JPWO2006006477A1 - Polypeptide and DNA involved in bone disease or joint disease - Google Patents

Abstract

Measuring the expression level of at least one gene encoding a protein having an amino acid sequence selected from the group consisting of SEQ ID NOs: 18 to 34 in a test sample derived from an individual to be tested, wherein the test When the expression level in the sample is different from the expression level in a control sample derived from a normal individual, the test sample is an indicator that the sample is derived from an individual suspected of having bone disease or joint disease. Provided is a method for detecting or selecting a bone disease or joint disease easily and rapidly.

Description

  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 is 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, The present invention relates to a kit used for the screening method, a pharmaceutical composition, and a method for diagnosing a bone disease or a joint disease.

  A joint is a movable joint between bones, and the bone in this part is covered with articular cartilage to make the movement smooth. In the joint, synovial fluid is secreted from the synovium, which smoothes the movement of the joint.

  Osteoarthritis (OA) is a debilitating disease with chronic arthritis and degenerative degeneration of the articular cartilage of the synovial joint. OA is known to increase its incidence with aging and is one of the most frequent diseases for the elderly. In today's aging society, it has become one of the major challenges, but the cause is not clear and there is no effective treatment. For a long time, OA has been considered to be caused by inevitable aging, but genetic studies have revealed that genetic factors are greatly 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 it is thought that the disease is caused by the interaction of these genes with environmental factors such as exercise and nutrition. Research to date has listed a large number of candidate genes (see Patent Documents 1 and 2) and SNPs (single nucleotide polymorphisms) as genes related to OA (see Non-Patent Document 2). However, the onset mechanism of OA has not been completely elucidated, and there is a possibility that an unknown gene may be involved.

  Rheumatoid arthritis (RA) is an unexplained chronic inflammatory disease whose main symptom is polyarthritis that makes small and medium joints symmetrical. Local production of inflammatory cytokines and rheumatoid factors, formation of immune complexes consisting of IgG and rheumatoid factors in synovial fluid, neutrophil migration, immune complex phagocytosis, lysosomal enzyme release, articular cartilage destruction, synovial proliferation, Chronic inflammation and joint destruction progress by a series of reactions such as osteoclast activation. Research to date has listed a large number of candidate genes (see Patent Documents 3 and 4) and SNPs as genes related to RA (see Non-Patent Document 3). However, the onset mechanism of RA has not been completely elucidated, and it is considered that an unknown gene may be involved.

  In addition, there are many causative diseases that exhibit polyarthritis (symptoms), and among them, the frequency of rheumatoid arthritis (RA) and OA is high. Both OA and RA are common in that joints become painful and need to be identified. Currently, when OA or RA is diagnosed, examinations for knowing the degree of inflammation, X-ray examinations, and the like are performed. However, early diagnosis may be difficult, and clinical examination and follow-up are required (see Non-Patent Document 4). Therefore, there is a demand for the development of a diagnostic method that can easily distinguish between OA and RA and a simple diagnostic method so that a disease can be discovered earlier.

  Under the circumstances as described above, identification of a gene involved in the development of OA or RA and a method for diagnosing a disease using the gene have been desired.

  N1032 (SEQ ID NO: 1) is a tumor suppressor gene candidate 1 (bcsc-1) (SEQ ID NO: 18) for human breast cancer. However, there is no report on the relationship between N1032 and OA.

  N1108 (SEQ ID NO: 2) is a human gene encoding a protein phosphatase 2A B′α1 regulatory subunit (SEQ ID NO: 19) (see Non-Patent Document 5). However, there is no report on the relationship between N1108 and OA.

  N1112 (SEQ ID NO: 3) is a human gene that encodes a novel human zinc finger transcription factor (SEQ ID NO: 20) that is homologous to the rodent gene Kid1 and is expressed mainly in the kidney. (See Patent Document 6). However, there is no report on the relationship between N1112 and OA.

  N1123 (SEQ ID NO: 4) is a human gene encoding phosphoglycerate kinase (SEQ ID NO: 21) encoded on the X chromosome (see Non-Patent Document 7). However, there is no report on the relationship between N1123 and OA.

  N0154 (SEQ ID NO: 5) is a human gene encoding L-iditol-2 dehydrogenase (SEQ ID NO: 22) (see Non-Patent Document 8). However, there is no report on the relationship between N0154 and OA.

  N0281 (SEQ ID NO: 6) is the causative gene EXT1 (SEQ ID NO: 23) of 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). However, there is no report on the relationship between N0439 and OA.

  N0446 (SEQ ID NO: 8) is a human gene having homology with 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, there is no report on the relationship between N0513 and OA.

  N0553 (SEQ ID NO: 10) is a human gene encoding serine / threonine kinase (SEQ ID NO: 27) having two LIM motifs at the N ′ end (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.

  N0160 (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 is no report on the relationship between N0160 and OA.

  E0215 (SEQ ID NO: 13) is a human gene encoding EGF-containing fibrin-like extracellular matrix protein 1 (EFEMP1, Fibulin-3) (SEQ ID NO: 30) (see Non-Patent Document 15). However, there is no report on the relationship between E0215 and OA.

  E0739 (SEQ ID NO: 14) is a human gene encoding a cell poly (rC) -binding protein (SEQ ID NO: 31) having three K-homology domains (see Non-Patent Document 16). However, there is no report on the relationship between E0739 and OA.

  C0052 (SEQ ID NO: 15) is a human gene encoding AP-endonuclease (SEQ ID NO: 32) (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-2α (SEQ ID NO: 33) (see Non-Patent Document 18). However, there is no report on 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.
JP 2003-183177 A JP 2004-75675 A JP 2002-51782 A JP 2003-204790 A Specter, TD, et al., British Medical Journal (BMJ), 312, 940-943 (1996) Ikegawa, S. et al., Journal of Bone and Mineral Research (JBMR), 17, 1290-1296 (2002) Yamamoto, K. et al., Nature Genetics, 35, 341-348 (2003). Yoshida Hiroshi "RA or other inflammatory polyarthritis (DRG / PPS-compliant clinical guidelines)" Japan Society for Clinical Laboratory Medicine, September 2002, p.85-88 Kamibayashi, C. et al., The Journal of Biological Chemistry, 271, 5164-5170 (1996). Nakamura, Y. et al., Cytogenet. Cell Genet, 78, 285-288 (1997) Orkin, SH et al., Proceedings of the National Academy of Science (Proc. Natl. Acad. Sci. USA), 80, 472-476 (1983) Carper, D. et al., Genomics, 26, 55-62 (1995). Wells, DE, et al., Nat. Genet., 11, 137-143 (1995) Kimata, K. et al., The Journal of Biological Chemistry, 273, 9208-9213 (1998). Miller, FW et al., Biochemical and Biophysical Research Communications, Vol. 210, pp. 556-566 (1995) Cowan, NJ et al., Cell, 86, 287-296 (1996). Mizuno, K. et al., The Journal of Biological Chemistry, 270, 31321-31330 (1995) Goetzl, EJ, et al., Biochemical. Biophys. Res. Commun., 193, 546-553 (1993) Goldstein, S. et al., Molecular and Cellular Biology, 15, 120-128 (1995). Celis, JE et al., European Journal of Biochem., 230, 447-453 (1995). Hickson, ID et al., Nucleic Acids Res., 19: 5519-5523 (1991) Cerami, A. et al., The Journal of Experimental Med., 172, 911-919 (1990) Taylor, JB et al., Biochemical Journal (Biochem. J.), 300, 271-276 (1994)

  The present invention relates to expression of a protein gene having an amino acid sequence selected from the group consisting of SEQ ID NOs: 18 to 34, or a protein having an amino acid sequence in which one or several amino acids are deleted, substituted or added in the amino acid sequence. A method for simple and rapid detection or selection of a sample from an individual suspected of suffering from a bone disease or joint disease by measuring a level, a simple and rapid diagnostic method for bone disease or joint disease, and the method It aims at providing the diagnostic agent which can be performed efficiently. It is another object of the present invention 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 relates to a protein gene having an amino acid sequence selected from the group consisting of SEQ ID NOs: 18 to 34 or a protein having an amino acid sequence in which one or several amino acids are deleted, substituted or added in the amino acid sequence It is an object of the present invention to provide an efficient screening method for a therapeutic or prophylactic agent for bone diseases or joint diseases by detecting or measuring the kinetics of the above, and a screening kit that can perform the screening method simply and rapidly.

  Furthermore, the present invention provides a compound or salt thereof suitably used as a therapeutic or prophylactic agent for bone disease or joint disease obtained by the screening method, the compound suitable for treatment or prevention of bone disease or joint disease, or a salt thereof. It aims at providing the pharmaceutical composition which uses the salt as an active ingredient.

  The inventors collected and analyzed the synovium excised at the time of surgery with the consent of the OA patient or RA patient. As a result of intensive studies, a gene whose expression was changed in the synovial tissue of OA patients as compared with non-OA patients was found. Moreover, the gene by which an expression change was seen in the synovial tissue of RA patient compared with non-RA patient was discovered. In OA knee tissue, it is considered that various inflammatory substances are secreted from the augmented synovium stimulated by debris of cartilage tissue destroyed by mechanical stress. Therefore, it was considered that expression of genes encoding inflammatory substances and substances that can serve as markers for joint tissue destruction was enhanced in the synovium that received such stimulation. In the synovial tissue, undifferentiated fibroblast synovial cells having the ability to differentiate into mesenchymal cells such as adipocyte-like cells, osteoblasts, and chondrocytes are present. As a result of further research, the present inventors have found that the gene can be used as a novel diagnostic agent for bone diseases or joint diseases caused by abnormal synovial tissue abnormalities, thereby completing the present invention.

