WO2005056791A1 - 新規PlexinポリペプチドとそれをコードするDNA、及びその用途 - Google Patents
新規PlexinポリペプチドとそれをコードするDNA、及びその用途 Download PDFInfo
- Publication number
- WO2005056791A1 WO2005056791A1 PCT/JP2004/015997 JP2004015997W WO2005056791A1 WO 2005056791 A1 WO2005056791 A1 WO 2005056791A1 JP 2004015997 W JP2004015997 W JP 2004015997W WO 2005056791 A1 WO2005056791 A1 WO 2005056791A1
- Authority
- WO
- WIPO (PCT)
- Prior art keywords
- seq
- polypeptide
- amino acid
- dna
- gene
- Prior art date
Links
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P9/00—Drugs for disorders of the cardiovascular system
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
Definitions
- Novel Plexin polypeptide DNA encoding it, and its uses
- the present invention has a Plexin-like sequence, is specifically expressed in blood vessels throughout the angiogenesis stage from the early stage of angiogenesis during ontogeny, and is strong in the adult stomach / stratified squamous epithelium after growth.
- Novel DNA a gene containing the DNA, a gene containing the DNA, a novel polypeptide encoded by the DNA, and the polypeptide, which are slightly expressed in the uterus and expressed in various organs including the brain ,
- the present invention relates to a kit for measuring the activity of differentiating regulators and compounds for controlling vascular proliferation.
- the human KIAA0620 gene is present on chromosome 3q21.3, and several SNPs of the human KIAA0620 gene have been reported.
- the human KIAA0620 gene Healing fracture healing, vascular occlusion and collateral circulation, periodic vascular network formation in uterine mucosa (Neoplastic, luteal formation), etc., which are involved in the angiogenesis process and are undesirable, such as cancer growth, rheumatoid arthritis, diabetic retinopathy, endometriosis, obesity, and heart attacks where the angiogenesis process is undesirable
- human KIAA0620 gene to express mRNA of human KIAA0620-related gene in vascular endothelial cells or central nervous system (CNS) during mouse development. More detail on human KIAA0620 gene and its relationship. In order to conduct research, research using laboratory animals, not humans, is essential. However, a gene corresponding to the human KIAA0620 gene in rodents, especially mice, which is an important model animal for elucidation of human pathological conditions, has not been obtained, and studies using this gene cannot be carried out. Helped.
- Non-patent Document 1 1.Shibuya Masashi, 1999, Experimental Medicine, 17: 712-715, Current status of angiogenesis research and molecular regulation mechanism
- Non-Patent Document 2 2.Massunori Hirashima, Shinichi Nishikawa, 1999, Experimental Medicine, 17: 716-720, Embryology of vascular endothelial cells
- Non-patent document 3 3.Jun Yamashita, 2001, Experimental Medicine, 19: 830-835, Angiogenesis from ES cells
- Non-patent document 4 4.Nobuyuki Takakura, 2001, Experimental Medicine, 19: 836-840, Vascular cells
- Nonpatent Literature 5 5.Chisa Sukuminami, Hiroyuki Shibata, Yuji Kai, 2001, Experimental Medicine, 19: 841-846, Cartilage 'Bone Formation and Vascular Invasion
- Non-Patent Document 6 6.Matsuura Shigeaki, Okazaki Nohisa, Tani Naoyuki, Ero Hidetoshi, 1999, Experimental Medicine
- Non-Patent Document 7 7. Jane, R.K. & Carmely, P.F., 2002, Nikkei Science, March, 22-29, Science of Angiogenesis Changing Medical Care
- Non-Patent Document 8 8.van der Zwaag, B. et al, Dev.Dyn., 2002, 225: 336-343,
- PLEXIN-Dl a novel plexin family member, is expressed in vascular endothelium and central nervous system during mouse embryogenesis.
- Non-Patent Document 9 9.Tamagnone L, Artigiani S, Chen H, He Z, Ming GI, Song H, Chedotal A, Winberg ML, Goodman CS, Poo M, Tessier—Lavigne M, Comoglio PM., 2001, Cell, 99: 71--80, Plexins are a large family of receptors for transmembrane, secreted, and GPI— anchored semaphorins in vertebrates.
- Non-Patent Document 10 10.Manahan, D., 1997, Science, 277: 48-50, Signaling vascular morphogenesis and maintenance.
- Non-Patent Document 11 11Barinaga, M., 1997, Science, 275: 482-484, Desigining therapies that target tumor blood vessels.
- Non-Patent Document 12 12. Asahara T, Murohara ⁇ , Sullivan A, Silver M, van der Zee R, Li T, Witzenoichler B, Schatteman G, Isner JM. Asahara, T. et al., 1997, science, 275: 964-967, Isolation of putative progenitor endothelial cells for angiogenesis.Non-patent document 13: 13Risau, W., 1997, Nature, 386: 671-674, Mechanisms of angiogenesis.
- Patent Document 14 14Yamashita J, Itoh H, Hirashima M, Ogawa M, Nishikawa S, Yurugi T, Naito M, Nakao K, Nishikawa S. Flkl— positive cells derived from embryonic stem cells serve as vascular progenitors.
- Non-Patent Document 16 16Tamagnone L, & Comoglio PM., 2000, Trends in Cell Biology, 10: 377-383, Signalling by semaphorin receptors: cell guidance and beyond.
- Non-Patent Document 17 17Shimizu M, Murakami Y, Suto F, Fujisawa H, 2000, J. Cell Biol., 148: 1283-1293, Determination of cell adhesion sites of neuropilin-1.
- the human KIAA0620 gene had been obtained in the human long-chain cDNA project, but the information on its function, which is not complete, is incomplete. That is, in an experiment for detecting the expression of a human KIAA0620-related gene during mouse development using the human KIAA0620 gene as a probe, mRNA that reacts with the human KIAA0620 gene is expressed in vascular endothelial cells or the central nervous system (CNS). was reported. Power and human The KIAA0620 gene, which is a model animal that is important for elucidating human pathology, requires research using laboratory animals instead of humans in order to study the functions of the genes related to it in more detail. It was difficult to obtain the gene corresponding to the human KIAA0620 gene in rats, mice, hamsters, etc., especially in mice, and the gene was left unacquired, making it impossible to carry out research using this gene. Helped.
- obtaining a novel gene derived from a rodent such as a rat, a mouse or a hamster, particularly a mouse derived from a rodent related to the human KIAA0620 gene, and obtaining information on the function of the protein encoded by the novel gene of the present invention.
- a rodent such as a rat, a mouse or a hamster
- obtaining information on the function of the protein encoded by the novel gene of the present invention Is a very important means for searching for specific binding proteins, agonists and antagonists of the polypeptide of the present invention.
- the inactivation experiment of the polypeptide of the present invention (for example, production of a recombinant animal cell or a recombinant animal overexpressing or knocking out the gene)
- an agonist or an antagosto for the polypeptide of the present invention By analyzing the physiological action of the polypeptide from, it is possible to prepare an agonist or an antagosto for the polypeptide of the present invention, and a specific binding protein, agonist or antago for the polypeptide of the present invention.
- the second strike can be used as a preventive, therapeutic or diagnostic agent for diseases related to dysfunction of patients with various organ-related diseases.
- a decrease or increase in the function of the polypeptide of the present invention in a living body may be a cause of diseases relating to various organs.
- administration of the polypeptide antibody of the present invention targeting various organs of the polypeptide which can be achieved only by administration of a ligand / ligand inhibitor or an antagonist or agonist for the protein.
- the present invention can also be applied to the administration of antisense nucleic acid to the gene encoding the polypeptide or the administration of a short double-stranded RNA (RNAi) synthesized based on the sequence information of the gene, or gene therapy using the gene.
- RNAi short double-stranded RNA
- the nucleotide sequence encoding the polypeptide of the present invention is information necessary for examining the presence or absence of a gene deletion or mutation in a patient with a disease involving various organs of the polypeptide of the present invention.
- the gene encoding the polypeptide of the present invention can be applied to an agent for preventing, treating or diagnosing a disease associated with dysfunction of the polypeptide of the present invention.
- the present inventors have conducted intensive studies by using completely different materials and screening methods.
- the human KIAA0620 gene was found to be highly homologous to the human KIAA0620 gene from a mouse fetal tail bud-derived cDNA library.
- the gene obtained is expressed in a blood vessel-specific manner throughout the angiogenesis period from the early stages of angiogenesis during ontogeny, and in mature adults, it is strongly expressed in the stomach and stratified squamous epithelium. Slightly strong expression in the uterus In addition, it was found that expression was observed in various organs including the brain.
- the polypeptide sequence of the present invention was found to have domains characteristic of the Plexin family, They showed that they are involved in important functions such as proliferation, differentiation and guidance of the cells constituting the tissue, and based on the genetic information, obtained novel polypeptides and specific antibodies. Furthermore, they have found that the region defining the extramembrane portion of the protein encoded by the human or mouse KIAA0620 gene inhibits angiogenesis, and particularly inhibits the normal construction of the retinal vascular network. The present invention has been completed based on the above findings.
- the present invention provides a DNA comprising a base sequence encoding the following polypeptide (a) or (b): (a) SEQ ID NO: 1, SEQ ID NO: 15 or SEQ ID NO: A part of the amino acid sequence identical or substantially identical to the amino acid sequence represented by 18 (for example, the following novel Plexin family members, the second in the amino acid sequence represented by SEQ ID NO: 1 (methionine)) A polypeptide consisting of 1,745 amino acids at the 1,746th position (aranine), or a polypeptide having a power of 1,996 amino acids at the 2nd (methionine) to 1,997th position (aranine) in the amino acid sequence represented by SEQ ID NO: 15 ) Or a polypeptide that is all-powerful; or (b) an amino acid sequence represented by SEQ ID NO: 1, SEQ ID NO: 15, or SEQ ID NO: 18, which comprises an amino acid sequence in which some amino acids have been deleted, substituted, or added
- the amino acid sequence represented by SEQ ID NO: 18 and a part thereof for example, those having at least one motif found in the novel Plexin sequence shown below, or SEQ ID NO: 1
- the second (methionine)-1,746-position (alanine) polypeptide having 1,745 amino acid power or in the amino acid sequence shown in SEQ ID NO: 15, the second (methionine)-1,997th (aranine) Out of 1,996 amino acids
- B a DNA that hybridizes under stringent conditions to a DNA consisting of a base sequence complementary to the DNA of (a); or
- (c) a DNA that hybridizes under a stringent condition relates to a DNA which hybridizes with a DNA consisting of a complementary base sequence under stringent conditions and encodes a protein having substantially the same biological activity as a part or all of the polypeptide (a).
- the DNA according to the first and second aspects of the present invention described above is collectively referred to as “the DNA of the present invention” hereinafter.
- the DNA of the present invention encodes a novel polypeptide.
- the present invention also relates to genes derived from rodents such as rats, mice or hamsters, and particularly mouse genes, containing these DNAs.
- rodents such as rats, mice or hamsters
- mouse genes containing these DNAs.
- mouse gene is also referred to as “mouse (m) KIAA0620 gene” or “mplD0920 gene”! / ⁇ ⁇ .
- the present invention relates to a polypeptide (hereinafter, also referred to as “polypeptide of the present invention”) encoded by the DNA or gene of the present invention (hereinafter, also referred to as “KIAA0620 gene”), for example, the DNA of the present invention.
- a polypeptide hereinafter, also referred to as “KIAA0620 protein” which is a recombinant protein produced in a host cell into which a gene has been introduced.
- vascular endothelial cells Flk-1 positive cells: Literatures 1, 10, 12, and 14
- Useful as a novel molecular marker for is there.
- the polypeptide of the present invention is useful for providing a biomarker relating to angiogenesis, an amino acid sequence required for producing an antibody, an angiogenesis inhibitor and the like.
- the present invention relates to a recombinant vector containing the DNA or gene of the present invention, and a polypeptide of the present invention or a partial peptide thereof (for example, several dozens of C-terminal regions as described in Examples below).
- the present invention relates to an antibody that specifically binds to a recombinant protein containing the same amino acid sequence, or a recombinant protein containing the polypeptide, or a salt thereof.
- a substance (ligand) or ligand inhibitor that specifically binds to the polypeptide or the recombinant protein of the present invention is used.
- a screening method for a factor a compound that changes the expression level of the protein, a compound (antagonist, agonist) that changes the binding to a protein that binds to the protein, and a screening kit.
- transgenic cell or a transgenic animal into which the recombinant vector of the present invention has been introduced is prepared to provide an in vitro or in vivo disease state model.
- the DNA, gene, polypeptide or recombinant protein of the present invention can be prepared by using a recombinant animal or a recombinant animal cell or an antibody to control vascular growth / differentiation or a control compound (including a synthetic compound). ), And various kits for the screening.
- the present invention provides a DNA of the present invention, a recombinant vector containing the DNA of the present invention, An expression vector, a transformant carrying the vector, and the transformant are cultured to produce and accumulate a recombinant protein containing a part of the polypeptide of the present invention or a full-length polypeptide, and collect this.
- a recombinant protein containing a peptide or a salt thereof are provided in this manner! / Provided is a recombinant protein containing a peptide or a salt thereof.
- the present invention also relates to a pharmaceutical comprising the DNA of the present invention, a polypeptide of the present invention or a partial peptide thereof, or a base substantially complementary to a DNA encoding a recombinant protein containing the polypeptide.
