatcac aagcattttc ttcactgaaa agcctgaaaa ttctgcggat cagaggatat
1140 gtctttaaag agttgaaaag ctttaacctc tcgccattac ataatcttca
aaatcttgaa 1200 gttcttgatc ttggcactaa ctttataaaa attgctaacc
tcagcatgtt taaacaattt 1260 aaaagactga aagtcataga tctttcagtg
aataaaatat caccttcagg agattcaagt 1320 gaagttggct tctgctcaaa
tgccagaact tctgtagaaa gttatgaacc ccaggtcctg 1380 gaacaattac
attatttcag atatgataag tatgcaagga gttgcagatt caaaaacaaa 1440
gaggcttctt tcatgtctgt taatgaaagc tgctacaagt atgggcagac cttggatcta
1500 agtaaaaata gtatattttt tgtcaagtcc tctgattttc agcatctttc
tttcctcaaa 1560 tgcctgaatc tgtcaggaaa tctcattagc caaactctta
atggcagtga attccaacct 1620 ttagcagagc tgagatattt ggacttctcc
aacaaccggc ttgatttact ccattcaaca 1680 gcatttgaag agcttcacaa
actggaagtt ctggatataa gcagtaatag ccattatttt 1720 caatcagaag
gaattactca tatgctaaac tttaccaaga acctaaaggt tctgcagaaa 1800
ctgatgatga acgacaatga catctcttcc tccaccagca ggaccatgga gagtgagtct
1860 cttagaactc tggaattcag aggaaatcac ttagatgttt tatggagaga
aggtgataac 1920 agatacttac aattattcaa gaatctgcta aaattagagg
aattagacat ctctaaaaat 1980 tccctaagtt tcttgccttc tggagttttt
gatggtatgc ctccaaatct aaagaatctc 2040 tctttggcca aaaatgggct
caaatctttc agttggaaga aactccagtg tctaaagaac 2100 ctggaaactt
tggacctcag ccacaaccaa ctgaccactg tccctgagag attatccaac 2160
tgttccagaa gcctcaagaa tctgattctt aagaataatc aaatcaggag tctgacgaag
2220 tattttctac aagatgcctt ccagttgcga tatctggatc tcagctcaaa
taaaatccag 2280 atgatccaaa agaccagctt cccagaaaat gtcctcaaca
atctgaagat gttgcttttg 2340 catcataatc ggtttctgtg cacctgtgat
gctgtgtggt ttgtctggtg ggttaaccat 2400 acggaggtga ctattcctta
cctggccaca gatgtgactt gtgtggggcc aggagcacac 2460 aagggccaaa
gtgtgatctc cctggatctg tacacctgtg agttagatct gactaacctg 2520
attctgttct cactttccat atctgtatct ctctttctca tggtgatgat gacagcaagt
2580 cacctctatt tctgggatgt gtggtatatt taccatttct gtaaggccaa
gataaagggg 2640 tatcagcgtc taatatcacc agactgttgc tatgatgctt
ttattgtgta tgacactaaa 2700 gacccagctg tgaccgagtg ggttttggct
gagctggtgg ccaaactgga agacccaaga 2760 gagaaacatt ttaatttatg
tctcgaggaa agggactggt taccagggca gccagttctg 2820 gaaaaccttt
cocagagcat acagcttagc aaaaagacag tgtttgtgat gacagacaag 2880
tatgcaaaga ctgaaaattt taagatagca ttttacttgt cccatcagag gctcatggat
2940 gaaaaagttg atgtgattat cttgatattt cttgagaagc cttttcagaa
gtccaagttc 3000 ctccagctcc ggaaaaggct ctgtgggagt tctgtccttg
agtggccaac aaacccgcaa 3060 gctcacccat acttctggca gtgtctaaag
aacgccctgg ccacagacaa tcatgtggcc 3120 tatagtcagg tgttcaagga aacggtc
3147
TABLE 8 Amino Acid Sequence of Human TLR7 . : . : . : . : . : . :
60 AF240467.pep
MVFPMWTLKRQILILFNIILISKLLGARWFPKTLPCDVTLDVPKNHVIVDCTDKHLTEIP 60
hTLR7.pep
MVFPMWTLKRQILILFNIILISKLLGARWFPKTLPCDVTLDVPKNHVIVDCTDKHLTEIP 60
AF245702.pep
MVFPMWTLKRQILILFNIILISKLLGARWFPKTLPCDVTLDVPKNHVIVDCTDKHLTEIP 60 . :
. : . : . : . : . : 120 AF240467.pep
GGIPTNTTNLTLTINHIPDISPASFHRLDHLVEIDFRCNCVPIPLGSKNNMCIKRLQIKP 120
hTLR7.pep
GGIPTNTTNLTLTINHIPDISPASFHRLDHLVEIDFRCNCVPIPLGSKNNMCIKRLQIKP 120
AF245702.pep
GGIPTNTTNLTLTINHIPDISPASFHRLDHLVEIDFRCNCVPIPLGSKNNMCIKRLQIKP 120 .
: . : . : . : . : . : 180 AF240467.pep
RSFSGLTYLKSLYLDGNQLLEIPQGLPPSLQLLSLEANNIFSIRKENLTELANIEILYLG 180
hTLR7.pep
RSFSGLTYLKSLYLDGNQLLEIPQGLPPSLQLLSLEANNIFSIRKENLTELANIEILYLG 180
AF245702.pep
RSFSGLTYLKSLYLDGNQLLEIPQGLPPSLQLLSLEANNIFSIRKENLTELANIEILYLG 180 .
: . : . : . : . : . : 240 AF240467.pep
QNCYYRNPCYVSYSIEKDAFLNLTKLKVLSLKDNNVTAVPTVLPSTLTELYLYNNMIAKI 240
hTLR7.pep
QNCYYRNPCYVSYSIEKDAFLNLTKLKVLSLKDNNVTAVPTVLPSTLTELYLYNNMIAKI 240
AF245702.pep
QNCYYRNPCYVSYSIEKDAFLNLTKLKVLSLKDNNVTAVPTVLPSTLTELYLYNNMIAKI 240 .
: . : . : . : . : . : 300 AF240467.pep
QEDDFNNLNQLQILDLSGNCPRCYNAPFPCAPCKNNSPLQIPVNAFDALTELKVLRLHSN 300
hTLR7.pep
QEDDFNNLNQLQILDLSGNCPRCYNAPFPCAPCKNNSPLQIPVNAFDALTELKVLRLHSN 300
AF245702.pep
QEDDFNNLNQLQILDLSGNCPRCYNAPFPCAPCKNNSPLQIPVNAFDALTELKVLRLHSN 300 .
: . : . : . : . : . : 360 AF240467.pep
SLQHVPPRWFKNINKLQELDLSQNFLAKEIGDAKFLHFLPSLIQLDLSFNFELQVYRASM 360
hTLR7.pep
SLQHVPPRWFKNINKLQELDLSQNFLAKEIGDAKFLHFLPSLIQLDLSFNFELQVYRASM 360
AF245702.pep
SLQHVPPRWFKNINKLQELDLSQNFLAKEIGDAKFLHFLPSLIQLDLSFNFELQVYRASM 360 .
: . : . : . : . : . : 420 AF240467.pep
NLSQAFSSLKSLKILRIRGYVFKELKSFNLSPLHNLQNLEVLDLGTNFIKIANLSMFKQF 420
hTLR7.pep
NLSQAFSSLKSLKILRIRGYVFKELKSFNLSPLHNLQNLEVLDLGTNFIKIANLSMFKQF 420
AF245702.pep
NLSQAFSSLKSLKILRIRGYVFKELKSFNLSPLHNLQNLEVLDLGTNFIKIANLSMFKQF 420 .
: . : . : . : . : . : 480 AF240467.pep
KRLKVIDLSVNKISPSGDSSEVGFCSNARTSVESYEPQVLEQLHYFRYDKYARSCRFKNK 480
hTLR7.pep
KRLKVIDLSVNKISPSGDSSEVGFCSNARTSVESYEPQVLEQLHYFRYDKYARSCRFKNK 480
AF245702.pep
KRLKVIDLSVNKISPSGDSSEVGFCSNARTSVESYEPQVLEQLHYFRYDKYARSCRFKNK 480 .
: . : . : . : . : . : 540 AF240467.pep
EASFMSVNESCYKYGQTLDLSKNSIFFVKSSDFQHLSFLKCLNLSGNLISQTLNGSEFQP 540
hTLR7.pep
EASFMSVNESCYKYGQTLDLSKNSIFFVKSSDFQHLSFLKCLNLSGNLISQTLNGSEFQP 540
AF245702.pep
EASFMSVNESCYKYGQTLDLSKNSIFFVKSSDFQHLSFLKCLNLSGNLISQTLNGSEFQP 540 .
: . : . : . : . : . : AF240467.pep
LAELRYLDFSNNRLDLLHSTAFEELHKLEVLDISSNSHYFQSEGITHMLNFTKNLKVLQK 600
hTLR7.pep
LAELRYLDFSNNRLDLLHSTAFEELHKLEVLDISSNSHYFQSEGITHMLNFTKNLKVLQK 600
AF245702.pep
LAELRYLDFSNNRLDLLHSTAFEELHKLEVLDISSNSHYFQSEGITHMLNFTKNLKVLQK 600 .
: . : . : . : . : . : AF240467.pep
LMMNDNDISSSTSRTMESESLRTLEFRGNHLDVLWREGDNRYLQLFKNLLKLEELDISKN 660
hTLR7.pep
LMMNDNDISSSTSRTMESESLRTLEFRGNHLDVLWREGDNRYLQLFKNLLKLEELDISKN 660
AF245702.pep
LMMNDNDISSSTSRTMESESLRTLEFRGNHLDVLWREGDNRYLQLFKNLLKLEELDISKN 660 .
: . : . : . : . : . : AF240467.pep
SLSFLPSGVFDGMPPNLKNLSLAKNGLKSFSWKKLQCLKNLETLDLSHNQLTTVPERLSN 720
hTLR7.pep
SLSFLPSGVFDGMPPNLKNLSLAKNGLKSFSWKKLQCLKNLETLDLSHNQLTTVPERLSN 720
AF245702.pep
SLSFLPSGVFDGMPPNLKNLSLAKNGLKSFSWKKLQCLKNLETLDLSHNQLTTVPERLSN 720 .
: . : . : . : . : . : AF240467.pep
CSRSLKNLILKNNQIRSLTKYFLQDAFQLRYLDLSSNKIQMIQKTSFPENVLNNLKMLLL 780
hTLR7.pep
CSRSLKNLILKNNQIRSLTKYFLQDAFQLRYLDLSSNKIQMIQKTSFPENVLNNLKMLLL 780
AF245702.pep
CSRSHKNLILKNNQIRSPTKYFLQDAFQLRYLDLSSNKIQMIQKTSFPENVLNNLKMLLL 780 .
: . : . : . : . : . : AF240467.pep
HHNRFLCTCDAVWFVWWVNHTEVTIPYLATDVTCVGPGAHKGQSVISLDLYTCELDLTNL 840
hTLR7.pep
HHNRFLCTCDAVWFVWWVNHTEVTIPYLATDVTCVGPGAHKGQSVISLDLYTCELDLTNL 840
AF245702.pep
HHNRFLCTCDAVWFVWWVNHTEVTIPYLATDVTCVGPGAHKGQSVISLDLYTCELDLTNL 840 .
: . : . : . : . : . : AF240467.pep
ILFSLSISVSLFLMVMMTASHLYFWDVWYIYHFCKAKIKGYQRLISPDCCYDAFIVYDTK 900
hTLR7.pep
ILFSLSISVSLFLMVMMTASHLYFWDVWYIYHFCKAKIKGYQRLISPDCCYDAFIVYDTK 900
AF245702.pep
ILFSLSISVSLFLMVMMTASHLYFWDVWYIYHFCKAKIKGYQRLISPDCCYDAFIVYDTK 900 .
: . : . : . : . : . : AF240467.pep
DPAVTEWVLAELVAKLEDPREKHFNLCLEERDWLPGQPVLENLSQSIQLSKKTVFVMTDK 960
hTLR7.pep
DPAVTEWVLAELVAKLEDPREKHFNLCLEERDWLPGQPVLENLSQSIQLSKKTVFVMTDK 960
AF245702.pep
DPAVTEWVLAELVAKLEDPREKHFNLCLEERDWLPGQPVLENLSQSIQLSKKTVFVMTDK 960 .
: . : . : . : . : . : AF240467.pep
YAKTENFKIAFYLSHQRLMDEKVDVIILIFLEKPFQKSKFLQLRKRLCGSSVLEWPTNPQ 1020
hTLR7.pep
YAKTENFKIAFYLSHQRLMDEKVDVIILIFLEKPFQKSKFLQLRKRLCGSSVLEWPTNPQ 1020
AF245702.pep
YAKTENFKIAFYLSHQRLMDEKVDVIILIFLEKPFQKSKFLQLRKRLCGSSVLEWPTNPQ 1020 .
: . : . : . : . : . : 1080 AF240467.pep
AHPYFWQCLKNALATDNHVAYSQVFKETV 1049 hTLR7.pep
AHPYFWQCLKNALATDNHVAYSQVFKETV 1049 AF245702.pep
AHPYFWQCLKNALATDNHVAYSQVFKETV 1049
In Table 8 the sequences are assigned as follows: hTLR7.pep, SEQ ID
NO:170; AF240467.pep, SEQ ID NO:171; AF245702.pep, SEQ ID
NO:172.
Example 17
Method of Cloning the Murine TLR7
Alignment of human TLR7 protein sequence with mouse EST database
using tfasta yielded 4 hits with mouse EST sequences bb116163,
aa266744, bb210780 and aa276879. Two primers were designed that
bind to aa266744 sequence for use in a RACE-PCR to amplify 5' and
3' ends of the murine TLR7 cDNA. The library used for the RACE PCR
was a mouse spleen marathon-ready cDNA commercially available from
Clontech. A 5' fragment with a length of 3000 bp obtained by this
method was cloned into Promega pGEM-T Easy vector. After sequencing
of the 5' end, additional primers were designed for amplification
of the complete murine TLR7 cDNA. The primer for the 5' end was
obtained from the sequence of the 5' RACE product whereas the
primer for the 3' end was selected from the mouse EST sequence
aa266744.
Three independent PCR reactions were set up using a murine
macrophage RAW264.7 (ATCC TIB-71) cDNA as a template with the
primers 5'-CTCCTCCACCAGACCTCTTGATTCC-3' (SEQ ID NO:208) and
5'-CAAGGCATGTCCTAGGTGGTGACATTC-3' (SEQ ID NO:209). The resulting
amplification products were cloned into pGEM-T Easy vector and
fully sequenced (SEQ ID NO:173; Table 9). The open reading frame of
mTLR7 (SEQ ID NO:174; Table 10) starts at base 49, ends at base
3201 and codes for a protein of 1050 amino acids (SEQ ID NO:175;
Table11). To create an expression vector for murine TLR7 cDNA,
pGEM-T Easy vector plus mTLR7 insert was cut with NotI, the
fragment isolated and ligated into a NotI digested pcDNA3.1
expression vector (Invitrogen).
TABLE 9 cDNA Sequence for Murine TLR7 (5' to 3'; SEQ ID NO:173)
ATTCTCCTCC ACCAGACCTC TTGATTCCAT TTTGAAAGAA AACTGAAAAT GGTGTTTTCG
60 ATGTGGACAC GGAAGAGACA AATTTTGATC TTTTTAAATA TGCTCTTAGT
TTCTAGAGTC 120 TTTGGGTTTC GATGGTTTCC TAAAACTCTA CCTTGTGAAG
TTAAAGTAAA TATCCCAGAG 180 GCCCATGTGA TCGTGGACTG CACAGACAAG
CATTTGACAG AAATCCCTGA GGGCATTCCC 240 ACTAACACCA CCAATCTTAC
CCTTACCATC AACCACATAC CAAGCATCTC TCCAGATTCC 300 TTCCGTAGGC
TGAACCATCT GGAAGAAATC GATTTAAGAT GCAATTGTGT ACCTGTTCTA 360
CTGGGGTCCA AAGCCAATGT GTGTACCAAG AGGCTGCAGA TTAGACCTGG AAGCTTTAGT
420 GGACTCTCTG ACTTAAAAGC CCTTTACCTG GATGGAAACC AACTTCTGGA
GATACCACAG 480 GATCTGCCAT CCAGCTTACA TCTTCTGAGC CTTGAGGCTA
ACAACATCTT CTCCATCACG 540 AAGGAGAATC TAACAGAACT GGTCAACATT
GAAACACTCT ACCTGGGTCA AAACTGTTAT 600 TATCGAAATC CTTGCAATGT
TTCCTATTCT ATTGAAAAAG ATGCTTTCCT AGTTATGAGA 660 AATTTGAAGG
TTCTCTCACT AAAAGATAAC AATGTCACAG CTGTCCCCAC CACTTTGCCA 720
CCTAATTTAC TAGAGCTCTA TCTTTATAAC AATATCATTA AGAAAATCCA AGAAAATGAT
780 TTTAATAACC TCAATGAGTT GCAAGTTCTT GACCTAAGTG GAAATTGCCC
TCGATGTTAT 840 AATGTCCCAT ATCCGTGTAC ACCGTGTGAA AATAATTCCC
CCTTACAGAT CCATGACAAT 900 GCTTTCAATT CATTGACAGA ATTAAAAGTT
TTACGTTTAC ACAGTAATTC TCTTCAGCAT 960 GTGCCCCCAA CATGGTTTAA
AAACATGAGA AACCTCCAGG AACTAGACCT CTCCCAAAAC 1020 TACTTGGCCA
GAGAAATTGA GGAGGCCAAA TTTTTGCATT TTCTTCCCAA CCTTGTTGAG 1080
TTGGATTTTT CTTTCAATTA TGAGCTGCAG GTCTACCATG CATCTATAAC TTTACCACAT
1140 TCACTCTCTT CATTGGAAAA CTTGAAAATT CTGCGTGTCA AGGGGTATGT
CTTTAAAGAG 1200 CTGAAAAACT CCAGTCTTTC TGTATTGCAC AAGCTTCCCA
GGCTGGAAGT TCTTGACCTT 1260 GGCACTAACT TCATAAAAAT TGCTGACCTC
AACATATTCA AACATTTTGA AAACCTCAAA 1320 CTCATAGACC TTTCAGTGAA
TAAGATATCT CCTTCAGAAG AGTCAAGAGA AGTTGGCTTT 1380 TGTCCTAATG
CTCAAACTTC TGTAGACCGT CATGGGCCCC AGGTCCTTGA GGCCTTACAC 1440
TATTTCCGAT ACGATGAATA TGCACGGAGC TGCAGGTTCA AAAACAAAGA GCCACCTTCT
1500 TTCTTGCCTT TGAATGCAGA CTGCCACATA TATGGGCAGA CCTTAGACTT
AAGTAGAAAT 1560 AACATATTTT TTATTAAACC TTCTGATTTT CAGCATCTTT
CATTCCTCAA ATGCCTCAAC 1620 TTATCAGGAA ACACCATTGG CCAAACTCTT
AATGGCAGTG AACTCTGGCC GTTGAGAGAG 1680 TTGCGGTACT TAGACTTCTC
CAACAACCGG CTTGATTTAC TCTACTCAAC AGCCTTTGAA 1740 GAGCTCCAGA
GTCTTGAAGT TCTGGATCTA AGTAGTAACA GCCACTATTT TCAAGCAGAA 1800
GGAATTACTC ACATGCTAAA CTTTACCAAG AAATTACGGC TTCTGGACAA ACTCATGATG
1860 AATGATAATG ACATCTCTAC TTCGGCCAGC AGGACCATGG AAAGTGACTC
TCTTCGAATT 1920 CTGGAGTTCA GAGGCAACCA TTTAGATGTT CTATGGAGAG
CCGGTGATAA CAGATACTTG 1980 GACTTCTTCA AGAATTTGTT CAATTTAGAG
GTATTAGATA TCTCCAGAAA TTCCCTGAAT 2040 TCCTTGCCTC CTGAGGTTTT
TGAGGGTATG CCGCCAAATC TAAAGAATCT CTCCTTGGCC 2100 AAAAATGGGC
TCAAATCTTT CTTTTGGGAC AGACTCCAGT TACTGAAGCA TTTGGAAATT 2160
TTGGACCTCA GCCATAACCA GCTGACAAAA GTACCTGAGA GATTGGCCAA CTGTTCCAAA
2220 AGTCTCACAA CACTGATTCT TAAGCATAAT CAAATCAGGC AATTGACAAA
ATATTTTCTA 2280 GAAGATGCTT TGCAATTGCG CTATCTAGAC ATCAGTTCAA
ATAAAATCCA GGTCATTCAG 2340 AAGACTAGCT TCCCAGAAAA TGTCCTCAAC
AATCTGGAGA TGTTGGTTTT ACATCACAAT 2400 CGCTTTCTTT GCAACTGTGA
TGCTGTGTGG TTTGTCTGGT GGGTTAACCA TACAGATGTT 2460 ACTATTCCAT
ACCTGGCCAC TGATGTGACT TGTGTAGGTC CAGGAGCACA CAAAGGTCAA 2520
AGTGTCATAT CCCTTGATCT GTATACGTGT GAGTTAGATC TCACAAACCT GATTCTGTTC
2580 TCAGTTTCCA TATCATCAGT CCTCTTTCTT ATGGTAGTTA TGACAACAAG
TCACCTCTTT 2640 TTCTGGGATA TGTGGTACAT TTATTATTTT TGGAAAGCAA
AGATAAAGGG GTATCAGCAT 2700 CTGCAATCCA TGGAGTCTTG TTATGATGCT
TTTATTGTGT ATGACACTAA AAACTCAGCT 2760 GTGACAGAAT GGGTTTTGCA
GGAGCTGGTG GCAAAATTGG AAGATCCAAG AGAAAAACAC 2820 TTCAATTTGT
GTCTAGAAGA AAGAGACTGG CTACCAGGAC AGCCAGTTCT AGAAAACCTT 2880
TCCCAGAGCA TACAGCTCAG CAAAAAGACA GTGTTTGTGA TGACACAGAA ATATGCTAAG
2940 ACTGAGAGTT TTAAGATGGC ATTTTATTTG TCTCATCAGA GGCTCCTGGA
TGAAAAAGTG 3000 GATGTGATTA TCTTGATATT CTTGGAAAAG CCTCTTCAGA
AGTCTAAGTT TCTTCAGCTC 3060 AGGAAGAGAC TCTGCAGGAG CTCTGTCCTT
GAGTGGCCTG CAAATCCACA GGCTCACCCA 3120 TACTTCTGGC AGTGCCTGAA
AAATGCCCTG ACCACAGACA ATCATGTGGC TTATAGTCAA 3180 ATGTTCAAGG
AAACAGTCTA GCTCTCTGAA GAATGTCACC ACCTAGGACA TGCCTTGAAT 3240 CGA
3243
TABLE 10 Coding Region for Murine TLR7 (5' to 3'; SEQ ID NO:174)
ATGGTGTTTT CGATGTGGAC ACGGAAGAGA CAAATTTTGA TCTTTTTAAA TATGCTCTTA
60 GTTTCTAGAG TCTTTGGGTT TCGATGGTTT CCTAAAACTC TACCTTGTGA
AGTTAAAGTA 120 AATATCCCAG AGGCCCATGT GATCGTGGAC TGCACAGACA
AGCATTTGAC AGAAATCCCT 180 GAGGGCATTC CCACTAACAC CACCAATCTT
ACCCTTACCA TCAACCACAT ACCAAGCATC 240 TCTCCAGATT CCTTCCGTAG
GCTGAACCAT CTCGAAGAAA TCGATTTAAG ATGCAATTGT 300 GTACCTGTTC
TACTGGGGTC CAAAGCCAAT GTGTGTACCA AGAGGCTGCA GATTAGACCT 360
GGAAGCTTTA GTGGACTCTC TGACTTAAAA GCCCTTTACC TGGATGGAAA CCAACTTCTG
420 GAGATACCAC AGGATCTGCC ATCCAGCTTA CATCTTCTGA GCCTTGAGGC
TAACAACATC 480 TTCTCCATCA CGAAGGAGAA TCTAACAGAA CTGGTCAACA
TTGAAACACT CTACCTGGGT 540 CAAAACTGTT ATTATCGAAA TCCTTGCAAT
GTTTCCTATT CTATTGAAAA AGATGCTTTC 600 CTAGTTATGA GAAATTTGAA
GGTTCTCTCA CTAAAAGATA ACAATGTCAC AGCTGTCCCC 660 ACCACTTTGC
CACCTAATTT ACTAGAGCTC TATCTTTATA ACAATATCAT TAAGAAAATC 720
CAAGAAAATG ATTTTAATAA CCTCAATGAG TTGCAAGTTC TTGACCTAAG TGGAAATTGC
780 CCTCGATGTT ATAATGTCCC ATATCCGTGT ACACCGTGTG AAAATAATTC
CCCCTTACAG 840 ATCCATGACA ATGCTTTCAA TTCATTGACA GAATTAAAAG
TTTTACGTTT ACACAGTAAT 900 TCTCTTCAGC ATGTGCCCCC AACATGGTTT
AAAAACATGA GAAACCTCCA GGAACTAGAC 960 CTCTCCCAAA ACTACTTGGC
CAGAGAAATT GAGGAGGCCA AATTTTTGCA TTTTCTTCCC 1020 AACCTTGTTG
AGTTGGATTT TTCTTTCAAT TATGAGCTGC AGGTCTACCA TGCATCTATA 1080
ACTTTACCAC ATTCACTCTC TTCATTGGAA AACTTGAAAA TTCTGCGTGT CAAGGGGTAT
1140 GTCTTTAAAG AGCTGAAAAA CTCCAGTCTT TCTGTATTGC ACAAGCTTCC
CAGGCTGGAA 1200 GTTCTTGACC TTGGCACTAA CTTCATAAAA ATTGCTGACC
TCAACATATT CAAACATTTT 1260 GAAAACCTCA AACTCATAGA CCTTTCAGTG
AATAAGATAT CTCCTTCAGA AGAGTCAAGA 1320 GAAGTTGGCT TTTGTCCTAA
TGCTCAAACT TCTGTAGACC GTCATGGGCC CCAGGTCCTT 1380 GAGGCCTTAC
ACTATTTCCG ATACGATGAA TATGCACGGA GCTGCAGGTT CAAAAACAAA 1440
GAGCCACCTT CTTTCTTGCC TTTGAATGCA GACTGCCACA TATATGGGCA GACCTTAGAC
1500 TTAAGTAGAA ATAACATATT TTTTATTAAA CCTTCTGATT TTCAGCATCT
TTCATTCCTC 1560 AAATGCCTCA ACTTATCAGG AAACACCATT GGCCAAACTC
TTAATGGCAG TGAACTCTGG 1620 CCGTTGAGAG AGTTGCGGTA CTTAGACTTC
TCCAACPACC GGCTTGATTT ACTCTACTCA 1680 ACAGCCTTTG AAGAGCTCCA
GAGTCTTGAA GTTCTGGATC TAAGTAGTAA CAGCCACTAT 1740 TTTCAAGCAG
AAGGAATTAC TCACATGCTA AACTTTACCA AGAAATTACG GCTTCTGGAC 1800
AAACTCATGA TGAATGATAA TGACATCTCT ACTTCGGCCA GCAGGACCAT GGAAAGTGAC
1860 TCTCTTCGAA TTCTGGAGTT CAGAGGCAAC CATTTAGATG TTCTATGGAG
AGCCGGTGAT 1920 AACAGATACT TGGACTTCTT CAAGAATTTG TTCAATTTAG
AGGTATTAGA TATCTCCAGA 1980 AATTCCCTGA ATTCCTTGCC TCCTGAGGTT
TTTGAGGGTA TGCCGCCAAA TCTAAAGAAT 2040 CTCTCCTTGG CCAAAAATGG
GCTCAAATCT TTCTTTTGGG ACAGACTCCA GTTACTGAAG 2100 CATTTGGAAA
TTTTGGACCT CAGCCATAAC CAGCTGACAA AAGTACCTGA GAGATTGGCC 2160
AACTGTTCCA AAAGTCTCAC AACACTGATT CTTAAGCATA ATCAAATCAG GCAATTGACA
2220 AAATATTTTC TAGAAGATGC TTTGCAATTG CGCTATCTAG ACATCAGTTC
AAATAAAATC 2280 CAGGTCATTC AGAAGACTAG CTTCCCAGAA AATGTCCTCA
ACAATCTGGA GATGTTGGTT 2340 TTACATCACA ATCGCTTTCT TTGCAACTGT
GATGCTGTGT GGTTTGTCTG GTGGGTTAAC 2400 CATACAGATG TTACTATTCC
ATACCTGGCC ACTGATGTGA CTTGTGTAGG TCCAGGAGCA 2460 CACAAAGGTC
AAAGTGTCAT ATCCCTTGAT CTGTATACGT GTGAGTTAGA TCTCACAAAC 2520
CTGATTCTGT TCTCAGTTTC CATATCATCA GTCCTCTTTC TTATGGTAGT TATGACAACA
2580 AGTCACCTCT TTTTCTGGGA TATGTGGTAC ATTTATTATT TTTGGAAAGC
AAAGATAAAG 2640 GGGTATCAGC ATCTGCAATC CATGGAGTCT TGTTATGATG
CTTTTATTGT GTATGACACT 2700 AAAAACTCAG CTGTGACAGA ATGGGTTTTG
CAGGAGCTGG TGGCAAAATT GGAAGATCCA 2760 AGAGAAAAAC ACTTCAATTT
GTGTCTAGAA GAAAGAGACT GGCTACCAGG ACAGCCAGTT 2820 CTAGAAAACC
TTTCCCAGAG CATACAGCTC AGCAAAAAGA CAGTGTTTGT GATGACACAG 2880
AAATATGCTA AGACTGAGAG TTTTAAGATG GCATTTTATT TGTCTCATCA GAGGCTCCTG
2940 GATGAAAAAG TGGATGTGAT TATCTTGATA TTCTTGGAAA AGCCTCTTCA
GAAGTCTAAG 3000 TTTCTTCAGC TCAGGAAGAG ACTCTGCAGG AGCTCTGTCC
TTGAGTGGCC TGCAAATCCA 3060 CAGGCTCACC CATACTTCTG GCAGTGCCTG
AAAAATGCCC TGACCACAGA CAATCATGTG 3120 GCTTATAGTC AAATGTTCAA
GGAAACAGTC 3150
TABLE 11 Amino Acid Sequences of Murine TLR7 and Human TLR7 . : . :
. : . : . : . : 60 hTLR7.pep
MVFPMWTLKRQILILFNIILISKLLGARWFPKTLPCDVTLDVPKNHVIVDCTDKHLTEIP 60
mTLR7.pep
MVFSMWTRKRQILIFLNMLLVSRVFGFRWFPKTLPCEVKVNIPEAHVIVDCTDKHLTEIP 60 . :
. : . : . : . : . : 120 hTLR7.pep
GGIPTNTTNLTLTINHIPDISPASFHRLDHLVEIDFRCNCVPIPLGSKNNMCIKRLQIKP 120
mTLR7.pep
EGIPTNTTNLTLTINHIPSISPDSFRRLNHLEEIDLRCNCVPVLLGSKANVCTKRLQIRP 120 .
: . : . : . : . : . : 180 hTLR7.pep
RSFSGLTYLKSLYLDGNQLLEIPQGLPPSLQLLSLEANNIFSIRKENLTELANIEILYLG 180
mTLR7.pep
GSFSGLSDLKALYLDGNQLLEIPQDLPSSLHLLSLEANNIFSITKENLTELVNIETLYLG 180 .
: . : . : . : . : . : 240 hTLR7.pep
QNCYYRNPCYVSYSIEKDAFLNLTKLKVLSLKDNNVTAVPTVLPSTLTELYLYNNMIAKI 240
mTLP7.pep
QNCYYRNPCNVSYSIEKDAFLVMRNLKVLSLKDNNVTAVPTTLPPNLLELYLYNNIIKKI 240 .
: . : . : . : . : . : 300 hTLR7.pep
QEDDFNNLNQLQILDLSGNCPRCYNAPFPCAPCKNNSPLQIPVNAFDALTELKVLRLHSN 300
mTLR7.pep
QENDFNNLNELQVLDLSGNCPRCYNVPYPCTPCENNSPLQIHDNAFNSLTELKVLRLHSN 300 .
: . : . : . : . : . : 360 hTLR7.pep
SLQHVPPRWFKNINKLQELDLSQNFLAKEIGDAKFLHFLPSLIQLDLSFNFELQVYRASM 360
mTLR7.pep
SLQHVPPTWFKNMRNLQELDLSQNYLAREIEEAKFLHFLPNLVELDFSFNYELQVYHASI 360 .
: . : . : . : . : . : 420 hTLR7.pep
NLSQAFSSLKSLKILRIRGYVFKELKSFNLSPLHNLQNLEVLDLGTNFIKIANLSMFKQF 420
mTLR7.pep
TLPHSLSSLENLKILRVKGYVFKELKNSSLSVLHKLPRLEVLDLGTNFIKIADLNIFKHF 420 .
: . : . : . : . : . : 480 hTLR7.pep
KRLKVIDLSVNKISPSGDSSEVGFCSNARTSVESYEPQVLEQLHYFRYDKYARSCRFKNK 480
mTLR7.pep
ENLKLIDLSVNKISPSEESREVGFCPNAQTSVDRHGPQVLEALHYFRYDEYARSCRFKNK 480 .
: . : . : . : . : . : 540 hTLR7.pep
EA-SFMSVNESCYKYGQTLDLSKNSIFFVKSSDFQHLSFLKCLNLSGNLISQTLNGSEFQ 539
mTLR7.pep
EPPSFLPLNADCHIYGQTLDLSRNNIFFIKPSDFQHLSFLKCLNLSGNTIGQTLNGSELW 540 .
: . : . : . : . : . : 600 hTLR7.pep
PLAELRYLDFSNNRLDLLHSTAFEELHKLEVLDISSNSHYFQSEGITHMLNFTKNLKVLQ 599
mTLR7.pep
PLRELRYLDFSNNRLDLLYSTAFEELQSLEVLDLSSNSHYFQAEGITHMLNFTKKLRLLD 600 .
: . : . : . : . : . : 660 hTLR7.pep
KLMMNDNDISSSTSRTMESESLRTLEFRGNHLDVLWREGDNRYLQLFKNLLKLEELDISK 659
mTLR7.pep
KLMMNDNDISTSASRTMESDSLRILEFRGNHLDVLWRAGDNRYLDFFKNLFNLEVLDISR 660 .
: . : . : . : . : . : 720 hTLR7.pep
NSLSFLPSGVFDGMPPNLKNLSLAKNGLKSFSWKKLQCLKNLETLDLSHNQLTTVPERLS 719
mTLR7.pep
NSLNSLPPEVFEGMPPNLKNLSLAKNGLKSFFWDRLQLLKHLEILDLSHNQLTKVPERLA 720 .
: . : . : . : . : . : 780 hTLR7.pep
NCSRSLKNLILKNNQIRSLTKYFLQDAFQLRYLDLSSNKIQMIQKTSFPENVLNNLKMLL 779
mTLR7.pep
NCSKSLTTLILKHNQIRQLTKYFLEDALQLRYLDISSNKIQVIQKTSFPENVLNNLEMLV 780 .
: . : . : . : . : . : 840 hTLR7.pep
LHHNRFLCTCDAVWFVWWVNHTEVTIPYLATDVTCVGPGAHKGQSVISLDLYTCELDLTN 839
mTLR7.pep
LHHNRFLCNCDAVWFVWWVNHTDVTIPYLATDVTCVGPGAHKGQSVISLDLYTCELDLTN 840 .
: . : . : . : . : . : 900 hTLR7.pep
LILFSLSISVSLFLMVMMTASHLYFWDVWYIYHFCKAKIKGYQRLISPDCCYDAFIVYDT 899
mTLR7.pep
LILFSVSISSVLFLMVVMTTSHLFFWDMMYIYYFWKAKIKGYQHLQSMESCYDAFIVYDT 900 .
: . : . : . : . : . : 960 hTLR7.pep
KDPAVTEWVLAELVAKLEDPREKHFNLCLEERDWLPGQPVLENLSQSIQLSKKTVFVMTD 959
mTLR7.pep
KNSAVTEWVLQELVAKLEDPREKHFNLCLEERDWLPGQPVLENLSQSIQLSKKTVFVMTQ 960 .
: . : . : . : . : . : 1020 bb210788.pep
VDVIILIFLVKPFQKFNFL*LRKRISRSSVLECPPNP 37 aa276879.pep
QKSKFLQLRKRLCRSSVLEWPANP 24 aa266744.pep
LGKPLQKSKFLQLRKRLCRSSVLEWPANP 29 bb116163.pep
IETFQMPSFLSIQRLLDDKVDVIILIFLE*PL*KSKFLQLRKRFCRSSVLEWPANP 56
hTLR7.pep
KYAKTENFKIAFYLSHQRLMDEKVDVIILIFLEKPFQKSKFLQLRKRLCGSSVLEWPTNP 1019
mTLR7.pep
KYAKTESFKMAFYLSHQRLLDEKVDVIILIFLEKPLQKSKFLQLRKRLCRSSVLEWPANP 1020 .
: . : . : . : . : . : 1080 bb210788.pep
QAHPYFCQCLKNALTTDNHVAYSQMFKETV 67 aa276879.pep
QAHPYFWQCLKNALTTDNHVAYSQMFKETV 54 aa266744.pep
QAHPYFWQCLKNALTTDNHVAYSQMFKETV 59 bb116163.pep
QAHPYFWQCLKNALTTDNHVAYSQMFKETV 86 hTLR7.pep
QAHPYFWQCLKNALATDNHVAYSQVFKETV 1049 mTLR7.pep
QAHPYFWQCLKNALTTDNHVAYSQMFKETV 1050
In Table 11 the sequences are assigned as follows: mTLR7.pep, SEQ
ID NO:175; hTLR7.pep, SEQ ID NO:170; bb210788.pep, SEQ ID NO:176;
aa276879.pep, SEQ ID NO:177; aa266744.pep, SEQ ID NO:178; and
bb116163.pep, SEQ ID NO:179.
Example 18
Method of Cloning Human TLR8
Two accession numbers in the GenBank database, AF245703 and
AF246971, describe the DNA sequence for human TLR8. To create an
expression vector for human TLR8, human TLR8 cDNA was amplified
from a cDNA made from human peripheral mononuclear blood cells
(PBMC) using the primers 5'-CTGCGCTGCTGCAAGTTACGGAATG-3' (SEQ ID
NO:180) and 5'-GCGCGAAATCATGACTTAACGTCAG-3 (SEQ ID NO:181). The
fragment was cloned into pGEM-T Easy vector (Promega), cut with the
restriction enzyme NotI and ligated into a NotI-digested pcDNA3.1
expression vector (Invitrogen). The insert was fully sequenced and
translated into protein. The cDNA sequence for hTLR8 is SEQ ID
NO:182, is presented in Table 12. The open reading frame starts at
base 83, ends at base 3208, and codes for a protein of 1041 amino
acids. SEQ ID NO:183 (Table 13), corresponding to bases 83-3205 of
SEQ ID NO:182 (Table 12), is the coding region for the polypeptide
of SEQ ID NO:184 (Table 14).
The protein sequence of the cloned hTLR8 cDNA matches the sequence
described under the GenBank accession number AF245703. The sequence
deposited under GenBank accession number AF246971 contains an
insertion at the N-terminus of 15 amino acids (MKESSLQNSSCSLGKETKK;
SEQ ID NO:185) and three single amino acid changes at positions 217
(P to S), 266 (L to P) and 867 (V to I).
