JPWO1998002543A1 - Novel VEGF-like factor - Google Patents

Abstract

(57) [Abstract] A novel human gene with significant homology to the VEGF-C gene was isolated by PCR using primers designed based on the sequence of an EST predicted to be homologous to the C-terminal portion of VEGF-C, a member of the VEGF family. Mouse and rat genes were also isolated based on the isolated human gene. Furthermore, the protein encoded by the human gene was isolated by introducing and expressing it in Escherichia coli. The isolated protein and gene are expected to be useful in gene therapy for VEGF-D gene deficiency, wound treatment, and promotion of collateral circulation formation. Furthermore, VEGF-D protein inhibitors are expected to be used as novel anticancer drugs.

Description

[Detailed Description of the Invention] Novel VEGF-like Factor Technical Field The present invention relates to a protein factor involved in human angiogenesis and belongs to the field of genetic engineering. Background Art The process of angiogenesis, in which endothelial cells present in the inner walls of animal blood vessels create new blood vessels, is triggered by the transmission of specific signals. Various factors have been reported to be involved in this signal transduction. The substance that has attracted the most attention is vascular endothelial growth factor (hereinafter referred to as "VEGF"). VEGF is a protein factor that has been purified and isolated as a substance that promotes vascular endothelial cell proliferation and vascular permeability (Senger, D.R. et al., Science, 219:983-985 (1983); Ferrara, N. and Henzel, W.J. Biochem. Biophys. Res. Commun., 161:851-858 (1989)). The human VEGF gene contains eight exons, and differential splicing of these exons results in the formation of four subtypes consisting of 121, 165, 189, and 206 amino acids, which are reported to result in different secretion patterns of VEGF (Houck, K.A. et al. Mol. Endocrinol. 5, 1806-1814 (1991)). VEGF also has a specific receptor, flt-1, and binding of VEGF to flt-1 has been reported to be important for signal transduction (Vries, C.D. et al. Science, 255:989-991 (1992)).

VEGF-related factors, such as placental growth factor (PlGF) and platelet-derived growth factor (PDGF), have been isolated and shown to have growth-promoting activity on vascular endothelial cells (Maglione, D. et al., Proc. Natl. Acad. Sci. U.S.A. 88, 9267-9271 (1991); Betsholtz, C. et al., Nature 320, 695-699 (1986)). More recently, 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, 290-298 (1996)) have been isolated.

These factors are thought to form a family, and it is possible that other unknown factors may also be included as members.

VEGF has been suggested to play a role not only in angiogenesis during development but also in pathological angiogenesis observed in diabetes, rheumatoid arthritis, retinopathy, and solid tumor growth. Furthermore, in addition to the aforementioned effect of promoting vascular endothelial cell proliferation, the vascular permeability-increasing activity of VEGF has been suggested to be involved in the formation of edema resulting from various causes. Furthermore, these VEGF family members have been suggested to act not only on blood vessels but also on blood cells and lymphatic vessels, and to be involved in the differentiation and proliferation of blood cells and lymphatic vessel formation. Therefore, the VEGF family is currently attracting considerable attention as a useful target for the development of new drugs. Disclosure of the Invention : The objective of the present invention is to isolate novel proteins belonging to the VEGF family and the genes encoding said proteins.

The present inventors searched the GenBank database for genes homologous to VEGF-C, a recently cloned member of the VEGF family. The GenBank database contained ESTs (Expressed Sequence Tags) and STSs (Sequence Tagged Sites). The inventors identified an EST that predicted homology to the C-terminal portion of VEGF-C. Based on this sequence, primers were designed to amplify and isolate the corresponding cDNA using 5' and 3' RACE. The nucleotide sequence of the isolated cDNA was determined, and the deduced amino acid sequence was found to share significant homology with VEGF-C throughout its entirety. Based on this homology, the inventors believe the isolated human clone is the fourth member of the VEGF family (hereafter referred to as "VEGF-D"). Furthermore, the inventors successfully expressed the protein encoded by the isolated human VEGF-D gene in Escherichia coli, purified it, and isolated it. Furthermore, the present inventors have succeeded in isolating mouse and rat VEGF-D genes based on the isolated human VEGF-D gene.

