WO2006006477A1 - Polypeptide participating in bone disease or joint disease and dna thereof - Google Patents

Polypeptide participating in bone disease or joint disease and dna thereof Download PDF

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Publication number
WO2006006477A1
WO2006006477A1 PCT/JP2005/012536 JP2005012536W WO2006006477A1 WO 2006006477 A1 WO2006006477 A1 WO 2006006477A1 JP 2005012536 W JP2005012536 W JP 2005012536W WO 2006006477 A1 WO2006006477 A1 WO 2006006477A1
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Prior art keywords
protein
amino acid
acid sequence
seq
expression level
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PCT/JP2005/012536
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French (fr)
Japanese (ja)
Inventor
Atsumasa Uchida
Akihiko Matsumine
Hikaru Sonoda
Satoshi Orita
Takenobu Tasaki
Nobuyuki Ide
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Shionogi & Co., Ltd.
Mie University
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Priority to JP2006528956A priority Critical patent/JPWO2006006477A1/en
Publication of WO2006006477A1 publication Critical patent/WO2006006477A1/en

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    • 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
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • 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
    • C07K14/475—Growth factors; Growth regulators
    • C07K14/51—Bone morphogenetic factor; Osteogenins; Osteogenic factor; Bone-inducing factor
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/158—Expression markers
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/10—Musculoskeletal or connective tissue disorders

Definitions

  • the present invention relates to a means for diagnosis, treatment, prevention and the like of bone disease or joint disease. More specifically, the present invention relates to a method for detecting or selecting a sample derived from an individual suspected of having a bone disease or joint disease, or a method for screening a diagnostic agent, a therapeutic agent or a preventive agent for bone disease or joint disease. Further, the present invention relates to a kit used for the screening method, a pharmaceutical composition, and a diagnostic method for bone disease or joint disease.
  • a joint is a movable joint between bones, and the bone in this part is covered with articular cartilage in order to make the movement smooth.
  • synovial fluid is secreted from the synovium, smoothing the joint movement.
  • OA Osteoarthritis
  • OA is a debilitating disease with chronic arthritis and degenerative degeneration of articular cartilage in synovial joints.
  • OA is known to increase in incidence with age and is one of the most frequent diseases for the elderly. It is one of the major challenges in the present age facing an aging society, but the cause is not clear and there is no effective treatment.
  • OA has been considered to be caused by unavoidable aging, but genetic studies have revealed that genetic factors are largely involved in the development of OA (see Non-Patent Document 1). .
  • OA is a multifactorial genetic disease, and multiple genetic factors are involved in its onset and spread, and the disease is thought to be due to the interaction of these genes with environmental factors such as exercise and nutrition.
  • Patent Documents 1 and 2 research to date has listed a large number of candidate genes (see Patent Documents 1 and 2) and SNPs (-nucleotide polymorphisms) as genes related to OA (see Non-Patent Document 2).
  • SNPs nucleotide polymorphisms
  • Rheumatoid arthritis is an unexplained chronic inflammatory disease whose main symptom is polyarthritis with symmetrical small and medium joints. Inflammatory site force-in and local production of rheumatoid factor, formation of immune complex consisting of IgG and rheumatoid factor in synovial fluid, neutrophil migration Chronic inflammation and joint destruction progress through a series of reactions such as running, immune complex phagocytosis, lysosomal enzyme release, articular cartilage destruction, synovial proliferation, and osteoclast activation. Studies to date have listed a large number of candidate genes (see Patent Documents 3 and 4) and SNPs (see Non-Patent Document 3) as genes related to RA. However, the pathogenesis of RA has not been fully elucidated, and it is possible that unknown genes may be involved.
  • N1032 (SEQ ID NO: 1) is a tumor suppressor gene candidate 1 (bcsc— 1) for human breast cancer (SEQ ID NO: 1).
  • N1108 (SEQ ID NO: 2) is a human gene encoding a protein phosphatase 2A B ′ a 1 regulatory subunit (SEQ ID NO: 19) (see Non-Patent Document 5).
  • N1112 (SEQ ID NO: 3) is a human that encodes a novel human zinc finger transcription factor (SEQ ID NO: 20) that is homologous to the rodent gene Kidl and is mainly expressed in the kidney. It is a gene (see Non-Patent Document 6). However, there is no report on the relationship between N1112 and OA.
  • N1123 (SEQ ID NO: 4) is a human gene that encodes phosphodariserate kinase (SEQ ID NO: 21) encoded by the X chromosome (see Non-Patent Document 7). However, there is no report on the relationship between Ni 23 and OA!
  • N0154 (SEQ ID NO: 5) is a human gene that codes for L-iditol-2 dehydrogenase (SEQ ID NO: 22) (see Non-Patent Document 8). In connection with N0154 and OA There is no report about it.
  • N0281 (SEQ ID NO: 6) is the gene EXT1 (SEQ ID NO: 23) that causes human hereditary multiple exostosis (see Non-Patent Document 9). However, there is no report on the relationship between N0281 and OA.
  • N0439 (SEQ ID NO: 7) is a human gene encoding heparan-sulfate-6-sulfotransferase (SEQ ID NO: 24) (see Non-Patent Document 10).
  • SEQ ID NO: 24 heparan-sulfate-6-sulfotransferase
  • N0446 (SEQ ID NO: 8) is a human gene having homology to histidyl-tRNA synthetase (SEQ ID NO: 25) (see Non-patent Document 11). However, there is no report on the relationship between N0446 and OA.
  • N0513 (SEQ ID NO: 9) is a human gene encoding tubulin-folding cofactor C (SEQ ID NO: 26) (see Non-Patent Document 12). However, with N0513
  • N0553 (SEQ ID NO: 10) is a human gene encoding N, serine Z threonine kinase (SEQ ID NO: 27) having two LIM motifs at the ends (see Non-patent Document 13). However, there is no report on the relationship between N0553 and OA!
  • N0065 (SEQ ID NO: 11) is a human gene encoding LAK-4 (SEQ ID NO: 28), which is a membrane protein. However, there is no report on the relationship between N0065 and OA.
  • NO 160 (SEQ ID NO: 12) is a human gene encoding a vasoactive intestinal peptide receptor (SEQ ID NO: 29) (see Non-Patent Document 14). However, there has been no report on the relationship between N0160 and OA!
  • E0215 (SEQ ID NO: 13) is an EGF-containing fibrin-like extracellular matrix protein 1 (SEQ ID NO: 13) is an EGF-containing fibrin-like extracellular matrix protein 1 (SEQ ID NO: 13) is an EGF-containing fibrin-like extracellular matrix protein 1 (SEQ ID NO: 13) is an EGF-containing fibrin-like extracellular matrix protein 1 (SEQ ID NO: 13) is an EGF-containing fibrin-like extracellular matrix protein 1 (SEQ ID NO: 13) is an EGF-containing fibrin-like extracellular matrix protein 1 (SEQ ID NO: 13) is an EGF-containing fibrin-like extracellular matrix protein 1 (SEQ ID NO: 13) is an EGF-containing fibrin-like extracellular matrix protein 1 (SEQ ID NO: 13) is an EGF-containing fibrin-like extracellular matrix protein 1 (SEQ ID NO: 13) is an EGF-containing fibrin-like extracellular matrix protein 1 (SEQ ID NO: 13)
  • E0739 (SEQ ID NO: 14) is a human gene encoding a poly (rC) _binding protein (SEQ ID NO: 31) of a cell having three K-homology domains (Non-patent Document 16). reference). However, there was no report on the relationship between E0739 and OA!
  • C0052 (SEQ ID NO: 15) is a human encoding AP-endonuclease (SEQ ID NO: 32) It is a gene (see Non-Patent Document 17). However, there is no report on the relationship between C0052 and OA.
  • D0032 (SEQ ID NO: 16) is a human gene encoding macrophage inflammatory protein-2 ⁇ (SEQ ID NO: 33) (see Non-Patent Document 18). However, there is no report about the relationship between D0032 and OA!
  • E0083 (SEQ ID NO: 17) is a human gene encoding glutathione S-transferase (SEQ ID NO: 34) (see Non-Patent Document 19). However, there is no report on the relationship between E0083 and OA.
  • Patent Document 1 Japanese Patent Laid-Open No. 2003-183177
  • Patent Document 2 Japanese Patent Laid-Open No. 2004-75675
  • Patent Document 3 Japanese Patent Application Laid-Open No. 2002-51782
  • Patent Document 4 Japanese Patent Laid-Open No. 2003-204790
  • Non-Patent Document 1 Spector, T. D. et al., British Medical Journal (BMJ), 312 ⁇ , 940–943 (1996)
  • Non-Patent Document 2 Ikegawa, S. et al., Journal of Bone and Mineral Research (door R), 17 ⁇ , 1290–1296 (2002)
  • Non-Patent Document 3 Yamamoto, K. et al., Nat. Genet., 35 ⁇ , 341-348 (2003)
  • Non-Patent Document 4 Hiroshi Yoshida “RA or other inflammatory polyarthritis (DRG / PPS guidelines for clinical testing)” Japan Society for Clinical Laboratory Medicine, September 2002, P.85—88
  • Non-Patent Document 5 Kam3 ⁇ 4ayashi, C. et al., The Journal of Biological Chemistry (J. Biol. Chem.), 271, 5164-5170 (1996)
  • Non-Patent Document 6 Nakamura, Y. et al., Cytogenet. Cell Genet, 78 ⁇ , 285-288 (1997)
  • Non-Patent Document 7 Orkin, S.H. et al., Proceedings of the National Academy of Science (Proc. Natl. Acad. Sci. U.S.A.), 80 ⁇ , pp. 472-476 (
  • Non-Patent Document 8 Carper, D. et al., Genomics, 26 ⁇ , 55-62
  • Non-Patent Document 9 Wells, DE et al., Nat. Genet., 11 ⁇ , 137-143 (1995)
  • Non-Patent Document 10 Kimata, K. et al., The Journal of Biological Chemistry, J. Biol. Chem., 273, 9208–9213 (1998)
  • Non-Patent Document 11 Miller, F.W. et al., Biochemical and Biophysical Research Communications (Biochem. Biophys. Res. Commun.), 210 ⁇ , 556-5 pp. 66 (1995)
  • Non-Patent Document 12 Cowan, NJ et al., Cell, 86, 287-296 (1996)
  • Non-Patent Document 13 Mizuno, K., et al., The Journal of Biological Chemistry ⁇ Lee (J. Biol. Chem.), 270 ⁇ , 31321–31330 (1995)
  • Non-Patent Document 14 Goetzl, E.J. et al., Biochemical and Biophysical Research Communications (Biochem. Biophys. Res. Commun.), 193 ⁇ , 546 — 553 (1993)
  • Non-Patent Document 15 Goldstein, S. et al., Molecular and Cellular Neurology (Mol. Cell. Biol.), 15 ⁇ , 120-128 (1995)
  • Non-Patent Document 16 Celis, J.E. et al., Eguchi. J. Biochem., 230 ⁇ , 447-453 (1995)
  • Non-Patent Document 17 Hickson, I.D. et al., Nucleic Acids Res., 19 ⁇ , 5519-5523 (1991)
  • Non-Patent Document 18 Cerami, ⁇ . Et al., The Journal of Medical Medicine (J. Exp. Med.), 172 ⁇ , 911– 919 (1990)
  • Non-Patent Document 19 Taylor, J.B. et al., Biochemical Journal (Biochem. J.), 300 ⁇ , 271–276 (1994)
  • the present invention relates to a protein having an amino acid sequence selected from the group consisting of SEQ ID NOs: 18 to 34, or one or several amino acids in the amino acid sequence deleted, substituted or added
  • a method for simple and quick detection or selection of a sample derived from an individual suspected of having bone disease or joint disease, bone disease or joint, by measuring the expression level of a gene of a protein having a defined amino acid sequence It is an object of the present invention to provide a simple and rapid diagnosis method of a disease and a diagnostic agent capable of efficiently performing the method. Another object of the present invention is to provide a diagnostic agent that is a typical joint disease and can distinguish between RA and OA showing similar symptoms.
  • the present invention also provides a protein having an amino acid sequence selected from the group consisting of SEQ ID NOs: 18 to 34, or an amino acid sequence in which one or several amino acids have been deleted, substituted or added in the amino acid sequence.
  • the present invention provides an efficient screening method for a therapeutic or prophylactic agent for bone disease or joint disease by detecting or measuring the dynamics of a gene having a protein having a protein, and a screening kit capable of performing the screening method simply and rapidly. For the purpose.
  • the present invention is a compound or salt thereof suitably used as a therapeutic or prophylactic agent for bone disease or joint disease obtained by the screening method, and suitable for the treatment or prevention of bone disease or joint disease.
  • Another object of the present invention is to provide a pharmaceutical composition comprising the above compound or a salt thereof as an active ingredient.
  • the present inventors collected and analyzed the synovium excised at the time of surgery with the consent of OA patients or RA patients. As a result of intensive research, we found a gene whose expression was changed in the synovial tissue of OA patients compared to non-OA patients. In addition, we found genes whose expression was altered in the synovial tissue of RA patients compared to non-RA patients. In OA knee tissue, various inflammatory substances are thought to be secreted from the proliferative synovium stimulated by debris of cartilage tissue destroyed by mechanical-stress. Therefore, it was considered that in the synovium subjected to such stimulation, expression of genes encoding inflammatory substances and substances that can serve as markers for joint tissue destruction was enhanced.
  • fibroblast synovial cells having the ability to differentiate into mesenchymal cells such as adipocyte-like cells, osteoblasts, and chondrocytes are present.
  • mesenchymal cells such as adipocyte-like cells, osteoblasts, and chondrocytes.
  • the gene can be used as a novel diagnostic agent for bone diseases or joint diseases caused by abnormal synovial tissue abnormalities.
  • the present invention has been completed.
  • the bone disease or joint disease is cartilage dysplasia, bone dysplasia, osteoporosis, osteoarthritis, rheumatoid arthritis, arthritis, synovitis, metabolic arthropathy or arthropathy due to sports (1) Described method,
  • a protein having an amino acid sequence selected from the group consisting of SEQ ID NO: 18, 21, 23, 25, 29 in a test sample derived from an individual to be tested or one or several amino acids in the amino acid sequence Measuring the expression level of at least one gene encoding a protein having a deleted, substituted or added amino acid sequence, wherein the expression level in the test sample is expressed in a control sample from a normal individual
  • a sample derived from an individual suspected of suffering from osteoarthritis if it is higher than the level and lower than the expression level in a control sample from a patient with rheumatoid arthritis
  • a method for detecting or selecting a sample derived from an individual who is suspected of suffering from osteoarthritis
  • a method for detecting or sorting a sample from an individual
  • a protein having the amino acid sequence of SEQ ID NO: 34 in a test sample derived from an individual to be tested, or having an amino acid sequence in which one or several amino acids are deleted, substituted or added in the amino acid sequence Measuring the expression level of the gene encoding the protein, wherein the expression level in the test sample is lower than the expression level in a control sample from a normal individual and from an individual with rheumatoid arthritis If it is higher than the expression level in the control sample, the test sample suffers from osteoarthritis, which is an indicator that the sample is from an individual suspected of suffering from osteoarthritis! /,
  • osteoarthritis which is an indicator that the sample is from an individual suspected of suffering from osteoarthritis! /
  • a method for detecting or selecting a sample derived from an individual
  • a sample derived from an individual suspected of suffering from rheumatoid arthritis if it is higher than the level and higher than the expression level in a control sample from a patient with osteoarthritis A method for detecting or selecting a sample derived from an individual suspected of having rheumatoid arthritis,
  • a protein having the amino acid sequence of SEQ ID NO: 34 in a test sample derived from an individual to be tested, or an amino acid sequence in which one or several amino acids have been deleted, substituted or added in the amino acid sequence A step of measuring the expression level of a gene encoding a protein, wherein the expression level in the test sample is lower than the expression level in a control sample derived from a normal individual and derived from an individual with osteoarthritis If the expression level is lower than the expression level in the control sample, the test sample is suffering from rheumatoid arthritis, which is an indicator that the sample is derived from an individual suspected of suffering from rheumatoid arthritis A method for detecting or sorting a sample from a suspect individual, (8) Expression level ability SEQ ID NO: 18-34 or a protein having an amino acid sequence selected from the group consisting of 18 to 34, or a protein having an amino acid sequence in which one or several amino acids are deleted, substituted or added in the amino acid sequence The method according to any one of (1) to
  • SEQ ID NO A protein having an amino acid sequence selected from the group consisting of 18 to 34 or at least an amino acid sequence in which one or several amino acids have been deleted, substituted or added in the amino acid sequence Measured using one protein or a partial peptide thereof or a salt thereof as an index, (1) to (7) V, the method according to any one of
  • the present invention relates to a preventive agent for bone disease or joint disease and a screening method for Z or therapeutic agent, characterized by using the partial peptide or a salt thereof.
  • the present invention also provides: (24) A protein having an amino acid sequence selected from the group consisting of SEQ ID NOs: 18 to 34 in the presence of a test substance, or an amino acid sequence in which one or several amino acids are deleted, substituted or added in the amino acid sequence A prophylactic agent for a therapeutic agent for bone disease or joint disease and a screening method for Z or therapeutic agent, characterized by detecting or measuring the dynamics of at least one gene encoding a protein having
  • step (II) After the step (I), the binding between the protein or a partial peptide thereof and a test substance is detected, whereby the test substance binding to the protein or the partial peptide is detected as a bone disease or a joint disease. Selected as a candidate compound for prophylactic and Z or therapeutic agents
  • step (B) expressing the nucleic acid in the presence of the test substance in the cells obtained in step (A), and
  • step (B) expressing the nucleic acid in the presence of the test substance in the cells obtained in step (A), and
  • step (B) expressing the nucleic acid in the presence of the test substance in the cells obtained in step (A), and
  • the bone disease or joint disease is cartilage dysplasia, bone dysplasia, osteoporosis, osteoarthritis, rheumatoid arthritis, arthritis, synovitis, metabolic arthropathy or arthropathy due to sports (24) ⁇ (28) V, the method described in
  • a pharmaceutical composition comprising the compound or salt thereof according to (34) as an active ingredient,
  • Measuring the expression level of at least one gene encoding a protein having a specific amino acid sequence, wherein the expression level in the test sample is different from the expression level in a control sample from a normal individual or RA patient A diagnostic method for bone disease or joint disease, which is an indicator that the test sample is a sample derived from an individual suspected of having bone disease or joint disease,
  • SEQ ID NO: 18-34 Protein having an amino acid sequence selected from the group consisting of amino acids or one or several amino acids deleted, substituted or added in the amino acid sequence The method according to (37), wherein at least one protein having the above or a partial peptide thereof or a salt thereof is measured as an index,
  • Bone disease or joint disease is cartilage dysplasia, bone dysplasia, osteoporosis, degenerative joint (37) to (39) V, which is a method described in any of the above, which is an arthritis, rheumatoid arthritis, arthritis, synovitis, metabolic arthropathy or sports joint disorder.
  • the protein used in the present invention changes in expression in OA synovium or RA synovium, it is useful, for example, as a diagnostic index for bone disease or joint disease caused by abnormal synovial tissue.
  • the protein used in the present invention is useful as a diagnostic index for OA or RA, which distinguishes OA and RA which are difficult to discriminate because arthralgia is one of the symptoms.
  • a compound or salt thereof that regulates the activity of the protein, a compound or salt thereof that regulates the expression of the protein gene, a neutralizing antibody against the protein, or an antisense nucleotide of the gene encoding the protein comprises It can be safely used as a prophylactic or therapeutic agent for bone diseases or joint diseases.
  • the present invention relates to a method for detecting or selecting a sample derived from an individual suspected of having a bone disease or joint disease, a method for diagnosing a bone disease or joint disease, and a diagnostic agent used for the detection, selection and diagnosis method. Further, the present invention relates to a screening method for a therapeutic or prophylactic agent for bone disease or joint disease, a kit used for the screening method, and a pharmaceutical composition.
  • Bone disease or joint disease means, for example, cartilage dysplasia, bone dysplasia, osteoporosis, osteoarthritis, rheumatoid arthritis, arthritis, synovitis, metabolic arthropathy or sports Examples include joint disorders.
  • the present invention can be suitably used particularly for osteoarthritis or rheumatoid arthritis.
  • the "method for detecting or selecting a sample derived from an individual suffering from bone disease or joint disease and suspected” refers to a protein in a test sample derived from an individual to be tested or One characteristic is to measure the expression level of a gene encoding a partial peptide. Therefore, according to the detection or selection method of the present invention, it is possible to easily and quickly detect or select whether or not the subject individual is suspected of having bone disease or joint disease. The effect is demonstrated.
  • OA and RA which are typical joint diseases and exhibit similar symptoms, can be distinguished, and samples from OA patients or RA patients can be detected or selected.
  • the "individual” is not particularly limited, but includes mammals, particularly humans, mice, mice, monkeys, and the like.
  • Test sample refers to, for example, tissue (eg, periosteum, cartilage, etc.), cells, synovial fluid, blood, urine, etc. It can be prepared by conventional methods. It is particularly preferable to prepare it from joint fluid, blood and urine that can be easily examined. Collect joint fluid, blood and urine in the same way as normal joint fluid, blood and urine tests.
  • the control sample can be prepared from the same site as the test sample, for example, as long as it is prepared from the same part as the normal individual, RA patient or OA patient test sample .
  • tissue for example, periosteum, cartilage, etc.
  • the tissue should be adjusted and used by a conventional method. You can also.
  • the "detection or selection method" of the present invention is a protein having an amino acid sequence selected from the group consisting of SEQ ID NOs: 18 to 34 in a test sample derived from an individual to be tested, or 1 or more in the amino acid sequence. Measuring the expression level of at least one gene encoding a protein having an amino acid sequence in which several amino acids have been deleted, substituted or added, wherein the expression level in the test sample is expressed in the control sample When it is different from the level, it becomes an indicator that the test sample is a sample derived from an individual suspected of having bone disease or joint disease.
  • a gene showing an expression increase of 2 times or more compared to normal individuals is preferred as an indicator.
  • SEQ ID NO: 2 consisting of a base sequence selected from the group consisting of 2, 3, 7, 9-11 and 13-16 10
  • An individual gene can be determined to be an OA patient if its expression level is higher than the expression level in normal individuals and RA patients.
  • Six genes having a nucleotide sequence ability selected from the group consisting of SEQ ID NOs: 1, 4-6, 8, 12 have higher expression levels in test samples than expression levels in normal individuals, and RA patients If it is lower than the expression level in, it can be determined that the patient is OA.
  • the gene consisting of the nucleotide sequence of SEQ ID NO: 17 can be judged to be an OA patient when the expression level in the test sample is lower than the expression level in normal individuals and higher than the expression level in RA patients. is there.
  • a gene having a nucleotide sequence ability of SEQ ID NO: 6 or 13 is preferable as an index.
  • genes having a nucleotide sequence ability selected from the group consisting of SEQ ID NOs: 1, 4-6, 8, 12 are expressed in the expression level in normal individuals and OA patients. If it is above the threshold, it can be determined that the patient is RA.
  • the gene having the nucleotide sequence of SEQ ID NO: 17 can be determined to be an RA patient when the expression level in the test sample is lower than the expression level in normal individuals and OA patients.
  • a "protein having an amino acid sequence selected from the group consisting of SEQ ID NOs: 18 to 34" encoded by a gene whose expression level is measured in the “detection or selection method” of the present invention (hereinafter referred to as "the present invention”).
  • Is sometimes referred to as a protein of human warm-blooded animals eg, guinea pigs, rats, mice, rabbits, rabbits, pigs, hidges, rabbits, monkeys, etc.
  • Cells neurons, glial cells, spleen j8 cells, bone marrow cells, mesangial cells, Langerhans cells, epidermal cells, epithelial cells, goblet cells, endothelial cells, smooth muscle cells, fibroblasts, fibroblasts, muscle cells, adipocytes, Immune cells (eg, macrophages, T cells, B cells, natural killer cells, mast cells, neutrophils, basophils, eosinophils, monocytes), megakaryocytes, synovial cells, chondrocytes, bone cells, Osteoblast Cysts, osteoclasts, mammary cells, hepatocytes or stromal cells, or precursor cells of these cells, stem cells or cancer cells, etc.) or
  • amino acid sequence in which one or several amino acids are deleted, substituted or added is, for example, (A) 1 or 2 in an amino acid sequence selected from the group consisting of SEQ ID NOs: 18 to 34 An amino acid sequence in which one or more (preferably about 1 to 30, preferably about 1 to 10, more preferably (1 to 5)) amino acids have been deleted, (B) from SEQ ID NOs: 18 to 34 1 or 2 (preferably about 1 to 30, preferably about 1 to 10, more preferably a number (1 to 5)) of amino acids are added to the amino acid sequence selected from the group consisting of (C) 1 or 2 or more amino acid sequences selected from the group consisting of SEQ ID NOs: 18 to 34 (preferably about 1 to 30, preferably about 1 to about LO, more preferably numbers) An amino acid sequence in which (1-5) amino acids are inserted; (D) from the group consisting of SEQ ID NOs: 18-34 One or more amino acids in the selected amino acid sequence (preferably about 1 to 30, preferably about 1 to 10, more
  • the "gene expression level" is preferably measured using the amount of mRNA or protein of the gene as an index. Any method may be used as long as it is a molecular biological measurement method for detecting mRNA or an immunological measurement method for detecting protein. Examples of molecular biological measurement methods include polymerase chain reaction (PCR), Northern plot method, dot plot method, analysis method using microarray or macroarray, and immunological measurement methods include microtiter plates. Examples include ELISA method using RIA, RIA method, fluorescent antibody method, Western plot method, immunohistochemical staining method and the like.
  • an expression level force protein having an amino acid sequence selected from the group consisting of SEQ ID NOs: 18 to 34, or an amino acid in which one or several amino acids are deleted, substituted or added in the amino acid sequence
  • a method in which the amount of mRNA of at least one gene encoding a protein having a sequence is measured as an index. This corresponds to the molecular biological measurement method described above.
  • a protein having an amino acid sequence selected from the group consisting of SEQ ID NOs: 18 to 34 Or a method using a nucleic acid capable of detecting at least one gene encoding a protein having an amino acid sequence in which one or several amino acids have been deleted, substituted or added in the amino acid sequence, A method that is a nucleic acid consisting of a base sequence selected from the group consisting of SEQ ID NOs: 1 to 17, a method wherein the gene is a nucleic acid consisting of a base sequence of SEQ ID NOs: 6 and Z, or 13, SEQ ID NOs: 35 and Z or A method using a nucleic acid having a nucleotide sequence of 36 is also included. Specific description will be given below.
  • a test sample derived from an individual to be tested is, for example, a protein having an amino acid sequence selected from the group consisting of SEQ ID NOs: 18 to 34, or one or several amino acids in the amino acid sequence.
  • the sample is subjected to PCR using a primer pair that can specifically amplify a nucleic acid encoding a protein having a deleted, substituted or added amino acid sequence or a partial sequence characteristic of the nucleic acid.
  • the expression level of the gene of the protein of the present invention can be evaluated by measuring the formation or the amount of the hybrid or the appearance or the amount of the amplification product and comparing it with the result in the control sample.
  • the "primer pair" used in PCR includes a nucleic acid encoding the protein of the present invention or a primer corresponding to an antisense sequence at the 5 'end of a nucleic acid comprising a sequence portion characteristic of the nucleic acid and 3'.
  • a pair of primers consisting of a primer corresponding to the antisense sequence at the end.
  • Such a primer pair can be appropriately selected based on an appropriate Tm value, secondary structure, etc. in consideration of operability during use. Specifically, it is preferable that the entire base sequence described in SEQ ID NOs: 1 to 17 or a characteristic partial sequence thereof can be amplified.
  • primer pairs selected from the group consisting of nucleic acids having the base sequences described in SEQ ID NOs: 1 to 17 and the like can be mentioned.
  • Specific examples of the primer pair include, but are not limited to, a primer pair of SEQ ID NOs: 35 and 36, and the like.
  • Such a primer may be a primer labeled with a conventional fluorescent dye or radioactive substance.
  • the amplification product can be detected by visualizing with a fluorescent substance, detection based on a labeled primer, etc. in conventional agarose gel electrophoresis or the like.
  • the “partial sequence characteristic to (nucleic acid)” means, for example, a base of a gene other than the gene encoding the protein of the present invention among sequences registered in a database.
  • a partial sequence not substantially found in a sequence for example, sequence identity with a sequence registered in a database, usually 20% or less, preferably 10% or less, more preferably 5% or less, Particularly preferred is a sequence that is 0%.
  • a test sample derived from an individual to be tested is, for example, one or a number in the protein having the amino acid sequence selected from the group consisting of the aforementioned SEQ ID NOs: 18 to 34 or the amino acid sequence.
  • a probe that detects a nucleic acid encoding a protein having an amino acid sequence in which a single amino acid has been deleted, substituted, or added the formation of the hybrid or the amount thereof, or the appearance or amount of the amplification product is measured, and a control sample It is possible to evaluate the expression level of the gene of the protein of the present invention by comparing with the results in.
  • a test sample derived from an individual to be tested is, for example, a protein having an amino acid sequence selected from the group consisting of SEQ ID NOs: 18 to 34 or the amino acid
  • a microarray or macroarray containing at least one probe for detecting a nucleic acid encoding a protein having an amino acid sequence in which one or several amino acids are deleted, substituted or added in the sequence a hybrid is formed or
  • the expression level of the gene of the protein of the present invention can be evaluated by measuring the amount or the appearance of the amplification product or the amount thereof and comparing it with the result in the control sample.
  • the “probe” used in the Northern blot method or the analysis method using a microarray or macroarray is used to detect a nucleic acid having a base sequence selected from the group consisting of SEQ ID NOs: 1 to 17.
  • a nucleic acid used as a probe can be appropriately selected based on an appropriate Tm value, secondary structure, etc. in consideration of operability during use.
  • Such a nucleic acid may be labeled with a conventional fluorescent dye, radioactive substance or the like.
  • the present invention includes a protein having an amino acid sequence selected from the group consisting of SEQ ID NOs: 18 to 34, or an amino acid in which one or several amino acids have been deleted, substituted or added in the amino acid sequence. At least one protein having a sequence or part thereof A method of measuring using a partial peptide or a salt thereof as an index is also included. This corresponds to the above-mentioned method of immunological measurement.
  • the "partial peptide of the protein” is a partial peptide of the protein of the present invention, and may be any one as long as it has the same properties as the protein of the present invention.
  • the amino acid sequence of the protein of the present invention has at least 20 or more, preferably 50 or more, more preferably 70 or more, more preferably 100 or more, most preferably 200 or more amino acid sequences. Peptides and the like are used.
  • the "partial peptide" has one or more (preferably about 1 to: LO, more preferably several (1 to 5)) amino acids in its amino acid sequence, Or 1 or 2 or more (preferably about 1 to 20, more preferably about 1 to 10, more preferably a number (1 to 5)) of amino acids are added to the amino acid sequence, or 1 or 2 or more (preferably about 1 to 20, more preferably about 1 to 10, more preferably a number (1 to 5)) of amino acids are inserted into the amino acid sequence, or the amino acid One or more amino acids in the sequence (preferably about 1 to: about LO, more preferably about several, and even more preferably about 1 to 5) may be substituted with other amino acids. ,.
  • salts with a physiologically acceptable acid eg, inorganic acid, organic acid
  • base eg, alkali metal salt
  • Acid addition salts that are acceptable are preferred.
  • examples of such salts include salts with inorganic acids (for example, hydrochloric acid, phosphoric acid, hydrobromic acid, sulfuric acid), or organic acids (for example, acetic acid, formic acid, propionic acid, fumaric acid, maleic acid, Salts with succinic acid, tartaric acid, succinic acid, malic acid, succinic acid, benzoic acid, methanesulfonic acid, benzenesulfonic acid, etc. are used.
  • inorganic acids for example, hydrochloric acid, phosphoric acid, hydrobromic acid, sulfuric acid
  • organic acids for example, acetic acid, formic acid, propionic acid, fumaric acid, maleic acid
  • a protein having an amino acid sequence selected from the group consisting of SEQ ID NOs: 18 to 34, or at least 1 having an amino acid sequence in which one or several amino acids are deleted, substituted, or added in the amino acid sequence A method using an antibody against one protein or a partial peptide thereof or a salt thereof or a fragment thereof, a protein having an amino acid sequence of SEQ ID NO: 23 and Z or 30, or a deletion of or substitution of one or several amino acids in the amino acid sequence Or a method using an antibody or fragment thereof against at least one protein having an added amino acid sequence or a partial peptide thereof or a salt thereof.
  • the Specific description will be given below.
  • each of a test sample and a control sample derived from an individual to be tested is subjected to polyacrylamide gel electrophoresis, Western plot analysis using an antibody against the protein of the present invention or a fragment thereof,
  • the expression level of the gene can be evaluated by subjecting it to an immunoassay or the like using an antibody or a fragment thereof, and measuring and comparing the amount of the protein.
  • the "antibody” includes an antibody against the protein of the present invention, a fragment thereof, and the like. That is, the antibody may be a polyclonal antibody or a monoclonal antibody as long as it has an ability to specifically bind to the protein of the present invention.
  • the antibody may be, for example, an antibody that can specifically bind to a specific partial fragment in the protein of the present invention.
  • the method for producing the antibody is specifically described below. Further, the obtained antibody can be purified and then treated with peptidase to obtain antibody fragments.
  • derivatives of the above-described antibodies for example, chimeric antibodies, humanized antibodies, Fab fragments, single chain antibodies and the like, and antibodies modified by known techniques can also be used.
  • Such an antibody or a fragment thereof may be labeled with a conventional enzyme (for example, peroxidase), a fluorescent dye, a radioactive substance, avidin or piotin.
  • a polyclonal antibody can be prepared by administering a peptide having a partial amino acid sequence of the protein of the present invention to an animal as an antigen.
  • Usagi, goat, rat, mouse, hamster, etc. can be used as the animal to be administered.
  • the dose of the antigen is preferably 50 to: LOO / zg per animal.
  • a carrier protein such as keyhole limpet haemocyanin or bovine thyroglobulin brin.
  • the peptide used as an antigen can be synthesized with a peptide synthesizer.
  • the antigen is administered 3 to 10 times every 1 to 2 weeks after the first administration.
  • Serum can be obtained from a non-human mammal whose serum showed a sufficient antibody titer against the antigen used for immunization, and polyclonal antibodies can be separated and purified from the serum by the following method.
  • Methods for separating and purifying antibodies include centrifugation, salting out with 40-50% saturated ammonium sulfate, force prillic acid precipitation (Antibodies, A Laboratory manual. Cold Spring Harbor Laboratory, (1988)), or Examples include a single or combination treatment of DEAE-sepharose column, anion exchange column, chromatography using protein A or G-force ram or gel filtration column, and the like.
  • Rats whose sera showed sufficient antibody titers against the polypeptide fragment fragment polypeptide of the present invention used for immunization are used as a source of antibody-producing cells.
  • the spleen is removed 3 to 7 days after the final administration of the antigenic substance to the rat showing the antibody titer.
  • the spleen is shredded in MEM medium (Nissui Pharmaceutical Co., Ltd.), loosened with forceps, centrifuged at 1,200 rpm for 5 minutes, and the supernatant is discarded.
  • myeloma cells cell lines obtained from mice or rats are used.
  • 8-azaguanine resistant mouse BALB / c-derived myeloma cell line P3-X63Ag8-U1 (hereinafter abbreviated as P3—U1) [Curr. Topics. Microbiol. Immunol, 81, 1 (1978), Europ. J Immunol, 6, 511 (1976)], SP2 / 0-Agl4 (SP-2) [Nature, 276,269 (1978)], P3—X63—Ag8653 (653) [J.
  • the antibody can be used, for example, in a method for detecting a sample from an OA or RA patient. Such methods include using antibodies to detect the presence or absence of a protein of the invention described herein in a suitable biological sample.
  • suitable biological samples for use herein include synovial fluid or normal thread and tissue biopsies obtained from patients, homogenates or extracts thereof.
  • the detection method involves the use of an antibody immobilized on a solid support to bind to the protein and remove it from the rest of the sample.
  • the binding protein can then be detected using a secondary antibody or reagent having a reporter group.
  • a competitive assembly can be used, in which the protein is labeled with a reporter group and allowed to bind to the immobilized antibody after incubation of the antibody with the sample. The extent to which the sample components inhibit the binding of labeled protein to the antibody indicates the reactivity of the sample with the immobilized antibody and, as a result, the concentration of the protein in the sample.
  • the solid support can be any material known to those of skill in the art to which antibodies can be attached.
  • the solid support can be, for example, a test well of a microtiter plate, or a -trocellulose filter, or other suitable membrane.
  • the support can be a bead or disk (eg, glass, glass fiber, latex or a plastic material (eg, polystyrene or polyvinyl chloride)).
  • the support can also be a magnetic particle or fiber optic sensor (eg, disclosed in US Pat. No. 5,359,681).
  • the antibody may be immobilized on a solid support using various techniques known in the art and described in detail in the patent and scientific literature.
  • immobilization refers to non-covalent association (eg adsorption) and covalent attachment (force that can be a direct link between an antigen and a functional group on a support, or by a cross-linking agent. Both). It is preferable to fix the microtiter plate by adsorption to a well or a membrane. In such cases, adsorption can be achieved by contacting the antibody in a suitable buffer with the support for a suitable time. The contact time varies with temperature, but is typically between about 1 hour and 1 day.
  • the contact force between a wall of a plastic microtiter plate eg, polystyrene or polychlorinated butyl
  • an amount of antibody in the range of about 10 ng to about 1 ⁇ g, and preferably about 100-200 ng Enough to fix the amount of protein.
  • Covalent attachment of an antibody to a solid support also generally involves the attachment of a bifunctional agent and support that reacts with both the support and a functional group (eg, a hydroxyl group or an amino group) on the antibody. This can be achieved by first reacting.
  • an antibody can be covalently attached to a support having a suitable polymer coat by using benzoquinone or by condensation of an aldehyde group on the support with an amine and an active hydrogen on a binding partner (e.g., Pierce Immunotechnology Catalog and Handbook (199 1) A12-A13).
  • a binding partner e.g., Pierce Immunotechnology Catalog and Handbook (199 1) A12-A13.
  • the assay for the detection of a protein in a sample is a two-antibody sandwich assay.
  • the assembly begins with binding of an antibody immobilized on a solid support (usually a microtiter plate well) and a biological sample to the antibody to which the polypeptide in the sample is immobilized. It can be done by contacting.
  • the unbound sample is then removed of immobilized polypeptide-antibody complex force and a secondary antibody (containing a reporter group) that can bind to a different site on the polypeptide is added.
  • the amount of secondary antibody that remains bound to the solid support is then determined using a method appropriate to the particular reporter group.
  • the remaining polypeptide binding sites on the support are typically blocked.
  • Any suitable blocking agent eg, ushi serum albumin or Tween 20 TM (Sigma Chemical Co., St. Louis, MO)
  • the immobilized antibody is then incubated with the sample and the polypeptide is allowed to bind to the antibody.
  • the sample can be diluted with an appropriate diluent (eg, phosphate buffered saline (PBS)) prior to incubation.
  • PBS phosphate buffered saline
  • a suitable contact time ie, incubation time
  • incubation time is sufficient to detect the presence of the polypeptide in a sample obtained from an individual having breast cancer.
  • the contact time is a time sufficient to achieve a level of binding that is at least 95% of the level of binding achieved at equilibrium between the bound and unbound polypeptides.
  • the time required to achieve equilibrium is readily determined by assessing the level of binding that occurs over a period of time. An incubation time of about 30 minutes at room temperature is generally sufficient.
  • the sample should be loaded with a solid support in an appropriate buffer (eg, 0.1% T It can be removed by washing with PBS containing ween 20TM.
  • a secondary antibody having a reporter group can then be added to the solid support.
  • Preferred reporter groups include enzymes (eg, horse radish peroxidase), substrates, cofactors, inhibitors, dyes, radionuclides, chromophores, fluorescent groups, and piotin. Coupling of the antibody to the reporter group can be accomplished using standard methods known to those skilled in the art.
  • the secondary antibody is then incubated with the immobilized antibody-polypeptide complex for a time sufficient to detect the bound polypeptide.
  • the appropriate time can generally be determined by assessing the level of binding that occurs over a given time.
  • binding! /,! / The secondary antibody is removed, and the bound secondary antibody is detected using a reporter group.
  • the method used to detect the reporter group depends on the nature of the reporter group. For radioactive groups, scintillation counting or autoradiographic methods are generally appropriate. Spectroscopy can be used to detect dyes, chromophores and fluorescent groups. Piotin can be detected using avidin coupled to different reporter groups (usually radioactive groups or fluorescent groups or enzymes). Enzyme reporter groups can generally be detected by addition of a substrate (generally for a specific time) followed by spectroscopic analysis of the reaction product or other analysis.
  • the reporter stays attached to the solid support.
  • the force detected is generally determined in advance when normal tissue force is also established. Compared to the signal corresponding to the cutoff value.
  • the cut-off value is the average signal obtained when the immobilized antibody is incubated with a sample from a patient without bone or joint disease. In general, a sample that produces a signal that is 3 standard deviations above the predetermined cutoff value can be considered positive for bone or joint disease.
  • the cut-off value is determined using a Receiver Oparator Curve according to the method of Sackett et al., Linical Epidemiology: A Basic science for linical Medicene 10 page 7, (Little Brown and Co, 1985).
  • the cutoff value is the true positive rate (ie sensitivity) and false positive rate (100% specificity) corresponding to each possible cutoff value for diagnostic test results. Can be determined by plotting the pair with. On the plot Cut-off value closest to the upper left corner (ie the value that encloses the largest area) force The sample that is the most accurate cut-off value and produces a signal higher than the cut-off value determined by this method is positive Can be considered. Alternatively, the cut-off value can be shifted to the left along the plot to minimize the false positive rate, or to the right to minimize the false negative rate.
  • the detection of the protein of the present invention is performed in a flow-through test or strip test format.
  • the antibody is immobilized on a membrane (for example, a nitrocell mouth).
  • the polypeptide in the sample binds to the immobilized antibody as it passes through the sample canvas.
  • the labeled secondary antibody then flows through the solution canvas containing the secondary antibody and binds to the antibody polypeptide complex. Detection of bound secondary antibody can then be performed as described above.
  • the strip test format one end of the membrane to which the antibody is bound is immersed in a solution containing the sample. The sample moves along the membrane through the area containing the secondary antibody and into the area of the immobilized antibody.
