VEGF-LIKE FACTOR ANTIBODIES AND METHODS OF USE THEREOF

Abstract

A novel human gene having a significant homology with a VEGF-C gene, a member of the VEGF family, has been isolated by the PCR method using primers designed based on the sequence of EST that is assumed to be homologous with the C-terminal region of the VEGF-C gene. Mouse and rat genes have been isolated based on the human gene isolated as above. A protein encoded by the above human gene has been isolated by introducing the gene into Escherichia coli and expressing it. The isolated protein and genes can be applied to, for example, gene therapy for the VEGF-D deficiency, wound healing, and promotion of collateral vessel formation. Furthermore, VEGF-D protein inhibitors can be used as a novel anticancer drug, etc.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application is a divisional of U.S. patent application Ser. No. 11/397,289, filed Apr. 4, 2006, which is a continuation of U.S. patent application Ser. No. 10/978,107, filed Oct. 29, 2004, which, in turn, is a divisional of U.S. patent application Ser. No. 09/214,982, filed Jan. 14, 1999, now U.S. Pat. No. 6,828,426, which is the U.S. national stage application of International Application No. PCT/JP97/02456, filed Jul. 15, 1997, which, in turn, claims the benefit of Japanese Patent Application No. 8/185216, filed Jul. 15, 1996.

TECHNICAL FIELD

[0002] The present invention relates to a protein factor involved in angiogenesis in humans and falls in the field of genetic engineering.

BACKGROUND ART

[0003] The process of angiogenesis, in which endothelial cells existing in the inner wall of blood vessels of animals generate new blood vessels, is triggered by transduction of a specific signal. A variety of substances are reportedly involved in this signal transduction. The most notable substance among them is the vascular endothelial growth factor (VEGF). VEGF is a protein factor which was isolated and purified, and can increase the proliferation of endothelial cells and the permeability of blood vessels (Senger, D. R. et al., Science 219: 983-985 (1983); Ferrara, N. and Henzel, W. J., Biochem. Biophys. Res. Commun. 161: 851-858 (1989)). It has been reported that the human VEGF gene contains eight exons and produces four subtypes consisting of 121, 165, 189, or 206 amino acid residues, depending on the difference in splicing, which causes different secretion patterns (Houck, K. A. et al., Mol. Endocrinol. 5: 1806-1814 (1991)). It has also been reported that there is a VEGF-specific receptor, flt-1, and that the binding of VEGF to flt-1 is important for the signal transduction (Vries, C. D. et al., Science 255: 989-991 (1992)).

[0004] Placental growth factor (PlGF) and platelet-derived growth factor (PDGF) have thus far been isolated and are factors related to VEGF. These factors are found to promote proliferation activities of vascular endothelial cells (Maglione, D. et al., Proc. Natl. Acad. Sci. USA 88: 9267-9271 (1991); Betsholtz, C. et al., Nature 320: 695-699 (1986)). In addition, VEGF-B (Olofsson, B. et al., Proc. Natl. Acad. Sci. USA 93: 2576-2581 (1996)) and VEGF-C (Lee, J. et al., Proc. Natl. Acad. Sci. USA 93: 1988-1992 (1996); Joukov, V. et al., EMBO J. 15, 290-299 (1996)) have recently been isolated.

[0005] These factors appear to constitute a family, and this may contain additional unknown factors.

[0006] It has been suggested that VEGF is involved in not only vascular formation at the developmental stage but also in the pathological neovascularization associated with diabetes, rheumatoid arthritis, retinopathy, and the growth of solid tumors. Furthermore, in addition to its vascular endothelial cell growth-promoting effects listed above, VEGF's ability to increase vascular permeability was suggested to be involved in the edema formation resulting from various causes. Also, these VEGF family factors may act on not only the blood vessels but also the blood cells and the lymphatic vessels. They may thus play a role in the differentiation and proliferation of blood cells and the formation of lymphatic vessels. Consequently, the VEGF family factors are presently drawing extraordinary attention for developing useful, novel drugs.

DISCLOSURE OF THE INVENTION

[0007] An objective of the present invention is to isolate a novel protein belonging to the VEGF family and a gene encoding the protein. We searched for genes having homology to VEGF-C, which is a recently cloned VEGF family gene, against Expressed Sequence Tags (EST) and Sequence Tagged Sites (STS) in the GenBank database. As a result, we found an EST that was assumed to have homology to the C-terminal portion of VEGF-C. We then designed primers based on the sequence, and amplified and isolated the corresponding cDNA using the 5' RACE method and the 3' RACE method. The nucleotide sequence of the isolated cDNA was determined, and the deduced amino acid sequence therefrom revealed that the amino acid sequence had significant homology to that of VEGF-C. Based on the homology, we have assumed that the isolated human clone is a fourth member of the VEGF family (hereinafter designated as VEGF-D). We have also succeeded in expressing the protein encoded by the isolated human VEGF-D gene in E. coli cells, and have also purified and isolated it. Furthermore, we have succeeded in isolating the mouse and rat VEGF-D genes using the isolated human VEGF-D gene.

[0008] In particular, the present invention relates to a novel protein belonging to the VEGF family and a gene encoding the protein. More specifically it relates to

(1) A protein shown by SEQ ID NO. 1 or having the amino acid sequence derived therefrom in which one or more amino acids are substituted, deleted, or added; (2) A protein encoded by a DNA that hybridizes with the DNA shown by SEQ ID NO. 2; (3) A DNA encoding the protein of (1); (4) A DNA hybridizing with the DNA shown by SEQ ID NO. 2; (5) A vector containing the DNA of (3) or (4); (6) A transformant carrying the vector of (5); (7) A method of producing the protein of (1) or (2), which comprises culturing the transformant of (6); (8) An antibody binding to the protein of (1) or (2); (9) A method of screening a compound binding to the protein of (1) or (2), which comprises a step of detecting the activity of the protein of (1) or (2) to bind to a test sample; and (10) A compound binding to the protein of (1) or (2), wherein said compound has been isolated by the method of (9).

[0009] The protein of the present invention (VEGF-D) has significant homology to VEGF-C and can be considered to be a fourth factor of the VEGF family. Since the major function of VEGF is vascular formation at the developmental stage and VEGF is considered to be involved in the pathological neovascularization associated with diabetes, rheumatoid arthritis, retinopathy, and the growth of solid tumors, the protein of the present invention is thought to have similar functions.

[0010] A person skilled in the art could prepare functionally equivalent proteins through modifying VEGF-D of the present invention by adding, deleting, or substituting one or more of the amino acids of VEGF-D shown by SEQ ID NO. 1 using known methods. Modifications of the protein can also occur naturally in addition to the artificial modifications described above. These modified proteins are also included in the present invention. Known methods for adding, deleting, or substituting amino acids include the overlap extension polymerase chain reaction (OE-PCR) method (Gene, 1989, 77 (1): 51).

[0011] The DNA encoding VEGF-D of the present invention, shown by SEQ ID NO. 2, is useful for isolating DNAs encoding the proteins having similar functions to VEGF-D in other organisms. For example, a person skilled in the art could routinely isolate homologs of human VEGF-D of the present invention from other organisms by allowing the DNA shown by SEQ ID NO. 2, or part thereof, as a probe, to hybridize with the DNA derived from other organisms. The DNA that hybridizes with the DNA shown by SEQ ID NO. 2 is also included in the present invention. The other organisms include mice, rats, and rabbits.

[0012] The DNA encoding a protein that is functionally equivalent to VEGF-D usually has high homology to the DNA shown by SEQ ID NO. 2. The high homology used herein means at least 70% or higher, more preferably 80% or higher, and still more preferably 90% or higher of sequence homology.

[0013] An example of the hybridization conditions for isolating the DNA having high homology will be given below. Prehybridization is performed in ExpressHyb Solution at 68° C. for 30 minutes. The probe labeled with a radioisotope is denatured at 95° C. to 100° C. for 2 to 5 minutes and rapidly chilled on ice. The probe is added to a new ExpressHyb Solution. The blot is transferred to the solution containing the probe and allowed to hybridize under a temperature gradient of 68° C. to 55° C. for 2 hours. The blot is washed four times, for 10 minute each, with a 2×SSC solution containing 0.05% SDS at room temperature. The blot is then washed with a 0.1×SSC solution containing 0.1% SDS at 45° C. for 3 minutes. The blot is subjected to autoradiography.

[0014] An example of the hybridization conditions for isolating the DNA having very high homology will be given below. Prehybridization is performed in ExpressHyb Solution at 68° C. for 30 minutes. The probe labeled with a radioisotope is denatured at 95° C. to 100° C. for 2 to 5 minutes and rapidly chilled on ice. The probe is added into a new ExpressHyb Solution. The blot is transferred into the solution containing the probe, and allowed to hybridize at 68° C. for 1 hour. The blot was washed four times, for 10 minute each, with a 2×SSC solution containing 0.05% SDS at room temperature. The blot was then washed with a 0.1×SSC solution containing 0.1% SDS at 50° C. for 40 minutes, during which the solution was replaced once. The blot was then subjected to autoradiography.

[0015] Note that the hybridization condition can vary depending on the length of the probe (whether it is an oligomer or a probe with more than several hundred bases), the labeling method (whether the probe is radioisotopically labeled or non-radioisotopically labeled), and the type of the target gene to be cloned. A person skilled in the art would properly select the suitable hybridization conditions. In the present invention, it is especially desirable that the condition does not allow the probe to hybridize with the DNA encoding VEGF-C.