TABLE 12 cDNA Sequence for Human TLR8 (5' to 3' ; SEQ ID NO:182)
gctcccggcc gccatggcgg ccgcgggaat tcgattctgc gctgctgcaa gttacggaat
60 gaaaaattag aacaacagaa acatggaaaa catgttcctt cagtcgtcaa
tgctgacctg 120 cattttcctg ctaatatctg gttcctgtga gttatgcgcc
gaagaaaatt tttctagaag 180 ctatccttgt gatgagaaaa agcaaaatga
ctcagttatt gcagagtgca gcaatcgtcg 240 actacaggaa gttccccaaa
cggtgggcaa atatgtgaca gaactagacc tgtctgataa 300 tttcatcaca
cacataacga atgaatcatt tcaagggctg caaaatctca ctaaaataaa 360
tctaaaccac aaccccaatg tacagcacca gaacggaaat cccggtatac aatcaaatgg
420 cttgaatatc acagacgggg cattcctcaa cctaaaaaac ctaagggagt
tactgcttga 480 agacaaccag ttaccccaaa taccctctgg tttgccagag
tctttgacag aacttagtct 540 aattcaaaac aatatataca acataactaa
agagggcatt tcaagactta taaacttgaa 600 aaatctctat ttggcctgga
actgctattt taacaaagtt tgcgagaaaa ctaacataga 660 agatggagta
tttgaaacgc tgacaaattt ggagttgcta tcactatctt tcaattctct 720
ttcacacgtg ccacccaaac tgccaagctc cctacgcaaa ctttttctga gcaacaccca
780 gatcaaatac attagtgaag aagatttcaa gggattgata aatttaacat
tactagattt 840 aagcgggaac tgtccgaggt gcttcaatgc cccatttcca
tgcgtgcctt gtgatggtgg 900 tgcttcaatt aatatagatc gttttgcttt
tcaaaacttg acccaacttc gatacctaaa 960 cctctctagc acttccctca
ggaagattaa tgctgcctgg tttaaaaata tgcctcatct 1020 gaaggtgctg
gatcttgaat tcaactattt agtgggagaa atagcctctg gggcattttt 1080
aacgatgctg ccccgcttag aaatacttga cttgtctttt aactatataa aggggagtta
1140 tccacagcat attaatattt ccagaaactt ctctaaactt ttgtctctac
gggcattgca 1200 tttaagaggt tatgtgttcc aggaactcag agaagatgat
ttccagcccc tgatgcagct 1260 tccaaactta tcgactatca acttgggtat
taattttatt aagcaaatcg atttcaaact 1320 tttccaaaat ttctccaatc
tggaaattat ttacttgtca gaaaacagaa tatcaccgtt 1380 ggtaaaagat
acccggcaga gttatgcaaa tagttcctct tttcaacgtc atatccggaa 1440
acgacgctca acagattttg agtttgaccc acattcgaac ttttatcatt tcacccgtcc
1500 tttaataaag ccacaatgtg ctgcttatgg aaaagcctta gatttaagcc
tcaacagtat 1560 tttcttcatt gggccaaacc aatttgaaaa tcttcctgac
attgcctgtt taaatctgtc 1620 tgcaaatagc aatgctcaag tgttaagtgg
aactgaattt tcagccattc ctcatgtcaa 1680 atatttggat ttgacaaaca
atagactaga ctttgataat gctagtgctc ttactgaatt 1740 gtccgacttg
gaagttctag atctcagcta taattcacac tatttcagaa tagcaggcgt 1800
aacacatcat ctagaattta ttcaaaattt cacaaatcta aaagttttaa acttgagcca
1860 caacaacatt tatactttaa cagataagta taacctggaa agcaagtccc
tggtagaatt 1920 agttttcagt ggcaatcgcc ttgacatttt gtggaatgat
gatgacaaca ggtatatctc 1980 cattttcaaa ggtctcaaga atctgacacg
tctggattta tcccttaata ggctgaagca 2040 catcccaaat gaagcattcc
ttaatttgcc agcgagtctc actgaactac atataaatga 2100 taatatgtta
aagtttttta actggacatt actccagcag tttcctcgtc tcgagttgct 2160
tgacttacgt ggaaacaaac tactcttttt aactgatagc ctatctgact ttacatcttc
2220 ccttcggaca ctgctgctga gtcataacag gatttcccac ctaccctctg
gctttctttc 2280 tgaagtcagt agtctgaagc acctcgattt aagttccaat
ctgctaaaaa caatcaacaa 2340 atccgcactt gaaactaaga ccaccaccaa
attatctatg ttggaactac acggaaaccc 2400 ctttgaatgc acctgtgaca
ttggagattt ccgaagatgg atggatgaac atctgaatgt 2460 caaaattccc
agactggtag atgtcatttg tgccagtcct ggggatcaaa gagggaagag 2520
tattgtgagt ctggagctaa caacttgtgt ttcagatgtc actgcagtga tattattttt
2580 cttcacgttc tttatcacca ccatggttat gttggctgcc ctggctcacc
atttgtttta 2640 ctgggatgtt tggtttatat ataatgtgtg tttagctaag
gtaaaaggct acaggtctct 2700 ttccacatcc caaactttct atgatgctta
catttcttat gacaccaaag acgcctctgt 2760 tactgactgg gtgataaatg
agctgcgcta ccaccttgaa gagagccgag acaaaaacgt 2820 tctcctttgt
ctagaggaga gggattggga cccgggattg gccatcatcg acaacctcat 2880
gcagagcatc aaccaaagca agaaaacagt atttgtttta accaaaaaat atgcaaaaag
2940 ctggaacttt aaaacagctt tttacttggc tttgcagagg ctaatggatg
agaacatgga 3000 tgtgattata tttatcctgc tggagccagt gttacagcat
tctcagtatt tgaggctacg 3060 gcagcggatc tgtaagagct ccatcctcca
gtggcctgac aacccgaagg cagaaggctt 3120 gttttggcaa actctgagaa
atgtggtctt gactgaaaat gattcacggt ataacaatat 3180 gtatgtcgat
tccattaagc aatactaact gacgttaagt catgatttcg cgcaatcact 3240
agtgaattcg cggccgcctg caggtcgacc atatgggaga gctcccaacg cgttggatgc
3300 atagcttgag 3310
TABLE 13 Coding Region for Human TLR8 (5' to 3'; SEQ ID NO:183)
atggaaaaca tgttccttca gtcgtcaatg ctgacctgca ttttcctgct aatatctggt
60 tcctgtgagt tatgcgccga agaaaatttt tctagaagct atccttgtga
tgagaaaaag 120 caaaatgact cagttattgc agagtgcagc aatcgtcgac
tacaggaagt tccccaaacg 180 gtgggcaaat atgtgacaga actagacctg
tctgataatt tcatcacaca cataacgaat 240 gaatcatttc aagggctgca
aaatctcact aaaataaatc taaaccacaa ccccaatgta 300 cagcaccaga
acggaaatcc cggtatacaa tcaaatggct tgaatatcac agacggggca 360
ttcctcaacc taaaaaacct aagggagtta ctgcttgaag acaaccagtt accccaaata
420 ccctctggtt tgccagagtc tttgacagaa cttagtctaa ttcaaaacaa
tatatacaac 480 ataactaaag agggcatttc aagacttata aacttgaaaa
atctctattt ggcctggaac 540 tgctatttta acaaagtttg cgagaaaact
aacatagaag atggagtatt tgaaacgctg 600 acaaatttgg agttgctatc
actatctttc aattctcttt cacacgtgcc acccaaactg 660 ccaagctccc
tacgcaaact ttttctgagc aacacccaga tcaaatacat tagtgaagaa 720
gatttcaagg gattgataaa tttaacatta ctagatttaa gcgggaactg tccqaggtgc
780 ttcaatgccc catttccatg cgtgccttgt gatggtggtg cttcaattaa
tatagatcgt 840 tttgcttttc aaaacttgac ccaacttcga tacctaaacc
tctctagcac ttccctcagg 900 aagattaatg ctgcctggtt taaaaatatg
cctcatctga aggtgctgga tcttgaattc 960 aactatttag tgggagaaat
agcctctggg gcatttttaa cgatgctgcc ccgcttagaa 1020 atacttgact
tgtcttttaa ctatataaag gggagttatc cacagcatat taatatttcc 1080
agaaacttct ctaaactttt gtctctacgg gcattgcatt taagaggtta tgtgttccag
1140 gaactcagag aagatgattt ccagcccctg atgcagcttc caaacttatc
gactatcaac 1200 ttgggtatta attttattaa gcaaatcgat ttcaaacttt
tccaaaattt ctccaatctg 1260 gaaattattt acttgtcaga aaacagaata
tcaccgttgg taaaagatac ccggcagagt 1320 tatgcaaata gttcctcttt
tcaacgtcat atccggaaac gacgctcaac agattttgag 1380 tttgacccac
attcgaactt ttatcatttc acccgtcctt taataaagcc acaatgtgct 1440
gcttatggaa aagccttaga tttaagcctc aacagtattt tcttcattgg gccaaaccaa
1500 tttgaaaatc ttcctgacat tgcctgttta aatctgtctg caaatagcaa
tgctcaagtg 1560 ttaagtggaa ctgaattttc agccattcct catgtcaaat
atttggattt gacaaacaat 1620 agactagact ttgataatgc tagtgctctt
actgaattgt ccgacttgga agttctagat 1680 ctcagctata attcacacta
tttcagaata gcaggcgtaa cacatcatct agaatttatt 1740 caaaatttca
caaatctaaa agttttaaac ttgagccaca acaacattta tactttaaca 1800
gataagtata acctggaaag caagtccctg gtagaattag ttttcagtgg caatcgcctt
1860 gacattttgt ggaatgatga tgacaacagg tatatctcca ttttcaaagg
tctcaagaat 1920 ctgacacgtc tggatttatc ccttaatagg ctgaagcaca
tcccaaatga agcattcctt 1980 aatttgccag cgagtctcac tgaactacat
ataaatgata atatgttaaa gttttttaac 2040 tggacattac tccagcagtt
tcctcgtctc gagttgcttg acttacgtgg aaacaaacta 2100 ctctttttaa
ctgatagcct atctgacttt acatcttccc ttcggacact gctgctgagt 2160
cataacagga tttcccacct accctctggc tttctttctg aagtcagtag tctgaagcac
2220 ctcgatttaa gttccaatct gctaaaaaca atcaacaaat ccgcacttga
aactaagacc 2280 accaccaaat tatctatgtt ggaactacac ggaaacccct
ttgaatgcac ctgtgacatt 2340 ggagatttcc gaagatggat ggatgaacat
ctgaatgtca aaattcccag actggtagat 2400 gtcatttgtg ccagtcctgg
ggatcaaaga gggaagagta ttgtgagtct ggagctaaca 2460 acttgtgttt
cagatgtcac tgcagtgata ttatttttct tcacgttctt tatcaccacc 2520
atggttatgt tggctgccct ggctcaccat ttgttttact gggatgtttg gtttatatat
2580 aatgtgtgtt tagctaaggt aaaaggctac aggtctcttt ccacatccca
aactttctat 2640 gatgcttaca tttcttatga caccaaagac gcctctgtta
ctgactgggt gataaatgag 2700 ctgcgctacc accttgaaga gagccgagac
aaaaacgttc tcctttgtct agaggagagg 2760 gattgggacc cgggattggc
catcatcgac aacctcatgc agagcatcaa ccaaagcaag 2820 aaaacagtat
ttgttttaac caaaaaatat gcaaaaagct ggaactttaa aacagctttt 2880
tacttggctt tgcagaggct aatggatgag aacatggatg tgattatatt tatcctgctg
2940 gagccagtgt tacagcattc tcagtatttg aggctacggc agcggatctg
taagagctcc 3000 atcctccagt ggcctgacaa cccgaaggca gaaggcttgt
tttggcaaac tctgagaaat 3060 gtggtcttga ctgaaaatga ttcacggtat
aacaatatgt atgtcgattc cattaagcaa 3120 tac 3123
TABLE 14 Amino Acid Sequence of Human TLR8 . : . : . : . : . : . :
60 AF245703 .pep MENMFLQSSMLTCIFLLISGSCELCAEENFSRSYPCDEKKQN 42
hTLR8.pep MENMFLQSSMLTCIFLLISGSCELCAEENFSRSYPCDEKKQN 42
AF246971.pep
MKESSLQNSSCSLGKETKKENMFLQSSMLTCIFLLISGSCELCAEENFSRSYPCDEKKQN 60 . :
. : . : . : . : . : 120 AF245703.pep
DSVIAECSNRRLQEVPQTVGKYVTELDLSDNFITHITNESFQGLQNLTKINLNHNPNVQH 102
hTL28.pep
DSVIAECSNRRLQEVPQTVGKYVTELDLSDNFITHITNESFQGLQNLTKINLNHNPNVQH 102
AF246971.pep
DSVIAECSNRRLQEVPQTVGKYVTELDLSDNFITHITNESFQGLQNLTKINLNHNPNVQH 120 .
: . : . : . : . : . : 180 AF245703.pep
QNGNPGIQSNGLNITDGAFLNLKNLRELLLEDNQLPQIPSGLPESLTELSLIQNNIYNIT 162
hTLR8.pep
QNGNPGIQSNGLNITDGAFLNLKNLRELLLEDNQLPQIPSGLPESLTELSLIQNNIYNIT 162
AF246971.pep
QNGNPGIQSNGLNITDGAFLNLKNLRELLLEDNQLPQIPSGLPESLTELSLIQNNIYNIT 180 .
: . : . : . : . : . : 240 AF245703.pep
KEGISRLINLKNLYLAWNCYFNKVCEKTNIEDGVFETLTNLELLSLSFNSLSHVPPKLPS 222
hTLR8.pep
KEGISRLINLKNLYLAWNCYFNKVCEKTNIEDGVFETLTNLELLSLSFNSLSHVPPKLPS 222
AF246971.pep
KEGISRLINLKNLYLAWNCYFNKVCEKTNIEDGVFETLTNLELLSLSFNSLSHVSPKLPS 240 .
: . : . : . : . : . : 300 AF245703.pep
SLRKLFLSNTQIKYISEEDFKGLINLTLLDLSGNCPRCFNAPFPCVPCDGGASINIDRFA 282
hTLR8.pep
SLRKLFLSNTQIKYISEEDFKGLINLTLLDLSGNCPRCFNAPFPCVPCDGGASINIDRFA 282
AF246971.pep
SLRKLFLSNTQIKYISEEDFKGLINLTLLDLSGNCPRCFNAPFPCVPCDGGASINIDRFA 300 .
: . : . : . : . : . : 360 AF245703.pep
FQNLTQLRYLNLSSTSLRKINAAWFKNMPHLKVLDLEFNYLVGEIASGAFLTMLPRLEIL 342
hTLR8.pep
FQNLTQLRYLNLSSTSLRKINAAWFKNMPHLKVLDLEFNYLVGEIASGAFLTMLPRLEIL 342
AF246971.pep
FQNLTQLRYLNLSSTSLRKINAAWFKNMPHLKVLDLEFNYLVGEIASGAFLTMLPRLEIL 360 .
: . : . : . : . : . : 420 AF245703.pep
DLSFNYIKGSYPQHINISRNFSKLLSLRALHLRGYVFQELREDDFQPLMQLPNLSTINLG 402
hTLR8.pep
DLSFNYIKGSYPQHINISRNFSKLISLRALHLRGYVFQELREDDFQPLMQLPNLSTINLG 402
AF246971.pep
DLSFNYIKGSYPQHINISRNFSKPLSLRALHLRGYVFQELREDDFQPLMQLPNLSTINLG 420 .
: . : . : . : . : . : 480 AF245703.pep
INFIKQIDFKLFQNFSNLEIIYLSENRISPLVKDTRQSYANSSSFQRHIRKRRSTDFEFD 462
hTLR8.pep
INFIKQIDFKLFQNFSNLEIIYLSENRISPLVKDTRQSYANSSSFQRHIRKRRSTDFEFD 462
AF246971.pep
INFIKQIDFKLFQNFSNLEIIYLSENRISPLVKDTRQSYANSSSFQRHIRKRRSTDFEFD 480 .
: . : . : . : . : . : 540 AF245703.pep
PHSNFYHFTRPLIKPQCAAYGKALDLSLNSIFFIGPNQFENLPDIACLNLSANSNAQVLS 522
hTLR8.pep
PHSNFYHFTRPLIKPQCAAYGKALDLSLNSIFFIGPNQFENLPDIACLNLSANSNAQVLS 522
AF246971.pep
PHSNFYHFTRPLIKPQCAAYGKALDLSLNSIFFIGPNQFENLPDIACLNLSANSNAQVLS 540 .
: . : . : . : . : . : 600 AF245703.pep
GTEFSAIPHVKYLDLTNNRLDFDNASALTELSDLEVLDLSYNSHYFRIAGVTHHLEFIQN 582
hTLR8.pep
GTEFSAIPHVKYLDLTNNRLDFDNASALTELSDLEVLDLSYNSHYFRIAGVTHHLEFIQN 582
AF246971.pep
GTEFSAIPHVKYLDLTNNRLDFDNASALTELSDLEVLDLSYNSHYFRIAGVTHHLEFIQN 600 .
: . : . : . : . : . : 660 AF245703.pep
FTNLKVLNLSHNNIYTLTDKYNLESKSLVELVFSGNRLDILWNDDDNRYISIFKGLKNLT 642
hTLR8.pep
FTNLKVLNLSHNNIYTLTDKYNLESKSLVELVFSGNRLDILWNDDDNRYISIFKGLKNLT 642
AF246971.pep
FTNLKVLNLSHNNIYTLTDKYNLESKSLVELVFSGNRLDILWNDDDNRYISIFKGLKNLT 660 .
: . : . : . : . : . : 720 AF245703.pep
RLDLSLNRLKHIPNEAFLNLPASLTELHINDNMLKFFNWTLLQQFPRLELLDLRGNKLLF 702
hTLR8.pep
RLDLSLNRLKHIPNEAFLNLPASLTELHINDNMLKFFNWTLLQQFPRLELLDLRGNKLLF 702
AF246971.pep
RLDLSLNRLKHIPNEAFLNLPASLTELHINDNMLKFFNWTLLQQFPRLELLDLRGNKLLF 720 .
: . : . : . : . : . : 780 AF245703.pep
LTDSLSDFTSSLRTLLLSHNRISHLPSGFLSEVSSLKHLDLSSNLLKTINKSALETKTTT 762
hTLR8.pep
LTDSLSDFTSSLRTLLLSHNRISHLPSGFLSEVSSLKHLDLSSNLLKTINKSALETKTTT 762
AF246971.pep
LTDSLSDFTSSLRTLLLSHNRISHLPSGFLSEVSSLKHLDLSSNLLKTINKSALETKTTT 780 .
: . : . : . : . : . : 840 AF245703.pep
KLSMLELHGNPFECTCDIGDFRRWMDEHLNVKIPRLVDVICASPGDQRGKSIVSLELTTC 822
hTLR8.pep
KLSMLELHGNPFECTCDIGDFRRWMDEHLNVKIPRLVDVICASPGDQRGKSIVSLELTTC 822
AF246971.pep
KLSMLELHGNPFECTCDIGDFRRWMDEHLNVKIPRLVDVICASPGDQRGKSIVSLELTTC 840 .
: . : . : . : . : . : 900 AF245703.pep
VSDVTAVILFFFTFFITTMVMLAALAHHLFYWDVWFIYNVCLAKVKGYRSLSTSQTFYDA 882
hTLR8.pep
VSDVTAVILFFFTFFITTMVMLAALAHHLFYWDVWFIYNVCLAKVKGYRSLSTSQTFYDA 882
AF246971.pep
VSDVTAVILFFFTFFITTMVMLAALAHHLFYWDVWFIYNVCLAKIKGYRSLSTSQTFYDA 900 .
: . : . : . : . : . : 960 AF245703.pep
YISYDTKDASVTDWVINELRYHLEESRDKNVLLCLEERDWDPGLAIIDNLMQSINQSKKT 942
hTLR8.pep
YISYDTKDASVTDWVINELRYHLEESRDKNVLLCLEERDWDPGLAIIDNLMQSINQSKKT 942
AF246971.pep
YISYDTKDASVTDWVINELRYHLEESRDKNVLLCLEERDWDPGLAIIDNLMQSINQSKKT 960 .
: . : . : . : . : . : 1020 AF245703.pep
VFVLTKKYAKSWNFKTAFYLALQRLMDENMDVIIFILLEPVLQHSQYLRLRQRICKSSIL 1002
hTLR8.pep
VFVLTKKYAKSWNFKTAFYLALQRLMDENMDVIIFILLEPVLQHSQYLRLRQRICKSSIL 1002
AF246971.pep
VFVLTKKYAKSWNFKTAFYLALQRLMDENMDVIIFILLEPVLQHSQYLRLRQRICKSSIL 1020 .
: . : . : . : . : . : 1080 AF245703.pep
QWPDNPKAEGLFWQTLRNVVLTENDSRYNNMYVDSIKQY 1041 hTLR8.pep
QWPDNPKAEGLFWQTLRNVVLTENDSRYNNMYVDSIKQY 1041 AF246971.pep
QWPDNPKAEGLFWQTLRNVVLTENDSRYNNMYVDSIKQY 1059
In Table 14 the sequences are assigned as follows: hTLR8.pep, SEQ
ID NO:184; AF245703.pep, SEQ ID NO:186; and AF246971.pep, SEQ ID
NO:187.
Example 19
Method of Cloning the Murine TLR8
Alignment of human TLR8 protein sequence with mouse EST database
using tfasta yielded 1 hit with mouse EST sequence bf135656. Two
primers were designed that bind to bf135656 sequence for use in a
RACE-PCR to amplify 5' and 3' ends of the murine TLR8 cDNA. The
library used for the RACE PCR was a mouse spleen marathon-ready
cDNA commercially available from Clontech. A 5' fragment with a
length of 2900 bp and a 3' fragment with a length of 2900 bp
obtained by this method were cloned into Promega pGEM-T Easy
vector. After sequencing of the 5' end and 3' end of each fragment,
partial sequences of mTLR8 were obtained and allowed the design of
primers for amplification of the complete murine TLR8 cDNA.
Three independent PCR reactions were set up using a spleen murine
cDNA from Clontech as a template with the primers
5'-GAGAGAAACAAACGTTTTACCTTC-3' (SEQ ID NO:188) and
5'-GATGGCAGAGTCGTGACTTCCC-3' (SEQ ID NO:189). The resulting
amplification products were cloned into pGEM-T Easy vector, fully
sequenced, translated into protein, and aligned to the human TLR8
protein sequence (GenBank accession number AF245703). The cDNA
sequence for mTLR8 is SEQ ID NO:190, presented in Table 15. The
open reading frame of mTLR8 starts at base 59, ends at base 3157,
and codes for a protein of 1032 amino acids. SEQ ID NO:191 (Table
16), corresponding to bases 59-3154 of SEQ ID NO:190 (Table 15), is
the coding region for the polypeptide of SEQ ID NO:192 (Table 17).
To create an expression vector for murine TLR8, cDNA pGEM-T Easy
vector with the mTLR8 insert was cut with NotI, the fragment
isolated, and ligated into a NotI-digested pcDNA3.1 expression
vector (Invitrogen).
TABLE 15 cDNA Sequence for Murine TLR8 (5' to 3'; SEQ ID NO:190)
attcagagtt ggatgttaag agagaaacaa acgttttacc ttcctttgtc tatagaacat
60 ggaaaacatg ccccctcagt catggattct gacgtgcttt tgtctgctgt
cctctggaac 120 cagtgccatc ttccataaag cgaactattc cagaagctat
ccttgtgacg agataaggca 180 caactccctt gtgattgcag aatgcaacca
tcgtcaactg catgaagttc cccaaactat 240 aggcaagtat gtgacaaaca
tagacttgtc agacaatgcc attacacata taacgaaaga 300 gtcctttcaa
aagctgcaaa acctcactaa aatcgatctg aaccacaatg ccaaacaaca 360
gcacccaaat gaaaataaaa atggtatgaa tattacagaa ggggcacttc tcagcctaag
420 aaatctaaca gttttactgc tggaagacaa ccagttatat actatacctg
ctgggttgcc 480 tgagtctttg aaagaactta gcctaattca aaacaatata
tttcaggtaa ctaaaaacaa 540 cacttttggg cttaggaact tggaaagact
ctatttgggc tggaactgct attttaaatg 600 taatcaaacc tttaaggtag
aagatggggc atttaaaaat cttatacact tgaaggtact 660 ctcattatct
ttcaataacc ttttctatgt gccccccaaa ctaccaagtt ctctaaggaa 720
actttttctg agtaatgcca aaatcatgaa catcactcag gaagacttca aaggactgga
780 aaatttaaca ttactagatc tgagtggaaa ctgtccaagg tgttacaatg
ctccatttcc 840 ttgcacacct tgcaaggaaa actcatccat ccacatacat
cctctggctt ttcaaagtct 900 cacccaactt ctctatctaa acctttccag
cacttccctc aggacgattc cttctacctg 960 gtttgaaaat ctgtcaaatc
tgaaggaact ccatcttgaa ttcaactatt tagttcaaga 1020 aattgcctcg
ggggcatttt taacaaaact acccagttta caaatccttg atttgtcctt 1080
caactttcaa tataaggaat atttacaatt tattaatatt tcctcaaatt tctctaagct
1140 tcgttctctc aagaagttgc acttaagagg ctatgtgttc cgagaactta
aaaagaagca 1200 tttcgagcat ctccagagtc ttccaaactt ggcaaccatc
aacttgggca ttaactttat 1260 tgagaaaatt gatttcaaag ctttccagaa
tttttccaaa ctcgacgtta tctatttatc 1320 aggaaatcgc atagcatctg
tattagatgg tacagattat tcctcttggc gaaatcgtct 1380 tcggaaacct
ctctcaacag acgatgatga gtttgatcca cacgtgaatt tttaccatag 1440
caccaaacct ttaataaagc cacagtgtac tgcttatggc aaggccttgg atttaagttt
1500 gaacaatatt ttcattattg ggaaaagcca atttgaaggt tttcaggata
tcgcctgctt 1560 aaatctgtcc ttcaatgcca atactcaagt gtttaatggc
acagaattct cctccatgcc 1620 ccacattaaa tatttggatt taaccaacaa
cagactagac tttgatgata acaatgcttt 1680 cagtgatctt cacgatctag
aagtgctgga cctgagccac aatgcacact atttcagtat 1740 agcaggggta
acgcaccgtc taggatttat ccagaactta ataaacctca gggtgttaaa 1800
cctgagccac aatggcattt acaccctcac agaggaaagt gagctgaaaa gcatctcact
1860 gaaagaattg gttttcagtg gaaatcgtct tgaccatttg tggaatgcaa
atgatggcaa 1920 atactggtcc atttttaaaa gtctccagaa tttgatacgc
ctggacttat catacaataa 1980 ccttcaacaa atcccaaatg gagcattcct
caatttgcct cagagcctcc aagagttact 2040 tatcagtggt aacaaattac
gtttctttaa ttggacatta ctccagtatt ttcctcacct 2100 tcacttgctg
gatttatcga gaaatgagct gtattttcta cccaattgcc tatctaagtt 2160
tgcacattcc ctggagacac tgctactgag ccataatcat ttctctcacc taccctctgg
2220 cttcctctcc gaagccagga atctggtgca cctggatcta agtttcaaca
caataaagat 2280 gatcaataaa tcctccctgc aaaccaagat gaaaacgaac
ttgtctattc tggagctaca 2340 tgggaactat tttgactgca cgtgtgacat
aagtgatttt cgaagctggc tagatgaaaa 2400 tctgaatatc acaattccta
aattggtaaa tgttatatgt tccaatcctg gggatcaaaa 2460 atcaaagagt
atcatgagcc tagatctcac gacttgtgta tcggatacca ctgcagctgt 2520
cctgtttttc ctcacattcc ttaccacctc catggttatg ttggctgctc tggttcacca
2580 cctgttttac tgggatgttt ggtttatcta tcacatgtgc tctgctaagt
taaaaggcta 2640 caggacttca tccacatccc aaactttcta tgatgcttat
atttcttatg acaccaaaga 2700 tgcatctgtt actgactggg taatcaatga
actgcgctac caccttgaag agagtgaaga 2760 caaaagtgtc ctcctttgtt
tagaggagag ggattgggat ccaggattac ccatcattga 2820 taacctcatg
cagagcataa accagagcaa gaaaacaatc tttgttttaa ccaagaaata 2880
tgccaagagc tggaacttta aaacagcttt ctacttggcc ttgcagaggc taatggatga
2940 gaacatggat gtgattattt tcatcctcct ggaaccagtg ttacagtact
cacagtacct 3000 gaggcttcgg cagaggatct gtaagagctc catcctccag
tggcccaaca atcccaaagc 3060 agaaaacttg ttttggcaaa gtctgaaaaa
tgtggtcttg actgaaaatg attcacggta 3120 tgacgatttg tacattgatt
ccattaggca atactagtga tgggaagtca cgactctgcc 3180 atcataaaaa
cacacagctt ctccttacaa tgaaccgaat 3220
TABLE 16 Coding Region for Murine TLR8 (5' to 3'; SEQ ID NO:191)
atggaaaaca tgccccctca gtcatggatt ctgacgtgct tttgtctgct gtcctctgga
60 accagtgcca tcttccataa agcgaactat tccagaagct atccttgtga
cgagataagg 120 cacaactccc ttgtgattgc agaatgcaac catcgtcaac
tgcatgaagt tccccaaact 180 ataggcaagt atgtgacaaa catagacttg
tcagacaatg ccattacaca tataacgaaa 240 gagtcctttc aaaagctgca
aaacctcact aaaatcgatc tgaaccacaa tgccaaacaa 300 cagcacccaa
atgaaaataa aaatggtatg aatattacag aaggggcact tctcagccta 360
agaaatctaa cagttttact gctggaagac aaccagttat atactatacc tgctgggttg
420 cctgagtctt tgaaagaact tagcctaatt caaaacaata tatttcaggt
aactaaaaac 480 aacacttttg ggcttaggaa cttggaaaga ctctatttgg
gctggaactg ctattttaaa 540 tgtaatcaaa cctttaaggt agaagatggg
gcatttaaaa atcttataca cttgaaggta 600 ctctcattat ctttcaataa
ccttttctat gtgcccccca aactaccaag ttctctaagg 660 aaactttttc
tgagtaatgc caaaatcatg aacatcactc aggaagactt caaaggactg 720
gaaaatttaa cattactaga tctgagtgga aactgtccaa ggtgttacaa tgctccattt
780 ccttgcacac cttgcaagga aaactcatcc atccacatac atcctctggc
ttttcaaagt 840 ctcacccaac ttctctatct aaacctttcc agcacttccc
tcaggacgat tccttctacc 900 tggtttgaaa atctgtcaaa tctgaaggaa
ctccatcttg aattcaacta tttagttcaa 960 gaaattgcct cgggggcatt
tttaacaaaa ctacccagtt tacaaatcct tgatttgtcc 1020 ttcaactttc
aatataagga atatttacaa tttattaata tttcctcaaa tttctctaag 1080
cttcgttctc tcaagaagtt gcacttaaga ggctatgtgt tccgagaact taaaaagaag
1140 catttcgagc atctccagag tcttccaaac ttggcaacca tcaacttggg
cattaacttt 1200 attgagaaaa ttgatttcaa agctttccag aatttttcca
aactcgacgt tatctattta 1260 tcaggaaatc gcatagcatc tgtattagat
ggtacagatt attcctcttg gcgaaatcgt 1320 cttcggaaac ctctctcaac
agacgatgat gagtttgatc cacacgtgaa tttttaccat 1380 agcaccaaac
ctttaataaa gccacagtgt actgcttatg gcaaggcctt ggatttaagt 1440
ttgaacaata ttttcattat tgggaaaagc caatttgaag gttttcagga tatcgcctgc
1500 ttaaatctgt ccttcaatgc caatactcaa gtgtttaatg gcacagaatt
ctcctccatg 1560 ccccacatta aatatttgga tttaaccaac aacagactag
actttgatga taacaatgct 1620 ttcagtgatc ttcacgatct agaagtgctg
gacctgagcc acaatgcaca ctatttcagt 1680 atagcagggg taacgcaccg
tctaggattt atccagaact taataaacct cagggtgtta 1740 aacctgagcc
acaatggcat ttacaccctc acagaggaaa gtgagctgaa aagcatctca 1800
ctgaaagaat tggttttcag tggaaatcgt cttgaccatt tgtggaatgc aaatgatggc
1860 aaatactggt ccatttttaa aagtctccag aatttgatac gcctggactt
atcatacaat 1920 aaccttcaac aaatcccaaa tggagcattc ctcaatttgc
ctcagagcct ccaagagtta 1980 cttatcagtg gtaacaaatt acgtttcttt
aattggacat tactccagta ttttcctcac 2040 cttcacttgc tggatttatc
gagaaatgag ctgtattttc tacccaattg cctatctaag 2100 tttgcacatt
ccctggagac actgctactg agccataatc atttctctca cctaccctct 2160
ggcttcctct ccgaagccag gaatctggtg cacctggatc taagtttcaa cacaataaag
2220 atgatcaata aatcctccct gcaaaccaag atgaaaacga acttgtctat
tctggagcta 2280 catgggaact attttgactg cacgtgtgac ataagtgatt
ttcgaagctg gctagatgaa 2340 aatctgaata tcacaattcc taaattggta
aatgttatat gttccaatcc tggggatcaa 2400 aaatcaaaga gtatcatgag
cctagatctc acgacttgtg tatcggatac cactgcagct 2460 gtcctgtttt
tcctcacatt ccttaccacc tccatggtta tgttggctgc tctggttcac 2520
cacctgtttt actgggatgt ttggtttatc tatcacatgt gctctgctaa gttaaaaggc
2580 tacaggactt catccacatc ccaaactttc tatgatgctt atatttctta
tgacaccaaa 2640 gatgcatctg ttactgactg ggtaatcaat gaactgcgct
accaccttga agagagtgaa 2700 gacaaaagtg tcctcctttg tttagaggag
agggattggg atccaggatt acccatcatt 2760 gataacctca tgcagagcat
aaaccagagc aagaaaacaa tctttgtttt aaccaagaaa 2820 tatgccaaga
gctggaactt taaaacagct ttctacttgg ccttgcagag gctaatggat 2880
gagaacatgg atgtgattat tttcatcctc ctggaaccag tgttacagta ctcacagtac
2940 ctgaggcttc ggcagaggat ctgtaagagc tccatcctcc agtggcccaa
caatcccaaa 3000 gcagaaaact tgttttggca aagtctgaaa aatgtggtct
tgactgaaaa tgattcacgg 3060 tatgacgatt tgtacattga ttccattagg caatac
3096
TABLE 17 Amino Acid Sequences of Murine TLR8 and Human TLR8 . : . :
. : . : . : . : 60 mTLR8.pep
MENMPPQSWILTCFCLLSSGTSAIFHKANYSRSYPCDEIRHNSLVIAECNHRQLHEVPQT 60
hTLR8.pep
MENMFLQSSMLTCIFLLISGSCELCAEENFSRSYPCDEKKQNDSVIAECSNRRLQEVPQT 60 . :
. : . : . : . : . : 120 mTLR8.pep
IGKYVTNIDLSDNAITHITKESFQKLQNLTKIDLNHNAKQQH----PNENKNGMNITEGA 116
hTLR8.pep
VGKYVTELDLSDNFITHITNESFQGLQNLTKINLNHNPNVQHQNGNPGIQSNGLNITDGA 120 .
: . : . : . : . : . : 180 mTLR8.pep
LLSLRNLTVLLLEDNQLYTIPAGLPESLKELSLIQNNIFQVTKNNTFGLRNLERLYLGWN 176
hTLR8.pep
FLNLKNLRELLLEDNQLPQIPSGLPESLTELSLIQNNIYNITKEGISRLINLKNLYLAWN 180 .
: . : . : . : . : . : 240 mTLR8.pep
CYFK--CNQTFKVEDGAFKNLIHLKVLSLSFNNLFYVPPKLPSSLRKLFLSNAKIMNITQ 234
hTLR8.pep
CYFNKVCEKT-NIEDGVFETLTNLELLSLSFNSLSHVPPKLPSSLRKLFLSNTQIKYISE 239 .
: . : . : . : . : . : 300 mTLR8.pep
EDFKGLENLTLLDLSGNCPRCYNAPFPCTPCKENSSIHIHPLAFQSLTQLLYLNLSSTSL 294
hTLR8.pep
EDFKGLINLTLLDLSGNCPRCFNAPFPCVPCDGGASINIDRFAFQNLTQLRYLNLSSTSL 299 .
: . : . : . : . : . : 360 mTLR8.pep
RTIPSTWFENLSNLKELHLEFNYLVQEIASGAFLTKLPSLQILDLSFNFQYKEYLQFINI 354
hTLR8.pep
RKINAAWFKNMPHLKVLDLEFNYLVGEIASGAFLTMLPRLEILDLSFNYIKGSYPQHINI 359 .
: . : . : . : . : . : 420 mTLR8.pep
SSNFSKLRSLKKLHLRGYVFRELKKKHFEHLQSLPNLATINLGINFIEKIDFKAFQNFSK 414
hTLR8.pep
SRNFSKLLSLRALHLRGYVFQELREDDFQPLMQLPNLSTINLGINFIKQIDFKLFQNFSN 419 .
: . : . : . : . : . : 480 mTLR8.pep
LDVIYLSGNRIASVLDGT--DY---SSWRNRLRKPLSTDDDEFDPHVNFYHSTKPLIKPQ 469
hTLR8.pep
LEIIYLSENRISPLVKDTRQSYANSSSFQRHIRKRRSTDF-EFDPHSNFYHFTRPLIKPQ 478 .
: . : . : . : . : . : 540 mTLR8.pep
CTAYGKALDLSLNNIFIIGKSQFEGFQDIACLNLSFNANTQVFNGTEFSSMPHIKYLDLT 529
hTLR8.pep
CAAYGKALDLSLNSIFFIGPNQFENLPDIACLNLSANSNAQVLSGTEFSAIPHVKYLDLT 538 .
: . : . : . : . : . : 600 mTLR8.pep
NNRLDFDDNNAFSDLHDLEVLDLSHNAHYFSIAGVTHRLGFIQNLINLRVLNLSHNGIYT 589
hTLR8.pep
NNRLDFDNASALTELSDLEVLDLSYNSHYFRIAGVTHHLEFIQNFTNLKVLNLSHNNIYT 598 .
: . : . : . : . : . : 660 mTLR8.pep
LTEESELKSISLKELVFSGNRLDHLWNANDGKYWSIFKSLQNLIRLDLSYNNLQQIPNGA 649
hTLR8.pep
LTDKYNLESKSLVELVFSGNRLDILWNDDDNRYISIFKGLKNLTRLDLSLNRLKHIPNEA 658 .
: . : . : . : . : . : 720 mTLR8.pep
FLNLPQSLQELLISGNKLRFFNWTLLQYFPHLIILLDLSRNELYFLPNCLSKFAHSLETLL 709
hTLP8.pep
FLNLPASLTELHINDNMLKFFNWTLLQQFPRLELLDLRGNKLLFLTDSLSDFTSSLRTLL 718 .
: . : . : . : . : . : 780 bf135656.pep
NHFSHLPSGFLSEARNLVHLDLSFNTIKMINKSSLQTKMKTNLSILELHGNYFDCTC 57
mTLR8.pep
LSHNHFSHLPSGFLSEARNLVHLDLSFNTIKMINKSSLQTKMKTNLSILELHGNYFDCTC 769
hTLR8.pep
LSHNRISHLPSGFLSEVSSLKHLDLSSNLLKTINKSALETKTTTKLSMLELHGNPFECTC 778 .
: . : . : . : . : . : 840 bf135656.pep
DISDFRSWLDENLNITIPKLVNVICSNPGDQKSKSIMSLDLTTCVSDTTAAVLFFLTFLT 117
mTLR8.pep
DISDFRSWLDENLNITIPKLVNVICSNPGDQKSKSIMSLDLTTCVSDTTAAVLFFLTFLT 829
hTLR8.pep
DIGDFRRWMDEHLNVKIPRLVDVICASPGDQRGKSIVSLELTTCVSDVTAVILFFFTFFI 838 .
: . : . : . : . : . : 900 bf135656.pep
TSMVMLAALVHHLFYWDVWFIYHMCSAKLKGYRTSSTSQTFYDAYISYDTKDASVTDWVI 177
mTLR8.pep
TSMVMLAALVHHLFYWDVWFIYHMCSAKLKGYRTSSTSQTFYDAYISYDTKDASVTDWVI 889
hTLR8.pep
TTMVMLAALAHHLFYWDVWFIYNVCLAKVKGYRSLSTSQTFYDAYISYDTKDASVTDWVI 898 .
: . : . : . : . : . : 960 bf135656.pep NELRYHLE 185 mTLR8.pep
NELRYHLEESEDKSVLLCLEERDWDPGLPIIDNLMQSINQSKKTIFVLTKKYAKSWNFKT 949
hTLR8.pep
NELRYHLEESRDKNVLLCLEERDWDPGLAIIDNLMQSINQSKKTVFVLTKKYAKSWNFKT 958 .
: . : . : . : . : . : 1020 mTLR8.pep
AFYLALQRLMDENMDVIIFLLEPVLQYSQYLRLRQRICKSSILQWPNNPKAENLFWQSL 1009
hTLR8.pep
AFYLALQRLMDENMDVIIFILLEPVLQHSQYLRLRQRICKSSILQWPDNPKAEGLFWQTL 1018 .
: . : . : . : . : . : 1080 mTLR8.pep KNVVLTENDSRYDDLYIDSIRQY 1032
hTLR8.pep RNVVLTENDSRYNNMYVDSIKQY 1041
In Table 17 the sequences are assigned as follows: mTLR8.pep, SEQ
ID NO:192; hTLR8.pep, SEQ ID NO:184; and bf135656.pep, SEQ ID
NO:193.
