U.S. patent number RE39,015 [Application Number 09/935,366] was granted by the patent office on 2006-03-14 for dna encoding interleukin-b30.
This patent grant is currently assigned to Schering Corporation. Invention is credited to J. Fernando Bazan.
United States Patent |
RE39,015 |
Bazan |
March 14, 2006 |
DNA encoding interleukin-B30
Abstract
Purified genes encoding cytokines from a mammal, reagents
related thereto including purified proteins, specific antibodies,
and nucleic acids encoding this molecule are provided. Methods of
using the reagents and diagnostic kits are also provided.
Inventors: |
Bazan; J. Fernando (Palo Alto,
CA) |
Assignee: |
Schering Corporation
(Kenilworth, NJ)
|
Family
ID: |
26732218 |
Appl.
No.: |
09/935,366 |
Filed: |
August 22, 2001 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
Issue Date |
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60053765 |
Jul 25, 1997 |
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Reissue of: |
09122443 |
Jul 24, 1998 |
06060284 |
May 9, 2000 |
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Current U.S.
Class: |
435/69.52;
435/252.3; 435/254.11; 435/320.1; 435/325; 435/471; 435/71.1;
435/71.2; 530/351; 536/23.1; 536/23.5 |
Current CPC
Class: |
C07H
21/04 (20130101); C07K 14/54 (20130101); A61K
38/00 (20130101); A61K 2039/505 (20130101); Y10T
436/143333 (20150115) |
Current International
Class: |
C12N
15/24 (20060101); C07K 14/54 (20060101); C12N
15/63 (20060101); C12N 5/10 (20060101) |
Field of
Search: |
;435/69.52,70.1,71.1,71.2,725,421,320.1,252.3,254.11
;536/23.1,23.5,24.3,24.31 ;530/350,357 |
Other References
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126-128 and 228-234. cited by examiner .
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prediction. Trends in Genetics. Jun. 1998, vol. 14, No. 6, pp.
248-250. cited by examiner .
Brenner et al. Errors in Genome Annotation. Trends in Genetics
1999, 15:132-133. cited by examiner .
Bork et al. Go Hunting in sequence databases but watch out for the
traps. Trends in Genetics 1996, 12:425-427. cited by examiner .
Bork et al., Current Opinion in Structural Biology (1998)
8:331-332. cited by other .
Ngo et al., The Protein Folding Problem and Tertiary Structure
Prediction (1994) pp. 492-495. cited by other .
Schafer et al., Gene (1993) 136:295-300. cited by other .
Skolnick et al., Tibtech (2000) 18:34-39. cited by other .
Wells, Aditivity of Mutational Effects in Proteins, Biochemistry
(1990) 26(37):8509-8517. cited by other .
F.R. Blattner, et al., GenPept, Accession No. 2506659, Nov. 1,
1997. Definition: "Hypothetical 55.1 KD Protein in OGT-DBPA
Intergenic Region." cited by examiner .
T.D. Gaffney, et al., GenPept, Accession No. 1172467, Nov. 1, 1995.
Definition: "CDP-Diacylglycerol--Glycerol-3-Phosphate
3-Phosphatidyltransferase (Phosphatidylglycerophosphate Synthase)
(PGP Synthase)." cited by examiner .
Toshio Hirano, The Cytokine Handbook, Angus W. Thompson, Editor,
2nd Ed., Ch. 8:145-168, Academic Press, San Diego, 1994.
"Interleukin-6". cited by examiner .
L. Hillier, et al., GenBank, Accession No. AA418955, Oct. 16, 1997.
Definition: "zw01c10.r1 Soars NhHMPu S1 Homo sapiens cDNA clone
768018 5'." cited by examiner .
Y. Ito, GenBank, Accession No. AB004061, Jun. 2, 1997. Definition:
"domestic pig mRNA for STAT2, complete cds." cited by examiner
.
Shigekazu Nagata, The Cytokine Handbook, Angus W. Thompson, Editor,
2nd Ed., Ch. 20:371-385-168, Academic Press, San Diego, 1994.
"Granulocyte Colony Stimulation Factor and its Receptor". cited by
examiner .
W. Northemann, et al., GenPept, Accession No. 111880, Jun. 18,
1993. Definition: "interleukin-6-precursor--rat." cited by examiner
.
W. Northemann, et al., GenPept, Accession No. 124350, Jul. 15,
1998. Definition: "Interleukin-6 Precursor (IL-6)". cited by
examiner .
J. Takeda, GenBank, Accession No. C06368, Oct. 11, 1996.
Definition: "similar to none". cited by examiner .
M. Tsuchiya, et al., GenPept, Accession No. 117565, Jul. 15, 1998.
Definition: "Granulocyte Colony-Stimulating Factor Precursor
(G-CSF)." cited by examiner .
M. Tsuchiya, et al., GenPept, Accession No. 90543, Jun. 7, 1996.
Definition: "granulocyte colony-stimulating factor
precursor--mouse." cited by examiner .
J. Van Snick, et al., GenPept, Accession No. 124348, Jul. 15, 1998.
Definition: Interleukin-6 Precursor (IL-6) (Interleukin HP-1)
(B-Cell Hybridoma Growth Factor). cited by examiner .
J. Van Snick, et al., GenPept, Accession No. 69691, Jun. 7, 1996.
Definition "interleukin-6 precursor--mouse." cited by
examiner.
|
Primary Examiner: Mertz; Prema
Attorney, Agent or Firm: Hill; Laurie L. Ching; Edwin P.
Parent Case Text
This application is a conversion of provisional U.S. patent
application U.S. Ser. No. 60/053,765, filed Jul. 25, 1997, which is
incorporated herein by reference.
Claims
What is claimed is:
.[.1. An isolated or recombinant polynucleotide encoding an
antigenic polypeptide comprising: a) at least 17 contiguous amino
acids from a mature coding portion of SEQ ID NO: 2, b) at least 17
contiguous amino acids from a mature coding portion of SEQ ID NO:
4; or c) at least 17 contiguous amino acids from a mature coding
portion of SEQ ID NO: 5..].
.[.2. The polynucleotide of claim 1, encoding a mature polypeptide
of: a) SEQ ID NO: 2; b) SEQ ID NO: 4; or c) SEQ ID NO: 5..].
.[.3. The polynucleotide of claim 1, which encodes amino residues
155-164 of SEQ ID NO: 2 and hybridizes under stringent wash
conditions of at least 65.degree. C., less than about 150 mM salt
to the complement of: a) the open reading frame of SEQ ID NO: 1; or
b) the open reading frame of SEQ ID NO: 3..].
.[.4. The polynucleotide of claim 3, comprising: a) at least 67
contiguous nucleotides of a coding portion of SEQ ID NO: 1, wherein
said contiguous nucleotides are from nucleotides 466-555 of SEQ ID
NO: 1; or b) at least 67 contiguous nucleotides of a coding portion
of SEQ ID NO: 3, wherein said contiguous nucleotides are from
nucleotides 580-670 of SEQ ID NO: 3..].
.[.5. A recombinant or expression vector comprising said
polynucleotide of claim 1..].
.[.6. An isolated host cell comprising said expression vector of
claim 5..].
.[.7. A method of making an antigenic polypeptide comprising
expressing said recombinant polynucleotide of claim 1 and isolating
said polypeptide, thereby making said antigenic polypeptide..].
.[.8. Said polynucleotide of claim 1, wherein said contiguous amino
acids number 20..].
.[.9. Said polynucleotide of claim 1, wherein said contiguous amino
acids number 30..].
.[.10. Said polynucleotide of claim 1, wherein said contiguous
amino acids number 35..].
.[.11. Said polynucleotide of claim 1, wherein said contiguous
amino acids number 40..].
.[.12. Said polynucleotide of claim 2, that is a variant due to the
degeneracy of the genetic code..].
.[.13. The polynucleotide of claim 8, wherein said wash conditions
are a) at least 70.degree. C.; b) less than about 100 mM salt; or
c) both a) and b)..].
.[.14. The polynucleotide of claim 3, wherein said wash conditions
a) are at least 50% formamide; b) are less than about 100 mM salt;
or c) are both a) and b)..].
.[.15. The polynucleotide of claim 1, that: a) encodes the mature
polypeptide of SEQ ID NO:2,4, or 5; or b) comprises the mature
coding portion of SEQ ID NO: 1 or 3..].
.[.16. The polynucleotide of claim 2, wherein said polynucleotide:
a) encodes a polypeptide with a natural sequence of the mature
coding portion of SEQ ID NO: 2 or 4; b) is isolated from nature; c)
encodes a polypeptide comprising five or fewer conservative
substitutions from a natural sequence of SEQ ID NO: 2 or 4; d)
encodes a polypeptide comprising five or fewer conservative
substitutions from a natural sequence of SEQ ID NO: 4..].
.[.17. The polynucleotide of claim 8, which: a) is attached to a
solid substrate; b) is detectably labeled; c) is in a sterile
composition; d) encodes an antigenic polypeptide having at least 12
amino acid residues; or e) is synthetically produced..].
.Iadd.18. An isolated or recombinant polynucleotide encoding a
polypeptide of: a) SEQ ID NO:2; b) SEQ ID NO:4; or c) SEQ ID
NO:5..Iaddend.
.Iadd.19. A recombinant or expression vector comprising said
polynucleotide of claim 18..Iaddend.
.Iadd.20. An isolated host cell comprising said expression vector
of claim 19..Iaddend.
.Iadd.21. A method of making a polypeptide comprising expressing
said recombinant polynucleotide of claim 18 and isolating said
polypeptide, thereby making said polypeptide..Iaddend.
.Iadd.22. The polynucleotide of claim 18, that is a variant due to
the degeneracy of the genetic code..Iaddend.
.Iadd.23. An isolated or recombinant polynucleotide that: a)
encodes the mature polypeptide of SEQ ID NO: 2, 4, or 5; or b)
comprises the mature coding portion of SEQ ID NO: 1 or
3..Iaddend.
.Iadd.24. The polynucleotide of claim 18, wherein said
polynucleotide is isolated from a human or a mouse..Iaddend.
.Iadd.25. The polynucleotide of claim 18, which: a) is attached to
a solid substrate; b) is detectably labeled; c) is in sterile
composition; or d) is synthetically produced..Iaddend.
.Iadd.26. A recombinant or expression vector comprising said
polynucleotide of claim 23..Iaddend.
.Iadd.27. An isolated host cell comprising said expression vector
of claim 26..Iaddend.
.Iadd.28. A method of making a polypeptide comprising expressing
said recombinant polynucleotide of claim 23 and isolating said
polypeptide, thereby making said polypeptide..Iaddend.
.Iadd.29. The polynucleotide of claim 23, that is a variant due to
the degeneracy of the genetic code..Iaddend.
.Iadd.30. The polynucleotide of claim 23, wherein said
polynucleotide is isolated from a human or a mouse..Iaddend.
.Iadd.31. The polynucleotide of claim 23, which: a) is attached to
a solid substrate; b) is detectably labeled; c) is in sterile
composition; or d) is synthetically produced..Iaddend.
Description
FIELD OF THE INVENTION
The present invention pertains the compositions related to proteins
which function in controlling biology and physiology of mammalian
cells, e.g., cells of a mammalian immune system. In particular, it
provides purified genes, proteins, antibodies, and related reagents
useful, e.g., to regulate activation, development, differentiation,
and function of various cell types, including hematopoietic
cells.
BACKGROUND OF THE INVENTION
Recombinant DNA technology refers generally to the technique of
integrating genetic information from a donor source into vectors
for subsequent processing, such as through introduction into a
host, whereby the transferred genetic information is copied and/or
expressed in the new environment. Commonly, the genetic information
exists in the form of complementary DNA (cDNA) derived from
messenger RNA (mRNA) coding for a desired protein product. The
carrier is frequently a plasmid having the capacity to incorporate
cDNA for later replication in a host and, in some cases, actually
to control expression of the cDNA and thereby direct synthesis of
the encoded product in the host.
For some time, it has been known that the mammalian immune response
is based on a series of complex cellular interactions, called the
"immune network". Recent research has provided new insights into
the inner workings of this network. While it remains clear that
much of the response does, in fact, revolve around the network-like
interactions of lymphocytes, macrophages, granulocytes, and other
cells, immunologists now generally hold the opinion that soluble
proteins, known as lymphokines, cytokines, or monokines, play a
critical role in controlling these cellular interactions. Thus,
there is considerable interest in the isolation, characterization,
and mechanisms of action of cell modulatory factors, an
understanding of which will lead to significant advancements in the
diagnosis and therapy of numerous medical abnormalities, e.g.,
immune system disorders. Some of these factors are hematopoietic
growth factors, e.g., granulocyte colony stimulatory factor
(G-CSF). See, e.g., Thomson (1994; ed.) The Cytokine Handbook (2d
ed.) Academic Press, San Diego; Metcalf and Nicola (1995) The
Hematopoietic Colony Stimulatory Factors Cambridge University
Press; and Aggarwal and Gutterman (1991) Human Cytokines Blackwell
Pub.
