U.S. patent application number 10/979303 was filed with the patent office on 2005-06-02 for novel proteins and methods for producing the proteins.
Invention is credited to Goto, Masaaki, Higashio, Kanji, Kobayashi, Fumie, Mochizuki, Shin'ichi, Morinaga, Tomonori, Nakagawa, Nobuaki, Shima, Nobuyuki, Tsuda, Eisuke, Ueda, Masatsugu, Yano, Kazuki, Yasuda, Hisataka.
Application Number | 20050118682 10/979303 |
Document ID | / |
Family ID | 26395808 |
Filed Date | 2005-06-02 |
United States Patent
Application |
20050118682 |
Kind Code |
A1 |
Goto, Masaaki ; et
al. |
June 2, 2005 |
Novel proteins and methods for producing the proteins
Abstract
A protein which inhibits osteoclast differentiation and/or
maturation and a method for producing the protein. The protein is
produced by human embryonic lung fibroblasts and has a molecular
weight of about 60 kD and about 120 kD under non-reducing
conditions and about 60 kD under reducing conditions on
SDS-polyacrylamide gel electrophoresis. The protein can be isolated
and purified from the culture medium of fibroblasts. Furthermore,
the protein can be produced by gene engineering. The present
invention includes cDNA for producing the protein by gene
engineering, antibodies having specific affinity for the protein or
a method for determining protein concentration using these
antibodies.
Inventors: |
Goto, Masaaki;
(Shimotsuga-gun, JP) ; Tsuda, Eisuke;
(Shimotsuga-gun, JP) ; Mochizuki, Shin'ichi;
(Kawachi-gun, JP) ; Yano, Kazuki; (Shimotsuga-gun,
JP) ; Kobayashi, Fumie; (Kawachi-gun, JP) ;
Shima, Nobuyuki; (Kawachi-gun, JP) ; Yasuda,
Hisataka; (Kawachi-gun, JP) ; Nakagawa, Nobuaki;
(Shimotsuga-gun, JP) ; Morinaga, Tomonori;
(Shimotsuga-gun, JP) ; Ueda, Masatsugu;
(Kawagoe-shi, JP) ; Higashio, Kanji; (Kawagoe-shi,
JP) |
Correspondence
Address: |
ARNOLD & PORTER LLP
ATTN: IP DOCKETING DEPT.
555 TWELFTH STREET, N.W.
WASHINGTON
DC
20004-1206
US
|
Family ID: |
26395808 |
Appl. No.: |
10/979303 |
Filed: |
November 3, 2004 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10979303 |
Nov 3, 2004 |
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10232858 |
Sep 3, 2002 |
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6855808 |
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10232858 |
Sep 3, 2002 |
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08915004 |
Aug 20, 1997 |
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08915004 |
Aug 20, 1997 |
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PCT/JP96/00374 |
Feb 20, 1996 |
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Current U.S.
Class: |
435/69.2 ;
435/184; 435/320.1; 435/325; 435/7.2; 530/388.26; 536/23.2 |
Current CPC
Class: |
A61P 19/10 20180101;
C07K 14/70578 20130101 |
Class at
Publication: |
435/069.2 ;
435/184; 435/320.1; 435/325; 536/023.2; 530/388.26; 435/007.2 |
International
Class: |
G01N 033/53; G01N
033/567; C07H 021/04; C12N 009/99 |
Foreign Application Data
Date |
Code |
Application Number |
Feb 20, 1995 |
JP |
54977/1995 |
Jul 21, 1995 |
JP |
207508/1995 |
Claims
What is claimed:
1-31. (canceled)
32. A polypeptide, having at least one biological activity selected
from the group consisting of improving decreased bone mass,
inhibiting differentiation of osteoclasts, inhibiting maturation of
osteoclasts, and inhibiting formation of osteoclasts, wherein said
polypeptide is encoded by a nucleic acid selected from the group
consisting of: a) the nucleic acid of SEQ ID NO:6; and b) nucleic
acids which hybridize with the nucleic acid of SEQ ID NO:6 at
65.degree. C. overnight in a hybridization buffer.
33. A purified and isolated polypeptide comprising human OCIF.
34. A purified and isolated polypeptide comprising human OCIF and
having the amino acid sequence as shown in SEQ ID NO:5, or a
substitution or deletion mutant thereof.
35. The polypeptide of claim 33 having the amino acid sequence as
shown in SEQ ID NO:5 from residues 22-401.
36. The polypeptide of claim 33 which is characterized by being a
product of expression of an exogenous DNA sequence.
37. The polypeptide of claim 36 wherein the DNA is cDNA or genomic
DNA.
38. A pharmaceutical composition comprising a therapeutically
effective amount of OCIF in a pharmaceutically acceptable carrier,
solubilizer, and/or stabilizer.
39. A pharmaceutical composition comprising an isolated polypeptide
consisting of the amino acid sequence as shown in SEQ ID NO:4 from
residues 1-380, and a pharmaceutically acceptable carrier,
solubilizer and/or stabilizer.
40. A pharmaceutical composition comprising an isolated polypeptide
consisting of the amino acid sequence as shown in SEQ ID NO:5 from
residues 1-401, and a pharmaceutically acceptable carrier,
solubilizer and/or stabilizer.
41. The pharmaceutical composition of claim 38 wherein the OCIF is
human OCIF.
42. An osteoclastogenesis inhibitory factor dimer consisting of
osteoclastogenesis inhibitory factor monomers.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This is a continuation of U.S. application Ser. No.
10/232,858, filed Sep. 3, 2002, which is a continuation of U.S.
application Ser. No. 08/915,004, filed Aug. 20, 1997, which is a
continuation-in-part of international application PCT/JP96/00374,
filed Feb. 20, 1996, which claims priority to Japanese applications
JP/054977, filed Feb. 20, 1995 and JP/207508, filed Jul. 21,
1995.
INCORPORATION OF SEQUENCE LISTING
[0002] Herein incorporated by reference is the Sequence Listing,
which has been submitted on paper and diskette as a file named
"SubSeq16991027.txt" which is 136,653 bytes in size (measured in
MS-DOS), and which was created on Nov. 2, 2004.
FIELD OF THE INVENTION
[0003] This invention relates to a novel protein,
osteoclastogenesis inhibitory factor (OCIF), and methods for
producing the protein.
BACKGROUND OF THE INVENTION
[0004] Human bones are always remodelling by the repeated process
of resorption and reconstitution. Osteoblasts and osteoclasts are
considered to be the cells mainly responsible for bone formation
and bone resorption, respectively. A typical example of a disease
caused by abnormal bone metabolism is osteoporosis. Osteoporosis is
known to develop when bone resorption by osteoclasts exceeds bone
formation by osteoblasts, but the mechanism of osteoporosis has not
yet been completely elucidated. Osteoporosis causes bone pain and
makes bones fragile, leading to fracture, particularly in elderly
patients. Osteoporosis has therefore become a social issue with the
increasing number of elderly people in the population. Therefore,
effective drugs for the treatment of the disease are expected to be
developed. Bone mass reduction caused by abnormal bone metabolism
is thought to be prevented by inhibiting bone resorption, improving
bone formation, or improving the balance of bone metabolism.
[0005] Bone formation is promoted by stimulating growth,
differentiation, or activation of osteoblasts. Many cytokines
reportedly stimulate growth or differentiation of osteoblasts, i.e.
fibroblast growth factor (FGF) (Rodan S. B. et al., Endocrinology
vol. 121, p1917, 1987), insulin-like growth factor-I (IGF-I) (Hock
J. M. et al., Endocrinology vol. 122, p254, 1988), insulin-like
growth factor-II (IGF-II) (McCarthy T. et al., Endocrinology vol.
124, p301, 1989), Activin A (Centrella M. et al., Mol. Cell Biol.
vol. 11, p250, 1991), Vasculotropin (Varonique M. et al., Biochem.
Biophys. Res. Commun. vol. 199, p380, 1994), and bone morphogenetic
protein (BMP) (Yamaguchi, A. et al., J. Cell Biol. vol. 113, p682,
1991, Sampath T. K. et al., J. Biol. Chem. vol. 267, p20532, 1992,
and Knutsen R. et al., Biochem. Biophys. Res. Commun. vol.
194,p1352, 1993).
[0006] On the other hand, cytokines which inhibit differentiation
and/or maturation of osteoclasts have also been intensively
studied. Transforming growth factor-.beta. (Chenu C. et al., Proc.
Natl. Acad. Sci. USA, vol. 85, p5683, 1988) and interleukin-4
(Kasano K. et al., Bone-Miner., vol. 21, p179, 1993) inhibit the
differentiation of osteoclasts. Calcitonin (Bone-Miner., vol. 17,
p347, 1992), macrophage colony-stimulating factor (Hattersley G. et
al., J. Cell. Physiol. vol. 137, p199, 1988), interleukin-4
(Watanabe, K. et al., Biochem. Biophys. Res. Commun. vol. 172,
p1035, 1990), and interferon-.gamma. (Gowen M. et al., J.
Bone-Miner. Res., vol. 1, p469, 1986) inhibit bone resorption by
osteoclasts.
[0007] These cytokines are expected to be effective drugs for
improving bone mass reduction by stimulating bone formation and/or
by inhibiting bone resorption. Cytokines such as insulin like
growth factor-I and bone morphogenetic proteins have been
investigated in clinical trials for their effectiveness for
treating patients with bone diseases. Calcitonin is already used
for osteoporosis and to diminish pain in osteoporosis patients.
[0008] Examples of drugs now clinically utilized for the treatment
of bone diseases and for shortening the treatment period are
dihydroxyvitamine D.sub.3, vitamin K.sub.2, calcitonin and its
derivatives, hormones such as estradiol, ipriflavon, and calcium
preparations. However, these drugs do not provide satisfactory
therapeutic effects, and novel drug substances are expected to be
developed. Since bone metabolism is manifest in the balance between
bone resorption and bone formation, cytokines which inhibit
osteoclast differentiation and/or maturation are expected to be
developed as drugs for the treatment of bone diseases such as
osteoporosis.
SUMMARY OF THE INVENTION
[0009] The purpose of this invention is to offer both a novel
factor, termed osteoclastogenesis inhibitory factor (OCIF), and a
procedure to produce the factor efficiently.
[0010] The inventors have intensively searched for
osteoclastogenesis inhibitory factors in human embryonic fibroblast
IMR-90 (ATCC CCL 186) conditioned medium and have found a novel
osteoclastogenesis inhibitory factor (OCIF) which inhibits
differentiation and/or maturation of osteoclasts.
[0011] The inventors have established a method for accumulating the
protein to a high concentration by culturing IMR-90 cells on
alumina ceramic pieces, which function as cell adherence
matrices.
[0012] The inventors have also established an efficient method for
isolating the protein, OCIF, from the IMR-90 conditioned medium
using the following sequential column chromatography: ion-exchange,
heparin affinity, cibacron-blue affinity, and reverse phase.
[0013] After determining the amino acid sequence of the purified
natural OCIF, a cDNA encoding this protein was successfully cloned.
A procedure for producing this protein was also established. The
invention concerns a protein which is produced by human lung
fibroblast cells, has a molecular weight by SDS-PAGE of 60 kD under
reducing conditions and molecular weights of 60 kD and 120 kD under
non-reducing conditions, and has affinity for both cation-exchange
resins and heparin. The protein's ability to inhibit the
differentiation and maturation of osteoclasts is reduced when
treated for 10 minutes at 70.degree. C. or for 30 minutes at
56.degree. C., and its ability to inhibit differentiation and
maturation of osteoclasts is lost when treated for 10 minutes at
90.degree. C. The amino acid sequence of the OCIF protein of the
present invention is clearly different from any other factors known
to inhibit the formation of osteoclasts.
[0014] The invention includes a method for purifying OCIF protein,
comprising: (1) culturing human fibroblasts, (2) applying the
conditioned medium to a heparin column to obtain the adsorbed
fraction, (3) purifying the OCIF protein using a cation-exchange
column, (4) purifying the OCIF protein using a heparin affinity
column, (5) purifying the OCIF protein using a Cibacron blue
affinity column, and (6) isolating the OCIF protein using
reverse-phase column chromatography. Cibacron blue F3GA dye may be
coupled to a carrier made of synthetic hydrophilic polymers, for
example, to form columns conventionally called "blue columns".
[0015] The invention includes a method for producing OCIF protein
in high concentration by culturing human fibroblasts using alumina
ceramic pieces as the cell-adherence matrices.
[0016] Moreover, the inventors determined the amino acid sequences
of peptides derived from OCIF, designed the oligonucleotide primers
based on these amino acid sequences, and obtained cDNA fragments
encoding OCIF from a cDNA library of IMR-90 human fetal lung
fibroblast cells. The full length OCIF cDNA encoding the OCIF
protein is cloned from a cDNA library using an OCIF DNA fragment as
a probe. The OCIF cDNA containing the entire coding region is
inserted into an expression vector. Recombinant OCIF can be
produced by expressing the OCIF cDNA, containing the entire coding
region, in mammalian cells or bacteria.
BRIEF DESCRIPTION OF THE FIGURES
[0017] FIGS. 1A and 1B show the elution pattern of crude OCIF
protein (HILOAD.TM.-Q/FF pass-through fraction; sample 3) from a
HILOAD.TM.-S/HP column.
[0018] FIG. 2 shows the elution pattern of crude OCIF protein
(heparin-5PW fraction; sample 5) from a blue-5PW column.
[0019] FIG. 3 shows the elution pattern of OCIF protein (blue-5PW
fraction 49 to 50) from a reverse-phase column.
[0020] FIGS. 4A and 4B show the SDS-PAGE results of isolated OCIF
proteins under reducing or non-reducing conditions. Description of
the lanes:
[0021] lane 1, 4: molecular weight marker proteins;
[0022] lane 2, 5: OCIF protein of peak 6 in FIG. 3;
[0023] lane 3, 6: OCIF protein of peak 7 in FIG. 3.
[0024] FIG. 5 shows the elution pattern of peptides (peak 7)
obtained by the digestion of pyridyl ethylated OCIF protein
digested with lysylendopeptidase, on a reverse-phase column.
[0025] FIG. 6 shows the SDS-PAGE results of isolated natural (n)
OCIF protein and recombinant (r) OCIF proteins under non-reducing
conditions. rOCIF (E) and rOCIF (C) proteins were produced by
293/EBNA cells and by CHO cells, respectively. Description of the
lanes:
[0026] lane 1: molecular weight marker proteins;
[0027] lane 2: a monomer type nOCIF protein;
[0028] lane 3: a dimer type nOCIF protein;
[0029] lane 4: a monomer type rOCIF (E) protein;
[0030] lane 5: a dimer type rOCIF (E) protein;
[0031] lane 6: a monomer type rOCIF (C) protein;
[0032] lane 7: a dimer type rOCIF (C) protein.
[0033] FIG. 7 shows the SDS-PAGE results of isolated natural (n)
OCIF proteins and recombinant (r) OCIF proteins under reducing
conditions. rOCIF (E) and rOCIF (C) were produced by 293/EBNA cells
and by CHO cells, respectively. Description of the lanes:
[0034] lane 8: molecular weight marker proteins;
[0035] lane 9: a monomer type nOCIF protein;
[0036] lane 10: a dimer type nOCIF protein;
[0037] lane 11: a monomer type rOCIF (E) protein;
[0038] lane 12: a dimer type rOCIF (E) protein;
[0039] lane 13: a monomer type rOCIF (C) protein;
[0040] lane 14: a dimer type rOCIF (C) protein.
[0041] FIG. 8 shows the SDS-PAGE results of isolated natural (n)
OCIF proteins and recombinant (r) OCIF proteins from which N-linked
sugar chains were removed under reducing conditions. rOCIF (E) and
rOCIF (C) are rOCIF proteins produced by 293/EBNA cells and by CHO
cells, respectively. Description of the lanes:
[0042] lane 15: molecular weight marker proteins;
[0043] lane 16: a monomer type nOCIF protein;
[0044] lane 17: a dimer type nOCIF protein;
[0045] lane 18: a monomer type rOCIF (E) protein;
[0046] lane 19: a dimer type rOCIF (E) protein;
[0047] lane 20: a monomer type rOCIF (C) protein;
[0048] lane 21: a dimer type rOCIF (C) protein.
[0049] FIG. 9 shows a comparison of OCIF (SEQ ID NO: 5) and OCIF2
(SEQ ID NO: 9) amino acid sequences.
[0050] FIG. 10 shows a comparison of OCIF (SEQ ID NO: 5) and OCIF3
(SEQ ID NO: 11) amino acid sequences.
[0051] FIG. 11 shows a comparison of OCIF (SEQ ID NO: 5) and OCIF4
(SEQ ID NO: 13) amino acid sequences.
[0052] FIG. 12 shows a comparison of OCIF (SEQ ID NO: 5) and OCIF5
(SEQ ID NO: 15) amino acid sequences.
[0053] FIG. 13 shows a standard curve determining OCIF protein
concentration by an EIA employing anti-OCIF polyclonal
antibodies.
[0054] FIG. 14 shows a standard curve determining OCIF protein
concentration by an EIA employing anti-OCIF monoclonal
antibodies.
[0055] FIG. 15 shows the effect of rOCIF protein on model rats with
osteoporosis.
DETAILED DESCRIPTION OF THE INVENTION
[0056] The OCIF protein of the present invention can be isolated
from human fibroblast conditioned medium with high yield. The
procedure to isolate OCIF is based on ordinary techniques for
purifying proteins from biomaterials, in accordance with the
physical and chemical properties of OCIF protein. For example,
concentrating procedures include ordinary biochemical techniques
such as ultrafiltration, lyophilization, and dialysis. Purifying
procedures include combinations of several chromatographic
techniques for purifying proteins such as ion-exchange column
chromatography, affinity column chromatography, gel filtration
column chromatography, hydrophobic column chromatography, reverse
phase column chromatography, and preparative gel electrophoresis.
The human fibroblasts used for the production of OCIF protein are
preferably IMR-90 cells. A method for producing IMR-90 conditioned
medium is preferably a process comprising adhering human embryonic
fibroblast IMR-90 cells to alumina ceramic pieces in roller-bottles
in DMEM medium supplemented with 5% newborn calf serum, and
cultivating the cells in roller-bottles for 7 to 10 days by stand
cultivation. CHAPS (3-[(3-cholamidopropyl)-dimethylam-
monio]-1-propanesulfonate) is preferably added to the buffer as a
detergent in the protein purification procedure.
[0057] The OCIF protein of the instant invention can be obtained
initially as a basic heparin binding OCIF fraction by applying the
culture medium to a heparin column (Heparin-SEPHAROSE.TM. CL-6B,
Pharmacia), eluting with 10 mM Tris-HCl buffer, pH 7.5, containing
2 M NaCl, and applying the OCIF fraction to a
Q.cndot.anion-exchange column (HILOAD.TM.-Q/FF, Pharmacia), and
collecting the non-adsorbed fraction. OCIF protein can be purified
by subjecting the obtained OCIF fraction to purification on an
S.cndot.cation-exchange column (HILOAD.TM.-S/FF, Pharmacia), a
heparin column (Heparin-5PW, TOSOH), a Cibacron Blue column
(Blue-5PW, TOSOH), and a reverse-phase column (BU-300 C4, Perkin
Elmer).
[0058] The present invention relates to a method of cloning cDNA
encoding the OCIF protein based on the amino acid sequence of
natural OCIF and a method for obtaining recombinant OCIF protein.
The OCIF protein is purified according to the method described in
the present invention and is treated with endopeptidase (for
example, lysylendopeptidase). The amino acid sequences of the
peptides produced by the digestion are determined and the mixture
of oligonucleotides that can encode each internal amino acid
sequence is synthesized. The OCIF cDNA fragment is obtained by PCR
(preferably RT-PCR, reverse transcriptase PCR) using the
oligonucleotide mixtures described above as primers. The full
length OCIF cDNA encoding the OCIF protein is cloned from a cDNA
library using an OCIF DNA fragment as a probe. The OCIF cDNA
containing the entire coding region is inserted into an expression
vector. Recombinant OCIF can be produced by expressing the OCIF
cDNA, containing the entire coding region, in mammalian cells or
bacteria.
[0059] The present invention relates to the novel proteins OCIF2,
OCIF3, OCIF4, and OCIF5 that are variants of OCIF and have the
activity described above. These OCIF variants are obtained from the
cDNA library constructed with IMR-90 poly(A)+RNA using the OCIF
cDNA fragment as a hybridization probe. Each of the OCIF variant
cDNAs containing the entire coding region is inserted into an
expression vector. Each recombinant OCIF variant protein can be
produced by expressing each of the OCIF variant cDNAs, containing
the entire coding region, in conventional hosts. Each recombinant
OCIF variant protein can be purified according to the method
described in this invention. Each recombinant OCIF variant protein
has the ability to inhibit osteoclastogenesis.
[0060] The present invention further includes OCIF mutants. They
are substitution mutants comprising the replacement of one cysteine
residue, possibly involved in dimer formation, with a serine
residue or various deletion mutants of OCIF. Substitutions or
deletions are introduced into the OCIF cDNA using polymerase chain
reaction (PCR) or restriction enzyme digestion. Each of these
mutated OCIF cDNAs is inserted into a vector having an appropriate
promoter for gene expression. The resultant expression vector for
each of the OCIF mutants is introduced into eukaryotic cells such
as mammalian cells. Each of OCIF mutants can be obtained and
purified from the conditioned media of the transfected cells.
[0061] The present invention provides polyclonal antibodies and a
method to quantitatively determine OCIF concentration using these
polyclonal antibodies.
[0062] Natural OCIF obtained from IMR-90 conditioned medium,
recombinant OCIF produced by such hosts as microorganisms and
eukaryotes using OCIF cDNA, synthetic peptides based on the amino
acid sequence of OCIF, or peptides obtained from OCIF by partial
digestion can be used as antigens. Anti-OCIF polyclonal antibodies
are obtained by immunizing appropriate mammals with the antigens,
in combination with adjuvants if necessary, and purifying the
antibodies from the serum by ordinary purification methods.
Anti-OCIF polyclonal antibodies which are labelled with
radioisotopes or enzymes can be used in radio-immunoassay (RIA)
systems or enzyme-immunoassay (EIA) systems. Using these assay
systems, the concentration of OCIF in biological materials such as
blood, ascites and cell-culture medium can be easily
determined.
[0063] The present invention provides novel monoclonal antibodies
and a method for quantitatively determining OCIF concentration
using these monoclonal antibodies.
[0064] Anti-OCIF monoclonal antibodies can be produced by
conventional methods using OCIF as an antigen. Native OCIF obtained
from the culture medium of IMR-90 cells and recombinant OCIF
produced by such hosts as microorganisms and eukaryotes transfected
with OCIF cDNA can be used as antigens. Alternatively, synthetic
peptides based on the amino acid sequence of OCIF and peptides
obtained from OCIF by partial digestion can be also used as
antigens. Immunized lymphocytes obtained by immunizing mammals such
as mice or rats with the antigen or by an in vitro immunization
method were fused with mammalian myeloma cells to obtain
hybridomas. The hybridoma clones secreting antibodies which
recognize OCIF were selected and cultured to obtain the desired
antibodies. For immunizations, OCIF is suitably diluted with a
saline solution (0.15 M NaCl), and is intravenously or
intraperitoneally administered with an adjuvant to animals 2-5
times every 2-20 days. The immunized animal was killed three days
after the final immunization, the spleen was removed and the
splenocytes were used as immunized B lymphocytes.
[0065] Mouse myeloma cell lines useful for cell fusion with
immunized B lymphocytes include, for example, p3/.times.63-Ag8,
p3-U1, NS-1, MPC-11, SP-2/0, FO, p3.times.63 Ag8.653, and S194
cells. The rat cell line R-210 may also be used. Alternatively
human B lymphocytes immunized by an in vitro immunization method
are fused with human myeloma cells or EB virus transformed human B
lymphocytes to produce human type antibodies.
[0066] Cell fusion of immunized B lymphocytes and myeloma cells is
carried out principally by conventional methods. For example, the
method of Koehler G. et al. (Nature 256, 495-497, 1975) is
generally used. Alternatively, an electric pulse method can be
used. The immunized B lymphocytes and transformed B cells are mixed
at conventional ratios and a cell culture medium without FBS
containing polyethylene glycol is generally used to fuse the cells.
The fusions products are cultured in HAT selection medium
containing FBS to select hybridomas.
[0067] An EIA, plaque assay, Ouchterlony, or agglutination assay
can be used to screen for hybridomas producing anti-OCIF
antibodies. EIA is a simple assay which is easy to perform with
sufficient accuracy and is therefore generally used. The desired
antibody can be selected easily and accurately using EIA and
purified OCIF. Hybridomas obtained thereby can be cultured by
conventional methods of cell culture and frozen for stock if
necessary. The antibody can be produced by culturing hybridoma
cells using ordinary cell culture methods or by transplanting
hybridoma cells intraperitoneally into live animals. The antibody
can be purified by ordinary purification methods such as salt
precipitation, gel filtration, and affinity chromatography. The
antibody obtained specifically reacts with OCIF and can be used to
determine OCIF concentration and to purify OCIF protein. The
antibodies of the present invention recognize epitopes of OCIF and
have high affinity for OCIF. Therefore, they can be used for the
construction of EIA. This assay system is useful for determining
the concentration of OCIF in biological materials such as blood and
ascites.
[0068] The present invention provides agents containing OCIF as an
effective ingredient, that are useful for treating bone diseases.
Rats were subjected to denervation of the left forelimb. Test
compounds were administered daily after surgery for 14 days. After
2 weeks of treatment, the animals were sacrificed and their
forelimbs were dissected. Thereafter bones were tested for
mechanical strength by the three point bending method. OCIF
improved the mechanical strength of bone in a dose dependent
manner.
[0069] The OCIF protein of the invention is useful as a
pharmaceutical ingredient for treating or improving decreased bone
mass in bone diseases such as osteoporosis, rheumatism,
osteoarthritis, and abnormal bone metabolism in multiple myeloma.
OCIF protein is also useful as an antigen in the immunological
diagnosis of bone diseases. Pharmaceutical preparations containing
OCIF protein as an active ingredient are formulated and can be
orally or parenterally administered. The preparation contains the
OCIF protein of the present invention as an effective ingredient
and is safely administered to humans and animals. Examples of
pharmaceutical preparations include compositions for injection or
intravenous drip, suppositories, nasal preparations, sublingual
preparations, and tapes for percutaneous absorption. The
pharmaceutical preparation for injection can be prepared by mixing
a pharmacologically effective amount of OCIF protein and a
pharmaceutically acceptable carrier. The carriers are vehicles
and/or activators, e.g. amino acids, saccharides, cellulose
derivatives, and other organic and inorganic compounds, which are
generally added to active ingredients. When the OCIF protein is
mixed with the vehicles and/or activators for injection, pH
adjusters, buffers, stabilizers, solubilizing agents, etc. can be
added by conventional methods, if necessary.
BEST MODE FOR CARRYING OUT THE INVENTION
[0070] The present invention will be further explained by the
following examples, though the scope of the invention is not
restricted thereto.
EXAMPLE 1
[0071] Preparation of a Conditioned Medium of Human Fibroblast
IMR-90
[0072] Human fetal lung fibroblast IMR-90 (ATCC-CCL186) cells were
cultured on alumina ceramic pieces (80 g) (alumina: 99.5%,
manufactured by Toshiba Ceramic K. K.) in DMEM medium (manufactured
by Gibco BRL Co.) supplemented with 5% CS and 10 mM HEPES buffer
(500 ml/roller bottle) at 37.degree. C. in the presence of 5%
CO.sub.2 for 7 to 10 days using 60 roller bottles (490 cm.sup.2,
110.times.171 mm, manufactured by Corning Co.) in static culture.
The conditioned medium was harvested and a fresh medium was added
to the roller bottles. About 30 L of IMR-90 conditioned medium per
batch culture was obtained. The conditioned medium was designated
as sample 1.
EXAMPLE 2
[0073] Assay Method for Osteoclast Development Inhibitory
Activity
[0074] Osteoclast development inhibitory activity was assayed by
measuring tartrate-resistant acid phosphatase (TRAP) activity
according to the methods of M. Kumegawa et al. (Protein Nucleic
Acid Enzyme, vol. 34 p999, 1989) and N. Takahashi et al.
(Endocrinology, vol. 122, p1373, 1988 ) with modifications.
Briefly, bone marrow cells obtained from a 17 day-old mouse were
suspended in .alpha.-MEM.TM. (manufactured by GIBCO BRL Co.)
containing 10% FBS, 2.times.10.sup.-8 M of activated vitamin
D.sub.3 and a test sample and were inoculated into each well of a
96-well plate at a cell density of 3.times.10.sup.5 cells/0.2
ml/well. The plates were incubated for 7 days at 37.degree. C. in
humidified 5% CO.sub.2. Cultures were maintained by replacing 0.16
ml of old medium with the same volume of fresh medium on day 3 and
day 5 after cultivation began. On day 7, the plates were washed
with phosphate buffered saline, and the cells were fixed with
ethanol/acetone (1: 1) for 1 min. at room temperature. Osteoclast
development was tested by determining acid phosphatase activity
using a kit (Acid Phosphatase, Leucocyte, Catalog No. 387-A,
manufactured by Sigma Co.). A decrease in the number of TRAP
positive cells was taken as an indication of OCIF activity.
EXAMPLE 3
[0075] Purification of OCIF
[0076] i) Heparin SEPHAROSE.TM. CL-6B Column Chromatography
[0077] 90 L of IMR-90 conditioned medium (sample 1) was filtered
using a 0.22 .mu. membrane filter (hydrophilic MILLIDISK.TM. 2000
cm.sup.2, Millipore Co.), and was divided into three 30 liter
portions. Each portion was applied to a heparin SEPHAROSE CL-6B
column (5.times.4.1 cm, Pharmacia Co.) equilibrated with 10 mM
Tris-HCl containing 0.3 M NaCl, pH 7.5. After washing the column
with 10 mM Tris-HCl, pH 7.5 at a flow rate of 500 ml/hr., the
heparin SEPHAROSE.TM. CL-6B adsorbent protein fraction was eluted
with 10 mM Tris-HCl, pH 7.5, containing 2 M NaCl. The fraction was
designated sample 2.
[0078] ii) HILOAD.TM.-Q/FF Column Chromatography
[0079] The heparin SEPHAROSE.TM.-adsorbent fraction (sample 2) was
dialyzed against 10 mM Tris-HCl, pH 7.5, supplemented with CHAPS to
a final concentration of 0.1%, incubated at 4.degree. C. overnight
and divided into two portions. Each portion was then applied to an
anion-exchange column (HILOAD.TM.-Q/FF, 2.6.times.10 cm, Pharmacia
Co.) which was equilibrated with 50 mM Tris-HCl, 0.1% CHAPS, pH 7.5
to obtain a non-adsorbent fraction (1000 ml). The fraction was
designated sample 3.
[0080] iii) HILOAD.TM.-S/HP Column Chromatography
[0081] The HILOAD.TM.-Q non-adsorbent fraction (sample 3) was
applied to a cation-exchange column (HILOAD.TM.-S/HP, 2.6.times.10
cm, Pharmacia Co.) which was equilibrated with 50 mM Tris-HCl, 0.1%
CHAPS, pH 7.5. After washing the column with 50 mM Tris-HCl, 0.1%
CHAPS, pH 7.5, the adsorbed protein was eluted with a linear
gradient from 0 to 1 M NaCl at a flow rate of 8 ml/min for 100 min.
and fractions (12 ml) were collected. Every ten fractions from
numbers 1 to 40 were pooled to form one portion. 100 .mu.L each of
the four portions was tested for OCIF activity. OCIF activity was
observed in fractions 11 to 30 (as shown in FIGS. 1A and 1B).
Fractions 21 to 30, which had higher specific activity, were pooled
and designated sample 4.
[0082] iv) Heparin-5PW Affinity Column Chromatography
[0083] One hundred and twenty ml of HILOAD.TM.-S fractions 21 to 30
(sample 4) was diluted with 240 ml of 50 mM Tris-HCl, 0.1% CHAPS,
pH 7.5, and applied to a heparin-5PW affinity column (0.8.times.7.5
cm, Tosoh Co.) which was equilibrated with 50 mM Tris-HCl, 0.1%
CHAPS, pH 7.5. After washing the column with 50 mM Tris-HCl, 0.1%
CHAPS, pH 7.5, the adsorbed protein was eluted with a linear
gradient from 0 to 2 M NaCl at a flow rate of 0.5 ml/min for 60
min. and fractions (0.5 ml) were collected. Fifty .mu.L were
removed from each fraction to test for OCIF activity. The active
fractions, eluted with 0.7 to 1.3 M NaCl were pooled and designated
sample 5.
[0084] v) Blue 5PW Affinity Column Chromatography
[0085] Ten ml of sample 5 were diluted with 190 ml of 50 mM
Tris-HCl, 0.1% CHAPS, pH 7.5 and applied to a blue-5PW affinity
column, (0.5.times.5 cm, Tosoh Co.) which was equilibrated with 50
mM Tris-HCl, 0.1% CHAPS, pH 7.5. After washing the column with 50
mM Tris-HCl, 0.1% CHAPS, pH 7.5, the adsorbed protein was eluted
with a 30 ml linear gradient from 0 to 2 M NaCl at a flow rate of
0.5 ml/min. and fractions (0.5 ml) were collected. Using 25 .mu.L
of each fraction, OCIF activity was evaluated. Fractions 49 to 70,
eluted with 1.0-1.6 M NaCl, had OCIF activity.
[0086] vi) Reverse Phase Column Chromatography
[0087] The blue 5PW fraction obtained by collecting fractions 49
and 50 was acidified with 10 .mu.L of 25% TFA and applied to a
reverse phase C4 column (BU-300, 2.1.times.220 mm, manufactured by
Perkin-Elmer) which was equilibrated with 0.1% of TFA and 25%
acetonitrile. The adsorbed protein was eluted with a linear
gradient from 25 to 55% acetonitrile at a flow rate of 0.2 ml/min.
for 60 min., and each protein peak was collected (FIG. 3). One
hundred .mu.L of each peak fraction was tested for OCIF activity,
and peaks 6 and 7 had OCIF activity. The result was shown in Table
1.
1 TABLE 1 Dilution Sample 1/40 1/120 1/360 1/1080 Peak 6 ++ ++ + -
Peak 7 ++ + - - [++ means OCIF activity inhibiting osteoclast
development more than 80%, + means OCIF activity inhibiting
osteoclast development between 30% and 80%, and - means no OCIF
activity.]
EXAMPLE 4
[0088] Molecular Weight of OCIF Protein
[0089] The two protein peaks with OCIF activity (peaks 6 and 7)
were subjected to SDS-polyacrylamide gel electrophoresis under
reducing and non-reducing conditions. Briefly, 20 .mu.L of each
peak fraction was concentrated under vacuum and dissolved in 1.5
.mu.L of 10 mM Tris-HCl, pH 8, 1 mM EDTA, 2.5% SDS, 0.01%
bromophenol blue, and incubated at 37.degree. C. overnight under
non-reducing conditions or under reducing conditions (with 5% of
2-mercaptoethanol). Each 1.0 .mu.L of sample was then analyzed by
SDS-polyacrylamide gel electrophoresis with a gradient gel of
10-15% acrylamide (Pharmacia Co.) and an electrophoresis-device
(Fast System, Pharmacia Co.). The following molecular weight marker
proteins were used to calculate molecular weight: phosphorylase b
(94 kD), bovine serum albumin (67 kD), ovalbumin (43 kD), carbonic
anhydrase (30 kD), trypsin inhibitor (20.0 kD), and lactalbumin
(14.4 kD). After electrophoresis, protein bands were visualized by
silver stain using Phast Silver Stain Kit. The results are shown in
FIG. 4.
[0090] A protein band with an apparent molecular weight of 60 kD
was detected in the peak 6 sample under both reducing and
non-reducing conditions. A protein band with an apparent 60 kD was
detected under reducing conditions and a protein band with an
apparent 120 kD was detected under non-reducing conditions in the
peak 7 sample. Therefore, the protein of peak 7 was considered to
be a homodimer of the protein of peak 6.
EXAMPLE 5
[0091] Thermostability of OCIF
[0092] Twenty .mu.L of sample from the blue-5PW fractions 51 and 52
was diluted to 30 .mu.L with 10 mM phosphate buffered saline, pH
7.2, and incubated for 10 min. at 70.degree. C. or 90.degree. C.,
or for 30 min. at 56.degree. C. The heat-treated samples were
tested for OCIF activity. The results are shown in Table 2.
