Chemistry: natural resins or derivatives; peptides or proteins; – Proteins – i.e. – more than 100 amino acid residues – Hormones – e.g. – prolactin – thymosin – growth factors – etc.
Reexamination Certificate
1999-01-14
2004-12-07
Saoud, Christine J. (Department: 1647)
Chemistry: natural resins or derivatives; peptides or proteins;
Proteins, i.e., more than 100 amino acid residues
Hormones, e.g., prolactin, thymosin, growth factors, etc.
C536S023510
Reexamination Certificate
active
06828426
ABSTRACT:
TECHNICAL FIELD
The present invention relates to a protein factor involved in angiogenesis in humans and falls in the field of genetic engineering.
BACKGROUND ART
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.
DISCLOSURE OF THE INVENTION
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 Solu
Hirata Yuichi
Nezu Jun-ichi
Chugai Seiyaku Kabushiki Kaisha
Clark & Elbing LLP
Saoud Christine J.
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