Lymphokines apparently mediate cellular activities in a variety of
ways. They have been shown to support the proliferation, growth,
and differentiation of pluripotential hematopoietic stem cells into
vast numbers of progenitors comprising diverse cellular lineages
making up a complex immune system. Proper and balanced interactions
between the cellular components are necessary for a healthy immune
response. The different cellular lineages often respond in a
different manner when lymphokines are administered in conjunction
with other agents.
Cell lineages especially important to the immune response include
two classes of lymphocytes: B-cells, which can produce and secrete
immunoglobulins (proteins with the capability of recognizing and
binding to foreign matter to effect its removal), and T-cells of
various subsets that secrete lymphokines and induce or suppress the
B-cells and various other cells (including other T-cells) making up
the immune network. These lymphocytes interact with many other cell
types.
Another important cell lineage is the mast cell (which has not been
positively identified in all mammalian species), which is a
granule-containing connective tissue cell located proximal to
capillaries throughout the body. These cells are found in
especially high concentrations in the lungs, skin, and
gastrointestinal and genitourinary tracts. Mast cells play a
central role in allergy-related disorders, particularly anaphylaxis
as follows: when selected antigens crosslink one class of
immunoglobulins bound to receptors on the mast cell surface, the
mast cell degranulates and releases mediators, e.g., histamine,
serotonin, heparin, and prostaglandins, which cause allergic
reactions, e.g., anaphylaxis.
Research to better understand and treat various immune disorders
has been hampered by the general inability to maintain cells of the
immune system in vitro. Immunologists have discovered that
culturing these cells can be accomplished through the use of T-cell
and other cell supernatants, which contain various growth factors,
including many of the lymphokines.
From the foregoing, it is evident that the discovery and
development of new lymphokines, e.g., related to G-CSF and/or IL-6,
could contribute to new therapies for a wide range of degenerative
or abnormal conditions which directly or indirectly involve the
immune system and/or hematopoietic cells. In particular, the
discovery and development of lymphokines which enhance or
potentiate the beneficial activities of known lymphokines would be
highly advantageous. The present invention provides new interleukin
compositions and related compounds, and methods for their use.
SUMMARY OF THE INVENTION
The present invention is directed to mammalian, e.g., rodent,
canine, feline, primate, interleukin-B30 (IL-B30) and its
biological activities. It includes nucleic acids coding for
polypeptides themselves and methods for their production and use.
The nucleic acids of the invention are characterized, in part, by
their homology to cloned complementary DNA (cDNA) sequences
enclosed herein, and/or by functional assays for growth factor- or
cytokine-like activities, e.g., G-CSF (see Nagata (1994) in Thomson
The Cytokine Handbook 2d ed., Academic Press, San Diego) and/or
IL-6 (see Hirano (1994) in Thomson The Cytokine Handbook 2d ed.,
Academic Press, San Diego), applied to the polypeptides, which are
typically encoded by these nucleic acids. Methods for modulating or
intervening in the control of a growth factor dependent physiology
or an immune response are provided.
The present invention is based, in part, upon the discovery of a
new cytokine sequence exhibiting significant sequence and
structural similarity to G-CSF and IL-6. In particular, it provides
primate, e.g., human, gene encoding a protein whose mature size is
about 168 amino acids, and pig and murine, e.g., mouse, sequences.
Functional equivalents exhibiting significant sequence homology
will be available from other mammalian, e.g., cow, horse, and rat,
and non-mammalian species.
In various protein embodiments, the invention provides: a
substantially pure or recombinant IL-B30 protein or peptide
exhibiting at least about 85% sequence identity over a length of at
least about 12 amino acids to SEQ ID NO: 2; a natural sequence
IL-B30 of SEQ ID NO: 2; and a fusion protein comprising IL-B30
sequence. In certain embodiments, the homology is at least about
90% identity and the portion is at least about 9 amino acids; the
homology is at least about 80% identity and the portion is at least
about 17 amino acids; or the homology is at least about 70%
identity and the portion is at least about 25 amino acids. In other
embodiments, the IL-B30: comprises a mature sequence of Table 1; or
exhibits a post-translational modification pattern distinct from
natural IL-B30; or the protein or peptide: is from a warm blooded
animal selected from a mammal, including a primate; comprises at
least one polypeptide segment of SEQ ID NO: 2; exhibits a plurality
of portions exhibiting the identity; is a natural allelic variant
of IL-B30; has a length at least about 30 amino acids; exhibits at
least two non-overlapping epitopes which are specific for a
mammalian IL-B30; exhibits a sequence identity at least about 90%
over a length of at least about 20 amino acids to mammalian IL-B30;
is glycosylated; has a molecular weight of at least 10 kD with
natural glycosylation; is a synthetic polypeptide; is attached to a
solid substrate; is conjugated to another chemical moiety; is a
5-fold or less substitution from natural sequence; or is a deletion
or insertion variant from a natural sequence. Preferred embodiments
include a composition comprising: a sterile IL-B30 protein or
peptide; or the IL-B30 protein or peptide and a carrier, wherein
the carrier is: an aqueous compound, including water, saline,
and/or buffer; and/or formulated for oral, rectal, nasal, topical,
or parenteral administration. In fusion protein embodiments, the
protein can have: mature protein sequence of Table 1; a detection
or purification tag, including a FLAG, His6, or Ig sequence; and/or
sequence of another cytokine or chemokine.
Kit embodiments include those with an IL-B30 protein or
polypeptide, and: a compartment comprising the protein or
polypeptide; and/or instructions for use or disposal of reagents in
the kit.
In binding compound embodiments, the compound may have an antigen
binding site from an antibody, which specifically binds to a
natural IL-B30 protein, wherein: the IL-B30 is a mammalian protein;
the binding compound is an Fv, Fab, or Fab2 fragment; the binding
compound is conjugated to another chemical moiety; or the antibody:
is raised against a peptide sequence of a mature polypeptide of
Table 1; is raised against a mature IL-B30; is raised to a purified
rodent IL-B30; is immunoselected; is a polyclonal antibody; binds
to a denatured IL-B30; exhibits a Kd of at least 30 .mu.M; is
attached to a solid substrate, including a bead or plastic
membrane; is in a sterile composition; or is detectably labeled,
including a radioactive or fluorescent label. Kits containing
binding compounds include those with: a compartment comprising the
binding compound; and/or instructions for use or disposal of
reagents in the kit. Often the kit is capable of making a
qualitative or quantitative analysis. Preferred compositions will
comprise: a sterile binding compound; or the binding compound and a
carrier, wherein the carrier is: an aqueous compound, including
water, saline, and/or buffer; and/or formulated for oral, rectal,
nasal, topical, or parenteral administration.
Nucleic acid embodiments include an isolation or recombinant
nucleic acid encoding an IL-B30 protein or peptide or fusion
protein, wherein: the IL-B30 is from a mammal; and/or the nucleic
acid: encodes an antigenic peptide sequence of Table 1; encodes a
plurality of antigenic peptide sequences of Table 1; exhibits at
least about 80% identity to a natural cDNA encoding the segment; is
an expression vector; further comprises an origin of replication;
is from a natural source; comprises a detectable label; comprises
synthetic nucleotide sequence; is less than 6 kb, preferably less
than 3 kb; is from a mammal, including a primate; comprises a
natural full length coding sequence; is a hybridization probe for a
gene encoding the IL-B30; or is a PCR primer, PCR product, or
mutagenesis primer. The invention also provides a cell, tissue, or
organ comprising such a recombinant nucleic acid, and preferably
the cell will be: a prokaryotic cell; a eukaryotic cell; a
bacterial cell; a yeast cell; an insect cell; a mammalian cell; a
mouse cell; a primate cell; or a human cell.
Kit embodiments include those with such nucleic acids, and: a
compartment comprising the nucleic acid; a compartment further
comprising the IL-B30 protein or polypeptide; and/or instructions
for use or disposal of reagents in the kit. Typically, the kit is
capable of making a qualitative or quantitative analysis.
In certain embodiments, the nucleic acid: hybridizes under wash
conditions of 30.degree. C. and less than 2M salt, or of 45.degree.
C. and/or 500 mM salt, or 55.degree. C. and/or 150 mM salt, to SEQ
ID NO: 1; or exhibits at least about 85% identity and/or the
stretch is at least about 30 nucleotides, or exhibits at least 90%
identity and/or the stretch is at least 55 nucleotides, or exhibits
at least 95% and/or the stretch is at least 75 nucleotides, to a
primate IL-B30.
The invention embraces a method of modulating physiology or
development of a cell or tissue culture cells comprising contacting
the cell with an agonist or antagonist of a mammalian IL-B30. The
method may be where: the contacting is in combination with an
agonist or antagonist of G-CSF and/or IL-6; or the contacting is
with an antagonist, including a binding composition comprising an
antibody binding site which specifically binds an IL-B30.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
All references cited herein are incorporated herein by reference to
the same extent as if each individual publication or patent
application was specifically and individually indicated to be
incorporated by reference.
OUTLINE
I. General
II. Purified IL-B30
A. physical properties
B. biological properties
III. Physical Variants
A. sequence variants, fragments
B. post-translational variants 1. glycosylation 2. others IV.
Functional Variants
A. analogs, fragments 1. agonists 2. antagonists
B. mimetics 1. protein 2. chemicals
C. species variants
V. Antibodies
A. polyclonal
B. monoclonal
C. fragments, binding compositions
VI. Nucleic Acids
A. natural isolates; methods
B. synthetic genes
C. methods to isolate
VII. Making IL-B30, mimetics
A. recombinant methods
B. synthetic methods
C. natural purification
VIII. Uses
A. diagnostic
B. therapeutic
IX. Kits
A. nucleic acid reagents
B. protein reagents
C. antibody reagents
X. Isolating receptors for IL-B30
I. GENERAL
The present invention provides amino acid sequences and DNA
sequences encoding various mammalian proteins which are cytokines,
e.g., which are secreted molecules which can mediate a signal
between immune or other cells. See, e.g., Paul (1994) Fundamental
Immunology (3d ed.) Raven Press, N.Y. The full length cytokines,
and fragments, or antagonists will be useful in physiological
modulation of cells expressing a receptor. It is likely that IL-B30
has either stimulatory or inhibitory effects on hematopoietic
cells, including, e.g., lymphoid cells, such as T-cells, B-cells,
natural killer (NK) cells, macrophages, dendritic cells,
hematopoietic progenitors, etc. The proteins will also be useful as
antigens, e.g., immunogens, for raising antibodies to various
epitopes on the protein, both linear and conformational
epitopes.
A cDNA encoding IL-B30 was identified from a human cell line. The
molecule was designated huIL-B30. A related gene corresponding to a
pig sequence was also identified. A rodent sequence, e.g., from
mouse, is also described.
The human gene encodes a small soluble cytokine-like protein, of
about 168 amino acids. The signal sequence probably is about 21
residues, and would run from the Met to about Ala. See Table 1 and
SEQ. ID. NO: 1 and 2. IL-B30 exhibits structural motifs
characteristic of a member of the long chain cytokines. Compare,
e.g., IL-B30, G-CSF, and IL-6, sequences available from GenBank.
See also Table 2.