That is, the present invention
(1) A protein having an amino acid sequence selected from the group consisting of SEQ ID NOs: 18 to 34 in a test sample derived from an individual to be tested, or one or several amino acids are deleted, substituted or added in the amino acid sequence Measuring the expression level of at least one gene encoding a protein having a specific amino acid sequence, wherein the expression level in the test sample is different from the expression level in a control sample from a normal individual. Method for detecting or selecting a sample derived from an individual suspected of having a bone disease or joint disease, which is an indicator that the sample is a sample derived from an individual suspected of having a bone disease or joint disease ,
(2) The bone disease or joint disease is cartilage dysplasia, bone dysplasia, osteoporosis, osteoarthritis, rheumatoid arthritis, arthritis, synovitis, metabolic arthropathy or arthropathy due to sports (1) the method of,
(3) A protein having an amino acid sequence selected from the group consisting of SEQ ID NO: 18, 21-23, 25, and 29 in a test sample derived from an individual to be tested, or lacking 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 greater than the expression level in a control sample from a normal individual Is higher and is lower than the expression level in a control sample derived from a patient with rheumatoid arthritis, an indicator that the test sample is a sample derived from an individual suspected of suffering from osteoarthritis A method for detecting or selecting a sample from an individual suspected of suffering from osteoarthritis,
(4) A protein having an amino acid sequence selected from the group consisting of SEQ ID NOs: 19, 20, 24, 26-28, 30-33 in a test sample derived from an individual to be tested, or one or several in the amino acid sequence Measuring the expression level of at least one gene encoding a protein having an amino acid sequence in which the amino acid is deleted, substituted or added, wherein the expression level in the test sample is a control sample derived from a normal individual A sample derived from an individual suspected of suffering from osteoarthritis if the expression level is higher than the expression level in a control sample from an individual with rheumatoid arthritis A method for detecting or selecting a sample derived from an individual suspected of suffering from osteoarthritis, which is an indicator of being
(5) A protein having the amino acid sequence of SEQ ID NO: 34 in a test sample derived from an individual to be tested or a protein 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 encoding gene, wherein the expression level in the test sample is lower than the expression level in a control sample from a normal individual and in a control sample from a rheumatoid arthritis patient individual If the expression level is higher, the test sample is suspected of suffering from osteoarthritis, which is an indicator that the sample is derived from an individual suspected of suffering from osteoarthritis A method for detecting or selecting an individual-derived sample;
(6) A protein having an amino acid sequence selected from the group consisting of SEQ ID NOs: 18, 21-23, 25, and 29 in a test sample derived from an individual to be tested, or lacking 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 greater than the expression level in a control sample from a normal individual The test sample is a sample from an individual suspected of having rheumatoid arthritis. A method for detecting or selecting a sample from an individual suspected of suffering from rheumatoid arthritis,
(7) A protein having the amino acid sequence of SEQ ID NO: 34 in a test sample derived from an individual to be tested or a protein 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 encoding gene, wherein the expression level in the test sample is lower than the expression level in a control sample from a normal individual and the control sample from an osteoarthritis patient individual An individual suspected of suffering from rheumatoid arthritis, wherein the test sample is an indicator that the test sample is derived from an individual suspected of suffering from rheumatoid arthritis A method of detecting or sorting a sample derived from,
(8) A protein having an amino acid sequence selected from the group consisting of SEQ ID NOs: 18 to 34, or a protein having an amino acid sequence in which one or several amino acids are deleted, substituted or added in the amino acid sequence The method according to any one of (1) to (7), wherein the amount of mRNA of at least one gene encoded is measured as an index,
(9) A protein having an amino acid sequence selected from the group consisting of SEQ ID NOs: 18 to 34, or at least one having an amino acid sequence in which one or several amino acids are deleted, substituted or added in the amino acid sequence The method according to any one of (1) to (7), which is measured using one protein or a partial peptide thereof or a salt thereof as an index,
(10) At least one encoding a protein having an amino acid sequence selected from the group consisting of SEQ ID NOs: 18 to 34 or a protein having an amino acid sequence in which one or several amino acids are deleted, substituted or added in the amino acid sequence The method according to (8), wherein a nucleic acid capable of detecting one gene is used,
(11) The method according to (10), wherein the gene is a nucleic acid comprising a base sequence selected from the group consisting of SEQ ID NOs: 1 to 17,
(12) The method according to (10), wherein the gene is a nucleic acid comprising the base sequence of SEQ ID NO: 6 and / or 13.
(13) The method according to (12), wherein a nucleic acid comprising the nucleotide sequence of SEQ ID NO: 35 and / or 36 is used.
(14) a protein having an amino acid sequence selected from the group consisting of SEQ ID NOs: 18 to 34, or at least one protein having an amino acid sequence in which one or several amino acids are deleted, substituted or added in the amino acid sequence, or a protein thereof The method according to (9), wherein an antibody against a partial peptide or a salt thereof or a fragment thereof is used,
(15) a protein having the amino acid sequence of SEQ ID NO: 23 and / or 30, or at least one protein having an amino acid sequence in which one or several amino acids are deleted, substituted or added in the amino acid sequence, or a partial peptide thereof; The method according to (9), wherein an antibody against the salt or a fragment thereof is used,
(16) At least one encoding a protein having an amino acid sequence selected from the group consisting of SEQ ID NOs: 18 to 34 or a protein having an amino acid sequence in which one or several amino acids are deleted, substituted or added in the amino acid sequence A diagnostic agent for use in the method according to any one of (10) to (15), comprising a nucleic acid capable of detecting one gene;
(17) The diagnostic agent according to (16), wherein the gene is a nucleic acid comprising a base sequence selected from the group consisting of SEQ ID NOs: 1 to 17,
(18) The diagnostic agent according to (16), wherein the gene is a nucleic acid comprising the nucleotide sequence of SEQ ID NO: 6 and / or 13,
(19) The diagnostic agent according to (18), comprising a nucleic acid comprising the nucleotide sequence of SEQ ID NO: 35 and / or 36,
(20) a protein having an amino acid sequence selected from the group consisting of SEQ ID NOs: 18 to 34, or at least one protein having an amino acid sequence in which one or several amino acids are deleted, substituted or added in the amino acid sequence, or a protein thereof A diagnostic agent for use in the method according to (14) or (15), comprising an antibody against a partial peptide or a salt thereof or a fragment thereof,
(21) a protein having the amino acid sequence of SEQ ID NO: 23 and / or 30, or at least one protein having an amino acid sequence in which one or several amino acids are deleted, substituted or added in the amino acid sequence, or a partial peptide thereof; A diagnostic agent for use in the method according to (14) or (15), comprising an antibody against the salt or a fragment thereof,
(22) At least one encoding a protein having an amino acid sequence selected from the group consisting of SEQ ID NOs: 18 to 34, or a protein having an amino acid sequence in which one or several amino acids are deleted, substituted or added in the amino acid sequence Screening method for preventive and / or therapeutic agent for bone disease or joint disease, characterized by using two genes,
(23) a protein having an amino acid sequence selected from the group consisting of SEQ ID NOs: 18 to 34, or at least one protein having an amino acid sequence in which one or several amino acids are deleted, substituted or added in the amino acid sequence, or a protein thereof The present invention relates to a screening method for a prophylactic and / or therapeutic agent for bone disease or joint disease, characterized by using a 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 method for screening a prophylactic agent and / or therapeutic agent for a bone disease or joint disease therapeutic agent, comprising detecting or measuring the dynamics of at least one gene encoding a protein having
(25) (I) A protein having an amino acid sequence selected from the group consisting of SEQ ID NOs: 18 to 34 in the presence of a test substance, or one or several amino acids in the amino acid sequence have been deleted, substituted or added A step of bringing a protein having an amino acid sequence or a partial peptide thereof or a salt thereof into contact with a test substance; and (II) detecting the binding between the protein or a partial peptide thereof and the test substance after the step (I). And thereby selecting a test substance that binds to the protein or a partial peptide thereof as a candidate compound for a prophylactic and / or therapeutic agent for bone disease or joint disease,
(26) (A) A protein having an amino acid sequence selected from the group consisting of SEQ ID NOs: 18 to 34 in the presence of a test substance, or one or several amino acids in the amino acid sequence have been deleted, substituted or added Introducing a nucleic acid encoding a protein having an amino acid sequence into a cell;
(B) a step of expressing the nucleic acid in the cell obtained in the step (A) in the presence of the test substance, and (C) expression of the nucleic acid as compared with the case where the test substance is absent. Or a change in the expression of the nucleic acid is measured, whereby a test substance that causes a change in the expression of the nucleic acid is selected as a candidate compound for a prophylactic and / or therapeutic agent for bone disease or joint disease The method according to (24), including the step of:
(27) (A) In the presence of a test substance, a protein having an amino acid sequence selected from the group consisting of SEQ ID NOs: 18 to 33, or one or several amino acids in the amino acid sequence is deleted, substituted or added Introducing a nucleic acid encoding a protein having an amino acid sequence into a cell;
(B) a step of expressing the nucleic acid in the cell obtained in the step (A) in the presence of the test substance, and (C) expression of the nucleic acid as compared with the case where the test substance is absent. The method according to (24), comprising the step of measuring the increase in the expression of the nucleic acid, thereby selecting a test substance that causes a change in the expression of the nucleic acid as a candidate compound for a preventive and / or therapeutic agent for bone disease or joint disease. ,
(28) (A) a protein having an amino acid sequence selected from the group consisting of SEQ ID NO: 34 in the presence of a test substance, or an amino acid sequence in which one or several amino acids are deleted, substituted or added in the amino acid sequence Introducing into a cell a nucleic acid encoding a protein having
(B) a step of expressing the nucleic acid in the cell obtained in the step (A) in the presence of the test substance, and (C) expression of the nucleic acid as compared with the case where the test substance is absent. The method according to (24), comprising the step of measuring a decrease in the amount of the test substance, thereby selecting a test substance that causes a change in the expression of the nucleic acid as a candidate compound for a preventive and / or therapeutic agent for bone disease or joint disease. ,
(29) The bone disease or joint disease is cartilage dysplasia, bone dysplasia, osteoporosis, osteoarthritis, rheumatoid arthritis, arthritis, synovitis, metabolic arthropathy or arthropathy due to sports (24) to ( 28) The method according to any one of
(30) a protein having an amino acid sequence selected from the group consisting of SEQ ID NOs: 18 to 34, a protein having an amino acid sequence in which one or several amino acids are deleted, substituted or added in the amino acid sequence, or a partial peptide thereof A kit for use in the method according to any one of (24), (25) and (29), comprising the salt;
(31) A protein having an amino acid sequence selected from the group consisting of SEQ ID NOs: 18 to 34 in the presence of a test substance, or an amino acid sequence in which one or several amino acids are deleted, substituted or added in the amino acid sequence A kit for use in the method according to any one of (24) and (26) to (29), comprising a nucleic acid construct comprising a nucleic acid encoding a protein having
(32) A nucleic acid encoding a protein having an amino acid sequence selected from the group consisting of SEQ ID NOs: 18 to 34, or a protein having an amino acid sequence in which one or several amino acids are deleted, substituted or added in the amino acid sequence A kit for use in the method according to any one of (24) and (26) to (29), comprising the introduced cells;
(33) a protein having an amino acid sequence selected from the group consisting of SEQ ID NOs: 18 to 34, or a protein or partial peptide having an amino acid sequence in which one or several amino acids are deleted, substituted or added in the amino acid sequence, or a peptide thereof A kit for use in the method according to any one of (24) to (29), comprising an antibody against a salt;
(34) The compound obtained by the method according to any one of (24) to (29) or a salt thereof,
(35) A pharmaceutical composition comprising the compound or salt thereof according to (34) as an active ingredient,
(36) The pharmaceutical composition according to (35), which is used for treatment or prevention of bone disease or joint disease,
(37) A protein having an amino acid sequence selected from the group consisting of SEQ ID NOs: 18 to 34 in a test sample derived from an individual to be tested, or one or several amino acids are deleted, substituted or added in the amino acid sequence Measuring the expression level of at least one gene encoding a protein having a specific amino acid sequence, wherein the expression level in the test sample is different from the expression level in a control sample from a normal individual or from an RA patient , A method for diagnosing bone disease or joint disease, wherein the test sample is an indicator that the sample is derived from an individual suspected of having bone disease or joint disease,
(38) A protein having an amino acid sequence selected from the group consisting of SEQ ID NOs: 18 to 34, or a protein having an amino acid sequence in which one or several amino acids are deleted, substituted or added in the amino acid sequence The method according to (37), wherein the mRNA amount of at least one gene encoded is measured as an index,
(39) 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 are deleted, substituted or added in the amino acid sequence The method according to (37), wherein the measurement is performed using two proteins or partial peptides or salts thereof as an index,
(40) The bone disease or joint disease is cartilage dysplasia, bone dysplasia, osteoporosis, osteoarthritis, rheumatoid arthritis, arthritis, synovitis, metabolic arthropathy or arthropathy due to sports (37) to (39) The method according to any one of the above.

  Since the protein used in the present invention changes in expression in OA synovium or RA synovium, it is useful, for example, as a diagnostic index for bone disease or joint disease caused by abnormality of synovial tissue. In addition, the protein used in the present invention is useful as a diagnostic index for OA or RA that distinguishes between OA and RA, which have arthralgia as one of the symptoms and are difficult to distinguish. Further, a compound or salt thereof that regulates the activity of the protein, a compound or salt thereof that regulates the expression of the protein gene, a neutralizing antibody against the protein, an antisense nucleotide of the gene encoding the protein, It can be safely used as a prophylactic or therapeutic agent for joint diseases.

Fibulin-3 gene expression

  The present invention relates to a method for detecting or selecting a sample derived from an individual suspected of having a bone disease or a joint disease, a method for diagnosing a bone disease or a joint disease, a diagnostic agent used for the detection / selection / diagnostic method, a bone disease Or it is related with the screening method of the therapeutic agent or preventive agent of a joint disease, the kit used for this screening method, and a pharmaceutical composition.

  The “bone disease or joint disease” includes, for example, cartilage dysplasia, bone dysplasia, osteoporosis, osteoarthritis, rheumatoid arthritis, arthritis, synovitis, metabolic arthropathy, or joint disorders due to sports. The present invention can be suitably used particularly for osteoarthritis or rheumatoid arthritis.

  Hereinafter, the present invention will be described in detail. In this specification, unless otherwise specified, gene recombination technology, recombinant protein production technology in cells, separation and purification method of expressed protein, analysis method, molecular biology method, immunological method, etc. A well-known method is employ | adopted.

  The “method for detecting or selecting a sample derived from an individual suspected of having bone disease or joint disease” according to the present invention is the expression of a gene encoding a protein or partial peptide in a test sample derived from an individual to be tested. One characteristic is to measure the level. Therefore, according to the detection or selection method of the present invention, it is possible to easily and quickly detect or select whether or not the subject individual is suspected of having bone disease or joint disease. The effect is demonstrated. In addition, OA and RA, which are representative 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.

  A “test sample” is prepared from a tissue suspected of having bone disease or joint disease (eg, periosteum, cartilage, etc.), cells, joint fluid, blood, urine, etc., by a conventional technique. Can be done. It is particularly preferable to prepare it from joint fluid, blood and urine that can be easily examined. Synovial fluid, blood, and urine are collected in the same manner as normal joint fluid testing, blood testing, and urinalysis. The control sample may be prepared from the same site as the test sample from a normal individual, RA patient, or OA patient, and can be prepared from the same site in the same manner as the test sample, for example. In addition, if there is a tissue (eg, periosteum, cartilage, etc.) removed at the time of surgery from an individual suspected of suffering from a bone disease or joint disease, the tissue should be adjusted by a conventional method and used. You can also.

  The “detection or selection method” of the present invention includes a protein having an amino acid sequence selected from the group consisting of SEQ ID NOs: 18 to 34 in a test sample derived from an individual to be tested, or one or several amino acids 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 different from the expression level in the control sample The test sample is an indicator that the sample is derived from an individual suspected of having bone disease or joint disease. Seventeen genes having a base sequence selected from the group consisting of SEQ ID NOs: 1 to 17 serve as indices for bone disease or joint disease only by the expression of one gene. In particular, a gene showing an expression increase of 2 times or more compared to a normal individual is preferable as an index.

  Ten genes having a base sequence selected from the group consisting of SEQ ID NOs: 2, 3, 7, 9-11, and 13-16 have higher expression levels in the test sample than those in normal individuals and RA patients. If so, it can be determined that the patient is an OA patient. Six genes consisting of a base sequence selected from the group consisting of SEQ ID NOs: 1, 4-6, 8, 12 have an expression level in the test sample higher than that in normal individuals, and in RA patients When it becomes lower than the expression level, it can be determined that the patient is OA. A gene consisting of the nucleotide sequence of SEQ ID NO: 17 can be determined to be an OA patient when the expression level in the test sample is lower than the expression level in a normal individual and higher than the expression level in an RA patient. . In particular, the gene consisting of the nucleotide sequence of SEQ ID NO: 6 or 13 is preferable as an index.

