U.S. patent application number 11/068882 was filed with the patent office on 2005-08-25 for dendritic cell receptor.
This patent application is currently assigned to The Corporation of the Trustees of the Order of the Sisters of Mercy in Queensland. Invention is credited to Hart, Derek N..
Application Number | 20050186612 11/068882 |
Document ID | / |
Family ID | 19925770 |
Filed Date | 2005-08-25 |
United States Patent
Application |
20050186612 |
Kind Code |
A1 |
Hart, Derek N. |
August 25, 2005 |
Dendritic cell receptor
Abstract
The invention provides isolated human DEC-205, its extracellular
domain and functionally equivalent fragments thereof. Also provided
are polynucleotides encoding same and vectors which include such
polynucleotides. Further provided are methods of recombinantly
producing human DEC-205, an extracellular domain thereof or a
functionally equivalent fragment, and ligands that bind to human
DEC-205 or a fragment thereof. Also provided are constructs for use
in prophylaxis or therapy comprising such a ligand, human DEC-205
or an extracellular domain thereof coupled to a toxin or to an
antigen capable of inducing a protective immune response in a
patient.
Inventors: |
Hart, Derek N.;
(Christchurch, NZ) |
Correspondence
Address: |
NIXON & VANDERHYE, PC
901 NORTH GLEBE ROAD, 11TH FLOOR
ARLINGTON
VA
22203
US
|
Assignee: |
The Corporation of the Trustees of
the Order of the Sisters of Mercy in Queensland
Queensland
AU
|
Family ID: |
19925770 |
Appl. No.: |
11/068882 |
Filed: |
March 2, 2005 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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11068882 |
Mar 2, 2005 |
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10141956 |
May 10, 2002 |
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10141956 |
May 10, 2002 |
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09194612 |
Mar 18, 1999 |
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6432666 |
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Current U.S.
Class: |
435/6.16 ;
435/320.1; 435/325; 435/69.1; 530/350; 536/23.5 |
Current CPC
Class: |
A61K 2039/6031 20130101;
A61P 37/02 20180101; C07K 2319/00 20130101; A61K 39/385 20130101;
C07K 14/705 20130101; G01N 33/56972 20130101 |
Class at
Publication: |
435/006 ;
435/069.1; 435/320.1; 435/325; 530/350; 536/023.5 |
International
Class: |
C12Q 001/68; C07H
021/04; C07K 014/705 |
Foreign Application Data
Date |
Code |
Application Number |
May 29, 1996 |
NZ |
286692 |
May 29, 1997 |
WO |
PCT/NZ97/00068 |
Claims
1-13. (canceled)
14. A ligand that binds to human DEC-205 which has an approximate
molecular weight of 198-205 kDa and which includes the following
amino acid sequences:
9 (i) TVDCNDNQPGAICYYSGNETEKEVKPVDSVKCPSPVLNTPWIPF
QNCCYNFIITKNRHMATTQDEVQSTCEKLHPKSHILSIRDEKE
NNFVLEQLLYFNYMASWVMLGITYRNNSL; and (ii)
SQHRLFHLHSQKCLGLDITKSVNELRMFSCDSSAML;
or a functionally equivalent fragment thereof.
15. A ligand that binds to an extracellular domain of human DEC-205
or fragment thereof as defined in claim 14.
16. A ligand as claimed in claim 14 which is an antibody, or
antibody binding fragment.
17. A construct for use in prophylaxis or therapy comprising a
ligand as claimed in claim 14 coupled to an antigen capable of
inducing protective immune response in a patient.
18. A construct for use in prophylaxis or therapy comprising a
ligand as claimed in claim 14 coupled to a toxin.
19. A construct for use in prophylaxis or therapy comprising human
DEC-205 or an extracellular domain thereof as defined in claim 14
coupled to an antigen capable of inducing a protective immune
response in a patient.
20. A construct for use in prophylaxis or therapy comprising human
DEC-205 or an extracellular domain thereof as defined in claim 14
coupled to a toxin.
21. A method of prophylaxis or therapy which comprises
administering to a patient in need of the same human DEC-205 as
defined in claim 14, an extracellular domain as claimed in as
defined above, a ligand as defined above, or a construct as defined
above.
22. A process for isolating activated dendritic cells expressing
human DEC-205 on the surface thereof comprising the step of
contacting a sample containing said cells with a ligand as claimed
in claim 16, and isolating those cells to which the ligand has
bound.
Description
FIELD OF THE INVENTION
[0001] This invention relates to dendritic cell receptors. In
particular, it relates to human DEC-205, to the production and use
thereof, and to ligands which bind to it. Human DEC-205 and its
ligands are useful in prophylaxis and therapy.
BACKGROUND OF THE INVENTION
[0002] Dendritic cells perform important immunoregulatory functions
by presenting antigens in the form of peptides bound to
cell-surface major histocompatibility complex (MHC) molecules to T
cells. Identification of the mechanism by which this antigen
presentation function is achieved therefore has important
implications in manipulating immune response in prophylaxis and
therapy, particularly in humans.
[0003] Jiang et al, Nature 375: 151-155 (1995) disclose a murine
dendritic cell receptor having a molecular weight of 205 kDa
(murine DEC-205). However, they do not disclose a receptor on human
dendritic cells.
[0004] The applicant has now identified a receptor on human
dendritic cells. It is broadly to this receptor (likely to be the
human homolog of murine DEC-205) that the present invention is
directed.
SUMMARY OF THE INVENTION
[0005] The present invention has a number of aspects. In a first
aspect, the invention provides isolated human DEC-205 which has an
approximate molecular weight of 198-205 kDa and which includes the
following amino acid sequences:
1 (i) TVDCNDNQPGAICYYSGNETEKEVKPVDSVKCPSPVLNTPWIPF
QNCCYNFIITKNRHMATTQDEVQSTCEKLHPKSHILSIRDEKE
NNFVLEQLLYFNYMASWVMLGITYRNNSL; and (ii)
SQHRLFHLHSQKCLGLDITKSVNELRMFSCDSSAML;
[0006] or a functionally equivalent fragment thereof.
[0007] In a further aspect the invention provides isolated human
DEC-205 which comprises the amino acid sequence shown in FIG. 11 or
a functionally equivalent fragment thereof.
[0008] In a still further aspect, the invention provides isolated
mature human DEC-205, which comprises the amino acids 27 to 1722
shown for human DEC-205 in FIG. 11.
[0009] In yet a further aspect, the invention provides an
extracellular domain of human DEC-205 or a functionally-equivalent
fragment thereof.
[0010] In a preferred embodiment, the extracellular domain fragment
includes at least a portion of carbohydrate recognition domain
(CRD7), spacer, and a portion of carbohydrate recognition domain
(CRD8) of human DEC-205 (amino acids 1208 to 1323 of the amino acid
sequence of FIG. 11).
[0011] In a still further aspect, the invention provides a
polynucleotide encoding human DEC-205 or its extracellular domain
as defined above. This polynucleotide is preferably DNA, more
preferably cDNA, but can also be RNA.
[0012] In a specific embodiment, the polynucleotide coding for
human DEC-205 includes the following nucleotide sequences:
2 (iii) A ACA GTT GAT TGC AAT GAC AAT CAA CCA GGT GCT ATT TGC TAC
TAT TCA GGA AAT GAG ACT GAA AAA GAG GTC AAA CCA GTT GAC AGT GTT AAA
TGT CCA TCT CCT GTT CTA AAT ACT CCG TGG ATA CCA TTT CAG AAC TGT TGC
TAC AAT TTC ATA ATA ACA AAG AAT AGG CAT ATG GCA ACA ACA CAG GAT GAA
GTT CAT ACT AAA TGC CAG AAA CTG AAT CCA AAA TCA CAT ATT CTG AGT ATT
CGA GAT GAA AAG GAG AAT AAC TTT GTT CTT GAG CAA CTG CTG TAC TTC AAT
TAT ATG GCT TCA TGG GTC ATG TTA GGA ATA ACT TAT AGA AAT AAX TCT
CTT; and (iv) ATT AAT ATG CTG TGG AAG TGG GTG TCC GAG CAT CGG CTC
TTT CAT TTG CAC TCC CAA AAG TGC CTT GGC CTC GAT ATT ACC AAA TCG GTA
AAT GAG CTG AGA ATG TTC AGC TGT GAC TCC AGT GCC ATG CTG TGG TGG AAA
TGC GAG CAC CA
[0013] wherein X is T or G.
[0014] In a further embodiment, the polynucleotide comprises part
or all of the nucleotide sequence of FIG. 10.
[0015] In yet a further aspect, the invention provides a vector
including a polynucleotide as defined above.
[0016] In still a further aspect, the invention provides a method
of producing human DEC-205, the extracellular domain thereof or a
functional fragment comprising the steps of:
[0017] (a) culturing a host cell which has been transformed or
transfected with a vector as defined above to express the encoded
human DEC-205, extracellular domain or fragment; and
[0018] (b) recovering the expressed human DEC-205, extracellular
domain or fragment.
[0019] As yet an additional aspect, the invention provides a ligand
that binds to human DEC-205 or its extracellular domain as defined
above.
[0020] Preferably, the ligand is an antibody or antibody binding
fragment or carbohydrate bearing protein.
[0021] The antibody or antibody binding fragment can be used in
methods for extracting or isolating activated dendritic cells.
[0022] In still a further aspect, the invention provides a
construct for use in therapy or prophylaxis. The construct will
usually be a ligand-antigen construct or a DEC-205-antigen
construct although ligand-toxin and DEC-205-toxin constructs are
also contemplated. The ligand-antigen construct preferably consists
of an antibody or antibody binding fragment which binds to human
DEC-205 and a host-protective antigen. The DEC-205-antigen
construct preferably consists of at least the extra-cellular domain
of human DEC-205 and a host-protective antigen.
[0023] In yet further aspects, the invention contemplates methods
of therapy or prophylaxis which employ human DEC-205, ligands or
constructs containing them.
[0024] In yet a further aspect, the invention provides a molecule
(hapten) which may be used to generate antibodies for identifying
or purifying human dendritic cells, which includes a peptide based
upon part or all of the sequence of FIG. 11.
DESCRIPTION OF THE DRAWINGS
[0025] While the invention is broadly as defined above, it will be
appreciated by those persons skilled in this art that it is not
limited thereto and that it includes embodiments more particularly
described below. It will also be better understood by reference to
the accompanying drawings, in which
[0026] FIG. 1 shows the structure of human DEC-205;
[0027] FIG. 2 shows the strategy for isolation of human DEC-205
cDNA.
[0028] A. A schematic presentation of human DEC-205 mRNA with the
regions corresponding to DEC-205 domains. The positions of the
primers used for the cDNA cloning and analysis are indicated with
arrows. The positions of reverse transcriptase-polymerase chain
reaction (RT-PCR) fragments 1 to 6 and the clone pBK14-1 are
indicated with bars: B. RT-PCR amplification of fragment 1 and 2
from L428 and HEL cell line RNA. L428 and HEL cells were subjected
to RT-PCR with two pairs of degenerate primers (DEC-a/-b, and
DEC-d/-e), fractionated by electrophoresis through 2% agarose gel,
and stained with ethidium bromide. C. RT-PCR and 3'-RACE
amplification of fragment 3 and 4 from L428 cells using the primers
028/023 and 029/019, respectively. A cDNA pool of L428 cells was
subjected to 3'-RACE and RT-PCR, electrophoresed through 0.8%
agarose gel, and stained with ethidium bromide. The numbers on the
top correspond to the name of fragment in FIG. 2A. The positions of
DNA molecular size standard are indicateds to the right. The
estimated molecular size of the RT-PCR products are indicated to
the left;
[0029] FIG. 3 shows protein similarity between human and mouse
DEC-205.
[0030] A. The predicted amino acid sequence of human DEC-205 is
aligned with the mouse homolog. The regions corresponding to
DEC-205 domain structure are bracketed. The positions of amino
acids are shaded where there are identical or conservatively
replaced amino acids between the sequences, and the asterisks
indicates conserved cysteines. The diamonds indicates potential
N-glycosylation sites conserved between the sequences. The arrow
indicates one amino acid deletion in CRD-5 of human DEC-205. The
circles indicate conserved potential serine-phosphorylation sites
by protein kinase C (open circle) or casein kinase (closed circle).
B. The % identity between human and mouse DEC-205 is indicated
above each domain (boxed, See FIG. 2A for key);
[0031] FIG. 4 shows that human DEC-205 is probably a one-copy gene.
Genomic DNA isolated from the peripheral blood of four individuals
was digested with the restriction enzymes BglII BamHI, HindIII or
EcoRI and subjected to Southern blot analysis with the
[.sup.32P]cysteine-rich domain probe. The final wash was
0.3.times.SSC at 65.degree. C. The positions of the DNA molecular
size standards are indicated to the right;
[0032] FIG. 5 shows that human DEC-205 gene localizes on chromosome
2.
[0033] A somatic cell hybrid panel blot (restriction-digested with
PstI) was subjected to Southern blot analysis with the
[.sup.32P]cysteine-rich domain probe. The final wash was
0.3.times.SSC at 65.degree. C. The positions of the DNA molecular
size standards are indicated to the right The estimated molecular
size of the probe-specific bands are indicated to the left. The
asterisk indicates weakly hybridized bands. M, male; F, female;
[0034] FIG. 6 shows that human DEC-205 gene maps to chromosome band
2q24. A. A metaphase spread of human chromosomes were subjected to
fluorescent in situ hybridization (FISH) with 6.6 kb human DEC-205
cDNA probe. The final wash was 0.1.times.SSC at 60.degree. C. The
FISH image was overlaid with a DAPI-stained chromosome image. The
DEC-205 specific signals are indicated by the arrowheads. B. An
inverted image of chromosome 2 containing DEC-205-specific signal
(see FIG. 6A) is aligned with an ideogram of chromosome 2. The
chromosome band corresponding to DEC-205 gene is indicated to the
right;
[0035] FIG. 7 shows that expression of DEC-205 transcripts within
human hematopoetic cell lines. Total RNA prepared from the cell
lines were subjected to Northern blot analysis with the
[.sup.32P]fragment 3 (A and B), or [.sup.32P]-actin (C) probes. The
final wash was 0.1.times.SSC at 65.degree. C. The positions of the
RNA molecular size standards are indicated to the right The
estimated molecular size of DEC-205 transcripts are indicated to
the left. A, 24 h exposure; B, 72 h exposure;
[0036] FIG. 8 shows RT-PCR analysis of DEC-205 mRNA in human DC
preparations. Specific product is seen using lineage negative;
fresh DC (lane 2) and a stronger signal with CMRF44* low density
cultured DC (lane 3). CD8* T lymphocytes (lane 1) contain no
DEC-205 mRNA Ethidium stain.
[0037] FIG. 9 represents the result of an ELISA assay showing a
monoclonal antibody binding specifically to DEC-205 peptide 1 and
not peptide 3. Positive control binding of a hyperimmunized rabbit
anti-DEC-205-peptide 1 serum and hyperimmunized rabbit
anti-DEC-205-peptide 2 serum are shown;
[0038] FIG. 10 gives the DNA sequence for human DEC-205 (coding
region only);
[0039] FIG. 11 gives the human DEC-205 amino acid sequence.