That is, the present invention relates to novel proteins belonging to the VEGF family and genes encoding said proteins. More specifically, the present invention relates to: (1) a protein set forth in SEQ ID NO: 1, or a protein having an amino acid sequence in which one or more amino acids have been substituted, deleted, or added; (2) a protein encoded by DNA that hybridizes with DNA set forth in SEQ ID NO: 2; (3) DNA encoding the protein set forth in (1); (4) DNA that hybridizes with DNA set forth in SEQ ID NO: 2; (5) a vector containing the DNA set forth in (3) or (4); (6) a transformant harboring the vector set forth in (5); (7) a method for producing the protein set forth in (1) or (2), comprising culturing the transformant set forth in (6); (8) an antibody that binds to the protein set forth in (1) or (2); (9) (10) A method for screening for a compound that binds to the protein described in (1) or (2), comprising a step of detecting the binding activity between the protein described in (1) or (2) and a test sample; and (11) A compound that binds to the protein described in (1) or (2), isolated by the method described in (9).

The protein of the present invention (VEGF-D) shares significant homology with VEGF-C and is believed to be the fourth member of the VEGF family. VEGF's primary function is angiogenesis during development, and it is also thought to be involved in pathological angiogenesis observed in diabetes, rheumatoid arthritis, retinopathy, and solid tumor growth. The protein of the present invention is also thought to play a similar role.

Those skilled in the art can modify the VEGF-D of the present invention by adding, deleting, or substituting one or more amino acids of the VEGF-D set forth in SEQ ID NO: 1 using known methods to prepare functionally equivalent proteins. In addition to such artificial modifications, protein modifications can also occur naturally. Such modified proteins are also within the scope of the present invention. Known methods for adding, deleting, or substituting amino acids include, for example, overlap extension polymerase chain reaction (OE-PCR) (Gene 1989 77(1)p51).

Furthermore, the DNA set forth in SEQ ID NO:2, which encodes the VEGF-D of the present invention, can be used to isolate DNA encoding proteins with functions similar to those of VEGF-D in other organisms. For example, those skilled in the art can routinely isolate homologs of the human VEGF-D of the present invention from other organisms by hybridizing the DNA sequence set forth in SEQ ID NO:2 or a portion thereof as a probe to DNA from other organisms. Therefore, DNA that hybridizes with the DNA set forth in SEQ ID NO:2 is also within the scope of the present invention. Examples of other organisms include mice, rats, and rabbits.

DNA encoding a protein functionally equivalent to VEGF-D typically has high homology to the DNA set forth in SEQ ID NO: 2. Here, "high homology" refers to at least 70% or more, preferably 80% or more, and more preferably 90% or more sequence identity.

An example of hybridization conditions for isolating highly homologous DNA is as follows: Prehybridization is performed in ExpressHyb Solution at 68°C for 30 minutes. Radioisotope-labeled probes are denatured at 95°C to 100°C for 2 to 5 minutes and then rapidly cooled on ice. The probe is added to fresh ExpressHyb Solution. The solution is replaced with the probe-containing solution, and hybridization is performed for 2 hours under a temperature gradient from 68°C to 55°C. Wash four times, 10 minutes each, with 2x SSC, 0.05% SDS at room temperature. Wash three times with 0.1x SSC, 0.1% SDS at 45°C for 3 minutes. Autoradiography is performed.

Furthermore, an example of hybridization conditions for isolating DNA with very high homology is as follows: Prehybridization is performed in ExpressHyb Solution at 68°C for 30 minutes. Radioisotope-labeled probes are denatured at 95°C–100°C for 2–5 minutes and then rapidly cooled on ice. The probe is added to fresh ExpressHyb Solution. The solution is replaced with the probe-containing solution, and hybridization is performed at 68°C for 1 hour. Wash four times for 10 minutes each with 2x SSC, 0.05% SDS solution at room temperature. Wash four times for 10 minutes with 0.1x SSC, 0.1% SDS solution at 50°C for 40 minutes, changing the solution once during the wash. Take an autoradiograph.