2TABLE 2 Thermostability of OCIF Dilution Sample 1/300 1/900 1/2700
Untreated ++ + - 70.degree. C., 10 min + - - 56.degree. C., 30 min
+ - - 90.degree. C., 10 min - - - [++ means OCIF activity
inhibiting osteoclast development more than 80%, +means OCIF
activity inhibiting osteoclast development between 30% and 80%, and
- means no OCIF activity.]
EXAMPLE 6
[0093] Internal Amino Acid Sequence of OCIF Protein
[0094] Each 2 fractions (1 ml) from fractions 51 to 70 of the
blue-5PW fractions were acidified with 10 .mu.L of 25% TFA, and
applied to a reverse phase C4 column (BU-300, 2.1.times.220 mm,
manufactured by Perkin-Elmer Co.) equilibrated with 25%
acetonitrile containing 0.1% TFA. The adsorbed protein was eluted
with a 12 ml linear gradient of 25 to 55% acetonitrile at a flow
rate of 0.2 ml/min, and the protein fractions corresponding to
peaks 6 and 7 were collected, respectively. The protein from each
peak was applied to a protein sequencer (PROCISE.TM. 494,
Perkin-Elmer Co.). However, the N-terminal sequence of the protein
of each peak could not be analyzed. Therefore, the N-terminus of
the protein of each peak was considered to be blocked. Internal
amino acid sequences of these proteins were therefore analyzed.
[0095] The protein from peak 6 or 7 purified by C4-HPLC, was
concentrated by centrifugation and pyridilethylated under reducing
conditions. Briefly, 50 .mu.L of 0.5 M Tris-HCl, pH 8.5, containing
100 .mu.g of dithiothreitol, 10 mM EDTA, 7 M guanidine-HCl, and 1%
CHAPS was added to each of the samples, and the mixtures were
incubated overnight in the dark at room temperature. Each mixture
was acidified with 25% TFA (a final concentration 0.1%) and applied
to a reverse phase C4 column (BU300, 2.1.times.30 mm, Perkin-Elmer
Co.) equilibrated with 20% acetonitrile containing 0.1% TFA. The
pyridil-ethylated OCIF protein was eluted with a 9 ml linear
gradient from 20 to 50% acetonitrile at a flow rate of 0.3 ml/min,
and each protein peak was collected. The pyridil-ethylated OCIF
protein was concentrated under vacuum and dissolved in 25 .mu.L of
0.1 M Tris-HCl, pH 9, containing 8 M Urea, and 0.1% TWEEN.TM.80.
Seventy-three .mu.L of 0.1 M Tris-HCl, pH 9, and 0.02 .mu.g of
lysyl endopeptidase (Wako Pure Chemical, Japan) were added to the
tube, and incubated at 37.degree. C. for 15 hours. Each digest was
acidified with 1 .mu.L of 25% TFA and was applied to a reverse
phase C8 column (RP-300, 2.1.times.220 mm, Perkin-Elmer Co.)
equilibrated with 0.1% TFA.
[0096] The peptide fragments were eluted from the column with a
linear gradient of 0 to 50% acetonitrile at a flow rate of 0.2
m/min for 70 min., and each peptide peak was collected. Each
peptide fragment (P1-P3) was applied to the protein sequencer. The
sequences of the peptides are shown in SEQ ID NOs: 1-3,
respectively.
EXAMPLE 7
[0097] Determination of the Nucleotide Sequence of OCIF cDNA
[0098] i) Isolation of poly(A)+RNA from IMR-90 Cells
[0099] About 10 .mu.g of poly(A)+RNA was isolated from
1.times.10.sup.8 cells of IMR-90 using a FASTTRACK.TM. mRNA
isolation kit (Invitrogen) according to the manufacturer's
instructions.
[0100] ii) Preparation of Mixed Primers
[0101] The following two mixed primers were synthesized based on
the amino acid sequences of two peptides (peptide P2 and peptide
P3, SEQ ID NOs: 2 and 3, respectively). All the oligonucleotides in
the mixed primers No. 2F (SEQ ID NO: 107) can code for the amino
acid sequence from the sixth residue, glutamine (Gln) to the
twelfth residue, leucine (Leu), in peptide P2. All the
oligonucleotides in the mixed primers No. 3R (SEQ ID NO: 108) can
code for the amino acid sequence from the sixth residue, histidine
(His), to the twelfth residue, lysine (Lys), in peptide P3. The
sequences of the mixed primers No. 2F and No. 3R were shown in
Table 3.
3 TABLE 3 No. 2F (SEQ ID NO: 107) 5'-CAAGAACAAA CTTTTCAATT-3' G G G
C C GC A G No. 3R (SEQ ID NO: 108) 5'-TTTATACATT GTAAAAGAAT G-3' C
G C G GCTG A C G T
[0102] iii) Amplification of an OCIF cDNA Fragment by PCR
(Polymerase Chain Reaction)
[0103] First strand cDNA was generated using a SUPERSCRIPT.TM. II
cDNA synthesis kit 23 (Gibco BRL) and 1 .mu.g of poly(A)+RNA
obtained in EXAMPLE 7-i), according to the manufacturer's
instructions. The DNA fragment encoding OCIF was obtained by PCR
using cDNA template and the primers shown in EXAMPLE 7-ii).
[0104] PCR was performed using the following conditions:
4 10.times. Ex Taq Buffer (Takara Shuzo) 5 .mu.L 2.5 mM solution of
dNTPs 4 .mu.L cDNA solution 1 .mu.L Ex Taq (Takara Shuzo) 0.25
.mu.L sterile distilled water 29.75 .mu.L 40 .mu.M solution of
primers No. 2F 5 .mu.L 40 .mu.M solution of primers No. 3R 5
.mu.L
[0105] The components of the reaction were mixed in a
microcentrifuge tube. An initial denaturation step at 95.degree. C.
for 3 min was followed by 30 cycles of denaturation at 95.degree.
C. for 30 sec, annealing at 50.degree. C. for 30 sec and extention
at 70.degree. C. for 2 min. After the amplification, a final
extention step was performed at 70.degree. C. for 5 min. The sizes
of the PCR products were determined on a 1.5% agarose gel
electrophoresis. An approximately 400 bp OCIF DNA fragment was
obtained.
EXAMPLE 8
[0106] Cloning of the OCIF cDNA Fragment Amplified by PCR and
Determination of its DNA Sequence
[0107] The OCIF cDNA fragment amplified by PCR in EXAMPLE 7-iii)
was inserted into the plasmid pBLUESCRIPT II SK.TM. using a DNA
ligation kit ver. 2 (Takara Shuzo) according to the method of
Marchuk, D. et al. (Nucleic Acids Res., vol 19, p 1154, 1991). E.
coli strain DH5 a (Gibco BRL) was transformed with the ligation
mixture. The transformants were grown and a plasmid containing the
OCIF cDNA (about 400 bp) was purified using commonly used methods.
This plasmid was called pBSOCIF. The sequence of the OCIF cDNA in
pBSOCIF was determined using a TAQ DYE DEOXY TERMINATER CYCLE
SEQUENCING.TM. kit (Perkin Elmer). The size of the OCIF cDNA is 397
bp. The OCIF cDNA encodes an amino acid sequence containing 132
residues. The amino acid sequences of the internal peptides
(peptide P2 and peptide P3, SEQ ID NOs: 2 and 3, respectively) that
were used to design the primers were found at the amino or carboxyl
terminus of the 132 amino acid sequence predicted by the 397 bp
OCIF cDNA. In addition, the amino acid sequence of the internal
peptide P1 (SEQ ID NO: 1) was also found in the predicted amino
acid sequence of OCIF. These data show that the 397 bp OCIF cDNA is
a portion of the full length OCIF cDNA.
EXAMPLE 9
[0108] Preparation of the DNA Probe
[0109] The 397 bp OCIF cDNA was prepared according to the
conditions described in EXAMPLE 7-iii). The OCIF cDNA was subjected
to a preparative agarose gel electrophoresis. The OCIF cDNA was
purified from the gel using a QIAEX.TM. gel extraction kit
(QIAGEN), labeled with [.alpha..sup.32P]dCTP using Megaprime DNA
labeling system (Amersham) and used to select a phage containing
the full length OCIF cDNA.
EXAMPLE 10
[0110] Preparation of the cDNA Library
[0111] cDNA was generated using a Great Lengths cDNA synthesis kit
(Clontech), oligo (dT) primer, [.alpha..sup.32P]dCTP and 2.5 .mu.g
of poly(A)+RNA obtained in EXAMPLE 7-i), according to the
manufacturer's instructions. An EcoRI-SalI-NotI adaptor was ligated
to the cDNA. The cDNA was separated from free adaptor DNA and
unincorporated free [.alpha..sup.32P]dCTP. The purified cDNA was
precipitated with ethanol and dissolved in 10 .mu.L of TE buffer
(10 mM Tris-HCl (pH 8.0), 1 mM EDTA). The cDNA comprising the
adaptor was ligated into .lambda.ZAP EXPRESS.TM. vector
(Stratagene) at the EcoRI site. The recombinant .lambda.ZAP
EXPRESS.TM. phage DNA containing the cDNA was in vitro packaged
using a GIGAPACK.TM. gold # II packaging extract (Stratagene)
yielding a recombinant .lambda.ZAP EXPRESS.TM. phage library.
EXAMPLE 11
[0112] Screening of Recombinant Phage
[0113] Recombinant phages obtained in EXAMPLE 10 were used to
infect E. coli strain, XL1-Blue MRF' (Stratagene) at 37.degree. C.
for 15 min. The infected E. coli cells were added to NZY medium
containing 0.7% agar at 50.degree. C. and plated onto NZY agar
plates. After the plates were incubated at 37.degree. C. overnight,
HYBOND.TM. N (Amersham) membranes were placed on the surface of the
plates containing plaques. The membranes were denatured in alkali
solution, neutralized, and washed in 2.times.SSC according to
standard methods. The phage DNA was immobilized onto the membranes
using UV CROSSLINK.TM. (Stratagene). The membranes were incubated
in hybridization buffer (Amersham) containing 100 .mu.g/ml salmon
sperm DNA at 65.degree. C. for 4 hours and then incubated at
65.degree. C. overnight in the same buffer containing
2.times.10.sup.5 cpm/ml of denatured OCIF DNA probe. The membranes
were washed twice with 2.times.SSC and twice with a solution
containing 0.1.times.SSC and 0.1% SDS at 65.degree. C. for 10 min
each time. The positive clones were purified by repeating the
screening twice. The purified .lambda.ZAP EXPRESS.TM. phage clone
containing a DNA insert of about 1.6 kb was used in the experiments
described below. This phage was called .lambda. OCIF. The purified
.lambda. OCIF was used to infect E. coli strain XL-1 blue MRF'
(Stratagene) according to the protocol in the .lambda.ZAP
EXPRESS.TM. cloning kit (Stratagene). The culture broth of infected
XL-1 blue MRF' was prepared. Purified .lambda. OCIF and
EXASSIST.TM. helper phage (Stratagene) were coinfected into E. coli
strain XL-1 blue MRF', according to the protocol supplied with the
kit. The culture broth of the coinfected XL-1 blue MRF' was added
to a culture of E. coli strain XLOR (Stratagene) to transform them.
Thus we obtained a Kanamycin-resistant transformant harboring a
plasmid designated pBKOCIF which is a pBKCMV (Stratagene) vector
containing the 1.6 kb insert fragment.
[0114] The transformant including the plasmid containing about 1.6
kb OCIF cDNA was obtained by lifting the Kanamycin-resistant
colonies. The plasmid was called pBKOCIF. The transformant has been
deposited in the National Institute of Bioscience and
Human-Technology (NIBH), Agency of Industrial Science and
Technology as "FERM BP-5267" as pBK/01F10. A national deposit
(Accession number, FERM P-14998) was transferred to the
international deposit, on Oct. 25, 1995 according to the Budapest
treaty. The transformant pBK/01F10 was grown and the plasmid
pBKOCIF was purified according to standard methods.
EXAMPLE 12
[0115] Determination of the Nucleotide Sequence of OCIF cDNA
Containing the Full Coding Region.
[0116] The nucleotide sequence of OCIF cDNA obtained in EXAMPLE 11
was determined using a TAQ DYE DEOXY TERMINATER CYCLE
SEQUENCING.TM. kit (Perkin Elmer). The primers used were T3, T7
(Stratagene) and synthetic primers designed according to the OCIF
cDNA sequence. The sequences of these primers are shown in SEQ ID
NOs: 16 to 29. The nucleotide sequence of the OCIF cDNA is shown in
SEQ ID NO: 6 and the amino acid sequence predicted by the cDNA
sequence is shown in SEQ ID NO: 5.
EXAMPLE 13
[0117] Production of Recombinant OCIF by 293/EBNA Cells
[0118] i) Construction of the Plasmid for Expressing OCIF cDNA
[0119] pBKOCIF, containing about 1.6 kb OCIF cDNA, was prepared as
described in EXAMPLE 11 and digested with restriction enzymes BamHI
and XhoI. The OCIF cDNA insert was cut out, isolated by an agarose
gel electrophoresis and purified using a QIAEX.TM. gel extraction
kit (QIAGEN). The purified OCIF cDNA insert was ligated into the
expression vector pCEP4 (Invitrogen) using DNA ligation kit ver. 2
(Takara Shuzo) digested with restriction enzymes BamHI and XhoI. E.
coli strain DH5.alpha. (Gibco BRL) was transformed with the
ligation mixture.
[0120] The transformants were grown and the plasmid containing the
OCIF cDNA (about 1.6 kb) was purified using a QIAGEN.TM. column
(QIAGEN). The expression plasmid pCEPOCIF was precipitated with
ethanol and dissolved in sterile distilled water for use in the
experiments described below.
[0121] ii) Transient Expression of OCIF cDNA and Analysis of OCIF
Biological Activity
[0122] Recombinant OCIF was produced using the expression plasmid
pCEPOCIF (prepared in EXAMPLE 13-i) according to the method
described below. 8.times.10.sup.5 cells of 293/EBNA (Invitrogen)
were inoculated into each well of a 6-well plate using IMDM
containing 10% fetal bovine serum (FBS; Gibco BRL). After the cells
were incubated for 24 hours, the culture medium was removed and the
cells were washed with serum free IMDM. The expression plasmid
pCEPOCIF and lipofectamine (Gibco BRL) were diluted with
OPTI-MEM.TM. (Gibco BRL), mixed, and added to the cells in each
well according to the manufacturer's instructions. Three .mu.g of
pCEPOCIF and 12 .mu.L of lipofectamine were used for each
transfection. After the cells were incubated with pCEPOCIF and
lipofectamine for 38 hours, the medium was replaced with 1 ml of
OPTI-MEM.TM.. After incubation for 30 hours, the conditioned medium
was harvested and used for the biological assay. The biological
activity of OCIF was analyzed according to the method described
below. Bone marrow cells obtained from 17 day old mice were
suspended in .alpha.-MEM.TM. (manufactured by GIBCO BRL Co.)
containing 10% FBS, 1.times.10.sup.-8M activated vitamin D.sub.3
and a test sample, and were inoculated and cultured for 7 days at
37.degree. C. in humidified 5% CO.sub.2 as described in EXAMPLE 2.
During incubation, 160 .mu.L of old medium in each well was
replaced with the same volume of the fresh medium containing test
sample diluted with 1.times.10.sup.-8 M of activated vitamin
D.sub.3 and .alpha.-MEM.TM. containing FBS on day 3 and day 5. On
day 7, after washing the wells with phosphate buffered saline,
cells were fixed with ethanol/acetone (1:1) for 1 min. and
osteoclast development was tested using an acid phosphatase
activity measuring kit (Acid Phosphatase, Leucocyte, Catalog No.
387A, Sigma Co.). A decrease in the number of TRAP positive cells
was taken as an OCIF activity. The conditioned medium showed the
same OCIF activity as natural OCIF protein from IMR-90 conditioned
medium (Table 4).
5TABLE 4 OCIF activity of 293/EBNA conditioned medium. Dilution
Cultured Cell 1/20 1/40 1/80 1/160 1/320 1/640 1/1280 OCIF
expression ++ ++ ++ ++ ++ + - vector transfected vector - - - - - -
- transfected untreated - - - - - - - [++; OCIF activity inhibiting
osteoclast development more than 80%, +; OCIF activity inhibiting
osteoclast development between 30% and 80%, and -; no OCIF
activity.]
[0123] iii) Isolation of Recombinant OCIF Protein from
293/EBNA-Conditioned Medium
[0124] 293/EBNA-conditioned medium (1.8 L) obtained by cultivating
the cells described in EXAMPLE 13-ii) was supplemented with 0.1%
CHAPS and filtrated using a 0.22 .mu.m membrane filter (Sterivex
GS, Millipore Co.). The conditioned medium was applied to a 50 ml
heparin SEPHAROSE.TM. CL-6B column (2.6.times.10 cm, Pharmacia Co.)
equilibrated with 10 mM Tris-HCl, pH 7.5. After washing the column
with 10 mM Tris-HCl, pH 7.5, the adsorbed protein was eluted from
the column with a linear gradient from 0 to 2 M NaCl at a flow rate
of 4 ml/min for 100 min. and 8 ml fractions were collected. Using
150 .mu.L of each fraction, OCIF activity was assayed according to
the method described in EXAMPLE 2. An OCIF active 112 ml fraction,
eluted with approximately 0.6 to 1.2 M NaCl, was obtained.
[0125] One hundred twelve ml of the active fraction was diluted to
1000 ml with 10 mM Tris-HCl, 0.1% CHAPS, pH 7.5, and applied to a
heparin affinity column (heparin-5PW, 0.8.times.7.5 cm, Tosoh Co.)
equilibrated with 10 mM Tris-HCl, 0.1% CHAPS, pH 7.5. After washing
the column with 10 mM Tris-HCl, 0.1% CHAPS, pH 7.5, the adsorbed
protein was eluted from the column with a linear gradient from 0 to
2 M NaCl at a flow rate of 0.5 ml/min for 60 min. and 0.5 ml
fractions were collected. Four .mu.L of each fraction was analyzed
by SDS-polyacrylamide gel electrophoresis under reducing and
non-reducing conditions as described in EXAMPLE 4. A single band of
rOCIF protein with an apparent molecular weight of 60 kD was
detected in fractions from 30 to 32 by SDS-PAGE under reducing
conditions. Bands of rOCIF protein with apparent molecular weights
of 60 kD and 120 kD were also detected in fractions from 30 to 32
under non-reducing conditions. The isolated rOCIF from fractions 30
to 32 was designated as recombinant OCIF derived from 293/EBNA
(rOCIF(E)). 1.5 ml of the rOCIF(E) (535 .mu.g/ml) was obtained when
determined by the method of Lowry, using bovine serum albumin as a
standard protein.
EXAMPLE 14
[0126] Production of Recombinant OCIF using CHO Cells
[0127] i) Construction of the Plasmid for Expressing OCIF
[0128] pBKOCIF containing about 1.6 kb OCIF cDNA was prepared as
described in EXAMPLE 11, and digested with restriction enzymes SalI
and EcoRV. About 1.4 kb OCIF cDNA insert was separated by agarose
gel electrophoresis and purified from the gel using a QIAEX.TM. gel
extraction kit (QIAGEN). The expression vector, pcDL-SR .alpha. 296
(Molecular and Cellular Biology, vol 8, p466-472, 1988) was
digested with restriction enzymes PstI and KpnI. About 3.4 kb of
the expression vector fragment was cut out, separated by agarose
gel electrophoresis and purified from the gel using a QIAEX.TM. gel
extraction kit (QIAGEN). The ends of the purified OCIF cDNA insert
and the expression vector fragment were blunted using a DNA
blunting kit (Takara Shuzo). The purified OCIF cDNA insert and the
expression vector fragment were ligated using a DNA ligation kit
ver. 2 (Takara Shuzo). E. coli strain DH5a .alpha. (Gibco BRL) was
transformed with the ligation mixture. A transformant containing
the OCIF expression plasmid, pSR .alpha.OCIF was obtained.
[0129] ii) Preparation of the Expression Plasmid
[0130] The transformant containing the OCIF expression plasmid, pSR
.alpha.OCIF prepared in EXAMPLE 13-i) and the transformant
containing the mouse DHFR expression plasmid, pBAdDSV shown in WO
92/01053 were grown according to standard methods. Both plasmids
were purified by alkali treatment, polyethylene glycol
precipitation, and cesium chloride density gradient ultra
centrifugation according to the method of Maniatis et al.
(Molecular Cloning, 2nd edition).
[0131] iii) Adaptation of CHOdhFr.sup.- Cells to the Protein Free
Medium
[0132] CHOdhFr.sup.- cells (ATCC, CRL 9096) were cultured in IMDM
containing 10% fetal bovine serum. The cells were adapted to
EXCELL.TM. 301 (JRH Bioscience) and then adapted to EXCELL.TM. PF
CHO (JRH Bioscience) according to the manufacturer's
instructions.
[0133] iv) Transfection of the OCIF Expression Plasmid, and the
Mouse DHFR Expression Plasmid, into CHOdhFr.sup.- Cells.
[0134] CHOdhFr.sup.- cells prepared in EXAMPLE 14-iii) were
transfected by electroporation with pSR .alpha.OCIF and pBAdDSV
prepared in EXAMPLE 14-ii). Two hundred .mu.g of pSR .alpha.OCIF
and 20 .mu.g of pBAdDSV were dissolved under sterile conditions in
0.8 ml of IMDM (Gibco BRL) containing 10% fetal bovine serum.
CHOdhFr.sup.- cells (2.times.10.sup.7) were suspended in 0.8 ml of
this medium. The cell suspension was transferred to a cuvette (Bio
Rad) and the cells were transfected by electroporation using a GENE
PULSER.TM.(Bio Rad) under the conditions of 360 V and 960 .mu.F.
The suspension of electroporated cells was transferred to T-flasks
(Sumitomo Bakelite) containing 10 ml of EXCELL.TM. PF-CHO, and
incubated in the CO.sub.2 incubator for 2 days. The transfected
cells were then inoculated into each well of a 96 well plate
(Sumitomo Bakelite) at a density of 5000 cells/well and cultured
for about 2 weeks. The transformants expressing DHFR are selected
since EXCELL.TM. PF-CHO does not contain nucleotides and the
parental cell line CHOdhFr.sup.- can not grow in this medium. Most
of the transformants expressing DHFR express OCIF since the OCIF
expression plasmid was used ten times as much as the mouse DHFR
expression plasmid. The transformants whose conditioned medium had
high OCIF activity were selected from among the transformants
expressing DHFR according to the method described in EXAMPLE 2. The
transformants that express large amounts of OCIF were cloned by the
limiting dilution method. The clones whose conditioned medium had
high OCIF activity were selected as described above and a
transformant expressing large amounts of OCIF named, 5561, was
obtained.
[0135] v) Production of Recombinant OCIF
[0136] To produce recombinant OCIF (rOCIF), clone 5561 was
inoculated into a 3 L spinner flask with EXCELL.TM. 301 medium (3
L) at a cell density of 1.times.10.sup.5 cells/ml. The 5561 cells
were cultured in a spinner flask at 37.degree. C. for 4 to 5 days.
When the concentration of the 5561 cells reached 1.times.10.sup.6
cells/ml, about 2.7 L of the conditioned medium was harvested. Then
about 2.7 L of EXCELL.TM. 301 was added to the spinner flask and
the 5561 cells were cultured repeatedly.
[0137] About 20 L of the conditioned medium was harvested using the
three spinner flasks.
[0138] vi) Isolation of Recombinant OCIF Protein from CHO
Cell-Conditioned Medium
[0139] CHO cell-conditioned medium (1.0 L) described in EXAMPLE
14-v) was supplemented with 1.0 g CHAPS and filtrated with a 0.22
.mu.m membrane filter (Sterivex-GS, Millipore Co.). The conditioned
medium was applied to a heparin SEPHAROSE.TM.-FF column
(2.6.times.10 cm, Pharmacia Co.) equilibrated with 10 mM Tris-HCl,
pH 7.5. After washing the column with 10 mM Tris-HCl, 0.1% CHAPS,
pH 7.5, the adsorbed protein was eluted from the column with a
linear gradient from 0 to 2 M NaCl at a flow rate of 4 ml/min for
100 min. and 8 ml fractions were collected. Using 150 .mu.L of each
fraction, OCIF activity was assayed according to the method
described in EXAMPLE 2. An active fraction (112 ml) eluted with
approximately 0.6 to 1.2 M NaCl was obtained.
[0140] The 112 ml active fraction was diluted to 1200 ml with 10 mM
Tris-HCl, 0.1% CHAPS, pH 7.5, and applied to an affinity column
(Blue-5PW, 0.5.times.5.0 cm, Tosoh Co.) equilibrated with 10 mM
Tris-HCl, 0.1% CHAPS, pH 7.5. After washing the column with 10 mM
Tris-HCl, 0.1% CHAPS, pH 7.5, the adsorbed protein was eluted from
the column with a linear gradient from 0 to 3 M NaCl at a flow rate
of 0.5 ml/min for 60 min. and fractions (0.5 ml) were collected.
Four .mu.L of each fraction were subjected to SDS-polyacrylamide
gel electrophoresis under reducing and non-reducing conditions as
described in EXAMPLE 4. A single band of rOCIF protein with an
apparent molecular weight of 60 kD was detected in fractions 30 to
38 using SDS-PAGE under reducing conditions. Bands of rOCIF protein
with apparent molecular weights of 60 kD and 120 kD using SDS-PAGE
under non-reducing conditions were also detected in fractions 30 to
38. The isolated rOCIF fraction, from fractions 30 to 38, was
designated as purified recombinant OCIF derived from CHO cells
(rOCIF(C)). 4.5 ml of the rOCIF(C) (113 .mu.g/ml) was obtained, as
determined by the method of Lowry using bovine serum albumin as a
standard protein.
EXAMPLE 15
[0141] Determination of N-Terminal Amino Acid Sequence of
rOCIFs
[0142] Three .mu.g of the isolated rOCIF(E) and rOCIF(C) were
adsorbed to polyvinylidene difluoride (PVDF) membranes with
PROSPIN.TM. (PERKIN ELMER Co.). The membranes were washed with 20%
ethanol and the N-terminal amino acid sequences of the adsorbed
proteins were analyzed by protein sequencer (PROCISE.TM. 492,
PERKIN ELMER Co.). The determined N-terminal amino acid sequence is
shown in SEQ ID NO: 7.
[0143] The N-terminal amino acid of rOCIF(E) and rOCIF(C) was
glutamic acid located at position 22 from Met of the translation
start site, as shown in SEQ ID NO: 5. The 21 amino acids from Met
to Gln were identified as a signal peptide. The N-terminal amino
acid sequence of OCIF isolated from IMR-90 conditioned medium could
not be determined. Accordingly, the N-terminal glutamic acid of
OCIF may be blocked by the conversion of glutamic acid to
pyroglutamine within cell culture or purification steps.
EXAMPLE 16
[0144] Biological Activity of Recombinant (r) OCIF and Natural (n)
OCIF
[0145] i) Inhibition of Vitamin D.sub.3 Induced Osteoclast
Formation in Murine Bone Marrow Cells
[0146] Each of the rOCIF(E) and nOCIF samples were diluted with
.alpha.-MEM.TM. (GIBCO BRL Co.) containing 10% FBS and
2.times.10.sup.-8 M of activated vitamin D.sub.3 (a final
concentration of 250 ng/ml). Each sample was serially diluted with
the same medium, and 100 .mu.L of each diluted sample was added to
each well of a 96-well plate. Bone marrow cells obtained from 17
day old mice were inoculated at a cell density of 3.times.10.sup.5
cells/100 .mu.L/well into each well of a 96-well plate and cultured
for 7 days at 37.degree. C. in humidified 5% CO.sub.2. On day 7,
the cells were fixed and stained with an acid phosphatase measuring
kit (Acid Phosphatase, Leucocyte, No. 387-A, Sigma) according to
the method described in EXAMPLE 2. A decrease in acid phosphatase
activity (TRAP) was taken as an indication of OCIF activity. A
decrease in acid phosphatase-positive cells was evaluated by
solubilizing the pigment of dye and measuring absorbance. Briefly,
100 .mu.L of a mixture of 0.1 N NaOH and dimethylsulfoxide (1:1)
was added to each well and the well was vibrated to solubilize the
dye. After solubilizing the dye completely, an absorbance of each
well was measured at 590 nm, subtracting the absorbance at 490 nm
using a microplate reader (IMMUNOREADER.TM. NJ-2000, InterMed). The
microplate reader was adjusted to 0 absorbance using a well with
monolayered bone marrow cells which were cultured in the medium
without activated vitamin D.sub.3. A decrease in TRAP activity was
expressed as a percentage of the control absorbance value (=100%)
(measured for wells with bone marrow cells cultured in the absence
of OCIF). The results are shown in Table 5.
6TABLE 5 Inhibition of vitamin D.sub.3-induced osteoclast formation
from murine bone marrow cells OCIF concentration(ng/ml) 250 125 63
31 16 0 rOCIF(E) 0 0 3 62 80 100 nOCIF 0 0 27 27 75 100 (%)
[0147] Both nOCIF and rOCIF(E) inhibited osteoclast formation in a
dose dependent manner at concentrations of 16 ng/ml or greater.
[0148] ii) Inhibition of Vitamin D.sub.3-Induced Osteoclast
Formation in Co-Cultures of Stromal Cells and Mouse Spleen
Cells.
[0149] The effect of OCIF on osteoclast formation induced by
Vitamin D.sub.3 in co-cultures of stromal cells and mouse spleen
cells was tested according to the method of N. Udagawa et al.
(Endocrinology, vol. 125, p 1805-1813, 1989). Briefly, samples of
each of rOCIF(E), rOCIF(C), and nOCIF were serially diluted with
.alpha.-MEM.TM. (GIBCO BRL Co.) containing 10% FBS,
2.times.10.sup.-8 M activated vitamin D.sub.3 and 2.times.10.sup.-7
M dexamethasone and 100 .mu.L of each of the diluted samples was
added to each well of 96 well-microwell plates. Murine bone
marrow-derived stromal ST2 cells (RIKEN Cell Bank RCB0224) at
5.times.10.sup.3 cells per 100 .mu.L of .alpha.-MEM.TM. containing
10% FBS and spleen cells from 8 week old ddy mice at
1.times.10.sup.5 cells per 100 .mu.L in the same medium, were
inoculated into each well of a 96-well plate and cultured for 5
days at 37.degree. C. in humidified 5% CO.sub.2. On day 5, the
cells were fixed and stained using an acid phosphatase kit (Acid
Phosphatase, Leucocyte, No. 387-A, Sigma). A decrease in acid
phosphatase-positive cells was taken as an indication of OCIF
activity. The decrease in acid phosphatase-positive cells was
evaluated according to the method described in EXAMPLE 16-i). The
results are shown in Table 6 (rOCIF(E) and rOCIF(C)) and Table 7
(rOCIF(E) and nOCIF).
7TABLE 6 Inhibition of osteoclast formation in co-cultures of
stromal cells and mouse spleen cells. OCIF concentration (ng/ml) 50
25 13 6 0 rOCIF(E) 3 22 83 80 100 rOCIF(C) 13 19 70 96 100 (%)
[0150]
8TABLE 7 Inhibition of osteoclast formation in co-cultures of
stromal cells and mouse spleen cells. OCIF concentration(ng/ml) 250
63 16 0 rOCIF(E) 7 27 37 100 rOCIF(C) 13 23 40 100 (%)
[0151] nOCIF, rOCIF(E) and rOCIF(C) inhibited osteoclast formation
in a dose dependent manner at concentrations of 6-16 ng/ml or
greater.
[0152] iii) Inhibition of PTH-Induced Osteoclast Formation in
Murine Bone Marrow Cells.
[0153] The effect of OCIF on osteoclast formation induced by PTH
was tested according to the method of N. Takahashi et al.
(Endocrinology, vol. 122, p1373-1382, 1988). Briefly, samples of
each of rOCIF(E) and nOCIF (125 ng/ml) were serially diluted with
.alpha.-MEM.TM. (manufactured by GIBCO BRL Co.) containing 10% FBS
and 2.times.10.sup.-8 M PTH, and 100 .mu.L of each of the diluted
samples was added to the wells of 96 well-plates. Bone marrow cells
from 17 day old ddy mice at a cell density of 3.times.10.sup.5
cells per 100 .mu.L of .alpha.-MEM.TM. containing 10% FBS were
inoculated into each well of a 96-well plate and cultured for 5
days at 37.degree. C. in humidified 5% CO.sub.2. On day 5, the
cells were fixed with ethanol/acetone (1:1) for 1 min. at room
temperature and stained with an acid phosphatase kit (Acid
Phosphatase, Leucocyte, No. 387-A, Sigma) according to the method
described in EXAMPLE 2. A decrease in acid phosphatase-positive
cells was taken as an indication of OCIF activity. The decrease in
acid phosphatase-positive cells was evaluated according to the
method described in EXAMPLE 16-i). The results are shown in Table
8.
9TABLE 8 Inhibition of PTH-induced osteoclast formation from murine
bone marrow cells. OCIF concentration(ng/ml) 125 63 31 16 8 0
rOCIF(E) 6 58 58 53 88 100 nOCIF 18 47 53 56 91 100
[0154] nOCIF and rOCIF(E) inhibited osteoclast formation in a dose
dependent manner at concentrations of 16 ng/ml or greater.
[0155] iv) Inhibition of IL-11-Induced Osteoclast Formation
[0156] The effect of OCIF on osteoclast formation induced by IL-11
was tested according to the method of T. Tamura et al. (Proc. Natl.
Acad. Sci. USA, vol. 90, p11924-11928, 1993). Briefly, samples of
each of rOCIF(E) and nOCIF were serially diluted with
.alpha.-MEM.TM. (GIBCO BRL Co.) containing 10% FBS and 20 ng/ml
IL-11 and 100 .mu.l of each diluted sample was added to each well
in a 96-well plate. Newborn mouse calvaria-derived pre-adipocyte
MC3T3-G2/PA6 cells (RIKEN Cell Bank RCB1127) at 5.times.10.sup.3
cells per 100 .mu.l of .alpha.-MEM.TM. containing 10% FBS, and
spleen cells from 8 week old ddy mouse, at 1.times.10.sup.5 cells
per 100 .mu.L in the same medium, were inoculated into each well of
a 96-well plate and cultured for 5 days at 37.degree. C. in
humidified 5% CO.sub.2. On day 5, the cells were fixed and stained
with an acid phosphatase kit (Acid Phosphatase, Leucocyte, No.
387-A, Sigma). Acid phosphatase positive cells were counted under a
microscope and a decrease of the cell numbers was taken as an
indication of OCIF activity. The results are shown in Table 9.
10TABLE 9 OCIF concentration(ng/ml) 500 125 31 7.8 2.0 0.5 0 nOCIF
0 0 1 4 13 49 31 rOCIF(E) 0 0 1 3 10 37 31
[0157] Both nOCIF and rOCIF(E) inhibited osteoclast formation in a
dose dependent manner at concentrations of 2 ng/ml or greater.
[0158] The results shown in Tables 4-8 indicated that OCIF inhibits
all the vitamin D.sub.3, PTH, and IL-11-induced osteoclast
formations at almost the same doses. Accordingly, OCIF could be
used for treating different types of bone disorders due to
decreased bone mass, that are caused by different substances that
induce bone resorption.
EXAMPLE 17
[0159] Isolation of Monomer-Type OCIF and Dimer-Type OCIF
[0160] Each rOCIF(E) and rOCIF(C) sample containing 100 .mu.g of
OCIF protein, was supplemented with {fraction (1/100)} volume of
25% trifluoro acetic acid and applied to a reverse phase column
(PROTEIN-RP.TM., 2.0.times.250 mm, YMC Co.) equilibrated with 30%
acetonitrile containing 0.1% trifluoro acetic acid. OCIF protein
was eluted from the column with a linear gradient from 30 to 55%
acetonitrile at a flow rate of 0.2 ml/min for 50 min. and each OCIF
peak was collected. The monomer-type OCIF peak fraction and
dimer-type OCIF peak fraction were each lyophilized.
EXAMPLE 18
[0161] Determination of the Molecular Weight of Recombinant
OCIFs
[0162] Each 1 .mu.g of the isolated monomer-type and dimer-type
nOCIF purified using a reverse phase column according to EXAMPLE
3-iv) and each 1 .mu.g of monomer-type and dimer-type rOCIF
described in EXAMPLE 17 was concentrated under vacuum. Each sample
was incubated in the buffer for SDS-PAGE, subjected to
SDS-polyacrylamide gel electrophoresis, and protein bands on the
gel were stained with silver according to the method described in
EXAMPLE 4. Results of electrophoresis under non-reducing conditions
and reducing conditions are shown in FIGS. 6 and 7,
respectively.