  • the concentration of secondary antibody at this site Indicates the presence of bone or joint disease. Typically, the concentration of secondary antibody at this site produces a pattern (eg, a line) that can be read visually. The absence of such a pattern indicates a negative result.
  • the amount of antibody immobilized on the membrane is sufficient if it contains a sufficient level of polypeptide to produce a positive signal in a two-antibody sandwich assembly of the type discussed above in terms of biological sample power. Chosen to produce a visually discernable pattern.
  • the amount of antibody immobilized on the membrane ranges from about 25 ng to about 1 ⁇ g, and more preferably from about 50 ng to about 1 ⁇ g. Such tests can typically be performed using very small biological samples.
  • the method for detecting or selecting a sample derived from an individual suspected of suffering from a bone disease or joint disease of the present invention can also be used as a diagnostic method for bone disease or joint disease.
  • a diagnostic method is also encompassed by the present invention.
  • a diagnostic agent containing the probe and Z or primer pair, or an antibody against the protein or a fragment thereof, which can perform the diagnostic method of the present invention simply, rapidly, with high throughput and high reliability Diagnostics are provided.
  • SEQ ID NO : at least one gene encoding a protein having an amino acid sequence selected from the group consisting of 18 to 34, or a protein having an amino acid sequence in which one or several amino acids are deleted, substituted or added in the amino acid sequence
  • a diagnostic agent for bone disease or joint disease containing a nuclear acid capable of detecting
  • a diagnostic agent for bone disease or joint disease wherein the gene is a nucleic acid comprising a base sequence selected from the group consisting of SEQ ID NOs: 1 to 17,
  • a diagnostic agent for a diagnostic agent for bone disease or joint disease wherein the gene is a nucleic acid comprising the nucleotide sequence of SEQ ID NO: 6 and Z or 13;
  • a diagnostic agent for bone disease or joint disease comprising a nucleic acid having a nucleotide sequence of SEQ ID NO: 35 and Z or 36,
  • a diagnostic agent for bone disease or joint disease comprising an antibody against or a fragment thereof.
  • the diagnostic agent of the present invention may further contain a detection reagent, a buffer solution, a control sample, a description of the diagnostic method of the present invention, and the like.
  • the present invention also includes the following screening methods.
  • SEQ ID NO : at least one gene encoding a protein having an amino acid sequence selected from the group consisting of 18 to 34, or a protein having an amino acid sequence in which one or several amino acids are deleted, substituted or added in the amino acid sequence
  • a method for screening a preventive agent and a Z or therapeutic agent for bone diseases or joint diseases characterized by using a protein having an amino acid sequence selected from the group consisting of SEQ ID NOs: 18 to 34, or Use of at least one protein having an amino acid sequence in which one or several amino acids are deleted, substituted or added in the amino acid sequence, a partial peptide thereof, or a salt thereof, And Z or therapeutic agent screening method.
  • the protein used for the screening method may be a protein derived from human warm-blooded animal cells or any tissue in which those cells are present, or may be a synthetic protein.
  • a protein having an “amino acid sequence in which one or several amino acids have been deleted, substituted or added” has the same meaning as the protein whose expression is measured in the “detection or selection method” described above.
  • the protein of the present invention can be obtained by using the method described in Molecular Cloning 2nd Edition, Current 'Protocols' in 'Molecular' Biology Supplement 1-38, etc., for example, by the following method.
  • the encoded gene can be expressed and produced in the host cell.
  • a DNA fragment of an appropriate length containing a portion encoding the protein is prepared as necessary.
  • a DNA is prepared by substituting the base sequence of the portion encoding the protein so that the codon is optimal for host expression. The DNA is useful for improving the production rate of the protein.
  • a recombinant DNA (recombinant vector) is prepared by inserting the DNA fragment or full-length DNA downstream of the promoter of an appropriate expression vector.
  • a transformant producing a protein can be obtained by introducing the recombinant vector into a host cell suitable for the expression vector.
  • any prokaryotic cell, yeast, animal cell, plant cell, insect cell, etc. can be used as long as it can express the target gene.
  • the expression vector a vector that can replicate autonomously in the host cell or can be integrated into a chromosome and contains a promoter at a position suitable for transcription of the gene of the protein of the present invention is used.
  • the protein expression vector of the present invention is preferably composed of a promoter, a ribosome binding sequence, a protein of the present invention, and a transcription termination sequence as well as being capable of autonomous replication in prokaryotes! ⁇ . Includes genes that control the promoter!
  • expression vectors include pBTrp2, pBTacl, pBTac2 (all sold by Boehringer Mannheim), PKK233-2 (Falmacia), pSE280 (Invitrogen), pGEMEX-EX (manufactured by Promega), pQE -8 (QIAGEN), pKYPIO (JP-A 58-110600), pKYP200 [Agric. Biol. Chem., 48, 669 (1984)], pLSAl [Agric. Biol. Chem., 53, 277 (1989)], pGELl [Proc. Natl. Acad. Sci.
  • promoters derived from E. coli and phage such as trp promoter (Ptrp), lac promoter (Plac), PL promoter, PR promoter, PSE promoter, SP01 promoter, SP02 promoter, penP promoter, etc. Can do. It is also possible to use an artificially designed and modified promoter such as a promoter in which two Ptrps are connected in series (Ptrp x2), tac promoter, lacT 7 promoter, let I promoter, etc.
  • a plasmid in which the distance between the Shine-Dalgarno sequence, which is a ribosome binding sequence, and the initiation codon is adjusted to an appropriate distance (eg, 6 to 18 bases).
  • an appropriate distance eg, 6 to 18 bases.
  • the transcription termination sequence is not necessarily required for the expression of the gene of the protein of the present invention, it is preferable to place the transcription termination sequence directly under the structural gene.
  • Host cells include Escherichia, Serratia, Bacillus, Brevibacterium, Microorganisms belonging to the genus Corynebacterium, Microbacterium, Syudomonas, etc., such as Escherichia coli XLl-Blue, Escherichia coli XL2-Blue, Escherichia coli DH1, Escherichia coli MC1000, Escherichia coli KY3276, Eschericnia coli W1485, Escher icnia coli JM109, Escherichia coli HB101, Escherichia coli No.49, Escherichia coli W3110, Escherichia coli NY49, Escherichia coli BL21 (DE3), Escherichia coli BL21 (DE3) pLysS, Escherichia coli HMS174 (DE3), Escherichia coli HMS174 (DE3) pLysS , Ser
  • any method can be used as long as it is a method for introducing DNA into the host cell.
  • the Elect Mouth Position Method [Nucleic Acids Res., 16, 6127 (1988)], calcium ion [Proc. Natl. Acad. Sci. USA, 69, 2 110 (1972)], protoplast method (JP-A 63-2483942), Gene, 17, 107 (1982) and Molecular & General Genetics, 168 , 111 (1979).
  • yeast strains When yeast strains are used as host cells, examples of expression vectors include YEpl3 (ATCC37115), YEp24 (ATCC37051), YCp50 (ATCC37419), pHS19, pHS15 and the like. Any promoter can be used as long as it can be expressed in yeast strains.For example, PH05 promoter, PGK promoter, GAP promoter 1, ADH promoter, gal 1 promoter, gal 10 promoter, heat shock protein promoter, Examples of host cells that can include MF a 1 promoter, CUP 1 promoter and the like include yeast strains belonging to the genus Saccharomyces, Schizosaccharomyces, Kluybe mouth genus, Trichosporon genus, Schizophyllum genus, Pichia genus, etc.
  • any method can be used as long as it introduces DNA into yeast.
  • the electopore position method [Methods Enzymol, 194, 182 (1990)]
  • Spheroplast Proc. Natl. Acad. Sci. USA. 84, 1929 (1978)
  • lithium acetate method J. BacterioL, 153, 163 (1983)
  • examples of expression vectors include pcDN Al / Amp, pcDNAI, pCDM8 (all commercially available from Funakoshi), pAGE107 (JP-A-3-22979, Cytotechnology, 3, 133 (1990). )], PCR—Bluntll—TOPO, pR 4 (manufactured by Invitrogen), pAGE103 (J. Biochem., 101, 1307 (1987)), pAMo, pAMoA [J. Biol. Chem., 268, 22782-22787 (1993) ), Also known as pAMoPRSA (Japanese Patent Laid-Open No.
  • pAS3-3 Japanese Patent Laid-Open No. 2-227075
  • pHM6, pHB6 Roche Diagnostatus
  • PKK223-3 pGEX (Amersham Biotech)
  • pET-3 pET-11
  • pBluescriptll registered trademark
  • SK (+) ⁇ pBluescriptll registered trademark
  • SK (—) Stratagene
  • pUC19 pTrxFus
  • pSTV28 Teakara Bio
  • pMAL-c2X manufactured by New England Biolabs.
  • Any vector may be used as long as it is a vector that can be expressed in animal cells by incorporating the gene.
  • Any promoter can be used as long as it can be expressed in animal cells.
  • cytomegalovirus (human CMV) IE immediateearly gene promoter
  • SV40 early promoter SV40 early promoter
  • Moro- ⁇ ⁇ ⁇ Long terminal repeat promoter long terminal repeat promoter of Moloney Murine Leukemia Virus
  • retrowinores promoter heat shock promoter
  • SRa promoter meta-mouth tinee promoter
  • Host cells include mouse 'myeloma cells, rat' myeloma cells, mouse 'hybrids. Dormer cells, Chinese's cells, CHO cells that are Muster cells, BHK cells, African monkey kidney cells, Namalwa cells or Namalwa KJM-1 cells that are human cells, human fetal kidney cells, human leukemia cells, HBT5637 Sho 63-299), and human colorectal cancer cell lines.
  • Mouse 'myeloma cells include SP2 / 0, NSO, etc.
  • rat' myeloma cells include YB2 / 0, human fetal kidney cells such as HEK293 (ATCC: CRL-1573), and human leukemia cells such as BALL-1.
  • Examples of African green monkey kidney cells include COS-1 and COS-7, and examples of human colon cancer cell lines include HCT-15.
  • any method can be used as long as it is a method for introducing DNA into animal cells.
  • the ribofusion method [Proc. Natl. Acad. Sci. USA, 84, 7413 (1987)], virology, 52, 456 (1973), and the like.
  • a tank is prepared according to a known method [tissue culture, 20 (1994), tissue culture, 21 (1995), Trends in Biotechnology, 15, 45 (1997)]. Can produce quality.
  • expression vectors include Ti plasmid and tobacco mosaic virus vector.
  • Any promoter can be used as long as it can be expressed in plant cells, and examples thereof include a cauliflower mosaic virus (CaMV) 35S promoter, an actin-1 promoter, and the like.
  • gene expression efficiency can be increased by inserting intron 1 of the maize alcohol dehydrogenase gene between the promoter and the gene to be expressed.
  • host cells include plant cells such as potato, tobacco, corn, rice, rape, soybean, tomato, carrot, wheat, barley, rye, alfalfa and flax.
  • any method can be used as long as it is a method for introducing DNA into plant cells.
  • Agrobacterium JP 59-140 885, JP 60-70080, WO94 / 00977
  • Elect Mouth Position Method Japanese Patent Laid-Open No. 60-251887)
  • a method using a particle gun Gene gun
  • Patent No. 2606856, Patent No. 2517813 Patent No. 2606856, Patent No. 2517813
  • a recombinant gene transfer vector and a baculovirus are co-introduced into insect cells to obtain a recombinant virus in an insect cell culture supernatant, and then the insect cells are further infected with the recombinant virus to express a protein.
  • Examples of the gene transfer vector used in the method include pVL1392, pVL1393, pBlueBacIII (all manufactured by Invitrogen) and the like.
  • baculovirus for example, use of the autographa californica nu clear polyhedrosis virus; an autographa californica force, which is a virus that infects the night stealing insects.
  • Spodoptera frugiperda ovarian cells As insect cells, Spodoptera frugiperda ovarian cells, Trichoplusia ni ovarian cells, silkworm ovary-derived cultured cells, and the like can be used. Spodoptera frugiperda ovarian cells are S19, S11 (Baculovirus 'Expression' Vectorz Laboratories Manual), Trichoplusia ni ovary cells are High 5, BTI-TN-5Bl-4 (Invitrogen) Bombyx mori N4 etc. can be mentioned as cultured cells derived from silkworm ovary.
  • Examples of a method for co-introducing the recombinant gene introduction vector and the baculovirus into insect cells for preparing a recombinant virus include, for example, the calcium phosphate method (JP-A-2-227075), the lipofuxion method [Proc. Natl. Acad Sci. USA, 84,7413 (1987)].
  • DNA can be introduced into insect cells using a method similar to the method for introducing DNA into animal cells. For example, the electopore position method [Cytotechnology, 3, 133 (1990)], the calcium phosphate method ( JP-A-2-227075), ribofusion method [Proc. Natl. Acad. Sci. USA, 84, 7413 (1987)] and the like.
  • the transformant having a recombinant vector incorporating the DNA encoding the protein of the present invention is a cell such as Escherichia coli, yeast, animal cells or plant cells
  • the cells are cultured according to a normal culture method suitable for various hosts.
  • the protein can be produced by accumulating and collecting the protein and recovering the protein from the transformant or culture medium.
  • the transformant is an animal or plant individual, it is bred or cultivated according to a normal growth method suitable for various hosts, the protein is produced and accumulated, and the protein is recovered from the animal or plant individual.
  • the protein can be produced.
  • the host is an animal individual, for example, a non-human transgenic animal carrying the polynucleotide of the present invention is bred, and the protein of the protein of the present invention encoded by the recombinant DNA is produced and accumulated in the animal.
  • the protein of the present invention can be produced by recovering the protein from the individual animal. Production in animal individual • Examples of the accumulation location include milk, saliva, eggs and the like of the animal.
  • a transgenic plant carrying the polynucleotide of the protein of the present invention is cultivated, and the protein of the protein of the present invention encoded by the recombinant DNA is produced in the plant individual.
  • the protein of the protein of the present invention can be produced by accumulating and recovering the protein from the plant individual.
  • the transformant harboring the polynucleotide of the present invention is cultured in a medium and the recombinant DNA is encoded.
  • the protein of the present invention can be produced by producing and accumulating the protein in the culture solution and recovering the culture solution force.
  • the method for culturing the transformant of the protein of the present invention of the present invention in a medium can be carried out according to a usual method used for culturing a host.
  • a medium for culturing a transformant obtained by using a prokaryote such as E. coli or a eukaryote such as yeast as a host contains a carbon source, a nitrogen source, inorganic salts, etc. that the organism can assimilate.
  • Either a natural medium or a synthetic medium may be used as long as it can efficiently culture the body.
  • the medium for culturing the obtained transformant contains a carbon source, nitrogen source, inorganic salts, etc. that can be assimilated by the host.
  • the medium can efficiently culture the transformant, the difference between the natural medium and the synthetic medium may be used.
  • a medium for culturing a transformant whose host is Escherichia coli for example, a YT medium containing butatotryptone, yeast etastruct and sodium chloride sodium is preferable.
  • each microorganism can assimilate, glucose, slakedose, sucrose, molasses containing them, carbohydrates such as starch or starch hydrolysate, organic acids such as acetic acid and propionic acid, Alcohols such as ethanol and propanol can be used.
  • Nitrogen sources include ammonia, ammonium chloride, ammonium sulfate, ammonium acetate, ammonium phosphates of various inorganic and organic acids, and other nitrogen-containing substances.
  • peptone, meat extract, yeast extract, corn steep liquor, caseincaro hydrolyzate, soybean meal and soybean meal hydrolyzate, various fermented cells and digested products thereof can be used.
  • monopotassium phosphate dipotassium phosphate
  • magnesium phosphate magnesium sulfate
  • sodium chloride salt ferrous sulfate
  • manganese sulfate copper sulfate
  • calcium carbonate calcium carbonate
  • Culturing is performed under aerobic conditions such as shaking culture or deep aeration stirring culture.
  • the culture temperature is 15-40 ° C, and the culture time is usually 5-7 days.
  • the pH is maintained at 3.0 to 9.0.
  • the pH is adjusted using inorganic or organic acids, alkaline solutions, urea, calcium carbonate, ammonia, etc. Further, antibiotics such as ampicillin and tetracycline may be added to the medium as needed during the culture.
  • Microtransformed with an expression vector using an inducible promoter as the promoter When the organism is cultured, an inducer may be added to the medium as necessary. For example, when cultivating a microorganism transformed with an expression vector using the lac promoter, cultivate a microorganism transformed with an expression vector using trp promoter, such as isopropyl ⁇ -D-thiogalatatopyranoside. When doing so, indoleacrylic acid or the like may be added to the medium. Plant cells and organs into which a gene has been introduced can be cultured in large quantities using a jar mentor. As a culture medium, it is possible to use a commonly used Murashige and Stag (MS) medium, White medium, or a medium supplemented with plant hormones such as auxin and cytokinin. it can.
  • MS Murashige and Stag
  • White medium or a medium supplemented with plant hormones such as auxin and cytokinin. it can.
  • a culture medium for culturing the cell is a generally used RPMI1640 medium (The Journal of the American Medica 1 Association, 199, 519 (1967)), Eagle's MEM medium [Science, 122,501 (1952)], DME M medium [Virology, 8, 396 (1959)], 199 medium [Proceeding of the Society for the Biologi cal Medicine, 73, 1 (1950)] or these media
  • RPMI1640 medium The Journal of the American Medica 1 Association, 199, 519 (1967)
  • Eagle's MEM medium Science, 122,501 (1952)]
  • DME M medium Virology, 8, 396 (1959)]
  • 199 medium Proceeding of the Society for the Biologi cal Medicine, 73, 1 (1950)
  • medium supplemented with fetal calf serum is used.
  • Cultivation is usually carried out for 1 to 7 days under conditions such as pH 6-8, 25-40 ° C, and 5% CO.
  • antibiotics such as kanamycin, penicillin, streptomycin and the like may be added to the medium as needed during culture.
  • the transformant for protein production of the present invention is an insect cell
  • a medium for culturing the cell a commonly used TNM-FH medium (manufactured by Pharmingen), Sf-900 II SFM medium ( Gibco BRL), ExCell400, ExCell405 [all manufactured by JRH Biosciences], Grace's InsectMedium [Nature, 195, 788 (1962)] and the like can be used.
  • the usual enzyme isolation and purification methods can be used.
  • the culture is treated by a method such as centrifugation to obtain a soluble fraction. .
  • a solvent extraction method from the soluble fraction, a solvent extraction method, a salting-out method using ammonium sulfate, a desalting method, an organic solvent Precipitation method, anion-exchange chromatography using resin such as Jetylaminoethyl (DEAE) -Sepharose, DIAION HPA-75 (manufactured by Mitsubishi Kasei), resin such as S- Sepharose FF (manufactured by Pharmacia) Cation exchange chromatography method used, hydrophobic chromatography method using resins such as butyl sepharose and ferrule sepharose, gel filtration method using molecular sieve, affinity chromatography method, chromatofocusing method, isoelectric
  • a purified sample can be obtained using a method such as electrophoresis such as point electrophoresis.
  • culture is performed.
  • the cells in the culture are collected by centrifuging the product, and after washing the cells, an ultrasonic crusher, a French press, a Mantongauri Homogenizer one, the cells were disrupted by Dyno mill or the like to obtain a cell-free extract.
  • the protein when expressed in an insoluble form in the cell, the protein is similarly collected, disrupted and centrifuged from the precipitate fraction obtained by the usual method. After recovering the quality, the insoluble matter of the protein is solubilized with a protein denaturant.
  • the solubilized solution may not contain a protein denaturant! Is diluted to a dilute solution so that the protein denaturant concentration does not denature the protein, or dialyzed to form the protein into a normal three-dimensional structure. Thereafter, a purified preparation can be obtained by the same isolation and purification method as described above.
  • the protein can be purified according to a conventional protein purification method [J. Evan. Sadler et al .: Methods in Enzymology, 83, 458].
  • the tamper of the present invention The protein can be produced as a fusion protein with other proteins and purified using affinity chromatography using a substance that has an affinity for the fused protein.
  • the protein of the present invention can be produced as a fusion protein with protein A and purified by affinity chromatography using immunoglobulin G.
  • the protein of the present invention can be produced as a fusion protein with a FLAG peptide and purified by affinity chromatography using an anti-FLAG antibody [Pro Natl. Acad. Sci "USA, 86, 8227. (1989), Genes Develop., 4, 1288 (1990)].
  • the protein of the present invention is produced by in vitro transcription according to a known method [J. Biomolecular NMR, 6, 129-134, Science, 242, 1162-1164, J. Biochem., 110, 166-168 (1991)].
  • ⁇ Can be produced using a translation system.
  • the structural analysis of the purified protein of the present invention is carried out by a method generally used in protein chemistry, for example, the method described in Protein Structural Analysis for Gene Cloning (published by Hisashi Hirano, Tokyo Kagaku Dojin, 1993) Is possible.
  • Partial peptide or salt thereof can be produced according to the above protein synthesis method or by cleaving the protein of the present invention with an appropriate peptidase.
  • the partial peptide obtained by the above method is a free form, there is a known method, which can be converted to an appropriate salt by a method analogous thereto, and conversely,
  • the "gene encoding the protein" used in the screening method may be any nucleic acid containing a base sequence encoding the protein used in the present invention described above! Preferably it is DNA.
  • the DNA may be any of genomic DNA, genomic DNA library, cDNA derived from the above-mentioned cell 'tissue, cDNA library derived from the above-mentioned cell' tissue, and synthetic DNA.
  • a “gene encoding a protein” can be obtained as follows.
  • a cDNA library is prepared from human brain, heart, skeletal muscle, spleen, kidney, liver, small intestine, placenta, normal human cells derived from these tissues, human umbilical vein endothelial cells, human ovarian cancer, or human colon cancer by a conventional method To do.
  • DNA obtained by the method include DNA having a base sequence represented by SEQ ID NOs: 1 to 17 and the like.
  • a plasmid containing DNA of sequence number: 1-17 the plasmid described in the below-mentioned Example can be mention
  • any vector can be used as long as it can be expressed in animal cells by incorporating the cDNA.
  • pcDNAI / Amp, pcDNAI, and pCDM8 all available from Funakoshi PAGE107 (JP-A-3-22979, Cytotechnology, 3, 133 (1990)
  • pREP4 manufactured by Invitrogene
  • pAGE103 J. Biochem., 101, 1307 (1987)
  • p AMo, pAMoA J. Biol. Chem., 268, 22782-22787 (1993)
  • pAS3-3 JP-A-2-227075
  • An expression vector containing cDNA is introduced into a selectable animal cell to obtain a transformed cell.
  • any method can be used as long as it is a method for introducing DNA into animal cells.
  • the electoral position method [Cytotechnology, 3, 133 (1990)]
  • the calcium phosphate method Japanese Patent Laid-Open No. 2-22 7075
  • the lipofusion method [Proc. Natl. Acad. Sci. USA, 84, 7413 (1987)]
  • Animal cells include Namalwa cells that are human cells, Namalwa KJM-1 cells that are sublines of Namalwa cells, COS cells that are monkey cells, CHO cells that are Chinese hamster cells, HBT5637 ( JP-A-63-299), HCT-15, which is a colon cancer cell line, can be mentioned, and preferably, Namalwa cells, Namalwa KJM-1 cells or HCT-15 can be used.
  • the obtained transformed cells are cultured by a conventional method. Specifically, it can be cultured by the following culture method for transformants.
  • the culture medium for culturing the cell may be a commonly used RPMI1640 medium [The Journal of the American Medical Association, 199, 519 (1967)], Eagle's MEM medium [Science, 122,501 (1952)], DMEM medium [Virology, 8, 396 (1959)], 199 medium [Proceeding of the Society for the Biological Medicine, 73, 1 (1950)], or fetal calf serum etc. Dwarf medium etc. are used
  • Cultivation is usually carried out for 1 to 7 days under conditions such as pH 6 to 8, 25 to 40 ° C, and the presence of 5% CO.
  • antibiotics such as kanamycin, penicillin, streptomycin and the like may be added to the medium as needed during culture.
  • DNA encoding the protein of the present invention can be obtained.
  • DNA can be prepared by chemically synthesizing DNA encoding the protein of the present invention based on the amino acid sequence.
  • Chemical synthesis of DNA can be carried out using a DNA synthesizer manufactured by Shimadzu Corporation using the thiophosphite method, a DNA synthesizer model 392 manufactured by Perkin Elmer Company using the phosphoramidite method, and the like.
  • the oligonucleotides described below were used as sense primers (SEQ ID NO: 35) and antisense primers (SEQ ID NO: 36) to express mRNA complementary to these DNAs.
  • the target DNA can also be prepared by performing PCR using cDNA prepared from the mRNA of the cells as a saddle.
  • the "gene encoding the partial peptide” may be any polynucleotide as long as it contains a nucleotide sequence encoding the partial peptide used in the present invention, and DNA is preferred.
  • the DNA may be any of genomic DNA, genomic DNA library, cDNA derived from the aforementioned cell 'tissue, cDNA library derived from the aforementioned cell' tissue, and synthetic DNA. It can be obtained according to the above-mentioned method for obtaining a “gene encoding a protein”.
  • the screening method for a therapeutic or prophylactic agent for bone disease or joint disease of the present invention comprises detecting or measuring the dynamics of the protein of the present invention or a gene encoding the protein in the presence of a test substance.
  • test substance by examining the influence of the test substance on the dynamics of the protein of the present invention, which is associated with the onset of bone disease or joint disease, it is effective as a therapeutic or prophylactic agent for bone disease or joint disease. Effective screening of substances.
  • the influence of the test substance on the dynamics of the protein of the present invention is preferably examined using the dynamics in the absence of the test substance as a control.
  • the screening method of the present invention comprises a prophylactic agent for bone disease or joint disease, and
  • test substance examples include a compound or a salt thereof.
  • the compound or a salt thereof includes a low molecular compound, a high molecular compound, a polypeptide or a derivative thereof, a nucleic acid or a derivative thereof, and the like.
  • the strong test substance may be a natural substance or a non-natural substance.
  • the derivative of the polypeptide include a modified polypeptide obtained by adding a modifying group, a noriant polypeptide obtained by altering an amino acid residue, and the like.
  • nucleic acid derivatives include modified nucleic acids obtained by adding modifying groups, variant nucleic acids obtained by modifying bases, peptide nucleic acids, and the like.
  • the "kinetics of the protein of the present invention" to be measured or detected in the screening method of the present invention includes, for example, the presence or absence of binding between the protein and its ligand; Expression, specifically, the presence or absence of the expression may include fluctuations in the expression.
  • the screening method of the present invention has roughly two embodiments depending on the "kinetics of the protein of the present invention" to be measured or detected.
  • step (II) After the step (I), the binding between the protein and the test substance is detected, whereby the test substance binding to the protein is converted into a prophylactic agent and a Z or therapeutic agent for bone disease or joint disease. Selecting as a candidate compound
  • the method containing is mentioned. According to a powerful method, a substance that acts on the function of the protein can be selected through binding to the protein. Therefore, the selected candidate compound can be directly and specifically selected. It has the characteristic property of binding to and regulating its function.
  • the contact between the protein and the test substance is, for example, in the solution without interfering with the original function of the protein. And maintained under appropriate reaction conditions (eg, reaction temperature, reaction time, etc.) (ie, reacting both).
  • solutions such as phosphate buffered physiological saline (PBS), HEPES buffer, and Tris buffer.
  • PBS phosphate buffered physiological saline
  • HEPES buffer HEPES buffer
  • Tris buffer Tris buffer
  • reaction conditions in step (I) are not particularly limited.
  • the solution usually pH 6.0 to: LO. 0, preferably pH 7.0 to 9.0, more preferably pH 7. 5 to 8.5, more preferably pH 8.0, usually 10 ° C to 50 ° C, preferably 20 ° C to 40 ° C, more preferably 25 ° C to 37 ° C, more preferably 25 It may be maintained at ° C, usually 1 minute to 1 hour, preferably 3 to 30 minutes, more preferably 5 to 20 minutes, and even more preferably 10 minutes.
  • step (ii) the binding between the protein and the test substance is detected, and the presence or absence of the binding is examined.
  • the binding is performed by, for example, reducing the radiation activity of the protein contained in the solution after reacting the protein in Step (I) with a test substance labeled with, for example, a radioactive substance as described above. It can be detected by analyzing in competition with a large excess of non-radioactive test substance.
  • the test substance used is selected as a prophylactic agent for bone disease or joint disease and a candidate compound for Z or therapeutic agent.
  • step (I) and step (i) may be performed by a continuous process. Such an embodiment can be carried out, for example, by utilizing a nodding assembly on a carrier holding a test substance.
  • step (A) in order to introduce a gene encoding the protein of the present invention into a cell, for example, downstream of and under the control of an expression regulatory element known as an expression regulatory region of the protein, It is preferable to use a nucleic acid construct in which the gene encoding the protein is operably linked.
  • an expression regulatory element known as an expression regulatory region of the protein. Examples of the expression regulatory element are described in Niimi T et al., Molecular Endocrinology, 2001, Vol. 15, 2021-2136.
  • the "nucleic acid construct" can be easily constructed by inserting the expression regulatory element and the gene encoding the protein of the present invention into a cloning site of a conventional expression vector.
  • the vector include viral vectors and plasmid vectors.
  • the gene encoding the protein of the present invention includes the gene itself; A gene that hybridizes to the antisense strand of a gene under stringent conditions and encodes a protein having an action equivalent to that of the protein; has a mutation of at least one base in the base sequence of the gene; A gene encoding a protein having the same action as the protein; having at least 60%, preferably 80% or more, more preferably 90% or more sequence identity with the base sequence of the gene; A gene encoding a protein having the same action as the above is also included.
  • the “stringent condition” refers to a high stringency condition, for example, a condition of 1 ⁇ SSC / 0.2% SDS.
  • the lower strength for example, f column, 0.5 X SSC, preferably 0.2 X SSC, more preferably 0.1 X SSC, etc. and Z or Washing or the like may be performed at a higher temperature, for example, a force of 50 ° C or higher, preferably 60 ° C or higher, more preferably 65 ° C or higher, which varies depending on the Tm value of the nucleic acid used.
  • the "mutation” refers to base substitution, deletion, addition and insertion. Such a mutation may be a naturally occurring variation or a mutation artificially introduced by a conventional site-specific mutation method or the like.
  • sequence identity means that at least two sequences to be compared are appropriately aligned, the same residue present in each sequence is determined, and the number of matching sites is determined. Next, it is a value that can be calculated by dividing the number of matching sites by the total number of residues in the sequence region to be compared and multiplying the obtained value by 100. Specifically, such sequence identity can be calculated by, for example, a BLAST algorithm that is generally available at a homepage address http: ZZwww.ncbi.nlm.nih.gov/BLA STZ.
  • the vector used for the expression of the gene and the host cell into which the nucleic acid construct is introduced As long as expression of the gene can be achieved by any combination thereof, etc., they may be derived from any species. Specific examples, a method for introducing the nucleic acid construct into cells, a method for culturing a transformant into which the nucleic acid construct has been introduced, and the like are the same as described for the transformant in the protein production method.
  • the gene encoding the protein of the present invention is derived from a eukaryotic organism. In view of the fact that the therapeutic agent or the preventive agent provided in the present invention can be suitably used in eukaryotes, particularly mammals, particularly humans, the expression of the gene
  • the host cell used in the above is preferably an animal cell.
  • step (A) when a stable cell line into which the nucleic acid construct has been introduced is used, step (A) may be omitted.
  • step (B) a gene encoding the protein of the present invention is expressed in the cells obtained in step (A) in the presence of the test substance.
  • the step can be performed, for example, by culturing the cells obtained in the step (A) using a medium containing a test substance.
  • the culture conditions are the same as the transformant culture conditions in the above protein production method.
  • step (C) the presence or absence of the expression of the gene is detected or the variation in the expression of the gene is measured as compared to the case where the test substance is absent.
  • a test substance that causes a change in the expression of the gene is selected as a prophylactic agent for bone disease or joint disease and a candidate compound for Z or therapeutic agent.
  • the "in the absence of the test substance” means that in the step (B), the cell obtained in the step (A) in the absence of the test substance is compared with the above.
  • the gene When the gene is expressed, it becomes a standard for grasping the change in the expression of the gene in the presence of the test substance. Specifically, a cell when exerted or an extract of the cell is used as a control.
  • the expression of the gene in the control is When observed, the expression of the gene is not detected in the cells contacted with the test substance or the expression level is decreased compared to the subject, i.e., the expression of the gene is decreased compared to the control. Then, it is determined that the test substance is a candidate compound for a prophylactic and Z or therapeutic agent for bone disease or joint disease, and the test substance is selected as a candidate compound.
  • the selected candidate compound is considered to have a characteristic property when the expression of the gene at the cellular level is suppressed at the transcriptional level, and specifically suppresses the expression of the gene. It exhibits the excellent effect of being able to. [0133] Detection of the presence or absence of gene expression, or measurement of fluctuations in expression thereof can be performed according to the molecular biological measurement method or immunological measurement method described above.
  • Examples of the compound obtained by the screening method of the present invention or a salt thereof include, for example, a compound that inhibits the function of the protein by binding to the protein of the present invention, such as a low molecular weight compound, a high molecular weight compound, and the like.
  • Examples include a molecular compound, an antibody against the protein of the present invention, a nucleic acid such as an antisense strand of a nucleic acid capable of encoding the protein of the present invention, a peptide, and the like.
  • the protein of the present invention used in the screening method of the present invention a nucleic acid construct having a gene encoding the protein of the present invention, a cell into which the gene has been introduced, the protein of the present invention
  • a kit for carrying out the screening method of the present invention is provided. Powerful screening kits are also included in the present invention.
  • the screening kit of the present invention includes:
  • a kit comprising the protein of the present invention
  • kits comprising a nucleic acid construct comprising a gene encoding the protein of the present invention
  • a kit comprising a cell into which a gene encoding the protein of the present invention has been introduced, an antibody against the protein of the present invention or a fragment thereof Kits, etc.
  • kits may contain the probe and Z or a primer pair that can be suitably used in the screening method of the present invention, if desired.
  • detection reagents may be included.
  • the compound or salt thereof obtained by the screening method of the present invention can exert a therapeutic or prophylactic effect on bone diseases or joint diseases in which the expression of the protein of the present invention is involved in the onset thereof. . Therefore, the compound obtained by the screening method or a salt thereof is used for treatment or prevention of bone disease or joint disease, for example, 0 A or RA.
  • a pharmaceutical composition is provided.
  • the pharmaceutical composition of the present invention is characterized in that it contains a compound or a salt thereof obtained by the screening method of the present invention as an active ingredient. Therefore, the pharmaceutical composition of the present invention exerts a therapeutic or prophylactic effect on the bone disease or joint disease through the action on the expression of the protein of the present invention (for example, it works to suppress the expression).
  • the content of the compound or the salt thereof in the pharmaceutical composition of the present invention can be adjusted as appropriate depending on the disease to be treated, the age of the patient, the body weight, etc., and may be a therapeutically effective amount.
  • molecular compounds or polymer compounds for example, 0.0001 to: LOOOmg, preferably 0.001 to 100 mg, and in the case of positive peptides or derivatives thereof, for example, 0.0001 to 10 OOmg, preferably ⁇ is 0. 001-100 mg, in the case of nucleic acids or derivatives thereof, for example, 0.001 01: L00 mg, preferably 0.0001-10 mg!
  • the pharmaceutical composition of the present invention may further contain various auxiliaries capable of stably holding the compound or a salt thereof.
  • auxiliaries capable of stably holding the compound or a salt thereof.
  • auxiliaries capable of stably holding the compound or a salt thereof.
  • auxiliaries capable of stably holding the compound or a salt thereof.
  • pharmacologically acceptable auxiliaries, excipients, binders, and stabilizers exhibiting the property of inhibiting the active ingredient from degrading before reaching the site where the active ingredient is to be delivered.
  • the dosage form of the pharmaceutical composition of the present invention is appropriately selected according to the type of active ingredient; individual, organ, local site, tissue to be administered; age, weight, etc. of the individual to be administered.
  • Examples of the administration form include subcutaneous injection, intramuscular injection, intravenous injection, and local administration.
  • the dosage of the pharmaceutical composition of the present invention is appropriately determined according to the type of active ingredient; the individual to be administered, the organ, the local site, the tissue; the age, weight, etc. of the individual to be administered. Selected.
  • the administration method is not particularly limited, but when the active ingredient is a low molecular weight compound or a high molecular weight compound, the amount of the active ingredient is, for example, 0.0001 to: LOOOmgZkg body weight, preferably 0.001 to 100 mgZkg.
  • polypeptide or derivative thereof for example, 0.0001 to 1000 mg Zkg body weight, preferably ⁇ 0.001 to 100 mg Zkg body weight, in the case of nucleic acid or derivative thereof, for example, 0.0001 to 100 mg Zkg body weight, preferably 0. 000 1 ⁇ : One dose or multiple doses per day, etc., so as to be a single dose of LOmgZkg body weight.
  • the administration period is not particularly limited.
  • the pharmacological evaluation of the pharmaceutical composition of the present invention is carried out, for example, by administering the pharmaceutical composition of the present invention to a bone disease or joint disease model mouse and comparing the bone disease or joint disease in the administered animal compared to the non-administered animal. This can be done by a method that evaluates the improvement as an index.
  • the present invention also provides a method for diagnosing a bone disease or a joint disease.
  • One feature of the diagnostic method of the present invention is that the expression level of the gene of the protein of the present invention in each of the test sample and the control sample derived from the individual to be tested is measured. Therefore, according to the diagnostic method of the present invention, it is possible to easily and quickly diagnose whether or not a test subject has a bone disease or a joint disease! ! /, An excellent effect is exhibited.
  • the individual to be tested when the expression level force in the test sample is different from the expression level in the control sample (for example, when it becomes higher or lower), the individual to be tested becomes a bone disease. Or it becomes an indicator of suspected to have joint disease.
  • MRNA was prepared from human mesenchymal stem cells (BIO WHITTAKER) using QuickPrep Micro mRNA Purification Kit (Amersham Biosciences) according to the attached manual. The obtained mRNA was converted to cDNA by Superscript Choice System for cDNA Synthesis (Invitrogen) according to the attached manual to obtain a cDNA clone.
  • the frozen synovial tissue is crushed using a cryopulverizer CP-1 00W (Microtech's-Thion Co., Ltd.) cooled with liquid nitrogen, and immediately homogenized in a TRIzol reagent (Invitrogen Corp.). Suspension was performed using Polytron 2100 (Kinematica), and total RNA was extracted according to the formulation of TRI zol reagent.
  • RNA described in Example 2 was recovered after DNase I treatment using RNeasy mini kit (Qiagen) according to the attached manual.
  • RNA amplified RNA
  • aRNA amplified RNA
  • Hybridization is a protocol of Type 7star slide (Amersham Neoscience). The pretreatment was followed for 12 hours at 48 ° C with the probe. Hybri dizaton Buffer Ver. 2 (Amersham Bio-Signs) was used as the buffer. After washing, reading was performed with a Microarray System Generation III Scanner (Molecular Dynamics). In accordance with this method, 78 slide experiments were conducted using 62 cases of knee OA, RA, and non-OA non-RA, including duplicate experiments.
  • 3DR is one of the check genes included in Lucidea Universal ScoreCard (trade name, Amersham Biosciences) described in Example 1. Also, in some cases, the force measured by multiple slides. Unless otherwise noted, the slide with the smallest slide number was used for analysis in cases with multiple slides. As described in Example 1, SET1 and SET2 data exist in one slide.
  • Condition A In either SET1 or SET2, there is a significant difference between OA and normal, and between OA and RA in either the average value comparison or the average absolute difference comparison.
  • Condition B For both SET1 and SET2, there is a significant difference between OA and normal or between OA and RA when comparing the mean values.
  • the significant difference here refers to the significance of the regression coefficient test corresponding to the comparison between two pairs of OA and normal and OA and RA.
  • Condition A is met.
  • Second selection criteria The first selection criteria are not met, but Condition B and Condition C are met.
  • the expression level in OA is + based on the expression level in RA patients and is one based on the expression level in normal individuals
  • the expression level of the gene is normal individuals> OA patients> This suggests that he is an RA patient.
  • the expression level in an OA patient is one based on the expression level in an RA patient and is + based on the expression level in a normal individual
  • the expression level of the gene is RA patient> OA Patient> normal, suggesting that it is an individual.
  • Example 4 For each of the 26 OA-related genes described in Example 4, the base sequence of the PCR fragment described in Example 1 was converted to the DNA sequencing device MegaBACElOOO (Amaci (Ambi Biosciences). Except for the two genes from which PCR fragments were not obtained, the base sequences obtained for the remaining 24 genes were subjected to homology search using the gene information analysis software bioSCOUT (LION). As a result, the gene names were found for all 24 genes, and 3 were duplicated with Fibulin-3. We searched PubMED (National Center for Biotechnology Information) and PATENTWeb (MicroPatent) for 22 genes excluding duplicates, and examined whether the relationship between each gene and OA is known, With the exception of five genes that have already been shown to be related, the remaining 17 genes are involved in OA. Table 3 shows the 17 genes.
  • Fibulin-3 SEQ ID NO: 13
  • the expression profile of Fibulin-3 gene in membrane tissue was examined and compared with the results of DNA chip. All samples used RNA used for DNA chip analysis. Sample number 1 is normal, 2-12 are synovial RNA from OA patients.
  • RNA prepared from clinical samples of human knee osteoarthritis synovium described in Example 2 was treated with DNase I using RNeasy mini kit (Qiagen) according to the attached manual, and then recovered.
  • RNA 2.5; z g, cDNA was synthesized using the Superscript First-strand Synthesis System for RT-PCR (Invitrogen) according to the attached manual.
  • probe search software Primer based on the sequence of human Fibulin-3 (SEQ ID NO: 13)
  • Primers for real-time quantitative PCR were selected by Express (Applied Biosystems). Primers were synthesized by Qiagen. Using this primer, real-time quantitative PCR with SYBR Green I was performed using the synthesized cDNA as a saddle type, and the amount of mRNA was quantified.
  • Example 4 the expression level is normal individuals> OA patients> RA patients, or It was suggested that rheumatoid arthritis can be diagnosed using the mRNA expression as an index for the seven genes that showed a relationship between RA patients> OA patients> normal individuals.
  • a sample derived from an individual suspected of suffering from a bone disease or joint disease, particularly osteoarthritis or rheumatoid arthritis can be detected or selected easily and rapidly. it can.
  • diagnostic agents necessary for detection or selection are provided.