- Antisense nucleotides having a sequence or a drug containing them, a short double-stranded RNA (RNAi) synthesized based on the sequence information of the DNA of the present invention or a drug containing them, and a drug of the present invention It is intended to provide a medicine comprising a polypeptide, a partial peptide thereof, or a recombinant protein containing the polypeptide.
- the present invention comprehensively prepares the DNA of the present invention, the polypeptide of the present invention, a partial polypeptide thereof, a recombinant protein containing the polypeptide, or an antibody against the DNA or gene of the present invention.
- the present invention also relates to so-called DNA chips (arrays) and protein chips obtained by integrating them.
- the present inventor has used a cDNA library derived from mouse fetal tail buds and applied a special screening method to improve the human KIAA0620 gene that could not be obtained until now.
- the polypeptide encoded by the novel DNA sequence is 1,746 or 1,997 amino acids long, and its polypeptide ri3 ⁇ , bemaphorin / CD100 antigen domain, and three Plexin / inemaphornvintegrin proteins.
- TM transmembrane
- the gene sequence of the present invention is specifically expressed in blood vessels throughout the angiogenesis stage from the early stage of angiogenesis during ontogeny, and is strongly expressed in the stomach and stratified squamous epithelium in adult humans after growth, In the brain and other various organs It has been found for the first time that the polypeptide encoded by the gene of the present invention is used in angiogenesis or in various organs including the brain, in which growth and function are maintained, and blood vessels in various organs are used. It was suggested that newborns may be involved in important functions related to diseases related to the newborn.
- a novel polypeptide corresponding to the extramembrane portion of the mouse KIAA0620 protein which is expressed in various organs including the brain, inhibits angiogenesis, and in particular, normal construction of the retinal vascular network was found to inhibit. That is, it was confirmed that the recombinant Plexin is a membrane protein and that the recombinant Plexin extramembrane region obtained by expressing the extramembrane region inhibits angiogenesis by a genetic recombination method using mammalian cells as a host.
- FIG. 1 Semaphorin / CDIOO antigen domain, three Plexin / Semapnornv integral domains, three elisurface receptor IPT / TIG domains, and transmembrane (TM) observed in the novel Plexin polypeptide of the present invention Indicates the segment (aa: amino acid sequence, the number is the number of amino acids, Bottom: domain and transmembrane (TM) segment identified by different search methods such as HMMPfam search method, HMMSmart search method, and Sosui search method from the top From these results, a new Plexin polypeptide was newly identified by the HMMPfam search method, the HMMSmart search method, and the Sosui search method.
- FIG. 2A shows the expression site of the mplD0920 (mouse KIAA0620) gene at each developmental stage 10.5d.p.c. in the mouse by whole mount in situ method. The image detected by the negative control using the sense probe is shown (the deeply stained site along the blood vessel is the expression site).
- FIG. 2B shows the expression site of the mplD0920 (mouse KIAA0620) gene at each developmental stage 10.5d.p.c. in mice by whole mount in situ method.
- the expression intensity of the mplD0920 gene by the antisense probe is shown (the deeply stained site along the blood vessel is the expression site).
- FIG. 2C The expression site of the mplD0920 (mouse KIAA0620) gene at each developmental stage of 8 dpc by whole mount in situ method (the deep staining site along the blood vessel is the expression site)
- FIG. 2D shows the expression site of mplD0920 (mouse KIAA0620) gene at each developmental stage 9.5d.p.c in mice by whole mount in situ method (dense staining site is observed along blood vessels).
- FIG. 3A shows the in situ expression site of the mplD0920 (mouse KIAA0620) gene in a sagittal frozen section of a fetus 14.5 days after fertilization. Above shows the detection image due to the negative control by the sense Burobu, below shows the expression strength of mp F00920 gene by antisense probe (deep dyeing sites expression site).
- FIG. 3B shows a strongly magnified detection image of the expression intensity of the mplD0920 gene by an antisense probe in the brain (the deeply stained site is the expression site).
- FIG. 4A shows the site of expression of the mplD0920 (mouse KIAA0620) gene at the mRNA level by in situ for paraffin sections of the stomach in adults. The squamous epithelium of the stomach is deeply stained.
- FIG. 4B shows the expression site of the mplD0920 (mouse KIAA0620) gene at the mRNA level by in situ method for cerebral paraffin sections in adults. Glial cells in the cerebrum are stained.
- FIG. 4C shows the expression site of the mpiD0920 (mouse KIAA0620) gene at the mRNA level by the in situ method for paraffin sections of the cerebellum in adults.
- the Purkinje cell layer of the cerebellum is deeply stained.
- FIG. 5 is a photograph of electrophoresis showing a comparison of the expression frequency of the mouse KIAA0620 gene at the mRNA level in the process of mouse embryo development by RT-PCR.
- the upper row shows the expression intensity of the FM gene used as a control, and the lower row shows the expression intensity of the mouse KIAA0620 gene with ethidium bromide staining pattern after fractionation by agarose electrophoresis.
- M high range marker ⁇ 1: HEK293 total proteins
- 4 HEK293 total proteins
- 9 mKIAA0620—cds—V5 proteins, (the above 5-8 lanes are stained with anti-mKIAA0620 antibody).
- the figures in the figures indicate dilution ratios.
- FIG. 7 Photomicrographs showing the results of detection of mouse KIAA0620 gene expression in neonatal retinal blood vessels using the in situ and hydridization methods (left figure: 2 days after birth, center: 4 days) Eyes, right: Day 7, dark spots are observed along the neovascularization).
- FIG. 8 Using HEK293 cells as a host, the mouse KIAA0620 full length-V5 fusion protein and the mouse extramembrane region including the transmembrane region (TM), that is, the mouse KIAA0620 extramembrane region (TM) -V5 fusion protein were expressed.
- FIG. 4 is a photograph showing the results of analyzing the protein fraction extracted and the hydrophobic protein extracted from the total protein fraction using Western blotting.
- 1. is the total protein fraction prepared from the control cells
- 2. is the hydrophobic protein fraction prepared from the control cells
- 3. is the total protein fraction prepared from the mouse KIAA0620 full-length V5 fusion protein expressing cells.
- 4 is a hydrophobic protein fraction prepared from mouse KIAA0620 full length-V5 fusion protein expressing cells
- 5 is a total protein fraction prepared from mouse KIAA0620 extramembrane domain (TM) -V5 fusion protein expressing cells
- 6. Shows the results of analysis of the hydrophobic protein fraction prepared from expression of the mouse KIAA0620 extramembrane domain (TM) -V5 fusion protein.
- the left picture shows the detection image by the colorimetric method
- the right picture shows the detection image by the chemiluminescence method
- the ladder and the numerical value (kDa) in the center show the size marker and the molecular weight by it.
- FIG. 10 is a micrograph showing the results of an experiment (3 days after administration on the first day after birth) of the inhibition experiment of neonatal mouse retinal vascular development using the mouse KIAA0620 extramembrane-IgGlFc fusion protein.
- Three days after ocular injection normal construction of the retinal vascular network was markedly impaired (right figure: mKIAA0620 extramembrane-Fc).
- the intraocular injection of IgGlFc protein alone showed no change Was not observed (Fig. Left: Fc).
- the DNA of the present invention is isolated from the cDNA library prepared by the present inventors as a cDNA fragment, using the mRNA derived from the mouse fetal tail bud collected by the present inventors as a starting material. Was determined and identified. That is, specifically, (DNA
- amino acid sequence from the 1st to the 1746th amino acid (1,746 amino acids in length) of the amino acid sequence represented by SEQ ID NO: 1 (1,746 amino acids in length) is an amino acid sequence (1,985 amino acids in length) encoded by the human KIAA0620 gene.
- the plasmid containing the DNA of the present invention having the nucleotide sequence shown in SEQ ID NO: 2 is a 1-1, Tsukuba East Higashi, Ibaraki Prefecture, Japan, the sixth independent administrative corporation. It was deposited at the National Institute of Advanced Industrial Science and Technology Patent Organism Depositary on September 10, 2003, and has the accession number FERM P-19518. The plasmid was further transferred to an international deposit under the Budapest Convention on the International Recognition of the Deposit of Microorganisms for Patent Procedure on October 25, 2004, and given the accession number FERM ABP-10154.
- the sequence of the human KIAA0620 gene is 6,754 base pairs in length, and the amino acid sequence of the encoded polypeptide is 1,985 amino acids in length (DNA Research, 1998, 5: 169-176, http: // (www.kazusa.or.jp/huge/glpage/KIAA0620/, NCB GenBank Accession No. AB014520) o
- additional polypeptides are encoded on the N-terminal side. Possibility may be verified.
- a suitable primer for example, the base of SEQ ID NO: 2
- a suitable primer for example, the base of SEQ ID NO: 2
- Pur rodent-derived mRNA is a rodent-derived tissue power
- the upstream side of the DNA of the present invention (5 ′ side of the gene) is obtained by performing a reverse transcription reaction after performing hybridization with the prepared mRNA.
- a new cDNA fragment containing the region can be specifically synthesized.
- the present invention is carried out by homologous cloning such as colony hybridization using a part of SEQ ID NO: 2 as a probe. It is possible to prepare the entire region of the rodent-derived KIAA0620 gene containing the DNA of the present invention. Alternatively, other methods, such as using the DNA of the present invention as a probe, By using homologous cloning such as colony hybridization, the 5 terminal region of the KIAA0620 gene derived from various rodents including mice can be prepared.
- Using the completed massively parallel genomic comparator perform a large-scale 'high-speed' nucleotide sequence search for the mouse mKIAA0620 from the published mouse genome sequence and add it to the mKIAA0620 nucleotide sequence based on the power of the published mouse genome sequence.
- a primer sequence necessary for obtaining an amino acid sequence newly added to the N-terminal side of the amino acid sequence shown in SEQ ID NO: 1 of the present invention was designed.
- a PCR method such as 5′RACE using these primers, as described specifically in the Examples of the present specification, 251 newly added N-terminals are added. The amino acid sequence could be determined.
- the DNA of the present invention may be any DNA as long as it comprises a base sequence encoding the polypeptide of the present invention described above.
- Various rodent-derived organs such as brain, heart, lung, liver, spleen, kidney, testis, thymus, muscle, bone marrow, etc., or cDNA libraries derived from various tissues and cells at each stage of development From cDNA isolated or synthetic DNA.
- the vector used for the library construction may be any of noctoriophage, plasmid, cosmid, phagemid and the like.
- RNA fraction or an mRNA fraction prepared from their cytoplasms were used for straightforward Reverse Transcriptase Polymerase and a hein Reaction (hereinafter, abbreviated as “RT-PCR method”). ) Positive amplification is also possible.
- amino acid sequence substantially identical to the amino acid sequence represented by SEQ ID NO: 1, SEQ ID NO: 15 or SEQ ID NO: 18 is the entire amino acid sequence represented by SEQ ID NO: 1, SEQ ID NO: 15 or SEQ ID NO: 18. Degree of homology with amino acid sequence Average of about 92.5% or more, preferably about 95% Above, more preferably about 98% or more of the amino acid sequence.
- the polypeptide of the present invention having substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 1, SEQ ID NO: 15 or SEQ ID NO: 18 includes, for example, the aforementioned SEQ ID NO: 1, SEQ ID NO: Having the above-mentioned homology to the amino acid sequence represented by SEQ ID NO: 15 or SEQ ID NO: 18, and substantially having the amino acid sequence represented by SEQ ID NO: 1, SEQ ID NO: 15 or SEQ ID NO: 18; And polypeptides having the same biological activity.
- substantially the same quality means that their activities are the same in nature.
- polypeptide of the present invention includes, for example, a part (preferably about 120, more preferably 1) in the amino acid sequence represented by SEQ ID NO: 1, SEQ ID NO: 15 or SEQ ID NO: 18.
- Polypeptides having substantially the same biological activity as the polypeptide having the indicated amino acid sequence are also included.
- the DNA of the present invention encodes, for example, the amino acid sequence represented by SEQ ID NO: 1, SEQ ID NO: 15 or SEQ ID NO: 18 in the nucleotide sequence represented by SEQ ID NO: 2, SEQ ID NO: 16 or SEQ ID NO: 19 That hybridizes under stringent conditions to a DNA having the same amino acid sequence as that shown in SEQ ID NO: 1 or SEQ ID NO: 15 Any DNA that encodes a polypeptide (protein) having biological activity! Under these conditions, the amino acid sequence represented by SEQ ID NO: 1, SEQ ID NO: 15 or SEQ ID NO: 18 in the nucleotide sequence represented by SEQ ID NO: 2, SEQ ID NO: 16 or SEQ ID NO: 19 is encoded.
- a DNA that can hybridize with a DNA consisting of a base sequence complementary to the base DNA is, for example, about 87.5% or more, preferably about 90 % Or more, more preferably about 95% or more.
- Hybridization can be performed according to a method known in the art or a method analogous thereto, such as the method described in Molecular cloning third.ed. (Cold Spring Harbor Lab. Press, 2001). When a commercially available library is used, it can be performed according to the method described in the attached instruction manual.
- "stringing The ⁇ unconditional conditions '' are, for example, when the probe is labeled with DIG DNA Labeling (Boehringer-I Mannheim Cat No.
- Cloning of the DNA of the present invention may be carried out by a PCR method using a synthetic DNA primer having an appropriate base sequence such as a portion of the polypeptide of the present invention, or by incorporating the DNA into an appropriate vector.