[0163] A protein band with an apparent molecular weight of 60 kD
was detected in each monomer-type OCIF sample, and a protein band
with an apparent molecular weight of 120 kD was detected in each
dimer-type OCIF sample under non-reducing conditions. A protein
band with an apparent molecular weight of 60 kD was detected in
each monomer-type OCIF sample under reducing conditions.
Accordingly, the molecular weights of monomer-type nOCIF from
IMR-90 cells, rOCIF from 293/EBNA cells and rOCIF from CHO cells
were almost the same (60 kD). Molecular weights of dimer-type nOCIF
from IMR-90 cells, rOCIF from 293/EBNA cells, and rOCIF from CHO
cells were also the same (120 kD).
EXAMPLE 19
[0164] Removal of the N-Linked Oligosaccharide Chain and Measuring
the Molecular Weight of Natural and Recombinant OCIF
[0165] Each sample containing 5 .mu.g of the isolated monomer-type
and dimer-type nOCIF purified using a reverse phase column
according to EXAMPLE 3-iv) and each sample containing 5 .mu.g of
monomer-type and dimer-type rOCIF described in EXAMPLE 17 were
concentrated under vacuum. Each sample was dissolved in 9.5 .mu.L
of 50 mM sodium phosphate buffer, pH 8.6, containing 100 mM
2-mercaptoethanol, supplemented with 0.5 .mu.L of 250 U/ml
N-glycanase (Seikagaku kogyo Co.) and incubated for one day at
37.degree. C. Each sample was supplemented with 10 .mu.L of 20 mM
Tris-HCl, pH 8.0 containing 2 mM EDTA, 5% SDS, and 0.02%
bromo-phenol blue and heated for 5 min at 100 .degree. C. Each 1
.mu.L of the samples was subjected to SDS-polyacrylamide gel
electrophoresis, and protein bands on the gel were stained with
silver as described in EXAMPLE 4. The patterns of electrophoresis
are shown in FIG. 8.
[0166] An apparent molecular weight of each of the deglycosylated
nOCIF from IMR-90 cells, rOCIF from CHO cells, and rOCIF from
293/EBNA cells was 40 kD under reducing conditions. An apparent
molecular weight of each of the untreated nOCIF from IMR-90 cells,
rOCIF from 293/EBNA cells, and rOCIF from CHO cells was 60 kD under
reducing conditions. Accordingly, the results indicate that the
OCIF proteins are glycoproteins with N-linked sugar chains.
EXAMPLE 20
[0167] Cloning of OCIF Variant cDNAs and Determination of their DNA
Sequences
[0168] The plasmid pBKOCIF, comprising OCIF cDNA inserted into
plasmid pBKCMV (Stratagene), was obtained as in EXAMPLES 10 and 11.
Further, during the screening of the cDNA library with the 397 bp
OCIF cDNA probe, the transformants containing plasmids whose insert
sizes were different from that of pBKOCIF were obtained. These
transformants containing the plasmids were grown and the plasmids
were purified according to the standard method. The sequence of the
insert DNA in each plasmid was determined using a TAQ DYE DEOXY
TERMINATER CYCLE SEQUENCING.TM. kit (Perkin Elmer). The primers
used were T3, T7, (Stratagene) and synthetic primers prepared based
on the nucleotide sequence of OCIF cDNA. There are four OCIF
variants (OCIF2, 3, 4, and 5) in addition to OCIF. The nucleotide
sequence of OCIF2 is shown in SEQ ID NO: 8 and the amino acid
sequence of OCIF2 predicted by the nucleotide sequence is shown in
SEQ ID NO: 9. The nucleotide sequence of OCIF3 is shown in SEQ ID
NO: 10 and the amino acid sequence of OCIF3 predicted by the
nucleotide sequence is shown in SEQ ID NO: 11. The nucleotide
sequence of OCIF4 is shown in SEQ ID NO: 12 and the amino acid
sequence of OCIF4 predicted by the nucleotide sequence is shown in
SEQ ID NO: 13. The nucleotide sequence of OCIF5 is shown in SEQ ID
NO: 14 and the amino acid sequence of OCIF5 predicted by the
nucleotide sequence is shown in SEQ ID NO: 15. The structures of
OCIF variants are shown in FIGS. 9 to 12 and are briefly described
below.
[0169] OCIF2
[0170] The OCIF2 cDNA has a deletion of 21 bp from guanine at
nucleotide number 265 to guanine at nucleotide number 285 in the
OCIF cDNA (SEQ ID NO: 6).
[0171] Accordingly, OCIF2 has a deletion of 7 amino acids from
glutamic acid (Glu) at amino acid number 89 to glutamine (Gln) at
amino acid number 95 in OCIF (SEQ ID NO: 5).
[0172] OCIF3
[0173] The OCIF3 cDNA has a point mutation at nucleotide number 9
in the OCIF cDNA (SEQ ID NO: 6) where cytidine is replaced with
guanine. Accordingly, OCIF3 has a mutation where asparagine (Asn)
at amino acid number 3 in OCIF (SEQ ID NO: 5) is replaced with
lysine (Lys). The mutation seems to be located in the signal
sequence and has no essential effect on the secretion of OCIF3.
OCIF3 cDNA has a deletion of 117 bp from guanine at nucleotide
number 872 to cytidine at nucleotide number 988 in the OCIF cDNA
(SEQ ID NO: 6).
[0174] Accordingly, OCIF3 has a deletion of 39 amino acids from
threonine (Thr) at amino acid number 291 to leucine (Leu) at amino
acid number 329 in OCIF (SEQ ID NO: 5).
[0175] OCIF4
[0176] The OCIF4 cDNA has two point mutations in the OCIF cDNA (SEQ
ID NO: 6). Cytidine at nucleotide number 9 is replaced with guanine
and guanine at nucleotide number 22 is replaced with thymidine in
the OCIF cDNA (SEQ ID NO: 6).
[0177] Accordingly, OCIF4 has two mutations. Asparagine (Asn) at
amino acid number 3 in OCIF (SEQ ID NO: 5) is replaced with lysine
(Lys), and alanine (Ala) at amino acid number 8 is replaced with
serine (Ser). These mutations seem to be located in the signal
sequence and have no essential effect on the secreted OCIF4.
[0178] The OCIF4 cDNA has about 4 kb DNA, comprising intron 2 of
the OCIF gene, inserted between nucleotide number 400 and
nucleotide number 401 in the OCIF cDNA (SEQ ID NO: 6). The open
reading frame stops in intron 2.
[0179] Accordingly, OCIF4 has an additional novel amino acid
sequence containing 21 amino acids after alanine (Ala) at amino
acid number 133 in OCIF (SEQ ID NO: 5).
[0180] OCIF5
[0181] The OCIF5 cDNA has a point mutation at nucleotide number 9
in the OCIF cDNA (SEQ ID NO: 6) where cytidine is replaced with
guanine.
[0182] Accordingly, OCIF5 has a mutation where asparagine (Asn) at
amino acid number 3 in OCIF (SEQ ID NO: 5) is replaced with lysine
(Lys). The mutation seems to be located in the signal sequence and
has no essential effect on the secretion of OCIF5.
[0183] The OCIF5 cDNA has the latter portion (about 1.8 kb) of
intron 2 between nucleotide number 400 and nucleotide number 401 in
OCIF cDNA (SEQ ID NO: 6). The open reading frame stops in the
latter portion of intron 2.
[0184] Accordingly, OCIF5 has an additional novel amino acid
sequence containing 12 amino acids after alanine (Ala) at amino
acid number 133 in OCIF (SEQ ID NO: 5).
EXAMPLE 21
[0185] Production of OCIF Variants
[0186] i) Construction of the Plasmid for Expressing OCIF
Variants
[0187] Plasmids containing OCIF2 or OCIF3 cDNA were obtained as
described in EXAMPLE 20 and called pBKOCIF2 and pBKOCIF3,
respectively. pBKOCIF2 and pBKOCIF3 were digested with restriction
enzymes BamHI and XhoI. The OCIF2 and OCIF3 cDNA inserts were
separated by agarose gel electrophoresis and purified from the gel
using a QIAEX.TM. gel extraction kit (QIAGEN). The purified OCIF2
and OCIF3 cDNA inserts were individually ligated using a DNA
ligation kit ver. 2 (Takara Shuzo) to the expression vector pCEP4
(Invitrogen) that had been digested with restriction enzymes BamHI
and XhoI. E. coli strain DH5 .alpha. (Gibco BRL) was transformed
with the ligation mixture.
[0188] The plasmid containing OCIF4 cDNA was obtained as described
in EXAMPLE 20 and called pBKOCIF4. pBKOCIF4 was digested with
restriction enzymes Spel and XhoI (Takara Shuzo). The OCIF4 cDNA
insert was separated by agarose gel electrophoresis, and purified
from the gel using a QIAEX.TM. gel extraction kit (QIAGEN). The
purified OCIF4 cDNA insert was ligated using a DNA ligation kit
ver. 2 (Takara Shuzo) to an expression vector pCEP4 (Invitrogen)
that had been digested with restriction enzymes NheI and XhoI
(Takara Shuzo). E. coli strain DH5 a (Gibco BRL) was transformed
with the ligation mixture.
[0189] The plasmid containing OCIF5 cDNA was obtained as described
in EXAMPLE 20 and called pBKOCIF5. pBKOCIF5 was digested with the
restriction enzyme HindIII (Takara Shuzo). The 5' portion of the
coding region in the OCIF5 cDNA insert was separated by agarose gel
electrophoresis and purified from the gel using QIAEX.TM. gel
extraction kit (QIAGEN). The OCIF expression plasmid, pCEPOCIF,
obtained in EXAMPLE 13-i) was digested with the restriction enzyme
HindIII (Takara Shuzo). The 5' portion of the coding region in the
OCIF cDNA was removed. The rest of the plasmid that contains pCEP
vector and the 3' portion of the coding region of OCIF cDNA was
called pCEPOCIF-3'. pCEPOCIF3' was separated by agarose gel
electrophoresis and purified from the gel using a QIAEX.TM. gel
extraction kit (QIAGEN). The OCIF5 cDNA HindIII fragment and
pCEPOCIF-3' were ligated using a DNA ligation kit ver. 2 (Takara
Shuzo). E. coli strain DH5 .alpha. (Gibco BRL) was transformed with
the ligation mixture.
[0190] The transformants obtained were grown at 37.degree. C.
overnight and the OCIF variant expression plasmids (pCEPOCIF2,
pCEPOCIF3, pCEPOCIF4, and pCEPOCIF5) were purified using QIAGEN.TM.
columns (QIAGEN). These OCIF-variant-expression plasmids were
precipitated with ethanol, dissolved in sterile distilled water,
and used in the experiments described below.
[0191] ii) Transient Expression of OCIF Variant cDNAs and Analysis
of the Biological Activity of Recombinant OCIF Variants.
[0192] Recombinant OCIF variants were produced using the expression
plasmids, pCEPOCIF2, pCEPOCIF3, pCEPOCIF4, and pCEPOCIF5 as
described in EXAMPLE 21-i) according to the method described in
EXAMPLE 13-ii). The biological activities of recombinant OCIF
variants were analyzed. The results were that these OCIF variants
(OCIF2, OCIF3, OCIF4, and OCIF5) had weak activity.
EXAMPLE 22
[0193] Preparation of OCIF Mutants
[0194] i) Construction of a Plasmid Vector for Subcloning cDNAs
Encoding OCIF Mutants
[0195] The plasmid vector (5 .mu.g) described in EXAMPLE 11 was
digested with restriction enzymes BamHI and XhoI (Takara Shuzo).
The digested DNA was subjected to preparative agarose gel
electrophoresis. A DNA fragment with an approximate size of 1.6
kilobase pairs (kb) that contained the entire coding sequence for
OCIF was purified from the gel using a QIAEX.TM. gel extraction kit
(QIAGEN). The purified DNA was dissolved in 20 .mu.L of sterile
distilled water. This solution was designated DNA solution 1.
pBLUESCRIPT II SK+.TM. (3 .mu.g) (Stratagene) was digested with
restriction enzymes BamHI and XhoI (Takara Shuzo). The digested DNA
was subjected to preparative agarose gel electrophoresis. A DNA
fragment with an approximate size of 3.0 kb was purified from the
gel using a QIAEX.TM. DNA extraction kit (QIAGEN). The purified DNA
was dissolved in 20 .mu.L of sterile distilled water. This solution
was designated DNA solution 2. One microliter of DNA solution 2, 4
.mu.L of DNA solution I and 5 .mu.L of ligation buffer I from a DNA
ligation kit ver. 2 (Takara Shuzo) were mixed and incubated at
16.degree. C. for 30 min. (The ligation mixture was used in the
transformation of E. coli in the manner described below).
Conditions for transformation of E. coli were as follows. One
hundred microliters of competent E. coli strain DH5 .alpha. cells
(GIBCO BRL) and 5 .mu.L of the ligation mixture were mixed in a
sterile 15-ml tube (IWAKI glass). The tube was kept on ice for 30
min. After incubation for 45 sec at 42.degree. C., 250 .mu.L of L
broth (1% Tryptone, 0.5% yeast extract, 1% NaCl) was added to the
cells. The cell suspension was then incubated for 1 hr. at
37.degree. C. with shaking. Fifty microliters of the cell
suspension was plated onto an L-agar plate containing 5 .mu.g/ml of
ampicillin. The plate was incubated overnight at 37.degree. C.
[0196] Six colonies which grew on the plate were each incubated in
2 ml of L-broth containing 50 .mu.g/ml ampicillin overnight at
37.degree. C. with shaking. The structure of the plasmids in the
colonies was analyzed. A plasmid in which the 1.6-kb DNA fragment
containing the entire OCIF cDNA is inserted between the digestion
sites of BamHI and XhoI of pBLUESCRIPT II SK+.TM. was obtained and
designated as pSK.+-.OCIF.
[0197] ii) Preparation of Mutants in which One of the Cys Residues
in OCIF is Replaced with a Ser Residue
[0198] 1) Introduction of Mutations into OCIF cDNA
[0199] OCIF mutants were prepared in which one of the five Cys
residues present in OCIF at positions 174, 181, 256, 298 and 379
(in SEQ ID NO: 4) was replaced with a Ser residue and were
designated OCIF-C19S (174Cys to Ser), OCIF-C20S (181Cys to Ser),
OCIF-C21S (256Cys to Ser), OCIF-C22S (298Cys to Ser) and OCIF-C23S
(379Cys to Ser), respectively. The amino acid sequences of these
mutants are provided in the sequence listing as SEQ ID NOs:62, 63,
64, 65, and 66, respectively.
[0200] To prepare the mutants, nucleotides encoding the
corresponding Cys residues were replaced with those encoding Ser.
Mutagenesis was carried out by a two-step polymerase chain reaction
(PCR). The first step of the PCRs consisted of two reactions, PCR 1
and PCR 2.
11 PCR 1 10.times. Ex Taq Buffer (Takara Shuzo) 10 .mu.L 2.5 mM
solution of dNTPs 8 .mu.L the plasmid vector described in 2 .mu.L
EXAMPLE 11 (8 ng/ml) sterile distilled water 73.5 .mu.L 20 .mu.M
solution of primer 1 5 .mu.L 100 .mu.M solution of primer 2 1 .mu.L
(for mutagenesis) Ex Taq (Takara Shuzo) 0.5 .mu.L PCR 2 10.times.
Ex Taq Buffer (Takara Shuzo) 10 .mu.L 2.5 mM solution of dNTPs 8
.mu.L the plasmid vector described in 2 .mu.L EXAMPLE 11 (8 ng/ml)
sterile distilled water 73.5 .mu.L 20 .mu.M solution of primer 3 5
.mu.L 100 .mu.M solution of primer 4 1 .mu.L (for mutagenesis) Ex
Taq (Takara Shuzo) 0.5 .mu.L
[0201] Specific sets of primers were used for each mutation and
other components were unchanged. Primers used for the reactions are
shown in Table 10. The nucleotide sequences of the primers are
shown in SEQ ID NOs: 20, 23, 27 and 30-40. The PCRs were performed
under the following conditions. An initial denaturation step at
97.degree. C. for 3 min was followed by 25 cycles of denaturation
at 95.degree. C. for 1 min, annealing at 55.degree. C. for 1 min
and extension at 72.degree. C. min. After these amplification
cycles, final extension was performed at 70.degree. C. for 5 min.
The sizes of the PCR products were confirmed by agarose gel
electrophoresis of the reaction solutions. After the first PCR,
excess primers were removed using an Amicon MICROCON.TM. (Amicon).
The final volume of the solutions that contained the PCR products
were made to 50 .mu.L with sterile distilled water. These purified
PCR products were used for the second PCR (PCR 3).
12 PCR 3 10.times. Ex Taq Buffer (Takara Shuzo) 10 .mu.L 2.5 mM
solution of DNTPs 8 .mu.L solution containing DNA 5 .mu.L fragment
obtained from PCR 1 solution containing DNA 5 .mu.L fragment
obtained from PCR 2 sterile distilled water 61.5 .mu.L 20 .mu.M
solution of primer 1 5 .mu.L 20 .mu.M solution of primer 3 5 .mu.L
Ex Taq (Takara Shuzo) 0.5 .mu.L
[0202]
13TABLE 10 mutants primer-1 primer-2 primer-3 primer-4 OCIF-C19S IF
10 C19SR IF 3 C19SF OCIF-C20S IF 10 C20SR IF 3 C20SF OCIF-C21S IF
10 C21SR IF 3 C21SF OCIF-C22S IF 10 C22SR IF 14 C22SF OCIF-C23S IF
6 C23SR IF 14 C23SF
[0203] The reaction conditions were exactly the same as those for
PCR 1 or PCR 2. The sizes of the PCR products were confirmed by
1.0% or 1.5% agarose gel electrophoresis. The DNA fragments were
precipitated with ethanol, dried under vacuum and dissolved in 40
.mu.L of sterile distilled water. The solutions containing DNA
fragments with mutations C19S, C20S, C21S, C22S and C23S were
designated as DNA solution A, DNA solution B, DNA solution C, DNA
solution D and DNA solution E, respectively.
[0204] The DNA fragment which is contained in solution A (20 .mu.L)
was digested with restriction enzymes NdeI and SphI (Takara Shuzo).
A DNA fragment with an approximate size of 400 base pairs (bp) was
extracted from a preparative agarose gel and dissolved in 20 .mu.L
of sterile distilled water. This DNA solution was designated DNA
solution 3. Two micrograms of pSK+-OCIF were digested with
restriction enzymes NdeI and SphI. A DNA fragment with an
approximate size of 4.2 kb was purified from a preparative agarose
gel using a QIAEX.TM. gel extraction kit and dissolved in 20 .mu.L
of sterile distilled water. This DNA solution was designated DNA
solution 4. Two microliters of DNA solution 3, 3 .mu.L of DNA
solution 4 and 5 .mu.L of ligation buffer I from a DNA ligation kit
ver. 2 were mixed and the ligation reaction was carried out.
Competent E. coli strain DH5.alpha. cells were transformed with 5
.mu.L of the ligation mixture. Ampicillin-resistant transformants
were screened for a clone containing plasmid DNA. DNA structure was
analyzed by restriction enzyme mapping and by DNA sequencing. The
plasmid thus obtained was named pSK-OCIF-C19S.
[0205] The DNA fragment contained in solution B (20 .mu.L) was
digested with restriction enzymes NdeI and SphI. A DNA fragment
with an approximate size of 400 bp was extracted from a preparative
agarose gel using a QIAEX.TM. gel extraction kit and dissolved in
20 .mu.L of sterile distilled water. This DNA solution was
designated DNA solution 5. Two microliters of DNA solution 5, 3
.mu.L of DNA solution 4 and 5 .mu.L of ligation buffer I from a DNA
ligation kit ver. 2 were mixed and the ligation reaction was
carried out. Competent E. coli strain DH5 .alpha. cells were
transformed with 5 .mu.L of the ligation mixture.
Ampicillin-resistant transformants were screened for a clone
containing plasmid DNA. DNA structure was analyzed by restriction
enzyme mapping and by DNA sequencing. The plasmid thus obtained was
named pSK-OCIF-C20S.
[0206] The DNA fragment which is contained in solution C (20 .mu.L)
was digested with restriction enzymes NdeI and SphI. A DNA fragment
with an approximate size of 400 bp was extracted from a preparative
agarose gel using a QIAEX.TM. gel extraction kit and dissolved in
20 .mu.L of sterile distilled water. This DNA solution was
designated DNA solution 6. Two microliters of DNA solution 6, 3
.mu.L of DNA solution 4 and 5 .mu.L of ligation buffer I from a
ligation kit ver. 2 were mixed and the ligation reaction was
carried out. Competent E. coli strain DH5 .alpha. cells were
transformed with 5 .mu.L of the ligation mixture.
Ampicillin-resistant transformants were screened for a clone
containing plasmid DNA. DNA structure was analyzed by restriction
enzyme mapping and by DNA sequencing. The plasmid thus obtained was
named pSK-OCIF-C21S.
[0207] The DNA fragment which is contained in solution D (20 .mu.L)
was digested with restriction enzymes NdeI and BstPI. A DNA
fragment with an approximate size of 600 bp was extracted from a
preparative agarose gel using a QIAEX.TM. gel extraction kit and
dissolved in 20 .mu.L of sterile distilled water. This DNA solution
was designated DNA solution 7. Two micrograms of pSK+-OCIF were
digested with restriction enzymes NdeI and BstPI. A DNA fragment
with an approximate size of 4.0 kb was extracted from a preparative
agarose gel using a QIAEX.TM. gel extraction kit and dissolved in
20 .mu.L of sterile distilled water. This DNA solution was
designated DNA solution 8. Two microliters of DNA solution 7, 3
.mu.L of DNA solution 8 and 5 .mu.L of ligation buffer I from a DNA
ligation kit ver. 2 were mixed and the ligation reaction was
carried out. Competent E. coli strain DH5 .alpha. cells were
transformed with 5 .mu.L of the ligation mixture.
Ampicillin-resistant transformants were screened for a clone
containing plasmid DNA in which the 600-bp NdeI-BstPI fragment with
the mutation (the C22S mutation) is substituted for the 600-bp
NdeI-BstPI fragment of pSK+-OCIF by analyzing the DNA structure.
DNA structure was analyzed by restriction enzyme mapping and by DNA
sequencing. The plasmid thus obtained was named pSK-OCIF-C22S.
[0208] The DNA fragment which is contained in solution E (20 .mu.L)
was digested with restriction enzymes BstPI and EcoRV. A DNA
fragment with an approximate size of 120 bp was extracted from a
preparative agarose gel using a QIAEX.TM. gel extraction kit and
dissolved in 20 .mu.L of sterile distilled water. This DNA solution
was designated DNA solution 9. Two micrograms of pSK+-OCIF were
digested with restriction enzymes BstEII and EcoRV. A DNA fragment
with an approximate size of 4.5 kb was extracted from a preparative
agarose gel using a QIAEX.TM. gel extraction kit and dissolved in
20 .mu.L of sterile distilled water. This DNA solution was
designated DNA solution 10. Two microliters of DNA solution 9, 3
.mu.L of DNA solution 10 and 5 .mu.L of ligation buffer I from a
DNA ligation kit ver. 2 were mixed and the ligation was carried
out. Competent E. coli strain DH5 .alpha. cells were transformed
with 5 .mu.L of the ligation mixture. Ampicillin-resistant
transformants were screened for a clone containing plasmid DNA. DNA
structure was analyzed by restriction enzyme mapping and by DNA
sequencing. The plasmid thus obtained was named pSK-OCIF-C23S.
[0209] 2) Construction of Vectors for Expressing the OCIF
Mutants
[0210] pSK-OCIF-C19S, pSK-OCIF-C20S, pSK-OCIF-C21S, pSK-OCIF-C22S
and pSK-OCIF-C23S were digested with restriction enzymes BamHI and
XhoI. The 1.6 kb BamHI-XhoI DNA fragment encoding each OCIF mutant
was isolated and dissolved in 20 .mu.L of sterile distilled water.
The DNA solutions that contain 1.6 kb cDNA fragments derived from
pSK-OCIF-C19S, pSK-OCIF-C20S, pSK-OCIF-C21S, pSK-OCIF-C22S and
pSK-OCIF-C23S were designated C19S DNA solution, C20S DNA solution,
C21S DNA solution, C22S DNA solution and C23S DNA solution,
respectively. Five micrograms of expression vector pCEP 4
(Invitrogen) were digested with restriction enzymes BamHI and XhoI.
A DNA fragment with an approximate size of 10 kb was purified and
dissolved in 40 .mu.L of sterile distilled water. This DNA solution
was designated as pCEP 4 DNA solution. One microliter of pCEP 4 DNA
solution and 6 .mu.L of either C19S DNA solution, C20S DNA
solution, C21S DNA solution, C22S DNA solution or C23S DNA solution
were independently mixed with 7 .mu.L of ligation buffer I from a
DNA ligation kit ver. 2 and the ligation reactions were carried
out. Competent E. coli strain DH5 .alpha. cells (100 .mu.L) were
transformed with 7 .mu.L of each ligation mixture.
Ampicillin-resistant transformants were screened for clones
containing plasmid in which a 1.6-kb cDNA fragment is inserted
between the recognition sites of BamHI and XhoI of pCEP 4 by
analyzing the DNA structure. The plasmids which were obtained
containing the cDNA encoding OCIF-C19S, OCIF-C20S, OCIF-C21 S,
OCIF-C22S and OCIF-C23S (SEQ ID NOs: 83, 84, 85, 86, and 87,
respectively) were designated pCEP4-OCIF-C19S, pCEP4-OCIF-C20S,
pCEP4-OCIF-C21S, pCEP4-OCIF-C22S and pCEP4-OCIF-C23S,
respectively.
[0211] iii) Preparation of Domain-Deletion Mutants of OCIF
[0212] (1) deletion Mutagenesis of OCIF cDNA
[0213] A series of OCIF mutants with deletions from Thr 2 to Ala
42, from Pro 43 to Cys 84, from Glu 85 to Lys 122, from Arg 123 to
Cys 164, from Asp 177 to Gln 251 or from Ile 252 to His 326 were
prepared (positions of the amino acid residues are shown in SEQ ID
NO: 4). These mutants were designated as OCIF-DCR1, OCIF-DCR2,
OCIF-DCR3, OCIF-DCR4, OCIF-DDD1 and OCIF-DDD2, respectively, and
assigned SEQ ID NOs: 67, 68, 69, 70, 71, and 72, respectively.
[0214] Mutagenesis was performed by two-step PCR as described in
EXAMPLE 22-ii). The primer sets for the reactions are shown in
Table 11 and the nucleotide sequences of the primers are shown in
SEQ ID NOs: 19, 25, 40-53 and 54.
14TABLE 11 mutants primer-1 primer-2 primer-3 primer-4 OCIF-DCR1
XhoI F DCR1R IF 2 DCR1F OCIF-DCR2 XhoI F DCR2R IF 2 DCR2F OCIF-DCR3
XhoI F DCR3R IF 2 DCR3F OCIF-DCR4 XhoI F DCR4R IF 16 DCR4F
OCIF-DDD1 IF 8 DDD1R IF 14 DDD1F OCIF-DDD2 IF 8 DDD2R IF 14
DDD2F
[0215] The final PCR products were precipitated with ethanol, dried
under vacuum and dissolved in 40 .mu.L of sterile distilled water.
Solutions of DNA fragments coding for portions of OCIF-DCR1,
OCIF-DCR2, OCIF-DCR3, OCIF-DCR4, OCIF-DDD1 and OCIF-DDD2 were
designated DNA solutions F, G, H, I, J and K, respectively.
[0216] The DNA fragment contained in solution F (20 .mu.L) was
digested with restriction enzymes NdeI and XhoI. A DNA fragment
with an approximate size of 500 bp was extracted from a preparative
agarose gel using a QIAEX.TM. gel extraction kit and dissolved. in
20 .mu.L of sterile distilled water. This DNA solution was
designated DNA solution 11. Two micrograms of pSK+-OCIF were
digested with restriction enzymes NdeI and XhoI. A DNA fragment
with an approximate size of 4.0 kb was extracted from a preparative
agarose gel using a QIAEX.TM. gel extraction kit and dissolved in
20 .mu.L of sterile distilled water. This DNA solution was
designated DNA solution 12. Two microliters of DNA solution 11, 3
.mu.L of DNA solution 12 and 5 .mu.L of ligation buffer I from a
DNA ligation kit ver. 2 were mixed and the ligation was carried
out. Competent E. coli strain DH5 a cells were transformed with 5
.mu.L of the ligation mixture. Ampicillin-resistant transformants
were screened for a clone containing plasmid DNA. DNA structure was
analyzed by restriction enzyme mapping and by DNA sequencing. The
plasmid thus obtained was named pSK-OCIF-DCR1.
[0217] The DNA fragment which is contained in solution G (20 .mu.L)
was digested with restriction enzymes NdeI and XhoI. A DNA fragment
with an approximate size of 500 bp was extracted from a preparative
agarose gel using a QIAEX.TM. gel extraction kit and dissolved in
20 .mu.L of sterile distilled water. This DNA solution was
designated DNA solution 13. Two microliters of DNA solution 13, 3
.mu.L of DNA solution 12 and 5 .mu.L of ligation buffer I from a
DNA ligation kit ver. 2 were mixed and ligation was carried out.
Competent E. coli strain DH5 .alpha. cells were transformed with 5
.mu.L of the ligation mixture. Ampicillin-resistant transformants
were screened for a clone containing a plasmid DNA. DNA structure
was analyzed by restriction enzyme mapping and by DNA sequencing.
The plasmid thus obtained was named pSK-OCIF-DCR2.
[0218] The DNA fragment contained in solution H (20 .mu.L) was
digested with restriction enzymes NdeI and XhoI. A DNA fragment
with an approximate size of 500 bp was extracted from a preparative
agarose gel using a QIAEX.TM. gel extraction kit and dissolved in
20 .mu.L of sterile distilled water. This DNA solution was
designated DNA solution 14. Two microliters of DNA solution 14, 3
.mu.L of DNA solution 12 and 5 .mu.L of ligation buffer I from a
DNA ligation kit ver. 2 were mixed and the ligation reaction was
carried out. Competent E. coil strain DH5 .alpha. cells were
transformed with 5 .mu.L of the ligation mixture.
Ampicillin-resistant transformants were screened for a clone
containing a plasmid DNA. DNA structure was analyzed by restriction
enzyme mapping and by DNA sequencing. The plasmid thus obtained was
named pSK-OCIF-DCR3.
[0219] The DNA fragment contained in solution 1 (20 .mu.L) was
digested with restriction enzymes XhoI and SphI. A DNA fragment
with an approximate size of 900 bp was extracted from a preparative
agarose gel using a QIAEX.TM. gel extraction kit and dissolved in
20 .mu.L of sterile distilled water. This DNA solution was
designated DNA solution 15. Two micrograms of pSK+-OCIF were
digested with restriction enzymes XhoI and SphI. A DNA fragment
with an approximate size of 3.6 kb was extracted from a preparative
agarose gel using a QIAEX.TM. gel extraction kit and dissolved in
20 .mu.L of sterile distilled water. This DNA solution was
designated DNA solution 16. Two microliters of DNA solution 15, 3
.mu.L of DNA solution 16 and 5 .mu.L of ligation buffer I from a
DNA ligation kit ver. 2 were mixed and the ligation reaction was
carried out. Competent E. coli strain DH5 .alpha. cells were
transformed with 5 .mu.L of the ligation mixture.
Ampicillin-resistant transformants were screened for a clone
containing plasmid DNA. DNA structure was analyzed by restriction
enzyme mapping and by DNA sequencing. The plasmid thus obtained was
named pSK-OCIF-DCR4.
[0220] The DNA fragment contained in solution J (20 .mu.L) was
digested with restriction enzymes BstPI and NdeI. A DNA fragment
with an approximate size of 400 bp was extracted from a preparative
agarose gel using a QIAEX.TM. gel extraction kit and dissolved in
20 .mu.L of sterile distilled water. This DNA solution was
designated DNA solution 17. Two microliters of DNA solution 17, 3
.mu.L of DNA solution 8 and 5 .mu.L of ligation buffer I from a DNA
ligation kit ver. 2 were mixed and the ligation reaction was
carried out. Competent E. coli strain DH5 .alpha. cells were
transformed with 5 .mu.L of the ligation mixture.
Ampicillin-resistant transformants were screened for a clone
containing plasmid DNA. DNA structure was analyzed by restriction
enzyme mapping and by DNA sequencing. The plasmid thus obtained was
named pSK-OCIF-DDD1.
[0221] The DNA fragment contained in solution K (20 .mu.L) was
digested with restriction enzymes NdeI and BstPI. A DNA fragment
with an approximate size of 400 bp was extracted from a preparative
agarose gel using a QIAEX.TM. gel extraction kit and dissolved in
20 .mu.L of sterile distilled water. This DNA solution was
designated DNA solution 18. Two microliters of DNA solution 18, 3
.mu.L of DNA solution 8 and 5 .mu.L of ligation buffer I from a DNA
ligation kit ver. 2 were mixed and the ligation reaction was
carried out. Competent E. coli strain DH5 .alpha. cells were
transformed with 5 .mu.L of the ligation mixture.
Ampicillin-resistant transformants were screened for a clone
containing plasmid DNA. DNA structure was analyzed by restriction
enzyme mapping and by DNA sequencing. The plasmid thus obtained was
named pSK-OCIF-DDD2.
[0222] 2) Construction of Vectors for Expressing the OCIF
Mutants
[0223] pSK-OCIF-DCR1, pSK-OCIF-DCR2, pSK-OCIF-DCR3, pSK-OCIF-DCR4,
pSK-OCIF-DDD1 and pSK-OCIF-DDD2 were digested with restriction
enzymes BamHI and XhoI. The BamHI-XhoI DNA fragment containing the
entire coding sequence for each OCIF mutant was isolated and
dissolved in 20 .mu.L of sterile distilled water. These DNA
solutions that contain the BamHI-XhoI fragment derived from
pSK-OCIF-DCR1, pSK-OCIF-DCR2, pSK-OCIF-DCR3, pSK-OCIF-DCR4,
pSK-OCIF-DDD1 and pSK-OCIF-DDD2 were designated DCR1 DNA solution,
DCR2 DNA solution, DCR3 DNA solution, DCR4 DNA solution, DDD1 DNA
solution and DDD2 DNA solution, respectively. One microliter of
pCEP 4 DNA solution and 6 .mu.L of either DCR1 DNA solution, DCR2
DNA solution, DCR3 DNA solution, DCR4 DNA solution, DDD1 DNA
solution or DDD2 DNA solution were independently mixed with 7 .mu.L
of ligation buffer I from a DNA ligation kit ver. 2 and the
ligation reactions were carried out. Competent E. coli strain DH5
.alpha. cells (100 .mu.L) were transformed with 7 .mu.L of each
ligation mixture. Ampicillin-resistant transformants were screened
for a clone containing plasmid DNA in which the DNA fragment with
deletions is inserted between the recognition sites of BamHI and
XhoI of pCEP 4 by analyzing the DNA structure. The plasmids
containing the cDNA encoding OCIF-DCR1, OCIF-DCR2, OCIF-DCR3,
OCIF-DCR4, OCIF-DDD1 and OCIF-DDD2 (SEQ ID NOs: 88, 89, 90, 91, 92,
and 93, respectively) were designated pCEP4-OCIF-DCR1,
pCEP4-OCIF-DCR2, pCEP4-OCIF-DCR3, pCEP4-OCIF-DCR4, pCEP4-OCIF-DDD1
and pCEP4-OCIF-DDD2, respectively.
[0224] iv) Preparation of OCIF with C-Terminal Domain
Truncation
[0225] (1) mutagenesis of OCIF cDNA
[0226] A series of OCIF mutants with deletions from Cys at amino
acid residue 379 to Leu 380, from Ser 331 to Leu 380, from Asp 252
to Leu 380, from Asp 177 to Leu 380, from Arg 123 to Leu 380 and
from Cys 86 to Leu 380 was prepared. Positions of the amino acid
residues are shown in SEQ ID NO: 4. These mutants were designated
as OCIF-CL, OCIF-CC, OCIF-CDD2, OCIF-CDD1, OCIF-CCR4 and OCIF-CCR3,
respectively, and assigned SEQ ID NOs:73, 74, 75, 76, 77, and 78,
respectively.
