WO2002064769A1 - Novel disease marker - Google Patents

Abstract

A marker for cancer, cancer metastasis and bone diseases, a method of detecting cancer and bone diseases and a diagnosis kit are provided. Also, drugs aiming at preventing and treating cancer, arthritis deformans, rheumatoid arthritis and osteoporosis are provided. CO126 is expressed in mesenchymal stem cells and shows an increase in the expression in gastric cancer. Thus, it is useful as a gastric cancer marker. Also, CO126 has an activity of inducing the differentiation of cartilage cells and, therefore, is useful as a marker for arthritis deformans and rheumatoid arthritis. Moreover, a CO126 polypeptide, a polynucleotide encoding this polypeptide and an antibody against this polypeptide are useful in screening a substance controlling the activity of inducing the cartilage cell differentiation. A substance obtained by the screening is useful as a drug such as a preventive or a remedy for gastric cancer, arthritis deformans and rheumatoid arthritis.

Description

 Description New disease marker Technical field

 The present invention relates to a pre-bovine synth having an amino acid sequence represented by SEQ ID NO: 2, a transmembrane protein C0126 having homology to c-MET and TEM7, cancer metastasis, and bone disease, The present invention relates to the use as a marker for gastric cancer, osteoarthritis, rheumatoid arthritis and osteoporosis. Also, a C0126 polypeptide and a fragment thereof, a polynucleotide encoding the polypeptide and a fragment thereof, and a cancer, cancer metastasis and bone using an antibody recognizing the polypeptide and the fragment thereof The present invention relates to medicaments, preparations, and methods for diagnosis, prevention, and treatment of diseases, particularly osteoarthritis, rheumatoid arthritis, and osteoporosis. Background art

 A polypeptide represented by the amino acid sequence of SEQ ID NO: 4 was isolated as a transmembrane polypeptide, and then a polynucleotide encoding the polypeptide was isolated and its sequence (SEQ ID NO: 3) Was determined (WO 00/77037). The polypeptide is a polypeptide consisting of 507 amino acids, but its function has not been elucidated yet.

Plexin is a receptor for semaphorin, a single transmembrane protein with a cystin-rich domain highly homologous to c-MET in the extracellular region, and is widely expressed in vivo (Neuron, 1 4, 1189 (1995).)). Semaphorin has been found as a factor involved in neural circuit formation (Cell, 75, 1389 (1993).). Neural circuits are formed by cell adhesion factors and repulsive factors. Semaphorin acts as a repulsive factor in nervous system tissues and repulsively induces axons to target cells. In recent years, peripheral tissue The muff-year-old Lin is angiogenesis (Journal of Cell Biology, 146, 233 (19999).), Cancer cell proliferation and cancer metastasis (Cancer Research, 58, 1238 (1998)). ) Has also been found to be involved. Plexin has a site related to the oral synkinase activity in the intracellular domain, and transmits the action of semaphorin into the cell (CelΊ, 99, 71-80 (19999).). . c-MET is a receptor for hepatocyte growth factor (HGF) (Proceeding of National Academy of Sciences USA, 84, 6379 (1987).), and is a single transmembrane protein that enters the intracellular region. It has an oral synkinase region in Thailand and is also expressed in nerve cells, and is involved in axonal outgrowth and induction (N euro η, 17, 117 (1996).). T EM 7 (Tumor Endothelial Masker 7) was discovered as a marker gene for colorectal cancer, and its expression was also increased in sarcoma, metastatic liver cancer, lung cancer, 脬 cancer, breast cancer and brain tumor, It is thought to be involved (Science, 289, 1197 (2000).) _C Cancer is a disease that develops in most tissues of the body, and has been a target gene for canceration. A number of oncogenes and tumor suppressor genes have been discovered, and the mechanism of human carcinogenesis is described in multiple steps at a specific gene level (Molecular Medicine, 36, 424 (1999)). ). For example, gastric cancer is classified into poorly differentiated adenocarcinoma and well-differentiated adenocarcinoma with adenoma and intestinal metaplasia. Long shortening and genetic instability occur. By early cancer, (2) telomerase activation and telomerase reverse transcriptase expression, (3) abnormal CD44 transcript, cyclin E overexpression, and c-met expression were induced. I have. After that, it becomes advanced cancer, and (4) abnormalities such as chromosome 7q deletion, cyclin E gene amplification, decreased p27 expression, decreased nm23 expression, overexpression of growth factor, and abnormal CD44 transcript ing. Genetic abnormalities characteristic of well-differentiated types include (1) D1S191 instability, DNA methylation, (2) inactivation of APC gene, inactivation of p53 gene, K-ras gene activation, _P S 2 expression mourning Loss, (3) reduced p27 expression, c-erb B2 gene amplification. Genetic abnormalities characteristic of poorly differentiated are (1) deletion of chromosome 17p12-21, mutation of cadherin gene, amplification of K-sam gene, and amplification of c-met gene. . c-met is a receptor for hepatocyte growth factor, which stimulates hepatocyte growth factor to phosphorylate catenin, thereby reducing the function of force helin and promoting cell dissociation. Therefore, it is thought that hepatocyte growth factor acts as a cell-dispersing factor due to an increase in c-met protein, and is involved in diffuse invasion. However, the mechanism of carcinogenesis has not yet been fully elucidated, and it is thought that unknown genes may be involved. Under such circumstances, identification of a novel gene involved in the development of cancer and a method of diagnosing cancer using the gene have been desired.

 Osteoarthritis and rheumatoid arthritis are diseases in which the cartilage tissue of the joints is degenerated and worn. Currently, the main treatment methods are symptomatic treatments centering on analgesia and anti-inflammatory. However, there is a need for the development of treatments that promote cartilage destruction and regeneration for fundamental treatment. Chondrocytes are formed when mesenchymal stem cells differentiate into cartilage. Mesenchymal stem cells are cells contained in bone marrow, have pluripotency, and are stem cells derived from mesodermal tissue such as bone, cartilage, tendons, ligaments, fat, and skeletal muscle. In particular, it can be easily differentiated into fat cells, chondrocytes, and osteoblasts (Science, 284, 144 (1999).). Therefore, elucidation of the mechanism controlling cartilage differentiation of mesenchymal stem cells is important for developing drugs and methods for the prevention and treatment of osteoarthritis, rheumatoid arthritis and osteoporosis. The mechanism has not yet been fully elucidated, and it is thought that unknown genes may be involved. Under these circumstances, the identification of genes involved in cartilage differentiation and the use of the genes or their products for the diagnosis, prevention and treatment of osteoarthritis, rheumatoid arthritis and osteoporosis have been anticipated. It had been. Disclosure of the invention

The present invention relates to cancer, cancer metastasis and bone disease, particularly gastric cancer, osteoarthritis, rheumatism An object of the present invention is to provide a novel marker useful for diagnosing osteoarthritis and osteoporosis. Another object of the present invention is to provide polypeptides and polynucleotides useful for the prevention and treatment of cancer, cancer metastasis and bone diseases, particularly osteoarthritis, rheumatoid arthritis and osteoporosis.

 The present inventors have conducted intensive studies for the purpose of isolating genes involved in angiogenesis and differentiation of mesenchymal stem cells, and as a result, SEQ ID NO: 1 has been identified as a novel cancer and cancer metastasis marker. The represented polynucleotide was found. In addition, they found that the expression of this marker was increased in gastric cancer tissues, and confirmed that it was useful as a marker for cancer or cancer metastasis. Furthermore, as a result of further research, we found a C0126 polypeptide represented by SEQ ID NO: 2 as a novel bone disease marker. In addition, when mesenchymal stem cells are differentiated into chondrocytes with chondrocyte differentiation-inducing factors, expression of C 0 126 is increased, and when C 0 126 is expressed in mesenchymal stem cell lines, They found that differentiation into cells was promoted, and confirmed that the marker of the present invention was useful as a marker for bone diseases associated with chondrocyte differentiation. In addition, they also confirmed that osteoarthritis, rheumatoid arthritis, and osteoporosis were present. The present invention was suggested to be useful as a marker of the present invention, and the present invention was completed.

 That is, the present invention

 (1) a cancer, cancer metastasis or bone disease containing a polypeptide comprising an amino acid sequence represented by A sp from position 1 Sp to position 45 A rg of SEQ ID NO: 2;

 (2) a cancer, cancer metastasis or bone disease comprising a polypeptide comprising an amino acid sequence represented by Asn at position 46 to Tyr at position 43 of SEQ ID NO: 2;

 (3) a marker according to (1) or (2), comprising a polypeptide comprising an amino acid sequence represented by Asp at position 1 to Cys at position 507 in SEQ ID NO: 2;

 (4) The marker according to (1) or (2), comprising a polypeptide consisting of an amino acid sequence represented by Met at position 122 of SEQ ID NO: 2 to Cys at position 507;

(5) Ars at position 45 from Asp at position 1 of SEQ ID NO: 2, Tyr at position 43 from Asn at position 46, Cys at position 507 from Asp at position 1 2 1 2 2nd Met Cancer or cancer metastasis, including a polypeptide having one or several amino acid mutations selected from deletion, substitution or insertion in any of the polypeptides consisting of the amino acid sequence represented by Cys from position 507 to position 507; Or a marker of bone disease;

 (6) The marker according to (1) to (5), wherein the cancer is gastric cancer;

 (7) the marker according to any one of (1) to (5), wherein the bone disease is osteoarthritis, rheumatoid arthritis or osteoporosis;

 (8) a marker for cancer, cancer metastasis or bone disease, comprising a polynucleotide consisting of a nucleotide sequence represented by A from position 43 to position A of position C of SEQ ID NO: 1;

(9) a marker for cancer, cancer metastasis, or bone disease, comprising a polynucleotide consisting of the nucleotide sequence represented by 288th G to 422th A of SEQ ID NO: 1;

 (10) a marker according to (8) or (9), including a polynucleotide consisting of a base sequence represented by 288th G to 1808th C of SEQ ID NO: 1;

(11) the method according to (8) or (9), which comprises a polynucleotide consisting of a base sequence represented by A from position 222 to position C of position 208 of SEQ ID NO: 1; (1 2) SEQ ID NO: 1 from 4 2 3rd A to 180 8th C, 2 8 8th G to 4 2 2nd A, 2 8 8th G from 1808 Of cancer, cancer metastasis, or bone disease containing a polynucleotide that hybridizes under stringent conditions to any of the nucleotide sequences represented by C or 222 of the 2nd A to 1808th C One;

 (13) The marker according to (8) to (12), wherein the cancer is gastric cancer;

 (14) the marker according to any one of (8) to (12), wherein the bone disease is osteoarthritis, rheumatoid arthritis or osteoporosis;

 (15) The method according to any one of (1) to (7), comprising using an antibody that recognizes a polypeptide consisting of the amino acid sequence of SEQ ID NO: 2 or a part thereof, comprising the following steps: How to detect the power of

 (a) contacting a test substance with the antibody; and

(b) detecting the binding between the test substance and the antibody; (16) The marker according to any one of (8) to (15), wherein a polynucleotide comprising the nucleotide sequence of SEQ ID NO: 1 or a part thereof comprising the following steps is used. How to detect one;

 (a) contacting a test substance with the polynucleotide, and

 (b) detecting the binding between the test substance and the polynucleotide;

 (17) A kit for diagnosing cancer, cancer metastasis, or bone disease, comprising using the detection method according to (15) or (16);

 (18) The diagnostic kit according to (17), wherein the cancer is gastric cancer;

 (19) The diagnostic kit according to (17), wherein the bone disease is osteoarthritis, rheumatoid arthritis or osteoporosis;

 (20) A method for screening for a chondrocyte-inducing activity regulating substance, comprising using a transformant that expresses a polynucleotide containing the nucleotide sequence represented by SEQ ID NO: 1 or a part thereof, comprising the following steps;

 (a) culturing a transformant expressing the polynucleotide in the presence or absence of the test substance, and

 (b) comparing the amount of cartilage matrix produced in the transformant expressing the polynucleotide in the presence or absence of the test substance;

 (21) a kit for screening a chondrocyte differentiation-inducing activity regulating substance, comprising a transformant expressing a polynucleotide comprising the nucleotide sequence represented by SEQ ID NO: 1 or a part thereof;

 (22) A chondrocyte differentiation-inducing activity regulating substance obtained by the screening method according to (21);

(23) a chondrocyte differentiation-inducing activity control according to (22), which is a polypeptide comprising an amino acid sequence represented by A sn at position 46 to A 1a at position 43 2 of SEQ ID NO: 2 (24) The compound according to (22) or (23) or a salt thereof, a polynucleotide comprising the nucleotide sequence described in SEQ ID NO: 1 or a part thereof, a nucleotide sequence described in SEQ ID NO: 1 or Expression vector for human gene therapy containing a polynucleotide consisting of a part thereof Evening, a polypeptide comprising the amino acid sequence of SEQ ID NO: 2 or a part thereof, or a pharmaceutical composition containing, as an active ingredient, an antibody against the polypeptide; (25) prevention of cancer or cancer metastasis Or a therapeutic agent according to (24); (26) a pharmaceutical composition according to (24), which is a prophylactic or therapeutic agent for bone disease; (27) osteoarthritis, The pharmaceutical composition according to (24), which is a prophylactic or therapeutic agent for rheumatoid arthritis or osteoporosis;

 (28) A method for preventing or treating cancer, cancer metastasis, or bone disease, comprising administering the pharmaceutical composition according to any one of (24) to (27);

About.

 The terms used in the present specification are used in a meaning commonly used in the art, unless otherwise specified. Hereinafter, terms used particularly in the present specification will be described.

 In the present invention, the amino acid sequence represented by Arg at position 45 from Asp at position 1 of SEQ ID NO: 2 or 432 from Asn at position 46 of SEQ ID NO: 2 An amino acid sequence represented by T yr at position 1); an amino acid sequence represented by Cys at position 507 from Asp at position 1 in SEQ ID NO: 2; and the amino acid sequence represented by Gys at position 507 from et. Furthermore, the marker according to the present invention may be a “base sequence represented by A to C at position 808 of SEQ ID NO: 1” or “4 to G at position 288 of SEQ ID NO: 1”. 2 The nucleotide sequence represented by A at the second position '', and the `` nucleotide sequence represented by G from the 288th position of SEQ ID NO: 1 to C at the position of 1808th position '' and `` 22nd position A of the sequence number 1 '' And the base sequence represented by the 180th C-position ”.

 `` Cancer '' refers to malignant tumors and malignant neoplasms classified as carcinoma, sarcoma, leukemia, malignant lymphoma, germ cell tumor, etc., including gastric cancer, and including these cancer metastases I do. “Bone disease” includes, for example, osteoarthritis, rheumatoid arthritis, osteoporosis and the like.

As used herein, the term “marker” refers to a cancer, cancer metastasis or bone disease in a living body. It is a substance used to detect a disease. Certain mRNAs or proteins can be markers if their expression is increased or decreased in cancer, cancer metastasis or bone disease, for example, from Met at position 22 to position 507 of SEQ ID NO: 2. A polypeptide consisting of the amino acid sequence represented by Cys; a polynucleotide consisting of the nucleotide sequence represented by C from the 22nd A to the 208th C of SEQ ID NO: 1; and a base sequence complementary thereto And some of them.

 "Part of the nucleotide sequence of SEQ ID NO: 1" refers to at least 10 or more consecutive nucleotide sequences of the nucleotide sequence of SEQ ID NO: 1, preferably It is at least 10 or more consecutive nucleotide sequences of the nucleotide sequence represented by C at the 1808th position from A in FIG.

 “Part of the amino acid sequence of SEQ ID NO: 2” refers to an amino acid sequence of at least 5 consecutive amino acids of the amino acid sequence of SEQ ID NO: 2, and preferably one amino acid sequence of SEQ ID NO: 2. An amino acid sequence of at least 5 consecutive amino acid sequences represented by Cys at position 507 from position Met, more preferably from amino acid sequence at position 1 of SEQ ID NO: 2 from Asp at position 1 An amino acid sequence of at least 5 contiguous residues of the amino acid sequence represented by Cys at position 5, more preferably an amino acid sequence from position 1 Asp of SEQ ID NO: 2 to Arg at position 45 or Arg at position 46. It is an amino acid sequence of at least 5 consecutive amino acid sequences represented by A la at position 432 from A sn.

 The term "one or several amino acids" refers to a number of amino acids that can be deleted, substituted and added by site-directed mutagenesis or the like, and is 50 or less, preferably 30 or less, more preferably It means 20 or less, more preferably 10 or less amino acids. Further, the polypeptide is a polypeptide having a function as a marker by deletion, substitution, and addition.