- Hybridization can be performed according to, for example, the method described in d, in Molecular Cloning third. Ed. (Cola Spring Harbor Lao. Press, 2001 J.
- the DNA sequence can be converted using a known kit, for example, the Superscript II reverse transcriptase kit (Gibco BRL) or the like, using the Gapped duplex method or the Kunkel method.
- the DNA encoding the cloned polypeptide may be used as it is, or may be digested with a restriction enzyme or added with a linker, if desired, depending on the purpose, or a method similar thereto.
- the DNA has an ATG as a translation initiation codon at its 5 'end and TAA, TGA or the like as a translation stop codon at its 3' end. May have a TAG. These translation initiation and termination codons may Chi possible to Caro with by using an appropriate synthetic DNA adapter.
- the expression vector of the polypeptide of the present invention can be prepared according to a method known in the art. For example, (1) a DNA fragment containing the DNA of the present invention or a rodent-derived gene containing the DNA of the present invention is cut out, and (2) the DNA fragment is ligated downstream of a promoter in an appropriate expression vector. It can be manufactured by the following.
- expression vectors include Escherichia coli-derived plasmids (eg, pBR322, pBR325, pUC18, pUC118), Bacillus subtilis-derived plasmids (eg, pUBllO, pTP5, pC194), yeast-derived plasmids (eg, p SH19, pSH15), ⁇ phage and other batteriophage, or expression vectors using sequences derived from animal viruses such as SV40, CMV virus, retrovirus, vaccinia virus, baculovirus, and paciperoma virus. it can.
- the promoter used in the present invention may be any promoter as long as it is appropriate for the host used for gene expression.
- the promoter includes the SRa promoter, the SV40 promoter, the LTR promoter, the CMV promoter, the HSV-TK promoter, the HSP promoter, and the metamouth thionein promoter.
- expression vectors include, if desired, an enhancer, a splicing signal, a cascade signal with poly A, a selection marker, and an SV40 replication origin (hereinafter sometimes referred to as SV40ori), which are known in the art. ) Can be added.
- the protein encoded by the DNA of the present invention may be expressed as a fusion protein with another protein (for example, daltathione S transferase, histidine tag, calmodulin binding protein, protein A, etc.). Is also possible.
- a fusion protein can be cleaved using an appropriate protease and separated into respective proteins.
- Bluescript SK (+) known in the art may be used as a gene transfer expression vector targeting animal cells for the purpose of producing recombinant animal cells.
- / _) It is preferable to use a vector linked to a Tie2 promoter (vascular endothelial cell-specific promoter) for vascular endothelial cell-specific expression.
- Tie2 promoter vascular endothelial cell-specific promoter
- a cloning system known as (Invitrogen: Cat. No. ll821-014) can also be used.
- Examples of host cells include Escherichia, Bacillus, yeast, insect cells, insects, animal cells, and the like.
- Escherichia bacteria include DH1 derived from Escherichia coli U (Escherichia coli) K12 (Proc. Natl. Acad. Sci. USA, 60, 160 (1968)), JM103 (Nucleic Acids Research, 9 (309) (1981)), JA221 (Journal of Molecular Biology, 120, 517 (1978)), and HB101 (Journal of Molecular Biology, 41, 459 (1969)), or Escherichia coli.
- Bacillus subtilis M II 14 Gene, 24, 255 (1983)
- 207-21 Journal of Biochemistry, 95, 87 (1984)] and the like are used.
- yeast for example, Saccharomyces cerevisiae (
- animal cells examples include monkey kidney cell-derived COS-1, COS-7, Vero cells, Chinese nomster CHO cells (hereinafter abbreviated as CHO cells), and dhfr gene-deficient Chinese hamster cells CHO (hereinafter, CHO (dhfr- ) Cells), mouse L cells, mouse AtT-20, mouse myeloma cells, rat GH3 cells, HEK293T cells, human FL cells, human Hela cells, human myeloma cells, and the like.
- CHO cells Chinese nomster CHO cells
- mouse L cells mouse AtT-20
- mouse myeloma cells rat GH3 cells
- HEK293T cells human FL cells
- human Hela cells human myeloma cells
- Cells for gene transfer targeting animal cells for the purpose of producing recombinant animal cells include, for example, cultured mouse ES cells, mouse fertilized eggs, mouse NIH3T3 cell line, human fetuses. A kidney cell-derived 293 cell line or the like is used.
- Transformation of these host cells can be performed according to methods known in the art.
- the following documents can be referred to. Proc. Natl. Acad. Sci. US A (69, 2110—, 1972), Gene (17, 107—, 1982), Molecular & General Genetics (168, pill—, 1979), Methods in Enzymology ( Natl. Acad. Sci. USA (Vol. 75, 1929—, 1978), Cell Engineering Separate Volume 8 New Cell Engineering Experiment Protocol (P263—267, 1995, Shujunsha) Issue), and Virology (52, 456—, 1973).
- the transformation of host animal cells for the purpose of transforming a transgenic animal may be carried out in accordance with the present invention. It can be performed according to a method known in the technical field. For example, one can refer to the documents described below. Proc. Natl. Acad. Sci. USA (Volume 77, p7380—7384, 1980), Separate Volume on Experimental Medicine, Genetargain's Latest Technology (p34—41, 2000, published by Yodosha), Mouse Operation Manual Second Edition (P225-252, p279-285, 1994, published by Modern Publishing), Special Issue on Experimental Medicine, Developmental Engineering Experiments (196 pages, 1994, published by Yodosha), and Trend in Genetics (5 volumes, p70-76, 1989)
- mice a microinjection method is used. Intraperitoneal administration of pregnant horse serum (5 units) to mice for egg collection (mouse strains C57BLZ6, C3H, BDF1) and 48 hours later, intraperitoneal administration of human chorionic gonadotropin (5 units) to induce ovulation . Ovulation occurs 14 hours after hormone injection, during which time they are mated with male mice. After confirming copulation by the presence or absence of a female vaginal plug, female mice are sacrificed, and fertilized eggs are collected from the ampulla of the fallopian tubes.
- the collected fertilized eggs are transferred to a Petri dish containing the culture solution, and the target gene is injected into the pronucleus of the fertilized eggs using a micromanipulator under a microscope. After injection of the target gene, the survived fertilized eggs are transferred into the fallopian tubes of pseudopregnant mice. Newborns (transgenic mice) are obtained approximately 20 days after transfer of the fertilized egg.
- a target gene is introduced into cultured mouse ES cells by electroporation, cells that have undergone homologous recombination of the target gene are selected, and the ES cells are then transferred to another blastocyst. Are injected into the fallopian tubes of the foster mother to produce chimeric mice.
- this method first, it is necessary to select ES cells that have been transfected, or ES cells or ES cells that have been transfected, but have randomly entered ES cells that have undergone homologous recombination. Utilizes the neomycin resistance gene. This gene is inserted between the homologous regions.
- homologous recombination is confirmed by the ability to detect DNA fragments by Southern blotting and PCR.
- a positive / negative selection method can be used as another method.
- a herpesvirus-derived thymidine kinase gene or diphtheria toxin fragment A gene is used as a second selection marker gene.
- Non-homologous recombinant cells of the target gene can be killed by adding ganciclovir or expressing diphtheria toxin fragment A.
- the thus obtained transformant transformed with the expression vector containing the rodent-derived gene containing the DNA of the present invention can be cultured according to a method known in the art. it can.
- culturing is usually performed at about 15 to 43 ° C for about 3 to 24 hours, and if necessary, aeration and stirring may be added.
- the cultivation is usually performed at about 30 to 40 ° C for about 6 to 24 hours, and if necessary, aeration and stirring can be applied.
- culturing a transformant in which the host is yeast When culturing a transformant in which the host is yeast, culturing is usually performed at about 20 to 35 ° C for about 24 to 72 hours using a medium adjusted to pH about 5 to 8, and if necessary. Aeration and stirring can also be added.
- the cultivation is usually performed for about 15 to 60 hours at about 30 to 40 ° C using a medium adjusted to a pH of about 6 to 8, and if necessary, aeration or the like may be performed. Stirring can be performed.
- the polypeptide of the present invention for example, after culturing, cells or cells are collected by a known method, suspended in an appropriate buffer, and subjected to ultrasonic wave, lysozyme, or the like. After the cells are destroyed by freezing and thawing, etc., the cells are destroyed, and a crude extract of the protein is obtained by centrifugation or filtration.
- the buffer may contain a protein denaturant such as urea or guanidine hydrochloride, or a surfactant such as Triton X-100 TM.
- the protein contained in the thus obtained culture supernatant or extract can be purified by appropriately combining known separation and purification methods.
- the polypeptide (protein) of the present invention thus obtained can be converted to a salt by a known method or a method analogous thereto, and conversely, if the polypeptide (protein) is obtained as a salt, a known method or analogous method Thus, it can be converted into a free form or another salt.
- the N-terminal methionine is removed with methionine aminopeptidase, or the myristoylation of the N-terminal amino acid is performed with myristoyltransferase.
- N using cetyltransferase Amino acids can be arbitrarily modified by acetylating the terminal amino acid or by using other modifying enzymes.
- the C-terminal amino acid can be modified by the action of a processing carboxyl peptidase that modifies the C-terminal, a C-terminal amidase, or the like.
- the polypeptide can be partially removed by the action of an appropriate protease such as trypsin, chymotrypsin, FactorXa, thrombin, or KEX2 protease.
- unnecessary polypeptide portions can also be removed using an appropriate proteolytic enzyme.
- polypeptide (protein) of the present invention or a salt thereof can be measured by various binding assays and enzyme immunoassays using specific antibodies.
- the polypeptide (protein) of the present invention has a force at which the C-terminus is usually a carboxyl group (one COOH) or carboxylate (one COO-), and the C-terminus is an amide (one CONH) or ester.
- R in the ester is, for example, a C1-6 alkyl group such as methyl, ethyl, n-propyl, isopropyl or n-butyl, for example, a C3-8 cycloalkyl group such as cyclopentyl, cyclohexyl, and the like.
- C6-12 aralkyl groups such as C6-12 aryl groups such as naphthyl and the like, for example, phenyl C1-2 alkyl groups such as benzyl and phenethyl or ⁇ -naphthyl-C1-2 alkyl groups such as ⁇ -naphthylmethyl.
- Bivaloyl oxymethyl ester which is widely used as an ester for power and oral use, is used.
- the polypeptide of the present invention (the existence state of which is a protein) has a carboxyl group (or carboxylate) other than the C-terminus
- the carboxyl group is amidated or esterified.
- the ester in this case, for example, the above-mentioned C-terminal ester and the like are used.
- the amino group of the methionine residue at the ⁇ -terminal is protected by a protecting group (for example, a C1-6 acyl group such as a formyl group or an acetyl group).
- a protecting group for example, a C1-6 acyl group such as a formyl group or an acetyl group.
- the peptide comprising a part of the polypeptide of the present invention is a partial peptide of the above-mentioned polypeptide of the present invention (the existence state of which is a protein), and has substantially the same activity.
- Any material may be used.
- a peptide having an amino acid sequence and having substantially the same biological activity as the recombinant protein of the present invention can be used.
- partial peptide examples include those having a motif characteristic of the novel Plexin in the amino acid sequence represented by SEQ ID NO: 1, or the second (methionine) in the amino acid sequence represented by SEQ ID NO: 1.
- a polypeptide consisting of 1,745 amino acids at the 1,746th position (alanine) can be mentioned.
- the partial peptide of the present invention usually has a carboxyl group (one COOH) or carboxylate (COO-) at the C-terminus, but has an amide (one CONH) at the C-terminus as in the protein of the present invention described above.
- the partial peptide of the present invention has a structure in which the amino group of the N-terminal methionine residue is protected with a protecting group, similarly to the above-described protein of the present invention.
- a protecting group similarly to the above-described protein of the present invention.
- a physiologically acceptable acid addition salt is particularly preferable.
- examples of such salts include salts with inorganic acids (eg, hydrochloric acid, phosphoric acid, hydrobromic acid, sulfuric acid) and organic acids (eg, acetic acid, formic acid, propionic acid, fumaric acid, maleic acid) And succinic acid, tartaric acid, citric acid, malic acid, oxalic acid, benzoic acid, methanesulfonic acid and benzenesulfonic acid).
- the polypeptide of the present invention (the existence state of which is a protein), a peptide consisting of a part thereof, a salt thereof, or an amide thereof may be prepared by a chemical synthesis method known in the art. You can also. For example, use a commercially available resin for protein synthesis. An amino acid having an ⁇ -amino group and a side chain functional group appropriately protected is condensed on a resin according to the sequence of a target protein according to various condensation methods known per se in the art.
- the protein is cleaved from the resin and, at the same time, various protecting groups are removed.
- an intramolecular disulfide bond formation reaction is carried out in a highly diluted solution to obtain the target protein, its partial peptide or an amide thereof.
- the condensation of the protected amino acids described above is used, for example, in the synthesis of proteins represented by carpoimides such as DCC, ⁇ , ⁇ , -diisopropylcarbodiimide, and ⁇ -ethyl- ⁇ ,-(3-dimethylaminoprolyl) carboimide.
- Various types of active reagents that can be used can be used.
- the ability to directly add a protected amino acid to a resin together with a racemization inhibitor eg, HOBt, HOOBt
- a racemization inhibitor eg, HOBt, HOOBt
- a protected amino acid as a control acid anhydride or HOBt ester or HOOBt ester is used.