[0227] Mutagenesis for OCIF-CL was performed by the two-step PCR as
described in EXAMPLE 22-ii). The primer set for the reaction is
shown in Table 12. The nucleotide sequences of the primers are
shown in SEQ ID NOs: 23, 40, 55, and 66. The final PCR products
were precipitated with ethanol, dried under vacuum and dissolved in
40 .mu.L of sterile distilled water. This DNA solution was
designated solution L.
[0228] The DNA fragment contained in solution L (20 .mu.L) was
digested with restriction enzymes BstPI and EcoRV. A DNA fragment
with an approximate size of 100 bp was extracted from a preparative
agarose gel using a QIAEX.TM. gel extraction kit and dissolved in
20 .mu.L of sterile distilled water. This DNA solution was
designated DNA solution 19. Two microliters of DNA solution 19, 3
.mu.L of DNA solution 10 (described in EXAMPLE 22-ii)) and 5 .mu.L
of ligation buffer I from a DNA ligation kit ver. 2 were mixed and
ligation reaction was carried out. Competent E. coli strain DH5
.alpha. cells were transformed with 5 .mu.L of the ligation
mixture. Ampicillin-resistant transformants were screened for a
clone containing plasmid DNA. DNA structure was analyzed by
restriction enzyme mapping and by DNA sequencing. The plasmid thus
obtained was named pSK-OCIF-CL. Mutagenesis of OCIF cDNA to prepare
OCIF-CC, OCIF-CDD2, OCIF-CDD1, OCIF-CCR4 and OCIF-CCR3 was
performed by a one-step PCR reaction.
[0229] PCR reactions for mutagenesis to prepare OCIF-CC, OCIF-CDD2,
OCIF-CDD1, OCIF-CCR4 and OCIF-CCR3 were as follows:
15 10.times. Ex Taq Buffer (Takara Shuzo) 10 .mu.L 2.5 mM solution
of dNTPs 8 .mu.L the plasmid vector containing the entire OCIF cDNA
2 .mu.L described in EXAMPLE 11 (8 ng/ml) sterile distilled water
73.5 .mu.L 20 .mu.M solution of primer OCIF Xho F 5 .mu.L 100 .mu.M
solution of primer (for mutagenesis) 1 .mu.L Ex Taq (Takara Shuzo)
0.5 .mu.L
[0230]
16TABLE 12 mutants primer-1 primer-2 primer-3 primer-4 OCIF-CL IF 6
CL R IF 14 CL F
[0231] Specific primers were used for each mutagenesis and other
components were unchanged.
[0232] Primers used for the mutagenesis are shown in Table 13.
Their nucleotide sequences are shown in SEQ ID NOs: 57-61. The
components of each PCR were mixed in a microcentrifuge tube and PCR
was performed as follows. The microcentrifuge tubes were treated
for 3 minutes at 97.degree. C. and then incubated sequentially, for
30 seconds at 95.degree. C., 30 seconds at 50.degree. C. and 3
minutes at 70.degree. C. This three-step incubation procedure was
repeated 25 times, and after that, the tubes were incubated for 5
minutes at 70.degree. C. An aliquot of the reaction mixture was
removed from each tube and analyzed by agarose gel electrophoresis
to confirm the size of each product.
[0233] Excess primers in the PCRs were removed using an Amicon
MICROCON.TM. (Amicon) after completion of the reaction. The DNA
fragments were precipitated with ethanol, dried under vacuum and
dissolved in 40 .mu.L of sterile distilled water. The DNA fragment
in each DNA solution was digested with restriction enzymes XhoI and
BamHI. After the reactions, DNA was precipitated with ethanol,
dried under vacuum and dissolved in 20 .mu.L of sterile distilled
water.
[0234] The solutions containing the DNA fragment with the CC
deletion, the CDD2 deletion, the CDD1 deletion, the CCR4 deletion
and the CCR3 deletion were designated CC DNA solution, CDD2 DNA
solution, CDD1 DNA solution, CCR4 DNA solution and CCR3 DNA
solution, respectively.
17 TABLE 13 mutants primers for the mutagenesis OCIF-CC CC R
OCIF-CDD2 CDD2 R OCIF-CDD1 CDD1 R OCIF-CCR4 CCR4 R OCIF-CCR3 CCR3
R
[0235] (2) Construction of Vectors for Expressing the OCIF
Mutants
[0236] pSK-OCIF-CL was digested with restriction enzymes BamHI and
XhoI. The BamHI-XhoI DNA fragment containing the entire coding
sequence for OCIF-CL was isolated and dissolved in 20 .mu.L of
sterile distilled water. This DNA solution was designated CL DNA
solution. One microliter of pCEP 4 DNA solution and 6 .mu.L of
either CL DNA solution, CC DNA solution, CDD2 DNA solution, CDD1
DNA solution, CCR4 DNA solution or CCR3 DNA solution were
independently mixed with 7 .mu.L of ligation buffer I from a DNA
ligation kit ver. 2 and the ligation reactions were carried out.
Competent E. coli strain DH5 .alpha. cells (100 .mu.L) were
transformed with 7 .mu.L of each ligation mixture.
Ampicillin-resistant transformants were screened for clones
containing plasmids which have the desirable mutations in the OCIF
cDNA by analyzing the DNA structure. In each plasmid, the OCIF cDNA
fragment having a deletion was inserted between the recognition
sites of XhoI and BamHI of pCEP 4. The plasmids containing the cDNA
encoding OCIF-CL, OCIF-CC, OCIF-CDD 1, OCIF-CDD2, OCIF-CCR4 and
OCIF-CCR3 (SEQ ID NOs: 94, 95, 96, 97, 98, and 99, respectively)
were designated pCEP4-OCIF-CL, pCEP4-OCIF-CC, pCEP4-OCIF-CDD2,
pCEP4-OCIF-CDD1, pCEP4-OCIF-CCR4 and pCEP4-OCIF-CCR3,
respectively.
[0237] v) Preparation of OCIF Mutants with C-Terminal
Truncations
[0238] (1) Introduction of C-Terminal Truncations to OCIF
[0239] A series of OCIF mutants with C-terminal truncations was
prepared. An OCIF mutant in which 10 residues from Gln at 371 to
Leu at 380 were replaced with 2 residues (Leu-Val) was designated
OCIF-CBst (SEQ ID NO: 79). An OCIF mutant in which 83 residues from
Cys 298 to Leu 380 were replaced with 3 residues (Ser-Leu-Asp) was
designated OCIF-CSph (SEQ ID NO: 80). An OCIF mutant in which 214
residues from Asn 167 to Leu 380 were removed was designated
OCIF-CBsp (SEQ ID NO: 81). An OCIF mutant in which 319 residues
from Asp 62 to Leu 380 were replaced with 2 residues (Leu-Val) was
designated OCIF-CPst (SEQ ID NO:82). Positions of the amino acid
residues are shown in SEQ ID NO: 4.
[0240] Two micrograms each of pSK'-OCIF were digested with BstPI,
SphI, PstI (Takara Shuzo) or BspEI (New England Biolabs) followed
by phenol extraction and ethanol precipitation. The precipitated
DNA was dissolved in 10 .mu.L of sterile distilled water. The ends
of the DNAs in 2 .mu.L of each solution were blunted using a DNA
blunting kit in a final volume of 5 .mu.L. To the reaction
mixtures, 1 .mu.g (1 .mu.L) of an Amber codon-containing XbaI
linker (5'-CTAGTCTAGACTAG-3') and 6 .mu.L of ligation buffer I from
a DNA ligation kit ver. 2 were added.
[0241] After the ligation reactions, 6 .mu.L each of the reaction
mixtures was used to transform E. Coli strain DH5 .alpha..
Ampicillin-resistant transformants were screened for clones
containing plasmids. DNA structure was analyzed by restriction
enzyme mapping and by DNA sequencing. The plasmids thus obtained
were named pSK-OCIF-CBst, pSK-OCIF-CSph, pSK-OCIF-CBsp and
pSK-OCIF-CPst, respectively.
[0242] (2) Construction of Vectors Expressing the OCIF Mutants
[0243] pSK-OCIF-CBst, pSK-OCIF-CSph, pSK-OCIF-CBsp and
pSK-OCIF-CPst were digested with restriction enzymes BamHI and
XhoI. The 1.5 kb DNA fragment containing the entire coding sequence
for each OCIF mutant was isolated and dissolved in 20 .mu.L of
sterile distilled water. These DNA solutions that contained the
BamHI-XhoI fragment derived from pSK-OCIF-CBst, pSK-OCIF-CSph,
pSK-OCIF-CBsp or pSK-OCIF-CPst were designated CBst DNA solution,
CSph DNA solution, CBsp DNA solution and CPst DNA solution,
respectively. One microliter of pCEP 4 DNA solution (described in
EXAMPLE 22-ii)) and 6 .mu.L of either CBst DNA solution, CSph DNA
solution, CBsp DNA solution or CPst DNA solution were independently
mixed with 7 .mu.L of ligation buffer I from a DNA ligation kit
ver. 2 and the ligation reactions were carried out. Competent E.
coli strain DH5 .alpha. cells (100 .mu.L) were transformed with 7
.mu.L of each ligation mixture. Ampicillin-resistant transformants
were screened for clones containing plasmids in which the cDNA
fragment was inserted between the recognition sites of BamHI and
XhoI of pCEP 4 by analyzing the DNA structure. The plasmids
containing the cDNA encoding OCIF-CBst, OCIF-CSph, OCIF-CBsp or
OCIF-CPst (SEQ ID NOs: 100, 101, 102, and 103, respectively) were
designated pCEP4-OCIF-CBst, pCEP4-OCIF-CSph, pCEP4-OCIF-CBsp and
pCEP4-OCIF-CPst, respectively.
[0244] vi) Preparation of Vectors for Expressing the OCIF
Mutants
[0245] E. coli clones harboring the expression vectors for OCIF
mutants (a total of 21 clones) were grown and the vectors were
purified by using QIAGEN.TM. columns (QIAGEN). All the expression
vectors were precipitated with ethanol and dissolved in appropriate
volumes of sterile distilled water and used for further
manipulations shown below.
[0246] vii) Transient Expression of the cDNAs for OCIF Mutants and
Biological Activities of the Mutants
[0247] OCIF mutants were produced using the expression vectors
prepared in EXAMPLE 22-vi). The method was essentially the same as
described in EXAMPLE 13. Only the modified points are described
below. 2.times.10.sup.5 cells of 293/EBNA suspended in IMDM
containing 10% fetal bovine serum were seeded into each well of a
24 well plate. One microgram of purified vector DNA and 4 .mu.L of
lipofectamine were used for each transfection. A mixture of the
expression vector and lipofectamine in OPTI-MEM.TM. (GIBCO BRL) in
a final volume of 0.5 ml was added to the cells in a well. After
the cells were incubated at 37.degree. C. for 24 hr in 5% CO.sub.2,
the medium was replaced with 0.5 ml of EXCELL.TM. 301 medium (JSR).
The cells were incubated at 37.degree. C. for a further 48 hours in
5% CO.sub.2. The conditioned medium was collected and used in
assays for in vitro biological activity. The nucleotide sequences
of cDNAs for the OCIF mutants are shown in SEQ ID NOs: 83-103. The
deduced amino acid sequences for the OCIF mutants are shown in SEQ
ID NOs: 62-82. The assay for in vitro biological activity was
performed as described in EXAMPLE 13. The antigen concentration of
each conditioned medium was determined by ELISA as described in
EXAMPLE 24. Table 14 shows the activity of each mutant relative to
that of the unaltered OCIF.
18 TABLE 14 mutants activity the unaltered OCIF ++ OCIF-C19S +
OCIF-C20S .+-. OCIF-C21S .+-. OCIF-C22S + OCIF-C23S ++ OCIF-DCR1
.+-. OCIF-DCR2 .+-. OCIF-DCR3 .+-. OCIF-DCR4 .+-. OCIF-DDD1 +
OCIF-DDD2 .+-. OCIF-CL ++ OCIF-CC ++ OCIF-CDD2 ++ OCIF-CDD1 +
OCIF-CCR4 .+-. OCIF-CCR3 .+-. OCIF-CBst ++ OCIF-CSph ++ OCIF-CBsp
.+-. OCIF-CPst .+-. ++ indicates relative activity more than 50% of
that of the unaltered OCIF; + indicates relative activity between
10% and 50%; .+-. indicates relative activity less than 10%, or
production level too low to determine the accurate biological
activity.
[0248] viii) Western Blot Analysis
[0249] Ten microliters of the final conditioned medium was used for
western blot analysis. Ten microliters of each sample were mixed
with 10 .mu.L of SDS-PAGE sample buffer (0.5 M Tris-HCl, 20%
glycerol, 4% SDS, 20 .mu.g/ml bromophenol blue, pH 6.8), boiled for
3 min. and subjected to 10% SDS polyacrylamide gel electrophoresis
under non-reducing conditions. After the electrophoresis, the
separated proteins were blotted to PVDF membrane (PROBLOTT.TM.,
Perkin Elmer) using a semi-dry electroblotter (BIO-RAD). The
membrane was incubated at 37.degree. C. with horseradish
peroxidase-labeled anti-OCIF antibodies for 2 hr. After the
membrane was washed, protein bands which react with the labeled
antibodies were detected using an ECL.TM. system (Amersham). Two
protein bands with approximate molecular weights of 60 kD and 120
kD were detected for the unaltered OCIF. On the other hand, almost
exclusively a 60 kD protein band was detected for the OCIF-C23S,
OCIF-CL and OCIF-CC mutants. Protein bands with approximate weights
of 40 kD-50 kD and 30 kD-40 kD were the major ones for OCIF-CDD2
and OCIF-CDD1, respectively. These results indicate that Cys at 379
is responsible for the dimer formation, both the monomers and the
dimers maintain the biological activity and a deletion of residues
from Asp at 177 to Leu at 380 does not abolish the biological
activity of OCIF (positions of the amino acid residues are shown in
SEQ ID NO: 4).
EXAMPLE 23
[0250] Isolation of Human Genomic OCIF Gene
[0251] i) Screening of a Human Genomic Library
[0252] An amplified human placenta genomic library in LAMBDA
FIX.TM. II vector (Stratagene) was screened for the gene encoding
human OCIF using the human OCIF cDNA as a probe. Essentially,
screening was done according to the instruction manual supplied
with the genomic library. The basic protocols described in
Molecular Cloning: A Laboratory Manual were also employed to
manipulate phage, E. coli, and DNA.
[0253] The library was titered, and 1.times.10.sup.6 pfu of phage
was mixed with XL1-Blue MRA host E. coli cells and plated onto 20
plates (9 cm.times.13 cm) with 9 ml per plate of top agarose. The
plates were incubated overnight at 37.degree. C. Filter plaque
lifts were prepared using HYBOND.TM.-N nylon membranes (Amersham).
The membranes were processed by denaturation in a solution
containing 1.5 M NaCl and 0.5 M NaOH for 1 minute at room
temperature. The membranes were then neutralized by placing each
one in 1 M Tris-HCl (pH 7.5) and a solution containing 1.5 M NaCl
and 0.5 M Tris-HCl (pH 7.5) successively for one minute each. The
membranes were then transferred onto a filter paper wetted with
2.times.SSC. Phage DNA was fixed onto the membranes with 1200
microjoules of UV energy using a STRATALINKER UV CROSSLINKER.TM.
2400 (STRATAGENE) and the membranes were air dried. The membranes
were immersed in Rapid Hybridization buffer (Amersham) and
incubated for one hour at 65.degree. C. before hybridization with
.sup.32P-labeled cDNA probe in the same buffer overnight at
65.degree. C. Screening probe was prepared by labeling the OCIF
cDNA with .sup.32P using the Megaprime DNA labeling system
(Amersham). Approximately, 5.times.10.sup.5 cpm probe was used for
each ml of hybridization buffer. After the hybridization, the
membranes were rinsed in 2.times.SSC for five minutes at room
temperature. The membranes were then washed four times, 20 minutes
each time, in 0.5.times.SSC containing 0.1% SDS at 65.degree. C.
After the final wash, the membranes were dried and subjected to
autoradiography at -80.degree. C. with SUPER HR-H.TM. X-ray film
(FUJI PHOTO FILM Co., Ltd.) and an intensifying screen.
[0254] Upon examination of the autoradiograms, six positive signals
were detected. Agar plugs were picked from the regions
corresponding to these signals for phage purification. Each agar
plug was soaked overnight in 0.5 ml of SM buffer containing 1%
chloroform to extract phage. Each extract containing phage was
diluted 1000 fold with SM buffer and an aliquot of 1 .mu.L or 20
.mu.L was mixed with host E. coli described above. The mixture was
plated onto agar plates with top agarose as described above. The
plates were incubated overnight at 37.degree. C., and filter lifts
were prepared, prehybridized, hybridized, washed and
autoradiographed as described above. This process of phage
purification was applied to all six positive signals initially
detected on the autoradiograms and was repeated until all phage
plaques on agar plates hybridize with the cDNA probe. After
purification, agar plugs of each phage isolate were soaked in SM
buffer containing 1% chloroform and stored at 4.degree. C. Six
individual phage isolates were designated .lambda.0IF3,
.lambda.0IF8, .lambda.0IF9, .lambda.0IF11, .lambda.0IF12 and
.lambda.0IFI7, respectively.
[0255] ii) Analysis of the Genomic Clones by Restriction Enzyme
Digestion and Southern Blot Hybridization
[0256] DNA was prepared from each phage isolate by the plate lysate
method as described in Molecular Cloning: A Laboratory Manual. DNA
prepared from each phage was digested with restriction enzymes and
the fragments derived from the digestion were separated on agarose
gels. The fragments were then transferred to nylon membranes and
subjected to Southern blot hybridization using OCIF cDNA as a
probe. The results of the analysis revealed that the six phage
isolates are individual clones. Among these fragments derived from
restriction enzyme digestion, those fragments which hybridized with
the OCIF cDNA probe were subcloned into plasmid vectors and
subjected to nucleotide sequence analysis as described below.
[0257] iii) Subcloning Restriction Fragments Derived from Genomic
Clones into Plasmid Vectors and Determining their Nucleotide
Sequence.
[0258] .lambda.0IF8 DNA was digested with restriction enzymes EcoRI
and NotI and the DNA fragments derived therefrom were separated on
a 0.7% agarose gel. The 5.8 kilobase pair (kb) EcoRI/NotI fragment
was extracted from the gel using a QIAEX.TM. II Gel Extraction Kit
(QIAGEN) according to the procedure recommended by the
manufacturer. The 5.8 kb EcoRI/NotI fragment was ligated with
pBLUESCRIPT II SK+.TM. vector (STRATAGENE), which had been
linearized with restriction enzymes EcoRI and NotI, using
READY-TO-GO.TM. T4 DNA Ligase (Pharmacia) according to the
procedure recommended by the manufacturer. Competent DH5 .alpha.E.
coli cells (Amersham) were transformed with the recombinant plasmid
and transformants were selected on L-plates containing 50 .mu.g/ml
of ampicillin.
[0259] A clone harboring the recombinant plasmid containing the 5.8
kb EcoRI/NotI fragment was isolated and this plasmid was termed
pBSG8-5.8. pBSG8-5.8 was digested with HindIII and a 0.9 kb DNA
fragment derived from this digestion was isolated in the same
manner as described above. This 0.9 kb fragment was then cloned
into pBLUESCRIPT II SK-.TM. at the HindIlI site as described above.
This recombinant plasmid containing 0.9 kb HindIII fragment was
denoted PBS8H0.9.
[0260] .lambda.0F11 DNA was digested with EcoRI and 6 kb, 3.6 kb,
2.6 kb EcoRI fragments were isolated in the same manner as
described above and cloned into a pBLUESCRIPT II SK+.TM. vector at
the EcoRI site as described above. These recombinant plasmids were
termed pBSG11-6, pBSG11-3.6, and pBSG11-2.6, respectively. pBSG11-6
was digested with HindIII and the digest was separated on a 0.7%
agarose gel. Three fragments, 2.2 kb, 1.1 kb, and 1.05 kb in
length, were extracted from the gel and cloned independently into
pBLUESCRIPT II SK-.TM. vector at the HindIII site in the same
manner as described above. These recombinant plasmids were termed
PBS6H2.2, PBS6H1.1 and PBS6H1.05, respectively.
[0261] The nucleotide sequence of the cloned genomic DNA was
determined using a ABI DYEDEOXY TERMINATOR CYCLE SEQUENCING READY
REACTION.TM. Kit (PERKIN ELMER) and a 373A DNA Sequencing system
(Applied Biosystems). Plasmids pBSG8-5.8, pBS8H0.9, pBSG11-6,
pBSG11-3.6, pBSG11-2.6, pBS6H2.2, pBS6H1.1 and pBS6H1.05 were
prepared according to the alkaline-SDS procedure as described in
Molecular Cloning: A Laboratory Manual and used as templates for
DNA sequence analysis. The nucleotide sequence of the human OCIF
gene is presented in SEQ ID NO: 104 and SEQ ID NO: 105. The
nucleotide sequence of the DNA, between exon 1 and exon 2, was not
entirely determined. There is a stretch of approximately 17 kb
between the sequences given in SEQ ID NO: 104 and SEQ ID NO:
105.
EXAMPLE 24
[0262] Quantitation of OCIF by EIA
[0263] i) Preparation of anti-OCIF antibody
[0264] Male Japanese white rabbits (Kitayama Labs Co., LTD)
weighing 2.5-3.0 kg were used in immunization for preparing
antisera. For immunization, an emulsion was prepared by mixing an
equal volume of rOCIF (200 .mu.g/ml) and complete Freund's adjuvant
(Difco, Cat. 0638-60-7). Three rabbits were immunized
subcutaneously six times at one week intervals with 1 ml of
emulsion per injection. Whole blood was obtained ten days after the
final immunization and serum was isolated. Antibody was purified
from serum as follows. Antiserum was diluted two-fold with PBS.
After adding ammonium sulfate at a final concentration of 40% w/v,
the antiserum was allowed to stand at 4.degree. C. for 1 hr. The
precipitate obtained by centrifugation at 8000.times.g for 20 min.
was dissolved in a small volume of PBS and was dialyzed against
PBS. The resultant solution was loaded onto a Protein
G-SEPHAROSE.TM. column (Pharmacia). After washing with PBS,
absorbed immunoglobulin G was eluted with 0.1 M glycine-HCl buffer
(pH 3.0). The eluate was immediately neutralized with 1.5 M
Tris-HCl buffer (pH 8.7) and dialyzed against PBS. Protein
concentration was determined by absorbance at 280 nm (E.sup.1%
13.5).
[0265] Horseradish peroxidase-labeled antibody was prepared using
an IMMUNOPURE.TM. Maleimide Activated Horseradish Peroxidase Kit
(Pierce, Cat. 31494). Briefly, one mg of IgG was incubated with 80
.mu.g of N-succinimidyl-S-acetylthioacetate for 30 min. After
deacetylation with 5 mg of hydroxylamine HCl, modified IgG was
separated using a polyacrylamide desalting column. The protein pool
was mixed with one mg of maleimide-activated horseradish peroxidase
and incubated at room temperature for 1 hr.
[0266] ii) Quantitation of OCIF by Sandwich EIA
[0267] Microtiter plates (Nunc MaxiSorp Immunoplate) were coated
with rabbit anti-OCIF IgG by incubating 0.2 .mu.g in 100 .mu.L of
50 mM sodium bicarbonate buffer pH 9.6 at 4.degree. C. overnight.
After blocking the plates by incubating for 1 hour at 37.degree. C.
with 300 .mu.L of 25% BLOCKACE.TM./PBS (Snow Brand Milk Products),
100 .mu.L samples were incubated for 2 hours at room temperature.
After washing the plates three times with PBST (PBS containing
0.05% TWEEN.TM. 20), 100 .mu.L of 1:10000 diluted horseradish
peroxidase-labeled anti-OCIF IgG was added and incubated for 2
hours at room temperature. The amount of OCIF was determined by
incubation with 100 .mu.L of a substrate solution (TMB, ScyTek
Lab., Cat. TM4999) and measurement of the absorbance at 450 nm
using an IMMUNOREADER.TM. (Nunc NJ2000). Purified recombinant OCIF
was used as a standard protein and a typical standard curve is
shown in FIG. 13.
EXAMPLE 25
[0268] Anti-OCIF Monoclonal Antibody
[0269] i) Preparation of a Hybridoma Producing Anti-OCIF Monoclonal
Antibody.
[0270] OCIF was purified to homogeneity from the culture medium of
human fibroblasts, IMR-90 cells by the purification method
described in EXAMPLE 11. Purified OCIF was dissolved in PBS at a
concentration of 10 .mu.g/100 .mu.L. BALB/c mice were immunized by
administering this solution intraperitoneally three times every two
weeks. In the first and the second immunizations, the emulsion was
composed of an equal volume of OCIF and Freund's complete adjuvant.
Three days after the final immunization, the spleen was removed and
lymphocytes isolated and fused with mouse myeloma
p3.times.63-Ag8.653 cells according to conventional methods using
polyethyleneglycol. Then the fused cells were cultured in HAT
medium to select hybridomas. The presence of anti-OCIF antibody in
the culture medium of each hybridoma was determined by solid phase
ELISA. Briefly, each well of a 96-well immunoplate (Nunc) was
coated with 100 .mu.L of purified OCIF (10 .mu.g/ml in 0.1 M
NaHCO.sub.3) and blocked with 50% BLOCKACE.TM. (Snow Brand Milk
Products Co. Ltd.). The hybridoma clones secreting anti-OCIF
antibody were established by limit dilution cloning 3-5 times and
by solid phase ELISA screening. Several hybridoma clones producing
high levels of anti-OCIF antibody were selected.
[0271] ii) Production of Anti-OCIF Monoclonal Antibodies.
[0272] Each hybridoma clone secreting anti-OCIF antibody obtained
in EXAMPLE 25-i) was transplanted intraperitoneally into mice given
Pristane (Aldrich) at a cell density of 1.times.10.sup.6
cells/mouse. The accumulated ascites was collected 10-14 days after
transplantation, thereby obtaining anti-OCIF specific monoclonal
antibody of the present invention. Purified antibodies were
obtained by Affigel protein A SEPHAROSE.TM. chromatography (BioRad)
according to the manufacturer's manual. Briefly, the ascites fluid
was diluted with an equal volume of a binding buffer (BioRad) and
applied to a protein A column. The column was washed with a
sufficient volume of binding buffer and eluted with an elution
buffer (BioRad). After neutralizing, the eluate obtained was
dialyzed in water and subsequently lyophilized. The purity of the
antibody thereby obtained was analyzed by SDS/PAGE and a homogenous
band with a molecular weight of about 150,000 was detected.
[0273] iii) Selection of Monoclonal Antibodies having High Affinity
for OCIF
[0274] Each antibody obtained in EXAMPLE 25-ii) was dissolved in
PBS and the protein concentration was determined by the method of
Lowry. Each antibody solution was diluted to the same concentration
and then serially diluted with PBS. Monoclonal antibodies, which
can recognize OCIF even at highly dilute concentrations, were
selected by solid phase ELISA described in EXAMPLE 25-ii). Thus,
three monoclonal antibodies A1G5, E3H8 and D2F4 were selected.
[0275] iv) Determination of Class and Subclass of Antibodies
[0276] The class and subclass of the antibodies of the present
invention obtained in EXAMPLE 25-iii) were analyzed using an
immunoglobulin class and subclass analysis kit (Amersham). The
procedure was carried out according to the kit directions. The
results are shown in Table 15. The antibodies of the present
invention, E3H8, A1G5 and D2F4 belong to the IgG.sub.1, IgG.sub.2a
and IgG.sub.2b subclasses, respectively.
19TABLE 15 Analysis of class and subclass of the antibodies of the
present invention. Antibody IgG.sub.1 IgG.sub.2a IgG.sub.2b
IgG.sub.3 IgA IgM .kappa. A1G5 - + - - - - + E3H8 + - - - - - +
D2F4 - - + - - - +
[0277] v) Quantitation of OCIF by ELISA
[0278] Three kinds of monoclonal antibodies, A1G5, E3H8 and D2F4
obtained in EXAMPLE 25-iv), were used as solid phase antibodies and
enzyme-labeled antibodies, respectively. Sandwich ELISA was
constructed by different combinations of solid phase antibody and
labeled antibody. The labeled antibody was prepared using an
IMMUNOPURE.TM. Maleimide-Activated Horseradish Peroxidase Kit
(Pierce, Cat. No. 31494). Each monoclonal antibody was dissolved in
0.1 M NaHCO.sub.3 at a concentration of 10 .mu.g/ml, and 100 .mu.L
of the solution was added to each well of a 96-well immunoplate
(Nunc, MaxiSorp Cat. No. 442404) followed by allowing them to stand
at room temperature overnight. Subsequently, each well of the plate
was blocked with 50% BLOCKACE.TM. (Snow Brand Milk Products, Co.,
Ltd.) at room temperature for 50 minutes, and washed three times
with PBS containing 0.1% TWEEN.TM. 20 (washing buffer).
[0279] A series of concentrations of OCIF was prepared by diluting
OCIF with 1st reaction buffer (0.2 M Tris-HCl buffer, pH 7.4,
containing 40% BLOCKACE.TM. and 0.1% TWEEN.TM. 20). Each well of a
96-well immunoplate was filled with 100 .mu.L of the prepared OCIF
solution with each concentration, allowed to stand at 37.degree. C.
for 3 hours, and subsequently washed three times with washing
buffer. The POD-labeled antibody was diluted 400-fold with 2nd
reaction buffer (0.1 M Tris-HCl buffer, pH 7.4, containing 25%
BLOCKACE.TM. and 0.1% TWEEN.TM. 20), and 100 .mu.L of the diluted
solution was added to each well of the immunoplates. Each
immunoplate was allowed to stand at 37.degree. C. for 2 hours, and
subsequently washed three times with washing buffer. After washing,
100 .mu.L of a substrate solution (0.1 M citrate-phosphate buffer,
pH 4.5, containing 0.4 mg/ml of o-phenylenediamine HCl and 0.006%
H.sub.2O.sub.2) was added to each well of the immunoplates and the
immunoplates incubated at 37.degree. C. for 15 min. The enzyme
reaction was terminated by adding 50 .mu.L of 6 N H.sub.2SO.sub.4
to each well. The optical density of each well was determined at
492 nm using an immunoreader (IMMUNOREADER.TM. NJ 2000, Nunc).
[0280] Using three different monoclonal antibodies of the present
invention, each combination of solid phase and POD-labeled
antibodies leads to an accurate determination of OCIF
concentration. Each monoclonal antibody of the present invention
was confirmed to recognize a different epitope of OCIF. A typical
standard curve of OCIF using a combination of solid phase antibody,
A1G5, and POD-labeled antibody, E3H8, is shown in FIG. 14.
[0281] vi) Determination of OCIF in Human Serum
[0282] The concentration of OCIF in five samples of normal human
serum was determined using an EIA system described in EXAMPLE
25-v). The immunoplates were coated with A1G5 as described in
EXAMPLE 25-v), and 50 .mu.L of the 1.sup.st reaction buffer was
added to each well of the immunoplates. Subsequently, 50 .mu.L of
each human serum was added to each well of the immunoplates. The
immunoplates were incubated at 37.degree. C. for 3 hours and washed
three times with washing buffer. After washing, each well of the
immunoplates was filled with 100 .mu.L of POD-E3H8 antibody diluted
400-fold with the 2.sup.nd reaction buffer and incubated at
37.degree. C. for 2 hours. After washing the immunoplates three
times with washing buffer, 100 .mu.L of the substrate solution
described in EXAMPLE 25-v) was added to each well and incubated at
37.degree. C. for 15 min. The enzyme reaction was terminated by
adding 50 .mu.L of 6 N H.sub.2SO.sub.4 to each well of the
immunoplates. The optical density of each well was determined at
492 nm using an immunoreader (IMMUNOREADER.TM. NJ 2000, Nunc).
[0283] 1.sup.st reaction buffer containing the known amount of OCIF
was treated in the same way and a standard curve of OCIF as shown
in FIG. 2 was obtained. Using the standard curve of OCIF, the
amount of OCIF in human serum sample was determined. The results
were shown in Table 16.
20TABLE 16 The amount of OCIF in normal human serum Serum Sample
OCIF Concentration (ng/ml) 1 5.0 2 2.0 3 1.0 4 3.0 5 1.5
EXAMPLE 26
[0284] Therapeutic Effect on Osteoporosis
[0285] (1) Method
[0286] Six week old male Fischer rats were subjected to denervation
of the left forelimb. These rats were assigned to four groups (10
rats/group) and treated as follows: group A, sham operated rats
without administration; group B, denerved rats with the vehicle
administered intravenously; group C, denerved rats with OCIF
administered intravenously at a dose of 5 .mu.g/kg twice a day;
group D, denerved rats with OCIF administered intravenously at a
dose of 50 .mu.g/kg twice a day. After denervation, OCIF was
administered daily for 14 days. After 2 weeks treatment, the
animals were sacrificed and their forelimbs were dissected.
Thereafter bones were tested for mechanical strength.
[0287] (2) Results
[0288] A decrease in bone strength was observed in control animals
as compared to those animals of the normal groups while bone
strength was increased in the group of animals that received 50 mg
of OCIF per kg body weight.
[0289] Samples of the hybridomas that produce the claimed
monoclonal antibodies were deposited in the National Institute of
Bioscience and Human Technology National Institute of Advanced
Industrial Science and Technology. The National Institute of
Bioscience and Human Technology National Institute of Advanced
Industrial Science and Technology accession numbers for the
deposited hybridomas are:
21 Hybridoma Antibody Designation Deposit Date Accession No. A1G5
Feb. 5, 2001 FERM BP-7441 D2F4 Feb. 5, 2001 FERM BP-7442 E3H8 Feb.
5, 2001 FERM BP-7443
[0290] These deposits were made under the Budapest Treaty and will
be maintained and made accessible to others in accordance with the
provisions thereof.
[0291] The hybridomas will be maintained in a public depository for
a term of at least 30 years and at least five years after the most
recent request for the furnishing of a sample of the deposit is
received by the depository. In any case, samples will be stored
under agreements that would make them available beyond the
enforceable life of any patent issuing from the above-referenced
application.
[0292] Industrial Availability
[0293] The present invention provides both a novel protein which
inhibits the formation of osteoclasts and an efficient procedure
for producing the protein. The protein of the present invention
inhibits the formation of osteoclasts. The protein will be useful
for the treatment of many diseases accompanied by bone loss, such
as osteoporosis, and as an antigen to prepare antibodies useful for
the immunological diagnosis of such diseases.
[0294] A national deposit of the microorganism (accession number
Bikkoken No. P-14998) was made on Jun. 21, 1995, and transferred to
the international depository named "National Institute of
Bioscience and Human-Technology (NIBH), Agency of Industrial
Science and Technology, Ministry of International Trade and
Industry" having an address of 1-3, Higashi 1-chome, Tsukuba-shi,
Ibaraki-ken, 305, JAPAN on Oct. 25, 1995 as accession number FERM
BP-5267, pursuant to the Budapest Treaty. The deposit is a Budapest
Treaty deposit and will be maintained and made accessible to others
in accordance with the provisions thereof. The international
depository is currently known as "International Patent Organism
Depositary, National Institute of Advanced Industrial Science and
Technology".