"Hybridizing under stringent conditions" refers to a method known and used in the art, for example, a colony hybridization method, a plaque hybridization method, or a Southern blot hybridization method. Means that hybridization is performed using a membrane on which polynucleotides derived from colonies or plaques are immobilized, in the presence of 0.7 to 1 μM NaCl at 65 ° C. After hybridization, use 0.1 to 2 times concentration of SSC (Saline Sodium Citrate; 150 mM sodium chloride, 15 mM sodium citrate) solution at 65 ° C. Even if the membrane is washed with C, it means that it has hybridized. Hybridization: Μ ο Ί ecu Ί ar CI oning: AL aboratory M anual, S econd Edition (1 989) (Cold Spring Spring L aboratory Press), Current Protocolsin Molecular Biology (1 9 94) (Wi1ey-Interscience) DNACloning 1: Core Technology, AP ractical Approach, S econd Edition (1995) (Oxford U niversity Press) It can be performed according to it.

 The term “antibody” is used in the sense commonly used in the art, and includes all or fragments, derivatives, conjugates, and modified forms of the antibody. Preferably, it is an antibody that recognizes C 0 126 or a fragment thereof, more preferably, it is an antibody that specifically recognizes, and still more preferably, it is an antibody that recognizes monospecifically. Such antibodies may be either polyclonal or monoclonal.

 The “detection method” may be any method as long as it is a molecular biology measurement method for detecting mRNA or an immunology measurement method for detecting polypeptide. Examples of molecular biological measurement methods include Northern hybridization method, dot blot method, and RT-PCR method. Examples of immunological measurement methods include ELISA (Enzyme Linked Immunosorbent Assay), RIA (RadioImmuno Assay), and the like: a fluorescent antibody method, a stamplot method, and an immunohistochemical staining method.

“Diagnostic kit” means that C 0 126 is a marker for cancer, cancer metastasis, and bone disease. Therefore, at least a polynucleotide or a part thereof for detecting the marker and a polynucleotide of C 0 126 as a standard reagent, or an antibody against C 0 126 and C 0 12 as a standard reagent 6 is a kit for measuring the marker by molecular biology or immunologically based on the detection method described above.

 The “chondrocyte differentiation-inducing activity” is an activity that promotes the differentiation of stem cells into chondrocytes. The chondrocyte differentiation-inducing activity can be measured by detecting the formation of cartilage matrix, which is generated when stem cells are differentiated into chondrocytes, for example, various collagens, aggrecan, and open theoglycans such as chondroitin sulfate.

 The “chondrocyte differentiation-inducing activity regulating substance” is a substance that regulates the differentiation of stem cells into chondrocytes, and is a substance that can regulate the chondrocyte differentiation-inducing activity described above. Such substances screen for substances that increase or decrease, based on the cartilage matrix such as various collagens generated when stem cells are differentiated into chondrocytes and protein-derived glycans such as aglycan and chondroitin sulfate. Can be obtained by As a chondrocyte differentiation-inducing activity regulating substance, a “binding substance” that binds to C 0 126, a “binding activity regulator” that regulates the binding of C 0 126 to the binding substance, and the expression of C 0 126 Any substance can be used as long as it can regulate the induction of chondrocyte differentiation, such as an "expression regulating substance". Examples of such substances include low molecular substances, polynucleotides, and polypeptides. No. Preferably, it is a polypeptide consisting of an amino acid sequence represented by Me from position 122 of SEQ ID NO: 2 to A1a at position 432, more preferably Asp at position 1 of SEQ ID NO: 2. And a polypeptide comprising an amino acid sequence represented by A1a at position 432, more preferably an amino acid represented by A1a at position 43 from Asn at position 46 of SEQ ID NO: 2. It is a polypeptide consisting of an acid sequence.

The “screening method” is a method for screening a binding substance, a binding activity regulating substance, and an expression regulating substance for C 0 126, and the above-described molecular biological measurement method, immunological measurement method, Publicly available in the field, such as bioassays and coupled This can be achieved by appropriately adapting known techniques.

 The “screening kit” is used to screen a chondrocyte differentiation-inducing activity modulator, and at least a polynucleotide or a part thereof and a standard reagent for detecting CO126. Contains an antibody against C0126, and an antibody against C0126 and a C0126 polypeptide as a standard reagent. It is a kit for screening regulatory substances by molecular biological assay, immunological assay, bioassay and binding assay.

 “Pharmaceutical composition” means at least a C 0 126 polypeptide or a part thereof, a C 0 126 polynucleotide or a part thereof, or an antibody against the C 0 126 or a part thereof Any substance that regulates chondrocyte differentiation-inducing activity may be used, and is used for the prevention or treatment of specific diseases, for example, cancer, bone diseases, particularly gastric cancer, osteoarthritis, rheumatoid arthritis, osteoporosis, etc. sell. Examples of such a substance include substances such as low molecular weight substances, polynucleotides, and polypeptides. Preferably, it is a polypeptide comprising an amino acid sequence represented by A 位 a at position 43 2 from Me at position 22 of SEQ ID NO: 2, and more preferably a polypeptide comprising an amino acid sequence represented by AΊa at position 42 A polypeptide comprising an amino acid sequence represented by AΊa at position 432 from Asp, and more preferably A, a at position 43, from Asn at position 46 of SEQ ID NO: 2 A polypeptide comprising the represented amino acid sequence. The pharmaceutical composition of the present invention also includes "expression vector for gene therapy". An “expression vector for gene therapy” is an expression vector in which a part or all of the polynucleotide is incorporated, and the gene is supplemented to cells by introducing it into cells and tissues. Or a vector that can repair and correct gene defects. As such a vector, those in which part or all of the sequence of a virus lacking replication ability is replaced with a therapeutic gene are used. Because the markers are associated with cancer, cancer metastasis, and bone disease, the polynucleotides are useful for their gene therapy.

“Prophylactic or therapeutic agent” refers to an increase in C 0 126 in cancer or bone disease. Therefore, a substance that regulates the expression level or activity of C 0 126 is useful, and is a preparation containing at least the above-mentioned pharmaceutical composition.

 "Prophylaxis or treatment method" refers to a specific disease, for example, cancer, cancer metastasis and bone disease, preferably gastric cancer, osteoarthritis, rheumatoid arthritis or osteoporosis, by administering the above-mentioned pharmaceutical composition. Is a method of preventing or treating. BRIEF DESCRIPTION OF THE FIGURES

 FIG. 1 is a schematic diagram showing a schematic diagram of a C 0 126 polypeptide.

 The C0126 polypeptide is a secreted protein consisting of 529 amino acids. The predicted signal peptide region (Met from position 122 to Thr-1 in the amino acid sequence) SP; Signal peptide), from Cyc at position 306 to Cys at position 33 are conserved by Het receptor Met and seamaph 0r1n receptor p1eXin A region with similarity to the Met-related sequences (MRS), from the Gly at position 433 to the Met at position 455, is a predicted transmembrane region (TM; T). ransmembranedomain).

 FIG. 2 shows the expression amount and the ratio of C 0 126 mA in human gastric cancer tissue and the corresponding normal tissue.

 FIG. 3 shows a change in the expression level of C 0 126 polypeptide mRNA during cartilage differentiation induction.

 Northern blot analysis of human mesenchymal stem cells treated with IGF-1 having chondrocyte differentiation-inducing activity, IL-11 and FGF-2 having bone differentiation-inducing activity was performed, and the control group (untreated group) was analyzed. The expression level of C 0 126 polypeptide mRNA is shown as a relative expression level with respect to 1.

 FIG. 4 shows the cartilage differentiation-inducing activity of CO 126 (Alcian blue staining).

Using the Alcian blue staining property as a cartilage differentiation marker as an index, C 0 126 and B It shows cartilage differentiation-inducing activity when treated with MP-2, BMP-4, and retinoic acid (RA). Alcian blue staining in the control group (untreated) is shown as a relative activity, assuming that it is 1.

 FIG. 5 shows the cartilage differentiation inhibitory activity (alcian blue-stainability) of the C 0 126 extracellular region.

 When the extracellular region of C◦ 126 was added to cultured cells treated with BM P-2, BMP-4 and retinoic acid (RA) using the Alcian Blue monostaining which is a cartilage differentiation marker as an index (C It shows the activity of inhibiting the induction of cartilage differentiation when the N-terminal (+) and the C-126 extracellular region are not added (C-126 N-terminal (1)). The relative activity is shown by setting the Alcian blue staining in the control group (untreated) to 1. BEST MODE FOR CARRYING OUT THE INVENTION

 A method for detecting cancer, cancer metastasis or / and bone disease using the marker of the present invention, a diagnostic kit, a chondrocyte differentiation-inducing activity regulating substance, a method for screening a chondrocyte differentiation-inducing activity regulating substance, gene therapy The expression vector, prophylactic or therapeutic agent, and prophylactic or therapeutic method are described. In the present specification, unless otherwise specified, a gene recombination technique, a recombinant protein production technique, a separation and purification method of an expressed protein, an analysis method, and an immunological technique known in the art are employed.

(1) Acquire C 0 1 2 6

 From human mesenchymal stem cells, normal human cells derived from brain, stomach, heart, skeletal muscle, spleen, liver, small intestine, placenta, lung and kidney, or human cancer cells derived from stomach, according to standard methods c Create a DNA library.

As a method for preparing a cDNA library, Molecular C 1 oning: AL aboratory Manual, S Econd Edition (1989) (Cold Spring Harbor Laboratory) P ress), C urrent P rotocolsin Mo 1 ecu 1 ar Biology (1994) (Green P publishing A ssociates and Wi'ley—Interscience), DNACT oning 1: Core T echniques, AP ractical A pproach, Methods described in S econd Edition (1995) (Oxford University Press), or commercially available kits, for example, Superscript Plasmid Systemforc DNASynthesis and Plasmid Cloning (Invitrogen) And ZAP-c DNAS Synthesis K its (manufactured by Stratagene). Also, Superscript First-strand Synthesis System for RT-PCR was performed using mRNA prepared from human cancer cell-derived strain C0L0205 (ATCC: CCL-222; colon adenocarcinoma-derived strain). CDNA can also be synthesized using a cDNA synthesis kit such as (manufactured by Invitrogen).

 After adding SfiI linkers to both ends of the prepared cDNA, a clone vector, for example, pAMo (TheJournalofBiologyca 1C hemistry, 268 (30), 2 278 2- 2 2787 (1 93 93).). The plasmid is used to transform Escherichia coli strain Bacteria1StRainLE3992, GlycerolStock (promega) to produce a cDNA library. A clone containing the desired DNA is selected from the prepared cDNA library by the following method. Plasmid is prepared from the above-prepared cDNA library by a method using a conventional method or a commercially available kit, for example, QIAGENPasmidaxiKit (manufactured by Qiagen).

A plasmid having a DNA fragment encoding an amino acid sequence having homology with the amino acid sequence of TEM 7 is selected from one of the cDNA libraries prepared above. Such a DNA fragment contains the amino acid sequence in TEM 7, plexin and c-Met. Find two or more regions that are well conserved, design degenerate primers corresponding to the DNA sequence encoding the amino acid sequence of the region, and amplify them by the polymerase chain reaction (PCR) method. Is obtained. The method for preparing the degenerate primers was PCR Primer: AL aboratory anua 1 (19995) (Cold Spring Harbor Laboratory Press), and the protocol series "cDNA cloning" (19996). (Edited by Junichiro Inoue and Kentaro Senba; Yodosha) and Science, 241, 42 (1988). The PCR method is Mo 1 ecu 1 ar Cloning: AL aboratory Manual, S Econd Edition (1 989) (Cold Spring Spring Laboratory Press) and PCR protocol (1 989) (Academic P ress). The DNA fragment thus amplified by the PCR method is inserted into an appropriate plasmid and subcloned. Subcloning can be performed by treating the amplified DNA fragment as it is or with a restriction enzyme or DNA polymerase, and then incorporating it into a plasmid vector by a conventional method. Such vectors include pBluescripts. IISK (10), pBluescript IISK (-), pPCR—ScriptAmp SK (+) (Stratagene), pDIRECT (Nucreic Acids Research, 18, 60 69 (1 990).), PT7Blue (Novagen), pCRII (Invitro Jen), pCR—TRAP (Genehan Yuichi) , P No TAT 7 (Eppendorf 5 prime) and the like.

Screening the base sequence of the amplified PCR fragment to encode an amino acid sequence that is homologous to the known TEM 7 amino acid sequence but does not completely match DNA fragments can be selected. Nucleotide sequences can be obtained by a commonly used nucleotide sequence analysis method, for example, the Sanger et al. Science USA, 74, 5463 (1977).) Or 373 ADNA Sequencer (manufactured by Applied Biosystems). The DNA fragment selected in this manner is used as a probe. A known TEM is obtained by performing hybridization analysis such as colony hybridization and plaque hybridization on the cDNA library prepared above. CDNA encoding a polypeptide having homozygosity with 7 can be obtained. Probes can be used after labeling the DNA fragment radioactive isotopes such as ^{3 2} P, digoxigenin (digo X igenin :), etc. enzymes such as Western Wasabiperu old Kishida over zero. Hybridization is performed by a commonly used method, for example, as described in Mo 1 ecu 1 ar Cloning: AL aboratory Manual, S econd Edition (1 989) (Cold Spring Harbor L aboratory Press). Can be performed in the following manner.

 The cDNA fragment obtained by hybridization is digested as it is or after digestion with an appropriate restriction enzyme, and then inserted into a plasmid vector by a conventional method, and a commonly used nucleotide sequence analysis method, such as the didexy method of Sanger et al. Procceedingsofthe National Academy of Sciences USA, 74, 546 3 (1977).) And base sequence analysis using a base sequence analyzer such as 373 ADNA Sequencer (manufactured by Applied Dubai Systems Inc.). Thus, the desired DNA can be obtained. Examples of the DNA obtained in this manner include a DNA encoding the polypeptide represented by SEQ ID NO: 2, and more specifically, the DNA having the base sequence represented by SEQ ID NO: 1 DNA can be mentioned. Examples of the plasmid containing DNA of SEQ ID NO: 1 include the plasmid described in Examples below.

The DNA obtained as described above is inserted into an expression vector to construct an expression plasmid. After introducing the obtained expression plasmid into appropriate animal cells, the cells are separated into chondrocytes. Using the activation-inducing activity as an index, it can be examined whether or not the DNA has a physiological activity involved in the formation of cartilage in bone tissue.

 As the expression vector, any vector can be used as long as it can incorporate the cDNA and can be expressed in animal cells. For example, pCR2.1TOP0, pcDNA1.1, pcDNA 1.1 / Amp, pCDM8, pREP (manufactured by Invitrogen), pHM6, pHB6 (manufactured by Roche Diagnostics), pKK2 23-3, p GEX (Amersham Pharmacia Biotech), pET-3, pET-11, pBluescriptll SK (10), pBluescriptll SK (-) (Stratagene), pUC19 , PTrxFus, pREP4 (manufactured by Invitrogen), pUC118, pSTV2

8 (manufactured by Takara Shuzo), pMA L—c 2 X (manufactured by New Engla n d B i O L abs), pAGE 107 (Cy t o t e c h n o l o g y, 3 (2), 1 3 3

JP-A-3-22997), pAGE 103 (The Journa 1 of Biochemistry, 101 (5), 1307—1310 (198). 7).) _S p AM op AM o A (T he J ournalof B io 1 ogyca 1 C hemistry, 2 6 8 (30), 2 278 2

2-2787 (1993).), PAMoPRSA (JP-A-5-336963), pAS3-3 (JP-A-2-227075) and the like.

 As a method for introducing the expression vector into a host, any method can be used as long as it is a method for introducing DNA. When the host is an animal cell, the electroporation method (Cytotec hnolology, 3 (2), 13 3-1 40 (1 9

90).), The calcium phosphate method (Japanese Patent Laid-Open No. 2-227075), the lipofection method (ProceedingsoftheNationaAcademyofSciencesUSA, 84, 7413 (1987) .; Vilology, 52). , 456 (1973).).

As the host, an appropriate cell or tissue corresponding to the expression vector can be used, and examples include animal cells. Animal cells used as hosts Nama wa (Burkit's lymphoma, ATGC: CRL-1432) and its sublines Nam wa KJM-1 and HCT-15 (human colon cancer cells, ATCC: CCL-22) 5), monkey-derived cell line C 0 S-1 (African green monkey kidney cells (SV40-transformed cells), ATCC: CRL-1650) and COS-7 (African green monkey kidney cells (SV40-transformed cells) Cells), ATCC: CRL—165 1), CHO—K 1 (Chinese hamster ovary cells, ATCC: CCL—61) and HBT 5637 (Japanese Unexamined Patent Publication No. 9 9), C3H / 10T1 / 2 of mouse-derived cell lines (embryonic cells, ATCC: CC-226), and the like, but preferably C3H / 10T1 / 2. Use cells.