  In addition, in the case of six genes having a base sequence selected from the group consisting of SEQ ID NOs: 1, 4-6, 8, and 12, the expression level in the test sample is higher than the expression level in normal individuals and OA patients It can be determined that the patient is an RA patient. The gene consisting of the base 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.

  "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 "protein of the present invention") May be referred to as cells of humans and warm-blooded animals (eg, guinea pigs, rats, mice, chickens, rabbits, pigs, sheep, cows, monkeys, etc.) (eg, hepatocytes, splenocytes, neurons, glial cells) , Pancreatic β cells, bone marrow cells, mesangial cells, Langerhans cells, epidermal cells, epithelial cells, epithelial cells, goblet cells, endothelial cells, smooth muscle cells, fibroblasts, fiber cells, 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, osteoblasts , Osteoclasts, mammary cells, hepatocytes or stromal cells, or precursor cells of these cells, stem cells, cancer cells, etc.) or any tissue in which those cells exist, such as the brain, each part of the brain (eg, olfactory bulb) , Amygdala, basal sphere, hippocampus, thalamus, hypothalamus, cerebral cortex, medulla, cerebellum), spinal cord, pituitary gland, stomach, pancreas, kidney, liver, gonad, thyroid, gallbladder, bone marrow, adrenal gland, skin, muscle, Protein derived from lung, gastrointestinal tract (eg, large intestine, small intestine), blood vessel, heart, thymus, spleen, submandibular gland, peripheral blood, prostate, testis, ovary, placenta, uterus, bone, joint, skeletal muscle, etc. May be.

  The “amino acid sequence in which one or several amino acids have been deleted, substituted or added” is, for example, (A) one or more amino acid sequences selected from the group consisting of SEQ ID NOs: 18 to 34 (preferably Is selected from the group consisting of (B) SEQ ID NO: 18-34, an amino acid sequence from which about 1-30, preferably about 1-10, more preferably (1-5) amino acids have been deleted. An amino acid sequence in which one or more (preferably about 1 to 30, preferably about 1 to 10, more preferably several (1 to 5)) amino acids are added to the amino acid sequence to be obtained, (C) sequence 1 or 2 (preferably about 1 to 30, preferably about 1 to 10, more preferably a number (1 to 5)) of amino acid sequences selected from the group consisting of numbers: 18 to 34 Is inserted No acid sequence, (D) 1 or 2 or more in an amino acid sequence selected from the group consisting of SEQ ID NOs: 18 to 34 (preferably about 1 to 30, preferably about 1 to 10, more preferably a number A protein containing an amino acid sequence in which (1-5) amino acids are substituted with other amino acids, or (E) an amino acid sequence combining them. When the amino acid sequence is inserted, deleted or substituted, the position of the insertion, deletion or substitution is not particularly limited.

  The “gene expression level” is preferably measured using the mRNA amount or protein amount 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 blotting, dot blotting, microarray or macroarray analysis methods, and immunological measurement methods include microtiter plates. Examples include ELISA method using RIA, RIA method, fluorescent antibody method, Western blot method, immunohistochemical staining method and the like.

  In the present invention, the expression level has 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 are deleted, substituted or added in the amino acid sequence Also included is a method in which the amount of mRNA of at least one gene encoding a protein is used as an index. This corresponds to the molecular biological measurement method described above.

  For example, at least one encoding a protein having an amino acid sequence selected from the group consisting of SEQ ID NOs: 18 to 34 or a protein having an amino acid sequence in which one or several amino acids are deleted, substituted or added in the amino acid sequence A method using a nucleic acid capable of detecting a gene, a method wherein the gene is a nucleic acid comprising a base sequence selected from the group consisting of SEQ ID NOs: 1 to 17, and the gene is SEQ ID NO: 6 and / or 13 And a method using a nucleic acid comprising the nucleotide sequence of SEQ ID NO: 35 and / or 36. Specific description will be given below.

Molecular Biological Measurement Method In the case of PCR, a test sample derived from an individual to be tested is, for example, a protein having an amino acid sequence selected from the group consisting of SEQ ID NOs: 18 to 34, or 1 or a number 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 an amino acid sequence in which one amino acid is deleted, substituted, or added, 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 for 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 an antisense at the 3 ′ end. A primer pair consisting of a primer corresponding to the sequence is exemplified. Such a primer pair can be appropriately selected based on an appropriate Tm value, secondary structure, etc. in consideration of operability during use. Specifically, it is preferable that the entire base sequence described in SEQ ID NOs: 1 to 17 or a characteristic partial sequence thereof can be amplified. More specifically, for example, a primer pair selected from the group consisting of nucleic acids having the base sequences described in SEQ ID NOs: 1 to 17 and the like. Specific examples of the primer pair include, but are not particularly limited to, a primer pair of SEQ ID NOs: 35 and 36. Such a primer may be a primer labeled with a conventional fluorescent dye or radioactive substance. The amplification product can be detected by visualization with ethidium bromide or a fluorescent substance, detection based on a labeled primer, or the like in conventional agarose gel electrophoresis or the like.

  In the present specification, the “partial sequence characteristic to (nucleic acid)” means, for example, a nucleotide sequence of a gene other than the gene encoding the protein of the present invention among sequences registered in a database. A partial sequence not found, for example, a sequence whose sequence identity with a sequence registered in a database is usually 20% or less, preferably 10% or less, more preferably 5% or less, particularly preferably 0% Say.

  In the case of Northern blotting, 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. Using a probe that detects a nucleic acid that encodes a protein having a deleted, substituted, or added amino acid sequence, and measuring the amount of hybrid formation or its amount or the appearance or amount of an amplification product and comparing it with the result in a control sample Thus, the gene expression level of the protein of the present invention can be evaluated.

  In the case of the analysis method using a microarray or a macroarray, a test sample derived from an individual to be tested is, for example, a protein having an amino acid sequence selected from the group consisting of SEQ ID NOs: 18 to 34 or 1 in the amino acid sequence. Alternatively, using a microarray or macroarray containing at least one probe for detecting a nucleic acid encoding a protein having an amino acid sequence in which several amino acids have been deleted, substituted, or added, formation of a hybrid or its amount or amplification product The expression level of the gene of the protein of the present invention can be evaluated by measuring the appearance or the amount thereof and comparing with the result in the control sample.

  Examples of the “probe” used in the Northern blot method or the analysis method using a microarray or a macroarray include nucleic acids used for detecting a nucleic acid having a base sequence selected from the group consisting of SEQ ID NOs: 1 to 17. It is done. 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.

  In the present invention, the expression level has 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 are deleted, substituted or added in the amino acid sequence A method in which at least one protein or a partial peptide thereof or a salt thereof is used as an index is also included. This corresponds to the immunological measurement method described above.

  The “protein partial peptide” is a partial peptide of the protein of the present invention, and any protein may be used as long as it has the same properties as the protein of the present invention. For example, among the constituent amino acid sequences of the protein of the present invention, a peptide having an amino acid sequence of at least 20, preferably 50 or more, more preferably 70 or more, more preferably 100 or more, and most preferably 200 or more. Is used.

  In addition, the “partial peptide” is a deletion of one or more (preferably about 1 to 10, more preferably several (1 to 5)) amino acids in the amino acid sequence, or an amino acid thereof. One or more (preferably about 1 to 20, more preferably about 1 to 10, more preferably several (1 to 5)) amino acids are added to the sequence, or 1 is added to the amino acid sequence. Or two or more (preferably about 1 to 20, more preferably about 1 to 10, more preferably several (1 to 5)) amino acids are inserted, or 1 or 2 in the amino acid sequence One or more (preferably about 1 to 10, more preferably several, and further preferably about 1 to 5) amino acids may be substituted with other amino acids.

  As the “salt of protein or partial peptide”, a salt with a physiologically acceptable acid (eg, inorganic acid, organic acid) or base (eg, alkali metal salt) is used, and particularly physiologically acceptable. The acid addition salts are preferred. Such salts include, for example, salts with inorganic acids (eg hydrochloric acid, phosphoric acid, hydrobromic acid, sulfuric acid) or organic acids (eg acetic acid, formic acid, propionic acid, fumaric acid, maleic acid, succinic acid). Acid, tartaric acid, citric acid, malic acid, succinic acid, benzoic acid, methanesulfonic acid, benzenesulfonic acid) and the like.

  For example, a protein having an amino acid sequence selected from the group consisting of SEQ ID NOs: 18 to 34, or at least one protein having an amino acid sequence in which one or several amino acids are deleted, substituted or added in the amino acid sequence, or a portion thereof A method using an antibody against a peptide or a salt thereof or a fragment thereof, a protein having the amino acid sequence of SEQ ID NO: 23 and / or 30, or an amino acid sequence in which one or several amino acids are deleted, substituted or added in the amino acid sequence Examples include a method using an antibody or a fragment thereof against at least one protein or a partial peptide thereof or a salt thereof. Specific description will be given below.

Immunological measurement method When the amount of protein is used as an index, each of a test sample and a control sample derived from an individual to be tested is subjected to Western chromatography using polyacrylamide gel electrophoresis, 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 blot analysis, immunoassay using the antibody or fragment thereof, etc., and measuring and comparing the amount of protein.

  Examples of the “antibody” include 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, after purification of the obtained antibody, treatment with a peptidase or the like yields an antibody fragment. Furthermore, derivatives of the above-mentioned 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 fragment thereof may be labeled with a conventional enzyme (for example, peroxidase), a fluorescent dye, a radioactive substance, avidin or biotin.

(I) Preparation of polyclonal antibody A polyclonal antibody can be prepared by administering a peptide having a partial amino acid sequence of the protein of the present invention as an antigen to an animal.
Rabbits, goats, rats, mice, hamsters and the like can be used as animals to be administered. The dose of the antigen is preferably 50 to 100 μg per animal. When a peptide is used, it is desirable that the peptide is covalently bound to a carrier protein such as keyhole limpet haemocyanin or bovine thyroglobulin as an antigen. 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. Three to seven days after each administration, blood was collected from the fundus venous plexus, and the reaction of the serum with the antigen used for immunization was determined by enzyme immunoassay [enzyme immunoassay (ELISA): 1976, published by Medical Shoin. Antibodies-A Laboratory Manual, Cold Spring Harbor Lavoratory (1988)].
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, caprylic acid precipitation (Antibodies, A Laboratory manual, Cold Spring Harbor Laboratory, (1988)), or DEAE-Sepharose column, Examples thereof include a method of treating chromatography or the like using an ion exchange column, a protein A or G-column or a gel filtration column alone or in combination.

(Ii) Preparation of monoclonal antibody (a) Preparation of antibody-producing cells Rats whose serum showed a sufficient antibody titer against the partial fragment polypeptide of the protein of the present invention used for immunization were used as a source of antibody-producing cells. Provide.
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 tweezers, centrifuged at 1,200 rpm for 5 minutes, and the supernatant is discarded. The spleen cells of the obtained precipitate fraction were treated with Tris-ammonium chloride buffer (pH 7.65) for 1 to 2 minutes to remove erythrocytes and then washed 3 times with MEM medium. Used as a cell.
(B) Preparation of myeloma cells As myeloma cells, cell lines obtained from mice or rats are used. For example, 8-azaguanine resistant mouse (BALB / c-derived) myeloma cell line P3-X63Ag8-U1 (hereinafter abbreviated as P3-U1) [Curr. Topics. Microbiol. Immunol., 81, 1 (1978), Europ. J. Immunol., 6, 511 (1976)], SP2 / 0-Ag14 (SP-2) (Nature, 276,269 (1978)), P3-X63-Ag8653 (653) [J. Immunol., 123, 1548 ( 1979)], P3-X63-Ag8 (X63) [Nature, 256, 495 (1975)] and the like. These cell lines consisted of 8-azaguanine medium [RPMI-1640 medium with glutamine (1.5 mM), 2-mercaptoethanol (5 × 10 −5 M), gentamicin (10 μg / ml) and fetal calf serum (FCS) ( The medium is further subcultured with a medium supplemented with CSL (10%) (hereinafter referred to as normal medium) and further added with 8-azaguanine (15 μg / ml). in cultured using the cell less than 2 × 10 ⁷ to fusion.
(C) Production of hybridoma The antibody-producing cells obtained in (a) and the myeloma cells obtained in (b) were mixed with MEM medium or PBS (1.83 g of disodium phosphate, 0.21 g of monopotassium phosphate, sodium chloride). Wash well with 65 g, 1 liter of distilled water, pH 7.2), mix so that the number of cells is antibody-producing cells: myeloma cells = 5 to 10: 1, and centrifuge at 1,200 rpm for 5 minutes. Discard the supernatant.
The cell group of the obtained precipitate fraction is thoroughly loosened, and with stirring, at 37 ° C., 2 g of polyethylene glycol-1000 (PEG-1000), 2 ml of MEM and dimethyl sulfoxide ( Add 0.2 to 1 ml of a solution mixed with 0.7 ml of DMSO), and add 1-2 ml of MEM medium several times every 1 to 2 minutes. After the addition, MEM medium is added to prepare a total volume of 50 ml. The preparation is centrifuged at 900 rpm for 5 minutes, and the supernatant is discarded. The cells of the obtained precipitate fraction are loosened gently and then sucked with a pipette and blown gently to give a HAT medium [normal medium with hypoxanthine (10 ⁻⁴ M), thymidine (1.5 × 10 ⁻⁵ M ) And aminopterin (4 × 10 ⁻⁷ M)] in 100 ml.
The suspension is dispensed into a 96-well culture plate at 100 μl / well and cultured in a 5% CO ₂ incubator at 37 ° C. for 7 to 14 days. After culturing, a part of the culture supernatant is removed and the partial fragment of the protein of the present invention is obtained by enzyme immunoassay described in Antibodies, A Laboratory Manual, Cold Spring Harbor Laboratory, Chapter 14 (1988). A hybridoma that specifically reacts with the polypeptide is selected.