Sequence CWU 1
1
108 1 6 PRT Homo sapiens MISC_FEATURE (1)..(1) X = unknown 1 Xaa
Tyr His Phe Pro Lys 1 5 2 14 PRT Homo sapiens MISC_FEATURE (1)..(1)
X = unknown 2 Xaa Gln His Ser Xaa Gln Glu Gln Thr Phe Gln Leu Xaa
Lys 1 5 10 3 12 PRT Homo sapiens MISC_FEATURE (1)..(1) X = unknown
3 Xaa Ile Arg Phe Leu His Ser Phe Thr Met Tyr Lys 1 5 10 4 380 PRT
Homo sapiens 4 Glu Thr Phe Pro Pro Lys Tyr Leu His Tyr Asp Glu Glu
Thr Ser His 1 5 10 15 Gln Leu Leu Cys Asp Lys Cys Pro Pro Gly Thr
Tyr Leu Lys Gln His 20 25 30 Cys Thr Ala Lys Trp Lys Thr Val Cys
Ala Pro Cys Pro Asp His Tyr 35 40 45 Tyr Thr Asp Ser Trp His Thr
Ser Asp Glu Cys Leu Tyr Cys Ser Pro 50 55 60 Val Cys Lys Glu Leu
Gln Tyr Val Lys Gln Glu Cys Asn Arg Thr His 65 70 75 80 Asn Arg Val
Cys Glu Cys Lys Glu Gly Arg Tyr Leu Glu Ile Glu Phe 85 90 95 Cys
Leu Lys His Arg Ser Cys Pro Pro Gly Phe Gly Val Val Gln Ala 100 105
110 Gly Thr Pro Glu Arg Asn Thr Val Cys Lys Arg Cys Pro Asp Gly Phe
115 120 125 Phe Ser Asn Glu Thr Ser Ser Lys Ala Pro Cys Arg Lys His
Thr Asn 130 135 140 Cys Ser Val Phe Gly Leu Leu Leu Thr Gln Lys Gly
Asn Ala Thr His 145 150 155 160 Asp Asn Ile Cys Ser Gly Asn Ser Glu
Ser Thr Gln Lys Cys Gly Ile 165 170 175 Asp Val Thr Leu Cys Glu Glu
Ala Phe Phe Arg Phe Ala Val Pro Thr 180 185 190 Lys Phe Thr Pro Asn
Trp Leu Ser Val Leu Val Asp Asn Leu Pro Gly 195 200 205 Thr Lys Val
Asn Ala Glu Ser Val Glu Arg Ile Lys Arg Gln His Ser 210 215 220 Ser
Gln Glu Gln Thr Phe Gln Leu Leu Lys Leu Trp Lys His Gln Asn 225 230
235 240 Lys Asp Gln Asp Ile Val Lys Lys Ile Ile Gln Asp Ile Asp Leu
Cys 245 250 255 Glu Asn Ser Val Gln Arg His Ile Gly His Ala Asn Leu
Thr Phe Glu 260 265 270 Gln Leu Arg Ser Leu Met Glu Ser Leu Pro Gly
Lys Lys Val Gly Ala 275 280 285 Glu Asp Ile Glu Lys Thr Ile Lys Ala
Cys Lys Pro Ser Asp Gln Ile 290 295 300 Leu Lys Leu Leu Ser Leu Trp
Arg Ile Lys Asn Gly Asp Gln Asp Thr 305 310 315 320 Leu Lys Gly Leu
Met His Ala Leu Lys His Ser Lys Thr Tyr His Phe 325 330 335 Pro Lys
Thr Val Thr Gln Ser Leu Lys Lys Thr Ile Arg Phe Leu His 340 345 350
Ser Phe Thr Met Tyr Lys Leu Tyr Gln Lys Leu Phe Leu Glu Met Ile 355
360 365 Gly Asn Gln Val Gln Ser Val Lys Ile Ser Cys Leu 370 375 380
5 401 PRT Homo sapiens 5 Met Asn Asn Leu Leu Cys Cys Ala Leu Val
Phe Leu Asp Ile Ser Ile 1 5 10 15 Lys Trp Thr Thr Gln Glu Thr Phe
Pro Pro Lys Tyr Leu His Tyr Asp 20 25 30 Glu Glu Thr Ser His Gln
Leu Leu Cys Asp Lys Cys Pro Pro Gly Thr 35 40 45 Tyr Leu Lys Gln
His Cys Thr Ala Lys Trp Lys Thr Val Cys Ala Pro 50 55 60 Cys Pro
Asp His Tyr Tyr Thr Asp Ser Trp His Thr Ser Asp Glu Cys 65 70 75 80
Leu Tyr Cys Ser Pro Val Cys Lys Glu Leu Gln Tyr Val Lys Gln Glu 85
90 95 Cys Asn Arg Thr His Asn Arg Val Cys Glu Cys Lys Glu Gly Arg
Tyr 100 105 110 Leu Glu Ile Glu Phe Cys Leu Lys His Arg Ser Cys Pro
Pro Gly Phe 115 120 125 Gly Val Val Gln Ala Gly Thr Pro Glu Arg Asn
Thr Val Cys Lys Arg 130 135 140 Cys Pro Asp Gly Phe Phe Ser Asn Glu
Thr Ser Ser Lys Ala Pro Cys 145 150 155 160 Arg Lys His Thr Asn Cys
Ser Val Phe Gly Leu Leu Leu Thr Gln Lys 165 170 175 Gly Asn Ala Thr
His Asp Asn Ile Cys Ser Gly Asn Ser Glu Ser Thr 180 185 190 Gln Lys
Cys Gly Ile Asp Val Thr Leu Cys Glu Glu Ala Phe Phe Arg 195 200 205
Phe Ala Val Pro Thr Lys Phe Thr Pro Asn Trp Leu Ser Val Leu Val 210
215 220 Asp Asn Leu Pro Gly Thr Lys Val Asn Ala Glu Ser Val Glu Arg
Ile 225 230 235 240 Lys Arg Gln His Ser Ser Gln Glu Gln Thr Phe Gln
Leu Leu Lys Leu 245 250 255 Trp Lys His Gln Asn Lys Asp Gln Asp Ile
Val Lys Lys Ile Ile Gln 260 265 270 Asp Ile Asp Leu Cys Glu Asn Ser
Val Gln Arg His Ile Gly His Ala 275 280 285 Asn Leu Thr Phe Glu Gln
Leu Arg Ser Leu Met Glu Ser Leu Pro Gly 290 295 300 Lys Lys Val Gly
Ala Glu Asp Ile Glu Lys Thr Ile Lys Ala Cys Lys 305 310 315 320 Pro
Ser Asp Gln Ile Leu Lys Leu Leu Ser Leu Trp Arg Ile Lys Asn 325 330
335 Gly Asp Gln Asp Thr Leu Lys Gly Leu Met His Ala Leu Lys His Ser
340 345 350 Lys Thr Tyr His Phe Pro Lys Thr Val Thr Gln Ser Leu Lys
Lys Thr 355 360 365 Ile Arg Phe Leu His Ser Phe Thr Met Tyr Lys Leu
Tyr Gln Lys Leu 370 375 380 Phe Leu Glu Met Ile Gly Asn Gln Val Gln
Ser Val Lys Ile Ser Cys 385 390 395 400 Leu 6 1206 DNA Homo sapiens
6 atgaacaact tgctgtgctg cgcgctcgtg tttctggaca tctccattaa gtggaccacc
60 caggaaacgt ttcctccaaa gtaccttcat tatgacgaag aaacctctca
tcagctgttg 120 tgtgacaaat gtcctcctgg tacctaccta aaacaacact
gtacagcaaa gtggaagacc 180 gtgtgcgccc cttgccctga ccactactac
acagacagct ggcacaccag tgacgagtgt 240 ctatactgca gccccgtgtg
caaggagctg cagtacgtca agcaggagtg caatcgcacc 300 cacaaccgcg
tgtgcgaatg caaggaaggg cgctaccttg agatagagtt ctgcttgaaa 360
cataggagct gccctcctgg atttggagtg gtgcaagctg gaaccccaga gcgaaataca
420 gtttgcaaaa gatgtccaga tgggttcttc tcaaatgaga cgtcatctaa
agcaccctgt 480 agaaaacaca caaattgcag tgtctttggt ctcctgctaa
ctcagaaagg aaatgcaaca 540 cacgacaaca tatgttccgg aaacagtgaa
tcaactcaaa aatgtggaat agatgttacc 600 ctgtgtgagg aggcattctt
caggtttgct gttcctacaa agtttacgcc taactggctt 660 agtgtcttgg
tagacaattt gcctggcacc aaagtaaacg cagagagtgt agagaggata 720
aaacggcaac acagctcaca agaacagact ttccagctgc tgaagttatg gaaacatcaa
780 aacaaagacc aagatatagt caagaagatc atccaagata ttgacctctg
tgaaaacagc 840 gtgcagcggc acattggaca tgctaacctc accttcgagc
agcttcgtag cttgatggaa 900 agcttaccgg gaaagaaagt gggagcagaa
gacattgaaa aaacaataaa ggcatgcaaa 960 cccagtgacc agatcctgaa
gctgctcagt ttgtggcgaa taaaaaatgg cgaccaagac 1020 accttgaagg
gcctaatgca cgcactaaag cactcaaaga cgtaccactt tcccaaaact 1080
gtcactcaga gtctaaagaa gaccatcagg ttccttcaca gcttcacaat gtacaaattg
1140 tatcagaagt tatttttaga aatgataggt aaccaggtcc aatcagtaaa
aataagctgc 1200 ttataa 1206 7 15 PRT Homo sapiens 7 Glu Thr Phe Pro
Pro Lys Tyr Leu His Tyr Asp Glu Glu Thr Ser 1 5 10 15 8 1185 DNA
Homo sapiens 8 atgaacaact tgctgtgctg cgcgctcgtg tttctggaca
tctccattaa gtggaccacc 60 caggaaacgt ttcctccaaa gtaccttcat
tatgacgaag aaacctctca tcagctgttg 120 tgtgacaaat gtcctcctgg
tacctaccta aaacaacact gtacagcaaa gtggaagacc 180 gtgtgcgccc
cttgccctga ccactactac acagacagct ggcacaccag tgacgagtgt 240
ctatactgca gccccgtgtg caaggagtgc aatcgcaccc acaaccgcgt gtgcgaatgc
300 aaggaagggc gctaccttga gatagagttc tgcttgaaac ataggagctg
ccctcctgga 360 tttggagtgg tgcaagctgg aaccccagag cgaaatacag
tttgcaaaag atgtccagat 420 gggttcttct caaatgagac gtcatctaaa
gcaccctgta gaaaacacac aaattgcagt 480 gtctttggtc tcctgctaac
tcagaaagga aatgcaacac acgacaacat atgttccgga 540 aacagtgaat
caactcaaaa atgtggaata gatgttaccc tgtgtgagga ggcattcttc 600
aggtttgctg ttcctacaaa gtttacgcct aactggctta gtgtcttggt agacaatttg
660 cctggcacca aagtaaacgc agagagtgta gagaggataa aacggcaaca
cagctcacaa 720 gaacagactt tccagctgct gaagttatgg aaacatcaaa
acaaagacca agatatagtc 780 aagaagatca tccaagatat tgacctctgt
gaaaacagcg tgcagcggca cattggacat 840 gctaacctca ccttcgagca
gcttcgtagc ttgatggaaa gcttaccggg aaagaaagtg 900 ggagcagaag
acattgaaaa aacaataaag gcatgcaaac ccagtgacca gatcctgaag 960
ctgctcagtt tgtggcgaat aaaaaatggc gaccaagaca ccttgaaggg cctaatgcac
1020 gcactaaagc actcaaagac gtaccacttt cccaaaactg tcactcagag
tctaaagaag 1080 accatcaggt tccttcacag cttcacaatg tacaaattgt
atcagaagtt atttttagaa 1140 atgataggta accaggtcca atcagtaaaa
ataagctgct tataa 1185 9 394 PRT Homo sapiens 9 Met Asn Asn Leu Leu
Cys Cys Ala Leu Val Phe Leu Asp Ile Ser Ile 1 5 10 15 Lys Trp Thr
Thr Gln Glu Thr Phe Pro Pro Lys Tyr Leu His Tyr Asp 20 25 30 Glu
Glu Thr Ser His Gln Leu Leu Cys Asp Lys Cys Pro Pro Gly Thr 35 40
45 Tyr Leu Lys Gln His Cys Thr Ala Lys Trp Lys Thr Val Cys Ala Pro
50 55 60 Cys Pro Asp His Tyr Tyr Thr Asp Ser Trp His Thr Ser Asp
Glu Cys 65 70 75 80 Leu Tyr Cys Ser Pro Val Cys Lys Glu Cys Asn Arg
Thr His Asn Arg 85 90 95 Val Cys Glu Cys Lys Glu Gly Arg Tyr Leu
Glu Ile Glu Phe Cys Leu 100 105 110 Lys His Arg Ser Cys Pro Pro Gly
Phe Gly Val Val Gln Ala Gly Thr 115 120 125 Pro Glu Arg Asn Thr Val
Cys Lys Arg Cys Pro Asp Gly Phe Phe Ser 130 135 140 Asn Glu Thr Ser
Ser Lys Ala Pro Cys Arg Lys His Thr Asn Cys Ser 145 150 155 160 Val
Phe Gly Leu Leu Leu Thr Gln Lys Gly Asn Ala Thr His Asp Asn 165 170
175 Ile Cys Ser Gly Asn Ser Glu Ser Thr Gln Lys Cys Gly Ile Asp Val
180 185 190 Thr Leu Cys Glu Glu Ala Phe Phe Arg Phe Ala Val Pro Thr
Lys Phe 195 200 205 Thr Pro Asn Trp Leu Ser Val Leu Val Asp Asn Leu
Pro Gly Thr Lys 210 215 220 Val Asn Ala Glu Ser Val Glu Arg Ile Lys
Arg Gln His Ser Ser Gln 225 230 235 240 Glu Gln Thr Phe Gln Leu Leu
Lys Leu Trp Lys His Gln Asn Lys Asp 245 250 255 Gln Asp Ile Val Lys
Lys Ile Ile Gln Asp Ile Asp Leu Cys Glu Asn 260 265 270 Ser Val Gln
Arg His Ile Gly His Ala Asn Leu Thr Phe Glu Gln Leu 275 280 285 Arg
Ser Leu Met Glu Ser Leu Pro Gly Lys Lys Val Gly Ala Glu Asp 290 295
300 Ile Glu Lys Thr Ile Lys Ala Cys Lys Pro Ser Asp Gln Ile Leu Lys
305 310 315 320 Leu Leu Ser Leu Trp Arg Ile Lys Asn Gly Asp Gln Asp
Thr Leu Lys 325 330 335 Gly Leu Met His Ala Leu Lys His Ser Lys Thr
Tyr His Phe Pro Lys 340 345 350 Thr Val Thr Gln Ser Leu Lys Lys Thr
Ile Arg Phe Leu His Ser Phe 355 360 365 Thr Met Tyr Lys Leu Tyr Gln
Lys Leu Phe Leu Glu Met Ile Gly Asn 370 375 380 Gln Val Gln Ser Val
Lys Ile Ser Cys Leu 385 390 10 1089 DNA Homo sapiens 10 atgaacaagt
tgctgtgctg cgcgctcgtg tttctggaca tctccattaa gtggaccacc 60
caggaaacgt ttcctccaaa gtaccttcat tatgacgaag aaacctctca tcagctgttg
120 tgtgacaaat gtcctcctgg tacctaccta aaacaacact gtacagcaaa
gtggaagacc 180 gtgtgcgccc cttgccctga ccactactac acagacagct
ggcacaccag tgacgagtgt 240 ctatactgca gccccgtgtg caaggagctg
cagtacgtca agcaggagtg caatcgcacc 300 cacaaccgcg tgtgcgaatg
caaggaaggg cgctaccttg agatagagtt ctgcttgaaa 360 cataggagct
gccctcctgg atttggagtg gtgcaagctg gaaccccaga gcgaaataca 420
gtttgcaaaa gatgtccaga tgggttcttc tcaaatgaga cgtcatctaa agcaccctgt
480 agaaaacaca caaattgcag tgtctttggt ctcctgctaa ctcagaaagg
aaatgcaaca 540 cacgacaaca tatgttccgg aaacagtgaa tcaactcaaa
aatgtggaat agatgttacc 600 ctgtgtgagg aggcattctt caggtttgct
gttcctacaa agtttacgcc taactggctt 660 agtgtcttgg tagacaattt
gcctggcacc aaagtaaacg cagagagtgt agagaggata 720 aaacggcaac
acagctcaca agaacagact ttccagctgc tgaagttatg gaaacatcaa 780
aacaaagacc aagatatagt caagaagatc atccaagata ttgacctctg tgaaaacagc
840 gtgcagcggc acattggaca tgctaacctc agtttgtggc gaataaaaaa
tggcgaccaa 900 gacaccttga agggcctaat gcacgcacta aagcactcaa
agacgtacca ctttcccaaa 960 actgtcactc agagtctaaa gaagaccatc
aggttccttc acagcttcac aatgtacaaa 1020 ttgtatcaga agttattttt
agaaatgata ggtaaccagg tccaatcagt aaaaataagc 1080 tgcttataa 1089 11
362 PRT Homo sapiens 11 Met Asn Lys Leu Leu Cys Cys Ala Leu Val Phe
Leu Asp Ile Ser Ile 1 5 10 15 Lys Trp Thr Thr Gln Glu Thr Phe Pro
Pro Lys Tyr Leu His Tyr Asp 20 25 30 Glu Glu Thr Ser His Gln Leu
Leu Cys Asp Lys Cys Pro Pro Gly Thr 35 40 45 Tyr Leu Lys Gln His
Cys Thr Ala Lys Trp Lys Thr Val Cys Ala Pro 50 55 60 Cys Pro Asp
His Tyr Tyr Thr Asp Ser Trp His Thr Ser Asp Glu Cys 65 70 75 80 Leu
Tyr Cys Ser Pro Val Cys Lys Glu Leu Gln Tyr Val Lys Gln Glu 85 90
95 Cys Asn Arg Thr His Asn Arg Val Cys Glu Cys Lys Glu Gly Arg Tyr
100 105 110 Leu Glu Ile Glu Phe Cys Leu Lys His Arg Ser Cys Pro Pro
Gly Phe 115 120 125 Gly Val Val Gln Ala Gly Thr Pro Glu Arg Asn Thr
Val Cys Lys Arg 130 135 140 Cys Pro Asp Gly Phe Phe Ser Asn Glu Thr
Ser Ser Lys Ala Pro Cys 145 150 155 160 Arg Lys His Thr Asn Cys Ser
Val Phe Gly Leu Leu Leu Thr Gln Lys 165 170 175 Gly Asn Ala Thr His
Asp Asn Ile Cys Ser Gly Asn Ser Glu Ser Thr 180 185 190 Gln Lys Cys
Gly Ile Asp Val Thr Leu Cys Glu Glu Ala Phe Phe Arg 195 200 205 Phe
Ala Val Pro Thr Lys Phe Thr Pro Asn Trp Leu Ser Val Leu Val 210 215
220 Asp Asn Leu Pro Gly Thr Lys Val Asn Ala Glu Ser Val Glu Arg Ile
225 230 235 240 Lys Arg Gln His Ser Ser Gln Glu Gln Thr Phe Gln Leu
Leu Lys Leu 245 250 255 Trp Lys His Gln Asn Lys Asp Gln Asp Ile Val
Lys Lys Ile Ile Gln 260 265 270 Asp Ile Asp Leu Cys Glu Asn Ser Val
Gln Arg His Ile Gly His Ala 275 280 285 Asn Leu Ser Leu Trp Arg Ile
Lys Asn Gly Asp Gln Asp Thr Leu Lys 290 295 300 Gly Leu Met His Ala
Leu Lys His Ser Lys Thr Tyr His Phe Pro Lys 305 310 315 320 Thr Val
Thr Gln Ser Leu Lys Lys Thr Ile Arg Phe Leu His Ser Phe 325 330 335
Thr Met Tyr Lys Leu Tyr Gln Lys Leu Phe Leu Glu Met Ile Gly Asn 340
345 350 Gln Val Gln Ser Val Lys Ile Ser Cys Leu 355 360 12 465 DNA
Homo sapiens 12 atgaacaagt tgctgtgctg ctcgctcgtg tttctggaca
tctccattaa gtggaccacc 60 caggaaacgt ttcctccaaa gtaccttcat
tatgacgaag aaacctctca tcagctgttg 120 tgtgacaaat gtcctcctgg
tacctaccta aaacaacact gtacagcaaa gtggaagacc 180 gtgtgcgccc
cttgccctga ccactactac acagacagct ggcacaccag tgacgagtgt 240
ctatactgca gccccgtgtg caaggagctg cagtacgtca agcaggagtg caatcgcacc
300 cacaaccgcg tgtgcgaatg caaggaaggg cgctaccttg agatagagtt
ctgcttgaaa 360 cataggagct gccctcctgg atttggagtg gtgcaagctg
gtacgtgtca atgtgcagca 420 aaattaatta ggatcatgca aagtcagata
gttgtgacag tttag 465 13 154 PRT Homo sapiens 13 Met Asn Lys Leu Leu
Cys Cys Ser Leu Val Phe Leu Asp Ile Ser Ile 1 5 10 15 Lys Trp Thr
Thr Gln Glu Thr Phe Pro Pro Lys Tyr Leu His Tyr Asp 20 25 30 Glu
Glu Thr Ser His Gln Leu Leu Cys Asp Lys Cys Pro Pro Gly Thr 35 40
45 Tyr Leu Lys Gln His Cys Thr Ala Lys Trp Lys Thr Val Cys Ala Pro
50 55 60 Cys Pro Asp His Tyr Tyr Thr Asp Ser Trp His Thr Ser Asp
Glu Cys 65 70 75 80 Leu Tyr Cys Ser Pro Val Cys Lys Glu Leu Gln Tyr
Val Lys Gln Glu 85 90 95 Cys Asn Arg Thr His Asn Arg Val Cys Glu
Cys Lys Glu Gly Arg Tyr 100 105 110 Leu Glu Ile Glu Phe Cys Leu Lys
His Arg Ser Cys Pro Pro Gly Phe 115 120 125 Gly Val Val Gln Ala Gly
Thr Cys Gln Cys Ala Ala Lys Leu Ile Arg 130 135 140 Ile Met
Gln Ser Gln Ile Val Val Thr Val 145 150 14 438 DNA Homo sapiens 14
atgaacaagt tgctgtgctg cgcgctcgtg tttctggaca tctccattaa gtggaccacc
60 caggaaacgt ttcctccaaa gtaccttcat tatgacgaag aaacctctca
tcagctgttg 120 tgtgacaaat gtcctcctgg tacctaccta aaacaacact
gtacagcaaa gtggaagacc 180 gtgtgcgccc cttgccctga ccactactac
acagacagct ggcacaccag tgacgagtgt 240 ctatactgca gccccgtgtg
caaggagctg cagtacgtca agcaggagtg caatcgcacc 300 cacaaccgcg
tgtgcgaatg caaggaaggg cgctaccttg agatagagtt ctgcttgaaa 360
cataggagct gccctcctgg atttggagtg gtgcaagctg gatgcaggag aagacccaag
420 ccacagatat gtatctga 438 15 145 PRT Homo sapiens 15 Met Asn Lys
Leu Leu Cys Cys Ala Leu Val Phe Leu Asp Ile Ser Ile 1 5 10 15 Lys
Trp Thr Thr Gln Glu Thr Phe Pro Pro Lys Tyr Leu His Tyr Asp 20 25
30 Glu Glu Thr Ser His Gln Leu Leu Cys Asp Lys Cys Pro Pro Gly Thr
35 40 45 Tyr Leu Lys Gln His Cys Thr Ala Lys Trp Lys Thr Val Cys
Ala Pro 50 55 60 Cys Pro Asp His Tyr Tyr Thr Asp Ser Trp His Thr
Ser Asp Glu Cys 65 70 75 80 Leu Tyr Cys Ser Pro Val Cys Lys Glu Leu
Gln Tyr Val Lys Gln Glu 85 90 95 Cys Asn Arg Thr His Asn Arg Val
Cys Glu Cys Lys Glu Gly Arg Tyr 100 105 110 Leu Glu Ile Glu Phe Cys
Leu Lys His Arg Ser Cys Pro Pro Gly Phe 115 120 125 Gly Val Val Gln
Ala Gly Cys Arg Arg Arg Pro Lys Pro Gln Ile Cys 130 135 140 Ile 145
16 20 DNA Artificial Sequence Synthetic Sequence 16 aattaaccct
cactaaaggg 20 17 22 DNA Artificial Sequence Synthetic Sequence 17
gtaatacgac tcactatagg gc 22 18 20 DNA Artificial Sequence Synthetic
Sequence 18 acatcaaaac aaagaccaag 20 19 20 DNA Artificial Sequence
Synthetic Sequence 19 tcttggtctt tgttttgatg 20 20 20 DNA Artificial
Sequence Synthetic Sequence 20 ttattcgcca caaactgagc 20 21 20 DNA
Artificial Sequence Synthetic Sequence 21 ttgtgaagct gtgaaggaac 20
22 20 DNA Artificial Sequence Synthetic Sequence 22 gctcagtttg
tggcgaataa 20 23 20 DNA Artificial Sequence Synthetic Sequence 23
gtgggagcag aagacattga 20 24 20 DNA Artificial Sequence Synthetic
Sequence 24 aatgaacaac ttgctgtgct 20 25 20 DNA Artificial Sequence
Synthetic Sequence 25 tgacaaatgt cctcctggta 20 26 20 DNA Artificial
Sequence Synthetic Sequence 26 aggtaggtac caggaggaca 20 27 20 DNA
Artificial Sequence Synthetic Sequence 27 gagctgccct cctggatttg 20
28 20 DNA Artificial Sequence Synthetic Sequence 28 caaactgtat
ttcgctctgg 20 29 20 DNA Artificial Sequence Synthetic Sequence 29
gtgtgaggag gcattcttca 20 30 32 DNA Artificial Sequence Synthetic
Sequence 30 gaatcaactc aaaaaagtgg aatagatgtt ac 32 31 32 DNA
Artificial Sequence Synthetic Sequence 31 gtaacatcta ttccactttt
ttgagttgat tc 32 32 30 DNA Artificial Sequence Synthetic Sequence
32 atagatgtta ccctgagtga ggaggcattc 30 33 30 DNA Artificial
Sequence Synthetic Sequence 33 gaatgcctcc tcactcaggg taacatctat 30
34 31 DNA Artificial Sequence Synthetic Sequence 34 caagatattg
acctcagtga aaacagcgtg c 31 35 31 DNA Artificial Sequence Synthetic
Sequence 35 gcacgctgtt ttcactgagg gcaatatctt g 31 36 31 DNA
Artificial Sequence Synthetic Sequence 36 aaaacaataa aggcaagcaa
acccagtgac c 31 37 31 DNA Artificial Sequence Synthetic Sequence 37
ggtcactggg tttgcttgcc tttattgttt t 31 38 31 DNA Artificial Sequence
Synthetic Sequence 38 tcagtaaaaa taagcagctt ataactggcc a 31 39 31
DNA Artificial Sequence Synthetic Sequence 39 tggccagtta taagctgctt
atttttactg a 31 40 22 DNA Artificial Sequence Synthetic Sequence 40
ttggggttta ttggaggaga tg 22 41 36 DNA Artificial Sequence Synthetic
Sequence 41 accacccagg aaccttgccc tgaccactac tacaca 36 42 36 DNA
Artificial Sequence Synthetic Sequence 42 gtcagggcaa ggttcctggg
tggtccactt aatgga 36 43 36 DNA Artificial Sequence Synthetic
Sequence 43 accgtgtgcg ccgaatgcaa ggaagggcgc tacctt 36 44 36 DNA
Artificial Sequence Synthetic Sequence 44 ttccttgcat tcggcgcaca
cggtcttcca ctttgc 36 45 36 DNA Artificial Sequence Synthetic
Sequence 45 aaccgcgtgt gcagatgtcc agatgggttc ttctca 36 46 36 DNA
Artificial Sequence Synthetic Sequence 46 atctggacat ctgcacacgc
ggttgtgggt gcgatt 36 47 36 DNA Artificial Sequence Synthetic
Sequence 47 acagtttgca aatccggaaa cagtgaatca actcaa 36 48 36 DNA
Artificial Sequence Synthetic Sequence 48 actgtttccg gatttgcaaa
ctgtatttcg ctctgg 36 49 36 DNA Artificial Sequence Synthetic
Sequence 49 aatgtggaat agatattgac ctctgtgaaa acagcg 36 50 36 DNA
Artificial Sequence Synthetic Sequence 50 agaggtcaat atctattcca
catttttgag ttgatt 36 51 36 DNA Artificial Sequence Synthetic
Sequence 51 agatcatcca agacgcacta aagcactcaa agacgt 36 52 36 DNA
Artificial Sequence Synthetic Sequence 52 gctttagtgc gtcttggatg
atcttcttga ctatat 36 53 29 DNA Artificial Sequence Synthetic
Sequence 53 ggctcgagcg cccagccgcc gcctccaag 29 54 20 DNA Artificial
Sequence Synthetic Sequence 54 tttgagtgct ttagtgcgtg 20 55 30 DNA
Artificial Sequence Synthetic Sequence 55 tcagtaaaaa taagctaact
ggaaatggcc 30 56 30 DNA Artificial Sequence Synthetic Sequence 56
ggccatttcc agttagctta tttttactga 30 57 29 DNA Artificial Sequence
Synthetic Sequence 57 ccggatcctc agtgctttag tgcgtgcat 29 58 29 DNA
Artificial Sequence Synthetic Sequence 58 ccggatcctc attggatgat
cttcttgac 29 59 29 DNA Artificial Sequence Synthetic Sequence 59
ccggatcctc atattccaca tttttgagt 29 60 29 DNA Artificial Sequence
Synthetic Sequence 60 ccggatcctc atttgcaaac tgtatttcg 29 61 29 DNA
Artificial Sequence Synthetic Sequence 61 ccggatcctc attcgcacac
gcggttgtg 29 62 401 PRT Homo sapiens 62 Met Asn Asn Leu Leu Cys Cys
Ala Leu Val Phe Leu Asp Ile Ser Ile 1 5 10 15 Lys Trp Thr Thr Gln
Glu Thr Phe Pro Pro Lys Tyr Leu His Tyr Asp 20 25 30 Glu Glu Thr
Ser His Gln Leu Leu Cys Asp Lys Cys Pro Pro Gly Thr 35 40 45 Tyr
Leu Lys Gln His Cys Thr Ala Lys Trp Lys Thr Val Cys Ala Pro 50 55
60 Cys Pro Asp His Tyr Tyr Thr Asp Ser Trp His Thr Ser Asp Glu Cys
65 70 75 80 Leu Tyr Cys Ser Pro Val Cys Lys Glu Leu Gln Tyr Val Lys
Gln Glu 85 90 95 Cys Asn Arg Thr His Asn Arg Val Cys Glu Cys Lys
Glu Gly Arg Tyr 100 105 110 Leu Glu Ile Glu Phe Cys Leu Lys His Arg
Ser Cys Pro Pro Gly Phe 115 120 125 Gly Val Val Gln Ala Gly Thr Pro
Glu Arg Asn Thr Val Cys Lys Arg 130 135 140 Cys Pro Asp Gly Phe Phe
Ser Asn Glu Thr Ser Ser Lys Ala Pro Cys 145 150 155 160 Arg Lys His
Thr Asn Cys Ser Val Phe Gly Leu Leu Leu Thr Gln Lys 165 170 175 Gly
Asn Ala Thr His Asp Asn Ile Cys Ser Gly Asn Ser Glu Ser Thr 180 185
190 Gln Lys Ser Gly Ile Asp Val Thr Leu Cys Glu Glu Ala Phe Phe Arg
195 200 205 Phe Ala Val Pro Thr Lys Phe Thr Pro Asn Trp Leu Ser Val
Leu Val 210 215 220 Asp Asn Leu Pro Gly Thr Lys Val Asn Ala Glu Ser
Val Glu Arg Ile 225 230 235 240 Lys Arg Gln His Ser Ser Gln Glu Gln
Thr Phe Gln Leu Leu Lys Leu 245 250 255 Trp Lys His Gln Asn Lys Asp
Gln Asp Ile Val Lys Lys Ile Ile Gln 260 265 270 Asp Ile Asp Leu Cys
Glu Asn Ser Val Gln Arg His Ile Gly His Ala 275 280 285 Asn Leu Thr
Phe Glu Gln Leu Arg Ser Leu Met Glu Ser Leu Pro Gly 290 295 300 Lys
Lys Val Gly Ala Glu Asp Ile Glu Lys Thr Ile Lys Ala Cys Lys 305 310
315 320 Pro Ser Asp Gln Ile Leu Lys Leu Leu Ser Leu Trp Arg Ile Lys
Asn 325 330 335 Gly Asp Gln Asp Thr Leu Lys Gly Leu Met His Ala Leu
Lys His Ser 340 345 350 Lys Thr Tyr His Phe Pro Lys Thr Val Thr Gln
Ser Leu Lys Lys Thr 355 360 365 Ile Arg Phe Leu His Ser Phe Thr Met
Tyr Lys Leu Tyr Gln Lys Leu 370 375 380 Phe Leu Glu Met Ile Gly Asn
Gln Val Gln Ser Val Lys Ile Ser Cys 385 390 395 400 Leu 63 401 PRT
Homo sapiens 63 Met Asn Asn Leu Leu Cys Cys Ala Leu Val Phe Leu Asp
Ile Ser Ile 1 5 10 15 Lys Trp Thr Thr Gln Glu Thr Phe Pro Pro Lys
Tyr Leu His Tyr Asp 20 25 30 Glu Glu Thr Ser His Gln Leu Leu Cys
Asp Lys Cys Pro Pro Gly Thr 35 40 45 Tyr Leu Lys Gln His Cys Thr
Ala Lys Trp Lys Thr Val Cys Ala Pro 50 55 60 Cys Pro Asp His Tyr
Tyr Thr Asp Ser Trp His Thr Ser Asp Glu Cys 65 70 75 80 Leu Tyr Cys
Ser Pro Val Cys Lys Glu Leu Gln Tyr Val Lys Gln Glu 85 90 95 Cys
Asn Arg Thr His Asn Arg Val Cys Glu Cys Lys Glu Gly Arg Tyr 100 105
110 Leu Glu Ile Glu Phe Cys Leu Lys His Arg Ser Cys Pro Pro Gly Phe
115 120 125 Gly Val Val Gln Ala Gly Thr Pro Glu Arg Asn Thr Val Cys
Lys Arg 130 135 140 Cys Pro Asp Gly Phe Phe Ser Asn Glu Thr Ser Ser
Lys Ala Pro Cys 145 150 155 160 Arg Lys His Thr Asn Cys Ser Val Phe
Gly Leu Leu Leu Thr Gln Lys 165 170 175 Gly Asn Ala Thr His Asp Asn
Ile Cys Ser Gly Asn Ser Glu Ser Thr 180 185 190 Gln Lys Cys Gly Ile
Asp Val Thr Leu Ser Glu Glu Ala Phe Phe Arg 195 200 205 Phe Ala Val
Pro Thr Lys Phe Thr Pro Asn Trp Leu Ser Val Leu Val 210 215 220 Asp
Asn Leu Pro Gly Thr Lys Val Asn Ala Glu Ser Val Glu Arg Ile 225 230
235 240 Lys Arg Gln His Ser Ser Gln Glu Gln Thr Phe Gln Leu Leu Lys
Leu 245 250 255 Trp Lys His Gln Asn Lys Asp Gln Asp Ile Val Lys Lys
Ile Ile Gln 260 265 270 Asp Ile Asp Leu Cys Glu Asn Ser Val Gln Arg
His Ile Gly His Ala 275 280 285 Asn Leu Thr Phe Glu Gln Leu Arg Ser
Leu Met Glu Ser Leu Pro Gly 290 295 300 Lys Lys Val Gly Ala Glu Asp
Ile Glu Lys Thr Ile Lys Ala Cys Lys 305 310 315 320 Pro Ser Asp Gln
Ile Leu Lys Leu Leu Ser Leu Trp Arg Ile Lys Asn 325 330 335 Gly Asp
Gln Asp Thr Leu Lys Gly Leu Met His Ala Leu Lys His Ser 340 345 350
Lys Thr Tyr His Phe Pro Lys Thr Val Thr Gln Ser Leu Lys Lys Thr 355
360 365 Ile Arg Phe Leu His Ser Phe Thr Met Tyr Lys Leu Tyr Gln Lys
Leu 370 375 380 Phe Leu Glu Met Ile Gly Asn Gln Val Gln Ser Val Lys