Example 20
Transient Transfectants Expressing TLR8 and TLR7
The cloned human TLR7 and human TLR8 cDNA (our result) were cloned
into the expression vector pcDNA3.1 (-) from Invitrogen using the
NotI site. Utilizing a "gain of function" assay, hTLR7 and hTLR8
expression vectors were transiently expressed in human 293
fibroblasts (ATCC, CRL-1573) using the calcium phosphate method.
Activation was monitored by IL-8 production after stimulus with
CpG-ODN (2006 or 1668, 2 .mu.M) or LPS (100 ng/ml). None of the
stimuli used activated 293 cells transfected with either hTLR7 or
hTLR8.
Example 21
Screening for TLR9, 8 and 7 Modulators
Human TLR receptors 9, 8 and 7 are expressed differentially among
tissues which may be due to differences in promoter structure. Du X
et al., Eur Cytokine Netw 11:362-71 (2000); Chuang T H et al., Eur
Cytokine Netw 11:372-8 (2000). For the human Toll-like receptors 9,
8 and 7 the genomic locus has been defined and sequenced. TLR9 is
located on chromosome 3 (GenBank accession numbers NT.sub.--
005985, AC006252), TLR7 on chromosome X (GenBank accession numbers
NT.sub.-- 011774, AC005859, AC003046) and TLR8 close to TLR7 also
on chromosome X (GenBank accession numbers NT.sub.-- 011774,
AC005859). To verify differences in the promoter regions the
putative promoter region of each gene are cloned in reporter
vectors like pGL2-Basic (Promega, Madison, Wis., USA) which
contains the luciferase gene (luc) adjacent to a multiple cloning
site. After transient transfection of these constructs in various
cell lines, different stimuli can be tested for the activation of
the inserted promoter region which is detected by luciferase
activity. The promoter regions defined by the cloning of mTLR9,
mTLR8 and mTLR7 can be utilized in the same manner. Definition of
compounds that agonize or antagonize TLR9, 8, or 7 expression can
be used to enhance or dampen responses to nucleic acid ligands or
to any TLR9, 8 or 7 ligand defined by screening. These constructs
can be adapted to high throughput screening after stable
transfection similar to the use of TLR9 stable transfectants.
Each of the foregoing patents, patent applications and references
is hereby incorporated by reference. While the invention has been
described with respect to certain embodiments, it should be
appreciated that many modifications and changes may be made by
those of ordinary skill in the art without departing from the
spirit of the invention. It is intended that such modification,
changes and equivalents fall within the scope of the following
claims.
Example 22
Method Cloning the Murine and Human Extracellular TLR9 Domain Fused
to Human IgG1 Fc
Human IgG1 Fc was amplified from human B cell cDNA using the sense
and antisense primers 5' TATGGATCCTCTTGTGACAAAACTCACACATGC (SEQ ID
NO:216) and 5' ATA AAGCTTTCATTTACCCGGAGACAGGGAGAG (SEQ ID NO:217)
and ligated into pcDNA3.1(-) (Invitrogen) after digestion with the
restriction endonucleases BamHI and HindIII creating the vector
pcDNA-IgGFc. The extracellular domain of human TLR9 (amino acids 1
to 815) was amplified with the sense and antisense primers 5'
TATGAATTCCCACCATGGGTTTCTGCCGCAG (SEQ ID NO:218) and 5'
ATAGGATCCCCGGGGCACCAGGCCGCCGCCGCGGCCGCCGGAGAGGGCCTCAT CCAGGC (SEQ
ID NO:219). The primers amplify the extracellular domain of human
TLR9 and create adjacent to amino acid 815 an additional NotI
restriction site, a glycine linker and thrombin protease
recognition site. The translated sequence of this region starting
at amino acid 812 is DEALSGGRGGGLVPRGS (SEQ ID NO:220). The
fragment was cut with EcoRI and BamHI and cloned into pcDNA-IgGFc,
creating the vector coding for the fusion protein of the
extracellular domain of human TLR9 fused to the Fc part of human
IgG1 (pcDNAhTLR9IgGFc). Expressed extracellular TLR9 protein can be
separated from the IgG1 Fc fragment by digestion with Thrombin (see
figure).
The extracellular part of murine TLR9 (amino acids 1 to 816) was
amplified with the sense and antisense primers 5'
TATATGCGGCCGCCCACCATGGTTCTCCGTCGAAG (SEQ ID NO:221) and 5'
TATATGCGGCCGCCAGAGAGGACCTCATCCAGGC (SEQ ID NO:222) and cloned into
pcDNAhTLR9IgGFc after NotI digestion of PCR fragment and vector.
This procedure exchanged the human extracellular part of TLR9 with
the murine counterpart.
Example 23
Method of Expression and Purification of the Extracellular Domain
of TLR9 Fused to Human IgG1 Fc
Vector DNA coding for the human or murine TLR9 human IgGFc fusion
protein was transfected by Ca.sub.2 PO.sub.4 method into 293
fibroblast cells. Transfected cells were selected with 0.7 mg/ml
G418 and cloned. Expression of fusion protein was monitored by
enzyme-linked immunosorbent assay (ELISA). Cells were lysed in
lysis buffer (PBS, 1% Triton X-100) and supernatant was applied to
ELISA plates coated with polyclonal antibody against human IgG-Fc.
Bound fusion protein was detected by incubation with biotinylated
polyclonal antibodies against human IgG-Fc and
streptavidin-horseradish peroxidase conjugate.
For purification of the fusion protein cell lysates from 10.sup.9
cells were produced and incubated with Protein A sepharose which
binds tightly to human IgG-Fc. Incubation with the protease
thrombin releases the soluble extracellular domain of human TLR9.
FIG. 27 shows an example of the TLR9 fusion protein visualized by a
silver stained SDS-gel. FIG. 27 demonstrates that lysates of
transfected cells included a strong band travelling between 100 and
150 kD which was not present either in lysates of mock-transfected
cells or in supernatants transfected or mock-transfected cells. The
apparent molecular weight of the band decreased following thrombin
treatment, consistent with cleavage at the thrombin protease
recognition site interposed between the extracellular TLR9 domain
and the Fc fragment.
Example 24
Method of Cloning the Murine and Human Extracellular TLR7 and TLR8
Domain Fused to Human IgG1 Fc and Its Expression in 293 Cells
The extracellular domains of murine TLR7 (amino acids 1 to 837),
human TLR7 (amino acids 1 to 836), murine TLR8 (amino acids 1 to
816) and human TLR8 (amino acids 1 to 825) were amplified with the
primer pairs 5'TATATGCGGCCGCCCACCATGGTGTTTTCGATGTGGACACG (SEQ ID
NO:223) and 5' TATATGCGGCCGCCATCTAACTCACACGTATACAGATC (SEQ ID
NO:224); 5' TATATGCGGCCGCCCACCATGGTGTTTCCAATGTGGACACTG (SEQ ID
NO:225) and 5' TATATGCGGCCGCCATCTAACTCACAGGTGTACAGATC (SEQ ID
NO:226); 5'TATATGCGGCCGCCCACCATGGAAAACATGCCCCCTCAG (SEQ ID NO:227)
and 5' TATATGCGGCCGCCATCCGATACACAAGTCGTGAGATC (SEQ ID NO:228); and
5' TATATGCGGCCGCCCACCATGGAAAACATGTTCCTTCAGTC (SEQ ID NO:229) and 5'
TATATGCGGCCGCCATCTGAAACACAAGTTGTTAGCTC (SEQ ID NO:230),
respectively. Fragments were cloned into pcDNA-IgGFc after NotI
digestion.
Vector DNA coding for the extracellular domain of human or murine
TLR7 or TLR8 fused to human IgGFc fusion protein was transfected by
Ca.sub.2 PO.sub.4 method into 293 fibroblast cells. Transfected
cells were selected with 0.7 mg/ml G418 and cloned. Expression of
fusion protein was monitored by ELISA. Cells were lysed in lysis
buffer (PBS, 1% Triton X-100) and supernatant was applied to ELISA
plates coated with polyclonal antibody against human IgG-Fc. Bound
fusion protein was detected by incubation with biotinylated
polyclonal antibodies against human IgG-Fc and
Streptavidin-horseradish peroxidase conjugate.
Example 25
Method of Antibody Production Against Murine and Human TLR9 and
Characterization of Activity
C57/B6 mice were immunized three times by intraperitoneal
administration of 20 .mu.g of the extracellular domain of human
TLR9 mixed with 10 mmol of the CpG-ODN 1668. B cells taken from
immunized mice were fused with a non antibody producing B-cell
hybridoma P3XAG8 using standard protocols. Hybridoma supernatants
were screened for reactivity in ELISA using murine and human TLR9
fusion proteins. For identification of positive hybridomas ELISA
plates were coated with polyclonal antibody against human IgG-Fc
and incubated with lysate containing murine or human TLR9 IgG-Fc
fusion protein. Plates were then incubated with individual
hybridoma supernatants, and bound TLR9-specific antibodies were
detected by incubation with biotinylated polyclonal antibodies
against murine IgG and Streptavidin-horseradish peroxidase
conjugate.
Ten antibodies have been isolated which are of IgG1, IgG2a and
IgG2b isotype. They have been tested for reactivity against human
and murine TLR9 and their performance in western blotting or
intracellular staining. Table 18 shows the names (ID), isotypes,
reactivity and performance in western blotting and intracellular
staining.
All isolated antibodies were readily purified using standard
protein A affinity chromatography.
TABLE 18 Monclonal Antibodies Raised Against Murine and Human TLR9
Reactivity in ELISA Western Intracellular # ID Isotype mTLR9 hTLR9
Blotting Staining 1 1-3A11 G1 YES YES YES NO 2 1-1B1 G1 YES YES YES
NO 3 1-2A9 G2a NO YES YES YES 4 1-3F2 G1 YES YES YES NO 5 2-1E2 G2a
NO YES YES YES 6 1-5G5 G2a YES YES YES YES 7 1-2F1 G1 YES YES YES
NO 8 1-5F12 G2b NO YES NO NO 9 1-3C9 G2a NO YES YES YES 10 1-3F5
G2b NO YES NO NO
Example 26
Method for Intracellular Staining
Mock transfected 293 cells and human TLR9 transfected 293 cells
were seeded on cover slips and cultured overnight. The following
day cells were washed in PBS and fixed with 2% formalin for 10
minutes at room temperature. Cells were permeabilized with 0.2%
saponin in PBS and incubated with 2 .mu.g/ml anti human
TLR9-specific antibody 2-1E2 for 1 h. After two wash steps cells
were incubated with Alexis488-conjugated goat anti-mouse IgG
antibody and TLR9 was visualized utilizing confocal microscopy on a
Zeiss LSM510microscope. Results indicated that cytoplasms of human
TLR9 transfected 293 cells, but not mock transfected 293 cells,
stained positive for human TLR9.
Example 27
Method for Western Blotting
Lysates of 293 cells transfected with murine TLR9, human TLR9 or
murine TLR2IgG1-Fc fusion protein were separated by SDS-PAGE.
Proteins were transferred to a nylon membrane utilizing a BioRad
semi dry blotter according to the manufacturer's protocol. The
membrane was incubated with 2 .mu.g/ml of the human TLR9-specific
antibody 2-1E2, and human TLR9 was detected by polyclonal goat
anti-mouse peroxidase conjugate. Peroxidase activity was monitored
with ECL reagent (Amersham) and incubation of the membrane on film
(see FIG. 29).
SEQUENCE LISTING <100> GENERAL INFORMATION: <160>
NUMBER OF SEQ ID NOS: 230 <200> SEQUENCE CHARACTERISTICS:
<210> SEQ ID NO 1 <211> LENGTH: 3200 <212> TYPE:
DNA <213> ORGANISM: Mus musculus <220> FEATURE:
<221> NAME/KEY: misc_feature <222> LOCATION: (0)...(0)
<223> OTHER INFORMATION: Murine TLR9 cDNA <400>
SEQUENCE: 1 tgtcagaggg agcctcggga gaatcctcca tctcccaaca tggttctccg
tcgaaggact 60 ctgcacccct tgtccctcct ggtacaggct gcagtgctgg
ctgagactct ggccctgggt 120 accctgcctg ccttcctacc ctgtgagctg
aagcctcatg gcctggtgga ctgcaattgg 180 ctgttcctga agtctgtacc
ccgtttctct gcggcagcat cctgctccaa catcacccgc 240 ctctccttga
tctccaaccg tatccaccac ctgcacaact ccgacttcgt ccacctgtcc 300
aacctgcggc agctgaacct caagtggaac tgtccaccca ctggccttag ccccctgcac
360 ttctcttgcc acatgaccat tgagcccaga accttcctgg ctatgcgtac
actggaggag 420 ctgaacctga gctataatgg tatcaccact gtgccccgac
tgcccagctc cctggtgaat 480 ctgagcctga gccacaccaa catcctggtt
ctagatgcta acagcctcgc cggcctatac 540 agcctgcgcg ttctcttcat
ggacgggaac tgctactaca agaacccctg cacaggagcg 600 gtgaaggtga
ccccaggcgc cctcctgggc ctgagcaatc tcacccatct gtctctgaag 660
tataacaacc tcacaaaggt gccccgccaa ctgcccccca gcctggagta cctcctggtg
720 tcctataacc tcattgtcaa gctggggcct gaagacctgg ccaatctgac
ctcccttcga 780 gtacttgatg tgggtgggaa ttgccgtcgc tgcgaccatg
cccccaatcc ctgtatagaa 840 tgtggccaaa agtccctcca cctgcaccct
gagaccttcc atcacctgag ccatctggaa 900 ggcctggtgc tgaaggacag
ctctctccat acactgaact cttcctggtt ccaaggtctg 960 gtcaacctct
cggtgctgga cctaagcgag aactttctct atgaaagcat caaccacacc 020
aatgcctttc agaacctaac ccgcctgcgc aagctcaacc tgtccttcaa ttaccgcaag
080 aaggtatcct ttgcccgcct ccacctggca agttccttca agaacctggt
gtcactgcag 140 gagctgaaca tgaacggcat cttcttccgc tcgctcaaca
agtacacgct cagatggctg 200 gccgatctgc ccaaactcca cactctgcat
cttcaaatga acttcatcaa ccaggcacag 260 ctcagcatct ttggtacctt
ccgagccctt cgctttgtgg acttgtcaga caatcgcatc 320 agtgggcctt
caacgctgtc agaagccacc cctgaagagg cagatgatgc agagcaggag 380
gagctgttgt ctgcggatcc tcacccagct ccactgagca cccctgcttc taagaacttc
440 atggacaggt gtaagaactt caagttcacc atggacctgt ctcggaacaa
cctggtgact 500 atcaagccag agatgtttgt caatctctca cgcctccagt
gtcttagcct gagccacaac 560 tccattgcac aggctgtcaa tggctctcag
ttcctgccgc tgactaatct gcaggtgctg 620 gacctgtccc ataacaaact
ggacttgtac cactggaaat cgttcagtga gctaccacag 680 ttgcaggccc
tggacctgag ctacaacagc cagcccttta gcatgaaggg tataggccac 740
aatttcagtt ttgtggccca tctgtccatg ctacacagcc ttagcctggc acacaatgac
800 attcataccc gtgtgtcctc acatctcaac agcaactcag tgaggtttct
tgacttcagc 860 ggcaacggta tgggccgcat gtgggatgag gggggccttt
atctccattt cttccaaggc 920 ctgagtggcc tgctgaagct ggacctgtct
caaaataacc tgcatatcct ccggccccag 980 aaccttgaca acctccccaa
gagcctgaag ctgctgagcc tccgagacaa ctacctatct 2040 ttctttaact
ggaccagtct gtccttcctg cccaacctgg aagtcctaga cctggcaggc 2100
aaccagctaa aggccctgac caatggcacc ctgcctaatg gcaccctcct ccagaaactg
2160 gatgtcagca gcaacagtat cgtctctgtg gtcccagcct tcttcgctct
ggcggtcgag 2220 ctgaaagagg tcaacctcag ccacaacatt ctcaagacgg
tggatcgctc ctggtttggg 2280 cccattgtga tgaacctgac agttctagac
gtgagaagca accctctgca ctgtgcctgt 2340 ggggcagcct tcgtagactt
actgttggag gtgcagacca aggtgcctgg cctggctaat 2400 ggtgtgaagt
gtggcagccc cggccagctg cagggccgta gcatcttcgc acaggacctg 2460
cggctgtgcc tggatgaggt cctctcttgg gactgctttg gcctttcact cttggctgtg
2520 gccgtgggca tggtggtgcc tatactgcac catctctgcg gctgggacgt
ctggtactgt 2580 tttcatctgt gcctggcatg gctacctttg ctggcccgca
gccgacgcag cgcccaagct 2640 ctcccctatg atgccttcgt ggtgttcgat
aaggcacaga gcgcagttgc ggactgggtg 2700 tataacgagc tgcgggtgcg
gctggaggag cggcgcggtc gccgagccct acgcttgtgt 2760 ctggaggacc
gagattggct gcctggccag acgctcttcg agaacctctg ggcttccatc 2820
tatgggagcc gcaagactct atttgtgctg gcccacacgg accgcgtcag tggcctcctg
2880 cgcaccagct tcctgctggc tcagcagcgc ctgttggaag accgcaagga
cgtggtggtg 2940 ttggtgatcc tgcgtccgga tgcccaccgc tcccgctatg
tgcgactgcg ccagcgtctc 3000 tgccgccaga gtgtgctctt ctggccccag
cagcccaacg ggcagggggg cttctgggcc 3060 cagctgagta cagccctgac
tagggacaac cgccacttct ataaccagaa cttctgccgg 3120 ggacctacag
cagaatagct cagagcaaca gctggaaaca gctgcatctt catgcctggt 3180
tcccgagttg ctctgcctgc 3200 <200> SEQUENCE CHARACTERISTICS:
<210> SEQ ID NO 2 <211> LENGTH: 3096 <212> TYPE:
DNA <213> ORGANISM: Mus musculus <220> FEATURE:
<221> NAME/KEY: misc_feature <222> LOCATION: (0)...(0)
<223> OTHER INFORMATION: Murine TLR9 ORF <400>
SEQUENCE: 2 atggttctcc gtcgaaggac tctgcacccc ttgtccctcc tggtacaggc
tgcagtgctg 60 gctgagactc tggccctggg taccctgcct gccttcctac
cctgtgagct gaagcctcat 120 ggcctggtgg actgcaattg gctgttcctg
aagtctgtac cccgtttctc tgcggcagca 180 tcctgctcca acatcacccg
cctctccttg atctccaacc gtatccacca cctgcacaac 240 tccgacttcg
tccacctgtc caacctgcgg cagctgaacc tcaagtggaa ctgtccaccc 300
actggcctta gccccctgca cttctcttgc cacatgacca ttgagcccag aaccttcctg
360 gctatgcgta cactggagga gctgaacctg agctataatg gtatcaccac
tgtgccccga 420 ctgcccagct ccctggtgaa tctgagcctg agccacacca
acatcctggt tctagatgct 480 aacagcctcg ccggcctata cagcctgcgc
gttctcttca tggacgggaa ctgctactac 540 aagaacccct gcacaggagc
ggtgaaggtg accccaggcg ccctcctggg cctgagcaat 600 ctcacccatc
tgtctctgaa gtataacaac ctcacaaagg tgccccgcca actgcccccc 660
agcctggagt acctcctggt gtcctataac ctcattgtca agctggggcc tgaagacctg
720 gccaatctga cctcccttcg agtacttgat gtgggtggga attgccgtcg
ctgcgaccat 780 gcccccaatc cctgtataga atgtggccaa aagtccctcc
acctgcaccc tgagaccttc 840 catcacctga gccatctgga aggcctggtg
ctgaaggaca gctctctcca tacactgaac 900 tcttcctggt tccaaggtct
ggtcaacctc tcggtgctgg acctaagcga gaactttctc 960 tatgaaagca
tcaaccacac caatgccttt cagaacctaa cccgcctgcg caagctcaac 1020
ctgtccttca attaccgcaa gaaggtatcc tttgcccgcc tccacctggc aagttccttc
1080 aagaacctgg tgtcactgca ggagctgaac atgaacggca tcttcttccg
ctcgctcaac 1140 aagtacacgc tcagatggct ggccgatctg cccaaactcc
acactctgca tcttcaaatg 1200 aacttcatca accaggcaca gctcagcatc
tttggtacct tccgagccct tcgctttgtg 1260 gacttgtcag acaatcgcat
cagtgggcct tcaacgctgt cagaagccac ccctgaagag 1320 gcagatgatg
cagagcagga ggagctgttg tctgcggatc ctcacccagc tccactgagc 1380
acccctgctt ctaagaactt catggacagg tgtaagaact tcaagttcac catggacctg
1440 tctcggaaca acctggtgac tatcaagcca gagatgtttg tcaatctctc
acgcctccag 1500 tgtcttagcc tgagccacaa ctccattgca caggctgtca
atggctctca gttcctgccg 1560 ctgactaatc tgcaggtgct ggacctgtcc
cataacaaac tggacttgta ccactggaaa 1620 tcgttcagtg agctaccaca
gttgcaggcc ctggacctga gctacaacag ccagcccttt 1680 agcatgaagg
gtataggcca caatttcagt tttgtggccc atctgtccat gctacacagc 1740
cttagcctgg cacacaatga cattcatacc cgtgtgtcct cacatctcaa cagcaactca
1800 gtgaggtttc ttgacttcag cggcaacggt atgggccgca tgtgggatga
ggggggcctt 1860 tatctccatt tcttccaagg cctgagtggc ctgctgaagc
tggacctgtc tcaaaataac 1920 ctgcatatcc tccggcccca gaaccttgac
aacctcccca agagcctgaa gctgctgagc 1980 ctccgagaca actacctatc
tttctttaac tggaccagtc tgtccttcct gcccaacctg 2040 gaagtcctag
acctggcagg caaccagcta aaggccctga ccaatggcac cctgcctaat 2100
ggcaccctcc tccagaaact ggatgtcagc agcaacagta tcgtctctgt ggtcccagcc
2160 ttcttcgctc tggcggtcga gctgaaagag gtcaacctca gccacaacat
tctcaagacg 2220 gtggatcgct cctggtttgg gcccattgtg atgaacctga
cagttctaga cgtgagaagc 2280 aaccctctgc actgtgcctg tggggcagcc
ttcgtagact tactgttgga ggtgcagacc 2340 aaggtgcctg gcctggctaa
tggtgtgaag tgtggcagcc ccggccagct gcagggccgt 2400 agcatcttcg
cacaggacct gcggctgtgc ctggatgagg tcctctcttg ggactgcttt 2460
ggcctttcac tcttggctgt ggccgtgggc atggtggtgc ctatactgca ccatctctgc
2520 ggctgggacg tctggtactg ttttcatctg tgcctggcat ggctaccttt
gctggcccgc 2580 agccgacgca gcgcccaagc tctcccctat gatgccttcg
tggtgttcga taaggcacag 2640 agcgcagttg cggactgggt gtataacgag
ctgcgggtgc ggctggagga gcggcgcggt 2700 cgccgagccc tacgcttgtg
tctggaggac cgagattggc tgcctggcca gacgctcttc 2760 gagaacctct
gggcttccat ctatgggagc cgcaagactc tatttgtgct ggcccacacg 2820
gaccgcgtca gtggcctcct gcgcaccagc ttcctgctgg ctcagcagcg cctgttggaa
2880 gaccgcaagg acgtggtggt gttggtgatc ctgcgtccgg atgcccaccg
ctcccgctat 2940 gtgcgactgc gccagcgtct ctgccgccag agtgtgctct
tctggcccca gcagcccaac 3000 gggcaggggg gcttctgggc ccagctgagt
acagccctga ctagggacaa ccgccacttc 3060 tataaccaga acttctgccg
gggacctaca gcagaa 3096 <200> SEQUENCE CHARACTERISTICS:
<210> SEQ ID NO 3 <211> LENGTH: 1032 <212> TYPE:
PRT <213> ORGANISM: Mus musculus <400> SEQUENCE: 3 Met
Val Leu Arg Arg Arg Thr Leu His Pro Leu Ser Leu Leu Val Gln 1 5 10
15 Ala Ala Val Leu Ala Glu Thr Leu Ala Leu Gly Thr Leu Pro Ala Phe
20 25 30 Leu Pro Cys Glu Leu Lys Pro His Gly Leu Val Asp Cys Asn
Trp Leu 35 40 45 Phe Leu Lys Ser Val Pro Arg Phe Ser Ala Ala Ala
Ser Cys Ser Asn 50 55 60 Ile Thr Arg Leu Ser Leu Ile Ser Asn Arg
Ile His His Leu His Asn 65 70 75 80 Ser Asp Phe Val His Leu Ser Asn
Leu Arg Gln Leu Asn Leu Lys Trp 85 90 95 Asn Cys Pro Pro Thr Gly
Leu Ser Pro Leu His Phe Ser Cys His Met 100 105 110 Thr Ile Glu Pro
Arg Thr Phe Leu Ala Met Arg Thr Leu Glu Glu Leu 115 120 125 Asn Leu
Ser Tyr Asn Gly Ile Thr Thr Val Pro Arg Leu Pro Ser Ser 130 135 140
Leu Val Asn Leu Ser Leu Ser His Thr Asn Ile Leu Val Leu Asp Ala 145
150 155 160 Asn Ser Leu Ala Gly Leu Tyr Ser Leu Arg Val Leu Phe Met
Asp Gly 165 170 175 Asn Cys Tyr Tyr Lys Asn Pro Cys Thr Gly Ala Val
Lys Val Thr Pro 180 185 190 Gly Ala Leu Leu Gly Leu Ser Asn Leu Thr
His Leu Ser Leu Lys Tyr 195 200 205 Asn Asn Leu Thr Lys Val Pro Arg
Gln Leu Pro Pro Ser Leu Glu Tyr 210 215 220 Leu Leu Val Ser Tyr Asn
Leu Ile Val Lys Leu Gly Pro Glu Asp Leu 225 230 235 240 Ala Asn Leu
Thr Ser Leu Arg Val Leu Asp Val Gly Gly Asn Cys Arg 245 250 255 Arg
Cys Asp His Ala Pro Asn Pro Cys Ile Glu Cys Gly Gln Lys Ser 260 265
270 Leu His Leu His Pro Glu Thr Phe His His Leu Ser His Leu Glu Gly
275 280 285 Leu Val Leu Lys Asp Ser Ser Leu His Thr Leu Asn Ser Ser
Trp Phe 290 295 300 Gln Gly Leu Val Asn Leu Ser Val Leu Asp Leu Ser
Glu Asn Phe Leu 305 310 315 320 Tyr Glu Ser Ile Asn His Thr Asn Ala
Phe Gln Asn Leu Thr Arg Leu 325 330 335 Arg Lys Leu Asn Leu Ser Phe
Asn Tyr Arg Lys Lys Val Ser Phe Ala 340 345 350 Arg Leu His Leu Ala
Ser Ser Phe Lys Asn Leu Val Ser Leu Gln Glu 355 360 365 Leu Asn Met
Asn Gly Ile Phe Phe Arg Ser Leu Asn Lys Tyr Thr Leu 370 375 380 Arg
Trp Leu Ala Asp Leu Pro Lys Leu His Thr Leu His Leu Gln Met 385 390
395 400 Asn Phe Ile Asn Gln Ala Gln Leu Ser Ile Phe Gly Thr Phe Arg
Ala 405 410 415 Leu Arg Phe Val Asp Leu Ser Asp Asn Arg Ile Ser Gly
Pro Ser Thr 420 425 430 Leu Ser Glu Ala Thr Pro Glu Glu Ala Asp Asp
Ala Glu Gln Glu Glu 435 440 445 Leu Leu Ser Ala Asp Pro His Pro Ala
Pro Leu Ser Thr Pro Ala Ser 450 455 460 Lys Asn Phe Met Asp Arg Cys
Lys Asn Phe Lys Phe Thr Met Asp Leu 465 470 475 480 Ser Arg Asn Asn
Leu Val Thr Ile Lys Pro Glu Met Phe Val Asn Leu 485 490 495 Ser Arg
Leu Gln Cys Leu Ser Leu Ser His Asn Ser Ile Ala Gln Ala 500 505 510
Val Asn Gly Ser Gln Phe Leu Pro Leu Thr Asn Leu Gln Val Leu Asp 515
520 525 Leu Ser His Asn Lys Leu Asp Leu Tyr His Trp Lys Ser Phe Ser
Glu 530 535 540 Leu Pro Gln Leu Gln Ala Leu Asp Leu Ser Tyr Asn Ser
Gln Pro Phe 545 550 555 560 Ser Met Lys Gly Ile Gly His Asn Phe Ser
Phe Val Ala His Leu Ser 565 570 575 Met Leu His Ser Leu Ser Leu Ala
His Asn Asp Ile His Thr Arg Val 580 585 590 Ser Ser His Leu Asn Ser
Asn Ser Val Arg Phe Leu Asp Phe Ser Gly 595 600 605 Asn Gly Met Gly
Arg Met Trp Asp Glu Gly Gly Leu Tyr Leu His Phe 610 615 620 Phe Gln
Gly Leu Ser Gly Leu Leu Lys Leu Asp Leu Ser Gln Asn Asn 625 630 635
640 Leu His Ile Leu Arg Pro Gln Asn Leu Asp Asn Leu Pro Lys Ser Leu
645 650 655 Lys Leu Leu Ser Leu Arg Asp Asn Tyr Leu Ser Phe Phe Asn
Trp Thr 660 665 670 Ser Leu Ser Phe Leu Pro Asn Leu Glu Val Leu Asp
Leu Ala Gly Asn 675 680 685 Gln Leu Lys Ala Leu Thr Asn Gly Thr Leu
Pro Asn Gly Thr Leu Leu 690 695 700 Gln Lys Leu Asp Val Ser Ser Asn
Ser Ile Val Ser Val Val Pro Ala 705 710 715 720 Phe Phe Ala Leu Ala
Val Glu Leu Lys Glu Val Asn Leu Ser His Asn 725 730 735 Ile Leu Lys
Thr Val Asp Arg Ser Trp Phe Gly Pro Ile Val Met Asn 740 745 750 Leu
Thr Val Leu Asp Val Arg Ser Asn Pro Leu His Cys Ala Cys Gly 755 760
765 Ala Ala Phe Val Asp Leu Leu Leu Glu Val Gln Thr Lys Val Pro Gly
770 775 780 Leu Ala Asn Gly Val Lys Cys Gly Ser Pro Gly Gln Leu Gln
Gly Arg 785 790 795 800 Ser Ile Phe Ala Gln Asp Leu Arg Leu Cys Leu
Asp Glu Val Leu Ser 805 810 815 Trp Asp Cys Phe Gly Leu Ser Leu Leu
Ala Val Ala Val Gly Met Val 820 825 830 Val Pro Ile Leu His His Leu
Cys Gly Trp Asp Val Trp Tyr Cys Phe 835 840 845 His Leu Cys Leu Ala
Trp Leu Pro Leu Leu Ala Arg Ser Arg Arg Ser 850 855 860 Ala Gln Ala
Leu Pro Tyr Asp Ala Phe Val Val Phe Asp Lys Ala Gln 865 870 875 880
Ser Ala Val Ala Asp Trp Val Tyr Asn Glu Leu Arg Val Arg Leu Glu 885
890 895 Glu Arg Arg Gly Arg Arg Ala Leu Arg Leu Cys Leu Glu Asp Arg
Asp 900 905 910 Trp Leu Pro Gly Gln Thr Leu Phe Glu Asn Leu Trp Ala
Ser Ile Tyr 915 920 925
Gly Ser Arg Lys Thr Leu Phe Val Leu Ala His Thr Asp Arg Val Ser 930
935 940 Gly Leu Leu Arg Thr Ser Phe Leu Leu Ala Gln Gln Arg Leu Leu
Glu 945 950 955 960 Asp Arg Lys Asp Val Val Val Leu Val Ile Leu Arg
Pro Asp Ala His 965 970 975 Arg Ser Arg Tyr Val Arg Leu Arg Gln Arg
Leu Cys Arg Gln Ser Val 980 985 990 Leu Phe Trp Pro Gln Gln Pro Asn
Gly Gln Gly Gly Phe Trp Ala Gln 995 1000 1005 Leu Ser Thr Ala Leu
Thr Arg Asp Asn Arg His Phe Tyr Asn Gln Asn 1010 1015 1020 Phe Cys
Arg Gly Pro Thr Ala Glu 1025 1030 <200> SEQUENCE
CHARACTERISTICS: <210> SEQ ID NO 4 <211> LENGTH: 3352
<212> TYPE: DNA <213> ORGANISM: Homo sapiens
<220> FEATURE: <221> NAME/KEY: misc_feature <222>
LOCATION: (0)...(0) <223> OTHER INFORMATION: Human TLR9 per
GenBank AF245704 <400> SEQUENCE: 4 aggctggtat aaaaatctta
cttcctctat tctctgagcc gctgctgccc ctgtgggaag 60 ggacctcgag
tgtgaagcat ccttccctgt agctgctgtc cagtctgccc gccagaccct 120
ctggagaagc ccctgccccc cagcatgggt ttctgccgca gcgccctgca cccgctgtct
180 ctcctggtgc aggccatcat gctggccatg accctggccc tgggtacctt
gcctgccttc 240 ctaccctgtg agctccagcc ccacggcctg gtgaactgca
actggctgtt cctgaagtct 300 gtgccccact tctccatggc agcaccccgt
ggcaatgtca ccagcctttc cttgtcctcc 360 aaccgcatcc accacctcca
tgattctgac tttgcccacc tgcccagcct gcggcatctc 420 aacctcaagt
ggaactgccc gccggttggc ctcagcccca tgcacttccc ctgccacatg 480
accatcgagc ccagcacctt cttggctgtg cccaccctgg aagagctaaa cctgagctac
540 aacaacatca tgactgtgcc tgcgctgccc aaatccctca tatccctgtc
cctcagccat 600 accaacatcc tgatgctaga ctctgccagc ctcgccggcc
tgcatgccct gcgcttccta 660 ttcatggacg gcaactgtta ttacaagaac
ccctgcaggc aggcactgga ggtggccccg 720 ggtgccctcc ttggcctggg
caacctcacc cacctgtcac tcaagtacaa caacctcact 780 gtggtgcccc
gcaacctgcc ttccagcctg gagtatctgc tgttgtccta caaccgcatc 840
gtcaaactgg cgcctgagga cctggccaat ctgaccgccc tgcgtgtgct cgatgtgggc
900 ggaaattgcc gccgctgcga ccacgctccc aacccctgca tggagtgccc
tcgtcacttc 960 ccccagctac atcccgatac cttcagccac ctgagccgtc
ttgaaggcct ggtgttgaag 1020 gacagttctc tctcctggct gaatgccagt
tggttccgtg ggctgggaaa cctccgagtg 1080 ctggacctga gtgagaactt
cctctacaaa tgcatcacta aaaccaaggc cttccagggc 1140 ctaacacagc
tgcgcaagct taacctgtcc ttcaattacc aaaagagggt gtcctttgcc 1200
cacctgtctc tggccccttc cttcgggagc ctggtcgccc tgaaggagct ggacatgcac
1260 ggcatcttct tccgctcact cgatgagacc acgctccggc cactggcccg
cctgcccatg 1320 ctccagactc tgcgtctgca gatgaacttc atcaaccagg
cccagctcgg catcttcagg 1380 gccttccctg gcctgcgcta cgtggacctg
tcggacaacc gcatcagcgg agcttcggag 1440 ctgacagcca ccatggggga
ggcagatgga ggggagaagg tctggctgca gcctggggac 1500 cttgctccgg
ccccagtgga cactcccagc tctgaagact tcaggcccaa ctgcagcacc 1560
ctcaacttca ccttggatct gtcacggaac aacctggtga ccgtgcagcc ggagatgttt
1620 gcccagctct cgcacctgca gtgcctgcgc ctgagccaca actgcatctc
gcaggcagtc 1680 aatggctccc agttcctgcc gctgaccggt ctgcaggtgc
tagacctgtc ccgcaataag 1740 ctggacctct accacgagca ctcattcacg
gagctaccgc gactggaggc cctggacctc 1800 agctacaaca gccagccctt
tggcatgcag ggcgtgggcc acaacttcag cttcgtggct 1860 cacctgcgca
ccctgcgcca cctcagcctg gcccacaaca acatccacag ccaagtgtcc 1920
cagcagctct gcagtacgtc gctgcgggcc ctggacttca gcggcaatgc actgggccat
1980 atgtgggccg agggagacct ctatctgcac ttcttccaag gcctgagcgg
tttgatctgg 2040 ctggacttgt cccagaaccg cctgcacacc ctcctgcccc
aaaccctgcg caacctcccc 2100 aagagcctac aggtgctgcg tctccgtgac
aattacctgg ccttctttaa gtggtggagc 2160 ctccacttcc tgcccaaact
ggaagtcctc gacctggcag gaaaccggct gaaggccctg 2220 accaatggca
gcctgcctgc tggcacccgg ctccggaggc tggatgtcag ctgcaacagc 2280
atcagcttcg tggcccccgg cttcttttcc aaggccaagg agctgcgaga gctcaacctt
2340 agcgccaacg ccctcaagac agtggaccac tcctggtttg ggcccctggc
gagtgccctg 2400 caaatactag atgtaagcgc caaccctctg cactgcgcct
gtggggcggc ctttatggac 2460 ttcctgctgg aggtgcaggc tgccgtgccc
ggtctgccca gccgggtgaa gtgtggcagt 2520 ccgggccagc tccagggcct
cagcatcttt gcacaggacc tgcgcctctg cctggatgag 2580 gccctctcct
gggactgttt cgccctctcg ctgctggctg tggctctggg cctgggtgtg 2640