DETAILED DESCRIPTION OF THE INVENTION
[0040] A. Human DEC-205
[0041] The human DEC-205 of the invention is believed to be the
human homolog of murine DEC-205 and has an approximate molecular
weight of 198 to 205 kDa. It has the structure shown in FIGS. 1 and
2A. It also has the deduced amino acid sequence shown in FIG.
11.
[0042] Human DEC-205 can usefully be provided in a number of
different forms. These include human DEC-205 itself the "mature"
form of human DEC-205, and the extracellular receptor domain of
human DEC-205.
[0043] The "mature" form of human DEC-205 of the invention is human
DEC-205 less its native amino-terminus leader or signal sequence,
whereas the extracellular receptor domain is human DEC-205 lacking
the signal sequence, the transmembrane region and cytoplasmic
domain (where present).
[0044] The extracellular domain may be identified through commonly
recognised criteria of extracellular amino acid sequences. The
determination of appropriate criteria is known to those skilled in
the art, and has been described, for example by Hopp et al., Proc.
Natl. Acad. Sci. USA 78, 3824-3828 (1991); Kyte et al., J. Mol.
Biol. 157, 105-132 (1982); Emini, J. Virol 55, 836-839 (1985);
Jameson et al. CA BIOS 4, 181-186 (1988); and Karplus et al.
Naturwissenschaften 72, 212-213 (1985). Amino acid domains
predicted by these criteria to be surface exposed are
characteristic of extracellular domains.
[0045] The amino acid sequences of the predicted regions for human
DEC-205 are shown in FIG. 3A. These include the amino acid
sequences for the signal peptide, cysteine-rich domain, fibronectin
type II domain, Carbohydrate Recognition Domain-1, (CRD-1), CRD-2,
CRD-3, CRD-4, CRD-5. CRD-6, CRD-7, CRD-8, CRD-9, CRD-10,
transmembrane domain and cytoplasmic domain.
[0046] Human DEC-205 of the invention or its extracellular receptor
domain (or parts thereof) may be prepared by methods known in the
art. Such methods include protein synthesis from individual amino
acids as described by Stuart and Young in "Solid Phase Peptide
Synthesis", Second Edition, Pierce Chemical Company (1984). It is
however preferred that human DEC-205 and/or its extracellular
receptor domain or parts thereof be prepared by recombinant methods
as will be detailed hereinafter.
[0047] Example 1 provides further details of human DEC-205.
EXAMPLE 1
[0048] Langerhans cells were prepared from human skin. Epidermal
cell suspensions were prepared from split thickness normal human
breast skin by 30 min dispase (Boehringer-Mannheim, Mannheim,
Germany; 0.5% in PBS) treatment at 37.degree. C., followed by 10
min disaggregation in the presence of trypsin (0.25% in PBS), DNase
I (5 U/ml in PBS) and 5 mM EDTA at room temperature. Langerhans
cells were then enriched by Ficoll/Metrizoate gradient separation
(d=1.077 g/cm.sup.3). Final cell suspensions contained 3-15%
Langerhans cells as determined by HLA-DR positivity. Total RNA was
extracted using Trizol reagent according to the manufacturer's
instructions.
[0049] Degenerate primers were prepared on an Applied Biosystems
DNA Synthesizer with the primer sequences (d) and (e) as set out
below:
3 (d) 5'-GAX ACY GAX GGY TTX TGG AA-3' (e) 3'-GCY GTX TTZ TCZ AAC
CAC AT-5'
[0050] wherein X is C or T, Y is A, C, G or T, and Z is G or A.
[0051] Single stranded cDNA was prepared using total RNA and
reverse transcribed by AMV reverse transcriptase using the 3'
primer (e). Subsequently, the cDNA was amplified using the 5'(d)
and 3'(e) primer using PCR amplification according to techniques
known in the art.
[0052] The amplified products were run on a 2% agarose gel and
visualized with ethidium bromide staining.
[0053] The DNA was purified and ligated into the T tailed pGEM
vector (available from Promega) using standard techniques. The
ligation mixture was transformed into competent E. coli JM 109
bacteria (available from Promega) which were grown on agar plates
with appropriate antibiotic selection. Two colonies were isolated.
DNA was prepared and digested with restriction enzymes. Two inserts
of the same size as the PCR product were sequenced by
double-stranded DNA sequencing techniques using a Sequence Kit
(Sequence 2.0 USB Lab Supply, Pierce). The two clones corresponded
to human DEC-205.
[0054] The amino acid sequence of human DEC-205 was determined to
include the following amino acid sequences:
4 (i) TVDCNDNQPGAICYYSGNETEKEVKPVDSVKCPSPVLNTPWIPF
QNCCYNFIITKNRHMATTQDEVQSTCEKLHPKSHILSIRDEKE
NNFFVLEQLLYFNYMASWVMLGITYRNNSL; and (ii)
SQHRLFHLHSQKCLGLDITKSVNELRMFSCDSSAML
[0055] Determination of these sequences was fundamental to
isolating the cDNA for human DEC-205 detailed below.
[0056] In the partial sequences given above, individual amino acids
are represented by the single letter code as follows:
5 Three-letter One-letter Amino Acid abbreviation symbol Alanine
Ala A Arginine Arg R Asparagine Asn N Aspartic acid Asp D
Asparagine or aspartic acid Asx B Cysteine Cys C Glutamine Gln Q
Glutamic Acid Glu E Glutamine or glutamic acid Glx Z Glycine Gly G
Histidine His H Isoleucine Ile I Leucine Leu L Lysine Lys K
Methionine Met M Phenylalanine Phe F Proline Pro P Serine Ser S
Threonine Thr T Tryptophan Trp W Tyrosine Tyr Y Valine Val V
Unidentified X
[0057] This code also applies to the predicted full sequence of
FIG. 11, deduced from the cDNA encoding human DEC-205 isolated as
described below.
[0058] B. Polynucleotides Encoding Human DEC-205
[0059] In another aspect of this invention, the applicants provide
polynucleotides encoding human DEC-205 or its extracellular domain.
These polynucleotides may be DNA (isolated from nature, synthesised
or cDNA) or RNA. Most often, the polynucleotides will be DNA.
[0060] The polynucleotides of the invention specifically include
those which include the nucleotides
6 (iii) A ACA GTT GAT TGC AAT GAC AAT CAA CCA GGT GCT ATT TGC TAC
TAT TCA GGA AAT GAG ACT GAA AAA GAG GTC AAA CCA GTT GAC AGT GTT AAA
TGT CCA TCT CCT GTT CTA AAT ACT CCG TGG ATA CCA TTT CAG AAC TGT TGC
TAC AAT TTC ATA ATA ACA AAG AAT AGG CAT ATG GCA ACA ACA CAG GAT GAA
GTT CAT ACT AAA TGC CAG AAA CTG AAT CCA AAA TCA CAT ATT CTG AGT ATT
CGA GAT GAA AAG GAG AAT AAC TTT GTT CTT GAG CAA CTG CTG TAC TTC AAT
TAT ATG GCT TCA TGG GTC ATG TTA GGA ATA ACT TAT AGA AAT AAX TCT
CTT; and (iv) ATT AAT ATG CTG TGG AAG TGG GTG TCC GAG CAT CGG CTC
TTT CAT TTG CAC TCC CAA AAG TGC CTT GGC CTC GAT ATT ACC AAA TCG GTA
AAT GAG CTG AGA ATG TTC AGC TGT GAC TCC AGT GCC ATG CTG TGG TGG AAA
TGC GAG CAC CA
[0061] wherein X is T or G.
[0062] as well as the full nucleotide sequence shown in FIG.
10,
[0063] but are not limited thereto.
[0064] The invention also includes within its scope functional
equivalents of these polynucleotides.
[0065] This aspect of the invention will now be illustrated by the
following Examples.
EXAMPLE 2
Experimental Procedures
[0066] Cell culture--The cell lines, HEL, K562, KG-1, THP-1, U937,
Mann and Jurkat were obtained from the American Type Culture
Collection (Rockville, Md.). L428 cells were provided by V. Diehl
(Klinik for Innere Medizin, Cologne, Germany). HDLM2 and KMH2 cells
were obtained from the German Collection of Micro-organisms and
Cell Culture (Braunscfweig, Germany). Mono Mac 6 cells (Bufler et
al (1995) Eur. J. Immunol. 25, 604-610) were provided by H.
Engelmann (Institute for Immunology, Munchen, Germany). All cell
lines were maintained in RPMI 1640, 10% fetal calf serum 100 U/ml
penicillin, 100 ug/ml streptomycin except that HDLM2 cells were
with 20% fetal calf serum.
[0067] Isolation of leukocytes-Leukocyte populations were isolated
using standard laboratory procedures.
[0068] Isolation of cDNA encoding for human DEC-205--A set of
degenerate oligonucleotide primers were designed based on the
published amino acid sequence of mouse DEC-205 (Jiang et al (1995),
above) and synthesized in house or by Life Technologies (Auckland,
New Zealand) (see FIG. 2A). These primers were:
7 DEC-a (5'-AAYATGCTNTGGAARTGGGT-3'), DEC-b
(5'-TGRTGYTCRCAYTTCCACCA-3'), DEC-d (5'-GAYACNGAYGGNTTYTGGAA-3')
and DEC-e (5'-GCNGTYTTRTCRAACCACAT-3'),
[0069] where Y=C or T, R=A or G, N=A or C or G or T. Total RNA
isolated from L428 or HEL cells was reverse transcribed with avian
myeloblastosis virus reverse transcriptase (Promega, Madison, Wis.)
at 55.degree. C. for 1 h using the primers DEC-b or DEC-e. PCR was
performed using the resultant cDNA and Taq polymerase (Boehnringer
Mannheim, Auckland, New Zealand) with the primers DEC-a/-b for
DEC-b-primed or DEC-di-e for DEC-e-primed cDNAs. The PCR conditions
used were the initial denaturation at 94.degree. C. for 5 min 35
cycles of denaturation at 94.degree. C. for 1 min annealing at
54.degree. C. for 1 min, extension at 72.degree. C. for 1 min, and
the final extension at 72.degree. C. for 5 min. The PCR reactions
were fractionated with 2% agarose gel in 40 mM Tris-acetate, pH
8.3, 1 mM EDTA (TAE) buffer, and stained with 0.5 ug/ml ethidium
bromide. The PCR fragments (fragment 1 and 2, see FIGS. 2A and 2E)
were cloned into pGEM-T vector (Promega), and sequenced manually
using Sequenase DNA sequencing kit (Amersham Life Science,
Auckland, New Zealand).
[0070] A set of oligonucleotide primers nested within the DNA
sequence of fragment 1 and 2 were synthesized (see FIG. 2A). These
primers were:
[0071] 023 (5'-GCTCTAGAAACATGACCCATGAAGCC-3' containing a Xbal
site),
[0072] 028 (5'-GCTCTAGACATCGGCTCTTTCATTTGT-3' containing a Xbal
site) and
[0073] 029 (5'-CGGGATTCACAGTTGATTGCAATGACA-3' containing a EcoRI
site)
[0074] where incorporated restriction sites are underlined. Two ug
of poly(A) RNA from L428 cells was reverse transcribed with 200 U
of SuperScriptII (LifeTechnoloies) at 45.degree. C. for 1 h using
an oligo d(T) adaptor primer
[0075] 018 (5'-GACTAGTCTGCAGAATTCTTTTTTTTTTTTTTTTT-3',
[0076] containing a SpeI PstI and EcoRI sites). After
heat-inactivation at 70.degree. C. for 15 min, the reaction was
incubated with 1 U RNaseH (Life Technologies) at 37.degree. C. for
30 min, heat-inactivated at 70.degree. C. for 15 mil, and diluted
to 1 ml with 10 mM Tris-HCl, pH 8.0, 1 nmM EDTA (LA28 cDNA pool).
In order to isolate the fragment 3 (connecting the fragment 1 and
2) (see FIG. 2A), PCR was performed with 5 ul of L428 cDNA pool the
primers 028 and 023, and 2.5 U of Expand enzyme mix (Boehringer
Mannheim). The PCR conditions were the initial denaturation at
94.degree. C. for 2 min, 10 cycles of 10 cycles of denaturation at
94.degree. C. for 15 sec, annealing at 53.degree. C. for 30 sec,
and extension at 68.degree. C. for 4 min, followed by 20 cycles of
denaturation at 94.degree. C. for 15 sec, annealing at 53.degree.
C. for 30 sec, and extension at 68.degree. C. for 4 min plus
additional 20 sec for each cycle, and the final extension at
68.degree. C. for 15 min. 3'-rapid amplification of cDNA ends
(3'-RACE) (Frohman et al (1988) Proc. Natl. Acad. Sci. USA 85,
8998-9002) was performed in order to isolate the fragment 4
(connecting the fragment 1 and the 3'-untranslated region of
DEC-205) (see FIG. 2A). PCR was performed with 5 ul of L428 cDNA
pool and the primer 029 and an adaptor primer 019
(5'-GACTAGTCTGCAGAATTC, containing a SpeI, PstI and EcoRI site), in
the same conditions for the fragment 3. The PCR reactions were
fractionated with 0.8% agarose gel in TAE buffer, and stained with
ethidium bromide. Both the fragment 3 and 4 were restriction
digested with XbaI and EcoRI, respectively, and cloned into
pBluescript II (Stratagene, La Jolla, Calif.). The representative
clones from the fragment 3 (pB38fl) and 4 (pb30-3) were sequenced
with a LI-COR automated sequencer (LI-COR, Lincoln, Nebr.) using
SequiTherm cycle sequecing kit (Epicentre Technologies, Madison,
Wis.). If required, these plasmids were subjected to
exonucleaseIII-nested deletion using Erase-A-Base system (Promega),
and used for sequencing.
[0077] An oligo dT-primed L428 cDNA library was prepared using ZAP
Express cDNA Gigapack Cloning kit (Stratagene) according to
manufacturers instruction. The fragment 3 was labeled with
[.alpha.-32P]dCTP (NEN) using Multiprime system (Amersham Life
Science). The library was screened by plaque hybridization with the
[.sup.32P]fragment 3 using standard techniques (Sambrook, J.,
Fritsch, E. F., and Maniatis, T. (1989) Molecular Cloning: A
Laboratory Manual, 2Ed., Cold Spring Harbour Laboratory, New York,
USA). The specific activity of the probe was 0.8.times.10.sup.9
cpm/ug DNA and used at 1.times.10.sup.6 cpm/mil. The final wash was
in 0.1.times.SSC, 0.5% SDS at 65.degree. C. (1.times.SSC is 0.15 M
NaCl, 15 mMM Na-citrate, pH7.0). Positive clones were converted to
phagemid pBK-CMV (Stratagene) and sequenced using an automated
sequencer.