The transformant expressing C0126 is cultured by a conventional method well known in the art. The culture can be performed using a medium suitable for the transformed host, and a liquid medium is suitable as the medium used for the culture. Specifically, MEM medium (Science, 130, 432 (1959)), D-MEM medium (Viroogy, 8, 396 (1959)), RPMI 1640 A medium (The Journal of America Medical Association, 199, 519 (1966).), A YT medium, a BEM medium and the like are used. As a medium for culturing a transformant in which the host is an animal cell, for example, MEM medium, DMEM medium, RPIM medium, or the like to which an appropriate amount of fetal bovine serum (FCS) is added is used. In order to enhance the transcription activity of the promoter of the expression vector, a substance that promotes the transcription activity may be contained. For example, isopropyl-111-thio-1D-galactovyranosin (IPTG) can be used. The medium contains nutrients necessary for the growth of the transformants, such as glucose, amino acids, peptides, vitamins, hormones, serum, preferably FCS, chloride, magnesium chloride, etc. Culture medium having any composition can be used as long as such a medium can be used, and a commercially available medium can also be used. . 0~8. 0, 2 5 ~ 40 ° C, 5% C 0 2 Article of such presence To do.

 The desired DNA can be selected by screening the protein produced by the transformant using the chondrocyte differentiation-inducing activity of C126 as an index. The chondrocyte differentiation-inducing activity refers to an activity of increasing the cell growth rate of chondrocytes and promoting a morphological change of the cells. The activity can be detected by, for example, observing the morphology of chondrocytes with a microscope or measuring a cartilage matrix such as a chondrocyte differentiation marker, for example, aggrecan, collagen, or proteoglycan (such as chondroitin sulfate). Is possible. For example, the cartilage matrix is stained with an Alcian blue stain solution, and the cartilage matrix is extracted from the cells using guanidine hydrochloride, and the amount is measured using a spectrophotometer.

 The cells obtained by the culturing are transformed into the cells into which the expression plasmid has been introduced, by using the DNA as a marker for the nerve cell axon projection extension activity or inducing activity, or the angiogenesis activity. To examine whether it has a bioactivity involved in the formation or angiogenesis in peripheral tissues, cancer cell proliferation or cancer metastasis. If the above physiological activity is detected, it can be considered that the DNA encodes a novel TEM7 involved in the formation of neural circuits in central tissues or angiogenesis in peripheral tissues, cancer cell proliferation or cancer metastasis. it can.

As described above, in human mesenchymal stem cells, brain, heart, skeletal muscle, spleen, liver, small intestine, placenta and lung, or in human gastric cancer cells, formation of neural circuits in central tissues or peripheral tissues Thus, DNA encoding C0126 having a physiological activity involved in angiogenesis, cancer cell growth or cancer metastasis can be obtained. That is, against a cDNA library derived from a non-human animal, for example, a monkey, a lion, a sheep, a goat, a pig, a tiger, a nit, a mouse, a rat, a hamster, a guinea pig, etc. By performing cleaning, the target DNA can be obtained. The target DNA can also be prepared by chemically synthesizing a DNA encoding the polypeptide based on the amino acid sequence of the screened polypeptide having TEM 7 activity. Chemical synthesis of DNA was performed using the thiophosphosilicate method. It can be carried out using a DNA synthesizer manufactured by Shimadzu Corporation, a DA synthesizer mode1392 manufactured by Perkin-Elma using the phosphoramidite method, or the like. In addition, PCR is performed using ligated nucleotides described below as sense primers and antisense primers, and using cDNA prepared from mRNA of cells expressing mRNA complementary to these DNAs as type III. In this case, the target DNA can be prepared.

 At present, sequences of many human chromosomal genes whose functions are unknown are registered in the database. Therefore, C1026 is coded by comparing the sequence of the human cDNA coding C0126 with the sequence of the human chromosome gene registered in the database overnight. It may be possible to identify human chromosome genes and clarify the structure of the genes. If a chromosomal gene sequence that matches the cDNA sequence is registered, the cDNA sequence and the chromosomal gene sequence are compared to determine the promoter region, exon, and exon of the chromosomal gene encoding C0126. It is possible to screen thetron structure.

(2) Production of C 0 1 2 6 polypeptide

 The C0126 polypeptide can be prepared by the method described in Molecular 'Cloning 2nd Edition, Current-Protocols' in Molecular Molecular Biology Supplement 1-38, etc. C0126 polynucleotides can be expressed and produced in host cells.

Based on the full-length DNA encoding the C0126 polypeptide, a DNA fragment of an appropriate length containing a portion encoding the polypeptide is prepared, if necessary. In addition, a DNA is prepared in which the base is substituted so that the nucleotide sequence of the portion encoding the polypeptide is an optimal codon for expression in a host. The DNA is useful for improving the production rate of the polypeptide. The recombinant DNA (recombinant vector) is prepared by inserting the DNA fragment or the full-length DNA downstream of the promoter of an appropriate expression vector. The recombinant vector is transformed into a host suitable for the expression vector. By introducing the transformant into the main cell, a transformant producing C0126 polypeptide can be obtained.

 As the host, any prokaryotic cells, yeast, animal cells, plant cells, insect cells, and the like can be used as long as they can express the gene of interest. As the expression vector, those which can replicate autonomously in the host or can be integrated into the chromosome and which contain the promoter at a position suitable for transcription of the C〇126 gene are used.

(i) When using prokaryotes as hosts

 The expression vector of C 0 126 CDNA is capable of autonomous replication in prokaryotes, as well as a promoter, a ribosomal binding sequence, cDNA encoding C 0 126, and a transcription termination sequence. It is preferable to be composed of A gene for controlling the promoter may be included.

Examples of expression vectors include pcDNAI.1, pcDNA1.1 / Amp, pCDM8, pREP (manufactured by Invitrogen), pHM6, and pHB6 (□ KK 223-3, p GEX (Amersham Pharmacia Biotech), p ET-3, p ET-11, p B luescriptll SK (ten), p B luescriptll SK (-) (Trade Gene), pUC19, pTrxFus (invitrogen), pUC118, pSTV28 (manufactured by Takara Shuzo), pETS ystem (product of Novagen) , PMALC-X (manufactured by New England Bio Labs), pAGE107 (Cytotechnology, 3 (2), 133-140 (1990) .; 299 9), p KYP 200 (A grical tural and Biological Chemistry, stry, 48, 69 (1 984).), P LSA 1 (A grica 1 tural and Biological Chemistry, 53, 277 (1 98 9).), P GEL 1 (ProceedingoftheNational Academyof Scien ces USA, 82, 4306 (19985).), pAGE103 (The Journa 1 of Biochemistry, 101 (5), 1307-1313 (11987). ), P AM o, p AM o A (T he J ournalof B io Ί ogyca 1 C hemistry, 2 68 (30), 2 2 78 2 — 2 2787 (1 993).), P EG 400 (J ournalof B acteriology, 17 2, 2 39 2 (1 990).) _N p T rs 30 (FERBP—5407), p T s 32 (FERM BP-5408), p GHA 2 (FERMBP—400) , P GKA 2 (FERM B-6798), p AMo PRSA (Japanese Patent Laid-Open No. 5-336963), p AS3-3 (Japanese Patent Laid-Open No. 2-227705), pKYP 10 (Japanese Patent No. Akira 5 8- 1 1 0600), p T erm 2 ( JP-A 3 - 2 2 97 9, US 4686 1 9 1 s US 493 9094, US 5 1 607 3 5), p PA 1 ( JP 6 3 Examples of the promoter include any promoter that can be expressed in host cells such as Escherichia coli, for example, the trp promoter (P trp), 1 & 0 Promoter (PI ac), PL promoter Examples include a promoter derived from E. coli phage, such as a PR promoter and a PSE promoter, a SP 01 promoter, a SP 02 promoter, a pen P promoter, etc. A promoter in which two P trps are connected in series (P trpx 2), tac promoter —, lac T7 promoter, and artificially designed and modified promoters such as “let I promoter” can also be used.

 It is preferable to use a plasmid in which the distance between the Shine-Dalgarno (Shine-Dalgarno) sequence, which is a ribosome binding sequence, and the initiation codon is adjusted to an appropriate distance (for example, 6 to 18 bases). Although a transcription termination sequence is not necessarily required for the expression of C0126 polynucleotide, it is preferable to arrange a transcription termination sequence immediately below a structural gene.

The main genus is Escherichia, Serratla, Bacillus lus, Brevibacterium, Corynebacterium A microorganism belonging to the genus, Microbacterium, Pseudomonas, etc., for example, as the genus Escherichia, E.co1i XL 1 — B1tte strain, XL2—Bue strain, DH1 strain, MC1 000 shares, KY327 6 shares, W1485 shares, JM109 shares, HB101 shares, No. 49 shares, W3110 shares, NY49 shares, BL21 (DE3) shares, B and 2 1 (DE 3) p Lys S strains, HMS 174 (DE 3) strains, HMS 174 (DE 3) p Lys S strains, etc., are S. ficaria strains and S. fontico 1a strain, S.1 iquefacien si vermillion, S. marcescens vermillion, etc., as Bacillus 11us genus, B. subtiis strain, B. amy! oliquefaciens strain, and B. revibacterium genus a mmmoniagenes strain, B. imm ariophilum (ATCC: 14068) strain, B, saccharolyticum (ATCC: 14066) strain, etc., as C. orynebacterium genus, C. g1 ut am icum (ATCC: 1). Glutam icum (ATCC: 14067), C. g1 utamicum (ATCC: 13386), C. acetoacidophi 1 um (ATCC: 13870), Mi The genus crobacterium includes M. ammoniaphi 1 um (ATCC: 15354), and the genus Pseudomonas includes P. sp. D-0110 strain.

Any method for introducing the recombinant vector can be used as long as it is a method for introducing DNA into the above host cells. For example, the electroporation method (Nuc 1 eic Acids Research, 16, 61) 27 (1988).) A method using _N- calcium ion (Proceedingoftheationa1AcademyofSciencesUSA, 69, 2110 (1972).), A protoplast method (Japanese Patent Laid-Open No. 63-2483942). ), Gene, 17, 107 (1 982). And Molecular & Genera 1 Genetics, 168, 1 1 1 (1979). . (ii) When yeast is used as a host

When yeast is used as a host, as expression vectors, for example, YEp13 (ATCC: 37115), YEp24 (ATCC: 37051), YCp50 (ATCC: 37419), p HS 19, p HS 15 and the like can be exemplified. Any promoter can be used as long as it can be expressed in yeast. For example, the ADH1 (alcohol dehydrogenase) promoter, PH〇5 (acid phosphatase) promoter, PGK1 (phosphoglycerate) Kinase) promoter, GAPDH (glyceraldehyde 3-phosphate dehydrogenase) promoter, GAL 1 (galactose kinase) promoter, GAL 10 (UDP galactose 4-epimerase) promoter, MF Hi 1 (Hypheromone) promoter, CUP 1 (Meta-Machine Nine) promoter, etc. Examples of the host include S accharomyces, S. cerevisiae, S chizosaccharomyces, S. pom be, K luyveromyces, K. 1 actis, T richosporon, T. pu 1 "lulans, S. As a method for introducing a _c- recombinant vector such as a genus ch wa nniomyces, S. a11 uvius and a genus Pichia or P. pastoris, either a method for introducing DNA into a host or the like may be used. For example, the electroporation method (Methodsin Enzymology, 194, 182 (1992).), The spheroplast method (Proceedingsofthe National Academy of Sciences USA, 84, 192) 9 (1 978)), the lithium acetate method (Journalof B acteriology, 15 3, 16 3 (1983)) and ProceedingsoftheNationa 1 Academy of Sciences USA, 75, 192. 9 (1978).).

(iii) When using animal cells as hosts When an animal cell is used as a host, for example, pcDNA1 / Amp, pcDNA1, pCDM8, pREP4 (manufactured by Invitrogen) as an expression vector. PAGE107 (Cytotechnology, P, AGE 103 (The J ournalof Biochemistry, 101, 1307 (1987).), PAMo, pAMoA (pAMoPRSA) (The Journal of Biologycal C hemistry: 2688, 22782-22-2787 (1993).), PAS3-3 (JP-A-2-222705) and the like can be used.

 Any promoter can be used as long as it can be expressed in the host. For example, the promoter of the IE (Imed 1 ate-ear 1 y) gene of human cytomegalovirus (hCMV), SV Forty early promoters, Moloney Murine 'Longemia Murine Leu 1emia Virus' Long 'Terminal' Repeat-Promoter (Long Terminal Repeat Promoter), Letrowill Promoter, HSP promoter, SR promoter and metallothione promoter. Further, the enhancer of the IE gene of human CMV may be used together with the promoter.

Animal cells used as hosts include human-derived cell lines HEK293 (human embryonic cell, ATCC: CRL-1573), Namal wa (Burkitt lymphoma, ATCC: CRL-1432), HeLa (cervical cancer cells, ATCC: CCL12), HBT5637 (leukemia cells, JP-A-63-299), BAL-shi-1 (leukemia cells) and HCT- 15 (colorectal cancer cells), mouse-derived cell lines Sp2 0-Ag14 (mouse myeloid cells, ATCC: CRL—1581), C3H / 10T1 / 2 ( Mouse fetal cells ATCC: CCL-226) and NS0 (mouse bone marrow cells), monkey-derived cell line C 細胞 S-1 (African green monkey kidney cells (SV40 transformed cells), ATCC: CRL— 1650) and COS-7 (African green monkey kidney cells (SV40-transformed cells), ATCC: CRL-1651), Hamster-derived cell lines CH 0—K 1 (Chinese hamster ovary cells, ATCC: CCL—61) and BHK—21 (C—13) (Sicilian hamus pups kidney cells, ATCC: CCL—10) And rat-derived cell lines PC12 (adrenal pheochromocytoma, ATCC: CRL—1721) and YB2 / 0 (rat myeloid cells, ATCC: CRL—1662). can do.

As a method for introducing a recombinant vector, any method can be used as long as it introduces DNA into a host. Examples thereof include an electroporation method (Cytotechnology, 3, 133, (1990)), The calcium phosphate method (Japanese Patent Application Laid-Open No. 2-2705), the Lipofexion method (Proceedings of National Academy of Sciences, USA, 84, 7413 (1987)), and Vilology, 52, 456 (1973). ) _c

(i) When insect cells are used as a host

When insect cells are used as host, as the transfer one vector, for example, p VL 1 39 2, p VL 1 3 9 3 s p B lue B ac III ( manufactured by Lee Nbi Bok Rozhen Inc.), as an infection for virus Examples thereof include baculovirus (Acuovirus) Autographaca 1 ifornianuclear polyhed rosisvirus (AcMNPV) Bac-N-Blue DNA, which infects the insects of the family Aspergillus. Transformation of insect cells) 'The rest is!) For example, Baculovirus Expression Vector: AL aboratory Manual (1992) (W.H. Freemanand Company), Molecular Cloning: AL aboratory M anual, S econd Edition (1 989) (Cold Spring Harbor Laboratory Press), C urrent P rotocolsin Molecular Biology (1 994) (Wi iey-Interscience) _v BioT The method described in ecnology, 6, 47 (1988). A transfer vector containing the target gene and baculovirus DA for infection of insect cells are added to the insect cell culture solution, and the recombinantly expressed virus expressing the target gene is used to infect the insect cells. It can express peptides.

 Insect cells used as hosts include S podopterafrugiperda (spodoptera) -derived strain cells and T richoplusiani (Erythrophora persica) -derived strain cells. Specifically, S. frugiperda-derived cells include S f 9 (ATCC: CRL—1711, ovarian cells), Sf21 (ovarian cells), and other T. ni-derived cell lines, High Five, BTI—TN—5B1—4 (Egg cells, manufactured by Invitrogen).

 As a method for introducing a recombinant vector, any method can be used as long as it can be introduced into a host. For example, the calcium phosphate method (Japanese Patent Application Laid-Open No. 2-27055), lipofection; r'oceedingsoftheNationalAcacemyofSciencesUSA, 84, 7413 (19987). In addition, as in the case of animal cells, an electro-revolution method (Cytotechnique, 3, 1333 (1990).) Can also be used.

(V) When using plant cells as host cells

When a plant cell or a plant individual is used as a host, known methods (tissue culture, 20 (1994)., Tissue culture, 21 (1995)., Trendsin Biotechnology, 15, 4) 5 (19997).) Can be used to produce polypeptides. Examples of the expression vector include T i plasmid and Tapaco mosaic virus vector. As a promoter used for gene expression, any promoter can be used as long as it can be expressed in plant cells. For example, the 35S promoter of force reflex mosaic virus (CaMV) can be used. Cineactin 1 promotion Also promote Gene expression efficiency can also be increased by inserting a maize alcohol dehydrogenase gene, intron 1, etc., between the gene to be expressed in the evening.