However, hybridization conditions may vary depending on the length of the probe used (oligomer or probe of several hundred bases or more), the labeling method (radioisotope-labeled or non-radioisotope-labeled probe), and the type of target gene to be cloned. Those skilled in the art can appropriately select suitable hybridization conditions. In the present invention, conditions that do not hybridize with DNA encoding VEGF-C are particularly preferred.

The DNA of the present invention can also be used to produce the VEGF-D of the present invention as a recombinant protein. That is, the DNA encoding VEGF-D (e.g., the DNA set forth in SEQ ID NO: 2) can be inserted into an appropriate expression vector, the vector is introduced into a host, and the transformant is cultured to express the recombinant protein, thereby enabling mass production of the recombinant protein.

There are no particular limitations on the vectors used to produce recombinant proteins; however, vectors such as pGEMEX-1 (Promega) and pEF-BOS (Nucleic Acids. Res. 1990 18(17)p5322) are preferably used. Furthermore, E. coli, CHO cells, COS cells, etc. are preferably used as hosts into which the vectors are introduced.

The VEGF-D protein expressed in the transformant can be purified by an appropriate combination of techniques, such as solubilization using a homogenizer or ultrasonic cell disruption, extraction with various buffer solutions, solubilization or precipitation with acids or alkalis, extraction or precipitation with organic solvents, salting out with ammonium sulfate or other suitable methods, dialysis, ultrafiltration using membrane filters, gel filtration chromatography, ion exchange chromatography, reverse-phase chromatography, countercurrent distribution chromatography, high-performance liquid chromatography, isoelectric focusing or gel electrophoresis, and affinity chromatography using immobilized antibodies or receptors.

Once the recombinant protein is obtained, antibodies can be prepared using known methods. Examples include immunizing rabbits or sheep with the purified protein to produce polyclonal antibodies, or immunizing mice or rats to produce monoclonal antibodies from their antibody-producing cells. The resulting antibodies can then be used to quantify VEGF. While the antibodies can be used directly, they are more effectively humanized to reduce immunogenicity. Examples of antibody humanization methods include CDR grafting, in which antibody genes are cloned from monoclonal antibody-producing cells and the antigenic region of the cloned antibody is grafted onto an existing human antibody, and immunizing mice with a humanized immune system to directly produce human antibodies in the same manner as conventional monoclonal antibodies. The resulting VEGF-D protein or its antibodies can be administered intravenously via subcutaneous injection or other methods.

Furthermore, those skilled in the art can screen for compounds that bind to the proteins of the present invention using known techniques.