TABLE-US-00001 TABLE 1 Nucleic acid (SEQ ID NO: 1) encoding IL-B30
from a primate, e.g., human. Translated amino acid sequence is SEQ
ID NO: 2. ATG CTG GGG AGC AGA GCT GTA ATG CTG CTG TTG CTG CTG CCC
TGG ACA 48 Met Leu Gly Ser Arg Ala Val Met Leu Leu Leu Leu Leu Pro
Trp Thr -21 -20 -15 -10 GCT CAG GGC AGA GCT GTG CCT GGG GGC AGC AGC
CCT GCC TGG ACT CAG 96 Ala Gln Gly Arg Ala Val Pro Gly Gly Ser Ser
Pro Ala Trp Thr Gln -5 1 5 10 TGC CAG CAG CTT TCA CAG AAG CTC TGC
ACA CTG GCC TGG AGT GCA CAT 144 Cys Gln Gln Leu Ser Gln Lys Leu Cys
Thr Leu Ala Trp Ser Ala His 15 20 25 CCA CTA GTG GGA CAC ATG GAT
CTA AGA GAA GAG GGA GAT GAA GAG ACT 192 Pro Leu Val Gly His Met Asp
Leu Arg Glu Glu Gly Asp Glu Glu Thr 30 35 40 ACA AAT GAT GTT CCC
CAT ATC CAG TGT GGA GAT GGC TGT GAC CCC CAA 240 Thr Asn Asp Val Pro
His Ile Gln Cys Gly Asp Gly Cys Asp Pro Gln 45 50 55 GGA CTC AGG
GAC AAC AGT CAG TTC TGC TTG CAA AGG ATC CAC CAG GGT 288 Gly Leu Arg
Asp Asn Ser Gln Phe Cys Leu Gln Arg Ile His Gln Gly 60 65 70 75 CTG
ATT TTT TAT GAG AAG CTG CTA GGA TCG GAT ATT TTC ACA GGG GAG 336 Leu
Ile Phe Tyr Glu Lys Leu Leu Gly Ser Asp Ile Phe Thr Gly Glu 80 85
90 CCT TCT CTG CTC CCT GAT AGC CCT GTG GCG CAG CTT CAT GCC TCC CTA
384 Pro Ser Leu Leu Pro Asp Ser Pro Val Ala Gln Leu His Ala Ser Leu
95 100 105 CTG GGC CTC AGC CAA CTC CTG CAG CCT GAG GGT CAC CAC TGG
GAG ACT 432 Leu Gly Leu Ser Gln Leu Leu Gln Pro Glu Gly His His Trp
Glu Thr 110 115 120 CAG CAG ATT CCA AGC CTC AGT CCC AGC CAG CCA TGG
CAG CGT CTC CTT 480 Gln Gln Ile Pro Ser Leu Ser Pro Ser Gln Pro Trp
Gln Arg Leu Leu 125 130 135 CTC CGC TTC AAA ATC CTT CGC AGC CTC CAG
GCC TTT GTG GCT GTA GCC 528 Leu Arg Phe Lys Ile Leu Arg Ser Leu Gln
Ala Phe Val Ala Val Ala 140 145 150 155 GCC CGG GTC TTT GCC CAT GGA
GCA GCA ACC CTG AGT CCC TAA 570 Ala Arg Val Phe Ala His Gly Ala Ala
Thr Leu Ser Pro 160 165 coding sequence: ATGCTGGGGA GCAGAGCTGT
AATGCTGCTG TTGCTGCTGC CCTGGACAGC TCAGGGCAGA GCTGTGCCTG GGGGCAGCAG
CCCTGCCTGG ACTCAGTGCC AGCAGCTTTC ACAGAAGCTC TGCACACTGG CCTGGAGTGC
ACATCCACTA GTGGGACACA TGGATCTAAG AGAAGAGGGA GATGAAGAGA CTACAAATGA
TGTTCCCCAT ATCCAGTGTG GAGATGGCTG TGACCCCCAA GGACTCAGGG ACAACAGTCA
GTTCTGCTTG CAAAGGATCC ACCAGGGTCT GATTTTTTAT GAGAAGCTGC TAGGATCGGA
TATTTTCACA GGGGAGCCTT CTCTGCTCCC TGATAGCCCT GTGGCGCAGC TTCATGCCTC
CCTACTGGGC CTCAGCCAAC TCCTGCAGCC TGAGGGTCAC CACTGGGAGA CTCAGCAGAT
TCCAAGCCTC AGTCCCAGCC AGCCATGGCA GCGTCTCCTT CTCCGCTTCA AAATCCTTCG
CAGCCTCCAG GCCTTTGTGG CTGTAGCCGC CCGGGTCTTT GCCCATGGAG CAGCAACCCT
GAGTCCCTAA Rodent, e.g., mouse, IL-B30 (SEQ ID NO: 3 and 4):
CGCTTAGAAG TCGGACTACA GAGTTAGACT CAGAACCAAA GGAGGTGGAT AGGGGGTCCA
60 CAGGCCTGGT GCAGATCACA GAGCCAGCCA GATCTGAGAA GCAGGAACA AG ATG 115
Met -21 CTG GAT TGC AGA GCA GTA ATA ATG CTA TGG CTG TTG CCC TGG GTC
ACT 163 Leu Asp Cys Arg Ala Val Ile Met Leu Trp Leu Leu Pro Trp Val
Thr -20 -15 -10 -5 CAG GGC CTG GCT GTG CCT AGG AGT AGC AGT CCT GAC
TGG GCT CAG TGC 211 Gln Gly Leu Ala Val Pro Arg Ser Ser Ser Pro Asp
Trp Ala Gln Cys 1 5 10 CAG CAG CTC TCT CGG AAT CTC TGC ATG CTA GCC
TGG AAC GCA CAT GCA 259 Gln Gln Leu Ser Arg Asn Leu Cys Met Leu Ala
Trp Asn Ala His Ala 15 20 25 CCA GCG GGA CAT ATG AAT CTA CTA AGA
GAA GAA GAG GAT GAA GAG ACT 307 Pro Ala Gly His Met Asn Leu Leu Arg
Glu Glu Glu Asp Glu Glu Thr 30 35 40 AAA AAT AAT GTG CCC CGT ATC
CAG TGT GAA GAT GGT TGT GAC CCA CAA 355 Lys Asn Asn Val Pro Arg Ile
Gln Cys Glu Asp Gly Cys Asp Pro Gln 45 50 55 60 GGA CTC AAG GAC AAC
AGC CAG TTC TGC TTG CAA AGG ATC CGC CAA GGT 403 Gly Leu Lys Asp Asn
Ser Gln Phe Cys Leu Gln Arg Ile Arg Gln Gly 65 70 75 CTG GCT TTT
TAT AAG CAC CTG CTT GAC TCT GAC ATC TTC AAA GGG GAG 451 Leu Ala Phe
Tyr Lys His Leu Leu Asp Ser Asp Ile Phe Lys Gly Glu 80 85 90 CCT
GCT CTA CTC CCT GAT AGC CCC ATG GAG CAA CTT CAC ACC TCC CTA 499 Pro
Ala Leu Leu Pro Asp Ser Pro Met Glu Gln Leu His Thr Ser Leu 95 100
105 CTA GGA CTC AGC CAA CTC CTC CAG CCA GAG GAT CAC CCC CGG GAG ACC
547 Leu Gly Leu Ser Gln Leu Leu Gln Pro Glu Asp His Pro Arg Glu Thr
110 115 120 CAA CAG ATG CCC AGC CTG ATG TCT AGT CAG CAG TGG CAG CGC
CCC CTT 595 Gln Gln Met Pro Ser Leu Ser Ser Ser Gln Gln Trp Gln Arg
Pro Leu 125 130 135 140 CTC CGT TCC AAG ATC CTT CGA AGC CTC CAG GCC
TTT TTG GCC ATA GCT 643 Leu Arg Ser Lys Ile Leu Arg Ser Leu Gln Ala
Phe Leu Ala Ile Ala 145 150 155 GCC CGG GTC TTT GCC CAC GGA GCA GCA
ACT CTG ACT GAG CCC TTA GTG 691 Ala Arg Val Phe Ala His Gly Ala Ala
Thr Leu Thr Glu Pro Leu Val 160 165 170 CCA ACA GCT TAAGGATGCC
CAGGTTCCCA TGGCTACCAT GATAAGACTA 740 Pro Thr Ala 175 ATCTATCAGC
CCAGACATCT ACCAGTTAAT TAACCCATTA GGACTTGTGC TGTTCTTGTT 800
TCGTTTGTTT TGCGTGAAGG GCAAGGACAC CATTATTAAA GAGAAAAGAA ACAAACCCCA
860 GAGCAGGCAG CTGGCTAGAG AAAGGAGCTG GAGAAGAAGA ATAAAGTCTC
GAGCCCTTGG 920 CCTTGGAAGC GGGCAAGCAG CTGCGTGGCC TGAGGGGAAG
GGGGCGGTGG CATCGAGAAA 980 CTGTGAGAAA ACCCAGAGCA TCAGAAAAAG
TGAGCCCAGG CTTTGGCCAT TATCTGTAAG 1040 AAAAACAAGA AAAGGGGAAC
ATTATACTTT CCTGGGTGGC TCAGGGAAAT GTGCAGATGC 1100 ACAGTACTCC
AGACAGCAGC TCTGTACCTG CCTGCTCTGT CCCTCAGTTC TAACAGAATC 1160
TAGTCACTAA GAACTAACAG GACTACCAAT ACGAACTGAC AAA 1203
MLDCRAVIMLWLLFWVTQGLAVPRSSSPDWAQCQQLSRNLCMLAWNAHAPAGHMNLLREEEDEETKNNV
PRIQCEDGCDPQGLKDNSQFCLQRIRQGLAFYKHLLDSDIFKGEPALLPDSPMEQLHTSLLGLSQLLQP
EDHPRETQQNPSLSSSQQWQRPLLRSKILRSLQAFLAIAARVFAHGAATLTEPLVPTA
TABLE-US-00002 TABLE 2 Comparison of various IL-6 and G-CSF
embodiments compared to IL-B30. Human IL-B30 is SEQ ID NO: 2; mouse
IL-B30 is SEQ ID NO: 4; pig IL-B30 is SEQ ID NO: 5; bovine G-CSF is
SEQ ID NO: 6; feline G-CSF is SEQ ID NO: 7; human G-CSF is SEQ ID
NO: 8; mouse G-CSF is SEQ ID NO: 9; otter IL-6 is SEQ ID NO: 10;
feline IL-6 is SEQ ID NO: 11; human IL-6 is SEQ ID NO: 12; sheep
IL-6 is SEQ ID NO: 13; mouse IL-6 is SEQ ID NO: 14; chicken MGF is
SEQ ID NO: 15; and KSHV, kaposi's sarcoma herpes virus, a viral
IL-6, is SEQ ID NO: 16. i130_human .......... ......VPGG SSPVWTQCQQ
LSQKLCT.LA WSAHPLVG.. i130_mouse .......... ......VPRS SSPDWAQCQQ
LSRNLCM.LA WNAHAPAG.. i130_pig .......... .......... ..........
.......... .......... gcsf_bovin ......TPLG P.......AR SLPQSFLLKC
LEQVRKIQAD GAELQERL.. gcsf_felca ......TPLG P.......TS SLPQSFLLKC
LEQVRKVQAD GTALQERL.. gcsf_human ......TPLG P.......AS SLPQSFLLKC
LEQVRKIQGD GAALQEKLVS gcsf_mouse VPLVTVSALP P.......SL PLPRSFLLKS
LEQVRKIQAS GSVLLEQL.. i16_otter .AFPTPGPLP GDSKDDATSN RPPLTSADKM
EDFIKFILGK ISALRNEM.. i16_felca .AFPTPGPLG G....DATSN RLPLTPADKM
EELIKYILGK ISALKKEM.. i16_human .AFPAPVPPG EDSKDVAAPH RQPLTSSERI
DKQIRYILDG ISALRKET.. i16_sheep .AFPTPGPLG EDFKNDTTPS RLLLTTPEKT
EALIKHIVDK ISAIRKEI.. i16_mouse .AFPTSQVRR GDFTEDTTPN R.PVYTTSQV
GGLITHVLWE IVEMRKEL.. mgf_chick .......... .APLAELSGD HDFQLFLHKN
LEFTRKIRGD VAALQRAV.. i16_khsv .......... .......TRG KLPDAPEFEK
DLLIQRLNWM LWVIDECFRD i130_human .HMD.LREEG DEETTNDVPH I...QCGDGC
DPQGLRDNSQ FCLQRIHQGL i130_mouse .HMNLLREEE DEETKNNVPR I...QCEDGC
DPQGLKDNSQ FCLQRIRQGL i130_pig .......... .......... ..........