 Examples of the host include plant cells such as potato, tobacco, corn, rice, rape, soybean, tomato, carrot, wheat, barley, rye, Alfalpha, flax and the like.

 As a method for introducing a recombinant vector, any method for introducing DNA into a host can be used. For example, a method using Agrobacterium (Japanese Patent Application Laid-Open No. 59-140885) JP-A-60-70080, WO 94/00977), election-portion method (JP-A-60-251888), particle gun (gene gun) method (Japanese Patent No. 2606856, Patent No. 2517813).

(Vi) Culture method

 If the transformant carrying the recombinant vector incorporating the cDNA encoding C0126 is a cell such as Escherichia coli, yeast, animal cells, or plant cells, ordinary culture suitable for various hosts The polypeptide can be produced by culturing according to the method, producing and accumulating the polypeptide, and recovering the polypeptide from a transformant or a culture solution. When the transformant is an animal individual or a plant individual, it is bred or cultivated according to a normal growth method suitable for various hosts to produce and accumulate the polypeptide, and the polypeptide is produced from the animal individual or plant individual. By collecting, the polypeptide can be produced.

When the host is an animal individual, for example, a non-human transgenic animal having a polynucleotide encoding C0126 is bred, and chondrocyte differentiation encoding the recombinant DA is performed. By producing and accumulating a polypeptide having an inducing activity in the animal and recovering the polypeptide from the animal individual, a polypeptide having a chondrocyte differentiation-inducing activity can be produced. Production and storage in animals Examples thereof include cell membranes of the animal.

 When the host is a plant individual, for example, a transgenic plant having a polynucleotide encoding C0126 is cultivated, and the recombinant DNA encodes a chondrocyte differentiation-inducing activity. By producing and accumulating the polypeptide in the individual plant, and collecting the polypeptide from the individual plant, a polypeptide having an activity of inhibiting chondrocyte differentiation induction can be produced.

 When the host is a prokaryote such as Escherichia coli or a eukaryote such as yeast, for example, a transformant having a polynucleotide encoding C0126 is cultured in a medium, and the recombinant DNA By producing and accumulating a polypeptide having an activity of inhibiting chondrocyte differentiation induction in a culture solution, and recovering the polypeptide from the culture solution, C 0 126 can be produced.

 The above-mentioned method of culturing the transformant in a medium can be performed according to a usual method used for culturing a host.

 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, and the like that can be used by the host. Either a natural medium or a synthetic medium can be used as long as the medium can efficiently culture the transformant. As a medium for culturing a transformant in which the host is Escherichia coli, for example, a YT medium containing paktripton, yeast extract and sodium chloride is preferable.

 The carbon source may be any one that can be assimilated by each host, such as glucose, fructose, sucrose, molasses containing these, carbohydrates such as starch or starch hydrolysate, and organic acids such as acetic acid and propionic acid. Alcohols such as acids, ethanol, and propanol can be used.

Nitrogen sources include ammonia, ammonium chloride, ammonium sulfate, ammonium acetate, ammonium phosphate, and other ammonium salts of inorganic and organic acids, other nitrogen-containing substances, peptone, meat extract, yeast extract, corn steep. Liquor, casein hydrolyzate, soybean meal and soybean meal hydrolyzate, And its digests can be used.

 Examples of the inorganic salts include potassium potassium phosphate, potassium potassium phosphate, magnesium phosphate, magnesium sulfate, sodium chloride, ferrous sulfate, manganese sulfate, copper sulfate, and calcium carbonate. .

 The cultivation method is performed under aerobic conditions such as shaking culture or deep aeration and stirring culture. Culture temperature, culture time, and pH of the culture solution are set in the range suitable for each host, and culture is usually performed at 15 to 40 ° C, 5 hours to 7 days, and pH 3.0 to 9.0. . The pH can be adjusted using an inorganic or organic acid, an alkaline solution, urea, calcium carbonate, ammonia, or the like. If necessary, an antibiotic such as ampicillin tetracycline may be added to the medium. When culturing a microorganism transformed in an expression vector using an inducible promoter as a promoter, an inducer may be added to the medium, if necessary. For example, when culturing a host transformed with an expression vector using the 1 ac promoter, isopropyl-1-^-D-chi-galactovilanoside was transformed with an expression vector using the trp promoter. When the host is cultured, indoleacrylic acid or the like may be added to the medium.

When the transformant is a plant cell or tissue, it can be cultured in large quantities using a jar armmenter. As the culture medium, use commonly used Murashige-and-Square (MS) medium, White medium, or a medium supplemented with plant hormones such as Saichixin or cytokinin. be able to. When the transformant is an animal cell or tissue, a commonly used culture medium is RPMI 1640 medium (The Journal of American Medical Association, 199, 519 (1966)). ), MEM medium (Science, 130, 432 (1959)) and D-MEM medium (Virology, 8, 396 (1959)). 199 medium (ProceedingsoftheS) ocietyforthe Biological Medicine, 73, 1 (1950).) A medium obtained by adding fetal calf serum (FCS) or the like to these mediums is used.

Culture is carried out usually p H 6 ~ 8, 2 5~40 ° C, 5% C 0 1 ~ 7 days in conditions such as ₂ presence. If necessary, antibiotics such as kanamycin, penicillin, and streptomycin may be added to the medium during the culture.

 When the transformant is an insect cell, as a culture medium, commonly used TNM-FH medium (manufactured by Pharmingen), Sf-90OIIS FM medium (manufactured by Invitrogen), Ex Cell 400, Ex Ce 1 1405 (manufactured by JRH Biosciences), Grace's Insect Media (Nature, 1995, 788 (1962).), Etc. Can be.

(vii) Manufacturing method

 C 0 126 can be produced by culturing the transformant and isolating and purifying C 0 126 from the culture solution. Isolation and purification of C 0 126 can be carried out by conventional methods well known in the art. For example, enzyme isolation. Purification methods and purification of glycosyltransferases of S and 1er et al. The method (Methodsin Enzymology, 83, 458) can be used.

 When C 0 126 is produced and accumulated as a soluble polypeptide, the culture solution obtained by culturing the transformant as described above is separated into cells or cells and a medium by, for example, centrifugation. When C0126 is present in the host cells, the collected cells or cells are washed with an appropriate buffer such as STE solution, and then washed with ultrasonic waves, French press, Mantongaulin homogenizer, Dynomill, etc. The cells can be disrupted and obtained as a cell-free solution by centrifugation or filtration.

 The buffer used for the separation and purification of C 0 126 may contain an appropriate amount of a surfactant, such as sodium lauryl sulfate (SDS) or N-lauroyl sarcosine sodium (sarkosyl). May be.

The method of separating and purifying the target protein contained in the obtained crudely purified product can be carried out by a combination of various known separation and purification methods. These known methods include: For example, solvent extraction, salting out with ammonium sulfate, dialysis, precipitation with organic solvents, ultrafiltration, gel filtration, getylaminoethyl (DEAE) —Sepharose chromatography, DIAIONHPA—75 ( Anion chromatography and ion-exchange chromatography using lysine such as Mitsubishi Chemical Corporation, positive ion chromatography using lysine such as S-Sepharose FF (manufactured by Pharmacia), and Petil Sepha Examples include various chromatographic methods, such as hydrophobic chromatography and affinity chromatography, and various electrophoresis methods, such as SDS-polyacrylamide gel electrophoresis and isoelectric focusing. . Affinity chromatography can also be performed by using an antibody against C0126.

 When C 0 126 is produced and accumulated as an insoluble polypeptide, the cells or cells are separated in the same manner as described above, disrupted by an appropriate method, and the fraction containing the polypeptide is recovered. The collected sample is solubilized with a solubilizing agent such as sodium lauryl sulfate (SDS) or a surfactant such as N-lauroyl sarcosine sodium (sarkosyl). The solubilized solution is diluted or dialyzed to a concentration containing no or almost no solubilizing agent to form the polypeptide into a normal three-dimensional structure, followed by the same separation / purification method as described above. Thus, a purified sample can be obtained.

Alternatively, C0126 can be produced as a fusion protein with another protein and purified using affinity chromatography using a substance having affinity for the fused protein (Akio Yamakawa, Experimental Medicine, 13, 469-474 (1995). Examples of additional proteins used in the fusion protein include protein A and FLAG (Proceedings of Nationa 1 Academy of Sciences USA, 86, 8227 (1989)., Genes Development, 4). , 1288 (1990)., JP-A-5-336963, JP-A-6-823021). If protein A is used, a fusion protein of CO126 and protein A can be produced and purified by affinity chromatography using immunoglobulin G. it can. When a FLAG peptide is used, it can be purified by producing a fusion protein of C0126 and FLAG and performing affinity chromatography using an anti-FLAG antibody.

 C 0 126 can be produced by using an in vitro transcription / translation system according to a known method (Journal of Biomolecular NMR, 6, 129-134 (1995)). , Science, 242, 116-1-164 (1988)., The Journal of Biochemistry, 110, 166-168 (1991).

 Based on the amino acid sequence, C 0 126 can be synthesized by a chemical synthesis method such as the Fmoc method (full-year phenylmethyloxycarbonyl method) or the tBoc method (t-butyl-yearly carbonyl method), or commercially available peptide synthesis. Equipment, for example, APEX 396 (manufactured by Advanced Tochemtech), 433 A (manufactured by Applied Dubai Systems), PS3 (manufactured by Protein Technologies), 9050 (manufactured by Perceptive), PSSM-8 (Shimazu) (Manufactured by Seisakusho Co., Ltd.).

 Structural analysis of C 0 126 is carried out by a method commonly used in protein chemistry, for example, the method described in Protein Structural Analysis for Gene Cloning (Hisashi Hirano, Tokyo Chemical Dojin, 1993) Can be implemented. The chondrocyte differentiation-inducing activity of C0126 can be determined by known methods (Cell, 75, 138 (1993)., Journa 1 of Cell Biology. 146, 233 (1 , 999), Cancer Research, 58, 1238 (1998), Neuron, 17, 1157 (1996), Science, 289, 1197 (2000) .) Can be measured. (3) Preparation of antibody recognizing C 0 126

 (i) Preparation of polyclonal antibody

Total length of C 0 126, its partial peptide or polypeptide containing its partial peptide Antibodies can be prepared by administering the peptide as an antigen to a mammal. The antigen may be the antigen itself, or may be a carrier, for example, one bound to a serum albumin (BSA), a keyhole limpethemocyanin (KLH) from sika deer, or a substance bound to bovine thyroglobulin (BTG). Good, In order to enhance the immune response by the antigen, for example, complete adjuvant (CFA) and incomplete adjuvant (IFA) of oral infusion may be administered. As mammals used for immunization, mice, rats, porcupines, goats, hamsters and the like can be used.

 Polyclonal antibodies can be prepared, for example, according to the method of Reyn et al. (Antibodies: AL aboratory Manual, S Econd Edition (19989) (Cold Spring Harbor Laboratory Press)). Can be.

 Immunized mammals are obtained by administering the antigen 3 to 10 times every 1 to 2 weeks after the first administration of the antigen to mammals, and serum is collected from those mammals and purified. it can.

 The administration of the antigen is performed 3 to 10 times every 1 to 2 weeks after the first administration. The dose of the antigen is preferably 50 to 100 μ.9 per animal per administration. When a peptide is used, it is preferable to use the antigen covalently bonded to an appropriate carrier as the antigen. Peptides used as antigens can be synthesized by genetic engineering techniques or peptide synthesizers. Blood is collected from the fundus venous plexus 3 to 7 days after each administration, and the reaction of the serum with the antigen used for immunization is determined by enzyme immunoassay (enzyme immunoassay (ELIS II)): Medical Books (1976), Antibodies: AL aboratory Manual, S Econd Edition (19989) (Cold Spring Harbor L aboratory Press), etc. .

Blood is collected from the immunized mammal and the antibody titer is measured. Blood is collected at the time of immunization until a sufficient antibody titer is obtained, and serum is prepared to obtain a polyclonal antibody. Can be obtained. For separation and purification of polyclonal antibodies, centrifugation. Salting out with ammonium sulfate, caprylic acid precipitation (Antibodies: AL aboratory Manual, Ecological Edition (, 1998 9) (Cold Spring Harbor) This can be done alone or in combination with various chromatographic methods such as Laboatory Press) or DEAE-Sepharose columns, anion exchange columns, protein A columns, G-force columns or gel filtration columns.

(ii) Production of monoclonal antibody

 (a) Preparation of antibody-producing cells

 For the monoclonal antibody, after sufficient antibody titer is obtained in (i), spleen or lymph node is collected from those mammals, and the antibody-producing cells obtained therefrom are transferred to myeloma (myeoma). By fusing with cells, a hybridoma producing a monoclonal antibody can be obtained. As myeloid cells, a cell line established from a mouse or rat is used. The cell fusion can be performed by a known method, for example, according to the method of Koehler and Milsin (Nature, 256, 495-497 (1975).). .

 C 01 26, after administering the partial peptide or the polypeptide containing the partial peptide, and finally administering the antigenic substance to a rat showing a sufficient antibody titer, 3 to 7 The spleen is removed as cells. The spleen is shredded in MEM medium (manufactured by Nissui Pharmaceutical Co., Ltd.), loosened with forceps, and subjected to eccentric separation at 1,200 rpm for 5 minutes to obtain a precipitate fraction. 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 red blood cells, and then washed three times with MEM medium. Splenocytes are used as antibody producing cells.

(b) Preparation of myeloma cells

Cell lines derived from mice or rats are used as myeloma cells. Such cells include, for example, 8-azaguanine-resistant mouse (derived from BALB / c) myeloma cell line P3-X63Ag8-U1 (hereinafter abbreviated as P3-U1) (Current Topics) M icrobiologycal I mm unology 8 1, 1 (1 978). _N E uropian J ournalof I mm uno 1 ogy, 6, 5 11 (1 976).), SP 2 / 0—Ag 14 , SP—2) (Nature, 276, 269 (1978).) P3-X63—Ag8653 strain (hereinafter abbreviated as 653) (Journalof Immunology, 1 23, 1548 (19797).), P3-X63-Ag8 (hereinafter abbreviated as X63) (Nature, 2556, 495 (19755).), Etc. Can be used. These cell lines, 8- Azaguanin medium (normal medium containing 1 5 mu g 1 8- Azaguanin (1. 5 m M glutamine, ⁵ x 1 0- 5 M 2 one mercaptoethanol, l O g / ml RPMI 1640 medium containing gentamicin and 10% FCS (manufactured by CSL)), and subculture in normal medium 3-4 days before cell fusion. Cell fusion is used that way cells prepared with 2 X 1 0 ⁷ or more.

(c) Preparation of Hypri-Dorma

The antibody-producing cells prepared in (a) and the myeloma cells prepared in (b) were mixed with MEM medium or PBS (per liter; 1.83 g of sodium sodium phosphate, 0.21 g of lipase). Wash with 7.65 g NaCl, pH 7.2), and mix so that the number of antibody-producing cells is 5 to 10 times the number of myeloma cells. After centrifugation at 200 rpm for 5 minutes, obtain a sediment fraction. I The resulting precipitated fraction of cell groups Kuhogushi, antibody-producing cells 1 0 ⁸ per to said cell population, polyethylene glycol solution (2 g polyethylene glycol one Roux 1 000 (PEG- 1 000), 2 m 1 M EM medium, 0.7 ml dimethylsulfoxide (DMS0)) was added with stirring at 0.2 to 1 m1 at 37 ° C, and further for 1 to 2 minutes, 1 to 2 ml of MEM medium the total volume _c MEM medium is added several times and prepared to become 50 m Ί for 5 minutes違心min at 900 rpm for After separation, a sediment fraction is obtained. HAT medium precipitation fraction (normal medium 1 0- ⁴ Micromax hypoxanthine, 1.5 containing X 1 0 ⁵ Micromax thymidine and 4 X 1 0- ⁷ Μ Aminoputeri down) 1 00 m 1 was added, slowly Dissolve and suspend.

Dispensed at 1 00 "1 hole minute to each well of Bok play for 96 six culturing the suspension, 3 7 ° C, 5% 00 2 presence in 7 cultured ~ 1 4 days. Enzyme Immunoassay ( Antibodies: AL aboratory Manual, S Econd Edition (1989) (Cold Spring Harbor Laboratory Press)), etc. Select a hybridoma that produces an antibody that specifically reacts with 126.