- Solvents used for the condensation of the protected amino acid with the activated liposide include acid amides, halogenated hydrocarbons, alcohols, sulfoxides, ethers, and the like. It can be appropriately selected from solvents known to be usable for the condensation reaction.
- the reaction temperature is appropriately selected from a range known to be usable for a protein bond formation reaction, and a force in a range of usually about -20 to 50 ° C is also appropriately selected.
- the activated amino acid derivative is usually used in a 1.5-4 fold excess. As a result of the test using the ninhydrin reaction, when the condensation is insufficient, sufficient condensation can be performed by repeating the condensation reaction without removing the protecting group.
- the unreacted amino acid can be acetylated using acetic anhydride or acetylimidazole so as not to affect the subsequent reaction.
- the protecting group such as each amino group, carboxyl group, and serine hydroxyl group of the raw material, a group usually used in the technical field can be used.
- the protection of the functional group that should not be involved in the reaction of the raw material, the protective group, the elimination of the protective group, the activation of the functional group involved in the reaction, and the like can be appropriately selected from known groups or known means.
- Peptides or salts thereof which also partially act according to the present invention can be produced according to peptide synthesis methods known per se in the art, or by cleaving the protein of the present invention with an appropriate protease. Can be.
- a method for peptide synthesis For example, any of a solid phase synthesis method and a liquid phase synthesis method may be used. Examples of known condensation methods and elimination of protecting groups include the methods described in the following (1)-(3).
- a peptide consisting of a part can be obtained by, for example, using a well-known peptide synthesizer or the like to convert a ten-residue portion at an arbitrary position in the amino acid sequence represented by SEQ ID NO: Can be synthesized.
- a polypeptide having an appropriate site having a length of about 50 to 500 amino acids or a polypeptide having an appropriate site having a length of about 500 amino acids to its full length is selected.
- it may be produced as a recombinant protein using the above-mentioned gene recombination technology.
- the partial peptide of the present invention can also be purified and isolated by a method known per se for purification after the reaction, for example, by a combination of solvent extraction 'distillation' column chromatography, liquid chromatography and recrystallization.
- a method known per se for purification after the reaction for example, by a combination of solvent extraction 'distillation' column chromatography, liquid chromatography and recrystallization.
- the partial peptide obtained by the above method is a free form, it can be converted to an appropriate salt by a known method, and conversely, when it is obtained by a salt, it can be converted to a free form by a known method. Can be.
- An antibody that specifically binds to the polypeptide of the present invention (the existence state of which is a protein), a peptide composed of a part thereof, or a salt thereof is not limited as long as it can specifically recognize them. Any of a polyclonal antibody and a monoclonal antibody may be used.
- Antibodies against the polypeptide (protein), its partial peptide or a salt thereof of the present invention can be prepared by using the polypeptide (protein) or its partial peptide of the present invention as an antigen and producing antibodies or antisera known to those skilled in the art. Can be manufactured.
- partial peptides that can be used as an immunogen in this manner include, as specifically described in the Examples of the present specification, more than a dozen individual peptides in the C-terminal region of the polypeptide of the present invention shown in SEQ ID NO: 1. Peptides consisting of about 100 consecutive amino acids can be mentioned.
- polyclonal antibody the presence of an adjuvant is determined by binding the above antigen alone or to an appropriate carrier such as cellulose, polymerized amino acid, albumin and keyhole limpet hemocyanin (KLH). Or in the absence, for example, rats, egrets, sheep It can be easily obtained by immunizing appropriate animals such as goats and goats.
- Polyclonal antibodies can also be recovered and purified by various methods known in the serum of immunized animals.
- antibody-producing cells for example, spleen or lymph node-derived
- a known immortalized growth cell for example, Hybridomas are produced by cell fusion with mouse myeloma strain P3X63Ag8 or Sp2 / OAg-14 derived therefrom or myeloma cell strain such as rat myeloma strain Y3-Agl.2.3, YB2 / 0, IR983F).
- a clone of a hybridoma producing an antibody that specifically recognizes the polypeptide of the present invention is selected, and a culture solution of the hybridoma is recovered and purified. Obtainable.
- references regarding production of antibodies to the synthetic peptide and purification of the antibodies include, for example, New Cell Engineering Experimental Protocol, The University of Tokyo Medical Science Laboratory Cancer Research Division, Shujunsha, 1993, 2- 2-2, Preparation of antibody against synthetic peptide, p210-217.
- various chimeric antibodies such as humanized antibodies, containing the antigenic determinants of the thus obtained antibodies can be easily produced by various genetic engineering techniques known to those skilled in the art. Wear.
- the antibody of the present invention can be used for detecting the polypeptide (protein) of the present invention present in a subject such as a body fluid or a tissue.
- the antibody of the present invention can be used for quantification of the polypeptide (protein) of the present invention in a test solution by a known method, in particular, for quantification by a sandwich immunoassay using a monoclonal antibody, and for tissue analysis. It can be used for detection by staining or the like. Thereby, for example, a disease involving the polypeptide (protein) of the present invention or the like can be diagnosed.
- the antibody molecule itself may be used, or F (ab ') 2, Fab', or Fab fraction of the antibody molecule may be used.
- the method for quantifying the protein or the like of the present invention using the antibody of the present invention is not particularly limited.
- the amount of antibody, antigen or antibody-antigen complex corresponding to) is detected by chemical or physical means, and this is calculated from a standard curve prepared using a standard solution containing a known amount of antigen. If so, any measurement method may be used. For example, it is preferable to use a sandwich method described later in terms of sensitivity and specificity in which nephrometry, a competitive method, an immunometric method and a sandwich method are suitably used.
- a labeling agent used in a measurement method using a labeling substance for example, a radioisotope, an enzyme, a fluorescent substance, a luminescent substance and the like known in the art can be used.
- the antisense nucleic acid having a nucleotide sequence substantially complementary to DNA encoding the polypeptide (protein) of the present invention or a peptide comprising a part thereof is substantially complementary to the nucleotide sequence of the DNA.
- Any antisense nucleic acid may be used as long as it has a simple base sequence and an action capable of suppressing the expression of the DNA.
- the substantially complementary nucleotide sequence is, for example, a nucleotide sequence having about 95% or more, most preferably 100% homology with the entire nucleotide sequence or a partial nucleotide sequence of the nucleotide sequence complementary to the DNA of the present invention. And so on.
- Nucleic acid sequences (DNA, RNA or modified forms of these nucleic acids) having the same action as these antisense nucleic acids are also included in the present invention. Also, Short double-stranded RNA (RNAi) synthesized based on the base sequence information of the DNA can also be produced using a known nucleic acid synthesizer or the like.
- RNAi Short double-stranded RNA synthesized based on the base sequence information of the DNA
- the polypeptides (proteins) and the like of the present invention are useful as reagents for screening compounds that inhibit the activity of these substances or salts thereof. That is, the present invention provides a polypeptide (protein) of the present invention, a peptide capable of partially acting on the polypeptide or a salt thereof, and a compound that inhibits the activity of the substance or a salt thereof (hereinafter referred to as “the compound”). Inhibitors), and a screening kit therefor.
- the compound or a salt thereof obtained by using the screening method or the screening kit of the present invention is a compound selected from the above-mentioned test compounds, and the biological activity of the polypeptide (protein) or the like of the present invention.
- Is a compound that inhibits The compound or a salt thereof may directly inhibit the activity of the protein of the present invention or may indirectly inhibit the expression of the polypeptide (protein) of the present invention. It may be one that inhibits the activity of a peptide (protein) or the like.
- the salt of the compound for example, a pharmaceutically acceptable salt and the like are used. Examples include salts with inorganic bases, salts with organic bases, salts with inorganic acids, salts with organic acids, salts with basic or acidic amino acids, and the like.
- Compounds that inhibit biological activities such as the polypeptides (proteins) of the present invention may also be used as medicaments such as therapeutic and prophylactic agents for the above-mentioned various diseases.
- the polypeptide of the present invention or a peptide partially comprising the same in a mouse, a rodent, and a human Abnormality (genetic abnormality) of DNA or mRNA that encodes can be detected, for example, damage, mutation or decreased expression of the DNA or mRNA, or increase or decrease of the DNA or mRNA can cause overexpression. It is useful as a gene diagnostic agent.
- the above-described genetic diagnosis using the DNA of the present invention can be performed, for example, by the known Northern hybridization or PCR-SSCP method (Genomics, Vol. 5, pp. 874-879 (1989), Proceedings of the National Academy).
- the human KIAA0620 gene which is a homologous gene of the present invention, is abnormal, defective, or has a reduced expression level, it can exhibit an extremely normal function in vivo.
- gene therapy using an appropriate vector such as a retrovirus vector, adenovirus vector, or adenovirus associated virus vector as a vehicle can be performed according to known means.
- the gene of interest is introduced into the body of the patient and expressed, or (2) the polypeptide (protein) of the present invention or the polypeptide (protein) of human origin or the antibody of the present invention is injected into the patient.
- the function of the protein of the present invention can be exhibited in patients.
- the DNA of the present invention or a human-derived gene is used as an appropriate vector as a vehicle, and the DNA in the form of a vector is used alone or in combination with an auxiliary agent for promoting uptake. It can also be administered by a catheter such as a nodular mouth gel catheter.
- SEQ ID NO: 1 Shows the amino acid sequence (amino acid number: 1,746) of the polypeptide of the present invention.
- SEQ ID NO: 2 This shows the entire base sequence (6,178 base pairs) of clone mplD0920, including the base sequence of DNA encoding the polypeptide of the present invention having the amino acid sequence represented by SEQ ID NO: 1.
- SEQ ID NO: 15 This shows the amino acid sequence (amino acid number: 1,997) of the polypeptide of the present invention represented by SEQ ID NO: 1 with an additional 251 amino acids (SEQ ID NO: 11) added to the N-terminal side.
- SEQ ID NO: 16 SEQ ID NO: Shows the entire nucleotide sequence (6,931 base pairs) including the nucleotide sequence of the DNA encoding the polypeptide of the present invention having the amino acid sequence represented by SEQ ID NO: 15. 18] This shows the amino acid sequence (amino acid number: 1,337) of the extramembrane region of the polypeptide of the present invention derived from mouse.
- SEQ ID NO: 19 This shows the entire base sequence (4,011 base pairs) including the base sequence of DNA encoding the extra-membrane region of the polypeptide of the present invention having the amino acid sequence represented by SEQ ID NO: 18.
- g and c represent a fluorescein group, a phosphorothioate-modified G residue and a phosphorothioate-modified C residue, respectively, as primers, and mouse embryonic tail buds (ICR mouse fertilization at day 11.5 SI, SO, S Double-strand cDNA was synthesized using SuperScriptll reverse transcriptase kit (manufactured by Invitrogen) using mRNA derived from pre-segment mesoderm and somite mesoderm relating to -1, and S-2. An adapter having an attBl site was ligated to the cDNA.
- cDNA was size-fractionated into lkb-2kb, 2kb-3kb, 3kb_4kb, 4kb_5kb, and 5kb_7kb using agarose gel. These were transferred to a reduced-size attP pSPORT-1 entry vector (manufactured by Invitrogen) by a BP reaction, and then introduced into Escherichia coli ElectoroMax DH10B (manufactured by Invitrogen) by an electoral-portion method. More than 10 6 transformants appeared on the plate were collected and cultured in a liquid medium at 37 ° C for 2-3 hours to prepare a plasmid.
- the cDNA was transferred to an attR pBC destination vector (Invitrogen) by LR reaction. After purification of this plasmid, it was introduced into the DH10B strain by the electoral poration method, and the above-described fractionation operation was repeated 2-3 times until each fraction became an expected size. Finally, plasmid was introduced into the DH10B strain for each fraction.
- the cloning system using a homologous recombination reaction in a test tube used here is based on the method of Ohara et al. (Nucreic Acids Res., 29, e22 (2001) and DNA Research Vol. 9, 47-57 (2002)). )).
- DNA sequence of the 3 ′ end of about 16,608 clones contained in all the fractions was determined. From these, the entire nucleotide sequence of the cDNA of a clone having high homology to human KIAA0620 was determined.
- a DNA sequencer (ABI PRISM3700) manufactured by Applied Biosystems and a reaction kit manufactured by the company were used. Most arrays are shotgun black was determined using the dye terminator method.
- oligonucleotides were synthesized based on the determined base sequences and determined by the primer walking method.
- Comparison of the amino acid sequence and base sequence of KIAA0620 revealed that, at the amino acid sequence level, the 238th position of the amino acid sequence (1,985 amino acids long) encoded by the human KIAA0620 gene, the 1,985th position (1,748 amino acids long), and SEQ ID NO: It was found that about 91.88% homology was found in the amino acid sequence (1,746 amino acids long) from the 1st to the 1,746th amino acid sequence of the mplD0920 amino acid sequence (1,746 amino acids long) shown in 1.
- amino acid sequence of the polypeptide encoded by the human KIAA0620 gene between the 974th position and the 975th position of the amino acid sequence (1,746 amino acids in length) represented by SEQ ID NO: 1 of the present invention two amino acids, And insertion of alanine (V, A) was found.
- WO200157188-A2's Human Plexin-Bl / SEP receptor homologue SEQ ID NO: 2079D1 (1,992 amino acids long) is derived from humans, and the amino acid sequence of the polypeptide fragment is described for use as a diagnostic protein, therapeutic or biopharmaceutical. The power of its function is unknown.