Ile Ser Cys 385 390 395 400 Leu 64 401 PRT Homo sapiens 64 Met Asn
Asn Leu Leu Cys Cys Ala Leu Val Phe Leu Asp Ile Ser Ile 1 5 10 15
Lys Trp Thr Thr Gln Glu Thr Phe Pro Pro Lys Tyr Leu His Tyr Asp 20
25 30 Glu Glu Thr Ser His Gln Leu Leu Cys Asp Lys Cys Pro Pro Gly
Thr 35 40 45 Tyr Leu Lys Gln His Cys Thr Ala Lys Trp Lys Thr Val
Cys Ala Pro 50 55 60 Cys Pro Asp His Tyr Tyr Thr Asp Ser Trp His
Thr Ser Asp Glu Cys 65 70 75 80 Leu Tyr Cys Ser Pro Val Cys Lys Glu
Leu Gln Tyr Val Lys Gln Glu 85 90 95 Cys Asn Arg Thr His Asn Arg
Val Cys Glu Cys Lys Glu Gly Arg Tyr 100 105 110 Leu Glu Ile Glu Phe
Cys Leu Lys His Arg Ser Cys Pro Pro Gly Phe 115 120 125 Gly Val Val
Gln Ala Gly Thr Pro Glu Arg Asn Thr Val Cys Lys Arg 130 135 140 Cys
Pro Asp Gly Phe Phe Ser Asn Glu Thr Ser Ser Lys Ala Pro Cys 145 150
155 160 Arg Lys His Thr Asn Cys Ser Val Phe Gly Leu Leu Leu Thr Gln
Lys 165 170 175 Gly Asn Ala Thr His Asp Asn Ile Cys Ser Gly Asn Ser
Glu Ser Thr 180 185 190 Gln Lys Cys Gly Ile Asp Val Thr Leu Cys Glu
Glu Ala Phe Phe Arg 195 200 205 Phe Ala Val Pro Thr Lys Phe Thr Pro
Asn Trp Leu Ser Val Leu Val 210 215 220 Asp Asn Leu Pro Gly Thr Lys
Val Asn Ala Glu Ser Val Glu Arg Ile 225 230 235 240 Lys Arg Gln His
Ser Ser Gln Glu Gln Thr Phe Gln Leu Leu Lys Leu 245 250 255 Trp Lys
His Gln Asn Lys Asp Gln Asp Ile Val Lys Lys Ile Ile Gln 260 265 270
Asp Ile Asp Leu Ser Glu Asn Ser Val Gln Arg His Ile Gly His Ala 275
280 285 Asn Leu Thr Phe Glu Gln Leu Arg Ser Leu Met Glu Ser Leu Pro
Gly 290 295 300 Lys Lys Val Gly Ala Glu Asp Ile Glu Lys Thr Ile Lys
Ala Cys Lys 305 310 315 320 Pro Ser Asp Gln Ile Leu Lys Leu Leu Ser
Leu Trp Arg Ile Lys Asn 325 330 335 Gly Asp Gln Asp Thr Leu Lys Gly
Leu Met His Ala Leu Lys His Ser 340 345 350 Lys Thr Tyr His Phe Pro
Lys Thr Val Thr Gln Ser Leu Lys Lys Thr 355 360 365 Ile Arg Phe Leu
His Ser Phe Thr Met Tyr Lys Leu Tyr Gln Lys Leu 370 375 380 Phe Leu
Glu Met Ile Gly Asn Gln Val Gln Ser Val Lys Ile Ser Cys 385 390 395
400 Leu 65 401 PRT Homo sapiens 65 Met Asn Asn Leu Leu Cys Cys Ala
Leu Val Phe Leu Asp Ile Ser Ile 1 5 10 15 Lys Trp Thr Thr Gln Glu
Thr Phe Pro Pro Lys Tyr Leu His Tyr Asp 20 25 30 Glu Glu Thr Ser
His Gln Leu Leu Cys Asp Lys Cys Pro Pro Gly Thr 35 40 45 Tyr Leu
Lys Gln His Cys Thr Ala Lys Trp Lys Thr Val Cys Ala Pro 50 55 60
Cys Pro Asp His Tyr Tyr Thr Asp Ser Trp His Thr Ser Asp Glu Cys 65
70 75 80 Leu Tyr Cys Ser Pro Val Cys Lys Glu Leu Gln Tyr Val Lys
Gln Glu 85 90 95 Cys Asn Arg Thr His Asn Arg Val Cys Glu Cys Lys
Glu Gly Arg Tyr 100 105 110 Leu Glu Ile Glu Phe Cys Leu Lys His Arg
Ser Cys Pro Pro Gly Phe 115 120 125 Gly Val Val Gln Ala Gly Thr Pro
Glu Arg Asn Thr Val Cys Lys Arg 130 135 140 Cys Pro Asp Gly Phe Phe
Ser Asn Glu Thr Ser Ser Lys Ala Pro Cys 145 150 155 160 Arg Lys His
Thr Asn Cys Ser Val Phe Gly Leu Leu Leu Thr Gln Lys 165 170 175 Gly
Asn Ala Thr His Asp Asn Ile Cys Ser Gly Asn Ser Glu Ser Thr 180 185
190 Gln Lys Cys
Gly Ile Asp Val Thr Leu Cys Glu Glu Ala Phe Phe Arg 195 200 205 Phe
Ala Val Pro Thr Lys Phe Thr Pro Asn Trp Leu Ser Val Leu Val 210 215
220 Asp Asn Leu Pro Gly Thr Lys Val Asn Ala Glu Ser Val Glu Arg Ile
225 230 235 240 Lys Arg Gln His Ser Ser Gln Glu Gln Thr Phe Gln Leu
Leu Lys Leu 245 250 255 Trp Lys His Gln Asn Lys Asp Gln Asp Ile Val
Lys Lys Ile Ile Gln 260 265 270 Asp Ile Asp Leu Cys Glu Asn Ser Val
Gln Arg His Ile Gly His Ala 275 280 285 Asn Leu Thr Phe Glu Gln Leu
Arg Ser Leu Met Glu Ser Leu Pro Gly 290 295 300 Lys Lys Val Gly Ala
Glu Asp Ile Glu Lys Thr Ile Lys Ala Ser Lys 305 310 315 320 Pro Ser
Asp Gln Ile Leu Lys Leu Leu Ser Leu Trp Arg Ile Lys Asn 325 330 335
Gly Asp Gln Asp Thr Leu Lys Gly Leu Met His Ala Leu Lys His Ser 340
345 350 Lys Thr Tyr His Phe Pro Lys Thr Val Thr Gln Ser Leu Lys Lys
Thr 355 360 365 Ile Arg Phe Leu His Ser Phe Thr Met Tyr Lys Leu Tyr
Gln Lys Leu 370 375 380 Phe Leu Glu Met Ile Gly Asn Gln Val Gln Ser
Val Lys Ile Ser Cys 385 390 395 400 Leu 66 401 PRT Homo sapiens 66
Met Asn Asn Leu Leu Cys Cys Ala Leu Val Phe Leu Asp Ile Ser Ile 1 5
10 15 Lys Trp Thr Thr Gln Glu Thr Phe Pro Pro Lys Tyr Leu His Tyr
Asp 20 25 30 Glu Glu Thr Ser His Gln Leu Leu Cys Asp Lys Cys Pro
Pro Gly Thr 35 40 45 Tyr Leu Lys Gln His Cys Thr Ala Lys Trp Lys
Thr Val Cys Ala Pro 50 55 60 Cys Pro Asp His Tyr Tyr Thr Asp Ser
Trp His Thr Ser Asp Glu Cys 65 70 75 80 Leu Tyr Cys Ser Pro Val Cys
Lys Glu Leu Gln Tyr Val Lys Gln Glu 85 90 95 Cys Asn Arg Thr His
Asn Arg Val Cys Glu Cys Lys Glu Gly Arg Tyr 100 105 110 Leu Glu Ile
Glu Phe Cys Leu Lys His Arg Ser Cys Pro Pro Gly Phe 115 120 125 Gly
Val Val Gln Ala Gly Thr Pro Glu Arg Asn Thr Val Cys Lys Arg 130 135
140 Cys Pro Asp Gly Phe Phe Ser Asn Glu Thr Ser Ser Lys Ala Pro Cys
145 150 155 160 Arg Lys His Thr Asn Cys Ser Val Phe Gly Leu Leu Leu
Thr Gln Lys 165 170 175 Gly Asn Ala Thr His Asp Asn Ile Cys Ser Gly
Asn Ser Glu Ser Thr 180 185 190 Gln Lys Cys Gly Ile Asp Val Thr Leu
Cys Glu Glu Ala Phe Phe Arg 195 200 205 Phe Ala Val Pro Thr Lys Phe
Thr Pro Asn Trp Leu Ser Val Leu Val 210 215 220 Asp Asn Leu Pro Gly
Thr Lys Val Asn Ala Glu Ser Val Glu Arg Ile 225 230 235 240 Lys Arg
Gln His Ser Ser Gln Glu Gln Thr Phe Gln Leu Leu Lys Leu 245 250 255
Trp Lys His Gln Asn Lys Asp Gln Asp Ile Val Lys Lys Ile Ile Gln 260
265 270 Asp Ile Asp Leu Cys Glu Asn Ser Val Gln Arg His Ile Gly His
Ala 275 280 285 Asn Leu Thr Phe Glu Gln Leu Arg Ser Leu Met Glu Ser
Leu Pro Gly 290 295 300 Lys Lys Val Gly Ala Glu Asp Ile Glu Lys Thr
Ile Lys Ala Cys Lys 305 310 315 320 Pro Ser Asp Gln Ile Leu Lys Leu
Leu Ser Leu Trp Arg Ile Lys Asn 325 330 335 Gly Asp Gln Asp Thr Leu
Lys Gly Leu Met His Ala Leu Lys His Ser 340 345 350 Lys Thr Tyr His
Phe Pro Lys Thr Val Thr Gln Ser Leu Lys Lys Thr 355 360 365 Ile Arg
Phe Leu His Ser Phe Thr Met Tyr Lys Leu Tyr Gln Lys Leu 370 375 380
Phe Leu Glu Met Ile Gly Asn Gln Val Gln Ser Val Lys Ile Ser Ser 385
390 395 400 Leu 67 360 PRT Homo sapiens 67 Met Asn Asn Leu Leu Cys
Cys Ala Leu Val Phe Leu Asp Ile Ser Ile 1 5 10 15 Lys Trp Thr Thr
Gln Glu Pro Cys Pro Asp His Tyr Tyr Thr Asp Ser 20 25 30 Trp His
Thr Ser Asp Glu Cys Leu Tyr Cys Ser Pro Val Cys Lys Glu 35 40 45
Leu Gln Tyr Val Lys Gln Glu Cys Asn Arg Thr His Asn Arg Val Cys 50
55 60 Glu Cys Lys Glu Gly Arg Tyr Leu Glu Ile Glu Phe Cys Leu Lys
His 65 70 75 80 Arg Ser Cys Pro Pro Gly Phe Gly Val Val Gln Ala Gly
Thr Pro Glu 85 90 95 Arg Asn Thr Val Cys Lys Arg Cys Pro Asp Gly
Phe Phe Ser Asn Glu 100 105 110 Thr Ser Ser Lys Ala Pro Cys Arg Lys
His Thr Asn Cys Ser Val Phe 115 120 125 Gly Leu Leu Leu Thr Gln Lys
Gly Asn Ala Thr His Asp Asn Ile Cys 130 135 140 Ser Gly Asn Ser Glu
Ser Thr Gln Lys Cys Gly Ile Asp Val Thr Leu 145 150 155 160 Cys Glu
Glu Ala Phe Phe Arg Phe Ala Val Pro Thr Lys Phe Thr Pro 165 170 175
Asn Trp Leu Ser Val Leu Val Asp Asn Leu Pro Gly Thr Lys Val Asn 180
185 190 Ala Glu Ser Val Glu Arg Ile Lys Arg Gln His Ser Ser Gln Glu
Gln 195 200 205 Thr Phe Gln Leu Leu Lys Leu Trp Lys His Gln Asn Lys
Asp Gln Asp 210 215 220 Ile Val Lys Lys Ile Ile Gln Asp Ile Asp Leu
Cys Glu Asn Ser Val 225 230 235 240 Gln Arg His Ile Gly His Ala Asn
Leu Thr Phe Glu Gln Leu Arg Ser 245 250 255 Leu Met Glu Ser Leu Pro
Gly Lys Lys Val Gly Ala Glu Asp Ile Glu 260 265 270 Lys Thr Ile Lys
Ala Cys Lys Pro Ser Asp Gln Ile Leu Lys Leu Leu 275 280 285 Ser Leu
Trp Arg Ile Lys Asn Gly Asp Gln Asp Thr Leu Lys Gly Leu 290 295 300
Met His Ala Leu Lys His Ser Lys Thr Tyr His Phe Pro Lys Thr Val 305
310 315 320 Thr Gln Ser Leu Lys Lys Thr Ile Arg Phe Leu His Ser Phe
Thr Met 325 330 335 Tyr Lys Leu Tyr Gln Lys Leu Phe Leu Glu Met Ile
Gly Asn Gln Val 340 345 350 Gln Ser Val Lys Ile Ser Cys Leu 355 360
68 359 PRT Homo sapiens 68 Met Asn Asn Leu Leu Cys Cys Ala Leu Val
Phe Leu Asp Ile Ser Ile 1 5 10 15 Lys Trp Thr Thr Gln Glu Thr Phe
Pro Pro Lys Tyr Leu His Tyr Asp 20 25 30 Glu Glu Thr Ser His Gln
Leu Leu Cys Asp Lys Cys Pro Pro Gly Thr 35 40 45 Tyr Leu Lys Gln
His Cys Thr Ala Lys Trp Lys Thr Val Cys Ala Glu 50 55 60 Cys Lys
Glu Gly Arg Tyr Leu Glu Ile Glu Phe Cys Leu Lys His Arg 65 70 75 80
Ser Cys Pro Pro Gly Phe Gly Val Val Gln Ala Gly Thr Pro Glu Arg 85
90 95 Asn Thr Val Cys Lys Arg Cys Pro Asp Gly Phe Phe Ser Asn Glu
Thr 100 105 110 Ser Ser Lys Ala Pro Cys Arg Lys His Thr Asn Cys Ser
Val Phe Gly 115 120 125 Leu Leu Leu Thr Gln Lys Gly Asn Ala Thr His
Asp Asn Ile Cys Ser 130 135 140 Gly Asn Ser Glu Ser Thr Gln Lys Cys
Gly Ile Asp Val Thr Leu Cys 145 150 155 160 Glu Glu Ala Phe Phe Arg
Phe Ala Val Pro Thr Lys Phe Thr Pro Asn 165 170 175 Trp Leu Ser Val
Leu Val Asp Asn Leu Pro Gly Thr Lys Val Asn Ala 180 185 190 Glu Ser
Val Glu Arg Ile Lys Arg Gln His Ser Ser Gln Glu Gln Thr 195 200 205
Phe Gln Leu Leu Lys Leu Trp Lys His Gln Asn Lys Asp Gln Asp Ile 210
215 220 Val Lys Lys Ile Ile Gln Asp Ile Asp Leu Cys Glu Asn Ser Val
Gln 225 230 235 240 Arg His Ile Gly His Ala Asn Leu Thr Phe Glu Gln
Leu Arg Ser Leu 245 250 255 Met Glu Ser Leu Pro Gly Lys Lys Val Gly
Ala Glu Asp Ile Glu Lys 260 265 270 Thr Ile Lys Ala Cys Lys Pro Ser
Asp Gln Ile Leu Lys Leu Leu Ser 275 280 285 Leu Trp Arg Ile Lys Asn
Gly Asp Gln Asp Thr Leu Lys Gly Leu Met 290 295 300 His Ala Leu Lys
His Ser Lys Thr Tyr His Phe Pro Lys Thr Val Thr 305 310 315 320 Gln
Ser Leu Lys Lys Thr Ile Arg Phe Leu His Ser Phe Thr Met Tyr 325 330
335 Lys Leu Tyr Gln Lys Leu Phe Leu Glu Met Ile Gly Asn Gln Val Gln
340 345 350 Ser Val Lys Ile Ser Cys Leu 355 69 363 PRT Homo sapiens
69 Met Asn Asn Leu Leu Cys Cys Ala Leu Val Phe Leu Asp Ile Ser Ile
1 5 10 15 Lys Trp Thr Thr Gln Glu Thr Phe Pro Pro Lys Tyr Leu His
Tyr Asp 20 25 30 Glu Glu Thr Ser His Gln Leu Leu Cys Asp Lys Cys
Pro Pro Gly Thr 35 40 45 Tyr Leu Lys Gln His Cys Thr Ala Lys Trp
Lys Thr Val Cys Ala Pro 50 55 60 Cys Pro Asp His Tyr Tyr Thr Asp
Ser Trp His Thr Ser Asp Glu Cys 65 70 75 80 Leu Tyr Cys Ser Pro Val
Cys Lys Glu Leu Gln Tyr Val Lys Gln Glu 85 90 95 Cys Asn Arg Thr
His Asn Arg Val Cys Arg Cys Pro Asp Gly Phe Phe 100 105 110 Ser Asn
Glu Thr Ser Ser Lys Ala Pro Cys Arg Lys His Thr Asn Cys 115 120 125
Ser Val Phe Gly Leu Leu Leu Thr Gln Lys Gly Asn Ala Thr His Asp 130
135 140 Asn Ile Cys Ser Gly Asn Ser Glu Ser Thr Gln Lys Cys Gly Ile
Asp 145 150 155 160 Val Thr Leu Cys Glu Glu Ala Phe Phe Arg Phe Ala
Val Pro Thr Lys 165 170 175 Phe Thr Pro Asn Trp Leu Ser Val Leu Val
Asp Asn Leu Pro Gly Thr 180 185 190 Lys Val Asn Ala Glu Ser Val Glu
Arg Ile Lys Arg Gln His Ser Ser 195 200 205 Gln Glu Gln Thr Phe Gln
Leu Leu Lys Leu Trp Lys His Gln Asn Lys 210 215 220 Asp Gln Asp Ile
Val Lys Lys Ile Ile Gln Asp Ile Asp Leu Cys Glu 225 230 235 240 Asn
Ser Val Gln Arg His Ile Gly His Ala Asn Leu Thr Phe Glu Gln 245 250
255 Leu Arg Ser Leu Met Glu Ser Leu Pro Gly Lys Lys Val Gly Ala Glu
260 265 270 Asp Ile Glu Lys Thr Ile Lys Ala Cys Lys Pro Ser Asp Gln
Ile Leu 275 280 285 Lys Leu Leu Ser Leu Trp Arg Ile Lys Asn Gly Asp
Gln Asp Thr Leu 290 295 300 Lys Gly Leu Met His Ala Leu Lys His Ser
Lys Thr Tyr His Phe Pro 305 310 315 320 Lys Thr Val Thr Gln Ser Leu
Lys Lys Thr Ile Arg Phe Leu His Ser 325 330 335 Phe Thr Met Tyr Lys
Leu Tyr Gln Lys Leu Phe Leu Glu Met Ile Gly 340 345 350 Asn Gln Val
Gln Ser Val Lys Ile Ser Cys Leu 355 360 70 359 PRT Homo sapiens 70
Met Asn Asn Leu Leu Cys Cys Ala Leu Val Phe Leu Asp Ile Ser Ile 1 5
10 15 Lys Trp Thr Thr Gln Glu Thr Phe Pro Pro Lys Tyr Leu His Tyr
Asp 20 25 30 Glu Glu Thr Ser His Gln Leu Leu Cys Asp Lys Cys Pro
Pro Gly Thr 35 40 45 Tyr Leu Lys Gln His Cys Thr Ala Lys Trp Lys
Thr Val Cys Ala Pro 50 55 60 Cys Pro Asp His Tyr Tyr Thr Asp Ser
Trp His Thr Ser Asp Glu Cys 65 70 75 80 Leu Tyr Cys Ser Pro Val Cys
Lys Glu Leu Gln Tyr Val Lys Gln Glu 85 90 95 Cys Asn Arg Thr His
Asn Arg Val Cys Glu Cys Lys Glu Gly Arg Tyr 100 105 110 Leu Glu Ile
Glu Phe Cys Leu Lys His Arg Ser Cys Pro Pro Gly Phe 115 120 125 Gly
Val Val Gln Ala Gly Thr Pro Glu Arg Asn Thr Val Cys Lys Ser 130 135
140 Gly Asn Ser Glu Ser Thr Gln Lys Cys Gly Ile Asp Val Thr Leu Cys
145 150 155 160 Glu Glu Ala Phe Phe Arg Phe Ala Val Pro Thr Lys Phe
Thr Pro Asn 165 170 175 Trp Leu Ser Val Leu Val Asp Asn Leu Pro Gly
Thr Lys Val Asn Ala 180 185 190 Glu Ser Val Glu Arg Ile Lys Arg Gln
His Ser Ser Gln Glu Gln Thr 195 200 205 Phe Gln Leu Leu Lys Leu Trp
Lys His Gln Asn Lys Asp Gln Asp Ile 210 215 220 Val Lys Lys Ile Ile
Gln Asp Ile Asp Leu Cys Glu Asn Ser Val Gln 225 230 235 240 Arg His
Ile Gly His Ala Asn Leu Thr Phe Glu Gln Leu Arg Ser Leu 245 250 255
Met Glu Ser Leu Pro Gly Lys Lys Val Gly Ala Glu Asp Ile Glu Lys 260
265 270 Thr Ile Lys Ala Cys Lys Pro Ser Asp Gln Ile Leu Lys Leu Leu
Ser 275 280 285 Leu Trp Arg Ile Lys Asn Gly Asp Gln Asp Thr Leu Lys
Gly Leu Met 290 295 300 His Ala Leu Lys His Ser Lys Thr Tyr His Phe
Pro Lys Thr Val Thr 305 310 315 320 Gln Ser Leu Lys Lys Thr Ile Arg
Phe Leu His Ser Phe Thr Met Tyr 325 330 335 Lys Leu Tyr Gln Lys Leu
Phe Leu Glu Met Ile Gly Asn Gln Val Gln 340 345 350 Ser Val Lys Ile
Ser Cys Leu 355 71 326 PRT Homo sapiens 71 Met Asn Asn Leu Leu Cys
Cys Ala Leu Val Phe Leu Asp Ile Ser Ile 1 5 10 15 Lys Trp Thr Thr
Gln Glu Thr Phe Pro Pro Lys Tyr Leu His Tyr Asp 20 25 30 Glu Glu
Thr Ser His Gln Leu Leu Cys Asp Lys Cys Pro Pro Gly Thr 35 40 45
Tyr Leu Lys Gln His Cys Thr Ala Lys Trp Lys Thr Val Cys Ala Pro 50
55 60 Cys Pro Asp His Tyr Tyr Thr Asp Ser Trp His Thr Ser Asp Glu
Cys 65 70 75 80 Leu Tyr Cys Ser Pro Val Cys Lys Glu Leu Gln Tyr Val
Lys Gln Glu 85 90 95 Cys Asn Arg Thr His Asn Arg Val Cys Glu Cys
Lys Glu Gly Arg Tyr 100 105 110 Leu Glu Ile Glu Phe Cys Leu Lys His
Arg Ser Cys Pro Pro Gly Phe 115 120 125 Gly Val Val Gln Ala Gly Thr
Pro Glu Arg Asn Thr Val Cys Lys Arg 130 135 140 Cys Pro Asp Gly Phe
Phe Ser Asn Glu Thr Ser Ser Lys Ala Pro Cys 145 150 155 160 Arg Lys
His Thr Asn Cys Ser Val Phe Gly Leu Leu Leu Thr Gln Lys 165 170 175
Gly Asn Ala Thr His Asp Asn Ile Cys Ser Gly Asn Ser Glu Ser Thr 180
185 190 Gln Lys Cys Gly Ile Asp Ile Asp Leu Cys Glu Asn Ser Val Gln
Arg 195 200 205 His Ile Gly His Ala Asn Leu Thr Phe Glu Gln Leu Arg
Ser Leu Met 210 215 220 Glu Ser Leu Pro Gly Lys Lys Val Gly Ala Glu
Asp Ile Glu Lys Thr 225 230 235 240 Ile Lys Ala Cys Lys Pro Ser Asp
Gln Ile Leu Lys Leu Leu Ser Leu 245 250 255 Trp Arg Ile Lys Asn Gly
Asp Gln Asp Thr Leu Lys Gly Leu Met His 260 265 270 Ala Leu Lys His
Ser Lys Thr Tyr His Phe Pro Lys Thr Val Thr Gln 275 280 285 Ser Leu
Lys Lys Thr Ile Arg Phe Leu His Ser Phe Thr Met Tyr Lys 290 295 300
Leu Tyr Gln Lys Leu Phe Leu Glu Met Ile Gly Asn Gln Val Gln Ser 305
310 315 320 Val Lys Ile Ser Cys Leu 325 72 327 PRT Homo sapiens 72
Met Asn Asn Leu Leu Cys Cys Ala Leu Val Phe Leu Asp Ile Ser Ile 1 5
10 15 Lys Trp Thr Thr Gln Glu Thr Phe Pro Pro Lys Tyr Leu His Tyr
Asp 20 25 30 Glu Glu Thr Ser His Gln Leu Leu Cys Asp Lys Cys Pro
Pro Gly Thr 35 40 45 Tyr Leu Lys Gln His Cys Thr Ala Lys Trp Lys
Thr
Val Cys Ala Pro 50 55 60 Cys Pro Asp His Tyr Tyr Thr Asp Ser Trp
His Thr Ser Asp Glu Cys 65 70 75 80 Leu Tyr Cys Ser Pro Val Cys Lys
Glu Leu Gln Tyr Val Lys Gln Glu 85 90 95 Cys Asn Arg Thr His Asn
Arg Val Cys Glu Cys Lys Glu Gly Arg Tyr 100 105 110 Leu Glu Ile Glu
Phe Cys Leu Lys His Arg Ser Cys Pro Pro Gly Phe 115 120 125 Gly Val
Val Gln Ala Gly Thr Pro Glu Arg Asn Thr Val Cys Lys Arg 130 135 140
Cys Pro Asp Gly Phe Phe Ser Asn Glu Thr Ser Ser Lys Ala Pro Cys 145
150 155 160 Arg Lys His Thr Asn Cys Ser Val Phe Gly Leu Leu Leu Thr
Gln Lys 165 170 175 Gly Asn Ala Thr His Asp Asn Ile Cys Ser Gly Asn
Ser Glu Ser Thr 180 185 190 Gln Lys Cys Gly Ile Asp Val Thr Leu Cys
Glu Glu Ala Phe Phe Arg 195 200 205 Phe Ala Val Pro Thr Lys Phe Thr
Pro Asn Trp Leu Ser Val Leu Val 210 215 220 Asp Asn Leu Pro Gly Thr
Lys Val Asn Ala Glu Ser Val Glu Arg Ile 225 230 235 240 Lys Arg Gln
His Ser Ser Gln Glu Gln Thr Phe Gln Leu Leu Lys Leu 245 250 255 Trp
Lys His Gln Asn Lys Asp Gln Asp Ile Val Lys Lys Ile Ile Gln 260 265
270 Asp Ala Leu Lys His Ser Lys Thr Tyr His Phe Pro Lys Thr Val Thr
275 280 285 Gln Ser Leu Lys Lys Thr Ile Arg Phe Leu His Ser Phe Thr
Met Tyr 290 295 300 Lys Leu Tyr Gln Lys Leu Phe Leu Glu Met Ile Gly
Asn Gln Val Gln 305 310 315 320 Ser Val Lys Ile Ser Cys Leu 325 73
399 PRT Homo sapiens 73 Met Asn Asn Leu Leu Cys Cys Ala Leu Val Phe
Leu Asp Ile Ser Ile 1 5 10 15 Lys Trp Thr Thr Gln Glu Thr Phe Pro
Pro Lys Tyr Leu His Tyr Asp 20 25 30 Glu Glu Thr Ser His Gln Leu
Leu Cys Asp Lys Cys Pro Pro Gly Thr 35 40 45 Tyr Leu Lys Gln His
Cys Thr Ala Lys Trp Lys Thr Val Cys Ala Pro 50 55 60 Cys Pro Asp
His Tyr Tyr Thr Asp Ser Trp His Thr Ser Asp Glu Cys 65 70 75 80 Leu
Tyr Cys Ser Pro Val Cys Lys Glu Leu Gln Tyr Val Lys Gln Glu 85 90
95 Cys Asn Arg Thr His Asn Arg Val Cys Glu Cys Lys Glu Gly Arg Tyr
100 105 110 Leu Glu Ile Glu Phe Cys Leu Lys His Arg Ser Cys Pro Pro
Gly Phe 115 120 125 Gly Val Val Gln Ala Gly Thr Pro Glu Arg Asn Thr
Val Cys Lys Arg 130 135 140 Cys Pro Asp Gly Phe Phe Ser Asn Glu Thr
Ser Ser Lys Ala Pro Cys 145 150 155 160 Arg Lys His Thr Asn Cys Ser
Val Phe Gly Leu Leu Leu Thr Gln Lys 165 170 175 Gly Asn Ala Thr His
Asp Asn Ile Cys Ser Gly Asn Ser Glu Ser Thr 180 185 190 Gln Lys Cys
Gly Ile Asp Val Thr Leu Cys Glu Glu Ala Phe Phe Arg 195 200 205 Phe
Ala Val Pro Thr Lys Phe Thr Pro Asn Trp Leu Ser Val Leu Val 210 215
220 Asp Asn Leu Pro Gly Thr Lys Val Asn Ala Glu Ser Val Glu Arg Ile
225 230 235 240 Lys Arg Gln His Ser Ser Gln Glu Gln Thr Phe Gln Leu
Leu Lys Leu 245 250 255 Trp Lys His Gln Asn Lys Asp Gln Asp Ile Val
Lys Lys Ile Ile Gln 260 265 270 Asp Ile Asp Leu Cys Glu Asn Ser Val
Gln Arg His Ile Gly His Ala 275 280 285 Asn Leu Thr Phe Glu Gln Leu
Arg Ser Leu Met Glu Ser Leu Pro Gly 290 295 300 Lys Lys Val Gly Ala
Glu Asp Ile Glu Lys Thr Ile Lys Ala Cys Lys 305 310 315 320 Pro Ser
Asp Gln Ile Leu Lys Leu Leu Ser Leu Trp Arg Ile Lys Asn 325 330 335
Gly Asp Gln Asp Thr Leu Lys Gly Leu Met His Ala Leu Lys His Ser 340
345 350 Lys Thr Tyr His Phe Pro Lys Thr Val Thr Gln Ser Leu Lys Lys
Thr 355 360 365 Ile Arg Phe Leu His Ser Phe Thr Met Tyr Lys Leu Tyr
Gln Lys Leu 370 375 380 Phe Leu Glu Met Ile Gly Asn Gln Val Gln Ser
Val Lys Ile Ser 385 390 395 74 351 PRT Homo sapiens 74 Met Asn Asn
Leu Leu Cys Cys Ala Leu Val Phe Leu Asp Ile Ser Ile 1 5 10 15 Lys
Trp Thr Thr Gln Glu Thr Phe Pro Pro Lys Tyr Leu His Tyr Asp 20 25
30 Glu Glu Thr Ser His Gln Leu Leu Cys Asp Lys Cys Pro Pro Gly Thr
35 40 45 Tyr Leu Lys Gln His Cys Thr Ala Lys Trp Lys Thr Val Cys
Ala Pro 50 55 60 Cys Pro Asp His Tyr Tyr Thr Asp Ser Trp His Thr
Ser Asp Glu Cys 65 70 75 80 Leu Tyr Cys Ser Pro Val Cys Lys Glu Leu
Gln Tyr Val Lys Gln Glu 85 90 95 Cys Asn Arg Thr His Asn Arg Val
Cys Glu Cys Lys Glu Gly Arg Tyr 100 105 110 Leu Glu Ile Glu Phe Cys
Leu Lys His Arg Ser Cys Pro Pro Gly Phe 115 120 125 Gly Val Val Gln
Ala Gly Thr Pro Glu Arg Asn Thr Val Cys Lys Arg 130 135 140 Cys Pro
Asp Gly Phe Phe Ser Asn Glu Thr Ser Ser Lys Ala Pro Cys 145 150 155
160 Arg Lys His Thr Asn Cys Ser Val Phe Gly Leu Leu Leu Thr Gln Lys
165 170 175 Gly Asn Ala Thr His Asp Asn Ile Cys Ser Gly Asn Ser Glu
Ser Thr 180 185 190 Gln Lys Cys Gly Ile Asp Val Thr Leu Cys Glu Glu
Ala Phe Phe Arg 195 200 205 Phe Ala Val Pro Thr Lys Phe Thr Pro Asn
Trp Leu Ser Val Leu Val 210 215 220 Asp Asn Leu Pro Gly Thr Lys Val
Asn Ala Glu Ser Val Glu Arg Ile 225 230 235 240 Lys Arg Gln His Ser
Ser Gln Glu Gln Thr Phe Gln Leu Leu Lys Leu 245 250 255 Trp Lys His
Gln Asn Lys Asp Gln Asp Ile Val Lys Lys Ile Ile Gln 260 265 270 Asp
Ile Asp Leu Cys Glu Asn Ser Val Gln Arg His Ile Gly His Ala 275 280
285 Asn Leu Thr Phe Glu Gln Leu Arg Ser Leu Met Glu Ser Leu Pro Gly
290 295 300 Lys Lys Val Gly Ala Glu Asp Ile Glu Lys Thr Ile Lys Ala
Cys Lys 305 310 315 320 Pro Ser Asp Gln Ile Leu Lys Leu Leu Ser Leu
Trp Arg Ile Lys Asn 325 330 335 Gly Asp Gln Asp Thr Leu Lys Gly Leu
Met His Ala Leu Lys His 340 345 350 75 272 PRT Homo sapiens 75 Met
Asn Asn Leu Leu Cys Cys Ala Leu Val Phe Leu Asp Ile Ser Ile 1 5 10
15 Lys Trp Thr Thr Gln Glu Thr Phe Pro Pro Lys Tyr Leu His Tyr Asp
20 25 30 Glu Glu Thr Ser His Gln Leu Leu Cys Asp Lys Cys Pro Pro
Gly Thr 35 40 45 Tyr Leu Lys Gln His Cys Thr Ala Lys Trp Lys Thr
Val Cys Ala Pro 50 55 60 Cys Pro Asp His Tyr Tyr Thr Asp Ser Trp
His Thr Ser Asp Glu Cys 65 70 75 80 Leu Tyr Cys Ser Pro Val Cys Lys
Glu Leu Gln Tyr Val Lys Gln Glu 85 90 95 Cys Asn Arg Thr His Asn
Arg Val Cys Glu Cys Lys Glu Gly Arg Tyr 100 105 110 Leu Glu Ile Glu
Phe Cys Leu Lys His Arg Ser Cys Pro Pro Gly Phe 115 120 125 Gly Val
Val Gln Ala Gly Thr Pro Glu Arg Asn Thr Val Cys Lys Arg 130 135 140
Cys Pro Asp Gly Phe Phe Ser Asn Glu Thr Ser Ser Lys Ala Pro Cys 145
150 155 160 Arg Lys His Thr Asn Cys Ser Val Phe Gly Leu Leu Leu Thr
Gln Lys 165 170 175 Gly Asn Ala Thr His Asp Asn Ile Cys Ser Gly Asn
Ser Glu Ser Thr 180 185 190 Gln Lys Cys Gly Ile Asp Val Thr Leu Cys
Glu Glu Ala Phe Phe Arg 195 200 205 Phe Ala Val Pro Thr Lys Phe Thr
Pro Asn Trp Leu Ser Val Leu Val 210 215 220 Asp Asn Leu Pro Gly Thr
Lys Val Asn Ala Glu Ser Val Glu Arg Ile 225 230 235 240 Lys Arg Gln
His Ser Ser Gln Glu Gln Thr Phe Gln Leu Leu Lys Leu 245 250 255 Trp
Lys His Gln Asn Lys Asp Gln Asp Ile Val Lys Lys Ile Ile Gln 260 265
270 76 197 PRT Homo sapiens 76 Met Asn Asn Leu Leu Cys Cys Ala Leu
Val Phe Leu Asp Ile Ser Ile 1 5 10 15 Lys Trp Thr Thr Gln Glu Thr
Phe Pro Pro Lys Tyr Leu His Tyr Asp 20 25 30 Glu Glu Thr Ser His
Gln Leu Leu Cys Asp Lys Cys Pro Pro Gly Thr 35 40 45 Tyr Leu Lys
Gln His Cys Thr Ala Lys Trp Lys Thr Val Cys Ala Pro 50 55 60 Cys
Pro Asp His Tyr Tyr Thr Asp Ser Trp His Thr Ser Asp Glu Cys 65 70
75 80 Leu Tyr Cys Ser Pro Val Cys Lys Glu Leu Gln Tyr Val Lys Gln
Glu 85 90 95 Cys Asn Arg Thr His Asn Arg Val Cys Glu Cys Lys Glu
Gly Arg Tyr 100 105 110 Leu Glu Ile Glu Phe Cys Leu Lys His Arg Ser
Cys Pro Pro Gly Phe 115 120 125 Gly Val Val Gln Ala Gly Thr Pro Glu
Arg Asn Thr Val Cys Lys Arg 130 135 140 Cys Pro Asp Gly Phe Phe Ser
Asn Glu Thr Ser Ser Lys Ala Pro Cys 145 150 155 160 Arg Lys His Thr
Asn Cys Ser Val Phe Gly Leu Leu Leu Thr Gln Lys 165 170 175 Gly Asn
Ala Thr His Asp Asn Ile Cys Ser Gly Asn Ser Glu Ser Thr 180 185 190
Gln Lys Cys Gly Ile 195 77 143 PRT Homo sapiens 77 Met Asn Asn Leu
Leu Cys Cys Ala Leu Val Phe Leu Asp Ile Ser Ile 1 5 10 15 Lys Trp
Thr Thr Gln Glu Thr Phe Pro Pro Lys Tyr Leu His Tyr Asp 20 25 30
Glu Glu Thr Ser His Gln Leu Leu Cys Asp Lys Cys Pro Pro Gly Thr 35
40 45 Tyr Leu Lys Gln His Cys Thr Ala Lys Trp Lys Thr Val Cys Ala
Pro 50 55 60 Cys Pro Asp His Tyr Tyr Thr Asp Ser Trp His Thr Ser
Asp Glu Cys 65 70 75 80 Leu Tyr Cys Ser Pro Val Cys Lys Glu Leu Gln
Tyr Val Lys Gln Glu 85 90 95 Cys Asn Arg Thr His Asn Arg Val Cys
Glu Cys Lys Glu Gly Arg Tyr 100 105 110 Leu Glu Ile Glu Phe Cys Leu
Lys His Arg Ser Cys Pro Pro Gly Phe 115 120 125 Gly Val Val Gln Ala