[0016] The DNA of the present invention is also used to produce VEGF-D of the present invention as a recombinant protein. Specifically, the recombinant protein can be produced in large quantity by incorporating the DNA encoding VEGF-D (for example, the DNA shown by SEQ ID NO. 2) into a suitable expression vector, introducing the resulting vector into a host, and culturing the transformant to allow the recombinant protein to be expressed.

[0017] The vector to be used for producing the recombinant protein is not particularly restricted. However, vectors such as pGEMEX-1 (Promega) or pEF-BOS (Nucleic Acids Res. 1990, 18 (17): p. 5322) are preferable. Suitable examples of the host into which the vector is introduced include E. coli cells, CHO cells, and COS cells.

[0018] The VEGF-D protein expressed by the transformant can be purified by suitably combining purification treatments such as solubilization with a homogenizer or a sonicator, extraction by various buffers, solubilization or precipitation by acid or alkali, extraction or precipitation with organic solvents, salting out by ammonium sulfate and other agents, dialysis, ultrafiltration using membrane filters, gel filtration, ion exchange chromatography, reversed-phase chromatography, counter-current distribution chromatography, high-performance liquid chromatography, isoelectric focusing, gel electrophoresis, or affinity chromatography in which antibodies or receptors are immobilized.

[0019] Once the recombinant protein is obtained, antibodies against it can be prepared using known methods. The known methods include preparing polyclonal antibodies by immunizing rabbits, sheep, or other animals with the purified protein, and preparing monoclonal antibodies from the antibody-producing cells of immunized mice or rats. These antibodies will make it possible to quantify VEGF. Although the antibodies thus obtained can be used as they are, it will be more effective to use the humanized antibodies to reduce the immunogenicity. The methods of humanizing the antibodies include the CDR graft method and the method of directly producing a human antibody. In the CDR Graft method, the antibody gene is cloned from the monoclonal antibody-producing cells and its antigenic determinant portion is transplanted into an existing human antibody. In the method of directly producing a human antibody, a mouse whose immune system has been replaced by the human immune system is immunized, similar to ordinary monoclonal antibodies. The VEGF-D protein or its antibody thus obtained can be administered into the body by subcutaneous injection or a similar method.

[0020] A person skilled in the art could screen compounds that bind to the protein of the present invention by known methods.

[0021] For example, such compounds can be obtained by making a cDNA library on a phage vector (such as λgt11 and ZAP) from the cells expected to express the protein that binds to the protein of the present invention (such as lung, small intestine, and heart cells of mammals), expressing the cDNAs on LB-agarose, fixing the expressed proteins onto a filter, preparing the purified protein of the present invention as a biotin-labeled or a fusion protein with the GST protein, and reacting this protein with the above filter. The desired compounds could then be detected by west western blotting using streptavidin or an anti-GST antibody (Skolnik, E. Y., Margolis, B., Mohammadi, M., Lowenstein, E., Fischer, R., Drepps, A., Ullrich, A., and Schlessinger, J. (1991) Cloning of P13 kinase-associated p85 utilizing a novel method for expression/cloning of target proteins for receptor tyrosine kinases, Cell 65: 83-90). Another method comprises the following steps. First, express the protein of the present invention fused with the SRF binding domain or the GAL4 binding domain in yeast cells. Second, prepare a cDNA library which expresses cDNAs fused with the transcription activation domain of VP16 or GAL4 from the cells expected to express a protein that binds to the protein of the present invention. Third, introduce the cDNA into the above yeast cells. Fourth, isolate the library-derived cDNA from the positive clones. Finally, introduce the isolated cDNA into E. coli to allow it to be expressed. (When a protein that binds to the protein of the present invention is expressed in yeast cells, the reporter gene is activated and the positive clone can be detected.) This method can be performed using the two-hybrid system (MATCHMAKER Two-Hybrid System, Mammalian MATCHMAKER Two-Hybrid Assay Kit, or MATCHMAKER One-Hybrid System (all by Clontech) or the HybriZAP Two-Hybrid Vector System (Stratagene) (Dalton, S. and Treisman, R. (1992) Characterization of SAP-1, a protein recruited by serum response factor to the c-fos serum response element, Cell 68: 597-612). Alternatively, the binding proteins can be screened by preparing a cDNA library from the cells expected to express a substance, such as a receptor, which binds to the protein of the present invention (for example, vascular endothelial cells, bone marrow cells, or lymph duct cells), introducing it into such cells as COS, detecting the binding of the protein of the present invention by itself or labeled with a radioisotope or a fluorescence, and cloning proteins that bind to the protein of the present invention (Yamasaki, K., Taga, T., Hirata, Y., Yawata, H., Kawanishi, Y., Seed, B., Taniguchi, T., Hirano, T., and Kishimoto, T. (1988) Cloning and expression of human interleukin-6 (BSF-2/IFN beta2) receptor, Science 241: 825-828, Fukunaga, R., Ishizaka-Ikeda, E., Seto, Y., and Nagata, S. (1990) Expression cloning of a receptor for murine granulocyte colony-stimulating factor, Cell 61: 341-350). Still another method comprises applying the culture supernatant or the cellular extract of the cells expected to express a protein that binds to the protein of the present invention onto an affinity column to which the protein of the present invention has been immobilized, and purifying the proteins specifically bound to the column. In addition, a DNA encoding the protein that binds to the protein of the present invention can be obtained by determining the amino acid sequence of the binding protein, synthesizing oligonucleotides based on the sequence, and screening a cDNA library with the oligonucleotides as probes.

[0022] Furthermore, compounds that bind to the protein of the present invention can be screened by contacting compounds, a natural substance bank, or a random phage peptide display library with the immobilized protein of the present invention and detecting the molecules bound to the protein. These compounds can also be screened by high throughput screening utilizing combinatorial chemistry technology (Wrighton, N. C., Farrell, F. X., Chang, R., Kashyap, A. K., Barbone, F. P., Mulcahy, L. S., Johnson, D. L., Barrett, R. W., Jolliffe, L. K., and Dower, W. J., Small peptides as potent mimetics of the protein hormone erythropoietin, Science (United States) Jul. 26, 1996, 273: 458-464, Verdine, G. L., The combinatorial chemistry of nature, Nature (England) Nov. 7, 1996, 384: 11-13, Hogan, J. C. Jr. Directed combinatorial chemistry, Nature (England) Nov. 7, 1996, 384: 17-19).

[0023] VEGF-D of the present invention may be used for gene therapy by introducing the VEGF-D gene into the body of the patient with the VEGF-D deficiency, or expressing the gene in the body. An anti-sense DNA of the VEGF-D gene may also be used to inhibit the expression of the gene itself, thereby suppressing the pathological neovascularization.

[0024] Among the many available methods to introduce the VEGF-D gene or its antisense DNA into the body, the retrovirus method, the liposome method, the cationic liposome method, and the adenovirus method are preferable.

[0025] In order to express these genes in the body, the genes can be incorporated into a suitable vector and introduced into the body by the retrovirus method, the liposome method, the cationic liposome method, or the adenovirus method. Although the vectors to be used are not particularly limited, such vectors as pAdexlcw and pZIPneo are preferable.

[0026] The present invention may also be applied for diagnosing disorders caused by abnormalities of the VEGF-D gene, for example, by PCR to detect an abnormality of the nucleotide sequence of the VEGF-D gene.

[0027] Furthermore, according to the present invention, the VEGF-D protein or its agonists can be used to heal wounds, promote collateral, vessel formation, and aid hematopoiesis by the hematopoietic stem cells, by taking advantage of the angiogenic effect of the VEGF-D protein. The antibodies against the VEGF-D protein or its antagonists can be used as the therapeutic agents for pathological neovascularization, lymphatic dysplasia, dyshematopoiesis, or edemas arising from various causes. The anti-VEGF-D antibodies can be used for diagnosing diseases resulting from abnormal production of VEGF-D by quantifying VEGF-D.

BRIEF DESCRIPTION OF THE DRAWINGS

[0028] FIG. 1 shows the relationship among the VEGF-D gene, the EST sequences, and the primers used for cloning.

[0029] FIG. 2 compares the amino acid sequences of EST (H24828) and VEGF-C.

[0030] FIG. 3 compares the amino acid sequences deduced from the VEGF-D gene and from the known genes of the VEGF family proteins.

[0031] FIG. 4A shows the hydrophobicity plot of VEGF-D. FIG. 4b shows the prediction of the cleavage site of the VEGF-D signal peptide.

BEST MODE FOR IMPLEMENTING THE INVENTION

[0032] The following examples illustrate the present invention in detail, but are not to be construed to limit the scope of the invention.

Example 1

Homology Search by TFASTA Method

[0033] The sequence CGPNKELDENTCQCVC (SEQ ID NO. 3) was designed based on the consensus sequence found in the BR3P (Balbiani ring 3 protein) repeat at the C-terminus of VEGF-C. The entire ESTs and STS sequences in the Genbank database (as of 29 Feb. 1996) were then searched by the TFASTA method (Person and Lipman, Proc. Natl. Acad. Sci. USA 85: 2444-2448 (1988)). The searching conditions used are shown below (Table 1).

TABLE-US-00001 TABLE 1 Sequences 392,210 Symbols 135,585,305 Word Size 2 Gap creation penalty 12.0 Gap extension penalty 4.0

[0034] As a result, an EST (Accession No. H29828) that is considered to code the consensus sequence was found. The sequence is one of the ESTs registered by The WashU-Merck EST Project, and nine out of 16 amino acid residues were identical. Further searching for UniGene by NCBI based on this sequence revealed that five registered sequences (T64199, H24780, H24633, H24828, and T64277 (as of 1 Mar. 1996)), including the above EST, were considered to be derived from the same gene. T69277 and T64199, as well as H24828 and H24780, are the combination of the 5' sequence and the 3' sequence of the same clones, and the length of the insert in both of these clones was 0.9 kb (FIG. 1).