.......... SCLQRIHQGL gcsf_bovin .CAA.HKLCH PEELMLLRHS LGIP.QAPLS
SCSSQSLQLR GCLNQLHGGL gcsf_felca .CAA.HKLCH PEELVLLGHA LGIP.QAPLS
SCSSQALQLT GCLRQLHSGL gcsf_human ECAT.YKLCH PEELVLLGHS LGIP.WAPLS
SCPSQALQLA GCLSQLHSGL gcsf_mouse .CAT.YKLCH PEELVLLGHS LGIP.KASLS
GCSSQALQQT QCLSQLHSGL i16_otter .CDK.YNKCE DSKEVLAENN LNLPKLAEKD
RCFQSRFNQE TCLTRITTGL i16_felca .CDN.YNKCE DSKEALAENN LNLPKLAEKD
GCFQSGFNQE TCLTRITTGL i16_human .CNK.SNMCE SSKEALAENN LNLPKMAEKD
GCFQSGFNEE TCLVKIITGL i16_sheep .CEK.NDECE NSKETLAENK LKLPKMEEKD
GCFQSGFNQA ICLIKTTAGL i16_mouse .CNG.NSDCM NNDDALAENN LKLPEIQRND
GCYQTGYNQE ICLLKISSGL mgf_chick .CDT.FQLCT EEELQLVQPD PHLV.QAPLD
QCHKRGFQAE VCFTQIRAGL i16_khsv LCYR.TGICK GILEPAAIFH LKLPAINDTD
HCGLIGFNET SCLKKLADGF i130_human IFYEKLLGSD IFTGE..... .PSLLPDSPV
AQLHASLLGL SQLLQPE..C i130_mouse AFYKHLLDSD IFKGE..... .PALLPDSPM
EQLHTSLLGL SQLLQPE..D i130_pig VFYEKLLGSD IFTGE..... .PSLHPDGSV
GQLHASLLGL RQLLQPE..G gcsf_bovin FLYQGLLQAL AGIS...... .PELAPTLDT
LQLDVTDFAT NIWLQMEDLG gcsf_felca FLYQGLLQAL AGIS...... .PELAPTLDM
LQLDITDFAI NIWQQMEDVG gcsf_human FLYQGLLQAL EGIS...... .PELGFTLDT
LQLDVADFAT TIWQQMEELG gcsf_mouse CLYQGLLQAL SGIS...... .PALAPTLDL
LQLDVANFAT TIWQQMENLG i16_otter QEFQIHLKYL ESNYEG...N KDNAHSVYIS
TKHLLQTLRP M..NQIEVTT i16_felca QEFQIYLKFL QDKYEG...D KENAKSVYTS
TNVLLQMLKR KGKNQDEVTI i16_human LEFEVYLEYL QNRFES...S EEQARAVQMS
TKVLIQFLQK KAKNLDAITT i16_sheep LEYQIYLDFL QNEFEG...N QETVMELQSS
IRTLIQILKE KIAGL....I i16_mouse LEYHSYLEYM KNNLKDN..K KDKARVLQRD
TETLIHIFNQ EVKDLHKIVL mgf_chick HAYHDSLGAV LRLLP..... ..NHTTLVET
LQLDAANLSS NIQQQMEDLG i16_khsv FEFEVLFKFL TTEFGKSVIN VDVMELLTKT
LGWDIQEELN KLTKTHY..S i130_human HHWETQQIP. .SLSPSQ..P WQRLLLRFKI
LRSLQAFVAV AARVFAHGAA i130_mouse HPRETQQMP. .SLSSSQ..Q WQRPLLRSKI
LRSLQAFLAI AARVFAHGAA i130_pig HHWETEQTP. .SPSPSQ..P WQRLLLRLKI
LRSLQAFVAV AARVFAHGAA gcsf_bovin AAPAVQPTQ. .GAMPTFTSA FQRRAGGVLV
ASQLHRFLEL AYRGLRYLAE gcsf_felca MAPAVPPTQ. .GTMPTFTSA FQRRAGGTLV
ASNLQSFLEV AYRALRHFTK gcsf_human MAPALQPTQ. .GAMPAFASA FQRRAGGVLV
ASHLQSFLEV SYRVLRHLAQ gcsf_mouse VAPTVQPTQ. .SAMPAFTSA FQRRAGGVLA
ISYLQGFLET ARLALHHLA. i16_otter PDPTTDASL. .QALFKSQDK WLKHTTIHLI
LRRLEDFLQF SLRAIRIM.. i16_felca PVPTVEVGL. .QLSCSHR.R VAEAHNNHLT
LRRLEDFLQL RLRAVRIM.. i16_human PDPTTNASL. .LTKLQAQNQ WLQDHTTHLI
LRSPKEFLQS SLRALRQM.. i16_sheep TTPATHTDM. .LEKMQSSNE WVKNAKVIII
LRSLENFLQF SLRAIRMK.. i16_mouse PTPISNALL. .TDKLESQKE WLRTKTIQFI
LKSLEEFLKV TLRSTRQT.. mgf_chick LDTVTLPAEQ RSPPPTFSGP FQQQVGGFFI
LANFQRFLET AYRALRHLAR i16_khsv P.PKFDRG.. LLGRLQGLKY WVRHFASFYV
LSAMEKPAGQ AVRVLDSIPD i130_human TLSP.... i130_mouse TLTEPLVPTA
i130_pig TLSQ.... gcsf_bovin P....... gcsf_felca P.......
gcsf_human P....... gcsf_mouse ........ i16_otter ........
i16_felca ........ i16_human ........ i16_sheep ........ i16_mouse
........ mgf_chick L....... i16_khsv VTPDVHDK
I. General Methods
Many of the standard methods below are described or referenced,
e.g., in Maniatis, et al. (1982) Molecular Cloning, A Laboratory
Manual Cold Spring Harbor Laboratory, Cold Spring Harbor Press, NY;
Sambrook, et al. (1989) Molecular Cloning: A Laboratory Manual (2d
ed.) Vols. 1-3, CSH Press, NY; Ausubel, et al., Biology Greene
Publishing Associates, Brooklyn, N.Y.; or Ausubel, et al. (1987 and
Supplements) Current Protocols in Molecular Biology Wiley/Greene,
NY; Innis, et al. (eds. 1990) PCR Protocols: A Guide to Methods and
Applications Academic Press, NY. Methods for protein purification
include such methods as ammonium sulfate precipitation, column
chromatography, electrophoresis, centrifugation, crystallization,
and others. See, e.g., Ausubel, et al. (1987 and periodic
supplements); Deutscher (1990) "Guide to Protein Purification,"
Methods in Enzymology vol. 182, and other volumes in this series;
Coligan, et al. (1995 and supplements) Current Protocols in Protein
Science John Wiley and Sons, New York, N.Y.; P. Matsudaira (ed.
1993) A Practical Guide to Protein and Peptide Purification for
Microsequencing, Academic Press, San Diego, Calif.; and
manufacturer's literature on use of protein purification products,
e.g., Pharmacia, Piscataway, N.J., or Bio-Rad, Richmond, Calif.
Combination with recombinant techniques allow fusion to appropriate
segments (epitope tags), e.g., to a FLAG sequence or an equivalent
which can be fused, e.g., via a protease-removable sequence. See,
e.g., Hochuli (1989) Chemische Industrie 12:69-70; Hochuli (1990)
"Purification of Recombinant Proteins with Metal Chelate Absorbent"
in Setlow (ed.) Genetic Engineering Principle and Methods 12:87-98,
Plenum Press, NY; and Crowe, et al. (1992) OIAexpress: The High
Level Expression & Protein Purification System QUIAGEN, Inc.,
Chatsworth, Calif.
Standard immunological techniques are described, e.g., in
Hertzenberg, et al. (eds. 1996) Weir's Handbook of Experimental
Immunology vols. 1-4, Blackwell Science; Coligan (1991) Current
Protocols in Immunology Wiley/Greene, NY; and Methods in Enzymology
vols. 70, 73, 74, 84, 92, 93, 108, 116, 121, 132, 150, 162, and
163. Cytokine assays are described, e.g., in Thomson (ed. 1994) The
Cytokine Handbook (2d ed.) Academic Press, San Diego; Metcalf and
Nicola (1995) The Hematopoietic Colony Stimulating Factors
Cambridge University Press; and Aggarwal and Gutterman (1991) Human
Cytokines Blackwell Pub. Assays for vascular biological activities
are well known in the art. They will cover angiogenic and
angiostatic activities in tumor, or other tissues, e.g., arterial
smooth muscle proliferation (see, e.g., Koyoma, et al. (1996) Cell
87:1069-1078), monocyte adhesion to vascular epithelium (see
McEvoy, et al. (1997) J. Exp. Med. 185:2069-2077), etc. See also
Ross (1993) Nature 362:801-809; Rekhter and Gordon (1995) Am. J.
Pathol. 147:668-677; Thyberg, et al. (1990) Atherosclerosis
10:966-990; and Gumbiner (1996) Cell 84:345-357.
Assays for neural cell biological activities are described, e.g.,
in Wouterlood (ed. 1995) Neuroscience Protocols modules 10,
Elsevier; Methods in Neuroscieces Academic Press; and Neuromethods
Humana Press, Totowa, N.J. Methodology of development systems is
described, e.g., in Meisami (ed.) Handbook of Human Growth and
Developmental Bioloy CRC Press; and Chrispeels (ed.) Molecular
Techniques and Approaches in Developmental Biology
Interscience.
FACS analyses are described in Melamed, et al. (1990) Flow
Cytometry and Sortina Wiley-Liss, Inc., New York, N.Y.; Shapiro
(1988) Practical Flow Cytometry Liss, New York, N.Y.; and Robinson,
et al. (1993) Handbook of Flow Cytometry Methods Wiley-Liss, New
York, N.Y.
II. Cloning of Human IL-B30
The sequence of the gene is provided in Table 1. The sequence is
derived from a cDNA library made from melanocyte, fetal heart, and
pregnant uterus. It is also found from a cDNA library sequence
derived from a pancreatic islet. These sequences allow preparation
of PCR primers, or probes, to determine cellular distribution of
the gene. The sequences allow isolation of genomic DNA which encode
the message.
Using the probe or PCR primers, various tissues or cell types are
probed to determine cellular distribution. PCR products are cloned
using, e.g., a TA cloning kit (Invitrogen). The resulting cDNA
plasmids are sequenced from both termini on an automated sequencer
(Applied Biosystems).
III. Cellular Expression of IL-B30
An appropriate probe or primers specific for cDNA encoding primate
IL-B30 are prepared. Typically, the probe is labeled, e.g., by
random priming. The expression is probably in the cell types
described, and perhaps also in pancreatic islets. Southern
Analysis: DNA (5 .mu.g) from a primary amplified cDNA library was
digested with appropriate restriction enzymes to release the
inserts, run on a 1% agarose gel and transferred to a nylon
membrane (Schleicher and Schuell, Keene, N.H.).
Samples for human mRNA isolation include: peripheral blood
mononuclear cells (monocytes, T cells, NK cells, granulocytes, B
cells), resting (T100); peripheral blood mononuclear cells,
activated with anti-CD3 for 2, 6, 12 h pooled (T101); T cell, TH0
clone Mot 72, resting (T102); T cell, TH0 clone Mot 72, activated
with anti-CD28 and anti-CD3 for 3, 6, 12 h pooled (T103); T cell,
TH0 clone Mot 72, anergic treated with specific peptide for 2, 7,
12 h pooled (T104); T cell, TH1 clone HY06, resting (T107); T cell,
TH1 clone HY06, activated with anti-CD28 and anti-CD3 for 3, 6, 12
h pooled (T108); T cell, TH1 clone HY06, anergic treated with
specific peptide for 2, 6, 12 h pooled (T109); T cell, TH2 clone
HY935, resting (T110); T cell, TH2 clone HY935, activated with
anti-CD28 and anti-CD3 for 2, 7, 12 h pooled (T111); T cell tumor
lines Jurkat and Hut78, resting (T117); T cell clones, pooled
AD130.2, Tc783.12, Tc783.13, Tc783.58, Tc782.69, resting (T118); T
cell random .gamma..delta. T cell random .gamma..delta. T cell
clones, resting (Tll9); CD28-T cell clone; Splenocytes, resting
(B100); Splenocytes, activated with anti-CD40 and IL-4 (B100); B
cell EBV lines pooled WT49, RSB, JY, CVIR, 721.221, RM3, HSY,
resting (B102); B cell line JY, activated with PMA and ionomycin
for 1, 6 h pooled (B103); NK 20 clones pooled, resting (K100); NK
20 clones pooled, activated with PMA and ionomycin for 6 h (K101);
NKL clone, derived from peripheral blood of LGL leukemia patient,
IL-2 treated (K106); hematopoietic precursor line TF1, activated
with PMA and ionomycin for 1, 6 h pooled (C100); U937 premonocytic
line, resting (M100); U937 premonocytic line, activated with PMA
and ionomycin for 1, 6 h pooled (M101); elutriated monocytes,
activated with LPS, IFN.gamma., anti-IL-10 for 1, 2, 6, 12, 24 h
pooled (M102); elutriated monocytes, activated with LPS,
IFN.gamma., IL-10 for 1, 2, 6, 12, 24 h pooled (M103); elutriated
monocytes, activated with LPS, IFN.gamma., anti-IL-10 for 4, 16 h
pooled (M106); elutriated monocytes, activated with LPS,
IFN.gamma., IL-10 for 4, 16 h pooled (M107); elutriated monocytes,
activated LPS for 1 h (M108); elutriated monocytes, activated LPS
for 6 h (M109); DC 70% CD1a+, from CD34+ GM-CSF, TNF.alpha. 12
days, resting (D101); DC 70% CD1a+, from CD34+ GM-CSF, TNF.alpha.
12 days, activated with PMA and ionomycin for 1 hr (D102); DC 70%
CD1a+, from CD34+ GM-CSF, TNF.alpha. 12 days, activated with PMA
and ionomycin for 6 hr (D103); DC 95% CD1a+, from CD34+ GM-CSF,
TNF.alpha. 12 days FACS sorted, activated with PMA and ionomycin
for 1, 6 h pooled (D104); DC 95% CD14+, ex CD34+ GM-CSF, TNF.alpha.
12 days FACS sorted, activated with PMA and ionomycin 1, 6 hr
pooled (D105); DC CD1a+ CD86+, from CD34+ GM-CSF, TNF.alpha. 12
days FACS sorted, activated with PMA and ionomycin for 1, 6 h
pooled (D106); DC from monocytes GM-CSF, IL-4 5 days, resting
(D107); DC from monocytes GM-CSF, IL-4 5 days, resting (D108); DC
from monocytes GM-CSF, IL-4 5 days, activated LPS 4, 16 h pooled
(D109); DC from monocytes GM-CSF, IL-4 5 days, activated
TNF.alpha., monocyte supe for 4, 16 h pooled (D110); epithelial
cells, unstimulated; epithelial cells, IL-1.beta. activated; lung
fibroblast sarcoma line MRC5, activated with PMA and ionomycin for
1, 6 h pooled (C101); kidney epithelial carcinoma cell line CHA,
activated with PMA and ionomycin for 1, 6 h pooled (C102).