- the mouse-derived 1,746 amino acid sequence of the present invention is a novel sequence and has a useful function that can be described in terms of function, even though the amino acid sequence has a similar length of 1,925 amino acids.
- Coding sequence SEQ ID 118 downregulated in International Publication No.WO200281745-A2 (Title of Invention: Coding sequence SEQ ID 118, downregulated in osteogenesis, Applicant: Aventis Pharma SA., Publication date: 17, Oct. 2002) Approximately 86.95% comparison of 5,249 bases from 710th to 5,958th base of in osteogenesis (6,754 bases) with 5,243 bases from 1st to 5,243th base of the nucleotide sequence of the present invention (6,178 bases) High homology was observed.
- Coding sequence SEQ ID 118 is a human-derived nucleotide sequence whose expression is suppressed during bone formation, and has a relatively high homology to the mplD0920 nucleotide sequence of the present invention (SEQ ID NO: 2, 6, 178 nucleotides in length).
- the base sequence of the present invention Are different arrays.
- Plexin-Dl Applicant: Univ.Torino, Univ. Aliforma, Publication date: 01, Mar. 2001
- Human cDNA encoding Plexin-Dl (5,892 bp) from 5,542 to 5,790 bp with 5,249 bp.
- a somewhat high homology of about 86.91% was observed between the first and fifth 5,243 nucleotides in the nucleotide sequence (6,178 nucleotides) of the present invention.
- Plexin-Dl (6,178 bases in length) is derived from humans, and its amino acid sequence of the polypeptide fragment encoded by it is described as a diagnostic protein, therapeutic, or biopharmaceutical. The function is unknown.
- the nucleotide sequence of 6,178 nucleotides having a slightly higher homology the nucleotide sequence of 6,178 nucleotides derived from the mouse of the present invention is a novel sequence, and its function can be described and is useful.
- Human Plexin-Bl / SEP receptor homologue-encoding cDNA, SEQ ID NO: 729 (7,080 base length) The 5,270 base length from SEQ ID NO: 710 to 5,979 and the 5,243 base length from SEQ ID NO: 1 to 5,243 of the nucleotide sequence of the present invention (6,178 base) are slightly higher homology of about 85.50%. Was observed.
- Human Plexin-Bl / SEP receptor homologue-encoding cDNA, SEQ ID NO: 729 (7,080 nucleotides in length) is derived from humans.
- Coding sequence SEQ ID 43 is a human-derived nucleotide sequence whose expression is suppressed during osteogenesis, and has a strong homology to the mplD0920 nucleotide sequence (6,178 nucleotides) of the present invention Sl, 073 nucleotides long It can be said that the sequence has only homology with a part of the nucleotide sequence of the present invention in the non-coding region on the three prime side of the nucleotide sequence of the present invention, which is considerably short.
- sequences filed in 1), 2) and 3 slightly higher homology was observed as described above, as compared with the nucleotide sequence of the present invention (6,178 nucleotides in length).
- the sequence of the gene of the present invention is judged to be a completely different novel sequence although a somewhat high homology is recognized.
- the sequence filed in 4) includes a part of the base sequence of the present invention in the non-coding region on the 3 prime side, which does not cover the entire length of the base sequence of the present invention, that is, the mplD0920 gene sequence (6,178 base length). There is a relationship in which high homology is partially observed.
- the sequence of 4) and the sequence of the gene of the present invention are judged to be completely different novel sequences, although high homology is partially observed.
- the candidate clone mplD0920 showed a relatively high homology with the human KIAA0620 gene, and is similar to several known genes that have already been reported. It was found to be a novel gene from a new rodent.
- a protein analysis program pftoois (Bairoch A, Bucher P, Ho & iann K, Nucleic Acids Res. 1997 Jan 1; 25 (1): 217-21), which is a protein analysis program for searching the PROSITE database, and Pfam database
- a protein analysis program hmmer 2.1 (Sonnhammer, ELL, Eddy, SR, Birney, E., Bateman'A., And Durbin, R., Nucleic Acids Res 1998; 26, 320-322) to search for —A search was performed (Suyama et. Al. 1999 Nucleic Acids Res. 27: 338—339).
- the Semaphorin / CDIOO antigen domain was found at the N-terminal side at the 3-352nd position in the amino acid sequence shown in SEQ ID NO: 1.
- the Plexin / Semaphorin / integrin domain was found at the 371-424th position from the N-terminal side of the amino acid sequence shown in SEQ ID NO: 1. It was.
- Plexin / Semaphorin / integrin domains could be detected at positions 524-576 and 671-712.
- the cell surface receptor IPT / TIG domain is located at the 713-802th position from the N-terminal side, according to the HMMPfam search method, at the 714-802th position from the N-terminal side, and according to the HMMSmart search method.
- the N-terminal force is 803-889
- the cell surface receptor IPT / TIG domain is 804-889 from the N-terminal according to the HMMPfam search method
- the N-terminal force is 89 ton according to the HMMSmart search method.
- the HMMPfam search method revealed the 892-978th cell surface IPT / TIG domain from the N-terminal side.
- the Semaphorin / CDIOO antigen domain is a characteristic domain whose sequence is highly conserved and found outside the cell membrane of the Plexin family, and is thought to be involved in specific binding to Semaphorin (Reference 9). , 16).
- the Plexin / Semaphorin / integrin domain refers to a specific domain consisting of a characteristic sequence found in Plexin, Semaphorin and Integrin. Although the function of this cysteine-rich repeating structure found in several extracellular receptors is unknown, it is a novel neuronal cell surface molecule that mediates cell adhesion via homophilicity. It is known.
- Plexin is present in brain and epithelial tissues and is involved in Semaphorin-induced collapse and loss of growth canes, and Integrin is involved in completing epithelial cell migration (Reference 9, 16).
- the IPT / TIG domain has an immunoglobulin-like fold, and is found in cell membrane surface receptors such as Met and Ron, as well as intracellular transcription factors involved in DNA binding.
- Ron tyrosine kinase receptors share characteristic functions with members of the subfamily such as Met and Sea. For example, its function is to control cell separation and dissociation, cell movement, intracellular invasion of the extracellular skeleton, and phenomena (References 9, 16).
- the amino acid sequence of the polypeptide encoded by the mplD0920 gene of the present invention is 1,746 amino acids long and 237 amino acids shorter than human KIAA0620, a Semaphorin / CDIOO antigen domain, three Plexin / Semaphorin / integrin domains, and 3 cell surfaces It has a structure that is 239 amino acids shorter than human KIAA0620 protein (1,985 amino acids) with receptor IPT / TIG and a transmembrane (TM) segment at the C-terminal side (Fig. 1). Can be estimated.
- the human KIAA0620 gene is derived from the Plexin family (Tamagnone, L., et at., 2001, Cell,
- the mouse mplD0920 gene was amplified by PCR to prepare a probe and a reverse primer necessary for preparing a Probe using a commercially available automatic DNA synthesizer.
- the sequence of the forward primer was 5, -CCCCGGAACTTGAACGTGTC-3 '(SEQ ID NO: 3)
- the sequence of the reverse primer was 5, -CCACCTGTTCAAACTTGTGCTG-3' (SEQ ID NO: 4)
- the primers were used for the mouse mplD0920 gene.
- the design was such that when the transcript was amplified by the PCR method, a DNA fragment of about 1,069 bases (bp) each serving as an antisense or sense probe was obtained.
- the desired PCR product that is, about 1,069 (bp) DNA fragment for antisense or sense probe was cloned into T-vector supplied by Promega.
- the l, 069 (bp) DNA fragment cloned into the T-vector was converted into type I, and an antisense cRNA probe and a negative probe capable of detecting the mouse mplD0920 gene expression product using T7 or SP6 RNA polymerase according to the method shown by Promega.
- a sense cRNA probe to be used as a control was prepared and subjected to the following experiments for the purpose of tissue-specific expression analysis of the same gene at each stage of ontogeny.
- the mouse mplD0920 gene is specifically expressed in vascular endothelial cells only at the time of angiogenesis (Vasculogenesis) at the time of ontogeny, and particularly at the time when vascular progenitor cells (Flkl-positive cells) appear at the time of ontogeny (7.5-8.0). Consistent with (dpc), a mplD0920 positive signal was detected in the vascular site, indicating a vascular-specific expression pattern throughout the angiogenesis period.
- Vasculogenesis angiogenesis
- Flkl-positive cells vascular progenitor cells
- the expression of the mplD0920 gene is simply recognized as vascular endothelial cell-specific expression, so the early stages of angiogenesis during the mouse embryo development process (10.5, 8.0 ,; 9.5 dpc) and 9.5 endothelial cells (Fig. 2A, Fig. 2B, Fig. 2C, Fig. 2D).
- vascular endothelial cell-specific expression was not observed in the period from late ontogenesis to adulthood.
- the upper panel in Fig. 2A shows the detection of non-specific gene expression using the sense cRNA fragment used as a negative control
- the lower panel in Fig. 2A shows the expression pattern of the mplD0920 gene detected using the antisense cRNA fragment. This shows the result of the detection attempt. Based on these results, the mplD0920 gene expression is detected in the antisense cRNA fragment.
- the DNA fragment of about 1,069 nucleotides (bp) used is not detected in the sense cRNA fragment and can signal non-specific gene expression. Is low.
- the mouse mplD0920 gene which shows homology to the human KIAA0620 gene, was expressed at the mRNA level in mice using the in situ hybridization method. Analyzing specific tissue-specific expression Searched by.
- the forward and reverse primers required to generate a probe by amplifying the mouse mplD0920 gene by PCR were synthesized using a commercially available DNA synthesizer. Change the sequence of the forward primer to 5,
- Antisense! / ⁇ was designed to obtain a DNA fragment of about 1,069 bases (bp) each serving as a sense probe.
- the desired PCR product that is, about 1,069 (bp) DNA fragment for antisense or sense probe was cloned into a T-vector supplied by Promega.
- the expression intensity showed strong expression in the brain, cerebral cortex, and submandibular gland. Somewhat strong expression in the trigeminal ganglia, bone and ribs, latissimus dorsum, stomach and intestine, but also in the skin, dermis, brain, cerebellum, bone, spine, heart, atrial vessels, heart, ventricular myocardium. Expression was observed in various organs such as the stratum corneum and the heart and ventricle. The signals of the epidermis of the skin, the blood vessels of the lungs, and the myocardium of the lung 'alveolar cells, heart' and atria were weak and could not be confirmed. See FIGS. 3A and 3B.
- the polypeptide encoded by the gene of the present invention was found to be 14.5 after mouse fertilization.
- strong expression was observed in the brain, cerebral cortex, and submandibular gland, and rather strong expression was observed in the ganglia, bones, ribs, latissimus dorsi, stomach, and intestine of the trigeminal nerve. It is widely found in various organs including the lower glands, skin, bones, muscles, heart, etc., and is thought to be involved in the development of related organs and tissues and maintenance of function.
- Tissue Array Slide (Lot: ZEl supplied by SuperBioChips Lab; supplied by Funakoshi Co., Ltd.) on which paraffin tissue sections prepared from adult mouse tissues were placed was used as a subject. After fixation including treatment at ° C for 15 minutes,
- Super BioChips Lab's DIG label method shows in situ hybridization According to the method described in the sectioning method, 500 ng of DIG label RNA was used as a probe for the fibrous tissue on one slide glass, and hybridization was performed at 50 ° C for 16 hours to analyze tissue-specific expression. (Ref: Cell Engineering Separate Volume, De-Isotope Combat Protocol, 192-223, 1998).
- the signal was weak in the dermis of the skin, red pulp of the spleen, skeletal muscle, lung, lung blood vessels, heart blood vessels, salivary glands, liver, spleen, small intestine, large intestine, ovaries, and thymus, and could not be confirmed. See FIGS. 4A, 4B and 4C.
- the polypeptide encoded by the gene of the present invention is widely found in various organs including the stomach, the ovary and the brain in adults, whereby the generation of related organs and tissues is recognized. ⁇ ⁇ ⁇ It can be said that they are involved in functional maintenance. In particular, it is presumed that the polypeptide of the present invention is involved in the extension of nerve cells and maintenance of networks in the brain's nervous system, and the maintenance of structures in organs having a luminal structure such as the heart 'blood vessels and kidneys. Further, the function of the polypeptide of the present invention when involved in angiogenesis in the early stage of development and the function involved in, for example, the guidance of nerve cells in various tissues including the brain in adults, are molecular. May work differently. For example, in neurons, Plexin, in cooperation with Neuropilin, binds to dimerized Semaphorin to form a complex, which regulates the elongation of neurons and eliminates them (Reference 16).
- the desired PCR product that is, about 1,069 (bp) DNA fragment for antisense or sense probe was cloned into a T-vector supplied by Promega.
- a sense cRNA probe was prepared for use, and subjected to the following experiment aimed at analyzing the expression intensity of the gene corresponding to the mouse embryo development process. That is, the 1st strand cDNA in each mouse embryo development process and the above primers were mixed, and PCR was performed for each,
- DNA fragment of the KIAA0620 gene transcript was amplified.
- TaKaRa Ex Taq (Takara Shuzo, # RP001A) was used as the Taq polymerase, and the above DNA mixture was heated at 95 ° C (2.5 minutes), and then 95 ° C (30 seconds) Z60 ° C (30 seconds) A cycle of Z72 ° C (30 seconds) was repeated 35 times.