  The antibodies can be used, for example, in methods for detecting samples from OA or RA patients. 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 tissue biopsies obtained from patients, homogenates or extracts thereof.

  There are a variety of assay formats known to those of skill in the art for the use of antibodies to detect proteins in a sample. See, for example, Harlow and Lane, Anti bodies: A Laboratory Manual, Cold Spring Harbor Laboratory, 1988. Examples of the immunological detection method include an ELISA method, a fluorescent antibody method, a Western blot method, and an immunohistochemical staining method. When performed in a Western blot format, a polypeptide preparation from a biological sample is subjected to gel electrophoresis, transferred to a suitable membrane, and reacted with an antibody. The presence of the antibody on the membrane can then be detected using a suitable detection reagent as described below.

  In another embodiment, the detection method involves the use of an antibody immobilized on a solid support to bind to the protein and remove it from the rest of the sample. The bound protein can then be detected using a secondary antibody or reagent having a reporter group. Alternatively, competition assays 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 and antibody indicates the reactivity of the sample with the immobilized antibody and, as a result, indicates 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 nitrocellulose filter, or other suitable membrane. Alternatively, the support can be a bead or disk (eg, glass, glass fiber, latex or a plastic material (eg, polystyrene or polyvinyl chloride)). The support can also be a magnetic particle or fiber optic sensor (eg, as disclosed in US Pat. No. 5,359,681).

  The antibody can be immobilized on a solid support using a variety of techniques known in the art and described in detail in the patent and scientific literature. In the context of the present invention, the term “immobilization” can be a non-covalent association (eg adsorption) and covalent attachment (direct linkage between an antigen and a functional group on a support or by a crosslinker. Both). Fixation by adsorption to a well or membrane of a microtiter plate is preferred. In such cases, adsorption can be accomplished by contacting the antibody in a suitable buffer with the support for a suitable time. The contact time varies with temperature, but is typically between about 1 hour and 1 day. In general, contacting a well of a plastic microtiter plate (eg, polystyrene or polyvinyl chloride) with an amount of antibody in the range of about 10 ng to about 1 μg, and preferably about 100-200 ng, results in an appropriate amount of protein. Enough to fix.

  Covalent attachment of an antibody to a solid support also generally first reacts the support with a bifunctional agent that reacts with both the support and a functional group on the antibody (eg, a hydroxyl group or an amino group). Can be achieved. For example, an antibody can be covalently attached to a support having a suitable polymer coat using benzoquinone or by condensation of an aldehyde group on the support with an amine and active hydrogen on a binding partner (eg, Pierce). Immunotechnology Catalog and Handbook (199 1) A12-A13).

  In certain embodiments, the assay for detection of protein in a sample is a two-antibody sandwich assay. This assay involves first contacting an antibody immobilized on a solid support (usually a well of a microtiter plate) and a biological sample so that the polypeptide in the sample binds to the immobilized antibody. Can be performed. The unbound sample is then removed from the immobilized polypeptide-antibody complex 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 for the particular reporter group.

  More particularly, once the antibody is immobilized on the support as described above, the remaining polypeptide binding sites on the support are typically blocked. Any suitable blocking agent (eg, bovine serum albumin or Tween 20 ™ (Sigma Chemical Co., St. Louis, MO)) is known to those skilled in the art. The immobilized antibody is then incubated with the sample and the polypeptide is allowed to bind to the antibody. The sample can be diluted with a suitable diluent (eg, phosphate buffered saline (PBS)) prior to incubation. In general, a suitable contact time (ie, incubation time) is a time sufficient to detect the presence of the polypeptide in a sample obtained from an individual having breast cancer. Preferably, the contact time is sufficient to achieve a level of binding that is at least 95% of the level of binding achieved at equilibrium between the bound and unbound polypeptides. One skilled in the art understands that the time required to achieve equilibrium is readily determined by assaying the level of binding that occurs over a period of time. An incubation time of about 30 minutes at room temperature is generally sufficient.

  Unbound sample can then be removed by washing the solid support with a suitable buffer (eg, PBS containing 0.1% Tween 20 ™). A secondary antibody having a reporter group can then be added to the solid support. Preferred reporter groups include enzymes (eg, horseradish peroxidase), substrates, cofactors, inhibitors, dyes, radionuclides, chromogenic groups, fluorescent groups, and biotin. Binding 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 assaying the level of binding that occurs over a given time. The unbound secondary antibody is then 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. Biotin can be detected using avidin coupled to different reporter groups (usually radioactive or fluorescent groups or enzymes). Enzyme reporter groups can generally be detected by the addition of a substrate (generally for a specific time) followed by spectroscopic or other analysis of the reaction product.

  In order to determine the presence or absence of disease, the signal detected from the reporter group that remains attached to the solid support generally corresponds to a predetermined cutoff value established from normal tissue. To be compared with the signal to In one preferred embodiment, the cutoff value is the average signal obtained when the fixed antibody is incubated with a sample from a patient who does not have bone or joint disease. In general, a sample that produces a signal that is 3 standard deviations above a predetermined cut-off value can be considered positive for a bone or joint disease. In another preferred embodiment, the cutoff value is determined using a Receiver Oparator Curve according to the method of Sackett et al., Clinical Epidemiology: A Basic Science for Clinical Medicene, pages 106-7, (Little Brown and Co, 1985). Briefly, in this embodiment, the cutoff value is the true positive rate (ie sensitivity) and false positive rate (100% specificity) corresponding to each possible cutoff value for diagnostic test results. Can be determined by plotting pairs of The cutoff value closest to the upper left corner of the plot (i.e., the value surrounding the largest area) is the most accurate cutoff value, and the sample that produces a signal higher than the cutoff value determined by this method is It can be considered positive. 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.

  In a related embodiment, the detection of the protein of the invention is performed in a flow-through test or strip test format. Here, the antibody is immobilized on a membrane (for example, nitrocellulose). In the flow-through test, the polypeptide in the sample binds to the immobilized antibody as the sample passes through the membrane. The labeled secondary antibody then binds to the antibody-polypeptide complex as the solution containing the secondary antibody flows through the membrane. Detection of bound secondary antibody can then be performed as described above. In the strip test format, one end of the membrane to which the antibody is bound is immersed in a solution containing the sample. The sample moves along the membrane through the area containing the secondary antibody and into the area of the immobilized antibody. The concentration of secondary antibody in the range of fixed antibodies indicates the presence of bone disease or joint disease. Typically, the concentration of secondary antibody at this site produces a visually readable pattern (eg, a line). The absence of such a pattern indicates a negative result. In general, the amount of antibody immobilized on the membrane is visible when the biological sample contains a sufficient level of polypeptide to produce a positive signal in the two-antibody sandwich assay of the format discussed above. Is selected so as to produce a pattern that can be discriminated automatically. Preferably, the amount of antibody immobilized on the membrane ranges from about 25 ng to about 1 μg, and more preferably from about 50 ng to about 1 μg. Such a test can typically be performed using a very small amount of biological sample.

  Furthermore, the method for detecting or selecting a sample derived from an individual suspected of suffering from a bone disease or joint disease of the present invention can also be used as a method for diagnosing a bone disease or joint disease. Such a diagnostic method is also encompassed by the present invention. A diagnostic agent containing the probe and / or primer pair, or a diagnostic agent containing an antibody against the protein or a fragment thereof, capable of performing the diagnostic method of the present invention simply, rapidly, with high throughput and high reliability Is provided.

For example, the following diagnostic agents are also within the scope of the present invention.
-At least one gene encoding a protein having an amino acid sequence selected from the group consisting of SEQ ID NOs: 18 to 34 or a protein having an amino acid sequence in which one or several amino acids have been deleted, substituted or added in the amino acid sequence A diagnostic agent for bone disease or joint disease comprising a nucleic 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 / or 13,
-A diagnostic agent for bone disease or joint disease, comprising a nucleic acid comprising the nucleotide sequence of SEQ ID NO: 35 and / or 36,
-A protein having an amino acid sequence selected from the group consisting of SEQ ID NOs: 18 to 34, or at least one protein having an amino acid sequence in which one or several amino acids are deleted, substituted or added in the amino acid sequence, or a partial peptide thereof Or a diagnostic agent for bone disease or joint disease, comprising an antibody against a salt thereof or a fragment thereof,
-A protein having the amino acid sequence of SEQ ID NO: 23 and / or 30, or at least one protein having a deleted, substituted or added amino acid sequence in the amino acid sequence, or a partial peptide thereof, or a salt thereof A diagnostic agent for bone disease or joint disease, comprising an antibody against the above or a fragment thereof.

  The diagnostic agent of the present invention may further contain a detection reagent, a buffer solution, a control sample, instructions for the diagnostic method of the present invention, and the like.

The present invention also includes the following screening methods.
-At least one gene encoding a protein having an amino acid sequence selected from the group consisting of SEQ ID NOs: 18 to 34 or a protein having an amino acid sequence in which one or several amino acids have been deleted, substituted or added in the amino acid sequence A method for screening a prophylactic and / or therapeutic agent for bone disease or joint disease, characterized by using
-A protein having an amino acid sequence selected from the group consisting of SEQ ID NOs: 18 to 34, or at least one protein having an amino acid sequence in which one or several amino acids are deleted, substituted or added in the amino acid sequence or a partial peptide thereof Or a method for screening a prophylactic and / or therapeutic agent for bone disease or joint disease, characterized by using a salt thereof.

  The protein used in the screening method may be a protein derived from a human or warm-blooded animal cell, any tissue in which those cells are present, or 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.

Method for Producing Protein of the Present Invention The protein of the present invention is prepared by using the method described in Molecular Cloning 2nd Edition, Current Protocols in Molecular Biology Supplements 1-38, etc., for example, by the following method: The gene encoding the protein can be expressed and produced in the host cell.

  Based on the full-length DNA encoding the protein, if necessary, a DNA fragment having an appropriate length containing a portion encoding the protein is prepared. In addition, a base-substituted DNA is prepared so that the base sequence of the protein-encoding portion becomes an optimal codon 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 host cell can be used as long as it can express the target gene, such as prokaryotic cells, yeast, animal cells, plant cells, and insect cells. As the expression vector, a vector that can replicate autonomously in the host cell or can be integrated into a chromosome and contains a promoter at a position suitable for transcription of the gene of the protein of the present invention is used.

(I) When a prokaryotic organism such as a bacterium is used as a host cell The protein expression vector of the present invention is capable of autonomous replication in a prokaryotic organism, and at the same time a promoter, a ribosome binding sequence, a protein of the present invention, a transcription termination sequence, It is preferable that it is comprised. A gene that controls the promoter may also be included.
Examples of expression vectors include pBTrp2, pBTac1, pBTac2 (all available from Boehringer Mannheim), pKK233-2 (Pharmacia), pSE280 (Invitrogen), pGEMEX-1 (manufactured by Promega), pQE- 8 (QIAGEN), pKYP10 (JP 58-110600), pKYP200 [Agric. Biol. Chem., 48, 669 (1984)], pLSA1 [Agric. Biol. Chem., 53, 277 ( 1989)], pGEL1 [Proc. Natl. Acad. Sci. USA, 82, 4306 (1985)], pBluescript II SK + (Stratagene), pBluescript II SK (-) (Stratagene), pTrs30 (FERM BP-5407), pTrs32 (FERM BP-5408), pGHA2 (FERM BP-400), pGKA2 (FERM B-6798), pTerm2 (JP-A-3-22979, US4686191, US4939094, US5160735), pEG400 [J. Bacteriol. , 172, 2392 (1990)], pGEX (Pharmacia), pET system (Novagen), pSupex, pUB110, pTP5, pC194, pTrxFus (Invitrogen), pMAL-c2 (New England Biolabs), pUC19 [ Gene, 33, 103 (1985)], pSTV28 (manufactured by Takara Shuzo), pUC118 (manufactured by Takara Shuzo), pPA1 (Japanese Patent Laid-Open No. Sho 63-233798), and the like.
Any promoter can be used as long as it can be expressed in a host cell such as Escherichia coli. For example, promoters derived from Escherichia coli, phage, etc., such as trp promoter (Ptrp), lac promoter (Plac), PL promoter, PR promoter, PSE promoter, SPO1 promoter, SPO2 promoter, penP promoter, and the like can be mentioned. An artificially designed and modified promoter such as a promoter in which two Ptrps are connected in series (Ptrp x2), a tac promoter, a lacT7 promoter, and a let I promoter can also be used.
It is preferable to use a plasmid in which the distance between the Shine-Dalgarno sequence, which is a ribosome binding sequence, and the start codon is adjusted to an appropriate distance (for example, 6 to 18 bases). Although the transcription termination sequence is not necessarily required for the expression of the gene of the protein of the present invention, it is preferable to arrange the transcription termination sequence immediately below the structural gene.
Examples of host cells include Escherichia, Serratia, Bacillus, Brevibacterium, Corynebacterium, Microbacterium, Pseudomonas, and other microorganisms such as Escherichia coli XL1-Blue, Escherichia coli XL2-Blue, Escherichia coli DH1, Escherichia coli MC1000, Escherichia coli KY3276, Escherichia coli W1485, Escherichia coli JM109, Escherichia coli HB101, Escherichia coli No.49, Escherichia coli W3110, Escherichia coli NY49, Escherichia coli BL21 (DEcher3a BL21DE ) pLysS, Escherichia coli HMS174 (DE3), Escherichia coli HMS174 (DE3) pLysS, Serratia ficaria, Serratia fonticola, Serratia liquefaciens, Serratia marcescens, Bacillus subtilis, Bacillus amyloliquefaciens, Brevibacterium ameviliquemevici Corynebacterium glutamicum ATCC14067, Corynebacteriu Examples thereof include m glutamicum ATCC 13869, Corynebacterium acetoacidophilum ATCC 13870, Microbacterium ammoniaphilum ATCC 15354, Pseudomonas sp. D-0110 and the like.
As a method for introducing a recombinant vector, any method can be used as long as it is a method for introducing DNA into the host cell. For example, electroporation [Nucleic Acids Res., 16, 6127 (1988)], calcium ion [Proc. Natl. Acad. Sci. USA, 69, 2110 (1972)], protoplast method (JP 63-2483942), Gene, 17, 107 (1982), Molecular & General Genetics, 168, 111 (1979) and the like.