Expression of IL-B30 transcript was very high in elutriated
monocytes, activated with LPS, IFN.gamma., anti-IL-10 for 4, 16 h
pooled (M106); elutriated monocytes, activated with LPS,
IFN.gamma., anti-IL-10 for 1, 2, 6, 12, 24 h pooled (M102);
elutriated monocytes, activated LPS for 6 h (M109); and elutriated
monocytes, activated LPS for 1 h (M108). Expression was high in DC
95% CD1a+, from CD34+ GM-CSF, TNF.alpha. 12 days FACS sorted,
activated with PMA and ionomycin for 1, 6 h pooled (D104); and NK
20 clones pooled, activated with PMA and ionomycin for 6 h (K101).
Lesser expression was detected in DC 70% CD1a+, from CD34+ GM-CSF,
TNF.alpha. 12 days, activated with PMA and ionomycin for 6 hr
(D103); DC 70% CD1a+, from CD34+ GM-CSF, TNF.alpha. 12 days,
activated with PMA and ionomycin for 1 hr (D102) ; T cell, TH1
clone HY06, anergic treated with specific peptide for 2, 6, 12 h
pooled (T109); peripheral blood mononuclear cells, activated with
anti-CD3 for 2, 6, 12 h pooled (T101); T cell, TH0 clone Mot 72,
activated with anti-CD28 and anti-CD3 for 3, 6, 12 h pooled (T103);
Splenocytes, activated with anti-CD40 and IL-4 (B101); T cell, TH0
clone Mot 72, anergic treated with specific peptide for 2, 7, 12 h
pooled (T104); Splenocytes, resting (B100); T cell, TH1 clone HY06,
activated with anti-CD28 and anti-CD3 for 3, 6, 12 h pooled (T108);
epithelial cells, IL-1.beta. activated; elutriated monocytes,
activated with LPS, IFN.gamma., IL-10 for 4, 16 h pooled (M107);
and B cell line JY, activated with PMA and ionomycin for 1, 6 h
pooled (B103). Detectable expression was observed in DC from
monocytes GM-CSF, IL-4 5 days, activated LPS 4, 16 h pooled (D109);
T cell, TH0 clone Mot 72, resting (T102); peripheral blood
mononuclear cells (monocytes, T cells, NK cells, granulocytes, B
cells), resting (T100); T cells CD4+ CD45RO- T cells polarized 27
days in anti-CD28, IL-4, and anti IFN-.gamma., TH2 polarized,
activated with anti-CD3 and anti-CD28 4 h (T116); T cell clones,
pooled AD130.2, Tc783.12, Tc783.13, Tc783.58, Tc782.69, resting
(T118); U937 premonocytic line, resting (M100); hematopoietic
precursor line TF1, activated with PMA and ionomycin for 1, 6 h
pooled (C100); T cell, Th2 clone HY935, activated with anti-CD28
and anti-CD3 for 2, 7, 12 h pooled (T111); DC Cd1a+ CD86+, from
CD34+ GM-CSF, TNF.alpha. 12 days FACS sorted, activated with PMA
and ionomycin for 1, 6 h pooled (D106); elutriated monocytes,
activated with LPS, IFN.gamma., IL-10 for 1, 2, 6, 12, 24 h pooled
(M103); DC from monocytes GM-CSF, IL-4 5 days, activated
TNF.alpha., monocyte supe for 4, 16 h pooled (D110); DC from
monocytes GM-CSF, IL-4 5 days, resting (D108); U937 premonocytic
line, activated with PMA and ionomycin for 1, 6 h pooled (M101); T
cell random .gamma..delta. cell clones, resting (T119); and T cell,
TH1 clone HY06, activated with anti-CD28 and anti-CD3 for 3, 6, 12
h pooled (T108). No signal was detected in the other samples.
In summary, the distribution shows IL-B30 elevated in activated
macrophages, suggesting a role in inflammation; activated Th1
cells, suggesting a regulation or effector role in T helper
subsets, particularly Th1 immune responses; and activated dendritic
cells, suggesting a role in antigen presentation or germinal center
T or B cell interactions with DC.
Samples for mouse mRNA isolation include: resting mouse
fibroblastic L cell line (C200); Braf:ER (Braf fusion to estrogen
receptor) transfected cells, control (C201); Me114+ naive T cells
from spleen, resting (T209); Mel14+ naive T cells from spleen,
stimulated with IFN.gamma., IL-12, and anti IL-4 to polarize to TH1
cells, exposed to IFN.gamma. and IL-4 for 6, 12, 24 h, pooled
(T210); Mel14+ naive T cells from spleen, stimulated with IL-4 and
anti IFN.gamma. to polarize to Th2 cells, exposed to IL-4 and anti
IFN.gamma. for 6, 13, 24 h, pooled (T211); T cells, TH1 polarized
(Mel14 bright, CD4+ cells from spleen, polarized for 7 days with
IFN-.gamma. and anti IL-4; T200); T cells, TH2 polarized (Mel14
bright, CD4+ cells from spleen, polarized for 7 days with IL-4 and
anti-IFN-.gamma.; T201); T cells, highly TH1 polarized 3.times.
from transgenic Balb/C (see Openshaw, et al. (1995) J. Exp. Med.
182:1357-1367; activated with anti-CD3 for 2, 6, 24 h pooled;
T202); T cells, highly TH2 polarized 3.times. from transgenic
Balb/C (activated with anti-CD3 for 2, 6, 24 h pooled (T203); T
cells, highly TH1 polarized 3.times. from transgenic C57 bl/6
(activated with anti-CD3 for 2, 6, 24 h pooled; T212); T cells,
highly TH2 polarized 3.times. from transgenic C57 bl/6 (activated
with anti-CD3 for 2, 6, 24 h pooled; T213); T cells, highly TH1
polarized (naive CD4+ T cells from transgenic Balb/C, polarized
3.times. with IFN.gamma., IL-12, and anti-IL-4; stimulated with
IGIF, IL-12, and anti IL-4 for 6, 12, 24 h, pooled); CD44- CD25+
pre T cells, sorted from thymus (T204); TH1 T cell clone D1.1,
resting for 3 weeks after last stimulation with antigen (T205); TH1
T cell clone D1.1, 10 .mu.g/ml ConA stimulated 15 h (T206); TH2 T
cell clone CDC35, resting for 3 weeks after last stimulation with
antigen (T207); TH2 T cell clone CDC35, 10 .mu.g/ml ConA stimulated
15 h (T208); unstimulated B cell line CH12 (B201); unstimulated
mature B cell leukemia cell line A20 (B200); unstimulated large B
cells from spleen (B202); B cells from total spleen, LPS activated
(B203); metrizamide enriched dendritic cells from spleen, resting
(D200); dendritic cells from bone marrow, resting (D201);
unstimulated bone marrow derived dendritic cells depleted with anti
B220, anti CD3, and anti Class II, cultured in GM-CSF and IL-4
(D202); bone marrow derived dendritic cells depleted with anti
B220, anti CD3, and anti Class II, cultured in GM-CSF and IL-4,
stimulated with anti CD40 for 1,5 d, pooled (D203); monocyte cell
line RAW 264.7 activated with LPS 4 h (M200); bone-marrow
macrophages derived with GM and M-CSF (M201); bone-marrow
macrophages derived with GM-CSF, stimulated with LPS, IFN.gamma.,
and IL-10 for 24 h (M205); bone-marrow macrophages derived with
GM-CSF, stimulated with LPS, IFN.gamma., and anti IL-10 for 24 h
(M206); peritoneal macrophages (M207); macrophage cell line J774,
resting (M202); macrophage cell line J774+LPS+anti-IL-10 at 0.5, 1,
3, 6, 12 h pooled (M203); macrophage cell line J774+LPS+IL-10 at
0.5, 1, 3, 5, 12 h pooled (M204); unstimulated mast cell lines MC-9
and MCP-12 (M208); immortalized endothelial cell line derived from
brain microvascular endothelial cells, unstimulated (E200);
immortalized endothelial cell line derived from brain microvascular
endothelial cells, stimulated overnight with TNF.alpha. (E201);
immortalized endothelial cell line derived from brain microvascular
endothelial cells, stimulated overnight with TNF.alpha. (E202);
immortalized endothelial cell line derived from brain microvascular
endothelial cells, stimulated overnight with TNF.alpha. and IL-10
(E203); total aorta from wt C57 bl/6 mouse; total aorta from 5
month ApoE KO mouse (X207); total aorta from 12 month ApoE KO mouse
(X207); wt thymus (O214); total thymus rag-1 (O208); total kidney,
rag-1 (O209); total kidney, NZ B/W mouse; and total heart, rag-1
(O202). High signal was detected in the monocyte cell line RAW
264.7 activated with LPS 4 h (M200); T cells, highly TH1 polarized
3.times. from transgenic C57 bl/6 (activated with anti-CD3 for 2,
6, 24 h pooled; T212); and T cells, highly TH1 polarized (naive
CD4+ T cells from transgenic Balb/C, polarized 3.times. with
IFN.gamma., IL-12, and anti-IL-4; stimulated with IGIF, IL-12, and
anti IL-4 for 6, 12, 24 h, pooled). Detectable signals were
detected in T cells, highly TH1 polarized 3.times. from transgenic
Balb/C (see Openshaw, et al. (1995) J. Exp. Med. 182:1357-1367;
activated with anti-CD3 for 2, 6, 24 h pooled; T202); T cells, TH2
polarized (Mel14 bright, CD4+ cells from spleen, polarized for 7
days with IL-4 and anti-IFN-.gamma., T201); T cells, TH1 polarized
(Mel14 bright, CD4+ cells from spleen, polarized for 7 days with
IFN-.gamma. and anti IL-4; T200); macrophage cell line
J774+LPS+anti-IL-10 at 0.5, 1, 3, 6, 12 h pooled (M203); macrophage
cell line J774, resting (M202); macrophage cell line J774+LPS+IL-10
at 0.5, 1, 3, 5, 12 h pooled (M204); immortalized endothelial cell
line derived from brain microvascular endothelial cells, stimulated
overnight with TNF.alpha. (E201); and bone-marrow macrophages
derived with GM-CSF, stimulated with LPS, IFN.gamma., and anti
IL-10 for 24 h (M206). Other samples showed no signal. The
expression in the RAW 264.7 mouse monocyte cell line suggests a
natural source for protein.
IV. Chromosome mapping of IL-B30
An isolated cDNA encoding the IL-B30 is used. Chromosome mapping is
a standard technique. See, e.g., BIOS Laboratories (New Haven,
Conn.) and methods for using a mouse somatic cell hybrid panel with
PCR. Circumstantial evidence suggests that the mouse gene is
localized on chromosome 10.
V. Purification of IL-B30 Protein
Multiple transfected cell lines are screened for one which
expresses the cytokine at a high level compared with other cells.
Various cell lines are screened and selected for their favorable
properties in handling. Natural IL-B30 can be isolated from natural
sources, or by expression from a transformed cell using an
appropriate expression vector. Purification of the expressed
protein is achieved by standard procedures, or may be combined with
engineered means for effective purification at high efficiency from
cell lysates or supernatants, FLAG or His.sub.6 segments can be
used for such purification features. Alternatively, affinity
chromatography may be used with specific antibodies, see below.
Protein is produced in coli, insect cell, or mammalian expression
systems, as desired.
VI. Isolation of Homologous IL-B30 Genes
The IL-B30 cDNA, or other species counterpart sequence, can be used
as a hybridization probe to screen a library from a desired source,
e.g., a primate cell cDNA library. Many different species can be
screened both for stringency necessary for easy hybridization, and
for presence using a probe. Appropriate hybridization conditions
will be used to select for clones exhibiting specificity of cross
hybridization.
Screening by hybridization using degenerate probes based upon the
peptide sequences will also allow isolation of appropriate clones.
Alternatively, use of appropriate primers for PCR screening will
yield enrichment of appropriate nucleic acid clones.
Similar methods are applicable to isolate either species,
polymorphic, or allelic variants. Species variants are isolated
using cross-species hybridization techniques based upon isolation
of a full length isolate or fragment from one species as a
probe.
Alternatively, antibodies raised against human IL-B30 will be used
to screen for cells which express cross-reactive proteins from an
appropriate, e.g., cDNA library. The purified protein or defined
peptides are useful for generating antibodies by standard methods,
as described above. Synthetic peptides or purified protein are
presented to an immune system to generate monoclonal or polyclonal
antibodies. See, e.g., Coligan (1991) Current Protocols in
Immunology Wiley/Greene; and Harlow and Lane (1989) Antibodies: A
Laboratory Manual Cold Spring Harbor Press. The resulting
antibodies are used for screening, purification, or diagnosis, as
described.