[0078] In order to verify the DNA sequence obtained from the PCR
clones, pB38f for fragment 3 and pB30-3 for fragment 4, the
fragment 5 was PCR-amplified from a L428 cDNA pool using primers
058 (5'-CGGGATCCCTCTGGCCGCGCACTAATGA-3' containing a BamHI site)
and 050 (5'-CCGCTCGAGCTGTGGATACCAGCACATGCCT-3' containing a XhoI
site) (see FIG. 2A). The PCR conditions were identical to that for
the fragment 3 except using longer extension period (6 min) for
cycling. The fragment 5 was sequenced directly using the
IRD.sub.40-labeled custom primers (MWG-Biotech, Ebersberg, Germany)
and a LI-COR automated sequencer without cloning. These primers
were:
8 IRD001 (5'-GATGGGAACTCTTATGGGAGACCT-3' at nucleotide 523-555),
IRD002 (5'-TGATGCAGGCTGGGTGCCAAATAA-3' at nucleotide 1134-1157),
IRD003 (5'-AACTGGGCAACTGTTGGTGGAAGA-3' at nucleotide 1759-1782),
IRD004 (5'-ATGGCGAAGAGGCTGGCATTTCT- A-3' at nucleotide 2334-2357),
IRD005 (5'-CTCAAGCAAGCGATACCTGTCACT-3' at nucleotide 2972-2995),
IRD006 (5'-TGGGCAACTCGAAGACTGTGTAGT-3' at nucleotide 3624-3647),
IRD007 (5'-CACCAGCACAGCATTCTTGCTTGT-3' at nucleotide 4168-4191) and
IRD008 (5'-ATTTGTGAGCAGACTGATGAGGGA-3' at nucleotide
4797-4820).
[0079] The sequences of these primers were based on those of pb38fl
and pb30-3, and they were positioned as 540-650 bp apart, ensuring
the generation of contigs overlapping by at least 100 bp after
automated sequencing.
[0080] Southern blot analysis--Genomic DNA was prepared from
peripheral blood of patients with hematological disorders (each
patient was karyotyped at Canterbury Health Laboratories,
Christchurch, New Zealand). Approximately 8 ug of genomic DNA was
digested with BglII, BarnHI, EcoRI, or HindIII, fractionated in
0.8% agarose gel in 89 mM Tris-borate, pH 8.3, 2 mM EDTA, and
transfered to Hybond N+ by capillary reaction. A PCR-fragment
corresponding to the cyteine-rich domain was PCR-amplified from
pBK14-1 using the primers 058 and 059
(5'-CGGAATTCGATCTCATGATAAGGCTG- GTCACA-3' containing a EcoRI site)
(see FIG. 2). Briefly, PCR was performed with 2 ng of pBK14-1, the
primer 058 and 059, and Taq polymerase. The PCR conditions used
were the initial denaturation at 94.degree. C. for 2 min, 30 cycles
of denaturation at 94.degree. C. for 15 sec, annealing at
55.degree. C. for 15 sec, extension at 72.degree. C. for 30 sec,
and the final extension at 72.degree. C. for 5 min. The 450 bp PCR
product was labeled with [.alpha.-.sup.32P]dCTP using Multiprime
labeling system (Amersham Life Science). The blot was hybridized
with the probe using standard technique (Sambrook et al, (1989),
above). The specific activity of the probe was 0.8.times.10.sup.9
cpm/ug DNA and used at 1.times.10.sup.6 cpm/ml. The final wash was
in 0.3.times.SSC, 0.5% SDS at 65.degree. C., and exposed to X-OMAT
AR film (Kodak) with an intensifying screen at -70.degree. C.
[0081] A blot containing PstI-digested genomic DNA from a
human-rodent somatic hybrid cell panel was obtained from Oncor
(Gaithersburg, Md.), and probed with the [.sup.32P]cysteine-rich
domain fragment as described above.
[0082] Fluorescent in situ hybridization-Metaphase spreads were
prepared from phytohaemagluttnin-stimulated peripheral blood
lymphocytes of a 46,XY male donor using standard cytogenetic
procedures. The fragment 6 was amplified by recombinant PCR with
the fragment 3 and 4 (see FIG. 2A). PCR was performed with each of
the fragment 3 and 4 and the primers 028 and 019 in the same
conditions for the fragment 3 except using longer extension period
(7 min) for cycling. The fragment 6 was labelled with
biotin-14-dCTP using a BioPrime random prime labelling kit
(Bethesda Research Laboratories, Gathersberg, Md.), and hybridized
to metaphase cells on slides.
[0083] Conditions for hybridization and immunofluorescent detection
were essentially as described (Morris et al, (1993) Human Genetics,
91, 31-36), except that Cot 1 suppression was not required, slides
were washed to a stringency of 0.1.times.SSC, 60.degree. C. after
hybridization, and an additional amplification step was needed
because of the small size of the probe. For precise chromosome band
localization, DAPI and FITC images were captured separately for
each metaphase from the fluorescent microscope to computer using a
Photometrics KAF1400 CCD camera and IPLAB Spectrum software (Signal
Analytics, VA), and colour-joined using Multiprobe extension
software.
[0084] Northern blot analysis--Approximately 10 ug of total RNA
from cultured cells were fractionated in formaldehyde-denatured 1%
agarose gel and transferred to Hybond N+ (Amersham) using 3 M NaCl,
8 mM NaOH, 2 mM sarkosyl with Turboblotter (Schleicher &
Schuell, Keene, N.H.) for 3 h. The membrane was UV-crosslinked
(Stratalinker, Stratagene), and hybridized with [.sup.32P]fragment
3 or [.sup.32P]human .sctn.-actin probe using standard techniques
(Sambrook et al (1989), above). The specific activity of the probes
were 0.9-1.1.times.10.sup.9 cpm/ug DNA and used at
0.7-1.1.times.10.sup.6 cpm/ml. The final wash was in 0.1.times.SSC,
0.5% SDS at 68.degree. C., and exposed to X-OMAT AR film (Kodak)
with intensifying screen at -70.degree. C.
[0085] Reverse transcription--PCR analysis-Total RNA from isolated
leukocytes was incubated with RNase-free DNasel (Life
Technologies), and was reverse transcribed using SuperscriptII with
the oligo dT adaptor primer 018. PCR was performed using a pair of
DEC-205 specific primers 060 (GTGGATCCAGTACAAGGGTCA at nucleotide
46554686) and 056 (ACCAAATCAGTCCGCCCATGA at nucleotide 5116-5096)
with Taq polymerase in the presence of a PCR additive, Q buffer
(Qiagen) by touch down PCR (Don, R. H., Cox, P. T., Wainwright, B.
J., Baker, K., and Mattick, J. S., (1991) Nucleic Acid Res. 19,
4008). PCR conditions used were the initial denaturation at
92.degree. C. for 2 min, 21 cycles of denaturation at 92.degree. C.
for 15 sec, annealing at 60.degree. C. minus 0.5.degree. C./cycle
for 15 sec, extension at 68.degree. C. for 30 sec, 15 cycles of
denaturation at 92.degree. C., annealing at 50.degree. C.,
extension at 68.degree. C. for 1 min and the final extension at
68.degree. C. for 5 min. Human glycelaldehyde-3-phosphate
dehydrogenase (GAPDH) (Tokunaga, K., Nakamura, Y., Sakata, K.,
Fujimori, K., Ohkubo, M., Sawada, K., and Sakiyama, S. (1987)
Cancer Res. 47, 5616-5619) was used for normalization. The primers
for GAPDH were 053 (ATGGGGAAGGTGAAGGTCGGA-3' at nucleotide 61-81),
and 055 (AGGGGCCATCCACAGTCTTCT-3' at nucleotide 634-614). The PCR
reactions were fractionated with 1.5% agarose gel in TAE buffer,
and stained with 0.5 ug/ml ethidium bromide.
[0086] Sequence data analysis--The National Center of Biotechnology
Information (NCBI) Center electronic mail server BLAST was used to
search for homologous sequences. Sequence alignments and motif
search were done using Bestfit and Motifs programs, respectively,
of GCG computer package (Madison, Wis.).
[0087] Results
[0088] Isolation of cDNA for human DEC-205. Based on the amino acid
sequence of mouse DEC-205, a set of degenerate primers were
synthesized and used to perform RT-PCR using the Hodgkin's
disease-derived 1428 cell line and the myeloid HEL cell lines (FIG.
2). The two pair of primers (DEC-d/-e, and DEC-a/-b) gave rise to
the specific RT-PCR products, fragment 1 (390 bp) and 2 (150 bp),
respectively (FIGS. 2A and 2B). These specific fragments were
cloned and sequenced (data not shown). The deduced amino acid
sequences of fragment 1 and 2 were .about.80% identical to that of
mouse DEC-205, indicating that these fragments were derived from
the cDNA of human DEC-205.
[0089] Primers nested within these fragments were synthesized and
further RT-PCR and 3'-RACE performed using a L428 cDNA pool reverse
transcribed with an oligo dT adapter primer 018. A 3.8 kb RT-PCR
product (fragment 3) was obtained using primer 028 and 023 (FIGS.
2A and 2C). A 3.2 kb 3'-RACE product (fragment 4) was obtained
using primer 029 and an adaptor primer 019 (FIGS. 2A and 2C). The
fragment 3 was cloned and several identical clones were identified
by restriction enzyme map analysis (data not shown), and one of
which, pb38fl, was fully sequenced: The DNA sequence of the
fragment 3 (pB38fl) extending from the middle of cysteine-rich
domain to the middle of CRD-8 (FIG. 2A), was 82% identical to the
published mouse DEC-205 cDNA sequence. The fragment 4 was cloned
and two distinct clones identified by restriction enzyme map
analysis. Both clones were partially sequenced and the 3' end DNA
sequence of one clone (eg. pb30-3) was found to contain a poly A
tail and with 72% identical to 3'-untranslated region of mouse
DEC-205 (data not shown). Therefore, the pb30-3 was sequenced to
obtain the DNA sequence of the coding region of DEC-205 plus
partial 3'-untranslated region. The resulting DNA sequence for the
coding region was .about.80% identical to that of mouse DEC-205
spanning from the middle of CRD-8 to the end of cytoplasmic domain
(FIG. 2A). The DNA sequences obtained from pb38fl and pb30-3
overlapped by 320 bp, covering 95% of human DEC-205 coding
region.
[0090] In order to complete the 5' end of the DEC-205 cDNA
sequences a L428 cDNA library was screened by plaque hybridization
using .sup.32P-labeled fragment 3 as a probe. A clone (pBK14-1) was
isolated, and the 1.5 kb insert of this clone was sequenced (FIG.
2A). The sequence was .about.80% identical to the mouse sequence
and corresponded to the signal peptide, cysteine-rich domain,
fibronectin type II domain CRD-1 and part of the CRD-2. The pBK14-1
contained 51 bp 5'-untranslated region, and overlapped with
fragment 3 by .about.1.2 kb.
[0091] To validate the DNA sequencer obtained from the PCR clones,
a further RT-PCR fragment (fragment 5) amplified with primers 058
(nested in the cysteine-rich domain) and 050 (located .about.130 bp
downstream of the stop codon) was prepared (FIG. 2A). The fragment
5 PCR product was sequenced directly using IRD.sub.41-labeled
custom primers without cloning. A total of 10 point mutations,
presumably generated because of the low fidelity of thermostable
polymerases were found and corrected in the PCR clone-derived DNA
sequence. The complete cDNA sequence for human DEC-205 is 5166 bp
in size, and encodes for a predicted 198 kDa type I transmembrane
protein with 1722 amino acids before post translational
modification.
[0092] The deduced amino acid sequence of human DEC-205 showed 77%
overall identity with the homologous mouse protein (FIG. 3A). All
the cysteines, and putative N-glycosylation sites in the
extracellular domain of mouse DEC-205, were conserved in the human
sequence. In the cytoplasmic domain the putative serine
phosphorylation sites by protein kinase C or casein kinase, and a
tyrosine, which appears to be important for coated pit-mediated
internalization (Ezekowitz, R. A. B., Sastry, K., Bailly, P., and
Warner, A. (1990) J. Exp. Med. 172, 1785-1794; and Zvaritch, E.,
Lambeau, G., and Lazdunski M. (1996) J. Biol. Chem. 271, 250-257),
were also conserved. There was one amino acid deletion within the
CRD-5 in human DEC-205. All the extracelluar domains, including the
cysteine-rich domain, fibronectin type II domain, and CRD 1-10 were
74-87% identical between human and mouse sequences (FIG. 3B),
suggesting the importance of these domains for the function of
DEC-205. In contrast, the two hydrophobic domains, including the
signal peptide and transmembrane domain, showed much lower identity
(57% and 52%, respectively (FIG. 3B)) with the mouse protein,
confirming the observation that these hydrohobic domains are more
variable, and rapidly evolved structures (Von Heijne, G. (1990) J.
Membrane Biol. 115, 195-201).
[0093] DEC-205 is a single copy gene with polymorphism-Peripheral
blood-derived genomic DNA from 4 individuals was restriction
enzyme-digested with BglII, BamHI, HindIII or EcoRI and subjected
to Southern blot analysis. The cysteine-rich domain of the
macrophage mannose receptor (Kim, S. J., Ruiz, N., Bezouska, K, and
Drickamer, K. (1992) Genomics 14, 721-727; and Harris, N., Peters,
L. L., Eicher, E. M., Rits, M., Raspberry, D., Eichbaum, Q. G.,
Super, M., and Ezekowitz, R. A. B. (1994) Biochem. Biophys. Res.
Com. 198, 682-692) and phospholipase A2 receptor (Ancian, P.,
Lambeau, G., Mattei, M. G., and Lazdunski, M. (1995) 270,
8963-8970) is encoded by one exon. Therefore, we amplified the
cysteine-rich domain of human DEC-205 using primers 058 and 059 as
a potential single exon probe (450 bp), and used this to probe the
Southern blot in high stringency. A single band appeared in BglII-
, BamHI- or HindIII-digested genomic DNA from all individuals,
indicating that DEC-205 is a single copy gene (FIG. 4). The EcoRI
digests, however, produced a single band in two individuals and
double bands in another, indicating that the DEC-205 gene is
polymorphic. Further Southern blot analysis with larger panel of
individuals showed identical results (data not shown). Therefore,
DEC-205 is a single copy gene with at least one polymorphic
site.
[0094] DEC-205 gene maps to chromosome band 2q24--In order to map
the human DEC-205 gene, a somatic cell hybrid panel Southern blot
(PstI-digested) was probed with the [.sup.32P]cysteine-rich domain
as described above (FIG. 5). A 3.0 kb band in human genomic DNA was
found to hybridize strongly, and the identical band appeared in
chromosome 2-containing somatic human-mouse hybrid cells,
indicating that DEC-205 gene localizes on chromosome 2. The probe
also hybridized weakly with hamster DNA, suggesting the presence of
DEC-205 homolog in hamster as well as in the mouse (which also
hybridized strongly). The origin of the weakly hybridized bands
with apparent polymorphism in the human DNA-containing lanes is not
known. The identical band appeared in chromosome 2, and may either
be related to an alternative exon structure for this region of
DEC-205 or result from weak cross hybridization to another gene on
chromosome 2.