(Ii) When yeast is used as a host cell When a yeast strain is used as a host cell, examples of expression vectors include YEp13 (ATCC37115), YEp24 (ATCC37051), YCp50 (ATCC37419), pHS19, pHS15 and the like. Can do. Any promoter can be used as long as it can be expressed in yeast strains.For example, PHO5 promoter, PGK promoter, GAP promoter, ADH promoter, gal 1 promoter, gal 10 promoter, heat shock protein promoter, MFα1 promoter, CUP 1 Promoter can be listed.
Examples of host cells include yeast strains belonging to the genus Saccharomyces, Schizosaccharomyces, Kluyveromyces, Trichosporon, Siwaniomyces, Pichia, etc., specifically, Saccharomyces cerevisiae, Schizosaccharomyces pombe, Kluyveromyces lactis , Trichosporon pullulans, Schwanniomyces alluvius, Pichia pastoris and the like. As a method for introducing the recombinant vector, any method can be used as long as it is a method for introducing DNA into yeast. For example, electroporation method [Methods. Enzymol., 194, 182 (1990)], spheroplast method (Proc. Natl. Acad. Sci. USA, 84, 1929 (1978)), lithium acetate method (J. Bacteriol., 153, 163 (1983)), Proc. Natl. Acad. Sci. USA, 75, 1929 ( 1978) and the like.

(Iii) When animal cells are used as host cells When animal cells are used as host cells, examples of expression vectors include pcDNAI / Amp, pcDNAI, and pCDM8 (all of which are commercially available from Funakoshi), pAGE107 [Japanese Patent Laid-Open No. Hei 3- 22979, Cytotechnology, 3, 133 (1990)], pCR-BluntII-TOPO, pREP4 (manufactured by Invitrogen), pAGE103 [J. Biochem., 101, 1307 (1987)], pAMo, pAMoA [J. Biol. Chem. , 268, 22782-22787 (1993), also known as pAMoPRSA (Japanese Patent Laid-Open No. 05-336963)], pAS3-3 (Japanese Patent Laid-Open No. 2-227075), pHM6, pHB6 (manufactured by Roche Diagnostics), pKK223-3, pGEX (Amersham Biotech), pET-3, pET-11, pBluescript ptII (registered trademark) SK (+), pBluescript II (registered trademark) SK (-) (manufactured by Stratagene), pUC19, pTrxFus (manufactured by Invitrogen), pUC118, pSTV28 (manufactured by Takara Bio), pMAL-c2X (new England Biolabs) and the like. The vector used may be any vector as long as it 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. For example, cytomegalovirus (human CMV) IE (immediateearly) gene promoter, SV40 early promoter, Moloney Mullin Leukemia, Examples thereof include a long terminal repeat promoter of virus (Moloney Murine Leukemia Virus), a retrovirus promoter, a heat shock promoter, an SRα promoter, and a metallothionein promoter. In addition, an enhancer of human CMV IE gene may be used together with a promoter.
Host cells include mouse myeloma cells, rat myeloma cells, mouse hybridoma cells, Chinese hamster cells, CHO cells, BHK cells, African green monkey kidney cells, human cells, Namalwa cells or Namalwa KJM-1. Examples thereof include cells, human fetal kidney cells, human leukemia cells, HBT5637 (Japanese Patent Laid-Open No. 63-299), and human colon cancer cell lines. As mouse myeloma cells, SP2 / 0, NSO, etc., as rat myeloma cells, YB2 / 0, etc., as human fetal kidney cells, as HEK293 (ATCC: CRL-1573), as human leukemia cells, as BALL-1 etc. Examples of African green monkey kidney cells include COS-1 and COS-7, and examples of human colon cancer cell lines include HCT-15.
As a method for introducing a recombinant vector, any method can be used as long as it is a method for introducing DNA into animal cells. For example, electroporation method [Cytotechnology, 3, 133 (1990)], calcium phosphate method (Japanese Patent Laid-Open No. Hei 2). -227075), the lipofection method [Proc. Natl. Acad. Sci. USA, 84, 7413 (1987)], virology, 52, 456 (1973), and the like.

(Iv) When plant cells are used as host cells When plant cells or plant individuals are used as hosts, known methods [tissue culture, 20 (1994), tissue culture, 21 (1995), Trends in Biotechnology, 15, 45 (1997)] can be produced. Examples of the expression vector include Ti plasmid and tobacco mosaic virus vector.
Any promoter can be used as long as it can be expressed in plant cells, for example, cauliflower mosaic virus (CaMV) 35S promoter, rice actin 1 promoter and the like. In addition, gene expression efficiency can be increased by inserting intron 1 of a maize alcohol dehydrogenase gene between a promoter and a gene to be expressed.
Examples of host cells include plant cells such as potato, tobacco, corn, rice, rape, soybean, tomato, carrot, wheat, barley, rye, alfalfa and flax.
As a method for introducing the recombinant vector, any method can be used as long as it is a method for introducing DNA into plant cells. For example, Agrobacterium (JP 59-140885, JP 60-70080, WO94 / 00977), electroporation method (Japanese Patent Laid-Open No. 60-251887), a method using a particle gun (gene gun) (Patent No. 2606856, Patent No. 2517813), and the like.

(V) When insect cells are used as host cells When insect cells are used as hosts, for example, Baculovirus Expression Vectors, A Laboratory Manual [Baculovirus Expression Vectors, A Laboratory Manual, WH Freeman and Company, NewYork ( 1992)], Molecular Biology, A Laboratory Manual, Current Protocols in Molecular Biology Supplements 1-38 (Current Protocols in Molecular Biology), Bio / Technology, 6 , 47 (1988), etc., can be used to express the protein.
That is, the recombinant gene transfer vector and baculovirus are co-introduced into insect cells to obtain the recombinant virus in the insect cell culture supernatant, and then the recombinant virus is further infected into the insect cells to express the protein. it can. Examples of gene transfer vectors used in the method include pVL1392, pVL1393, pBlueBacIII (all manufactured by Invitrogen) and the like.
As the baculovirus, for example, an autographa californica nuclear polyhedrosis virus, which is a virus that infects the night stealing insects, can be used.
As insect cells, Spodoptera frugiperda ovary cells, Trichoplusia ni ovary cells, silkworm ovary-derived cultured cells, and the like can be used. Spodoptera frugiperda ovarian cells include Sf9, Sf21 (Baculovirus Expression Vectors A Laboratory Manual), Trichoplusia ni ovarian cells High 5, BTI-TN-5B1-4 (Invitrogen), etc. Examples of cultured cells derived from the ovary include Bombyx mori N4.
Examples of the 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-2-27075), the lipofection method [Proc. Natl. Acad Sci. USA, 84,7413 (1987)].
In addition, DNA can be introduced into insect cells using a method similar to the method for introducing DNA into animal cells. For example, electroporation (Cytotechnology, 3, 133 (1990)), calcium phosphate method (special Kaihei 2-227075), lipofection method [Proc. Natl. Acad. Sci. USA, 84, 7413 (1987)] and the like.

(Vi) Culture method When the transformant carrying the recombinant vector into which the DNA encoding the protein of the present invention is incorporated is a cell such as Escherichia coli, yeast, animal cell or plant cell, it is suitable for various hosts. The protein can be produced by culturing according to a culture method, producing and accumulating the protein, and recovering the protein from a transformant or a culture solution. When the transformant is an individual 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 individual or plant individual. The protein can be produced.
When the host is an animal individual, for example, a non-human transgenic animal carrying the polynucleotide of the present invention is bred, and the protein of the protein of the present invention encoded by the recombinant DNA is produced and accumulated in the animal. By collecting the protein from the individual animal, the protein of the protein of the present invention can be produced. Examples of the production / accumulation location in an animal individual include milk, saliva, eggs and the like of the animal.
When the host is a plant individual, for example, a transgenic plant carrying the polynucleotide of the protein of the present invention is cultivated, and the protein of the protein of the present invention encoded by the recombinant DNA is produced and accumulated in the plant individual. The protein of the present invention can be produced by recovering the protein from a plant individual.
When the host is a prokaryote such as Escherichia coli or a eukaryote such as yeast, for example, a transformant carrying the polynucleotide of the present invention is cultured in a medium, and the protein of the present invention encoded by the recombinant DNA Is produced and accumulated in a culture solution, and the protein of the present invention can be produced by recovering the protein from the culture solution.

The method for culturing the transformant of the protein of the present invention in a medium can be carried out according to a usual method used for culturing a host.
As a medium for culturing a transformant obtained by using a prokaryote such as E. coli or a eukaryote such as yeast as a host, the medium contains a carbon source, a nitrogen source, inorganic salts and the like that can be assimilated by the organism. Any natural or synthetic medium may be used as long as the medium can efficiently be cultured.
When the transformant is a prokaryote such as Escherichia coli or a eukaryote such as yeast, the medium for culturing the obtained transformant contains a carbon source, a nitrogen source, inorganic salts, etc. that can be assimilated by the host. As long as the medium can efficiently culture the transformant, either a natural medium or a synthetic medium may be used. As a medium for culturing a transformant whose host is Escherichia coli, for example, a YT medium containing bactotryptone, yeast extract and sodium chloride is preferable.
Any carbon source may be used as long as it can be assimilated by each microorganism. Glucose, fructose, sucrose, molasses containing these, carbohydrates such as starch or starch hydrolysate, organic acids such as acetic acid and propionic acid, ethanol Alcohols such as propanol can be used.
Nitrogen sources include ammonia, ammonium chloride, ammonium sulfate, ammonium acetate, ammonium salts of organic acids such as ammonium salts, other nitrogen-containing substances, peptone, meat extract, yeast extract, corn steep liquor, casein A hydrolyzate, soybean meal, soybean meal hydrolyzate, various fermented cells, digested products thereof, and the like can be used.
As the inorganic salt, monopotassium phosphate, dipotassium phosphate, magnesium phosphate, magnesium sulfate, sodium chloride, ferrous sulfate, manganese sulfate, copper sulfate, calcium carbonate and the like can be used. The culture is performed under aerobic conditions such as shaking culture or deep aeration stirring culture.
The culture temperature is preferably 15 to 40 ° C., and the culture time is usually 5 hours to 7 days. During the culture, the pH is maintained at 3.0 to 9.0. The pH is adjusted using an inorganic or organic acid, an alkaline solution, urea, calcium carbonate, ammonia or the like. Moreover, you may add antibiotics, such as an ampicillin and a tetracycline, to a culture medium as needed during culture | cultivation.

When culturing a microorganism transformed with an expression vector using an inducible promoter as a promoter, an inducer may be added to the medium as necessary. For example, isopropyl-β-D-thiogalactopyranoside is used when cultivating a microorganism transformed with an expression vector using the lac promoter, and indole acrylic is used when a microorganism transformed with an expression vector using the trp promoter is cultured. An 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 fermenter. As a medium for culturing, generally used Murashige and Skoog (MS) medium, White medium, or a medium obtained by adding plant hormones such as auxin and cytokinin to these mediums can be used.
When the transformant for protein production of the present invention is an animal cell, the culture medium for culturing the cell is a commonly used RPMI1640 medium [The Journal of the American Medical Association, 199, 519 (1967)], Eagle 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)) A medium supplemented with serum or the like is used.
The culture is usually performed for 1 to 7 days under conditions such as pH 6 to 8, 25 to 40 ° C., and 5% CO ₂ . Moreover, you may add antibiotics, such as kanamycin, penicillin, streptomycin, to a culture medium as needed during culture | cultivation.
When the transformant for protein production of the present invention is an insect cell, the culture medium for culturing the cell includes a commonly used TNM-FH medium (Pharmingen), Sf-900 II SFM medium (Gibco). BRL), ExCell400, ExCell405 [all manufactured by JRH Biosciences], Grace's InsectMedium [Nature, 195, 788 (1962)] and the like can be used.