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Abstract

It is intended to provide a method of conveniently and quickly detecting or screening a bone disease or a joint disease which comprises the step of measuring the expression level of at least one gene encoding a protein having an amino acid sequence selected from the group consisting of SEQ ID NOS:18 to 34 in a test sample originating in a subject individual, wherein a difference, if any, in the expression level between the test sample and a control sample originating in a normal individual serves as an indication showing a risk that the test sample might originate in an individual suffering from a bone disease or a joint disease.

Description

明 細 書  Specification
骨疾患又は関節疾患に関与するポリペプチド及びその DNA  Polypeptide and its DNA involved in bone disease or joint disease
技術分野  Technical field
[0001] 本発明は、骨疾患又は関節疾患の診断、治療、予防等のための手段に関する。よ り詳しくは、本発明は、骨疾患若しくは関節疾患に罹患している疑いがある個体由来 の試料の検出又は選別方法、又は診断薬、骨疾患若しくは関節疾患の治療剤又は 予防剤のスクリーニング方法、該スクリーニング方法に用いるキット、医薬組成物、骨 疾患又は関節疾患の診断方法に関する。  [0001] The present invention relates to a means for diagnosis, treatment, prevention and the like of bone disease or joint disease. More specifically, the present invention relates to a method for detecting or selecting a sample derived from an individual suspected of having a bone disease or joint disease, or a method for screening a diagnostic agent, a therapeutic agent or a preventive agent for bone disease or joint disease. Further, the present invention relates to a kit used for the screening method, a pharmaceutical composition, and a diagnostic method for bone disease or joint disease.
背景技術  Background art
[0002] 関節とは、骨相互間の可動結合部であり、この部分の骨は動きをスムーズにするた めに関節軟骨で覆われている。また、関節の中では滑膜から関節液が分泌されてお り、関節の運動を滑らかにしている。  A joint is a movable joint between bones, and the bone in this part is covered with articular cartilage in order to make the movement smooth. In the joints, synovial fluid is secreted from the synovium, smoothing the joint movement.
[0003] 変形性関節症 (OA)は、慢性の関節炎及び滑膜性関節の関節軟骨の退行変性を 伴う消耗性疾患である。 OAは、加齢と共にその発症率が増加することが知られてお り、高齢者にとって最も頻度の高い疾患の一つである。高齢ィ匕社会に直面している 現代において、大きな課題の一つとなっているが、その原因は明らかでなぐ有効な 治療法がない。長い間、 OAは不可避な加齢に起因するものとして考えられてきたが 、遺伝学的研究により OAの発症には遺伝的要因が大きく関わることが明らかになつ てきた (非特許文献 1参照)。 OAは多因子遺伝病であり、複数の遺伝的要因がその 発症、伸展に関与しており、これらの遺伝子と、運動、栄養等の環境因子の相互作 用により病気が成り立っていると考えられる。現在までの研究により、 OAに関連する 遺伝子として、多数の候補遺伝子 (特許文献 1及び 2参照)や SNPs (—塩基多型）が 挙げられている (非特許文献 2参照)。しかし、 OAの発症機構は完全には解明されて おらず、未知の遺伝子の関与しうる可能性が考えられている。  [0003] Osteoarthritis (OA) is a debilitating disease with chronic arthritis and degenerative degeneration of articular cartilage in synovial joints. OA is known to increase in incidence with age and is one of the most frequent diseases for the elderly. It is one of the major challenges in the present age facing an aging society, but the cause is not clear and there is no effective treatment. For a long time, OA has been considered to be caused by unavoidable aging, but genetic studies have revealed that genetic factors are largely involved in the development of OA (see Non-Patent Document 1). . OA is a multifactorial genetic disease, and multiple genetic factors are involved in its onset and spread, and the disease is thought to be due to the interaction of these genes with environmental factors such as exercise and nutrition. . Research to date has listed a large number of candidate genes (see Patent Documents 1 and 2) and SNPs (-nucleotide polymorphisms) as genes related to OA (see Non-Patent Document 2). However, the pathogenesis of OA has not been completely elucidated, and it is possible that unknown genes may be involved.
[0004] 慢性関節リウマチ (RA)は、中小関節を対称性におかす多発性関節炎を主症状と する原因不明の慢性炎症性疾患である。炎症性サイト力インとリウマトイド因子の局所 的産生、関節液中での IgGとリウマトイド因子よりなる免疫複合体の形成、好中球遊 走、免疫複合体貪食、リソソーム酵素放出、関節軟骨破壊、滑膜増殖、破骨細胞活 性化など一連の反応により炎症の慢性化と関節破壊が進行する。現在までの研究に より、 RAに関連する遺伝子として、多数の候補遺伝子 (特許文献 3及び 4参照）や SN Psが挙げられている (非特許文献 3参照)。しかし、 RAの発症機構は完全には解明さ れておらず、未知の遺伝子の関与しうる可能性が考えられて 、る。 [0004] Rheumatoid arthritis (RA) is an unexplained chronic inflammatory disease whose main symptom is polyarthritis with symmetrical small and medium joints. Inflammatory site force-in and local production of rheumatoid factor, formation of immune complex consisting of IgG and rheumatoid factor in synovial fluid, neutrophil migration Chronic inflammation and joint destruction progress through a series of reactions such as running, immune complex phagocytosis, lysosomal enzyme release, articular cartilage destruction, synovial proliferation, and osteoclast activation. Studies to date have listed a large number of candidate genes (see Patent Documents 3 and 4) and SNPs (see Non-Patent Document 3) as genes related to RA. However, the pathogenesis of RA has not been fully elucidated, and it is possible that unknown genes may be involved.
[0005] また、多関節炎 (症)を呈する原因疾患は多数あり、その中では関節リウマチ (RA) と OAの頻度が高い。 OAと RAは共に関節が痛くなるという点で共通しており、識別 の必要がある。現在、 OA又は RAの診断の際には、炎症の程度を知るための検査や X線検査等が行われる。ただし、早期では診断困難な場合があり、臨床検査と経過観 察が必要となる（非特許文献 4参照)。そのため、 OAと RAを容易に区別が出来る診 断方法、かつ、より早期に疾患が発見できるよう簡便な診断法の開発が求められてい る。 [0005] In addition, there are many causative diseases that exhibit polyarthritis (symptoms), and among them, rheumatoid arthritis (RA) and OA are frequent. Both OA and RA are common in that joints become painful and need to be identified. Currently, in order to diagnose OA or RA, examinations to determine the degree of inflammation, X-ray examinations, etc. are performed. However, early diagnosis may be difficult, and clinical examination and follow-up are required (see Non-Patent Document 4). Therefore, there is a need for the development of diagnostic methods that can easily distinguish OA and RA, and simple diagnostic methods that can detect diseases earlier.
[0006] 上記の様な状況の下、 OA又は RAの発症に関わる遺伝子の同定及び同遺伝子を 用いた疾患の診断方法が待望されて!ヽた。  [0006] Under the circumstances as described above, identification of a gene involved in the onset of OA or RA and a method for diagnosing a disease using the gene have been expected.
[0007] N1032 (配列番号： 1)は、ヒトの乳癌の癌抑制遺伝子候補 1 (bcsc— 1) (配列番号 [0007] N1032 (SEQ ID NO: 1) is a tumor suppressor gene candidate 1 (bcsc— 1) for human breast cancer (SEQ ID NO: 1).
: 18)である。しかしながら、 N1032と OAとの関連についての報告はない。  : 18). However, there is no report on the relationship between N1032 and OA.
[0008] N1108 (配列番号： 2)は、タンパク質フォスファターゼ 2A B' a 1調節サブユニット（ 配列番号： 19)をコードするヒト遺伝子である（非特許文献 5参照)。しかしながら、 N1N1108 (SEQ ID NO: 2) is a human gene encoding a protein phosphatase 2A B ′ a 1 regulatory subunit (SEQ ID NO: 19) (see Non-Patent Document 5). However, N1
108と OAとの関連につ!、ての報告はな!/、。 The relationship between 108 and OA!
[0009] N1112 (配列番号： 3)は、げっ歯動物の遺伝子 Kidlと相同性を持ち、主として腎 臓で発現する、新規ヒト亜鉛フィンガー型の転写因子 (配列番号： 20)をコードするヒ ト遺伝子である（非特許文献 6参照)。し力しながら、 N1112と OAとの関連について の報告はない。 [0009] N1112 (SEQ ID NO: 3) is a human that encodes a novel human zinc finger transcription factor (SEQ ID NO: 20) that is homologous to the rodent gene Kidl and is mainly expressed in the kidney. It is a gene (see Non-Patent Document 6). However, there is no report on the relationship between N1112 and OA.
[0010] N1123 (配列番号: 4)は、 X染色体にコードされたホスホダリセリン酸キナーゼ (配 列番号: 21)をコードするヒト遺伝子である（非特許文献 7参照)。しかしながら、 Ni l 23と OAとの関連につ!、ての報告はな!/、。  [0010] N1123 (SEQ ID NO: 4) is a human gene that encodes phosphodariserate kinase (SEQ ID NO: 21) encoded by the X chromosome (see Non-Patent Document 7). However, there is no report on the relationship between Ni 23 and OA!
[0011] N0154 (配列番号： 5)は、 L-イジトール- 2デヒドロゲナーゼ（配列番号： 22)をコー ドするヒト遺伝子である（非特許文献 8参照)。し力しながら、 N0154と OAとの関連に ついての報告はない。 N0154 (SEQ ID NO: 5) is a human gene that codes for L-iditol-2 dehydrogenase (SEQ ID NO: 22) (see Non-Patent Document 8). In connection with N0154 and OA There is no report about it.
[0012] N0281 (配列番号: 6)は、ヒトの遺伝性多発性外骨症の原因遺伝子 EXT1 (配列番 号： 23)である（非特許文献 9参照）。し力しながら、 N0281と OAとの関連について の報告はない。  [0012] N0281 (SEQ ID NO: 6) is the gene EXT1 (SEQ ID NO: 23) that causes human hereditary multiple exostosis (see Non-Patent Document 9). However, there is no report on the relationship between N0281 and OA.
[0013] N0439 (配列番号： 7)は、へパラン-サルフェート- 6-スルホトランスフェラーゼ（配 列番号： 24)をコードするヒト遺伝子である（非特許文献 10参照)。しかしながら、 NO [0013] N0439 (SEQ ID NO: 7) is a human gene encoding heparan-sulfate-6-sulfotransferase (SEQ ID NO: 24) (see Non-Patent Document 10). However, NO
439と OAとの関連につ!、ての報告はな!/、。 The relationship between 439 and OA!
[0014] N0446 (配列番号： 8)は、ヒスチジル -tRNA合成酵素（配列番号： 25)と相同性の あるヒト遺伝子である（非特許文献 11参照)。し力しながら、 N0446と OAとの関連に ついての報告はない。 [0014] N0446 (SEQ ID NO: 8) is a human gene having homology to histidyl-tRNA synthetase (SEQ ID NO: 25) (see Non-patent Document 11). However, there is no report on the relationship between N0446 and OA.
[0015] N0513 (配列番号： 9)は、チューブリン-フォールディング コファクター C (配列番 号： 26)をコードするヒト遺伝子である（非特許文献 12参照)。しかしながら、 N0513と [0015] N0513 (SEQ ID NO: 9) is a human gene encoding tubulin-folding cofactor C (SEQ ID NO: 26) (see Non-Patent Document 12). However, with N0513
OAとの関連につ 、ての報告はな 、。 No report on the relationship with OA.
[0016] N0553 (配列番号： 10)は、 N，端に 2つの LIMモチーフを持つセリン Zスレオニン キナーゼ (配列番号 : 27)をコードするヒト遺伝子である（非特許文献 13参照)。し力 ながら、 N0553と OAとの関連につ!、ての報告はな!/、。 N0553 (SEQ ID NO: 10) is a human gene encoding N, serine Z threonine kinase (SEQ ID NO: 27) having two LIM motifs at the ends (see Non-patent Document 13). However, there is no report on the relationship between N0553 and OA!
[0017] N0065 (配列番号： 11)は、膜タンパク質である LAK- 4 (配列番号： 28)をコードす るヒト遺伝子である。しかしながら、 N0065と OAとの関連についての報告はない。 [0017] N0065 (SEQ ID NO: 11) is a human gene encoding LAK-4 (SEQ ID NO: 28), which is a membrane protein. However, there is no report on the relationship between N0065 and OA.
[0018] NO 160 (配列番号： 12)は、血管作用性小腸ペプチドレセプター（配列番号： 29) をコードするヒト遺伝子である（非特許文献 14参照)。し力しながら、 N0160と OAと の関連につ!、ての報告はな 、。 [0018] NO 160 (SEQ ID NO: 12) is a human gene encoding a vasoactive intestinal peptide receptor (SEQ ID NO: 29) (see Non-Patent Document 14). However, there has been no report on the relationship between N0160 and OA!
[0019] E0215 (配列番号： 13)は、 EGF含有フイブリン様細胞外マトリックスタンパク質 1 ([0019] E0215 (SEQ ID NO: 13) is an EGF-containing fibrin-like extracellular matrix protein 1 (
EFEMP1、 Fibulin— 3) (配列番号： 30)をコードするヒト遺伝子である（非特許文献It is a human gene encoding EFEMP1, Fibulin-3) (SEQ ID NO: 30) (Non-patent literature)
15参照）。しかしながら、 E0215と OAとの関連についての報告はない。 15). However, there is no report on the relationship between E0215 and OA.
[0020] E0739 (配列番号： 14)は、 3つの K-相同ドメインを持つ細胞の poly(rC)_結合タン ノ ク質 (配列番号 : 31)をコードするヒト遺伝子である (非特許文献 16参照)。しかしな がら、 E0739と OAとの関連につ!、ての報告はな!/、。 [0020] E0739 (SEQ ID NO: 14) is a human gene encoding a poly (rC) _binding protein (SEQ ID NO: 31) of a cell having three K-homology domains (Non-patent Document 16). reference). However, there was no report on the relationship between E0739 and OA!
[0021] C0052 (配列番号： 15)は、 AP-エンドヌクレアーゼ（配列番号： 32)をコードするヒト 遺伝子である（非特許文献 17参照)。しかしながら、 C0052と OAとの関連について の報告はない。 [0021] C0052 (SEQ ID NO: 15) is a human encoding AP-endonuclease (SEQ ID NO: 32) It is a gene (see Non-Patent Document 17). However, there is no report on the relationship between C0052 and OA.
[0022] D0032 (配列番号： 16)は、マクロファージ炎症性タンパク質 - 2 α (配列番号： 33) をコードするヒト遺伝子である（非特許文献 18参照)。し力しながら、 D0032と OAと の関連につ!、ての報告はな 、。  [0022] D0032 (SEQ ID NO: 16) is a human gene encoding macrophage inflammatory protein-2α (SEQ ID NO: 33) (see Non-Patent Document 18). However, there is no report about the relationship between D0032 and OA!
[0023] E0083 (配列番号： 17)は、グルタチオン S-トランスフェラーゼ（配列番号： 34)をコ ードするヒト遺伝子である（非特許文献 19参照)。しカゝしながら、 E0083と OAとの関 連についての報告はない。 [0023] E0083 (SEQ ID NO: 17) is a human gene encoding glutathione S-transferase (SEQ ID NO: 34) (see Non-Patent Document 19). However, there is no report on the relationship between E0083 and OA.
特許文献 1 :特開 2003— 183177号公報  Patent Document 1: Japanese Patent Laid-Open No. 2003-183177
特許文献 2：特開 2004 - 75675号公報  Patent Document 2: Japanese Patent Laid-Open No. 2004-75675
特許文献 3：特開 2002— 51782号公報  Patent Document 3: Japanese Patent Application Laid-Open No. 2002-51782
特許文献 4：特開 2003 - 204790号公報  Patent Document 4: Japanese Patent Laid-Open No. 2003-204790
非特許文献 1 :スベクター（Spector, T. D.)ら、ブリティッシュ メディカル ジャーナル (BMJ)、 312卷、 940— 943頁（1996)  Non-Patent Document 1: Spector, T. D. et al., British Medical Journal (BMJ), 312 卷, 940–943 (1996)
非特許文献 2 :池川（Ikegawa, S.)ら、ジャーナル ォブ ボーン アンド ミネラル リ サーチ（扉 R)、 17卷、 1290— 1296頁（2002)  Non-Patent Document 2: Ikegawa, S. et al., Journal of Bone and Mineral Research (door R), 17 卷, 1290–1296 (2002)
非特許文献 3：山本（Yamamoto, K.)ら、ネイチヤー ジェネティックス (Nat. Genet.)、 35卷、 341— 348頁（2003)  Non-Patent Document 3: Yamamoto, K. et al., Nat. Genet., 35 卷, 341-348 (2003)
非特許文献 4：吉田 浩著「RA又は他の炎症性多発性関節症 (DRG/PPS対応臨床 検査のガイドライン)」日本臨床検査医学会、平成 14年 9月、 P.85— 88  Non-Patent Document 4: Hiroshi Yoshida “RA or other inflammatory polyarthritis (DRG / PPS guidelines for clinical testing)” Japan Society for Clinical Laboratory Medicine, September 2002, P.85—88
非特許文献 5 :カミバヤシ（Kam¾ayashi, C.)ら、ザ ジャーナル ォブ バイオロジカ ル ケミストリー（J. Biol. Chem.)、 271卷、 5164— 5170頁（1996)  Non-Patent Document 5: Kam¾ayashi, C. et al., The Journal of Biological Chemistry (J. Biol. Chem.), 271, 5164-5170 (1996)
非特許文献 6 :ナカムラ（Nakamura, Y.)ら、サイトジェネティックス アンド セル ジェ ネテイツタス（Cytogenet. Cell Genet)、 78卷、 285— 288頁（1997)  Non-Patent Document 6: Nakamura, Y. et al., Cytogenet. Cell Genet, 78 卷, 285-288 (1997)
非特許文献 7 :ォーキン（Orkin, S.H.)ら、プロシーディングス ォブ ザ ナショナル アカデミー ォブ サイエンス（Proc. Natl. Acad. Sci. U.S.A.)、 80卷、 472— 476頁（ Non-Patent Document 7: Orkin, S.H. et al., Proceedings of the National Academy of Science (Proc. Natl. Acad. Sci. U.S.A.), 80 卷, pp. 472-476 (
1983) (1983)
非特許文献 8 :カーパー（Carper, D.)ら、ゲノミックス（Genomics)、 26卷、 55— 62頁（ 非特許文献 9：ウエノレズ (Wells, D.E.)ら、ネイチヤー ジェネティックス（Nat. Genet.)、 11卷、 137— 143頁（1995) Non-Patent Document 8: Carper, D. et al., Genomics, 26 卷, 55-62 ( Non-Patent Document 9: Wells, DE et al., Nat. Genet., 11 卷, 137-143 (1995)
非特許文献 10 :キマタ（Kimata, K.)ら、ザ ジャーナル ォブ バイオロジカル ケミス 卜リー（J. Biol. Chem.)、 273卷、 9208— 9213頁（1998) Non-Patent Document 10: Kimata, K. et al., The Journal of Biological Chemistry, J. Biol. Chem., 273, 9208–9213 (1998)
非特許文献 11 :ミラー（Miller, F.W.)ら、バイオケミカル アンド バイオフィジカル リ サーチ コミュニケーションズ（Biochem. Biophys. Res. Commun.)、 210卷、 556— 5 66頁（1995) Non-Patent Document 11: Miller, F.W. et al., Biochemical and Biophysical Research Communications (Biochem. Biophys. Res. Commun.), 210 卷, 556-5 pp. 66 (1995)
非特許文献 12 :コーワン（Cowan, N.J.)ら、セル（Cell)、 86卷、 287— 296頁（1996) 非特許文献 13 :ミズノ（Mizuno, K.)ら、ザ ジャーナル ォブ バイオロジカル ケミス 卜リー（J. Biol. Chem.)、 270卷、 31321— 31330頁（1995) Non-Patent Document 12: Cowan, NJ et al., Cell, 86, 287-296 (1996) Non-Patent Document 13: Mizuno, K., et al., The Journal of Biological Chemistry 卜Lee (J. Biol. Chem.), 270 卷, 31321–31330 (1995)
非特許文献 14 :ゴッツェル（Goetzl, E.J.)ら、バイオケミカル アンド バイオフィジカル リサーチ コミュニケーションズ（Biochem. Biophys. Res. Commun.)、 193卷、 546 — 553頁（1993) Non-Patent Document 14: Goetzl, E.J. et al., Biochemical and Biophysical Research Communications (Biochem. Biophys. Res. Commun.), 193 卷, 546 — 553 (1993)
非特許文献 15 :ゴールドスティン（Goldstein, S.)ら、モレキュラー アンド セルラー ノィォロジー（Mol. Cell. Biol.) , 15卷、 120— 128頁（1995) Non-Patent Document 15: Goldstein, S. et al., Molecular and Cellular Neurology (Mol. Cell. Biol.), 15 卷, 120-128 (1995)
非特許文献 16 :セリス（Celis, J.E.)ら、ョ一口ビアン ジャーナル ォブ バイオケミスト リー（Eur. J. Biochem.)、 230卷、 447— 453頁（1995) Non-Patent Document 16: Celis, J.E. et al., Eguchi. J. Biochem., 230 卷, 447-453 (1995)
非特許文献 17 :ヒクソン（Hickson, I.D.)ら、ヌクレイック ァシッズ リサーチ（Nucleic A cids Res.) , 19卷、 5519— 5523頁（1991) Non-Patent Document 17: Hickson, I.D. et al., Nucleic Acids Res., 19 卷, 5519-5523 (1991)
非特許文献 18 :セラミ（Cerami, Α.)ら、ザ ジャーナル ォブ ェクスペリメンタル メ ディスン (J. Exp. Med.) , 172卷、 911— 919頁（1990) Non-Patent Document 18: Cerami, Α. Et al., The Journal of Medical Medicine (J. Exp. Med.), 172 頁, 911– 919 (1990)
非特許文献 19：ティラー（Taylor, J.B.)ら、バイオケミカル ジャーナル (Biochem. J.) 、 300卷、 271— 276頁（1994) Non-Patent Document 19: Taylor, J.B. et al., Biochemical Journal (Biochem. J.), 300 卷, 271–276 (1994)
発明の開示 Disclosure of the invention
発明が解決しょうとする課題 Problems to be solved by the invention
本発明は、配列番号： 18〜34からなる群より選ばれるアミノ酸配列を有するタンパ ク質又は該アミノ酸配列において 1若しくは数個のアミノ酸が欠失、置換若しくは付加 されたアミノ酸配列を有するタンパク質の遺伝子の発現レベルを測定することによる、 骨疾患若しくは関節疾患に罹患している疑いがある個体由来の試料の簡便かつ迅 速な検出又は選別方法、骨疾患又は関節疾患の簡便かつ迅速な診断方法、該方 法を効率的に行ないうる診断薬を提供することを目的とする。また、代表的な関節疾 患であり、似た症状を示す RAと OAの識別が可能な診断薬を提供することを目的と する。 The present invention relates to a protein having an amino acid sequence selected from the group consisting of SEQ ID NOs: 18 to 34, or one or several amino acids in the amino acid sequence deleted, substituted or added A method for simple and quick detection or selection of a sample derived from an individual suspected of having bone disease or joint disease, bone disease or joint, by measuring the expression level of a gene of a protein having a defined amino acid sequence It is an object of the present invention to provide a simple and rapid diagnosis method of a disease and a diagnostic agent capable of efficiently performing the method. Another object of the present invention is to provide a diagnostic agent that is a typical joint disease and can distinguish between RA and OA showing similar symptoms.
[0025] また、本発明は、配列番号： 18〜34からなる群より選ばれるアミノ酸配列を有するタ ンパク質又は該アミノ酸配列において 1若しくは数個のアミノ酸が欠失、置換若しくは 付加されたアミノ酸配列を有するタンパク質の遺伝子の動態を検出又は測定すること による、骨疾患若しくは関節疾患の治療剤又は予防剤の効率的なスクリーニング方 法、前記スクリーニング方法を簡便かつ迅速に行ないうるスクリーニング用キットを提 供することを目的とする。  [0025] The present invention also provides a protein having an amino acid sequence selected from the group consisting of SEQ ID NOs: 18 to 34, or an amino acid sequence in which one or several amino acids have been deleted, substituted or added in the amino acid sequence. The present invention provides an efficient screening method for a therapeutic or prophylactic agent for bone disease or joint disease by detecting or measuring the dynamics of a gene having a protein having a protein, and a screening kit capable of performing the screening method simply and rapidly. For the purpose.
[0026] さらに、本発明は、前記スクリーニング方法により得られる、骨疾患若しくは関節疾 患の治療剤又は予防剤として好適に使用される化合物又はその塩、骨疾患若しくは 関節疾患の治療又は予防に好適な前記化合物又はその塩を有効成分とする医薬組 成物を提供することを目的とする。  [0026] Further, the present invention is a compound or salt thereof suitably used as a therapeutic or prophylactic agent for bone disease or joint disease obtained by the screening method, and suitable for the treatment or prevention of bone disease or joint disease. Another object of the present invention is to provide a pharmaceutical composition comprising the above compound or a salt thereof as an active ingredient.
課題を解決するための手段  Means for solving the problem
[0027] 本発明者らは、 OA患者又は RA患者の同意の下に、手術時に摘出された滑膜を 採取し、解析を行った。鋭意研究の結果、非 OA患者と比較し OA患者の滑膜組織 において発現に変化が見られる遺伝子を見出した。また、非 RA患者と比較し RA患 者の滑膜組織において発現に変化が見られる遺伝子を見出した。 OA膝組織におい てはメカ-カル 'ストレスによって破壊された軟骨組織のデブリスによる刺激を受けた 増生滑膜から様々な炎症性物質が分泌されると考えられている。従って、そのような 刺激を受けた滑膜においては、炎症性物質や関節組織破壊のマーカーとなりうる物 質をコードする遺伝子の発現が増強されると考えられた。また、滑膜組織には、脂肪 細胞様細胞、骨芽細胞、軟骨細胞等の間葉系細胞へ分化する能力を有する未分ィ匕 な線維芽滑膜細胞が存在する。そこで、更に研究を進めた結果、該遺伝子が、滑膜 組織の異常が一因となる骨疾患又は関節疾患の新規な診断薬として使用可能であ ることを見出し、本発明を完成させた。 [0027] The present inventors collected and analyzed the synovium excised at the time of surgery with the consent of OA patients or RA patients. As a result of intensive research, we found a gene whose expression was changed in the synovial tissue of OA patients compared to non-OA patients. In addition, we found genes whose expression was altered in the synovial tissue of RA patients compared to non-RA patients. In OA knee tissue, various inflammatory substances are thought to be secreted from the proliferative synovium stimulated by debris of cartilage tissue destroyed by mechanical-stress. Therefore, it was considered that in the synovium subjected to such stimulation, expression of genes encoding inflammatory substances and substances that can serve as markers for joint tissue destruction was enhanced. In synovial tissue, undistinct fibroblast synovial cells having the ability to differentiate into mesenchymal cells such as adipocyte-like cells, osteoblasts, and chondrocytes are present. As a result of further research, the gene can be used as a novel diagnostic agent for bone diseases or joint diseases caused by abnormal synovial tissue abnormalities. The present invention has been completed.
すなわち、本発明は、  That is, the present invention
(1)被検対象の個体由来の被検試料における配列番号： 18〜34からなる群より選 ばれるアミノ酸配列を有するタンパク質又は該アミノ酸配列において 1若しくは数個の アミノ酸が欠失、置換若しくは付加されたアミノ酸配列を有するタンパク質をコードす る少なくとも 1つの遺伝子の発現レベルを測定するステップを含み、ここで、被検試料 における発現レベルが、正常個体由来の対照試料における発現レベルと異なる場合 、該被検試料が、骨疾患又は関節疾患に罹患している疑いがある個体由来の試料 であることの指標となる、骨疾患若しくは関節疾患に罹患している疑いがある個体由 来の試料の検出又は選別方法、  (1) A protein having an amino acid sequence selected from the group consisting of SEQ ID NOs: 18 to 34 in a test sample derived from an individual to be tested, or one or several amino acids are deleted, substituted or added in the amino acid sequence Measuring the expression level of at least one gene encoding a protein having a specific amino acid sequence, wherein the expression level in the test sample is different from the expression level in a control sample derived from a normal individual. Detection of a sample derived from an individual suspected of having a bone disease or joint disease, which is an indicator that the test sample is a sample derived from an individual suspected of having a bone disease or joint disease, or Sorting method,
(2)骨疾患又は関節疾患が、軟骨形成異常、骨形成異常、骨粗鬆症、変形性関節 症、慢性関節リウマチ、関節炎、滑膜炎、代謝性関節症又はスポーツによる関節障 害である（1)記載の方法、  (2) The bone disease or joint disease is cartilage dysplasia, bone dysplasia, osteoporosis, osteoarthritis, rheumatoid arthritis, arthritis, synovitis, metabolic arthropathy or arthropathy due to sports (1) Described method,
(3)被検対象の個体由来の被検試料における配列番号： 18、 21〜23、 25、 29から なる群より選ばれるアミノ酸配列を有するタンパク質又は該アミノ酸配列において 1若 しくは数個のアミノ酸が欠失、置換若しくは付加されたアミノ酸配列を有するタンパク 質をコードする少なくとも 1つの遺伝子の発現レベルを測定するステップを含み、ここ で、被検試料における発現レベル力 正常個体由来の対照試料における発現レべ ルよりも高くなり、かつ、慢性関節リウマチ患者由来の対照試料における発現レベル よりも低くなる場合、該被検試料が、変形性関節症に罹患している疑いがある個体由 来の試料であることの指標となる、変形性関節症に罹患して 、る疑 、がある個体由 来の試料の検出又は選別方法、  (3) A protein having an amino acid sequence selected from the group consisting of SEQ ID NO: 18, 21, 23, 25, 29 in a test sample derived from an individual to be tested or one or several amino acids in the amino acid sequence Measuring the expression level of at least one gene encoding a protein having a deleted, substituted or added amino acid sequence, wherein the expression level in the test sample is expressed in a control sample from a normal individual A sample derived from an individual suspected of suffering from osteoarthritis if it is higher than the level and lower than the expression level in a control sample from a patient with rheumatoid arthritis A method for detecting or selecting a sample derived from an individual who is suspected of suffering from osteoarthritis,
(4)被検対象の個体由来の被検試料における配列番号： 19、 20、 24、 26〜28、 30 〜33からなる群より選ばれるアミノ酸配列を有するタンパク質又は該アミノ酸配列に おいて 1若しくは数個のアミノ酸が欠失、置換若しくは付加されたアミノ酸配列を有す るタンパク質をコードする少なくとも 1つの遺伝子の発現レベルを測定するステップを 含み、ここで、被検試料における発現レベル力 正常個体由来の対照試料における 発現レベルよりも高くなり、かつ、慢性関節リウマチ患者個体由来の対照試料におけ る発現レベルよりも高くなる場合、該被検試料が、変形性関節症に罹患している疑い がある個体由来の試料であることの指標となる、変形性関節症に罹患している疑いが ある個体由来の試料の検出又は選別方法、 (4) a protein having an amino acid sequence selected from the group consisting of SEQ ID NOs: 19, 20, 24, 26-28, 30-33 in a test sample derived from an individual to be tested, or 1 or Measuring the expression level of at least one gene encoding a protein having an amino acid sequence in which several amino acids have been deleted, substituted or added, wherein the expression level in the test sample is derived from a normal individual In control samples from individuals with rheumatoid arthritis and higher than the expression level in The test sample is suspected of suffering from osteoarthritis, which is an indicator that the test sample is derived from an individual suspected of suffering from osteoarthritis. A method for detecting or sorting a sample from an individual,
(5)被検対象の個体由来の被検試料における配列番号： 34のアミノ酸配列を有する タンパク質又は該アミノ酸配列において 1若しくは数個のアミノ酸が欠失、置換若しく は付加されたアミノ酸配列を有するタンパク質をコードする遺伝子の発現レベルを測 定するステップを含み、ここで、被検試料における発現レベル力 正常個体由来の対 照試料における発現レベルよりも低くなり、かつ、慢性関節リウマチ患者個体由来の 対照試料における発現レベルよりも高くなる場合、該被検試料が、変形性関節症に 罹患している疑いがある個体由来の試料であることの指標となる、変形性関節症に 罹患して!/、る疑 、がある個体由来の試料の検出又は選別方法、  (5) A protein having the amino acid sequence of SEQ ID NO: 34 in a test sample derived from an individual to be tested, or having an amino acid sequence in which one or several amino acids are deleted, substituted or added in the amino acid sequence Measuring the expression level of the gene encoding the protein, wherein the expression level in the test sample is lower than the expression level in a control sample from a normal individual and from an individual with rheumatoid arthritis If it is higher than the expression level in the control sample, the test sample suffers from osteoarthritis, which is an indicator that the sample is from an individual suspected of suffering from osteoarthritis! /, A method for detecting or selecting a sample derived from an individual,
(6)被検対象の個体由来の被検試料における配列番号： 18、 21〜23、 25、 29から なる群より選ばれるアミノ酸配列を有するタンパク質又は該アミノ酸配列において 1若 しくは数個のアミノ酸が欠失、置換若しくは付加されたアミノ酸配列を有するタンパク 質をコードする少なくとも 1つの遺伝子の発現レベルを測定するステップを含み、ここ で、被検試料における発現レベル力 正常個体由来の対照試料における発現レべ ルよりも高くなり、かつ、変形性関節症患者由来の対照試料における発現レベルより も高くなる場合、該被検試料が、慢性関節リウマチに罹患している疑いがある個体由 来の試料であることの指標となる、慢性関節リウマチに罹患している疑いがある個体 由来の試料の検出又は選別方法、  (6) a protein having an amino acid sequence selected from the group consisting of SEQ ID NO: 18, 21, 23, 25, 29 in a test sample derived from an individual to be tested, or one or several amino acids in the amino acid sequence Measuring the expression level of at least one gene encoding a protein having a deleted, substituted or added amino acid sequence, wherein the expression level in the test sample is expressed in a control sample from a normal individual A sample derived from an individual suspected of suffering from rheumatoid arthritis if it is higher than the level and higher than the expression level in a control sample from a patient with osteoarthritis A method for detecting or selecting a sample derived from an individual suspected of having rheumatoid arthritis,
(7)被検対象の個体由来の被検試料における配列番号： 34のアミノ酸配列を有する タンパク質又は該アミノ酸配列において 1若しくは数個のアミノ酸が欠失、置換若しく は付加されたアミノ酸配列を有するタンパク質をコードする遺伝子の発現レベルを測 定するステップを含み、ここで、被検試料における発現レベル力 正常個体由来の対 照試料における発現レベルよりも低くなり、かつ、変形性関節症患者個体由来の対 照試料における発現レベルよりも低くなる場合、該被検試料が、慢性関節リウマチに 罹患している疑いがある個体由来の試料であることの指標となる、慢性関節リウマチ に罹患している疑いがある個体由来の試料の検出又は選別方法、 (8)発現レベル力 配列番号： 18〜34からなる群より選ばれるアミノ酸配列を有する タンパク質又は該アミノ酸配列において 1若しくは数個のアミノ酸が欠失、置換若しく は付加されたアミノ酸配列を有するタンパク質をコードする少なくとも 1つの遺伝子の mRNA量を指標として測定される、 (1)〜（7) Vヽずれかに記載の方法、 (7) A protein having the amino acid sequence of SEQ ID NO: 34 in a test sample derived from an individual to be tested, or an amino acid sequence in which one or several amino acids have been deleted, substituted or added in the amino acid sequence A step of measuring the expression level of a gene encoding a protein, wherein the expression level in the test sample is lower than the expression level in a control sample derived from a normal individual and derived from an individual with osteoarthritis If the expression level is lower than the expression level in the control sample, the test sample is suffering from rheumatoid arthritis, which is an indicator that the sample is derived from an individual suspected of suffering from rheumatoid arthritis A method for detecting or sorting a sample from a suspect individual, (8) Expression level ability SEQ ID NO: 18-34 or a protein having an amino acid sequence selected from the group consisting of 18 to 34, or a protein having an amino acid sequence in which one or several amino acids are deleted, substituted or added in the amino acid sequence The method according to any one of (1) to (7) V, wherein the amount of mRNA of at least one gene encoding
(9)発現レベル力 配列番号： 18〜34からなる群より選ばれるアミノ酸配列を有する タンパク質又は該アミノ酸配列において 1若しくは数個のアミノ酸が欠失、置換若しく は付加されたアミノ酸配列を有する少なくとも 1つのタンパク質又はその部分ペプチド 又はその塩を指標として測定される、 (1)〜（7) V、ずれかに記載の方法、  (9) Expression level ability SEQ ID NO :: A protein having an amino acid sequence selected from the group consisting of 18 to 34 or at least an amino acid sequence in which one or several amino acids have been deleted, substituted or added in the amino acid sequence Measured using one protein or a partial peptide thereof or a salt thereof as an index, (1) to (7) V, the method according to any one of
(10)配列番号： 18〜34からなる群より選ばれるアミノ酸配列を有するタンパク質又 は該アミノ酸配列において 1若しくは数個のアミノ酸が欠失、置換若しくは付加された アミノ酸配列を有するタンパク質をコードする少なくとも 1つの遺伝子を検出しうる核 酸を用いる（8)記載の方法、  (10) at least encoding a protein having an amino acid sequence selected from the group consisting of SEQ ID NOs: 18 to 34, or a protein having an amino acid sequence in which one or several amino acids are deleted, substituted or added in the amino acid sequence The method according to (8), wherein a nuclear acid capable of detecting one gene is used,
(11)該遺伝子が、配列番号： 1〜17からなる群より選ばれる塩基配列からなる核酸 である（10)記載の方法、  (11) The method according to (10), wherein the gene is a nucleic acid comprising a base sequence selected from the group consisting of SEQ ID NOs: 1 to 17,
( 12)該遺伝子が、配列番号： 6及び Z又は 13の塩基配列からなる核酸である（ 10) 記載の方法、  (12) The method according to (10), wherein the gene is a nucleic acid comprising the nucleotide sequence of SEQ ID NO: 6 and Z or 13.
(13)配列番号： 35及び Z又は 36の塩基配列力もなる核酸を用 、るものである（ 12) 記載の方法、  (13) The method according to (12), wherein a nucleic acid having the nucleotide sequence of SEQ ID NO: 35 and Z or 36 is used.
(14)配列番号： 18〜34からなる群より選ばれるアミノ酸配列を有するタンパク質又 は該アミノ酸配列において 1若しくは数個のアミノ酸が欠失、置換若しくは付加された アミノ酸配列を有する少なくとも 1つのタンパク質又はその部分ペプチド又はその塩 に対する抗体又はその断片を用いる（9)記載の方法、  (14) a protein having an amino acid sequence selected from the group consisting of SEQ ID NOs: 18 to 34, or at least one protein having an amino acid sequence in which one or several amino acids have been deleted, substituted or added in the amino acid sequence, or The method according to (9), wherein an antibody against the partial peptide or a salt thereof or a fragment thereof is used.
(15)配列番号： 23及び Z又は 30のアミノ酸配列を有するタンパク質又は該アミノ酸 配列において 1若しくは数個のアミノ酸が欠失、置換若しくは付加されたアミノ酸配列 を有する少なくとも 1つのタンパク質又はその部分ペプチド又はその塩に対する抗体 又はその断片を用いる（9)記載の方法、  (15) SEQ ID NO: at least one protein having an amino acid sequence of 23 and Z or 30, or at least one protein having an amino acid sequence in which one or several amino acids are deleted, substituted or added in the amino acid sequence, or a partial peptide thereof The method according to (9), wherein an antibody against the salt or a fragment thereof is used,
(16)配列番号： 18〜34からなる群より選ばれるアミノ酸配列を有するタンパク質又 は該アミノ酸配列において 1若しくは数個のアミノ酸が欠失、置換若しくは付加された アミノ酸配列を有するタンパク質をコードする少なくとも 1つの遺伝子を検出しうる核 酸を含有してなる、（ 10)〜（ 15) 、ずれかに記載の方法に使用するための診断薬、(16) SEQ ID NO: A protein having an amino acid sequence selected from the group consisting of 18 to 34, or one or several amino acids have been deleted, substituted or added in the amino acid sequence A diagnostic agent for use in the method according to any one of (10) to (15), comprising a nuclear acid capable of detecting at least one gene encoding a protein having an amino acid sequence,
(17)該遺伝子が、配列番号： 1〜17からなる群より選ばれる塩基配列からなる核酸 である（16)記載の診断薬、 (17) The diagnostic agent according to (16), wherein the gene is a nucleic acid comprising a base sequence selected from the group consisting of SEQ ID NOs: 1 to 17,
(18)該遺伝子が、配列番号： 6及び Z又は 13の塩基配列からなる核酸である（ 16) 記載の診断薬、  (18) The diagnostic agent according to (16), wherein the gene is a nucleic acid comprising the nucleotide sequence of SEQ ID NO: 6 and Z or 13;
(19)配列番号： 35及び Z又は 36の塩基配列からなる核酸を含有してなる、（ 18)記 載の診断薬、  (19) The diagnostic agent according to (18), comprising a nucleic acid consisting of the nucleotide sequence of SEQ ID NO: 35 and Z or 36,
(20)配列番号： 18〜34からなる群より選ばれるアミノ酸配列を有するタンパク質又 は該アミノ酸配列において 1若しくは数個のアミノ酸が欠失、置換若しくは付加された アミノ酸配列を有する少なくとも 1つのタンパク質又はその部分ペプチド又はその塩 に対する抗体又はその断片を含有してなる、（14)又は（15)記載の方法に使用する ための診断薬、  (20) a protein having an amino acid sequence selected from the group consisting of SEQ ID NOs: 18 to 34, or at least one protein having an amino acid sequence in which one or several amino acids are deleted, substituted or added in the amino acid sequence, or A diagnostic agent for use in the method according to (14) or (15), comprising an antibody against the partial peptide or a salt thereof or a fragment thereof,
(21)配列番号： 23及び Z又は 30のアミノ酸配列を有するタンパク質又は該アミノ酸 配列において 1若しくは数個のアミノ酸が欠失、置換若しくは付加されたアミノ酸配列 を有する少なくとも 1つのタンパク質又はその部分ペプチド又はその塩に対する抗体 又はその断片を含有してなる、（14)又は（15)記載の方法に使用するための診断薬  (21) a protein having the amino acid sequence of SEQ ID NO: 23 and Z or 30, or at least one protein having an amino acid sequence in which one or several amino acids are deleted, substituted or added in the amino acid sequence, or a partial peptide thereof A diagnostic agent for use in the method according to (14) or (15), comprising an antibody against the salt or a fragment thereof
(22)配列番号： 18〜34からなる群より選ばれるアミノ酸配列を有するタンパク質又 は該アミノ酸配列において 1若しくは数個のアミノ酸が欠失、置換若しくは付加された アミノ酸配列を有するタンパク質をコードする少なくとも 1つの遺伝子を用いることを特 徴とする骨疾患又は関節疾患の予防剤及び Z又は治療剤のスクリーニング方法、(22) at least encoding a protein having an amino acid sequence selected from the group consisting of SEQ ID NOs: 18 to 34 or a protein having an amino acid sequence in which one or several amino acids are deleted, substituted or added in the amino acid sequence A screening method for a preventive agent and a Z or therapeutic agent for bone disease or joint disease, characterized by using one gene,
(23)配列番号： 18〜34からなる群より選ばれるアミノ酸配列を有するタンパク質又 は該アミノ酸配列において 1若しくは数個のアミノ酸が欠失、置換若しくは付加された アミノ酸配列を有する少なくとも 1つのタンパク質若しくはその部分ペプチド又はその 塩を用いることを特徴とする骨疾患又は関節疾患の予防剤及び Z又は治療剤のスク リーニング方法、に関する。 (23) SEQ ID NO: A protein having an amino acid sequence selected from the group consisting of 18 to 34, or at least one protein having an amino acid sequence in which one or several amino acids are deleted, substituted or added in the amino acid sequence, or The present invention relates to a preventive agent for bone disease or joint disease and a screening method for Z or therapeutic agent, characterized by using the partial peptide or a salt thereof.
また、本発明は、 (24)被検物質の存在下、配列番号： 18〜34からなる群より選ばれるアミノ酸配列を 有するタンパク質又は該アミノ酸配列において 1若しくは数個のアミノ酸が欠失、置換 若しくは付加されたアミノ酸配列を有するタンパク質をコードする少なくとも 1つの遺伝 子の動態を検出又は測定することを特徴とする、骨疾患又は関節疾患治療剤の予防 剤及び Z又は治療剤のスクリーニング方法、 The present invention also provides: (24) A protein having an amino acid sequence selected from the group consisting of SEQ ID NOs: 18 to 34 in the presence of a test substance, or an amino acid sequence in which one or several amino acids are deleted, substituted or added in the amino acid sequence A prophylactic agent for a therapeutic agent for bone disease or joint disease and a screening method for Z or therapeutic agent, characterized by detecting or measuring the dynamics of at least one gene encoding a protein having
(25) (I)被検物質の存在下、配列番号： 18〜34からなる群より選ばれるアミノ酸配 列を有するタンパク質又は該アミノ酸配列において 1若しくは数個のアミノ酸が欠失、 置換若しくは付加されたアミノ酸配列を有するタンパク質又はその部分ペプチド又は その塩と、被検物質とを接触させるステップ、並びに  (25) (I) A protein having an amino acid sequence selected from the group consisting of SEQ ID NOs: 18 to 34 in the presence of a test substance, or one or several amino acids in the amino acid sequence are deleted, substituted or added Contacting a protein having a specific amino acid sequence or a partial peptide thereof or a salt thereof with a test substance, and
(II)前記ステップ (I)の後、該タンパク質又はその部分ペプチドと被検物質との結合 を検出し、それにより、該タンパク質又はその部分ペプチドと結合する被検物質を、 骨疾患又は関節疾患の予防剤及び Z又は治療剤の候補化合物として選択するステ ップ  (II) After the step (I), the binding between the protein or a partial peptide thereof and a test substance is detected, whereby the test substance binding to the protein or the partial peptide is detected as a bone disease or a joint disease. Selected as a candidate compound for prophylactic and Z or therapeutic agents
を含む、（24)記載の方法、 Including the method according to (24),
(26) (A)被検物質の存在下、配列番号： 18〜34からなる群より選ばれるアミノ酸配 列を有するタンパク質又は該アミノ酸配列において 1若しくは数個のアミノ酸が欠失、 置換若しくは付加されたアミノ酸配列を有するタンパク質をコードする核酸を細胞に 導入するステップ、  (26) (A) A protein having an amino acid sequence selected from the group consisting of SEQ ID NOs: 18 to 34 in the presence of a test substance, or one or several amino acids in the amino acid sequence are deleted, substituted or added Introducing a nucleic acid encoding a protein having a specific amino acid sequence into a cell,
(B)被検物質の存在下、前記ステップ (A)で得られた細胞にぉ ヽて前記核酸を発現 させるステップ、並びに  (B) expressing the nucleic acid in the presence of the test substance in the cells obtained in step (A), and
(C)被検物質の非存在下の場合と比較して、前記核酸の発現の有無を検出し、又は 前記核酸の発現の変動を測定し、それにより、該核酸の発現の変化をもたらす被検 物質を骨疾患又は関節疾患の予防剤及び Z又は治療剤の候補化合物として選択 するステップ  (C) Compared with the absence of the test substance, the presence or absence of the expression of the nucleic acid is detected, or the change in the expression of the nucleic acid is measured, thereby causing a change in the expression of the nucleic acid. Step of selecting a test substance as a prophylactic agent for bone disease or joint disease and candidate compound for Z or therapeutic agent
を含む、（24)記載の方法、 Including the method according to (24),
(27) (A)被検物質の存在下、配列番号： 18〜33からなる群より選ばれるアミノ酸配 列を有するタンパク質又は該アミノ酸配列において 1若しくは数個のアミノ酸が欠失、 置換若しくは付加されたアミノ酸配列を有するタンパク質をコードする核酸を細胞に 導入するステップ、 (27) (A) In the presence of a test substance, a protein having an amino acid sequence selected from the group consisting of SEQ ID NOs: 18 to 33, or one or several amino acids in the amino acid sequence are deleted, substituted or added A nucleic acid encoding a protein having a specific amino acid sequence Introducing steps,
(B)被検物質の存在下、前記ステップ (A)で得られた細胞にぉ ヽて前記核酸を発現 させるステップ、並びに  (B) expressing the nucleic acid in the presence of the test substance in the cells obtained in step (A), and
(C)被検物質の非存在下の場合と比較して、前記核酸の発現の上昇を測定し、それ により、該核酸の発現の変化をもたらす被検物質を骨疾患又は関節疾患の予防剤 及び Z又は治療剤の候補化合物として選択するステップ  (C) Compared to the absence of the test substance, the increase in the expression of the nucleic acid is measured, whereby the test substance that causes a change in the expression of the nucleic acid is used as a prophylactic agent for bone disease or joint disease. And selecting as a candidate compound for Z or therapeutic agent
を含む、（24)記載の方法、 Including the method according to (24),
(28) (A)被検物質の存在下、配列番号： 34からなる群より選ばれるアミノ酸配列を 有するタンパク質又は該アミノ酸配列において 1若しくは数個のアミノ酸が欠失、置換 若しくは付加されたアミノ酸配列を有するタンパク質をコードする核酸を細胞に導入 するステップ、  (28) (A) A protein having an amino acid sequence selected from the group consisting of SEQ ID NO: 34 in the presence of a test substance, or an amino acid sequence in which one or several amino acids have been deleted, substituted or added in the amino acid sequence Introducing into a cell a nucleic acid encoding a protein having
