VEGF-like factor

This application is the National stage application of PCT/JP97/02456, filed Jul. 15, 1997, which claims priority to Japanese application 8/185216, filed Jul. 15, 1996.

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

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 secretionpatterns (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)).

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.

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

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.

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 51 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. 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).

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.

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).

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.

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.

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.

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.

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.

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.

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.

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.

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.

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

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) orthe 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.

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).

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.

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.

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.

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.

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.

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

FIG. 2 compares the amino acid sequences of EST (H24828) and VEGF-C SEQ ID NOs: 28 and 29, respectively.

FIG. 3 compares the amino acid sequences deduced from the VEGF-D gene and from the known genes of the VEGF family proteins SEQ ID NOs:1 and 29-34, respectively

FIG. 4A shows the hydrophobicity plot of VEGF-D. FIG. 4B shows the prediction of the cleavage site of the VEGF-D signal peptide.

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

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 diagnose 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.

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<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