(Vii) Protein production method In order to isolate and purify the protein of the present invention from the culture of the transformant for protein production of the present invention, the usual enzyme isolation and purification methods can be used. For example, when the protein of the present invention is secreted outside the transformant for protein production of the present invention, the culture is treated by a technique such as centrifugation to obtain a soluble fraction. From the soluble fraction, anion using a resin such as solvent extraction method, salting out method using ammonium sulfate, precipitation method using organic solvent, diethylaminoethyl (DEAE) -Sepharose, DIAION HPA-75 (manufactured by Mitsubishi Kasei) Exchange chromatography method, cation exchange chromatography method using resin such as S-Sepharose FF (Pharmacia), hydrophobic chromatography method using resin such as butyl sepharose and phenyl sepharose, gel filtration using molecular sieve The purified sample can be obtained by using a method such as electrophoresis, affinity chromatography, chromatofocusing, electrophoresis such as isoelectric focusing.
When the protein of the present invention accumulates in the dissolved state in the cells of the transformant for protein production of the present invention, the culture is centrifuged to collect the cells in the culture, and after washing the cells Then, the cells are crushed with an ultrasonic crusher, a French press, a Manton Gaurin homogenizer, a dyno mill or the like to obtain a cell-free extract. From the supernatant obtained by centrifuging the cell-free extract, a solvent extraction method, a salting-out method using ammonium sulfate, a desalting method, a precipitation method using an organic solvent, diethylaminoethyl (DEAE) -Sepharose, DIAION HPA-75 (Mitsubishi Kasei Co., Ltd.) Anion exchange chromatography method using resin, S-Sepharose FF (Pharmacia Co., Ltd.) cation exchange chromatography method, resin such as butyl sepharose, phenyl sepharose The purified sample can be obtained by using a method such as hydrophobic chromatography, gel filtration using molecular sieve, affinity chromatography, chromatofocusing, and electrophoresis such as isoelectric focusing.

In addition, when the protein is expressed by forming an insoluble substance in the cell, the protein is recovered by a conventional method from the precipitate fraction obtained by crushing the cell and then performing centrifugation in the same manner. Thereafter, the insoluble material of the protein is solubilized with a protein denaturant. The solubilized solution is diluted or dialyzed into a solution that does not contain a protein denaturant or the protein denaturant has such a concentration that the protein is not denatured. A purified sample can be obtained by the same isolation and purification method.
Further, the protein can be purified according to a conventional protein purification method [J. Evan. Sadler et al .: Methods in Enzymology, 83,458]. In addition, the protein of the present invention can be produced as a fusion protein with other proteins and purified using affinity chromatography using a substance having an affinity for the fused protein [Akio Yamakawa, Experimental Medicine, 13 , 469-474 (1995)]. For example, the protein of the present invention is converted to protein A according to the method described by Law et al. [Proc. Natl. Acad. Sci., USA, 86, 8227 (1989), GenesDevelop., 4, 1288 (1990)]. As a fusion protein and purified by affinity chromatography using immunoglobulin G.

Moreover, 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 [Proc. Natl. Acad. Sci., USA, 86, 8227 (1989), Genes Develop., 4, 1288 (1990)].
Furthermore, it can also be purified by affinity chromatography using an antibody against the protein itself. The protein of the present invention can be transcribed in vitro according to a known method [J. Biomolecular NMR, 6, 129-134, Science, 242, 1162-1164, J. Biochem., 110, 166-168 (1991)].・ It is possible to produce using a translation system.
Production of the protein of the present invention also by chemical synthesis methods such as Fmoc method (fluorenylmethyloxycarbonyl method) and tBoc method (t-butyloxycarbonyl method) based on the amino acid information of the protein obtained above. Can do. Advanced ChemTech, Perkin Elmer, Pharmacia Biotech, Protein Technology Instrument, Synthecell-Vega, PerSeptive, It can also be chemically synthesized using a peptide synthesizer such as Shimadzu Corporation.
The structural analysis of the purified protein of the present invention can be carried out by a method usually used in protein chemistry, for example, a method described in Protein Structural Analysis for Gene Cloning (Hirano Hisashi, Tokyo Kagaku Dojin, 1993). .

  The “partial peptide or salt thereof” can be produced according to the above protein synthesis method or by cleaving the protein of the present invention with an appropriate peptidase. When the partial peptide obtained by the above method is a free form, it can be converted to an appropriate salt by a known method or a method similar thereto, and conversely, when obtained as a salt, the known method or a method equivalent thereto. It can be converted to the free form or other salts by methods.

  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 cells / tissues described above, cDNA library derived from the cells / tissues described above, and synthetic DNA.

Method for Acquiring a Gene Encoding the Protein of the Present Invention A “gene encoding a protein” can be obtained as follows.
A cDNA library is prepared by a conventional method from human brain, heart, skeletal muscle, spleen, kidney, liver, small intestine, placenta, human normal cells derived from these tissues, human umbilical vein endothelial cells, human ovarian cancer or human colon cancer. .

  Methods for preparing cDNA libraries include Molecular Cloning 2nd Edition, Current Protocols in Molecular Biology Supplements 1-38, A Laboratory Manual, 2nd Ed. (1989), DNA Cloning 1: Core Techniques, A Methods described in Practical Approach, Second Edition, Oxford University Press (1995), etc., or commercially available kits such as SuperScript Plasmid System for cDNA Synthesis and Plasmid Cloning [SuperScript Plasmid System for cDNA Synthesis and Plasmid Cloning (manufactured by Gibco BRL)] and Zap-cDNA synthesis kit [ZAP-cDNA Synthesis Kit, manufactured by Stratagene].

  Specific examples of the DNA obtained by the method include DNA having the base sequence represented by SEQ ID NOs: 1 to 17 and the like. As a plasmid containing DNA of sequence number: 1-17, the plasmid described in the below-mentioned Example can be mention | raise | lifted, for example.

  As the expression vector, any vector can be used as long as it can be expressed in animal cells by incorporating the cDNA. For example, pcDNAI / Amp, pcDNAI, pCDM8 (all available from Funakoshi), pAGE107 [ 3-22979, Cytotechnology, 3, 133 (1990)], pREP4 (manufactured by Invitrogen), pAGE103 [J. Biochem., 101, 1307 (1987)], pAMo, pAMoA [J. Biol. Chem., 268, 22782 -22787 (1993)], pAS3-3 (JP-A-2-27075) and the like can be used.

  An expression vector incorporating the cDNA is introduced into a selectable animal cell to obtain the transformed cell. As the method for introducing the expression vector, any method can be used as long as it is a method for introducing DNA into animal cells. For example, the electroporation method [Cytotechnology, 3, 133 (1990)], the calcium phosphate method (specifically, Kaihei 2-227075), lipofection method [Proc. Natl. Acad. Sci. USA, 84, 7413 (1987)], Virology, 52, 456 (1973).

  As animal cells, 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 299), HCT-15 which is a colorectal cancer cell line, and the like, 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 transformant culture method. When the transformant is an animal cell, the culture medium for culturing the cell is generally 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 media obtained by adding fetal calf serum etc. to these media, etc. Is used

The culture is usually performed for 1 to 7 days under conditions such as pH 6 to 8, 25 to 40 ° C., and 5% CO ₂ . Moreover, you may add antibiotics, such as kanamycin, penicillin, streptomycin, to a culture medium as needed during culture | cultivation.

As described above, DNA encoding the protein of the present invention can be obtained.
DNA can also be prepared by chemically synthesizing DNA encoding the protein of the present invention based on the amino acid sequence. The chemical synthesis of DNA can be performed using a Shimadzu DNA synthesizer using the thiophosphite method, a Perkin Elmer DNA synthesizer model 392 using the phosphoramidite method, or the like.

  Furthermore, using oligonucleotides described below as sense primers (SEQ ID NO: 35) and antisense primers (SEQ ID NO: 36), cDNA prepared from mRNA of cells expressing mRNA complementary to these DNAs as a template The target DNA can also be prepared by performing PCR.

  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 is preferably DNA. The DNA may be any of genomic DNA, genomic DNA library, cDNA derived from the cells / tissues described above, cDNA library derived from the cells / tissues described above, and synthetic DNA. It can be obtained according to the method for obtaining the “gene encoding protein” described above.

  One feature of the screening method for a therapeutic agent or preventive agent for bone disease or joint disease of the present invention is to detect or measure the dynamics of the protein of the present invention or a gene encoding the protein in the presence of a test substance. To do.

  That is, by examining the effect 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, screening for a substance effective as a therapeutic or prophylactic agent for bone disease or joint disease is performed. It is efficient. 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 can be used for pharmacological evaluation of candidate compounds for preventive and / or therapeutic agents for bone diseases or joint diseases.

  Examples of the test substance include a compound or a salt thereof. In addition, in this specification, the said compound or its salt contains a low molecular compound, a high molecular compound, polypeptide or its derivative (s), a nucleic acid or its derivative (s), etc. Such a test substance may be a natural substance or a non-natural substance. Examples of polypeptide derivatives include modified polypeptides obtained by adding modifying groups, variant polypeptides obtained by altering amino acid residues, and the like. Examples of nucleic acid derivatives include modified nucleic acids obtained by adding modifying groups, variant nucleic acids obtained by modifying bases, peptide nucleic acids, and the like.

  Examples of the “kinetics of the protein of the present invention” to be measured or detected in the screening method of the present invention include, for example, the presence or absence of binding between the protein and its ligand; the expression of the protein, specifically the presence or absence of the expression, Examples thereof include fluctuations in expression.

  As the screening method of the present invention, there are roughly two embodiments depending on the “kinetics of the protein of the present invention” to be measured or detected.

As a first embodiment of the screening method of the present invention,
(I) a step of bringing the protein of the present invention into contact with a test substance; and (II) after the step (I), detecting the binding between the protein and the test substance, thereby binding to the protein. And a method comprising selecting a test substance as a candidate compound for a prophylactic and / or therapeutic agent for bone disease or joint disease. According to such a method, a substance that affects the function of the protein can be selected through binding to the protein. Therefore, the selected candidate compound binds directly and specifically to the protein and It has the characteristic property of regulating function.

  In the step (I), the contact between the protein and the test substance is performed by, for example, mixing the protein and the test substance in a solution that does not interfere with the original function of the protein, and performing appropriate reaction conditions (for example, , Reaction temperature, reaction time, etc.) (ie, reacting both).

  Examples of the “solution that does not interfere with the original function of the protein” include solutions such as phosphate buffered physiological saline (PBS), HEPES buffer, Tris buffer, and the like.

  Although it does not specifically limit as reaction conditions in step (I), For example, pH 6.0-10.0 normally in the said solution, Preferably it is pH 7.0-9.0, More preferably, it is pH 7.5-8. 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 ° C., usually 1 minute to 1 hour, preferably For 3 to 30 minutes, more preferably 5 to 20 minutes, and even more preferably 10 minutes. More specifically, 10 mM Hepes, 80 mM KCl, 5% glycerol, 10 mM DTT, 0.1 mg / ml polydC, 50 ng / ml herring sperm DNA, 1 mg / ml BSA, 10% reticulocyte lysate are maintained under the above conditions. Can be mentioned.

  Next, in step (II), the binding between the protein and the test substance is detected, and the presence or absence of the binding is examined.

  The binding is performed, for example, in such a manner that the radioactivity of the protein contained in the solution after reacting the protein in step (I) with, for example, a test substance labeled with a radioactive substance as described above is greatly excessive. It can detect by analyzing in the competition with a non-radioactive test substance. When binding is detected, the test substance used is selected as a candidate compound for a prophylactic and / or therapeutic agent for bone disease or joint disease.

  Moreover, you may implement step (I) and step (II) by the continuous process. Such an embodiment can be carried out by using, for example, a binding assay to a carrier holding a test substance.

As a second embodiment of the screening method of the present invention,
(A) introducing a gene encoding the protein of the present invention into a cell;
(B) a step of expressing the gene in the cell obtained in the step (A) in the presence of the test substance; and (C) expression of the gene as compared to the case where the test substance is absent. Or a change in expression of the gene is measured to thereby select a test substance that causes a change in the expression of the gene as a candidate compound for a prophylactic and / or therapeutic agent for bone disease or joint disease A method including the step of:

  In step (A), in order to introduce a gene encoding the protein of the present invention into a cell, for example, the protein is encoded 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 genes to be operably linked are used. 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 a gene encoding the protein of the present invention into a cloning site of a conventional expression vector. Examples of the vector include viral vectors and plasmid vectors.

  The gene encoding the protein of the present invention includes the gene itself; a gene that hybridizes to the antisense strand of the gene under stringent conditions and encodes a protein having an action equivalent to that of the protein; A gene encoding a protein having a mutation of at least one base in the base sequence of the gene and having an action equivalent to that of the protein; at least 60%, preferably 80% or more, more preferably 90% with the base sequence of the gene; A gene encoding a protein having a sequence identity of at least% and having an action equivalent to that of the protein is also included.