(B)被検物質の存在下、前記ステップ (A)で得られた細胞にぉ ヽて前記核酸を発現 させるステップ、並びに  (B) expressing the nucleic acid in the presence of the test substance in the cells obtained in step (A), and
(C)被検物質の非存在下の場合と比較して、前記核酸の発現の減少を測定し、それ により、該核酸の発現の変化をもたらす被検物質を骨疾患又は関節疾患の予防剤 及び Z又は治療剤の候補化合物として選択するステップ  (C) Compared to the absence of the test substance, the decrease in the expression of the nucleic acid is measured, whereby the test substance that causes a change in the expression of the nucleic acid is used as a preventive agent for bone disease or joint disease. And selecting as a candidate compound for Z or therapeutic agent
を含む、（24)記載の方法、 Including the method according to (24),
(29)骨疾患又は関節疾患が軟骨形成異常、骨形成異常、骨粗鬆症、変形性関節 症、慢性関節リウマチ、関節炎、滑膜炎、代謝性関節症又はスポーツによる関節障 害である（24)〜（28) V、ずれかに記載の方法、  (29) The bone disease or joint disease is cartilage dysplasia, bone dysplasia, osteoporosis, osteoarthritis, rheumatoid arthritis, arthritis, synovitis, metabolic arthropathy or arthropathy due to sports (24) ~ (28) V, the method described in
(30)配列番号： 18〜34からなる群より選ばれるアミノ酸配列を有するタンパク質又 は該アミノ酸配列において 1若しくは数個のアミノ酸が欠失、置換若しくは付加された アミノ酸配列を有するタンパク質又はその部分ペプチド又はその塩を含有してなる（2 4)、 (25)及び（29) V、ずれかに記載の方法に使用するためのキット、  (30) A protein having an amino acid sequence selected from the group consisting of SEQ ID NOs: 18 to 34, a protein having an amino acid sequence in which one or several amino acids are deleted, substituted or added in the amino acid sequence, or a partial peptide thereof Or a kit for use in the method according to any one of (2 4), (25) and (29) V, which comprises a salt thereof,
(31)被検物質の存在下、配列番号： 18〜34からなる群より選ばれるアミノ酸配列を 有するタンパク質又は該アミノ酸配列において 1若しくは数個のアミノ酸が欠失、置換 若しくは付加されたアミノ酸配列を有するタンパク質をコードする核酸を含む核酸構 築物を含有してなる（24)、 (26)〜（29) V、ずれかに記載の方法に使用するためのキ ッ卜、 (31) A protein having an amino acid sequence selected from the group consisting of SEQ ID NOs: 18 to 34 in the presence of a test substance, or an amino acid sequence in which one or several amino acids are deleted, substituted or added in the amino acid sequence (24), (26) to (29) V, comprising a nucleic acid construct containing a nucleic acid encoding a protein having a key for use in the method according to any one of Ah,
(32)配列番号： 18〜34からなる群より選ばれるアミノ酸配列を有するタンパク質又 は該アミノ酸配列において 1若しくは数個のアミノ酸が欠失、置換若しくは付加された アミノ酸配列を有するタンパク質をコードする核酸が導入された細胞を含有してなる（ 24)、 (26)〜（29) V、ずれかに記載の方法に使用するためのキット、  (32) Nucleic acid encoding a protein having an amino acid sequence selected from the group consisting of SEQ ID NOs: 18 to 34 or a protein having an amino acid sequence in which one or several amino acids have been deleted, substituted or added in the amino acid sequence (24), (26) to (29) V, a kit for use in the method according to any one of the above,
(33)配列番号： 18〜34からなる群より選ばれるアミノ酸配列を有するタンパク質又 は該アミノ酸配列において 1若しくは数個のアミノ酸が欠失、置換若しくは付加された アミノ酸配列を有するタンパク質又は部分ペプチド又はその塩に対する抗体を含有 してなる（24)〜（29) V、ずれかに記載の方法に使用するためのキット、  (33) a protein having an amino acid sequence selected from the group consisting of SEQ ID NO: 18 to 34, or a protein or partial peptide having an amino acid sequence in which one or several amino acids are deleted, substituted or added in the amino acid sequence, or (24) to (29) V, a kit for use in the method according to any one of the above, comprising an antibody against the salt,
(34) (24)〜（29) V、ずれかに記載の方法により得られる化合物又はその塩、 (34) (24) to (29) V, a compound obtained by the method according to any one or a salt thereof,
(35) (34)に記載の化合物又はその塩を有効成分として含有してなる医薬組成物、(35) A pharmaceutical composition comprising the compound or salt thereof according to (34) as an active ingredient,
(36)骨疾患若しくは関節疾患の治療又は予防に用いる、（35)記載の医薬組成物、(36) The pharmaceutical composition according to (35), which is used for treatment or prevention of bone disease or joint disease,
(37)被検対象の個体由来の被検試料における配列番号： 18〜34からなる群より選 ばれるアミノ酸配列を有するタンパク質又は該アミノ酸配列において 1若しくは数個の アミノ酸が欠失、置換若しくは付加されたアミノ酸配列を有するタンパク質をコードす る少なくとも 1つの遺伝子の発現レベルを測定するステップを含み、ここで、被検試料 における発現レベル力 正常個体由来又は RA患者由来の対照試料における発現 レベルと異なる場合、該被検試料が、骨疾患又は関節疾患に罹患している疑いがあ る個体由来の試料であることの指標となる、骨疾患又は関節疾患の診断方法、(37) A protein having an amino acid sequence selected from the group consisting of SEQ ID NOs: 18 to 34 in a test sample derived from an individual to be tested, or one or several amino acids are deleted, substituted or added in the amino acid sequence Measuring the expression level of at least one gene encoding a protein having a specific amino acid sequence, wherein the expression level in the test sample is different from the expression level in a control sample from a normal individual or RA patient A diagnostic method for bone disease or joint disease, which is an indicator that the test sample is a sample derived from an individual suspected of having bone disease or joint disease,
(38)発現レベル力 配列番号： 18〜34からなる群より選ばれるアミノ酸配列を有す るタンパク質又は該アミノ酸配列において 1若しくは数個のアミノ酸が欠失、置換若し くは付加されたアミノ酸配列を有するタンパク質をコードする少なくとも 1つの遺伝子 の mRNA量を指標として測定される、 (37)記載の方法、 (38) Expression level ability SEQ ID NO: 18-34 or a protein having an amino acid sequence selected from the group consisting of 18 to 34, or an amino acid sequence in which one or several amino acids are deleted, substituted or added in the amino acid sequence The method according to (37), wherein the mRNA level of at least one gene encoding a protein having
(39)発現レベル力 配列番号： 18〜34からなる群より選ばれるアミノ酸配列を有す るタンパク質又は該アミノ酸配列において 1若しくは数個のアミノ酸が欠失、置換若し くは付加されたアミノ酸配列を有する少なくとも 1つのタンパク質又はその部分べプチ ド又はその塩を指標として測定される、 (37)記載の方法、  (39) Expression level ability SEQ ID NO: 18-34 Protein having an amino acid sequence selected from the group consisting of amino acids or one or several amino acids deleted, substituted or added in the amino acid sequence The method according to (37), wherein at least one protein having the above or a partial peptide thereof or a salt thereof is measured as an index,
(40)骨疾患又は関節疾患が、軟骨形成異常、骨形成異常、骨粗鬆症、変形性関節 症、慢性関節リウマチ、関節炎、滑膜炎、代謝性関節症又はスポーツによる関節障 害である（37)〜（39) V、ずれか記載の方法、に関する。 (40) Bone disease or joint disease is cartilage dysplasia, bone dysplasia, osteoporosis, degenerative joint (37) to (39) V, which is a method described in any of the above, which is an arthritis, rheumatoid arthritis, arthritis, synovitis, metabolic arthropathy or sports joint disorder.
発明の効果  The invention's effect
[0030] 本発明で用いられるタンパク質は OA滑膜又は RA滑膜で発現が変化するため、例 えば、滑膜組織の異常が一因となる骨疾患又は関節疾患の診断指標として有用で ある。また、本発明で用いられるタンパク質は、関節痛を症状の一つとし、判別しにく い OAと RAを区別する、 OA又は RAの診断指標として有用である。また、該タンパク 質の活性を調節する化合物又はその塩、該タンパク質遺伝子の発現を調節する化 合物又はその塩、該タンパク質に対する中和抗体、該タンパク質をコードする遺伝子 のアンチセンスヌクレオチドは、該骨疾患若しくは関節疾患の予防剤又は治療剤とし て安全に使用することができる。  [0030] Since the protein used in the present invention changes in expression in OA synovium or RA synovium, it is useful, for example, as a diagnostic index for bone disease or joint disease caused by abnormal synovial tissue. In addition, the protein used in the present invention is useful as a diagnostic index for OA or RA, which distinguishes OA and RA which are difficult to discriminate because arthralgia is one of the symptoms. Further, a compound or salt thereof that regulates the activity of the protein, a compound or salt thereof that regulates the expression of the protein gene, a neutralizing antibody against the protein, or an antisense nucleotide of the gene encoding the protein comprises It can be safely used as a prophylactic or therapeutic agent for bone diseases or joint diseases.
図面の簡単な説明  Brief Description of Drawings
[0031] [図 l]Fibulin— 3遺伝子の発現 [0031] [Fig. L] Fibulin-3 gene expression
発明を実施するための最良の形態  BEST MODE FOR CARRYING OUT THE INVENTION
[0032] 本発明は、骨疾患若しくは関節疾患に罹患している疑いがある個体由来の試料の 検出又は選別方法、骨疾患又は関節疾患の診断方法、該検出 ·選別 ·診断方法に 用いる診断薬、骨疾患若しくは関節疾患の治療剤又は予防剤のスクリーニング方法 、該スクリーニング方法に用いるキット、医薬組成物に関するものである。  [0032] The present invention relates to a method for detecting or selecting a sample derived from an individual suspected of having a bone disease or joint disease, a method for diagnosing a bone disease or joint disease, and a diagnostic agent used for the detection, selection and diagnosis method. Further, the present invention relates to a screening method for a therapeutic or prophylactic agent for bone disease or joint disease, a kit used for the screening method, and a pharmaceutical composition.
[0033] 「骨疾患又は関節疾患」とは、例えば、軟骨形成異常、骨形成異常、骨粗鬆症、変 形性関節症、慢性関節リウマチ、関節炎、滑膜炎、代謝性関節症又はスポーツによ る関節障害が挙げられる。本発明は、特に、変形性関節症又は慢性関節リウマチに 対して好適に使用され得る。  [0033] "Bone disease or joint disease" means, for example, cartilage dysplasia, bone dysplasia, osteoporosis, osteoarthritis, rheumatoid arthritis, arthritis, synovitis, metabolic arthropathy or sports Examples include joint disorders. The present invention can be suitably used particularly for osteoarthritis or rheumatoid arthritis.
[0034] 以下、本発明を詳細に説明する。なお、本明細書において、特に指示のない限り、 遺伝子組換え技術、細胞での組換えタンパク質の生産技術、発現タンパク質の分離 精製法、分析法、分子生物学手法及び免疫学的手法等は、公知の方法が採用され る。  [0034] Hereinafter, the present invention will be described in detail. In this specification, unless otherwise specified, gene recombination technology, recombinant protein production technology in cells, separation and purification methods of expressed proteins, analysis methods, molecular biology methods, immunological methods, etc. A known method is employed.
[0035] 本発明の「骨疾患若しくは関節疾患に罹患して 、る疑いがある個体由来の試料の 検出又は選別方法」は、被検対象の個体由来の被検試料におけるタンパク質又は 部分ペプチドをコードする遺伝子の発現レベルを測定することを 1つの特徴とする。 したがって、本発明の検出又は選別方法によれば、被検対象の個体が骨疾患又は 関節疾患に罹患している疑いがある力否かを簡便かつ迅速に検出又は選別すること ができるという優れた効果が発揮される。また、代表的な関節疾患であり、似た症状 を示す OAと RAを区別し、 OA患者又は RA患者由来の試料の検出又は選別するこ とがでさる。 [0035] The "method for detecting or selecting a sample derived from an individual suffering from bone disease or joint disease and suspected" according to the present invention refers to a protein in a test sample derived from an individual to be tested or One characteristic is to measure the expression level of a gene encoding a partial peptide. Therefore, according to the detection or selection method of the present invention, it is possible to easily and quickly detect or select whether or not the subject individual is suspected of having bone disease or joint disease. The effect is demonstrated. In addition, OA and RA, which are typical joint diseases and exhibit similar symptoms, can be distinguished, and samples from OA patients or RA patients can be detected or selected.
[0036] 前記「個体」としては、特に限定されるものではないが、哺乳動物、特にヒト、マウス 、マウス、サル等が挙げられる。  [0036] The "individual" is not particularly limited, but includes mammals, particularly humans, mice, mice, monkeys, and the like.
[0037] 「被検試料」は、例えば、骨疾患又は関節疾患に罹患して 、る疑 、のある個体由来 の組織 (例えば、骨膜、軟骨等)、細胞、関節液、血液、尿等力 慣用の手法により調 製されうる。特に簡便に検査可能な関節液、血液及び尿から調製するのが好ましい。 関節液、血液及び尿は、通常の関節液検査、血液検査及び尿検査と同様の方法で 採取する。対照試料は、正常個体、 RA患者又は OA患者力 被検試料と同様の部 位カゝら調製されたものであればよぐ例えば、被検試料と同様にして同部位より調製 することができる。また、骨疾患又は関節疾患に罹患している疑いのある個体から、 手術時に摘出された組織 (例えば、骨膜、軟骨等)が存在する場合、該組織を慣用 の手法により調整し、使用することもできる。  [0037] "Test sample" refers to, for example, tissue (eg, periosteum, cartilage, etc.), cells, synovial fluid, blood, urine, etc. It can be prepared by conventional methods. It is particularly preferable to prepare it from joint fluid, blood and urine that can be easily examined. Collect joint fluid, blood and urine in the same way as normal joint fluid, blood and urine tests. The control sample can be prepared from the same site as the test sample, for example, as long as it is prepared from the same part as the normal individual, RA patient or OA patient test sample . In addition, if there is a tissue (for example, periosteum, cartilage, etc.) extracted at the time of surgery from an individual suspected of having a bone disease or joint disease, the tissue should be adjusted and used by a conventional method. You can also.
[0038] 本発明の「検出又は選別方法」は、被検対象の個体由来の被検試料における配列 番号： 18〜34からなる群より選ばれるアミノ酸配列を有するタンパク質又は該ァミノ 酸配列において 1若しくは数個のアミノ酸が欠失、置換若しくは付加されたアミノ酸配 列を有するタンパク質をコードする少なくとも 1つの遺伝子の発現レベルを測定する ステップを含み、ここで、被検試料における発現レベル力 対照試料における発現レ ベルと異なる場合、該被検試料が、骨疾患又は関節疾患に罹患している疑いがある 個体由来の試料であることの指標となる。配列番号： 1〜17からなる群より選ばれる 塩基配列からなる 17個の遺伝子は、 1個の遺伝子の発現のみにより骨疾患又は関 節疾患の指標となる。特に、正常個体と比べ、 2倍以上の発現の上昇を表す遺伝子 が指標として好ましい。  [0038] The "detection or selection method" of the present invention is a protein having an amino acid sequence selected from the group consisting of SEQ ID NOs: 18 to 34 in a test sample derived from an individual to be tested, or 1 or more in the amino acid sequence. Measuring the expression level of at least one gene encoding a protein having an amino acid sequence in which several amino acids have been deleted, substituted or added, wherein the expression level in the test sample is expressed in the control sample When it is different from the level, it becomes an indicator that the test sample is a sample derived from an individual suspected of having bone disease or joint disease. SEQ ID NOs: 17 genes having a nucleotide sequence selected from the group consisting of 1 to 17 serve as indices of bone diseases or related diseases only by the expression of one gene. In particular, a gene showing an expression increase of 2 times or more compared to normal individuals is preferred as an indicator.
[0039] 配列番号： 2、 3、 7、 9〜11、 13〜16からなる群より選ばれる塩基配列からなる 10 個の遺伝子は、被検試料における発現レベル力 正常個体及び RA患者における発 現レベルより高くなる場合、 OA患者であると判断可能である。配列番号： 1、 4〜6、 8 、 12からなる群より選ばれる塩基配列力もなる 6個の遺伝子は、被検試料における発 現レベルが、正常個体における発現レベルより高くなり、かつ、 RA患者における発 現レベルより低くなる場合、 OA患者であると判断可能である。配列番号： 17の塩基 配列からなる遺伝子は、被検試料における発現レベル力 正常個体における発現レ ベルより低くなり、かつ、 RA患者における発現レベルより高くなる場合、 OA患者であ ると判断可能である。特に、配列番号: 6又は 13の塩基配列力もなる遺伝子が指標と して好ましい。 [0039] SEQ ID NO: 2, consisting of a base sequence selected from the group consisting of 2, 3, 7, 9-11 and 13-16 10 An individual gene can be determined to be an OA patient if its expression level is higher than the expression level in normal individuals and RA patients. Six genes having a nucleotide sequence ability selected from the group consisting of SEQ ID NOs: 1, 4-6, 8, 12 have higher expression levels in test samples than expression levels in normal individuals, and RA patients If it is lower than the expression level in, it can be determined that the patient is OA. The gene consisting of the nucleotide sequence of SEQ ID NO: 17 can be judged to be an OA patient when the expression level in the test sample is lower than the expression level in normal individuals and higher than the expression level in RA patients. is there. In particular, a gene having a nucleotide sequence ability of SEQ ID NO: 6 or 13 is preferable as an index.
[0040] また、配列番号： 1、 4〜6、 8、 12からなる群より選ばれる塩基配列力もなる 6個の遺 伝子は、被検試料における発現レベル力 正常個体及び OA患者における発現レべ ルより高くなる場合、 RA患者であると判断可能である。配列番号： 17の塩基配列力 なる遺伝子は、被検試料における発現レベル力 正常個体及び OA患者における発 現レベルより低くなる場合、 RA患者であると判断可能である。  [0040] In addition, 6 genes having a nucleotide sequence ability selected from the group consisting of SEQ ID NOs: 1, 4-6, 8, 12 are expressed in the expression level in normal individuals and OA patients. If it is above the threshold, it can be determined that the patient is RA. The gene having the nucleotide sequence of SEQ ID NO: 17 can be determined to be an RA patient when the expression level in the test sample is lower than the expression level in normal individuals and OA patients.
[0041] 本発明の「検出又は選別方法」において発現レベルを測定する遺伝子にコードさ れている「配列番号： 18〜34からなる群より選ばれるアミノ酸配列を有するタンパク質 」（以下、「本発明のタンパク質」と称することもある）は、ヒトゃ温血動物（例えば、モル モット、ラット、マウス、 -ヮトリ、ゥサギ、ブタ、ヒッジ、ゥシ、サル等）の細胞（例えば、 肝細胞、脾細胞、神経細胞、グリア細胞、脾臓 j8細胞、骨髄細胞、メサンギゥム細胞 、ランゲルハンス細胞、表皮細胞、上皮細胞、杯細胞、内皮細胞、平滑筋細胞、繊維 芽細胞、繊維細胞、筋細胞、脂肪細胞、免疫細胞 (例、マクロファージ、 T細胞、 B細 胞、ナチュラルキラー細胞、肥満細胞、好中球、好塩基球、好酸球、単球)、巨核球、 滑膜細胞、軟骨細胞、骨細胞、骨芽細胞、破骨細胞、乳腺細胞、肝細胞若しくは間 質細胞、又はこれら細胞の前駆細胞、幹細胞若しくはガン細胞等)若しくはそれらの 細胞が存在するあらゆる組織、例えば、脳、脳の各部位 (例、嗅球、扁桃核、大脳基 底球、海馬、視床、視床下部、大脳皮質、延髄、小脳)、脊髄、下垂体、胃、脾臓、腎 臓、肝臓、生殖腺、甲状腺、胆のう、骨髄、副腎、皮膚、筋肉、肺、消化管 (例、大腸 、小腸)、血管、心臓、胸腺、脾臓、顎下腺、末梢血、前立腺、睾丸、卵巣、胎盤、子 宫、骨、関節、骨格筋等に由来するタンパク質であってもよい。 [0041] A "protein having an amino acid sequence selected from the group consisting of SEQ ID NOs: 18 to 34" encoded by a gene whose expression level is measured in the "detection or selection method" of the present invention (hereinafter referred to as "the present invention"). Is sometimes referred to as a protein of human warm-blooded animals (eg, guinea pigs, rats, mice, rabbits, rabbits, pigs, hidges, rabbits, monkeys, etc.) Cells, neurons, glial cells, spleen j8 cells, bone marrow cells, mesangial cells, Langerhans cells, epidermal cells, epithelial cells, goblet cells, endothelial cells, smooth muscle cells, fibroblasts, fibroblasts, muscle cells, adipocytes, Immune cells (eg, macrophages, T cells, B cells, natural killer cells, mast cells, neutrophils, basophils, eosinophils, monocytes), megakaryocytes, synovial cells, chondrocytes, bone cells, Osteoblast Cysts, osteoclasts, mammary cells, hepatocytes or stromal cells, or precursor cells of these cells, stem cells or cancer cells, etc.) or any tissue in which those cells are present (e.g., brain, brain regions (e.g., Olfactory bulb, amygdala, basal ganglia, hippocampus, thalamus, hypothalamus, cerebral cortex, medulla, cerebellum), spinal cord, pituitary gland, stomach, spleen, kidney, liver, gonad, thyroid, gall bladder, bone marrow, adrenal gland, skin , Muscle, lung, gastrointestinal tract (eg, large intestine, small intestine), blood vessel, heart, thymus, spleen, submandibular gland, peripheral blood, prostate, testis, ovary, placenta, child Proteins derived from heels, bones, joints, skeletal muscles and the like may be used.
[0042] 「1若しくは数個のアミノ酸が欠失、置換若しくは付加されたアミノ酸配列」とは、例え ば、（A)配列番号： 18〜34からなる群より選ばれるアミノ酸配列中の 1又は 2個以上（ 好ましくは、 1〜30個程度、好ましくは 1〜10個程度、さらに好ましくは数（1〜5)個） のアミノ酸が欠失したアミノ酸配列、（B)配列番号： 18〜34からなる群より選ばれるァ ミノ酸配列に 1又は 2個以上 (好ましくは、 1〜30個程度、好ましくは 1〜10個程度、さ らに好ましくは数（1〜5)個）のアミノ酸が付加したアミノ酸配列、（C)配列番号： 18〜 34からなる群より選ばれるアミノ酸配列に 1又は 2個以上 (好ましくは、 1〜30個程度 、好ましくは 1〜： LO個程度、さらに好ましくは数（1〜5)個）のアミノ酸が挿入されたァ ミノ酸配列、（D)配列番号： 18〜34からなる群より選ばれるアミノ酸配列中の 1又は 2 個以上 (好ましくは、 1〜30個程度、好ましくは 1〜10個程度、さらに好ましくは数（1 〜5)個）のアミノ酸が他のアミノ酸で置換されたアミノ酸配列、又は（E)それらを組み 合わせたアミノ酸配列を含有するタンパク質が挙げられる。アミノ酸配列が挿入、欠 失又は置換されている場合、その挿入、欠失又は置換の位置としては、特に限定さ れない。  [0042] The "amino acid sequence in which one or several amino acids are deleted, substituted or added" is, for example, (A) 1 or 2 in an amino acid sequence selected from the group consisting of SEQ ID NOs: 18 to 34 An amino acid sequence in which one or more (preferably about 1 to 30, preferably about 1 to 10, more preferably (1 to 5)) amino acids have been deleted, (B) from SEQ ID NOs: 18 to 34 1 or 2 (preferably about 1 to 30, preferably about 1 to 10, more preferably a number (1 to 5)) of amino acids are added to the amino acid sequence selected from the group consisting of (C) 1 or 2 or more amino acid sequences selected from the group consisting of SEQ ID NOs: 18 to 34 (preferably about 1 to 30, preferably about 1 to about LO, more preferably numbers) An amino acid sequence in which (1-5) amino acids are inserted; (D) from the group consisting of SEQ ID NOs: 18-34 One or more amino acids in the selected amino acid sequence (preferably about 1 to 30, preferably about 1 to 10, more preferably a number (1 to 5)) are substituted with other amino acids. Examples thereof include an amino acid sequence or (E) a protein containing an amino acid sequence obtained by combining them. When the amino acid sequence is inserted, deleted or substituted, the position of the insertion, deletion or substitution is not particularly limited.
[0043] 「遺伝子の発現レベル」は、該遺伝子の mRNA量又はタンパク質の量を指標として 測定するのが好適である。 mRNAの検出を行う分子生物学的測定方法又はタンパ ク質の検出を行う免疫学的測定方法であればいかなる方法でもよい。例えば、分子 生物学的測定方法としてポリメラーゼ連鎖反応法 (PCR)、ノーザンプロット法、ドット プロット法、マイクロアレイ又はマクロアレイを用いた解析方法等が例示され、免疫学 的測定方法としては、マイクロタイタープレートを用いる ELISA法、 RIA法、蛍光抗 体法、ウエスタンプロット法、免疫組織染色法等が例示される。  [0043] The "gene expression level" is preferably measured using the amount of mRNA or protein of the gene as an index. Any method may be used as long as it is a molecular biological measurement method for detecting mRNA or an immunological measurement method for detecting protein. Examples of molecular biological measurement methods include polymerase chain reaction (PCR), Northern plot method, dot plot method, analysis method using microarray or macroarray, and immunological measurement methods include microtiter plates. Examples include ELISA method using RIA, RIA method, fluorescent antibody method, Western plot method, immunohistochemical staining method and the like.
[0044] 本発明には、発現レベル力 配列番号： 18〜34からなる群より選ばれるアミノ酸配 列を有するタンパク質又は該アミノ酸配列において 1若しくは数個のアミノ酸が欠失、 置換若しくは付加されたアミノ酸配列を有するタンパク質をコードする少なくとも 1つ の遺伝子の mRNA量を指標として測定される方法も包含される。これは、上記の分 子生物学的測定方法にあたる。  [0044] In the present invention, an expression level force protein having an amino acid sequence selected from the group consisting of SEQ ID NOs: 18 to 34, or an amino acid in which one or several amino acids are deleted, substituted or added in the amino acid sequence Also included is a method in which the amount of mRNA of at least one gene encoding a protein having a sequence is measured as an index. This corresponds to the molecular biological measurement method described above.
[0045] 例えば、配列番号： 18〜34からなる群より選ばれるアミノ酸配列を有するタンパク 質又は該アミノ酸配列において 1若しくは数個のアミノ酸が欠失、置換若しくは付加さ れたアミノ酸配列を有するタンパク質をコードする少なくとも 1つの遺伝子を検出しうる 核酸を用いる方法、さらには、該遺伝子が、配列番号： 1〜17からなる群より選ばれる 塩基配列からなる核酸である方法、該遺伝子が、配列番号: 6及び Z又は 13の塩基 配列からなる核酸である方法、配列番号： 35及び Z又は 36の塩基配列力 なる核 酸を用いる方法も包含される。以下に、具体的に記載する。 [0045] For example, a protein having an amino acid sequence selected from the group consisting of SEQ ID NOs: 18 to 34 Or a method using a nucleic acid capable of detecting at least one gene encoding a protein having an amino acid sequence in which one or several amino acids have been deleted, substituted or added in the amino acid sequence, A method that is a nucleic acid consisting of a base sequence selected from the group consisting of SEQ ID NOs: 1 to 17, a method wherein the gene is a nucleic acid consisting of a base sequence of SEQ ID NOs: 6 and Z, or 13, SEQ ID NOs: 35 and Z or A method using a nucleic acid having a nucleotide sequence of 36 is also included. Specific description will be given below.
[0046] 分子生物学的測定方法 [0046] Molecular biological measurement method
PCRの場合、被検対象の個体由来の被検試料を、例えば、前記配列番号： 18〜3 4からなる群より選ばれるアミノ酸配列を有するタンパク質又は該アミノ酸配列におい て 1若しくは数個のアミノ酸が欠失、置換若しくは付加されたアミノ酸配列を有するタ ンパク質をコードする核酸若しくは該核酸に特徴的な部分配列からなる核酸をそれ ぞれ特異的に増幅しうるプライマー対を用いた PCR等に供し、ハイブリッドの形成若 しくはその量又は増幅産物の出現若しくはその量を測定し、対照試料における結果 と比較することにより、本発明のタンパク質の遺伝子の発現レベルを評価することが できる。  In the case of PCR, a test sample derived from an individual to be tested is, for example, a protein having an amino acid sequence selected from the group consisting of SEQ ID NOs: 18 to 34, or one or several amino acids in the amino acid sequence. The sample is subjected to PCR using a primer pair that can specifically amplify a nucleic acid encoding a protein having a deleted, substituted or added amino acid sequence or a partial sequence characteristic of the nucleic acid. Then, the expression level of the gene of the protein of the present invention can be evaluated by measuring the formation or the amount of the hybrid or the appearance or the amount of the amplification product and comparing it with the result in the control sample.
[0047] PCRに用いられる「プライマー対」としては、本発明のタンパク質をコードする核酸 若しくは該核酸に特徴的な配列部分からなる核酸の 5 '末端のアンチセンス配列に対 応するプライマーと 3'末端のアンチセンス配列に対応するプライマーとからなるブラ イマ一対等が挙げられる。かかるプライマー対は、使用時における操作性を考慮し、 適切な Tm値、二次構造等に基づき、適宜選択されうる。具体的には、配列番号： 1 〜 17に記載の塩基配列の全部又はその特徴的な部分配列を増幅可能なものである のが好適である。より具体的には、例えば、配列番号： 1〜 17に記載の塩基配列を有 する核酸力 なる群より選ばれたプライマー対等が挙げられる。プライマー対の具体 例としては、特に限定はないが、配列番号： 35と 36とのプライマー対等が挙げられる 。かかるプライマーは、慣用の蛍光色素、放射性物質等で標識されたプライマーであ つてもよい。増幅産物の検出は、慣用のァガロースゲル電気泳動等において、臭化 ェチジゥムゃ蛍光物質による可視化、標識プライマーに基づく検出等を行なうこと等 により行なわれうる。 [0048] 本明細書にぉ 、て、「 (核酸に）特徴的な部分配列」とは、例えば、データベースに 登録された配列のうち、前記本発明のタンパク質をコードする遺伝子以外の遺伝子 の塩基配列には実質的に見出されない部分配列をいい、例えば、データベースに登 録された配列との配列同一性力 通常、 20%以下、好ましくは 10%以下、より好まし くは 5%以下、特に好ましくは 0%である配列を 、う。 [0047] The "primer pair" used in PCR includes a nucleic acid encoding the protein of the present invention or a primer corresponding to an antisense sequence at the 5 'end of a nucleic acid comprising a sequence portion characteristic of the nucleic acid and 3'. One example is a pair of primers consisting of a primer corresponding to the antisense sequence at the end. Such a primer pair can be appropriately selected based on an appropriate Tm value, secondary structure, etc. in consideration of operability during use. Specifically, it is preferable that the entire base sequence described in SEQ ID NOs: 1 to 17 or a characteristic partial sequence thereof can be amplified. More specifically, for example, primer pairs selected from the group consisting of nucleic acids having the base sequences described in SEQ ID NOs: 1 to 17 and the like can be mentioned. Specific examples of the primer pair include, but are not limited to, a primer pair of SEQ ID NOs: 35 and 36, and the like. Such a primer may be a primer labeled with a conventional fluorescent dye or radioactive substance. The amplification product can be detected by visualizing with a fluorescent substance, detection based on a labeled primer, etc. in conventional agarose gel electrophoresis or the like. In the present specification, the “partial sequence characteristic to (nucleic acid)” means, for example, a base of a gene other than the gene encoding the protein of the present invention among sequences registered in a database. A partial sequence not substantially found in a sequence, for example, sequence identity with a sequence registered in a database, usually 20% or less, preferably 10% or less, more preferably 5% or less, Particularly preferred is a sequence that is 0%.
[0049] ノーザンプロット法の場合、被検対象の個体由来の被検試料を、例えば、前記配列 番号： 18〜34からなる群より選ばれるアミノ酸配列を有するタンパク質又は該ァミノ 酸配列において 1若しくは数個のアミノ酸が欠失、置換若しくは付加されたアミノ酸配 列を有するタンパク質をコードする核酸を検出するプローブを用い、ハイブリッドの形 成若しくはその量又は増幅産物の出現若しくはその量を測定し、対照試料における 結果と比較することにより、本発明のタンパク質の遺伝子の発現レベルを評価するこ とがでさる。  [0049] In the case of the Northern plotting method, a test sample derived from an individual to be tested is, for example, one or a number in the protein having the amino acid sequence selected from the group consisting of the aforementioned SEQ ID NOs: 18 to 34 or the amino acid sequence. Using a probe that detects a nucleic acid encoding a protein having an amino acid sequence in which a single amino acid has been deleted, substituted, or added, the formation of the hybrid or the amount thereof, or the appearance or amount of the amplification product is measured, and a control sample It is possible to evaluate the expression level of the gene of the protein of the present invention by comparing with the results in.
[0050] マイクロアレイ又はマクロアレイを用いた解析方法の場合、被検対象の個体由来の 被検試料を、例えば、前記配列番号： 18〜34からなる群より選ばれるアミノ酸配列を 有するタンパク質又は該アミノ酸配列において 1若しくは数個のアミノ酸が欠失、置換 若しくは付加されたアミノ酸配列を有するタンパク質をコードする核酸を検出するプロ ーブを少なくとも 1つ以上含んだマイクロアレイ又はマクロアレイを用い、ハイブリッド の形成若しくはその量又は増幅産物の出現若しくはその量を測定し、対照試料にお ける結果と比較することにより、本発明のタンパク質の遺伝子の発現レベルを評価す ることがでさる。  [0050] In the case of an analysis method using a microarray or a macroarray, a test sample derived from an individual to be tested is, for example, a protein having an amino acid sequence selected from the group consisting of SEQ ID NOs: 18 to 34 or the amino acid Using a microarray or macroarray containing at least one probe for detecting a nucleic acid encoding a protein having an amino acid sequence in which one or several amino acids are deleted, substituted or added in the sequence, a hybrid is formed or The expression level of the gene of the protein of the present invention can be evaluated by measuring the amount or the appearance of the amplification product or the amount thereof and comparing it with the result in the control sample.
[0051] ノーザンブロット法やマイクロアレイ又はマクロアレイを用いた解析方法に用いられ る「プローブ」としては、配列番号： 1〜17からなる群より選ばれる塩基配列からなる核 酸を検出するために使用される核酸が挙げられる。プローブとして用いられる核酸は 、使用時における操作性を考慮し、適切な Tm値、二次構造等に基づき、適宜選択さ れうる。かかる核酸は、慣用の蛍光色素、放射性物質等で標識されていてもよい。  [0051] The “probe” used in the Northern blot method or the analysis method using a microarray or macroarray is used to detect a nucleic acid having a base sequence selected from the group consisting of SEQ ID NOs: 1 to 17. Nucleic acid to be used. A nucleic acid used as a probe can be appropriately selected based on an appropriate Tm value, secondary structure, etc. in consideration of operability during use. Such a nucleic acid may be labeled with a conventional fluorescent dye, radioactive substance or the like.
[0052] 本発明には、発現レベル力 配列番号： 18〜34からなる群より選ばれるアミノ酸配 列を有するタンパク質又は該アミノ酸配列において 1若しくは数個のアミノ酸が欠失、 置換若しくは付加されたアミノ酸配列を有する少なくとも 1つのタンパク質又はその部 分ペプチド又はその塩を指標として測定される方法も包含される。これは、上記の免 疫学的測定方法にあたる。 [0052] The present invention includes a protein having an amino acid sequence selected from the group consisting of SEQ ID NOs: 18 to 34, or an amino acid in which one or several amino acids have been deleted, substituted or added in the amino acid sequence. At least one protein having a sequence or part thereof A method of measuring using a partial peptide or a salt thereof as an index is also included. This corresponds to the above-mentioned method of immunological measurement.
[0053] 「タンパク質の部分ペプチド」としては、前記本発明のタンパク質の部分ペプチドで あって、好ましくは、前記本発明のタンパク質と同様の性質を有するものであればい ずれのものでもよい。例えば、本発明のタンパク質の構成アミノ酸配列のうち少なくと も 20個以上、好ましくは 50個以上、さらに好ましくは 70個以上、より好ましくは 100個 以上、最も好ましくは 200個以上のアミノ酸配列を有するペプチド等が用いられる。  [0053] The "partial peptide of the protein" is a partial peptide of the protein of the present invention, and may be any one as long as it has the same properties as the protein of the present invention. For example, the amino acid sequence of the protein of the present invention has at least 20 or more, preferably 50 or more, more preferably 70 or more, more preferably 100 or more, most preferably 200 or more amino acid sequences. Peptides and the like are used.
[0054] また、「部分ペプチド」は、そのアミノ酸配列中の 1又は 2個以上 (好ましくは、 1〜： LO 個程度、さらに好ましくは数（1〜5)個）のアミノ酸が欠失し、又は、そのアミノ酸配列 に 1又は 2個以上 (好ましくは、 1〜20個程度、より好ましくは 1〜10個程度、さらに好 ましくは数（1〜5)個）のアミノ酸が付加し、又は、そのアミノ酸配列に 1又は 2個以上（ 好ましくは、 1〜20個程度、より好ましくは 1〜10個程度、さらに好ましくは数（1〜5) 個）のアミノ酸が挿入され、又は、そのアミノ酸配列中の 1又は 2個以上 (好ましくは、 1 〜： LO個程度、より好ましくは数個、さらに好ましくは 1〜5個程度）のアミノ酸が他のァ ミノ酸で置換されて 、てもよ 、。  [0054] Further, the "partial peptide" has one or more (preferably about 1 to: LO, more preferably several (1 to 5)) amino acids in its amino acid sequence, Or 1 or 2 or more (preferably about 1 to 20, more preferably about 1 to 10, more preferably a number (1 to 5)) of amino acids are added to the amino acid sequence, or 1 or 2 or more (preferably about 1 to 20, more preferably about 1 to 10, more preferably a number (1 to 5)) of amino acids are inserted into the amino acid sequence, or the amino acid One or more amino acids in the sequence (preferably about 1 to: about LO, more preferably about several, and even more preferably about 1 to 5) may be substituted with other amino acids. ,.
[0055] 「タンパク質又は部分ペプチドの塩」としては、生理学的に許容される酸 (例、無機 酸、有機酸)や塩基 (例、アルカリ金属塩)等との塩が用いられ、とりわけ生理学的に 許容される酸付加塩が好ましい。この様な塩としては、例えば、無機酸 (例えば、塩酸 、リン酸、臭化水素酸、硫酸）との塩、あるいは有機酸 (例えば、酢酸、ギ酸、プロピオ ン酸、フマル酸、マレイン酸、コハク酸、酒石酸、クェン酸、リンゴ酸、蓚酸、安息香酸 、メタンスルホン酸、ベンゼンスルホン酸）との塩等が用いられる。  [0055] As the "salt of protein or partial peptide", a salt with a physiologically acceptable acid (eg, inorganic acid, organic acid) or base (eg, alkali metal salt) is used. Acid addition salts that are acceptable are preferred. Examples of such salts include salts with inorganic acids (for example, hydrochloric acid, phosphoric acid, hydrobromic acid, sulfuric acid), or organic acids (for example, acetic acid, formic acid, propionic acid, fumaric acid, maleic acid, Salts with succinic acid, tartaric acid, succinic acid, malic acid, succinic acid, benzoic acid, methanesulfonic acid, benzenesulfonic acid, etc. are used.
[0056] 例えば、配列番号： 18〜34からなる群より選ばれるアミノ酸配列を有するタンパク 質又は該アミノ酸配列において 1若しくは数個のアミノ酸が欠失、置換若しくは付加さ れたアミノ酸配列を有する少なくとも 1つのタンパク質又はその部分ペプチド又はそ の塩に対する抗体又はその断片を用いる方法、配列番号： 23及び Z又は 30のァミノ 酸配列を有するタンパク質又は該アミノ酸配列において 1若しくは数個のアミノ酸が 欠失、置換若しくは付加されたアミノ酸配列を有する少なくとも 1つのタンパク質又は その部分ペプチド又はその塩に対する抗体又はその断片を用いる方法が挙げられ る。以下に、具体的に記載する。 [0056] For example, a protein having an amino acid sequence selected from the group consisting of SEQ ID NOs: 18 to 34, or at least 1 having an amino acid sequence in which one or several amino acids are deleted, substituted, or added in the amino acid sequence A method using an antibody against one protein or a partial peptide thereof or a salt thereof or a fragment thereof, a protein having an amino acid sequence of SEQ ID NO: 23 and Z or 30, or a deletion of or substitution of one or several amino acids in the amino acid sequence Or a method using an antibody or fragment thereof against at least one protein having an added amino acid sequence or a partial peptide thereof or a salt thereof. The Specific description will be given below.
[0057] 免疫学的測定方法 [0057] Immunological measurement method
タンパク質の量を指標とする場合、被検対象の個体由来の被検試料及び対照試料 のそれぞれを、ポリアクリルアミドゲル電気泳動、本発明のタンパク質に対する抗体又 はその断片を用いたウェスタンプロット解析、前記抗体又はその断片を用いたィムノ アツセィ等に供し、タンパク質の量を測定し、比較することにより、遺伝子の発現レべ ルを評価することができる。  When the amount of protein is used as an index, each of a test sample and a control sample derived from an individual to be tested is subjected to polyacrylamide gel electrophoresis, Western plot analysis using an antibody against the protein of the present invention or a fragment thereof, The expression level of the gene can be evaluated by subjecting it to an immunoassay or the like using an antibody or a fragment thereof, and measuring and comparing the amount of the protein.
[0058] 「抗体」とは、本発明のタンパク質に対する抗体、その断片等が挙げられる。すなわ ち、前記抗体は、前記本発明のタンパク質に特異的に結合する能力を有するもので あれば、ポリクローナル抗体であってもよぐモノクローナル抗体であってもよい。前記 抗体は、例えば、前記本発明のタンパク質中における特定の部分断片に特異的に 結合しうる抗体であってもよい。前記抗体の作製方法は、下記に具体的に記載する。 また、得られた抗体を精製後、ぺプチダーゼ等により処理することにより、抗体の断片 が得られる。さらに、前記抗体の誘導体、例えば、キメラ抗体、ヒト化抗体、 Fabフラグ メント、単鎖抗体等や公知技術により修飾された抗体等を使用することもできる。かか る抗体又はその断片は、慣用の酵素（例えば、ペルォキシダーゼ等）、蛍光色素、放 射性物質、アビジン若しくはピオチン等で標識されて 、てもよ 、。  [0058] The "antibody" includes an antibody against the protein of the present invention, a fragment thereof, and the like. That is, the antibody may be a polyclonal antibody or a monoclonal antibody as long as it has an ability to specifically bind to the protein of the present invention. The antibody may be, for example, an antibody that can specifically bind to a specific partial fragment in the protein of the present invention. The method for producing the antibody is specifically described below. Further, the obtained antibody can be purified and then treated with peptidase to obtain antibody fragments. Furthermore, derivatives of the above-described antibodies, for example, chimeric antibodies, humanized antibodies, Fab fragments, single chain antibodies and the like, and antibodies modified by known techniques can also be used. Such an antibody or a fragment thereof may be labeled with a conventional enzyme (for example, peroxidase), a fluorescent dye, a radioactive substance, avidin or piotin.
[0059] (i)ポリクローナル抗体の作製  [0059] (i) Preparation of polyclonal antibody
本発明のタンパク質の一部のアミノ酸配列を有するペプチドを抗原として用い、動 物に投与することによりポリクローナル抗体を作製することができる。  A polyclonal antibody can be prepared by administering a peptide having a partial amino acid sequence of the protein of the present invention to an animal as an antigen.
投与する動物として、ゥサギ、ャギ、ラット、マウス、ハムスター等を用いることができ る。該抗原の投与量は動物 1匹当たり 50〜： LOO /z gが好ましい。ペプチドを用いる場 合は、ペプチドをスカシガイへモシァ-ン（keyhole limpet haemocyanin)ゃ牛チログ口 ブリン等のキャリアタンパク質に共有結合させたものを抗原とするのが望まし 、。抗原 とするペプチドは、ペプチド合成機で合成することができる。該抗原の投与は、 1回目 の投与の後 1〜2週間おきに 3〜10回行う。各投与後、 3〜7日目に眼底静脈叢より 採血し、該血清が免疫に用いた抗原と反応することを酵素免疫測定法〔酵素免疫測 定法（ELISA法）：医学書院刊 1976年、 Antibodies- A Laboratory Manual, Cold Sp ring Harbor Lavoratory (1988)〕等で確認する。 Usagi, goat, rat, mouse, hamster, etc. can be used as the animal to be administered. The dose of the antigen is preferably 50 to: LOO / zg per animal. In the case of using a peptide, it is desirable that the peptide is covalently bound to a carrier protein such as keyhole limpet haemocyanin or bovine thyroglobulin brin. The peptide used as an antigen can be synthesized with a peptide synthesizer. The antigen is administered 3 to 10 times every 1 to 2 weeks after the first administration. On the 3rd to 7th day after each administration, blood was collected from the fundus venous plexus, and the reaction of the serum with the antigen used for immunization was confirmed by enzyme immunoassay [Enzyme immunoassay (ELISA): 1976, published by Medical Shoin. Antibodies- A Laboratory Manual, Cold Sp ring Harbor Lavoratory (1988)].
免疫に用いた抗原に対し、その血清が充分な抗体価を示した非ヒトほ乳動物より血 清を取得し、該血清より、下記方法によりポリクローナル抗体を分離、精製することが できる。抗体を分離、精製する方法としては、遠心分離、 40〜50%飽和硫酸アンモ -ゥムによる塩析、力プリル酸沈殿 [Antibodies, A Laboratory manual.Cold Spring Ha rbor Laboratory, (1988)〕、又は DEAE—セファロースカラム、陰イオン交換カラム、 プロテイン A又は G—力ラムあるいはゲル濾過カラム等を用いるクロマトグラフィー等 を、単独又は組み合わせて処理する方法があげられる。  Serum can be obtained from a non-human mammal whose serum showed a sufficient antibody titer against the antigen used for immunization, and polyclonal antibodies can be separated and purified from the serum by the following method. Methods for separating and purifying antibodies include centrifugation, salting out with 40-50% saturated ammonium sulfate, force prillic acid precipitation (Antibodies, A Laboratory manual. Cold Spring Harbor Laboratory, (1988)), or Examples include a single or combination treatment of DEAE-sepharose column, anion exchange column, chromatography using protein A or G-force ram or gel filtration column, and the like.
(ii)モノクローナル抗体の作製 (ii) Production of monoclonal antibodies
(a)抗体産生細胞の調製  (a) Preparation of antibody-producing cells
免疫に用いた本発明のタンパク質の部分断片ポリペプチドに対し、その血清が十 分な抗体価を示したラットを抗体産生細胞の供給源として供する。  Rats whose sera showed sufficient antibody titers against the polypeptide fragment fragment polypeptide of the present invention used for immunization are used as a source of antibody-producing cells.
該抗体価を示したラットに抗原物質を最終投与した後 3〜7日目に、脾臓を摘出す る。 該脾臓を MEM培地（日水製薬社製）中で細断し、ピンセットでほぐし、 1, 200rp mで 5分間遠心分離した後、上清を捨てる。得られた沈殿画分の脾細胞をトリス一塩 化アンモ-ゥム緩衝液 (pH7. 65)で 1〜2分間処理し赤血球を除去した後、 MEM 培地で 3回洗浄し、得られた脾細胞を抗体産生細胞として用いる。  The spleen is removed 3 to 7 days after the final administration of the antigenic substance to the rat showing the antibody titer. The spleen is shredded in MEM medium (Nissui Pharmaceutical Co., Ltd.), loosened with forceps, centrifuged at 1,200 rpm for 5 minutes, and the supernatant is discarded. Treat the spleen cells of the obtained precipitate fraction with Tris monochloride ammonium buffer (pH 7.65) for 1-2 minutes to remove erythrocytes, and then wash with MEM medium 3 times. Cells are used as antibody producing cells.
(b)骨髄腫細胞の調製  (b) Preparation of myeloma cells