cccatgctgc atcacctctg tggctgggac ctctggtact gcttccacct gtgcctggcc
2700 tggcttccct ggcgggggcg gcaaagtggg cgagatgagg atgccctgcc
ctacgatgcc 2760 ttcgtggtct tcgacaaaac gcagagcgca gtggcagact
gggtgtacaa cgagcttcgg 2820 gggcagctgg aggagtgccg tgggcgctgg
gcactccgcc tgtgcctgga ggaacgcgac 2880 tggctgcctg gcaaaaccct
ctttgagaac ctgtgggcct cggtctatgg cagccgcaag 2940 acgctgtttg
tgctggccca cacggaccgg gtcagtggtc tcttgcgcgc cagcttcctg 3000
ctggcccagc agcgcctgct ggaggaccgc aaggacgtcg tggtgctggt gatcctgagc
3060 cctgacggcc gccgctcccg ctacgtgcgg ctgcgccagc gcctctgccg
ccagagtgtc 3120 ctcctctggc cccaccagcc cagtggtcag cgcagcttct
gggcccagct gggcatggcc 3180 ctgaccaggg acaaccacca cttctataac
cggaacttct gccagggacc cacggccgaa 3240 tagccgtgag ccggaatcct
gcacggtgcc acctccacac tcacctcacc tctgcctgcc 3300 tggtctgacc
ctcccctgct cgcctccctc accccacacc tgacacagag ca 3352 <200>
SEQUENCE CHARACTERISTICS: <210> SEQ ID NO 5 <211>
LENGTH: 3868 <212> TYPE: DNA <213> ORGANISM: Homo
sapiens <220> FEATURE: <221> NAME/KEY: misc_feature
<222> LOCATION: (0)...(0) <223> OTHER INFORMATION:
Human TLR9 per GenBank NM_017442 <400> SEQUENCE: 5 ggaggtcttg
tttccggaag atgttgcaag gctgtggtga aggcaggtgc agcctagcct 60
cctgctcaag ctacaccctg gccctccacg catgaggccc tgcagaactc tggagatggt
120 gcctacaagg gcagaaaagg acaagtcggc agccgctgtc ctgagggcac
cagctgtggt 180 gcaggagcca agacctgagg gtggaagtgt cctcttagaa
tggggagtgc ccagcaaggt 240 gtacccgcta ctggtgctat ccagaattcc
catctctccc tgctctctgc ctgagctctg 300 ggccttagct cctccctggg
cttggtagag gacaggtgtg aggccctcat gggatgtagg 360 ctgtctgaga
ggggagtgga aagaggaagg ggtgaaggag ctgtctgcca tttgactatg 420
caaatggcct ttgactcatg ggaccctgtc ctcctcactg ggggcagggt ggagtggagg
480 gggagctact aggctggtat aaaaatctta cttcctctat tctctgagcc
gctgctgccc 540 ctgtgggaag ggacctcgag tgtgaagcat ccttccctgt
agctgctgtc cagtctgccc 600 gccagaccct ctggagaagc ccctgccccc
cagcatgggt ttctgccgca gcgccctgca 660 cccgctgtct ctcctggtgc
aggccatcat gctggccatg accctggccc tgggtacctt 720 gcctgccttc
ctaccctgtg agctccagcc ccacggcctg gtgaactgca actggctgtt 780
cctgaagtct gtgccccact tctccatggc agcaccccgt ggcaatgtca ccagcctttc
840 cttgtcctcc aaccgcatcc accacctcca tgattctgac tttgcccacc
tgcccagcct 900 gcggcatctc aacctcaagt ggaactgccc gccggttggc
ctcagcccca tgcacttccc 960 ctgccacatg accatcgagc ccagcacctt
cttggctgtg cccaccctgg aagagctaaa 1020 cctgagctac aacaacatca
tgactgtgcc tgcgctgccc aaatccctca tatccctgtc 1080 cctcagccat
accaacatcc tgatgctaga ctctgccagc ctcgccggcc tgcatgccct 1140
gcgcttccta ttcatggacg gcaactgtta ttacaagaac ccctgcaggc aggcactgga
1200 ggtggccccg ggtgccctcc ttggcctggg caacctcacc cacctgtcac
tcaagtacaa 1260 caacctcact gtggtgcccc gcaacctgcc ttccagcctg
gagtatctgc tgttgtccta 1320 caaccgcatc gtcaaactgg cgcctgagga
cctggccaat ctgaccgccc tgcgtgtgct 1380 cgatgtgggc ggaaattgcc
gccgctgcga ccacgctccc aacccctgca tggagtgccc 1440 tcgtcacttc
ccccagctac atcccgatac cttcagccac ctgagccgtc ttgaaggcct 1500
ggtgttgaag gacagttctc tctcctggct gaatgccagt tggttccgtg ggctgggaaa
1560 cctccgagtg ctggacctga gtgagaactt cctctacaaa tgcatcacta
aaaccaaggc 1620 cttccagggc ctaacacagc tgcgcaagct taacctgtcc
ttcaattacc aaaagagggt 1680 gtcctttgcc cacctgtctc tggccccttc
cttcgggagc ctggtcgccc tgaaggagct 1740 ggacatgcac ggcatcttct
tccgctcact cgatgagacc acgctccggc cactggcccg 1800 cctgcccatg
ctccagactc tgcgtctgca gatgaacttc atcaaccagg cccagctcgg 1860
catcttcagg gccttccctg gcctgcgcta cgtggacctg tcggacaacc gcatcagcgg
1920 agcttcggag ctgacagcca ccatggggga ggcagatgga ggggagaagg
tctggctgca 1980 gcctggggac cttgctccgg ccccagtgga cactcccagc
tctgaagact tcaggcccaa 2040 ctgcagcacc ctcaacttca ccttggatct
gtcacggaac aacctggtga ccgtgcagcc 2100 ggagatgttt gcccagctct
cgcacctgca gtgcctgcgc ctgagccaca actgcatctc 2160 gcaggcagtc
aatggctccc agttcctgcc gctgaccggt ctgcaggtgc tagacctgtc 2220
ccacaataag ctggacctct accacgagca ctcattcacg gagctaccac gactggaggc
2280 cctggacctc agctacaaca gccagccctt tggcatgcag ggcgtgggcc
acaacttcag 2340 cttcgtggct cacctgcgca ccctgcgcca cctcagcctg
gcccacaaca acatccacag 2400 ccaagtgtcc cagcagctct gcagtacgtc
gctgcgggcc ctggacttca gcggcaatgc 2460 actgggccat atgtgggccg
agggagacct ctatctgcac ttcttccaag gcctgagcgg 2520 tttgatctgg
ctggacttgt cccagaaccg cctgcacacc ctcctgcccc aaaccctgcg 2580
caacctcccc aagagcctac aggtgctgcg tctccgtgac aattacctgg ccttctttaa
2640 gtggtggagc ctccacttcc tgcccaaact ggaagtcctc gacctggcag
gaaaccagct 2700 gaaggccctg accaatggca gcctgcctgc tggcacccgg
ctccggaggc tggatgtcag 2760 ctgcaacagc atcagcttcg tggcccccgg
cttcttttcc aaggccaagg agctgcgaga 2820 gctcaacctt agcgccaacg
ccctcaagac agtggaccac tcctggtttg ggcccctggc 2880 gagtgccctg
caaatactag atgtaagcgc caaccctctg cactgcgcct gtggggcggc 2940
ctttatggac ttcctgctgg aggtgcaggc tgccgtgccc ggtctgccca gccgggtgaa
3000 gtgtggcagt ccgggccagc tccagggcct cagcatcttt gcacaggacc
tgcgcctctg 3060 cctggatgag gccctctcct gggactgttt cgccctctcg
ctgctggctg tggctctggg 3120 cctgggtgtg cccatgctgc atcacctctg
tggctgggac ctctggtact gcttccacct 3180 gtgcctggcc tggcttccct
ggcgggggcg gcaaagtggg cgagatgagg atgccctgcc 3240 ctacgatgcc
ttcgtggtct tcgacaaaac gcagagcgca gtggcagact gggtgtacaa 3300
cgagcttcgg gggcagctgg aggagtgccg tgggcgctgg gcactccgcc tgtgcctgga
3360 ggaacgcgac tggctgcctg gcaaaaccct ctttgagaac ctgtgggcct
cggtctatgg 3420 cagccgcaag acgctgtttg tgctggccca cacggaccgg
gtcagtggtc tcttgcgcgc 3480 cagcttcctg ctggcccagc agcgcctgct
ggaggaccgc aaggacgtcg tggtgctggt 3540 gatcctgagc cctgacggcc
gccgctcccg ctacgtgcgg ctgcgccagc gcctctgccg 3600 ccagagtgtc
ctcctctggc cccaccagcc cagtggtcag cgcagcttct gggcccagct 3660
gggcatggcc ctgaccaggg acaaccacca cttctataac cggaacttct gccagggacc
3720 cacggccgaa tagccgtgag ccggaatcct gcacggtgcc acctccacac
tcacctcacc 3780 tctgcctgcc tggtctgacc ctcccctgct cgcctccctc
accccacacc tgacacagag 3840 caggcactca ataaatgcta ccgaaggc 3868
<200> SEQUENCE CHARACTERISTICS: <210> SEQ ID NO 6
<211> LENGTH: 1032 <212> TYPE: PRT <213>
ORGANISM: Homo sapiens <400> SEQUENCE: 6 Met Gly Phe Cys Arg
Ser Ala Leu His Pro Leu Ser Leu Leu Val Gln 1 5 10 15 Ala Ile Met
Leu Ala Met Thr Leu Ala Leu Gly Thr Leu Pro Ala Phe 20 25 30 Leu
Pro Cys Glu Leu Gln Pro His Gly Leu Val Asn Cys Asn Trp Leu 35 40
45 Phe Leu Lys Ser Val Pro His Phe Ser Met Ala Ala Pro Arg Gly Asn
50 55 60 Val Thr Ser Leu Ser Leu Ser Ser Asn Arg Ile His His Leu
His Asp 65 70 75 80 Ser Asp Phe Ala His Leu Pro Ser Leu Arg His Leu
Asn Leu Lys Trp 85 90 95 Asn Cys Pro Pro Val Gly Leu Ser Pro Met
His Phe Pro Cys His Met 100 105 110 Thr Ile Glu Pro Ser Thr Phe Leu
Ala Val Pro Thr Leu Glu Glu Leu 115 120 125 Asn Leu Ser Tyr Asn Asn
Ile Met Thr Val Pro Ala Leu Pro Lys Ser 130 135 140 Leu Ile Ser Leu
Ser Leu Ser His Thr Asn Ile Leu Met Leu Asp Ser 145 150 155 160 Ala
Ser Leu Ala Gly Leu His Ala Leu Arg Phe Leu Phe Met Asp Gly 165 170
175 Asn Cys Tyr Tyr Lys Asn Pro Cys Arg Gln Ala Leu Glu Val Ala Pro
180 185 190 Gly Ala Leu Leu Gly Leu Gly Asn Leu Thr His Leu Ser Leu
Lys Tyr 195 200 205 Asn Asn Leu Thr Val Val Pro Arg Asn Leu Pro Ser
Ser Leu Glu Tyr 210 215 220 Leu Leu Leu Ser Tyr Asn Arg Ile Val Lys
Leu Ala Pro Glu Asp Leu 225 230 235 240 Ala Asn Leu Thr Ala Leu Arg
Val Leu Asp Val Gly Gly Asn Cys Arg 245 250 255 Arg Cys Asp His Ala
Pro Asn Pro Cys Met Glu Cys Pro Arg His Phe 260 265 270 Pro Gln Leu
His Pro Asp Thr Phe Ser His Leu Ser Arg Leu Glu Gly 275 280 285 Leu
Val Leu Lys Asp Ser Ser Leu Ser Trp Leu Asn Ala Ser Trp Phe 290 295
300 Arg Gly Leu Gly Asn Leu Arg Val Leu Asp Leu Ser Glu Asn Phe Leu
305 310 315 320 Tyr Lys Cys Ile Thr Lys Thr Lys Ala Phe Gln Gly Leu
Thr Gln Leu 325 330 335 Arg Lys Leu Asn Leu Ser Phe Asn Tyr Gln Lys
Arg Val Ser Phe Ala 340 345 350 His Leu Ser Leu Ala Pro Ser Phe Gly
Ser Leu Val Ala Leu Lys Glu 355 360 365 Leu Asp Met His Gly Ile Phe
Phe Arg Ser Leu Asp Glu Thr Thr Leu 370 375 380 Arg Pro Leu Ala Arg
Leu Pro Met Leu Gln Thr Leu Arg Leu Gln Met 385 390 395 400 Asn Phe
Ile Asn Gln Ala Gln Leu Gly Ile Phe Arg Ala Phe Pro Gly 405 410 415
Leu Arg Tyr Val Asp Leu Ser Asp Asn Arg Ile Ser Gly Ala Ser Glu 420
425 430 Leu Thr Ala Thr Met Gly Glu Ala Asp Gly Gly Glu Lys Val Trp
Leu 435 440 445 Gln Pro Gly Asp Leu Ala Pro Ala Pro Val Asp Thr Pro
Ser Ser Glu 450 455 460 Asp Phe Arg Pro Asn Cys Ser Thr Leu Asn Phe
Thr Leu Asp Leu Ser 465 470 475 480 Arg Asn Asn Leu Val Thr Val Gln
Pro Glu Met Phe Ala Gln Leu Ser 485 490 495 His Leu Gln Cys Leu Arg
Leu Ser His Asn Cys Ile Ser Gln Ala Val 500 505 510 Asn Gly Ser Gln
Phe Leu Pro Leu Thr Gly Leu Gln Val Leu Asp Leu 515 520 525 Ser Arg
Asn Lys Leu Asp Leu Tyr His Glu His Ser Phe Thr Glu Leu 530 535 540
Pro Arg Leu Glu Ala Leu Asp Leu Ser Tyr Asn Ser Gln Pro Phe Gly 545
550 555 560 Met Gln Gly Val Gly His Asn Phe Ser Phe Val Ala His Leu
Arg Thr 565 570 575 Leu Arg His Leu Ser Leu Ala His Asn Asn Ile His
Ser Gln Val Ser 580 585 590 Gln Gln Leu Cys Ser Thr Ser Leu Arg Ala
Leu Asp Phe Ser Gly Asn 595 600 605 Ala Leu Gly His Met Trp Ala Glu
Gly Asp Leu Tyr Leu His Phe Phe 610 615 620 Gln Gly Leu Ser Gly Leu
Ile Trp Leu Asp Leu Ser Gln Asn Arg Leu 625 630 635 640 His Thr Leu
Leu Pro Gln Thr Leu Arg Asn Leu Pro Lys Ser Leu Gln 645 650 655 Val
Leu Arg Leu Arg Asp Asn Tyr Leu Ala Phe Phe Lys Trp Trp Ser 660 665
670 Leu His Phe Leu Pro Lys Leu Glu Val Leu Asp Leu Ala Gly Asn Arg
675 680 685 Leu Lys Ala Leu Thr Asn Gly Ser Leu Pro Ala Gly Thr Arg
Leu Arg 690 695 700 Arg Leu Asp Val Ser Cys Asn Ser Ile Ser Phe Val
Ala Pro Gly Phe 705 710 715 720
Phe Ser Lys Ala Lys Glu Leu Arg Glu Leu Asn Leu Ser Ala Asn Ala 725
730 735 Leu Lys Thr Val Asp His Ser Trp Phe Gly Pro Leu Ala Ser Ala
Leu 740 745 750 Gln Ile Leu Asp Val Ser Ala Asn Pro Leu His Cys Ala
Cys Gly Ala 755 760 765 Ala Phe Met Asp Phe Leu Leu Glu Val Gln Ala
Ala Val Pro Gly Leu 770 775 780 Pro Ser Arg Val Lys Cys Gly Ser Pro
Gly Gln Leu Gln Gly Leu Ser 785 790 795 800 Ile Phe Ala Gln Asp Leu
Arg Leu Cys Leu Asp Glu Ala Leu Ser Trp 805 810 815 Asp Cys Phe Ala
Leu Ser Leu Leu Ala Val Ala Leu Gly Leu Gly Val 820 825 830 Pro Met
Leu His His Leu Cys Gly Trp Asp Leu Trp Tyr Cys Phe His 835 840 845
Leu Cys Leu Ala Trp Leu Pro Trp Arg Gly Arg Gln Ser Gly Arg Asp 850
855 860 Glu Asp Ala Leu Pro Tyr Asp Ala Phe Val Val Phe Asp Lys Thr
Gln 865 870 875 880 Ser Ala Val Ala Asp Trp Val Tyr Asn Glu Leu Arg
Gly Gln Leu Glu 885 890 895 Glu Cys Arg Gly Arg Trp Ala Leu Arg Leu
Cys Leu Glu Glu Arg Asp 900 905 910 Trp Leu Pro Gly Lys Thr Leu Phe
Glu Asn Leu Trp Ala Ser Val Tyr 915 920 925 Gly Ser Arg Lys Thr Leu
Phe Val Leu Ala His Thr Asp Arg Val Ser 930 935 940 Gly Leu Leu Arg
Ala Ser Phe Leu Leu Ala Gln Gln Arg Leu Leu Glu 945 950 955 960 Asp
Arg Lys Asp Val Val Val Leu Val Ile Leu Ser Pro Asp Gly Arg 965 970
975 Arg Ser Arg Tyr Val Arg Leu Arg Gln Arg Leu Cys Arg Gln Ser Val
980 985 990 Leu Leu Trp Pro His Gln Pro Ser Gly Gln Arg Ser Phe Trp
Ala Gln 995 1000 1005 Leu Gly Met Ala Leu Thr Arg Asp Asn His His
Phe Tyr Asn Arg Asn 1010 1015 1020 Phe Cys Gln Gly Pro Thr Ala Glu
1025 1030 <200> SEQUENCE CHARACTERISTICS: <210> SEQ ID
NO 7 <211> LENGTH: 557 <212> TYPE: DNA <213>
ORGANISM: Mus musculus <400> SEQUENCE: 7 ggctttcaac
ctaaccgctg gcactcaacc tgtccttcaa ttaccgcaag aaggtatcct 60
ttgcccgcct ccacctggca agttccttta agaacctggt gtcactgcag gagctgaaca
120 tgaacggcat cttcttccgc ttgctcaaca agtacacgct cagatggctg
gccgatctgc 180 ccaaactcca cactctgcat cttcaaatga acttcatcaa
ccaggcacag ctcagcatct 240 ttggtacctt ccgagccctt cgctttgtgg
acttgtcaga caatcgcatc agtgggcctt 300 caacgctgtc agaagccacc
cctgaagagg cagatgatgc agagcaggag gagctgttgt 360 ctgcggatcc
tcacccagct ccgctgagca cccctgcttc taagaacttc atggacaggt 420
gtaagaactt caagttcaac atggacctgt ctcggaacaa cctggtgact atcacagcag
480 agatgtttgt aaatctctca cgcctccagt gtcttagcct gagccacaac
tcaattgcac 540 aggctgtcaa tggctct 557 <200> SEQUENCE
CHARACTERISTICS: <210> SEQ ID NO 8 <211> LENGTH: 178
<212> TYPE: PRT <213> ORGANISM: Mus musculus
<400> SEQUENCE: 8 Leu Asn Leu Ser Phe Asn Tyr Arg Lys Lys Val
Ser Phe Ala Arg Leu 1 5 10 15 His Leu Ala Ser Ser Phe Lys Asn Leu
Val Ser Leu Gln Glu Leu Asn 20 25 30 Met Asn Gly Ile Phe Phe Arg
Leu Leu Asn Lys Tyr Thr Leu Arg Trp 35 40 45 Leu Ala Asp Leu Pro
Lys Leu His Thr Leu His Leu Gln Met Asn Phe 50 55 60 Ile Asn Gln
Ala Gln Leu Ser Ile Phe Gly Thr Phe Arg Ala Leu Arg 65 70 75 80 Phe
Val Asp Leu Ser Asp Asn Arg Ile Ser Gly Pro Ser Thr Leu Ser 85 90
95 Glu Ala Thr Pro Glu Glu Ala Asp Asp Ala Glu Gln Glu Glu Leu Leu
100 105 110 Ser Ala Asp Pro His Pro Ala Pro Leu Ser Thr Pro Ala Ser
Lys Asn 115 120 125 Phe Met Asp Arg Cys Lys Asn Phe Lys Phe Asn Met
Asp Leu Ser Arg 130 135 140 Asn Asn Leu Val Thr Ile Thr Ala Glu Met
Phe Val Asn Leu Ser Arg 145 150 155 160 Leu Gln Cys Leu Ser Leu Ser
His Asn Ser Ile Ala Gln Ala Val Asn 165 170 175 Gly Ser <200>
SEQUENCE CHARACTERISTICS: <210> SEQ ID NO 9 <211>
LENGTH: 497 <212> TYPE: DNA <213> ORGANISM: Mus
musculus <220> FEATURE: <221> NAME/KEY: unknown
<222> LOCATION: (380)...(380) <223> OTHER INFORMATION:
<400> SEQUENCE: 9 gtgggtttgg tgtctatctt cactctcctg aaagatgcat
gggaagaaaa ctacccttta 60 cagccaacct ttgctccgtg ggcctggtgg
cttggtagca tatattgcgc acttgccaaa 120 tagcggtgta gtaagacaga
gcaaggcagg cagagcaact cgggaaccag acatgaagat 180 gcagctgttt
ccagctgttg ctctgagcta ttctgctgta ggtccccggc agaagttctg 240
gttatagaag tggcggttgt ccctagtcag ggctgtactc agctgggccc agaagccccc
300 ctgcccgttg ggtcgctggg gccagaagag cacactctgg cggcagagac
gctggcgcag 360 tcgcacatag cgggacggtn gggcatccgg acgcaggatc
accaacacca ccacgtcctt 420 gcggtcttcc aacaggcgct gctgagccag
caggaagctg gtgcgcagga ggccactgac 480 gcggtccgtg tgggcca 497
<200> SEQUENCE CHARACTERISTICS: <210> SEQ ID NO 10
<211> LENGTH: 95 <212> TYPE: PRT <213> ORGANISM:
Mus musculus <220> FEATURE: <221> NAME/KEY: UNSURE
<222> LOCATION: (39)...(39) <223> OTHER INFORMATION:
<400> SEQUENCE: 10 Ala His Thr Asp Arg Val Ser Gly Leu Leu
Arg Thr Ser Phe Leu Leu 1 5 10 15 Ala Gln Gln Arg Leu Leu Glu Asp
Arg Lys Asp Val Val Val Leu Val 20 25 30 Ile Leu Arg Pro Asp Ala
Xaa Pro Ser Arg Tyr Val Arg Leu Arg Gln 35 40 45 Arg Leu Cys Arg
Gln Ser Val Leu Phe Trp Pro Gln Arg Pro Asn Gly 50 55 60 Gln Gly
Gly Phe Trp Ala Gln Leu Ser Thr Ala Leu Thr Arg Asp Asn 65 70 75 80
Arg His Phe Tyr Asn Gln Asn Phe Cys Arg Gly Pro Thr Ala Glu 85 90
95 <200> SEQUENCE CHARACTERISTICS: <210> SEQ ID NO 11
<211> LENGTH: 373 <212> TYPE: DNA <213> ORGANISM:
Mus musculus <400> SEQUENCE: 11 tggaggaccg agattggctg
cctggccaga cgctcttcga gaacctctgg gcttccatct 60 atgggagccg
caagactcta tttgtgctgg cccacacgga ccgcgtcagt ggcctcctgc 120
gcaccagctt cctgctggct cagcagcgcc tgttggaaga ccgcaaggac gtggtggtgt
180 tggtgatcct gcgtccggat gcccaccgct cccgctatgt gcgactgcgc
cagcgtctct 240 gccgccagag tgtgctcttc tggccccagc agcccaacgg
gcaggggggc ttctgggccc 300 agctgagtac agccctgact agggacaacc
gccacttcta taaccagaac ttctgccggg 360 gacctacagc aga 373 <200>
SEQUENCE CHARACTERISTICS: <210> SEQ ID NO 12 <211>
LENGTH: 123 <212> TYPE: PRT <213> ORGANISM: Mus
musculus <400> SEQUENCE: 12 Glu Asp Arg Asp Trp Leu Pro Gly
Gln Thr Leu Phe Glu Asn Leu Trp 1 5 10 15 Ala Ser Ile Tyr Gly Ser
Arg Lys Thr Leu Phe Val Leu Ala His Thr 20 25 30 Asp Arg Val Ser
Gly Leu Leu Arg Thr Ser Phe Leu Leu Ala Gln Gln 35 40 45 Arg Leu
Leu Glu Asp Arg Lys Asp Val Val Val Leu Val Ile Leu Arg 50 55 60
Pro Asp Ala His Arg Ser Arg Tyr Val Arg Leu Arg Gln Arg Leu Cys 65
70 75 80 Arg Gln Ser Val Leu Phe Trp Pro Gln Gln Pro Asn Gly Gln
Gly Gly 85 90 95 Phe Trp Ala Gln Leu Ser Thr Ala Leu Thr Arg Asp
Asn Arg His Phe 100 105 110 Tyr Asn Gln Asn Phe Cys Arg Gly Pro Thr
Ala 115 120 <200> SEQUENCE CHARACTERISTICS: <210> SEQ
ID NO 13 <211> LENGTH: 489 <212> TYPE: DNA <213>
ORGANISM: Mus musculus <400> SEQUENCE: 13 gctacaacag
ccagcccttt agcatgaagg gtataggcca caatttcagt tttgtgaccc 60
atctgtccat gctacagagc cttagcctgg cacacaatga cattcatacc cgtgtgtcct
120 cacatctcaa cagcaactca gtgaggtttc ttgacttcag cggcaacggt
atgggccgca 180 tgtgggatga ggggggcctt tatctccatt tcttccaagg
cctgagtggc gtgctgaagc 240 tggacctgtc tcaaaataac ctgcatatcc
tccggcccca gaaccttgac aacctcccca 300 agagcctgaa gctgctgagc
ctccgagaca actacctatc tttctttaac tggaccagtc 360 tgtccttcct
acccaacctg gaagtcctag acctggcagg caaccagcta aaggccctga 420
ccaatggcac cctgcctaat ggcaccctcc tccagaaact cgatgtcagt agcaacagta
480 tcgtctctg 489 <200> SEQUENCE CHARACTERISTICS: <210>
SEQ ID NO 14 <211> LENGTH: 162 <212> TYPE: PRT
<213> ORGANISM: Mus musculus <400> SEQUENCE: 14 Tyr Asn
Ser Gln Pro Phe Ser Met Lys Gly Ile Gly His Asn Phe Ser 1 5 10 15
Phe Val Thr His Leu Ser Met Leu Gln Ser Leu Ser Leu Ala His Asn 20
25 30 Asp Ile His Thr Arg Val Ser Ser His Leu Asn Ser Asn Ser Val
Arg 35 40 45 Phe Leu Asp Phe Ser Gly Asn Gly Met Gly Arg Met Trp
Asp Glu Gly 50 55 60 Gly Leu Tyr Leu His Phe Phe Gln Gly Leu Ser
Gly Val Leu Lys Leu 65 70 75 80 Asp Leu Ser Gln Asn Asn Leu His Ile
Leu Arg Pro Gln Asn Leu Asp 85 90 95 Asn Leu Pro Lys Ser Leu Lys
Leu Leu Ser Leu Arg Asp Asn Tyr Leu 100 105 110 Ser Phe Phe Asn Trp
Thr Ser Leu Ser Phe Leu Pro Asn Leu Glu Val 115 120 125 Leu Asp Leu
Ala Gly Asn Gln Leu Lys Ala Leu Thr Asn Gly Thr Leu 130 135 140 Pro
Asn Gly Thr Leu Leu Gln Lys Leu Asp Val Ser Ser Asn Ser Ile 145 150
155 160 Val Ser <200> SEQUENCE CHARACTERISTICS: <210>
SEQ ID NO 15 <211> LENGTH: 462 <212> TYPE: DNA
<213> ORGANISM: Mus musculus <400> SEQUENCE: 15
gcggccgcgc cgctcctcca gccgcacccg cagctcgtta tacacccagt cggcaactgc
60 gctctgtgcc ttatcgaaca ccacgaaggc atcataaggg agagtttggg
cgctgcgtcg 120 gctgcgggct agcaaaggta gccatgccag gcacagatga
aaacagtacc agacgtccca 180 gccgcagaga tggtgcagta taggcaccac
catgcccacg gccacagcca agagtgaaag 240 gccaaagcag tcccaagaga
ggacctcatc caggcacagc cgcaggtcct gcgcgaagat 300 gctacggccc
tgcagctggc cggggctgcc acacttcaca ccattagcca ggccaggcac 360
cttggtctgc acctccaaca gtaagtctac gaaggctgcc ccacaggcac agtgcagagg
420 gttgcttctc acgtctagaa ctgtcaggtt catcacaatg gg 462 <200>
SEQUENCE CHARACTERISTICS: <210> SEQ ID NO 16 <211>
LENGTH: 154 <212> TYPE: PRT <213> ORGANISM: Mus
musculus <400> SEQUENCE: 16 Pro Ile Val Met Asn Leu Thr Val
Leu Asp Val Arg Ser Asn Pro Leu 1 5 10 15 His Cys Ala Cys Gly Ala
Ala Phe Val Asp Leu Leu Leu Glu Val Gln 20 25 30 Thr Lys Val Pro
Gly Leu Ala Asn Gly Val Lys Cys Gly Ser Pro Gly 35 40 45 Gln Leu
Gln Gly Arg Ser Ile Phe Ala Gln Asp Leu Arg Leu Cys Leu 50 55 60
Asp Glu Val Leu Ser Trp Asp Cys Phe Gly Leu Ser Leu Leu Ala Val 65
70 75 80 Ala Val Gly Met Val Val Pro Ile Leu His His Leu Cys Gly
Trp Asp 85 90 95 Val Trp Tyr Cys Phe His Leu Cys Leu Ala Trp Leu
Pro Leu Leu Ala 100 105 110 Arg Ser Arg Arg Ser Ala Gln Thr Leu Pro
Tyr Asp Ala Phe Val Val 115 120 125 Phe Asp Lys Ala Gln Ser Ala Val
Ala Asp Trp Val Tyr Asn Glu Leu 130 135 140 Arg Val Arg Leu Glu Glu
Arg Arg Gly Arg 145 150 <200> SEQUENCE CHARACTERISTICS:
<210> SEQ ID NO 17 <211> LENGTH: 17 <212> TYPE:
PRT <213> ORGANISM: Mus musculus <400> SEQUENCE: 17 Cys
Asp Leu Ser Asp Asn Arg Ile Ser Gly Pro Ser Thr Leu Ser Glu 1 5 10
15
Ala <200> SEQUENCE CHARACTERISTICS: <210> SEQ ID NO 18
<211> LENGTH: 14 <212> TYPE: PRT <213> ORGANISM:
Mus musculus <400> SEQUENCE: 18 His Phe Tyr Asn Gln Asn Phe
Cys Arg Gly Pro Thr Ala Glu 1 5 10 <200> SEQUENCE
CHARACTERISTICS: <210> SEQ ID NO 19 <211> LENGTH: 16
<212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 19 Pro Ala Pro Val Asp Thr Pro Ser Ser Glu
Asp Phe Arg Pro Asn Cys 1 5 10 15 <200> SEQUENCE
CHARACTERISTICS: <210> SEQ ID NO 20 <211> LENGTH: 14
<212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 20 His Phe Tyr Asn Arg Asn Phe Cys Gln Gly
Pro Thr Ala Glu 1 5 10 <200> SEQUENCE CHARACTERISTICS:
<210> SEQ ID NO 21 <211> LENGTH: 8 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic oligonucleotide
<400> SEQUENCE: 21 aacgttct 8 <200> SEQUENCE
CHARACTERISTICS: <210> SEQ ID NO 22 <211> LENGTH: 20
<212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
oligonucleotide <400> SEQUENCE: 22 aagcgaaaat gaaattgact 20
<200> SEQUENCE CHARACTERISTICS: <210> SEQ ID NO 23
<211> LENGTH: 24 <212> TYPE: DNA <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Synthetic oligonucleotide <400> SEQUENCE: 23
accatggacg aactgtttcc cctc 24 <200> SEQUENCE CHARACTERISTICS:
<210> SEQ ID NO 24 <211> LENGTH: 24 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic oligonucleotide
<400> SEQUENCE: 24 accatggacg acctgtttcc cctc 24 <200>
SEQUENCE CHARACTERISTICS: <210> SEQ ID NO 25 <211>
LENGTH: 24 <212> TYPE: DNA <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic oligonucleotide <400> SEQUENCE: 25 accatggacg
agctgtttcc cctc 24 <200> SEQUENCE CHARACTERISTICS:
<210> SEQ ID NO 26 <211> LENGTH: 24 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic oligonucleotide
<400> SEQUENCE: 26 accatggacg atctgtttcc cctc 24 <200>
SEQUENCE CHARACTERISTICS: <210> SEQ ID NO 27 <211>
LENGTH: 24 <212> TYPE: DNA <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic oligonucleotide <400> SEQUENCE: 27 accatggacg
gtctgtttcc cctc 24 <200> SEQUENCE CHARACTERISTICS:
<210> SEQ ID NO 28 <211> LENGTH: 24 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic oligonucleotide
<400> SEQUENCE: 28 accatggacg tactgtttcc cctc 24 <200>
SEQUENCE CHARACTERISTICS: <210> SEQ ID NO 29 <211>
LENGTH: 24 <212> TYPE: DNA <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic oligonucleotide <400> SEQUENCE: 29 accatggacg
ttctgtttcc cctc 24 <200> SEQUENCE CHARACTERISTICS:
<210> SEQ ID NO 30 <211> LENGTH: 20 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic oligonucleotide
<400> SEQUENCE: 30 agatttctag gaattcaatc 20 <200>
SEQUENCE CHARACTERISTICS: <210> SEQ ID NO 31 <211>
LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic oligonucleotide <400> SEQUENCE: 31 agcgggggcg
agcgggggcg 20 <200> SEQUENCE CHARACTERISTICS: <210> SEQ
ID NO 32 <211> LENGTH: 18 <212> TYPE: DNA <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic oligonucleotide <400> SEQUENCE:
32 agctatgacg ttccaagg 18 <200> SEQUENCE CHARACTERISTICS:
<210> SEQ ID NO 33 <211> LENGTH: 20 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic oligonucleotide
<400> SEQUENCE: 33 atcgactctc gagcgttctc 20 <200>
SEQUENCE CHARACTERISTICS: <210> SEQ ID NO 34 <211>
LENGTH: 17 <212> TYPE: DNA <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic oligonucleotide <400> SEQUENCE: 34 atgacgttcc
tgacgtt 17 <200> SEQUENCE CHARACTERISTICS: <210> SEQ ID
NO 35 <211> LENGTH: 20 <212> TYPE: DNA <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic oligonucleotide <400> SEQUENCE:
35 atggaaggtc caacgttctc 20 <200> SEQUENCE CHARACTERISTICS:
<210> SEQ ID NO 36 <211> LENGTH: 20 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic oligonucleotide
<400> SEQUENCE: 36 atggaaggtc cagcgttctc 20 <200>
SEQUENCE CHARACTERISTICS: <210> SEQ ID NO 37 <211>
LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic oligonucleotide <400> SEQUENCE: 37 atggactctc
cagcgttctc 20 <200> SEQUENCE CHARACTERISTICS: <210> SEQ
ID NO 38 <211> LENGTH: 20 <212> TYPE: DNA <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic oligonucleotide <400> SEQUENCE:
38 atggaggctc catcgttctc 20 <200> SEQUENCE CHARACTERISTICS:
<210> SEQ ID NO 39 <211> LENGTH: 7 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic oligonucleotide
<400> SEQUENCE: 39 caacgtt 7 <200> SEQUENCE
CHARACTERISTICS: <210> SEQ ID NO 40 <211> LENGTH: 15
<212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
oligonucleotide <400> SEQUENCE: 40 cacgttgagg ggcat 15
<200> SEQUENCE CHARACTERISTICS: <210> SEQ ID NO 41
<211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Synthetic oligonucleotide <400> SEQUENCE: 41
caggcataac ggttccgtag 20 <200> SEQUENCE CHARACTERISTICS:
<210> SEQ ID NO 42 <211> LENGTH: 8 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic oligonucleotide
<400> SEQUENCE: 42 ccaacgtt 8 <200> SEQUENCE
CHARACTERISTICS: <210> SEQ ID NO 43 <211> LENGTH: 21
<212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
oligonucleotide <400> SEQUENCE: 43 ctcctagtgg gggtgtccta t 21
<200> SEQUENCE CHARACTERISTICS: <210> SEQ ID NO 44
<211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Synthetic oligonucleotide <400> SEQUENCE: 44
ctgatttccc cgaaatgatg 20 <200> SEQUENCE CHARACTERISTICS:
<210> SEQ ID NO 45 <211> LENGTH: 15 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic oligonucleotide
<400> SEQUENCE: 45 ctgctgagac tggag 15 <200> SEQUENCE
CHARACTERISTICS:
<210> SEQ ID NO 46 <211> LENGTH: 20 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic oligonucleotide
<400> SEQUENCE: 46 gagaacgatg gaccttccat 20 <200>
SEQUENCE CHARACTERISTICS: <210> SEQ ID NO 47 <211>
LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic oligonucleotide <400> SEQUENCE: 47 gagaacgctc
cagcactgat 20 <200> SEQUENCE CHARACTERISTICS: <210> SEQ
ID NO 48 <211> LENGTH: 20 <212> TYPE: DNA <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic oligonucleotide <400> SEQUENCE:
48 gagaacgctc gaccttccat 20 <200> SEQUENCE CHARACTERISTICS:
<210> SEQ ID NO 49 <211> LENGTH: 20 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic oligonucleotide
<400> SEQUENCE: 49 gagaacgctc gaccttcgat 20 <200>
SEQUENCE CHARACTERISTICS: <210> SEQ ID NO 50 <211>
LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic oligonucleotide <400> SEQUENCE: 50 gagaacgctg
gaccttccat 20 <200> SEQUENCE CHARACTERISTICS: <210> SEQ
ID NO 51 <211> LENGTH: 20 <212> TYPE: DNA <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic oligonucleotide <400> SEQUENCE:
51 gagcaagctg gaccttccat 20 <200> SEQUENCE CHARACTERISTICS:
<210> SEQ ID NO 52 <211> LENGTH: 20 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic oligonucleotide
<400> SEQUENCE: 52 gattgcctga cgtcagagag 20 <200>
SEQUENCE CHARACTERISTICS: <210> SEQ ID NO 53 <211>
LENGTH: 15 <212> TYPE: DNA <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic oligonucleotide <400> SEQUENCE: 53 gcatgacgtt gagct
15 <200> SEQUENCE CHARACTERISTICS: <210> SEQ ID NO 54
<211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Synthetic oligonucleotide <400> SEQUENCE: 54
gcggcgggcg gcgcgcgccc 20 <200> SEQUENCE CHARACTERISTICS:
<210> SEQ ID NO 55 <211> LENGTH: 21 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic oligonucleotide
<400> SEQUENCE: 55 gcgtgcgttg tcgttgtcgt t 21 <200>
SEQUENCE CHARACTERISTICS: <210> SEQ ID NO 56 <211>
LENGTH: 15 <212> TYPE: DNA <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic oligonucleotide <400> SEQUENCE: 56 gctagacgtt agcgt
15 <200> SEQUENCE CHARACTERISTICS: <210> SEQ ID NO 57
<211> LENGTH: 15 <212> TYPE: DNA <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Synthetic oligonucleotide <400> SEQUENCE: 57
gctagacgtt agtgt 15 <200> SEQUENCE CHARACTERISTICS:
<210> SEQ ID NO 58 <211> LENGTH: 15 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic oligonucleotide
<400> SEQUENCE: 58 gctagatgtt agcgt 15 <200> SEQUENCE
CHARACTERISTICS: <210> SEQ ID NO 59 <211> LENGTH: 20
<212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
oligonucleotide <400> SEQUENCE: 59 gcttgatgac tcagccggaa 20
<200> SEQUENCE CHARACTERISTICS: <210> SEQ ID NO 60
<211> LENGTH: 18 <212> TYPE: DNA <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Synthetic oligonucleotide <400> SEQUENCE: 60
ggaatgacgt tccctgtg 18 <200> SEQUENCE CHARACTERISTICS:
<210> SEQ ID NO 61 <211> LENGTH: 19 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic oligonucleotide