For example, a cDNA library using a phage vector (e.g., λgt11, ZAP, etc.) is prepared from cells (e.g., mammalian lung, small intestine, or heart cells) expected to express a protein that binds to the protein of the present invention. The cDNA library is then expressed on LB-agarose, and the expressed protein is immobilized on a filter. The protein of the present invention is purified as a biotin-labeled or GST-fusion protein, and this is then reacted with the filter. Plaques expressing the binding protein are detected using streptavidin or anti-GST antibodies, using the "West-Western blotting" method (Skolnik EY, Margolis B, Mohammadi M, Lowenstein E, Fischer R, Drepps A, Ullrich A, and Schlessinger J (1991) Cloning of PI3 kinase-associated p85 utilizing a novel method for expression/cloning of target proteins for receptor tyrosine kinases. Cell 65, 83-90) can be prepared. Alternatively, a protein of the present invention may be fused to an SRF-binding domain or a GAL4-binding domain and expressed in yeast cells. From cells expected to express a protein that binds to the protein of the present invention, a cDNA library is constructed in which the protein is expressed in a form fused to a VP16 or GAL4 transcription activation domain. This library is then transformed into the yeast cells, and cDNA from the library is isolated from the detected positive clones and transformed into E. coli for expression. (When a protein that binds to the protein of the present invention is expressed in the yeast cells, binding between the two activates a reporter gene, allowing positive clones to be identified.) This system is described in the "two-hybrid system" ("MATCHMARKER Two-Hybrid System," "Mammalian MATCHMAKER Two-Hybrid Assay Kit," "MATCHMAKER One-Hybrid System" (all manufactured by Clontech), "HybriZAP Two-Hybrid Vector System" (manufactured by Stratagene), and the reference "Dalton S. and Treisman"). It can also be prepared according to the method described in "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, an expression cDNA library constructed from cells (e.g., vascular endothelial cells, bone marrow cells, or lymphatic cells) expected to express a substance that binds to the protein of the present invention, such as a receptor, can be introduced into cells such as COS cells, and the binding of the protein of the present invention can be detected using the protein itself or a radioactively or fluorescently labeled version. The binding of the protein of the present invention can then be cloned (Yamasaki K, Taga T, Hirata Y, Yawata H, Kawanishi Y, Seed B, Taniguchi T, Hirano T, Kishimoto T (1988) Cloning and expression of human interleukin-6 (BSF-2/IFN beta 2) receptor. Science, 241:825-828; Fukunaga R, Ishizaka-Ikeda E, Seto Y, Nagata S (1990) Expression cloning of a receptor for murine granulocyte colony-stimulating It is also possible to prepare a protein that specifically binds to the protein of the present invention by applying a culture supernatant or cell extract from cells expected to express a protein that binds to the protein of the present invention to an affinity column onto which the protein of the present invention has been immobilized, and purifying the protein that specifically binds to the column. Furthermore, DNA encoding a protein that binds to the protein of the present invention can be obtained by analyzing the amino acid sequence of the obtained protein, synthesizing oligo DNA based on the sequence, and screening a cDNA library using the DNA as a probe.

Alternatively, the immobilized protein of the present invention can be screened for binding molecules by reacting it with compounds, natural product libraries, or random phage peptide display libraries, or by high-throughput screening using combinatorial chemistry techniques (Wrighton NC; Farrell FX; Chang R; Kashyap AK; Barbone FP; Mulcahy LS; Johnson DL; Barrett RW; Jolliffe LK; Dower WJ., Small peptides as potent mimetics of the protein hormone erythropoietin, Science (UNITED STATES) July 26, 1996, 273 pp. 458-64; Verdine GL., The combinatorial chemistry of nature. Nature (ENGLAND) November 7, 1996, 384 pp. 11-13; Hogan JC Jr., Directed combinatorial chemistry. Nature (ENGLAND) November 7, 1996). It is also possible to screen for compounds that bind to the proteins of the present invention using the method described above.

Further possible uses of the VEGF-D of the present invention include gene therapy, in which the VEGF-D gene is introduced into or expressed in patients with VEGF-D gene deficiency. Alternatively, antisense sequences of the gene may be used to inhibit the expression of the gene itself and suppress pathological angiogenesis.

Various methods can be considered for introducing the VEGF-D gene or an antisense molecule to the gene into the body, and preferred methods include the retrovirus method, the liposome method, the cationic liposome method, and the adenovirus method.

To express these genes in vivo, the genes can be incorporated into an appropriate vector and then introduced into the body using the retrovirus method, liposome method, cationic liposome method, adenovirus method, or the like. There are no particular limitations on the vectors used, but vectors such as pAdexlcw and pZIPneo are preferred.

Furthermore, PCR detection of nucleotide sequence abnormalities in the VEGF-D gene may be useful for diagnosing diseases caused by VEGF-D gene abnormalities.

Further applications of the present invention include utilizing the angiogenic activity of VEGF-D protein to apply VEGF-D protein or its agonists to wound healing, promoting collateral circulation formation, or supporting hematopoiesis by hematopoietic stem cells, and using antibodies or antagonists to VEGF-D protein as therapeutic agents for pathological angiogenesis, lymphangiogenesis abnormalities, hematopoietic disorders, and edema caused by various causes. Furthermore, quantitative methods using VEGF-D antibodies may be used to diagnose diseases caused by abnormal VEGF-D production. BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 shows the relationship between the VEGF-D gene, each EST sequence, and the primers used for cloning.

FIG. 2 shows a comparison of the amino acid sequences of EST (H24828) and VEGF-C.