Gly Thr Pro Glu Arg Asn Thr Val Cys Lys 130 135 140 78 106 PRT Homo
sapiens 78 Met Asn Asn Leu Leu Cys Cys Ala Leu Val Phe Leu Asp Ile
Ser Ile 1 5 10 15 Lys Trp Thr Thr Gln Glu Thr Phe Pro Pro Lys Tyr
Leu His Tyr Asp 20 25 30 Glu Glu Thr Ser His Gln Leu Leu Cys Asp
Lys Cys Pro Pro Gly Thr 35 40 45 Tyr Leu Lys Gln His Cys Thr Ala
Lys Trp Lys Thr Val Cys Ala Pro 50 55 60 Cys Pro Asp His Tyr Tyr
Thr Asp Ser Trp His Thr Ser Asp Glu Cys 65 70 75 80 Leu Tyr Cys Ser
Pro Val Cys Lys Glu Leu Gln Tyr Val Lys Gln Glu 85 90 95 Cys Asn
Arg Thr His Asn Arg Val Cys Glu 100 105 79 393 PRT Homo sapiens 79
Met Asn Asn Leu Leu Cys Cys Ala Leu Val Phe Leu Asp Ile Ser Ile 1 5
10 15 Lys Trp Thr Thr Gln Glu Thr Phe Pro Pro Lys Tyr Leu His Tyr
Asp 20 25 30 Glu Glu Thr Ser His Gln Leu Leu Cys Asp Lys Cys Pro
Pro Gly Thr 35 40 45 Tyr Leu Lys Gln His Cys Thr Ala Lys Trp Lys
Thr Val Cys Ala Pro 50 55 60 Cys Pro Asp His Tyr Tyr Thr Asp Ser
Trp His Thr Ser Asp Glu Cys 65 70 75 80 Leu Tyr Cys Ser Pro Val Cys
Lys Glu Leu Gln Tyr Val Lys Gln Glu 85 90 95 Cys Asn Arg Thr His
Asn Arg Val Cys Glu Cys Lys Glu Gly Arg Tyr 100 105 110 Leu Glu Ile
Glu Phe Cys Leu Lys His Arg Ser Cys Pro Pro Gly Phe 115 120 125 Gly
Val Val Gln Ala Gly Thr Pro Glu Arg Asn Thr Val Cys Lys Arg 130 135
140 Cys Pro Asp Gly Phe Phe Ser Asn Glu Thr Ser Ser Lys Ala Pro Cys
145 150 155 160 Arg Lys His Thr Asn Cys Ser Val Phe Gly Leu Leu Leu
Thr Gln Lys 165 170 175 Gly Asn Ala Thr His Asp Asn Ile Cys Ser Gly
Asn Ser Glu Ser Thr 180 185 190 Gln Lys Cys Gly Ile Asp Val Thr Leu
Cys Glu Glu Ala Phe Phe Arg 195 200 205 Phe Ala Val Pro Thr Lys Phe
Thr Pro Asn Trp Leu Ser Val Leu Val 210 215 220 Asp Asn Leu Pro Gly
Thr Lys Val Asn Ala Glu Ser Val Glu Arg Ile 225 230 235 240 Lys Arg
Gln His Ser Ser Gln Glu Gln Thr Phe Gln Leu Leu Lys Leu 245 250 255
Trp Lys His Gln Asn Lys Asp Gln Asp Ile Val Lys Lys Ile Ile Gln 260
265 270 Asp Ile Asp Leu Cys Glu Asn Ser Val Gln Arg His Ile Gly His
Ala 275 280 285 Asn Leu Thr Phe Glu Gln Leu Arg Ser Leu Met Glu Ser
Leu Pro Gly 290 295 300 Lys Lys Val Gly Ala Glu Asp Ile Glu Lys Thr
Ile Lys Ala Cys Lys 305 310 315 320 Pro Ser Asp Gln Ile Leu Lys Leu
Leu Ser Leu Trp Arg Ile Lys Asn 325 330 335 Gly Asp Gln Asp Thr Leu
Lys Gly Leu Met His Ala Leu Lys His Ser 340 345 350 Lys Thr Tyr His
Phe Pro Lys Thr Val Thr Gln Ser Leu Lys Lys Thr 355 360 365 Ile Arg
Phe Leu His Ser Phe Thr Met Tyr Lys Leu Tyr Gln Lys Leu 370 375 380
Phe Leu Glu Met Ile Gly Asn Leu Val 385 390 80 321 PRT Homo sapiens
80 Met Asn Asn Leu Leu Cys Cys Ala Leu Val Phe Leu Asp Ile Ser Ile
1 5 10 15 Lys Trp Thr Thr Gln Glu Thr Phe Pro Pro Lys Tyr Leu His
Tyr Asp 20 25 30 Glu Glu Thr Ser His Gln Leu Leu Cys Asp Lys Cys
Pro Pro Gly Thr 35 40 45 Tyr Leu Lys Gln His Cys Thr Ala Lys Trp
Lys Thr Val Cys Ala Pro 50 55 60 Cys Pro Asp His Tyr Tyr Thr Asp
Ser Trp His Thr Ser Asp Glu Cys 65 70 75 80 Leu Tyr Cys Ser Pro Val
Cys Lys Glu Leu Gln Tyr Val Lys Gln Glu 85 90 95 Cys Asn Arg Thr
His Asn Arg Val Cys Glu Cys Lys Glu Gly Arg Tyr 100 105 110 Leu Glu
Ile Glu Phe Cys Leu Lys His Arg Ser Cys Pro Pro Gly Phe 115 120 125
Gly Val Val Gln Ala Gly Thr Pro Glu Arg Asn Thr Val Cys Lys Arg 130
135 140 Cys Pro Asp Gly Phe Phe Ser Asn Glu Thr Ser Ser Lys Ala Pro
Cys 145 150 155 160 Arg Lys His Thr Asn Cys Ser Val Phe Gly Leu Leu
Leu Thr Gln Lys 165 170 175 Gly Asn Ala Thr His Asp Asn Ile Cys Ser
Gly Asn Ser Glu Ser Thr 180 185 190 Gln Lys Cys Gly Ile Asp Val Thr
Leu Cys Glu Glu Ala Phe Phe Arg 195 200 205 Phe Ala Val Pro Thr Lys
Phe Thr Pro Asn Trp Leu Ser Val Leu Val 210 215 220 Asp Asn Leu Pro
Gly Thr Lys Val Asn Ala Glu Ser Val Glu Arg Ile 225 230 235 240 Lys
Arg Gln His Ser Ser Gln Glu Gln Thr Phe Gln Leu Leu Lys Leu 245 250
255 Trp Lys His Gln Asn Lys Asp Gln Asp Ile Val Lys Lys Ile Ile Gln
260 265 270 Asp Ile Asp Leu Cys Glu Asn Ser Val Gln Arg His Ile Gly
His Ala 275 280 285 Asn Leu Thr Phe Glu Gln Leu
Arg Ser Leu Met Glu Ser Leu Pro Gly 290 295 300 Lys Lys Val Gly Ala
Glu Asp Ile Glu Lys Thr Ile Lys Ala Ser Leu 305 310 315 320 Asp 81
187 PRT Homo sapiens 81 Met Asn Asn Leu Leu Cys Cys Ala Leu Val Phe
Leu Asp Ile Ser Ile 1 5 10 15 Lys Trp Thr Thr Gln Glu Thr Phe Pro
Pro Lys Tyr Leu His Tyr Asp 20 25 30 Glu Glu Thr Ser His Gln Leu
Leu Cys Asp Lys Cys Pro Pro Gly Thr 35 40 45 Tyr Leu Lys Gln His
Cys Thr Ala Lys Trp Lys Thr Val Cys Ala Pro 50 55 60 Cys Pro Asp
His Tyr Tyr Thr Asp Ser Trp His Thr Ser Asp Glu Cys 65 70 75 80 Leu
Tyr Cys Ser Pro Val Cys Lys Glu Leu Gln Tyr Val Lys Gln Glu 85 90
95 Cys Asn Arg Thr His Asn Arg Val Cys Glu Cys Lys Glu Gly Arg Tyr
100 105 110 Leu Glu Ile Glu Phe Cys Leu Lys His Arg Ser Cys Pro Pro
Gly Phe 115 120 125 Gly Val Val Gln Ala Gly Thr Pro Glu Arg Asn Thr
Val Cys Lys Arg 130 135 140 Cys Pro Asp Gly Phe Phe Ser Asn Glu Thr
Ser Ser Lys Ala Pro Cys 145 150 155 160 Arg Lys His Thr Asn Cys Ser
Val Phe Gly Leu Leu Leu Thr Gln Lys 165 170 175 Gly Asn Ala Thr His
Asp Asn Ile Cys Ser Gly 180 185 82 84 PRT Homo sapiens 82 Met Asn
Asn Leu Leu Cys Cys Ala Leu Val Phe Leu Asp Ile Ser Ile 1 5 10 15
Lys Trp Thr Thr Gln Glu Thr Phe Pro Pro Lys Tyr Leu His Tyr Asp 20
25 30 Glu Glu Thr Ser His Gln Leu Leu Cys Asp Lys Cys Pro Pro Gly
Thr 35 40 45 Tyr Leu Lys Gln His Cys Thr Ala Lys Trp Lys Thr Val
Cys Ala Pro 50 55 60 Cys Pro Asp His Tyr Tyr Thr Asp Ser Trp His
Thr Ser Asp Glu Cys 65 70 75 80 Leu Tyr Leu Val 83 1206 DNA Homo
sapiens 83 atgaacaact tgctgtgctg cgcgctcgtg tttctggaca tctccattaa
gtggaccacc 60 caggaaacgt ttcctccaaa gtaccttcat tatgacgaag
aaacctctca tcagctgttg 120 tgtgacaaat gtcctcctgg tacctaccta
aaacaacact gtacagcaaa gtggaagacc 180 gtgtgcgccc cttgccctga
ccactactac acagacagct ggcacaccag tgacgagtgt 240 ctatactgca
gccccgtgtg caaggagctg cagtacgtca agcaggagtg caatcgcacc 300
cacaaccgcg tgtgcgaatg caaggaaggg cgctaccttg agatagagtt ctgcttgaaa
360 cataggagct gccctcctgg atttggagtg gtgcaagctg gaaccccaga
gcgaaataca 420 gtttgcaaaa gatgtccaga tgggttcttc tcaaatgaga
cgtcatctaa agcaccctgt 480 agaaaacaca caaattgcag tgtctttggt
ctcctgctaa ctcagaaagg aaatgcaaca 540 cacgacaaca tatgttccgg
aaacagtgaa tcaactcaaa aaagtggaat agatgttacc 600 ctgtgtgagg
aggcattctt caggtttgct gttcctacaa agtttacgcc taactggctt 660
agtgtcttgg tagacaattt gcctggcacc aaagtaaacg cagagagtgt agagaggata
720 aaacggcaac acagctcaca agaacagact ttccagctgc tgaagttatg
gaaacatcaa 780 aacaaagacc aagatatagt caagaagatc atccaagata
ttgacctctg tgaaaacagc 840 gtgcagcggc acattggaca tgctaacctc
accttcgagc agcttcgtag cttgatggaa 900 agcttaccgg gaaagaaagt
gggagcagaa gacattgaaa aaacaataaa ggcatgcaaa 960 cccagtgacc
agatcctgaa gctgctcagt ttgtggcgaa taaaaaatgg cgaccaagac 1020
accttgaagg gcctaatgca cgcactaaag cactcaaaga cgtaccactt tcccaaaact
1080 gtcactcaga gtctaaagaa gaccatcagg ttccttcaca gcttcacaat
gtacaaattg 1140 tatcagaagt tatttttaga aatgataggt aaccaggtcc
aatcagtaaa aataagctgc 1200 ttataa 1206 84 1206 DNA Homo sapiens 84
atgaacaact tgctgtgctg cgcgctcgtg tttctggaca tctccattaa gtggaccacc
60 caggaaacgt ttcctccaaa gtaccttcat tatgacgaag aaacctctca
tcagctgttg 120 tgtgacaaat gtcctcctgg tacctaccta aaacaacact
gtacagcaaa gtggaagacc 180 gtgtgcgccc cttgccctga ccactactac
acagacagct ggcacaccag tgacgagtgt 240 ctatactgca gccccgtgtg
caaggagctg cagtacgtca agcaggagtg caatcgcacc 300 cacaaccgcg
tgtgcgaatg caaggaaggg cgctaccttg agatagagtt ctgcttgaaa 360
cataggagct gccctcctgg atttggagtg gtgcaagctg gaaccccaga gcgaaataca
420 gtttgcaaaa gatgtccaga tgggttcttc tcaaatgaga cgtcatctaa
agcaccctgt 480 agaaaacaca caaattgcag tgtctttggt ctcctgctaa
ctcagaaagg aaatgcaaca 540 cacgacaaca tatgttccgg aaacagtgaa
tcaactcaaa aatgtggaat agatgttacc 600 ctgagtgagg aggcattctt
caggtttgct gttcctacaa agtttacgcc taactggctt 660 agtgtcttgg
tagacaattt gcctggcacc aaagtaaacg cagagagtgt agagaggata 720
aaacggcaac acagctcaca agaacagact ttccagctgc tgaagttatg gaaacatcaa
780 aacaaagacc aagatatagt caagaagatc atccaagata ttgacctctg
tgaaaacagc 840 gtgcagcggc acattggaca tgctaacctc accttcgagc
agcttcgtag cttgatggaa 900 agcttaccgg gaaagaaagt gggagcagaa
gacattgaaa aaacaataaa ggcatgcaaa 960 cccagtgacc agatcctgaa
gctgctcagt ttgtggcgaa taaaaaatgg cgaccaagac 1020 accttgaagg
gcctaatgca cgcactaaag cactcaaaga cgtaccactt tcccaaaact 1080
gtcactcaga gtctaaagaa gaccatcagg ttccttcaca gcttcacaat gtacaaattg
1140 tatcagaagt tatttttaga aatgataggt aaccaggtcc aatcagtaaa
aataagctgc 1200 ttataa 1206 85 1206 DNA Homo sapiens 85 atgaacaact
tgctgtgctg cgcgctcgtg tttctggaca tctccattaa gtggaccacc 60
caggaaacgt ttcctccaaa gtaccttcat tatgacgaag aaacctctca tcagctgttg
120 tgtgacaaat gtcctcctgg tacctaccta aaacaacact gtacagcaaa
gtggaagacc 180 gtgtgcgccc cttgccctga ccactactac acagacagct
ggcacaccag tgacgagtgt 240 ctatactgca gccccgtgtg caaggagctg
cagtacgtca agcaggagtg caatcgcacc 300 cacaaccgcg tgtgcgaatg
caaggaaggg cgctaccttg agatagagtt ctgcttgaaa 360 cataggagct
gccctcctgg atttggagtg gtgcaagctg gaaccccaga gcgaaataca 420
gtttgcaaaa gatgtccaga tgggttcttc tcaaatgaga cgtcatctaa agcaccctgt
480 agaaaacaca caaattgcag tgtctttggt ctcctgctaa ctcagaaagg
aaatgcaaca 540 cacgacaaca tatgttccgg aaacagtgaa tcaactcaaa
aatgtggaat agatgttacc 600 ctgtgtgagg aggcattctt caggtttgct
gttcctacaa agtttacgcc taactggctt 660 agtgtcttgg tagacaattt
gcctggcacc aaagtaaacg cagagagtgt agagaggata 720 aaacggcaac
acagctcaca agaacagact ttccagctgc tgaagttatg gaaacatcaa 780
aacaaagacc aagatatagt caagaagatc atccaagata ttgacctcag tgaaaacagc
840 gtgcagcggc acattggaca tgctaacctc accttcgagc agcttcgtag
cttgatggaa 900 agcttaccgg gaaagaaagt gggagcagaa gacattgaaa
aaacaataaa ggcatgcaaa 960 cccagtgacc agatcctgaa gctgctcagt
ttgtggcgaa taaaaaatgg cgaccaagac 1020 accttgaagg gcctaatgca
cgcactaaag cactcaaaga cgtaccactt tcccaaaact 1080 gtcactcaga
gtctaaagaa gaccatcagg ttccttcaca gcttcacaat gtacaaattg 1140
tatcagaagt tatttttaga aatgataggt aaccaggtcc aatcagtaaa aataagctgc
1200 ttataa 1206 86 1206 DNA Homo sapiens 86 atgaacaact tgctgtgctg
cgcgctcgtg tttctggaca tctccattaa gtggaccacc 60 caggaaacgt
ttcctccaaa gtaccttcat tatgacgaag aaacctctca tcagctgttg 120
tgtgacaaat gtcctcctgg tacctaccta aaacaacact gtacagcaaa gtggaagacc
180 gtgtgcgccc cttgccctga ccactactac acagacagct ggcacaccag
tgacgagtgt 240 ctatactgca gccccgtgtg caaggagctg cagtacgtca
agcaggagtg caatcgcacc 300 cacaaccgcg tgtgcgaatg caaggaaggg
cgctaccttg agatagagtt ctgcttgaaa 360 cataggagct gccctcctgg
atttggagtg gtgcaagctg gaaccccaga gcgaaataca 420 gtttgcaaaa
gatgtccaga tgggttcttc tcaaatgaga cgtcatctaa agcaccctgt 480
agaaaacaca caaattgcag tgtctttggt ctcctgctaa ctcagaaagg aaatgcaaca
540 cacgacaaca tatgttccgg aaacagtgaa tcaactcaaa aatgtggaat
agatgttacc 600 ctgtgtgagg aggcattctt caggtttgct gttcctacaa
agtttacgcc taactggctt 660 agtgtcttgg tagacaattt gcctggcacc
aaagtaaacg cagagagtgt agagaggata 720 aaacggcaac acagctcaca
agaacagact ttccagctgc tgaagttatg gaaacatcaa 780 aacaaagacc
aagatatagt caagaagatc atccaagata ttgacctctg tgaaaacagc 840
gtgcagcggc acattggaca tgctaacctc accttcgagc agcttcgtag cttgatggaa
900 agcttaccgg gaaagaaagt gggagcagaa gacattgaaa aaacaataaa
ggcaagcaaa 960 cccagtgacc agatcctgaa gctgctcagt ttgtggcgaa
taaaaaatgg cgaccaagac 1020 accttgaagg gcctaatgca cgcactaaag
cactcaaaga cgtaccactt tcccaaaact 1080 gtcactcaga gtctaaagaa
gaccatcagg ttccttcaca gcttcacaat gtacaaattg 1140 tatcagaagt
tatttttaga aatgataggt aaccaggtcc aatcagtaaa aataagctgc 1200 ttataa
1206 87 1206 DNA Homo sapiens 87 atgaacaact tgctgtgctg cgcgctcgtg
tttctggaca tctccattaa gtggaccacc 60 caggaaacgt ttcctccaaa
gtaccttcat tatgacgaag aaacctctca tcagctgttg 120 tgtgacaaat
gtcctcctgg tacctaccta aaacaacact gtacagcaaa gtggaagacc 180
gtgtgcgccc cttgccctga ccactactac acagacagct ggcacaccag tgacgagtgt
240 ctatactgca gccccgtgtg caaggagctg cagtacgtca agcaggagtg
caatcgcacc 300 cacaaccgcg tgtgcgaatg caaggaaggg cgctaccttg
agatagagtt ctgcttgaaa 360 cataggagct gccctcctgg atttggagtg
gtgcaagctg gaaccccaga gcgaaataca 420 gtttgcaaaa gatgtccaga
tgggttcttc tcaaatgaga cgtcatctaa agcaccctgt 480 agaaaacaca
caaattgcag tgtctttggt ctcctgctaa ctcagaaagg aaatgcaaca 540
cacgacaaca tatgttccgg aaacagtgaa tcaactcaaa aatgtggaat agatgttacc
600 ctgtgtgagg aggcattctt caggtttgct gttcctacaa agtttacgcc
taactggctt 660 agtgtcttgg tagacaattt gcctggcacc aaagtaaacg
cagagagtgt agagaggata 720 aaacggcaac acagctcaca agaacagact
ttccagctgc tgaagttatg gaaacatcaa 780 aacaaagacc aagatatagt
caagaagatc atccaagata ttgacctctg tgaaaacagc 840 gtgcagcggc
acattggaca tgctaacctc accttcgagc agcttcgtag cttgatggaa 900
agcttaccgg gaaagaaagt gggagcagaa gacattgaaa aaacaataaa ggcatgcaaa
960 cccagtgacc agatcctgaa gctgctcagt ttgtggcgaa taaaaaatgg
cgaccaagac 1020 accttgaagg gcctaatgca cgcactaaag cactcaaaga
cgtaccactt tcccaaaact 1080 gtcactcaga gtctaaagaa gaccatcagg
ttccttcaca gcttcacaat gtacaaattg 1140 tatcagaagt tatttttaga
aatgataggt aaccaggtcc aatcagtaaa aataagcagc 1200 ttataa 1206 88
1083 DNA Homo sapiens 88 atgaacaact tgctgtgctg cgcgctcgtg
tttctggaca tctccattaa gtggaccacc 60 caggaacctt gccctgacca
ctactacaca gacagctggc acaccagtga cgagtgtcta 120 tactgcagcc
ccgtgtgcaa ggagctgcag tacgtcaagc aggagtgcaa tcgcacccac 180
aaccgcgtgt gcgaatgcaa ggaagggcgc taccttgaga tagagttctg cttgaaacat
240 aggagctgcc ctcctggatt tggagtggtg caagctggaa ccccagagcg
aaatacagtt 300 tgcaaaagat gtccagatgg gttcttctca aatgagacgt
catctaaagc accctgtaga 360 aaacacacaa attgcagtgt ctttggtctc
ctgctaactc agaaaggaaa tgcaacacac 420 gacaacatat gttccggaaa
cagtgaatca actcaaaaat gtggaataga tgttaccctg 480 tgtgaggagg
cattcttcag gtttgctgtt cctacaaagt ttacgcctaa ctggcttagt 540
gtcttggtag acaatttgcc tggcaccaaa gtaaacgcag agagtgtaga gaggataaaa
600 cggcaacaca gctcacaaga acagactttc cagctgctga agttatggaa
acatcaaaac 660 aaagaccaag atatagtcaa gaagatcatc caagatattg
acctctgtga aaacagcgtg 720 cagcggcaca ttggacatgc taacctcacc
ttcgagcagc ttcgtagctt gatggaaagc 780 ttaccgggaa agaaagtggg
agcagaagac attgaaaaaa caataaaggc atgcaaaccc 840 agtgaccaga
tcctgaagct gctcagtttg tggcgaataa aaaatggcga ccaagacacc 900
ttgaagggcc taatgcacgc actaaagcac tcaaagacgt accactttcc caaaactgtc
960 actcagagtc taaagaagac catcaggttc cttcacagct tcacaatgta
caaattgtat 1020 cagaagttat ttttagaaat gataggtaac caggtccaat
cagtaaaaat aagctgctta 1080 taa 1083 89 1080 DNA Homo sapiens 89
atgaacaact tgctgtgctg cgcgctcgtg tttctggaca tctccattaa gtggaccacc
60 caggaaacgt ttcctccaaa gtaccttcat tatgacgaag aaacctctca
tcagctgttg 120 tgtgacaaat gtcctcctgg tacctaccta aaacaacact
gtacagcaaa gtggaagacc 180 gtgtgcgccg aatgcaagga agggcgctac
cttgagatag agttctgctt gaaacatagg 240 agctgccctc ctggatttgg
agtggtgcaa gctggaaccc cagagcgaaa tacagtttgc 300 aaaagatgtc
cagatgggtt cttctcaaat gagacgtcat ctaaagcacc ctgtagaaaa 360
cacacaaatt gcagtgtctt tggtctcctg ctaactcaga aaggaaatgc aacacacgac
420 aacatatgtt ccggaaacag tgaatcaact caaaaatgtg gaatagatgt
taccctgtgt 480 gaggaggcat tcttcaggtt tgctgttcct acaaagttta
cgcctaactg gcttagtgtc 540 ttggtagaca atttgcctgg caccaaagta
aacgcagaga gtgtagagag gataaaacgg 600 caacacagct cacaagaaca
gactttccag ctgctgaagt tatggaaaca tcaaaacaaa 660 gaccaagata
tagtcaagaa gatcatccaa gatattgacc tctgtgaaaa cagcgtgcag 720
cggcacattg gacatgctaa cctcaccttc gagcagcttc gtagcttgat ggaaagctta
780 ccgggaaaga aagtgggagc agaagacatt gaaaaaacaa taaaggcatg
caaacccagt 840 gaccagatcc tgaagctgct cagtttgtgg cgaataaaaa
atggcgacca agacaccttg 900 aagggcctaa tgcacgcact aaagcactca
aagacgtacc actttcccaa aactgtcact 960 cagagtctaa agaagaccat
caggttcctt cacagcttca caatgtacaa attgtatcag 1020 aagttatttt
tagaaatgat aggtaaccag gtccaatcag taaaaataag ctgcttataa 1080 90 1092
DNA Homo sapiens 90 atgaacaact tgctgtgctg cgcgctcgtg tttctggaca
tctccattaa gtggaccacc 60 caggaaacgt ttcctccaaa gtaccttcat
tatgacgaag aaacctctca tcagctgttg 120 tgtgacaaat gtcctcctgg
tacctaccta aaacaacact gtacagcaaa gtggaagacc 180 gtgtgcgccc
cttgccctga ccactactac acagacagct ggcacaccag tgacgagtgt 240
ctatactgca gccccgtgtg caaggagctg cagtacgtca agcaggagtg caatcgcacc
300 cacaaccgcg tgtgcagatg tccagatggg ttcttctcaa atgagacgtc
atctaaagca 360 ccctgtagaa aacacacaaa ttgcagtgtc tttggtctcc
tgctaactca gaaaggaaat 420 gcaacacacg acaacatatg ttccggaaac
agtgaatcaa ctcaaaaatg tggaatagat 480 gttaccctgt gtgaggaggc
attcttcagg tttgctgttc ctacaaagtt tacgcctaac 540 tggcttagtg
tcttggtaga caatttgcct ggcaccaaag taaacgcaga gagtgtagag 600
aggataaaac ggcaacacag ctcacaagaa cagactttcc agctgctgaa gttatggaaa
660 catcaaaaca aagaccaaga tatagtcaag aagatcatcc aagatattga
cctctgtgaa 720 aacagcgtgc agcggcacat tggacatgct aacctcacct
tcgagcagct tcgtagcttg 780 atggaaagct taccgggaaa gaaagtggga
gcagaagaca ttgaaaaaac aataaaggca 840 tgcaaaccca gtgaccagat
cctgaagctg ctcagtttgt ggcgaataaa aaatggcgac 900 caagacacct
tgaagggcct aatgcacgca ctaaagcact caaagacgta ccactttccc 960
aaaactgtca ctcagagtct aaagaagacc atcaggttcc ttcacagctt cacaatgtac
1020 aaattgtatc agaagttatt tttagaaatg ataggtaacc aggtccaatc
agtaaaaata 1080 agctgcttat aa 1092 91 1080 DNA Homo sapiens 91
atgaacaact tgctgtgctg cgcgctcgtg tttctggaca tctccattaa gtggaccacc
60 caggaaacgt ttcctccaaa gtaccttcat tatgacgaag aaacctctca
tcagctgttg 120 tgtgacaaat gtcctcctgg tacctaccta aaacaacact
gtacagcaaa gtggaagacc 180 gtgtgcgccc cttgccctga ccactactac
acagacagct ggcacaccag tgacgagtgt 240 ctatactgca gccccgtgtg
caaggagctg cagtacgtca agcaggagtg caatcgcacc 300 cacaaccgcg
tgtgcgaatg caaggaaggg cgctaccttg agatagagtt ctgcttgaaa 360
cataggagct gccctcctgg atttggagtg gtgcaagctg gaaccccaga gcgaaataca
420 gtttgcaaat ccggaaacag tgaatcaact caaaaatgtg gaatagatgt
taccctgtgt 480 gaggaggcat tcttcaggtt tgctgttcct acaaagttta
cgcctaactg gcttagtgtc 540 ttggtagaca atttgcctgg caccaaagta
aacgcagaga gtgtagagag gataaaacgg 600 caacacagct cacaagaaca
gactttccag ctgctgaagt tatggaaaca tcaaaacaaa 660 gaccaagata
tagtcaagaa gatcatccaa gatattgacc tctgtgaaaa cagcgtgcag 720
cggcacattg gacatgctaa cctcaccttc gagcagcttc gtagcttgat ggaaagctta
780 ccgggaaaga aagtgggagc agaagacatt gaaaaaacaa taaaggcatg
caaacccagt 840 gaccagatcc tgaagctgct cagtttgtgg cgaataaaaa
atggcgacca agacaccttg 900 aagggcctaa tgcacgcact aaagcactca
aagacgtacc actttcccaa aactgtcact 960 cagagtctaa agaagaccat
caggttcctt cacagcttca caatgtacaa attgtatcag 1020 aagttatttt
tagaaatgat aggtaaccag gtccaatcag taaaaataag ctgcttataa 1080 92 981
DNA Homo sapiens 92 atgaacaact tgctgtgctg cgcgctcgtg tttctggaca
tctccattaa gtggaccacc 60 caggaaacgt ttcctccaaa gtaccttcat
tatgacgaag aaacctctca tcagctgttg 120 tgtgacaaat gtcctcctgg
tacctaccta aaacaacact gtacagcaaa gtggaagacc 180 gtgtgcgccc
cttgccctga ccactactac acagacagct ggcacaccag tgacgagtgt 240
ctatactgca gccccgtgtg caaggagctg cagtacgtca agcaggagtg caatcgcacc
300 cacaaccgcg tgtgcgaatg caaggaaggg cgctaccttg agatagagtt
ctgcttgaaa 360 cataggagct gccctcctgg atttggagtg gtgcaagctg
gaaccccaga gcgaaataca 420 gtttgcaaaa gatgtccaga tgggttcttc
tcaaatgaga cgtcatctaa agcaccctgt 480 agaaaacaca caaattgcag
tgtctttggt ctcctgctaa ctcagaaagg aaatgcaaca 540 cacgacaaca
tatgttccgg aaacagtgaa tcaactcaaa aatgtggaat agatattgac 600
ctctgtgaaa acagcgtgca gcggcacatt ggacatgcta acctcacctt cgagcagctt
660 cgtagcttga tggaaagctt accgggaaag aaagtgggag cagaagacat
tgaaaaaaca 720 ataaaggcat gcaaacccag tgaccagatc ctgaagctgc
tcagtttgtg gcgaataaaa 780 aatggcgacc aagacacctt gaagggccta
atgcacgcac taaagcactc aaagacgtac 840 cactttccca aaactgtcac
tcagagtcta aagaagacca tcaggttcct tcacagcttc 900 acaatgtaca
aattgtatca gaagttattt ttagaaatga taggtaacca ggtccaatca 960
gtaaaaataa gctgcttata a 981 93 984 DNA Homo sapiens 93 atgaacaact
tgctgtgctg cgcgctcgtg tttctggaca tctccattaa gtggaccacc 60
caggaaacgt ttcctccaaa gtaccttcat tatgacgaag aaacctctca tcagctgttg
120 tgtgacaaat gtcctcctgg tacctaccta aaacaacact gtacagcaaa
gtggaagacc 180 gtgtgcgccc cttgccctga ccactactac acagacagct
ggcacaccag tgacgagtgt 240 ctatactgca gccccgtgtg caaggagctg
cagtacgtca agcaggagtg caatcgcacc 300 cacaaccgcg tgtgcgaatg
caaggaaggg cgctaccttg agatagagtt ctgcttgaaa 360 cataggagct
gccctcctgg atttggagtg gtgcaagctg gaaccccaga gcgaaataca 420
gtttgcaaaa gatgtccaga tgggttcttc tcaaatgaga cgtcatctaa agcaccctgt
480 agaaaacaca caaattgcag tgtctttggt ctcctgctaa ctcagaaagg
aaatgcaaca 540 cacgacaaca tatgttccgg aaacagtgaa tcaactcaaa
aatgtggaat agatgttacc 600 ctgtgtgagg aggcattctt caggtttgct
gttcctacaa agtttacgcc taactggctt 660 agtgtcttgg tagacaattt
gcctggcacc aaagtaaacg cagagagtgt agagaggata 720 aaacggcaac
acagctcaca agaacagact ttccagctgc tgaagttatg gaaacatcaa 780
aacaaagacc aagatatagt caagaagatc atccaagacg cactaaagca ctcaaagacg
840 taccactttc ccaaaactgt cactcagagt ctaaagaaga ccatcaggtt
ccttcacagc 900 ttcacaatgt acaaattgta tcagaagtta tttttagaaa
tgataggtaa ccaggtccaa 960 tcagtaaaaa taagctgctt ataa 984 94 1200
DNA Homo sapiens 94 atgaacaact tgctgtgctg cgcgctcgtg tttctggaca
tctccattaa gtggaccacc 60 caggaaacgt ttcctccaaa
gtaccttcat tatgacgaag aaacctctca tcagctgttg 120 tgtgacaaat
gtcctcctgg tacctaccta aaacaacact gtacagcaaa gtggaagacc 180
gtgtgcgccc cttgccctga ccactactac acagacagct ggcacaccag tgacgagtgt
240 ctatactgca gccccgtgtg caaggagctg cagtacgtca agcaggagtg
caatcgcacc 300 cacaaccgcg tgtgcgaatg caaggaaggg cgctaccttg
agatagagtt ctgcttgaaa 360 cataggagct gccctcctgg atttggagtg
gtgcaagctg gaaccccaga gcgaaataca 420 gtttgcaaaa gatgtccaga
tgggttcttc tcaaatgaga cgtcatctaa agcaccctgt 480 agaaaacaca
caaattgcag tgtctttggt ctcctgctaa ctcagaaagg aaatgcaaca 540
cacgacaaca tatgttccgg aaacagtgaa tcaactcaaa aatgtggaat agatgttacc
600 ctgtgtgagg aggcattctt caggtttgct gttcctacaa agtttacgcc
taactggctt 660 agtgtcttgg tagacaattt gcctggcacc aaagtaaacg
cagagagtgt agagaggata 720 aaacggcaac acagctcaca agaacagact
ttccagctgc tgaagttatg gaaacatcaa 780 aacaaagacc aagatatagt
caagaagatc atccaagata ttgacctctg tgaaaacagc 840 gtgcagcggc
acattggaca tgctaacctc accttcgagc agcttcgtag cttgatggaa 900
agcttaccgg gaaagaaagt gggagcagaa gacattgaaa aaacaataaa ggcatgcaaa
960 cccagtgacc agatcctgaa gctgctcagt ttgtggcgaa taaaaaatgg
cgaccaagac 1020 accttgaagg gcctaatgca cgcactaaag cactcaaaga
cgtaccactt tcccaaaact 1080 gtcactcaga gtctaaagaa gaccatcagg
ttccttcaca gcttcacaat gtacaaattg 1140 tatcagaagt tatttttaga
aatgataggt aaccaggtcc aatcagtaaa aataagctaa 1200 95 1056 DNA Homo
sapiens 95 atgaacaact tgctgtgctg cgcgctcgtg tttctggaca tctccattaa
gtggaccacc 60 caggaaacgt ttcctccaaa gtaccttcat tatgacgaag
aaacctctca tcagctgttg 120 tgtgacaaat gtcctcctgg tacctaccta
aaacaacact gtacagcaaa gtggaagacc 180 gtgtgcgccc cttgccctga
ccactactac acagacagct ggcacaccag tgacgagtgt 240 ctatactgca
gccccgtgtg caaggagctg cagtacgtca agcaggagtg caatcgcacc 300
cacaaccgcg tgtgcgaatg caaggaaggg cgctaccttg agatagagtt ctgcttgaaa
360 cataggagct gccctcctgg atttggagtg gtgcaagctg gaaccccaga
gcgaaataca 420 gtttgcaaaa gatgtccaga tgggttcttc tcaaatgaga
cgtcatctaa agcaccctgt 480 agaaaacaca caaattgcag tgtctttggt
ctcctgctaa ctcagaaagg aaatgcaaca 540 cacgacaaca tatgttccgg
aaacagtgaa tcaactcaaa aatgtggaat agatgttacc 600 ctgtgtgagg
aggcattctt caggtttgct gttcctacaa agtttacgcc taactggctt 660
agtgtcttgg tagacaattt gcctggcacc aaagtaaacg cagagagtgt agagaggata