[0035] Translating the H24828 sequence into a protein sequence in a frame where homology is found suggested that this sequence codes 104 C-terminal amino acid residues. Comparing this amino acid sequence with the C-terminus of VEGF-C, 28 out of 104 amino acids (27%) were identical. Moreover, the amino acids that are important for maintaining the protein structure, such as cysteine and proline, were well conserved (FIG. 2). Conserved sequences are shown in a black box.

Example 2

cDNA Cloning from a Library

[0036] Primers for 5' RACE and 3' RACE (5' RACE primer: 5'-AGGGATGGGGAACTTGGAACGCTGAAT-3'(SEQ ID NO. 4), 3' RACE primer: 5'-GATCTAATCCAGCACCCCAAAAACTGC-3' (SEQ ID NO. 5)) were designed (FIG. 1). A double-stranded cDNA was synthesized from human lung-derived polyA.sup.+ RNA using reverse transcriptase. PCR was then performed using Marathon-Ready cDNA, Lung (Chlontech), having an adapter cDNA ligated to both ends as a template cDNA, and using the above primer and adapter primer (AP-1 primer) as primers. The above adapter cDNA contains the regions to which the adapter primers AP1 and AP-2 hybridize. The PCR was performed in a manner such that the system was exposed to treatment at 94° C. for 1 min; five cycles of treatment at 94° C. for 30 sec and at 72° C. for 4 min; five cycles of treatment at 94° C. for 30 sec and at 70° C. for 4 min; then 25 cycles of treatment at 94° C. for 20 sec and at 68° C. for 4 min. (TaKaRa Ex Taq (Takara Shuzo) and the attached buffer were used as Tag polymerase instead of Advantage KlenTaq Polymerase Mix.) As a result, 1.5 kb fragments were amplified at the 5' region and 0.9 kb fragments at the 3' region. These fragments were cloned with the pCR-Direct Cloning System (Clontech), CR-TRAP Cloning System (GenHunter), and PT7Blue-T vector (Novagen). When the 5'-RACE fragment was cloned into the pCR-Direct vector, the fragment was amplified again using 5'-CTGGTTCGGCCCAGAACTTGGAACGCTGAATCA-3' (SEQ No. 7) and 5'-CTCGCTCGCCACTAATACGACTCACTATAGG-3' (SEQ ID NO. 8) as primers.

Example 3

Nucleotide Sequence Analysis

[0037] ABI PRISM Dye Terminator Cycle Sequencing Ready Reaction Kit with Amplitaq DNA Polymerase FS and 377 A DNA Sequencer (ABI) were used for DNA sequencing. The primers used are the primers in the vectors (5'-AATTAACCCTCACTAAAGGG-3' (SEQ ID NO. 9), 5'-CCAGGGTTTTCCCAGTCACGAC-3' (SEQ ID NO. 10)), AP-2 primer (5'-ACTCACTATAGGGCTCGAGCGGC-3' (SEQ ID NO. 11)), and 10 primers in the sequence shown below (Table 2).

TABLE-US-00002 TABLE 2 SQ1 (SEQ ID NO. 12) 5'-AAGTCTGGAGACCTGCT-3' SQ2 (SEQ ID NO. 13) 5'-CAGCAGGTCTCCAGACT-3' SQ3 (SEQ ID NO. 14) 5'-CGCACCCAAGGAATGGA-3' SQ4 (SEQ ID NO. 15) 5'-TGACACCTGGCCATTCCA-3' SQ5 (SEQ ID NO. 16) 5'-CATCAGATGGTAGTTCAT-3' SQ6 (SEQ ID NO. 17) 5'-ATGCTGAGCGAGAGTCCATA-3' SQ7 (SEQ ID NO. 18) 5'-CACTAGGTTTGCGGCAACTT-3' SQ8 (SEQ ID NO. 19) 5'-GCTGTTGGCAAGCACTTACA-3' SQ9 (SEQ ID NO. 20) 5'-GATCCATCCAGATCCCTGAA-3' SQ10 (SEQ ID NO. 21) 5'-CAGATCAGGGCTGCTTCTA-3'

[0038] Determining the nucleotide sequence of the 1.5 kb fragment at the 5'-side and the 0.9 kb fragment at the 3'-side revealed that the sequence of the overlapping region was identical, confirming that 5'- and 3'-side cDNAs of the desired gene were obtained. Determining the entire nucleotide sequence of the cDNA revealed that this novel gene has the full length of 2 kb and can code a protein consisting of 354 amino acid residues (SEQ ID NO. 1 and SEQ ID NO. 2). FIG. 1 shows the relation between this gene and the EST sequences registered in the Genbank database. Comparing the amino acid sequence with other VEGF family proteins revealed that the amino acids that are well conserved between family proteins are also conserved in this novel gene, and therefore this gene is obviously a new member of the VEGF family (FIG. 3). In FIG. 3, HSVEGF indicates human VEGF; HSVEGF-D, HSVEGF-C, and HSVEGF-B indicate human VEGF homologues (human VEGF-D, human VEGF-C, and human VEGF-B, respectively); HSPDGF-A indicates human PDGF-A; HSPDGF-B indicates human PDGF-B; and HSP1GF2 indicates human P1GF2. The conserved sequences are shown in a black box. Since VEGF-D is highly homologous to VEGF-C that was cloned as the Flt4 ligand, it was presumed to be a ligand to a Flt-4-like receptor.

[0039] Deducing the signal peptide cleavage site (FIG. 9b) by hydrophobicity plot (FIG. 4A) and the method of von Heijne (von Heijne, G, Nucleic Acids Res. 14, 4683-4690 (1986)), N-terminal 21 amino acid residues may be cleaved as signal peptides, and they may also undergo additional processing like VEGF-C.

Example 4

Northern Blot Analysis

[0040] A 1 kb fragment, which had been cut out by digestion with EcoRI from the 5'-fragment subcloned into pCR-Direct vector, was labeled with [α-³²P]dCTP and used as a probe. Labeling was performed by random priming using Ready-to Go DNA labeling beads (Pharmacia). Hybridization was performed in ExpressHyb Hybridization Solution (Clontech) by the usual method using Multiple Tissue Northern (MTN) Blot-Human, Human II, Human Fetal, and Human Cell lines (Clontech). Significant expression was observed in lung, heart, and intestine. Weak expression was observed in skeletal muscle, ovary, colon, and pancreas. The apparent molecular weight of the mRNA was 2.2 kb, and the cloned fragment seemed to be almost the full length of the gene.

Example 5

VEGF-D Protein Expression in E. coli

[0041] Two primers, 5'-TCCAGATCTTTTGCGGCAACTTTCTATGACAT-3' (SEQ ID NO. 22) and 5'-CAGGTCGACTCAAACAGGCACTAATTCAGGTAC-3 (SEQ ID NO. 23), were synthesized to amplify the region corresponding to the 89th to 181st amino acid residues of human VEGF cDNA. The thus-obtained DNA fragment was digested with restriction enzymes BglII and SalI, and ligated using ligation kit II (Takara Shuzo Co., Ltd) to plasmid pQE42 (QIAGEN), which had been digested with restriction enzymes BamHI and SalI. The resulting plasmid was introduced into E. coli SG19003 [pREP4] (QIAGEN), and a plasmid, which was obtained as designed without any mutation, was selected (pQE42-BS3). Plasmid pQE42-BS3 was introduced into E. coli BL21 (Invitorogen) and cultured in 10 ml of L Broth containing 100 mg/l bicucilline (ampicillin sodium for injection, Meiji Seika Kaisha, Ltd.). 200 ml of fresh L Broth was then inoculated with the culture. After incubation at 37° C. for 1.5 hours, IPTG was added to 3 mM, and the culture was further incubated at 37° C. for 5 hours. After cells were harvested, a protein was purified with a Ni-NTA column following the protocol of QIAexpress TypeII kit.

Example 6

Expression of DHFR-VEGF-D Fusion Protein in E. coli

[0042] The region corresponding to the 89th to 181st amino acid residues of human VEGF cDNA was amplified with the same primers used in Example 5. The thus-obtained DNA fragment was digested with restriction enzymes BglI and SalI. The fragment was then ligated using ligation kit II (Takara Shuzo Co., Ltd.) to the plasmid pQE40 (QIAGEN), which had been digested with restriction enzymes BamHI and SalI. The resulting plasmid was introduced into E. coli SG19003 [pREP4] (QIAGEN), and a plasmid, which was obtained as designed without any mutation, was selected (pQE40-BS3). Plasmid pQE40-BS3 was introduced into E. coli BL21 (Invitrogen) and cultured in 10 ml of L Broth containing 100 mg/l bicucilline (ampicillin sodium for injection, Meiji Seika Kaisha, Ltd.). 200 ml of fresh L Broth was then inoculated with the culture. After incubation at 37° C. for 1.5 hours, IPTG was added to 3 mM, and the culture was further incubated at 37° C. for 5 hours. After cells were harvested, a DHFR-VEGF-D fusion protein was purified with a Ni-NTA column following the protocol of a QIAexpress TypeII kit.