FIG. 3 shows a comparison of the amino acid sequences of the VEGF-D gene and the genes constituting the VEGF family reported to date.

Figure 4a shows a hydrophobicity plot of VEGF-D. Figure 4b shows a predicted signal peptide cleavage site of VEGF-D. BEST MODE FOR CARRYING OUT THE INVENTION The present invention will be specifically explained below with reference to examples, but the present invention is not limited to these examples.

Example 1: Homology Search Using the TFASTA Method The sequence "CGPNKELDENTCQCVC (SEQ ID NO: 3)" was designed based on the consensus sequence found in the "BR3P (Balbiani ring 3 protein) repeat" located in the C-terminus of VEGF-C. All EST and STS sequences in the GenBank database (as of February 29, 1996) were searched using the TFASTA method (Pearson and Lipman. Proc. Natl. Acad. Sci. USA 85; 2444-2448 (1988)). The following search conditions were used (Table 1).

We identified an EST (Accession No. H24828) that appears to encode this consensus sequence. This sequence is one of the ESTs registered by the WashU-Herck EST Project, and 9 of the 16 amino acids used in the search matched this sequence. Further searches using NCBI's UniGene revealed five other ESTs, including this one, that appear to be derived from the same gene: T64149, H24780, H24633, H24828, and T64277 (as of March 1, 1996). Of these, T64277 and T64149, and H24828 and H24780, each represent a combination of the 5' and 3' sequences of the same clone, and the insert size of both clones was approximately 0.9 kb (Fig. 1).

When the H24828 sequence was translated into a protein sequence in the same frame where homology was found, it was predicted to encode the C-terminal 104 amino acids. Aligning this amino acid sequence with the VEGF-C sequence revealed that 28 of the 104 amino acids (2.7%) were identical, and amino acids important for maintaining protein structure, such as cysteine and proline, were highly conserved (Figure 2). Conserved sequences are shown in white.

Example 2 Cloning of cDNA from the Library Primers for 5' and 3' RACE (5'-AGGGATGGGGAACTTGGAACGCTGAAT-3' (SEQ ID NO: 4) for 5' RACE, and 5'-GATCTAATCCAGCACCCCAAAAACTGC-3' (SEQ ID NO: 5) for 3' RACE) were designed based on the sequence of the EST (H24828) found by the search (Figure 1). Double-stranded cDNA was synthesized from human lung polyA ⁺ RNA using reverse transcriptase. Furthermore, PCR was performed using "Marathon-Ready cDNA, Lung (Clontech)," a cDNA with adapter cDNA ligated to both ends, as a template, with the above primers and the adapter primer AP-1 primer (5'-CCATCCTAATACGACTCACTATAGGGC-3' (SEQ ID NO: 6)) (Figure 1). The adapter cDNA contains regions to which the adapter primers AP-1 and AP-2 hybridize. PCR was performed under the following conditions: 1 minute at 94°C, followed by 5 cycles of 94°C for 30 seconds and 72°C for 4 minutes, 5 cycles of 94°C for 30 seconds and 70°C for 4 minutes, and 25 cycles of 94°C for 20 seconds and 68°C for 4 minutes. (However, instead of Advantage K1 enTaq Polymerase Mix, TaKaRa Ex Taq (Takara Shuzo) and the accompanying buffer were used.) As a result, 1.5 kb and 0.9 kb fragments were amplified at the 5' and 3' ends, respectively. These fragments were cloned using the pCR-Direct Cloning System (Clontech), the pCR-TRAP Cloning System (GenHunter), and the PT7Blue-T vector (Novagen). When cloning the 5'RACE fragment into the pCR-Direct vector, re-amplification was performed using the primers 5'-CTGGTTCGGCCCAGAACTTGGAACGCTG AATCA-3' (SEQ ID NO: 7) and 5'-CTCGCTCGCCCACTAATACGACTCACTATAGG-3' (SEQ ID NO: 8).