[0095] Fluorescent in situ hybridization then was used to map the
DEC-205 gene in detail (FIGS. 6A and 6B). The 6.4 kb recombinant
PCR fragment (fragment 6) (FIG. 2A) was prepared from fragment 3
and 4, labeled with biotinylated nucleotides, and used as a probe
in a high stringency (FIG. 6A). Ninety-one (80%) of a combined
total 114 metaphase cells analysed from three experiments showed
fluorescent signals on one (27) or both (64) chromosomes 2 in the
middle of the long arm, specifically in band q24 (FIG. 6B). High
resolution banding analysis provided a more precise location of
signals (not shown). No additional site-specific signals were
detected on any other chromosome.
[0096] DEC-205 exhibits multiple transcripts in cell lines--A panel
of human cell lines, including myeloid, B lymphoid, T lymphoid and
Hodgkin's desease-derived cell lines, were analyzed for the
expression of DEC-205 transcripts by Northern blot analysis with
the [.sup.32P]fragment 3 as a probe (FIGS. 7A and 7B). Two DEC-205
transcripts, 7.8 and 9.5 kb in size, were detected, and the 7.8 kb
transcript was the most abundant. The expression level varied
between cell lines, however the myeloid cell line THP-1, the B
lymphoid cell line Mann and the Hodgkin's desease cell line KMH2
showed the highest level of expression. Even with longer exposure,
DEC-205 transcripts were not detectable in K562, KG-1, Monomac and
Jurkat cells, suggesting these cells are DEC-205 negative (FIG.
7B). Interestingly all Hodgkin's disease-derived cell lines tested
express the transcripts. Semiquantitative RT-PCR studies also
support these results (data not shown).
[0097] C. Recombinant Expression of Human DEC-205
[0098] In yet another aspect, the present invention relates to the
recombinant expression of human DEC-205 or of its extracellular
domain.
[0099] The Polynucleotides that encode human DEC-205 or the
extracellular domain of the invention may be inserted into known
vectors for use in standard recombinant DNA techniques. Standard
recombinant DNA techniques are those such as are described in
Sambrook et al.; "Molecular Cloning" 2nd Edition Cold Spring
Harbour Laboratory Press (1987) and by Ausubel et al., Eds,
"Current Protocols in Molecular Biology" Greene Publishing
Associates and Wiley-Interscience, New York (1987).
[0100] Vectors for expressing proteins in bacteria, especially E.
coli are known. Such vectors include the PATH vectors described by
Dieckmann and Tzagoloff in J. Biol. Chem. 260, 1513-1520 (1985).
These vectors contain DNA sequences that encode anthranilate
synthetase (TrpE) followed by a polylinker at the carboxy terminus.
Other expression vector systems are based on beta-galactosidase
(pGEX); lambda P maltose binding protein (pMAL); and gluthathione
S-transferase (pGST)--see Gene 67, 31 (1988) and Peptide Research
3, 167 (1990).
[0101] Vectors useful in yeast and insect cells are available and
well known. A suitable example of a yeast vector is the 2.mu.
plasmid.
[0102] Suitable vectors for use in mammalian cells are also known.
Such vectors include well-known derivatives of SV-40, adenovirus,
retrovirus-derived DNA sequences and vectors derived from
combination of plasmids and phage DNA.
[0103] Further eucaryotic expression vectors are known in the art
(e.g. P. J. Southern and P. Berg, J. Mol. Apol. Genet. 1, 327-341
(1982); S. Subramani et al, Mol. Cell. Biol. 1. 854-864 (1981); R.
J. Kaufmann and P. A. Sharp, "Amplification And Expression of
Sequences Cotransfected with a Modular Dihydrofolate Reductase
Complementary DNA Gene," J. Mol. Biol. 159, 601-621 (1982); RJ.
Kaufmann and P. A Sharp, Mol. Cell. Biol. 159, 601-664 (1982); S.
I. Scahill et at "Expression And Characterization Of The Product Of
A Human Immune Interferon DNA Gene In Chinese Hamster Ovary Cells,"
Proc. Natl. Acad. Sci. USA 80, 4654-4659 (1983); G. Urlaub and L. A
Chasin, Proc. Natl. Acad. Sci. USA 77, 42164220, (1980).
[0104] The expression vectors useful in the present invention
contain at least one expression control sequence that is
operatively linked to the DNA sequence or fragment to be expressed.
The control sequence is inserted in the vector in order to control
and to regulate the expression of the cloned DNA sequence. Examples
of useful expression control sequences are the lac system, the trp
system the tac system, the trc system, major operator and promoter
regions of phage lambda, the control region of fd coat protein, the
glycolytic promoters of yeast, e.g. the promoter for
3-phosphoglycerate kinase, the promoters of yeast acid phosphatase,
e.g. Pho5, the promoters of the yeast alpha-mating factors, and
promoters derived from polyoma, adenovirus, retrovirus, and simian
virus, e.g. the early and late promoters or SV40, and other
sequences known to control the expression of genes of prokaryotic
and eucaryotic cells and their viruses or combinations thereof.
[0105] Vectors containing the receptor-encoding DNA and control
signals are inserted into a host cell for expression of the
receptor. Some useful expression host cells include well-known
prokaryotic and eucaryotic cells. Some suitable prokaryotic hosts
include, for example, E. coli such as E. coli SG-936, E. coli HB
101, E. coli W3110, E. coli X 1776, E. coli X2282, E. coli DHT, and
E. coli MR01, Pseudomonas. Bacillus, such as Bacillus subtilis, and
Streptomyces. Suitable eucaryotic cells include yeast and other
fungi, insect, animal cells, such as COS cells and CHO cells, human
cells and plant cells in tissue culture.
[0106] D. Ligands
[0107] The invention also includes ligands that bind to human
DEC-205 of the invention.
[0108] The ligand will usually be an antibody or an antibody
binding fragment raised against human DEC-205 or its extracellular
domain, or against fragments thereof.
[0109] Such antibodies may be polyclonal but are preferably
monoclonal. Monoclonal antibodies may be produced by methods known
in the art. These methods include the immunological method
described by Kohler and Milstein in Nature 256, 495497 (1975) and
Campbell in "Monoclonal Antibody Technology, the Production and
Characterization of Rodent and Human Hybridomas" in Burdon et al.
Eds, Laboratory Techniques in Biochemistry and Molecular Biology,
Volume 13, Elsevier Science Publishers, Amsterdam (1985); as well
as by the recombinant DNA method described by Huse et al. in
Science 46, 1275-1281 (1989).
[0110] In yet another form, the ligand may also be a non-protein
probably carbohydrate containing, molecule that acts as a ligand
when it binds to, or otherwise comes into contact with, human
DEC-205.
[0111] In addition, ligands may be of two functional types. The
first functional type of ligand is a molecule which binds to human
DEC-205 and stimulates it in performing its normal function (a
"stimulant ligand"). The second functional type of ligand is a
molecule which binds to human DEC-205 and inhibits or prevents it
performing its normal function (an "antagonistic ligand").
[0112] Both types of ligand will find application in either
therapeutic or prophylactic treatments as described below.
[0113] Example 3 describes the production of anti-DEC-205
antibodies.
EXAMPLE 3
Production of Anti-DEC-205 Antibodies
[0114] A BALB/c mouse was immunized ip/sc with L428 cells and
boosted SC with two peptides derived from the DEC-205 cDNA
sequence. DEC-205 peptide 1 ATTQDEVHTKC (aa1267-aa1277) and
DEC-205-peptide 2 TEKEVKPVDSVKC (aa1227-aa1239) were synthesized by
Chiron Mimotopes Pty Ltd (Clayton, Victoria, Australia). After a
third immunization with the two DEC-205 peptides sc/ip/IV the mouse
was sacrificed and a spleen cell suspension prepared. The spleen
cells were fused with the NS-1 myeloma cell line using standard
techniques (Hock et al, Immunology 1994;83:573). A hybridoma was
subsequently isolated, 2F5, which produced monoclonal antibody
binding to the DEC-205-peptide 1 but not the DEC-205-peptide 2 or a
third control DEC-205-peptide 3 (KCLGLDITKSVNELR) (aa82-aa96). This
is shown by FIG. 9.
[0115] E. Constructs
[0116] The invention also provides constructs. The constructs will
generally include antigens against which an immune response is
desired but can also include other products to be delivered
specifically to dendritic cells. Toxins, such as the ricin A chain
are not excluded. The other component of the construct will vary,
being either a ligand as described above or at least the
extracellular domain of human DEC-205. Both constructs will have
the potential to manipulate the immune system of the host.
[0117] In the ligand-antigen constructs, ligands which bind to
human DEC-205 (usually antibodies, antibody-binding fragments or
carbohydrates expressing proteins) can be coupled or otherwise
associated with the antigen against which an immune response is
desired. An example of such antigens are sugar-coated antigens such
as tumour-associated antigens In use, the ligand component binds to
human DEC-205 and the dendritic cell is `primed` with the
associated antigen. This `priming` action will assist in the
induction of an immediate immune response against the antigen.
[0118] The ligand-antigen construct can take any appropriate form
for administration to the dendritic cells. Such forms may differ
depending upon whether the therapeutic protocol involves isolation
of the patients dendritic cells (so that the priming action can
take place in vitro) or whether the construct is to be administered
to a patient in vivo.
[0119] The construct can be directly administered to a patient for
in vivo treatment It can also be administered in a form which
allows the construct to be expressed within the patient.
[0120] One example of such a form for administration to a patient
in vivo is a live recombinant viral vaccine. Such a vaccine
includes a polynucleotide encoding the DEC-205 ligand (or a portion
thereof) and the antigen. The vaccine is administered to the
patient and, once within the patient, expresses the encoded ligand
and antigen to bind to the patients dendritic cells (via human
DEC-205).
[0121] A number of such live recombinant viral vaccine systems are
known. An example of such a system is the Vaccinia virus system
(U.S. Pat. No. 4,603,112; Brochier et al., Nature 354:520
(1991)).
[0122] Administration can be via intravenous, intramuscular,
subcutaneous, topical, oral, intra nasal, rectal or
intracerebroventriqular routes, as appropriate.
[0123] F. Applications
[0124] Human DEC-205, its ligands and the constructs discussed
above can be employed therapeutically or prophylactically in
accordance with this invention to promote or inhibit any of the
known actions of dendritic cells and/or to manipulate the immune
system.
[0125] Thus, the antagonistic ligands per se have potential
application inter alia blocking or inhibiting the immune response
during transplantation procedures.
[0126] Ligands also have application in delivering other products
with which they are associated directly to dendritic cells. This
can be for therapeutic purposes (where the delivered product is an
immunogenic antigen) as discussed above. It can also be to target a
toxin (such as the ricin A-chain specifically to dendritic cells to
selectively destroy them as part of an immunosuppressive
process.
[0127] G. The Use of Human DEC-205 to Detect Dendritic Cells in
Cell Suspensions on Tissues and to Purify Dendritic Cells
[0128] Monoclonal antibodies or other ligands binding to DEC-205
may be used to identify or isolate DC for scientific study or
therapeutic application. For this application, the antibodies or
ligands can be used in conjunction with conventional
identification/separation systems. An example of such a system is
the avidin-biotin immunoaffinity system available from Cell-Pro
Inc, Washington, USA (see U.S. Pat. No. 5,215,927, U.S. Pat. No.
5,225,353, U.S. Pat. No. 5,262,334 and U.S. Pat. No.
5,240,856).
[0129] This system employs directly or indirectly a biotinylated
monoclonal antibody directed against a target cell and a column
containing immunobilized avidin and can be readily adapted to
extract activated human dendritic cells, in this case from human
peripheral blood, using the anti-DEC-205 antibody as follows:
[0130] 1. A sample of human peripheral blood containing the human
dendritic cells is mixed with biotinylated anti-DEC-205 antibody
and incubated to allow formation of antibody/human DC
complexes.
[0131] 2. Following incubation, the mixture is introduced into a
CellPro continuous-flow immunoadsorption column filled with
avidin-coated beads, the strong affinity between biotin and avidin
causing the biotin-coated antibodies (together with the human DC to
which they have bound) to adhere to the avidin-coated beads.
[0132] 3. After unwanted cells present in the mixture are washed
away, captured activated human DC are removed from the column by
gentle agitation and are available for use.
[0133] Variations on this theme using the anti-DEC-205 antibody as
primary antibody (to bind to activated DC) and a biotinylated
secondary antibody (to bind to the anti-DEC-205 antibody) can also
be employed.
[0134] It will be appreciated that before admixture with the
anti-DEC-205 antibody in accordance with the above protocol the
human peripheral blood sample should be treated to ensure that the
DC the sample contains are activated. This can easily be achieved
by, for example, overnight incubation of the sample.
[0135] H. Functional Equivalents
[0136] The invention includes functional equivalents of human
DEC-205, extracellular domains and nucleic acid molecules described
above.
[0137] Human DEC-205 and its extracellular domain are or include
proteins. A protein is considered a functional equivalent of
another protein for a specific function if the equivalent protein
is immunologically cross-reactive with, and has the same function
as, the original protein. The equivalent may, for example, be a
fragment of the protein, or a substitution, addition or deletion
mutant of the protein.
[0138] For example, it is possible to substitute amino acids in a
sequence with equivalent amino acids using conventional techniques.
Groups of amino acids known normally to be equivalent are:
[0139] (a) Ala(A) Ser(S) Thr(T) Pro(P) Gly(G);
[0140] (b) Asn(N) Asp(D) Glu(E) Gln(O);
[0141] (c) His(H) Arg(R) Lys(K);
[0142] (d) Met(M) Leu(L) Ile(I) Val(V); and
[0143] (e) Phe(F) Tyr(Y) Trp(W).
[0144] Substitutions, additions and/or deletions in human DEC-205
may be made as long as the resulting equivalent protein is
immunologically cross-reactive with, and have the same function as,
the native human DEC-205.
[0145] The equivalent human DEC-205 will normally have
substantially the same amino acid sequence as the native human
DEC-205. An amino acid sequence that is substantially the same as
another sequence, but that differs from the other sequence by means
of one or more substitutions, additions and/or deletions is
considered to be an equivalent sequence. Preferably, less than
250/o, more preferably less than 10%, and most preferably less than
5% of the number of amino acid residues in the amino acid sequence
of the native human DEC-205 are substituted for, added to, or
deleted from.
[0146] Equivalent nucleic acid molecules include nucleic acid
sequences that encode proteins equivalent to human DEC-205 as
defined above. Equivalent nucleic acid molecules also include
nucleic acid sequences that, due to the degeneracy of the nucleic
acid code, differ from native nucleic acid sequences in ways that
do not affect the corresponding amino acid sequences.
[0147] Those persons skilled in the art will of course appreciate
that the above description is provided by way of example only and
that the invention is limited only by the lawful scope of the
appended claims.