Example 7

Cloning Mouse VEGF-D cDNA

[0043] Two Hybond-N+ (Amersham) filters (20 cm×22 cm) on which 1.5×10⁵ pfu of Mouse lung 5'-stretch cDNA library was transferred were prepared. Gradient hybridization from 68° C. to 55° C. was performed for 2 hours in ExpressHyb Hybridization Solution (Clontech) using as a probe an approximately 50 ng Pvu II fragment of human VEGF-D, which had been labeled with α³²P-dCTP (Amersham) using Ready-To-Go DNA Labeling Beads (-dCTP) (Pharmacia). The filters were washed four times in 2×SSC, 0.05% SDS at room temperature for 10 min, then washed in 0.1×SSC, 0.1% SDS at 45° C. for 3 min. The washed filters were exposed overnight at -80° C. using HyperFilm MP (Amersham) and intensifying paper. Positive clones were subjected to the second screening in the same manner as above to isolate a single clone. Isolated lambda DNAs were purified from the plate lysate using a QIAGEN Lambda MAX I Kit (Qiagen). Insert DNAs were cut out with EcoRI and subcloned into pUC118 EcoRI/BAP (Takara Shuzo Co., Ltd.). Its nucleotide sequence was then determined with ABI377 sequencer (Perkin Elmer). The cDNA coding the full length of mouse VRGF-D was reconstructed with two of the obtained clones that overlapped each other. SEQ ID NO. 24 shows the nucleotide sequence of mouse VEGF-D cDNA and the deduced amino acid sequence therefrom.

Example 8

Cloning Rat VEGF-D cDNA

[0044] Two Hybond-N+ (Amersham) filters (20 cm×22 cm), on which 1.5×10⁵ pfu of Rat lung 5'-stretch cDNA library had been transferred, were prepared. Gradient hybridization from 68° C. to 55° C. was performed for 2 hours in ExpressH.Fyb Hybridization Solution (Clontech) using as a probe an approximately 1 μg fragment containing 1-782 bp of the mouse VEGF-D cDNA which had been labeled with α³²P-dCTP (Amersham) using Ready-To-Go DNA Labeling Beads (-dCTP) (Pharmacia). The filters were washed four times in 2×SSC, 0.05% SDS at room temperature for 10 min, then washed in 0.1×SSC, 0.1% SDS at 45° C. for 3 min. The washed filters were exposed overnight at -80° C. using HyperFilm MP (Amersham) and intensifying paper. Positive clones were subjected to the second screening in the same manner as above to isolate a single clone. The isolated positive clone was excised into pBluescript using E. coli SOLAR (Stratagene) and helper phage ExAssist (Stratagene), then the sequence was determined with ABI377 sequencer (Perkin Elmer). The sequence seemed to be the rat VEGF-D cDNA but did not contain the termination codon.

[0045] To obtain the C-terminal cDNA which had not been obtained, PCR was performed using Marathon-Ready rat kidney cDNA (Clontech) as a template and 5' primerGCTGCGAGTGTGTCTGTAAA (SEQ ID NO. 26) and 3' primer GGGTAGTGGGCAACAGTGACAGCAA (SEQ ID NO. 27) with 40 cycles of 94° C. for 15 sec, 55° C. for 30 sec, and 72° C. for 2 min. After the thus-obtained fragment was subcloned into pGEM-T vector (promega), the nucleotide sequence was determined with ABI377 sequencer (Perkin Elmer). The resulting clone contained the C-terminus of rat VEGF-D. Based on the results of sequencing the clone obtained by plaque hybridization and the clone obtained by PCR, the full length of the rat VEGF-D sequence was determined. SEQ ID NO. 25 shows the determined nucleotide sequence and the deduced amino acid sequence therefrom.

INDUSTRIAL APPLICABILITY

[0046] In the present invention, a novel protein (VEGF-D) having significant homology to VEGF-C and its gene have been isolated. VEGF-D appears to be involved in the pathological neovascularization associated with diabetes, rheumatoid arthritis, the growth of solid tumors, differentiation and proliferation of blood cells, formation of lymphatic vessels, and formation of edema resulting from various causes as well as the normal neovascularization at the developmental stage. The gene of the present invention can be used to diagnos disorders caused by abnormalities of the VEGF-D gene and gene therapy for the VEGF-D deficiency. The VEGF-D protein, which is obtained by expressing the gene of the present invention, can be used for healing wounds, promoting collateral vessel formation, and aiding hematopoietic stem cell proliferation. The antibodies or inhibitors against the VEGF-D protein can be used for treating angiodysplasia and lymphangiodysplasia associated with inflammation, edemas arising from various causes, dyshematopoiesis, and, as a novel anticancer agent, for treating pathological neovascularization. The VEGF-D protein and its antibodies can be useful for diagnosing diseases resulting from abnormal production of VEGF-D.