Example 3: Nucleotide Sequence Analysis DNA sequences were determined using the ABI PRISM Dye Terminator Cycle Sequencing Ready Reaction Kit with Amplitaq DNA Polymerase FS and the 377A DNA Sequencer (ABI). Primers used included the vector primers (5'-AATTAA CCCTCACTAAAGGG-3' (SEQ ID NO: 9), 5'-CCAGGGTTTTCCCAGTCACGAC-3' (SEQ ID NO: 10)), the AP-2 primer (5'-ACTCACTATAGGGCTCGAGCGGC-3' (SEQ ID NO: 11)), and the following 10 internal primers (Table 2).

The nucleotide sequences of the cloned 5'-terminal fragment (approximately 1.5 kb) and the 3'-terminal fragment (approximately 0.9 kb) were determined, and the overlapping nucleotide sequences were identical, confirming the authenticity of the 5'-terminal and 3'-terminal cDNAs of the target gene. The entire nucleotide sequence of the cDNA was determined to reveal that this novel gene was approximately 2 kb long and capable of encoding a protein consisting of 354 amino acids (SEQ ID NO: 1 and SEQ ID NO: 2). The relationship between this gene and the EST sequences registered in the GenBank database is shown in Figure 1. Comparison of the amino acid sequence with other VEGF family members revealed that the amino acids highly conserved among family proteins were also conserved in this novel gene, demonstrating its identity as a novel member of the VEGF family (Figure 3). In Figure 3, "HSVEGF" refers to human VEGF, and "HSVEGF-D,""HSVEGF-C," and "HSVEGF-B" refer to the human VEGF homologs "VEGF-D,""VEGF-C," and "VEGF-B," respectively. Furthermore, "HSPDGF-A" refers to human "PDGF-A,""HSPDGF-B" refers to human "PDGF-B," and "HSPlGF2" refers to human "PlGF2." Conserved sequences are shown in white. VEGF-D, in particular, shows high homology with VEGF-C, which was cloned as a Flt4 ligand, and was therefore identified as a ligand for a receptor similar to Flt4.

Based on the hydrophobicity plot (Fig. 4a) and the prediction of the signal peptide cleavage site by the von Heijne method (von Heijne G., Nucleic Acids Res. 14, 4683-4690 (1986)) (Fig. 4b), it is believed that the 21 amino acids from the N-terminus are cleaved as a signal peptide, but it is also possible that further processing may occur, similar to VEGF-C.

Example 4: Northern Blot Analysis. An approximately 1 kbp fragment excised by EcoRV from the 5' fragment subcloned into the pCR-Direct vector was labeled with [α- ^32P ]dCTP and used as a probe. Labeling was performed by the random primer method using Ready-to-Go DNA labeling beads (Pharmacia). Hybridization was performed using Multiple Tissue Northern (MTN) Blot-Human, Human II, Human Fetal, and Human Cell line (Clontech) in ExpressHyb Hybridization Solution (Clontech) according to standard procedures. Strong expression was observed in the lung, heart, and small intestine. Weak expression was also observed in skeletal muscle, ovary, colon, and pancreas. The apparent molecular weight of the mRNA was approximately 2.2 kb, indicating that the cloned gene was nearly full-length.

Example 5: Expression of VEGF-D Protein in E. coli 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 amino acids 89 to 181 of human VEGF cDNA. The resulting DNA fragment was digested with restriction enzymes BglII and SalI and ligated to plasmid pQE42 (QIAGEN) digested with restriction enzymes BanmHI and SalI using the Ligation Kit II (Takara Shuzo). The resulting plasmid was introduced into E. coli SG19003[pREP4] (QIAGEN), and a mutation-free, completed plasmid (pQE42-BS3) was selected. Plasmid pQE42-BS3 was transformed into E. coli BL21 (Invitrogen) and cultured in 10 ml of L Broth containing 100 mg/L vicillin (injectable ampicillin sodium, Meiji Seika Kaisha). This culture was then inoculated into 200 ml of fresh L Broth. After culturing for 1.5 hours at 37°C, IPTG was added to the medium to a concentration of 3 mM, and the culture was continued for an additional 5 hours at 37°C. After harvesting the cells, the protein was purified using a Ni-NTA column according to the QIAexpress Type II kit protocol.