(4) Method for molecularly detecting the marker of the present invention

 Using the polynucleotide of the marker of the present invention or a ligated nucleotide prepared from the polynucleotide, a gene encoding the marker can be obtained by the Northern hybridization method, the dot plot method or the PCR method. The expression level can be measured at the mRNA level. Specifically, (a) a drug against a normal or disease model non-human mammal such as a mouse, a rat, a rabbit, a sheep, a pig, a pig, a cat, a dog, a monkey, etc. After a certain period of time after giving an anticancer agent or the like, blood or a specific organ, for example, a brain, a stomach, a kidney or the like, or a tissue or a cell isolated from the organ is obtained. The mRNA of the marker of the present invention contained in the obtained cells can be quantified by, for example, extracting mRNA from cells or the like by a usual method and, for example, using a technique such as PCR, which is known per se. The analysis can also be performed by performing Northern blotting by means.

(B) A transformant expressing the marker of the present invention is prepared according to the method described above, and mRNA of the marker contained in the transformant can be quantified and analyzed in the same manner.

Such molecular biological detection methods are used in methods for detecting cancer such as gastric cancer, or bone diseases such as osteoarthritis, rheumatoid arthritis, and osteoporosis. Can be.

(5) Immunological detection method of the marker of the present invention

Examples of the immunological detection method of the marker using an antibody against the marker of the present invention include an ELISA method using a microtiter plate, a fluorescent antibody method, a western plot method, and an immunohistological staining method. Moreover, San de I Tutsis ELISA method using an E 2 types having different peak Bok one flop of monospecific antibodies reactive to該Ma one car in the liquid phase, labeled with a radioisotope such as ^{1 25} I A quantification method such as radioimmunoassay using the marker-1 polypeptide and an antibody recognizing the marker-1 can be mentioned. In addition, various immunological detection methods described in Antibodies: AL aboratory Manual, Science Edition (1989) (Cold Spring Harbor Laboratory) are employed.

 Such an immunological detection method can be used in a method for detecting cancer such as gastric cancer, or a bone disease such as osteoarthritis, rheumatoid arthritis or osteoporosis.

 Such methods include the use of antibodies to detect the presence, absence, or amount of the present agents in a suitable biological sample. Suitable biological samples as used herein include cancer or normal tissue from patients, excised tissue, blood, lymph, urine, extracts from tissues or cells, and the like.

(6) Diagnostic kit

In the present invention, at least C0126 polynucleotide or a part thereof and C0126 as a standard reagent can be used, since C0126 can be the best for cancer, cancer metastasis and bone disease. It contains an antibody recognizing 126 polynucleotides or C 0 126 and a C 0 126 polypeptide or a part thereof as a standard reagent, and the molecular biological detection method according to (4). Or the immunological detection method according to (5). It is a kit for detecting the marker of the present invention based on the method.

 Such a diagnostic kit is useful as a diagnostic kit for cancer, cancer metastasis, or bone disease, and is particularly useful for detecting gastric cancer as a cancer, and osteoarthritis, rheumatoid arthritis, and osteoporosis as a bone disease. Can be used.

 When the stem cells are treated with a cartilage differentiation-inducing factor, the expression of the marker of the present invention is increased. When C0126-expressing cells using the stem cells as a host are treated with the bone differentiation-inducing factors BMP-2 or BMP-4, cartilage Expression of the marker is induced by inducing differentiation into cells, and by treating stem cells with the extracellular region of C0126 at the same time as BMP-2 or BMP-4, differentiation into chondrocytes is suppressed. By examining this, chondrocytes can be used for diagnosis and prediction of prognosis. In addition, it can be used for diagnosis of other bone diseases by examining the relationship between bone-related diseases and expression of the marker.

 In addition, since the expression of the marker of the present invention is increased in cancer cells of gastric cancer patients, by examining the polymorphism of this gene, it can be used for diagnosis of gastric cancer and prediction of prognosis.

 In addition, the polymorphism of this gene and the organ where this gene is expressed (brain, esophagus, stomach, lung, duodenum, ureter, small intestine cancer, colon, rectum, gallbladder thyroid, adrenal gland, bladder, prostate) By examining the association with a disease, it can be used to diagnose other diseases. The polymorphism analysis of the present gene can be performed using the gene sequence information of the present gene. Specifically, genetic polymorphisms can be analyzed using the Southern blot method, the direct sequence method, the PCR method, the DNA chip method, etc. (Clinical examination, 42, 1507—1517 ( 1998), Laboratory test, 42, 155-570 (1998).

() Screening method for modulator of chondrocyte differentiation-inducing activity

When human mesenchymal stem cells are treated with IGF-1 that has chondrocyte differentiation-inducing activity, induction of C0126 mRNA expression is observed, and mouse mesenchymal cells C3H / 10T1 / 2 cells express C0126 and are treated with BMP-2 or BMP-4, which have bone differentiation-inducing activity, because differentiation into chondrocytes is promoted. It is suggested that 126 is related to chondrocyte differentiation induction. Therefore, the CO 126 polypeptide and a part thereof, or the polynucleotide or a part thereof, are useful for screening substances that regulate chondrocyte differentiation-inducing activity. Examples of the chondrocyte differentiation-inducing activity regulating substance include (i) a binding substance that binds to CO 126. (Ii) a binding activity regulating substance that regulates the binding activity between the binding substance and the marker; (iii) C Examples include expression regulators that regulate expression such as transcription of the 126 gene or translation of mRNA. To screen for a chondrocyte differentiation-inducing activity modulator, a cell-stimulating activity mediated by the polypeptide, for example, various collagens and proteoglycans produced when stem cells are differentiated into chondrocytes, for example, aggrecan, chondroitin sulfate, etc. The amount of cartilage matrix produced can be measured using a known method or a commercially available kit.

(1) Screening method for binding substances

The C0126 polypeptide and a part thereof are useful as reagents for searching or screening for a binding substance of the polypeptide, for example, a ligand such as agonist. That is, the present invention provides a method for screening a binding substance to a C0126 polypeptide, which comprises contacting a C0126 polypeptide or a part thereof with a test substance. Test substances include, for example, proteins belonging to endocrine proteins such as semaphorin and HGF, humans and mammals, for example, tissue extracts such as mice, rats, pigs, mice, sheep, monkeys, etc., cells The culture supernatant is used. These test substances are added to CO 126, and fractionated while measuring the binding activity, etc., to finally obtain a single ligand. Specifically, the screening method for the binding substance is to use a C0126 polypeptide or a part thereof, or construct a recombinant polypeptide expression system and bind using the expression system. Screening can be performed by using Atsushi system or binding to CO 126 to measure cell stimulating activity, for example, the amount of cartilage matrix produced such as various collagens and proteoglycans, for example, aggrecan and chondroitin sulfate. it can. The polypeptide used in the method for screening a binding substance may be any one containing a C0126 polypeptide or a part thereof, but is preferably used. Polypeptides expressed in large amounts using animal cells are suitable. As the method for producing such a polypeptide, the above-described expression method is used, and it is preferable to use a polypeptide in which DNA encoding the polypeptide is expressed in mammalian cells or insect cells. Usually, complementary DNA is used as the DNA fragment encoding the target protein, but it is not necessarily limited to this. ₍ For example, a gene fragment or a synthetic DNA may be used. Inspection of the quantity and quality of the compounds can be carried out in a manner known per se, for example, in the case of The Journa 1 of Bological Chemistry, 26 7, 19 55 5 5-19 55 5 9 (19 Therefore, in the method of screening for a binding substance, a protein containing a C0126 polypeptide or a partial peptide thereof may be used as such. A polypeptide purified according to a known method or a partial peptide thereof, or a cell containing the polypeptide or a cell supernatant thereof may be used. 1 2 6 polypep When a cell containing a tide is used, the cell may be immobilized with glutaraldehyde, formalin, etc. The immobilization method can be carried out according to a method known per se. Examples of such cells include host cells that express the C0126 polypeptide, and Escherichia coli, Bacillus subtilis, yeast, insect cells, animal cells, and the like are used as the host cells. The fraction refers to a fraction contained in the cell culture supernatant.

In order to screen for a binding substance to C 0 126 polypeptide or its salt, an appropriate polypeptide fraction and a labeled test substance are required. The polypeptide fraction is preferably a natural polypeptide fraction or a recombinant polypeptide fraction having an activity equivalent thereto. Here, “equivalent activity” refers to the same ligand binding activity, signal transduction action and the like. Is a labeled test substance, labeled with a ^{3 H, 1 2 5 I,} 1 4 C, 3 5 S, for example, Semafori down, H Proteins belonging to GF can be mentioned.

Specifically, in order to carry out the screening method for the binding substance to the C0126 polypeptide or a salt thereof, first, the cell culture supernatant containing the C0126 polypeptide is subjected to the screening method. Prepare a polypeptide preparation by suspending in a buffer appropriate for the procedure. Buffers include ligands such as phosphate buffer, pH 4.0-10.0, preferably pH 6,0-8.0, and Tris-HC1 buffer and polypeptide. Any buffer may be used as long as it does not inhibit the binding to the enzyme. Also, in order to reduce non-specific binding, surfactants such as CHAPS, Tween-80 (Kao-Atlas), Triton X-100 digitonin, and dexylcoxolate, and serum albumin各種 Various proteins such as gelatin can be added to the buffer. Furthermore, protease inhibitors such as PMSF, leptin, E-64 (manufactured by Peptide Research Laboratories), and pepstatin can be added for the purpose of suppressing the degradation of receptors and binding substances by proteases. 0.0 to 1 ml ~1 0 m "l該Po Li peptidase de solution, fixed amount, preferably, 500 O cpm ~ 5 O 00 O 0 cpm of ^{3 H, 1 25 I, 1} 4 C, 35 Coexist with a test substance labeled with S. Prepare a reaction tube containing a large excess of unlabeled test substance to determine the non-specific binding amount (NSB). The reaction is carried out at 50 ° C., preferably 4 ° C. to 37 ° C., for 20 minutes to 24 hours, preferably 30 minutes to 3 hours.After the reaction, the reaction solution is filtered through a glass fiber filter and washed with an appropriate amount of the same buffer. The radioactivity remaining on the glass fiber filter paper is measured with a liquid scintillation counter or a counter.The count (B-NSB) obtained by subtracting the nonspecific binding amount (NSB) from the total binding amount (B) is Test substances above O cpm can be selected as binding substances for C 0 126 polypeptides or salts thereof.

In order to screen the binding substance to the C 0 126 polypeptide, cell stimulating activity via the CO 126 polypeptide, for example, various collagen glycans, proteoglycin such as chondroitin sulfate, etc. Can be measured using a known method or a commercially available measurement kit. Concrete Specifically, first, cells containing the polypeptide are cultured in a multiwell plate or the like. Before screening for the binding substance, replace the medium with a fresh medium or an appropriate buffer that is not toxic to the cells, add the test substance, etc., incubate for a certain period of time, and then remove the cells. Extract or collect the supernatant, and quantify the product produced according to each method. If the production of a substance that serves as an indicator of cell stimulating activity, such as collagen, aggrecan, or protein-specific glycans such as chondroitin sulfate, is difficult to assay with a cell-containing degrading enzyme, an inhibitor for the degrading enzyme is added Atsushi may be performed. (ii) Screening method for binding activity modulator

C 0 1 2 6 Poribe peptide or a portion thereof, C 0 1 2 6 Poripe _c CO 1 2 6 polypeptide is useful as a reagent for searching or disk re-learning the binding activity regulatory substances to Petit de As a method for screening a substance that modulates the binding activity between a substance and a binding substance, that is, a substance that changes the binding property, a C0126 polypeptide or a recombinant expression system that expresses the polypeptide is constructed. Substances that change the binding between the binding substance and the C0126 polypeptide by using bioassay or conjugated atsey systems, such as peptides, proteins, non-peptidic substances, synthetic substances, and fermentation production Objects can be screened efficiently. Such substances include cell stimulatory activity through the binding of the binding substance to the CO126 polypeptide, e.g., enhance or reduce the production of cartilage matrix such as various collagens, aggrecan, and proteoglycans such as chondroitin sulfate. Substances, substances that enhance or decrease the binding strength between the binding substance and the CO 126 polypeptide. That is, (a) when a binding agent is brought into contact with (a) a C0126 polypeptide, a part thereof or a salt thereof, and (b) a C0126 polypeptide, a part thereof or a salt thereof Comparing the binding amount of the binding substance with the binding substance and the test substance when the binding substance and the test substance are brought into contact with each other, and the C 0 126 polypeptide, a part thereof, or a salt thereof. Provided is a method for screening a binding activity modulator. The screening method for a binding activity regulating substance is characterized in that in (a) and (b), the amount of binding between the polypeptide and the binding substance is compared by measuring, for example, cell stimulating activity. And

 As a method for screening the binding activity-regulating substance of the present invention, specifically, (a) when a labeled binding substance is brought into contact with CO 126 polypeptide or the like, and a labeled binding substance and a test substance are used. The amount of binding of the labeled binding substance to the polypeptide or the like when contacted is measured and compared, and the binding property between the binding substance and the C 0 126 polypeptide or the like is changed. (B) when the labeled binding substance is brought into contact with cells or cell culture containing the C0126 polypeptide and the like, and the labeled binding substance and the test substance are The amount of binding of the labeled binding substance to the polypeptide or the like in the cell or cell culture when contacted is measured and compared with a binding substance and a C 0 126 polypeptide or the like. Result (C) Labeling with a polypeptide secreted into cell culture by culturing a transformant containing C126-polynucleotide, etc. The amount of binding of the labeled binding substance to the polypeptide and the like when the binding substance is brought into contact and when the labeled binding substance and the test substance are brought into contact with each other are measured and compared with the binding substance. Examples of the method include a method of screening for a binding activity-regulating substance that changes the binding property to C0126 polypeptide and the like.

(iiii) Screening method for expression regulator

The screening method for the expression regulator of C 0 126 polypeptide or a partial peptide thereof may be G 0 126 polypeptide or a part thereof, or C 0 126 polypeptide or a part thereof. The antibody can be used for screening C0126 polypeptide or a part of its expression regulator. For example, (a) blood of a non-human mammal, a specific organ, a tissue or cell isolated from an organ, or (b) C0126 polypeptide contained in a transformant or the like. Alternatively, by measuring the mRNA or protein content of a part thereof, screening of the CO 126 polypeptide or a part thereof can be carried out. (8) Screening kit for modulator of chondrocyte differentiation-inducing activity

 Examples of the screening kit for a chondrocyte differentiation-inducing activity regulating substance include: (i) a screening kit for a substance that binds to C0126 polypeptide or a salt thereof, or a salt thereof, (ii) A screening kit for a substance or a salt thereof that changes the binding property between a binding substance and a C0126 polypeptide or the like; (iii) a substance for regulating the expression of a C0126 polypeptide or a salt thereof. An example is a kit for screening.

 (i) Screening kit for binding substances

 The binding substance screening kit that binds to C 0 126 or its salt includes CO 126 polypeptide or its salt, a part of C 0 126 polypeptide or its salt, It contains cells containing a C0126 polypeptide or a cell supernatant fraction containing a C0126 polypeptide. Examples of binding substance screening kits include the following.

 (A) Reagent for binding substance screening

 (a) Screening and cleaning solutions

 A mixture of Hank's balanced salt solution (manufactured by Invitrogen) and 0.05% of serum serum albumin (manufactured by Sigma-Aldrich) was sterilized by filtration through a 0.45 μm pore size filter. Stored at 4 ° C or prepared at the time of use.

(b) Add C 3 H / 10 T 1/2 cells expressing the C 0 126 polypeptide to a 12-well plate at ⁵ × 10 ⁵ cells / well, and at 37 ° C for 1 day Cultured.

 (c) Labeled test substance

Commercially available ^{3 H, 1 25 I, 1} 4 C, 3 5 have in 4 ° C solution obtained by the test substance labeled with a S is stored at one 20 ° C, diluted with the measurement buffer 1 Then prepare it before use. For test substances that are poorly soluble in water, dissolve in dimethylformamide, DMSO, methanol, etc.

 (d) Unlabeled test substance

The same substance as the labeling substance was prepared at a concentration of 100 to 1000 times.

 (B) Measurement method

 (a) After washing C0126 cell-expressed C3H / 10T1 / 2 cells cultured in a 12-well tissue culture plate twice with 1 ml of measurement buffer, 4901 measurement buffer is added to each well.

 (b) Add 5 標識 of labeled test substance and react at room temperature for 1 hour. To measure the amount of non-specific binding, add 5 μl of unlabeled test substance.

 (c) Remove the reaction solution and wash 3 times with 1 ml of washing solution. The labeled test substance bound to the cells is dissolved in 0.2 M Na0H (containing 1% SDS), and mixed with 4 ml of liquid scintillator A (manufactured by Wako Pure Chemical Industries).