<400> SEQUENCE: 61 ggggtcaacg ttgacgggg 19 <200>
SEQUENCE CHARACTERISTICS: <210> SEQ ID NO 62 <211>
LENGTH: 19 <212> TYPE: DNA <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic oligonucleotide <400> SEQUENCE: 62 ggggtcagtc
ttgacgggg 19 <200> SEQUENCE CHARACTERISTICS: <210> SEQ
ID NO 63 <211> LENGTH: 20 <212> TYPE: DNA <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic oligonucleotide <400> SEQUENCE:
63 gtatttccca gaaaaggaac 20 <200> SEQUENCE CHARACTERISTICS:
<210> SEQ ID NO 64 <211> LENGTH: 20 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic oligonucleotide
<400> SEQUENCE: 64 gtccatttcc cgtaaatctt 20 <200>
SEQUENCE CHARACTERISTICS: <210> SEQ ID NO 65 <211>
LENGTH: 6 <212> TYPE: DNA <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic oligonucleotide <400> SEQUENCE: 65 gtcgct 6
<200> SEQUENCE CHARACTERISTICS: <210> SEQ ID NO 66
<211> LENGTH: 6 <212> TYPE: DNA <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Synthetic oligonucleotide <400> SEQUENCE: 66
gtcgtt 6 <200> SEQUENCE CHARACTERISTICS: <210> SEQ ID
NO 67 <211> LENGTH: 18 <212> TYPE: DNA <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic oligonucleotide <400> SEQUENCE:
67 taccgcgtgc gaccctct 18 <200> SEQUENCE CHARACTERISTICS:
<210> SEQ ID NO 68 <211> LENGTH: 20 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic oligonucleotide
<400> SEQUENCE: 68 tatgcatatt cctgtaagtg 20 <200>
SEQUENCE CHARACTERISTICS: <210> SEQ ID NO 69 <211>
LENGTH: 8 <212> TYPE: DNA <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic oligonucleotide <400> SEQUENCE: 69 tcaacgtc 8
<200> SEQUENCE CHARACTERISTICS: <210> SEQ ID NO 70
<211> LENGTH: 8 <212> TYPE: DNA <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Synthetic oligonucleotide <400> SEQUENCE: 70
tcaacgtt 8 <200> SEQUENCE CHARACTERISTICS: <210> SEQ ID
NO 71 <211> LENGTH: 8 <212> TYPE: DNA <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic oligonucleotide <400> SEQUENCE:
71 tcaagctt 8 <200> SEQUENCE CHARACTERISTICS: <210> SEQ
ID NO 72 <211> LENGTH: 8 <212> TYPE: DNA <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic oligonucleotide <400> SEQUENCE:
72 tcagcgct 8 <200> SEQUENCE CHARACTERISTICS: <210> SEQ
ID NO 73 <211> LENGTH: 12 <212> TYPE: DNA <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic oligonucleotide <400> SEQUENCE:
73 tcagcgtgcg cc 12
<200> SEQUENCE CHARACTERISTICS: <210> SEQ ID NO 74
<211> LENGTH: 8 <212> TYPE: DNA <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Synthetic oligonucleotide <400> SEQUENCE: 74
tcatcgat 8 <200> SEQUENCE CHARACTERISTICS: <210> SEQ ID
NO 75 <211> LENGTH: 20 <212> TYPE: DNA <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic oligonucleotide <400> SEQUENCE:
75 tccacgacgt tttcgacgtt 20 <200> SEQUENCE CHARACTERISTICS:
<210> SEQ ID NO 76 <211> LENGTH: 20 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic oligonucleotide
<400> SEQUENCE: 76 tccaggactt ctctcaggtt 20 <200>
SEQUENCE CHARACTERISTICS: <210> SEQ ID NO 77 <211>
LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic oligonucleotide <400> SEQUENCE: 77 tccataacgt
tcctgatgct 20 <200> SEQUENCE CHARACTERISTICS: <210> SEQ
ID NO 78 <211> LENGTH: 20 <212> TYPE: DNA <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic oligonucleotide <400> SEQUENCE:
78 tccatagcgt tcctagcgtt 20 <200> SEQUENCE CHARACTERISTICS:
<210> SEQ ID NO 79 <211> LENGTH: 20 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic oligonucleotide
<400> SEQUENCE: 79 tccatcacgt gcctgatgct 20 <200>
SEQUENCE CHARACTERISTICS: <210> SEQ ID NO 80 <211>
LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic oligonucleotide <400> SEQUENCE: 80 tccatgacgg
tcctgatgct 20 <200> SEQUENCE CHARACTERISTICS: <210> SEQ
ID NO 81 <211> LENGTH: 20 <212> TYPE: DNA <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic oligonucleotide <400> SEQUENCE:
81 tccatgacgt ccctgatgct 20 <200> SEQUENCE CHARACTERISTICS:
<210> SEQ ID NO 82 <211> LENGTH: 20 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic oligonucleotide
<400> SEQUENCE: 82 tccatgacgt gcctgatgct 20 <200>
SEQUENCE CHARACTERISTICS: <210> SEQ ID NO 83 <211>
LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic oligonucleotide <400> SEQUENCE: 83 tccatgacgt
tcctgacgtt 20 <200> SEQUENCE CHARACTERISTICS: <210> SEQ
ID NO 84 <211> LENGTH: 20 <212> TYPE: DNA <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic oligonucleotide <400> SEQUENCE:
84 tccatgacgt tcctgatgct 20 <200> SEQUENCE CHARACTERISTICS:
<210> SEQ ID NO 85 <211> LENGTH: 20 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic oligonucleotide
<400> SEQUENCE: 85 tccatgagct tcctgatgct 20 <200>
SEQUENCE CHARACTERISTICS: <210> SEQ ID NO 86 <211>
LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic oligonucleotide <400> SEQUENCE: 86 tccatgccgg
tcctgatgct 20 <200> SEQUENCE CHARACTERISTICS: <210> SEQ
ID NO 87 <211> LENGTH: 20 <212> TYPE: DNA <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic oligonucleotide <400> SEQUENCE:
87 tccatgcgtg cgtgcgtttt 20 <200> SEQUENCE CHARACTERISTICS:
<210> SEQ ID NO 88 <211> LENGTH: 20 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic oligonucleotide
<400> SEQUENCE: 88 tccatgcgtt gcgttgcgtt 20 <200>
SEQUENCE CHARACTERISTICS: <210> SEQ ID NO 89 <211>
LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic oligonucleotide <400> SEQUENCE: 89 tccatgctgg
tcctgatgct 20 <200> SEQUENCE CHARACTERISTICS: <210> SEQ
ID NO 90 <211> LENGTH: 20 <212> TYPE: DNA <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic oligonucleotide <400> SEQUENCE:
90 tccatggcgg tcctgatgct 20 <200> SEQUENCE CHARACTERISTICS:
<210> SEQ ID NO 91 <211> LENGTH: 20 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic oligonucleotide
<400> SEQUENCE: 91 tccatgtcga tcctgatgct 20 <200>
SEQUENCE CHARACTERISTICS: <210> SEQ ID NO 92 <211>
LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic oligonucleotide <400> SEQUENCE: 92 tccatgtcgc
tcctgatgct 20 <200> SEQUENCE CHARACTERISTICS: <210> SEQ
ID NO 93 <211> LENGTH: 20 <212> TYPE: DNA <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic oligonucleotide <400> SEQUENCE:
93 tccatgtcgg tcctgatgct 20 <200> SEQUENCE CHARACTERISTICS:
<210> SEQ ID NO 94 <211> LENGTH: 20 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic oligonucleotide
<400> SEQUENCE: 94 tccatgtcgg tcctgctgat 20 <200>
SEQUENCE CHARACTERISTICS: <210> SEQ ID NO 95 <211>
LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic oligonucleotide <400> SEQUENCE: 95 tccatgtcgt
ccctgatgct 20 <200> SEQUENCE CHARACTERISTICS: <210> SEQ
ID NO 96 <211> LENGTH: 20 <212> TYPE: DNA <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic oligonucleotide <400> SEQUENCE:
96 tccatgtcgt tcctgatgct 20 <200> SEQUENCE CHARACTERISTICS:
<210> SEQ ID NO 97 <211> LENGTH: 20 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic oligonucleotide
<400> SEQUENCE: 97 tccatgtcgt tcctgtcgtt 20 <200>
SEQUENCE CHARACTERISTICS: <210> SEQ ID NO 98 <211>
LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic oligonucleotide <400> SEQUENCE: 98 tccatgtcgt
ttttgtcgtt 20 <200> SEQUENCE CHARACTERISTICS: <210> SEQ
ID NO 99 <211> LENGTH: 19 <212> TYPE: DNA <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic oligonucleotide <400> SEQUENCE:
99 tcctgacgtt cctgacgtt 19 <200> SEQUENCE CHARACTERISTICS:
<210> SEQ ID NO 100 <211> LENGTH: 19 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic oligonucleotide
<400> SEQUENCE: 100 tcctgtcgtt cctgtcgtt 19 <200>
SEQUENCE CHARACTERISTICS: <210> SEQ ID NO 101 <211>
LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic oligonucleotide <400> SEQUENCE: 101
tcctgtcgtt ccttgtcgtt 20 <200> SEQUENCE CHARACTERISTICS:
<210> SEQ ID NO 102 <211> LENGTH: 20 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic oligonucleotide
<400> SEQUENCE: 102 tcctgtcgtt ttttgtcgtt 20 <200>
SEQUENCE CHARACTERISTICS: <210> SEQ ID NO 103 <211>
LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic oligonucleotide <400> SEQUENCE: 103 tccttgtcgt
tcctgtcgtt 20 <200> SEQUENCE CHARACTERISTICS: <210> SEQ
ID NO 104 <211> LENGTH: 20 <212> TYPE: DNA <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic oligonucleotide <400> SEQUENCE:
104 tcgatcgggg cggggcgagc 20 <200> SEQUENCE CHARACTERISTICS:
<210> SEQ ID NO 105 <211> LENGTH: 21 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic oligonucleotide
<400> SEQUENCE: 105 tcgtcgctgt ctccgcttct t 21 <200>
SEQUENCE CHARACTERISTICS: <210> SEQ ID NO 106 <211>
LENGTH: 27 <212> TYPE: DNA <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic oligonucleotide <400> SEQUENCE: 106 tcgtcgctgt
ctccgcttct tcttgcc 27 <200> SEQUENCE CHARACTERISTICS:
<210> SEQ ID NO 107 <211> LENGTH: 21 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic oligonucleotide
<400> SEQUENCE: 107 tcgtcgctgt ctgcccttct t 21 <200>
SEQUENCE CHARACTERISTICS: <210> SEQ ID NO 108 <211>
LENGTH: 21 <212> TYPE: DNA <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic oligonucleotide <400> SEQUENCE: 108 tcgtcgctgt
tgtcgtttct t 21 <200> SEQUENCE CHARACTERISTICS: <210>
SEQ ID NO 109 <211> LENGTH: 14 <212> TYPE: DNA
<213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic oligonucleotide
<400> SEQUENCE: 109 tcgtcgtcgt cgtt 14 <200> SEQUENCE
CHARACTERISTICS: <210> SEQ ID NO 110 <211> LENGTH: 20
<212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
oligonucleotide <400> SEQUENCE: 110 tcgtcgttgt cgttgtcgtt 20
<200> SEQUENCE CHARACTERISTICS: <210> SEQ ID NO 111
<211> LENGTH: 22 <212> TYPE: DNA <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Synthetic oligonucleotide <400> SEQUENCE: 111
tcgtcgttgt cgttttgtcg tt 22 <200> SEQUENCE CHARACTERISTICS:
<210> SEQ ID NO 112 <211> LENGTH: 24 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic oligonucleotide
<400> SEQUENCE: 112 tcgtcgtttt gtcgttttgt cgtt 24 <200>
SEQUENCE CHARACTERISTICS: <210> SEQ ID NO 113 <211>
LENGTH: 18 <212> TYPE: DNA <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic oligonucleotide <400> SEQUENCE: 113 tctcccagcg
cgcgccat 18 <200> SEQUENCE CHARACTERISTICS: <210> SEQ
ID NO 114 <211> LENGTH: 17 <212> TYPE: DNA <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic oligonucleotide <400> SEQUENCE:
114 tctcccagcg ggcgcat 17 <200> SEQUENCE CHARACTERISTICS:
<210> SEQ ID NO 115 <211> LENGTH: 18 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic oligonucleotide
<400> SEQUENCE: 115 tctcccagcg tgcgccat 18 <200>
SEQUENCE CHARACTERISTICS: <210> SEQ ID NO 116 <211>
LENGTH: 8 <212> TYPE: DNA <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic oligonucleotide <400> SEQUENCE: 116 tcttcgaa 8
<200> SEQUENCE CHARACTERISTICS: <210> SEQ ID NO 117
<211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Synthetic oligonucleotide <400> SEQUENCE: 117
tgcagattgc gcaatctgca 20 <200> SEQUENCE CHARACTERISTICS:
<210> SEQ ID NO 118 <211> LENGTH: 24 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic oligonucleotide
<400> SEQUENCE: 118 tgctgctttt gtgcttttgt gctt 24 <200>
SEQUENCE CHARACTERISTICS: <210> SEQ ID NO 119 <211>
LENGTH: 7 <212> TYPE: DNA <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic oligonucleotide <400> SEQUENCE: 119 tgtcgct 7
<200> SEQUENCE CHARACTERISTICS: <210> SEQ ID NO 120
<211> LENGTH: 7 <212> TYPE: DNA <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Synthetic oligonucleotide <400> SEQUENCE: 120
tgtcgtt 7 <200> SEQUENCE CHARACTERISTICS: <210> SEQ ID
NO 121 <211> LENGTH: 13 <212> TYPE: DNA <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic oligonucleotide <400> SEQUENCE:
121 tgtcgttgtc gtt 13 <200> SEQUENCE CHARACTERISTICS:
<210> SEQ ID NO 122 <211> LENGTH: 19 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic oligonucleotide
<400> SEQUENCE: 122 tgtcgttgtc gttgtcgtt 19 <200>
SEQUENCE CHARACTERISTICS: <210> SEQ ID NO 123 <211>
LENGTH: 25 <212> TYPE: DNA <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic oligonucleotide <400> SEQUENCE: 123 tgtcgttgtc
gttgtcgttg tcgtt 25 <200> SEQUENCE CHARACTERISTICS:
<210> SEQ ID NO 124 <211> LENGTH: 21 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic oligonucleotide
<400> SEQUENCE: 124 tgtcgtttgt cgtttgtcgt t 21 <200>
SEQUENCE CHARACTERISTICS: <210> SEQ ID NO 125 <211>
LENGTH: 31 <212> TYPE: PRT <213> ORGANISM: Unknown
<220> FEATURE: <221> NAME/KEY: UNSURE <222>
LOCATION: (2)...(8) <223> OTHER INFORMATION: <400>
SEQUENCE: 125 Arg Xaa Xaa Xaa Xaa Xaa Xaa Xaa Arg Xaa Asp Xaa Tyr
Xaa Xaa Xaa 1 5 10 15 Xaa Xaa Xaa Xaa Xaa Xaa Arg Ser Xaa Xaa Xaa
Xaa Xaa Xaa Tyr 20 25 30 <200> SEQUENCE CHARACTERISTICS:
<210> SEQ ID NO 126 <211> LENGTH: 31 <212> TYPE:
PRT <213> ORGANISM: Homo sapiens <220> FEATURE:
<221> NAME/KEY: UNSURE <222> LOCATION: (2)...(8)
<223> OTHER INFORMATION: <400> SEQUENCE: 126 Gln Xaa
Xaa Xaa Xaa Xaa Xaa Xaa Lys Xaa Asp Xaa Tyr Xaa Xaa Xaa 1 5 10 15
Xaa Xaa Xaa Xaa Xaa Xaa Arg Leu Xaa Xaa Xaa Xaa Xaa Xaa Tyr 20 25
30 <200> SEQUENCE CHARACTERISTICS: <210> SEQ ID NO 127
<211> LENGTH: 31 <212> TYPE: PRT <213> ORGANISM:
Mus musculus <220> FEATURE: <221> NAME/KEY: UNSURE
<222> LOCATION: (2)...(8) <223> OTHER INFORMATION:
<400> SEQUENCE: 127 Gln Xaa Xaa Xaa Xaa Xaa Xaa Xaa Lys Xaa
Asp Xaa Tyr Xaa Xaa Xaa 1 5 10 15 Xaa Xaa Xaa Xaa Xaa Xaa Gln Leu
Xaa Xaa Xaa Xaa Xaa Xaa Tyr 20 25 30 <200> SEQUENCE
CHARACTERISTICS:
<210> SEQ ID NO 128 <211> LENGTH: 24 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic oligonucleotide
<400> SEQUENCE: 128 tcgtcgtttt gtcgttttgt cgtt 24 <200>
SEQUENCE CHARACTERISTICS: <210> SEQ ID NO 129 <211>
LENGTH: 6 <212> TYPE: DNA <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic oligonucleotide <400> SEQUENCE: 129 gacgtt 6
<200> SEQUENCE CHARACTERISTICS: <210> SEQ ID NO 130
<211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Synthetic oligonucleotide <400> SEQUENCE: 130
tccatgacgt tcttgacgct 20 <200> SEQUENCE CHARACTERISTICS:
<210> SEQ ID NO 131 <211> LENGTH: 20 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic oligonucleotide
<400> SEQUENCE: 131 tccatgacgt tcttgacgtt 20 <200>
SEQUENCE CHARACTERISTICS: <210> SEQ ID NO 132 <211>
LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic oligonucleotide <400> SEQUENCE: 132 tccatgacgt
tcttgatgtt 20 <200> SEQUENCE CHARACTERISTICS: <210> SEQ
ID NO 133 <211> LENGTH: 20 <212> TYPE: DNA <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic oligonucleotide <400> SEQUENCE:
133 tccatgacgt ttttgatgtt 20 <200> SEQUENCE CHARACTERISTICS:
<210> SEQ ID NO 134 <211> LENGTH: 20 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic oligonucleotide
<400> SEQUENCE: 134 tccatgtcgt tcttgatgtt 20 <200>
SEQUENCE CHARACTERISTICS: <210> SEQ ID NO 135 <211>
LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic oligonucleotide <400> SEQUENCE: 135 tccatgtcgt
ttttgatgtt 20 <200> SEQUENCE CHARACTERISTICS: <210> SEQ
ID NO 136 <211> LENGTH: 20 <212> TYPE: DNA <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic oligonucleotide <400> SEQUENCE:
136 tccatgtcgt ttttgttgtt 20 <200> SEQUENCE CHARACTERISTICS:
<210> SEQ ID NO 137 <211> LENGTH: 20 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic oligonucleotide
<400> SEQUENCE: 137 tccatgacgt tattgatgtt 20 <200>
SEQUENCE CHARACTERISTICS: <210> SEQ ID NO 138 <211>
LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic oligonucleotide <400> SEQUENCE: 138 tccatgacgt
ccttgatgtt 20 <200> SEQUENCE CHARACTERISTICS: <210> SEQ
ID NO 139 <211> LENGTH: 20 <212> TYPE: DNA <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic oligonucleotide <400> SEQUENCE:
139 tccatgacgt cattgatgtt 20 <200> SEQUENCE CHARACTERISTICS:
<210> SEQ ID NO 140 <211> LENGTH: 20 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic oligonucleotide
<400> SEQUENCE: 140 hhhhhhhhhh hhhhwggggg 20 <200>
SEQUENCE CHARACTERISTICS: <210> SEQ ID NO 141 <211>
LENGTH: 45 <212> TYPE: DNA <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic oligonucleotide <400> SEQUENCE: 141 ctgcatggag
tgcggccaaa agtccctcca cctacatccc gatac 45 <200> SEQUENCE
CHARACTERISTICS: <210> SEQ ID NO 142 <211> LENGTH: 45
<212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
oligonucleotide <400> SEQUENCE: 142 gtatcgggat gtaggtggag
ggacttttgg ccgcactcca tgcag 45 <200> SEQUENCE
CHARACTERISTICS: <210> SEQ ID NO 143 <211> LENGTH: 45
<212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
oligonucleotide <400> SEQUENCE: 143 ctgtatagaa tgtcctcgtc
acttccccca gctgcaccct gagac 45 <200> SEQUENCE
CHARACTERISTICS: <210> SEQ ID NO 144 <211> LENGTH: 45
<212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
oligonucleotide <400> SEQUENCE: 144 gtctcagggt gcagctgggg
gaagtgacga ggacattcta tacag 45 <200> SEQUENCE
CHARACTERISTICS: <210> SEQ ID NO 145 <211> LENGTH: 14
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Mutated from
human <400> SEQUENCE: 145 Cys Xaa Xaa Cys Xaa Xaa Xaa Xaa Xaa
Xaa Cys Xaa Xaa Cys 1 5 10 <200> SEQUENCE CHARACTERISTICS:
<210> SEQ ID NO 146 <211> LENGTH: 20 <212> TYPE:
PRT <213> ORGANISM: Homo sapiens <400> SEQUENCE: 146
Cys Arg Arg Cys Asp His Ala Pro Asn Pro Cys Met Glu Cys Pro Arg 1 5
10 15 is Phe Pro Gln 20 <200> SEQUENCE CHARACTERISTICS:
<210> SEQ ID NO 147 <211> LENGTH: 20 <212> TYPE:
PRT <213> ORGANISM: Unknown <220> FEATURE: <223>
OTHER INFORMATION: Mutated from human <400> SEQUENCE: 147 Cys
Arg Arg Cys Asp His Ala Pro Asn Pro Cys Met Glu Cys Gly Gln 1 5 10
15 Lys Ser Leu His 20 <200> SEQUENCE CHARACTERISTICS:
<210> SEQ ID NO 148 <211> LENGTH: 20 <212> TYPE:
PRT <213> ORGANISM: Mus musculus <400> SEQUENCE: 148
Cys Arg Arg Cys Asp His Ala Pro Asn Pro Cys Met Ile Cys Gly Gln 1 5
10 15 Lys Ser Leu His 20 <200> SEQUENCE CHARACTERISTICS:
<210> SEQ ID NO 149 <211> LENGTH: 20 <212> TYPE:
PRT <213> ORGANISM: Unknown <220> FEATURE: <223>
OTHER INFORMATION: Mutated from mouse <400> SEQUENCE: 149 Cys
Arg Arg Cys Asp His Ala Pro Asn Pro Cys Met Ile Cys Pro Arg 1 5 10
15 His Phe Pro Gln 20 <200> SEQUENCE CHARACTERISTICS:
<210> SEQ ID NO 150 <211> LENGTH: 32 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic oligonucleotide
<400> SEQUENCE: 150 cacaataagc tggccctcgc ccacgagcac tc 32
<200> SEQUENCE CHARACTERISTICS: <210> SEQ ID NO 151
<211> LENGTH: 32 <212> TYPE: DNA <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Synthetic oligonucleotide <400> SEQUENCE: 151
gagtgctcgt gggcgagggc cagcttattg tg 32 <200> SEQUENCE
CHARACTERISTICS: <210> SEQ ID NO 152 <211> LENGTH: 32
<212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
oligonucleotide <400> SEQUENCE: 152 cataacaaac tggccttggc
ccactggaaa tc 32 <200> SEQUENCE CHARACTERISTICS: <210>
SEQ ID NO 153 <211> LENGTH: 32 <212> TYPE: DNA
<213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic oligonucleotide
<400> SEQUENCE: 153 gatttccagt gggccaaggc cagtttgtta tg 32
<200> SEQUENCE CHARACTERISTICS: <210> SEQ ID NO 154
<211> LENGTH: 30 <212> TYPE: DNA <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Synthetic oligonucleotide <400> SEQUENCE: 154
gcgactggct gcatggcaaa accctctttg 30
<200> SEQUENCE CHARACTERISTICS: <210> SEQ ID NO 155
<211> LENGTH: 30 <212> TYPE: DNA <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Synthetic oligonucleotide <400> SEQUENCE: 155
caaagagggt tttgccatgc agccagtcgc 30 <200> SEQUENCE
CHARACTERISTICS: <210> SEQ ID NO 156 <211> LENGTH: 30
<212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
oligonucleotide <400> SEQUENCE: 156 cgagattggc tgcatggcca
gacgctcttc 30 <200> SEQUENCE CHARACTERISTICS: <210> SEQ
ID NO 157 <211> LENGTH: 30 <212> TYPE: DNA <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic oligonucleotide <400> SEQUENCE:
157 gaagagcgtc tggccatgca gccaatctcg 30 <200> SEQUENCE
CHARACTERISTICS: <210> SEQ ID NO 158 <211> LENGTH: 15
<212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
oligonucleotide <400> SEQUENCE: 158 ggcctcagca tcttt 15
<200> SEQUENCE CHARACTERISTICS: <210> SEQ ID NO 159
<211> LENGTH: 15 <212> TYPE: DNA <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Synthetic oligonucleotide <400> SEQUENCE: 159
ggcctatcga ttttt 15 <200> SEQUENCE CHARACTERISTICS:
<210> SEQ ID NO 160 <211> LENGTH: 15 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic oligonucleotide
<400> SEQUENCE: 160 ggccgtagca tcttc 15 <200> SEQUENCE
CHARACTERISTICS: <210> SEQ ID NO 161 <211> LENGTH: 15
<212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
oligonucleotide <400> SEQUENCE: 161 ggcctatcga ttttt 15
<200> SEQUENCE CHARACTERISTICS: <210> SEQ ID NO 162
<211> LENGTH: 34 <212> TYPE: DNA <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Synthetic oligonucleotide <400> SEQUENCE: 162
cagctccagg gcctatcgat ttttgcacag gacc 34 <200> SEQUENCE
CHARACTERISTICS: <210> SEQ ID NO 163 <211> LENGTH: 34
<212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
oligonucleotide <400> SEQUENCE: 163 ggtcctgtgc aaaaatcgat
aggccctgga gctg 34 <200> SEQUENCE CHARACTERISTICS:
<210> SEQ ID NO 164 <211> LENGTH: 34 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic oligonucleotide
<400> SEQUENCE: 164 cagctgcagg gcctatcgat tttcgcacag gacc 34
<200> SEQUENCE CHARACTERISTICS: <210> SEQ ID NO 165
<211> LENGTH: 34 <212> TYPE: DNA <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Synthetic oligonucleotide <400> SEQUENCE: 165
ggtcctgtgc gaaaatcgat aggccctgca gctg 34 <200> SEQUENCE
CHARACTERISTICS: <210> SEQ ID NO 166 <211> LENGTH: 24
<212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
oligonucleotide <400> SEQUENCE: 166 cacctctcat gctctgctct
cttc 24 <200> SEQUENCE CHARACTERISTICS: <210> SEQ ID NO
167 <211> LENGTH: 25 <212> TYPE: DNA <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic oligonucleotide <400> SEQUENCE:
167 gctagaccgt ttccttgaac acctg 25 <200> SEQUENCE
CHARACTERISTICS: <210> SEQ ID NO 168 <211> LENGTH: 3373
<212> TYPE: DNA <213> ORGANISM: Homo sapiens
<220> FEATURE: <221> NAME/KEY: misc_feature <222>
LOCATION: (0)...(0) <223> OTHER INFORMATION: Human TLR7 cDNA
<400> SEQUENCE: 168 agctggctag cgtttaaacg ggccctctag
actcgagcgg ccgcgaattc actagtgatt 60 cacctctcat gctctgctct
cttcaaccag acctctacat tccattttgg aagaagacta 120 aaaatggtgt
ttccaatgtg gacactgaag agacaaattc ttatcctttt taacataatc 180
ctaatttcca aactccttgg ggctagatgg tttcctaaaa ctctgccctg tgatgtcact
240 ctggatgttc caaagaacca tgtgatcgtg gactgcacag acaagcattt
gacagaaatt 300 cctggaggta ttcccacgaa caccacgaac ctcaccctca
ccattaacca cataccagac 360 atctccccag cgtcctttca cagactggac
catctggtag agatcgattt cagatgcaac 420 tgtgtaccta ttccactggg
gtcaaaaaac aacatgtgca tcaagaggct gcagattaaa 480 cccagaagct
ttagtggact cacttattta aaatcccttt acctggatgg aaaccagcta 540
ctagagatac cgcagggcct cccgcctagc ttacagcttc tcagccttga ggccaacaac
600 atcttttcca tcagaaaaga gaatctaaca gaactggcca acatagaaat
actctacctg 660 ggccaaaact gttattatcg aaatccttgt tatgtttcat
attcaataga gaaagatgcc 720 ttcctaaact tgacaaagtt aaaagtgctc
tccctgaaag ataacaatgt cacagccgtc 780 cctactgttt tgccatctac
tttaacagaa ctatatctct acaacaacat gattgcaaaa 840 atccaagaag
atgattttaa taacctcaac caattacaaa ttcttgacct aagtggaaat 900
tgccctcgtt gttataatgc cccatttcct tgtgcgccgt gtaaaaataa ttctccccta
960 cagatccctg taaatgcttt tgatgcgctg acagaattaa aagttttacg
tctacacagt 1020 aactctcttc agcatgtgcc cccaagatgg tttaagaaca
tcaacaaact ccaggaactg 1080 gatctgtccc aaaacttctt ggccaaagaa
attggggatg ctaaatttct gcattttctc 1140 cccagcctca tccaattgga
tctgtctttc aattttgaac ttcaggtcta tcgtgcatct 1200 atgaatctat
cacaagcatt ttcttcactg aaaagcctga aaattctgcg gatcagagga 1260
tatgtcttta aagagttgaa aagctttaac ctctcgccat tacataatct tcaaaatctt
1320 gaagttcttg atcttggcac taactttata aaaattgcta acctcagcat
gtttaaacaa 1380 tttaaaagac tgaaagtcat agatctttca gtgaataaaa
tatcaccttc aggagattca 1440 agtgaagttg gcttctgctc aaatgccaga
acttctgtag aaagttatga accccaggtc 1500 ctggaacaat tacattattt
cagatatgat aagtatgcaa ggagttgcag attcaaaaac 1560 aaagaggctt
ctttcatgtc tgttaatgaa agctgctaca agtatgggca gaccttggat 1620
ctaagtaaaa atagtatatt ttttgtcaag tcctctgatt ttcagcatct ttctttcctc
1680 aaatgcctga atctgtcagg aaatctcatt agccaaactc ttaatggcag
tgaattccaa 1740 cctttagcag agctgagata tttggacttc tccaacaacc
ggcttgattt actccattca 1800 acagcatttg aagagcttca caaactggaa
gttctggata taagcagtaa tagccattat 1860 tttcaatcag aaggaattac
tcatatgcta aactttacca agaacctaaa ggttctgcag 1920 aaactgatga
tgaacgacaa tgacatctct tcctccacca gcaggaccat ggagagtgag 1980
tctcttagaa ctctggaatt cagaggaaat cacttagatg ttttatggag agaaggtgat
2040 aacagatact tacaattatt caagaatctg ctaaaattag aggaattaga
catctctaaa 2100 aattccctaa gtttcttgcc ttctggagtt tttgatggta
tgcctccaaa tctaaagaat 2160 ctctctttgg ccaaaaatgg gctcaaatct
ttcagttgga agaaactcca gtgtctaaag 2220 aacctggaaa ctttggacct
cagccacaac caactgacca ctgtccctga gagattatcc 2280 aactgttcca
gaagcctcaa gaatctgatt cttaagaata atcaaatcag gagtctgacg 2340
aagtattttc tacaagatgc cttccagttg cgatatctgg atctcagctc aaataaaatc
2400 cagatgatcc aaaagaccag cttcccagaa aatgtcctca acaatctgaa
gatgttgctt 2460 ttgcatcata atcggtttct gtgcacctgt gatgctgtgt
ggtttgtctg gtgggttaac 2520 catacggagg tgactattcc ttacctggcc
acagatgtga cttgtgtggg gccaggagca 2580 cacaagggcc aaagtgtgat
ctccctggat ctgtacacct gtgagttaga tctgactaac 2640 ctgattctgt
tctcactttc catatctgta tctctctttc tcatggtgat gatgacagca 2700
agtcacctct atttctggga tgtgtggtat atttaccatt tctgtaaggc caagataaag
2760 gggtatcagc gtctaatatc accagactgt tgctatgatg cttttattgt
gtatgacact 2820 aaagacccag ctgtgaccga gtgggttttg gctgagctgg
tggccaaact ggaagaccca 2880 agagagaaac attttaattt atgtctcgag
gaaagggact ggttaccagg gcagccagtt 2940 ctggaaaacc tttcccagag
catacagctt agcaaaaaga cagtgtttgt gatgacagac 3000 aagtatgcaa
agactgaaaa ttttaagata gcattttact tgtcccatca gaggctcatg 3060
gatgaaaaag ttgatgtgat tatcttgata tttcttgaga agccttttca gaagtccaag
3120 ttcctccagc tccggaaaag gctctgtggg agttctgtcc ttgagtggcc
aacaaacccg 3180 caagctcacc catacttctg gcagtgtcta aagaacgccc
tggccacaga caatcatgtg 3240 gcctatagtc aggtgttcaa ggaaacggtc
tagaatcgaa ttcccgcggc cgccactgtg 3300 ctggatatct gcagaattcc
accacactgg actagtggat ccgagctcgg taccaagctt 3360 aagtttaaac cgc
3373 <200> SEQUENCE CHARACTERISTICS: <210> SEQ ID NO
169 <211> LENGTH: 3147 <212> TYPE: DNA <213>
ORGANISM: Homo sapiens <220> FEATURE: <221> NAME/KEY:
misc_feature <222> LOCATION: (0)...(0) <223> OTHER
INFORMATION: Human TLR7 ORF <400> SEQUENCE: 169 atggtgtttc
caatgtggac actgaagaga caaattctta tcctttttaa cataatccta 60
atttccaaac tccttggggc tagatggttt cctaaaactc tgccctgtga tgtcactctg
120 gatgttccaa agaaccatgt gatcgtggac tgcacagaca agcatttgac
agaaattcct 180 ggaggtattc ccacgaacac cacgaacctc accctcacca
ttaaccacat accagacatc 240 tccccagcgt cctttcacag actggaccat
ctggtagaga tcgatttcag atgcaactgt 300 gtacctattc cactggggtc
aaaaaacaac atgtgcatca agaggctgca gattaaaccc 360 agaagcttta
gtggactcac ttatttaaaa tccctttacc tggatggaaa ccagctacta 420
gagataccgc agggcctccc gcctagctta cagcttctca gccttgaggc caacaacatc
480 ttttccatca gaaaagagaa tctaacagaa ctggccaaca tagaaatact
ctacctgggc 540 caaaactgtt attatcgaaa tccttgttat gtttcatatt
caatagagaa agatgccttc 600 ctaaacttga caaagttaaa agtgctctcc
ctgaaagata acaatgtcac agccgtccct 660 actgttttgc catctacttt
aacagaacta tatctctaca acaacatgat tgcaaaaatc 720 caagaagatg
attttaataa cctcaaccaa ttacaaattc ttgacctaag tggaaattgc 780
cctcgttgtt ataatgcccc atttccttgt gcgccgtgta aaaataattc tcccctacag