VII. Preparation of antibodies specific of IL-B30
Synthetic peptides or purified protein are presented to an immune
system to generate monoclonal or polyclonal antibodies. See, e.g.,
Coligan (1991) Current Protocols in Immunology Wiley/Greene; and
Harlow and Lane (1989) Antibodies: A Laboratory Manual Cold Spring
Harbor Press. Polyclonal serum, or hybridomas may be prepared. In
appropriate situations, the binding reagent is either labeled as
described above, e.g., fluorescence or otherwise, or immobilized to
a substrate for panning methods. Immunoselection and related
techniques are available to prepare selective reagents, as
desired.
VIII. Evaluation of Breadth of Biological Functions
Biological activities of IL-B30 were tested based on the sequence
and structural homology between IL-B30 and IL-6 and G-CSF.
Initially, assays that had shown biological activities of IL-6 or
G-CSF are examined.
A. Effects on proliferation of cells
The effect on proliferation of various cell types are evaluated
with various concentrations of cytokine. A dose response analysis
is performed, in combinations with the related cytokines IL-6,
G-CSF, etc.
B. Effects on the expression of cell surface molecules on human
monocytes
Monocytes are purified by negative selection from peripheral blood
mononuclear cells of normal healthy donors. Briefly,
3.times.10.sup.8 ficoll banded mononuclear cells are incubated on
ice with a cocktail of monoclonal antibodies (Becton-Dickinson;
Mountain View, Calif.) consisting, e.g., of 200 .mu.l Dickinson;
Mountain View, Calif.) consisting, e.g., of 200 .mu.l of .alpha.CD2
(Leu-5A), 200 .mu.l of .alpha.CD3 (Leu-4), 100 .mu.l of .alpha.CD8
(Leu 2a), 100 .mu.l of .alpha.CD19 (Leu-12), 100 .mu.l of
.alpha.CD20 (Leu-16), 100 .mu.l of .alpha.CD56 (Leu-19), 100 .mu.l
of .alpha.CD67 (IOM 67; Immunotech, Westbrook, Me.), and
anti-glycophorin antibody (10F7MN, ATCC, Rockville, Md.). Antibody
bound cells are washed and then incubated with sheep anti-mouse IgG
coupled magnetic beads (Dynal, Oslo, Norway) at a bead to cell
ratio of 20:1. Antibody bound cells are separated from monocytes by
application of a magnetic field. Subsequently, human monocytes are
cultured in Yssel's medium (Gemini Bioproducts, Calabasas, Calif.)
containing 1% human AB serum in the absence or presence of IL-B30,
IL-6, G-CSF or combinations.
Analyses of the expression of cell surface molecules can be
performed by direct immunoflorescence. For example,
2.times.10.sup.5 purified human monocytes are incubated in
phosphate buffered saline (PBS) containing 1% human serum on ice
for 20 minutes. Cells are pelleted at 200.times.g. Cells are
resuspended in 20 ml PE or FITC labeled mAb. Following an
additional 20 minute incubation on ice, cells are washed in PBS
containing 1% human serum followed by two washes in PBS alone.
Cells are fixed in PBS containing 1% paraformaldehyde and analyzed
on FACScan flow cytometer (Becton Dickinson; Mountain View,
Calif.). Exemplary mAbs are used, e.g.: CD11b (anti-mac1), CD11c (a
gp150/95), CD14 (Leu-M3), CD54 (Leu 54), CD80 (anti-BB1/B7), HLA-DR
(L243) from Becton-Dickinson and CD86 (FUN 1; Pharmingen), CD64
(32.2; Medarex), CD40 (mAb89; Schering-Plough France).
C. Effects of IL-B30 on cytokine production by human monocytes
Human monocytes are isolated as described and cultured in Yssel's
medium (Gemini Bioproducts, Calabasas, Calif.) containing 1% human
AB serum in the absence or presence of IL-B30 (1/100 dilution
baculovirus expressed material). In addition, monocytes are
stimulated with LPS (E. coli 0127:B8 Difco) in the absence or
presence of IL-B30 and the concentration of cytokines (IL-1.beta.,
IL-6, TNF.alpha., GM-CSF, and IL-10) in the cell culture
supernatant determined by ELISA.
For intracytoplasmic staining for cytokines, monocytes are cultured
(1 million/ml) in Yssel's medium in the absence or presence of
IL-B30 and LPS (E. coli 0127:B8 Difco) and 10 mg/ml Brefeldin A
(Epicentre technologies Madison Wis.) for 12 hrs. Cells are washed
in PBS and incubated in 2% formaldehyde/PBS solution for 20 minutes
at RT. Subsequently cells are washed, resuspended in
permeabilization buffer (0.5% saponin/(Sigma) in PBS/BSA
(0.5%)/Azide (1 mM)) and incubated for 20 minutes at RT. Cells
(2.times.10.sup.5) are centrifuged and resuspended in 20 ml
directly conjugated anti-cytokine mAbs diluted 1:10 in
permeabilization buffer for 20 minutes at RT. The following
antibodies can be used: IL-1.alpha.-PE (364-3B3-14); IL-6-PE
(MQ2-13A5); TNF.alpha.-PE (MAb11); GM-CSF-PE (BVD2-21C11); and
IL-12-PE (C11.5.14; Pharmingen San Diego, Calif.). Subsequently,
cells are washed twice in permeabilization buffer and once in
PBS/BSA/Azide and analyzed on FACScan flow cytometer (Becton
Dickinson; Mountain View, Calif.).
D. Effects of IL-B30 on proliferation of human peripheral blood
mononuclear cells (PBMC).
Total PBMC are isolated from buffy coats of normal healthy donors
by centrifugation through ficoll-hypaque as described (Boyum, et
al.). PBMC are cultured in 200 .mu.l Yssel's medium (Gemini
Bioproducts, Calabasas, Calif.) containing 1% human AB serum in 96
well plates (Falcon, Becton-Dickinson, N.J.) in the absence or
presence of IL-B30. Cells are cultured in medium alone or in
combination with 100 U/ml IL-2 (R&D Systems) for 120 hours.
3H-Thymidine (0.1 mCi) is added during the last six hours of
culture and 3H-Thymidine incorporation determined by liquid
scintillation counting.
The native, recombinant, and fusion proteins would be tested for
agonist and antagonist activity in many other biological assay
systems, e.g., on T-cells, B-cells, NK, macrophages, dendritic
cells, hematopoietic progenitors, etc. Because of the IL-6 and
G-CSF structural relationship, assays related to those activities
should be analyzed
IL-B30 is evaluated for agonist or antagonist activity on
transfected cells expressing IL-6 or G-CSF receptor and controls.
See, e.g., Ho, et al. (1993) Proc. Nat'l Acad. Sci. USA 90,
11267-11271; Ho, et al. (1995) Mol. Cell. Biol. 15:5043-5053; and
Liu, et al. (1994). J. Immunol. 152:1821-1829.
IL-B30 is evaluated for effect in macrophage/dendritic cell
activation and antigen presentation assays, T cell cytokine
production and proliferation in response to antigen or allogeneic
stimulus. See, e.g., de Waal Malefyt et al. (1991) J. Exp. Med.
174:1209-1220; de Waal Malefyt et al. (1991) J. Exp. Med.
174:915-924; Fiorentino, et al. (1991) J. Immunol. 147, 3815-3822;
Fiorentino, et al. (1991) J. Immunol. 146:3444-3451; and Groux, et
al. (1996) J. Exp. Med. 184:19-29.
IL-B30 will also be evaluated for effects on NK cell stimulation.
Assays may be based, e.g., on Hsu, et al. (1992) Internat. Immunol.
4:563-569; and Schwarz, et al. (1994) J. Immunother. 16:95-104.
B cell growth and differentiation effects will be analyzed, e.g.,
by the methodology described, e.g., in Defrance, et al. (1992). J.
Exp. Med. 175:671-682; Rousset, et al. (1992) Proc. Nat'l Acad.
Sci. USA 89:1890-1893; including IgG2 and IgA2 switch factor
assays. Note that, unlike COS7 supernatants, NIH3T3 and COP
supernatants apparently do not interfere with human B cell
assays.
IX. Generation and Analysis of Genetically Altered Animals
Transgenic mice can be generated by standard methods.
Such animals are useful to determine the effects of deletion of the
gene, in specific tissues, or completely throughout the organism.
Such may provide interesting insight into development of the animal
or particular tissues in various stages. Moreover, the effect on
various responses to biological stress can be evaluated. See, e.g.,
Hogan, et al. (1995) Manipulating the Mouse Embryo: A Laboratory
Manual (2d ed.) Cold Spring Harbor Laboratory Press.
A transgenic mouse has been generated, and while the animal seems
to survive birth, it fails to thrive, and typically dies within a
few weeks. The construct is based upon an actin promoter with a CMV
enhancer, which should lead to broad and high expression. The mice,
like IL-6 transgenic mice, are runted. Moreover, they exhibit a
bloated abdomen, inflammation of the stomach and intestines,
infiltration of cells into the liver, and typically die before day
50. The mice do not breed. A second subset of the transgenic mice
have a less severe phenotype, and attempts to breed them are taking
place.
The genomic structure for the mouse IL-B30 has been determined. A
strategy for the production of IL-B30 knockout mice has been
developed, and constructs have been started.
All references cited herein are incorporated herein by reference to
the same extent as if each individual publication or patent
application was specifically and individually indicated to be
incorporated by reference in its entirety for all purposes.
Many modifications and variations of this invention can be made
without departing from its spirit and scope, as will be apparent to
those skilled in the art. The specific embodiments described herein
are offered by way of example only, and the invention is to be
limited only by the terms of the appended claims, along with the
full scope of equivalents to which such claims are entitled.