Sequence CWU 1
1
31 1 1722 PRT Homo sapiens 1 Met Arg Thr Gly Trp Ala His Pro Ser
Pro Pro Gly Gly Ala Pro His 1 5 10 15 Ala Ala Leu Leu Val Leu Arg
Ser Arg Gly Ala Leu Trp Pro Arg Thr 20 25 30 Asn Asp Pro Phe Thr
Ile Val His Gly Asn Thr Gly Lys Cys Ile Lys 35 40 45 Pro Val Tyr
Gly Trp Ile Val Ala Asp Asp Cys Asp Glu Thr Glu Asp 50 55 60 Lys
Leu Trp Lys Trp Val Ser Gln His Arg Leu Phe His Leu His Ser 65 70
75 80 Gln Lys Cys Leu Gly Leu Asp Ile Thr Lys Ser Val Asn Glu Leu
Arg 85 90 95 Met Phe Ser Cys Asp Ser Ser Ala Met Leu Trp Trp Lys
Cys Glu His 100 105 110 His Ser Leu Tyr Gly Ala Ala Arg Tyr Trp Leu
Ala Leu Lys Asp Gly 115 120 125 His Gly Thr Ala Ile Ser Asn Ala Ser
Asp Val Trp Lys Lys Gly Gly 130 135 140 Ser Glu Glu Ser Leu Cys Asp
Gln Pro Tyr His Glu Ile Tyr Thr Arg 145 150 155 160 Asp Gly Asn Ser
Tyr Gly Arg Pro Cys Glu Phe Pro Phe Leu Ile Asp 165 170 175 Gly Thr
Trp His His Asp Cys Ile Leu Asp Glu Asp His Ser Gly Pro 180 185 190
Trp Cys Ala Thr Thr Leu Asn Tyr Glu Tyr Asp Arg Lys Trp Gly Ile 195
200 205 Cys Leu Lys Pro Glu Asn Gly Cys Glu Asp Asn Trp Glu Lys Asn
Glu 210 215 220 Gln Phe Gly Ser Cys Tyr Gln Phe Asn Thr Gln Thr Ala
Leu Ser Trp 225 230 235 240 Lys Glu Ala Tyr Val Ser Cys Gln Asn Gln
Gly Ala Asp Leu Leu Ser 245 250 255 Ile Asn Ser Ala Ala Glu Leu Thr
Tyr Leu Lys Glu Lys Glu Gly Ile 260 265 270 Ala Lys Ile Phe Trp Ile
Gly Leu Asn Gln Leu Tyr Ser Ala Arg Gly 275 280 285 Trp Glu Trp Ser
Asp His Lys Pro Leu Asn Phe Leu Asn Trp Asp Pro 290 295 300 Asp Arg
Pro Ser Ala Pro Thr Ile Gly Gly Ser Ser Cys Ala Arg Met 305 310 315
320 Asp Ala Glu Ser Gly Leu Trp Gln Ser Phe Ser Cys Glu Ala Gln Leu
325 330 335 Pro Tyr Val Cys Arg Lys Pro Leu Asn Asn Thr Val Glu Leu
Thr Asp 340 345 350 Val Trp Thr Tyr Ser Asp Thr Arg Cys Asp Ala Gly
Trp Leu Pro Asn 355 360 365 Asn Gly Phe Cys Tyr Leu Leu Val Asn Glu
Ser Asn Ser Trp Asp Lys 370 375 380 Ala His Ala Lys Cys Lys Ala Phe
Ser Ser Asp Leu Ile Ser Ile His 385 390 395 400 Ser Leu Ala Asp Val
Glu Val Val Val Thr Lys Leu His Asn Glu Asp 405 410 415 Ile Lys Glu
Glu Val Trp Ile Gly Leu Lys Asn Ile Asn Ile Pro Thr 420 425 430 Leu
Phe Gln Trp Ser Asp Gly Thr Glu Val Thr Leu Thr Tyr Trp Asp 435 440
445 Glu Asn Glu Pro Asn Val Pro Tyr Asn Lys Thr Pro Asn Cys Val Ser
450 455 460 Tyr Leu Gly Glu Leu Gly Gln Trp Lys Val Gln Ser Cys Glu
Glu Lys 465 470 475 480 Leu Lys Tyr Val Cys Lys Arg Lys Gly Glu Lys
Leu Asn Asp Ala Ser 485 490 495 Ser Asp Lys Met Cys Pro Pro Asp Glu
Gly Trp Lys Arg His Gly Glu 500 505 510 Thr Cys Tyr Lys Ile Tyr Glu
Asp Glu Val Pro Phe Gly Thr Asn Cys 515 520 525 Asn Leu Thr Ile Thr
Ser Arg Phe Glu Gln Glu Tyr Leu Asn Asp Leu 530 535 540 Met Lys Lys
Tyr Asp Lys Ser Leu Arg Lys Tyr Phe Trp Thr Gly Leu 545 550 555 560
Arg Asp Val Asp Ser Cys Gly Glu Tyr Asn Trp Ala Thr Val Gly Gly 565
570 575 Arg Arg Arg Ala Val Thr Phe Ser Asn Trp Asn Phe Leu Glu Pro
Ala 580 585 590 Ser Pro Gly Gly Cys Val Ala Met Ser Thr Gly Lys Ser
Val Gly Lys 595 600 605 Trp Glu Val Lys Asp Cys Arg Ser Phe Lys Ala
Leu Ser Ile Cys Lys 610 615 620 Lys Met Ser Gly Pro Leu Gly Pro Glu
Glu Ala Ser Pro Lys Pro Asp 625 630 635 640 Asp Pro Cys Pro Glu Gly
Trp Gln Ser Phe Pro Ala Ser Leu Ser Cys 645 650 655 Tyr Lys Val Phe
His Ala Glu Arg Ile Val Arg Lys Arg Asn Trp Glu 660 665 670 Glu Ala
Glu Arg Phe Cys Gln Ala Leu Gly Ala His Leu Ser Ser Phe 675 680 685
Ser His Val Asp Glu Ile Lys Glu Phe Leu His Phe Leu Thr Asp Gln 690
695 700 Phe Ser Gly Gln His Trp Leu Trp Ile Gly Leu Asn Lys Arg Ser
Pro 705 710 715 720 Asp Leu Gln Gly Ser Trp Gln Trp Ser Asp Arg Thr
Pro Val Ser Thr 725 730 735 Ile Ile Met Pro Asn Glu Phe Gln Gln Asp
Tyr Asp Ile Arg Asp Cys 740 745 750 Ala Ala Val Lys Val Phe His Arg
Pro Trp Arg Arg Gly Trp His Phe 755 760 765 Tyr Asp Asp Arg Glu Phe
Ile Tyr Leu Arg Pro Phe Ala Cys Asp Thr 770 775 780 Lys Leu Glu Trp
Val Cys Gln Ile Pro Lys Gly Arg Thr Pro Lys Thr 785 790 795 800 Pro
Asp Trp Tyr Asn Pro Asp Arg Ala Gly Ile His Gly Pro Pro Leu 805 810
815 Ile Ile Glu Gly Ser Glu Tyr Trp Phe Val Ala Asp Leu His Leu Asn
820 825 830 Tyr Glu Glu Ala Val Leu Tyr Cys Ala Ser Asn His Ser Phe
Leu Ala 835 840 845 Thr Ile Thr Ser Phe Val Gly Leu Lys Ala Ile Lys
Asn Lys Ile Ala 850 855 860 Asn Ile Ser Gly Asp Gly Gln Lys Trp Trp
Ile Arg Ile Ser Glu Trp 865 870 875 880 Pro Ile Asp Asp His Phe Thr
Tyr Ser Arg Tyr Pro Trp His Arg Phe 885 890 895 Pro Val Thr Phe Gly
Glu Glu Cys Leu Tyr Met Ser Ala Lys Thr Trp 900 905 910 Leu Ile Asp
Leu Gly Lys Pro Thr Asp Cys Ser Thr Lys Leu Pro Phe 915 920 925 Ile
Cys Glu Lys Tyr Asn Val Ser Ser Leu Glu Lys Tyr Ser Pro Asp 930 935
940 Ser Ala Ala Lys Val Gln Cys Ser Glu Gln Trp Ile Pro Phe Gln Asn
945 950 955 960 Lys Cys Phe Leu Lys Ile Lys Pro Val Ser Leu Thr Phe
Ser Gln Ala 965 970 975 Ser Asp Thr Cys His Ser Tyr Gly Gly Thr Leu
Pro Ser Val Leu Ser 980 985 990 Gln Ile Glu Gln Asp Phe Ile Thr Ser
Leu Leu Pro Asp Met Glu Ala 995 1000 1005 Thr Leu Trp Ile Gly Leu
Arg Trp Thr Ala Tyr Glu Lys Ile Asn Lys 1010 1015 1020 Trp Thr Asp
Asn Arg Glu Leu Thr Tyr Ser Asn Phe His Pro Leu Leu 1025 1030 1035
1040 Val Ser Gly Arg Leu Arg Ile Pro Glu Asn Phe Phe Glu Glu Glu
Ser 1045 1050 1055 Arg Tyr His Cys Ala Leu Ile Leu Asn Leu Gln Lys
Ser Pro Phe Thr 1060 1065 1070 Gly Thr Trp Asn Phe Thr Ser Cys Ser
Glu Arg His Phe Val Ser Leu 1075 1080 1085 Cys Gln Lys Tyr Ser Glu
Val Lys Ser Arg Gln Thr Leu Gln Asn Ala 1090 1095 1100 Ser Glu Thr
Val Lys Tyr Leu Asn Asn Leu Tyr Lys Ile Ile Pro Lys 1105 1110 1115
1120 Thr Leu Thr Trp His Ser Ala Lys Arg Glu Cys Leu Lys Ser Asn
Met 1125 1130 1135 Gln Leu Val Ser Ile Thr Asp Pro Tyr Gln Gln Ala
Phe Leu Ser Val 1140 1145 1150 Gln Ala Leu Leu His Asn Ser Ser Leu
Trp Ile Gly Leu Phe Ser Gln 1155 1160 1165 Asp Asp Glu Leu Asn Phe
Gly Trp Ser Asp Gly Lys Arg Leu His Phe 1170 1175 1180 Ser Arg Trp
Ala Glu Thr Asn Gly Gln Leu Glu Asp Cys Val Val Leu 1185 1190 1195
1200 Asp Thr Asp Gly Phe Trp Lys Thr Val Asp Cys Asn Asp Asn Gln
Pro 1205 1210 1215 Gly Ala Ile Cys Tyr Tyr Ser Gly Asn Glu Thr Glu
Lys Glu Val Lys 1220 1225 1230 Pro Val Asp Ser Val Lys Cys Pro Ser
Pro Val Leu Asn Thr Pro Trp 1235 1240 1245 Ile Pro Phe Gln Asn Cys
Cys Tyr Asn Phe Ile Ile Thr Lys Asn Arg 1250 1255 1260 His Met Ala
Thr Thr Gln Asp Glu Val His Thr Lys Cys Gln Lys Leu 1265 1270 1275
1280 Asn Pro Lys Ser His Ile Leu Ser Ile Arg Asp Glu Lys Glu Asn
Asn 1285 1290 1295 Phe Val Leu Glu Gln Leu Leu Tyr Phe Asn Tyr Met
Ala Ser Trp Val 1300 1305 1310 Met Leu Gly Ile Thr Tyr Arg Asn Asn
Ser Leu Met Trp Phe Asp Lys 1315 1320 1325 Thr Pro Leu Ser Tyr Thr
His Trp Arg Ala Gly Arg Pro Thr Ile Lys 1330 1335 1340 Asn Glu Lys
Phe Leu Ala Gly Leu Ser Thr Asp Gly Phe Trp Asp Ile 1345 1350 1355
1360 Gln Thr Phe Lys Val Ile Glu Glu Ala Val Tyr Phe His Gln His
Ser 1365 1370 1375 Ile Leu Ala Cys Lys Ile Glu Met Val Asp Tyr Lys
Glu Glu His Asn 1380 1385 1390 Thr Thr Leu Pro Gln Phe Met Pro Tyr
Glu Asp Gly Ile Tyr Ser Val 1395 1400 1405 Ile Gln Lys Lys Val Thr
Trp Tyr Glu Ala Leu Asn Met Cys Ser Gln 1410 1415 1420 Ser Gly Gly
His Leu Ala Ser Val His Asn Gln Asn Gly Gln Leu Phe 1425 1430 1435
1440 Leu Glu Asp Ile Val Lys Arg Asp Gly Phe Pro Leu Trp Val Gly
Leu 1445 1450 1455 Ser Ser His Asp Gly Ser Glu Ser Ser Phe Glu Trp
Ser Asp Gly Ser 1460 1465 1470 Thr Phe Asp Tyr Ile Pro Trp Lys Gly
Gln Thr Ser Pro Gly Asn Cys 1475 1480 1485 Val Leu Leu Asp Pro Lys
Gly Thr Trp Lys His Glu Lys Cys Asn Ser 1490 1495 1500 Val Lys Asp
Gly Ala Ile Cys Tyr Lys Pro Thr Lys Ser Lys Lys Leu 1505 1510 1515
1520 Ser Arg Leu Thr Tyr Ser Ser Arg Cys Pro Ala Ala Lys Glu Asn
Gly 1525 1530 1535 Ser Arg Trp Ile Gln Tyr Lys Gly His Cys Tyr Lys
Ser Asp Gln Ala 1540 1545 1550 Leu His Ser Phe Ser Glu Ala Lys Lys
Leu Cys Ser Lys His Asp His 1555 1560 1565 Ser Ala Thr Ile Val Ser
Ile Lys Asp Glu Asp Glu Asn Lys Phe Val 1570 1575 1580 Ser Arg Leu
Met Arg Glu Asn Asn Asn Ile Thr Met Arg Val Trp Leu 1585 1590 1595
1600 Gly Leu Ser Gln His Ser Val Asp Gln Ser Trp Ser Trp Leu Asp
Gly 1605 1610 1615 Ser Glu Val Thr Phe Val Lys Trp Glu Asn Lys Ser
Lys Ser Gly Val 1620 1625 1630 Gly Arg Cys Ser Met Leu Ile Ala Ser
Asn Glu Thr Trp Lys Lys Val 1635 1640 1645 Glu Cys Glu His Gly Phe
Gly Arg Val Val Cys Lys Val Pro Leu Gly 1650 1655 1660 Pro Asp Tyr
Thr Ala Ile Ala Ile Ile Val Ala Thr Leu Ser Ile Leu 1665 1670 1675
1680 Val Leu Met Gly Gly Leu Ile Trp Phe Leu Phe Gln Arg His Arg
Leu 1685 1690 1695 His Leu Ala Gly Phe Ser Ser Val Arg Tyr Ala Gln
Gly Val Asn Glu 1700 1705 1710 Asp Glu Ile Met Leu Pro Ser Phe His
Asp 1715 1720 2 5169 DNA Homo sapiens CDS (1)..(5166) 2 atg agg aca
ggc tgg gcg cac ccc tcg ccg ccc ggc ggg gct cct cat 48 Met Arg Thr
Gly Trp Ala His Pro Ser Pro Pro Gly Gly Ala Pro His 1 5 10 15 gct
gct ctt ctg gtt ctt cga tct cgc gga gcc ctc tgg ccg cgc act 96 Ala
Ala Leu Leu Val Leu Arg Ser Arg Gly Ala Leu Trp Pro Arg Thr 20 25
30 aat gac ccc ttc acc atc gtc cat gga aat acg ggc aag tgc atc aag
144 Asn Asp Pro Phe Thr Ile Val His Gly Asn Thr Gly Lys Cys Ile Lys
35 40 45 cca gtg tat ggc tgg ata gta gca gac gac tgt gat gaa act
gag gac 192 Pro Val Tyr Gly Trp Ile Val Ala Asp Asp Cys Asp Glu Thr