- amplification of an approximately 1129 bp DNA fragment derived from the FLkl transcript using a primer for detecting Flkl gene expression was also performed.
- the amplified PCR product was fractionated by electrophoresis using 2% agarose gel (Rockland # 50070) with a size marker, and the amount of the fractionated PCR product was compared semiquantitatively (Figure 5). ).
- Flkl was found to be expressed at 7.5 dpc when angiogenesis was initiated during mouse embryo development, and continued to be highly expressed up to 9.5 dp throughout the angiogenesis period
- mouse KIAA0620 gene expression was Angiogenesis was detected at 6.5 dp, slightly earlier than when it was observed, and high expression continued up to 9.5 dp throughout the angiogenesis period. This suggests that KIAA0620 will be expressed earlier and induce angiogenesis earlier than FM, which has traditionally been regarded as a marker gene for angiogenesis. I was able to understand.
- mouse mKIAA0620 (gene name: mplD0920) was shorter than human KIAA0620, and it was speculated that there might be a sequence added to the mKIAA0620 base sequence.
- sequence-related information (SEQ ID NO: 17) was searched, and as a result, as shown below, an amino acid sequence having a length of 251 amino acids added to the N-terminal of the amino acid sequence shown in SEQ ID NO: 1 of the present invention It has become possible to design a primer sequence necessary for obtaining a primer.
- a primer (AATGTTGTGTCCTTTGACCCTTAC, 24 bases, name GSP2, a complementary sequence to the 24 bases from the 1,524th to 1,547th 24 bases in sequence 2; SEQ ID NO: 7) was designed, and a predicted sequence fragment was obtained by PCR.
- amplification reaction did not occur under normal reaction conditions, and it was not possible to obtain a PCR product. It was speculated that a special sequence (sequence rich in GC) that inhibited the reaction was present in the region where amplification was expected.
- PCRx Enhancer solution Using Polymerase (lnvitrogen: Cat No.11708-013) and the attached PCRx Enhancer solution, conditions (1X PCRx Enhancer solution conditions: Denature: 94 ° C forl5s Anneal: 50 ° C for 30s Extend: 68 ° C for 3min30s, An extension reaction was performed with a new setting of 40 cycles). As a result, a DNA fragment of about 2,314 bases including the target fragment of about 2,300 bases was amplified by PCR.
- the obtained DNA fragment was used as the primary type II, and Platinum Pfx DNA was used as described above.
- Nested PCR was performed using Polymerase. That is, Forward primer (
- N 'force shown in the amino acid sequence of human PLEXIN-Dl reported in van der Zwaag et al. also corresponds to the signal sequence (cleavable signal sequence) from the 30th to 46th position.
- protein synthesis starts from the 73rd Met because it is recognized from the 120th position to the 120th position. That is, it was considered that both the 2nd and 73rd Met of SEQ ID NO: 11 may be the starting points of protein synthesis.
- Hyseq INC., Publication date: 11, Oct. 2001 5 'to the 177 amino acid sequence of human plexin-Dl (1,925 amino acids long) from the 1st to 177th amino acids and the mplD0920 amino acid sequence of the present invention.
- a slightly higher homology is observed in the 179 amino acid sequence from the 73rd to the 251st amino acid sequence of the 251 amino acid sequence (the amino acid sequence from the 1st to the 251st amino acid in the amino acid sequence shown in SEQ ID NO: 15) from the 251 amino acid sequence ( 86.03%).
- Coding sequence SEQ ID 118 downregulated in International Publication No.WO200281745-A2 (Title of Invention: Coding sequence SEQ ID 118, downregulated in osteogenesis, Applicant: Aventis Pharma SA., Publication date: 17, Oct. 2002)
- osteogenesis (6,754 bases in length) 284 bases from 284 to 709 bases or 206 bases from 25 to 230 bases and a 753 base length sequence added to 5 'of the present invention (base sequence shown in SEQ ID NO: 16) (The first force is also the base sequence at position 753) of which 426 bases from position 328 to position 753 or 205 bases from position 64 to position 268, approx. 89.28% or 95.15%! Matoko! Homology was recognized.
- a base sequence obtained by adding a base sequence of 753 bases to the 5 'of the previously described mplD0920 gene (base sequence shown in SEQ ID NO: 2) (base sequence shown in SEQ ID NO: 16) (Row) each show a relatively high homology with the human KIAA0620 gene, resembling several known genes for which several reports have already been made, but proved to be novel genes derived from entirely new rodents did.
- Expression constructs are prepared using Gateway Cloning Technology (Invitrogen: Cat.No. 11821-014) system, and the following target PCR products are incorporated into pENTR / D-TOPO Vector (Invitrogen :). Clones were made.
- the PCR product used for native protein was prepared by PCR reaction using the following primers designed and synthesized as full-length cDNA (start codon force up to the stop codon). That is, as a Forward primer
- the tagged PCR product used for the tagged fusion protein type was prepared by PCR reaction using the following primers designed, synthesized, and used as tagged full-length cDNA (start codon force up to the stop codon).
- the forward primer is the forward primer (CACCatgggctgtgggcgtggtct, 24 bases long, from the sixth position of sequence 16).
- Reverse primer is the reverse primer 2 (GGCCTCGCTGTAACACTCATAGA, 23 bases in length, including the 25 bases 20 bases long sequence including the 20 bases long sequence, 23 bases, the complementary sequence to the 23 bases from 5,218th to 5,240th bases in sequence 2 Entry clones were prepared using codon-free ⁇ : SEQ ID NO: 14).
- Each entry clone was prepared using an LR Clonase enzyme mix (Invitrogen: Cat. No. ll791-019), and the expression destination vector (
- pcDNA-DEST40 vector and pcDNA-DEST47 vector both are plasmid vectors that are expressed under the control of the CAG promoter) to construct expression vectors, mouse KIAA0620 native protein, mouse KIAA0620-GFP-HisTag, mouse KIAA0620- A vector expressing the V5 fusion protein in a mammalian cell was constructed.
- Transfection reagent (Fugene 6; Roche or TransIT-LT1 reagent; Mirus) was used to transduce genes into human embryonic kidney cells (HEK293T cells) to prepare transfected cells.
- a synthetic peptide obtained by adding a cysteine to the end of FLEEQAEKRGISDPD was carrier-coupled to a keyhole limpet (KLH), which was used as a carrier at the N-terminus, and bonded to the carrier.
- KLH keyhole limpet
- Synthetic peptides purified from 80% to 90% purity were used as antigens.
- Japanese white rabbits female were initially immunized intradermally with 0.15 mg of antigen, and then sensitized 4 times with 0.3 mg of antigen every 2 weeks for a total of 5 intradermal immunizations Immunization was performed.
- a test blood sample (3 ml) was prepared before immunization in order to secure a control serum.
- the absorbances (490 nm) at 1,000-fold dilution, 2,000-fold dilution, 4,000-fold dilution, 8,000-fold dilution, and 16,000-fold dilution were 0.171, 0.101, 0.089, 0.084, and 0.076, respectively. It was confirmed that the sequence did not react with pre-immune ephedra serum and was specific.
- HEK293T cells transfected with the mouse KIAA0620 gene forced expression vector were washed with a PBS (-) solution on a culture dish, and the pipetting operation was repeated using Lysis Buffer (TNE buffer) to dissolve the protein.
- the solution thus obtained was passed through a 28 G syringe needle, and then centrifuged (700 g, 10 min), and the supernatant was recovered as a sample solution.
- the sample solution contains a mouse KIAA0620 transgenic cell-derived protein.
- HEK293T cells into which an expression vector expressing the V5 fusion protein of mouse KIAA0620 protein was introduced were used, and a sample solution was prepared in the same manner as described above.
- Various sample solutions were fractionated by polyacrylamide gel electrophoresis (PAGE).
- PAGE polyacrylamide gel electrophoresis
- polyacrylamide gel electrophoresis about 20 micrograms of proteins derived from various transfected cells were applied to each lane of polyacrylamide gel, fractionated by electrophoresis, and the fractionated proteins were blotted on a membrane.
- An immunostaining reaction was performed on the blotted membrane using an antibody diluted 500- or 1000-fold.
- polyacrylamide gel electrophoresis was performed using 412% Tris-Glycine Gel (# No.EC6035Box) manufactured by Invitrogen, and the protein was transferred using a semi-dry transfer cell manufactured by BioRad and transferred to the membrane. Plotted. Biosource's Goat Anti-Rabbit IgG, HRP-conjugate antibody (# No.ALI0404) diluted 5,000-fold was used as the secondary antibody, and the company's ECL Western blotting Detection Reagents was used for detection. (# No. RPN2133) was used. For the purpose of knowing the molecular weight, a prestained protein marker (High Range, SDS-PAGE (#No. 26039-75) manufactured by Nacalai Testa) was electrophoresed together with each sample.
- a prestained protein marker High Range, SDS-PAGE (#No. 26039-75) manufactured by Nacalai Testa
- HEK293T recombinant cells transfected with an expression vector expressing the V5 fusion protein type of mouse KIAA0620 protein or a recombinant cell used as a control were used as a starting material for the membrane obtained above.
- Purified ⁇ A clear band was detected at a position corresponding to the size of the mouse KIAA0620-V5 fusion protein (approximately 220 kDa) in a detection system using a 500-fold or 1,000-fold dilution of the heron polyclonal antibody (anti-mKIAA0620 antibody). was done.
- several bands other than about 220 kDa were detected at lower molecular weight positions.
- mouse KIAA0620-V5 fusion protein contains a transmembrane part, it was not sufficiently solubilized in the sample preparation process and remained in the membrane fraction. May have been detected as an apparently low content.
- the prepared polyclonal antibody reacted with the target mKIAA0620 protein fusion protein, and it was determined that the polyclonal antibody (anti-mKIAA0620 antibody) of the present invention was useful for detecting the amino acid sequence of mKIAA0620 protein.
- Figure 6 shows the results.
- the desired PCR product that is, about 1,069 (bp) DNA fragment for antisense or sense probe was cloned into a T-vector supplied by Promega.
- the l, 069 (bp) DNA fragment cloned into the T-vector was transformed into type II, and the mouse mplD0920 gene expression product could be detected using RNA synthase (Roche, DIG RNA Labeling Kit) according to the method shown by Roche.
- Antisense cRNA probe to be used and a sense cRNA probe to be used as a negative control were prepared, and the expression of mouse KIAA0620 gene in retinas extracted from neonatal ICR mice was observed by retinal flat mount in situ hybridization method. .
- mouse KIAA0620 gene expression was observed along the blood vessels It was confirmed. From these experimental results, extremely strong expression of mouse KIAA0620 gene was observed in developing blood vessels in the retina of neonatal mice (2 to 4 days after birth), especially in endothelial cells at the apical site of neovascular vessels.
- LSL anti-mouse type 4 collagen antibody
- mice KIAA0620 gene expression was observed only at the site of neovascularization in the periphery of the retina, and in the center of the retina where blood vessels had already been formed, the expression of the gene significantly declined. (Figure 7).
- the Plexin family includes Plexin-A (type Al, type A2, type A3, and type A4), Plexin-B (type Bl, type B2, and type B3), Plexin-Cl, Plexin-Dl, and four subfamilies, PlexinA is a functional receptor for semaphorin la, and PlexinBl is a transmembrane As a receptor for semaphorin Sema4D (CD00), Plexin-C1 has been reported to act as a receptor for GPI-anchored semaphorin Sema7A (Sema-Kl)!
- the Plexin protein belonging to the Plexin family is a transmembrane protein having a large molecular weight and contains a characteristic cysteine-rich domain outside the membrane.
- semaphorin or -europhylline is known as its receptor. Semaphorins have been reported to affect neuronal axon guidance in the nervous system and to affect vascular and muscle development, immune response, angiogenesis, tumor growth and metastasis.
- Plexin-A is known to regulate motor nerves or nerve axon guidance in the CNS (Central Nervous System).
- the polypeptide of the present invention has a relatively high homology to human Plexin-D1, and is called an extramembrane sema domain, a cysteine-rich MRS motif, and an SP domain commonly found in Plexin families. All the intramembrane domains characteristic of plexin are observed. In particular, the consensus sequence of the cysteine-rich MRS motif is
- the polypeptide of the present invention is implicated not only in the role of angiogenesis during ontogenesis but also in the important function of cells in various tissues. By analogy. When these are combined, the novelty of the present invention
- Plexin polypeptides are mainly involved in the process of blood vessel development and shunting in the early stages of development, but also in adults, tissues and cells having a hollow structure such as cells such as gastric gland, uterine gland and tubule It is a novel polypeptide that plays an important role in the complex process, and is at the center of in vivo complex proteins in various organs related to these important functions.
- the probe for detecting gene expression derived from the mplD0920 gene specifically targets vascular endothelial cells (Flk-1 positive cells: References 1, 10, 12, and 14) in ontogenesis in rodents and the like.
- vascular endothelial cells Flk-1-positive cells
- angiogenesis the stage of endothelial cell proliferation and isolation.
- Flt-1 positive Flt-1 positive cells: References 1 and 10.
- the gene of the present invention has appeared.
- the polypeptide encoded by the gene of the present invention has a cell membrane receptor structure, it is predicted that the ligand may be a novel vascular inducing factor, and the polypeptide or the corresponding antibody is predicted. It can be used as a tool to search for vascular inducing factors.