SEQUENCE LISTINGS
1
16570 base pairsnucleic acidsinglelinearcDNACDS 1..567mat_peptide
64..567 1ATG CTG GGG AGC AGA GCT GTA ATG CTG CTG TTG CTG CTG CCC
TGG ACA 48Met Leu Gly Ser Arg Ala Val Met Leu Leu Leu Leu Leu Pro
Trp Thr-21 -20 -15 -10GCT CAG GGC AGA GCT GTG CCT GGG GGC AGC AGC
CCT GCC TGG ACT CAG 96Ala Gln Gly Arg Ala Val Pro Gly Gly Ser Ser
Pro Ala Trp Thr Gln -5 1 5 10TGC CAG CAG CTT TCA CAG AAG CTC TGC
ACA CTG GCC TGG AGT GCA CAT 144Cys Gln Gln Leu Ser Gln Lys Leu Cys
Thr Leu Ala Trp Ser Ala His 15 20 25CCA CTA GTG GGA CAC ATG GAT CTA
AGA GAA GAG GGA GAT GAA GAG ACT 192Pro Leu Val Gly His Met Asp Leu
Arg Glu Glu Gly Asp Glu Glu Thr 30 35 40ACA AAT GAT GTT CCC CAT ATC
CAG TGT GGA GAT GGC TGT GAC CCC CAA 240Thr Asn Asp Val Pro His Ile
Gln Cys Gly Asp Gly Cys Asp Pro Gln 45 50 55GGA CTC AGG GAC AAC AGT
CAG TTC TGC TTG CAA AGG ATC CAC CAG GGT 288Gly Leu Arg Asp Asn Ser
Gln Phe Cys Leu Gln Arg Ile His Gln Gly 60 65 70 75CTG ATT TTT TAT
GAG AAG CTG CTA GGA TCG GAT ATT TTC ACA GGG GAG 336Leu Ile Phe Tyr
Glu Lys Leu Leu Gly Ser Asp Ile Phe Thr Gly Glu 80 85 90CCT TCT CTG
CTC CCT GAT AGC CCT GTG GCG CAG CTT CAT GCC TCC CTA 384Pro Ser Leu
Leu Pro Asp Ser Pro Val Ala Gln Leu His Ala Ser Leu 95 100 105CTG
GGC CTC AGC CAA CTC CTG CAG CCT GAG GGT CAC CAC TGG GAG ACT 432Leu
Gly Leu Ser Gln Leu Leu Gln Pro Glu Gly His His Trp Glu Thr 110 115
120CAG CAG ATT CCA AGC CTC AGT CCC AGC CAG CCA TGG CAG CGT CTC CTT
480Gln Gln Ile Pro Ser Leu Ser Pro Ser Gln Pro Trp Gln Arg Leu Leu
125 130 135CTC CGC TTC AAA ATC CTT CGC AGC CTC CAG GCC TTT GTG GCT
GTA GCC 528Leu Arg Phe Lys Ile Leu Arg Ser Leu Gln Ala Phe Val Ala
Val Ala140 145 150 155GCC CGG GTC TTT GCC CAT GGA GCA GCA ACC CTG
AGT CCC TAA 570Ala Arg Val Phe Ala His Gly Ala Ala Thr Leu Ser Pro
160 165189 amino acidsamino acidlinearprotein 2Met Leu Gly Ser Arg
Ala Val Met Leu Leu Leu Leu Leu Pro Trp Thr-21 -20 -15 -10Ala Gln
Gly Arg Ala Val Pro Gly Gly Ser Ser Pro Ala Trp Thr Gln -5 1 5
10Cys Gln Gln Leu Ser Gln Lys Leu Cys Thr Leu Ala Trp Ser Ala His
15 20 25Pro Leu Val Gly His Met Asp Leu Arg Glu Glu Gly Asp Glu Glu
Thr 30 35 40Thr Asn Asp Val Pro His Ile Gln Cys Gly Asp Gly Cys Asp
Pro Gln 45 50 55Gly Leu Arg Asp Asn Ser Gln Phe Cys Leu Gln Arg Ile
His Gln Gly 60 65 70 75Leu Ile Phe Tyr Glu Lys Leu Leu Gly Ser Asp
Ile Phe Thr Gly Glu 80 85 90Pro Ser Leu Leu Pro Asp Ser Pro Val Ala
Gln Leu His Ala Ser Leu 95 100 105Leu Gly Leu Ser Gln Leu Leu Gln
Pro Glu Gly His His Trp Glu Thr 110 115 120Gln Gln Ile Pro Ser Leu
Ser Pro Ser Gln Pro Trp Gln Arg Leu Leu 125 130 135Leu Arg Phe Lys
Ile Leu Arg Ser Leu Gln Ala Phe Val Ala Val Ala140 145 150 155Ala
Arg Val Phe Ala His Gly Ala Ala Thr Leu Ser Pro 160 1651203 base
pairsnucleic acidsinglelinearcDNACDS 113..700mat_peptide 176..700
3CGCTTAGAAG TCGGACTACA GAGTTAGACT CAGAACCAAA GGAGGTGGAT AGGGGGTCCA
60CAGGCCTGGT GCAGATCACA GAGCCAGCCA GATCTGAGAA GCAGGGAACA AG ATG 115
Met -21CTG GAT TGC AGA GCA GTA ATA ATG CTA TGG CTG TTG CCC TGG GTC
ACT 163Leu Asp Cys Arg Ala Val Ile Met Leu Trp Leu Leu Pro Trp Val
Thr-20 -15 -10 -5CAG GGC CTG GCT GTG CCT AGG AGT AGC AGT CCT GAC
TGG GCT CAG TGC 211Gln Gly Leu Ala Val Pro Arg Ser Ser Ser Pro Asp
Trp Ala Gln Cys 1 5 10CAG CAG CTC TCT CGG AAT CTC TGC ATG CTA GCC
TGG AAC GCA CAT GCA 259Gln Gln Leu Ser Arg Asn Leu Cys Met Leu Ala
Trp Asn Ala His Ala 15 20 25CCA GCG GGA CAT ATG AAT CTA CTA AGA GAA
GAA GAG GAT GAA GAG ACT 307Pro Ala Gly His Met Asn Leu Leu Arg Glu
Glu Glu Asp Glu Glu Thr 30 35 40AAA AAT AAT GTG CCC CGT ATC CAG TGT
GAA GAT GGT TGT GAC CCA CAA 355Lys Asn Asn Val Pro Arg Ile Gln Cys
Glu Asp Gly Cys Asp Pro Gln 45 50 55 60GGA CTC AAG GAC AAC AGC CAG
TTC TGC TTG CAA AGG ATC CGC CAA GGT 403Gly Leu Lys Asp Asn Ser Gln
Phe Cys Leu Gln Arg Ile Arg Gln Gly 65 70 75CTG GCT TTT TAT AAG CAC
CTG CTT GAC TCT GAC ATC TTC AAA GGG GAG 451Leu Ala Phe Tyr Lys His
Leu Leu Asp Ser Asp Ile Phe Lys Gly Glu 80 85 90CCT GCT CTA CTC CCT
GAT AGC CCC ATG GAG CAA CTT CAC ACC TCC CTA 499Pro Ala Leu Leu Pro
Asp Ser Pro Met Glu Gln Leu His Thr Ser Leu 95 100 105CTA GGA CTC
AGC CAA CTC CTC CAG CCA GAG GAT CAC CCC CGG GAG ACC 547Leu Gly Leu
Ser Gln Leu Leu Gln Pro Glu Asp His Pro Arg Glu Thr 110 115 120CAA
CAG ATG CCC AGC CTG AGT TCT AGT CAG CAG TGG CAG CGC CCC CTT 595Gln
Gln Met Pro Ser Leu Ser Ser Ser Gln Gln Trp Gln Arg Pro Leu125 130
135 140CTC CGT TCC AAG ATC CTT CGA AGC CTC CAG GCC TTT TTG GCC ATA
GCT 643Leu Arg Ser Lys Ile Leu Arg Ser Leu Gln Ala Phe Leu Ala Ile
Ala 145 150 155GCC CGG GTC TTT GCC CAC GGA GCA GCA ACT CTG ACT GAG
CCC TTA GTG 691Ala Arg Val Phe Ala His Gly Ala Ala Thr Leu Thr Glu
Pro Leu Val 160 165 170CCA ACA GCT TAAGGATGCC CAGGTTCCCA TGGCTACCAT
GATAAGACTA 740Pro Thr Ala 175ATCTATCAGC CCAGACATCT ACCAGTTAAT
TAACCCATTA GGACTTGTGC TGTTCTTGTT 800TCGTTTGTTT TGCGTGAAGG
GCAAGGACAC CATTATTAAA GAGAAAAGAA ACAAACCCCA 860GAGCAGGCAG
CTGGCTAGAG AAAGGAGCTG GAGAAGAAGA ATAAAGTCTC GAGCCCTTGG
920CCTTGGAAGC GGGCAAGCAG CTGCGTGGCC TGAGGGGAAG GGGGCGGTGG
CATCGAGAAC 980CTGTGAGAAA ACCCAGAGCA TCAGAAAAAG TGAGCCCAGG
CTTTGGCCAT TATCTGTAAG 1040AAAAACAAGA AAAGGGGAAC ATTATACTTT
CCTGGGTGGC TCAGGGAAAT GTGCAGATGC 1100ACAGTACTCC AGACAGCAGC
TCTGTACCTG CCTGCTCTGT CCCTCAGTTC TAACAGAATC 1160TAGTCACTAA
GAACTAACAG GACTACCAAT ACGAACTGAC AAA 1203196 amino acidsamino
acidlinearprotein 4Met Leu Asp Cys Arg Ala Val Ile Met Leu Trp Leu
Leu Pro Trp Val-21 -20 -15 -10Thr Gln Gly Leu Ala Val Pro Arg Ser
Ser Ser Pro Asp Trp Ala Gln -5 1 5 10Cys Gln Gln Leu Ser Arg Asn
Leu Cys Met Leu Ala Trp Asn Ala His 15 20 25Ala Pro Ala Gly His Met
Asn Leu Leu Arg Glu Glu Glu Asp Glu Glu 30 35 40Thr Lys Asn Asn Val
Pro Arg Ile Gln Cys Glu Asp Gly Cys Asp Pro 45 50 55Gln Gly Leu Lys
Asp Asn Ser Gln Phe Cys Leu Gln Arg Ile Arg Gln 60 65 70 75Gly Leu
Ala Phe Tyr Lys His Leu Leu Asp Ser Asp Ile Phe Lys Gly 80 85 90Glu
Pro Ala Leu Leu Pro Asp Ser Pro Met Glu Gln Leu His Thr Ser 95 100
105Leu Leu Gly Leu Ser Gln Leu Leu Gln Pro Glu Asp His Pro Arg Glu
110 115 120Thr Gln Gln Met Pro Ser Leu Ser Ser Ser Gln Gln Trp Gln
Arg Pro 125 130 135Leu Leu Arg Ser Lys Ile Leu Arg Ser Leu Gln Ala
Phe Leu Ala Ile140 145 150 155Ala Ala Arg Val Phe Ala His Gly Ala
Ala Thr Leu Thr Glu Pro Leu 160 165 170Val Pro Thr Ala 175102 amino
acidsamino acidNot Relevantlinearpeptide 5Ser Cys Leu Gln Arg Ile
His Gln Gly Leu Val Phe Tyr Glu Lys Leu1 5 10 15Leu Gly Ser Asp Ile
Phe Thr Gly Glu Pro Ser Leu His Pro Asp Gly 20 25 30Ser Val Gly Gln
Leu His Ala Ser Leu Leu Gly Leu Arg Gln Leu Leu 35 40 45Gln Pro Glu
Gly His His Trp Glu Thr Glu Gln Thr Pro Ser Pro Ser 50 55 60Pro Ser
Gln Pro Trp Gln Arg Leu Leu Leu Arg Leu Lys Ile Leu Arg65 70 75
80Ser Leu Gln Ala Phe Val Ala Val Ala Ala Arg Val Phe Ala His Gly
85 90 95Ala Ala Thr Leu Ser Gln 100174 amino acidsamino acidNot
Relevantlinearpeptide 6Thr Pro Leu Gly Pro Ala Arg Ser Leu Pro Gln
Ser Phe Leu Leu Lys1 5 10 15Cys Leu Glu Gln Val Arg Lys Ile Gln Ala
Asp Gly Ala Glu Leu Gln 20 25 30Glu Arg Leu Cys Ala Ala His Lys Leu
Cys His Pro Glu Glu Leu Met 35 40 45Leu Leu Arg His Ser Leu Gly Ile
Pro Gln Ala Pro Leu Ser Ser Cys 50 55 60Ser Ser Gln Ser Leu Gln Leu
Arg Gly Cys Leu Asn Gln Leu His Gly65 70 75 80Gly Leu Phe Leu Tyr
Gln Gly Leu Leu Gln Ala Leu Ala Gly Ile Ser 85 90 95Pro Glu Leu Ala
Pro Thr Leu Asp Thr Leu Gln Leu Asp Val Thr Asp 100 105 110Phe Ala
Thr Asn Ile Trp Leu Gln Met Glu Asp Leu Gly Ala Ala Pro 115 120
125Ala Val Gln Pro Thr Gln Gly Ala Met Pro Thr Phe Thr Ser Ala Phe
130 135 140Gln Arg Arg Ala Gly Gly Val Leu Val Ala Ser Gln Leu His
Arg Phe145 150 155 160Leu Glu Leu Ala Tyr Arg Gly Leu Arg Tyr Leu
Ala Glu Pro 165 170174 amino acidsamino acidNot
Relevantlinearpeptide 7Thr Pro Leu Gly Pro Thr Ser Ser Leu Pro Gln
Ser Phe Leu Leu Lys1 5 10 15Cys Leu Glu Gln Val Arg Lys Val Gln Ala
Asp Gly Thr Ala Leu Gln 20 25 30Glu Arg Leu Cys Ala Ala His Lys Leu
Cys His Pro Glu Glu Leu Val 35 40 45Leu Leu Gly His Ala Leu Gly Ile
Pro Gln Ala Pro Leu Ser Ser Cys 50 55 60Ser Ser Gln Ala Leu Gln Leu
Thr Gly Cys Leu Arg Gln Leu His Ser65 70 75 80Gly Leu Phe Leu Tyr
Gln Gly Leu Leu Gln Ala Leu Ala Gly Ile Ser 85 90 95Pro Glu Leu Ala
Pro Thr Leu Asp Met Leu Gln Leu Asp Ile Thr Asp 100 105 110Phe Ala
Ile Asn Ile Trp Gln Gln Met Glu Asp Val Gly Met Ala Pro 115 120
125Ala Val Pro Pro Thr Gln Gly Thr Met Pro Thr Phe Thr Ser Ala Phe