Glu Asp 50 55 60 aag tta tgg aag tgg gtg tcc cag cat cgg ctc ttt
cat ttg cac tcc 240 Lys Leu Trp Lys Trp Val Ser Gln His Arg Leu Phe
His Leu His Ser 65 70 75 80 caa aag tgc ctt ggc ctc gat att acc aaa
tcg gta aat gag ctg aga 288 Gln Lys Cys Leu Gly Leu Asp Ile Thr Lys
Ser Val Asn Glu Leu Arg 85 90 95 atg ttc agc tgt gac tcc agt gcc
atg ctg tgg tgg aaa tgt gag cac 336 Met Phe Ser Cys Asp Ser Ser Ala
Met Leu Trp Trp Lys Cys Glu His 100 105 110 cac tct ctg tac gga gct
gcc cgg tac tgg ctg gct ctg aag gat gga 384 His Ser Leu Tyr Gly Ala
Ala Arg Tyr Trp Leu Ala Leu Lys Asp Gly 115 120 125 cat ggc aca gca
atc tca aat gca tct gat gtc tgg aag aaa gga ggc 432 His Gly Thr Ala
Ile Ser Asn Ala Ser Asp Val Trp Lys Lys Gly Gly 130 135 140 tca gag
gaa agc ctt tgt gac cag cct tat cat gag atc tat acc aga 480 Ser Glu
Glu Ser Leu Cys Asp Gln Pro Tyr His Glu Ile Tyr Thr Arg 145 150 155
160 gat ggg aac tct tat ggg aga cct tgt gaa ttt cca ttc tta att gat
528 Asp Gly Asn Ser Tyr Gly Arg Pro Cys Glu Phe Pro Phe Leu Ile Asp
165 170 175 ggg acc tgg cat cat gat tgc att ctt gat gaa gat cat agt
ggg cca 576 Gly Thr Trp His His Asp Cys Ile Leu Asp Glu Asp His Ser
Gly Pro 180 185 190 tgg tgt gcc acc acc tta aat tat gaa tat gac cga
aag tgg ggc atc 624 Trp Cys Ala Thr Thr Leu Asn Tyr Glu Tyr Asp Arg
Lys Trp Gly Ile 195 200 205 tgc tta aag cct gaa aac ggt tgt gaa gat
aat tgg gaa aag aac gag 672 Cys Leu Lys Pro Glu Asn Gly Cys Glu Asp
Asn Trp Glu Lys Asn Glu 210 215 220 cag ttt gga agt tgc tac caa ttt
aat act cag acg gct ctt tct tgg 720 Gln Phe Gly Ser Cys Tyr Gln Phe
Asn Thr Gln Thr Ala Leu Ser Trp 225 230 235 240 aaa gaa gct tat gtt
tca tgt cag aat caa gga gct gat tta ctg agc 768 Lys Glu Ala Tyr Val
Ser Cys Gln Asn Gln Gly Ala Asp Leu Leu Ser 245 250 255 atc aac agt
gct gct gaa tta act tac ctt aaa gaa aaa gaa ggc att 816 Ile Asn Ser
Ala Ala Glu Leu Thr Tyr Leu Lys Glu Lys Glu Gly Ile 260 265 270 gct
aag att ttc tgg att ggt tta aat cag cta tac tct gct aga ggc 864 Ala
Lys Ile Phe Trp Ile Gly Leu Asn Gln Leu Tyr Ser Ala Arg Gly 275 280
285 tgg gaa tgg tca gac cac aaa cca tta aac ttt ctc aac tgg gat cca
912 Trp Glu Trp Ser Asp His Lys Pro Leu Asn Phe Leu Asn Trp Asp Pro
290 295 300 gac agg ccc agt gca cct act ata ggt ggc tcc agc tgt gca
aga atg 960 Asp Arg Pro Ser Ala Pro Thr Ile Gly Gly Ser Ser Cys Ala
Arg Met 305 310 315 320 gat gct gag tct ggt ctg tgg cag agc ttt tcc
tgt gaa gct caa ctg 1008 Asp Ala Glu Ser Gly Leu Trp Gln Ser Phe
Ser Cys Glu Ala Gln Leu 325 330 335 ccc tat gtc tgc agg aaa cca tta
aat aat aca gtg gag tta aca gat 1056 Pro Tyr Val Cys Arg Lys Pro
Leu Asn Asn Thr Val Glu Leu Thr Asp 340 345 350 gtc tgg aca tac tca
gat acc cgc tgt gat gca ggc tgg ctg cca aat 1104 Val Trp Thr Tyr
Ser Asp Thr Arg Cys Asp Ala Gly Trp Leu Pro Asn 355 360 365 aat gga
ttt tgc tat ctg ctg gta aat gaa agt aat tcc tgg gat aag 1152 Asn
Gly Phe Cys Tyr Leu Leu Val Asn Glu Ser Asn Ser Trp Asp Lys 370 375
380 gca cat gcg aaa tgc aaa gcc ttc agt agt gac cta atc agc att cat
1200 Ala His Ala Lys Cys Lys Ala Phe Ser Ser Asp Leu Ile Ser Ile
His 385 390 395 400 tct cta gca gat gtg gag gtg gtt gtc aca aaa ctc
cat aat gag gat 1248 Ser Leu Ala Asp Val Glu Val Val Val Thr Lys
Leu His Asn Glu Asp 405 410 415 atc aaa gaa gaa gtg tgg ata ggc ctt
aag aac ata aac ata cca act 1296 Ile Lys Glu Glu Val Trp Ile Gly
Leu Lys Asn Ile Asn Ile Pro Thr 420 425 430 tta ttt cag tgg tca gat
ggt act gaa gtt act cta aca tat tgg gat 1344 Leu Phe Gln Trp Ser
Asp Gly Thr Glu Val Thr Leu Thr Tyr Trp Asp 435
440 445 gag aat gag cca aat gtt ccc tac aat aag acg ccc aac tgt gtt
tcc 1392 Glu Asn Glu Pro Asn Val Pro Tyr Asn Lys Thr Pro Asn Cys
Val Ser 450 455 460 tac tta gga gag cta ggt cag tgg aaa gtc caa tca
tgt gag gag aaa 1440 Tyr Leu Gly Glu Leu Gly Gln Trp Lys Val Gln
Ser Cys Glu Glu Lys 465 470 475 480 cta aaa tat gta tgc aag aga aag
gga gaa aaa ctg aat gac gca agt 1488 Leu Lys Tyr Val Cys Lys Arg
Lys Gly Glu Lys Leu Asn Asp Ala Ser 485 490 495 tct gat aag atg tgt
cct cca gat gag ggc tgg aag aga cat gga gaa 1536 Ser Asp Lys Met
Cys Pro Pro Asp Glu Gly Trp Lys Arg His Gly Glu 500 505 510 acc tgt
tac aag att tat gag gat gag gtc cct ttt gga aca aac tgc 1584 Thr
Cys Tyr Lys Ile Tyr Glu Asp Glu Val Pro Phe Gly Thr Asn Cys 515 520
525 aat ctg act atc act agc aga ttt gag caa gaa tac cta aat gat ttg
1632 Asn Leu Thr Ile Thr Ser Arg Phe Glu Gln Glu Tyr Leu Asn Asp
Leu 530 535 540 atg aaa aag tat gat aaa tct cta aga aaa tac ttc tgg
act ggc ctg 1680 Met Lys Lys Tyr Asp Lys Ser Leu Arg Lys Tyr Phe
Trp Thr Gly Leu 545 550 555 560 aga gat gta gat tct tgt gga gag tat
aac tgg gca act gtt ggt gga 1728 Arg Asp Val Asp Ser Cys Gly Glu
Tyr Asn Trp Ala Thr Val Gly Gly 565 570 575 aga agg cgg gct gta acc
ttt tcc aac tgg aat ttt ctt gag cca gct 1776 Arg Arg Arg Ala Val
Thr Phe Ser Asn Trp Asn Phe Leu Glu Pro Ala 580 585 590 tcc ccg ggc
ggc tgc gtg gct atg tct act gga aag tct gtt gga aag 1824 Ser Pro
Gly Gly Cys Val Ala Met Ser Thr Gly Lys Ser Val Gly Lys 595 600 605
tgg gag gtg aag gac tgc aga agc ttc aaa gca ctt tca att tgc aag
1872 Trp Glu Val Lys Asp Cys Arg Ser Phe Lys Ala Leu Ser Ile Cys
Lys 610 615 620 aaa atg agt gga ccc ctt ggg cct gaa gaa gca tcc cct
aag cct gat 1920 Lys Met Ser Gly Pro Leu Gly Pro Glu Glu Ala Ser
Pro Lys Pro Asp 625 630 635 640 gac ccc tgt cct gaa ggc tgg cag agt
ttc ccc gca agt ctt tct tgt 1968 Asp Pro Cys Pro Glu Gly Trp Gln
Ser Phe Pro Ala Ser Leu Ser Cys 645 650 655 tat aag gta ttc cat gca
gaa aga att gta aga aag agg aac tgg gaa 2016 Tyr Lys Val Phe His
Ala Glu Arg Ile Val Arg Lys Arg Asn Trp Glu 660 665 670 gaa gct gaa
cga ttc tgc caa gcc ctt gga gca cac ctt tct agc ttc 2064 Glu Ala
Glu Arg Phe Cys Gln Ala Leu Gly Ala His Leu Ser Ser Phe 675 680 685
agc cat gtg gat gaa ata aag gaa ttt ctt cac ttt tta acg gac cag
2112 Ser His Val Asp Glu Ile Lys Glu Phe Leu His Phe Leu Thr Asp
Gln 690 695 700 ttc agt ggc cag cat tgg ctg tgg att ggt ttg aat aaa
agg agc cca 2160 Phe Ser Gly Gln His Trp Leu Trp Ile Gly Leu Asn
Lys Arg Ser Pro 705 710 715 720 gat tta caa gga tcc tgg caa tgg agt
gat cgt aca cca gtg tct act 2208 Asp Leu Gln Gly Ser Trp Gln Trp
Ser Asp Arg Thr Pro Val Ser Thr 725 730 735 att atc atg cca aat gag
ttt cag cag gat tat gac atc aga gac tgt 2256 Ile Ile Met Pro Asn
Glu Phe Gln Gln Asp Tyr Asp Ile Arg Asp Cys 740 745 750 gct gct gtc
aag gta ttt cat agg cca tgg cga aga ggc tgg cat ttc 2304 Ala Ala
Val Lys Val Phe His Arg Pro Trp Arg Arg Gly Trp His Phe 755 760 765
tat gat gat aga gaa ttt att tat ttg agg cct ttt gct tgt gat aca
2352 Tyr Asp Asp Arg Glu Phe Ile Tyr Leu Arg Pro Phe Ala Cys Asp
Thr 770 775 780 aaa ctt gaa tgg gtg tgc caa att cca aaa ggc cgt act
cca aaa aca 2400 Lys Leu Glu Trp Val Cys Gln Ile Pro Lys Gly Arg
Thr Pro Lys Thr 785 790 795 800 cca gac tgg tac aat cca gac cgt gct
gga att cat gga cct cca ctt 2448 Pro Asp Trp Tyr Asn Pro Asp Arg
Ala Gly Ile His Gly Pro Pro Leu 805 810 815 ata att gaa gga agt gaa
tat tgg ttt gtt gct gat ctt cac cta aac 2496 Ile Ile Glu Gly Ser
Glu Tyr Trp Phe Val Ala Asp Leu His Leu Asn 820 825 830 tat gaa gaa
gcc gtc ctg tac tgt gcc agc aat cac agc ttt ctt gcg 2544 Tyr Glu
Glu Ala Val Leu Tyr Cys Ala Ser Asn His Ser Phe Leu Ala 835 840 845
act ata aca tct ttt gtg gga cta aaa gcc atc aaa aac aaa ata gca
2592 Thr Ile Thr Ser Phe Val Gly Leu Lys Ala Ile Lys Asn Lys Ile
Ala 850 855 860 aat ata tct ggt gat gga cag aag tgg tgg ata aga att
agc gag tgg 2640 Asn Ile Ser Gly Asp Gly Gln Lys Trp Trp Ile Arg
Ile Ser Glu Trp 865 870 875 880 cca ata gat gat cat ttt aca tac tca
cga tat cca tgg cac cgc ttt 2688 Pro Ile Asp Asp His Phe Thr Tyr
Ser Arg Tyr Pro Trp His Arg Phe 885 890 895 cct gtg aca ttt gga gag
gaa tgc ttg tac atg tct gcc aag act tgg 2736 Pro Val Thr Phe Gly
Glu Glu Cys Leu Tyr Met Ser Ala Lys Thr Trp 900 905 910 ctt atc gac
tta ggt aaa cca aca gac tgt agt acc aag ttg ccc ttc 2784 Leu Ile
Asp Leu Gly Lys Pro Thr Asp Cys Ser Thr Lys Leu Pro Phe 915 920 925
atc tgt gaa aaa tat aat gtt tct tcg tta gag aaa tac agc cca gat
2832 Ile Cys Glu Lys Tyr Asn Val Ser Ser Leu Glu Lys Tyr Ser Pro
Asp 930 935 940 tct gca gct aaa gtg caa tgt tct gag caa tgg att cct
ttt cag aat 2880 Ser Ala Ala Lys Val Gln Cys Ser Glu Gln Trp Ile
Pro Phe Gln Asn 945 950 955 960 aag tgt ttt cta aag atc aaa ccc gtg
tct ctc aca ttt tct caa gca 2928 Lys Cys Phe Leu Lys Ile Lys Pro
Val Ser Leu Thr Phe Ser Gln Ala 965 970 975 agc gat acc tgt cac tcc
tat ggt ggc acc ctt cct tca gtg ttg agc 2976 Ser Asp Thr Cys His
Ser Tyr Gly Gly Thr Leu Pro Ser Val Leu Ser 980 985 990 cag att gaa
caa gac ttt att aca tcc ttg ctt ccg gat atg gaa gct 3024 Gln Ile
Glu Gln Asp Phe Ile Thr Ser Leu Leu Pro Asp Met Glu Ala 995 1000
1005 act tta tgg att ggt ttg cgc tgg act gcc tat gaa aag ata aac
aaa 3072 Thr Leu Trp Ile Gly Leu Arg Trp Thr Ala Tyr Glu Lys Ile
Asn Lys 1010 1015 1020 tgg aca gat aac aga gag ctg acg tac agt aac
ttt cac cca tta ttg 3120 Trp Thr Asp Asn Arg Glu Leu Thr Tyr Ser
Asn Phe His Pro Leu Leu 1025 1030 1035 1040 gtt agt ggg agg ctg aga
ata cca gaa aat ttt ttt gag gaa gag tct 3168 Val Ser Gly Arg Leu
Arg Ile Pro Glu Asn Phe Phe Glu Glu Glu Ser 1045 1050 1055 cgc tac