720 aaacggcaac acagctcaca agaacagact ttccagctgc tgaagttatg
gaaacatcaa 780 aacaaagacc aagatatagt caagaagatc atccaagata
ttgacctctg tgaaaacagc 840 gtgcagcggc acattggaca tgctaacctc
accttcgagc agcttcgtag cttgatggaa 900 agcttaccgg gaaagaaagt
gggagcagaa gacattgaaa aaacaataaa ggcatgcaaa 960 cccagtgacc
agatcctgaa gctgctcagt ttgtggcgaa taaaaaatgg cgaccaagac 1020
accttgaagg gcctaatgca cgcactaaag cactga 1056 96 819 DNA Homo
sapiens 96 atgaacaact tgctgtgctg cgcgctcgtg tttctggaca tctccattaa
gtggaccacc 60 caggaaacgt ttcctccaaa gtaccttcat tatgacgaag
aaacctctca tcagctgttg 120 tgtgacaaat gtcctcctgg tacctaccta
aaacaacact gtacagcaaa gtggaagacc 180 gtgtgcgccc cttgccctga
ccactactac acagacagct ggcacaccag tgacgagtgt 240 ctatactgca
gccccgtgtg caaggagctg cagtacgtca agcaggagtg caatcgcacc 300
cacaaccgcg tgtgcgaatg caaggaaggg cgctaccttg agatagagtt ctgcttgaaa
360 cataggagct gccctcctgg atttggagtg gtgcaagctg gaaccccaga
gcgaaataca 420 gtttgcaaaa gatgtccaga tgggttcttc tcaaatgaga
cgtcatctaa agcaccctgt 480 agaaaacaca caaattgcag tgtctttggt
ctcctgctaa ctcagaaagg aaatgcaaca 540 cacgacaaca tatgttccgg
aaacagtgaa tcaactcaaa aatgtggaat agatgttacc 600 ctgtgtgagg
aggcattctt caggtttgct gttcctacaa agtttacgcc taactggctt 660
agtgtcttgg tagacaattt gcctggcacc aaagtaaacg cagagagtgt agagaggata
720 aaacggcaac acagctcaca agaacagact ttccagctgc tgaagttatg
gaaacatcaa 780 aacaaagacc aagatatagt caagaagatc atccaatga 819 97
594 DNA Homo sapiens 97 atgaacaact tgctgtgctg cgcgctcgtg tttctggaca
tctccattaa gtggaccacc 60 caggaaacgt ttcctccaaa gtaccttcat
tatgacgaag aaacctctca tcagctgttg 120 tgtgacaaat gtcctcctgg
tacctaccta aaacaacact gtacagcaaa gtggaagacc 180 gtgtgcgccc
cttgccctga ccactactac acagacagct ggcacaccag tgacgagtgt 240
ctatactgca gccccgtgtg caaggagctg cagtacgtca agcaggagtg caatcgcacc
300 cacaaccgcg tgtgcgaatg caaggaaggg cgctaccttg agatagagtt
ctgcttgaaa 360 cataggagct gccctcctgg atttggagtg gtgcaagctg
gaaccccaga gcgaaataca 420 gtttgcaaaa gatgtccaga tgggttcttc
tcaaatgaga cgtcatctaa agcaccctgt 480 agaaaacaca caaattgcag
tgtctttggt ctcctgctaa ctcagaaagg aaatgcaaca 540 cacgacaaca
tatgttccgg aaacagtgaa tcaactcaaa aatgtggaat atga 594 98 432 DNA
Homo sapiens 98 atgaacaact tgctgtgctg cgcgctcgtg tttctggaca
tctccattaa gtggaccacc 60 caggaaacgt ttcctccaaa gtaccttcat
tatgacgaag aaacctctca tcagctgttg 120 tgtgacaaat gtcctcctgg
tacctaccta aaacaacact gtacagcaaa gtggaagacc 180 gtgtgcgccc
cttgccctga ccactactac acagacagct ggcacaccag tgacgagtgt 240
ctatactgca gccccgtgtg caaggagctg cagtacgtca agcaggagtg caatcgcacc
300 cacaaccgcg tgtgcgaatg caaggaaggg cgctaccttg agatagagtt
ctgcttgaaa 360 cataggagct gccctcctgg atttggagtg gtgcaagctg
gaaccccaga gcgaaataca 420 gtttgcaaat ga 432 99 321 DNA Homo sapiens
99 atgaacaact tgctgtgctg cgcgctcgtg tttctggaca tctccattaa
gtggaccacc 60 caggaaacgt ttcctccaaa gtaccttcat tatgacgaag
aaacctctca tcagctgttg 120 tgtgacaaat gtcctcctgg tacctaccta
aaacaacact gtacagcaaa gtggaagacc 180 gtgtgcgccc cttgccctga
ccactactac acagacagct ggcacaccag tgacgagtgt 240 ctatactgca
gccccgtgtg caaggagctg cagtacgtca agcaggagtg caatcgcacc 300
cacaaccgcg tgtgcgaatg a 321 100 1182 DNA Homo sapiens 100
atgaacaact tgctgtgctg cgcgctcgtg tttctggaca tctccattaa gtggaccacc
60 caggaaacgt ttcctccaaa gtaccttcat tatgacgaag aaacctctca
tcagctgttg 120 tgtgacaaat gtcctcctgg tacctaccta aaacaacact
gtacagcaaa gtggaagacc 180 gtgtgcgccc cttgccctga ccactactac
acagacagct ggcacaccag tgacgagtgt 240 ctatactgca gccccgtgtg
caaggagctg cagtacgtca agcaggagtg caatcgcacc 300 cacaaccgcg
tgtgcgaatg caaggaaggg cgctaccttg agatagagtt ctgcttgaaa 360
cataggagct gccctcctgg atttggagtg gtgcaagctg gaaccccaga gcgaaataca
420 gtttgcaaaa gatgtccaga tgggttcttc tcaaatgaga cgtcatctaa
agcaccctgt 480 agaaaacaca caaattgcag tgtctttggt ctcctgctaa
ctcagaaagg aaatgcaaca 540 cacgacaaca tatgttccgg aaacagtgaa
tcaactcaaa aatgtggaat agatgttacc 600 ctgtgtgagg aggcattctt
caggtttgct gttcctacaa agtttacgcc taactggctt 660 agtgtcttgg
tagacaattt gcctggcacc aaagtaaacg cagagagtgt agagaggata 720
aaacggcaac acagctcaca agaacagact ttccagctgc tgaagttatg gaaacatcaa
780 aacaaagacc aagatatagt caagaagatc atccaagata ttgacctctg
tgaaaacagc 840 gtgcagcggc acattggaca tgctaacctc accttcgagc
agcttcgtag cttgatggaa 900 agcttaccgg gaaagaaagt gggagcagaa
gacattgaaa aaacaataaa ggcatgcaaa 960 cccagtgacc agatcctgaa
gctgctcagt ttgtggcgaa taaaaaatgg cgaccaagac 1020 accttgaagg
gcctaatgca cgcactaaag cactcaaaga cgtaccactt tcccaaaact 1080
gtcactcaga gtctaaagaa gaccatcagg ttccttcaca gcttcacaat gtacaaattg
1140 tatcagaagt tatttttaga aatgataggt aacctagtct ag 1182 101 966
DNA Homo sapiens 101 atgaacaact tgctgtgctg cgcgctcgtg tttctggaca
tctccattaa gtggaccacc 60 caggaaacgt ttcctccaaa gtaccttcat
tatgacgaag aaacctctca tcagctgttg 120 tgtgacaaat gtcctcctgg
tacctaccta aaacaacact gtacagcaaa gtggaagacc 180 gtgtgcgccc
cttgccctga ccactactac acagacagct ggcacaccag tgacgagtgt 240
ctatactgca gccccgtgtg caaggagctg cagtacgtca agcaggagtg caatcgcacc
300 cacaaccgcg tgtgcgaatg caaggaaggg cgctaccttg agatagagtt
ctgcttgaaa 360 cataggagct gccctcctgg atttggagtg gtgcaagctg
gaaccccaga gcgaaataca 420 gtttgcaaaa gatgtccaga tgggttcttc
tcaaatgaga cgtcatctaa agcaccctgt 480 agaaaacaca caaattgcag
tgtctttggt ctcctgctaa ctcagaaagg aaatgcaaca 540 cacgacaaca
tatgttccgg aaacagtgaa tcaactcaaa aatgtggaat agatgttacc 600
ctgtgtgagg aggcattctt caggtttgct gttcctacaa agtttacgcc taactggctt
660 agtgtcttgg tagacaattt gcctggcacc aaagtaaacg cagagagtgt
agagaggata 720 aaacggcaac acagctcaca agaacagact ttccagctgc
tgaagttatg gaaacatcaa 780 aacaaagacc aagatatagt caagaagatc
atccaagata ttgacctctg tgaaaacagc 840 gtgcagcggc acattggaca
tgctaacctc accttcgagc agcttcgtag cttgatggaa 900 agcttaccgg
gaaagaaagt gggagcagaa gacattgaaa aaacaataaa ggctagtcta 960 gactag
966 102 564 DNA Homo sapiens 102 atgaacaact tgctgtgctg cgcgctcgtg
tttctggaca tctccattaa gtggaccacc 60 caggaaacgt ttcctccaaa
gtaccttcat tatgacgaag aaacctctca tcagctgttg 120 tgtgacaaat
gtcctcctgg tacctaccta aaacaacact gtacagcaaa gtggaagacc 180
gtgtgcgccc cttgccctga ccactactac acagacagct ggcacaccag tgacgagtgt
240 ctatactgca gccccgtgtg caaggagctg cagtacgtca agcaggagtg
caatcgcacc 300 cacaaccgcg tgtgcgaatg caaggaaggg cgctaccttg
agatagagtt ctgcttgaaa 360 cataggagct gccctcctgg atttggagtg
gtgcaagctg gaaccccaga gcgaaataca 420 gtttgcaaaa gatgtccaga
tgggttcttc tcaaatgaga cgtcatctaa agcaccctgt 480 agaaaacaca
caaattgcag tgtctttggt ctcctgctaa ctcagaaagg aaatgcaaca 540
cacgacaaca tatgttccgg ctag 564 103 255 DNA Homo sapiens 103
atgaacaact tgctgtgctg cgcgctcgtg tttctggaca tctccattaa gtggaccacc
60 caggaaacgt ttcctccaaa gtaccttcat tatgacgaag aaacctctca
tcagctgttg 120 tgtgacaaat gtcctcctgg tacctaccta aaacaacact
gtacagcaaa gtggaagacc 180 gtgtgcgccc cttgccctga ccactactac
acagacagct ggcacaccag tgacgagtgt 240 ctatacctag tctag 255 104 1317
DNA Homo sapiens 104 ctggagacat ataacttgaa cacttggccc tgatggggaa
gcagctctgc agggactttt 60 tcagccatct gtaaacaatt tcagtggcaa
cccgcgaact gtaatccatg aatgggacca 120 cactttacaa gtcatcaagt
ctaacttcta gaccagggaa ttaatggggg agacagcgaa 180 ccctagagca
aagtgccaaa cttctgtcga tagcttgagg ctagtggaaa gacctcgagg 240
aggctactcc agaagttcag cgcgtaggaa gctccgatac caatagccct ttgatgatgg
300 tggggttggt gaagggaaca gtgctccgca aggttatccc tgccccaggc
agtccaattt 360 tcactctgca gattctctct ggctctaact accccagata
acaaggagtg aatgcagaat 420 agcacgggct ttagggccaa tcagacatta
gttagaaaaa ttcctactac atggtttatg 480 taaacttgaa gatgaatgat
tgcgaactcc ccgaaaaggg ctcagacaat gccatgcata 540 aagaggggcc
ctgtaatttg aggtttcaga acccgaagtg aaggggtcag gcagccgggt 600
acggcggaaa ctcacagctt tcgcccagcg agaggacaaa ggtctgggac acactccaac
660 tgcgtccgga tcttggctgg atcggactct cagggtggag gagacacaag
cacagcagct 720 gcccagcgtg tgcccagccc tcccaccgct ggtcccggct
gccaggaggc tggccgctgg 780 cgggaagggg ccgggaaacc tcagagcccc
gcggagacag cagccgcctt gttcctcagc 840 ccggtggctt ttttttcccc
tgctctccca ggggacagac accaccgccc cacccctcac 900 gccccacctc
cctgggggat cctttccgcc ccagccctga aagcgttaat cctggagctt 960
tctgcacacc ccccgaccgc tcccgcccaa gcttcctaaa aaagaaaggt gcaaagtttg
1020 gtccaggata gaaaaatgac tgatcaaagg caggcgatac ttcctgttgc
cgggacgcta 1080 tatataacgt gatgagcgca cgggctgcgg agacgcaccg
gagcgctcgc ccagccgccg 1140 cctccaagcc cctgaggttt ccggggacca
caatgaacaa gttgctgtgc tgcgcgctcg 1200 tggtaagtcc ctgggccagc
cgacgggtgc ccggcgcctg gggaggctgc tgccacctgg 1260 tctcccaacc
tcccagcgga ccggcgggga aaaaggctcc actcgctccc tcccaag 1317 105 10190
DNA Homo sapiens 105 gcttactttg tgccaaatct cattaggctt aaggtaatac
aggactttga gtcaaatgat 60 actgttgcac ataagaacaa acctattttc
atgctaagat gatgccactg tgttcctttc 120 tccttctagt ttctggacat
ctccattaag tggaccaccc aggaaacgtt tcctccaaag 180 taccttcatt
atgacgaaga aacctctcat cagctgttgt gtgacaaatg tcctcctggt 240
acctacctaa aacaacactg tacagcaaag tggaagaccg tgtgcgcccc ttgccctgac
300 cactactaca cagacagctg gcacaccagt gacgagtgtc tatactgcag
ccccgtgtgc 360 aaggagctgc agtacgtcaa gcaggagtgc aatcgcaccc
acaaccgcgt gtgcgaatgc 420 aaggaagggc gctaccttga gatagagttc
tgcttgaaac ataggagctg ccctcctgga 480 tttggagtgg tgcaagctgg
tacgtgtcaa tgtgcagcaa aattaattag gatcatgcaa 540 agtcagatag
ttgtgacagt ttaggagaac acttttgttc tgatgacatt ataggatagc 600
aaattgcaaa ggtaatgaaa cctgccaggt aggtactatg tgtctggagt gcttccaaag
660 gaccattgct cagaggaata ctttgccact acagggcaat ttaatgacaa
atctcaaatg 720 cagcaaatta ttctctcatg agatgcatga tggttttttt
tttttttttt aaagaaacaa 780 actcaagttg cactattgat agttgatcta
tacctctata tttcacttca gcatggacac 840 cttcaaactg cagcactttt
tgacaaacat cagaaatgtt aatttatacc aagagagtaa 900 ttatgctcat
attaatgaga ctctggagtg ctaacaataa gcagttataa ttaattatgt 960
aaaaaatgag aatggtgagg ggaattgcat ttcattatta aaaacaaggc tagttcttcc
1020 tttagcatgg gagctgagtg tttgggaggg taaggactat agcagaatct
cttcaatgag 1080 cttattcttt atcttagaca aaacagattg tcaagccaag
agcaagcact tgcctataaa 1140 ccaagtgctt tctcttttgc attttgaaca
gcattggtca gggctcatgt gtattgaatc 1200 ttttaaacca gtaacccacg
ttttttttct gccacatttg cgaagcttca gtgcagccta 1260 taacttttca
tagcttgaga aaattaagag tatccactta cttagatgga agaagtaatc 1320
agtatagatt ctgatgactc agtttgaagc agtgtttctc aactgaagcc ctgctgatat
1380 tttaagaaat atctggattc ctaggctgga ctcctttttg tgggcagctg
tcctgcgcat 1440 tgtagaattt tggcagcacc cctggactct agccactaga
taccaatagc agtccttccc 1500 ccatgtgaca gccaaaaatg tcttcagaca
ctgtcaaatg tcgccaggtg gcaaaatcac 1560 tcctggttga gaacagggtc
atcaatgcta agtatctgta actattttaa ctctcaaaac 1620 ttgtgatata
caaagtctaa attattagac gaccaatact ttaggtttaa aggcatacaa 1680
atgaaacatt caaaaatcaa aatctattct gtttctcaaa tagtgaatct tataaaatta
1740 atcacagaag atgcaaattg catcagagtc ccttaaaatt cctcttcgta
tgagtatttg 1800 agggaggaat tggtgatagt tcctactttc tattggatgg
tactttgaga ctcaaaagct 1860 aagctaagtt gtgtgtgtgt cagggtgcgg
ggtgtggaat cccatcagat aaaagcaaat 1920 ccatgtaatt cattcagtaa
gttgtatatg tagaaaaatg aaaagtgggc tatgcagctt 1980 ggaaactaga
gaattttgaa aaataatgga aatcacaagg atctttctta aataagtaag 2040
aaaatctgtt tgtagaatga agcaagcagg cagccagaag actcagaaca aaagtacaca
2100 ttttactctg tgtacactgg cagcacagtg ggatttattt acctctccct
ccctaaaaac 2160 ccacacagcg gttcctcttg ggaaataaga ggtttccagc
ccaaagagaa ggaaagacta 2220 tgtggtgtta ctctaaaaag tatttaataa
ccgttttgtt gttgctgttg ctgttttgaa 2280 atcagattgt ctcctctcca
tattttattt acttcattct gttaattcct gtggaattac 2340 ttagagcaag
catggtgaat tctcaactgt aaagccaaat ttctccatca ttataatttc 2400
acattttgcc tggcaggtta taatttttat atttccactg atagtaataa ggtaaaatca
2460 ttacttagat ggatagatct ttttcataaa aagtaccatc agttatagag
ggaagtcatg 2520 ttcatgttca ggaaggtcat tagataaagc ttctgaatat
attatgaaac attagttctg 2580 tcattcttag attctttttg ttaaataact
ttaaaagcta acttacctaa aagaaatatc 2640 tgacacatat gaacttctca
ttaggatgca ggagaagacc caagccacag atatgtatct 2700 gaagaatgaa
caagattctt aggcccggca cggtggctca catctgtaat ctcaagagtt 2760
tgagaggtca aggcgggcag atcacctgag gtcaggagtt caagaccagc ctggccaaca
2820 tgatgaaacc ctgcctctac taaaaataca aaaattagca gggcatggtg
gtgcatgcct 2880 gcaaccctag ctactcagga ggctgagaca ggagaatctc
ttgaaccctc gaggcggagg 2940 ttgtggtgag ctgagatccc tctactgcac
tccagcctgg gtgacagaga tgagactccg 3000 tccctgccgc cgcccccgcc
ttccccccca aaaagattct tcttcatgca gaacatacgg 3060 cagtcaacaa
agggagacct gggtccaggt gtccaagtca cttatttcga gtaaattagc 3120
aatgaaagaa tgccatggaa tccctgccca aatacctctg cttatgatat tgtagaattt
3180 gatatagagt tgtatcccat ttaaggagta ggatgtagta ggaaagtact
aaaaacaaac 3240 acacaaacag aaaaccctct ttgctttgta aggtggttcc
taagataatg tcagtgcaat 3300 gctggaaata atatttaata tgtgaaggtt
ttaggctgtg ttttcccctc ctgttctttt 3360 tttctgccag ccctttgtca
tttttgcagg tcaatgaatc atgtagaaag agacaggaga 3420 tgaaactaga
accagtccat tttgcccctt tttttatttt ctggttttgg taaaagatac 3480
aatgaggtag gaggttgaga tttataaatg aagtttaata agtttctgta gctttgattt
3540 ttctctttca tatttgttat cttgcataag ccagaattgg cctgtaaaat
ctacatatgg 3600 atattgaagt ctaaatctgt tcaactagct tacactagat
ggagatattt tcatattcag 3660 atacactgga atgtatgatc tagccatgcg
taatatagtc aagtgtttga aggtatttat 3720 ttttaatagc gtctttagtt
gtggactggt tcaagttttt ctgccaatga tttcttcaaa 3780 tttatcaaat
atttttccat catgaagtaa aatgcccttg cagtcaccct tcctgaagtt 3840
tgaacgactc tgctgtttta aacagtttaa gcaaatggta tatcatcttc cgtttactat
3900 gtagcttaac tgcaggctta cgcttttgag tcagcggcca actttattgc
caccttcaaa 3960 agtttattat aatgttgtaa atttttactt ctcaaggtta
gcatacttag gagttgcttc 4020 acaattagga ttcaggaaag aaagaacttc
agtaggaact gattggaatt taatgatgca 4080 gcattcaatg ggtactaatt
tcaaagaatg atattacagc agacacacag cagttatctt 4140 gattttctag
gaataattgt atgaagaata tggctgacaa cacggcctta ctgccactca 4200
gcggaggctg gactaatgaa caccctaccc ttctttcctt tcctctcaca tttcatgagc
4260 gttttgtagg taacgagaaa attgacttgc atttgcatta caaggaggag
aaactggcaa 4320 aggggatgat ggtggaagtt ttgttctgtc taatgaagtg
aaaaatgaaa atgctagagt 4380 tttgtgcaac ataatagtag cagtaaaaac
caagtgaaaa gtctttccaa aactgtgtta 4440 agagggcatc tgctgggaaa
cgatttgagg agaaggtact aaattgcttg gtattttccg 4500 taggaacccc
agagcgaaat acagtttgca aaagatgtcc agatgggttc ttctcaaatg 4560
agacgtcatc taaagcaccc tgtagaaaac acacaaattg cagtgtcttt ggtctcctgc
4620 taactcagaa aggaaatgca acacacgaca acatatgttc cggaaacagt
gaatcaactc 4680 aaaaatgtgg aataggtaat tacattccaa aatacgtctt
tgtacgattt tgtagtatca 4740 tctctctctc tgagttgaac acaaggcctc
cagccacatt cttggtcaaa cttacatttt 4800 ccctttcttg aatcttaacc
agctaaggct actctcgatg cattactgct aaagctacca 4860 ctcagaatct
ctcaaaaact catcttctca cagataacac ctcaaagctt gattttctct 4920
cctttcacac tgaaatcaaa tcttgcccat aggcaaaggg cagtgtcaag tttgccactg
4980 agatgaaatt aggagagtcc aaactgtaga attcacgttg tgtgttatta
ctttcacgaa 5040 tgtctgtatt attaactaaa gtatatattg gcaactaaga
agcaaagtga tataaacatg 5100 atgacaaatt aggccaggca tggtggctta
ctcctataat cccaacattt tggggggcca 5160 aggtaggcag atcacttgag
gtcaggattt caagaccagc ctgaccaaca tggtgaaacc 5220 ttgtctctac
taaaaataca aaaattagct gggcatggta gcaggcactt ctagtaccag 5280
ctactcaggg ctgaggcagg agaatcgctt gaacccagga gatggaggtt gcagtgagct
5340 gagattgtac cactgcactc cagtctgggc aacagagcaa gatttcatca
cacacacaca 5400 cacacacaca cacacacaca ttagaaatgt gtacttggct
ttgttaccta tggtattagt 5460 gcatctattg catggaactt ccaagctact
ctggttgtgt taagctcttc attgggtaca 5520 ggtcactagt attaagttca
ggttattcgg atgcattcca cggtagtgat gacaattcat 5580 caggctagtg
tgtgtgttca ccttgtcact cccaccacta gactaatctc agaccttcac 5640
tcaaagacac attacactaa agatgatttg cttttttgtg tttaatcaag caatggtata
5700 aaccagcttg actctcccca aacagttttt cgtactacaa agaagtttat
gaagcagaga 5760 aatgtgaatt gatatatata tgagattcta acccagttcc
agcattgttt cattgtgtaa 5820 ttgaaatcat
agacaagcca ttttagcctt tgctttctta tctaaaaaaa aaaaaaaaaa 5880
aatgaaggaa ggggtattaa aaggagtgat caaattttaa cattctcttt aattaattca
5940 tttttaattt tacttttttt catttattgt gcacttacta tgtggtactg
tgctatagag 6000 gctttaacat ttataaaaac actgtgaaag ttgcttcaga
tgaatatagg tagtagaacg 6060 gcagaactag tattcaaagc caggtctgat
gaatccaaaa acaaacaccc attactccca 6120 ttttctggga catacttact
ctacccagat gctctgggct ttgtaatgcc tatgtaaata 6180 acatagtttt
atgtttggtt attttcctat gtaatgtcta cttatatatc tgtatctatc 6240
tcttgctttg tttccaaagg taaactatgt gtctaaatgt gggcaaaaaa taacacacta
6300 ttccaaatta ctgttcaaat tcctttaagt cagtgataat tatttgtttt
gacattaatc 6360 atgaagttcc ctgtgggtac taggtaaacc tttaatagaa
tgttaatgtt tgtattcatt 6420 ataagaattt ttggctgtta cttatttaca
acaatatttc actctaatta gacatttact 6480 aaactttctc ttgaaaacaa
tgcccaaaaa agaacattag aagacacgta agctcagttg 6540 gtctctgcca
ctaagaccag ccaacagaag cttgatttta ttcaaacttt gcattttagc 6600
atattttatc ttggaaaatt caattgtgtt ggttttttgt ttttgtttgt attgaataga
6660 ctctcagaaa tccaattgtt gagtaaatct tctgggtttt ctaacctttc
tttagatgtt 6720 accctgtgtg aggaggcatt cttcaggttt gctgttccta
caaagtttac gcctaactgg 6780 cttagtgtct tggtagacaa tttgcctggc
accaaagtaa acgcagagag tgtagagagg 6840 ataaaacggc aacacagctc
acaagaacag actttccagc tgctgaagtt atggaaacat 6900 caaaacaaag
accaagatat agtcaagaag atcatccaag gtaattacat tccaaaatac 6960
gtctttgtac gattttgtag tatcatctct ctctctgagt tgaacacaag gcctccagcc
7020 acattcttgg tcaaacttac attttccctt tcttgaatct taaccagcta
aggctactct 7080 cgatgcatta ctgctaaagc taccactcag aatctctcaa
aaactcatct tctcacagat 7140 aacacctcaa agcttgattt tctctccttt
cacactgaaa tcaaatcttg cccataggca 7200 aagggcagtg tcaagtttgc
cactgagatg aaattaggag agtccaaact gtagaattca 7260 cgttgtgtgt
tattactttc acgaatgtct gtattattaa ctaaagtata tattggcaac 7320
taagaagcaa agtgatataa acatgatgac aaattaggcc aggcatggtg gcttactcct
7380 ataatcccaa cattttgggg ggccaaggta ggcagatcac ttgaggtcag
gatttcaaga 7440 ccagcctgac caacatggtg aaaccttgtc tctactaaaa
atacaaaaat tagctgggca 7500 tggtagcagg cacttctagt accagctact
cagggctgag gcaggagaat cgcttgaacc 7560 caggagatgg aggttgcagt
gagctgagat tgtaccactg cactccagtc tgggcaacag 7620 agcaagattt
catcacacac acacacacac acacacacac acacattaga aatgtgtact 7680
tggctttgtt acctatggta ttagtgcatc tattgcatgg aacttccaag ctactctggt
7740 tgtgttaagc tcttcattgg gtacaggtca ctagtattaa gttcaggtta
ttcggatgca 7800 ttccacggta gtgatgacaa ttcatcaggc tagtgtgtgt
gttcaccttg tcactcccac 7860 cactagacta atctcagacc ttcactcaaa
gacacattac actaaagatg atttgctttt 7920 ttgtgtttaa tcaagcaatg
gtataaacca gcttgactct ccccaaacag tttttcgtac 7980 tacaaagaag
tttatgaagc agagaaatgt gaattgatat atatatgaga ttctaaccca 8040
gttccagcat tgtttcattg tgtaattgaa atcatagaca agccatttta gcctttgctt
8100 tcttatctaa aaaaaaaaaa aaaaaaatga aggaaggggt attaaaagga
gtgatcaaat 8160 tttaacattc tctttaatta attcattttt aattttactt
tttttcattt attgtgcact 8220 tactatgtgg tactgtgcta tagaggcttt
aacatttata aaaacactgt gaaagttgct 8280 tcagatgaat ataggtagta
gaacggcaga actagtattc aaagccaggt ctgatgaatc 8340 caaaaacaaa
cacccattac tcccattttc tgggacatac ttactctacc cagatgctct 8400
gggctttgta atgcctatgt aaataacata gttttatgtt tggttatttt cctatgtaat
8460 gtctacttat atatctgtat ctatctcttg ctttgtttcc aaaggtaaac
tatgtgtcta 8520 aatgtgggca aaaaataaca cactattcca aattactgtt
caaattcctt taagtcagtg 8580 ataattattt gttttgacat taatcatgaa
gttccctgtg ggtactaggt aaacctttaa 8640 tagaatgtta atgtttgtat
tcattataag aatttttggc tgttacttat ttacaacaat 8700 atttcactct
aattagacat ttactaaact ttctcttgaa aacaatgccc aaaaaagaac 8760
attagaagac acgtaagctc agttggtctc tgccactaag accagccaac agaagcttga
8820 ttttattcaa actttgcatt ttagcatatt ttatcttgga aaattcaatt
gtgttggttt 8880 tttgtttttg tttgtattga atagactctc agaaatccaa
ttgttgagta aatcttctgg 8940 gttttctaac ctttctttag atattgacct
ctgtgaaaac agcgtgcagc ggcacattgg 9000 acatgctaac ctcaccttcg
agcagcttcg tagcttgatg gaaagcttac cgggaaagaa 9060 agtgggagca
gaagacattg aaaaaacaat aaaggcatgc aaacccagtg accagatcct 9120
gaagctgctc agtttgtggc gaataaaaaa tggcgaccaa gacaccttga agggcctaat
9180 gcacgcacta aagcactcaa agacgtacca ctttcccaaa actgtcactc
agagtctaaa 9240 gaagaccatc aggttccttc acagcttcac aatgtacaaa
ttgtatcaga agttattttt 9300 agaaatgata ggtaaccagg tccaatcagt
aaaaataagc tgcttataac tggaaatggc 9360 cattgagctg tttcctcaca
attggcgaga tcccatggat gagtaaactg tttctcaggc 9420 acttgaggct
ttcagtgata tctttctcat taccagtgac taattttgcc acagggtact 9480
aaaagaaact atgatgtgga gaaaggacta acatctcctc caataaaccc caaatggtta
9540 atccaactgt cagatctgga tcgttatcta ctgactatat tttcccttat
tactgcttgc 9600 agtaattcaa ctggaaatta aaaaaaaaaa actagactcc
actgggcctt actaaatatg 9660 ggaatgtcta acttaaatag ctttgggatt
ccagctatgc tagaggcttt tattagaaag 9720 ccatattttt ttctgtaaaa
gttactaata tatctgtaac actattacag tattgctatt 9780 tatattcatt
cagatataag atttggacat attatcatcc tataaagaaa cggtatgact 9840
taattttaga aagaaaatta tattctgttt attatgacaa atgaaagaga aaatatatat
9900 ttttaatgga aagtttgtag catttttcta ataggtactg ccatattttt
ctgtgtggag 9960 tatttttata attttatctg tataagctgt aatatcattt
tatagaaaat gcattattta 10020 gtcaattgtt taatgttgga aaacatatga
aatataaatt atctgaatat tagatgctct 10080 gagaaattga atgtacctta
tttaaaagat tttatggttt tataactata taaatgacat 10140 tattaaagtt
ttcaaattat tttttattgc tttctctgtt gcttttattt 10190 106 391 PRT Homo
sapiens 106 Phe Leu Asp Ile Ser Ile Lys Trp Thr Thr Gln Glu Thr Phe
Pro Pro 1 5 10 15 Lys Tyr Leu His Tyr Asp Glu Glu Thr Ser His Gln
Leu Leu Cys Asp 20 25 30 Lys Cys Pro Pro Gly Thr Tyr Leu Lys Gln
His Cys Thr Ala Lys Trp 35 40 45 Lys Thr Val Cys Ala Pro Cys Pro
Asp His Tyr Tyr Thr Asp Ser Trp 50 55 60 His Thr Ser Asp Glu Cys
Leu Tyr Cys Ser Pro Val Cys Lys Glu Leu 65 70 75 80 Gln Tyr Val Lys
Gln Glu Cys Asn Arg Thr His Asn Arg Val Cys Glu 85 90 95 Cys Lys
Glu Gly Arg Tyr Leu Glu Ile Glu Phe Cys Leu Lys His Arg 100 105 110
Ser Cys Pro Pro Gly Phe Gly Val Val Gln Ala Gly Thr Pro Glu Arg 115
120 125 Asn Thr Val Cys Lys Arg Cys Pro Asp Gly Phe Phe Ser Asn Glu
Thr 130 135 140 Ser Ser Lys Ala Pro Cys Arg Lys His Thr Asn Cys Ser
Val Phe Gly 145 150 155 160 Leu Leu Leu Thr Gln Lys Gly Asn Ala Thr
His Asp Asn Ile Cys Ser 165 170 175 Gly Asn Ser Glu Ser Thr Gln Lys
Cys Gly Ile Asp Val Thr Leu Cys 180 185 190 Glu Glu Ala Phe Phe Arg
Phe Ala Val Pro Thr Lys Phe Thr Pro Asn 195 200 205 Trp Leu Ser Val
Leu Val Asp Asn Leu Pro Gly Thr Lys Val Asn Ala 210 215 220 Glu Ser
Val Glu Arg Ile Lys Arg Gln His Ser Ser Gln Glu Gln Thr 225 230 235
240 Phe Gln Leu Leu Lys Leu Trp Lys His Gln Asn Lys Asp Gln Asp Ile
245 250 255 Val Lys Lys Ile Ile Gln Asp Ile Asp Leu Cys Glu Asn Ser
Val Gln 260 265 270 Arg His Ile Gly His Ala Asn Leu Thr Phe Glu Gln
Leu Arg Ser Leu 275 280 285 Met Glu Ser Leu Pro Gly Lys Lys Val Gly
Ala Glu Asp Ile Glu Lys 290 295 300 Thr Ile Lys Ala Cys Lys Pro Ser
Asp Gln Ile Leu Lys Leu Leu Ser 305 310 315 320 Leu Trp Arg Ile Lys
Asn Gly Asp Gln Asp Thr Leu Lys Gly Leu Met 325 330 335 His Ala Leu
Lys His Ser Lys Thr Tyr His Phe Pro Lys Thr Val Thr 340 345 350 Gln
Ser Leu Lys Lys Thr Ile Arg Phe Leu His Ser Phe Thr Met Tyr 355 360
365 Lys Leu Tyr Gln Lys Leu Phe Leu Glu Met Ile Gly Asn Gln Val Gln
370 375 380 Ser Val Lys Ile Ser Cys Leu 385 390 107 20 DNA
Artificial Sequence Synthetic Sequence 107 cargarcara cnttycaryt 20
108 21 DNA Artificial Sequence Synthetic Sequence 108 yttrtacatn
gtraanswrt g 21
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