Sequence CWU 1 SEQUENCE LISTING <160> NUMBER OF SEQ ID NOS: 34 <210> SEQ ID NO 1 <211> LENGTH: 354 <212> TYPE: PRT <213> ORGANISM: Homo sapiens <400> SEQUENCE: 1 Met Tyr Arg Glu Trp Val Val Val Asn Val Phe Met Met Leu Tyr Val 1 5 10 15 Gln Leu Val Gln Gly Ser Ser Asn Glu His Gly Pro Val Lys Arg Ser 20 25 30 Ser Gln Ser Thr Leu Glu Arg Ser Glu Gln Gln Ile Arg Ala Ala Ser 35 40 45 Ser Leu Glu Glu Leu Leu Arg Ile Thr His Ser Glu Asp Trp Lys Leu 50 55 60 Trp Arg Cys Arg Leu Arg Leu Lys Ser Phe Thr Ser Met Asp Ser Arg 65 70 75 80 Ser Ala Ser His Arg Ser Thr Arg Phe Ala Ala Thr Phe Tyr Asp Ile 85 90 95 Glu Thr Leu Lys Val Ile Asp Glu Glu Trp Gln Arg Thr Gln Cys Ser 100 105 110 Pro Arg Glu Thr Cys Val Glu Val Ala Ser Glu Leu Gly Lys Ser Thr 115 120 125 Asn Thr Phe Phe Lys Pro Pro Cys Val Asn Val Phe Arg Cys Gly Gly 130 135 140 Cys Cys Asn Glu Glu Ser Leu Ile Cys Met Asn Thr Ser Thr Ser Tyr 145 150 155 160 Ile Ser Lys Gln Leu Phe Glu Ile Ser Val Pro Leu Thr Ser Val Pro 165 170 175 Glu Leu Val Pro Val Lys Val Ala Asn His Thr Gly Cys Lys Cys Leu 180 185 190 Pro Thr Ala Pro Arg His Pro Tyr Ser Ile Ile Arg Arg Ser Ile Gln 195 200 205 Ile Pro Glu Glu Asp Arg Cys Ser His Ser Lys Lys Leu Cys Pro Ile 210 215 220 Asp Met Leu Trp Asp Ser Asn Lys Cys Lys Cys Val Leu Gln Glu Glu 225 230 235 240 Asn Pro Leu Ala Gly Thr Glu Asp His Ser His Leu Gln Glu Pro Ala 245 250 255 Leu Cys Gly Pro His Met Met Phe Asp Glu Asp Arg Cys Glu Cys Val 260 265 270 Cys Lys Thr Pro Cys Pro Lys Asp Leu Ile Gln His Pro Lys Asn Cys 275 280 285 Ser Cys Phe Glu Cys Lys Glu Ser Leu Glu Thr Cys Cys Gln Lys His 290 295 300 Lys Leu Phe His Pro Asp Thr Cys Ser Cys Glu Asp Arg Cys Pro Phe 305 310 315 320 His Thr Arg Pro Cys Ala Ser Gly Lys Thr Ala Cys Ala Lys His Cys 325 330 335 Arg Phe Pro Lys Glu Lys Arg Ala Ala Gln Gly Pro His Ser Arg Lys 340 345 350 Asn Pro <210> SEQ ID NO 2 <211> LENGTH: 2004 <212> TYPE: DNA <213> ORGANISM: Homo sapiens <220> FEATURE: <221> NAME/KEY: CDS <222> LOCATION: (403)...(1464) <400> SEQUENCE: 2 ccagctttct gtarctgtaa gcattggtgg ccacaccacc tccttacaaa gcaactagaa 60 cctgcggcat acattggaga gattttttta attttctgga caygaagtaa atttagagtg 120 ctttcyaatt tcaggtagaa gacatgtcca ccttctgatt atttttggag aacattttga 180 tttttttcat ctctctctcc ccacccctaa gattgtgcaa aaaaagcgta ccttgcctaa 240 ttgaaataat ttcattggat tttgatcaga actgatcatt tggttttctg tgtgaagttt 300 tgaggtttca aactttcctt ctggagaatg ccttttgaaa caattttctc tagctgcctg 360 atgtcaactg cttagtaatc agtggatatt gaaatattca aa atg tac aga gag 414 Met Tyr Arg Glu 1 tgg gta gtg gtg aat gtt ttc atg atg ttg tac gtc cag ctg gtg cag 462 Trp Val Val Val Asn Val Phe Met Met Leu Tyr Val Gln Leu Val Gln 5 10 15 20 ggc tcc agt aat gaa cat gga cca gtg aag cga tca tct cag tcc aca 510 Gly Ser Ser Asn Glu His Gly Pro Val Lys Arg Ser Ser Gln Ser Thr 25 30 35 ttg gaa cga tct gaa cag cag atc agg gct gct tct agt ttg gag gaa 558 Leu Glu Arg Ser Glu Gln Gln Ile Arg Ala Ala Ser Ser Leu Glu Glu 40 45 50 cta ctt cga att act cac tct gag gac tgg aag ctg tgg aga tgc agg 606 Leu Leu Arg Ile Thr His Ser Glu Asp Trp Lys Leu Trp Arg Cys Arg 55 60 65 ctg agg ctc aaa agt ttt acc agt atg gac tct cgc tca gca tcc cat 654 Leu Arg Leu Lys Ser Phe Thr Ser Met Asp Ser Arg Ser Ala Ser His 70 75 80 cgg tcc act agg ttt gcg gca act ttc tat gac att gaa aca cta aaa 702 Arg Ser Thr Arg Phe Ala Ala Thr Phe Tyr Asp Ile Glu Thr Leu Lys 85 90 95 100 gtt ata gat gaa gaa tgg caa aga act cag tgc agc cct aga gaa acg 750 Val Ile Asp Glu Glu Trp Gln Arg Thr Gln Cys Ser Pro Arg Glu Thr 105 110 115 tgc gtg gag gtg gcc agt gag ctg ggg aag agt acc aac aca ttc ttc 798 Cys Val Glu Val Ala Ser Glu Leu Gly Lys Ser Thr Asn Thr Phe Phe 120 125 130 aag ccc cct tgt gtg aac gtg ttc cga tgt ggt ggc tgt tgc aat gaa 846 Lys Pro Pro Cys Val Asn Val Phe Arg Cys Gly Gly Cys Cys Asn Glu 135 140 145 gag agc ctt atc tgt atg aac acc agc acc tcg tac att tcc aaa cag 894 Glu Ser Leu Ile Cys Met Asn Thr Ser Thr Ser Tyr Ile Ser Lys Gln 150 155 160 ctc ttt gag ata tca gtg cct ttg aca tca gta cct gaa tta gtg cct 942 Leu Phe Glu Ile Ser Val Pro Leu Thr Ser Val Pro Glu Leu Val Pro 165 170 175 180 gtt aaa gtt gcc aat cat aca ggt tgt aag tgc ttg cca aca gcc ccc 990 Val Lys Val Ala Asn His Thr Gly Cys Lys Cys Leu Pro Thr Ala Pro 185 190 195 cgc cat cca tac tca att atc aga aga tcc atc cag atc cct gaa gaa 1038 Arg His Pro Tyr Ser Ile Ile Arg Arg Ser Ile Gln Ile Pro Glu Glu 200 205 210 gat cgc tgt tcc cat tcc aag aaa ctc tgt cct att gac atg cta tgg 1086 Asp Arg Cys Ser His Ser Lys Lys Leu Cys Pro Ile Asp Met Leu Trp 215 220 225 gat agc aac aaa tgt aaa tgt gtt ttg cag gag gaa aat cca ctt gct 1134 Asp Ser Asn Lys Cys Lys Cys Val Leu Gln Glu Glu Asn Pro Leu Ala 230 235 240 gga aca gaa gac cac tct cat ctc cag gaa cca gct ctc tgt ggg cca 1182 Gly Thr Glu Asp His Ser His Leu Gln Glu Pro Ala Leu Cys Gly Pro 245 250 255 260 cac atg atg ttt gac gaa gat cgt tgc gag tgt gtc tgt aaa aca cca 1230 His Met Met Phe Asp Glu Asp Arg Cys Glu Cys Val Cys Lys Thr Pro 265 270 275 tgt ccc aaa gat cta atc cag cac ccc aaa aac tgc agt tgc ttt gag 1278 Cys Pro Lys Asp Leu Ile Gln His Pro Lys Asn Cys Ser Cys Phe Glu 280 285 290 tgc aaa gaa agt ctg gag acc tgc tgc cag aag cac aag cta ttt cac 1326 Cys Lys Glu Ser Leu Glu Thr Cys Cys Gln Lys His Lys Leu Phe His 295 300 305 cca gac acc tgc agc tgt gag gac aga tgc ccc ttt cat acc aga cca 1374 Pro Asp Thr Cys Ser Cys Glu Asp Arg Cys Pro Phe His Thr Arg Pro 310 315 320 tgt gca agt ggc aaa aca gca tgt gca aag cat tgc cgc ttt cca aag 1422 Cys Ala Ser Gly Lys Thr Ala Cys Ala Lys His Cys Arg Phe Pro Lys 325 330 335 340 gag aaa agg gct gcc cag ggg ccc cac agc cga aag aat cct 1464 Glu Lys Arg Ala Ala Gln Gly Pro His Ser Arg Lys Asn Pro 345 350 tgattcagcg ttccaagttc cccatccctg tcatttttaa cagcatgctg ctttgccaag 1524 ttgctgtcac tgtttttttc ccaggtgtta aaaaaaaaat ccattttaca cagcaccaca 1584 gtgaatccag accaaccttc cattcacacc agctaaggag tccctggttc attgatggat 1644 gtcttctagc tgcagatgcc tctgcgcacc aaggaatgga gaggagggga cccatgtaat 1704 ccttttgttt agttttgttt ttgttttttg gtgaatgaga aaggtgtgct ggtcatggaa 1764 tggcaggtgt catatgactg attactcaga gcagatgagg aaaactgtag tctctgagtc 1824 ctttgctaat cgcaactctt gtgaattatt ctgattcttt tttatgcaga atttgattcg 1884 tatgatcagt actgactttc tgattactgt ccagcttata gtcttccagt ttaatgaact 1944 accatctgat gtttcatatt taagtgtatt taaagaaaat aaacaccatt attcaagtct 2004 <210> SEQ ID NO 3 <211> LENGTH: 16 <212> TYPE: PRT <213> ORGANISM: Homo sapiens <400> SEQUENCE: 3 Cys Gly Pro Asn Lys Glu Leu Asp Glu Asn Thr Cys Gln Cys Val Cys 1 5 10 15 <210> SEQ ID NO 4 <211> LENGTH: 27 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic DNA <400> SEQUENCE: 4 agggatgggg aacttggaac gctgaat 27 <210> SEQ ID NO 5 <211> LENGTH: 27 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic DNA <400> SEQUENCE: 5 gatctaatcc agcaccccaa aaactgc 27 <210> SEQ ID NO 6 <211> LENGTH: 27 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic DNA <400> SEQUENCE: 6 ccatcctaat acgactcact atagggc 27 <210> SEQ ID NO 7 <211> LENGTH: 33 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic DNA <400> SEQUENCE: 7 ctggttcggc ccagaacttg gaacgctgaa tca 33 <210> SEQ ID NO 8 <211> LENGTH: 32 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic DNA <400> SEQUENCE: 8 ctcgctcgcc cactaatacg actcactata gg 32 <210> SEQ ID NO 9 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic DNA <400> SEQUENCE: 9 aattaaccct cactaaaggg 20 <210> SEQ ID NO 10 <211> LENGTH: 22 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic DNA <400> SEQUENCE: 10 ccagggtttt cccagtcacg ac 22 <210> SEQ ID NO 11 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic DNA <400> SEQUENCE: 11 actcactata gggctcgagc ggc 23 <210> SEQ ID NO 12 <211> LENGTH: 17 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic DNA <400> SEQUENCE: 12 aagtctggag acctgct 17 <210> SEQ ID NO 13 <211> LENGTH: 17 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic DNA <400> SEQUENCE: 13 cagcaggtct ccagact 17 <210> SEQ ID NO 14 <211> LENGTH: 17 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic DNA <400> SEQUENCE: 14 cgcacccaag gaatgga 17 <210> SEQ ID NO 15 <211> LENGTH: 18 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic DNA <400> SEQUENCE: 15 tgacacctgg ccattcca 18 <210> SEQ ID NO 16 <211> LENGTH: 18 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic DNA <400> SEQUENCE: 16 catcagatgg tagttcat 18 <210> SEQ ID NO 17 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic DNA <400> SEQUENCE: 17 atgctgagcg agagtccata 20 <210> SEQ ID NO 18 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic DNA <400> SEQUENCE: 18 cactaggttt gcggcaactt 20 <210> SEQ ID NO 19 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic DNA <400> SEQUENCE: 19 gctgttggca agcacttaca 20 <210> SEQ ID NO 20 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic DNA <400> SEQUENCE: 20 gatccatcca gatccctgaa 20 <210> SEQ ID NO 21 <211> LENGTH: 19 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic DNA <400> SEQUENCE: 21 cagatcaggg ctgcttcta 19 <210> SEQ ID NO 22 <211> LENGTH: 32 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic DNA <400> SEQUENCE: 22 tccagatctt ttgcggcaac tttctatgac at 32 <210> SEQ ID NO 23 <211> LENGTH: 33 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic DNA <400> SEQUENCE: 23 caggtcgact caaacaggca ctaattcagg tac 33 <210> SEQ ID NO 24 <211> LENGTH: 1581 <212> TYPE: DNA <213> ORGANISM: Mus musculus <220> FEATURE: <221> NAME/KEY: CDS <222> LOCATION: (96)...