130 135 140Gln Arg Arg Ala Gly Gly Thr Leu Val Ala Ser Asn Leu Gln
Ser Phe145 150 155 160Leu Glu Val Ala Tyr Arg Ala Leu Arg His Phe
Thr Lys Pro 165 170177 amino acidsamino acidNot
Relevantlinearpeptide 8Thr Pro Leu Gly Pro Ala Ser Ser Leu Pro Gln
Ser Phe Leu Leu Lys1 5 10 15Cys Leu Glu Gln Val Arg Lys Ile Gln Gly
Asp Gly Ala Ala Leu Gln 20 25 30Glu Lys Leu Val Ser Glu Cys Ala Thr
Tyr Lys Leu Cys His Pro Glu 35 40 45Glu Leu Val Leu Leu Gly His Ser
Leu Gly Ile Pro Trp Ala Pro Leu 50 55 60Ser Ser Cys Pro Ser Gln Ala
Leu Gln Leu Ala Gly Cys Leu Ser Glu65 70 75 80Leu His Ser Gly Leu
Met Ala Pro Ala Leu Gln Pro Thr Gln Gly Ala 85 90 95Met Pro Ala Phe
Ala Ser Ala Phe Gln Arg Arg Ala Gly Gly Val Leu 100 105 110Val Ala
Ser His Leu Gln Ser Phe Leu Glu Val Ser Tyr Arg Val Leu 115 120
125Arg His Leu Ala Gln Phe Leu Tyr Gln Gly Leu Leu Gln Ala Leu Glu
130 135 140Gly Ile Ser Pro Glu Leu Gly Pro Thr Leu Asp Thr Leu Gln
Leu Asp145 150 155 160Val Ala Asp Phe Ala Thr Thr Ile Trp Gln Gln
Met Glu Glu Leu Gly 165 170 175Pro178 amino acidsamino acidNot
Relevantlinearpeptide 9Val Pro Leu Val Thr Val Ser Ala Leu Pro Pro
Ser Leu Pro Leu Pro1 5 10 15Arg Ser Phe Leu Leu Lys Ser Leu Glu Gln
Val Arg Lys Ile Gln Ala 20 25 30Ser Gly Ser Val Leu Leu Glu Gln Leu
Cys Ala Thr Tyr Lys Leu Cys 35 40 45His Pro Glu Glu Leu Val Leu Leu
Gly His Ser Leu Gly Ile Pro Lys 50 55 60Ala Ser Leu Ser Gly Cys Ser
Ser Gln Ala Leu Gln Gln Thr Gln Cys65 70 75 80Leu Ser Gln Leu His
Ser Gly Leu Cys Leu Tyr Gln Gly Leu Leu Gln 85 90 95Ala Leu Ser Gly
Ile Ser Pro Ala Leu Ala Pro Thr Leu Asp Leu Leu 100 105 110Gln Leu
Asp Val Ala Asn Phe Ala Thr Thr Ile Trp Gln Gln Met Glu 115 120
125Asn Leu Gly Val Ala Pro Thr Val Gln Pro Thr Gln Ser Ala Met Pro
130 135 140Ala Phe Thr Ser Ala Phe Gln Arg Arg Ala Gly Gly Val Leu
Ala Ile145 150 155 160Ser Tyr Leu Gln Gly Phe Leu Glu Thr Ala Arg
Leu Ala Leu His His 165 170 175Leu Ala186 amino acidsamino acidNot
Relevantlinearpeptide 10Ala Phe Pro Thr Pro Gly Pro Leu Gly Gly Asp
Ser Lys Asp Asp Ala1 5 10 15Thr Ser Asn Arg Pro Pro Leu Thr Ser Ala
Asp Lys Met Glu Asp Phe 20 25 30Ile Lys Phe Ile Leu Gly Lys Ile Ser
Ala Leu Arg Asn Glu Met Cys 35 40 45Asp Lys Tyr Asn Lys Cys Glu Asp
Ser Lys Glu Val Leu Ala Glu Asn 50 55 60Asn Leu Asn Leu Pro Lys Leu
Ala Glu Lys Asp Arg Cys Phe Gln Ser65 70 75 80Arg Phe Asn Gln Glu
Thr Cys Leu Thr Arg Ile Thr Thr Gly Leu Gln 85 90 95Glu Phe Gln Ile
His Leu Lys Tyr Leu Glu Ser Asn Tyr Glu Gly Asn 100 105 110Lys Asp
Asn Ala His Ser Val Tyr Ile Ser Thr Lys His Leu Leu Gln 115 120
125Thr Leu Arg Pro Met Asn Gln Ile Glu Val Thr Thr Pro Asp Pro Thr
130 135 140Thr Asp Ala Ser Leu Gln Ala Leu Phe Lys Ser Gln Asp Lys
Trp Leu145 150 155 160Lys His Thr Thr Ile His Leu Ile Leu Arg Arg
Leu Glu Asp Phe Leu 165 170 175Gln Phe Ser Leu Arg Ala Ile Arg Ile
Met 180 185183 amino acidsamino acidNot Relevantlinearpeptide 11Ala
Phe Pro Thr Pro Gly Pro Leu Gly Gly Asp Ala Thr Ser Asn Arg1 5 10
15Leu Pro Leu Thr Pro Ala Asp Lys Met Glu Glu Leu Ile Lys Tyr Ile
20 25 30Leu Gly Lys Ile Ser Ala Leu Lys Lys Glu Met Cys Asp Asn Tyr
Asn 35 40 45Lys Cys Glu Asp Ser Lys Glu Ala Leu Ala Glu Asn Asn Leu
Asn Leu 50 55 60Pro Lys Leu Ala Glu Lys Asp Gly Cys Phe Gln Ser Gly
Phe Asn Gln65 70 75 80Glu Thr Cys Leu Thr Arg Ile Thr Thr Gly Leu
Gln Glu Phe Gln Ile 85 90 95Tyr Leu Lys Phe Leu Gln Asp Lys Tyr Glu
Gly Asp Lys Glu Asn Ala 100 105 110Lys Ser Val Tyr Thr Ser Thr Asn
Val Leu Leu Gln Met Leu Lys Arg 115 120 125Lys Gly Lys Asn Gln Asp
Glu Val Thr Ile Pro Val Pro Thr Val Glu 130 135 140Val Gly Leu Gln
Leu Ser Cys Ser His Arg Arg Val Ala Glu Ala His145 150 155 160Asn
Asn His Leu Thr Leu Arg Arg Leu Glu Asp Phe Leu Gln Leu Arg 165 170
175Leu Arg Ala Val Arg Ile Met 180188 amino acidsamino
acidsinglelinearpeptide 12Ala Phe Pro Ala Pro Val Pro Pro Gly Glu
Asp Ser Lys Asp Val Ala1 5 10 15Ala Pro His Arg Gln Pro Leu Thr Ser
Ser Glu Arg Ile Asp Lys Gln 20 25 30Ile Arg Tyr Ile Leu Asp Gly Ile
Ser Ala Leu Arg Lys Glu Thr Cys 35 40 45Asn Lys Ser Asn Met Cys Glu
Ser Ser Lys Glu Ala Leu Ala Glu Asn 50 55 60Asn Leu Asn Leu Pro Lys
Met Ala Glu Lys Asp Gly Cys Phe Gln Ser65 70 75 80Gly Phe Asn Glu
Glu Thr Cys Leu Val Lys Ile Ile Thr Gly Leu Leu 85
90 95Glu Phe Glu Val Tyr Leu Glu Tyr Leu Gln Asn Arg Phe Glu Ser
Ser 100 105 110Glu Glu Gln Ala Arg Ala Val Gln Met Ser Thr Lys Val
Leu Ile Gln 115 120 125Phe Leu Gln Lys Lys Ala Lys Asn Leu Asp Ala
Ile Thr Thr Pro Asp 130 135 140Pro Thr Thr Asn Ala Ser Leu Leu Thr
Lys Leu Gln Ala Gln Asn Gln145 150 155 160Trp Leu Gln Asp Met Thr
Thr His Leu Ile Leu Arg Ser Phe Lys Glu 165 170 175Phe Leu Gln Ser
Ser Leu Arg Ala Leu Arg Gln Met 180 185184 amino acidsamino acidNot
Relevantlinearpeptide 13Ala Phe Pro Thr Pro Gly Pro Leu Gly Glu Asp
Phe Lys Asn Asp Thr1 5 10 15Thr Pro Ser Arg Leu Leu Leu Thr Thr Pro
Glu Lys Thr Glu Ala Leu 20 25 30Ile Lys His Ile Val Asp Lys Ile Ser
Ala Ile Arg Lys Glu Ile Cys 35 40 45Glu Lys Asn Asp Glu Cys Glu Asn
Ser Lys Glu Thr Leu Ala Glu Asn 50 55 60Lys Leu Lys Leu Pro Lys Met
Glu Glu Lys Asp Gly Cys Phe Gln Ser65 70 75 80Gly Phe Asn Gln Ala
Ile Cys Leu Ile Lys Thr Thr Ala Gly Leu Leu 85 90 95Glu Tyr Gln Ile
Tyr Leu Asp Phe Leu Gln Asn Glu Phe Glu Gly Asn 100 105 110Gln Glu
Thr Val Met Glu Leu Gln Ser Ser Ile Arg Thr Leu Ile Gln 115 120
125Ile Leu Lys Glu Lys Ile Ala Gly Leu Ile Thr Thr Pro Ala Thr His
130 135 140Thr Asp Met Leu Glu Lys Met Gln Ser Ser Asn Glu Trp Val
Lys Asn145 150 155 160Ala Lys Val Ile Ile Ile Leu Arg Ser Leu Glu
Asn Phe Leu Gln Phe 165 170 175Ser Leu Arg Ala Ile Arg Met Lys
180188 amino acidsamino acidNot Relevantlinearpeptide 14Ala Phe Pro
Thr Ser Gln Val Arg Arg Gly Asp Phe Thr Glu Asp Thr1 5 10 15Thr Pro
Asn Arg Pro Val Tyr Thr Thr Ser Gln Val Gly Gly Leu Ile 20 25 30Thr
His Val Leu Trp Glu Ile Val Glu Met Arg Lys Glu Leu Cys Asn 35 40
45Gly Asn Ser Asp Cys Met Asn Asn Asp Asp Ala Leu Ala Glu Asn Asn
50 55 60Leu Lys Leu Pro Glu Ile Gln Arg Asn Asp Gly Cys Tyr Gln Thr
Gly65 70 75 80Tyr Asn Gln Glu Ile Cys Leu Leu Lys Ile Ser Ser Gly
Leu Leu Glu 85 90 95Tyr His Ser Tyr Leu Glu Tyr Met Lys Asn Asn Leu
Lys Asp Asn Lys 100 105 110Lys Asp Lys Ala Arg Val Leu Gln Arg Asp
Thr Glu Thr Leu Ile His 115 120 125Ile Phe Asn Gln Glu Val Lys Asp
Leu His Lys Ile Val Leu Pro Thr 130 135 140Pro Ile Ser Asn Ala Leu
Leu Thr Asp Lys Leu Glu Ser Gln Lys Glu145 150 155 160Trp Leu Arg
Thr Lys Thr Ile Gln Phe Ile Leu Lys Ser Leu Glu Glu 165 170 175Phe
Leu Lys Val Thr Leu Arg Ser Thr Arg Gln Thr 180 185178 amino
acidsamino acidNot Relevantlinearpeptide 15Ala Pro Leu Ala Glu Leu
Ser Gly Asp His Asp Phe Gln Leu Phe Leu1 5 10 15His Lys Asn Leu Glu
Phe Thr Arg Lys Ile Arg Gly Asp Val Ala Ala 20 25 30Leu Gln Arg Ala
Val Cys Asp Thr Phe Gln Leu Cys Thr Glu Glu Glu 35 40 45Leu Gln Leu
Val Gln Pro Asp Pro His Leu Val Gln Ala Pro Leu Asp 50 55 60Gln Cys
His Lys Arg Gly Phe Gln Ala Glu Val Cys Phe Thr Gln Ile65 70 75
80Arg Ala Gly Leu His Ala Tyr His Asp Ser Leu Gly Ala Val Leu Arg
85 90 95Leu Leu Pro Asn His Thr Thr Leu Val Glu Thr Leu Gln Leu Asp
Ala 100 105 110Ala Asn Leu Ser Ser Asn Ile Gln Gln Gln Met Glu Asp
Leu Gly Leu 115 120 125Asp Thr Val Thr Leu Pro Ala Glu Gln Arg Ser
Pro Pro Pro Thr Phe 130 135 140Ser Gly Pro Phe Gln Gln Gln Val Gly
Gly Phe Phe Ile Leu Ala Asn145 150 155 160Phe Gln Arg Phe Leu Glu
Thr Ala Tyr Arg Ala Leu Arg His Leu Ala 165 170 175Arg Leu185 amino
acidsamino acidNot Relevantlinearpeptide 16Thr Arg Gly Lys Leu Pro
Asp Ala Pro Glu Phe Glu Lys Asp Leu Leu1 5 10 15Ile Gln Arg Leu Asn
Trp Met Leu Trp Val Ile Asp Glu Cys Phe Arg 20 25 30Asp Leu Cys Tyr
Arg Thr Gly Ile Cys Lys Gly Ile Leu Glu Pro Ala 35 40 45Ala Ile Phe
His Leu Lys Leu Pro Ala Ile Asn Asp Thr Asp His Cys 50 55 60Gly Leu
Ile Gly Phe Asn Glu Thr Ser Cys Leu Lys Lys Leu Ala Asp65 70 75
80Gly Phe Phe Glu Phe Glu Val Leu Phe Lys Phe Leu Thr Thr Glu Phe
85 90 95Gly Lys Ser Val Ile Asn Val Asp Val Met Glu Leu Leu Thr Lys
Thr 100 105 110Leu Gly Trp Asp Ile Gln Glu Glu Leu Asn Lys Leu Thr
Lys Thr His 115 120 125Tyr Ser Pro Pro Lys Phe Asp Arg Gly Leu Leu
Gly Arg Leu Gln Gly 130 135 140Leu Lys Tyr Trp Val Arg His Phe Ala
Ser Phe Tyr Val Leu Ser Ala145 150 155 160Met Glu Lys Phe Ala Gly
Gln Ala Val Arg Val Leu Asp Ser Ile Pro 165 170 175Asp Val Thr Pro
Asp Val His Asp Lys 180 185
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