骨髄腫細胞としては、マウス又はラットから取得した株化細胞を使用する。例えば、 8—ァザグァニン耐性マウス（BALB/c由来）骨髄腫細胞株 P3- X63Ag8- U1 (以下、 P3 — U1と略す）〔Curr. Topics. Microbiol. Immunol, 81, 1 (1978)、 Europ. J. Immunol, 6, 511 (1976)] , SP2/0-Agl4(SP-2) [Nature, 276,269 (1978)〕、 P3— X63— Ag8653(653) [J. Immunol, 123, 1548 (1979)]、 P3-X63-Ag8(X63丌 Nature, 256, 495 (1975)〕等を 用いることができる。これらの細胞株は、 8—ァザグァニン培地〔RPMI-1640培地にグ ルタミン（1. 5mM)、 2—メルカプトエタノール（5 X 10- 5M)、ジェンタマイシン（10 gZml)及び牛胎児血清 (FCS) (CSL社製、 10%)を加えた培地 (以下、正常培地と いう）に、さらに 8—ァザグァニン（15 gZml)をカ卩えた培地〕で継代する力 細胞融 合の 3〜4日前に正常培地で培養し、融合には該細胞を 2 X 107個以上用いる。 (c)ハイプリドーマの作製 As myeloma cells, cell lines obtained from mice or rats are used. For example, 8-azaguanine resistant mouse (BALB / c-derived) myeloma cell line P3-X63Ag8-U1 (hereinafter abbreviated as P3—U1) [Curr. Topics. Microbiol. Immunol, 81, 1 (1978), Europ. J Immunol, 6, 511 (1976)], SP2 / 0-Agl4 (SP-2) [Nature, 276,269 (1978)], P3—X63—Ag8653 (653) [J. Immunol, 123, 1548 (1979)] P3-X63-Ag8 (X63 丌 Nature, 256, 495 (1975)), etc. These cell lines are composed of 8-azaguanine medium (RPMI-1640 medium with glutamine (1.5 mM), 2 -A medium containing mercaptoethanol (5 X 10-5M), gentamicin (10 gZml), and fetal calf serum (FCS) (CSL, 10%) (hereinafter referred to as normal medium) is further added with 8-azaguanine. The ability to subculture with (medium containing 15 gZml)] Culture in normal medium 3-4 days before cell fusion, and use 2 × 10 7 or more of these cells for fusion. (c) Production of High Pridoma
(a)で取得した抗体産生細胞と (b)で取得した骨髄腫細胞を MEM培地又は PBS ( リン酸ニナトリウム 1. 83g、リン酸一カリウム 0. 21g、食塩 7. 65g、蒸留水 1リットル 、 pH7. 2)でよく洗浄し、細胞数が、抗体産生細胞:骨髄腫細胞 = 5〜： LO : lになるよ う混合し、 1, 200rpmで 5分間遠心分離した後、上清を捨てる。  The antibody-producing cells obtained in (a) and the myeloma cells obtained in (b) are mixed with MEM medium or PBS (1.83 g disodium phosphate, 0.21 g monopotassium phosphate, 7.65 g sodium chloride, 1 liter distilled water) Wash thoroughly at pH 7.2), mix so that the number of cells is antibody-producing cells: myeloma cells = 5 to: LO: l, centrifuge at 1,200 rpm for 5 minutes, and discard the supernatant. .
得られた沈澱画分の細胞群をよくほぐし、該細胞群に、攪拌しながら、 37°Cで、 108 抗体産生細胞あたり、ポリエチレングライコ一ルー 1000 (PEG— 1000) 2g、 MEM 2ml及びジメチルスルホキシド（DMSO) O. 7mlを混合した溶液を 0. 2〜： Lml添カロし 、更に 1〜2分間毎に MEM培地 l〜2mlを数回添加する。添加後、 MEM培地をカロ えて全量が 50mlになるように調製する。該調製液を 900rpmで 5分間遠心分離後、 上清を捨てる。得られた沈殿画分の細胞を、ゆるやかにほぐした後、メスピペットによ る吸込み、吹出しでゆるやかに HAT培地〔正常培地にヒポキサンチン（10— 4M)、チミ ジン（1. 5 X 10— 5M)及びアミノプテリン (4 X 10— 7M)をカ卩えた培地〕 100ml中に懸濁 する。 Thoroughly disaggregate the cell group of the obtained precipitate fraction, and in the cell group, with stirring, at 37 ° C, 2 g of polyethylene glycoeluate 1000 (PEG-1000), MEM 2 ml and dimethyl sulfoxide (DMSO) O. 7 ml of the mixed solution is added with 0.2 to Lml, and 1-2 ml of MEM medium is added several times every 1 to 2 minutes. After the addition, prepare MEM medium so that the total volume is 50 ml. Centrifuge the preparation at 900 rpm for 5 minutes, and discard the supernatant. After the precipitated fraction of the cells obtained was gently loosened, suction that by the measuring pipette, to gently HAT medium [normal medium in blowing hypoxanthine (10- 4 M), thymidine (1. 5 X 10 - 5 M) and suspending aminopterin (4 X 10- 7 M) in Ca卩example media] 100 ml.
該懸濁液を 96穴培養用プレートに 100 1Z穴ずつ分注し、 5% COインキュベー  Dispense the suspension into a 96-well culture plate at 100 1Z holes, and add 5% CO incubator.
2  2
ター中、 37°Cで 7〜14日間培養する。培養後、培養上清の一部をとりアンチボディィ ズ L Antibodies, A Laboratorymanuai,し old Spring Harbor Laboratory, chapter 1 (l 988)〕等に述べられている酵素免疫測定法により、本発明のタンパク質の部分断片 ポリペプチドに特異的に反応するハイブリドーマを選択する。  Incubate at 37 ° C for 7-14 days. After culturing, a portion of the culture supernatant is removed and subjected to the enzyme immunoassay described in Antibodies L Antibodies, A Laboratorymanuai, old Spring Harbor Laboratory, chapter 1 (l 988)], etc. A hybridoma that specifically reacts with the polypeptide is selected.
[0061] 抗体は、例えば、 OA又は RA患者由来の試料を検出するための方法において使用 され得る。このような方法は、抗体を使用して、適切な生物学的サンプルにおける本 明細書に記載の本発明のタンパク質の存在又は非存在を検出することを包含する。 本明細書中に使用される適切な生物学的サンプルは、患者から取得した滑膜液又 は正常糸且織生検、これらのホモジェネート又は抽出物を含む。  [0061] The antibody can be used, for example, in a method for detecting a sample from an OA or RA patient. Such methods include using antibodies to detect the presence or absence of a protein of the invention described herein in a suitable biological sample. Suitable biological samples for use herein include synovial fluid or normal thread and tissue biopsies obtained from patients, homogenates or extracts thereof.
[0062] サンプル中のタンパク質を検出するための抗体の使用について、当業者に公知の 種々のアツセィ开式が存在する。例えば、 Harlow及び Lane, Anti bodies: A Laborator y Manual, Cold Spring Harbor Laboratory, 1988を参照のこと。免疫学的検出法とし ては、 ELISA法'蛍光抗体法、ウェスタンプロット法、免疫組織染色法等をあげること ができる。ウェスタンブロット形式において行う場合には、生物学的サンプル由来の ポリペプチド調製物は、ゲル電気泳動に供され、適切なメンブレンに移され、そして 抗体と反応させられる。次いで、メンブレン上の抗体の存在は、下記のように、適切な 検出試薬を用いて検出され得る。 [0062] There are a variety of access methods known to those of skill in the art for the use of antibodies to detect proteins in a sample. See, for example, Harlow and Lane, Anti bodies: A Laboratory Manual, Cold Spring Harbor Laboratory, 1988. Examples of immunological detection methods include ELISA method 'fluorescent antibody method, Western plot method, immunohistochemical staining method, etc. Can do. When performed in a Western blot format, a polypeptide preparation from a biological sample is subjected to gel electrophoresis, transferred to an appropriate membrane, and reacted with an antibody. The presence of the antibody on the membrane can then be detected using a suitable detection reagent as described below.
[0063] 別の実施態様にお!、て、検出方法は、タンパク質と結合し、そしてこれをサンプル の残りから取り出すための固相支持体に固定された抗体の使用を包含する。次いで 、結合タンパク質は、レポーター基を有する二次抗体又は試薬を用いて検出され得 る。あるいは、タンパク質がレポーター基で標識され、そして抗体とサンプルとのイン キュベーシヨン後、固定抗体と結合させられる、競合アツセィが使用され得る。サンプ ルの成分が標識タンパク質と抗体との結合を阻害する程度が、サンプルの固定抗体 との反応性を示し、そして結果として、サンプル中のタンパク質の濃度を示す。  [0063] In another embodiment, the detection method involves the use of an antibody immobilized on a solid support to bind to the protein and remove it from the rest of the sample. The binding protein can then be detected using a secondary antibody or reagent having a reporter group. Alternatively, a competitive assembly can be used, in which the protein is labeled with a reporter group and allowed to bind to the immobilized antibody after incubation of the antibody with the sample. The extent to which the sample components inhibit the binding of labeled protein to the antibody indicates the reactivity of the sample with the immobilized antibody and, as a result, the concentration of the protein in the sample.
[0064] 固相支持体は、抗体が付着し得る、当業者に公知の、任意の物質であり得る。固相 支持体は、例えば、マイクロタイタープレートの試験ゥエル、若しくは-トロセルロース フィルター、若しくは他の適切なメンブレンであり得る。あるいは、支持体は、ビーズ又 はディスク（例えば、ガラス、ガラス繊維、ラテックス又は可塑性物質 (例えば、ポリスチ レン又はポリ塩ィ匕ビ二ル)）であり得る。支持体はまた、磁性粒子又は繊維光学センサ 一（例えば、米国特許第 5,359,681号に開示される）であり得る。  [0064] The solid support can be any material known to those of skill in the art to which antibodies can be attached. The solid support can be, for example, a test well of a microtiter plate, or a -trocellulose filter, or other suitable membrane. Alternatively, the support can be a bead or disk (eg, glass, glass fiber, latex or a plastic material (eg, polystyrene or polyvinyl chloride)). The support can also be a magnetic particle or fiber optic sensor (eg, disclosed in US Pat. No. 5,359,681).
[0065] 抗体は、特許文献及び科学文献に詳細に記載されている、当業者に公知の種々 の技術を用いて、固相支持体に固定され得る。本発明の文脈において、用語「固定」 は、非共有的会合 (例えば、吸着)及び共有的付着 (抗原と支持体上の官能基との 間の直接的連結であり得る力、又は架橋剤による連結であり得る）の両方をいう。マイ クロタイタープレートのゥエル又はメンブレンへの吸着による固定が好ましい。このよう な場合において、吸着は、適切な緩衝液中の抗体を支持体と適切な時間の間接触 させることにより達成され得る。接触時間は、温度により変化するが、代表的には、約 1時間と 1日の間である。一般的に、ブラスティックマイクロタイタープレート（例えば、 ポリスチレン又はポリ塩化ビュル）のゥヱルと、約 10ngから約 1 μ g、及び好ましくは約 1 00〜200ngの範囲の量の抗体との接触力 適切な量のタンパク質を固定するに十分 である。 [0066] 抗体の固相支持体への共有的付着はまた、一般的に、支持体と抗体上の官能基（ 例えば、水酸基又はアミノ基)の両方に反応する二官能性薬剤と支持体をまず反応 させることにより達成され得る。例えば、抗体は、適切なポリマーコートを有する支持 体に、ベンゾキノンを用いるか又は支持体上のアルデヒド基と結合パートナー上のァ ミン及び活性水素との縮合により、共有的に付着され得る（例えば、 Pierce Immunote chnology Catalog and Handbook (199 1) A12〜A13を参照のこと）。 [0065] The antibody may be immobilized on a solid support using various techniques known in the art and described in detail in the patent and scientific literature. In the context of the present invention, the term “immobilization” refers to non-covalent association (eg adsorption) and covalent attachment (force that can be a direct link between an antigen and a functional group on a support, or by a cross-linking agent. Both). It is preferable to fix the microtiter plate by adsorption to a well or a membrane. In such cases, adsorption can be achieved by contacting the antibody in a suitable buffer with the support for a suitable time. The contact time varies with temperature, but is typically between about 1 hour and 1 day. In general, the contact force between a wall of a plastic microtiter plate (eg, polystyrene or polychlorinated butyl) and an amount of antibody in the range of about 10 ng to about 1 μg, and preferably about 100-200 ng Enough to fix the amount of protein. [0066] Covalent attachment of an antibody to a solid support also generally involves the attachment of a bifunctional agent and support that reacts with both the support and a functional group (eg, a hydroxyl group or an amino group) on the antibody. This can be achieved by first reacting. For example, an antibody can be covalently attached to a support having a suitable polymer coat by using benzoquinone or by condensation of an aldehyde group on the support with an amine and an active hydrogen on a binding partner (e.g., Pierce Immunotechnology Catalog and Handbook (199 1) A12-A13).
[0067] 特定の実施態様において、サンプル中のタンパク質の検出のためのアツセィは、二 抗体サンドイッチアツセィである。このアツセィは、まず、固相支持体 (通常、マイクロタ イタ一プレートのゥエル）に固定されている抗体と、生物学的サンプルとを、サンプル 中のポリペプチドが固定された抗体に結合するように接触させることにより行われ得る 。次いで、非結合サンプルは、固定されたポリペプチド 抗体複合体力 除去され、 そしてポリペプチド上の異なる部位に結合し得る二次抗体 (レポーター基を含む）が 添加される。次いで、固相支持体に結合したままの二次抗体の量は、特定のレポ一 ター基に適切な方法を用いて決定される。  [0067] In certain embodiments, the assay for the detection of a protein in a sample is a two-antibody sandwich assay. The assembly begins with binding of an antibody immobilized on a solid support (usually a microtiter plate well) and a biological sample to the antibody to which the polypeptide in the sample is immobilized. It can be done by contacting. The unbound sample is then removed of immobilized polypeptide-antibody complex force and a secondary antibody (containing a reporter group) that can bind to a different site on the polypeptide is added. The amount of secondary antibody that remains bound to the solid support is then determined using a method appropriate to the particular reporter group.
[0068] より詳細には、ー且抗体が支持体上に上記のように固定されると、支持体上の残り のポリペプチド結合部位は代表的にはブロックされる。任意の適切なブロッキング剤 ( 例えば、ゥシ血清アルブミン又は Tween 20TM(Sigma Chemical Co., St. Louis, MO)) は、当業者に公知である。次いで、固定抗体はサンプルと共にインキュベートされ、 そしてポリペプチドは抗体に結合させられる。サンプルは、インキュベーションの前に 適切な希釈剤 (例えば、リン酸緩衝化生理食塩水 (PBS))で希釈され得る。一般的に 、適切な接触時間（すなわち、インキュベーション時間）は、乳癌を有する個体から得 られたサンプル中のポリペプチドの存在を検出するに十分な時間である。好ましくは 、接触時間は、結合ポリペプチド及び非結合ポリペプチドとの間の平衡で達成される 結合レベルの少なくとも 95%の結合のレベルを達成するに十分な時間である。当業 者は、平衡を達成するに必要な時間は、一定時間にわたって生じる結合のレベルを アツセィすることにより容易に決定されることを理解する。室温にて、約 30分間のイン キュベーシヨン時間が一般的に十分である。  [0068] More specifically, and once the antibody is immobilized on the support as described above, the remaining polypeptide binding sites on the support are typically blocked. Any suitable blocking agent (eg, ushi serum albumin or Tween 20 ™ (Sigma Chemical Co., St. Louis, MO)) is known to those skilled in the art. The immobilized antibody is then incubated with the sample and the polypeptide is allowed to bind to the antibody. The sample can be diluted with an appropriate diluent (eg, phosphate buffered saline (PBS)) prior to incubation. In general, a suitable contact time (ie, incubation time) is sufficient to detect the presence of the polypeptide in a sample obtained from an individual having breast cancer. Preferably, the contact time is a time sufficient to achieve a level of binding that is at least 95% of the level of binding achieved at equilibrium between the bound and unbound polypeptides. One skilled in the art understands that the time required to achieve equilibrium is readily determined by assessing the level of binding that occurs over a period of time. An incubation time of about 30 minutes at room temperature is generally sufficient.
[0069] 次 、で、結合して!/、な 、サンプルは、固相支持体を適切な緩衝液 (例えば、 0.1 %T ween 20TMを含む PBS)で洗浄することにより除去され得る。次いで、レポーター基を 有する二次抗体が、固相支持体に添加され得る。好ましいレポーター基は、酵素（例 えば、西洋ヮサビペルォキシダーゼ）、基質、補因子、インヒビター、色素、放射性核 種、発色基、蛍光基、及びピオチンを含む。抗体とのレポーター基との結合は、当業 者に公知の標準的方法を用いて達成され得る。 [0069] Next, bind with! /, The sample should be loaded with a solid support in an appropriate buffer (eg, 0.1% T It can be removed by washing with PBS containing ween 20TM. A secondary antibody having a reporter group can then be added to the solid support. Preferred reporter groups include enzymes (eg, horse radish peroxidase), substrates, cofactors, inhibitors, dyes, radionuclides, chromophores, fluorescent groups, and piotin. Coupling of the antibody to the reporter group can be accomplished using standard methods known to those skilled in the art.
[0070] 次いで、二次抗体は、固定抗体-ポリペプチド複合体と共に、結合したポリペプチド を検出するに十分な時間インキュベートされる。適切な時間は、一般的に、所定の時 間にわたって生じる結合のレベルをアツセィすることにより決定され得る。次いで、結 合して!/、な!/、二次抗体が除去され、そして結合した二次抗体はレポーター基を用い て検出される。レポーター基を検出するために用いられる方法は、レポーター基の性 質に依存する。放射活性基については、シンチレーシヨン計数法又はオートラジオグ ラフィ一法が一般的に適切である。分光法は、色素、発色基及び蛍光基を検出する ために使用され得る。ピオチンは、異なるレポーター基 (通常は、放射活性基若しく は蛍光基又は酵素）に結合されたアビジンを用いて検出され得る。酵素レポーター 基は、一般的に、基質の添加 (一般的に特定の時間の間）、続く反応産物の分光分 析又は他の分析により検出され得る。  [0070] The secondary antibody is then incubated with the immobilized antibody-polypeptide complex for a time sufficient to detect the bound polypeptide. The appropriate time can generally be determined by assessing the level of binding that occurs over a given time. Then, binding! /,! /, The secondary antibody is removed, and the bound secondary antibody is detected using a reporter group. The method used to detect the reporter group depends on the nature of the reporter group. For radioactive groups, scintillation counting or autoradiographic methods are generally appropriate. Spectroscopy can be used to detect dyes, chromophores and fluorescent groups. Piotin can be detected using avidin coupled to different reporter groups (usually radioactive groups or fluorescent groups or enzymes). Enzyme reporter groups can generally be detected by addition of a substrate (generally for a specific time) followed by spectroscopic analysis of the reaction product or other analysis.
[0071] 疾患の存在又は非存在を決定するために、固相支持体に結合したままのレポータ 一基力 検出されるシグナルは、一般的に、正常組織力も確立された事前に決定さ れたカットオフ値に対応するシグナルと比較される。 1つの好ま 、実施態様にお!ヽ て、カットオフ値は、固定抗体が骨疾患又は関節疾患を有さない患者由来のサンプ ルとインキュベートされる場合に得られる平均シグナルである。一般的に、事前に決 定されるカットオフ値を 3標準偏差越えるシグナルを生じるサンプルは、骨疾患又は 関節疾患について陽性と考えられ得る。別の好ましい実施態様において、カットオフ 値は、 Sackettら、し linical Epidemiology: A Basic science forし linical Medicene 10り- 7 頁、（Little Brown and Co, 1985)の方法に従う Receiver Oparator Curveを用いて決 定される。簡潔に述べると、この実施態様においては、カットオフ値は、診断試験結 果についてそれぞれの可能なカットオフ値に対応する真の陽性率 (すなわち、感度） と偽陽性率（100%特異性）との対をプロットすることにより決定され得る。プロット上の 左上角に最も近いカットオフ値 (すなわち、最も大きな面積を囲む値）力 最も正確な カットオフ値であり、この方法により決定されるカットオフ値よりも高いシグナルを生じ るサンプルは、陽性であると考えられ得る。あるいは、カットオフ値は、偽陽性率を最 少にするためにプロットに沿って左に、又は偽陰性率を最少にするために右にシフト され得る。 [0071] To determine the presence or absence of the disease, the reporter stays attached to the solid support. The force detected is generally determined in advance when normal tissue force is also established. Compared to the signal corresponding to the cutoff value. In one preferred embodiment, the cut-off value is the average signal obtained when the immobilized antibody is incubated with a sample from a patient without bone or joint disease. In general, a sample that produces a signal that is 3 standard deviations above the predetermined cutoff value can be considered positive for bone or joint disease. In another preferred embodiment, the cut-off value is determined using a Receiver Oparator Curve according to the method of Sackett et al., Linical Epidemiology: A Basic science for linical Medicene 10 page 7, (Little Brown and Co, 1985). Determined. Briefly, in this embodiment, the cutoff value is the true positive rate (ie sensitivity) and false positive rate (100% specificity) corresponding to each possible cutoff value for diagnostic test results. Can be determined by plotting the pair with. On the plot Cut-off value closest to the upper left corner (ie the value that encloses the largest area) force The sample that is the most accurate cut-off value and produces a signal higher than the cut-off value determined by this method is positive Can be considered. Alternatively, the cut-off value can be shifted to the left along the plot to minimize the false positive rate, or to the right to minimize the false negative rate.
[0072] 関連する実施態様にお!、て、フロースルー試験又はストリップ試験形式にぉ 、て本 発明のタンパク質の検出が行われる。ここで、抗体はメンブレン (例えば、ニトロセル口 ース）に固定されている。フロースルー試験において、サンプル中のポリペプチドは、 サンプルカ^ンプレンを通り抜けると、固定抗体に結合する。次いで、標識した二次 抗体が、二次抗体を含む溶液カ^ンプレンを通って流れると抗体 ポリペプチド複合 体に結合する。次いで、結合した二次抗体の検出は、上記のように行われ得る。ストリ ップ試験形式においては、抗体が結合しているメンブレンの一方の端力 サンプルを 含む溶液に浸される。サンプルは、二次抗体を含む領域を通り、そして固定抗体の 範囲へメンブレンに沿って移動する。固定抗体の範囲での二次抗体の濃度力 骨疾 患又は関節疾患の存在を示す。代表的には、この部位での二次抗体の濃度は、視 覚的に読み取り得るパターン (例えば、線)を生じる。このようなパターンが存在しなけ れば、結果が陰性であることが示される。一般的に、メンブレン上に固定される抗体 の量は、生物学的サンプル力 上で議論した形式の二抗体サンドイッチアツセィにお いて陽性のシグナルを生じるに十分なレベルのポリペプチドを含む場合に、視覚的 に判別し得るパターンを生じるように選択される。好ましくは、メンブレン上に固定され る抗体の量は、約 25ng〜約 1 μ gの範囲であり、そしてより好ましくは約 50ng〜約 1 μ gの範囲である。このような試験は、代表的には、非常に少量の生物学的サンプルを 用いて行われ得る。  [0072] In a related embodiment, the detection of the protein of the present invention is performed in a flow-through test or strip test format. Here, the antibody is immobilized on a membrane (for example, a nitrocell mouth). In the flow-through test, the polypeptide in the sample binds to the immobilized antibody as it passes through the sample canvas. The labeled secondary antibody then flows through the solution canvas containing the secondary antibody and binds to the antibody polypeptide complex. Detection of bound secondary antibody can then be performed as described above. In the strip test format, one end of the membrane to which the antibody is bound is immersed in a solution containing the sample. The sample moves along the membrane through the area containing the secondary antibody and into the area of the immobilized antibody. Secondary antibody concentration in the range of fixed antibodies. Indicates the presence of bone or joint disease. Typically, the concentration of secondary antibody at this site produces a pattern (eg, a line) that can be read visually. The absence of such a pattern indicates a negative result. In general, the amount of antibody immobilized on the membrane is sufficient if it contains a sufficient level of polypeptide to produce a positive signal in a two-antibody sandwich assembly of the type discussed above in terms of biological sample power. Chosen to produce a visually discernable pattern. Preferably, the amount of antibody immobilized on the membrane ranges from about 25 ng to about 1 μg, and more preferably from about 50 ng to about 1 μg. Such tests can typically be performed using very small biological samples.
[0073] さらに、本発明の骨疾患若しくは関節疾患に罹患している疑いがある個体由来の 試料の検出又は選別方法は、骨疾患若しくは関節疾患の診断方法にも使用すること ができる。このような診断方法も、本発明に包含される。本発明の診断方法を、簡便、 迅速に、高処理量で、高い信頼性で行ないうる、前記プローブ及び Z又はプライマ 一対を含有した診断薬、又は前記タンパク質に対する抗体若しくはその断片を含有 した診断薬が提供される。 [0073] Furthermore, the method for detecting or selecting a sample derived from an individual suspected of suffering from a bone disease or joint disease of the present invention can also be used as a diagnostic method for bone disease or joint disease. Such a diagnostic method is also encompassed by the present invention. A diagnostic agent containing the probe and Z or primer pair, or an antibody against the protein or a fragment thereof, which can perform the diagnostic method of the present invention simply, rapidly, with high throughput and high reliability Diagnostics are provided.
[0074] 例えば、以下の診断薬も本発明の範囲内である。  [0074] For example, the following diagnostic agents are also within the scope of the present invention.
配列番号： 18〜34からなる群より選ばれるアミノ酸配列を有するタンパク質又 は該アミノ酸配列において 1若しくは数個のアミノ酸が欠失、置換若しくは付加された アミノ酸配列を有するタンパク質をコードする少なくとも 1つの遺伝子を検出しうる核 酸を含有してなる骨疾患若しくは関節疾患の診断薬、  SEQ ID NO :: at least one gene encoding a protein having an amino acid sequence selected from the group consisting of 18 to 34, or a protein having an amino acid sequence in which one or several amino acids are deleted, substituted or added in the amino acid sequence A diagnostic agent for bone disease or joint disease containing a nuclear acid capable of detecting
該遺伝子が、配列番号： 1〜17からなる群より選ばれる塩基配列からなる核酸 である骨疾患若しくは関節疾患の診断薬、  A diagnostic agent for bone disease or joint disease, wherein the gene is a nucleic acid comprising a base sequence selected from the group consisting of SEQ ID NOs: 1 to 17,
該遺伝子が、配列番号: 6及び Z又は 13の塩基配列からなる核酸である骨疾 患若しくは関節疾患の診断薬の診断薬、  A diagnostic agent for a diagnostic agent for bone disease or joint disease, wherein the gene is a nucleic acid comprising the nucleotide sequence of SEQ ID NO: 6 and Z or 13;
配列番号： 35及び Z又は 36の塩基配列力もなる核酸を含有してなる、骨疾患 若しくは関節疾患の診断薬、  A diagnostic agent for bone disease or joint disease, comprising a nucleic acid having a nucleotide sequence of SEQ ID NO: 35 and Z or 36,
配列番号： 18〜34からなる群より選ばれるアミノ酸配列を有するタンパク質又 は該アミノ酸配列において 1若しくは数個のアミノ酸が欠失、置換若しくは付加された アミノ酸配列を有する少なくとも 1つのタンパク質又はその部分ペプチド又はその塩 に対する抗体又はその断片を含有してなる、骨疾患若しくは関節疾患の診断薬、 A protein having an amino acid sequence selected from the group consisting of SEQ ID NO: 18 to 34, or at least one protein having an amino acid sequence in which one or several amino acids are deleted, substituted or added in the amino acid sequence, or a partial peptide thereof Or a diagnostic agent for bone disease or joint disease, comprising an antibody against a salt thereof or a fragment thereof,
- 配列番号： 23及び Z又は 30のアミノ酸配列を有するタンパク質又は該アミノ酸 配列において 1若しくは数個のアミノ酸が欠失、置換若しくは付加されたアミノ酸配列 を有する少なくとも 1つのタンパク質又はその部分ペプチド又はその塩に対する抗体 又はその断片を含有してなる、骨疾患若しくは関節疾患の診断薬。 -A protein having the amino acid sequence of SEQ ID NO: 23 and Z or 30, or at least one protein having an amino acid sequence in which one or several amino acids are deleted, substituted or added in the amino acid sequence, or a partial peptide thereof or a salt thereof A diagnostic agent for bone disease or joint disease, comprising an antibody against or a fragment thereof.
[0075] 本発明の診断薬には、検出用試薬、緩衝液、対照試料、本発明の診断方法の説 明書等をさらに含有させてもよい。 [0075] The diagnostic agent of the present invention may further contain a detection reagent, a buffer solution, a control sample, a description of the diagnostic method of the present invention, and the like.
[0076] また、本発明は、以下のスクリーニング方法も包含する。  [0076] The present invention also includes the following screening methods.
配列番号： 18〜34からなる群より選ばれるアミノ酸配列を有するタンパク質又 は該アミノ酸配列において 1若しくは数個のアミノ酸が欠失、置換若しくは付加された アミノ酸配列を有するタンパク質をコードする少なくとも 1つの遺伝子を用いることを特 徴とする骨疾患又は関節疾患の予防剤及び Z又は治療剤のスクリーニング方法、 配列番号： 18〜34からなる群より選ばれるアミノ酸配列を有するタンパク質又 は該アミノ酸配列において 1若しくは数個のアミノ酸が欠失、置換若しくは付加された アミノ酸配列を有する少なくとも 1つのタンパク質若しくはその部分ペプチド又はその 塩を用いることを特徴とする骨疾患又は関節疾患の予防剤及び Z又は治療剤のスク リーニング方法。 SEQ ID NO :: at least one gene encoding a protein having an amino acid sequence selected from the group consisting of 18 to 34, or a protein having an amino acid sequence in which one or several amino acids are deleted, substituted or added in the amino acid sequence A method for screening a preventive agent and a Z or therapeutic agent for bone diseases or joint diseases, characterized by using a protein having an amino acid sequence selected from the group consisting of SEQ ID NOs: 18 to 34, or Use of at least one protein having an amino acid sequence in which one or several amino acids are deleted, substituted or added in the amino acid sequence, a partial peptide thereof, or a salt thereof, And Z or therapeutic agent screening method.
[0077] スクリーニング方法に使用するタンパク質は、ヒトゃ温血動物の細胞若しくはそれら の細胞が存在するあらゆる組織等に由来するタンパク質であってもよぐ合成タンパ ク質であってもよい。「1若しくは数個のアミノ酸が欠失、置換若しくは付加されたアミ ノ酸配列」を有するタンパク質は、前記の「検出又は選別方法」において発現を測定 されるタンパク質と同意義である。  [0077] The protein used for the screening method may be a protein derived from human warm-blooded animal cells or any tissue in which those cells are present, or may be a synthetic protein. A protein having an “amino acid sequence in which one or several amino acids have been deleted, substituted or added” has the same meaning as the protein whose expression is measured in the “detection or selection method” described above.
[0078] 本発明のタンパク質の製造方法  [0078] Protein production method of the present invention
本発明のタンパク質は、モレキュラ^ ~ ·クロー-ング第 2版、カレント 'プロトコールズ' イン'モレキュラー 'バイオロジーサプルメント 1〜38等に記載された方法等を用い、 例えば以下の方法により、タンパク質をコードしている遺伝子を宿主細胞中で発現さ せ、製造することができる。  The protein of the present invention can be obtained by using the method described in Molecular Cloning 2nd Edition, Current 'Protocols' in 'Molecular' Biology Supplement 1-38, etc., for example, by the following method. The encoded gene can be expressed and produced in the host cell.
[0079] タンパク質をコードする全長 DNAを基にして、必要に応じて、該タンパク質をコード する部分を含む適当な長さの DNA断片を調製する。また、該タンパク質をコードする 部分の塩基配列を、宿主の発現に最適なコドンとなるように、塩基を置換した DNAを 調製する。該 DNAは該タンパク質の生産率を向上させる上で有用である。該 DNA 断片、又は全長 DNAを適当な発現ベクターのプロモーターの下流に挿入することに より、組換え体 DNA (組換えベクター）を作製する。該組換えベクターを、該発現べク ターに適合した宿主細胞に導入することにより、タンパク質を生産する形質転換体を 得ることができる。  [0079] Based on the full-length DNA encoding the protein, a DNA fragment of an appropriate length containing a portion encoding the protein is prepared as necessary. In addition, a DNA is prepared by substituting the base sequence of the portion encoding the protein so that the codon is optimal for host expression. The DNA is useful for improving the production rate of the protein. A recombinant DNA (recombinant vector) is prepared by inserting the DNA fragment or full-length DNA downstream of the promoter of an appropriate expression vector. A transformant producing a protein can be obtained by introducing the recombinant vector into a host cell suitable for the expression vector.
[0080] 宿主細胞としては、原核細胞、酵母、動物細胞、植物細胞、昆虫細胞等、 目的とす る遺伝子を発現できるものであればいずれも用いることができる。発現ベクターとして は、上記宿主細胞において自立複製が可能、又は染色体中への組込みが可能で、 本発明のタンパク質の遺伝子の転写に適した位置にプロモーターを含有しているも のが用いられる。  [0080] As the host cell, any prokaryotic cell, yeast, animal cell, plant cell, insect cell, etc. can be used as long as it can express the target gene. As the expression vector, a vector that can replicate autonomously in the host cell or can be integrated into a chromosome and contains a promoter at a position suitable for transcription of the gene of the protein of the present invention is used.
[0081] (i)細菌等の原核生物を宿主細胞として用いる場合 本発明のタンパク質の発現ベクターは、原核生物中で自立複製可能であると同時 に、プロモーター、リボソーム結合配列、本発明のタンパク質、転写終結配列、より構 成されて!/ヽることが好まし ヽ。プロモーターを制御する遺伝子が含まれて!/ヽてもよ!/、。 発現ベクターとしては、例えば、 pBTrp2、 pBTacl、 pBTac2 (いずれもベーリンガー マンハイム社より巿販）、 PKK233- 2 (フアルマシア社）、 pSE280 (インビトロジェン社）、 pGEMEX-Ι〔プロメガ (Promega)社製〕、 pQE- 8 (キアゲン (QIAGEN)社製）、 pKYPIO ( 特開昭 58- 110600)、 pKYP200〔Agric. Biol. Chem., 48, 669(1984)〕、 pLSAl [Agric. B iol. Chem., 53, 277 (1989)〕、 pGELl〔Proc. Natl. Acad. Sci. USA, 82, 4306 (1985)〕、 pBluescript II SK+ (ストラタジーン社製）、 pBluescript II SK (-) (ストラタジーン社製）、 p Trs30 (FERM BP- 5407)、 pTrs32(FERM BP- 5408)、 pGHA2(FERM BP- 400)、 pGKA2 (FERM B- 6798)、 pTerm2 (特開平 3- 22979、 US4686191、 US4939094, US5160735) , pEG400 [J. BacterioL, 172, 2392 (1990)〕、 pGEX (フアルマシア社製）、 pETシステム（ ノバジェン社製）、 pSupex、 pUB110、 pTP5、 pC194、 pTrxFus (Invitrogen社製）、 pMA L-c2 (New England Biolabs社製）、 pUC19〔Gene, 33, 103 (1985)〕、 pSTV28 (宝酒造 社製）、 pUC118 (宝酒造社製）、 pPAl (特開昭 63-233798)等を例示することができる プロモーターとしては、大腸菌等の宿主細胞中で発現できるものであればいかなる ものでもよい。例えば、 trpプロモーター（Ptrp)、 lacプロモーター（Plac)、 PLプロモー ター、 PRプロモーター、 PSEプロモーター等の、大腸菌やファージ等に由来するプロ モーター、 S P01プロモーター、 SP02プロモーター、 penPプロモーター等をあげるこ とができる。また Ptrpを 2つ直列させたプロモーター（Ptrp x2)、 tacプロモーター、 lacT 7プロモーター、 let Iプロモーターのように人為的に設計改変されたプロモーター等も 用!/、ることができる。 [0081] (i) When prokaryotes such as bacteria are used as host cells The protein expression vector of the present invention is preferably composed of a promoter, a ribosome binding sequence, a protein of the present invention, and a transcription termination sequence as well as being capable of autonomous replication in prokaryotes!ヽ. Includes genes that control the promoter! Examples of expression vectors include pBTrp2, pBTacl, pBTac2 (all sold by Boehringer Mannheim), PKK233-2 (Falmacia), pSE280 (Invitrogen), pGEMEX-EX (manufactured by Promega), pQE -8 (QIAGEN), pKYPIO (JP-A 58-110600), pKYP200 [Agric. Biol. Chem., 48, 669 (1984)], pLSAl [Agric. Biol. Chem., 53, 277 (1989)], pGELl [Proc. Natl. Acad. Sci. USA, 82, 4306 (1985)], pBluescript II SK + (Stratagene), pBluescript II SK (-) (Stratagene), p Trs30 (FERM BP-5407), pTrs32 (FERM BP-5408), pGHA2 (FERM BP-400), pGKA2 (FERM B-6798), pTerm2 (JP 3-22979, US4686191, US4939094, US5160735), pEG400 [J BacterioL, 172, 2392 (1990)], pGEX (manufactured by Pharmacia), pET system (manufactured by Novagen), pSupex, pUB110, pTP5, pC194, pTrxFus (manufactured by Invitrogen), pMA L-c2 (New England Biolabs), pUC19 [Gene, 33, 103 (1985)], pSTV28 (Takara Shuzo), pUC118 (Takara Shuzo), pPAl (Japanese Patent Laid-Open No. 63-233798) and the like. Any substance can be used as long as it can be expressed in a host cell such as Escherichia coli. For example, promoters derived from E. coli and phage, such as trp promoter (Ptrp), lac promoter (Plac), PL promoter, PR promoter, PSE promoter, SP01 promoter, SP02 promoter, penP promoter, etc. Can do. It is also possible to use an artificially designed and modified promoter such as a promoter in which two Ptrps are connected in series (Ptrp x2), tac promoter, lacT 7 promoter, let I promoter, etc.
リボソーム結合配列であるシャイン一ダルガノ（Shine-Dalgarno)配列と開始コドンと の間を適当な距離 (例えば 6〜18塩基）に調節したプラスミドを用いることが好ま Uヽ 。本発明のタンパク質の遺伝子の発現には転写終結配列は必ずしも必要ではな ヽ が、構造遺伝子直下に転写終結配列を配置することが好ま Uヽ。  It is preferable to use a plasmid in which the distance between the Shine-Dalgarno sequence, which is a ribosome binding sequence, and the initiation codon is adjusted to an appropriate distance (eg, 6 to 18 bases). Although the transcription termination sequence is not necessarily required for the expression of the gene of the protein of the present invention, it is preferable to place the transcription termination sequence directly under the structural gene.
宿主細胞としては、ェシエリヒア属、セラチア属、バチルス属、ブレビバクテリウム属、 コリネバクテリウム属、ミクロバクテリウム属、シユードモナス属等に属する微生物、例 は、 Escherichia coli XLl— Blue、 Escherichia coli XL2— Blue、 Escherichia coli DH1、 Escherichia coli MC1000、 Escherichia coli KY3276、 Eschericnia coli W1485、 Escher icnia coli JM109、 Escherichia coli HB101、 Escherichia coli No.49、 Escherichia coli W3110、 Escherichia coli NY49、 Escherichia coli BL21(DE3)、 Escherichia coli BL21( DE3)pLysS、 Escherichia coli HMS174(DE3)、 Escherichia coli HMS174(DE3)pLysS、 S erratia ficaria、 Serratia fonticola、 Serratia liquefaciens、 Serratia marcescens、 Bacillus subtilis、 Bacillus amyloliquefaciens、 Brevibacterium ammmoniagenes、 Brevibacteriu m immariophilum ATCC 14068、 Brevibacterium saccharolyticumATCC 1406b、 Coryn ebacterium glutamicum ATCC13032、 Corynebacterium glutamicum ATCC14067、 C orynebacterium glutamicum ATCC13869、 Corynebacterium acetoacidophilum ATCC 13870、 Microbacterium ammoniaphilum ATCC15354、 Pseudomonas sp. D- 0110等を あげることができる。 Host cells include Escherichia, Serratia, Bacillus, Brevibacterium, Microorganisms belonging to the genus Corynebacterium, Microbacterium, Syudomonas, etc., such as Escherichia coli XLl-Blue, Escherichia coli XL2-Blue, Escherichia coli DH1, Escherichia coli MC1000, Escherichia coli KY3276, Eschericnia coli W1485, Escher icnia coli JM109, Escherichia coli HB101, Escherichia coli No.49, Escherichia coli W3110, Escherichia coli NY49, Escherichia coli BL21 (DE3), Escherichia coli BL21 (DE3) pLysS, Escherichia coli HMS174 (DE3), Escherichia coli HMS174 (DE3) pLysS , Serratia ficaria, Serratia fonticola, Serratia liquefaciens, Serratia marcescens, Bacillus subtilis, Bacillus amyloliquefaciens, Brevibacterium ammmoniagenes, Brevibacteriu m immariophilum ATCC 14068, Brevibacterium saccharolyticum ATCC 1406b, Corynmic ATCC 1406b, Coryn emicum 13870, Microbac terium ammoniaphilum ATCC 15354, Pseudomonas sp. D-0110, and the like.
組換えベクターの導入方法としては、上記宿主細胞へ DNAを導入する方法であれ ばいずれも用いることができ、例えば、エレクト口ポレーシヨン法 [Nucleic Acids Res., 16, 6127 (1988)〕、カルシウムイオンを用いる方法〔Proc. Natl. Acad. Sci. USA, 69, 2 110 (1972)〕、プロトプラスト法（特開昭 63- 2483942)、 Gene, 17, 107 (1982)や Molecul ar & General Genetics, 168, 111 (1979)に記載の方法等をあげることができる。  As a method for introducing a recombinant vector, any method can be used as long as it is a method for introducing DNA into the host cell. For example, the Elect Mouth Position Method [Nucleic Acids Res., 16, 6127 (1988)], calcium ion [Proc. Natl. Acad. Sci. USA, 69, 2 110 (1972)], protoplast method (JP-A 63-2483942), Gene, 17, 107 (1982) and Molecular & General Genetics, 168 , 111 (1979).
(ii)酵母を宿主細胞として用いる場合 (ii) When yeast is used as a host cell
酵母菌株を宿主細胞として用いる場合には、発現ベクターとして、例えば、 YEpl3 ( ATCC37115)、 YEp24 (ATCC37051)、 YCp50 (ATCC37419)、 pHS19、 pHS15等を例 示することができる。プロモーターとしては、酵母菌株中で発現できるものであればい かなるものでもよぐ例えば、 PH05プロモーター、 PGKプロモーター、 GAPプロモータ 一、 ADHプロモーター、 gal 1プロモーター、 gal 10プロモーター、ヒートショックタンパ ク質プロモーター、 MF a 1プロモーター、 CUP 1プロモーター等をあげることができる 宿主細胞としては、サッカロマイセス属、シゾサッカロマイセス属、クルイべ口ミセス 属、トリコスポロン属、シヮ-ォミセス属、ピチア属等に属する酵母菌株をあげることが でき、具体的には、 Saccharomyces cerevisiae、 schizosaccharomyces pombe、 Kluyver omyces lactis、 richosporon pullulans、 Schwanniomyces alluvius、 Pichia pastoris等を あげることができる。組換えベクターの導入方法としては、酵母に DNAを導入する方 法であればいずれも用いることができ、例えば、エレクト口ポレーシヨン法 [Methods. E nzymol, 194, 182 (1990)〕、スフエロプラスト法〔Proc. Natl. Acad. Sci. USA.84, 1929 (1978)〕、酢酸リチウム法〔J. BacterioL, 153, 163(1983)〕、 Proc. Natl. Acad. Sci. USA , 75, 1929 (1978)記載の方法等をあげることができる。 When yeast strains are used as host cells, examples of expression vectors include YEpl3 (ATCC37115), YEp24 (ATCC37051), YCp50 (ATCC37419), pHS19, pHS15 and the like. Any promoter can be used as long as it can be expressed in yeast strains.For example, PH05 promoter, PGK promoter, GAP promoter 1, ADH promoter, gal 1 promoter, gal 10 promoter, heat shock protein promoter, Examples of host cells that can include MF a 1 promoter, CUP 1 promoter and the like include yeast strains belonging to the genus Saccharomyces, Schizosaccharomyces, Kluybe mouth genus, Trichosporon genus, Schizophyllum genus, Pichia genus, etc. Can give Specific examples include Saccharomyces cerevisiae, schizosaccharomyces pombe, Kluyveromyces lactis, richosporon pullulans, Schwanniomyces alluvius, Pichia pastoris, and the like. As a method for introducing a recombinant vector, any method can be used as long as it introduces DNA into yeast. For example, the electopore position method [Methods Enzymol, 194, 182 (1990)], Spheroplast (Proc. Natl. Acad. Sci. USA. 84, 1929 (1978)), lithium acetate method (J. BacterioL, 153, 163 (1983)), Proc. Natl. Acad. Sci. USA, 75, 1929 ( 1978) and the like.
(iii)動物細胞を宿主細胞として用いる場合 (iii) When animal cells are used as host cells
動物細胞を宿主細胞として用いる場合には、発現ベクターとして、例えば、 pcDN Al/Amp, pcDNAI、 pCDM8 (いずれもフナコシ社より市販）、 pAGE107〔特開 平 3— 22979、 Cytotechnology, 3, 133 (1990)〕、 pCR— Bluntll— TOPO、 pR ΕΡ4 (インビトロジェン社製）、 pAGE103〔J. Biochem., 101, 1307 (1987)〕、 pAMo、 pAMoA [J. Biol. Chem., 268, 22782-22787 (1993)、別名 pAMoPRSA (特開平 05- 336963)〕、 pAS3— 3 (特開平 2- 227075)、 pHM6、 pHB6 (ロシュダイァグノスティッ タス社製）、 PKK223— 3、 pGEX (アマシャムバイオテク社製）、 pET— 3、 pET— 11 、 pBluescriptll (登録商標） SK ( + )ゝ pBluescriptll (登録商標） SK (—）（ストラ タジーン社製）、 pUC19、 pTrxFus (インビトロジェン社製）、 pUC118、 pSTV28 (タ カラバイオ社製）、 pMAL— c2X (ニューイングランドバイオラボ社製)等を例示するこ とができる。使用されるベクターとしては、前記遺伝子を組み込んで動物細胞で発現 できるベクターであれば 、ずれでもよ 、。  When animal cells are used as host cells, examples of expression vectors include pcDN Al / Amp, pcDNAI, pCDM8 (all commercially available from Funakoshi), pAGE107 (JP-A-3-22979, Cytotechnology, 3, 133 (1990). )], PCR—Bluntll—TOPO, pR 4 (manufactured by Invitrogen), pAGE103 (J. Biochem., 101, 1307 (1987)), pAMo, pAMoA [J. Biol. Chem., 268, 22782-22787 (1993) ), Also known as pAMoPRSA (Japanese Patent Laid-Open No. 05-336963)], pAS3-3 (Japanese Patent Laid-Open No. 2-227075), pHM6, pHB6 (Roche Diagnostatus), PKK223-3, pGEX (Amersham Biotech), pET-3, pET-11, pBluescriptll (registered trademark) SK (+) ゝ pBluescriptll (registered trademark) SK (—) (Stratagene), pUC19, pTrxFus (Invitrogen), pUC118, pSTV28 (Takara Bio) And pMAL-c2X (manufactured by New England Biolabs). Any vector may be used as long as it is a vector that can be expressed in animal cells by incorporating the gene.
プロモーターとしては、動物細胞中で発現できるものであればいずれも用いることが でき、例えば、サイトメガロウィルス（ヒト CMV)の IE (immediateearly)遺伝子のプロモ 一ター、 SV40の初期プロモーター、モロ-^ ~ ·ミュリン'ロイケミア'ウィルス（Moloney Murine Leukemia Virus)のロング'ターミナノレ'リピート 'プロモーター (Long Terminal Repeat Promoter) ,レトロウイノレスのプロモーター、ヒートショックプロモーター、 SR a プロモーター、あるいはメタ口チォネインのプロモーター等をあげることができる。また 、ヒト CMVの IE遺伝子のェンハンサーをプロモーターと共に用いてもよ!、。  Any promoter can be used as long as it can be expressed in animal cells. For example, cytomegalovirus (human CMV) IE (immediateearly) gene promoter, SV40 early promoter, Moro- ^ ~ · Long terminal repeat promoter, long terminal repeat promoter of Moloney Murine Leukemia Virus, retrowinores promoter, heat shock promoter, SRa promoter, or meta-mouth tinee promoter Can do. You can also use the human CMV IE gene enhancer with a promoter!
宿主細胞としては、マウス'ミエローマ細胞、ラット 'ミエローマ細胞、マウス'ハイブリ ドーマ細胞、チャイニーズ'ノ、ムスターの細胞である CHO細胞、 BHK細胞、アフリカミ ドリザル腎臓細胞、ヒトの細胞である Namalwa細胞又は Namalwa KJM-1細胞、ヒト胎児 腎臓細胞、ヒト白血病細胞、 HBT5637 (特開昭 63— 299)、ヒト大腸癌細胞株等をあげ ることができる。マウス'ミエローマ細胞としては、 SP2/0、 NSO等、ラット 'ミエローマ細 胞としては YB2/0等、ヒト胎児腎臓細胞としては HEK293(ATCC: CRL-1573)等、ヒト 白血病細胞としては BALL-1等、アフリカミドリザル腎臓細胞としては COS-l、 COS-7 、ヒト大腸癌細胞株としては HCT-15等をあげることができる。 Host cells include mouse 'myeloma cells, rat' myeloma cells, mouse 'hybrids. Dormer cells, Chinese's cells, CHO cells that are Muster cells, BHK cells, African monkey kidney cells, Namalwa cells or Namalwa KJM-1 cells that are human cells, human fetal kidney cells, human leukemia cells, HBT5637 Sho 63-299), and human colorectal cancer cell lines. Mouse 'myeloma cells include SP2 / 0, NSO, etc., rat' myeloma cells include YB2 / 0, human fetal kidney cells such as HEK293 (ATCC: CRL-1573), and human leukemia cells such as BALL-1. Examples of African green monkey kidney cells include COS-1 and COS-7, and examples of human colon cancer cell lines include HCT-15.
組換えベクターの導入方法としては、動物細胞に DNAを導入する方法であれば ヽ ずれも用いることができ、例えば、エレクト口ポレーシヨン法〔Cytotechnology, 3, 133 ( 1990)〕、リン酸カルシウム法（特開平 2- 227075)、リボフヱクシヨン法〔Proc. Natl. Acad . Sci. USA, 84, 7413 (1987)〕、 virology, 52, 456 (1973)に記載の方法等をあげること ができる。  As a method for introducing a recombinant vector, any method can be used as long as it is a method for introducing DNA into animal cells. 2-227075), the ribofusion method [Proc. Natl. Acad. Sci. USA, 84, 7413 (1987)], virology, 52, 456 (1973), and the like.