  The “stringent condition” refers to a high stringency condition, for example, a condition of 1 × SSC / 0.2% SDS. Here, in order to improve stringency, lower ionic strength, for example, conditions such as 0.5 × SSC, preferably 0.2 × SSC, more preferably 0.1 × SSC and / or higher temperature, for example, Although it depends on the Tm value of the nucleic acid used, washing or the like may be performed under conditions of 50 ° C. or higher, preferably 60 ° C. or higher, more preferably 65 ° C. or higher.

  The “mutation” refers to base substitution, deletion, addition and insertion. Such a mutation may be a naturally occurring variation or may be a mutation artificially introduced by a conventional site-specific mutation method or the like.

  The “sequence identity” refers to appropriately aligning at least two sequences to be compared, determining the same residues present in each sequence, determining the number of matching sites, and then comparing A value that can be calculated by dividing the number of matching sites by the total number of residues in the target sequence region and multiplying the obtained value by 100. Specifically, such sequence identity is, for example, the homepage address http: // www. ncbi. nlm. nih. In gov / BLAST /, it can be calculated by a BLAST algorithm that is generally available.

  In the various aspects of the present invention, when the expression of the gene encoding the protein of the present invention is essential due to its constitution, the vector used in the expression of the gene, the host cell into which the nucleic acid construct is introduced, etc. As long as the expression of the gene can be achieved by any combination of the above, they may be derived from any species. Specific examples, the method for introducing the nucleic acid construct into cells, the method for culturing the transformant into which the nucleic acid construct has been introduced, and the like are the same as those described for the transformant in the protein production method. The gene encoding the protein of the present invention is derived from eukaryotes, and the therapeutic agents or preventive agents provided in the present invention can be suitably used in eukaryotes, particularly mammals, particularly humans. Considering this point, it is preferable that the host cell used for the expression of the gene is an animal cell.

  In this embodiment, when a stable cell line into which the nucleic acid construct is introduced is used, step (A) may be omitted.

  Next, in step (B), the gene encoding the protein of the present invention is expressed in the cells obtained in step (A) in the presence of the test substance.

  This 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 protein production method.

  Subsequently, in step (C), the presence or absence of the expression of the gene is detected or the variation in the expression of the gene is measured as compared with the case in the absence of the test substance. A test substance that causes a change in expression is selected as a candidate compound for a prophylactic and / or therapeutic agent for bone disease or joint disease.

  The “in the absence of the test substance” refers to the case where in step (B) the gene is expressed in the cell obtained in step (A) in the absence of the test substance, This is a reference for grasping the change in the expression of the gene in the presence of the test substance. Specifically, the cell in such a case or an extract of the cell is used as a control.

  When the presence or absence of the expression of the gene is detected or the change in the expression is measured, when a change in the expression of the gene is recognized compared to the control, for example, the expression of the gene is seen in the control In addition, when the expression of the gene is not detected in the cells contacted with the test substance or when the expression level is lower than that of the subject, that is, when the expression of the gene is decreased compared to the control, the subject The test substance is determined to be a candidate compound for a prophylactic and / 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 of suppressing the expression of the gene at the transcriptional level at the cellular level, and has an excellent effect of being able to specifically suppress the expression of the gene. Demonstrate.

  Detection of the presence / absence of gene expression or measurement of variation 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 compound, a high molecular compound, Examples thereof include an antibody against the protein of the invention, a nucleic acid such as an antisense strand of a nucleic acid comprising a gene encoding the protein of the invention, a peptide, and the like.

  In addition, 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, an antibody of the protein of the present invention, etc. A kit for carrying out the screening method of the present invention is provided. Such a screening kit is also included in the present invention.

As the screening kit of the present invention, specifically,
-A kit comprising the protein of the present invention,
A kit comprising a nucleic acid construct comprising a gene encoding the protein of the present invention,
-A kit comprising cells into which a gene encoding the protein of the present invention has been introduced,
-A kit comprising an antibody against the protein of the present invention or a fragment thereof,
Etc.

Each kit may contain the probe and / or primer pair that can be suitably used in the screening method of the present invention, if desired. Moreover,
If desired, detection reagents, buffer solutions, standard substances, instructions for carrying out the screening method of the present invention, and the like may be contained.

  According to the screening kit of the present invention, the excellent effect that the screening method can be carried out simply and rapidly at a high throughput is exhibited.

  The compound obtained by the screening method of the present invention or a salt thereof can exert a therapeutic or prophylactic effect on a bone disease or joint disease in which the expression of the protein of the present invention is involved in its onset. Therefore, a pharmaceutical composition for use in the treatment or prevention of bone diseases or joint diseases such as OA or RA is provided by the compound obtained by the screening method or a salt thereof.

  The pharmaceutical composition of the present invention is characterized by containing the compound obtained by the screening method of the present invention or a salt thereof 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 salt thereof in the pharmaceutical composition of the present invention can be appropriately adjusted depending on the disease to be treated, the age, weight, etc. of the patient, and may be a therapeutically effective amount. In the case of a polymer compound, for example, 0.0001 to 1000 mg, preferably 0.001 to 100 mg. In the case of a polypeptide or a derivative thereof, for example, 0.0001 to 1000 mg, preferably 0.001 to 100 mg, a nucleic acid or a derivative thereof. In this case, for example, it is desirable to be 0.00001 to 100 mg, preferably 0.0001 to 10 mg.

  The pharmaceutical composition of the present invention may further contain various auxiliaries capable of stably holding the compound or a salt thereof. Specifically, pharmaceutically acceptable auxiliaries, excipients, binders, and stabilizers that exhibit the property of inhibiting the active ingredient from degrading before reaching the site where the active ingredient is to be delivered. Agents, buffers, solubilizers, isotonic agents and the like.

  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 also 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. The administration method is not particularly limited, but when the active ingredient is a low molecular compound or a high molecular compound, the amount of the active ingredient is, for example, 0.0001 to 1000 mg / kg body weight, preferably 0.001 to 100 mg. / Kg body weight, in the case of a polypeptide or a derivative thereof, for example, 0.0001 to 1000 mg / kg body weight, preferably 0.001 to 100 mg / kg body weight, in the case of a nucleic acid or a derivative thereof, for example, 0.00001 to 100 mg / kg Examples include administration once or a plurality of times per day so as to obtain a single dose of body weight, preferably 0.0001 to 10 mg / kg body weight. The administration period is not particularly limited.

  The pharmacological evaluation of the pharmaceutical composition of the present invention can be carried out, for example, by administering the pharmaceutical composition of the present invention to a bone disease or joint disease model mouse and improving the bone disease or joint disease in the administered animal compared to the non-administered animal. It can be performed by a method of evaluating the case where it is seen as an index.

  The present invention also provides a method for diagnosing bone disease or joint disease. One feature of the diagnostic method of the present invention is to measure the expression level of the gene of the protein of the present invention in each of a test sample and a control sample derived from an individual to be tested. Therefore, according to the diagnostic method of the present invention, it is possible to easily and quickly diagnose whether or not an individual subject to be examined is suspected of having bone disease or joint disease. The

  In the diagnostic method of the present invention, when the expression level in the test sample is different from the expression level in the control sample (for example, when it is high or low), the individual to be tested has bone disease or joint disease. It is an indicator of suspected illness.

  The following examples are provided for purposes of illustration and not limitation. As a genetic manipulation method, the method described in Molecular Cloning 2nd edition was used unless otherwise specified.

Preparation of DNA chip mRNA was prepared from human mesenchymal stem cells (BIO WHITKER) using QuickPrep Micro mRNA Purification Kit (Amersham Biosciences) according to the attached manual. The obtained mRNA was converted to cDNA using SuperScript Choice System for cDNA Synthesis (Invitrogen) according to the attached manual to obtain a cDNA clone. Subsequently, 3265 human cDNA fragments were prepared from the cDNA clones by PCR, and together with Lucidea Universal ScoreCard (Amersham Biosciences), which is a control DNA for microarrays, a DNA chip production system Microarray System Generation III Spotter (Molecular Dynamics) was used. A DNA chip was prepared by spotting 2 sets on a Type 7 star slide glass (Molecular Dynamics) in a symmetrical manner (hereinafter referred to as SET1 on the left side and SET2 on the right side) per slide.

Preparation of RNA from human knee joint synovium 44 patients who were diagnosed with osteoarthritis of the knee (knee OA) based on symptoms, physical findings, and image findings in orthopedic surgery at Mie University School of Medicine and were treated for surgery. As control groups, 10 patients with rheumatoid arthritis (RA) and 8 non-OA non-RA patients (malignant bone tumors in the distal femur such as osteosarcoma and Ewing sarcoma) Patients who undergo joint surgery due to trauma) were also included. After giving sufficient explanations to these subjects in advance, the synovial tissue (approximately 0.5 g) of the knee joint is removed from the subjects who have obtained consent at the time of surgery, placed in a microtube, and immediately liquid Frozen with nitrogen. The frozen synovial tissue is pulverized using a sample pulverizer cryopress CP-100W (Microtech Nichion) cooled with liquid nitrogen, and immediately homogenizer Polytron 2100 (Kinematica) in TRIzol reagent (Invitrogen). The total RNA was extracted according to the TRIzol reagent prescription.

Search for genes whose expression fluctuates in osteoarthritis synovial specimens Using the DNA chip described in Example 1, gene expression fluctuation analysis in human osteoarthritis synovial clinical specimens was performed. The RNA described in Example 2 was recovered after DNase I treatment with RNeasy mini kit (Qiagen) according to the attached manual.
Using 2.5 μg of the total RNA thus obtained, amplified RNA (aRNA) was synthesized according to the manual of MessageAmp aRNA Kit (Ambion). Using the synthesized aRNA as a probe template, using Atlas Glass Fluorescent Labeling Kit (CLONTECH, currently BD BIOSCIENCE) or Atlas PowerScript Fluorescent Labeling Kit (currently BD BIOSCImC, using BD BIOSCIENCEC) 5 dCTP (Amersham Bioscience) was incorporated to fluorescently label cDNA. Using this as a probe, hybridization with a DNA chip was performed using an Automated Slide Processor (Amersham Bioscience). Hybridization was performed for 12 hours at 48 ° C. with the probe after pretreatment according to the protocol of Type 7star slide (Amersham Bioscience). As a buffer, Hybridizaton Buffer Ver. 2 (Amersham Biosigns) was used. After washing, reading was performed with a Microarray System Generation III Scanner (Molecular Dynamics). According to this method, 78 slide experiments including duplicate experiments were performed using 62 cases consisting of knee OA, RA, and non-OA non-RA.

Search for genes related to osteoarthritis by statistical analysis Using gene expression data obtained from the DNA chip experiment described in Example 3, compare genes involved in osteoarthritis with normal or rheumatoid arthritis And searched.

とし、３DR遺伝子の発現量がスライド内及びスライド間で同一になるように、（1）式の正規化を実施した。

なお、３ＤＲとは実施例１で述べたＬｕｃｉｄｅａ Ｕｎｉｖｅｒｓａｌ ＳｃｏｒｅＣａｒｄ（商品名、アマシャム バイオサイエンス社）に含まれるチェック用遺伝子のひとつである。また、症例によっては複数枚のスライドで測定がなされていたが、とくに断らない限り、複数のスライドがあった症例では、スライド番号の最も小さいスライドを解析に用いた。また、実施例１で述べたように、1スライドには、ＳＥＴ１とＳＥＴ２のデータが存在する。
正規化には統計解析パッケージソフトウェアＳＡＳ（ＳＡＳ Ｉｎｓｔｉｔｕｔｅ Ｊａｐａｎ社）を利用した。データ解析には、単変量解析のＣｌｕｓｔｅｒ＋ＬｏｇｉｓｔｉｃｓではＳＡＳを利用した。1. Normalized data for analysis Gene expression level

And normalization of the expression (1) was performed so that the expression level of the 3DR gene was the same within the slide and between the slides.

3DR is one of the check genes contained in Lucidea Universal ScoreCard (trade name, Amersham Bioscience) described in Example 1. In some cases, the measurement was performed on a plurality of slides. Unless otherwise specified, the slide with the smallest slide number was used for analysis in a case where there were a plurality of slides. Further, as described in the first embodiment, the data of SET1 and SET2 exist in one slide.
For normalization, statistical analysis package software SAS (SAS Institute Japan) was used. For data analysis, SAS was used in Cluster + Logistics of univariate analysis.

で表示する。また、スライドが正常、ＯＡ、ＲＡのいずれから得られたかを識別する場合は、それぞれ

で表示する。
全３２６５個の遺伝子の中から正常、ＯＡ、ＲＡの３群を識別するにあたって、とくに重要な遺伝子を選ぶことを目的として、１遺伝子ごとの解析（単変量解析）を行った。2. Univariate analysis as a gene unit
The expression level after normalization in the k (= 1,..., 3265) -th gene of the i (= 1,.

Is displayed. In addition, when identifying whether the slide was obtained from normal, OA, or RA,

Is displayed.
Analysis of each gene (univariate analysis) was performed for the purpose of selecting particularly important genes in distinguishing three groups of normal, OA, and RA from all 3265 genes.