cac tgt gcc cta ata ctc aac ctc caa aaa tca ccg ttt act 3216 Arg
Tyr His Cys Ala Leu Ile Leu Asn Leu Gln Lys Ser Pro Phe Thr 1060
1065 1070 ggg acg tgg aat ttt aca tcc tgc agt gaa cgc cac ttt gtg
tct ctc 3264 Gly Thr Trp Asn Phe Thr Ser Cys Ser Glu Arg His Phe
Val Ser Leu 1075 1080 1085 tgt cag aaa tat tca gaa gtt aaa agc aga
cag acg ttg cag aat gct 3312 Cys Gln Lys Tyr Ser Glu Val Lys Ser
Arg Gln Thr Leu Gln Asn Ala 1090 1095 1100 tca gaa act gta aag tat
cta aat aat ctg tac aaa ata atc cca aag 3360 Ser Glu Thr Val Lys
Tyr Leu Asn Asn Leu Tyr Lys Ile Ile Pro Lys 1105 1110 1115 1120 act
ctg act tgg cac agt gct aaa agg gag tgt ctg aaa agt aac atg 3408
Thr Leu Thr Trp His Ser Ala Lys Arg Glu Cys Leu Lys Ser Asn Met
1125 1130 1135 cag ctg gtg agc atc acg gac cct tac cag cag gca ttc
ctc agt gtg 3456 Gln Leu Val Ser Ile Thr Asp Pro Tyr Gln Gln Ala
Phe Leu Ser Val 1140 1145 1150 cag gcg ctc ctt cac aac tct tcc tta
tgg atc gga ctc ttc agt caa 3504 Gln Ala Leu Leu His Asn Ser Ser
Leu Trp Ile Gly Leu Phe Ser Gln 1155 1160 1165 gat gat gaa ctc aac
ttt ggt tgg tca gat ggg aaa cgt ctt cat ttt 3552 Asp Asp Glu Leu
Asn Phe Gly Trp Ser Asp Gly Lys Arg Leu His Phe 1170 1175 1180 agt
cgc tgg gct gaa act aat ggg caa ctc gaa gac tgt gta gta tta 3600
Ser Arg Trp Ala Glu Thr Asn Gly Gln Leu Glu Asp Cys Val Val Leu
1185 1190 1195 1200 gac act gat gga ttc tgg aaa aca gtt gat tgc aat
gac aat caa cca 3648 Asp Thr Asp Gly Phe Trp Lys Thr Val Asp Cys
Asn Asp Asn Gln Pro 1205 1210 1215 ggt gct att tgc tac tat tca gga
aat gag act gaa aaa gag gtc aaa 3696 Gly Ala Ile Cys Tyr Tyr Ser
Gly Asn Glu Thr Glu Lys Glu Val Lys 1220 1225 1230 cca gtt gac agt
gtt aaa tgt cca tct cct gtt cta aat act ccg tgg 3744 Pro Val Asp
Ser Val Lys Cys Pro Ser Pro Val Leu Asn Thr Pro Trp 1235 1240 1245
ata cca ttt cag aac tgt tgc tac aat ttc ata ata aca aag aat agg
3792 Ile Pro Phe Gln Asn Cys Cys Tyr Asn Phe Ile Ile Thr Lys Asn
Arg 1250 1255 1260 cat atg gca aca aca cag gat gaa gtt cat act aaa
tgc cag aaa ctg 3840 His Met Ala Thr Thr Gln Asp Glu Val His Thr
Lys Cys Gln Lys Leu 1265 1270 1275 1280 aat cca aaa tca cat att ctg
agt att cga gat gaa aag gag aat aac 3888 Asn Pro Lys Ser His Ile
Leu Ser Ile Arg Asp Glu Lys Glu Asn Asn 1285 1290 1295 ttt gtt ctt
gag caa ctg ctg tac ttc aat tat atg gct tca tgg gtc 3936 Phe Val
Leu Glu Gln Leu Leu Tyr Phe Asn Tyr Met Ala Ser Trp Val 1300 1305
1310 atg tta gga ata act tat aga aat aat tct ctt atg tgg ttt gat
aag 3984 Met Leu Gly Ile Thr Tyr Arg Asn Asn Ser Leu Met Trp Phe
Asp Lys 1315 1320 1325 acc cca ctg tca tat aca cat tgg aga gca gga
aga cca act ata aaa 4032 Thr Pro Leu Ser Tyr Thr His Trp Arg Ala
Gly Arg Pro Thr Ile Lys 1330 1335 1340 aat gag aag ttt ttg gct ggt
tta agt act gac ggc ttc tgg gat att 4080 Asn Glu Lys Phe Leu Ala
Gly Leu Ser Thr Asp Gly Phe Trp Asp Ile 1345 1350 1355 1360 caa acc
ttt aaa gtt att gaa gaa gca gtt tat ttt cac cag cac agc 4128 Gln
Thr Phe Lys Val Ile Glu Glu Ala Val Tyr Phe His Gln His Ser 1365
1370 1375 att ctt gct tgt aaa att gaa atg gtt gac tac aaa gaa gaa
cat aat 4176 Ile Leu Ala Cys Lys Ile Glu Met Val Asp Tyr Lys Glu
Glu His Asn 1380 1385 1390 act aca ctg cca cag ttt atg cca tat gaa
gat ggt att tac agt gtt 4224 Thr Thr Leu Pro Gln Phe Met Pro Tyr
Glu Asp Gly Ile Tyr Ser Val 1395 1400 1405 att caa aaa aag gta aca
tgg tat gaa gca tta aac atg tgt tct caa 4272 Ile Gln Lys Lys Val
Thr Trp Tyr Glu Ala Leu Asn Met Cys Ser Gln 1410 1415 1420 agt gga
ggt cac ttg gca agc gtt cac aac caa aat ggc cag ctc ttt 4320 Ser
Gly Gly His Leu Ala Ser Val His Asn Gln Asn Gly Gln Leu Phe 1425
1430 1435 1440 ctg gaa gat att gta aaa cgt gat gga ttt cca cta tgg
gtt ggg ctc 4368 Leu Glu Asp Ile Val Lys Arg Asp Gly Phe Pro Leu
Trp Val Gly Leu 1445 1450 1455 tca agt cat gat gga agt gaa tca agt
ttt gaa tgg tct gat ggt agt 4416 Ser Ser His Asp Gly Ser Glu Ser
Ser Phe Glu Trp Ser Asp Gly Ser 1460 1465 1470 aca ttt gac tat atc
cca tgg aaa ggc caa aca tct cct gga aat tgt 4464 Thr Phe Asp Tyr
Ile Pro Trp Lys Gly Gln Thr Ser Pro Gly Asn Cys 1475 1480 1485 gtt
ctc ttg gat cca aaa gga act tgg aaa cat gaa aaa tgc aac tct 4512
Val Leu Leu Asp Pro Lys Gly Thr Trp Lys His Glu Lys Cys Asn Ser
1490 1495 1500 gtt aag gat ggt gct att tgt tat aaa cct aca aaa tct
aaa aag ctg 4560 Val Lys Asp Gly Ala Ile Cys Tyr Lys Pro Thr Lys
Ser Lys Lys Leu 1505 1510 1515 1520 tcc cgt ctt aca tat tca tca aga
tgt cca gca gca aaa gag aat ggg 4608 Ser Arg Leu Thr Tyr Ser Ser
Arg Cys Pro Ala Ala Lys Glu Asn Gly 1525 1530 1535 tca cgg tgg atc
cag tac aag ggt cac tgt tac aag tct gat cag gca 4656 Ser Arg Trp
Ile Gln Tyr Lys Gly His Cys Tyr Lys Ser Asp Gln Ala 1540 1545 1550
ttg cac agt ttt tca gag gcc aaa aaa ttg tgt tca aaa cat gat cac
4704 Leu His Ser Phe Ser Glu Ala Lys Lys Leu Cys Ser Lys His Asp
His 1555 1560 1565 tct gca act atc gtt tcc ata aaa gat gaa gat gag
aat aaa ttt gtg 4752 Ser Ala Thr Ile Val Ser Ile Lys Asp Glu Asp
Glu Asn Lys Phe Val 1570 1575 1580 agc aga ctg atg agg gaa aat aat
aac att acc atg aga gtt tgg ctt 4800 Ser Arg Leu Met Arg Glu Asn
Asn Asn Ile Thr Met Arg Val Trp Leu 1585 1590 1595 1600 gga tta tct
caa cat tct gtt gac cag tct tgg agt tgg tta gat gga 4848 Gly Leu
Ser Gln His Ser Val Asp Gln Ser Trp Ser Trp Leu Asp Gly 1605 1610
1615 tca gaa gtg aca ttt gtc aaa tgg gaa aat aaa agt aag agt ggt
gtt 4896 Ser Glu Val Thr Phe Val Lys Trp Glu Asn Lys Ser Lys Ser
Gly Val 1620 1625 1630 gga aga tgt agc atg ttg ata gct tca aat gaa
act tgg aaa aaa gtt 4944 Gly Arg Cys Ser Met Leu Ile Ala Ser Asn
Glu Thr Trp Lys Lys Val 1635 1640 1645 gaa tgt gaa cat ggt ttt gga
aga gtt gtc tgc aaa gtg cct ctg ggc 4992 Glu Cys Glu His Gly Phe
Gly Arg Val Val Cys Lys Val Pro Leu Gly 1650 1655 1660 cct gat tac
aca gca ata gct atc ata gtt gcc aca cta agt atc tta 5040 Pro Asp
Tyr Thr Ala Ile Ala Ile Ile Val Ala Thr Leu Ser Ile Leu 1665 1670
1675 1680 gtt ctc atg ggc gga ctg att tgg ttc ctc ttc caa agg cac
cgt ttg 5088 Val Leu Met Gly Gly Leu Ile Trp Phe Leu Phe Gln Arg
His Arg Leu 1685 1690 1695 cac ctg gcg ggt ttc tca tca gtt cga tat
gca caa gga gtg aat gaa 5136 His Leu Ala Gly Phe Ser Ser Val Arg
Tyr Ala Gln Gly Val Asn Glu 1700 1705 1710 gat gag att atg ctt cct
tct ttc cat gac taa 5169 Asp Glu Ile Met Leu Pro Ser Phe His Asp
1715 1720 3 349 DNA Homo sapiens 3 aacagttgat tgcaatgaca atcaaccagg
tgctatttgc tactattcag gaaatgagac 60 tgaaaaagag gtcaaaccag
ttgacagtgt taaatgtcca tctcctgttc taaatactcc 120 gtggatacca
tttcagaact gttgctacaa tttcataata acaaagaata ggcatatggc 180
aacaacacag gatgaagttc atactaaatg ccagaaactg aatccaaaat cacatattct
240 gagtattcga gatgaaaagg agaataactt tgttcttgag caactgctgt
acttcaatta 300 tatggcttca tgggtcatgt taggaataac ttatagaaat
aaktctctt 349 4 152 DNA Homo sapiens 4 attaatatgc tgtggaagtg
ggtgtcccag catcggctct ttcatttgca ctcccaaaag 60 tgccttggcc
tcgatattac caaatcggta aatgagctga gaatgttcag ctgtgactcc 120
agtgccatgc tgtggtggaa atgcgagcac ca 152 5 20 DNA Artificial
Sequence Description of Artificial Sequence Primer 5 gaycangayg
gnttytggaa 20 6 20 DNA Artificial Sequence Description of
Artificial Sequence Primer 6 tacaccaarc trttytgncg 20 7 20 DNA
Artificial Sequence Description of Artificial Sequence Primer 7
aayatgctnt ggaartgggt 20 8 20 DNA Artificial Sequence Description
of Artificial Sequence Primer 8 tgrtgytcrc ayttccacca 20 9 20 DNA
Artificial Sequence Description of Artificial Sequence Primer 9
gayacngayg gnttytggaa 20 10 20 DNA Artificial Sequence Description
of Artificial Sequence Primer 10 gcngtyttrt craaccacat 20 11 26 DNA
Artificial Sequence Description of Artificial Sequence Primer 11
gctctagaaa catgacccat gaagcc 26 12 27 DNA Artificial Sequence
Description of Artificial Sequence Primer 12 gctctagaca tcggctcttt
catttgt 27 13 27 DNA Artificial Sequence Description of Artificial
Sequence Primer 13 cgggattcac agttgattgc aatgaca 27 14 35 DNA
Artificial Sequence Description of Artificial Sequence Oligo d(T)
adaptor primer 14 gactagtctg cagaattctt tttttttttt ttttt 35 15 18
DNA Artificial Sequence Description of Artificial Sequence Adaptor
primer 15 gactagtctg cagaattc 18 16 28 DNA Artificial Sequence
Description of Artificial Sequence Primer 16 cgggatccct ctggccgcgc
actaatga 28 17 31 DNA Artificial Sequence Description of Artificial
Sequence Primer 17 ccgctcgagc tgtggatacc agcacatgcc t 31 18 24 DNA
Artificial Sequence Description of Artificial Sequence Primer 18
gatgggaact cttatgggag acct 24 19 24 DNA Artificial Sequence
Description of Artificial Sequence Primer 19 tgatgcaggc tggctgccaa
ataa 24 20 24 DNA Artificial Sequence Description of Artificial
Sequence Primer 20 aactgggcaa ctgttggtgg aaga 24 21 24 DNA
Artificial Sequence Description of Artificial Sequence Primer 21
atggcgaaga ggctggcatt tcta 24 22 24 DNA Artificial Sequence
Description of Artificial Sequence Primer 22 ctcaagcaag cgatacctgt
cact 24 23 24 DNA Artificial Sequence Description of Artificial
Sequence Primer 23 tgggcaactc gaagactgtg tagt 24 24 24 DNA
Artificial Sequence Description of Artificial Sequence Primer 24
caccagcaca gcattcttgc ttgt 24 25 24 DNA Artificial Sequence
Description of Artificial Sequence Primer 25 atttgtgagc agactgatga