(iv)植物細胞を宿主細胞として用いる場合 (iv) When plant cells are used as host cells
植物細胞又は植物個体を宿主として用いる場合には、公知の方法〔組織培養， 20 ( 1994)、組織培養， 21 (1995)、 Trends in Biotechnology, 15, 45 (1997)]に準じてタン ノ ク質を生産することができる。発現ベクターとして、例えば、 Tiプラスミド、タバコモ ザイクウィルスベクター等をあげることができる。  When a plant cell or plant individual is used as a host, a tank is prepared according to a known method [tissue culture, 20 (1994), tissue culture, 21 (1995), Trends in Biotechnology, 15, 45 (1997)]. Can produce quality. Examples of expression vectors include Ti plasmid and tobacco mosaic virus vector.
遺伝子発現に用いるプロモーターとしては、植物細胞中で発現できるものであれば いずれも用いることができ、例えば、カリフラワーモザイクウィルス（CaMV)の 35Sプロ モーター、ィネアクチン 1プロモーター等をあげることができる。また、プロモーターと 発現させる遺伝子の間に、トウモロコシのアルコール脱水素酵素遺伝子のイントロン 1 などを挿入することにより、遺伝子の発現効率をあげることもできる。  Any promoter can be used as long as it can be expressed in plant cells, and examples thereof include a cauliflower mosaic virus (CaMV) 35S promoter, an actin-1 promoter, and the like. In addition, gene expression efficiency can be increased by inserting intron 1 of the maize alcohol dehydrogenase gene between the promoter and the gene to be expressed.
宿主細胞としては、ポテト、タバコ、トウモロコシ、イネ、アブラナ、大豆、トマト、ニン ジン、小麦、大麦、ライ麦、アルファルファ、亜麻等の植物細胞等をあげることができ る。  Examples of host cells include plant cells such as potato, tobacco, corn, rice, rape, soybean, tomato, carrot, wheat, barley, rye, alfalfa and flax.
組換えベクターの導入方法としては、植物細胞に DNAを導入する方法であれば ヽ ずれも用いることができ、例えば、ァグロバタテリゥム (Agrobacterium) (特開昭 59- 140 885、特開昭 60-70080、 WO94/00977)、エレクト口ポレーシヨン法（特開昭 60-251887 )、パーティクルガン (遺伝子銃）を用いる方法 (特許第 2606856、特許第 2517813)等 をあげることができる。 As a method for introducing a recombinant vector, any method can be used as long as it is a method for introducing DNA into plant cells. For example, Agrobacterium (JP 59-140 885, JP 60-70080, WO94 / 00977), Elect Mouth Position Method (Japanese Patent Laid-Open No. 60-251887) ), A method using a particle gun (gene gun) (Patent No. 2606856, Patent No. 2517813) and the like.
(V)昆虫細胞を宿主細胞として用いる場合 (V) When insect cells are used as host cells
昆虫細胞を宿主として用いる場合には、例えば、バキュロウィルス'イクスプレツショ ン'へグタ ~~ス /' ·フホフトリ ~~ 'マ二ユ ノレ LBaculovirus Expression Vectors, A Labor atory Manual, W. H. Freeman and Company, NewYork (1992)]、モレキュフ ~~ ·ノ ィ ォロジ一ァ'ラボラトリー 'マ-ユアノレ (Molecular Biology, A Laboratory Manual)、力 レント.プロトコ一ルズ.イン.モレキュラ^ ~ ·バイオロジーサプルメント 1〜38 (Current Protocols in Molecular Biology)、 Bio/Technology, 6, 47 (1988)等に記載された方法 によって、タンパク質を発現することができる。  When insect cells are used as hosts, for example, baculovirus 'expression' hetagus ~~ su / 'Fukhofori ~~' Many Yunole LBaculovirus Expression Vectors, A Laboratory Manual, WH Freeman and Company, NewYork ( 1992)], Molecule ~~ Norology 'Laboratory' Ma-Yuanore (Molecular Biology, A Laboratory Manual), Rent.Protocols.In.Molecular ^ ~ · Biology Supplements 1 to 38 (Current Protocols In Molecular Biology), Bio / Technology, 6, 47 (1988), etc., proteins can be expressed.
即ち、組換え遺伝子導入ベクター及びバキュロウィルスを昆虫細胞に共導入して昆 虫細胞培養上清中に組換えウィルスを得た後、さらに組換えウィルスを昆虫細胞に 感染させ、タンパク質を発現させることができる。該方法において用いられる遺伝子 導入ベクターとしては、例えば、 pVL1392、 pVL1393、 pBlueBacIII (すべてインビトロジ ェン社製)等をあげることができる。  That is, a recombinant gene transfer vector and a baculovirus are co-introduced into insect cells to obtain a recombinant virus in an insect cell culture supernatant, and then the insect cells are further infected with the recombinant virus to express a protein. Can do. Examples of the gene transfer vector used in the method include pVL1392, pVL1393, pBlueBacIII (all manufactured by Invitrogen) and the like.
バキュロウィルスとしては、例えば、夜盗蛾科昆虫に感染するウィルスであるアウトグ ラファ 'カリフオル-力'ヌクレア一'ポリへドロシス'ウィルス (Autographa californica nu clear polyhedrosis virus;等を用 ヽること »でさ 。  As a baculovirus, for example, use of the autographa californica nu clear polyhedrosis virus; an autographa californica force, which is a virus that infects the night stealing insects.
昆虫細胞としては、 Spodoptera frugiperdaの卵巣細胞、 Trichoplusia niの卵巣細胞 、カイコ卵巣由来の培養細胞等を用いることができる。 Spodoptera frugiperdaの卵巣 細胞としては S19、 S1 1 (バキュロウィルス'イクスプレツシヨン'ベクターズァ 'ラボラトリ ~ ·マニュアル）等、 Trichoplusia niの卵巣細胞としては High 5、 BTI-TN-5Bl-4 (イン ビトロジェン社製)等、カイコ卵巣由来の培養細胞としては Bombyx mori N4等をあげ ることがでさる。  As insect cells, Spodoptera frugiperda ovarian cells, Trichoplusia ni ovarian cells, silkworm ovary-derived cultured cells, and the like can be used. Spodoptera frugiperda ovarian cells are S19, S11 (Baculovirus 'Expression' Vectorz Laboratories Manual), Trichoplusia ni ovary cells are High 5, BTI-TN-5Bl-4 (Invitrogen) Bombyx mori N4 etc. can be mentioned as cultured cells derived from silkworm ovary.
組換えウィルスを調製するための、昆虫細胞への上記組換え遺伝子導入ベクター と上記バキュロウィルスの共導入方法としては、例えば、リン酸カルシウム法 (特開平 2 -227075)、リポフエクシヨン法〔Proc. Natl. Acad. Sci. USA, 84,7413 (1987)〕等をあげ ることがでさる。 また、動物細胞に DNAを導入する方法と同様の方法を用いて、昆虫細胞に DNA を導入することもでき、例えば、エレクト口ポレーシヨン法〔Cytotechnology, 3, 133 (19 90)〕、リン酸カルシウム法（特開平 2- 227075)、リボフヱクシヨン法〔Proc. Natl. Acad. S ci. USA, 84, 7413 (1987)〕等をあげることができる。 Examples of a method for co-introducing the recombinant gene introduction vector and the baculovirus into insect cells for preparing a recombinant virus include, for example, the calcium phosphate method (JP-A-2-227075), the lipofuxion method [Proc. Natl. Acad Sci. USA, 84,7413 (1987)]. In addition, DNA can be introduced into insect cells using a method similar to the method for introducing DNA into animal cells. For example, the electopore position method [Cytotechnology, 3, 133 (1990)], the calcium phosphate method ( JP-A-2-227075), ribofusion method [Proc. Natl. Acad. Sci. USA, 84, 7413 (1987)] and the like.
[0086] (vi)培養方法 [0086] (vi) Culture method
本発明のタンパク質をコードする DNAを組み込んだ組換え体ベクターを保有する 形質転換体が、大腸菌、酵母、動物細胞あるいは植物細胞等の細胞の場合、各種 宿主に適した通常の培養方法に従って培養し、該タンパク質を産生 '蓄積させ、形質 転換体又は培養液より該タンパク質を回収することにより、該タンパク質を製造するこ とができる。形質転換体が、動物個体又は植物個体の場合、各種宿主に適した通常 の生育方法に従って飼育又は栽培し、該タンパク質を産生 ·蓄積させ、該動物個体 又は植物個体より該タンパク質を回収することにより、該タンパク質を製造することが できる。  When the transformant having a recombinant vector incorporating the DNA encoding the protein of the present invention is a cell such as Escherichia coli, yeast, animal cells or plant cells, the cells are cultured according to a normal culture method suitable for various hosts. The protein can be produced by accumulating and collecting the protein and recovering the protein from the transformant or culture medium. When the transformant is an animal or plant individual, it is bred or cultivated according to a normal growth method suitable for various hosts, the protein is produced and accumulated, and the protein is recovered from the animal or plant individual. The protein can be produced.
宿主が動物個体の場合、例えば、本発明のポリヌクレオチドを保有する非ヒトトラン スジエニック動物を飼育し、該組換え体 DNAのコードする本発明のタンパク質のタン パク質を該動物中に産生 ·蓄積させ、該動物個体中から該タンパク質を回収すること により、本発明のタンパク質のタンパク質を製造することができる。動物個体中の産生 •蓄積場所としては、例えば、該動物のミルク、唾液、卵等を挙げることができる。 宿主が植物個体の場合、例えば、本発明のタンパク質のポリヌクレオチドを保有す るトランスジエニック植物を栽培し、該組換え体 DNAのコードする本発明のタンパク 質のタンパク質を該植物個体中に産生 ·蓄積させ、植物個体中から該タンパク質を 回収することにより、本発明のタンパク質のタンパク質を製造することができる。  When the host is an animal individual, for example, a non-human transgenic animal carrying the polynucleotide of the present invention is bred, and the protein of the protein of the present invention encoded by the recombinant DNA is produced and accumulated in the animal. The protein of the present invention can be produced by recovering the protein from the individual animal. Production in animal individual • Examples of the accumulation location include milk, saliva, eggs and the like of the animal. When the host is a plant individual, for example, a transgenic plant carrying the polynucleotide of the protein of the present invention is cultivated, and the protein of the protein of the present invention encoded by the recombinant DNA is produced in the plant individual. · The protein of the protein of the present invention can be produced by accumulating and recovering the protein from the plant individual.
宿主が大腸菌等の原核生物又は酵母等の真核生物である場合、例えば、本発明 のポリヌクレオチドを保有する形質転換体を培地中で培養し、該組換え体 DNAのコ ードする本発明のタンパク質を培養液に産生 ·蓄積させ、該培養液力 該タンパク質 を回収することにより、本発明のタンパク質を製造することができる。  When the host is a prokaryote such as Escherichia coli or a eukaryote such as yeast, for example, the transformant harboring the polynucleotide of the present invention is cultured in a medium and the recombinant DNA is encoded. The protein of the present invention can be produced by producing and accumulating the protein in the culture solution and recovering the culture solution force.
[0087] 本発明の本発明のタンパク質の形質転換体を培地で培養する方法は、宿主の培 養に用いられる通常の方法に従って行うことができる。 大腸菌等の原核生物あるいは酵母等の真核生物を宿主として得られた形質転換 体を培養する培地としては、該生物が資化し得る炭素源、窒素源、無機塩類等を含 有し、形質転換体の培養を効率的に行える培地であれば天然培地、合成培地のい ずれを用いてもよい。 [0087] The method for culturing the transformant of the protein of the present invention of the present invention in a medium can be carried out according to a usual method used for culturing a host. A medium for culturing a transformant obtained by using a prokaryote such as E. coli or a eukaryote such as yeast as a host contains a carbon source, a nitrogen source, inorganic salts, etc. that the organism can assimilate. Either a natural medium or a synthetic medium may be used as long as it can efficiently culture the body.
形質転換体が大腸菌等の原核生物あるいは酵母等の真核生物である場合、得ら れた形質転換体を培養する培地としては、宿主が資化し得る炭素源、窒素源、無機 塩類等を含有し、形質転換体の培養を効率的に行える培地であれば天然培地、合 成培地の 、ずれを用いてもょ 、。宿主が大腸菌である形質転換体を培養する際の培 地としては、例えば、バタトトリプトン、イーストエタストラクト及び塩ィ匕ナトリウムを含む YT培地が好ましい。  When the transformant is a prokaryote such as Escherichia coli or a eukaryote such as yeast, the medium for culturing the obtained transformant contains a carbon source, nitrogen source, inorganic salts, etc. that can be assimilated by the host. However, if the medium can efficiently culture the transformant, the difference between the natural medium and the synthetic medium may be used. As a medium for culturing a transformant whose host is Escherichia coli, for example, a YT medium containing butatotryptone, yeast etastruct and sodium chloride sodium is preferable.
炭素源としては、それぞれの微生物が資化し得るものであればよぐグルコース、フ ラタトース、スクロース、これらを含有する糖蜜、デンプンあるいはデンプン加水分解 物等の炭水化物、酢酸、プロピオン酸等の有機酸、エタノール、プロパノール等のァ ルコール類を用いることができる。  As the carbon source, as long as each microorganism can assimilate, glucose, slakedose, sucrose, molasses containing them, carbohydrates such as starch or starch hydrolysate, organic acids such as acetic acid and propionic acid, Alcohols such as ethanol and propanol can be used.
窒素源としては、アンモニア、塩化アンモ-ゥム、硫酸アンモ-ゥム、酢酸アンモ- ゥム、リン酸アンモ-ゥム等の各種無機酸や有機酸のアンモ-ゥム塩、その他含窒素 物質、並びに、ペプトン、肉エキス、酵母エキス、コーンスチープリカー、カゼインカロ 水分解物、大豆粕及び大豆粕加水分解物、各種発酵菌体及びその消化物等を用 いることがでさる。  Nitrogen sources include ammonia, ammonium chloride, ammonium sulfate, ammonium acetate, ammonium phosphates of various inorganic and organic acids, and other nitrogen-containing substances. In addition, peptone, meat extract, yeast extract, corn steep liquor, caseincaro hydrolyzate, soybean meal and soybean meal hydrolyzate, various fermented cells and digested products thereof can be used.
無機塩としては、リン酸第一カリウム、リン酸第二カリウム、リン酸マグネシウム、硫酸 マグネシウム、塩ィ匕ナトリウム、硫酸第一鉄、硫酸マンガン、硫酸銅、炭酸カルシウム 等を用いることができる。培養は、振盪培養又は深部通気攪拌培養等の好気的条件 下で行う。  As the inorganic salt, monopotassium phosphate, dipotassium phosphate, magnesium phosphate, magnesium sulfate, sodium chloride salt, ferrous sulfate, manganese sulfate, copper sulfate, calcium carbonate and the like can be used. Culturing is performed under aerobic conditions such as shaking culture or deep aeration stirring culture.
培養温度は 15〜40°Cがよぐ培養時間は、通常 5時間〜 7日間である。培養中 pH は、 3. 0〜9. 0に保持する。 pHの調整は、無機あるいは有機の酸、アルカリ溶液、 尿素、炭酸カルシウム、アンモニア等を用いて行う。また培養中必要に応じて、アンピ シリンやテトラサイクリン等の抗生物質を培地に添加してもよい。  The culture temperature is 15-40 ° C, and the culture time is usually 5-7 days. During the cultivation, the pH is maintained at 3.0 to 9.0. The pH is adjusted using inorganic or organic acids, alkaline solutions, urea, calcium carbonate, ammonia, etc. Further, antibiotics such as ampicillin and tetracycline may be added to the medium as needed during the culture.
プロモーターとして誘導性のプロモーターを用いた発現ベクターで形質転換した微 生物を培養するときには、必要に応じてインデューサーを培地に添加してもよい。例 えば、 lacプロモーターを用いた発現ベクターで形質転換した微生物を培養するとき にはイソプロピル一 β—D—チォガラタトピラノシド等を、 trpプロモーターを用いた発 現ベクターで形質転換した微生物を培養するときにはインドールアクリル酸等を培地 に添加してもよい。遺伝子を導入した植物の細胞や器官は、ジャーフアーメンターを 用いて大量培養することができる。培養する培地としては、一般に使用されているム ラシゲ.アンド'スターグ (MS)培地、ホワイト (White)培地、又はこれら培地にオーキシン 、サイトカイニン等、植物ホルモンを添カ卩した培地等を用いることができる。 Microtransformed with an expression vector using an inducible promoter as the promoter When the organism is cultured, an inducer may be added to the medium as necessary. For example, when cultivating a microorganism transformed with an expression vector using the lac promoter, cultivate a microorganism transformed with an expression vector using trp promoter, such as isopropyl β-D-thiogalatatopyranoside. When doing so, indoleacrylic acid or the like may be added to the medium. Plant cells and organs into which a gene has been introduced can be cultured in large quantities using a jar mentor. As a culture medium, it is possible to use a commonly used Murashige and Stag (MS) medium, White medium, or a medium supplemented with plant hormones such as auxin and cytokinin. it can.
本発明のタンパク質製造用形質転換体が動物細胞である場合、該細胞を培養する 培地は、一般に使用されている RPMI1640培地〔T he Journal of the American Medica 1 Association, 199, 519 (1967)〕、 Eagleの MEM培地 [Science, 122,501 (1952)〕、 DME M培地 [Virology, 8, 396 (1959)〕、 199培地 [Proceeding of the Society for the Biologi cal Medicine, 73, 1 (1950)〕又はこれら培地に牛胎児血清等を添加した培地等が用 いられる。  When the transformant for protein production of the present invention is an animal cell, a culture medium for culturing the cell is a generally used RPMI1640 medium (The Journal of the American Medica 1 Association, 199, 519 (1967)), Eagle's MEM medium [Science, 122,501 (1952)], DME M medium [Virology, 8, 396 (1959)], 199 medium [Proceeding of the Society for the Biologi cal Medicine, 73, 1 (1950)] or these media For example, medium supplemented with fetal calf serum is used.
培養は、通常 pH6〜8、 25〜40°C、 5%CO存在下等の条件下で 1〜7日間行う。  Cultivation is usually carried out for 1 to 7 days under conditions such as pH 6-8, 25-40 ° C, and 5% CO.
2  2
また培養中必要に応じて、カナマイシン、ペニシリン、ストレプトマイシン等の抗生物 質を培地に添加してもよい。 In addition, antibiotics such as kanamycin, penicillin, streptomycin and the like may be added to the medium as needed during culture.
本発明のタンパク質製造用形質転換体が昆虫細胞である場合、該細胞を培養する 培地としては、一般に使用されている TNM— FH培地（ファーミンジェン社製）、 Sf-9 00 II SFM培地（ギブコ BRL社製）、 ExCell400、 ExCell405〔いずれも JRHバイオ サイエンシーズ社製〕、 Grace's InsectMedium [Nature, 195, 788 (1962)〕等を用いる ことができる。  When the transformant for protein production of the present invention is an insect cell, as a medium for culturing the cell, a commonly used TNM-FH medium (manufactured by Pharmingen), Sf-900 II SFM medium ( Gibco BRL), ExCell400, ExCell405 [all manufactured by JRH Biosciences], Grace's InsectMedium [Nature, 195, 788 (1962)] and the like can be used.
(vii)タンパク質の製造方法 (vii) Protein production method
本発明のタンパク質製造用形質転換体の培養物から、本発明のタンパク質を単離' 精製するには、通常の酵素の単離 ·精製法を用いることができる。例えば、本発明の タンパク質が本発明のタンパク質製造用形質転換体の細胞外に該タンパク質が分泌 される場合には、該培養物を遠心分離等の手法により処理し、可溶性画分を取得す る。該可溶性画分から、溶媒抽出法、硫安等による塩析法脱塩法、有機溶媒による 沈殿法、ジェチルアミノエチル（DEAE)—セファロース、 DIAION HPA- 75 (三菱化 成社製）等レジンを用いた陰イオン交換クロマトグラフィー法、 S- Sepharose FF (ファ ルマシア社製)等のレジンを用いた陽イオン交換クロマトグラフィー法、プチルセファ ロース、フエ-ルセファロース等のレジンを用いた疎水性クロマトグラフィー法、分子 篩を用いたゲルろ過法、ァフィユティークロマトグラフィー法、クロマトフォーカシング 法、等電点電気泳動等の電気泳動法等の手法を用い、精製標品を得ることができる 本発明のタンパク質が本発明のタンパク質製造用形質転換体の細胞内に溶解状態 で蓄積する場合には、培養物を遠心分離することにより、培養物中の細胞を集め、該 細胞を洗浄した後に、超音波破砕機、フレンチプレス、マントンガウリンホモゲナイザ 一、ダイノミル等により細胞を破砕し、無細胞抽出液を得る。該無細胞抽出液を遠心 分離することにより得られた上清から、溶媒抽出法、硫安等による塩析法脱塩法、有 機溶媒による沈殿法、ジェチルアミノエチル（DEAE)—セファロース、 DIAION HPA -75 (三菱ィ匕成社製)等レジンを用いた陰イオン交換クロマトグラフィー法、 S-S印 haro se FF (フアルマシア社製)等のレジンを用いた陽イオン交換クロマトグラフィー法、ブ チルセファロース、フエ-ルセファロース等のレジンを用いた疎水性クロマトグラフィー 法、分子篩を用いたゲルろ過法、ァフィユティークロマトグラフィー法、クロマトフォー カシング法、等電点電気泳動等の電気泳動法等の手法を用い、精製標品を得ること ができる。 In order to isolate and purify the protein of the present invention from the culture of the transformant for protein production of the present invention, the usual enzyme isolation and purification methods can be used. For example, when the protein of the present invention is secreted outside the transformant for protein production of the present invention, the culture is treated by a method such as centrifugation to obtain a soluble fraction. . From the soluble fraction, a solvent extraction method, a salting-out method using ammonium sulfate, a desalting method, an organic solvent Precipitation method, anion-exchange chromatography using resin such as Jetylaminoethyl (DEAE) -Sepharose, DIAION HPA-75 (manufactured by Mitsubishi Kasei), resin such as S- Sepharose FF (manufactured by Pharmacia) Cation exchange chromatography method used, hydrophobic chromatography method using resins such as butyl sepharose and ferrule sepharose, gel filtration method using molecular sieve, affinity chromatography method, chromatofocusing method, isoelectric A purified sample can be obtained using a method such as electrophoresis such as point electrophoresis. When the protein of the present invention accumulates in a dissolved state in the cells of the transformant for protein production of the present invention, culture is performed. The cells in the culture are collected by centrifuging the product, and after washing the cells, an ultrasonic crusher, a French press, a Mantongauri Homogenizer one, the cells were disrupted by Dyno mill or the like to obtain a cell-free extract. From the supernatant obtained by centrifuging the cell-free extract, a solvent extraction method, a salting-out method using ammonium sulfate, a desalting method, a precipitation method using an organic solvent, jetylaminoethyl (DEAE) -Sepharose, DIAION Anion exchange chromatography using a resin such as HPA-75 (Mitsubishi Kosei Co., Ltd.), Cation exchange chromatography using a resin such as SS mark haro se FF (Falmacia), Butyl sepharose, Methods such as hydrophobic chromatography using resin such as ferrule sepharose, gel filtration using molecular sieve, affinity chromatography, chromatofocusing, electrophoresis such as isoelectric focusing etc. Used to obtain a purified sample.
また、該タンパク質が細胞内に不溶体を形成して発現した場合は、同様に細胞を回 収後破砕し、遠心分離を行うことにより得られた沈殿画分より、通常の方法により該タ ンパク質を回収後、該タンパク質の不溶体をタンパク質変性剤で可溶化する。該可 溶化液を、タンパク質変性剤を含まなヽある!、はタンパク質変性剤の濃度がタンパク 質が変性しない程度に希薄な溶液に希釈、あるいは透析し、該タンパク質を正常な 立体構造に構成させた後、上記と同様の単離精製法により精製標品を得ることがで きる。  In addition, when the protein is expressed in an insoluble form in the cell, the protein is similarly collected, disrupted and centrifuged from the precipitate fraction obtained by the usual method. After recovering the quality, the insoluble matter of the protein is solubilized with a protein denaturant. The solubilized solution may not contain a protein denaturant! Is diluted to a dilute solution so that the protein denaturant concentration does not denature the protein, or dialyzed to form the protein into a normal three-dimensional structure. Thereafter, a purified preparation can be obtained by the same isolation and purification method as described above.
また、通常のタンパク質の精製方法〔J. Evan. Sadlerら:メソッド'イン'ェンザィモロ ジー（Methods in Enzymology), 83,458〕に準じて精製できる。また、本発明のタンパ ク質を他のタンパク質との融合タンパク質として生産し、融合したタンパク質に親和性 を持つ物質を用いたァフィユティークロマトグラフィーを利用して精製することもできるIn addition, the protein can be purified according to a conventional protein purification method [J. Evan. Sadler et al .: Methods in Enzymology, 83, 458]. The tamper of the present invention The protein can be produced as a fusion protein with other proteins and purified using affinity chromatography using a substance that has an affinity for the fused protein.
〔山川彰夫，実験医学， 13, 469-474(1995)] o例えば、ロウらの方法〔Pro Natl.Acad. Sci.'USA, 86, 8227 (1989)、 GenesDevelop., 4, 1288 (1990)〕に記載の方法に準じて 、本発明のタンパク質をプロテイン Aとの融合タンパク質として生産し、ィムノグロプリ ン Gを用いるァフィユティークロマトグラフィーにより精製することができる。 [Akio Yamakawa, Experimental Medicine, 13, 469-474 (1995)] o For example, the method of Lowe et al. [Pro Natl. Acad. Sci. 'USA, 86, 8227 (1989), GenesDevelop., 4, 1288 (1990) In accordance with the method described in the above, the protein of the present invention can be produced as a fusion protein with protein A and purified by affinity chromatography using immunoglobulin G.
[0091] また、本発明のタンパク質を FLAGペプチドとの融合タンパク質として生産し、抗 F LAG抗体を用いるァフィユティークロマトグラフィーにより精製することができる〔Pro Natl. Acad. Sci" USA, 86, 8227 (1989)、 Genes Develop., 4, 1288 (1990)〕。  [0091] The protein of the present invention can be produced as a fusion protein with a FLAG peptide and purified by affinity chromatography using an anti-FLAG antibody [Pro Natl. Acad. Sci "USA, 86, 8227. (1989), Genes Develop., 4, 1288 (1990)].
更に、該タンパク質自身に対する抗体を用いたァフィユティークロマトグラフィーで 精製することもできる。本発明のタンパク質は、公知の方法〔J. Biomolecular NMR, 6, 129-134、 Science, 242, 1162-1164、 J.Biochem., 110, 166-168 (1991)〕に準じて、 in vitro転写 ·翻訳系を用いてを生産することができる。  Furthermore, it can be purified by affinity chromatography using an antibody against the protein itself. The protein of the present invention is produced by in vitro transcription according to a known method [J. Biomolecular NMR, 6, 129-134, Science, 242, 1162-1164, J. Biochem., 110, 166-168 (1991)]. · Can be produced using a translation system.
上記で取得されたタンパク質のアミノ酸情報を基に、 Fmoc法 (フルォレニルメチル ォキシカルボ-ル法）、 tBoc法（t ブチルォキシカルボ-ル法）等の化学合成法に よっても本発明のタンパク質を製造することができる。また、アドバンスト'ケムテック (A dvanced ChemTech )社、パーキン 'エルマ一社、フアルマシアバイオテク社、プロテ イン'テクノロジー 'インストウノレメント（Protein Technology Instrument)社、シンセセノレ •ベガ（Synthecell— Vega)社、パーセプティブ（PerSeptive)社、島津製作所等のぺプ チド合成機を利用しィ匕学合成することもできる。  Based on the amino acid information of the protein obtained above, chemical synthesis methods such as Fmoc method (fluorenylmethyloxycarbon method) and tBoc method (tbutyloxycarbon method) can also be used. Protein can be produced. Also, Advanced 'Advanced ChemTech', Perkin 'Elma I, Hurmasia Biotech, Protein' Technology 'Protein Technology Instrument, Synthecell-Vega, Perceptive (PerSeptive), Shimadzu Corporation and other peptide synthesizers can be used for chemical synthesis.
精製した本発明のタンパク質の構造解析は、タンパク質ィ匕学で通常用いられる方 法、例えば遺伝子クローユングのためのタンパク質構造解析 (平野久著、東京化学 同人発行、 1993年）に記載の方法により実施可能である。  The structural analysis of the purified protein of the present invention is carried out by a method generally used in protein chemistry, for example, the method described in Protein Structural Analysis for Gene Cloning (published by Hisashi Hirano, Tokyo Kagaku Dojin, 1993) Is possible.
[0092] 「部分ペプチド又はその塩」は、上記タンパク質の合成法に従って、あるいは本発 明のタンパク質を適当なぺプチダーゼで切断することによって製造することができる。 上記方法で得られる部分ペプチドが遊離体である場合は、公知の方法ある 、はそれ に準じる方法によって適当な塩に変換することができるし、逆に塩で得られた場合は [0092] "Partial peptide or salt thereof" can be produced according to the above protein synthesis method or by cleaving the protein of the present invention with an appropriate peptidase. When the partial peptide obtained by the above method is a free form, there is a known method, which can be converted to an appropriate salt by a method analogous thereto, and conversely,
、公知の方法あるいはそれに準じる方法によって遊離体又は他の塩に変換すること ができる。 , Converted into a free form or other salt by a known method or a method analogous thereto Can do.
[0093] スクリーニング方法に使用する「タンパク質をコードする遺伝子」は、前述した本発 明で用いられるタンパク質をコードする塩基配列を含有する核酸であれば!/ヽかなるも のであってもよい。好ましくは DNAである。 DNAとしては、ゲノム DNA、ゲノム DNA ライブラリー、前記した細胞'組織由来の cDNA、前記した細胞'組織由来の cDNA ライブラリー、合成 DNAのいずれでもよい。  [0093] The "gene encoding the protein" used in the screening method may be any nucleic acid containing a base sequence encoding the protein used in the present invention described above! Preferably it is DNA. The DNA may be any of genomic DNA, genomic DNA library, cDNA derived from the above-mentioned cell 'tissue, cDNA library derived from the above-mentioned cell' tissue, and synthetic DNA.
[0094] 本発明のタンパク質をコードする遺伝子の取得方法  [0094] Method for obtaining gene encoding protein of the present invention
「タンパク質をコードする遺伝子」は、以下のように取得することができる。 ヒト脳、心臓、骨格筋、ひ臓、腎臓、肝臓、小腸、胎盤、これら組織由来のヒト正常細 胞、ヒト臍帯静脈内皮細胞又はヒト卵巣癌あるいはヒト大腸癌より、常法により cDNA ライブラリーを作製する。  A “gene encoding a protein” can be obtained as follows. A cDNA library is prepared from human brain, heart, skeletal muscle, spleen, kidney, liver, small intestine, placenta, normal human cells derived from these tissues, human umbilical vein endothelial cells, human ovarian cancer, or human colon cancer by a conventional method To do.
[0095] cDNAライブラリ一作製法としては、モレキュラー ·クローユング第 2版やカレント ·プ ロトコールズ.イン.モレキュラ^ ~ ·バイオロジーサプノレメント 1〜38、 A Laboratory Ma nual, 2nd Ed.(1989 DNAし lonmg 1: Core Techniques, A Practical Approach, bee ond Edition, Oxford University Press (1995)等に記載された方法、あるいは市販のキ ット、例えばスーパースクリプト ·プラスミド ·システム ·フォ^ ~ · cDNA'シンセシス ·アン ド'プラスミド 'クローニング [Superscript Plasmid System for cDNA Synthesis and Plas mid Cloning;ギブコ BRL (Gibco BRL)社製〕やザップ— cDNA 'シンセシス'キット〔Z AP-cDNA Synthesis Kit,ストラタジーン社製〕を用いる方法等があげられる。  [0095] As a method for preparing a cDNA library, Molecular Cloning 2nd Edition and Current Protocols. In. Molecular ^ ~ Biology Supment 1-38, A Laboratory Manual, 2nd Ed. lonmg 1: Core Techniques, A Practical Approach, bee ond Edition, Oxford University Press (1995), etc., or commercially available kits such as superscripts, plasmids, systems, systems, cDNA synthesis And 'plasmid' cloning [Superscript Plasmid System for cDNA Synthesis and Plas mid Cloning; Gibco BRL (Gibco BRL)] or Zapp cDNA 'synthesis' kit (Z AP-cDNA Synthesis Kit, Stratagene) Methods and the like.
[0096] 該方法により取得される DNAとして、具体的には、配列番号： 1〜17で表される塩 基配列を有する DNA等をあげることができる。配列番号： 1〜17の DNAを含むプラ スミドとしては、例えば、後述の実施例に記載したプラスミドをあげることができる。  [0096] Specific examples of DNA obtained by the method include DNA having a base sequence represented by SEQ ID NOs: 1 to 17 and the like. As a plasmid containing DNA of sequence number: 1-17, the plasmid described in the below-mentioned Example can be mention | raise | lifted, for example.
[0097] 該発現べクタ一としては、該 cDNAを組み込んで動物細胞で発現できるベクターで あればいかなるものでも用いることができ、例えば、 pcDNAI/Amp、 pcDNAI、 pCDM8 ( いずれもフナコシ社より市販）、 PAGE107〔特開平 3- 22979、 Cytotechnology, 3, 133 ( 1990)〕、 pREP4 (インビトロジヱン社製）、 pAGE103〔J. Biochem., 101, 1307 (1987)〕、 p AMo、 pAMoA[J. Biol. Chem., 268, 22782-22787 (1993)〕、 pAS3- 3 (特開平 2- 22707 5)等を用いることができる。 [0098] cDNAを^ aみ込んだ発現ベクターは、 目的とする cDNAを選択可能な動物細胞に 導入し、形質転換細胞を取得する。該発現べクタ一の導入方法としては、動物細胞 に DNAを導入する方法であればいずれの方法も用いることができ、例えば、エレクト 口ポレーシヨン法〔Cytotechnology, 3, 133 (1990)〕、リン酸カルシウム法（特開平 2- 22 7075)、リポフエクシヨン法〔Proc. Natl. Acad. Sci. USA, 84, 7413 (1987)〕、 Virology, 52, 456 (1973)に記載の方法等の方法をあげることができる。 [0097] As the expression vector, any vector can be used as long as it can be expressed in animal cells by incorporating the cDNA. For example, pcDNAI / Amp, pcDNAI, and pCDM8 (all available from Funakoshi) PAGE107 (JP-A-3-22979, Cytotechnology, 3, 133 (1990)), pREP4 (manufactured by Invitrogene), pAGE103 (J. Biochem., 101, 1307 (1987)), p AMo, pAMoA [J. Biol. Chem., 268, 22782-22787 (1993)], pAS3-3 (JP-A-2-227075) and the like can be used. [0098] An expression vector containing cDNA is introduced into a selectable animal cell to obtain a transformed cell. As the method for introducing the expression vector, any method can be used as long as it is a method for introducing DNA into animal cells. For example, the electoral position method [Cytotechnology, 3, 133 (1990)], the calcium phosphate method (Japanese Patent Laid-Open No. 2-22 7075), the lipofusion method [Proc. Natl. Acad. Sci. USA, 84, 7413 (1987)], Virology, 52, 456 (1973), and the like. .
[0099] 動物細胞としては、ヒトの細胞である Namalwa細胞、 Namalwa細胞のサブラインであ る Namalwa KJM-1細胞、サルの細胞である COS細胞、チャイニーズ 'ハムスターの細 胞である CHO細胞、 HBT5637 (特開昭 63— 299)、大腸癌細胞株である HCT-15等を あげることができ、好ましくは、 Namalwa細胞、 Namalwa KJM-1細胞又は HCT- 15を用 いうる。  [0099] Animal cells include Namalwa cells that are human cells, Namalwa KJM-1 cells that are sublines of Namalwa cells, COS cells that are monkey cells, CHO cells that are Chinese hamster cells, HBT5637 ( JP-A-63-299), HCT-15, which is a colon cancer cell line, can be mentioned, and preferably, Namalwa cells, Namalwa KJM-1 cells or HCT-15 can be used.
[0100] 得られた形質転換細胞を常法により培養する。具体的には、以下の形質転換体の 培養方法により培養することができる。形質転換体が動物細胞である場合、該細胞を 培養する培地は、一般に使用されている RPMI1640培地〔The Journal of the America n Medical Association, 199, 519 (1967)〕、 Eagleの MEM培地 [Science, 122,501 (1952 )]、 DMEM培地 [Virology, 8, 396 (1959)〕、 199培地 [Proceeding of the Society for th e Biological Medicine, 73, 1 (1950)〕又はこれら培地に牛胎児血清等を添カ卩した培地 等が用いられる  [0100] The obtained transformed cells are cultured by a conventional method. Specifically, it can be cultured by the following culture method for transformants. When the transformant is an animal cell, the culture medium for culturing the cell may be a commonly used RPMI1640 medium [The Journal of the American Medical Association, 199, 519 (1967)], Eagle's MEM medium [Science, 122,501 (1952)], DMEM medium [Virology, 8, 396 (1959)], 199 medium [Proceeding of the Society for the Biological Medicine, 73, 1 (1950)], or fetal calf serum etc. Dwarf medium etc. are used
[0101] 培養は、通常 pH6〜8、 25〜40°C、 5%CO存在下等の条件下で 1〜7日間行う。  [0101] Cultivation is usually carried out for 1 to 7 days under conditions such as pH 6 to 8, 25 to 40 ° C, and the presence of 5% CO.
2  2
また培養中必要に応じて、カナマイシン、ペニシリン、ストレプトマイシン等の抗生物 質を培地に添加してもよい。  In addition, antibiotics such as kanamycin, penicillin, streptomycin and the like may be added to the medium as needed during culture.
[0102] 以上のようにして、本発明のタンパク質をコードする DNAを取得することができる。  [0102] As described above, DNA encoding the protein of the present invention can be obtained.
また、アミノ酸配列に基づいて、本発明のタンパク質をコードする DNAをィ匕学合成 することによつても DNAを調製することができる。 DNAの化学合成は、チォホスファ イト法を利用した島津製作所社製の DNA合成機、フォスフォアミダイト法を利用した パーキン.エルマ一社製の DNA合成機 model392等を用いて行うことができる。  Alternatively, DNA can be prepared by chemically synthesizing DNA encoding the protein of the present invention based on the amino acid sequence. Chemical synthesis of DNA can be carried out using a DNA synthesizer manufactured by Shimadzu Corporation using the thiophosphite method, a DNA synthesizer model 392 manufactured by Perkin Elmer Company using the phosphoramidite method, and the like.
[0103] さらに、後述のオリゴヌクレオチドをセンスプライマー（配列番号： 35)及びアンチセ ンスプライマー（配列番号： 36)として用い、これら DNAに相補的な mRNAを発現し て 、る細胞の mRNAから調製した cDNAを铸型として、 PCRを行うことによつても、 目的とする DNAを調製することができる。 [0103] Furthermore, the oligonucleotides described below were used as sense primers (SEQ ID NO: 35) and antisense primers (SEQ ID NO: 36) to express mRNA complementary to these DNAs. Thus, the target DNA can also be prepared by performing PCR using cDNA prepared from the mRNA of the cells as a saddle.
[0104] 「部分ペプチドをコードする遺伝子」は、前述した本発明で用いられる部分ペプチド をコードする塩基配列を含有するポリヌクレオチドであればいかなるものであってもよ く、 DNAが好ましい。 DNAとしては、ゲノム DNA、ゲノム DNAライブラリー、前記し た細胞'組織由来の cDNA、前記した細胞'組織由来の cDNAライブラリー、合成 D NAのいずれでもよい。上記、「タンパク質をコードする遺伝子」の取得方法に従って 、取得することがでさる。 [0104] The "gene encoding the partial peptide" may be any polynucleotide as long as it contains a nucleotide sequence encoding the partial peptide used in the present invention, and DNA is preferred. The DNA may be any of genomic DNA, genomic DNA library, cDNA derived from the aforementioned cell 'tissue, cDNA library derived from the aforementioned cell' tissue, and synthetic DNA. It can be obtained according to the above-mentioned method for obtaining a “gene encoding a protein”.
[0105] 本発明の骨疾患若しくは関節疾患の治療剤又は予防剤のスクリーニング方法は、 被検物質の存在下、本発明のタンパク質若しくは該タンパク質をコードする遺伝子の 動態を検出又は測定することを 1つの特徴とする。  [0105] The screening method for a therapeutic or prophylactic agent for bone disease or joint disease of the present invention comprises detecting or measuring the dynamics of the protein of the present invention or a gene encoding the protein in the presence of a test substance. With one feature.
[0106] すなわち、骨疾患又は関節疾患の発症との関連が認められる本発明のタンパク質 の動態に対する被検物質による影響を調べることにより、骨疾患若しくは関節疾患に 対する治療剤又は予防剤として有効な物質のスクリーニングを効率的に行なうもので ある。本発明のタンパク質の動態に対する被検物質による影響は、被検物質の非存 在下における該動態を対照として調べるのが好適である。  That is, by examining the influence of the test substance on the dynamics of the protein of the present invention, which is associated with the onset of bone disease or joint disease, it is effective as a therapeutic or prophylactic agent for bone disease or joint disease. Effective screening of substances. The influence of the test substance on the dynamics of the protein of the present invention is preferably examined using the dynamics in the absence of the test substance as a control.
[0107] また、本発明のスクリーニング方法は、骨疾患又は関節疾患に対する予防剤及び [0107] Further, the screening method of the present invention comprises a prophylactic agent for bone disease or joint disease, and
Z又は治療剤の候補化合物の薬理評価に用いることができる。 It can be used for pharmacological evaluation of candidate compounds for Z or therapeutic agents.
[0108] 前記被検物質としては、化合物又はその塩が挙げられる。なお、本明細書にぉ 、 ては、前記化合物又はその塩は、低分子化合物、高分子化合物、ポリペプチド又は その誘導体、核酸又はその誘導体等を含む。力かる被検物質は、天然物質であって もよぐ非天然物質であってもよい。ポリペプチドの誘導体としては、修飾基を付加し て得られた修飾ポリペプチド、アミノ酸残基を改変することにより得られたノリアントポ リペプチド等が挙げられる。また、核酸の誘導体としては、修飾基を付加して得られた 修飾核酸、塩基を改変することにより得られたバリアント核酸、ペプチド核酸等が挙げ られる。  [0108] Examples of the test substance include a compound or a salt thereof. In the present specification, the compound or a salt thereof includes a low molecular compound, a high molecular compound, a polypeptide or a derivative thereof, a nucleic acid or a derivative thereof, and the like. The strong test substance may be a natural substance or a non-natural substance. Examples of the derivative of the polypeptide include a modified polypeptide obtained by adding a modifying group, a noriant polypeptide obtained by altering an amino acid residue, and the like. Examples of nucleic acid derivatives include modified nucleic acids obtained by adding modifying groups, variant nucleic acids obtained by modifying bases, peptide nucleic acids, and the like.
[0109] 本発明のスクリーニング方法における測定又は検出対象の「本発明のタンパク質の 動態」としては、例えば、該タンパク質とそのリガンドとの結合の有無;該タンパク質の 発現、具体的には、該発現の有無ゃ該発現の変動等が挙げられる。 [0109] The "kinetics of the protein of the present invention" to be measured or detected in the screening method of the present invention includes, for example, the presence or absence of binding between the protein and its ligand; Expression, specifically, the presence or absence of the expression may include fluctuations in the expression.
[0110] 本発明のスクリーニング方法としては、測定又は検出対象の「本発明のタンパク質 の動態」に応じて、大きく 2つの実施態様がある。  [0110] The screening method of the present invention has roughly two embodiments depending on the "kinetics of the protein of the present invention" to be measured or detected.
[0111] 本発明のスクリーニング方法の第 1の実施態様としては、 [0111] As a first embodiment of the screening method of the present invention,
(I)本発明のタンパク質と、被検物質とを接触させるステップ、並びに  (I) a step of bringing the protein of the present invention into contact with a test substance; and
(II)前記ステップ (I)の後、該タンパク質と被検物質との結合を検出し、それにより、 該タンパク質と結合する被検物質を、骨疾患又は関節疾患の予防剤及び Z又は治 療剤の候補化合物として選択するステップ  (II) After the step (I), the binding between the protein and the test substance is detected, whereby the test substance binding to the protein is converted into a prophylactic agent and a Z or therapeutic agent for bone disease or joint disease. Selecting as a candidate compound
を含む方法が挙げられる。力かる方法によれば、前記タンパク質への結合を介して当 該タンパク質の機能に作用する物質を選択することができるため、選択された候補ィ匕 合物は、直接的かつ特異的に前記タンパク質に結合してその機能を調節するという 特徴的な性質を有する。  The method containing is mentioned. According to a powerful method, a substance that acts on the function of the protein can be selected through binding to the protein. Therefore, the selected candidate compound can be directly and specifically selected. It has the characteristic property of binding to and regulating its function.
[0112] 前記ステップ (I)において、前記タンパク質と被検物質との接触は、例えば、該タン パク質の本来の機能を妨げな 、溶液中にお!、て、前記タンパク質と被検物質とを混 合し、適切な反応条件 (例えば、反応温度、反応時間等)の下、維持する (すなわち、 両者を反応させる）ことにより行なわれうる。  [0112] In the step (I), the contact between the protein and the test substance is, for example, in the solution without interfering with the original function of the protein. And maintained under appropriate reaction conditions (eg, reaction temperature, reaction time, etc.) (ie, reacting both).
[0113] 前記「タンパク質の本来の機能を妨げない溶液」としては、例えば、リン酸緩衝化生 理的食塩水（PBS)、 HEPESバッファー、 Trisバッファ一等の溶液が挙げられる。  [0113] Examples of the "solution that does not interfere with the original function of the protein" include solutions such as phosphate buffered physiological saline (PBS), HEPES buffer, and Tris buffer.
[0114] ステップ (I)における反応条件としては、特に限定されないが、例えば、前記溶液中 、通常、 pH6. 0〜： LO. 0、好ましくは pH7. 0〜9. 0、より好ましくは pH7. 5〜8. 5、 さらに好ましくは pH8. 0で、通常、 10°Cから 50°C、好ましくは 20°C〜40°C、より好ま しくは 25°C〜37°C、さらに好ましくは 25°C、通常、 1分間〜 1時間、好ましくは 3〜30 分間、より好ましくは 5〜20分間、さらに好ましくは 10分間維持すること等が挙げられ る。より具体的には、 10mM Hepes、 80mM KC1、 5% グリセロール、 10mM D TT、 0. lmgZ ml polydIdC、 50ng, ml herring sperm DNA、 lmg/ ml B SA、 10% reticulocyte lysate中、前記条件下に維持することが挙げられる。  [0114] The reaction conditions in step (I) are not particularly limited. For example, in the solution, usually pH 6.0 to: LO. 0, preferably pH 7.0 to 9.0, more preferably pH 7. 5 to 8.5, more preferably pH 8.0, usually 10 ° C to 50 ° C, preferably 20 ° C to 40 ° C, more preferably 25 ° C to 37 ° C, more preferably 25 It may be maintained at ° C, usually 1 minute to 1 hour, preferably 3 to 30 minutes, more preferably 5 to 20 minutes, and even more preferably 10 minutes. More specifically, 10 mM Hepes, 80 mM KC1, 5% glycerol, 10 mM D TT, 0.1 mg mg ml polydIdC, 50 ng, ml herring sperm DNA, lmg / ml B SA, 10% reticulocyte lysate, maintained under the above conditions To do.
[0115] ついで、ステップ (Π)において、前記タンパク質と被検物質との結合を検出し、該結 合の有無を調べる。 [0116] 結合は、例えば、前記のようにしてステップ (I)で前記タンパク質と、例えば、放射性 物質により標識した被検物質とを反応させた後の溶液に含まれる該タンパク質の放 射活性を、大過剰の非放射性被検物質との競合下において解析することにより検出 することができる。結合が検出された場合、使用された被検物質を骨疾患又は関節 疾患の予防剤及び Z又は治療剤の候補化合物として選択する。 [0115] Next, in step (ii), the binding between the protein and the test substance is detected, and the presence or absence of the binding is examined. [0116] The binding is performed by, for example, reducing the radiation activity of the protein contained in the solution after reacting the protein in Step (I) with a test substance labeled with, for example, a radioactive substance as described above. It can be detected by analyzing in competition with a large excess of non-radioactive test substance. When binding is detected, the test substance used is selected as a prophylactic agent for bone disease or joint disease and a candidate compound for Z or therapeutic agent.
[0117] また、ステップ (I)とステップ (Π)を連続した工程により実施してもよい。かかる態様 は、例えば、被検物質を保持した担体へのノインデイングアツセィ等を利用すること により実施することができる。  [0117] Further, step (I) and step (i) may be performed by a continuous process. Such an embodiment can be carried out, for example, by utilizing a nodding assembly on a carrier holding a test substance.
[0118] 本発明のスクリーニング方法の第 2の実施態様としては、  [0118] As a second embodiment of the screening method of the present invention,
(A)本発明のタンパク質をコードする遺伝子を細胞に導入するステップ、  (A) introducing a gene encoding the protein of the present invention into a cell;