840 atccctgtaa atgcttttga tgcgctgaca gaattaaaag ttttacgtct
acacagtaac 900 tctcttcagc atgtgccccc aagatggttt aagaacatca
acaaactcca ggaactggat 960 ctgtcccaaa acttcttggc caaagaaatt
ggggatgcta aatttctgca ttttctcccc 1020 agcctcatcc aattggatct
gtctttcaat tttgaacttc aggtctatcg tgcatctatg 1080 aatctatcac
aagcattttc ttcactgaaa agcctgaaaa ttctgcggat cagaggatat 1140
gtctttaaag agttgaaaag ctttaacctc tcgccattac ataatcttca aaatcttgaa
1200 gttcttgatc ttggcactaa ctttataaaa attgctaacc tcagcatgtt
taaacaattt 1260 aaaagactga aagtcataga tctttcagtg aataaaatat
caccttcagg agattcaagt 1320 gaagttggct tctgctcaaa tgccagaact
tctgtagaaa gttatgaacc ccaggtcctg 1380 gaacaattac attatttcag
atatgataag tatgcaagga gttgcagatt caaaaacaaa 1440 gaggcttctt
tcatgtctgt taatgaaagc tgctacaagt atgggcagac cttggatcta 1500
agtaaaaata gtatattttt tgtcaagtcc tctgattttc agcatctttc tttcctcaaa
1560 tgcctgaatc tgtcaggaaa tctcattagc caaactctta atggcagtga
attccaacct 1620 ttagcagagc tgagatattt ggacttctcc aacaaccggc
ttgatttact ccattcaaca 1680 gcatttgaag agcttcacaa actggaagtt
ctggatataa gcagtaatag ccattatttt 1740 caatcagaag gaattactca
tatgctaaac tttaccaaga acctaaaggt tctgcagaaa 1800 ctgatgatga
acgacaatga catctcttcc tccaccagca ggaccatgga gagtgagtct 1860
cttagaactc tggaattcag aggaaatcac ttagatgttt tatggagaga aggtgataac
1920 agatacttac aattattcaa gaatctgcta aaattagagg aattagacat
ctctaaaaat 1980 tccctaagtt tcttgccttc tggagttttt gatggtatgc
ctccaaatct aaagaatctc 2040 tctttggcca aaaatgggct caaatctttc
agttggaaga aactccagtg tctaaagaac 2100 ctggaaactt tggacctcag
ccacaaccaa ctgaccactg tccctgagag attatccaac 2160 tgttccagaa
gcctcaagaa tctgattctt aagaataatc aaatcaggag tctgacgaag 2220
tattttctac aagatgcctt ccagttgcga tatctggatc tcagctcaaa taaaatccag
2280 atgatccaaa agaccagctt cccagaaaat gtcctcaaca atctgaagat
gttgcttttg 2340 catcataatc ggtttctgtg cacctgtgat gctgtgtggt
ttgtctggtg ggttaaccat 2400 acggaggtga ctattcctta cctggccaca
gatgtgactt gtgtggggcc aggagcacac 2460 aagggccaaa gtgtgatctc
cctggatctg tacacctgtg agttagatct gactaacctg 2520 attctgttct
cactttccat atctgtatct ctctttctca tggtgatgat gacagcaagt 2580
cacctctatt tctgggatgt gtggtatatt taccatttct gtaaggccaa gataaagggg
2640 tatcagcgtc taatatcacc agactgttgc tatgatgctt ttattgtgta
tgacactaaa 2700 gacccagctg tgaccgagtg ggttttggct gagctggtgg
ccaaactgga agacccaaga 2760 gagaaacatt ttaatttatg tctcgaggaa
agggactggt taccagggca gccagttctg 2820 gaaaaccttt cccagagcat
acagcttagc aaaaagacag tgtttgtgat gacagacaag 2880 tatgcaaaga
ctgaaaattt taagatagca ttttacttgt cccatcagag gctcatggat 2940
gaaaaagttg atgtgattat cttgatattt cttgagaagc cttttcagaa gtccaagttc
3000 ctccagctcc ggaaaaggct ctgtgggagt tctgtccttg agtggccaac
aaacccgcaa 3060 gctcacccat acttctggca gtgtctaaag aacgccctgg
ccacagacaa tcatgtggcc 3120 tatagtcagg tgttcaagga aacggtc 3147
<200> SEQUENCE CHARACTERISTICS: <210> SEQ ID NO 170
<211> LENGTH: 1049 <212> TYPE: PRT
<213> ORGANISM: Homo sapiens <400> SEQUENCE: 170 Met
Val Phe Pro Met Trp Thr Leu Lys Arg Gln Ile Leu Ile Leu Phe 1 5 10
15 Asn Ile Ile Leu Ile Ser Lys Leu Leu Gly Ala Arg Trp Phe Pro Lys
20 25 30 Thr Leu Pro Cys Asp Val Thr Leu Asp Val Pro Lys Asn His
Val Ile 35 40 45 Val Asp Cys Thr Asp Lys His Leu Thr Glu Ile Pro
Gly Gly Ile Pro 50 55 60 Thr Asn Thr Thr Asn Leu Thr Leu Thr Ile
Asn His Ile Pro Asp Ile 65 70 75 80 Ser Pro Ala Ser Phe His Arg Leu
Asp His Leu Val Glu Ile Asp Phe 85 90 95 Arg Cys Asn Cys Val Pro
Ile Pro Leu Gly Ser Lys Asn Asn Met Cys 100 105 110 Ile Lys Arg Leu
Gln Ile Lys Pro Arg Ser Phe Ser Gly Leu Thr Tyr 115 120 125 Leu Lys
Ser Leu Tyr Leu Asp Gly Asn Gln Leu Leu Glu Ile Pro Gln 130 135 140
Gly Leu Pro Pro Ser Leu Gln Leu Leu Ser Leu Glu Ala Asn Asn Ile 145
150 155 160 Phe Ser Ile Arg Lys Glu Asn Leu Thr Glu Leu Ala Asn Ile
Glu Ile 165 170 175 Leu Tyr Leu Gly Gln Asn Cys Tyr Tyr Arg Asn Pro
Cys Tyr Val Ser 180 185 190 Tyr Ser Ile Glu Lys Asp Ala Phe Leu Asn
Leu Thr Lys Leu Lys Val 195 200 205 Leu Ser Leu Lys Asp Asn Asn Val
Thr Ala Val Pro Thr Val Leu Pro 210 215 220 Ser Thr Leu Thr Glu Leu
Tyr Leu Tyr Asn Asn Met Ile Ala Lys Ile 225 230 235 240 Gln Glu Asp
Asp Phe Asn Asn Leu Asn Gln Leu Gln Ile Leu Asp Leu 245 250 255 Ser
Gly Asn Cys Pro Arg Cys Tyr Asn Ala Pro Phe Pro Cys Ala Pro 260 265
270 Cys Lys Asn Asn Ser Pro Leu Gln Ile Pro Val Asn Ala Phe Asp Ala
275 280 285 Leu Thr Glu Leu Lys Val Leu Arg Leu His Ser Asn Ser Leu
Gln His 290 295 300 Val Pro Pro Arg Trp Phe Lys Asn Ile Asn Lys Leu
Gln Glu Leu Asp 305 310 315 320 Leu Ser Gln Asn Phe Leu Ala Lys Glu
Ile Gly Asp Ala Lys Phe Leu 325 330 335 His Phe Leu Pro Ser Leu Ile
Gln Leu Asp Leu Ser Phe Asn Phe Glu 340 345 350 Leu Gln Val Tyr Arg
Ala Ser Met Asn Leu Ser Gln Ala Phe Ser Ser 355 360 365 Leu Lys Ser
Leu Lys Ile Leu Arg Ile Arg Gly Tyr Val Phe Lys Glu 370 375 380 Leu
Lys Ser Phe Asn Leu Ser Pro Leu His Asn Leu Gln Asn Leu Glu 385 390
395 400 Val Leu Asp Leu Gly Thr Asn Phe Ile Lys Ile Ala Asn Leu Ser
Met 405 410 415 Phe Lys Gln Phe Lys Arg Leu Lys Val Ile Asp Leu Ser
Val Asn Lys 420 425 430 Ile Ser Pro Ser Gly Asp Ser Ser Glu Val Gly
Phe Cys Ser Asn Ala 435 440 445 Arg Thr Ser Val Glu Ser Tyr Glu Pro
Gln Val Leu Glu Gln Leu His 450 455 460 Tyr Phe Arg Tyr Asp Lys Tyr
Ala Arg Ser Cys Arg Phe Lys Asn Lys 465 470 475 480 Glu Ala Ser Phe
Met Ser Val Asn Glu Ser Cys Tyr Lys Tyr Gly Gln 485 490 495 Thr Leu
Asp Leu Ser Lys Asn Ser Ile Phe Phe Val Lys Ser Ser Asp 500 505 510
Phe Gln His Leu Ser Phe Leu Lys Cys Leu Asn Leu Ser Gly Asn Leu 515
520 525 Ile Ser Gln Thr Leu Asn Gly Ser Glu Phe Gln Pro Leu Ala Glu
Leu 530 535 540 Arg Tyr Leu Asp Phe Ser Asn Asn Arg Leu Asp Leu Leu
His Ser Thr 545 550 555 560 Ala Phe Glu Glu Leu His Lys Leu Glu Val
Leu Asp Ile Ser Ser Asn 565 570 575 Ser His Tyr Phe Gln Ser Glu Gly
Ile Thr His Met Leu Asn Phe Thr 580 585 590 Lys Asn Leu Lys Val Leu
Gln Lys Leu Met Met Asn Asp Asn Asp Ile 595 600 605 Ser Ser Ser Thr
Ser Arg Thr Met Glu Ser Glu Ser Leu Arg Thr Leu 610 615 620 Glu Phe
Arg Gly Asn His Leu Asp Val Leu Trp Arg Glu Gly Asp Asn 625 630 635
640 Arg Tyr Leu Gln Leu Phe Lys Asn Leu Leu Lys Leu Glu Glu Leu Asp
645 650 655 Ile Ser Lys Asn Ser Leu Ser Phe Leu Pro Ser Gly Val Phe
Asp Gly 660 665 670 Met Pro Pro Asn Leu Lys Asn Leu Ser Leu Ala Lys
Asn Gly Leu Lys 675 680 685 Ser Phe Ser Trp Lys Lys Leu Gln Cys Leu
Lys Asn Leu Glu Thr Leu 690 695 700 Asp Leu Ser His Asn Gln Leu Thr
Thr Val Pro Glu Arg Leu Ser Asn 705 710 715 720 Cys Ser Arg Ser Leu
Lys Asn Leu Ile Leu Lys Asn Asn Gln Ile Arg 725 730 735 Ser Leu Thr
Lys Tyr Phe Leu Gln Asp Ala Phe Gln Leu Arg Tyr Leu 740 745 750 Asp
Leu Ser Ser Asn Lys Ile Gln Met Ile Gln Lys Thr Ser Phe Pro 755 760
765 Glu Asn Val Leu Asn Asn Leu Lys Met Leu Leu Leu His His Asn Arg
770 775 780 Phe Leu Cys Thr Cys Asp Ala Val Trp Phe Val Trp Trp Val
Asn His 785 790 795 800 Thr Glu Val Thr Ile Pro Tyr Leu Ala Thr Asp
Val Thr Cys Val Gly 805 810 815 Pro Gly Ala His Lys Gly Gln Ser Val
Ile Ser Leu Asp Leu Tyr Thr 820 825 830 Cys Glu Leu Asp Leu Thr Asn
Leu Ile Leu Phe Ser Leu Ser Ile Ser 835 840 845 Val Ser Leu Phe Leu
Met Val Met Met Thr Ala Ser His Leu Tyr Phe 850 855 860 Trp Asp Val
Trp Tyr Ile Tyr His Phe Cys Lys Ala Lys Ile Lys Gly 865 870 875 880
Tyr Gln Arg Leu Ile Ser Pro Asp Cys Cys Tyr Asp Ala Phe Ile Val 885
890 895 Tyr Asp Thr Lys Asp Pro Ala Val Thr Glu Trp Val Leu Ala Glu
Leu 900 905 910 Val Ala Lys Leu Glu Asp Pro Arg Glu Lys His Phe Asn
Leu Cys Leu 915 920 925 Glu Glu Arg Asp Trp Leu Pro Gly Gln Pro Val
Leu Glu Asn Leu Ser 930 935 940 Gln Ser Ile Gln Leu Ser Lys Lys Thr
Val Phe Val Met Thr Asp Lys 945 950 955 960 Tyr Ala Lys Thr Glu Asn
Phe Lys Ile Ala Phe Tyr Leu Ser His Gln 965 970 975 Arg Leu Met Asp
Glu Lys Val Asp Val Ile Ile Leu Ile Phe Leu Glu 980 985 990 Lys Pro
Phe Gln Lys Ser Lys Phe Leu Gln Leu Arg Lys Arg Leu Cys 995 1000
1005 Gly Ser Ser Val Leu Glu Trp Pro Thr Asn Pro Gln Ala His Pro
Tyr 1010 1015 1020 Phe Trp Gln Cys Leu Lys Asn Ala Leu Ala Thr Asp
Asn His Val Ala 1025 1030 1035 1040 Tyr Ser Gln Val Phe Lys Glu Thr
Val 1045 <200> SEQUENCE CHARACTERISTICS: <210> SEQ ID
NO 171 <211> LENGTH: 989 <212> TYPE: PRT <213>
ORGANISM: Homo sapiens <400> SEQUENCE: 171 Met Val Phe Pro
Met Trp Thr Leu Lys Arg Gln Ile Leu Ile Leu Phe 1 5 10 15 Asn Ile
Ile Leu Ile Ser Lys Leu Leu Gly Ala Arg Trp Phe Pro Lys 20 25 30
Thr Leu Pro Cys Asp Val Thr Leu Asp Val Pro Lys Asn His Val Ile 35
40 45 Val Asp Cys Thr Asp Lys His Leu Thr Glu Ile Pro Gly Gly Ile
Pro 50 55 60 Thr Asn Thr Thr Asn Leu Thr Leu Thr Ile Asn His Ile
Pro Asp Ile 65 70 75 80 Ser Pro Ala Ser Phe His Arg Leu Asp His Leu
Val Glu Ile Asp Phe 85 90 95 Arg Cys Asn Cys Val Pro Ile Pro Leu
Gly Ser Lys Asn Asn Met Cys 100 105 110 Ile Lys Arg Leu Gln Ile Lys
Pro Arg Ser Phe Ser Gly Leu Thr Tyr 115 120 125 Leu Lys Ser Leu Tyr
Leu Asp Gly Asn Gln Leu Leu Glu Ile Pro Gln 130 135 140 Gly Leu Pro
Pro Ser Leu Gln Leu Leu Ser Leu Glu Ala Asn Asn Ile 145 150 155 160
Phe Ser Ile Arg Lys Glu Asn Leu Thr Glu Leu Ala Asn Ile Glu Ile 165
170 175 Leu Tyr Leu Gly Gln Asn Cys Tyr Tyr Arg Asn Pro Cys Tyr Val
Ser 180 185 190 Tyr Ser Ile Glu Lys Asp Ala Phe Leu Asn Leu Thr Lys
Leu Lys Val 195 200 205 Leu Ser Leu Lys Asp Asn Asn Val Thr Ala Val
Pro Thr Val Leu Pro 210 215 220 Ser Thr Leu Thr Glu Leu Tyr Leu Tyr
Asn Asn Met Ile Ala Lys Ile 225 230 235 240 Gln Glu Asp Asp Phe Asn
Asn Leu Asn Gln Leu Gln Ile Leu Asp Leu 245 250 255 Ser Gly Asn Cys
Pro Arg Cys Tyr Asn Ala Pro Phe Pro Cys Ala Pro 260 265 270 Cys Lys
Asn Asn Ser Pro Leu Gln Ile Pro Val Asn Ala Phe Asp Ala 275 280 285
Leu Thr Glu Leu Lys Val Leu Arg Leu His Ser Asn Ser Leu Gln His 290
295 300 Val Pro Pro Arg Trp Phe Lys Asn Ile Asn Lys Leu Gln Glu Leu
Asp 305 310 315 320 Leu Ser Gln Asn Phe Leu Ala Lys Glu Ile Gly Asp
Ala Lys Phe Leu 325 330 335 His Phe Leu Pro Ser Leu Ile Gln Leu Asp
Leu Ser Phe Asn Phe Glu 340 345 350 Leu Gln Val Tyr Arg Ala Ser Met
Asn Leu Ser Gln Ala Phe Ser Ser 355 360 365 Leu Lys Ser Leu Lys Ile
Leu Arg Ile Arg Gly Tyr Val Phe Lys Glu 370 375 380 Leu Lys Ser Phe
Asn Leu Ser Pro Leu His Asn Leu Gln Asn Leu Glu 385 390 395 400 Val
Leu Asp Leu Gly Thr Asn Phe Ile Lys Ile Ala Asn Leu Ser Met 405 410
415 Phe Lys Gln Phe Lys Arg Leu Lys Val Ile Asp Leu Ser Val Asn Lys
420 425 430 Ile Ser Pro Ser Gly Asp Ser Ser Glu Val Gly Phe Cys Ser
Asn Ala 435 440 445 Arg Thr Ser Val Glu Ser Tyr Glu Pro Gln Val Leu
Glu Gln Leu His 450 455 460 Tyr Phe Arg Tyr Asp Lys Tyr Ala Arg Ser
Cys Arg Phe Lys Asn Lys 465 470 475 480 Glu Ala Ser Phe Met Ser Val
Asn Glu Ser Cys Tyr Lys Tyr Gly Gln 485 490 495 Thr Leu Asp Leu Ser
Lys Asn Ser Ile Phe Phe Val Lys Ser Ser Asp 500 505 510 Phe Gln His
Leu Ser Phe Leu Lys Cys Leu Asn Leu Ser Gly Asn Leu 515 520 525 Ile
Ser Gln Thr Leu Asn Gly Ser Glu Phe Gln Pro Leu Met Met Asn 530 535
540 Asp Asn Asp Ile Ser Ser Ser Thr Ser Arg Thr Met Glu Ser Glu Ser
545 550 555 560 Leu Arg Thr Leu Glu Phe Arg Gly Asn His Leu Asp Val
Leu Trp Arg 565 570 575 Glu Gly Asp Asn Arg Tyr Leu Gln Leu Phe Lys
Asn Leu Leu Lys Leu 580 585 590 Glu Glu Leu Asp Ile Ser Lys Asn Ser
Leu Ser Phe Leu Pro Ser Gly 595 600 605 Val Phe Asp Gly Met Pro Pro
Asn Leu Lys Asn Leu Ser Leu Ala Lys 610 615 620 Asn Gly Leu Lys Ser
Phe Ser Trp Lys Lys Leu Gln Cys Leu Lys Asn 625 630 635 640 Leu Glu
Thr Leu Asp Leu Ser His Asn Gln Leu Thr Thr Val Pro Glu 645 650 655
Arg Leu Ser Asn Cys Ser Arg Ser Leu Lys Asn Leu Ile Leu Lys Asn 660
665 670 Asn Gln Ile Arg Ser Leu Thr Lys Tyr Phe Leu Gln Asp Ala Phe
Gln 675 680 685 Leu Arg Tyr Leu Asp Leu Ser Ser Asn Lys Ile Gln Met
Ile Gln Lys 690 695 700 Thr Ser Phe Pro Glu Asn Val Leu Asn Asn Leu
Lys Met Leu Leu Leu 705 710 715 720 His His Asn Arg Phe Leu Cys Thr
Cys Asp Ala Val Trp Phe Val Trp 725 730 735 Trp Val Asn His Thr Glu
Val Thr Ile Pro Tyr Leu Ala Thr Asp Val 740 745 750 Thr Cys Val Gly
Pro Gly Ala His Lys Gly Gln Ser Val Ile Ser Leu 755 760 765 Asp Leu
Tyr Thr Cys Glu Leu Asp Leu Thr Asn Leu Ile Leu Phe Ser 770 775 780
Leu Ser Ile Ser Val Ser Leu Phe Leu Met Val Met Met Thr Ala Ser 785
790 795 800 His Leu Tyr Phe Trp Asp Val Trp Tyr Ile Tyr His Phe Cys
Lys Ala 805 810 815 Lys Ile Lys Gly Tyr Gln Arg Leu Ile Ser Pro Asp
Cys Cys Tyr Asp 820 825 830 Ala Phe Ile Val Tyr Asp Thr Lys Asp Pro
Ala Val Thr Glu Trp Val 835 840 845 Leu Ala Glu Leu Val Ala Lys Leu
Glu Asp Pro Arg Glu Lys His Phe 850 855 860 Asn Leu Cys Leu Glu Glu
Arg Asp Trp Leu Pro Gly Gln Pro Val Leu 865 870 875 880 Glu Asn Leu
Ser Gln Ser Ile Gln Leu Ser Lys Lys Thr Val Phe Val
885 890 895 Met Thr Asp Lys Tyr Ala Lys Thr Glu Asn Phe Lys Ile Ala
Phe Tyr 900 905 910 Leu Ser His Gln Arg Leu Met Asp Glu Lys Val Asp
Val Ile Ile Leu 915 920 925 Ile Phe Leu Glu Lys Pro Phe Gln Lys Ser
Lys Phe Leu Gln Leu Arg 930 935 940 Lys Arg Leu Cys Gly Ser Ser Val
Leu Glu Trp Pro Thr Asn Pro Gln 945 950 955 960 Ala His Pro Tyr Phe
Trp Gln Cys Leu Lys Asn Ala Leu Ala Thr Asp 965 970 975 Asn His Val
Ala Tyr Ser Gln Val Phe Lys Glu Thr Val 980 985 <200>
SEQUENCE CHARACTERISTICS: <210> SEQ ID NO 172 <211>
LENGTH: 1049 <212> TYPE: PRT <213> ORGANISM: Homo
sapiens <400> SEQUENCE: 172 Met Val Phe Pro Met Trp Thr Leu
Lys Arg Gln Ile Leu Ile Leu Phe 1 5 10 15 Asn Ile Ile Leu Ile Ser
Lys Leu Leu Gly Ala Arg Trp Phe Pro Lys 20 25 30 Thr Leu Pro Cys
Asp Val Thr Leu Asp Val Pro Lys Asn His Val Ile 35 40 45 Val Asp
Cys Thr Asp Lys His Leu Thr Glu Ile Pro Gly Gly Ile Pro 50 55 60
Thr Asn Thr Thr Asn Leu Thr Leu Thr Ile Asn His Ile Pro Asp Ile 65
70 75 80 Ser Pro Ala Ser Phe His Arg Leu Asp His Leu Val Glu Ile
Asp Phe 85 90 95 Arg Cys Asn Cys Val Pro Ile Pro Leu Gly Ser Lys
Asn Asn Met Cys 100 105 110 Ile Lys Arg Leu Gln Ile Lys Pro Arg Ser
Phe Ser Gly Leu Thr Tyr 115 120 125 Leu Lys Ser Leu Tyr Leu Asp Gly
Asn Gln Leu Leu Glu Ile Pro Gln 130 135 140 Gly Leu Pro Pro Ser Leu
Gln Leu Leu Ser Leu Glu Ala Asn Asn Ile 145 150 155 160 Phe Ser Ile
Arg Lys Glu Asn Leu Thr Glu Leu Ala Asn Ile Glu Ile 165 170 175 Leu
Tyr Leu Gly Gln Asn Cys Tyr Tyr Arg Asn Pro Cys Tyr Val Ser 180 185
190 Tyr Ser Ile Glu Lys Asp Ala Phe Leu Asn Leu Thr Lys Leu Lys Val
195 200 205 Leu Ser Leu Lys Asp Asn Asn Val Thr Ala Val Pro Thr Val
Leu Pro 210 215 220 Ser Thr Leu Thr Glu Leu Tyr Leu Tyr Asn Asn Met
Ile Ala Lys Ile 225 230 235 240 Gln Glu Asp Asp Phe Asn Asn Leu Asn
Gln Leu Gln Ile Leu Asp Leu 245 250 255 Ser Gly Asn Cys Pro Arg Cys
Tyr Asn Ala Pro Phe Pro Cys Ala Pro 260 265 270 Cys Lys Asn Asn Ser
Pro Leu Gln Ile Pro Val Asn Ala Phe Asp Ala 275 280 285 Leu Thr Glu
Leu Lys Val Leu Arg Leu His Ser Asn Ser Leu Gln His 290 295 300 Val
Pro Pro Arg Trp Phe Lys Asn Ile Asn Lys Leu Gln Glu Leu Asp 305 310
315 320 Leu Ser Gln Asn Phe Leu Ala Lys Glu Ile Gly Asp Ala Lys Phe
Leu 325 330 335 His Phe Leu Pro Ser Leu Ile Gln Leu Asp Leu Ser Phe
Asn Phe Glu 340 345 350 Leu Gln Val Tyr Arg Ala Ser Met Asn Leu Ser
Gln Ala Phe Ser Ser 355 360 365 Leu Lys Ser Leu Lys Ile Leu Arg Ile
Arg Gly Tyr Val Phe Lys Glu 370 375 380 Leu Lys Ser Phe Asn Leu Ser
Pro Leu His Asn Leu Gln Asn Leu Glu 385 390 395 400 Val Leu Asp Leu
Gly Thr Asn Phe Ile Lys Ile Ala Asn Leu Ser Met 405 410 415 Phe Lys
Gln Phe Lys Arg Leu Lys Val Ile Asp Leu Ser Val Asn Lys 420 425 430
Ile Ser Pro Ser Gly Asp Ser Ser Glu Val Gly Phe Cys Ser Asn Ala 435
440 445 Arg Thr Ser Val Glu Ser Tyr Glu Pro Gln Val Leu Glu Gln Leu
His 450 455 460 Tyr Phe Arg Tyr Asp Lys Tyr Ala Arg Ser Cys Arg Phe
Lys Asn Lys 465 470 475 480 Glu Ala Ser Phe Met Ser Val Asn Glu Ser
Cys Tyr Lys Tyr Gly Gln 485 490 495 Thr Leu Asp Leu Ser Lys Asn Ser
Ile Phe Phe Val Lys Ser Ser Asp 500 505 510 Phe Gln His Leu Ser Phe
Leu Lys Cys Leu Asn Leu Ser Gly Asn Leu 515 520 525 Ile Ser Gln Thr
Leu Asn Gly Ser Glu Phe Gln Pro Leu Ala Glu Leu 530 535 540 Arg Tyr
Leu Asp Phe Ser Asn Asn Arg Leu Asp Leu Leu His Ser Thr 545 550 555
560 Ala Phe Glu Glu Leu His Lys Leu Glu Val Leu Asp Ile Ser Ser Asn
565 570 575 Ser His Tyr Phe Gln Ser Glu Gly Ile Thr His Met Leu Asn
Phe Thr 580 585 590 Lys Asn Leu Lys Val Leu Gln Lys Leu Met Met Asn
Asp Asn Asp Ile 595 600 605 Ser Ser Ser Thr Ser Arg Thr Met Glu Ser
Glu Ser Leu Arg Thr Leu 610 615 620 Glu Phe Arg Gly Asn His Leu Asp
Val Leu Trp Arg Glu Gly Asp Asn 625 630 635 640 Arg Tyr Leu Gln Leu
Phe Lys Asn Leu Leu Lys Leu Glu Glu Leu Asp 645 650 655 Ile Ser Lys
Asn Ser Leu Ser Phe Leu Pro Ser Gly Val Phe Asp Gly 660 665 670 Met
Pro Pro Asn Leu Lys Asn Leu Ser Leu Ala Lys Asn Gly Leu Lys 675 680
685 Ser Phe Ser Trp Lys Lys Leu Gln Cys Leu Lys Asn Leu Glu Thr Leu
690 695 700 Asp Leu Ser His Asn Gln Leu Thr Thr Val Pro Glu Arg Leu
Ser Asn 705 710 715 720 Cys Ser Arg Ser His Lys Asn Leu Ile Leu Lys
Asn Asn Gln Ile Arg 725 730 735 Ser Pro Thr Lys Tyr Phe Leu Gln Asp
Ala Phe Gln Leu Arg Tyr Leu 740 745 750 Asp Leu Ser Ser Asn Lys Ile
Gln Met Ile Gln Lys Thr Ser Phe Pro 755 760 765 Glu Asn Val Leu Asn
Asn Leu Lys Met Leu Leu Leu His His Asn Arg 770 775 780 Phe Leu Cys
Thr Cys Asp Ala Val Trp Phe Val Trp Trp Val Asn His 785 790 795 800
Thr Glu Val Thr Ile Pro Tyr Leu Ala Thr Asp Val Thr Cys Val Gly 805
810 815 Pro Gly Ala His Lys Gly Gln Ser Val Ile Ser Leu Asp Leu Tyr
Thr 820 825 830 Cys Glu Leu Asp Leu Thr Asn Leu Ile Leu Phe Ser Leu
Ser Ile Ser 835 840 845 Val Ser Leu Phe Leu Met Val Met Met Thr Ala
Ser His Leu Tyr Phe 850 855 860 Trp Asp Val Trp Tyr Ile Tyr His Phe
Cys Lys Ala Lys Ile Lys Gly 865 870 875 880 Tyr Gln Arg Leu Ile Ser
Pro Asp Cys Cys Tyr Asp Ala Phe Ile Val 885 890 895 Tyr Asp Thr Lys
Asp Pro Ala Val Thr Glu Trp Val Leu Ala Glu Leu 900 905 910 Val Ala
Lys Leu Glu Asp Pro Arg Glu Lys His Phe Asn Leu Cys Leu 915 920 925
Glu Glu Arg Asp Trp Leu Pro Gly Gln Pro Val Leu Glu Asn Leu Ser 930
935 940 Gln Ser Ile Gln Leu Ser Lys Lys Thr Val Phe Val Met Thr Asp
Lys 945 950 955 960 Tyr Ala Lys Thr Glu Asn Phe Lys Ile Ala Phe Tyr
Leu Ser His Gln 965 970 975 Arg Leu Met Asp Glu Lys Val Asp Val Ile
Ile Leu Ile Phe Leu Glu 980 985 990 Lys Pro Phe Gln Lys Ser Lys Phe
Leu Gln Leu Arg Lys Arg Leu Cys 995 1000 1005 Gly Ser Ser Val Leu
Glu Trp Pro Thr Asn Pro Gln Ala His Pro Tyr 1010 1015 1020 Phe Trp
Gln Cys Leu Lys Asn Ala Leu Ala Thr Asp Asn His Val Ala 1025 1030
1035 1040 Tyr Ser Gln Val Phe Lys Glu Thr Val 1045 <200>
SEQUENCE CHARACTERISTICS: <210> SEQ ID NO 173 <211>
LENGTH: 3243 <212> TYPE: DNA <213> ORGANISM: Mus
musculus <220> FEATURE: <221> NAME/KEY: misc_feature
<222> LOCATION: (0)...(0) <223> OTHER INFORMATION:
Murine TLR7 cDNA <400> SEQUENCE: 173 attctcctcc accagacctc
ttgattccat tttgaaagaa aactgaaaat ggtgttttcg 60 atgtggacac
ggaagagaca aattttgatc tttttaaata tgctcttagt ttctagagtc 120
tttgggtttc gatggtttcc taaaactcta ccttgtgaag ttaaagtaaa tatcccagag
180 gcccatgtga tcgtggactg cacagacaag catttgacag aaatccctga
gggcattccc 240 actaacacca ccaatcttac ccttaccatc aaccacatac
caagcatctc tccagattcc 300 ttccgtaggc tgaaccatct ggaagaaatc
gatttaagat gcaattgtgt acctgttcta 360 ctggggtcca aagccaatgt
gtgtaccaag aggctgcaga ttagacctgg aagctttagt 420 ggactctctg
acttaaaagc cctttacctg gatggaaacc aacttctgga gataccacag 480
gatctgccat ccagcttaca tcttctgagc cttgaggcta acaacatctt ctccatcacg
540 aaggagaatc taacagaact ggtcaacatt gaaacactct acctgggtca
aaactgttat 600 tatcgaaatc cttgcaatgt ttcctattct attgaaaaag
atgctttcct agttatgaga 660 aatttgaagg ttctctcact aaaagataac
aatgtcacag ctgtccccac cactttgcca 720 cctaatttac tagagctcta
tctttataac aatatcatta agaaaatcca agaaaatgat 780 tttaataacc
tcaatgagtt gcaagttctt gacctaagtg gaaattgccc tcgatgttat 840
aatgtcccat atccgtgtac accgtgtgaa aataattccc ccttacagat ccatgacaat
900 gctttcaatt cattgacaga attaaaagtt ttacgtttac acagtaattc
tcttcagcat 960 gtgcccccaa catggtttaa aaacatgaga aacctccagg
aactagacct ctcccaaaac 1020 tacttggcca gagaaattga ggaggccaaa
tttttgcatt ttcttcccaa ccttgttgag 1080 ttggattttt ctttcaatta
tgagctgcag gtctaccatg catctataac tttaccacat 1140 tcactctctt
cattggaaaa cttgaaaatt ctgcgtgtca aggggtatgt ctttaaagag 1200
ctgaaaaact ccagtctttc tgtattgcac aagcttccca ggctggaagt tcttgacctt
1260 ggcactaact tcataaaaat tgctgacctc aacatattca aacattttga
aaacctcaaa 1320 ctcatagacc tttcagtgaa taagatatct ccttcagaag
agtcaagaga agttggcttt 1380 tgtcctaatg ctcaaacttc tgtagaccgt
catgggcccc aggtccttga ggccttacac 1440 tatttccgat acgatgaata
tgcacggagc tgcaggttca aaaacaaaga gccaccttct 1500 ttcttgcctt
tgaatgcaga ctgccacata tatgggcaga ccttagactt aagtagaaat 1560
aacatatttt ttattaaacc ttctgatttt cagcatcttt cattcctcaa atgcctcaac
1620 ttatcaggaa acaccattgg ccaaactctt aatggcagtg aactctggcc
gttgagagag 1680 ttgcggtact tagacttctc caacaaccgg cttgatttac
tctactcaac agcctttgaa 1740 gagctccaga gtcttgaagt tctggatcta
agtagtaaca gccactattt tcaagcagaa 1800 ggaattactc acatgctaaa
ctttaccaag aaattacggc ttctggacaa actcatgatg 1860 aatgataatg
acatctctac ttcggccagc aggaccatgg aaagtgactc tcttcgaatt 1920
ctggagttca gaggcaacca tttagatgtt ctatggagag ccggtgataa cagatacttg
1980 gacttcttca agaatttgtt caatttagag gtattagata tctccagaaa
ttccctgaat 2040 tccttgcctc ctgaggtttt tgagggtatg ccgccaaatc
taaagaatct ctccttggcc 2100 aaaaatgggc tcaaatcttt cttttgggac
agactccagt tactgaagca tttggaaatt 2160 ttggacctca gccataacca
gctgacaaaa gtacctgaga gattggccaa ctgttccaaa 2220 agtctcacaa
cactgattct taagcataat caaatcaggc aattgacaaa atattttcta 2280
gaagatgctt tgcaattgcg ctatctagac atcagttcaa ataaaatcca ggtcattcag
2340 aagactagct tcccagaaaa tgtcctcaac aatctggaga tgttggtttt
acatcacaat 2400 cgctttcttt gcaactgtga tgctgtgtgg tttgtctggt
gggttaacca tacagatgtt 2460 actattccat acctggccac tgatgtgact
tgtgtaggtc caggagcaca caaaggtcaa 2520 agtgtcatat cccttgatct
gtatacgtgt gagttagatc tcacaaacct gattctgttc 2580 tcagtttcca
tatcatcagt cctctttctt atggtagtta tgacaacaag tcacctcttt 2640
ttctgggata tgtggtacat ttattatttt tggaaagcaa agataaaggg gtatcagcat
2700 ctgcaatcca tggagtcttg ttatgatgct tttattgtgt atgacactaa
aaactcagct 2760 gtgacagaat gggttttgca ggagctggtg gcaaaattgg
aagatccaag agaaaaacac 2820 ttcaatttgt gtctagaaga aagagactgg
ctaccaggac agccagttct agaaaacctt 2880 tcccagagca tacagctcag
caaaaagaca gtgtttgtga tgacacagaa atatgctaag 2940 actgagagtt
ttaagatggc attttatttg tctcatcaga ggctcctgga tgaaaaagtg 3000
gatgtgatta tcttgatatt cttggaaaag cctcttcaga agtctaagtt tcttcagctc
3060 aggaagagac tctgcaggag ctctgtcctt gagtggcctg caaatccaca
ggctcaccca 3120 tacttctggc agtgcctgaa aaatgccctg accacagaca
atcatgtggc ttatagtcaa 3180 atgttcaagg aaacagtcta gctctctgaa
gaatgtcacc acctaggaca tgccttgaat 3240 cga 3243 <200> SEQUENCE
CHARACTERISTICS: <210> SEQ ID NO 174 <211> LENGTH: 3150
<212> TYPE: DNA <213> ORGANISM: Mus musculus
<220> FEATURE: <221> NAME/KEY: misc_feature <222>
LOCATION: (0)...(0) <223> OTHER INFORMATION: Murine TLR7 ORF
<400> SEQUENCE: 174 atggtgtttt cgatgtggac acggaagaga
caaattttga tctttttaaa tatgctctta 60 gtttctagag tctttgggtt
tcgatggttt cctaaaactc taccttgtga agttaaagta 120 aatatcccag
aggcccatgt gatcgtggac tgcacagaca agcatttgac agaaatccct 180
gagggcattc ccactaacac caccaatctt acccttacca tcaaccacat accaagcatc
240 tctccagatt ccttccgtag gctgaaccat ctggaagaaa tcgatttaag
atgcaattgt 300 gtacctgttc tactggggtc caaagccaat gtgtgtacca
agaggctgca gattagacct 360 ggaagcttta gtggactctc tgacttaaaa
gccctttacc tggatggaaa ccaacttctg 420 gagataccac aggatctgcc
atccagctta catcttctga gccttgaggc taacaacatc 480 ttctccatca
cgaaggagaa tctaacagaa ctggtcaaca ttgaaacact ctacctgggt 540
caaaactgtt attatcgaaa tccttgcaat gtttcctatt ctattgaaaa agatgctttc
600 ctagttatga gaaatttgaa ggttctctca ctaaaagata acaatgtcac
agctgtcccc 660 accactttgc cacctaattt actagagctc tatctttata
acaatatcat taagaaaatc 720 caagaaaatg attttaataa cctcaatgag
ttgcaagttc ttgacctaag tggaaattgc 780 cctcgatgtt ataatgtccc
atatccgtgt acaccgtgtg aaaataattc ccccttacag 840 atccatgaca
atgctttcaa ttcattgaca gaattaaaag ttttacgttt acacagtaat 900
tctcttcagc atgtgccccc aacatggttt aaaaacatga gaaacctcca ggaactagac
960 ctctcccaaa actacttggc cagagaaatt gaggaggcca aatttttgca
ttttcttccc 1020 aaccttgttg agttggattt ttctttcaat tatgagctgc
aggtctacca tgcatctata 1080 actttaccac attcactctc ttcattggaa
aacttgaaaa ttctgcgtgt caaggggtat 1140 gtctttaaag agctgaaaaa
ctccagtctt tctgtattgc acaagcttcc caggctggaa 1200 gttcttgacc
ttggcactaa cttcataaaa attgctgacc tcaacatatt caaacatttt 1260
gaaaacctca aactcataga cctttcagtg aataagatat ctccttcaga agagtcaaga
1320 gaagttggct tttgtcctaa tgctcaaact tctgtagacc gtcatgggcc
ccaggtcctt 1380 gaggccttac actatttccg atacgatgaa tatgcacgga
gctgcaggtt caaaaacaaa 1440 gagccacctt ctttcttgcc tttgaatgca
gactgccaca tatatgggca gaccttagac 1500
ttaagtagaa ataacatatt ttttattaaa ccttctgatt ttcagcatct ttcattcctc
1560 aaatgcctca acttatcagg aaacaccatt ggccaaactc ttaatggcag
tgaactctgg 1620 ccgttgagag agttgcggta cttagacttc tccaacaacc
ggcttgattt actctactca 1680 acagcctttg aagagctcca gagtcttgaa
gttctggatc taagtagtaa cagccactat 1740 tttcaagcag aaggaattac
tcacatgcta aactttacca agaaattacg gcttctggac 1800 aaactcatga
tgaatgataa tgacatctct acttcggcca gcaggaccat ggaaagtgac 1860
tctcttcgaa ttctggagtt cagaggcaac catttagatg ttctatggag agccggtgat
1920 aacagatact tggacttctt caagaatttg ttcaatttag aggtattaga
tatctccaga 1980 aattccctga attccttgcc tcctgaggtt tttgagggta
tgccgccaaa tctaaagaat 2040 ctctccttgg ccaaaaatgg gctcaaatct
ttcttttggg acagactcca gttactgaag 2100 catttggaaa ttttggacct
cagccataac cagctgacaa aagtacctga gagattggcc 2160 aactgttcca
aaagtctcac aacactgatt cttaagcata atcaaatcag gcaattgaca 2220
aaatattttc tagaagatgc tttgcaattg cgctatctag acatcagttc aaataaaatc
2280 caggtcattc agaagactag cttcccagaa aatgtcctca acaatctgga
gatgttggtt 2340 ttacatcaca atcgctttct ttgcaactgt gatgctgtgt
ggtttgtctg gtgggttaac 2400 catacagatg ttactattcc atacctggcc
actgatgtga cttgtgtagg tccaggagca 2460 cacaaaggtc aaagtgtcat
atcccttgat ctgtatacgt gtgagttaga tctcacaaac 2520 ctgattctgt
tctcagtttc catatcatca gtcctctttc ttatggtagt tatgacaaca 2580
agtcacctct ttttctggga tatgtggtac atttattatt tttggaaagc aaagataaag
2640 gggtatcagc atctgcaatc catggagtct tgttatgatg cttttattgt
gtatgacact 2700 aaaaactcag ctgtgacaga atgggttttg caggagctgg
tggcaaaatt ggaagatcca 2760 agagaaaaac acttcaattt gtgtctagaa
gaaagagact ggctaccagg acagccagtt 2820 ctagaaaacc tttcccagag
catacagctc agcaaaaaga cagtgtttgt gatgacacag 2880 aaatatgcta
agactgagag ttttaagatg gcattttatt tgtctcatca gaggctcctg 2940
gatgaaaaag tggatgtgat tatcttgata ttcttggaaa agcctcttca gaagtctaag
3000 tttcttcagc tcaggaagag actctgcagg agctctgtcc ttgagtggcc
tgcaaatcca 3060 caggctcacc catacttctg gcagtgcctg aaaaatgccc
tgaccacaga caatcatgtg 3120 gcttatagtc aaatgttcaa ggaaacagtc 3150
<200> SEQUENCE CHARACTERISTICS: <210> SEQ ID NO 175
<211> LENGTH: 1050 <212> TYPE: PRT <213>
ORGANISM: Mus musculus <400> SEQUENCE: 175 Met Val Phe Ser
Met Trp Thr Arg Lys Arg Gln Ile Leu Ile Phe Leu 1 5 10 15 Asn Met
Leu Leu Val Ser Arg Val Phe Gly Phe Arg Trp Phe Pro Lys 20 25 30
Thr Leu Pro Cys Glu Val Lys Val Asn Ile Pro Glu Ala His Val Ile 35
40 45 Val Asp Cys Thr Asp Lys His Leu Thr Glu Ile Pro Glu Gly Ile
Pro 50 55 60 Thr Asn Thr Thr Asn Leu Thr Leu Thr Ile Asn His Ile
Pro Ser Ile 65 70 75 80 Ser Pro Asp Ser Phe Arg Arg Leu Asn His Leu
Glu Glu Ile Asp Leu 85 90 95 Arg Cys Asn Cys Val Pro Val Leu Leu
Gly Ser Lys Ala Asn Val Cys 100 105 110 Thr Lys Arg Leu Gln Ile Arg
Pro Gly Ser Phe Ser Gly Leu Ser Asp 115 120 125 Leu Lys Ala Leu Tyr
Leu Asp Gly Asn Gln Leu Leu Glu Ile Pro Gln 130 135 140 Asp Leu Pro
Ser Ser Leu His Leu Leu Ser Leu Glu Ala Asn Asn Ile 145 150 155 160