 (d) The radioactivity is measured using a liquid scintillation counter (Beckman Coulter, Inc.).

(ii) Screening kit for binding activity modulator

 A screening kit for a binding activity-regulating substance that changes the binding property between a binding substance and C 0 126 polypeptide, etc., contains C 0 126 polypeptide and C 0 126 polypeptide. And cells containing a cell or a cell culture solution containing the C0126 polypeptide. The following are examples of such a screening kit.

 (A) Screening reagent for binding activity modulator

 (a) Screening and cleaning solutions

A mixture of Hank's balanced salt solution (manufactured by Invitrogen) and 0.05% of serum serum albumin (manufactured by Sigma-Aldrich) was sterilized by filtration through a 0.45 m filter. What was stored at 4 ° C or prepared at the time of use. (b) C 0 1 2 6 polypeptide

The C 0 1 26 polypeptide C 3 H / 1 0 T 1/2 cells expressing, was added 1 2 Anapu rate at 5 X 1 0 ⁵ cells / six, 37 ° C, in 1 day culture What you did.

 (c) Labeled test substance

Commercially available ^{3 H, 1 25 I 1 4} C, 35 have with the solution 4 ° C and was test substance labeled with a S are - stored at 20 ° C, diluted to 1 Myumyu with the assay buffer _{(When the} test substance is hardly soluble in water, dissolve in dimethylformamide, DMS〇, methanol, etc.)

 (d) Unlabeled test substance

 The same substance as the labeling substance was prepared at a concentration of 100 to 1,000 times.

 (B) Measurement method

 (a) After washing C0126 polypeptide-expressing C3H / 10T1 / 2 cells cultured in a 12-well tissue culture plate twice with 1 ml of a screening solution, Add 49 0 μl of measurement buffer to each well.

 (b) Add 51 to the labeled test substance and react at room temperature for 1 hour. To determine the amount of non-specific binding, add 5 I 1 of unlabeled test substance.

 (c) Remove the reaction solution and wash 3 times with 1 ml of washing solution. The labeled test substance bound to the cells is dissolved in 0.2 M Na0H (containing 1% SDS) and mixed with 4 ml of liquid scintillator A (Wako Pure Chemical Industries, Ltd.).

 (d) Measure the radioactivity using a liquid scintillation counter (Beckman Coal, Yuichi).

(ii) i) Screening kit for expression regulator

The screening kit for the C0126 polypeptide expression regulator or a salt thereof is a cell culture solution containing cells expressing the C0126 polypeptide or cells expressing the C0126 polypeptide. And the like. The following are examples of such screening kits. (a) Screening kit based on immunoassay

The expression amount of C0126 polypeptide can be quantified by an immunological technique using an antibody against C0126 or a part thereof. As a method for the quantification of C0126 polypeptide, a sandwich ELISA method using two types of monoclonal antibodies that are different from C0126 polypeptide in the liquid phase and have different epitopes is used. ., such as ^{1 26} C labeled with a radioisotope such as I 0 1 2 6 Poribe peptide radio I Takeno assay I method using an antibody that specifically recognizes it is exemplified. Therefore, in the case of diagnosis using an antibody, a C0126 polypeptide is included as a standard antigen in addition to an antibody against C0126 or a part thereof. Further, the kit may include a standard curve.

 (A) Screening reagent

 (a) Screening and cleaning solutions

 Hank balanced salt solution (manufactured by Invitrodin) supplemented with 0.05% of serum serum albumin (manufactured by Sigma-Aldrich), and sterilized by filtration through a 0.45 m filter. Stored at 4 ° C or prepared at the time of use.

 (b) Cells expressing C 0 126 polypeptide

C 0 1 2 6 polypeptide and the expressed 〇 31 "1/1 0 T 1/2 cells, dispensed in 1 2 Anapu rate Bok to 5 X 1 0 ⁵ cells / well, at 37 ° C for Cultured for 1 day, (c) Test substance

 Store the test substance in an aqueous solution at 4 ° C or 120 ° C and dilute with a screening solution before use. Test compounds that are poorly soluble in water should be dissolved in dimethylformamide, DMS, or methanol.

 (d) Labeled antibody

Commercially available ^{3 H, 1 25 I, 1} 4 C, 3 5 C 01 labeled with a S 2 6 polypeptide or antibodies to a portion thereof.

(e) Reference material C0126 Polypeptide prepared at various concentrations.

 (B) Measurement method

 (a) Wash C2H / 10T1 expression cells expressing C0126 polypeptide cultured in a 12-well tissue culture plate twice with 1 ml of a screening solution. After that, add the same solution of 490, to each 6.

(b) Add 10 μl of the test substance solution of 10-3-1-10 _ ¹ QM, and react at room temperature for 1 hour.

 (c) After adding the labeled antibody solution, perform a shaking reaction at 37 ° C for 2 hours.

 (d) After separation by eccentricity, the radioactivity of the residue is measured using an α-counter (manufactured by Beckman) to determine PMB.

(b) Screening kit based on molecular biological quantification method

Expression amount of DNA encoding C126 polypeptide by Northern hybridization method or PCR method using CO126 polynucleotide or oligonucleotide prepared from the polynucleotide. Can be quantified at the mRNA level. Specifically, (i) a drug against a normal or disease model non-human mammal, for example, a mouse, a rat, a rabbit, a sheep, a pig, a pig, a cat, a cat, a dog, a sal, etc. After a certain period of time after administration of an anticancer agent or the like, blood or a specific organ, such as the brain, stomach or kidney, or a tissue or cell isolated from the organ is obtained. The mRNA of the C0126 polypeptide or its partial peptide contained in the obtained cells can be obtained, for example, by extracting mRNA by an extraction method well-known in the art, and using, for example, a technique such as PCR. It can be quantified or analyzed by a known Northern blot method, or (ii) a transformant which expresses the C0126 polypeptide or its partial peptide can be subjected to the method described above. The thus prepared transformant can be quantified and analyzed for mRNA of the C126 polypeptide or its partial peptide in the same manner. In the case of diagnosis using polynucleotides, labeled C 0 126 polynucleotides are included in the kit. Includes the year.

 (A) Screening reagent

 (a) Cells expressing C 0 126 polypeptide

C_〇 the C 3 H / 1 0 T 1/2 cells expressing the 1 26 polypeptide, dispensed 1 2 Rokupu rate at 5 X 1 0 ⁵ cells / six min, 1 day incubation at 37 ° C for What you did.

 (b) Test substance

 Store the test substance in an aqueous solution at 4 ° C or -20 ° C, and dilute with a screening solution before use. Test compounds that are poorly soluble in water should be dissolved in dimethylformamide, DMS, or methanol.

 (c) labeled polynucleotide

C 0 126 labeled with ³ H, ¹²⁵ I, ¹⁴ C, ³⁵ S, etc., which are commercially available.

 (B) Measurement method

(A) 1 C 0 1 cultured played at Bok for 2-well tissue culture 2 6 polypeptide de expression C 3 H / 1 0 T 1 /2 test substance ^{1 0- 3 ~ 1 0- 1 0} Μ into cells After adding 5 μl of the solution, incubate at 37 ° C for 1 hour.

 (b) Extract all RNA with TRIzo1 reagent (manufactured by Invitrogen).

 (c) Transfer 10 g of RNA to the membrane after electrophoresis.

 (d) After immersing the transcribed nylon membrane in the labeled polynucleotide solution, perform a hybridization reaction at 65 ° C for 16 hours.

 (d) The nylon membrane after hybridization is exposed to an X-ray film, and the detected band is quantified.

(9) Pharmaceutical composition

The substance obtained by using the screening method or the screening kit of the present invention or a salt thereof includes a binding substance, a binding activity regulating substance, an expression regulating substance, and a C 0 126 polypeptide. Or a part thereof, a C0126 polynucleotide or a part thereof, and an antibody against the C0126 polypeptide or a part thereof, specifically, (a) a substance that enhances or reduces the amount of cell stimulating activity through binding of a binding substance to a C 0 126 polypeptide, for example, various collagens, proteoglycans such as aglycan and chondroitin sulfate, and (b) A substance that enhances or reduces the binding activity between a binding substance and a C0126 polypeptide, or (c) a substance that enhances or reduces the expression level of a C0126 polypeptide. Examples of the substance include low molecular weight compounds, peptides, proteins, non-peptidic substances, synthetic substances, and fermentation products. These substances may be novel substances or known substances. It can be either natural or non-natural. An agonist against a C0126 polypeptide or the like has the same action as the physiological activity of a binding substance against a C0126 polypeptide or the like. It is useful as a low toxic drug. Antagonists against C 0 126 polypeptides and the like can suppress the physiological activity of a binding substance to C 0 126 polypeptides and the like, and thus are safe and low toxic to suppress the activity of the binding substance. It is useful as a new medicine. A substance that enhances the binding strength between a binding substance and a C0126 polypeptide is useful as a safe and low-toxicity drug for enhancing the biological activity of the binding substance to C0126 polypeptide and the like. is there. Substances that decrease the binding force between the binding substance and the C0126 polypeptide are useful as safe and low-toxic drugs for reducing the biological activity of the binding substance to the C126 polypeptide and the like. is there.

Also included is an expression vector for gene therapy that expresses C 0 126. Since C 0 126 can be a marker of cancer, cancer metastasis, and bone disease, it is suggested that it is related to the disease, and diseases such as gastric cancer, osteoarthritis, rheumatoid arthritis, and osteoporosis It is useful for gene therapy for An expression vector for gene therapy is an expression vector that incorporates a part or all of a C0126 polynucleotide, and is introduced into cells or tissues to induce any disease, regardless of congenital or acquired disease. Is to normalize the abnormalities that cause it at the gene level. It is a vector that can supplement cells with normal genes and repair and correct gene defects. (10) Prophylactic or therapeutic agent

 A pharmaceutical composition containing an antibody, a binding substance, a binding regulator, an expression regulator or a gene therapy vector against CO 126 or a part thereof can be administered alone as a therapeutic agent. However, it is usually desirable to mix it with one or more pharmacologically acceptable carriers and provide it as a pharmaceutical preparation produced by any method well known in the field of pharmaceutics.

Dosage forms of the pharmaceutical composition, sprays, capsules, tablets, granules, syrups, emulsions, suppositories, injections, ointments, tapes, formulations suitable for _c oral administration such as ribosome preparations and the like Examples include emulsions, syrups, capsules, tablets, powders, and granules. For example, liquid preparations such as emulsions and syrups include water, sugars such as sucrose, sorbitol, fructose, glycols such as polyethylene glycol, propylene glycol, oils such as sesame oil, rapeseed oil, soybean oil, p — Can be manufactured using preservatives such as hydroxybenzoic acid esters, flavors such as strawberry flavor and ぺ permint as additives. Capsules, tablets, powders, granules, etc. include excipients such as lactose, glucose, sucrose, mannitol, disintegrants such as starch, sodium alginate, lubricants such as magnesium stearate, talc, etc. Binders such as polyvinyl alcohol, hydroxypropyl cellulose, gelatin, surfactants such as fatty acid esters, and plasticizers such as glycerin can be used as additives.

Formulations suitable for parenteral administration include injections, suppositories, sprays and the like. For example, an injection is prepared using a carrier comprising a salt solution, a glucose solution, or a mixture of both. Suppositories are prepared using carriers such as cocoa butter, hydrogenated fats or carboxylic acids. A spray is prepared using the substance itself or a carrier that does not irritate the oral and respiratory tract mucosa of the recipient and disperses the substance as fine particles to facilitate absorption. Specific examples of the carrier include lactose and glycerin. Formulations such as aerosols and dry powders are possible depending on the nature of the substance and the carrier used. In addition, in these parenteral preparations, oral preparations The components exemplified as additives can also be added.

 Expression vectors for gene therapy are classified into viral vector type and non-viral vector type.Viral vector type has good gene expression efficiency, and non-viral vector type has advantages such as low toxicity and low immunogenicity. It is excellent in large-scale preparation. In addition, there is a combination of both advantages, such as a virus / positively charged polymer / DNA complex, HVJ liposome and the like. Preparations for gene therapy differ depending on the virus vector type and non-viral vector type.

 A virus vector-type preparation is prepared by using a virus lacking replication ability as a virus vector and replacing a part or all of the virus polynucleotide sequence with a therapeutic gene as a gene therapy vector. Examples of such viruses include retrovirus, adenovirus, and adeno-associated virus (AAV). Viral vectors are widely used with efficient gene transfer.

 Examples of the non-viral vector type 1 preparation include a positively charged DDS agent prepared by using a carrier such as positively charged ribosomes for DNA alone. Positively charged ribosomes include quaternary ammonium surfactants, positively charged derivatives such as cholesterol and diacylglycerol, various positively charged fats such as lipid derivatives of polyamines, polyvinylpyrrolidone (PVP), polylysine, Examples include positively charged polymers such as polyethyleneimine (PEI).

In addition, as a preventive and therapeutic agent for various diseases containing a substance that alters the expression level of C0126 polypeptide, C0126 polypeptide is used as described above, for example, in vivo such as cartilage differentiation. Given that they may play some important role, expression modulators that alter the expression level of the C0126 polypeptide or its partial peptides may be associated with C0126 polypeptide dysfunction. It can be used as a prophylactic and / or therapeutic agent for diseases that occur. When the substance is used as a preventive and / or therapeutic agent for a disease associated with impaired function of C0126 polypeptide, it can be formulated according to a conventional method. For example, the substance is orally administered as tablets, capsules, elixirs, microcapsules, etc., which are sugar-coated as necessary. Alternatively, it can be used parenterally in the form of a sterile solution with water or another pharmaceutically acceptable liquid, or an injection such as a suspension. For example, the substance can be mixed with known physiologically acceptable carriers, flavoring agents, excipients, vehicles, preservatives, stabilizers, binders, etc. in the unit dosage form generally required for the practice of pharmaceutical preparations. It can be manufactured by doing. The amount of active ingredient in these preparations is such that an appropriate dose in the specified range can be obtained.

 Examples of additives that can be mixed with tablets and capsules include binders such as gelatin, corn starch, tragacanth, gum arabic, excipients such as crystalline cellulose, cone starch, and gelatin. , Bulking agents such as alginic acid, lubricating agents such as magnesium stearate, sweeteners such as sucrose, lactose or saccharine, flavoring agents such as peppermint, coconut oil or cherry. Used. When the unit dosage form is a capsule, the above type of material can further contain a liquid carrier such as an oil or fat. Sterile compositions for injection can be formulated according to standard pharmaceutical practice, such as dissolving or suspending the active substance in vehicles such as water for injection, and naturally occurring vegetable oils such as sesame oil and coconut oil. it can. Examples of aqueous liquids for injection include physiological saline, isotonic solutions such as D-sorbitol, D-mannitol, and sodium chloride containing glucose and adjuvants. Suitable solubilizing agents, for example, It may be used in combination with alcohols such as ethanol, propylene glycol and polyethylene glycol, and nonionic surfactants such as polysorbate 80 and HCO-50. As the oily liquid, for example, sesame oil, soybean oil and the like are used, and may be used in combination with a solubilizing agent such as benzyl benzoate or benzyl alcohol.

Examples of the prophylactic / therapeutic agents include buffer solutions such as phosphate buffer and sodium acetate buffer, soothing agents such as benzalkonium chloride and proforce hydrochloride, human serum albumin, and polyethylene glycol. And other preservatives such as benzyl alcohol and phenol, and antioxidants. The prepared injection solution is usually filled in a suitable ampoule. (11) Prevention or treatment method

 Since the above-mentioned pharmaceutical composition can be a preventive or therapeutic agent for cancer, cancer metastasis, and bone disease, cancer, cancer metastasis, bone disease, especially A method for preventing or treating a disease such as gastric cancer, osteoarthritis, rheumatoid arthritis, or osteoporosis is provided. As such a method, it is desirable to use the above-mentioned pharmaceutical composition in the most effective manner for prevention or treatment, and it is preferable to use it by oral administration or by buccal, respiratory, rectal, subcutaneous, or intramuscular administration. And parenteral administration such as intravenous.