ggga 24 26 32 DNA Artificial Sequence Description of Artificial
Sequence PCR-fragment 26 cggaattcga tctcatgata aggctggtca ca 32 27
21 DNA Artificial Sequence Description of Artificial Sequence
Primer 060 27 gtggatccag tacaagggtc a 21 28 21 DNA Artificial
Sequence Description of Artificial Sequence Primer 056 28
accaaatcag tccgcccatg a 21 29 21 DNA Artificial Sequence
Description of Artificial Sequence Primer 053 29 atggggaagg
tgaaggtcgg a 21 30 21 DNA Artificial Sequence Description of
Artificial Sequence Primer 053 30 aggggccatc cacagtcttc t 21 31
1723 PRT Murine sp. 31 Met Arg Thr Gly Arg Val Thr Pro Gly Leu Ala
Ala Gly Leu Leu Leu 1 5 10 15 Leu Leu Leu Arg Ser Phe Gly Leu Val
Glu Pro Ser Glu Ser Ser Gly 20 25 30 Asn Asp Pro Phe Thr Ile Val
His Glu Asn Thr Gly Lys Cys Ile Gln 35 40 45 Pro Leu Ser Asp Trp
Val Val Ala Gln Asp Cys Ser Gly Thr Asn Asn 50 55 60 Met Leu Trp
Lys Trp Val Ser Gln His Arg Leu Phe His Leu Glu Ser 65 70 75 80 Gln
Lys Cys Leu Gly Leu Asp Ile Thr Lys Ala Thr Asp Asn Leu Arg 85 90
95 Met Phe Ser Cys Asp Ser Thr Val Met Leu Trp Trp Lys Cys Glu His
100 105 110 His Ser Leu Tyr Thr Ala Ala Gln Tyr Arg Leu Ala Leu Lys
Asp Gly 115 120 125 Tyr Ala Val Ala Asn Thr Asn Thr Ser Asp Val Trp
Lys Lys Gly Gly 130 135 140 Ser Glu Glu Asn Leu Cys Ala Gln Pro Tyr
His Glu Ile Tyr Thr Arg 145 150 155 160 Asp Gly Asn Ser Tyr Gly Arg
Pro Cys Glu Phe Pro Phe Leu Ile Gly 165 170 175 Glu Thr Trp Tyr His
Asp Cys Ile His Asp Glu Asp His Ser Gly Pro 180 185 190 Trp Cys Ala
Thr Thr Leu Ser Tyr Glu Tyr Asp Gln Lys Trp Gly Ile 195 200 205 Cys
Leu Leu Pro Glu Ser Gly Cys Glu Gly Asn Trp Glu Lys Asn Glu 210 215
220 Gln Ile Gly Ser Cys Tyr Gln Phe Asn Asn Gln Glu Ile Leu Ser Trp
225 230 235 240 Lys Glu Ala Tyr Val Ser Cys Gln Asn Gln Gly Ala Asp
Leu Leu Ser 245 250 255 Ile His Ser Ala Ala Glu Leu Ala Tyr Ile Thr
Gly Lys Glu Asp Ile 260 265 270 Ala Arg Leu Val Trp Leu Gly Leu Asn
Gln Leu Tyr Ser Ala Arg Gly 275 280 285 Trp Glu Trp Ser Asp Phe Arg
Pro Leu Lys Phe Leu Asn Trp Asp Pro 290 295 300 Gly Thr Pro Val Ala
Pro Val Ile Gly Gly Ser Ser Cys Ala Arg Met 305 310 315 320 Asp Thr
Glu Ser Gly Leu Trp Gln Ser Val Ser Cys Glu Ser Gln Gln 325 330 335
Pro Tyr Val Cys Lys Lys Pro Leu Asn Asn Thr Leu Glu Leu Pro Asp 340
345 350 Val Trp Thr Tyr Thr Asp Thr His Cys His Val Gly Trp Leu Pro
Asn 355 360 365 Asn Gly Phe Cys Tyr Leu Leu Ala Asn Glu Ser Ser Ser
Trp Asp Ala 370 375 380 Ala His Leu Lys Cys Lys Ala Phe Gly Ala Asp
Leu Ile Ser Met His 385 390 395 400 Ser Leu Ala Asp Val Glu Val Val
Val Thr Lys Leu His Asn Gly Asp 405 410 415 Val Lys Lys Glu Ile Trp
Thr Gly Leu Lys Asn Thr Asn Ser Pro Ala 420 425 430 Leu Phe Gln Trp
Ser Asp Gly Thr Glu Val Thr Leu Thr Tyr Trp Asn 435 440 445 Glu Asn
Glu Pro Ser Val Pro Phe Asn Lys Thr Pro Asn Cys Val Ser 450 455 460
Tyr Leu Gly Lys Leu Gly Gln Trp Lys Val Gln Ser Cys Glu Lys Lys 465
470 475 480 Leu Arg Tyr Val Cys Lys Lys Lys Gly Glu Ile Thr Lys Asp
Ala Glu 485 490 495 Ser Asp Lys Leu Cys Pro Pro Asp Glu Gly Trp Lys
Arg His Gly Glu 500 505 510 Thr Cys Tyr Lys Ile Tyr Glu Lys Glu Ala
Pro Phe Gly Thr Asn Cys 515 520 525 Asn Leu Thr Ile Thr Ser Arg Phe
Glu Gln Glu Phe Leu Asn Tyr Met 530 535 540 Met Lys Asn Tyr Asp Lys
Ser Leu Arg Lys Tyr Phe Trp Thr Gly Leu 545 550 555 560 Arg Asp Pro
Asp Ser Arg Gly Glu Tyr Ser Trp Ala Val Ala Gln Gly 565 570 575 Val
Lys Gln Ala Val Thr Phe Ser Asn Trp Asn Phe Leu Glu Pro Ala 580 585
590 Ser Pro Gly Gly Cys Val Ala Met Ser Thr Gly Lys Thr Leu Gly Lys
595 600 605 Trp Glu Val Lys Asn Cys Arg Ser Phe Arg Ala Leu Ser Ile
Cys Lys 610 615 620 Lys Val Ser Glu Pro Gln Glu Pro Glu Glu Ala Ala
Pro Lys Pro Asp 625 630 635 640 Asp Pro Cys Pro Glu Gly Trp His Thr
Phe Pro Ser Ser Leu Ser Cys 645 650 655 Tyr Lys Val Phe His Ile Glu
Arg Ile Val Arg Lys Arg Asn Trp Glu 660 665 670 Glu Ala Glu Arg Phe
Cys Gln Ala Leu Gly Ala His Leu Pro Ser Phe 675 680 685 Ser Arg Arg
Glu Glu Ile Lys Asp Phe Val His Leu Leu Lys Asp Gln 690 695 700 Phe
Ser Gly Gln Arg Trp Leu Trp Ile Gly Leu Asn Lys Arg Ser Pro 705 710
715 720 Asp Leu Gln Gly Ser Trp Gln Trp Ser Asp Arg Thr Pro Val Ser
Ala 725 730 735 Val Met Met Glu Pro Glu Phe Gln Gln Asp Phe Asp Ile
Arg Asp Cys 740 745 750 Ala Ala Ile Lys Val Leu Asp Val Pro Trp Arg
Arg Val Trp His Leu 755 760 765 Tyr Glu Asp Lys Asp Tyr Ala Tyr Trp
Lys Pro Phe Ala Cys Asp Ala 770 775 780 Lys Leu Glu Trp Val Cys Gln
Ile Pro Lys Gly Ser Thr Pro Gln Met 785 790 795 800 Pro Asp Trp Tyr
Asn Pro Glu Arg Thr Gly Ile His Gly Pro Pro Val 805 810 815 Ile Ile
Glu Gly Ser Glu Tyr Trp Phe Val Ala Asp Pro His Leu Asn 820 825 830
Tyr Glu Glu Ala Val Leu Tyr Cys Ala Ser Asn His Ser Phe Leu Ala 835
840 845 Thr Ile Thr Ser Phe Thr Gly Leu Lys Ala Ile Lys Asn Lys Leu
Ala 850 855 860 Asn Ile Ser Gly Glu Glu Gln Lys Trp Trp Val Lys Thr
Ser Glu Asn 865 870 875 880 Pro Ile Asp Arg Tyr Phe Leu Gly Ser Arg
Arg Arg Leu Trp His His 885 890 895 Phe Pro Met Thr Phe Gly Asp Glu
Cys Leu His Met Ser Ala Lys Thr 900 905 910 Trp Leu Val Asp Leu Ser
Lys Arg Ala Asp Cys Asn Ala Lys Leu Pro 915 920 925 Phe Ile Cys Glu
Arg Tyr Asn Val Ser Ser Leu Glu Lys Tyr Ser Pro 930 935 940 Asp Pro
Ala Ala Lys Val Gln Cys Thr Glu Lys Trp Ile Pro Phe Gln 945 950 955
960 Asn Lys Cys Phe Leu Lys Val Asn Ser Gly Pro Val Thr Phe Ser Gln
965 970 975 Ala Ser Gly Ile Cys His Ser Tyr Gly Gly Thr Leu Pro Ser
Val Leu 980 985 990 Ser Arg Gly Glu Gln Asp Phe Ile Ile Ser Leu Leu
Pro Glu Met Glu 995 1000 1005 Ala Ser Leu Trp Ile Gly Leu Arg Trp
Thr Ala Tyr Glu Arg Ile Asn 1010 1015 1020 Arg Trp Thr Asp Asn Arg
Glu Leu Thr Tyr Ser Asn Phe His Pro Leu 1025 1030 1035 1040 Leu Val
Gly Arg Arg Leu Ser Ile Pro Thr Asn Phe Phe Asp Asp Glu 1045 1050
1055 Ser His Phe His Cys Ala Leu Ile Leu Asn Leu Lys Lys Ser Pro
Leu 1060 1065 1070 Thr Gly Thr Trp Asn Phe Thr Ser Cys Ser Glu Arg
His Ser Leu Ser 1075 1080 1085 Leu Cys Gln Lys Tyr Ser Glu Thr Glu
Asp Gly Gln Pro Trp Glu Asn 1090 1095 1100 Thr Ser Lys Thr Val Lys
Tyr Leu Asn Asn Leu Tyr Lys Ile Ile Ser 1105 1110 1115 1120 Lys Pro
Leu Thr Trp His Gly Ala Leu Lys Glu Cys Met Lys Glu Lys 1125 1130
1135 Met Arg Leu Val Ser Ile Thr Asp Pro Tyr Gln Gln Ala Phe Leu
Ala 1140 1145 1150 Val Gln Ala Thr Leu Arg Asn Ser Ser Phe Trp Ile
Gly Leu Ser Ser 1155 1160 1165 Gln Asp Asp Glu Leu Asn Phe Gly Trp
Ser Asp Gly Lys Arg Leu Gln 1170 1175 1180 Phe Ser Asn Trp Ala Gly
Ser Asn Glu Gln Leu Asp Asp Cys Val Ile 1185 1190 1195 1200 Leu Asp
Thr Asp Gly Phe Trp Lys Thr Ala Asp Cys Asp Asp Asn Gln 1205 1210
1215 Pro Gly Ala Ile Cys Tyr Tyr Pro Gly Asn Glu Thr Glu Glu Glu
Val 1220 1225 1230 Arg Ala Leu Asp Thr Ala Lys Cys Pro Ser Pro Val
Gln Ser Thr Pro 1235 1240 1245 Trp Ile Pro Phe Gln Asn Ser Cys Tyr
Phe Asn Met Ile Thr Asn Asn 1250 1255 1260 Arg His Lys Thr Val Thr
Pro Glu Glu Val Gln Ser Thr Cys Glu Lys 1265 1270 1275 1280 Leu His
Pro Lys Ala His Ser Leu Ser Ile Arg Asn Glu Glu Glu Asn 1285 1290
1295 Thr Phe Val Val Glu Gln Leu Leu Tyr Phe Asn Tyr Ile Ala Ser
Trp 1300 1305 1310 Val Met Leu Gly Ile Thr Tyr Glu Asn Asn Ser Leu
Met Trp Phe Asp 1315 1320 1325 Lys Thr Ala Leu Ser Tyr Thr His Trp
Arg Thr Gly Arg Pro Thr Val 1330 1335 1340 Lys Asn Gly Lys Phe Leu
Ala Gly Leu Ser Thr Asp Gly Phe Trp Asp 1345 1350 1355 1360 Ile Gln
Ser Phe Asn Val Ile Glu Glu Thr Leu His Phe Tyr Gln His 1365 1370
1375 Ser Ile Ser Ala Cys Lys Ile Lys Met Val Asp Tyr Glu Asp Lys
His 1380 1385 1390 Asn Gly Thr Leu Pro Gln Phe Ile Pro Tyr Lys Asp
Gly Val Tyr Ser 1395 1400 1405 Val Ile Gln Lys Lys Val Thr Trp Tyr
Glu Ala Leu Asn Ala Cys Ser 1410 1415 1420 Gln Ser Gly Gly Glu Leu
Ala Ser Val His Asn Pro Asn Gly Lys Leu 1425 1430 1435 1440 Phe Leu
Glu Asp Ile Val Asn Arg Asp Gly Phe Pro Leu Asn Val Gly 1445 1450
1455 Leu Ser Ser His Asp Gly Ser Glu Ser Ser Phe Glu Trp Ser Asp
Gly 1460 1465 1470 Arg Ala Phe Asp Tyr Val Pro Trp Gln Ser Leu Gln
Ser Pro Gly Asp 1475 1480 1485 Cys Val Val Leu Tyr Pro Lys Gly Ile
Trp Arg Arg Glu Lys Cys Leu 1490 1495 1500 Ser Val Lys Asp Gly Ala
Ile Cys Tyr Lys Pro Thr Lys Asp Lys Lys 1505 1510 1515 1520 Leu Ile
Phe His Val Lys Ser Ser Lys Cys Pro Val Ala Lys Arg Asp 1525 1530
1535 Gly Pro Gln Trp Val Gln Tyr Gly Gly His Cys Tyr Ala Ser Asp
Gln 1540 1545 1550 Val Leu His Ser Phe Ser Glu Ala Lys Gln Val Cys
Gln Glu Leu Asp 1555 1560 1565 His Ser Ala Thr Val Val Thr Ile Ala
Asp Glu Asn Glu Asn Lys Phe 1570 1575 1580 Val Ser Arg Leu Met Arg
Glu Asn Tyr Asn Ile Thr Met Arg Val Trp 1585 1590 1595 1600 Leu Gly
Leu Ser Gln His Ser Leu Asp Gln Ser Trp Ser Trp Leu Asp 1605 1610
1615 Gly Leu Asp Val Thr Phe Val Lys Trp Glu Asn Lys Thr Lys Asp
Gly 1620 1625 1630 Asp Gly Lys Cys Ser Ile Leu Ile Ala Ser Asn Glu
Thr Trp Arg Lys 1635 1640 1645 Val His Cys Ser Arg Gly Tyr Ala Arg
Ala Val Cys Lys Ile Pro Leu 1650 1655 1660 Ser Pro Asp Tyr Thr Gly
Ile Ala Ile Leu Phe Ala Val Leu Cys Leu 1665 1670 1675 1680 Leu Gly
Leu Ile Ser Leu Ala Ile Trp Phe Leu Leu Gln Arg Ser His 1685 1690
1695 Ile Arg Trp Thr Gly Phe Ser Ser Val Arg Tyr Glu His Gly Thr
Asn 1700 1705 1710 Glu Asp Glu Val Met Leu Pro Ser Phe His Asp 1715
1720
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