- Proteins encoded by genes are involved in an important role in regulating cell growth and proliferation in many tissues in angiogenesis from the early stage of angiogenesis to the entire angiogenesis stage, and in many tissues. The possibility was shown. [0125]
- the gene encoding the novel polypeptide of the present invention is specifically expressed in blood vessels in the early stages of angiogenesis and throughout the angiogenesis period, and in adult tissues after growth, and in various organs.
- the polypeptides of the present invention are novel polypeptides, and elucidate the pathology related to growth, function maintenance, or systemic organs (for example, dysfunction of aging of cancer) in those tissues. It is clear that the present invention is useful for the development of preventive drugs, therapeutic drugs, and test drugs, which is an excellent point of the present invention. Further, by using the recombinant protein of the present invention or a recombinant expressing the recombinant protein, it is excellent in that it can be used for screening of agosto and antagodist or for drug design research.
- the mplD0920 gene is a vascular endothelial cell (Flk-1 positive cell: Literatures 1, 2, 3, 10, 12, and 14) or a vascular endothelial cell progenitor cell at the time of ontogenesis in rodents and the like. It is useful as a novel molecular marker for cells in the process of differentiating into endothelial cells.Because the polypeptide encoded by the gene has a cell membrane receptor structure, its ligand may be a novel angiogenic factor. The polypeptide or the antibody corresponding thereto is useful as a tool for searching for a predicted angiogenic factor.
- the recombinant protein of the present invention or a recombinant expressing the recombinant protein, it can be used for screening of antagonists and antagonists or for drug design research.
- the human KIAA0620 gene which is extremely important for vascular endothelial proliferation and Rats, mice or hamsters from rodents, especially from mice
- the gene encoding the Plexin-like polypeptide itself can directly be a disease-causing gene. It is easily inferred that the Plexin-like polypeptide of the present invention can cause the following diseases.
- pathological conditions related to angiogenesis in various organs for example, wound healing, fracture healing, vascular occlusion and collateral circulation, etc., and also involve the angiogenesis process, which is not desirable for cancer growth, rheumatoid arthritis, etc.
- diabetic retinopathy, endometriosis, obesity, and a heart attack neurodegenerative disease, anomalous foot circulation, obstructive arteriosclerosis, psoriasis vulgaris, and the like, in which angiogenesis is desirable, can be caused.
- the medical field in which the utility can be exerted by applying the amino acid sequence of the mouse mKIAA0620 polypeptide of the present invention, the nucleic acid sequence encoding the same, and an antibody that specifically binds to the amino acid sequence is vascular.
- the following fields are related to newborns and pathological conditions. That is, wound healing, fracture healing, vascular occlusion and collateral circulation formation related to normal angiogenesis process after growth, periodic vascular network formation in uterine mucosa (transient, luteal formation), etc.
- Vascular proliferative activity in vascular growth and regulation of drugs or drugs involved in the angiogenesis process which is undesirable, rheumatoid arthritis, diabetic retinopathy, endometriosis, obesity, angiogenesis
- vascular growth / dilators or drugs in various diseases such as heart attack, neurodegenerative disease, impaired circulation of the feet, obstructive arteriosclerosis, and psoriasis vulgaris, which are desirable processes.
- Yes References 1, 4, 5, 7, 11
- endostatin and angiostatin can be used by using tools that can be produced by the present invention.
- a growth inhibitory factor that inhibits specific angiogenesis such as various growth inhibitory factors capable of stopping the growth of solid tumors formed in vivo, or a receptor blocker, a monoclonal antibody against the extracellular binding site, etc.
- a drug can be used as an anticancer agent, and a protein or antibody for isolating hemangioblasts in regenerative therapy, and proliferation of isolated hemangioblasts (References 1, 2, 3, and 14) It can be used for a new screening system for growth factors and growth inhibitors related to.
- the human KIAA0620 protein or the Plexin family D1 By using an amino acid sequence of the mouse mKIAA0620 polypeptide, a nucleic acid sequence encoding the same, and an antibody that specifically binds to the amino acid sequence, a target rodent gene such as a mouse or a gene product can be used.
- a screening method for vascular growth / differentiation control factor or regulatory synthetic compound, and a measurement kit in other words, an interaction detection system using a recombinant protein, and an agosto 'antagost' in vivo ligand search system.
- amino acid sequence of the polypeptide of the present invention the nucleic acid sequence encoding the same, the control of vascular cell proliferation / differentiation by using an antibody that specifically binds to the amino acid sequence, and the inhibition of vascular growth supporting cancer growth It is clear that it is useful for elucidation of the mechanism and the development of prophylactic drugs, therapeutic drugs and test drugs, which is an excellent point of the present invention.
- Reverse primer is Reverse primer2 (GGCCTCGCTGTAACACTCATAGA, 23 nucleotides, SEQ ID NO: 5) , 218th to 5,240th A complementary sequence to the 23-mer sequence of SEQ ID NO: 15, which does not include the termination codon, and a 23-mer sequence of SEQ ID NO: 15 from 5,971 to 5,993: SEQ ID NO: 14)
- an expression destination vector pcDNA-DEST40 vector, pEF5 / FRT / V5-DEST vector: By recombination into the CMV promoter, the latter being a plasmid vector expressed under the control of the EF-1a
- a forced expression vector for the KIAA0620 full-V5 fusion protein was constructed using mammalian cells as a host.
- mice KIAA0620 extramembrane region (TM) encoding a polypeptide comprising the extramembrane region and the transmembrane (TM) portion, which also has the amino acid sequence of the second 1,1,363th 1,362 amino acids in SEQ ID NO: 15
- the LR Clonase enzyme mix (Invitrogen: Cat. No.l 1791) was used in the same manner as in the forced expression vector using the mammalian cell of the mouse KIAA0620 full-V5 fusion protein as a host.
- mouse ⁇ 0620 full-length-V5 fusion protein expression plasmid vector and mouse ⁇ 0620 extramembrane region (TM) -V5 fusion protein expression plasmid vector both expression vectors for Flp-In cloned from the target gene, Invitrogen Company supplies
- PEF5 / FRT / V5-DEST was transfected into human fetal-derived kidney cells (HEK293T cells), and cells expressing KIAA0620 full-length polypeptide or the transmembrane domain gene were forcibly expressed.
- the target thread-recombinant protein was not detected in all the cell-derived total protein fractions, and in all the fractions of the total protein and the membrane protein fraction using the membrane protein fraction kit of PIERCE biotecnology.
- the cell-derived total protein fraction and the mouse KIAA0620 extramembrane region (TM) -V5 fusion protein expression vector were used. Even in the transformed recombinant cells, bands were detected at around 200 kDa or around 140 kDa of interest in the cell-derived total protein fraction, respectively.
- the two types of protein fractions were prepared using various cells prepared by the same method as described above as materials. After increasing the protein concentration in the solution by adding and preparing a concentration step by the chillon method, a detection experiment using the same Western plot method as described above was performed. As a result, in the control cells, no recombinant protein was detected in both the whole protein extract fraction and the hydrophobic protein fraction further separated and prepared therefrom. On the other hand, in the recombinant cells transformed with the mouse KIAA0620 full length-V5 fusion protein expression vector, a clear band was detected around 200 kDa of interest in the total protein fraction. In addition, a band was detected at about 200 kDa in the hydrophobic protein fraction separated and prepared from the whole protein extract fraction. Mouse with extra-membrane region and transmembrane (TM) region
- the mouse KIAA0620 full length-V5 fusion protein was detected in the total protein fraction and the hydrophobic protein fraction, so that the transmembrane (TM) segment of the KIAA0620 protein functioned reliably and was localized on the membrane as a membrane protein. It was confirmed that there is.
- the artificially produced polypeptide containing the extramembrane region and the transmembrane (TM) portion shown above as a recombinant protein contains the transmembrane (TM) portion, and as expected, And the presence of both hydrophobic and protein fractions were obtained.
- each entry clone was subjected to a site-specific recombination reaction using an LR Clonase enzyme mix (Invitrogen: Cat. No. 11791-019) to express an expression destination vector (pcDNA-DEST40 vector, pEF5 / FRT / V5-DEST: the former is a CMV promoter, the latter is a plasmid vector that is expressed under the control of the EF-1a promoter, and the expression vector is constructed to construct a mouse KIAA0620 full-length native protein and a mouse KIAA0620 full-length.
- pcDNA-DEST40 vector pEF5 / FRT / V5-DEST
- the former is a CMV promoter
- the latter is a plasmid vector that is expressed under the control of the EF-1a promoter
- the expression vector is constructed to construct a mouse KIAA0620 full-length native protein and a mouse KIAA0620 full-length.
- mice KIAA0620 extramembrane region-V5 mouse KIAA0620 extramembrane region-V5
- mouse KIAA0620 full length-GFP mouse KIAA0620 full length-GFP
- mouse KIAA0620 full length-DsRed mouse KIAA0620 extramembrane region-DsRed
- mouse KIAA0620 full length-human IgGlFc fragment mouse KIAA0620 membrane
- a forced expression vector of the outer region-human IgGlFc fragment fusion protein type in mammalian cells was constructed. These were transfected into human fetal-derived kidney cells (HEK293 cells) using Transfection reagent (Fugene 6; Roche) to prepare transfected cells, and the expression of each fusion protein was confirmed as follows.
- the mouse KIAA0620 full-length or extra-membrane domain protein is identical to the theoretical molecular weight of the mouse KIAA0620 full-length or extra-membrane domain-V5 fusion protein. It was confirmed that it was expressed as a V5 fusion protein.
- HEK293 recombinant cells into which an expression vector expressing the mouse KIAA0620 full-length or extramembrane domain protein GFP, DsRed, and a human IgGlFc fragment fusion protein type were introduced, or other recombinant cells were used as starting materials.
- Anti-GFP mouse monoclonal antibody (Nacalai Testa, part number 04363-24) or anti-DsRed ⁇ sagi polyclonal antibody (BD Biosciences Clontech, part number 632397), anti-human IgGlFc mouse monoclonal antibody (CHEMICON International, Inc., product number MAB1302) was used to perform a detection experiment by Western blotting, and a clear single band could be detected at a position corresponding to the size of each fusion protein with each antibody. Since the detected molecular weights of the bands correspond to the theoretical molecular weights of the respective fusion proteins, the mouse KIAA full-length 0620 protein and mouse KIAA0620 extramembrane region protein are expressed as fusion proteins of the respective tags. Was confirmed.
- the constructed mouse KIAA0620 full-length or extramembrane region-GFP fusion protein expression plasmid vector (Flp-In expression vector with the target gene cloned, derived from PEF5 / FRT / V5-DEST supplied by Invitrogen), and mouse KIAA0620 full-length or Flp-in supplied by Invitrogen, each of the extramembrane region-DsRed fusion protein expression plasmid vector (Flp-In expression vector containing the target gene closed, derived from PEF5 / FRT / V5-DEST supplied by Invitrogen)
- HEK293 cells human fetal-derived kidney cells
- HEK293 host cells Flp-in-293 cells supplied by Invitrogen: 293 human embryonic kidny cells, one Hp recombinase target site (FRT site) at the position of the transcribed genome ) Were cultured until they became subconfluents.
- mice KIAA0620 full-length or extramembrane region-GFP fusion protein expression plasmid vector mouse KIAA0620 full-length or extramembrane domain-DsRed fusion protein expression plasmid vector 2 ⁇ g of transfection solution (FuGENE6 Transfection Reagent solution supplied by Roche) per 2 ⁇ g of mouse ⁇ Processed quality conversion.
- transfection solution FuGENE6 Transfection Reagent solution supplied by Roche
- Two control wells were treated with a transfection solution without a plasmid vector (FuGENE6 Transfection Reagent solution) and culture was continued.
- each well was cultivated.
- Each of the grown transformed cells is sorted by FACS for those that have a fluorescent protein (GFP or DsRed) that has been fused, and a stable strain is created by collecting only cells that stably express the introduced gene. did.
- the amino acid sequence of 1,337 amino acids shown in SEQ ID NO: 18 (identical to the 2nd to 1,338th amino acids in the amino acid sequence shown in SEQ ID NO: 15). And a plasmid vector expressing this gene product under the control of a CAG promoter was prepared. This was transfected into 293T cells using Mirus TransIT-LTl Transfection Reagents, and cultured in a serum-free medium (Invitrogen Gibco CD293) for 3 days, followed by a Protein G column (Amersham Biosciences, HiTrap TM Protein G HP). ) was used to purify the mouse KIAA0620 extramembrane-IgGlFc fusion protein from the culture supernatant.
- fusion protein solution (2.3 mg / ml in phosphate buffer) was added to a microinjector equipped with a glass capillary (Drummond, NANOJECT II, Drummond).
- Axiopl a n2 fluorescence for observation The microscope and Axiovision3.0 software (Carl Zeiss Co., Ltd., Oberkochen, Germany) were used, and the images were viewed with a confocal microscope system using Axiovert 200M laser microscope and LSM510-V3.2 software (Carl Zeiss Co., Ltd., Oberkochen, Germany).
- the DNA of the present invention, the polypeptide of the present invention, or the antibody of the present invention are useful for the growth, functional preservation, or maintenance of angiogenesis or in various organs including the brain. Elucidation of the mechanism, diagnosis, and treatment of human diseases, including diseases associated with angiogenesis in various organs, aging, dysfunction, and congenital malformations, including congenital malformations, as well as function preservation of various organs and prevention of aging. It is considered to play an essential role.