Example 6: Expression of DHFR-VEGF-D Fusion Protein in Escherichia coli The region corresponding to amino acids 89 to 181 of human VEGF cDNA was amplified using the same primers as in Example 5. The resulting DNA fragment was digested with the restriction enzyme BglISalI and ligated to plasmid pQE40 (QIAGEN) digested with the restriction enzymes BamHI and SalI using the "Ligation Kit II" (Takara Shuzo). The resulting plasmid was introduced into E. coli SG19003 [pREP 4] (QIAGEN), and a mutation-free, completed plasmid (pQE40-BS3) was selected. Plasmid pQE40-BS3 was transformed into E. coli BL21 (Invitrogen) and cultured in 10 ml of L Broth containing 100 mg/L ampicillin (injectable ampicillin sodium, Meiji Seika Kaisha). This culture was then inoculated into 200 ml of fresh L Broth. After culturing at 37°C for 1.5 hours, IPTG was added to the medium to a final concentration of 3 mM, and the culture was continued for an additional 5 hours at 37°C. After harvesting the cells, the DHFR-VEGF-D fusion protein was purified using a Ni-NTA column according to the QIAexpress Type II kit protocol.

Example 7 Cloning of Mouse VEGF-D cDNA Two "Hybond-N+" (Amersham) filters (20 cm x 22 cm) were prepared by transcribing 1.5 x ¹⁰ pfu of the "Mouse lung 5'-stretch cDNA library" (Clontech). Approximately 50 ng of human VEGF-D PvuII fragment was labeled with α ³² P-dCTP (Amersham) using "Ready-To-Go DNA Labeling Beads (-dCTP)" (Pharmacia) as a probe. Gradient hybridization was performed from 68°C to 55°C for 2 hours using "ExpressHyb Hybridization Solution" (Clontech). The filters were washed four times for 10 minutes at room temperature with 2x SSC, 0.05% SDS, followed by 3 minutes at 45°C with 0.1x SSC, 0.1% SDS. The filter was exposed overnight at -80°C using a HyperFilm MP (Amersham) and an intensifying screen. Positive clones were similarly screened a second time to isolate single clones. The isolated lambda DNA was purified from the plate lysate using the QIAGEN Lambda MAXI Kit (Qiagen). The insert DNA was excised with EcoRI and subcloned into pUC118 EcoRI/BAP (Takara), after which the sequence was determined using an ABI377 sequencer (Perkin Elmer). A cDNA encoding full-length mouse VEGF-D was reconstructed from two overlapping clones obtained. The nucleotide sequence and deduced amino acid sequence of mouse VEGF-D cDNA are shown in SEQ ID NO: 24.

Example 8 Cloning of Rat VEGF-D cDNA Two Hybond-N+ (Amersham) filters (20 cm x 22 cm) were prepared by transcribing 1.5 x ¹⁰ pfu of the Rat Lung Lambda ZAP II Vector (Stratagene). Approximately 1 μg of the 1-782 bp fragment of mouse VEGF-D cDNA was labeled with α ³² P-dCTP (Amersham) using Ready-To-Go DNA Labeling Beads (-dCTP) (Pharmacia). Gradient hybridization was performed for 2 hours from 68°C to 55°C using ExpressHyb Hybridization Solution (Clontech). The filters were washed four times for 10 minutes at room temperature with 2x SSC, 0.05% SDS, followed by 5 minutes at 45°C with 0.1x SSC, 0.1% SDS. The filter was exposed overnight at -80°C using a HyperFilm MP (Amersham) and an intensifying screen. Positive clones were similarly screened a second time to isolate single clones. The isolated positive clones were excised into pBluescript using E. coli SOLAR (Stratagene) and the helper phage ExAssist (Stratagene), and then the base sequence was determined using an ABI377 sequencer (Perkin Elmer).

The result was a sequence that appeared to be rat VEGF-D cDNA, but did not include the stop codon.