 Because the prophylactic or therapeutic agents prepared above are safe and low toxic, they can be used, for example, in mammals such as humans, mice, rabbits, sheep, pigs, rabbits, cats, dogs, and monkeys. Can be administered. The single dose of a prophylactic or therapeutic agent varies depending on the subject of administration, target organ, symptoms, administration method, and the like.In the case of oral administration, for example, in a hypertensive patient weighing 60 kg, the daily dose is generally It is about 0.1 to 100 mg, preferably about 1.0 to 50 mg, more preferably about 1.0 to 20 mg, and is administered parenterally. In general, about 0.01 to 30 mg / day, preferably about 0.1 to 20 mg / day, more preferably about 0.1 to 10 mg / day of a patient with hypertension is administered by intravenous injection. Give. In the case of other animals, the dose can be administered in terms of the weight per kg of body weight. The dose or frequency of administration varies depending on the desired therapeutic effect, administration method, treatment period, age, body weight, etc., but is usually 1 Og to 8 mg / kg per adult. Example

Examples will be described below. Unless otherwise specified, genetic manipulation techniques are described in Molecu 1 ar C 1 oning: AL aboratory Manual, S econd Edition (1 998 9) (Cold Spring Harbour Laboratory Press). The method was used. Example 1

From C 0 1 2 6 a code sul c DNA black-learning human Bok mesenchymal stem cells (B i oW hittaker Co.) 5 X 1 0 ⁷ carbon atoms, using a TRL zo 1 reagent (manufactured by Lee Nbi Bok Rozhen Co.) Then, the attached manual (accordingly, the total RNA was extracted to obtain 500 ^ g of total RNA. The obtained total RNA was used as a QUICK P “ep MicroRNA RNA Purification Kit” (Amersham Pharmacia Biotech Co., Ltd.). Was prepared according to the attached manual to prepare a po y (A) + RNA fraction.

A cDNA fragment for cloning was prepared from the poly (A) + RNA using Superscript Choice System (manufactured by Invitrogen). The resulting poly (A) + RNA 3;. G a 01 igo (d T) _{1 2} was synthesized Firth toss Bok run de c DNA using an _{1 8} p "imer and S uperscript IIRT, E co 1 Second strand cDNA was synthesized using iDNA polymerase I and RNase H. The resulting cDNA was treated with T4 DNA Polymerase to generate blunt-ended cDNA, followed by EcoRI. (Notl) A dapter was added to both 5 'and 3' ends using T4 DA ligase The cDNA to which the adapter was added was phosphorylated with T4 po 1 ynucleotidekinase at the 5, A cDNA fragment of about 500 bp or more was recovered using DNA szefraction columns, and the vector ρ Ί asmidp S port 1 (manufactured by Invitro Jardin) was treated with restriction enzyme Eco RI (manufactured by Takara Shuzo), and T 4 po 1 After phosphorylation of the 5 'end using ynucleotidekinase, the cDNA fragment obtained using T4 DNALigase was incorporated into the obtained recombinant plasmid. After precipitation of DNA with ethanol, it was dissolved in 20 μ 緩衝 of TE buffer (10 mM MTris-HCl, pH 8.0, 1 mM EDTA), and the pores were removed from the Elect. esearch, 1 6, 6 1 27 (1988).), And introduced into ElectroMA XDH10B (manufactured by Invitrogen) to prepare a transformant.

 The transformant is cultured at 37 ° C for 1 hour in a medium of S.C.C. medium (manufactured by Invitrodin) at 37 ° C, spread on an LB agar medium containing 1 OO zg / ml of ampicillin, and plated at 37 ° C. Cultured overnight at C.

 As a result, about 1,000,000 (95% insertion rate) transformants showing ampicillin resistance were obtained as a cDNA library.

 The obtained transformant is spread on another LB agar medium, cultured overnight at 37 ° C, and attached using QIA prep Spin Miniprep Kit (Qiagen) from about 100 cells. The plasmid was prepared in accordance with the manual described in 1). The cDNA cloned from the cDNA library was used as a Mega BACE 500 DNA Analysis using the DYE namic ETD ye Terminator Kit (Mega BACE) (manufactured by Amersham Pharmacia Bay Saitec) according to the attached manual. The entire nucleotide sequence of the cDNA fragment was determined using System (Amersham Pharmacy Abayishiteku Inc.). The optimal alignment for comparing amino acid sequences with TEM 7 was the Lipman-Pearson method (Science, 2) using GENETYX (manufactured by Software), a genetic information processing software. 27, 1455-1441 (1 985).).

As a result, a clone having a base sequence (SEQ ID NO: 1) encoding an amino acid sequence having a homology with a known TEM amino acid sequence but not completely matching was obtained. The plasmid containing the cDNA fragment was digested with a restriction enzyme EcoRI (manufactured by Takara Shuzo), and a cDNA fragment was obtained using QIA quick Gel Extraction Kit (manufactured by Qiagen) according to the attached manual. According to 50 ng the c DNA fragment R ediprimer IIDNAL abelling S ystem (Amersham off Alma shear Biotech) was used attached ^{manual, [- 32 P] d C} Τ Ρ (6000 C i / mm ο Ί, 20 m C i / m 1) (NE It was labeled with in the ^{3 2} P N Co., Ltd.). ^{3 2} P-labeled c DNA fragments were colony hybridization analysis using as a probe.

 A human brain cDNA library (HumanBranin) (Clontech)

The cells were applied to 50 LB plates at about 1 × 10 ⁴ colonies per plate, and cultured at 37 ° C. overnight to form colonies. The thus obtained cone was transferred to a nylon membrane for DNA blocking Hybond-N (manufactured by Amersham Pharmacia Biotech), and then dissolved (10% SDS) and denatured solution (0.1%). 5 NN a〇H, 1.5 MN a C), neutralization solution (0.5 MT ris

-HClspH 7.0 (including 1.5 MNaCl)) and 2XSSC in order and air-dried. The transferred DNA was immobilized on the nickel film by irradiating the film with ultraviolet light.

The labeled probe prepared above was added to Express Hyb Hybridization Solution (manufactured by Clontech), the DNA-immobilized nylon membrane was immersed in the solution, and hybridization was performed at 65 ° C for 16 hours. _c Haipuridaize one cane after down Nai Kuchinmaku was subjected to 2 x SSC (20 XSSC: 3 MN a C l, 0. 3 M Kuen acid sodium, p H 7. 0) 5 minutes at room temperature, 1 After washing with XSSC at 50 ° C for 30 minutes and with 0.5 XSSC at room temperature for 5 minutes, it was air-dried. Next, this nylon film was attached to an imaging plate (manufactured by Fuji Photo Film Co., Ltd.) together with an imaging plate (manufactured by Fuji Photo Film Co., Ltd.). Image analysis was performed with G. Colonies and hybridizations were repeated until a single clone was obtained for the colony where the signal was detected. Plasmid DNA was prepared from the obtained single clone colony using QIA prep Spin Miniprep Kit (manufactured by Qiagen), and DYEnic ETD ye Terminator Kit (Mega BACE) (Amersham Algacia Biotech) and Mega BACE 500 DNAAnalysis System (Amersham Biotech) according to the attached manual. The entire nucleotide sequence was determined.

 As a result, cDNA (SEQ ID NO: 1) containing a cDNA (SEQ ID NO: 2) C0126 encoding a polypeptide consisting of 529 amino acids was obtained. C 0 126 is the predicted signal peptide (SP) region in the region from the amino-terminal Met at position 22 to Thr at position 11 in the amino terminus. A transmembrane domain (T ransmenbranedomain: T な る) consisting of hydrophobic amino acids was present at Met at position 4.55 from G 1 y. In addition, the Cys at position 333 from Cys at position 306 contains Met, an HGF receptor, and Met-re 1 ated sequence (MRS), which is conserved at p1exin, a seamaphorin receptor. (Fig. 1). Human homology with TEM7 was 49%. Example 2

Expression of C 0 126 mRNA in normal human tissues Plasmid containing cDNA (SEQ ID NO: 1) encoding CO 126 in one of the cDNA libraries obtained in Example 1 was subjected to restriction enzyme Eco. RI (Takara Shuzo Co., Ltd.) with and consumption I spoon, QIA quick G el E xtraction K it c DNA fragment 50 obtained _c to afford the c DNA fragment containing the C 0783 according (QIAGEN) was used attached manual the ng to use the R ediprimer IIDNAL abel 1 ing S ystem ( Amersham off Alma shear Biotech), according to the attached ^{manual, [a- 32 P] d CTP} (6000 C i / mmo 1,

Labeled with ³² P with 20 mCi / m1) (manufactured by NEN). Prepared in this way

The ^{3 2} P-labeled c DNA fragment was Roh Zanburodzu Bok analysis using as a probe. The labeled probe prepared above was added to EX press HybHybridization Solution (manufactured by Clontech), and the solution was added to a Multi-Tissue T issue N orthern (MTN) Blots Human 12—Lane TNB lot (manufactured by Clontech) was immersed and hybridized at 65 ° C. for 16 hours. After hybridization, the membrane was treated with 2 XSSC (20 XSSC: 3 MNaCl, 0.3 M sodium citrate, pH 7.0) at room temperature for 5 min at 1 XSSC. After washing at 50 ° C for 30 minutes and with 0.5 XSSC at room temperature for 5 minutes, it was air-dried. Next, this film was attached to an imaging plate cassette (manufactured by Fuji Photo Film Co., Ltd.) together with the imaging plate (manufactured by Fuji Photo Film Co., Ltd.). Image analysis was performed using the Image Analyzer-FLA 3000 G (manufactured by Fuji Photo Film Co., Ltd.). As a result, the mRNA of C 0 126 was approximately 2.7 kb in size, and the brain, heart, skeletal muscle, It was expressed in various tissues such as spleen, liver, small intestine, placenta and lung, and particularly strong expression was observed in kidney. Example 3

 Expression of C 0 126 mRNA in Human Gastric Cancer Tissue The expression level of the gene in normal and tumor tissues of various human-derived organs was examined.

A nylon membrane Matched Tumor / Normal Expression Array (Clontech) immobilized with total RNA extracted from 12 types of cancer tissues derived from 68 humans and corresponding normal tissues was hybridized. to Nbaffa one (0. 5 M phosphate buffer one, p H 7. 2; 1% (w / V) BSA, 1 mmo 1 / LEDTA N 7% ( including the w / v) SDS) immersed in, 6 5 Pre-hybridization was performed for 1 hour at ° C. Next, the plasmid containing C0783 cDNA prepared in the same manner as in Example 2 was digested with the restriction enzyme EcoRI (Takara Shuzo), and 50 ng of the C0783-containing cDNA fragment was digested with Rediprimer II DNAL. according to abelling S ystem (Amersham off Al Macia Biotech Co., Ltd.) using the attached manual, [shed one ³² P] d CT P (6000 C i mm ol, 20 mC i / m 1) and the labeled probe was prepared by-labeled at ^{3 2} P in (NEN Co.). The nickle membrane after the preliminary hybridization was immersed in a hybridization buffer to which the labeled probe was added, and hybridization was carried out at 65 ° C. for 16 hours. Thereafter, the nylon membrane was sequentially washed with 2 × SSC at room temperature for 5 minutes, 1 × SSC at 50 ° C. for 30 minutes, and 0.5 × SSC at 50 ° C. for 30 minutes, and then air-dried. This nylon film was attached to an imaging plate (Fuji Photo Film Co., Ltd.) together with an imaging plate (Fuji Photo Film Co., Ltd.), and after performing radiography, the image analyzer FLA3000 Image analysis was performed using G (Fuji Photo Film Co., Ltd.). Using human ubiquitin as a control, the nylon membrane was dehybridized and analyzed in the same manner as above using the Human U biquitin Control DNA Probe attached to Ar.ay. Analysis of the control was performed. After correcting the expression level in each tissue based on the results, the expression level ratio in the cancer tissue corresponding to the normal tissue was calculated, and the expression level in various cancer tissues was analyzed.

 As a result, in 6 out of 8 gastric cancer patients, an increase in the expression of the C0126 gene in gastric cancer tissue was observed as compared to normal stomach (Fig. 2). Example 4

Expression of C 0 126 mRNA in Human Mesenchymal Stem Cells The expression change of C 0 126 mRNA when human mesenchymal stem cells were induced to differentiate into bone cells was analyzed. Human mesenchymal stem cells (BioWhittaker) were cultured using Human Mesenchymal Stem Cell Basal Medium (BioWhittaker). As a test, human IL-11 (R & DS ystems) was added to the cell culture solution to a final concentration of 0.5 ηg / m 1, and human IGF-1 (R & DS ystems) was added. Was added to a final concentration of 10 ng / mΊ, human FGF-basic (FGF-2) (manufactured by R & DS ystems) was added to a final concentration of 10 ng / m1 and no control was added. It was performed with 5% C_〇 1 day in ₂ or 4 days of culture. Cells were collected from each cell culture, and total RNA was extracted using TRIZ 0 L reagent (manufactured by Invitrogen) according to the attached manual to obtain total RNA. After 10 g of the obtained total mRNA was fractionated by 1% agarose gel (containing 5.5% formalin) electrophoresis, it was transferred to a nylon membrane Hybond-N (Amersham Pharmacia Biotech). The membrane is placed in a hybridization buffer (0.5 M phosphate buffer, pH 7.2; containing 1% (wZv) BSA, 1 mM EDTA, 7% (w / v) SDS). Pre-hybridization was performed at 65 ° C for 1 hour. Next, the plasmid containing CO126 cDNA prepared in the same manner as in Example 2 was digested with restriction enzyme EcoRI (Takara Shuzo), and 50 ng of the cDNA fragment containing CO126 was digested. R ediprimer IIDMAL abe 1 1 ing S ystem to use the (Amersham off Alma shear Biotech), according to the attached manual, Non one ^{32 P] d CTP (6000 C} 1 mmo 1, 20 m C 1 / m 1) the labeled probe was prepared by labeling with ^{3 2} P in (NEN Co.). The nylon membrane after the preliminary hybridization was immersed in the hybridization buffer to which the labeled probe was added, and hybridization was performed at 65 ° C. for 16 hours. Thereafter, the nylon film was washed with 2 × SSC at room temperature for 5 minutes, 1 × SSC at 50 ° C. for 30 minutes, and 0.5 × SSC at room temperature for 5 minutes, and then air-dried. This nylon film was attached to an imaging plate (manufactured by Fuji Photo Film) together with an imaging plate (manufactured by Fuji Photo Film Co., Ltd.), and after performing radiography, an image analyzer FLA 3 O 00 G (wealth) Image analysis was carried out by Shiseido Film Corporation. Each signal intensity was measured, and the ratio of the analysis result of the test group to the analysis result of the control group was calculated.

As a result, expression of C 0 126 was observed in IGF-1 which has chondrocyte differentiation-inducing activity, but IL-11β and FGF-2 which have osteoblast differentiation inducing activity were observed. Did not induce expression (Fig. 3). Example 5

 Expression of C 0 126 in mammalian cells

P3 XFLA G-CM V-1 (Sigma Aldrich Japan) was used to amplify the DNA encoding the 3XFLAG peptide by PCR, and the cloning vector PCR 2.1-T0P0 (Invitrogen) To make plasmid pCR-FLAG.

Next, from the cDNA fragment obtained in Example 1, a cDNA coding for C0126 shown in SEQ ID NO: 1 was inserted into the Notl site of pCR-FLAG, so that 3 A vector to which a cDNA sequence encoding XFLAG polypeptide was added was constructed. This plasmid is digested with the restriction enzyme XhoI, and inserted into the Xhol site of the expression vector pLXSN (Clontech) to express the C0126 expression vector pLXNS—C01 26 FLAG was constructed.

 As a host for PLXNS-C0126FLAG, EcoPacck2-293Cel1Line (manufactured by Clontech) was used.

1 x 1 Eco Pack 2-293 Cell Line was added to a 35 mm multi-dish into which D-MEM medium (manufactured by Sigma-Aldrich Japan) containing 10% (w / V) FCS was dispensed. 0 ⁵ was added to give a cell / Ueru were 1 6 hr at 5% C 0 ₂ in 37 ° C. It was added 2 g of p LXNS- C 0 1 2 6 FLAG and 3 mu 1 of F ugene (Roche die § Diagnostics stay box Co.) to the cell culture, 2 in 5% C 0 ₂ in 3 7 ° C Culture was performed for 4 hours. Thereafter, the medium was changed, and the cells were further cultured at 37 ° C. in 5% { ₂ } for 24 hours to produce retrovirus.

A supernatant containing a retrovirus was recovered from the cell culture solution, and a mouse fetal cell line C 3 H / 10T 1./2 cell (ATCC: CCL-122), which is a mouse mesenchymal stem cell Was transformed.

 C 3 H / 10 T 1/2 cells were prepared using Minimum Essentia 1 Medium (M EM) alphaediurn (1 x) 11 qu 1 d (manufactured by Invitrogen) containing 10% FCS. After culturing at 37 ° C for 16 hours. The culture medium was replaced with a culture in which polyprene (Nacalai Tesque) was added to the culture supernatant containing retrovirus so that the final concentration was 10 g / mΊ. Virus infection was carried out by culturing at 37 ° C for 8 hours. Remove the medium from the culture and infect the infected C3H / 10T1 / 2 cells with 10% FCS Mini Inum Ess e n t

1 a 1 M edium (M EM ) alpha M edium (1 x) 1 iquid C 0 1 26 produced in this way _c subjected to 1 6 hours further incubation at 37 ° C for using (i Nbitorojen Co.) C 0 1 in FLAG protein expressing cell lines

The expression of the 26 FLAG protein was analyzed by the ブ stanblotte method according to the method of expressing C 0 126 and aem Ίi.