(B)被検物質の存在下、前記ステップ (A)で得られた細胞にぉ ヽて前記遺伝子を発 現させるステップ、並びに  (B) in the presence of the test substance, the step of expressing the gene in the cells obtained in the step (A), and
(C)被検物質の非存在下の場合と比較して、前記遺伝子の発現の有無を検出し、又 は前記遺伝子の発現の変動を測定し、それにより、該遺伝子の発現の変化をもたら す被検物質を骨疾患又は関節疾患の予防剤及び Z又は治療剤の候補化合物とし て選択するステップ  (C) Compared to the absence of the test substance, the presence or absence of the expression of the gene is detected, or the variation in the expression of the gene is measured, thereby changing the expression of the gene. Selecting the test substance to be treated as a prophylactic agent for bone disease or joint disease and a candidate compound for Z or therapeutic agent
を含む方法が挙げられる。  The method containing is mentioned.
[0119] 前記ステップ (A)において、本発明のタンパク質をコードする遺伝子を細胞に導入 するには、例えば、該タンパク質の発現調節領域として知られる発現調節性エレ メントの下流かつその支配下に、前記タンパク質をコードする遺伝子が動作可能に連 結されてなる核酸構築物を用いて行なうのが好適である。前記発現調節性エレメント としては、例えば、 Niimi T et al., Molecular Endocrinology, 2001, Vol.15, 2021—2136 に記載されている。 [0119] In step (A), in order to introduce a gene encoding the protein of the present invention into a cell, for example, downstream of and under the control of an expression regulatory element known as an expression regulatory region of the protein, It is preferable to use a nucleic acid construct in which the gene encoding the protein is operably linked. Examples of the expression regulatory element are described in Niimi T et al., Molecular Endocrinology, 2001, Vol. 15, 2021-2136.
[0120] 前記「核酸構築物」は、前記発現調節性エレメント及び本発明のタンパク質をコード する遺伝子を慣用の発現ベクターのクローユング部位に挿入することにより簡便に構 築されうる。当該ベクターとしては、例えば、ウィルスベクター、プラスミドベクター等が 挙げられる。  [0120] The "nucleic acid construct" can be easily constructed by inserting the expression regulatory element and the gene encoding the protein of the present invention into a cloning site of a conventional expression vector. Examples of the vector include viral vectors and plasmid vectors.
[0121] なお、本発明のタンパク質をコードする遺伝子には、当該遺伝子そのもの；前記遺 伝子のアンチセンス鎖にストリンジェントな条件下にハイブリダィズし、かつ前記タン パク質と同等の作用を有するタンパク質をコードする遺伝子;前記遺伝子の塩基配 列において、少なくとも 1塩基の変異を有し、かつ前記タンパク質と同等の作用を有 するタンパク質をコードする遺伝子;前記遺伝子の塩基配列と少なくとも 60%、好ま しくは 80%以上、より好ましくは 90%以上の配列同一性を有し、かつ前記タンパク質 と同等の作用を有するタンパク質をコードする遺伝子等も含まれる。 [0121] The gene encoding the protein of the present invention includes the gene itself; A gene that hybridizes to the antisense strand of a gene under stringent conditions and encodes a protein having an action equivalent to that of the protein; has a mutation of at least one base in the base sequence of the gene; A gene encoding a protein having the same action as the protein; having at least 60%, preferably 80% or more, more preferably 90% or more sequence identity with the base sequence of the gene; A gene encoding a protein having the same action as the above is also included.
[0122] 前記「ストリンジェントな条件」とは、高ストリンジエンシーな条件、例えば、 1 X SSC /0. 2%SDSの条件等をいう。ここで、ストリンジエンシーを向上させるため、より低ィ 才ン強度、 f列えば、 0. 5 X SSC、好ましくは 0. 2 X SSC、より好ましくは 0. 1 X SSC 等の条件及び Z又はより高温、例えば、用いられる核酸の Tm値により異なる力 50 °C以上、好ましくは 60°C以上、より好ましくは 65°C以上等の条件下で洗浄等を行な つてもよい。  The “stringent condition” refers to a high stringency condition, for example, a condition of 1 × SSC / 0.2% SDS. Here, in order to improve the stringency, the lower strength, for example, f column, 0.5 X SSC, preferably 0.2 X SSC, more preferably 0.1 X SSC, etc. and Z or Washing or the like may be performed at a higher temperature, for example, a force of 50 ° C or higher, preferably 60 ° C or higher, more preferably 65 ° C or higher, which varies depending on the Tm value of the nucleic acid used.
[0123] 前記「変異」とは、塩基の置換、欠失、付加及び挿入を 、う。かかる変異は、天然に 存在するバリエーションであってもよぐ慣用の部位特異的変異方法等により人為的 に導入された変異であってもよ ヽ。  [0123] The "mutation" refers to base substitution, deletion, addition and insertion. Such a mutation may be a naturally occurring variation or a mutation artificially introduced by a conventional site-specific mutation method or the like.
[0124] 前記「配列同一性」とは、比較対象の少なくとも 2つの配列について、適切に整列化 させ、それぞれの配列に存在する同一の残基を決定して、適合部位の数を決定し、 ついで、比較対象の配列領域内の残基の総数で、前記適合部位の数を割、得られ た数値に 100をかけることにより算出されうる値をいう。かかる配列同一性は、具体的 には、例えば、ホームページアドレス http:ZZwww. ncbi. nlm. nih. gov/BLA STZにおいて、一般に利用可能である BLASTアルゴリズム等により算出されうる。  [0124] The "sequence identity" means that at least two sequences to be compared are appropriately aligned, the same residue present in each sequence is determined, and the number of matching sites is determined. Next, it is a value that can be calculated by dividing the number of matching sites by the total number of residues in the sequence region to be compared and multiplying the obtained value by 100. Specifically, such sequence identity can be calculated by, for example, a BLAST algorithm that is generally available at a homepage address http: ZZwww.ncbi.nlm.nih.gov/BLA STZ.
[0125] なお、本発明の種々の態様において、その構成上、本発明のタンパク質をコードす る遺伝子の発現を必須とする場合、当該遺伝子の発現において用いられるベクター 、核酸構築物を導入する宿主細胞等は、それらの任意の組み合わせにより、前記遺 伝子の発現が達成されうるのであれば、それらはどのような生物種由来のものであつ てもよい。具体例及び細胞への前記核酸構築物の導入方法、前記核酸構築物が導 入された形質転換体の培養方法等は、上記タンパク質の製造方法の形質転換体に 関する記載と同様である。本発明のタンパク質をコードする遺伝子が真核生物に由 来するものである点、及び本発明にお ヽて提供される治療剤又は予防剤等が真核 生物、中でも哺乳動物、特にヒトにおいて好適に使用されうる点を考慮すれば、前記 遺伝子の発現に使用される宿主細胞としては動物細胞であるのが好適である。 [0125] In various embodiments of the present invention, when the expression of the gene encoding the protein of the present invention is essential due to its constitution, the vector used for the expression of the gene and the host cell into which the nucleic acid construct is introduced As long as expression of the gene can be achieved by any combination thereof, etc., they may be derived from any species. Specific examples, a method for introducing the nucleic acid construct into cells, a method for culturing a transformant into which the nucleic acid construct has been introduced, and the like are the same as described for the transformant in the protein production method. The gene encoding the protein of the present invention is derived from a eukaryotic organism. In view of the fact that the therapeutic agent or the preventive agent provided in the present invention can be suitably used in eukaryotes, particularly mammals, particularly humans, the expression of the gene The host cell used in the above is preferably an animal cell.
[0126] また、本実施態様においては、前記核酸構築物が導入された安定な細胞系を用い る場合、ステップ (A)は、省略してもよい。  [0126] In this embodiment, when a stable cell line into which the nucleic acid construct has been introduced is used, step (A) may be omitted.
[0127] っ 、で、ステップ (B)にお 、て、被検物質の存在下、前記ステップ (A)で得られた 細胞において本発明のタンパク質をコードする遺伝子を発現させる。 Thus, in step (B), a gene encoding the protein of the present invention is expressed in the cells obtained in step (A) in the presence of the test substance.
[0128] 当該工程は、例えば、被検物質を含有した培地を用いて、前記ステップ (A)で得ら れた細胞を培養することにより行なわれうる。培養条件は上記タンパク質の製造方法 の形質転換体培養条件と同様である。 [0128] The step can be performed, for example, by culturing the cells obtained in the step (A) using a medium containing a test substance. The culture conditions are the same as the transformant culture conditions in the above protein production method.
[0129] 続、て、ステップ (C)にお 、て、被検物質の非存在下の場合と比較して、前記遺伝 子の発現の有無を検出し、又は前記遺伝子の発現の変動を測定し、それにより、該 遺伝子の発現の変化をもたらす被検物質を骨疾患又は関節疾患の予防剤及び Z又 は治療剤の候補化合物として選択する。 [0129] Subsequently, in step (C), the presence or absence of the expression of the gene is detected or the variation in the expression of the gene is measured as compared to the case where the test substance is absent. Thus, a test substance that causes a change in the expression of the gene is selected as a prophylactic agent for bone disease or joint disease and a candidate compound for Z or therapeutic agent.
[0130] なお、「被検物質の非存在下の場合」とは、ステップ (B)にお 、て、被検物質の非 存在下にステップ (A)で得られた細胞にぉ ヽて前記遺伝子を発現させた場合を ヽ 、被検物質の存在下における前記遺伝子の発現の変化を把握するための基準とな る。具体的には、力かる場合の細胞、又は該細胞の抽出液等を対照として用いる。  [0130] The "in the absence of the test substance" means that in the step (B), the cell obtained in the step (A) in the absence of the test substance is compared with the above. When the gene is expressed, it becomes a standard for grasping the change in the expression of the gene in the presence of the test substance. Specifically, a cell when exerted or an extract of the cell is used as a control.
[0131] 前記遺伝子の発現の有無を検出し、又はその発現の変動を測定した結果、対照と 比較して該遺伝子の発現の変化が認められた時、例えば、対照において前記遺伝 子の発現が見られた場合に、被検物質と接触させた細胞において前記遺伝子の発 現が検出されないか、又は対象に比べて発現レベルが低下した時、すなわち、対照 と比較して前記遺伝子の発現が減少した時、該被検物質が骨疾患若しくは関節疾 患の予防剤及び Z又は治療剤の候補化合物であると判定され、該被検物質を候補 化合物として選択する。  [0131] As a result of detecting the presence or absence of the expression of the gene or measuring the variation in the expression, when a change in the expression of the gene is recognized as compared with the control, for example, the expression of the gene in the control is When observed, the expression of the gene is not detected in the cells contacted with the test substance or the expression level is decreased compared to the subject, i.e., the expression of the gene is decreased compared to the control. Then, it is determined that the test substance is a candidate compound for a prophylactic and Z or therapeutic agent for bone disease or joint disease, and the test substance is selected as a candidate compound.
[0132] 選択された候補ィ匕合物は、細胞レベルにおいて前記遺伝子の発現を転写レベル で抑制すると!ヽぅ特徴的な性質を有するものと考えられ、前記遺伝子の発現を特異 的に抑制することができるという優れた効果を発揮する。 [0133] 遺伝子の発現の有無の検出、又はその発現の変動の測定は、上記分子生物学的 測定方法又は免疫学的測定方法に従って行うことができる。 [0132] The selected candidate compound is considered to have a characteristic property when the expression of the gene at the cellular level is suppressed at the transcriptional level, and specifically suppresses the expression of the gene. It exhibits the excellent effect of being able to. [0133] Detection of the presence or absence of gene expression, or measurement of fluctuations in expression thereof can be performed according to the molecular biological measurement method or immunological measurement method described above.
[0134] 本発明のスクリーニング方法により得られる化合物又はその塩としては、例えば、前 記本発明のタンパク質等に結合することにより、該タンパク質の機能を阻害する化合 物、例えば、低分子化合物、高分子化合物、該本発明のタンパク質に対する抗体、 該本発明のタンパク質をコードする遺伝子力 なる核酸のアンチセンス鎖等の核酸、 ペプチド等が挙げられる。  [0134] Examples of the compound obtained by the screening method of the present invention or a salt thereof include, for example, a compound that inhibits the function of the protein by binding to the protein of the present invention, such as a low molecular weight compound, a high molecular weight compound, and the like. Examples include a molecular compound, an antibody against the protein of the present invention, a nucleic acid such as an antisense strand of a nucleic acid capable of encoding the protein of the present invention, a peptide, and the like.
[0135] また、本発明のスクリーニング方法に用いられる前記本発明のタンパク質、該本発 明のタンパク質をコードする遺伝子を保持してなる核酸構築物、該遺伝子が導入さ れた細胞、本発明のタンパク質の抗体等を含む、本発明のスクリーニング方法を行な うためのキットが提供される。力かるスクリーニング用キットも本発明に含まれる。  [0135] Furthermore, the protein of the present invention used in the screening method of the present invention, a nucleic acid construct having a gene encoding the protein of the present invention, a cell into which the gene has been introduced, the protein of the present invention A kit for carrying out the screening method of the present invention is provided. Powerful screening kits are also included in the present invention.
[0136] 本発明のスクリーニング用キットとしては、具体的には、  [0136] Specifically, the screening kit of the present invention includes:
本発明のタンパク質を含有してなるキット、  A kit comprising the protein of the present invention,
本発明のタンパク質をコードする遺伝子を含む核酸構築物を含有してなるキット 本発明のタンパク質をコードする遺伝子が導入された細胞を含有してなるキット、 本発明のタンパク質に対する抗体又はその断片を含有してなるキット、 等が挙げられる。  A kit comprising a nucleic acid construct comprising a gene encoding the protein of the present invention A kit comprising a cell into which a gene encoding the protein of the present invention has been introduced, an antibody against the protein of the present invention or a fragment thereof Kits, etc.
[0137] また、各キットには、所望により、本発明のスクリーニング方法に好適に使用されうる 前記プローブ及び Z又はプライマー対を含有させてもよい。さらには、  [0137] Each kit may contain the probe and Z or a primer pair that can be suitably used in the screening method of the present invention, if desired. Moreover,
所望により検出用試薬、緩衝液、標準物質、本発明のスクリーニング方法を実施する ための説明書等を含有させてもよい。  If desired, detection reagents, buffers, standards, and instructions for carrying out the screening method of the present invention may be included.
[0138] 本発明のスクリーニング用キットによれば、前記スクリーニング方法を簡便かつ迅速 に、高処理量で行なうことができるという優れた効果が発揮される。  [0138] According to the screening kit of the present invention, an excellent effect that the screening method can be carried out simply and rapidly at a high throughput is exhibited.
[0139] 本発明のスクリーニング方法により得られたィ匕合物又はその塩は、その発症に本発 明のタンパク質の発現が関与する骨疾患又は関節疾患に対して治療又は予防作用 を発揮しうる。したがって、前記スクリーニング方法により得られた化合物又はその塩 により、骨疾患又は関節疾患、例えば、 0 A又は RAの治療又は予防に用いるための 医薬組成物が提供される。 [0139] The compound or salt thereof obtained by the screening method of the present invention can exert a therapeutic or prophylactic effect on bone diseases or joint diseases in which the expression of the protein of the present invention is involved in the onset thereof. . Therefore, the compound obtained by the screening method or a salt thereof is used for treatment or prevention of bone disease or joint disease, for example, 0 A or RA. A pharmaceutical composition is provided.
[0140] 本発明の医薬組成物は、本発明のスクリーニング方法により得られたィ匕合物又はそ の塩を有効成分として含有することに 1つの特徴がある。したがって、本発明の医薬 組成物は、骨疾患又は関節疾患に対し、本発明のタンパク質の発現に対する作用（ 例えば、該発現に対して抑制的に働く）を介して治療又は予防効果を発揮する。  [0140] The pharmaceutical composition of the present invention is characterized in that it contains a compound or a salt thereof obtained by the screening method of the present invention as an active ingredient. Therefore, the pharmaceutical composition of the present invention exerts a therapeutic or prophylactic effect on the bone disease or joint disease through the action on the expression of the protein of the present invention (for example, it works to suppress the expression).
[0141] 本発明の医薬組成物中における前記化合物又はその塩の含有量は、治療目的の 疾患、患者の年齢、体重等により適宜調節することができ、治療上有効量であればよ ぐ低分子化合物又は高分子化合物の場合、例えば、 0. 0001〜： LOOOmg、好まし くは 0. 001〜100mg、ポジペプチド又はその誘導体の場合、例えば、 0. 0001〜10 OOmg、好まし <は 0. 001〜100mg、核酸又はその誘導体の場合、例えば、 0. 000 01〜： L00mg、好ましくは 0. 0001〜10mgであること力望まし!/、。  [0141] The content of the compound or the salt thereof in the pharmaceutical composition of the present invention can be adjusted as appropriate depending on the disease to be treated, the age of the patient, the body weight, etc., and may be a therapeutically effective amount. In the case of molecular compounds or polymer compounds, for example, 0.0001 to: LOOOmg, preferably 0.001 to 100 mg, and in the case of positive peptides or derivatives thereof, for example, 0.0001 to 10 OOmg, preferably <is 0. 001-100 mg, in the case of nucleic acids or derivatives thereof, for example, 0.001 01: L00 mg, preferably 0.0001-10 mg!
[0142] 本発明の医薬組成物は、前記化合物又はその塩を安定に保持しうる種々の助剤を さらに含有してもよい。具体的には、有効成分の送達対象となる部位に到達するまで の間に、有効成分が分解することを抑制する性質を呈する薬学的に許容されうる助 剤、賦形剤、結合剤、安定剤、緩衝剤、溶解補助剤、等張剤等が挙げられる。  [0142] The pharmaceutical composition of the present invention may further contain various auxiliaries capable of stably holding the compound or a salt thereof. Specifically, pharmacologically acceptable auxiliaries, excipients, binders, and stabilizers exhibiting the property of inhibiting the active ingredient from degrading before reaching the site where the active ingredient is to be delivered. Agents, buffers, solubilizers, isotonic agents and the like.
[0143] 本発明の医薬組成物の投与形態は、有効成分の種類;投与対象となる個体、器官 、局所部位、組織;投与対象となる個体の年齢、体重等に応じて、適宜選択される。 前記投与形態としては、皮下注射、筋肉内注射、静脈内注射、局所投与等が挙げら れる。  [0143] The dosage form of the pharmaceutical composition of the present invention is appropriately selected according to the type of active ingredient; individual, organ, local site, tissue to be administered; age, weight, etc. of the individual to be administered. . Examples of the administration form include subcutaneous injection, intramuscular injection, intravenous injection, and local administration.
[0144] また、本発明の医薬組成物の投与量も、有効成分の種類;投与対象となる個体、器 官、局所部位、組織;投与対象となる個体の年齢、体重等に応じて、適宜選択される 。投与方法としては、特に限定されないが、有効成分が、低分子化合物又は高分子 化合物である場合、前記有効成分の量として、例えば、 0. 0001〜： LOOOmgZkg体 重、好ましくは 0. 001〜100mgZkg体重、ポリペプチド又はその誘導体の場合、例 えば、 0. 0001〜1000mgZkg体重、好まし <は 0. 001〜100mgZkg体重、核酸 又はその誘導体の場合、例えば、 0. 00001〜100mgZkg体重、好ましくは 0. 000 1〜： LOmgZkg体重の 1回投与量となるように、 1日につき、 1回若しくは複数回投与 すること等が挙げられる。投与期間も特に限定されるものではない。 [0145] 本発明の医薬組成物の薬理評価は、例えば、骨疾患又は関節疾患モデルマウス に、本発明の医薬組成物を投与し、非投与動物に比べ、投与動物において、骨疾患 又は関節疾患の改善が見られた場合を指標として評価する方法により行なうことがで きる。 [0144] Also, the dosage of the pharmaceutical composition of the present invention is appropriately determined according to the type of active ingredient; the individual to be administered, the organ, the local site, the tissue; the age, weight, etc. of the individual to be administered. Selected. The administration method is not particularly limited, but when the active ingredient is a low molecular weight compound or a high molecular weight compound, the amount of the active ingredient is, for example, 0.0001 to: LOOOmgZkg body weight, preferably 0.001 to 100 mgZkg. In the case of body weight, polypeptide or derivative thereof, for example, 0.0001 to 1000 mg Zkg body weight, preferably <0.001 to 100 mg Zkg body weight, in the case of nucleic acid or derivative thereof, for example, 0.0001 to 100 mg Zkg body weight, preferably 0. 000 1 ~: One dose or multiple doses per day, etc., so as to be a single dose of LOmgZkg body weight. The administration period is not particularly limited. [0145] The pharmacological evaluation of the pharmaceutical composition of the present invention is carried out, for example, by administering the pharmaceutical composition of the present invention to a bone disease or joint disease model mouse and comparing the bone disease or joint disease in the administered animal compared to the non-administered animal. This can be done by a method that evaluates the improvement as an index.
[0146] また、本発明により、骨疾患又は関節疾患の診断方法が提供される。本発明の診 断方法は、被検対象の個体由来の被検試料及び対照試料それぞれにおける本発 明のタンパク質の遺伝子の発現レベルを測定することを 1つの特徴とする。したがつ て、本発明の診断方法によれば、被検対象の個体が骨疾患又は関節疾患に罹患し て!、る疑 、がある力否かを簡便かつ迅速に診断することができると!/、う優れた効果が 発揮される。  [0146] The present invention also provides a method for diagnosing a bone disease or a joint disease. One feature of the diagnostic method of the present invention is that the expression level of the gene of the protein of the present invention in each of the test sample and the control sample derived from the individual to be tested is measured. Therefore, according to the diagnostic method of the present invention, it is possible to easily and quickly diagnose whether or not a test subject has a bone disease or a joint disease! ! /, An excellent effect is exhibited.
[0147] 本発明の診断方法においては、被検試料における発現レベル力 対照試料にお ける発現レベルと異なる場合 (例えば、高くなる場合、低くなる場合)、該被検対象の 個体が、骨疾患又は関節疾患に罹患している疑いがあることの指標となる。  [0147] In the diagnostic method of the present invention, when the expression level force in the test sample is different from the expression level in the control sample (for example, when it becomes higher or lower), the individual to be tested becomes a bone disease. Or it becomes an indicator of suspected to have joint disease.
[0148] 以下実施例は、例示のために提供され、そして限定するためではな!/ヽ。遺伝子操 作的手法として、特に断らな 、限りモレキュラー ·クローユング第 2版に記載された方 法を用いた。  [0148] The following examples are provided for purposes of illustration and not limitation! / ヽ. As a genetic engineering method, the method described in Molecular Cloning 2nd edition was used unless otherwise specified.
実施例 1  Example 1
[0149] DNAチップの作成 [0149] Creation of DNA chip
ヒト間葉系幹細胞（BIO WHITTAKER社）より、 QuickPrep Micro mRNA Purification Kit (アマシャムバイオサイエンス社）を用い、添付のマニュアルに従 つて mRNAを調製した。得られた mRNAを Superscript Choice System for cDNA Synthesis (インビトロジェン社）にて添付マニュアルに従って cDNA化して 、 cDNAクローンを得た。その後、 cDNAクローンから PCR法によりヒト cDNA断片 3 265個を調製し、マイクロアレイ用コントロール DNAである Lucidea Universal Sc oreCard (アマシャムバイオサイエンス社）と共に、 DNAチップ作製システム Microa rray System Generation III Spotter (モレキュラー ダイナミクス社）を用いて 、 Type7starスライドグラス (モレキュラー ダイナミクス社）に 1スライドあたり、左右対 称になる形で（以下、左側を SET1、右側を SET2とする） 2セットをスポットして DNA チップを作製した。 MRNA was prepared from human mesenchymal stem cells (BIO WHITTAKER) using QuickPrep Micro mRNA Purification Kit (Amersham Biosciences) according to the attached manual. The obtained mRNA was converted to cDNA by Superscript Choice System for cDNA Synthesis (Invitrogen) according to the attached manual to obtain a cDNA clone. Subsequently, 3265 human cDNA fragments were prepared from the cDNA clones by PCR, and together with Lucidea Universal ScACard (Amersham Biosciences), a control DNA for microarrays, a DNA chip production system Microarray System Generation III Spotter (Molecular Dynamics) ) On a Type7star glass slide (Molecular Dynamics) in a symmetrical form (hereinafter referred to as SET1 on the left side and SET2 on the right side). A chip was produced.
実施例 2  Example 2
[0150] ヒト膝関節滑膜からの RNA調製 [0150] Preparation of RNA from human knee joint synovium
三重大学医学部整形外科において症状、理学所見、画像所見より変形性膝関節症 (膝 OA)と診断され、手術治療を行った患者 44例を対象とした。またコントロール群と して慢性関節リウマチ (RA) 10例、及び非 OA非 RA8例（骨肉腫、ユーイング肉腫等 大腿骨遠位部における悪性骨腫瘍で治療のために関節の合併切除を必要とする患 者及び外傷により関節手術を受ける患者)も対象とした。これら対象者に対して事前 に十分な説明を行った上で、同意を得られた対象者カゝら手術時に膝関節の滑膜組 織 (約 0. 5g)を摘出し、マイクロチューブに入れ、ただちに液体窒素を用いて凍結し た。凍結した滑膜組織は、液体窒素にて冷却した試料粉砕機クライオプレス CP—1 00W (マイクロテック' -チオン社)を用いて粉砕し、直ちに TRIzol試薬 (インビトロジ ェン社）中でホモゲナイザ一 ·ポリトロン 2100 (Kinematica社）を用いて懸濁し、 TRI zol試薬の処方に従って全 RN Aを抽出した。  Forty-four patients who were diagnosed with osteoarthritis of the knee (knee OA) based on symptoms, physical findings, and image findings at Mie University School of Medicine were treated. In addition, 10 patients with rheumatoid arthritis (RA) and 8 patients with non-OA non-RA (bone sarcoma, Ewing sarcoma, etc.) needed malignant bone tumor in the distal femur to require joint resection for treatment Patients and patients undergoing joint surgery due to trauma) were also included. After sufficient explanation in advance for these subjects, the synovial tissue (approximately 0.5 g) of the knee joint was removed and placed in a microtube at the time of surgery. Immediately, it was frozen using liquid nitrogen. The frozen synovial tissue is crushed using a cryopulverizer CP-1 00W (Microtech's-Thion Co., Ltd.) cooled with liquid nitrogen, and immediately homogenized in a TRIzol reagent (Invitrogen Corp.). Suspension was performed using Polytron 2100 (Kinematica), and total RNA was extracted according to the formulation of TRI zol reagent.
実施例 3  Example 3
[0151] 変形性関節症滑膜検体において発現が変動する遺伝子の検索  [0151] Search for genes whose expression varies in synovial specimens of osteoarthritis
実施例 1で述べた DNAチップを用いて、ヒト変形性膝関節症滑膜臨床検体での遺 伝子発現変動解析を実施した。実施例 2で述べた RNAを RNeasy mini kit (キア ゲン社）にて添付マニュアルに従い、 DNase I処理した後、回収した。  Using the DNA chip described in Example 1, gene expression variation analysis was performed in clinical samples of human knee osteoarthritis synovium. The RNA described in Example 2 was recovered after DNase I treatment using RNeasy mini kit (Qiagen) according to the attached manual.
これで得た全 RNA2.5 μ gを用い、 MessageAmp aRNA Kit (Ambion社）のマ- ュアルに従い、増幅 RNA(aRNA)を合成した。合成した aRNAをプローブの铸型と し、 Atlas Glass Fluorescent Labelling Kit (CLONTECH社現 BD BIOSCIE NCE社）若しくは Atlas PowerScript Fluorescent Labeling Kit (現 BD BIOSC IENCE)を用いて、マ-ユアルに従い Random 18merプライマーにより Cy— 3 d CTP又は Cy— 5 dCTP (アマシャムバイオサイエンス社）を取り込ませて cDNAを蛍 光標識した。これをプローブとし、 Automated Slide Processor (アマシャムバイ ォサイエンス社）を用いて DNAチップとのハイブリダィゼーシヨンを実施した。ハイブ リダィゼーシヨンは、 Type 7starスライド（アマシャムノィォサイエンス社）のプロトコ ールに従い、前処理の後にプローブと 48°Cで 12時間行った。バッファーには Hybri dizaton Buffer Ver. 2 (アマシャムバイオサインス社）を用いた。洗浄後、 Microa rray System Generation III Scanner (モレキュラー ダイナミクス社）により読み 取りを行なった。この方法に従い、膝 OA、 RA、非 OA非 RAからなる 62症例を用い て、重複して行なった実験も含め、 78スライドの実験を行なった。 Using 2.5 μg of the total RNA thus obtained, amplified RNA (aRNA) was synthesized according to the manual of MessageAmp aRNA Kit (Ambion). Using the synthesized aRNA as a probe cage, use the Atlas Glass Fluorescent Labeling Kit (CLONTECH, currently BD BIOSCIE NCE) or Atlas PowerScript Fluorescent Labeling Kit (currently, BD BIOSC IENCE), and use the Random 18mer primer according to the manual. — 3d CTP or Cy-5 dCTP (Amersham Biosciences) was incorporated and the cDNA was fluorescently labeled. Using this as a probe, hybridization with a DNA chip was performed using an Automated Slide Processor (Amersham Bioscience). Hybridization is a protocol of Type 7star slide (Amersham Neoscience). The pretreatment was followed for 12 hours at 48 ° C with the probe. Hybri dizaton Buffer Ver. 2 (Amersham Bio-Signs) was used as the buffer. After washing, reading was performed with a Microarray System Generation III Scanner (Molecular Dynamics). In accordance with this method, 78 slide experiments were conducted using 62 cases of knee OA, RA, and non-OA non-RA, including duplicate experiments.
実施例 4  Example 4
[0152] 統計解析による変形性関節症関連遺伝子の探索  [0152] Search for osteoarthritis-related genes by statistical analysis
実施例 3で述べた DNAチップ実験より得られた遺伝子発現量データを用いて、変 形性関節症に関与する遺伝子を、正常者あるいは慢性関節リウマチと比較して探索 した。  Using the gene expression data obtained from the DNA chip experiment described in Example 3, genes involved in deformed arthropathy were searched in comparison with normal individuals or rheumatoid arthritis.
[0153] 1. 解析用正規化データ  [0153] 1. Normalized data for analysis
遺伝子発現量を  Gene expression level
[数 1]  [Number 1]
VOL k ( = 1,· · · , 3265 ) とし、 3DR遺伝子の発現量がスライド内及びスライド間で同一になるように、（1)式の 正規化を実施した。 VOL k (= 1,..., 3265), and normalization of equation (1) was performed so that the expression level of the 3DR gene was the same within the slide and between slides.
[数 2]  [Equation 2]
¾ = = 1，'··，3265 ( 1)
Figure imgf000052_0001
なお、 3DRとは実施例 1で述べた Lucidea Universal ScoreCard (商品名、アマ シャムバイオサイエンス社）に含まれるチェック用遺伝子のひとつである。また、症例 によっては複数枚のスライドで測定がなされていた力 とくに断らない限り、複数のス ライドがあった症例では、スライド番号の最も小さいスライドを解析に用いた。また、実 施例 1で述べたように、 1スライドには、 SET1と SET2のデータが存在する。 ¾ = = 1, '·, 3265 (1)
Figure imgf000052_0001
3DR is one of the check genes included in Lucidea Universal ScoreCard (trade name, Amersham Biosciences) described in Example 1. Also, in some cases, the force measured by multiple slides. Unless otherwise noted, the slide with the smallest slide number was used for analysis in cases with multiple slides. As described in Example 1, SET1 and SET2 data exist in one slide.
正規化には統計解析パッケージソフトウェア SAS (SAS Institute Japan社）を 利用した。データ解析には、単変量解析の Cluster+Logisticsでは SASを利用し た。 Statistical analysis package software SAS (SAS Institute Japan) is used for normalization. used. For data analysis, SAS was used in Cluster + Logistics for univariate analysis.
2. 遺伝子単位としての単変量解析  2. Univariate analysis as a gene unit
i ( = l, · ··, 61)番目のスライドの k( = l, · ··, 3265)番目の遺伝子における正規ィ匕 後の発現量を単に  i (= l, ..., 61) The expression level after normalization in the k (= l, ..., 3265) th gene of the slide is simply
[数 3] [Equation 3]
で表示する。また、スライドが正常、 OA、 RAのいずれから得られたかを識別する場 合は、それぞれ Is displayed. Also, if you want to identify whether the slide is normal, OA, or RA,
画 normal /M Normal / M
Xik 、 ik Xik で表示する。 Display with Xik, ik X ik.
全 3265個の遺伝子の中力も正常、 OA、 RAの 3群を識別するにあたって、とくに重 要な遺伝子を選ぶことを目的として、 1遺伝子ごとの解析 (単変量解析)を行った。 2.1 1症例 1スライド解析  Analysis of each gene (univariate analysis) was performed with the aim of selecting particularly important genes when distinguishing the three groups of normal, OA, and RA among all 3265 genes. 2.1 1 case 1 slide analysis
SET1と SET2を対象とした解析をそれぞれ実施し、次の条件を満たした遺伝子を 求めた。  Analysis for SET1 and SET2 was performed, and genes that satisfy the following conditions were determined.
条件 A: SET1と SET2のどちらかで、平均値の比較と平均絶対差の比較のどちらか で、 OAと正常の間、 OAと RAの間で共に有意差が認められること。 Condition A: In either SET1 or SET2, there is a significant difference between OA and normal, and between OA and RA in either the average value comparison or the average absolute difference comparison.
条件 B : SET1と SET2で共に、平均値の比較で、 OAと正常の間、 OAと RAの間の どちらかで有意差が認められること。 Condition B: For both SET1 and SET2, there is a significant difference between OA and normal or between OA and RA when comparing the mean values.
ここに、平均絶対差の比較はデータ  Here, comparison of mean absolute difference is data
[数 5] normal [Equation 5] normal
m k m k
Figure imgf000053_0001
を用いた比較を指し、
Figure imgf000053_0001
Refers to the comparison using
[数 6] normal  [Equation 6] normal
は正常群での中央値を表す。平均値の比較はデータ Represents the median in the normal group. Average comparison is data
[数 7] ik を用いた比較を指す。  [Equation 7] Refers to the comparison using ik.
OAと正常、 OAと RAの 2通りのペア間比較には、全体有意水準を 0. 05として、 D unnettの多重比較法を用いた。  For comparison between two pairs of OA and normal, OA and RA, Dunnett's multiple comparison method was used with an overall significance level of 0.05.
[0156] 2.2 1症例複数スライド解析 [0156] 2.2 Multiple slide analysis of one case
スライド間変動と SET間変動を含む線形混合効果モデルに基づく解析で、次の条 件を満たした遺伝子を求めた。  Genes satisfying the following conditions were determined by analysis based on a linear mixed-effects model including inter-slide variation and inter-SET variation.
条件 C :平均値の比較と平均絶対差の比較とで、 OAと正常の間、 OAと RAの間で共 に有意差が認められること。  Condition C: Significant differences are observed between OA and normal and between OA and RA between the average value and the average absolute difference.
ここでの有意差は、 OAと正常、 OAと RAの 2通りのペア間比較に対応する回帰係 数の検定での有意性を指す。  The significant difference here refers to the significance of the regression coefficient test corresponding to the comparison between two pairs of OA and normal and OA and RA.
[0157] 2.3 単変量解析の結果の集約 [0157] 2.3 Aggregation of univariate results
以下の 2つの基準で遺伝子を選択した。  Genes were selected according to the following two criteria.
第 1次選択基準：条件 Aを満たす。  Primary selection criteria: Condition A is met.
第 2次選択基準：第 1次選択基準を満たさず、条件 Bかつ条件 Cを満たす。  Second selection criteria: The first selection criteria are not met, but Condition B and Condition C are met.
その結果、第 1次選択基準で 23個、第 2次選択基準で 3個の遺伝子を選択し、合 計 26個の遺伝子を OAに関与する遺伝子とした (表 1)。これらの遺伝子について、 O A患者における発現量を RA患者と正常個体それぞれにおける発現量と比べた (表 2 ) oある遺伝子において、 OA患者における発現量が RA患者及び正常個体における 発現量を基準としてともに +である場合、該遺伝子の発現レベルは、 OA患者 >RA 患者、かつ、 OA患者 >正常個体であることを示唆している。ある遺伝子において、 O Aにおける発現量が RA患者における発現量を基準として +であり、正常個体におけ る発現量を基準として一である場合、該遺伝子の発現レベルは、正常個体 > OA患 者 >RA患者であることを示唆している。また、ある遺伝子において、 OA患者におけ る発現量が RA患者における発現量を基準として一であり、正常個体における発現量 を基準として +である場合、該遺伝子の発現レベルは、 RA患者 >OA患者 >正常 個体であることを示唆して 、る。 As a result, 23 genes were selected based on the primary selection criteria and 3 genes were selected based on the secondary selection criteria, and a total of 26 genes were selected as genes involved in OA (Table 1). For these genes, the expression levels in OA patients were compared with the expression levels in RA patients and normal individuals, respectively (Table 2). O For certain genes, the expression levels in OA patients were both relative to the expression levels in RA patients and normal individuals. If +, the expression level of the gene is OA patients> RA It suggests that the patient and OA patient> normal individual. For a gene, if the expression level in OA is + based on the expression level in RA patients and is one based on the expression level in normal individuals, the expression level of the gene is normal individuals> OA patients> This suggests that he is an RA patient. In addition, when the expression level in an OA patient is one based on the expression level in an RA patient and is + based on the expression level in a normal individual, the expression level of the gene is RA patient> OA Patient> normal, suggesting that it is an individual.
[0158] [表 1] 単変量解析で選出した 26個の遺伝子 [0158] [Table 1] 26 genes selected by univariate analysis
第 1次選択基準 C0052 G0953 N1108  Primary selection criteria C0052 G0953 N1108
C0772 N0065 N1112  C0772 N0065 N1112
D0032 NO 154 N1123  D0032 NO 154 N1123
E0083 N0281  E0083 N0281
E0215* N0439  E0215 * N0439
E0447 N0446  E0447 N0446
E0721* N0458  E0721 * N0458
E0739 N0513  E0739 N0513
E0905* N0553  E0905 * N0553
E1019 N1032  E1019 N1032
第 2次選択基準 C0988  Second selection criteria C0988
E0639  E0639
NO 160  NO 160
[0159] [表 2] [0159] [Table 2]
OA患者における 26個の遺伝子の発現量 Expression levels of 26 genes in OA patients
OA患者における発現量  Expression levels in OA patients
RAを基準 正常を基準  RA standard Normal condition
C0052 + +  C0052 + +
C0772 + +  C0772 + +
C0988 + +  C0988 + +
D0032 + +  D0032 ++
E0083 + 一  E0083 + one
E0215* + +  E0215 * + +
E0447 + +  E0447 + +
E0639 一 +  E0639 One +
E0721* + +  E0721 * + +
E0739 + +  E0739 + +
E0905* + +  E0905 * + +
E1019 + +  E1019 + +
G0953 + +  G0953 + +
画 65 + +  Pic 65 + +
NO 154 一 +  NO 154 one +
NO 160 一 +  NO 160 one +
N0281 ― +  N0281 ― +
N0439 + +  N0439 + +
N0446 一 +  N0446 One +
N0458 + +  N0458 + +
N0513 + +  N0513 + +
N0553 + +  N0553 + +
画 32 一 +  Pic 32 one +
N1108 + +  N1108 + +
N1112 + +  N1112 + +
N1123 ― +  N1123 ― +
+：発現量が卨ぃ  +: Expression level
-：発現量が低い 実施例 5  -: Low expression level Example 5
OAに関与する遺伝子の塩基配列解析  Nucleotide sequence analysis of genes involved in OA
実施例 4で述べた 26個の OAに関連する遺伝子について、それぞれ実施例 1で述 ベた PCR断片の塩基配列を DNAシークェンシング装置 MegaBACElOOO (アマシ ャムバイオサイェンシズ社)を用いて解析した。 PCR断片が得られなカゝつた 2個の遺 伝子を除き、残り 24個の遺伝子について得られた塩基配列はそれぞれ遺伝子情報 解析ソフトウェア bioSCOUT(LION社)を用いて相同性検索を行った。その結果、 2 4個すベての遺伝子にっ 、て遺伝子名が判明し、 3個は Fibulin— 3が重複して 、た 。重複を除く 22個の遺伝子について、 PubMED (米国立医学図書館生物工学情報 センター）及び PATENTWeb (MicroPatent社）を検索し、それぞれの遺伝子と O Aとの関連性が公知である力否かを検討し、その関連性が既に示されている遺伝子 5個を除き、残り 17個の遺伝子が OAに関与していることが新たに明ら力となった。表 3にその 17個の遺伝子を示す。 For each of the 26 OA-related genes described in Example 4, the base sequence of the PCR fragment described in Example 1 was converted to the DNA sequencing device MegaBACElOOO (Amaci (Ambi Biosciences). Except for the two genes from which PCR fragments were not obtained, the base sequences obtained for the remaining 24 genes were subjected to homology search using the gene information analysis software bioSCOUT (LION). As a result, the gene names were found for all 24 genes, and 3 were duplicated with Fibulin-3. We searched PubMED (National Center for Biotechnology Information) and PATENTWeb (MicroPatent) for 22 genes excluding duplicates, and examined whether the relationship between each gene and OA is known, With the exception of five genes that have already been shown to be related, the remaining 17 genes are involved in OA. Table 3 shows the 17 genes.
[表 3] 新たに O Aとの関連性が明らかとなった 1 7個の遺伝子 [Table 3] 17 newly found associations with OA
Figure imgf000057_0001
Figure imgf000057_0001
実施例 6 リアルタイム定量 PCR法による Fibulin— 3mRNAの発現解析 Example 6 Fibulin-3 mRNA expression analysis by real-time quantitative PCR
統計解析の結果、最終的に選出した 17個の遺伝子において、 4個の遺伝子が Fib ulin— 3 (配列番号： 13)であったことから、 RT— PCR法により、ヒト変形性膝関節症 滑膜組織における Fibulin— 3遺伝子の発現プロファイルを調べ、 DNAチップの結 果と比較した。サンプルはすべて DNAチップ解析に用いた RNAを用いた。サンプ ル番号 1は正常、 2— 12は OA患者の滑膜 RNAである。  As a result of statistical analysis, among the 17 genes finally selected, 4 genes were Fibulin-3 (SEQ ID NO: 13). The expression profile of Fibulin-3 gene in membrane tissue was examined and compared with the results of DNA chip. All samples used RNA used for DNA chip analysis. Sample number 1 is normal, 2-12 are synovial RNA from OA patients.
なお、 RT—PCRには、配列番号： 35及び 36のプライマー対を用いた。実施例 2で 述べたヒト変形性膝関節症滑膜臨床検体より調製した RNAを RNeasy mini kit ( キアゲン社）にて添付マニュアルに従い、 DNase I処理した後、回収した。  For RT-PCR, primer pairs of SEQ ID NOs: 35 and 36 were used. RNA prepared from clinical samples of human knee osteoarthritis synovium described in Example 2 was treated with DNase I using RNeasy mini kit (Qiagen) according to the attached manual, and then recovered.
得られた全 RNA 2.5 ;z gより RT—PCR用 Superscript First -strand Synth esis System (インビトロジェン社）を用いて添付マニュアルに従!ヽ、 cDNAを合成し た。次に、ヒト Fibulin— 3 (配列番号： 13)の配列をもとにプローブ検索ソフト Primer From the obtained total RNA 2.5; z g, cDNA was synthesized using the Superscript First-strand Synthesis System for RT-PCR (Invitrogen) according to the attached manual. Next, the probe search software Primer based on the sequence of human Fibulin-3 (SEQ ID NO: 13)
Express (アプライドバイオシステムズ社）によりリアルタイム定量 PCR用プライマー（ 配列番号： 35及び 36)を選択した。プライマーは、キアゲン社に合成を依頼した。こ のプライマーを用いて、合成した cDNAを铸型として SYBR Green Iによるリアルタ ィム定量 PCRを行ない、 mRNA量を定量した。 Primers for real-time quantitative PCR (SEQ ID NOs: 35 and 36) were selected by Express (Applied Biosystems). Primers were synthesized by Qiagen. Using this primer, real-time quantitative PCR with SYBR Green I was performed using the synthesized cDNA as a saddle type, and the amount of mRNA was quantified.
反応試薬としては、 SYBR Green PCR Master Mix (アプライドバイオシステムズ 社）を使用し、 95°Cで 10分間反応した後、 95°Cで 10秒、 60°Cで 60秒 40サイクル行 なって蛍光強度を定量した。 PCR、及び蛍光測定は、 ABI PRISM 5700 Sequ ence Detection System (アプライドバイオシステムズ社）を用いて行なった。その 結果、 DNAチップ実験同様、リアルタイム定量 PCR実験によっても、 OA臨床検体 では Fibulin— 3遺伝子の発現が増強することが確認された（図 1)。図 1のリアルタイ ム定量 PCRデータのグラフは、正常滑膜臨床検体 (サンプル番号 1)における発現量 を 1とした相対量で示している。なお、 DNAチップ法とリアルタイム定量 PCR法とによ る遺伝子発現量は正の相関（Pearsonの相関係数:約 0. 69)を示すことが確認され た。従って、残りの遺伝子についても同様の結果であることが予想され、表 3に示した 17個の遺伝子の mRNA発現を指標として、変形性関節症が診断できることが示唆さ れた。また、実施例 4において、発現レベルが正常個体 > OA患者 >RA患者、又は RA患者 > OA患者 >正常個体と 、う関係を示した 7個の遺伝子につ 、ては、 mRN A発現を指標として、慢性関節リウマチが診断できることが示唆された。 SYBR Green PCR Master Mix (Applied Biosystems) was used as a reaction reagent. After reacting at 95 ° C for 10 minutes, perform fluorescence for 10 seconds at 95 ° C and 40 seconds at 60 ° C for 40 seconds. Was quantified. PCR and fluorescence measurement were performed using ABI PRISM 5700 Sequence Detection System (Applied Biosystems). As a result, real-time quantitative PCR experiments, as well as DNA chip experiments, confirmed that Fibulin-3 gene expression was enhanced in OA clinical specimens (Figure 1). The graph of real-time quantitative PCR data in Fig. 1 shows the relative amount when the expression level is 1 in a normal synovial clinical specimen (sample number 1). It was confirmed that the gene expression level by the DNA chip method and the real-time quantitative PCR method showed a positive correlation (Pearson's correlation coefficient: about 0.69). Therefore, the same results were expected for the remaining genes, suggesting that osteoarthritis can be diagnosed using the mRNA expression of the 17 genes shown in Table 3 as an index. In Example 4, the expression level is normal individuals> OA patients> RA patients, or It was suggested that rheumatoid arthritis can be diagnosed using the mRNA expression as an index for the seven genes that showed a relationship between RA patients> OA patients> normal individuals.
産業上の利用可能性 Industrial applicability
本発明の検出又は選別方法により、骨疾患又は関節疾患、特に変形性関節症、慢 性関節リウマチに罹患している疑いがある個体由来の試料を簡便かつ迅速に検出又 は選別をすることができる。また、検出又は選別に必要な診断薬を提供する。  By the detection or screening method of the present invention, a sample derived from an individual suspected of suffering from a bone disease or joint disease, particularly osteoarthritis or rheumatoid arthritis, can be detected or selected easily and rapidly. it can. In addition, diagnostic agents necessary for detection or selection are provided.