To obtain the cDNA for the C-terminal portion that could not be cloned, we performed PCR using "Marathon-Ready rat kidney cDNA" (Clontech) as a template, with the 5' primer "GCTGCGAGTGTGTCTGTAAA (SEQ ID NO: 26)" and the 3' primer "GGGTAGTGGGCAACAGTGACAGCA A (SEQ ID NO: 27)" and repeated 40 cycles of 94°C for 15 seconds, 55°C for 30 seconds, and 72°C for 2 minutes. The resulting fragment was subcloned into the pGEM-T vector (Promega) and sequenced using an ABI377 sequencer (Perkin Elmer). The resulting clone contained the C-terminal portion of rat VEGF-D. The full-length rat VEGF-D was determined from the results of the clones obtained by plaque hybridization and PCR. The determined nucleotide sequence and deduced amino acid sequence are shown in SEQ ID NO: 25. INDUSTRIAL APPLICABILITY: According to the present invention, a novel protein (VEGF-D) with significant homology to VEGF-C and its gene have been isolated. VEGF-D is thought to be involved not only in normal angiogenesis during development but also in pathological angiogenesis observed in diabetes, rheumatoid arthritis, and solid tumor growth, as well as in the differentiation and proliferation of blood cells, lymphatic vessel formation, and edema formation due to various causes. The genes of the present invention can be used for the diagnosis of VEGF-D gene abnormalities and gene therapy for VEGF-D gene deficiency. VEGF-D proteins obtained by expressing the genes of the present invention are expected to be useful in wound healing, promoting collateral circulation formation, and supporting the proliferation of hematopoietic stem cells. Antibodies and inhibitors against VEGF-D proteins are expected to be useful in the treatment of inflammatory angiogenesis and lymphatic vessel abnormalities, edema due to various causes, and hematopoietic disorders, as well as in the treatment of pathological angiogenesis as novel anticancer agents. VEGF-D proteins and their antibodies are also expected to be useful in the diagnosis of diseases caused by abnormal VEGF-D production.

─────────────────────────────────────────────────────────── Continued from the front page (51) Int.Cl. ⁶ identification symbols FIG G01N 33/50 (81) Designated States EP (AT, BE, CH, DE, DK, ES, FI, FR, GB, GR, IE, IT, LU, MC, NL, PT, SE), OA (BF, BJ, CF, CG, CI, CM, GA, GN, ML, MR, NE, SN, TD, TG), AP (GH, KE, LS, MW, SD, SZ, UG, ZW), UA (AM, AZ, BY, KG, KZ, MD, RU, TJ, TM), AL, AM, AT , AU, AZ, BA, BB, BG, BR, BY, CA, CH, CN, CU, CZ, DE, DK, EE, ES, FI, GB, GE, GH, HU, IL, IS, JP, KE, KG, KR, KZ, LC, LK, LR, LS, LT, LU, LV, MD, MG, MK, MN, MW, MX, NO, NZ, PL, PT, RO, RU, SD, SE, SG, SI, SK, SL, TJ, TM, TR, TT, UA, UG, US, UZ, VN, YU, ZW (Note) This publication is based on the gazette published internationally by the International Bureau (WIPO). The effect of the international publication of the Japanese patent application (Japanese utility model registration application) related to this publication arises pursuant to Article 184-10, Paragraph 1 of the Patent Act (Article 48-13, Paragraph 2 of the Utility Model Act), and is unrelated to this publication.

Claims (10)

[Claims] 1. A protein set forth in SEQ ID NO: 1, or a protein having an amino acid sequence in which one or more amino acids have been substituted, deleted, or added. 2. A protein encoded by DNA that hybridizes with the DNA set forth in SEQ ID NO: 2. 3. DNA encoding the protein of claim 1. 4. A DNA that hybridizes with the DNA set forth in SEQ ID NO: 2. 5. A vector comprising the DNA of claim 3 or 4. 6. A transformant harboring the vector according to claim 5. 7. A method for producing the protein according to claim 1 or 2, comprising culturing the transformant according to claim 6. 8. An antibody that binds to the protein of claim 1 or 2. 9. A method for screening for a compound that binds to the protein of claim 1 or 2, comprising the step of detecting the binding activity of the protein of claim 1 or 2 to a test sample. 10. A compound that binds to the protein of claim 1 or 2, isolated by the method of claim 9.