- the information on the expression of the expression at each tissue and at the developmental stage by the expression frequency analysis at the mRNA level is universally expressed in various organs and is universally expressed in each developmental process! / It was clarified that the gene of the present invention is important in the development and differentiation of various organs, the maintenance of their functions, and aging.
- the DNA of the present invention and a rodent-derived gene containing the DNA, a part or the full length of a polypeptide encoded by them, an antibody against a part or the full length of the polypeptide, an antisense DNA and the like are It can be used as a drug or test agent in the development of various treatment and prevention methods using mice, which are model animals for treating human diseases including cancer and congenital malformations.
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Medicinal Chemistry (AREA)
- General Health & Medical Sciences (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Pharmacology & Pharmacy (AREA)
- Animal Behavior & Ethology (AREA)
- General Chemical & Material Sciences (AREA)
- Public Health (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Veterinary Medicine (AREA)
- Biochemistry (AREA)
- Cell Biology (AREA)
- Molecular Biology (AREA)
- Engineering & Computer Science (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Immunology (AREA)
- Genetics & Genomics (AREA)
- Biophysics (AREA)
- Gastroenterology & Hepatology (AREA)
- Zoology (AREA)
- Toxicology (AREA)
- Heart & Thoracic Surgery (AREA)
- Cardiology (AREA)
- Peptides Or Proteins (AREA)
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
- Saccharide Compounds (AREA)
- Investigating Or Analysing Biological Materials (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
Description
Claims
Priority Applications (5)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US10/573,262 US7482322B2 (en) | 2003-10-30 | 2004-10-28 | Plexin family-like polypeptide, and uses thereof |
JP2005516065A JP4751202B2 (ja) | 2003-10-30 | 2004-10-28 | 新規PlexinポリペプチドとそれをコードするDNA、及びその用途 |
DE602004019556T DE602004019556D1 (de) | 2003-10-30 | 2004-10-28 | Neues plexin-polypeptid, dieses codierende dna und verwendung davon |
EP04793105A EP1679372B1 (en) | 2003-10-30 | 2004-10-28 | Novel plexin polypeptide, dna encoding the same and use thereof |
US12/318,086 US7816132B2 (en) | 2003-10-30 | 2008-12-22 | DNA encoding plexin polypeptides and kits thereof |
Applications Claiming Priority (4)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
JP2003-371040 | 2003-10-30 | ||
JP2003371040 | 2003-10-30 | ||
JP2004229871 | 2004-08-05 | ||
JP2004-229871 | 2004-08-05 |
Related Child Applications (2)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
US10/573,262 A-371-Of-International US7482322B2 (en) | 2003-10-30 | 2004-10-28 | Plexin family-like polypeptide, and uses thereof |
US12/318,086 Division US7816132B2 (en) | 2003-10-30 | 2008-12-22 | DNA encoding plexin polypeptides and kits thereof |
Publications (1)
Publication Number | Publication Date |
---|---|
WO2005056791A1 true WO2005056791A1 (ja) | 2005-06-23 |
Family
ID=34680588
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
PCT/JP2004/015997 WO2005056791A1 (ja) | 2003-10-30 | 2004-10-28 | 新規PlexinポリペプチドとそれをコードするDNA、及びその用途 |
Country Status (6)
Country | Link |
---|---|
US (2) | US7482322B2 (ja) |
EP (1) | EP1679372B1 (ja) |
JP (1) | JP4751202B2 (ja) |
AT (1) | ATE423200T1 (ja) |
DE (1) | DE602004019556D1 (ja) |
WO (1) | WO2005056791A1 (ja) |
Cited By (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2007009816A3 (en) * | 2005-07-21 | 2007-03-29 | Stichting Katholieke Univ | Plexin d1 as a target for tumor diagnosis and therapy |
Families Citing this family (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
EP2385121A1 (en) | 2010-05-06 | 2011-11-09 | Netris Pharma | Antagonists of Sema3E/PlexinD1 interaction as anti-cancer agents |
Citations (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2001014420A2 (en) * | 1999-08-25 | 2001-03-01 | University Of Torino | Novel members of the plexin family and uses thereof |
WO2001057188A2 (en) * | 2000-02-03 | 2001-08-09 | Hyseq, Inc. | Novel nucleic acids and polypeptides |
WO2002081745A2 (en) * | 2001-04-05 | 2002-10-17 | Proskelia Pharmaceuticals | Genes involved in osteogenesis, and methods of use |
Family Cites Families (11)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
JPS63203626A (ja) * | 1987-02-19 | 1988-08-23 | Ajinomoto Co Inc | 免疫抑制剤 |
JP4711520B2 (ja) | 2000-03-21 | 2011-06-29 | 日本ケミカルリサーチ株式会社 | 生理活性ペプチド含有粉末 |
US7892730B2 (en) * | 2000-12-22 | 2011-02-22 | Sagres Discovery, Inc. | Compositions and methods for cancer |
AU2002366951A1 (en) * | 2001-12-10 | 2003-07-09 | Nuvelo,Inc. | Novel nucleic acids and polypeptides |
JP2005523688A (ja) * | 2002-01-18 | 2005-08-11 | ブリストル−マイヤーズ スクイブ カンパニー | タンパク質チロシンキナーゼおよび/またはタンパク質チロシンキナーゼ経路と相互作用する化合物の活性を予測するためのポリヌクレオチドおよびポリペプチドの同定 |
WO2003068268A2 (en) * | 2002-02-14 | 2003-08-21 | Bioinvent International Ab | Treatment, diagnosis and imaging of disease |
AU2003223520A1 (en) * | 2002-04-12 | 2003-10-27 | Mitokor | Targets for therapeutic intervention identified in the mitochondrial proteome |
JP2006517785A (ja) * | 2002-10-29 | 2006-08-03 | ジェネンテック・インコーポレーテッド | 免疫関連疾患の治療のための新規組成物と方法 |
AU2003295401B2 (en) * | 2002-11-08 | 2010-04-29 | Genentech, Inc. | Compositions and methods for the treatment of natural killer cell related diseases |
AU2003292231A1 (en) * | 2002-12-12 | 2004-06-30 | Novartis Ag | Methods for diagnosing and treating schizophrenia |
CA2519414A1 (en) * | 2003-03-19 | 2004-09-30 | Dnavec Research Inc. | Methods of treating inflammatory diseases associated with bone destruction |
-
2004
- 2004-10-28 AT AT04793105T patent/ATE423200T1/de not_active IP Right Cessation
- 2004-10-28 EP EP04793105A patent/EP1679372B1/en not_active Not-in-force
- 2004-10-28 DE DE602004019556T patent/DE602004019556D1/de active Active
- 2004-10-28 WO PCT/JP2004/015997 patent/WO2005056791A1/ja active Application Filing
- 2004-10-28 US US10/573,262 patent/US7482322B2/en not_active Expired - Fee Related
- 2004-10-28 JP JP2005516065A patent/JP4751202B2/ja not_active Expired - Fee Related
-
2008
- 2008-12-22 US US12/318,086 patent/US7816132B2/en not_active Expired - Fee Related
Patent Citations (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2001014420A2 (en) * | 1999-08-25 | 2001-03-01 | University Of Torino | Novel members of the plexin family and uses thereof |
WO2001057188A2 (en) * | 2000-02-03 | 2001-08-09 | Hyseq, Inc. | Novel nucleic acids and polypeptides |
WO2002081745A2 (en) * | 2001-04-05 | 2002-10-17 | Proskelia Pharmaceuticals | Genes involved in osteogenesis, and methods of use |
Non-Patent Citations (2)
Title |
---|
OKAZAKI N. ET AL.: "Prediction of the coding sequence of mouse homologues of KIAA gene: III. the complete nucleotide sequence of 500 mouse KIAA-homologous cDNAs identified by screening of terminal sequence of cDNA clones randomly sampled from size-fractionated libraries", DNA RES., vol. 10, no. 4, August 2003 (2003-08-01), pages 167 - 180, XP002983198 * |
ZWAAG B.V.D. ET AL.: "PLEXIN-D1, a novel plexin family member, is expressed in vascular endothelium and the central nervous system during mouse emryogenesis", DEV. DYN., vol. 225, no. 3, 2002, pages 336 - 343, XP002983199 * |
Cited By (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2007009816A3 (en) * | 2005-07-21 | 2007-03-29 | Stichting Katholieke Univ | Plexin d1 as a target for tumor diagnosis and therapy |
JP2009506985A (ja) * | 2005-07-21 | 2009-02-19 | ステイヒテイング・カソリーケ・ユニベルシタイト | 腫瘍の診断及び治療のための標的としてのプレキシンd1 |
Also Published As
Publication number | Publication date |
---|---|
US7816132B2 (en) | 2010-10-19 |
EP1679372B1 (en) | 2009-02-18 |
US7482322B2 (en) | 2009-01-27 |
JPWO2005056791A1 (ja) | 2007-12-06 |
US20090215170A1 (en) | 2009-08-27 |
JP4751202B2 (ja) | 2011-08-17 |
EP1679372A1 (en) | 2006-07-12 |
US20070212365A1 (en) | 2007-09-13 |
ATE423200T1 (de) | 2009-03-15 |
EP1679372A4 (en) | 2006-11-08 |
DE602004019556D1 (de) | 2009-04-02 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
US8604162B2 (en) | Collectin | |
US7816132B2 (en) | DNA encoding plexin polypeptides and kits thereof | |
KR101083852B1 (ko) | 유전자 전사 조절제 및 히스톤 탈아세틸화효소 저해 화합물의 스크리닝 방법 | |
JP4530631B2 (ja) | 新規タンパク質および癌の予防・治療剤 | |
US20040019006A1 (en) | Novel genes relating to pain and use of the genes for pharmaceuticals | |
JP2003503062A (ja) | タンキラーゼ2物質および方法 | |
JPH06293800A (ja) | 新規生理活性物質エピモルフィン、それをコードする 遺伝子及びエピモルフィンに対する抗体 | |
JP2004283094A (ja) | イムノグロブリン様繰り返しドメイン及びプロリンに富む配列を有する新規ポリペプチド及びそれをコードするdna | |
JP2004147642A (ja) | 新規タンパク質およびその用途 | |
JP2004135617A (ja) | 新規遺伝子及びそれにコードされる蛋白質 | |
JP2004041003A (ja) | 新規タンパク質、そのdnaおよびその用途 | |
JPWO2003048358A1 (ja) | 脂肪細胞分化関連遺伝子およびタンパク質 | |
JP2004337122A (ja) | Fhaドメイン及び膜アンカー領域を有する新規ポリペプチド及びそれをコードするdna | |
JP2003325185A (ja) | Itimモチーフを持つ新規な免疫機能抑制分子 | |
JP2004187668A (ja) | HAT(Half−A−TPR)繰り返しモチ−フ及びプロリンに富む配列を有する新規ポリペプチド及びそれをコードするDNA | |
JP2002355063A (ja) | 新規疾患関連遺伝子およびその用途 | |
JP2004081204A (ja) | RasGEFモチーフを有する新規ポリペプチド及びそれをコードするDNA | |
JP2005052003A (ja) | Ubaドメインを有する新規ポリペプチド及びそれをコードするdna | |
JP2001258573A (ja) | 新規ファット3遺伝子及びそれにコードされる蛋白質 | |
JP2004081196A (ja) | 新規タンパク質およびそのdna | |
JP2004073076A (ja) | BTB/POZドメインとKelch繰り返し配列を有する新規ポリペプチド及びそれをコードするDNA | |
JP2004323405A (ja) | 腎疾患の予防・治療剤 | |
JP2004000117A (ja) | 新規タンパク質およびそのdna | |
JP2004313173A (ja) | 新規タンパク質およびそのdna | |
JP2001327295A (ja) | 新規ヒトダクサス遺伝子及びそれにコードされる蛋白質 |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
AK | Designated states |
Kind code of ref document: A1 Designated state(s): AE AG AL AM AT AU AZ BA BB BG BR BW BY BZ CA CH CN CO CR CU CZ DE DK DM DZ EC EE EG ES FI GB GD GE GH GM HR HU ID IL IN IS JP KE KG KP KR KZ LC LK LR LS LT LU LV MA MD MG MK MN MW MX MZ NA NI NO NZ OM PG PH PL PT RO RU SC SD SE SG SK SL SY TJ TM TN TR TT TZ UA UG US UZ VC VN YU ZA ZM ZW |
|
AL | Designated countries for regional patents |
Kind code of ref document: A1 Designated state(s): BW GH GM KE LS MW MZ NA SD SL SZ TZ UG ZM ZW AM AZ BY KG KZ MD RU TJ TM AT BE BG CH CY CZ DE DK EE ES FI FR GB GR HU IE IT LU MC NL PL PT RO SE SI SK TR BF BJ CF CG CI CM GA GN GQ GW ML MR NE SN TD TG |
|
121 | Ep: the epo has been informed by wipo that ep was designated in this application | ||
WWE | Wipo information: entry into national phase |
Ref document number: 2005516065 Country of ref document: JP |
|
WWE | Wipo information: entry into national phase |
Ref document number: 2004793105 Country of ref document: EP |
|
WWP | Wipo information: published in national office |
Ref document number: 2004793105 Country of ref document: EP |
|
WWE | Wipo information: entry into national phase |
Ref document number: 10573262 Country of ref document: US Ref document number: 2007212365 Country of ref document: US |
|
WWP | Wipo information: published in national office |
Ref document number: 10573262 Country of ref document: US |