Phe Ser Ile Thr Lys Glu Asn Leu Thr Glu Leu Val Asn Ile Glu Thr 165
170 175 Leu Tyr Leu Gly Gln Asn Cys Tyr Tyr Arg Asn Pro Cys Asn Val
Ser 180 185 190 Tyr Ser Ile Glu Lys Asp Ala Phe Leu Val Met Arg Asn
Leu Lys Val 195 200 205 Leu Ser Leu Lys Asp Asn Asn Val Thr Ala Val
Pro Thr Thr Leu Pro 210 215 220 Pro Asn Leu Leu Glu Leu Tyr Leu Tyr
Asn Asn Ile Ile Lys Lys Ile 225 230 235 240 Gln Glu Asn Asp Phe Asn
Asn Leu Asn Glu Leu Gln Val Leu Asp Leu 245 250 255 Ser Gly Asn Cys
Pro Arg Cys Tyr Asn Val Pro Tyr Pro Cys Thr Pro 260 265 270 Cys Glu
Asn Asn Ser Pro Leu Gln Ile His Asp Asn Ala Phe Asn Ser 275 280 285
Leu Thr Glu Leu Lys Val Leu Arg Leu His Ser Asn Ser Leu Gln His 290
295 300 Val Pro Pro Thr Trp Phe Lys Asn Met Arg Asn Leu Gln Glu Leu
Asp 305 310 315 320 Leu Ser Gln Asn Tyr Leu Ala Arg Glu Ile Glu Glu
Ala Lys Phe Leu 325 330 335 His Phe Leu Pro Asn Leu Val Glu Leu Asp
Phe Ser Phe Asn Tyr Glu 340 345 350 Leu Gln Val Tyr His Ala Ser Ile
Thr Leu Pro His Ser Leu Ser Ser 355 360 365 Leu Glu Asn Leu Lys Ile
Leu Arg Val Lys Gly Tyr Val Phe Lys Glu 370 375 380 Leu Lys Asn Ser
Ser Leu Ser Val Leu His Lys Leu Pro Arg Leu Glu 385 390 395 400 Val
Leu Asp Leu Gly Thr Asn Phe Ile Lys Ile Ala Asp Leu Asn Ile 405 410
415 Phe Lys His Phe Glu Asn Leu Lys Leu Ile Asp Leu Ser Val Asn Lys
420 425 430 Ile Ser Pro Ser Glu Glu Ser Arg Glu Val Gly Phe Cys Pro
Asn Ala 435 440 445 Gln Thr Ser Val Asp Arg His Gly Pro Gln Val Leu
Glu Ala Leu His 450 455 460 Tyr Phe Arg Tyr Asp Glu Tyr Ala Arg Ser
Cys Arg Phe Lys Asn Lys 465 470 475 480 Glu Pro Pro Ser Phe Leu Pro
Leu Asn Ala Asp Cys His Ile Tyr Gly 485 490 495 Gln Thr Leu Asp Leu
Ser Arg Asn Asn Ile Phe Phe Ile Lys Pro Ser 500 505 510 Asp Phe Gln
His Leu Ser Phe Leu Lys Cys Leu Asn Leu Ser Gly Asn 515 520 525 Thr
Ile Gly Gln Thr Leu Asn Gly Ser Glu Leu Trp Pro Leu Arg Glu 530 535
540 Leu Arg Tyr Leu Asp Phe Ser Asn Asn Arg Leu Asp Leu Leu Tyr Ser
545 550 555 560 Thr Ala Phe Glu Glu Leu Gln Ser Leu Glu Val Leu Asp
Leu Ser Ser 565 570 575 Asn Ser His Tyr Phe Gln Ala Glu Gly Ile Thr
His Met Leu Asn Phe 580 585 590 Thr Lys Lys Leu Arg Leu Leu Asp Lys
Leu Met Met Asn Asp Asn Asp 595 600 605 Ile Ser Thr Ser Ala Ser Arg
Thr Met Glu Ser Asp Ser Leu Arg Ile 610 615 620 Leu Glu Phe Arg Gly
Asn His Leu Asp Val Leu Trp Arg Ala Gly Asp 625 630 635 640 Asn Arg
Tyr Leu Asp Phe Phe Lys Asn Leu Phe Asn Leu Glu Val Leu 645 650 655
Asp Ile Ser Arg Asn Ser Leu Asn Ser Leu Pro Pro Glu Val Phe Glu 660
665 670 Gly Met Pro Pro Asn Leu Lys Asn Leu Ser Leu Ala Lys Asn Gly
Leu 675 680 685 Lys Ser Phe Phe Trp Asp Arg Leu Gln Leu Leu Lys His
Leu Glu Ile 690 695 700 Leu Asp Leu Ser His Asn Gln Leu Thr Lys Val
Pro Glu Arg Leu Ala 705 710 715 720 Asn Cys Ser Lys Ser Leu Thr Thr
Leu Ile Leu Lys His Asn Gln Ile 725 730 735 Arg Gln Leu Thr Lys Tyr
Phe Leu Glu Asp Ala Leu Gln Leu Arg Tyr 740 745 750 Leu Asp Ile Ser
Ser Asn Lys Ile Gln Val Ile Gln Lys Thr Ser Phe 755 760 765 Pro Glu
Asn Val Leu Asn Asn Leu Glu Met Leu Val Leu His His Asn 770 775 780
Arg Phe Leu Cys Asn Cys Asp Ala Val Trp Phe Val Trp Trp Val Asn 785
790 795 800 His Thr Asp Val Thr Ile Pro Tyr Leu Ala Thr Asp Val Thr
Cys Val 805 810 815 Gly Pro Gly Ala His Lys Gly Gln Ser Val Ile Ser
Leu Asp Leu Tyr 820 825 830 Thr Cys Glu Leu Asp Leu Thr Asn Leu Ile
Leu Phe Ser Val Ser Ile 835 840 845 Ser Ser Val Leu Phe Leu Met Val
Val Met Thr Thr Ser His Leu Phe 850 855 860 Phe Trp Asp Met Trp Tyr
Ile Tyr Tyr Phe Trp Lys Ala Lys Ile Lys 865 870 875 880 Gly Tyr Gln
His Leu Gln Ser Met Glu Ser Cys Tyr Asp Ala Phe Ile 885 890 895 Val
Tyr Asp Thr Lys Asn Ser Ala Val Thr Glu Trp Val Leu Gln Glu 900 905
910 Leu Val Ala Lys Leu Glu Asp Pro Arg Glu Lys His Phe Asn Leu Cys
915 920 925 Leu Glu Glu Arg Asp Trp Leu Pro Gly Gln Pro Val Leu Glu
Asn Leu 930 935 940 Ser Gln Ser Ile Gln Leu Ser Lys Lys Thr Val Phe
Val Met Thr Gln 945 950 955 960 Lys Tyr Ala Lys Thr Glu Ser Phe Lys
Met Ala Phe Tyr Leu Ser His 965 970 975 Gln Arg Leu Leu Asp Glu Lys
Val Asp Val Ile Ile Leu Ile Phe Leu 980 985 990 Glu Lys Pro Leu Gln
Lys Ser Lys Phe Leu Gln Leu Arg Lys Arg Leu 995 1000 1005 Cys Arg
Ser Ser Val Leu Glu Trp Pro Ala Asn Pro Gln Ala His Pro 1010 1015
1020 Tyr Phe Trp Gln Cys Leu Lys Asn Ala Leu Thr Thr Asp Asn His
Val 1025 1030 1035 1040 Ala Tyr Ser Gln Met Phe Lys Glu Thr Val
1045 1050 <200> SEQUENCE CHARACTERISTICS: <210> SEQ ID
NO 176 <211> LENGTH: 66 <212> TYPE: PRT <213>
ORGANISM: Mus musculus <400> SEQUENCE: 176 Val Asp Val Ile
Ile Leu Ile Phe Leu Val Lys Pro Phe Gln Lys Phe 1 5 10 15 Asn Phe
Leu Leu Arg Lys Arg Ile Ser Arg Ser Ser Val Leu Glu Cys 20 25 30
Pro Pro Asn Pro Gln Ala His Pro Tyr Phe Cys Gln Cys Leu Lys Asn 35
40 45 Ala Leu Thr Thr Asp Asn His Val Ala Tyr Ser Gln Met Phe Lys
Glu 50 55 60 Thr Val 65 <200> SEQUENCE CHARACTERISTICS:
<210> SEQ ID NO 177 <211> LENGTH: 54 <212> TYPE:
PRT <213> ORGANISM: Mus musculus <400> SEQUENCE: 177
Gln Lys Ser Lys Phe Leu Gln Leu Arg Lys Arg Leu Cys Arg Ser Ser 1 5
10 15 Val Leu Glu Trp Pro Ala Asn Pro Gln Ala His Pro Tyr Phe Trp
Gln 20 25 30 Cys Leu Lys Asn Ala Leu Thr Thr Asp Asn His Val Ala
Tyr Ser Gln 35 40 45 Met Phe Lys Glu Thr Val 50 <200>
SEQUENCE CHARACTERISTICS: <210> SEQ ID NO 178 <211>
LENGTH: 59 <212> TYPE: PRT <213> ORGANISM: Mus musculus
<400> SEQUENCE: 178 Leu Gly Lys Pro Leu Gln Lys Ser Lys Phe
Leu Gln Leu Arg Lys Arg 1 5 10 15 Leu Cys Arg Ser Ser Val Leu Glu
Trp Pro Ala Asn Pro Gln Ala His 20 25 30 Pro Tyr Phe Trp Gln Cys
Leu Lys Asn Ala Leu Thr Thr Asp Asn His 35 40 45 Val Ala Tyr Ser
Gln Met Phe Lys Glu Thr Val 50 55 <200> SEQUENCE
CHARACTERISTICS: <210> SEQ ID NO 179 <211> LENGTH: 84
<212> TYPE: PRT <213> ORGANISM: Mus musculus
<400> SEQUENCE: 179 Ile Glu Thr Phe Gln Met Pro Ser Phe Leu
Ser Ile Gln Arg Leu Leu 1 5 10 15 Asp Asp Lys Val Asp Val Ile Ile
Leu Ile Phe Leu Glu Pro Leu Lys 20 25 30 Ser Lys Phe Leu Gln Leu
Arg Lys Arg Phe Cys Arg Ser Ser Val Leu 35 40 45 Glu Trp Pro Ala
Asn Pro Gln Ala His Pro Tyr Phe Trp Gln Cys Leu 50 55 60 Lys Asn
Ala Leu Thr Thr Asp Asn His Val Ala Tyr Ser Gln Met Phe 65 70 75 80
Lys Glu Thr Val <200> SEQUENCE CHARACTERISTICS: <210>
SEQ ID NO 180 <211> LENGTH: 25 <212> TYPE: DNA
<213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic oligonucleotide
<400> SEQUENCE: 180 ctgcgctgct gcaagttacg gaatg 25
<200> SEQUENCE CHARACTERISTICS: <210> SEQ ID NO 181
<211> LENGTH: 25 <212> TYPE: DNA <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Synthetic oligonucleotide <400> SEQUENCE: 181
gcgcgaaatc atgacttaac gtcag 25 <200> SEQUENCE
CHARACTERISTICS: <210> SEQ ID NO 182 <211> LENGTH: 3310
<212> TYPE: DNA <213> ORGANISM: Homo sapiens
<220> FEATURE:
<221> NAME/KEY: misc_feature <222> LOCATION: (0)...(0)
<223> OTHER INFORMATION: Human TLR8 cDNA <400>
SEQUENCE: 182 gctcccggcc gccatggcgg ccgcgggaat tcgattctgc
gctgctgcaa gttacggaat 60 gaaaaattag aacaacagaa acatggaaaa
catgttcctt cagtcgtcaa tgctgacctg 120 cattttcctg ctaatatctg
gttcctgtga gttatgcgcc gaagaaaatt tttctagaag 180 ctatccttgt
gatgagaaaa agcaaaatga ctcagttatt gcagagtgca gcaatcgtcg 240
actacaggaa gttccccaaa cggtgggcaa atatgtgaca gaactagacc tgtctgataa
300 tttcatcaca cacataacga atgaatcatt tcaagggctg caaaatctca
ctaaaataaa 360 tctaaaccac aaccccaatg tacagcacca gaacggaaat
cccggtatac aatcaaatgg 420 cttgaatatc acagacgggg cattcctcaa
cctaaaaaac ctaagggagt tactgcttga 480 agacaaccag ttaccccaaa
taccctctgg tttgccagag tctttgacag aacttagtct 540 aattcaaaac
aatatataca acataactaa agagggcatt tcaagactta taaacttgaa 600
aaatctctat ttggcctgga actgctattt taacaaagtt tgcgagaaaa ctaacataga
660 agatggagta tttgaaacgc tgacaaattt ggagttgcta tcactatctt
tcaattctct 720 ttcacacgtg ccacccaaac tgccaagctc cctacgcaaa
ctttttctga gcaacaccca 780 gatcaaatac attagtgaag aagatttcaa
gggattgata aatttaacat tactagattt 840 aagcgggaac tgtccgaggt
gcttcaatgc cccatttcca tgcgtgcctt gtgatggtgg 900 tgcttcaatt
aatatagatc gttttgcttt tcaaaacttg acccaacttc gatacctaaa 960
cctctctagc acttccctca ggaagattaa tgctgcctgg tttaaaaata tgcctcatct
1020 gaaggtgctg gatcttgaat tcaactattt agtgggagaa atagcctctg
gggcattttt 1080 aacgatgctg ccccgcttag aaatacttga cttgtctttt
aactatataa aggggagtta 1140 tccacagcat attaatattt ccagaaactt
ctctaaactt ttgtctctac gggcattgca 1200 tttaagaggt tatgtgttcc
aggaactcag agaagatgat ttccagcccc tgatgcagct 1260 tccaaactta
tcgactatca acttgggtat taattttatt aagcaaatcg atttcaaact 1320
tttccaaaat ttctccaatc tggaaattat ttacttgtca gaaaacagaa tatcaccgtt
1380 ggtaaaagat acccggcaga gttatgcaaa tagttcctct tttcaacgtc
atatccggaa 1440 acgacgctca acagattttg agtttgaccc acattcgaac
ttttatcatt tcacccgtcc 1500 tttaataaag ccacaatgtg ctgcttatgg
aaaagcctta gatttaagcc tcaacagtat 1560 tttcttcatt gggccaaacc
aatttgaaaa tcttcctgac attgcctgtt taaatctgtc 1620 tgcaaatagc
aatgctcaag tgttaagtgg aactgaattt tcagccattc ctcatgtcaa 1680
atatttggat ttgacaaaca atagactaga ctttgataat gctagtgctc ttactgaatt
1740 gtccgacttg gaagttctag atctcagcta taattcacac tatttcagaa
tagcaggcgt 1800 aacacatcat ctagaattta ttcaaaattt cacaaatcta
aaagttttaa acttgagcca 1860 caacaacatt tatactttaa cagataagta
taacctggaa agcaagtccc tggtagaatt 1920 agttttcagt ggcaatcgcc
ttgacatttt gtggaatgat gatgacaaca ggtatatctc 1980 cattttcaaa
ggtctcaaga atctgacacg tctggattta tcccttaata ggctgaagca 2040
catcccaaat gaagcattcc ttaatttgcc agcgagtctc actgaactac atataaatga
2100 taatatgtta aagtttttta actggacatt actccagcag tttcctcgtc
tcgagttgct 2160 tgacttacgt ggaaacaaac tactcttttt aactgatagc
ctatctgact ttacatcttc 2220 ccttcggaca ctgctgctga gtcataacag
gatttcccac ctaccctctg gctttctttc 2280 tgaagtcagt agtctgaagc
acctcgattt aagttccaat ctgctaaaaa caatcaacaa 2340 atccgcactt
gaaactaaga ccaccaccaa attatctatg ttggaactac acggaaaccc 2400
ctttgaatgc acctgtgaca ttggagattt ccgaagatgg atggatgaac atctgaatgt
2460 caaaattccc agactggtag atgtcatttg tgccagtcct ggggatcaaa
gagggaagag 2520 tattgtgagt ctggagctaa caacttgtgt ttcagatgtc
actgcagtga tattattttt 2580 cttcacgttc tttatcacca ccatggttat
gttggctgcc ctggctcacc atttgtttta 2640 ctgggatgtt tggtttatat
ataatgtgtg tttagctaag gtaaaaggct acaggtctct 2700 ttccacatcc
caaactttct atgatgctta catttcttat gacaccaaag acgcctctgt 2760
tactgactgg gtgataaatg agctgcgcta ccaccttgaa gagagccgag acaaaaacgt
2820 tctcctttgt ctagaggaga gggattggga cccgggattg gccatcatcg
acaacctcat 2880 gcagagcatc aaccaaagca agaaaacagt atttgtttta
accaaaaaat atgcaaaaag 2940 ctggaacttt aaaacagctt tttacttggc
tttgcagagg ctaatggatg agaacatgga 3000 tgtgattata tttatcctgc
tggagccagt gttacagcat tctcagtatt tgaggctacg 3060 gcagcggatc
tgtaagagct ccatcctcca gtggcctgac aacccgaagg cagaaggctt 3120
gttttggcaa actctgagaa atgtggtctt gactgaaaat gattcacggt ataacaatat
3180 gtatgtcgat tccattaagc aatactaact gacgttaagt catgatttcg
cgcaatcact 3240 agtgaattcg cggccgcctg caggtcgacc atatgggaga
gctcccaacg cgttggatgc 3300 atagcttgag 3310 <200> SEQUENCE
CHARACTERISTICS: <210> SEQ ID NO 183 <211> LENGTH: 3123
<212> TYPE: DNA <213> ORGANISM: Homo sapiens
<220> FEATURE: <221> NAME/KEY: misc_feature <222>
LOCATION: (0)...(0) <223> OTHER INFORMATION: Human TLR8 ORF
<400> SEQUENCE: 183 atggaaaaca tgttccttca gtcgtcaatg
ctgacctgca ttttcctgct aatatctggt 60 tcctgtgagt tatgcgccga
agaaaatttt tctagaagct atccttgtga tgagaaaaag 120 caaaatgact
cagttattgc agagtgcagc aatcgtcgac tacaggaagt tccccaaacg 180
gtgggcaaat atgtgacaga actagacctg tctgataatt tcatcacaca cataacgaat
240 gaatcatttc aagggctgca aaatctcact aaaataaatc taaaccacaa
ccccaatgta 300 cagcaccaga acggaaatcc cggtatacaa tcaaatggct
tgaatatcac agacggggca 360 ttcctcaacc taaaaaacct aagggagtta
ctgcttgaag acaaccagtt accccaaata 420 ccctctggtt tgccagagtc
tttgacagaa cttagtctaa ttcaaaacaa tatatacaac 480 ataactaaag
agggcatttc aagacttata aacttgaaaa atctctattt ggcctggaac 540
tgctatttta acaaagtttg cgagaaaact aacatagaag atggagtatt tgaaacgctg
600 acaaatttgg agttgctatc actatctttc aattctcttt cacacgtgcc
acccaaactg 660 ccaagctccc tacgcaaact ttttctgagc aacacccaga
tcaaatacat tagtgaagaa 720 gatttcaagg gattgataaa tttaacatta
ctagatttaa gcgggaactg tccgaggtgc 780 ttcaatgccc catttccatg
cgtgccttgt gatggtggtg cttcaattaa tatagatcgt 840 tttgcttttc
aaaacttgac ccaacttcga tacctaaacc tctctagcac ttccctcagg 900
aagattaatg ctgcctggtt taaaaatatg cctcatctga aggtgctgga tcttgaattc
960 aactatttag tgggagaaat agcctctggg gcatttttaa cgatgctgcc
ccgcttagaa 1020 atacttgact tgtcttttaa ctatataaag gggagttatc
cacagcatat taatatttcc 1080 agaaacttct ctaaactttt gtctctacgg
gcattgcatt taagaggtta tgtgttccag 1140 gaactcagag aagatgattt
ccagcccctg atgcagcttc caaacttatc gactatcaac 1200 ttgggtatta
attttattaa gcaaatcgat ttcaaacttt tccaaaattt ctccaatctg 1260
gaaattattt acttgtcaga aaacagaata tcaccgttgg taaaagatac ccggcagagt
1320 tatgcaaata gttcctcttt tcaacgtcat atccggaaac gacgctcaac
agattttgag 1380 tttgacccac attcgaactt ttatcatttc acccgtcctt
taataaagcc acaatgtgct 1440 gcttatggaa aagccttaga tttaagcctc
aacagtattt tcttcattgg gccaaaccaa 1500 tttgaaaatc ttcctgacat
tgcctgttta aatctgtctg caaatagcaa tgctcaagtg 1560 ttaagtggaa
ctgaattttc agccattcct catgtcaaat atttggattt gacaaacaat 1620
agactagact ttgataatgc tagtgctctt actgaattgt ccgacttgga agttctagat
1680 ctcagctata attcacacta tttcagaata gcaggcgtaa cacatcatct
agaatttatt 1740 caaaatttca caaatctaaa agttttaaac ttgagccaca
acaacattta tactttaaca 1800 gataagtata acctggaaag caagtccctg
gtagaattag ttttcagtgg caatcgcctt 1860 gacattttgt ggaatgatga
tgacaacagg tatatctcca ttttcaaagg tctcaagaat 1920 ctgacacgtc
tggatttatc ccttaatagg ctgaagcaca tcccaaatga agcattcctt 1980
aatttgccag cgagtctcac tgaactacat ataaatgata atatgttaaa gttttttaac
2040 tggacattac tccagcagtt tcctcgtctc gagttgcttg acttacgtgg
aaacaaacta 2100 ctctttttaa ctgatagcct atctgacttt acatcttccc
ttcggacact gctgctgagt 2160 cataacagga tttcccacct accctctggc
tttctttctg aagtcagtag tctgaagcac 2220 ctcgatttaa gttccaatct
gctaaaaaca atcaacaaat ccgcacttga aactaagacc 2280 accaccaaat
tatctatgtt ggaactacac ggaaacccct ttgaatgcac ctgtgacatt 2340
ggagatttcc gaagatggat ggatgaacat ctgaatgtca aaattcccag actggtagat
2400 gtcatttgtg ccagtcctgg ggatcaaaga gggaagagta ttgtgagtct
ggagctaaca 2460 acttgtgttt cagatgtcac tgcagtgata ttatttttct
tcacgttctt tatcaccacc 2520 atggttatgt tggctgccct ggctcaccat
ttgttttact gggatgtttg gtttatatat 2580 aatgtgtgtt tagctaaggt
aaaaggctac aggtctcttt ccacatccca aactttctat 2640 gatgcttaca
tttcttatga caccaaagac gcctctgtta ctgactgggt gataaatgag 2700
ctgcgctacc accttgaaga gagccgagac aaaaacgttc tcctttgtct agaggagagg
2760 gattgggacc cgggattggc catcatcgac aacctcatgc agagcatcaa
ccaaagcaag 2820 aaaacagtat ttgttttaac caaaaaatat gcaaaaagct
ggaactttaa aacagctttt 2880 tacttggctt tgcagaggct aatggatgag
aacatggatg tgattatatt tatcctgctg 2940 gagccagtgt tacagcattc
tcagtatttg aggctacggc agcggatctg taagagctcc 3000 atcctccagt
ggcctgacaa cccgaaggca gaaggcttgt tttggcaaac tctgagaaat 3060
gtggtcttga ctgaaaatga ttcacggtat aacaatatgt atgtcgattc cattaagcaa
3120 tac 3123 <200> SEQUENCE CHARACTERISTICS: <210> SEQ
ID NO 184 <211> LENGTH: 1041 <212> TYPE: PRT
<213> ORGANISM: Homo sapiens <400> SEQUENCE: 184 Met
Glu Asn Met Phe Leu Gln Ser Ser Met Leu Thr Cys Ile Phe Leu 1 5 10
15 Leu Ile Ser Gly Ser Cys Glu Leu Cys Ala Glu Glu Asn Phe Ser Arg
20 25 30 Ser Tyr Pro Cys Asp Glu Lys Lys Gln Asn Asp Ser Val Ile
Ala Glu 35 40 45 Cys Ser Asn Arg Arg Leu Gln Glu Val Pro Gln Thr
Val Gly Lys Tyr 50 55 60 Val Thr Glu Leu Asp Leu Ser Asp Asn Phe
Ile Thr His Ile Thr Asn 65 70 75 80 Glu Ser Phe Gln Gly Leu Gln Asn
Leu Thr Lys Ile Asn Leu Asn His 85 90 95 Asn Pro Asn Val Gln His
Gln Asn Gly Asn Pro Gly Ile Gln Ser Asn 100 105 110 Gly Leu Asn Ile
Thr Asp Gly Ala Phe Leu Asn Leu Lys Asn Leu Arg 115 120 125 Glu Leu
Leu Leu Glu Asp Asn Gln Leu Pro Gln Ile Pro Ser Gly Leu 130 135 140
Pro Glu Ser Leu Thr Glu Leu Ser Leu Ile Gln Asn Asn Ile Tyr Asn 145
150 155 160 Ile Thr Lys Glu Gly Ile Ser Arg Leu Ile Asn Leu Lys Asn
Leu Tyr 165 170 175 Leu Ala Trp Asn Cys Tyr Phe Asn Lys Val Cys Glu
Lys Thr Asn Ile 180 185 190 Glu Asp Gly Val Phe Glu Thr Leu Thr Asn
Leu Glu Leu Leu Ser Leu 195 200 205 Ser Phe Asn Ser Leu Ser His Val
Pro Pro Lys Leu Pro Ser Ser Leu 210 215 220 Arg Lys Leu Phe Leu Ser
Asn Thr Gln Ile Lys Tyr Ile Ser Glu Glu 225 230 235 240 Asp Phe Lys
Gly Leu Ile Asn Leu Thr Leu Leu Asp Leu Ser Gly Asn 245 250 255 Cys
Pro Arg Cys Phe Asn Ala Pro Phe Pro Cys Val Pro Cys Asp Gly 260 265
270 Gly Ala Ser Ile Asn Ile Asp Arg Phe Ala Phe Gln Asn Leu Thr Gln
275 280 285 Leu Arg Tyr Leu Asn Leu Ser Ser Thr Ser Leu Arg Lys Ile
Asn Ala 290 295 300 Ala Trp Phe Lys Asn Met Pro His Leu Lys Val Leu
Asp Leu Glu Phe 305 310 315 320 Asn Tyr Leu Val Gly Glu Ile Ala Ser
Gly Ala Phe Leu Thr Met Leu 325 330 335 Pro Arg Leu Glu Ile Leu Asp
Leu Ser Phe Asn Tyr Ile Lys Gly Ser 340 345 350 Tyr Pro Gln His Ile
Asn Ile Ser Arg Asn Phe Ser Lys Leu Leu Ser 355 360 365 Leu Arg Ala
Leu His Leu Arg Gly Tyr Val Phe Gln Glu Leu Arg Glu 370 375 380 Asp
Asp Phe Gln Pro Leu Met Gln Leu Pro Asn Leu Ser Thr Ile Asn 385 390
395 400 Leu Gly Ile Asn Phe Ile Lys Gln Ile Asp Phe Lys Leu Phe Gln
Asn 405 410 415 Phe Ser Asn Leu Glu Ile Ile Tyr Leu Ser Glu Asn Arg
Ile Ser Pro 420 425 430 Leu Val Lys Asp Thr Arg Gln Ser Tyr Ala Asn
Ser Ser Ser Phe Gln 435 440 445 Arg His Ile Arg Lys Arg Arg Ser Thr
Asp Phe Glu Phe Asp Pro His 450 455 460 Ser Asn Phe Tyr His Phe Thr
Arg Pro Leu Ile Lys Pro Gln Cys Ala 465 470 475 480 Ala Tyr Gly Lys
Ala Leu Asp Leu Ser Leu Asn Ser Ile Phe Phe Ile 485 490 495 Gly Pro
Asn Gln Phe Glu Asn Leu Pro Asp Ile Ala Cys Leu Asn Leu 500 505 510
Ser Ala Asn Ser Asn Ala Gln Val Leu Ser Gly Thr Glu Phe Ser Ala 515
520 525 Ile Pro His Val Lys Tyr Leu Asp Leu Thr Asn Asn Arg Leu Asp
Phe 530 535 540 Asp Asn Ala Ser Ala Leu Thr Glu Leu Ser Asp Leu Glu
Val Leu Asp 545 550 555 560 Leu Ser Tyr Asn Ser His Tyr Phe Arg Ile
Ala Gly Val Thr His His 565 570 575 Leu Glu Phe Ile Gln Asn Phe Thr
Asn Leu Lys Val Leu Asn Leu Ser 580 585 590 His Asn Asn Ile Tyr Thr
Leu Thr Asp Lys Tyr Asn Leu Glu Ser Lys 595 600 605 Ser Leu Val Glu
Leu Val Phe Ser Gly Asn Arg Leu Asp Ile Leu Trp 610 615 620 Asn Asp
Asp Asp Asn Arg Tyr Ile Ser Ile Phe Lys Gly Leu Lys Asn 625 630 635
640 Leu Thr Arg Leu Asp Leu Ser Leu Asn Arg Leu Lys His Ile Pro Asn
645 650 655 Glu Ala Phe Leu Asn Leu Pro Ala Ser Leu Thr Glu Leu His
Ile Asn 660 665 670 Asp Asn Met Leu Lys Phe Phe Asn Trp Thr Leu Leu
Gln Gln Phe Pro 675 680 685 Arg Leu Glu Leu Leu Asp Leu Arg Gly Asn
Lys Leu Leu Phe Leu Thr 690 695 700 Asp Ser Leu Ser Asp Phe Thr Ser
Ser Leu Arg Thr Leu Leu Leu Ser 705 710 715 720 His Asn Arg Ile Ser
His Leu Pro Ser Gly Phe Leu Ser Glu Val Ser 725 730 735 Ser Leu Lys
His Leu Asp Leu Ser Ser Asn Leu Leu Lys Thr Ile Asn 740 745 750 Lys
Ser Ala Leu Glu Thr Lys Thr Thr Thr Lys Leu Ser Met Leu Glu 755 760
765 Leu His Gly Asn Pro Phe Glu Cys Thr Cys Asp Ile Gly Asp Phe Arg
770 775 780 Arg Trp Met Asp Glu His Leu Asn Val Lys Ile Pro Arg Leu
Val Asp 785 790 795 800 Val Ile Cys Ala Ser Pro Gly Asp Gln Arg Gly
Lys Ser Ile Val Ser 805 810 815 Leu Glu Leu Thr Thr Cys Val Ser Asp
Val Thr Ala Val Ile Leu Phe 820 825 830 Phe Phe Thr Phe Phe Ile Thr
Thr Met Val Met Leu Ala Ala Leu Ala 835 840 845 His His Leu Phe Tyr
Trp Asp Val Trp Phe Ile Tyr Asn Val Cys Leu 850 855 860 Ala Lys Val
Lys Gly Tyr Arg Ser Leu Ser Thr Ser Gln Thr Phe Tyr 865 870 875 880
Asp Ala Tyr Ile Ser Tyr Asp Thr Lys Asp Ala Ser Val Thr Asp Trp 885
890 895 Val Ile Asn Glu Leu Arg Tyr His Leu Glu Glu Ser Arg Asp Lys
Asn 900 905 910 Val Leu Leu Cys Leu Glu Glu Arg Asp Trp Asp Pro Gly
Leu Ala Ile 915 920 925 Ile Asp Asn Leu Met Gln Ser Ile Asn Gln Ser
Lys Lys Thr Val Phe 930 935 940 Val Leu Thr Lys Lys Tyr Ala Lys Ser
Trp Asn Phe Lys Thr Ala Phe 945 950 955 960 Tyr Leu Ala Leu Gln Arg
Leu Met Asp Glu Asn Met Asp Val Ile Ile 965 970 975
Phe Ile Leu Leu Glu Pro Val Leu Gln His Ser Gln Tyr Leu Arg Leu 980
985 990 Arg Gln Arg Ile Cys Lys Ser Ser Ile Leu Gln Trp Pro Asp Asn
Pro 995 1000 1005 Lys Ala Glu Gly Leu Phe Trp Gln Thr Leu Arg Asn
Val Val Leu Thr 1010 1015 1020 Glu Asn Asp Ser Arg Tyr Asn Asn Met
Tyr Val Asp Ser Ile Lys Gln 1025 1030 1035 1040 Tyr <200>
SEQUENCE CHARACTERISTICS: <210> SEQ ID NO 185 <211>
LENGTH: 19 <212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 185 Met Lys Glu Ser Ser Leu Gln Asn Ser Ser
Cys Ser Leu Gly Lys Glu 1 5 10 15 Thr Lys Lys <200> SEQUENCE
CHARACTERISTICS: <210> SEQ ID NO 186 <211> LENGTH: 1041
<212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 186 Met Glu Asn Met Phe Leu Gln Ser Ser Met
Leu Thr Cys Ile Phe Leu 1 5 10 15 Leu Ile Ser Gly Ser Cys Glu Leu
Cys Ala Glu Glu Asn Phe Ser Arg 20 25 30 Ser Tyr Pro Cys Asp Glu
Lys Lys Gln Asn Asp Ser Val Ile Ala Glu 35 40 45 Cys Ser Asn Arg
Arg Leu Gln Glu Val Pro Gln Thr Val Gly Lys Tyr 50 55 60 Val Thr
Glu Leu Asp Leu Ser Asp Asn Phe Ile Thr His Ile Thr Asn 65 70 75 80
Glu Ser Phe Gln Gly Leu Gln Asn Leu Thr Lys Ile Asn Leu Asn His 85
90 95 Asn Pro Asn Val Gln His Gln Asn Gly Asn Pro Gly Ile Gln Ser
Asn 100 105 110 Gly Leu Asn Ile Thr Asp Gly Ala Phe Leu Asn Leu Lys
Asn Leu Arg 115 120 125 Glu Leu Leu Leu Glu Asp Asn Gln Leu Pro Gln
Ile Pro Ser Gly Leu 130 135 140 Pro Glu Ser Leu Thr Glu Leu Ser Leu
Ile Gln Asn Asn Ile Tyr Asn 145 150 155 160 Ile Thr Lys Glu Gly Ile
Ser Arg Leu Ile Asn Leu Lys Asn Leu Tyr 165 170 175 Leu Ala Trp Asn
Cys Tyr Phe Asn Lys Val Cys Glu Lys Thr Asn Ile 180 185 190 Glu Asp
Gly Val Phe Glu Thr Leu Thr Asn Leu Glu Leu Leu Ser Leu 195 200 205
Ser Phe Asn Ser Leu Ser His Val Pro Pro Lys Leu Pro Ser Ser Leu 210
215 220 Arg Lys Leu Phe Leu Ser Asn Thr Gln Ile Lys Tyr Ile Ser Glu
Glu 225 230 235 240 Asp Phe Lys Gly Leu Ile Asn Leu Thr Leu Leu Asp
Leu Ser Gly Asn 245 250 255 Cys Pro Arg Cys Phe Asn Ala Pro Phe Pro
Cys Val Pro Cys Asp Gly 260 265 270 Gly Ala Ser Ile Asn Ile Asp Arg
Phe Ala Phe Gln Asn Leu Thr Gln 275 280 285 Leu Arg Tyr Leu Asn Leu
Ser Ser Thr Ser Leu Arg Lys Ile Asn Ala 290 295 300 Ala Trp Phe Lys
Asn Met Pro His Leu Lys Val Leu Asp Leu Glu Phe 305 310 315 320 Asn
Tyr Leu Val Gly Glu Ile Ala Ser Gly Ala Phe Leu Thr Met Leu 325 330
335 Pro Arg Leu Glu Ile Leu Asp Leu Ser Phe Asn Tyr Ile Lys Gly Ser
340 345 350 Tyr Pro Gln His Ile Asn Ile Ser Arg Asn Phe Ser Lys Leu
Leu Ser 355 360 365 Leu Arg Ala Leu His Leu Arg Gly Tyr Val Phe Gln
Glu Leu Arg Glu 370 375 380 Asp Asp Phe Gln Pro Leu Met Gln Leu Pro
Asn Leu Ser Thr Ile Asn 385 390 395 400 Leu Gly Ile Asn Phe Ile Lys
Gln Ile Asp Phe Lys Leu Phe Gln Asn 405 410 415 Phe Ser Asn Leu Glu
Ile Ile Tyr Leu Ser Glu Asn Arg Ile Ser Pro 420 425 430 Leu Val Lys
Asp Thr Arg Gln Ser Tyr Ala Asn Ser Ser Ser Phe Gln 435 440 445 Arg
His Ile Arg Lys Arg Arg Ser Thr Asp Phe Glu Phe Asp Pro His 450 455
460 Ser Asn Phe Tyr His Phe Thr Arg Pro Leu Ile Lys Pro Gln Cys Ala
465 470 475 480 Ala Tyr Gly Lys Ala Leu Asp Leu Ser Leu Asn Ser Ile
Phe Phe Ile 485 490 495 Gly Pro Asn Gln Phe Glu Asn Leu Pro Asp Ile
Ala Cys Leu Asn Leu 500 505 510 Ser Ala Asn Ser Asn Ala Gln Val Leu
Ser Gly Thr Glu Phe Ser Ala 515 520 525 Ile Pro His Val Lys Tyr Leu
Asp Leu Thr Asn Asn Arg Leu Asp Phe 530 535 540 Asp Asn Ala Ser Ala
Leu Thr Glu Leu Ser Asp Leu Glu Val Leu Asp 545 550 555 560 Leu Ser
Tyr Asn Ser His Tyr Phe Arg Ile Ala Gly Val Thr His His 565 570 575
Leu Glu Phe Ile Gln Asn Phe Thr Asn Leu Lys Val Leu Asn Leu Ser 580
585 590 His Asn Asn Ile Tyr Thr Leu Thr Asp Lys Tyr Asn Leu Glu Ser
Lys 595 600 605 Ser Leu Val Glu Leu Val Phe Ser Gly Asn Arg Leu Asp
Ile Leu Trp 610 615 620 Asn Asp Asp Asp Asn Arg Tyr Ile Ser Ile Phe
Lys Gly Leu Lys Asn 625 630 635 640 Leu Thr Arg Leu Asp Leu Ser Leu
Asn Arg Leu Lys His Ile Pro Asn 645 650 655 Glu Ala Phe Leu Asn Leu
Pro Ala Ser Leu Thr Glu Leu His Ile Asn 660 665 670 Asp Asn Met Leu
Lys Phe Phe Asn Trp Thr Leu Leu Gln Gln Phe Pro 675 680 685 Arg Leu
Glu Leu Leu Asp Leu Arg Gly Asn Lys Leu Leu Phe Leu Thr 690 695 700
Asp Ser Leu Ser Asp Phe Thr Ser Ser Leu Arg Thr Leu Leu Leu Ser 705
710 715 720 His Asn Arg Ile Ser His Leu Pro Ser Gly Phe Leu Ser Glu
Val Ser 725 730 735 Ser Leu Lys His Leu Asp Leu Ser Ser Asn Leu Leu
Lys Thr Ile Asn 740 745 750 Lys Ser Ala Leu Glu Thr Lys Thr Thr Thr
Lys Leu Ser Met Leu Glu 755 760 765 Leu His Gly Asn Pro Phe Glu Cys
Thr Cys Asp Ile Gly Asp Phe Arg 770 775 780 Arg Trp Met Asp Glu His
Leu Asn Val Lys Ile Pro Arg Leu Val Asp 785 790 795 800 Val Ile Cys
Ala Ser Pro Gly Asp Gln Arg Gly Lys Ser Ile Val Ser 805 810 815 Leu
Glu Leu Thr Thr Cys Val Ser Asp Val Thr Ala Val Ile Leu Phe 820 825
830 Phe Phe Thr Phe Phe Ile Thr Thr Met Val Met Leu Ala Ala Leu Ala
835 840 845 His His Leu Phe Tyr Trp Asp Val Trp Phe Ile Tyr Asn Val
Cys Leu 850 855 860 Ala Lys Val Lys Gly Tyr Arg Ser Leu Ser Thr Ser
Gln Thr Phe Tyr 865 870 875 880 Asp Ala Tyr Ile Ser Tyr Asp Thr Lys
Asp Ala Ser Val Thr Asp Trp 885 890 895 Val Ile Asn Glu Leu Arg Tyr
His Leu Glu Glu Ser Arg Asp Lys Asn 900 905 910 Val Leu Leu Cys Leu
Glu Glu Arg Asp Trp Asp Pro Gly Leu Ala Ile 915 920 925 Ile Asp Asn
Leu Met Gln Ser Ile Asn Gln Ser Lys Lys Thr Val Phe 930 935 940 Val
Leu Thr Lys Lys Tyr Ala Lys Ser Trp Asn Phe Lys Thr Ala Phe 945 950
955 960 Tyr Leu Ala Leu Gln Arg Leu Met Asp Glu Asn Met Asp Val Ile
Ile 965 970 975 Phe Ile Leu Leu Glu Pro Val Leu Gln His Ser Gln Tyr
Leu Arg Leu 980 985 990 Arg Gln Arg Ile Cys Lys Ser Ser Ile Leu Gln
Trp Pro Asp Asn Pro 995 1000 1005 Lys Ala Glu Gly Leu Phe Trp Gln
Thr Leu Arg Asn Val Val Leu Thr 1010 1015 1020 Glu Asn Asp Ser Arg
Tyr Asn Asn Met Tyr Val Asp Ser Ile Lys Gln 1025 1030 1035 1040 Tyr
<200> SEQUENCE CHARACTERISTICS: <210> SEQ ID NO 187
<211> LENGTH: 1059 <212> TYPE: PRT <213>
ORGANISM: Homo sapiens <400> SEQUENCE: 187 Met Lys Glu Ser
Ser Leu Gln Asn Ser Ser Cys Ser Leu Gly Lys Glu 1 5 10 15 Thr Lys
Lys Glu Asn Met Phe Leu Gln Ser Ser Met Leu Thr Cys Ile 20 25 30
Phe Leu Leu Ile Ser Gly Ser Cys Glu Leu Cys Ala Glu Glu Asn Phe 35
40 45 Ser Arg Ser Tyr Pro Cys Asp Glu Lys Lys Gln Asn Asp Ser Val
Ile 50 55 60 Ala Glu Cys Ser Asn Arg Arg Leu Gln Glu Val Pro Gln
Thr Val Gly 65 70 75 80 Lys Tyr Val Thr Glu Leu Asp Leu Ser Asp Asn
Phe Ile Thr His Ile 85 90 95 Thr Asn Glu Ser Phe Gln Gly Leu Gln
Asn Leu Thr Lys Ile Asn Leu 100 105 110 Asn His Asn Pro Asn Val Gln
His Gln Asn Gly Asn Pro Gly Ile Gln 115 120 125 Ser Asn Gly Leu Asn
Ile Thr Asp Gly Ala Phe Leu Asn Leu Lys Asn 130 135 140 Leu Arg Glu
Leu Leu Leu Glu Asp Asn Gln Leu Pro Gln Ile Pro Ser 145 150 155 160
Gly Leu Pro Glu Ser Leu Thr Glu Leu Ser Leu Ile Gln Asn Asn Ile 165
170 175 Tyr Asn Ile Thr Lys Glu Gly Ile Ser Arg Leu Ile Asn Leu Lys
Asn 180 185 190 Leu Tyr Leu Ala Trp Asn Cys Tyr Phe Asn Lys Val Cys
Glu Lys Thr 195 200 205 Asn Ile Glu Asp Gly Val Phe Glu Thr Leu Thr
Asn Leu Glu Leu Leu 210 215 220 Ser Leu Ser Phe Asn Ser Leu Ser His
Val Ser Pro Lys Leu Pro Ser 225 230 235 240 Ser Leu Arg Lys Leu Phe
Leu Ser Asn Thr Gln Ile Lys Tyr Ile Ser 245 250 255 Glu Glu Asp Phe
Lys Gly Leu Ile Asn Leu Thr Leu Leu Asp Leu Ser 260 265 270 Gly Asn
Cys Pro Arg Cys Phe Asn Ala Pro Phe Pro Cys Val Pro Cys 275 280 285
Asp Gly Gly Ala Ser Ile Asn Ile Asp Arg Phe Ala Phe Gln Asn Leu 290
295 300 Thr Gln Leu Arg Tyr Leu Asn Leu Ser Ser Thr Ser Leu Arg Lys
Ile 305 310 315 320 Asn Ala Ala Trp Phe Lys Asn Met Pro His Leu Lys
Val Leu Asp Leu 325 330 335 Glu Phe Asn Tyr Leu Val Gly Glu Ile Ala
Ser Gly Ala Phe Leu Thr 340 345 350 Met Leu Pro Arg Leu Glu Ile Leu