(1169) <221> NAME/KEY: misc_feature <222> LOCATION: (1)...(1581) <223> OTHER INFORMATION: n = A,T,C or G <400> SEQUENCE: 24 ttccgggctt tgctggagaa tgccttttgc aacacttttc agtagctgcc tggaaacaac 60 tgcttagtca tcggtagaca tttaaaatat tcaaa atg tat gga gaa tgg gga 113 Met Tyr Gly Glu Trp Gly 1 5 atg ggg aat atc ctc atg atg ttc cat gtg tac ttg gtg cag ggc ttc 161 Met Gly Asn Ile Leu Met Met Phe His Val Tyr Leu Val Gln Gly Phe 10 15 20 agg agc gaa cat gga cca gtg aag gat ttt tct ttt gag cga tca tcc 209 Arg Ser Glu His Gly Pro Val Lys Asp Phe Ser Phe Glu Arg Ser Ser 25 30 35 cgg tcc atg ttg gaa cga tct gaa caa cag atc cga gca gct tct agt 257 Arg Ser Met Leu Glu Arg Ser Glu Gln Gln Ile Arg Ala Ala Ser Ser 40 45 50 ttg gag gag ttg ctg caa atc gcg cac tct gag gac tgg aag ctg tgg 305 Leu Glu Glu Leu Leu Gln Ile Ala His Ser Glu Asp Trp Lys Leu Trp 55 60 65 70 cga tgc cgg ttg aag ctc aaa agt ctt gcc agt atg gac tca cgc tca 353 Arg Cys Arg Leu Lys Leu Lys Ser Leu Ala Ser Met Asp Ser Arg Ser 75 80 85 gca tcc cat cgc tcc acc aga ttt gcg gca act ttc tat gac act gaa 401 Ala Ser His Arg Ser Thr Arg Phe Ala Ala Thr Phe Tyr Asp Thr Glu 90 95 100 aca cta aaa gtt ata gat gaa gaa tgg cag agg acc caa tgc agc cct 449 Thr Leu Lys Val Ile Asp Glu Glu Trp Gln Arg Thr Gln Cys Ser Pro 105 110 115 aga gag aca tgc gta gaa gtc gcc agt gag ctg ggg aag aca acc aac 497 Arg Glu Thr Cys Val Glu Val Ala Ser Glu Leu Gly Lys Thr Thr Asn 120 125 130 aca ttc ttc aag ccc ccc tgt gta aat gtc ttc cgg tgt gga ggc tgc 545 Thr Phe Phe Lys Pro Pro Cys Val Asn Val Phe Arg Cys Gly Gly Cys 135 140 145 150 tgc aac gaa gag ggt gtg atg tgt atg aac aca agc acc tcc tac atc 593 Cys Asn Glu Glu Gly Val Met Cys Met Asn Thr Ser Thr Ser Tyr Ile 155 160 165 tcc aaa cag ctc ttt gag ata tca gtg cct ctg aca tca gtg ccc gag 641 Ser Lys Gln Leu Phe Glu Ile Ser Val Pro Leu Thr Ser Val Pro Glu 170 175 180 tta gtg cct gtt aaa att gcc aac cat acg ggt tgt aag tgc ttg ccc 689 Leu Val Pro Val Lys Ile Ala Asn His Thr Gly Cys Lys Cys Leu Pro 185 190 195 acg ggc ccc cgc cat cct tac tca att atc aga aga tcc att cag acc 737 Thr Gly Pro Arg His Pro Tyr Ser Ile Ile Arg Arg Ser Ile Gln Thr 200 205 210 cca gaa gaa gat gaa tgt cct cat tcc aag aaa ctc tgt cct att gac 785 Pro Glu Glu Asp Glu Cys Pro His Ser Lys Lys Leu Cys Pro Ile Asp 215 220 225 230 atg ctg tgg gat aac acc aaa tgt aaa tgt gtt ttg caa gac gag act 833 Met Leu Trp Asp Asn Thr Lys Cys Lys Cys Val Leu Gln Asp Glu Thr 235 240 245 cca ctg cct ggg aca gaa gac cac tct tac ctc cag gaa ccc act ctc 881 Pro Leu Pro Gly Thr Glu Asp His Ser Tyr Leu Gln Glu Pro Thr Leu 250 255 260 tgt gga ccg cac atg acg ttt gat gaa gat cgc tgt gag tgc gtc tgt 929 Cys Gly Pro His Met Thr Phe Asp Glu Asp Arg Cys Glu Cys Val Cys 265 270 275 aaa gca cca tgt ccg gga gat ctc att cag cac ccg gaa aac tgc agt 977 Lys Ala Pro Cys Pro Gly Asp Leu Ile Gln His Pro Glu Asn Cys Ser 280 285 290 tgc ttt gag tgc aaa gaa agt ctg gag agc tgc tgc caa aag cac aag 1025 Cys Phe Glu Cys Lys Glu Ser Leu Glu Ser Cys Cys Gln Lys His Lys 295 300 305 310 att ttt cac cca gac acc tgc agc tgt gag gac aga tgt cct ttt cac 1073 Ile Phe His Pro Asp Thr Cys Ser Cys Glu Asp Arg Cys Pro Phe His 315 320 325 acc aga aca tgt gca agt aga aag cca gcc tgt gga aag cac tgg cgc 1121 Thr Arg Thr Cys Ala Ser Arg Lys Pro Ala Cys Gly Lys His Trp Arg 330 335 340 ttt cca aag gag aca agg gcc cag gga ctc tac agc cag gag aac cct 1169 Phe Pro Lys Glu Thr Arg Ala Gln Gly Leu Tyr Ser Gln Glu Asn Pro 345 350 355 tgattcaact tcctttcaag tccccccatc tctgtcattt taaacagctc actgctttgt 1229 caagttgctg tcactgttgc ccactacccc tgcccccccc cccccccgcc tccaggtgtt 1289 agaaaagttg atttgaccta gtgtcatggt aaagccacat ttccatgcaa tggcggctag 1349 gtgattcccc agttcactga caaatgactt gtagcttcaa atgtctttgc gccatcanca 1409 ctcaaaaagg aaggggtctg aagaacccct tgtttgataa ataaaaacag gtgcctgaaa 1469 caaaatatta ggtgccactc gattgggtcc ctcgggctgg ccaaattcca agggcaatgc 1529 tcctgaattt attgtgcccc ttccttaatg cggaatttcc ttttgtttga tt 1581 <210> SEQ ID NO 25 <211> LENGTH: 1491 <212> TYPE: DNA <213> ORGANISM: Rat rattus <220> FEATURE: <221> NAME/KEY: CDS <222> LOCATION: (270)...(1247) <400> SEQUENCE: 25 gccacctctt gattatttgt gcagcgggaa actttgaaat agttttcatc tctttctccc 60 atactaagat tgtgtgtggc cgtgggggag tccttgacta actcaagtca tttcattgga 120 ttttgattac aactgatcat gtgatatttt tttccatgta aagttttggg gcttcaaact 180 ttgcttctgg agaatgcctt ttgcaacact tttcagtagc tgcctggaaa caactgctta 240 gccatcagtg gacatttgaa atattcaaa atg tat gga gag tgg gcc gca gtg 293 Met Tyr Gly Glu Trp Ala Ala Val 1 5 aat att ctc atg atg tcc tat gtg tac ctg gtg cag ggc ttc agt att 341 Asn Ile Leu Met Met Ser Tyr Val Tyr Leu Val Gln Gly Phe Ser Ile 10 15 20 gaa cac cga gca gtg aag gat gtt tct ctt gag cga tca tcc cgg tct 389 Glu His Arg Ala Val Lys Asp Val Ser Leu Glu Arg Ser Ser Arg Ser 25 30 35 40 gtg ttg gaa cgt tct gaa caa cag atc cgc gcg gct tct act ttg gaa 437 Val Leu Glu Arg Ser Glu Gln Gln Ile Arg Ala Ala Ser Thr Leu Glu 45 50 55 gag ttg ctg caa gtc gca cac tct gag gac tgg aag ctg tgg cgg tgc 485 Glu Leu Leu Gln Val Ala His Ser Glu Asp Trp Lys Leu Trp Arg Cys 60 65 70 cgg ttg aag ctt aaa agt ctt gcc aat gtg gac tcg cgc tca aca tcc 533 Arg Leu Lys Leu Lys Ser Leu Ala Asn Val Asp Ser Arg Ser Thr Ser 75 80 85 cat cgc tcc acc aga ttt gcg gca act ttc tat gat act gaa aca cta 581 His Arg Ser Thr Arg Phe Ala Ala Thr Phe Tyr Asp Thr Glu Thr Leu 90 95 100 aaa gtt ata gat gaa gaa tgg cag agg acc caa tgc agc cct aga gag 629 Lys Val Ile Asp Glu Glu Trp Gln Arg Thr Gln Cys Ser Pro Arg Glu 105 110 115 120 aca tgc gta gaa gtc gcc agt gag ctg ggg aag aca acc aac aca ttt 677 Thr Cys Val Glu Val Ala Ser Glu Leu Gly Lys Thr Thr Asn Thr Phe 125 130 135 ttc aag ccc cct tgt gta aat gtc ttc cgg tgt gga gga tgc tgc aat 725 Phe Lys Pro Pro Cys Val Asn Val Phe Arg Cys Gly Gly Cys Cys Asn 140 145 150 gaa gag agc gtg atg tgt atg aac aca agc acc tcc tac atc tcc aaa 773 Glu Glu Ser Val Met Cys Met Asn Thr Ser Thr Ser Tyr Ile Ser Lys 155 160 165 cag ctc ttt gag ata tca gtg cct ctg aca tca gtg ccc gag tta gtg 821 Gln Leu Phe Glu Ile Ser Val Pro Leu Thr Ser Val Pro Glu Leu Val 170 175 180 cct gtt aaa att gcc aac cat acg ggt tgt aag tgt ttg ccc acg ggc 869 Pro Val Lys Ile Ala Asn His Thr Gly Cys Lys Cys Leu Pro Thr Gly 185 190 195 200 ccc cgg cat cct tat tca att atc aga aga tcc att cag atc cca gaa 917 Pro Arg His Pro Tyr Ser Ile Ile Arg Arg Ser Ile Gln Ile Pro Glu 205 210 215 gaa gat caa tgt cct cat tcc aag aaa ctc tgt cct gtt gac atg ctg 965 Glu Asp Gln Cys Pro His Ser Lys Lys Leu Cys Pro Val Asp Met Leu 220 225 230 tgg gat aac acc aaa tgt aaa tgt gtt tta caa gat gag aat cca ctg 1013 Trp Asp Asn Thr Lys Cys Lys Cys Val Leu Gln Asp Glu Asn Pro Leu 235 240 245 cct ggg aca gaa gac cac tct tac ctc cag gaa ccc gct ctc tgt gga 1061 Pro Gly Thr Glu Asp His Ser Tyr Leu Gln Glu Pro Ala Leu Cys Gly 250 255 260 cca cac atg atg ttt gat gaa gat cgc tgc gag tgt gtc tgt aaa gca 1109 Pro His Met Met Phe Asp Glu Asp Arg Cys Glu Cys Val Cys Lys Ala 265 270 275 280 cca tgt cct gga gat ctc att cag cac ccg gaa aac tgc agt tgc ttt 1157 Pro Cys Pro Gly Asp Leu Ile Gln His Pro Glu Asn Cys Ser Cys Phe 285 290 295 gaa tgc aaa gaa agt ctg gaa agc tgt tgc caa aag cac aag atg ttt 1205 Glu Cys Lys Glu Ser Leu Glu Ser Cys Cys Gln Lys His Lys Met Phe 300 305 310 cac cct gac acc tgc aga tca atg gtc ttt tca ctg tcc cct 1247 His Pro Asp Thr Cys Arg Ser Met Val Phe Ser Leu Ser Pro 315 320 325 taatttggtt tactggtgac atttaaagga catactaacc tgatttattg gggctctttt 1307 ctctcagggc ccaagcacac tcttaaagga acacagacgt ttggcctcta agaaatacat 1367 ggaagtatta tagagtgatg attaaattgt cttcttgttt