Claims

請求の範囲 The scope of the claims
[1] 被検対象の個体由来の被検試料における配列番号： 18〜34からなる群より選ばれ るアミノ酸配列を有するタンパク質又は該アミノ酸配列において 1若しくは数個のアミ ノ酸が欠失、置換若しくは付加されたアミノ酸配列を有するタンパク質をコードする少 なくとも 1つの遺伝子の発現レベルを測定するステップを含み、ここで、被検試料に おける発現レベル力 正常個体由来の対照試料における発現レベルと異なる場合、 該被検試料が、骨疾患又は関節疾患に罹患して 、る疑 、がある個体由来の試料で あることの指標となる、骨疾患若しくは関節疾患に罹患している疑いがある個体由来 の試料の検出又は選別方法。  [1] A protein having an amino acid sequence selected from the group consisting of SEQ ID NO: 18 to 34 in a test sample derived from an individual to be tested, or one or several amino acids deleted or substituted in the amino acid sequence Or measuring the expression level of at least one gene encoding a protein having an added amino acid sequence, wherein the expression level power in the test sample is different from the expression level in a control sample from a normal individual In this case, the test sample is derived from an individual suspected of suffering from a bone disease or joint disease, which is an indicator that the sample is derived from a certain individual. A method for detecting or sorting samples.
[2] 骨疾患又は関節疾患が、軟骨形成異常、骨形成異常、骨粗鬆症、変形性関節症、 慢性関節リウマチ、関節炎、滑膜炎、代謝性関節症又はスポーツによる関節障害で ある請求項 1記載の方法。  [2] The bone disease or joint disease is cartilage dysplasia, bone dysplasia, osteoporosis, osteoarthritis, rheumatoid arthritis, arthritis, synovitis, metabolic arthropathy or arthropathy due to sports. the method of.
[3] 被検対象の個体由来の被検試料における配列番号： 18、 21〜23、 25、 29からなる 群より選ばれるアミノ酸配列を有するタンパク質又は該アミノ酸配列において 1若しく は数個のアミノ酸が欠失、置換若しくは付加されたアミノ酸配列を有するタンパク質を コードする少なくとも 1つの遺伝子の発現レベルを測定するステップを含み、ここで、 被検試料における発現レベルが、正常個体由来の対照試料における発現レベルより も高くなり、かつ、慢性関節リウマチ患者由来の対照試料における発現レベルよりも 低くなる場合、該被検試料が、変形性関節症に罹患している疑いがある個体由来の 試料であることの指標となる、変形性関節症に罹患している疑いがある個体由来の 試料の検出又は選別方法。  [3] A protein having an amino acid sequence selected from the group consisting of SEQ ID NO: 18, 21, 23, 25, 29 in a test sample derived from an individual to be tested, or one or several amino acids in the amino acid sequence Measuring the expression level of at least one gene encoding a protein having a deleted, substituted or added amino acid sequence, wherein the expression level in the test sample is expressed in a control sample from a normal individual The test sample is from an individual suspected of suffering from osteoarthritis if it is higher than the level and lower than the expression level in a control sample from a patient with rheumatoid arthritis A method for detecting or selecting a sample derived from an individual suspected of suffering from osteoarthritis, which is an indicator of the above.
[4] 被検対象の個体由来の被検試料における配列番号： 19、 20、 24、 26〜28、 30〜3 3からなる群より選ばれるアミノ酸配列を有するタンパク質又は該アミノ酸配列におい て 1若しくは数個のアミノ酸が欠失、置換若しくは付加されたアミノ酸配列を有するタ ンパク質をコードする少なくとも 1つの遺伝子の発現レベルを測定するステップを含 み、ここで、被検試料における発現レベルが、正常個体由来の対照試料における発 現レベルよりも高くなり、かつ、慢性関節リウマチ患者個体由来の対照試料における 発現レベルよりも高くなる場合、該被検試料が、変形性関節症に罹患している疑いが ある個体由来の試料であることの指標となる、変形性関節症に罹患している疑いがあ る個体由来の試料の検出又は選別方法。 [4] A protein having an amino acid sequence selected from the group consisting of SEQ ID NOs: 19, 20, 24, 26-28, 30-33 in a test sample derived from an individual to be tested, or 1 or 2 in the amino acid sequence Measuring the expression level of at least one gene encoding a protein having an amino acid sequence in which several amino acids have been deleted, substituted or added, wherein the expression level in the test sample is normal A test sample is suspected of suffering from osteoarthritis if it is higher than the expression level in a control sample from an individual and higher than the expression level in a control sample from an individual with rheumatoid arthritis But A method for detecting or selecting a sample derived from an individual suspected of suffering from osteoarthritis, which serves as an indicator that the sample is derived from an individual.
[5] 被検対象の個体由来の被検試料における配列番号： 34のアミノ酸配列を有するタン ノ ク質又は該アミノ酸配列において 1若しくは数個のアミノ酸が欠失、置換若しくは付 加されたアミノ酸配列を有するタンパク質をコードする遺伝子の発現レベルを測定す るステップを含み、ここで、被検試料における発現レベル力 正常個体由来の対照試 料における発現レベルよりも低くなり、かつ、慢性関節リウマチ患者個体由来の対照 試料における発現レベルよりも高くなる場合、該被検試料が、変形性関節症に罹患 している疑いがある個体由来の試料であることの指標となる、変形性関節症に罹患し ている疑いがある個体由来の試料の検出又は選別方法。  [5] A protein having the amino acid sequence of SEQ ID NO: 34 in a test sample derived from an individual to be tested, or an amino acid sequence in which one or several amino acids have been deleted, substituted or added in the amino acid sequence Measuring the expression level of a gene encoding a protein having the expression level, wherein the expression level in the test sample is lower than the expression level in a control sample derived from a normal individual, and the individual has rheumatoid arthritis. If the expression level is higher than the expression level in the control sample derived from, the test sample suffers from osteoarthritis, which is an indicator that the sample is derived from an individual suspected of suffering from osteoarthritis. A method for detecting or sorting a sample derived from an individual suspected of having
[6] 被検対象の個体由来の被検試料における配列番号： 18、 21〜23、 25、 29からなる 群より選ばれるアミノ酸配列を有するタンパク質又は該アミノ酸配列において 1若しく は数個のアミノ酸が欠失、置換若しくは付加されたアミノ酸配列を有するタンパク質を コードする少なくとも 1つの遺伝子の発現レベルを測定するステップを含み、ここで、 被検試料における発現レベルが、正常個体由来の対照試料における発現レベルより も高くなり、かつ、変形性関節症患者由来の対照試料における発現レベルよりも高く なる場合、該被検試料が、慢性関節リウマチに罹患している疑いがある個体由来の 試料であることの指標となる、慢性関節リウマチに罹患している疑いがある個体由来 の試料の検出又は選別方法。  [6] A protein having an amino acid sequence selected from the group consisting of SEQ ID NOs: 18, 21 to 23, 25 and 29 in a test sample derived from an individual to be tested, or one or several amino acids in the amino acid sequence Measuring the expression level of at least one gene encoding a protein having a deleted, substituted or added amino acid sequence, wherein the expression level in the test sample is expressed in a control sample from a normal individual The test sample is from an individual suspected of suffering from rheumatoid arthritis if it is higher than the level and higher than the expression level in a control sample from a patient with osteoarthritis A method for detecting or selecting a sample derived from an individual suspected of having rheumatoid arthritis, which is an index of
[7] 被検対象の個体由来の被検試料における配列番号： 34のアミノ酸配列を有するタン ノ ク質又は該アミノ酸配列において 1若しくは数個のアミノ酸が欠失、置換若しくは付 加されたアミノ酸配列を有するタンパク質をコードする遺伝子の発現レベルを測定す るステップを含み、ここで、被検試料における発現レベル力 正常個体由来の対照試 料における発現レベルよりも低くなり、かつ、変形性関節症患者個体由来の対照試 料における発現レベルよりも低くなる場合、該被検試料が、慢性関節リウマチに罹患 している疑いがある個体由来の試料であることの指標となる、慢性関節リウマチに罹 患して 、る疑 、がある個体由来の試料の検出又は選別方法。  [7] A protein having the amino acid sequence of SEQ ID NO: 34 in a test sample derived from an individual to be tested, or an amino acid sequence in which one or several amino acids have been deleted, substituted or added in the amino acid sequence Measuring the expression level of a gene encoding a protein having an expression level, wherein the expression level in the test sample is lower than the expression level in a control sample derived from a normal individual, and the patient has osteoarthritis. Rheumatoid arthritis affected if the test sample is lower than the expression level in an individual-derived control sample, indicating that the test sample is derived from an individual suspected of suffering from rheumatoid arthritis Thus, a method for detecting or selecting a sample derived from an individual who is suspected.
[8] 発現レベル力 配列番号： 18〜34からなる群より選ばれるアミノ酸配列を有するタン ノ ク質又は該アミノ酸配列において 1若しくは数個のアミノ酸が欠失、置換若しくは付 加されたアミノ酸配列を有するタンパク質をコードする少なくとも 1つの遺伝子の mR NA量を指標として測定される、請求項 1〜7いずれかに記載の方法。 [8] Power of expression level SEQ ID NO: Tan having an amino acid sequence selected from the group consisting of 18 to 34 The protein or at least one gene encoding a protein having an amino acid sequence in which one or several amino acids have been deleted, substituted or added in the amino acid sequence, is measured using the mRNA amount as an index. -7 method in any one.
[9] 発現レベル力 配列番号： 18〜34からなる群より選ばれるアミノ酸配列を有するタン ノ ク質又は該アミノ酸配列において 1若しくは数個のアミノ酸が欠失、置換若しくは付 カロされたアミノ酸配列を有する少なくとも 1つのタンパク質又はその部分ペプチド又は その塩を指標として測定される、請求項 1〜7いずれかに記載の方法。  [9] Expression level ability SEQ ID NO :: A protein having an amino acid sequence selected from the group consisting of 18 to 34, or an amino acid sequence in which one or several amino acids have been deleted, substituted or appended in the amino acid sequence The method according to any one of claims 1 to 7, wherein the method is measured using at least one protein or a partial peptide thereof or a salt thereof as an index.
[10] 配列番号： 18〜34からなる群より選ばれるアミノ酸配列を有するタンパク質又は該ァ ミノ酸配列において 1若しくは数個のアミノ酸が欠失、置換若しくは付加されたァミノ 酸配列を有するタンパク質をコードする少なくとも 1つの遺伝子を検出しうる核酸を用 いる請求項 8記載の方法。  [10] It encodes a protein having an amino acid sequence selected from the group consisting of SEQ ID NOs: 18 to 34, or a protein having an amino acid sequence in which one or several amino acids have been deleted, substituted or added in the amino acid sequence 9. The method according to claim 8, wherein a nucleic acid capable of detecting at least one gene is used.
[11] 該遺伝子が、配列番号： 1〜17からなる群より選ばれる塩基配列力 なる核酸である 請求項 10記載の方法。  11. The method according to claim 10, wherein the gene is a nucleic acid having a base sequence ability selected from the group consisting of SEQ ID NOs: 1 to 17.
[12] 該遺伝子が、配列番号: 6及び Z又は 13の塩基配列力もなる核酸である請求項 10 記載の方法。  12. The method according to claim 10, wherein the gene is a nucleic acid having the nucleotide sequence of SEQ ID NO: 6 and Z or 13.
[13] 配列番号： 35及び Z又は 36の塩基配列力もなる核酸を用いるものである請求項 12 記載の方法。  [13] The method according to [12], wherein a nucleic acid having the nucleotide sequence of SEQ ID NO: 35 and Z or 36 is used.
[14] 配列番号： 18〜34からなる群より選ばれるアミノ酸配列を有するタンパク質又は該ァ ミノ酸配列において 1若しくは数個のアミノ酸が欠失、置換若しくは付加されたァミノ 酸配列を有する少なくとも 1つのタンパク質又はその部分ペプチド又はその塩に対す る抗体又はその断片を用いる請求項 9記載の方法。  [14] Protein having an amino acid sequence selected from the group consisting of SEQ ID NOs: 18 to 34, or at least one amino acid sequence in which one or several amino acids are deleted, substituted or added in the amino acid sequence 10. The method according to claim 9, wherein an antibody against the protein or a partial peptide thereof or a salt thereof is used.
[15] 配列番号： 23及び Z又は 30のアミノ酸配列を有するタンパク質又は該アミノ酸配列 において 1若しくは数個のアミノ酸が欠失、置換若しくは付加されたアミノ酸配列を有 する少なくとも 1つのタンパク質又はその部分ペプチド又はその塩に対する抗体又は その断片を用いる請求項 9記載の方法。  [15] SEQ ID NO: 23, protein having at least Z or 30 amino acid sequence, or at least one protein having an amino acid sequence in which one or several amino acids are deleted, substituted or added, or a partial peptide thereof 10. The method according to claim 9, wherein an antibody against the salt or a fragment thereof is used.
[16] 配列番号： 18〜34からなる群より選ばれるアミノ酸配列を有するタンパク質又は該ァ ミノ酸配列において 1若しくは数個のアミノ酸が欠失、置換若しくは付加されたァミノ 酸配列を有するタンパク質をコードする少なくとも 1つの遺伝子を検出しうる核酸を含 有してなる、請求項 10〜15いずれかに記載の方法に使用するための診断薬。 [16] SEQ ID NO: encodes a protein having an amino acid sequence selected from the group consisting of 18 to 34, or a protein having an amino acid sequence in which one or several amino acids are deleted, substituted or added in the amino acid sequence A nucleic acid capable of detecting at least one gene A diagnostic agent for use in the method according to any one of claims 10 to 15.
[17] 該遺伝子が、配列番号： 1〜17からなる群より選ばれる塩基配列力 なる核酸である 請求項 16記載の診断薬。 17. The diagnostic agent according to claim 16, wherein the gene is a nucleic acid having a base sequence ability selected from the group consisting of SEQ ID NOs: 1 to 17.
[18] 該遺伝子が、配列番号: 6及び Z又は 13の塩基配列力 なる核酸である請求項 16 記載の診断薬。 18. The diagnostic agent according to claim 16, wherein the gene is a nucleic acid having a nucleotide sequence of SEQ ID NO: 6, Z or 13.
[19] 配列番号： 35及び Z又は 36の塩基配列力もなる核酸を含有してなる、請求項 18記 載の診断薬。  [19] The diagnostic agent according to claim 18, comprising a nucleic acid having the nucleotide sequence of SEQ ID NO: 35 and Z or 36.
[20] 配列番号： 18〜34からなる群より選ばれるアミノ酸配列を有するタンパク質又は該ァ ミノ酸配列において 1若しくは数個のアミノ酸が欠失、置換若しくは付加されたァミノ 酸配列を有する少なくとも 1つのタンパク質又はその部分ペプチド又はその塩に対す る抗体又はその断片を含有してなる、請求項 14又は 15記載の方法に使用するため の診断薬。  [20] A protein having an amino acid sequence selected from the group consisting of SEQ ID NOs: 18 to 34, or at least one amino acid sequence in which one or several amino acids are deleted, substituted or added in the amino acid sequence 16. A diagnostic agent for use in the method according to claim 14 or 15, comprising an antibody against the protein or a partial peptide thereof or a salt thereof or a fragment thereof.
[21] 配列番号： 23及び Z又は 30のアミノ酸配列を有するタンパク質又は該アミノ酸配列 において 1若しくは数個のアミノ酸が欠失、置換若しくは付加されたアミノ酸配列を有 する少なくとも 1つのタンパク質又はその部分ペプチド又はその塩に対する抗体又は その断片を含有してなる、請求項 14又は 15記載の方法に使用するための診断薬。  [21] SEQ ID NO: 23, a protein having an amino acid sequence of Z or 30, or at least one protein having an amino acid sequence in which one or several amino acids have been deleted, substituted or added, or a partial peptide thereof Or a diagnostic agent for use in the method according to claim 14 or 15, which comprises an antibody against a salt thereof or a fragment thereof.
[22] 配列番号： 18〜34からなる群より選ばれるアミノ酸配列を有するタンパク質又は該ァ ミノ酸配列において 1若しくは数個のアミノ酸が欠失、置換若しくは付加されたァミノ 酸配列を有するタンパク質をコードする少なくとも 1つの遺伝子を用いることを特徴と する骨疾患又は関節疾患の予防剤及び Z又は治療剤のスクリーニング方法。  [22] SEQ ID NO: encodes a protein having an amino acid sequence selected from the group consisting of 18 to 34, or a protein having an amino acid sequence in which one or several amino acids are deleted, substituted or added in the amino acid sequence A method for screening a preventive agent and a Z or therapeutic agent for bone disease or joint disease, characterized by using at least one gene.
[23] 配列番号： 18〜34からなる群より選ばれるアミノ酸配列を有するタンパク質又は該ァ ミノ酸配列において 1若しくは数個のアミノ酸が欠失、置換若しくは付加されたァミノ 酸配列を有する少なくとも 1つのタンパク質若しくはその部分ペプチド又はその塩を 用いることを特徴とする骨疾患又は関節疾患の予防剤及び Z又は治療剤のスクリー ユング方法。  [23] A protein having an amino acid sequence selected from the group consisting of SEQ ID NOs: 18 to 34 or at least one amino acid sequence in which one or several amino acids are deleted, substituted or added in the amino acid sequence A method for screening a prophylactic agent and Z or therapeutic agent for bone disease or joint disease, characterized by using a protein or a partial peptide thereof or a salt thereof.
PCT/JP2005/012536 2004-07-09 2005-07-07 Polypeptide participating in bone disease or joint disease and dna thereof WO2006006477A1 (en)

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Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP2131199A1 (en) * 2008-06-03 2009-12-09 Université de Liège Biomarker for osteoarthritis and/or other ageing-related diseases, and use thereof
WO2009146957A1 (en) * 2008-06-03 2009-12-10 Universite De Liege Biomarker for osteoarthritis and/or other ageing-related diseases, and use thereof.
EP2133432A1 (en) * 2007-03-08 2009-12-16 Consejo Superior De Investigaciones Científicas Use of snail1 activity inhibiting compounds in the preparation of pharmaceutical compositions intended for the treatment of chondrodystrophies, method for identifying inhibiting compounds, pharmaceutical compositions, chondrodystrophy diagnosis method and applications thereof
JP2016504422A (en) * 2013-04-02 2016-02-12 イーホン・ジョウＹｉｈｏｎｇ ＺＨＯＵ Fibulin protein variants and corresponding nucleic acid sequences

Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2002055696A1 (en) * 1999-01-13 2002-07-18 Medical & Biological Laboratories Co., Ltd. Mast cell surface antigen, dna thereof and antibody against the antigen
WO2003009814A2 (en) * 2001-07-25 2003-02-06 Millennium Pharmaceuticals, Inc. Novel genes, compositions, kits, and methods for identification, assessment, prevention, and therapy of prostate cancer
JP2004075675A (en) * 2002-06-19 2004-03-11 Takeda Chem Ind Ltd Prophylactic/remedy for osteal/arthral disease
JP2004121218A (en) * 2002-08-06 2004-04-22 Jenokkusu Soyaku Kenkyusho:Kk Method for testing bronchial asthma or chronic obstructive pulmonary disease
WO2004041170A2 (en) * 2002-11-01 2004-05-21 Genentech, Inc. Compositions and methods for the treatment of immune related diseases

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2002055696A1 (en) * 1999-01-13 2002-07-18 Medical & Biological Laboratories Co., Ltd. Mast cell surface antigen, dna thereof and antibody against the antigen
WO2003009814A2 (en) * 2001-07-25 2003-02-06 Millennium Pharmaceuticals, Inc. Novel genes, compositions, kits, and methods for identification, assessment, prevention, and therapy of prostate cancer
JP2004075675A (en) * 2002-06-19 2004-03-11 Takeda Chem Ind Ltd Prophylactic/remedy for osteal/arthral disease
JP2004121218A (en) * 2002-08-06 2004-04-22 Jenokkusu Soyaku Kenkyusho:Kk Method for testing bronchial asthma or chronic obstructive pulmonary disease
WO2004041170A2 (en) * 2002-11-01 2004-05-21 Genentech, Inc. Compositions and methods for the treatment of immune related diseases

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
JUSTEN H, ET AL.: "Differential gene expression in synovium of rheumatoid arthritis and osteoarthritis.", MOL CELL BIOL RES COMMUN, vol. 3, no. 3, 2000, pages 165 - 172, XP001121949 *

Cited By (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP2133432A1 (en) * 2007-03-08 2009-12-16 Consejo Superior De Investigaciones Científicas Use of snail1 activity inhibiting compounds in the preparation of pharmaceutical compositions intended for the treatment of chondrodystrophies, method for identifying inhibiting compounds, pharmaceutical compositions, chondrodystrophy diagnosis method and applications thereof
EP2133432A4 (en) * 2007-03-08 2010-05-26 Consejo Superior Investigacion Use of snail1 activity inhibiting compounds in the preparation of pharmaceutical compositions intended for the treatment of chondrodystrophies, method for identifying inhibiting compounds, pharmaceutical compositions, chondrodystrophy diagnosis method and applications thereof
EP2131199A1 (en) * 2008-06-03 2009-12-09 Université de Liège Biomarker for osteoarthritis and/or other ageing-related diseases, and use thereof
WO2009146957A1 (en) * 2008-06-03 2009-12-10 Universite De Liege Biomarker for osteoarthritis and/or other ageing-related diseases, and use thereof.
WO2009146956A1 (en) * 2008-06-03 2009-12-10 Universite De Liege Biomarker for osteoarthritis and/or other ageing-related diseases, and use thereof
US8771968B2 (en) 2008-06-03 2014-07-08 Universitè de Liège Biomarker for osteoarthritis and/or other ageing-related diseases, and use thereof
US9052313B2 (en) 2008-06-03 2015-06-09 Universite De Liege Biomarker for osteoarthritis and/or other ageing-related diseases, and use thereof
EP2975411A1 (en) * 2008-06-03 2016-01-20 Universite De Liege Biomarker for osteoarthritis and use thereof
JP2016504422A (en) * 2013-04-02 2016-02-12 イーホン・ジョウＹｉｈｏｎｇ ＺＨＯＵ Fibulin protein variants and corresponding nucleic acid sequences

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