を用いた比較を指し、

は正常群での中央値を表す。平均値の比較はデータ

を用いた比較を指す。
ＯＡと正常、ＯＡとＲＡの２通りのペア間比較には、全体有意水準を０．０５として、Ｄｕｎｎｅｔｔの多重比較法を用いた。2.1 1 Case 1 Slide Analysis Analysis was performed for SET1 and SET2, and genes that satisfy the following conditions were determined.
Condition A: Significant differences are observed between OA and normal and between OA and RA in either SET1 or SET2 in either the average value comparison or the average absolute difference comparison.
Condition B: Both SET1 and SET2 have a significant difference between OA and normal or between OA and RA in comparison of mean values.
Here, comparison of mean absolute difference is data

Refers to the comparison using

Represents the median in the normal group. Average comparison is data

Refers to comparison using.
For comparison between two pairs of OA and normal and OA and RA, Dunnett's multiple comparison method was used with an overall significance level of 0.05.

2.2 Single case multiple slide analysis Genes satisfying the following conditions were determined by analysis based on a linear mixed effect model including inter-slide and SET variations.
Condition C: Significant differences are observed between OA and normal and between OA and RA in the comparison of average values and average absolute difference.
Here, the significant difference indicates the significance in the test of the regression coefficient corresponding to the comparison between two pairs of OA and normal and OA and RA.

2.3 Aggregation of univariate analysis results Genes were selected according to the following two criteria.
First selection criterion: Condition A is satisfied.
Second selection criterion: The first selection criterion is not satisfied, and the condition B and the condition C are satisfied.
As a result, 23 genes were selected based on the primary selection criteria and 3 genes were selected based on the secondary selection criteria, and a total of 26 genes were selected as genes involved in OA (Table 1). For these genes, the expression levels in OA patients were compared with the expression levels in RA patients and normal individuals, respectively (Table 2). When the expression level in an OA patient is + on the basis of the expression level in an RA patient and a normal individual, the expression level of the gene is OA patient> RA patient and OA patient> normal individual. It suggests. In a certain gene, when the expression level in OA is + based on the expression level in RA patients and − based on the expression level in normal individuals, the expression level of the gene is normal individuals> OA patients> RA patients. It suggests that there is. In addition, in a certain gene, when the expression level in OA patients is − based on the expression level in RA patients and + based on the expression level in normal individuals, the expression level of the gene is RA patient> OA patient> This suggests that it is a normal individual.

Analysis of nucleotide sequences of genes involved in OA For the 26 genes related to OA described in Example 4, each of the PCR fragment base sequences described in Example 1 was subjected to DNA sequencing apparatus MegaBACE1000 (Amersham Biosciences). And analyzed. Except for the two genes for which PCR fragments were not obtained, the base sequences obtained for the remaining 24 genes were subjected to homology search using gene information analysis software bioSCOUT (LION). As a result, the gene names were found for all 24 genes, and 3 were duplicated in Fibulin-3. We searched PubMED (National Center for Biotechnology Information) and PATENTWeb (MicroPentent) for 22 genes excluding duplication, and examined whether the relationship between each gene and OA is known, It became newly clear that the remaining 17 genes are involved in OA, except for 5 genes whose association has already been shown. Table 17 shows the 17 genes.

Expression analysis of Fibulin-3 mRNA by real-time quantitative PCR method As a result of statistical analysis, among the 17 genes finally selected, 4 genes were Fibulin-3 (SEQ ID NO: 13), so RT-PCR By this method, the expression profile of Fibulin-3 gene in the synovial tissue of human knee osteoarthritis was examined and compared with the result of the DNA chip. All samples used RNA used for DNA chip analysis. Sample number 1 is normal, and 2-12 is synovial RNA of OA patients.
For RT-PCR, primer pairs of SEQ ID NOs: 35 and 36 were used. RNA prepared from the human knee osteoarthritis synovial clinical specimen described in Example 2 was treated with DNase I at RNeasy mini kit (Qiagen), and collected after treatment with DNase I.
CDNA was synthesized from 2.5 μg of the obtained total RNA according to the attached manual using SuperScript First-strand Synthesis System (Invitrogen) for RT-PCR. Next, primers for real-time quantitative PCR (SEQ ID NOs: 35 and 36) were selected by the probe search software Primer Express (Applied Biosystems) based on the sequence of human Fibulin-3 (SEQ ID NO: 13). Primers were synthesized by Qiagen. Using this primer, real-time quantitative PCR with SYBR Green I was performed using the synthesized cDNA as a template to quantify the amount of mRNA.
As a reaction reagent, SYBR Green PCR Master Mix (Applied Biosystems) was used. After reacting at 95 ° C. for 10 minutes, the fluorescence intensity was quantified by carrying out 40 cycles at 95 ° C. for 10 seconds and 60 ° C. for 60 seconds. PCR and fluorescence measurements were performed using an ABI PRISM 5700 Sequence Detection System (Applied Biosystems). As a result, it was confirmed that the expression of the Fibulin-3 gene was enhanced in the OA clinical specimen as well as in the DNA chip experiment in the real-time quantitative PCR experiment (FIG. 1). The graph of the real-time quantitative PCR data in FIG. 1 shows a relative amount with an expression level of 1 in a normal synovial clinical specimen (sample number 1). In addition, it was confirmed that the gene expression level by the DNA chip method and the real-time quantitative PCR method shows a positive correlation (Pearson's correlation coefficient: about 0.69). Therefore, the same results were expected for the remaining genes, suggesting that osteoarthritis can be diagnosed using mRNA expression of the 17 genes shown in Table 3 as an index. In addition, in Example 4, rheumatoid arthritis is expressed using mRNA expression as an index for 7 genes whose expression levels are normal individuals> OA patients> RA patients or RA patients> OA patients> normal individuals. It was suggested that it could be diagnosed.

  By the detection or selection method of the present invention, a sample derived from an individual suspected of suffering from a bone disease or joint disease, particularly osteoarthritis, or rheumatoid arthritis can be detected or selected easily and rapidly. In addition, diagnostic agents necessary for detection or selection are provided.

Claims (23)

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 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 at least one gene encoding a protein having: wherein the test sample is different from the expression level in a control sample from a normal individual, A method for detecting or selecting a sample derived from an individual suspected of suffering from a bone disease or joint disease, which serves as an indicator that the sample is derived from an individual suspected of suffering from a bone disease or joint disease. The method according to claim 1, wherein the bone disease or joint disease is cartilage dysplasia, bone dysplasia, osteoporosis, osteoarthritis, rheumatoid arthritis, arthritis, synovitis, metabolic arthropathy or arthropathy due to sports. A protein having an amino acid sequence selected from the group consisting of SEQ ID NO: 18, 21-23, 25, and 29 in a test sample derived from an individual to be tested, or one or several amino acids are deleted or substituted in the amino acid sequence Or measuring the expression level of at least one gene encoding a protein having an added amino acid sequence, wherein the expression level in the test sample is higher than the expression level in a control sample from a normal individual And when the expression level is lower than the expression level in a control sample derived from a patient with rheumatoid arthritis, the test sample is an indicator that the sample is derived from an individual suspected of suffering from osteoarthritis. A method for detecting or selecting a sample derived from an individual suspected of suffering from osteoarthritis. A protein having an amino acid sequence selected from the group consisting of SEQ ID NOs: 19, 20, 24, 26-28, 30-33 in a test sample derived from an individual to be tested, or one or several amino acids in the amino acid sequence Measuring the expression level of at least one gene encoding a protein having a deleted, substituted or added amino acid sequence, wherein the expression level in the test sample is expressed in a control sample from a normal individual The test sample is a sample derived from an individual suspected of suffering from osteoarthritis if the expression level is higher than that in a control sample derived from a patient with rheumatoid arthritis A method for detecting or selecting a sample derived from an individual suspected of suffering from osteoarthritis, which is an index of the above. A gene encoding a protein having the amino acid sequence of SEQ ID NO: 34 in a test sample derived from an individual to be tested, 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 Measuring the expression level of the test sample, wherein the expression level in the test sample is lower than the expression level in a control sample from a normal individual, and greater than the expression level in a control sample from a rheumatoid arthritis patient individual. The test sample is derived from an individual suspected of suffering from osteoarthritis, which is an indicator that the test sample is derived from an individual suspected of suffering from osteoarthritis Sample detection or sorting method. A protein having an amino acid sequence selected from the group consisting of SEQ ID NO: 18, 21-23, 25, and 29 in a test sample derived from an individual to be tested, or one or several amino acids are deleted or substituted in the amino acid sequence Or measuring the expression level of at least one gene encoding a protein having an added amino acid sequence, wherein the expression level in the test sample is higher than the expression level in a control sample from a normal individual And, when it becomes higher than the expression level in the control sample derived from osteoarthritis patient, the test sample is an indicator that it is a sample derived from an individual suspected of having rheumatoid arthritis. A method for detecting or selecting a sample derived from an individual suspected of having rheumatoid arthritis. A gene encoding a protein having the amino acid sequence of SEQ ID NO: 34 in a test sample derived from an individual to be tested, 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 expression level in the test sample is lower than the expression level in a control sample from a normal individual, and the expression level in a control sample from an osteoarthritis patient individual A sample derived from an individual suspected of suffering from rheumatoid arthritis, an indicator that the test sample is derived from an individual suspected of suffering from rheumatoid arthritis Detection or sorting method. At least encoding a protein having an amino acid sequence selected from the group consisting of SEQ ID NOs: 18 to 34 or a protein having an amino acid sequence in which one or several amino acids are deleted, substituted or added in the amino acid sequence The method according to any one of claims 1 to 7, which is measured using the mRNA level of one gene as an index. A protein having an amino acid sequence selected from the group consisting of SEQ ID NOs: 18 to 34, or at least one protein having an amino acid sequence in which one or several amino acids are deleted, substituted or added in the amino acid sequence; The method according to any one of claims 1 to 7, which is measured using the partial peptide or a salt thereof as an index. At least one gene encoding a protein having an amino acid sequence selected from the group consisting of SEQ ID NOs: 18 to 34 or a protein having an amino acid sequence in which one or several amino acids are deleted, substituted or added in the amino acid sequence The method according to claim 8, wherein a detectable nucleic acid is used. The method according to claim 10, wherein the gene is a nucleic acid comprising a base sequence selected from the group consisting of SEQ ID NOs: 1-17. The method according to claim 10, wherein the gene is a nucleic acid comprising the nucleotide sequence of SEQ ID NO: 6 and / or 13. The method according to claim 12, wherein a nucleic acid comprising the nucleotide sequence of SEQ ID NO: 35 and / or 36 is used. A protein having an amino acid sequence selected from the group consisting of SEQ ID NOs: 18 to 34, at least one protein having an amino acid sequence in which one or several amino acids are deleted, substituted or added in the amino acid sequence, or a partial peptide thereof; The method according to claim 9, wherein an antibody against the salt or a fragment thereof is used. To a protein having the amino acid sequence of SEQ ID NO: 23 and / or 30, or at least one protein having an amino acid sequence in which one or several amino acids are deleted, substituted or added in the amino acid sequence, or a partial peptide thereof or a salt thereof The method according to claim 9, wherein an antibody or a fragment thereof is used. At least one gene encoding a protein having an amino acid sequence selected from the group consisting of SEQ ID NOs: 18 to 34 or a protein having an amino acid sequence in which one or several amino acids are deleted, substituted or added in the amino acid sequence The diagnostic agent for using for the method in any one of Claims 10-15 containing the nucleic acid which can be detected. The diagnostic agent according to claim 16, wherein the gene is a nucleic acid having a base sequence selected from the group consisting of SEQ ID NOs: 1 to 17. The diagnostic agent according to claim 16, wherein the gene is a nucleic acid comprising the nucleotide sequence of SEQ ID NO: 6 and / or 13. The diagnostic agent according to claim 18, comprising a nucleic acid comprising the nucleotide sequence of SEQ ID NO: 35 and / or 36. A protein having an amino acid sequence selected from the group consisting of SEQ ID NOs: 18 to 34, at least one protein having an amino acid sequence in which one or several amino acids are deleted, substituted or added in the amino acid sequence, or a partial peptide thereof; The diagnostic for using for the method of Claim 14 or 15 containing the antibody with respect to the salt, or its fragment | piece. To a protein having the amino acid sequence of SEQ ID NO: 23 and / or 30, or at least one protein having an amino acid sequence in which one or several amino acids are deleted, substituted or added in the amino acid sequence, or a partial peptide thereof or a salt thereof A diagnostic agent for use in the method according to claim 14 or 15, comprising an antibody or a fragment thereof. At least one gene encoding a protein having an amino acid sequence selected from the group consisting of SEQ ID NOs: 18 to 34 or a protein having an amino acid sequence in which one or several amino acids are deleted, substituted or added in the amino acid sequence A screening method for a prophylactic and / or therapeutic agent for bone disease or joint disease, which is characterized by being used. A protein having an amino acid sequence selected from the group consisting of SEQ ID NOs: 18 to 34, at least one protein having an amino acid sequence in which one or several amino acids are deleted, substituted or added in the amino acid sequence, or a partial peptide thereof; A screening method for a prophylactic and / or therapeutic agent for bone disease or joint disease, characterized by using the salt.

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