caaacagggt ctcatgatta 1427 cagacccgta ttgccatgcc tgccgtcatg ctatcatgag cggaaaagaa tcactggcat 1487 ttaa 1491 <210> SEQ ID NO 26 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic DNA <400> SEQUENCE: 26 gctgcgagtg tgtctgtaaa 20 <210> SEQ ID NO 27 <211> LENGTH: 25 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic DNA <400> SEQUENCE: 27 gggtagtggg caacagtgac agcaa 25 <210> SEQ ID NO 28 <211> LENGTH: 104 <212> TYPE: PRT <213> ORGANISM: Homo sapiens <400> SEQUENCE: 28 His Leu Gln Glu Pro Ala Leu Cys Gly Pro His Met Met Phe Asp Glu 1 5 10 15 Asp Arg Cys Glu Cys Val Cys Lys Thr Pro Cys Pro Lys Asp Leu Ile 20 25 30 Gln His Pro Lys Asn Cys Ser Cys Phe Glu Cys Lys Glu Ser Leu Glu 35 40 45 Thr Cys Cys Gln Lys His Lys Leu Phe His Pro Asp Thr Cys Ser Cys 50 55 60 Glu Asp Arg Cys Pro Phe His Thr Arg Pro Cys Ala Ser Gly Lys Thr 65 70 75 80 Ala Cys Ala Lys His Cys Arg Phe Pro Lys Glu Lys Arg Ala Ala Gln 85 90 95 Gly Pro His Ser Arg Lys Asn Pro 100 <210> SEQ ID NO 29 <211> LENGTH: 419 <212> TYPE: PRT <213> ORGANISM: Homo sapiens <400> SEQUENCE: 29 Met His Leu Leu Gly Phe Phe Ser Val Ala Cys Ser Leu Leu Ala Ala 1 5 10 15 Ala Leu Leu Pro Gly Pro Arg Glu Ala Pro Ala Ala Ala Ala Ala Phe 20 25 30 Glu Ser Gly Leu Asp Leu Ser Asp Ala Glu Pro Asp Ala Gly Glu Ala 35 40 45 Thr Ala Tyr Ala Ser Lys Asp Leu Glu Phe Gln Leu Arg Ser Val Ser 50 55 60 Ser Val Asp Glu Leu Met Thr Val Leu Tyr Pro Glu Tyr Trp Lys Met 65 70 75 80 Tyr Lys Cys Gln Leu Arg Lys Gly Gly Trp Gln His Asn Arg Glu Gln 85 90 95 Ala Asn Leu Asn Ser Arg Thr Glu Glu Thr Ile Lys Phe Ala Ala Ala 100 105 110 His Tyr Asn Thr Glu Ile Leu Lys Ser Ile Asp Asn Glu Trp Arg Lys 115 120 125 Thr Gln Cys Met Pro Arg Glu Val Cys Ile Asp Val Gly Lys Glu Phe 130 135 140 Gly Val Ala Thr Asn Thr Phe Phe Lys Pro Pro Cys Val Ser Val Tyr 145 150 155 160 Arg Cys Gly Gly Cys Cys Asn Ser Glu Gly Leu Gln Cys Met Asn Thr 165 170 175 Ser Thr Ser Tyr Leu Ser Lys Thr Leu Phe Glu Ile Thr Val Pro Leu 180 185 190 Ser Gln Gly Pro Lys Pro Val Thr Ile Ser Phe Ala Asn His Thr Ser 195 200 205 Cys Arg Cys Met Ser Lys Leu Asp Val Tyr Arg Gln Val His Ser Ile 210 215 220 Ile Arg Arg Ser Leu Pro Ala Thr Leu Pro Gln Cys Gln Ala Ala Asn 225 230 235 240 Lys Thr Cys Pro Thr Asn Tyr Met Trp Asn Asn His Ile Cys Arg Cys 245 250 255 Leu Ala Gln Glu Asp Phe Met Phe Ser Ser Asp Ala Gly Asp Asp Ser 260 265 270 Thr Asp Gly Phe His Asp Ile Cys Gly Pro Asn Lys Glu Leu Asp Glu 275 280 285 Glu Thr Cys Gln Cys Val Cys Arg Ala Gly Leu Arg Pro Ala Ser Cys 290 295 300 Gly Pro His Lys Glu Leu Asp Arg Asn Ser Cys Gln Cys Val Cys Lys 305 310 315 320 Asn Lys Leu Phe Pro Ser Gln Cys Gly Ala Asn Arg Glu Phe Asp Glu 325 330 335 Asn Thr Cys Gln Cys Val Cys Lys Arg Thr Cys Pro Arg Asn Gln Pro 340 345 350 Leu Asn Pro Gly Lys Cys Ala Cys Glu Cys Thr Glu Ser Pro Gln Lys 355 360 365 Cys Leu Leu Lys Gly Lys Lys Phe His His Gln Thr Cys Ser Cys Tyr 370 375 380 Arg Arg Pro Cys Thr Asn Arg Gln Lys Ala Cys Glu Pro Gly Phe Ser 385 390 395 400 Tyr Ser Glu Glu Val Cys Arg Cys Val Pro Ser Tyr Trp Lys Arg Pro 405 410 415 Gln Met Ser <210> SEQ ID NO 30 <211> LENGTH: 211 <212> TYPE: PRT <213> ORGANISM: Homo sapiens <400> SEQUENCE: 30 Met Arg Thr Leu Ala Cys Leu Leu Leu Leu Gly Cys Gly Tyr Leu Ala 1 5 10 15 His Val Leu Ala Glu Glu Ala Glu Ile Pro Arg Glu Val Ile Glu Arg 20 25 30 Leu Ala Arg Ser Gln Ile His Ser Ile Arg Asp Leu Gln Arg Leu Leu 35 40 45 Glu Ile Asp Ser Val Gly Ser Glu Asp Ser Leu Asp Thr Ser Leu Arg 50 55 60 Ala His Gly Val His Ala Thr Lys His Val Pro Phe Lys Arg Pro Leu 65 70 75 80 Pro Ile Arg Arg Lys Arg Ser Ile Glu Glu Ala Val Pro Ala Val Cys 85 90 95 Lys Thr Arg Thr Val Ile Tyr Glu Ile Pro Arg Ser Gln Val Asp Pro 100 105 110 Thr Ser Ala Asn Phe Leu Ile Trp Pro Pro Cys Val Glu Val Lys Arg 115 120 125 Cys Thr Gly Cys Cys Asn Thr Ser Ser Val Lys Cys Gln Pro Ser Arg 130 135 140 Val His His Arg Ser Val Lys Val Ala Lys Val Glu Tyr Val Arg Lys 145 150 155 160 Lys Pro Lys Leu Lys Glu Val Gln Val Arg Leu Glu Glu His Leu Glu 165 170 175 Cys Ala Cys Ala Thr Thr Ser Leu Asn Pro Asp Tyr Arg Glu Glu Asp 180 185 190 Thr Gly Arg Pro Arg Glu Ser Gly Lys Lys Arg Lys Arg Lys Arg Leu 195 200 205 Lys Pro Thr 210 <210> SEQ ID NO 31 <211> LENGTH: 241 <212> TYPE: PRT <213> ORGANISM: Homo sapiens <400> SEQUENCE: 31 Met Asn Arg Cys Trp Ala Leu Phe Leu Ser Leu Cys Cys Tyr Leu Arg 1 5 10 15 Leu Val Ser Ala Glu Gly Asp Pro Ile Pro Glu Glu Leu Tyr Glu Met 20 25 30 Leu Ser Asp His Ser Ile Arg Ser Phe Asp Asp Leu Gln Arg Leu Leu 35 40 45 His Gly Asp Pro Gly Glu Glu Asp Gly Ala Glu Leu Asp Leu Asn Met 50 55 60 Thr Arg Ser His Ser Gly Gly Glu Leu Glu Ser Leu Ala Arg Gly Arg 65 70 75 80 Arg Ser Leu Gly Ser Leu Thr Ile Ala Glu Pro Ala Met Ile Ala Glu 85 90 95 Cys Lys Thr Arg Thr Glu Val Phe Glu Ile Ser Arg Arg Leu Ile Asp 100 105 110 Arg Thr Asn Ala Asn Phe Leu Val Trp Pro Pro Cys Val Glu Val Gln 115 120 125 Arg Cys Ser Gly Cys Cys Asn Asn Arg Asn Val Gln Cys Arg Pro Thr 130 135 140 Gln Val Gln Leu Arg Pro Val Gln Val Arg Lys Ile Glu Ile Val Arg 145 150 155 160 Lys Lys Pro Ile Phe Lys Lys Ala Thr Val Thr Leu Glu Asp His Leu 165 170 175 Ala Cys Lys Cys Glu Thr Val Ala Ala Ala Arg Pro Val Thr Arg Ser 180 185 190 Pro Gly Gly Ser Gln Glu Gln Arg Ala Lys Thr Pro Gln Thr Arg Val 195 200 205 Thr Ile Arg Thr Val Arg Val Arg Arg Pro Pro Lys Gly Lys His Arg 210 215 220 Lys Phe Lys His Thr His Asp Lys Thr Ala Leu Lys Glu Thr Leu Gly 225 230 235 240 Ala <210> SEQ ID NO 32 <211> LENGTH: 170 <212> TYPE: PRT <213> ORGANISM: Homo sapiens <400> SEQUENCE: 32 Met Pro Val Met Arg Leu Phe Pro Cys Phe Leu Gln Leu Leu Ala Gly 1 5 10 15 Leu Ala Leu Pro Ala Val Pro Pro Gln Gln Trp Ala Leu Ser Ala Gly 20 25 30 Asn Gly Ser Ser Glu Val Glu Val Val Pro Phe Gln Phe Val Trp Gly 35 40 45 Arg Ser Tyr Cys Arg Ala Leu Glu Arg Leu Val Asp Val Val Ser Glu 50 55 60 Tyr Pro Ser Glu Val Glu His Met Phe Ser Pro Ser Cys Val Ser Leu 65 70 75 80 Leu Arg Cys Thr Gly Cys Cys Gly Asp Glu Asn Leu His Cys Val Pro 85 90 95 Val Glu Thr Ala Asn Val Thr Met Gln Leu Leu Lys Ile Arg Ser Gly 100 105 110 Asp Arg Pro Ser Tyr Val Glu Leu Thr Phe Ser Gln His Val Arg Cys 115 120 125 Glu Cys Arg Pro Leu Arg Glu Lys Met Lys Pro Glu Arg Arg Arg Pro 130 135 140 Lys Gly Arg Gly Lys Arg Arg Arg Glu Lys Gln Arg Pro Thr Asp Cys 145 150 155 160 His Leu Cys Gly Asp Ala Val Pro Arg Arg 165 170 <210> SEQ ID NO 33 <211> LENGTH: 232 <212> TYPE: PRT <213> ORGANISM: Homo sapiens <400> SEQUENCE: 33 Met Asn Phe Leu Leu Ser Trp Val His Trp Ser Leu Ala Leu Leu Leu 1 5 10 15 Tyr Leu His His Ala Lys Trp Ser Gln Ala Ala Pro Met Ala Glu Gly 20 25 30 Gly Gly Gln Asn His His Glu Val Val Lys Phe Met Asp Val Tyr Gln 35 40 45 Arg Ser Tyr Cys His Pro Ile Glu Thr Leu Val Asp Ile Phe Gln Glu 50 55 60 Tyr Pro Asp Glu Ile Glu Tyr Ile Phe Lys Pro Ser Cys Val Pro Leu 65 70 75 80 Met Arg Cys Gly Gly Cys Cys Asn Asp Glu Gly Leu Glu Cys Val Pro 85 90 95 Thr Glu Glu Ser Asn Ile Thr Met Gln Ile Met Arg Ile Lys Pro His 100 105 110 Gln Gly Gln His Ile Gly Glu Met Ser Phe Leu Gln His Asn Lys Cys 115 120 125 Glu Cys Arg Pro Lys Lys Asp Arg Ala Arg Gln Glu Lys Lys Ser Val 130 135 140 Arg Gly Lys Gly Lys Gly Gln Lys Arg Lys Arg Lys Lys Ser Arg Tyr 145 150 155 160 Lys Ser Trp Ser Val Tyr Val Gly Ala Arg Cys Cys Leu Met Pro Trp 165 170 175 Ser Leu Pro Gly Pro His Pro Cys Gly Pro Cys Ser Glu Arg Arg Lys 180 185 190 His Leu Phe Val Gln Asp Pro Gln Thr Cys Lys Cys Ser Cys Lys Asn 195 200 205 Thr Asp Ser Arg Cys Lys Ala Arg Gln Leu Glu Leu Asn Glu Arg Thr 210 215 220 Cys Arg Cys Asp Lys Pro Arg Arg 225 230 <210> SEQ ID NO 34 <211> LENGTH: 188 <212> TYPE: PRT <213> ORGANISM: Homo sapiens <400> SEQUENCE: 34 Met Ser Pro Leu Leu Arg Arg Leu Leu Leu Ala Ala Leu Leu Gln Leu 1 5 10 15 Ala Pro Ala Gln Ala Pro Val Ser Gln Pro Asp Ala Pro Gly His Gln 20 25 30 Arg Lys Val Val Ser Trp Ile Asp Val Tyr Thr Arg Ala Thr Cys Gln 35 40 45 Pro Arg Glu Val Val Val Pro Leu Thr Val Glu Leu Met Gly Thr Val 50 55 60 Ala Lys Gln Leu Val Pro Ser Cys Val Thr Val Gln Arg Cys Gly Gly 65 70 75 80 Cys Cys Pro Asp Asp Gly Leu Glu Cys Val Pro Thr Gly Gln His Gln 85 90 95 Val Arg Met Gln Ile Leu Met Ile Arg Tyr Pro Ser Ser Gln Leu Gly 100 105 110 Glu Met Ser Leu Glu Glu His Ser Gln Cys Glu Cys Arg Pro Lys Lys 115 120 125 Lys Asp Ser Ala Val Lys Pro Asp Ser Pro Arg Pro Leu Cys Pro Arg 130 135 140 Cys Thr Gln His His Gln Arg Pro Asp Pro Arg Thr Cys Arg Cys Arg 145 150 155 160 Cys Arg Arg Arg Ser Phe Leu Arg Cys Gln Gly Arg Gly Leu Glu Leu 165 170 175 Asn Pro Asp Thr Cys Arg Cys Arg Lys Leu Arg Arg 180 185

