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WO2006006477A1 - Polypeptide participant dans une maladie osseuse ou une maladie articulaire et adn de celui-ci - Google Patents

Polypeptide participant dans une maladie osseuse ou une maladie articulaire et adn de celui-ci 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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WIPO (PCT)
Prior art keywords
protein
amino acid
acid sequence
seq
expression level
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PCT/JP2005/012536
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English (en)
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/ja
Publication of WO2006006477A1 publication Critical patent/WO2006006477A1/fr

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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

Il est prévu de fournir un procédé pour détecter ou cribler pratiquement et rapidement une maladie osseuse ou une maladie articulaire qui comprend l’étape consistant à mesurer le niveau d’expression d’au moins un gène encodant pour une protéine ayant une séquence d’acides aminés choisie parmi le groupe comprenant les SEQ ID NOS:18 à 34 dans un échantillon d’essai provenant d’un sujet, procédé au cours duquel une différence, le cas échéant, du niveau d’expression entre l’échantillon d’essai et l’échantillon témoin provenant d'un individu normal sert d’indication montrant la probabilité que l’échantillon d’essai peut provenir d’un individu souffrant d’une maladie osseuse ou d’une maladie articulaire.
PCT/JP2005/012536 2004-07-09 2005-07-07 Polypeptide participant dans une maladie osseuse ou une maladie articulaire et adn de celui-ci WO2006006477A1 (fr)

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EP2131199A1 (fr) * 2008-06-03 2009-12-09 Université de Liège Biomarqueur pour l'arthrose et/ou autres maladies liées à l'âge, et utilisation associée
WO2009146957A1 (fr) * 2008-06-03 2009-12-10 Universite De Liege Biomarqueur de l'ostéoarthrite et/ou d'autres maladies liées au vieillissement, et utilisation associée
EP2133432A1 (fr) * 2007-03-08 2009-12-16 Consejo Superior De Investigaciones Científicas Utilisation des composes inhibant l'activite de snail1 dans l'elaboration de compositions pharmaceutiques utilisees dans le traitement de chondrodysplasies, procede d'identification de composes inhibiteurs, compositions pharmaceutiques, methode de diagnostic de chondrs
JP2016504422A (ja) * 2013-04-02 2016-02-12 イーホン・ジョウＹｉｈｏｎｇ ＺＨＯＵ フィビュリンタンパク質変異体および対応する核酸配列

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Cited By (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP2133432A1 (fr) * 2007-03-08 2009-12-16 Consejo Superior De Investigaciones Científicas Utilisation des composes inhibant l'activite de snail1 dans l'elaboration de compositions pharmaceutiques utilisees dans le traitement de chondrodysplasies, procede d'identification de composes inhibiteurs, compositions pharmaceutiques, methode de diagnostic de chondrs
EP2133432A4 (fr) * 2007-03-08 2010-05-26 Consejo Superior Investigacion Utilisation des composes inhibant l'activite de snail1 dans l'elaboration de compositions pharmaceutiques utilisees dans le traitement de chondrodysplasies, procede d'identification de composes inhibiteurs, compositions pharmaceutiques, methode de diagnostic de chondrs
EP2131199A1 (fr) * 2008-06-03 2009-12-09 Université de Liège Biomarqueur pour l'arthrose et/ou autres maladies liées à l'âge, et utilisation associée
WO2009146957A1 (fr) * 2008-06-03 2009-12-10 Universite De Liege Biomarqueur de l'ostéoarthrite et/ou d'autres maladies liées au vieillissement, et utilisation associée
WO2009146956A1 (fr) * 2008-06-03 2009-12-10 Universite De Liege Biopmarqueur de l'ostéoarthrite et/ou d'autres maladies liées au vieillissement, et utilisation associée
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 (fr) * 2008-06-03 2016-01-20 Universite De Liege Biomarqueur pour l'arthrose et utilisation associée
JP2016504422A (ja) * 2013-04-02 2016-02-12 イーホン・ジョウＹｉｈｏｎｇ ＺＨＯＵ フィビュリンタンパク質変異体および対応する核酸配列

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