Asp Leu Ser Phe Asn Tyr Ile Lys 355 360 365 Gly Ser Tyr Pro Gln His
Ile Asn Ile Ser Arg Asn Phe Ser Lys Pro 370 375 380 Leu Ser Leu Arg
Ala Leu His Leu Arg Gly Tyr Val Phe Gln Glu Leu 385 390 395 400 Arg
Glu Asp Asp Phe Gln Pro Leu Met Gln Leu Pro Asn Leu Ser Thr 405 410
415 Ile Asn Leu Gly Ile Asn Phe Ile Lys Gln Ile Asp Phe Lys Leu Phe
420 425 430 Gln Asn Phe Ser Asn Leu Glu Ile Ile Tyr Leu Ser Glu Asn
Arg Ile 435 440 445 Ser Pro Leu Val Lys Asp Thr Arg Gln Ser Tyr Ala
Asn Ser Ser Ser 450 455 460 Phe Gln Arg His Ile Arg Lys Arg Arg Ser
Thr Asp Phe Glu Phe Asp 465 470 475 480 Pro His Ser Asn Phe Tyr His
Phe Thr Arg Pro Leu Ile Lys Pro Gln 485 490 495 Cys Ala Ala Tyr Gly
Lys Ala Leu Asp Leu Ser Leu Asn Ser Ile Phe 500 505 510 Phe Ile Gly
Pro Asn Gln Phe Glu Asn Leu Pro Asp Ile Ala Cys Leu 515 520 525 Asn
Leu Ser Ala Asn Ser Asn Ala Gln Val Leu Ser Gly Thr Glu Phe 530 535
540 Ser Ala Ile Pro His Val Lys Tyr Leu Asp Leu Thr Asn Asn Arg Leu
545 550 555 560 Asp Phe Asp Asn Ala Ser Ala Leu Thr Glu Leu Ser Asp
Leu Glu Val 565 570 575 Leu Asp Leu Ser Tyr Asn Ser His Tyr Phe Arg
Ile Ala Gly Val Thr 580 585 590 His His Leu Glu Phe Ile Gln Asn Phe
Thr Asn Leu Lys Val Leu Asn 595 600 605 Leu Ser His Asn Asn Ile Tyr
Thr Leu Thr Asp Lys Tyr Asn Leu Glu 610 615 620 Ser Lys Ser Leu Val
Glu Leu Val Phe Ser Gly Asn Arg Leu Asp Ile 625 630 635 640 Leu Trp
Asn Asp Asp Asp Asn Arg Tyr Ile Ser Ile Phe Lys Gly Leu 645 650 655
Lys Asn Leu Thr Arg Leu Asp Leu Ser Leu Asn Arg Leu Lys His Ile 660
665 670 Pro Asn Glu Ala Phe Leu Asn Leu Pro Ala Ser Leu Thr Glu Leu
His 675 680 685 Ile Asn Asp Asn Met Leu Lys Phe Phe Asn Trp Thr Leu
Leu Gln Gln 690 695 700 Phe Pro Arg Leu Glu Leu Leu Asp Leu Arg Gly
Asn Lys Leu Leu Phe 705 710 715 720
Leu Thr Asp Ser Leu Ser Asp Phe Thr Ser Ser Leu Arg Thr Leu Leu 725
730 735 Leu Ser His Asn Arg Ile Ser His Leu Pro Ser Gly Phe Leu Ser
Glu 740 745 750 Val Ser Ser Leu Lys His Leu Asp Leu Ser Ser Asn Leu
Leu Lys Thr 755 760 765 Ile Asn Lys Ser Ala Leu Glu Thr Lys Thr Thr
Thr Lys Leu Ser Met 770 775 780 Leu Glu Leu His Gly Asn Pro Phe Glu
Cys Thr Cys Asp Ile Gly Asp 785 790 795 800 Phe Arg Arg Trp Met Asp
Glu His Leu Asn Val Lys Ile Pro Arg Leu 805 810 815 Val Asp Val Ile
Cys Ala Ser Pro Gly Asp Gln Arg Gly Lys Ser Ile 820 825 830 Val Ser
Leu Glu Leu Thr Thr Cys Val Ser Asp Val Thr Ala Val Ile 835 840 845
Leu Phe Phe Phe Thr Phe Phe Ile Thr Thr Met Val Met Leu Ala Ala 850
855 860 Leu Ala His His Leu Phe Tyr Trp Asp Val Trp Phe Ile Tyr Asn
Val 865 870 875 880 Cys Leu Ala Lys Ile Lys Gly Tyr Arg Ser Leu Ser
Thr Ser Gln Thr 885 890 895 Phe Tyr Asp Ala Tyr Ile Ser Tyr Asp Thr
Lys Asp Ala Ser Val Thr 900 905 910 Asp Trp Val Ile Asn Glu Leu Arg
Tyr His Leu Glu Glu Ser Arg Asp 915 920 925 Lys Asn Val Leu Leu Cys
Leu Glu Glu Arg Asp Trp Asp Pro Gly Leu 930 935 940 Ala Ile Ile Asp
Asn Leu Met Gln Ser Ile Asn Gln Ser Lys Lys Thr 945 950 955 960 Val
Phe Val Leu Thr Lys Lys Tyr Ala Lys Ser Trp Asn Phe Lys Thr 965 970
975 Ala Phe Tyr Leu Ala Leu Gln Arg Leu Met Asp Glu Asn Met Asp Val
980 985 990 Ile Ile Phe Ile Leu Leu Glu Pro Val Leu Gln His Ser Gln
Tyr Leu 995 1000 1005 Arg Leu Arg Gln Arg Ile Cys Lys Ser Ser Ile
Leu Gln Trp Pro Asp 1010 1015 1020 Asn Pro Lys Ala Glu Gly Leu Phe
Trp Gln Thr Leu Arg Asn Val Val 1025 1030 1035 1040 Leu Thr Glu Asn
Asp Ser Arg Tyr Asn Asn Met Tyr Val Asp Ser Ile 1045 1050 1055 Lys
Gln Tyr <200> SEQUENCE CHARACTERISTICS: <210> SEQ ID NO
188 <211> LENGTH: 24 <212> TYPE: DNA <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic oligonucleotide <400> SEQUENCE:
188 gagagaaaca aacgttttac cttc 24 <200> SEQUENCE
CHARACTERISTICS: <210> SEQ ID NO 189 <211> LENGTH: 22
<212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
oligonucleotide <400> SEQUENCE: 189 gatggcagag tcgtgacttc cc
22 <200> SEQUENCE CHARACTERISTICS: <210> SEQ ID NO 190
<211> LENGTH: 3220 <212> TYPE: DNA <213>
ORGANISM: Mus musculus <220> FEATURE: <221> NAME/KEY:
misc_feature <222> LOCATION: (0)...(0) <223> OTHER
INFORMATION: Murine TLR8 cDNA <400> SEQUENCE: 190 attcagagtt
ggatgttaag agagaaacaa acgttttacc ttcctttgtc tatagaacat 60
ggaaaacatg ccccctcagt catggattct gacgtgcttt tgtctgctgt cctctggaac
120 cagtgccatc ttccataaag cgaactattc cagaagctat ccttgtgacg
agataaggca 180 caactccctt gtgattgcag aatgcaacca tcgtcaactg
catgaagttc cccaaactat 240 aggcaagtat gtgacaaaca tagacttgtc
agacaatgcc attacacata taacgaaaga 300 gtcctttcaa aagctgcaaa
acctcactaa aatcgatctg aaccacaatg ccaaacaaca 360 gcacccaaat
gaaaataaaa atggtatgaa tattacagaa ggggcacttc tcagcctaag 420
aaatctaaca gttttactgc tggaagacaa ccagttatat actatacctg ctgggttgcc
480 tgagtctttg aaagaactta gcctaattca aaacaatata tttcaggtaa
ctaaaaacaa 540 cacttttggg cttaggaact tggaaagact ctatttgggc
tggaactgct attttaaatg 600 taatcaaacc tttaaggtag aagatggggc
atttaaaaat cttatacact tgaaggtact 660 ctcattatct ttcaataacc
ttttctatgt gccccccaaa ctaccaagtt ctctaaggaa 720 actttttctg
agtaatgcca aaatcatgaa catcactcag gaagacttca aaggactgga 780
aaatttaaca ttactagatc tgagtggaaa ctgtccaagg tgttacaatg ctccatttcc
840 ttgcacacct tgcaaggaaa actcatccat ccacatacat cctctggctt
ttcaaagtct 900 cacccaactt ctctatctaa acctttccag cacttccctc
aggacgattc cttctacctg 960 gtttgaaaat ctgtcaaatc tgaaggaact
ccatcttgaa ttcaactatt tagttcaaga 1020 aattgcctcg ggggcatttt
taacaaaact acccagttta caaatccttg atttgtcctt 1080 caactttcaa
tataaggaat atttacaatt tattaatatt tcctcaaatt tctctaagct 1140
tcgttctctc aagaagttgc acttaagagg ctatgtgttc cgagaactta aaaagaagca
1200 tttcgagcat ctccagagtc ttccaaactt ggcaaccatc aacttgggca
ttaactttat 1260 tgagaaaatt gatttcaaag ctttccagaa tttttccaaa
ctcgacgtta tctatttatc 1320 aggaaatcgc atagcatctg tattagatgg
tacagattat tcctcttggc gaaatcgtct 1380 tcggaaacct ctctcaacag
acgatgatga gtttgatcca cacgtgaatt tttaccatag 1440 caccaaacct
ttaataaagc cacagtgtac tgcttatggc aaggccttgg atttaagttt 1500
gaacaatatt ttcattattg ggaaaagcca atttgaaggt tttcaggata tcgcctgctt
1560 aaatctgtcc ttcaatgcca atactcaagt gtttaatggc acagaattct
cctccatgcc 1620 ccacattaaa tatttggatt taaccaacaa cagactagac
tttgatgata acaatgcttt 1680 cagtgatctt cacgatctag aagtgctgga
cctgagccac aatgcacact atttcagtat 1740 agcaggggta acgcaccgtc
taggatttat ccagaactta ataaacctca gggtgttaaa 1800 cctgagccac
aatggcattt acaccctcac agaggaaagt gagctgaaaa gcatctcact 1860
gaaagaattg gttttcagtg gaaatcgtct tgaccatttg tggaatgcaa atgatggcaa
1920 atactggtcc atttttaaaa gtctccagaa tttgatacgc ctggacttat
catacaataa 1980 ccttcaacaa atcccaaatg gagcattcct caatttgcct
cagagcctcc aagagttact 2040 tatcagtggt aacaaattac gtttctttaa
ttggacatta ctccagtatt ttcctcacct 2100 tcacttgctg gatttatcga
gaaatgagct gtattttcta cccaattgcc tatctaagtt 2160 tgcacattcc
ctggagacac tgctactgag ccataatcat ttctctcacc taccctctgg 2220
cttcctctcc gaagccagga atctggtgca cctggatcta agtttcaaca caataaagat
2280 gatcaataaa tcctccctgc aaaccaagat gaaaacgaac ttgtctattc
tggagctaca 2340 tgggaactat tttgactgca cgtgtgacat aagtgatttt
cgaagctggc tagatgaaaa 2400 tctgaatatc acaattccta aattggtaaa
tgttatatgt tccaatcctg gggatcaaaa 2460 atcaaagagt atcatgagcc
tagatctcac gacttgtgta tcggatacca ctgcagctgt 2520 cctgtttttc
ctcacattcc ttaccacctc catggttatg ttggctgctc tggttcacca 2580
cctgttttac tgggatgttt ggtttatcta tcacatgtgc tctgctaagt taaaaggcta
2640 caggacttca tccacatccc aaactttcta tgatgcttat atttcttatg
acaccaaaga 2700 tgcatctgtt actgactggg taatcaatga actgcgctac
caccttgaag agagtgaaga 2760 caaaagtgtc ctcctttgtt tagaggagag
ggattgggat ccaggattac ccatcattga 2820 taacctcatg cagagcataa
accagagcaa gaaaacaatc tttgttttaa ccaagaaata 2880 tgccaagagc
tggaacttta aaacagcttt ctacttggcc ttgcagaggc taatggatga 2940
gaacatggat gtgattattt tcatcctcct ggaaccagtg ttacagtact cacagtacct
3000 gaggcttcgg cagaggatct gtaagagctc catcctccag tggcccaaca
atcccaaagc 3060 agaaaacttg ttttggcaaa gtctgaaaaa tgtggtcttg
actgaaaatg attcacggta 3120 tgacgatttg tacattgatt ccattaggca
atactagtga tgggaagtca cgactctgcc 3180 atcataaaaa cacacagctt
ctccttacaa tgaaccgaat 3220 <200> SEQUENCE CHARACTERISTICS:
<210> SEQ ID NO 191 <211> LENGTH: 3096 <212>
TYPE: DNA <213> ORGANISM: Mus musculus <220> FEATURE:
<221> NAME/KEY: misc_feature <222> LOCATION: (0)...(0)
<223> OTHER INFORMATION: Murine TLR8 ORF <400>
SEQUENCE: 191 atggaaaaca tgccccctca gtcatggatt ctgacgtgct
tttgtctgct gtcctctgga 60 accagtgcca tcttccataa agcgaactat
tccagaagct atccttgtga cgagataagg 120 cacaactccc ttgtgattgc
agaatgcaac catcgtcaac tgcatgaagt tccccaaact 180 ataggcaagt
atgtgacaaa catagacttg tcagacaatg ccattacaca tataacgaaa 240
gagtcctttc aaaagctgca aaacctcact aaaatcgatc tgaaccacaa tgccaaacaa
300 cagcacccaa atgaaaataa aaatggtatg aatattacag aaggggcact
tctcagccta 360 agaaatctaa cagttttact gctggaagac aaccagttat
atactatacc tgctgggttg 420 cctgagtctt tgaaagaact tagcctaatt
caaaacaata tatttcaggt aactaaaaac 480 aacacttttg ggcttaggaa
cttggaaaga ctctatttgg gctggaactg ctattttaaa 540 tgtaatcaaa
cctttaaggt agaagatggg gcatttaaaa atcttataca cttgaaggta 600
ctctcattat ctttcaataa ccttttctat gtgcccccca aactaccaag ttctctaagg
660 aaactttttc tgagtaatgc caaaatcatg aacatcactc aggaagactt
caaaggactg 720 gaaaatttaa cattactaga tctgagtgga aactgtccaa
ggtgttacaa tgctccattt 780 ccttgcacac cttgcaagga aaactcatcc
atccacatac atcctctggc ttttcaaagt 840 ctcacccaac ttctctatct
aaacctttcc agcacttccc tcaggacgat tccttctacc 900 tggtttgaaa
atctgtcaaa tctgaaggaa ctccatcttg aattcaacta tttagttcaa 960
gaaattgcct cgggggcatt tttaacaaaa ctacccagtt tacaaatcct tgatttgtcc
1020 ttcaactttc aatataagga atatttacaa tttattaata tttcctcaaa
tttctctaag 1080 cttcgttctc tcaagaagtt gcacttaaga ggctatgtgt
tccgagaact taaaaagaag 1140 catttcgagc atctccagag tcttccaaac
ttggcaacca tcaacttggg cattaacttt 1200 attgagaaaa ttgatttcaa
agctttccag aatttttcca aactcgacgt tatctattta 1260 tcaggaaatc
gcatagcatc tgtattagat ggtacagatt attcctcttg gcgaaatcgt 1320
cttcggaaac ctctctcaac agacgatgat gagtttgatc cacacgtgaa tttttaccat
1380 agcaccaaac ctttaataaa gccacagtgt actgcttatg gcaaggcctt
ggatttaagt 1440 ttgaacaata ttttcattat tgggaaaagc caatttgaag
gttttcagga tatcgcctgc 1500 ttaaatctgt ccttcaatgc caatactcaa
gtgtttaatg gcacagaatt ctcctccatg 1560 ccccacatta aatatttgga
tttaaccaac aacagactag actttgatga taacaatgct 1620 ttcagtgatc
ttcacgatct agaagtgctg gacctgagcc acaatgcaca ctatttcagt 1680
atagcagggg taacgcaccg tctaggattt atccagaact taataaacct cagggtgtta
1740 aacctgagcc acaatggcat ttacaccctc acagaggaaa gtgagctgaa
aagcatctca 1800 ctgaaagaat tggttttcag tggaaatcgt cttgaccatt
tgtggaatgc aaatgatggc 1860 aaatactggt ccatttttaa aagtctccag
aatttgatac gcctggactt atcatacaat 1920 aaccttcaac aaatcccaaa
tggagcattc ctcaatttgc ctcagagcct ccaagagtta 1980 cttatcagtg
gtaacaaatt acgtttcttt aattggacat tactccagta ttttcctcac 2040
cttcacttgc tggatttatc gagaaatgag ctgtattttc tacccaattg cctatctaag
2100 tttgcacatt ccctggagac actgctactg agccataatc atttctctca
cctaccctct 2160 ggcttcctct ccgaagccag gaatctggtg cacctggatc
taagtttcaa cacaataaag 2220 atgatcaata aatcctccct gcaaaccaag
atgaaaacga acttgtctat tctggagcta 2280 catgggaact attttgactg
cacgtgtgac ataagtgatt ttcgaagctg gctagatgaa 2340 aatctgaata
tcacaattcc taaattggta aatgttatat gttccaatcc tggggatcaa 2400
aaatcaaaga gtatcatgag cctagatctc acgacttgtg tatcggatac cactgcagct
2460 gtcctgtttt tcctcacatt ccttaccacc tccatggtta tgttggctgc
tctggttcac 2520 cacctgtttt actgggatgt ttggtttatc tatcacatgt
gctctgctaa gttaaaaggc 2580 tacaggactt catccacatc ccaaactttc
tatgatgctt atatttctta tgacaccaaa 2640 gatgcatctg ttactgactg
ggtaatcaat gaactgcgct accaccttga agagagtgaa 2700 gacaaaagtg
tcctcctttg tttagaggag agggattggg atccaggatt acccatcatt 2760
gataacctca tgcagagcat aaaccagagc aagaaaacaa tctttgtttt aaccaagaaa
2820 tatgccaaga gctggaactt taaaacagct ttctacttgg ccttgcagag
gctaatggat 2880 gagaacatgg atgtgattat tttcatcctc ctggaaccag
tgttacagta ctcacagtac 2940 ctgaggcttc ggcagaggat ctgtaagagc
tccatcctcc agtggcccaa caatcccaaa 3000 gcagaaaact tgttttggca
aagtctgaaa aatgtggtct tgactgaaaa tgattcacgg 3060 tatgacgatt
tgtacattga ttccattagg caatac 3096 <200> SEQUENCE
CHARACTERISTICS: <210> SEQ ID NO 192 <211> LENGTH: 1032
<212> TYPE: PRT <213> ORGANISM: Mus musculus
<400> SEQUENCE: 192 Met Glu Asn Met Pro Pro Gln Ser Trp Ile
Leu Thr Cys Phe Cys Leu 1 5 10 15 Leu Ser Ser Gly Thr Ser Ala Ile
Phe His Lys Ala Asn Tyr Ser Arg 20 25 30 Ser Tyr Pro Cys Asp Glu
Ile Arg His Asn Ser Leu Val Ile Ala Glu 35 40 45 Cys Asn His Arg
Gln Leu His Glu Val Pro Gln Thr Ile Gly Lys Tyr 50 55 60 Val Thr
Asn Ile Asp Leu Ser Asp Asn Ala Ile Thr His Ile Thr Lys 65 70 75 80
Glu Ser Phe Gln Lys Leu Gln Asn Leu Thr Lys Ile Asp Leu Asn His 85
90 95 Asn Ala Lys Gln Gln His Pro Asn Glu Asn Lys Asn Gly Met Asn
Ile 100 105 110 Thr Glu Gly Ala Leu Leu Ser Leu Arg Asn Leu Thr Val
Leu Leu Leu 115 120 125 Glu Asp Asn Gln Leu Tyr Thr Ile Pro Ala Gly
Leu Pro Glu Ser Leu 130 135 140 Lys Glu Leu Ser Leu Ile Gln Asn Asn
Ile Phe Gln Val Thr Lys Asn 145 150 155 160 Asn Thr Phe Gly Leu Arg
Asn Leu Glu Arg Leu Tyr Leu Gly Trp Asn 165 170 175 Cys Tyr Phe Lys
Cys Asn Gln Thr Phe Lys Val Glu Asp Gly Ala Phe 180 185 190 Lys Asn
Leu Ile His Leu Lys Val Leu Ser Leu Ser Phe Asn Asn Leu 195 200 205
Phe Tyr Val Pro Pro Lys Leu Pro Ser Ser Leu Arg Lys Leu Phe Leu 210
215 220 Ser Asn Ala Lys Ile Met Asn Ile Thr Gln Glu Asp Phe Lys Gly
Leu 225 230 235 240 Glu Asn Leu Thr Leu Leu Asp Leu Ser Gly Asn Cys
Pro Arg Cys Tyr 245 250 255 Asn Ala Pro Phe Pro Cys Thr Pro Cys Lys
Glu Asn Ser Ser Ile His 260 265 270 Ile His Pro Leu Ala Phe Gln Ser
Leu Thr Gln Leu Leu Tyr Leu Asn 275 280 285 Leu Ser Ser Thr Ser Leu
Arg Thr Ile Pro Ser Thr Trp Phe Glu Asn 290 295 300 Leu Ser Asn Leu
Lys Glu Leu His Leu Glu Phe Asn Tyr Leu Val Gln 305 310 315 320 Glu
Ile Ala Ser Gly Ala Phe Leu Thr Lys Leu Pro Ser Leu Gln Ile 325 330
335 Leu Asp Leu Ser Phe Asn Phe Gln Tyr Lys Glu Tyr Leu Gln Phe Ile
340 345 350 Asn Ile Ser Ser Asn Phe Ser Lys Leu Arg Ser Leu Lys Lys
Leu His 355 360 365 Leu Arg Gly Tyr Val Phe Arg Glu Leu Lys Lys Lys
His Phe Glu His 370 375 380 Leu Gln Ser Leu Pro Asn Leu Ala Thr Ile
Asn Leu Gly Ile Asn Phe 385 390 395 400 Ile Glu Lys Ile Asp Phe Lys
Ala Phe Gln Asn Phe Ser Lys Leu Asp 405 410 415 Val Ile Tyr Leu Ser
Gly Asn Arg Ile Ala Ser Val Leu Asp Gly Thr 420 425 430 Asp Tyr Ser
Ser Trp Arg Asn Arg Leu Arg Lys Pro Leu Ser Thr Asp 435 440 445 Asp
Asp Glu Phe Asp Pro His Val Asn Phe Tyr His Ser Thr Lys Pro 450 455
460
Leu Ile Lys Pro Gln Cys Thr Ala Tyr Gly Lys Ala Leu Asp Leu Ser 465
470 475 480 Leu Asn Asn Ile Phe Ile Ile Gly Lys Ser Gln Phe Glu Gly
Phe Gln 485 490 495 Asp Ile Ala Cys Leu Asn Leu Ser Phe Asn Ala Asn
Thr Gln Val Phe 500 505 510 Asn Gly Thr Glu Phe Ser Ser Met Pro His
Ile Lys Tyr Leu Asp Leu 515 520 525 Thr Asn Asn Arg Leu Asp Phe Asp
Asp Asn Asn Ala Phe Ser Asp Leu 530 535 540 His Asp Leu Glu Val Leu
Asp Leu Ser His Asn Ala His Tyr Phe Ser 545 550 555 560 Ile Ala Gly
Val Thr His Arg Leu Gly Phe Ile Gln Asn Leu Ile Asn 565 570 575 Leu
Arg Val Leu Asn Leu Ser His Asn Gly Ile Tyr Thr Leu Thr Glu 580 585
590 Glu Ser Glu Leu Lys Ser Ile Ser Leu Lys Glu Leu Val Phe Ser Gly
595 600 605 Asn Arg Leu Asp His Leu Trp Asn Ala Asn Asp Gly Lys Tyr
Trp Ser 610 615 620 Ile Phe Lys Ser Leu Gln Asn Leu Ile Arg Leu Asp
Leu Ser Tyr Asn 625 630 635 640 Asn Leu Gln Gln Ile Pro Asn Gly Ala
Phe Leu Asn Leu Pro Gln Ser 645 650 655 Leu Gln Glu Leu Leu Ile Ser
Gly Asn Lys Leu Arg Phe Phe Asn Trp 660 665 670 Thr Leu Leu Gln Tyr
Phe Pro His Leu His Leu Leu Asp Leu Ser Arg 675 680 685 Asn Glu Leu
Tyr Phe Leu Pro Asn Cys Leu Ser Lys Phe Ala His Ser 690 695 700 Leu
Glu Thr Leu Leu Leu Ser His Asn His Phe Ser His Leu Pro Ser 705 710
715 720 Gly Phe Leu Ser Glu Ala Arg Asn Leu Val His Leu Asp Leu Ser
Phe 725 730 735 Asn Thr Ile Lys Met Ile Asn Lys Ser Ser Leu Gln Thr
Lys Met Lys 740 745 750 Thr Asn Leu Ser Ile Leu Glu Leu His Gly Asn
Tyr Phe Asp Cys Thr 755 760 765 Cys Asp Ile Ser Asp Phe Arg Ser Trp
Leu Asp Glu Asn Leu Asn Ile 770 775 780 Thr Ile Pro Lys Leu Val Asn
Val Ile Cys Ser Asn Pro Gly Asp Gln 785 790 795 800 Lys Ser Lys Ser
Ile Met Ser Leu Asp Leu Thr Thr Cys Val Ser Asp 805 810 815 Thr Thr
Ala Ala Val Leu Phe Phe Leu Thr Phe Leu Thr Thr Ser Met 820 825 830
Val Met Leu Ala Ala Leu Val His His Leu Phe Tyr Trp Asp Val Trp 835
840 845 Phe Ile Tyr His Met Cys Ser Ala Lys Leu Lys Gly Tyr Arg Thr
Ser 850 855 860 Ser Thr Ser Gln Thr Phe Tyr Asp Ala Tyr Ile Ser Tyr
Asp Thr Lys 865 870 875 880 Asp Ala Ser Val Thr Asp Trp Val Ile Asn
Glu Leu Arg Tyr His Leu 885 890 895 Glu Glu Ser Glu Asp Lys Ser Val
Leu Leu Cys Leu Glu Glu Arg Asp 900 905 910 Trp Asp Pro Gly Leu Pro
Ile Ile Asp Asn Leu Met Gln Ser Ile Asn 915 920 925 Gln Ser Lys Lys
Thr Ile Phe Val Leu Thr Lys Lys Tyr Ala Lys Ser 930 935 940 Trp Asn
Phe Lys Thr Ala Phe Tyr Leu Ala Leu Gln Arg Leu Met Asp 945 950 955
960 Glu Asn Met Asp Val Ile Ile Phe Ile Leu Leu Glu Pro Val Leu Gln
965 970 975 Tyr Ser Gln Tyr Leu Arg Leu Arg Gln Arg Ile Cys Lys Ser
Ser Ile 980 985 990 Leu Gln Trp Pro Asn Asn Pro Lys Ala Glu Asn Leu
Phe Trp Gln Ser 995 1000 1005 Leu Lys Asn Val Val Leu Thr Glu Asn
Asp Ser Arg Tyr Asp Asp Leu 1010 1015 1020 Tyr Ile Asp Ser Ile Arg
Gln Tyr 1025 1030 <200> SEQUENCE CHARACTERISTICS: <210>
SEQ ID NO 193 <211> LENGTH: 185 <212> TYPE: PRT
<213> ORGANISM: Mus musculus <400> SEQUENCE: 193 Asn
His Phe Ser His Leu Pro Ser Gly Phe Leu Ser Glu Ala Arg Asn 1 5 10
15 Leu Val His Leu Asp Leu Ser Phe Asn Thr Ile Lys Met Ile Asn Lys
20 25 30 Ser Ser Leu Gln Thr Lys Met Lys Thr Asn Leu Ser Ile Leu
Glu Leu 35 40 45 His Gly Asn Tyr Phe Asp Cys Thr Cys Asp Ile Ser
Asp Phe Arg Ser 50 55 60 Trp Leu Asp Glu Asn Leu Asn Ile Thr Ile
Pro Lys Leu Val Asn Val 65 70 75 80 Ile Cys Ser Asn Pro Gly Asp Gln
Lys Ser Lys Ser Ile Met Ser Leu 85 90 95 Asp Leu Thr Thr Cys Val
Ser Asp Thr Thr Ala Ala Val Leu Phe Phe 100 105 110 Leu Thr Phe Leu
Thr Thr Ser Met Val Met Leu Ala Ala Leu Val His 115 120 125 His Leu
Phe Tyr Trp Asp Val Trp Phe Ile Tyr His Met Cys Ser Ala 130 135 140
Lys Leu Lys Gly Tyr Arg Thr Ser Ser Thr Ser Gln Thr Phe Tyr Asp 145
150 155 160 Ala Tyr Ile Ser Tyr Asp Thr Lys Asp Ala Ser Val Thr Asp
Trp Val 165 170 175 Ile Asn Glu Leu Arg Tyr His Leu Glu 180 185
<200> SEQUENCE CHARACTERISTICS: <210> SEQ ID NO 194
<211> LENGTH: 36 <212> TYPE: DNA <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Synthetic oligonucleotide <400> SEQUENCE: 194
atagaattca ataatgggtt tctgccgcag cgccct 36 <200> SEQUENCE
CHARACTERISTICS: <210> SEQ ID NO 195 <211> LENGTH: 29
<212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
oligonucleotide <400> SEQUENCE: 195 atatctagat ccaggcagag
gcgcaggtc 29 <200> SEQUENCE CHARACTERISTICS: <210> SEQ
ID NO 196 <211> LENGTH: 16 <212> TYPE: PRT <213>
ORGANISM: Unknown <220> FEATURE: <221> NAME/KEY: UNSURE
<222> LOCATION: (4)...(5) <223> OTHER INFORMATION:
<400> SEQUENCE: 196 Gly Asn Cys Xaa Xaa Cys Xaa Xaa Xaa Xaa
Xaa Xaa Cys Xaa Xaa Cys 1 5 10 15 <200> SEQUENCE
CHARACTERISTICS: <210> SEQ ID NO 197 <211> LENGTH: 16
<212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 197 Gly Asn Cys Arg Arg Cys Asp His Ala Pro
Asn Pro Cys Met Glu Cys 1 5 10 15 <200> SEQUENCE
CHARACTERISTICS: <210> SEQ ID NO 198 <211> LENGTH: 16
<212> TYPE: PRT <213> ORGANISM: Mus musculus
<400> SEQUENCE: 198 Gly Asn Cys Arg Arg Cys Asp His Ala Pro
Asn Pro Cys Met Ile Cys 1 5 10 15 <200> SEQUENCE
CHARACTERISTICS: <210> SEQ ID NO 199 <211> LENGTH: 16
<212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 199 Gly Asn Cys Pro Arg Cys Tyr Asn Ala Pro
Phe Pro Cys Ala Pro Cys 1 5 10 15 <200> SEQUENCE
CHARACTERISTICS: <210> SEQ ID NO 200 <211> LENGTH: 16
<212> TYPE: PRT <213> ORGANISM: Mus musculus
<400> SEQUENCE: 200 Gly Asn Cys Pro Arg Cys Tyr Asn Val Pro
Tyr Pro Cys Thr Pro Cys 1 5 10 15 <200> SEQUENCE
CHARACTERISTICS: <210> SEQ ID NO 201 <211> LENGTH: 16
<212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 201 Gly Asn Cys Pro Arg Cys Phe Asn Ala Pro
Phe Pro Cys Val Pro Cys 1 5 10 15 <200> SEQUENCE
CHARACTERISTICS: <210> SEQ ID NO 202 <211> LENGTH: 16
<212> TYPE: PRT <213> ORGANISM: Mus musculus
<400> SEQUENCE: 202 Gly Asn Cys Pro Arg Cys Tyr Asn Ala Pro
Phe Pro Cys Thr Pro Cys 1 5 10 15 <200> SEQUENCE
CHARACTERISTICS: <210> SEQ ID NO 203 <211> LENGTH: 31
<212> TYPE: PRT <213> ORGANISM: Homo sapiens
<220> FEATURE: <221> NAME/KEY: UNSURE <222>
LOCATION: (2)...(8) <223> OTHER INFORMATION: <400>
SEQUENCE: 203 Arg Xaa Xaa Xaa Xaa Xaa Xaa Xaa Arg Xaa Asp Xaa Leu
Xaa Xaa Xaa 1 5 10 15 Xaa Xaa Xaa Xaa Xaa Xaa Lys Leu Xaa Xaa Xaa
Xaa Xaa Xaa Ser 20 25 30 <200> SEQUENCE CHARACTERISTICS:
<210> SEQ ID NO 204 <211> LENGTH: 31 <212> TYPE:
PRT <213> ORGANISM: Mus musculus <220> FEATURE:
<221> NAME/KEY: UNSURE <222> LOCATION: (2)...(8)
<223> OTHER INFORMATION: <400> SEQUENCE: 204 Arg Xaa
Xaa Xaa Xaa Xaa Xaa Xaa Arg Xaa Asp Xaa Leu Xaa Xaa Xaa 1 5 10 15
Xaa Xaa Xaa Xaa Xaa Xaa Ser Leu Xaa Xaa Xaa Xaa Xaa Xaa Ser 20 25
30 <200> SEQUENCE CHARACTERISTICS: <210> SEQ ID NO 205
<211> LENGTH: 31 <212> TYPE: PRT <213> ORGANISM:
Homo sapiens <220> FEATURE: <221> NAME/KEY: UNSURE
<222> LOCATION: (2)...(8) <223> OTHER INFORMATION:
<400> SEQUENCE: 205 Lys Xaa Xaa Xaa Xaa Xaa Xaa Xaa Arg Xaa
Asp Xaa Asp Xaa Xaa Xaa 1 5 10 15 Xaa Xaa Xaa Xaa Xaa Xaa Asp Leu
Xaa Xaa Xaa Xaa Xaa Xaa Tyr 20 25 30 <200> SEQUENCE
CHARACTERISTICS: <210> SEQ ID NO 206 <211> LENGTH: 31
<212> TYPE: PRT <213> ORGANISM: Mus musculus
<220> FEATURE: <221> NAME/KEY: UNSURE <222>
LOCATION: (2)...(8) <223> OTHER INFORMATION: <400>
SEQUENCE: 206 Lys Xaa Xaa Xaa Xaa Xaa Xaa Xaa Arg Xaa Asp Xaa Asp
Xaa Xaa Xaa 1 5 10 15 Xaa Xaa Xaa Xaa Xaa Xaa Asp Leu Xaa Xaa Xaa
Xaa Xaa Xaa His 20 25 30 <200> SEQUENCE CHARACTERISTICS:
<210> SEQ ID NO 207 <211> LENGTH: 20 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic oligonucleotide
<400> SEQUENCE: 207 tccatgacgt tcctgatgct 20 <200>
SEQUENCE CHARACTERISTICS: <210> SEQ ID NO 208 <211>
LENGTH: 25 <212> TYPE: DNA <213> ORGANISM: Artificial
Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic oligonucleotide
<400> SEQUENCE: 208 ctcctccacc agacctcttg attcc 25
<200> SEQUENCE CHARACTERISTICS: <210> SEQ ID NO 209
<211> LENGTH: 27 <212> TYPE: DNA <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Synthetic oligonucleotide <400> SEQUENCE: 209
caaggcatgt cctaggtggt gacattc 27 <200> SEQUENCE
CHARACTERISTICS: <210> SEQ ID NO 210 <211> LENGTH: 31
<212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 210 Gln Val Leu Asp Leu Ser Arg Asn Lys Leu
Asp Leu Tyr His Glu His 1 5 10 15 Ser Phe Thr Glu Leu Pro Arg Leu
Glu Ala Leu Asp Leu Ser Tyr 20 25 30 <200> SEQUENCE
CHARACTERISTICS: <210> SEQ ID NO 211 <211> LENGTH: 31
<212> TYPE: PRT <213> ORGANISM: Mus musculus
<400> SEQUENCE: 211 Gln Val Leu Asp Leu Ser His Asn Lys Leu
Asp Leu Tyr His Trp Lys 1 5 10 15 Ser Phe Ser Glu Leu Pro Gln Leu
Gln Ala Leu Asp Leu Ser Tyr 20 25 30 <200> SEQUENCE
CHARACTERISTICS: <210> SEQ ID NO 212 <211> LENGTH: 31
<212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 212 Arg Tyr Leu Asp Phe Ser Asn Asn Arg Leu
Asp Leu Leu His Ser Thr 1 5 10 15 Ala Phe Glu Glu Leu His Lys Leu
Glu Val Leu Asp Ile Ser Ser 20 25 30 <200> SEQUENCE
CHARACTERISTICS: <210> SEQ ID NO 213 <211> LENGTH: 31
<212> TYPE: PRT <213> ORGANISM: Mus musculus
<400> SEQUENCE: 213 Arg Tyr Leu Asp Phe Ser Asn Asn Arg Leu
Asp Leu Leu Tyr Ser Thr 1 5 10 15 Ala Phe Glu Glu Leu Gln Ser Leu
Glu Val Leu Asp Leu Ser Ser 20 25 30 <200> SEQUENCE
CHARACTERISTICS: <210> SEQ ID NO 214 <211> LENGTH: 31
<212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 214 Lys Tyr Leu Asp Leu Thr Asn Asn Arg Leu
Asp Phe Asp Asn Ala Ser 1 5 10 15 Ala Leu Thr Glu Leu Ser Asp Leu
Glu Val Leu Asp Leu Ser Tyr 20 25 30 <200> SEQUENCE
CHARACTERISTICS: <210> SEQ ID NO 215 <211> LENGTH: 31
<212> TYPE: PRT <213> ORGANISM: Mus musculus
<400> SEQUENCE: 215 Lys Tyr Leu Asp Leu Thr Asn Asn Arg Leu
Asp Phe Asp Asp Asn Asn 1 5 10 15 Ala Phe Ser Asp Leu His Asp Leu
Glu Val Leu Asp Leu Ser His 20 25 30 <200> SEQUENCE
CHARACTERISTICS: <210> SEQ ID NO 216 <211> LENGTH: 33
<212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
oligonucleotide <400> SEQUENCE: 216 tatggatcct cttgtgacaa
aactcacaca tgc 33 <200> SEQUENCE CHARACTERISTICS: <210>
SEQ ID NO 217 <211> LENGTH: 33 <212> TYPE: DNA
<213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic oligonucleotide
<400> SEQUENCE: 217 ataaagcttt catttacccg gagacaggga gag 33
<200> SEQUENCE CHARACTERISTICS: <210> SEQ ID NO 218
<211> LENGTH: 31 <212> TYPE: DNA <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Synthetic oligonucleotide <400> SEQUENCE: 218
tatgaattcc caccatgggt ttctgccgca g 31 <200> SEQUENCE
CHARACTERISTICS: <210> SEQ ID NO 219 <211> LENGTH: 59
<212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
oligonucleotide <400> SEQUENCE: 219 ataggatccc cggggcacca
ggccgccgcc gcggccgccg gagagggcct catccaggc 59 <200> SEQUENCE
CHARACTERISTICS: <210> SEQ ID NO 220 <211> LENGTH: 17
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
oligopeptide <400> SEQUENCE: 220 Asp Glu Ala Leu Ser Gly Gly
Arg Gly Gly Gly Leu Val Pro Arg Gly 1 5 10 15 Ser <200>
SEQUENCE CHARACTERISTICS: <210> SEQ ID NO 221 <211>
LENGTH: 35 <212> TYPE: DNA <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic oligonucleotide <400> SEQUENCE: 221 tatatgcggc
cgcccaccat ggttctccgt cgaag 35 <200> SEQUENCE
CHARACTERISTICS: <210> SEQ ID NO 222 <211> LENGTH: 34
<212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
oligonucleotide <400> SEQUENCE: 222 tatatgcggc cgccagagag
gacctcatcc aggc 34 <200> SEQUENCE CHARACTERISTICS:
<210> SEQ ID NO 223 <211> LENGTH: 41 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic oligonucleotide
<400> SEQUENCE: 223 tatatgcggc cgcccaccat ggtgttttcg
atgtggacac g 41 <200> SEQUENCE CHARACTERISTICS: <210>
SEQ ID NO 224 <211> LENGTH: 38 <212> TYPE: DNA
<213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic oligonucleotide
<400> SEQUENCE: 224 tatatgcggc cgccatctaa ctcacacgta tacagatc
38 <200> SEQUENCE CHARACTERISTICS: <210> SEQ ID NO 225
<211> LENGTH: 42 <212> TYPE: DNA <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Synthetic oligonucleotide <400> SEQUENCE: 225
tatatgcggc cgcccaccat ggtgtttcca atgtggacac tg 42 <200>
SEQUENCE CHARACTERISTICS: <210> SEQ ID NO 226 <211>
LENGTH: 38 <212> TYPE: DNA <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic oligonucleotide <400> SEQUENCE: 226 tatatgcggc
cgccatctaa ctcacaggtg tacagatc 38 <200> SEQUENCE
CHARACTERISTICS: <210> SEQ ID NO 227 <211> LENGTH: 39
<212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
oligonucleotide <400> SEQUENCE: 227 tatatgcggc cgcccaccat
ggaaaacatg ccccctcag 39 <200> SEQUENCE CHARACTERISTICS:
<210> SEQ ID NO 228 <211> LENGTH: 38 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic oligonucleotide
<400> SEQUENCE: 228 tatatgcggc cgccatccga tacacaagtc gtgagatc
38 <200> SEQUENCE CHARACTERISTICS: <210> SEQ ID NO 229
<211> LENGTH: 41 <212> TYPE: DNA <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Synthetic oligonucleotide <400> SEQUENCE: 229
tatatgcggc cgcccaccat ggaaaacatg ttccttcagt c 41 <200>
SEQUENCE CHARACTERISTICS: <210> SEQ ID NO 230 <211>
LENGTH: 38 <212> TYPE: DNA <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic oligonucleotide <400> SEQUENCE: 230 tatatgcggc
cgccatctga aacacaagtt gttagctc 38
* * * * *
References