Claims

1. An antibody binding to a protein shown by SEQ ID NO: 1 or having the amino acid sequence derived therefrom in which one or more amino acids are substituted, deleted, or added.

2. The antibody of claim 1, wherein said protein is encoded by a nucleic acid hybridizing with the nucleic acid shown by SEQ ID NO: 2.

3. The antibody of claim 1, wherein said protein is an isolated mammalian VEGF-D protein encoded by an isolated nucleic acid hybridizing under highly stringent conditions to the complement of the sequence set forth in SEQ ID NO: 2, wherein said nucleic acid has about 90% or higher sequence homology with the sequence set forth in SEQ ID NO: 2, and said highly stringent conditions involve gradient hybridization from a temperature of about 68.degree. C. to about 55.degree. C. for about 2 hours and washing with 2.times.SSC solution at room temperature, and wherein said protein has a biological activity of a VEGF family protein.

4. The antibody of claim 1, wherein said protein is encoded by an isolated nucleic acid that is capable of hybridizing with the complement of the nucleic acid shown by SEQ ID NO: 2 under the conditions of hybridizing under a temperature gradient of 68.degree. C. to 55.degree. C. for 2 hours and washing with a 2.times.SSC solution containing 0.05% SDS at room temperature for 10 minutes for four times and with a 0.1.times.SSC solution containing 0.1% SDS at 45.degree. C. for 3 minutes, and wherein said protein has at least one activity selected from the group consisting of vascular formation, lymphatic vessel formation, pathological neovascularization, increasing vascular permeability, edema formation, wound healing, and promoting collateral vessel formation.

5. The antibody of claim 1, wherein said protein is encoded by an isolated nucleic acid having 90% or higher sequence homology to the nucleic acid shown by SEQ ID NO: 2, and wherein said protein has at least one activity selected from the group consisting of vascular formation, lymphatic vessel formation, pathological neovascularization, increasing vascular permeability, edema formation, wound healing, and promoting collateral vessel formation.

6. The antibody of claim 1, wherein the amino acid sequence of said protein comprises the amino acid sequence of SEQ ID NO: 1.

7. The antibody of claim 1, wherein said antibody is humanized.

8. A method for inhibiting angiogenesis or neovascularization comprising administering to a mammal an effective amount of an antibody according to any one of claims 1 to 7.

9. The method of claim 8, wherein the method is for treating pathological neovascularization, lymphatic dysplasia, dyshematopoiesis, or edemas.

Images