The transformed C 3 H / 1 OT 1/2 cells were lysed into Lysis Buffer (10 mM MT “is—HCl, pH 7.4; 1% (w / v) Triton X—100” , 0.15 MNaCl, 1 mM EDTA) After fractionating 5 μl of the cell lysate by SDS-PAGE, Immobi 1 on—P (Nippon Millipore) The membrane was immersed in a blocking solution (PBS, pH 7.4; containing 5% (V / V) skim milk), and shaken at room temperature for 1 hour. Labeled antibody solution (PBS, pH 7.4; containing 5% (v / v) skim milk, Anti-FLAGM 2 Monoclonal Antibody Peroxydase (HRP) Conjugate (Sigma Aldrich) was added. The nylon membrane was washed three times for 10 minutes with a washing solution (PBS, pH 7.4; containing 0.1% Tween 20). Later, the nylon membrane and ECLP lus Western B lotting D etection Reagents (Amersham (Manufactured by Pharmacyanotech) at room temperature, and immediately the nylon film and X-ray film X— 0 MATAR (manufactured by Kodak Company) was attached to an X-Matt Power Set (manufactured by Kodak Company) and exposed for several minutes. Untransformed C3H / 10T1 / 2 cells were treated in the same manner as a control.

 As a result, a band having a size of about 100 Kd was detected. Since the expected molecular weight is greater than about 60 Kd, it is considered that a sugar chain was added. Example 6

 Expression of C0126 in baculovirus Expression of Baculovirus in Baculovirus was performed using Bac-to-BacBaccu1ovirus Expression System (manufactured by Invitrogen) according to the attached manual. went.

 CDNA encoding 3XFLAG-added polypeptide at the carboxy terminus of the amino acid sequence represented by Ty at position 454 from Asp at position 1 in SEQ ID NO: 2, which is the extracellular region of C0126 Is expressed in insect cells.

From p3XFLAG-CMV-14 (manufactured by Sigma-Aldrich Japan), a DNA fragment encoding 3XFLAG peptide and a DNA fragment encoding the extracellular region of CO126 were amplified by PCR. It was inserted into the XhoI site of pFastBac1 to construct pFastBac—C0783 FLAG. The recombinant plasmid was introduced into MAX Efficiency DH10 Bac Competent Cells and transferred to a baculovirus genome (bacmid DA) to construct a recombinant bacmid DNA. The recombinant bacmid DNA using C ell FECTINR eagent S f 9 cells: - introducing into (ATCC CRL 1 7 1 1 _N ovary cells), to obtain a recombinant baculovirus. The baculovirus-infected Sf9 cell culture 62.5Ί, 125 μl, 250 μl, 500001 each of 2 × 10 ⁶ cells / m1 Sf9 cell culture 50 The resulting mixture was cultured at 28 ° C for 72 hours. Centrifuging the cell culture, Recombinant C 0 126 FLAG was obtained in the supernatant fraction.

 The expression of C0126FFLAG was confirmed by the Western plot method in the same manner as in Example 5. After the culture supernatant 10 was fractionated by SDS-PAGE, it was electrotransferred to Nitrocell Mouth I-Sumemrene (manufactured by Tefco). The nylon membrane is immersed in a blocking solution (TBS, pH 7.4; containing 5% (V / V) skim milk) and shaken at room temperature for 1 hour. H 7.4; containing 5% (V / V) skim milk and adding Anti-FLAG M2Monoclona 1 Antibody Peroxydase (, HRP) Conjugate (Sigma Aldrich) It was immersed and left at room temperature for 1 hour. After washing the membrane with a washing solution (TBS, pH 7.4; containing 0.05% Triton X—100) three times for 10 minutes, the membrane and ECLP 1 were washed. Us Western B lotting Detection Reagents (Amersham Pharmacia Biotech) were reacted at room temperature, and the nylon film and the ELC detection film Hyperfilm— ECL (Amersham Pharmacia Biotech) were immediately replaced with X. —Mounted on a power sensor (Kodak) and exposed for several minutes. The culture supernatant of Sf9 cells not infected with the recombinant virus was treated in the same manner as a control.

 As a result, a single band with a size of about 80 Kd was detected. The signal of each band was detected at an intensity dependent on the amount of added virus, and it was confirmed that the extracellular region of C0126 was secreted into the culture supernatant of Sf9 cells.

Next, this band was cut out from SDS-PAGE, eluted, and the amino acid sequence of the N-terminal part of this polypeptide was analyzed using an amino acid sequencer. It was confirmed that the sequence matched the amino acid sequence predicted from the sequence, and that it was truncated between amino acids 45 and 46 of SEQ ID NO: 2. Since the amino acid sequence at positions 42 to 45, which corresponds to the position immediately before this cleavage site, matches the recognition sequence of a membrane-bound processing protease such as furin, CO 126 is derived from such a proteinase derived from cells. By 4 between 5th and 46th place It was expected to be disconnected. Therefore, it is assumed that the peptide corresponding to positions 1 to 45 is released extracellularly as a result of cleavage of the signal sequence and processing with a furin-like protease following expression of C0126. . Example 7

 C0126 Induced activity of cartilage differentiation In Example 4, it was shown that C0126 is up-regulated during cartilage differentiation. Therefore, the effect of C0126 expression on cartilage differentiation was examined.

C3H / 10T1Z2 cells transformed with the C0126FLAG expression plasmid prepared in Example 5 and untransformed C3H / 10T1 / 2 cells as a control Was cultured at 37 ° C for 16 hours using Mini Essential Medium (MEM) alpha Medium (1x) iquid (manufactured by Invitrogen) containing 10% FCS. Each culture cells were dispensed to a 4 X 1 0 ⁴ cells / Ueru 24 Uerupure Doo (co twelve Ngusha) After 1 6 hours at 37 ° C, containing 5% FCS medium After replacing the medium, culture the cells at 37 ° C for 6 hours, and replace the cells with the final concentration of human BM P—2 by replacing the medium with MIN alpha Essential Medium (MEM alpha Medium (1X) 1 iquid (Invitrogen)). (Sawade one Techno Logistics Co. one company) is 400 ng / ml, human BM P- 4 (R & DS ystems Inc.) is (manufactured Shigumaaru Doritsuchi Co.) 200 ng / m 1, retinoic acid is 1 0- ⁶ M Then, the cells were cultured for 3 days at 37 ° C. The medium of the cell culture medium was mixed with the same concentration of the same test substance in Minimun Essentia 1 Medium (MEM) alpha. The medium was replaced with one (1) iquid, and the cells were further cultured at 37 ° C for 3 days.

As a method of quantifying differentiation into chondrocytes, cartilage matrix, a chondrocyte differentiation marker, was stained with Alcian blue, and the absorbance was measured. After removing the culture medium of each cell culture, 10% formalin was added, and the cells were fixed at room temperature for 10 minutes to fix the cells. 200 μl of Alcian Blue staining solution (pH 2.5) (manufactured by Nacalai Tesque, Inc.) was added to the fixed cells, and the reaction was carried out at 4 ° C. overnight. Guanidine hydrochloride was added, and the reaction was carried out at room temperature for 6 hours to extract a stained substance. The absorbance of the extract at 650 nm was measured using a spectrophotometer (manufactured by Amersham Pharmacia Biotech) to determine the amount of Alcian Blue staining.

 As a result, in C0126-expressing cells, differentiation into chondrocytes by BMP-2 and BMP-4 was promoted (Fig. 4). Example 8

 Induction activity of cartilage differentiation of C 0 126 extracellular domain protein

The effect of C0126 extracellular domain protein on cell differentiation of C3H / 10T1-2 cells was examined.

 The C 0 126 extracellular domain proteins include the C 0 126 extracellular domain expressed by the baculovirus expression system prepared in Example 6, which is the C 01 26 extracellular domain SEQ ID NO: 46 from position A sn to position 454 T A polypeptide having 3 XFLAG added to the carboxy terminus of the amino acid sequence represented by yr was used.

 C 3 HZ10 T 1/2 cells were transformed with 10% FCS containing M i n i m u n E s s se n t i a Ί M e d i u m (, EM) a l p h a M e d i um (1) 1

1 using a quid (manufactured by Lee Nbitorojen Co.), and _c each culture cells was carried out for 1 6 hours incubation at 37 ° C for

Dispense into a 24-well plate (Corning) at 4 × 10 ⁴ cells / well and incubate at 37 ° C for 16 hours. Then, place the medium in Minimal Essential Medium containing 5% FCS. edium (.iVI E fvij alpha M ediurn (1X) 1 quid (manufactured by Invitrogen). After culturing at 7 ° C for 6 hours, the cell culture solution containing the CO 126 extracellular region protein prepared in Example 6 at a final concentration of 10 g / mΊ and the C 0 126 extracellular region A cell culture without added protein was prepared. Each cell culture solution of it, human BM P- 2 at a final concentration (Sawade - Techno LOGIS made one company) is (manufactured by R & DS ystems _{Ltd.) 1 000 ng / ml N human} BM P- 4 is 400 ng / m 1, after the retinoic acid (manufactured by Shigumaaru drill Tutsi Co.) was added to so that such a 1 0- ⁶ M, was cultured for 3 days at 37 ° C. The medium of the cell culture solution was exchanged for Mi π 1 mun Essential Medium (MEM) alpha Medium (1) liquid containing the same test substance at the same concentration, and the cells were further cultured at 37 ° C for 3 days.

 The method for quantifying the differentiation into chondrocytes was performed using Alcian Blue monostain in the same manner as in Example 7.

 After removing the medium of each cell culture, 10% formalin was added and the cells were treated at room temperature for 10 minutes to fix the cells. 2001 of Alcian blue staining solution (pH 2.5) (manufactured by Nacalai Tesque) was added to the fixed cells, and the reaction was carried out at 4 ° C overnight. Then, the staining solution was removed and 6 M hydrochloric acid was removed. Guanidine was added, and the reaction was performed at room temperature for 6 hours to extract a stained substance. The absorbance of the extract at 650 nm was measured using a spectrophotometer (manufactured by Amersham Pharmacia Biotech) to determine the amount of Alcian Blue staining.

 As a result, the differentiation of chondrocytes by BMP-2 and BMP-14 was suppressed by the C0126 extracellular domain protein (Fig. 5). Industrial applicability

According to the present invention, C0126 polypeptide which is a marker for gastric cancer, osteoarthritis and rheumatoid arthritis, a polynucleotide encoding the polypeptide, a vector for gene therapy incorporating the DNA, and the polypeptide Recognizing antibody, method for quantifying C0126 polypeptide using the antibody, determination of mRNA for the polypeptide Quantitative method, kit for diagnosing cancer, osteoarthritis or rheumatoid arthritis using the quantitative method, the polypeptide, an antibody recognizing the polypeptide, a pharmaceutical composition containing the polynucleotide, the pharmaceutical composition For preventing and treating osteoarthritis, rheumatoid arthritis or osteoporosis, and a method for preventing and treating osteoarthritis, rheumatoid arthritis or osteoporosis using the pharmaceutical composition. .

Claims

The scope of the claims 1. A marker for cancer, cancer metastasis or bone disease, comprising a polypeptide consisting of an amino acid sequence represented by Asp at position 1 to Arg at position 45 of SEQ ID NO: 2.  2. A marker for cancer, cancer metastasis or bone disease, comprising a polypeptide consisting of an amino acid sequence represented by Asn at position 46 to Tyr at position 43 of SEQ ID NO: 2.  3. The marker according to claim 1 or 2, which comprises a polypeptide consisting of an amino acid sequence represented by Asp at position 1 to Cys at position 507 in SEQ ID NO: 2.  4. The marker according to claim 1 or 2, which comprises a polypeptide consisting of an amino acid sequence represented by Met at position 222 of SEQ ID NO: 2 to Cys at position 507. 5. Ars at position 1 to 5 from Asp of SEQ ID NO: 2, Tyr at position 432 from Asn at position 46, Cys at position 507 from Asp at position 1 22 A polypeptide having one or several amino acid mutations selected from deletion, substitution or insertion in any of the polypeptides comprising an amino acid sequence represented by Cys at position 507 to position 507 Including cancer, cancer metastasis or bone disease.  6. The marker according to any one of claims 1 to 5, wherein the cancer is gastric cancer.  7. The marker according to any one of claims 1 to 5, wherein the bone disease is osteoarthritis, rheumatoid arthritis or osteoporosis.  8. A marker for cancer, cancer metastasis, or bone disease containing a polynucleotide consisting of the nucleotide sequence represented by A from position 43 to position A of position C of SEQ ID NO: 1.  9. A marker for cancer, cancer metastasis, or bone disease containing a polynucleotide consisting of the nucleotide sequence represented by G from 288th position to 422nd A in SEQ ID NO: 1.  10. The marker according to claim 8 or 9, comprising a polynucleotide consisting of a nucleotide sequence represented by G from 288th G to 180th C in SEQ ID NO: 1. 11. The marker according to claim 8 or 9, which comprises a polynucleotide consisting of a nucleotide sequence represented by A to 182nd C from 222 to 222 of SEQ ID NO: 1. 1 2. SEQ ID NO: 1 from 4 2 3rd A to 1 8 08th C, 2 8 8th G 4 2 2nd A, 288 8th G to 1808th C or 228 2nd A to 1808th C and any of the base sequences Marker for cancer, cancer metastasis, or bone disease containing a polynucleotide that hybridizes under suitable conditions 1 3. The marker according to any one of claims 8 to 12, wherein the cancer is gastric cancer. 14. The marker according to any one of claims 8 to 12, wherein the bone disease is osteoarthritis, rheumatoid arthritis or osteoporosis.  15. The marker according to any one of claims 1 to 7, comprising using an antibody that recognizes a polypeptide consisting of the amino acid sequence of SEQ ID NO: 2 or a part thereof, comprising the following steps: How to detect;  (a) contacting a test substance with the antibody; and  (b) a step of detecting the binding between the test substance and the antibody.  16. A marker according to any one of claims 8 to 15, wherein a polynucleotide comprising the base sequence of SEQ ID NO: 1 or a part thereof comprising the following steps is used. How to detect;  (a) contacting a test substance with the polynucleotide; and  (b) detecting the binding between the test substance and the polynucleotide;  17. A kit for diagnosing cancer, cancer metastasis, or bone disease, comprising using the detection method according to claim 15 or 16.  18. The diagnostic kit according to claim 17, wherein the cancer is gastric cancer.  19. The diagnostic kit according to claim 17, wherein the bone disease is osteoarthritis, rheumatoid arthritis or osteoporosis.  20. A method for screening a chondrocyte-inducing activity regulating substance, comprising using a transformant that expresses a polynucleotide containing a nucleotide sequence represented by SEQ ID NO: 1 or a part thereof, comprising the following steps; (a) culturing a transformant expressing the polynucleotide in the presence or absence of the test substance; and (b) a step of comparing the amount of cartilage matrix produced in the transformant expressing the polynucleotide in the presence or absence of the test substance.  21. A screening kit for a chondrocyte differentiation-inducing activity regulating substance, comprising a transformant that expresses a polynucleotide comprising the nucleotide sequence represented by SEQ ID NO: 1 or a part thereof.  22. A chondrocyte differentiation-inducing activity regulating substance obtained by the screening method according to claim 21.  23. The chondrocyte differentiation induction according to claim 22, which is a polypeptide comprising an amino acid sequence represented by A sn at position 46 to A 1a at position 43 of SEQ ID NO: 2. Activity modulator.  24. The compound according to claims 22 or 23 or a salt thereof, a polynucleotide comprising the base sequence described in SEQ ID NO: 1 or a part thereof, or a base sequence described in SEQ ID NO: 1 or a part thereof A pharmaceutical composition comprising, as an active ingredient, any one of an expression vector for human gene therapy containing the polynucleotide, a polypeptide containing the amino acid sequence of SEQ ID NO: 2 or a part thereof, and an antibody against the polypeptide. . 25. The pharmaceutical composition according to claim 24, which is an agent for preventing or treating cancer or cancer metastasis.  26. The pharmaceutical composition according to claim 24, which is a prophylactic or therapeutic agent for bone diseases. 27. The pharmaceutical composition according to claim 24, which is a prophylactic or therapeutic agent for osteoarthritis, rheumatoid arthritis or osteoporosis.  28. A method for preventing or treating cancer, cancer metastasis, or bone disease, comprising administering the pharmaceutical composition according to any one of claims 24 to 27.