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WO2004039974A1 - Nouvelle proteine et son adn - Google Patents

Nouvelle proteine et son adn Download PDF

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Publication number
WO2004039974A1
WO2004039974A1 PCT/JP2003/013919 JP0313919W WO2004039974A1 WO 2004039974 A1 WO2004039974 A1 WO 2004039974A1 JP 0313919 W JP0313919 W JP 0313919W WO 2004039974 A1 WO2004039974 A1 WO 2004039974A1
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WO
WIPO (PCT)
Prior art keywords
protein
dna
present
salt
seq
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Ceased
Application number
PCT/JP2003/013919
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English (en)
Japanese (ja)
Inventor
Tatsuya Watanabe
Masakazu Inazuka
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Takeda Pharmaceutical Co Ltd
Original Assignee
Takeda Pharmaceutical Co Ltd
Takeda Chemical Industries Ltd
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Filing date
Publication date
Application filed by Takeda Pharmaceutical Co Ltd, Takeda Chemical Industries Ltd filed Critical Takeda Pharmaceutical Co Ltd
Priority to AU2003280641A priority Critical patent/AU2003280641A1/en
Publication of WO2004039974A1 publication Critical patent/WO2004039974A1/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/136—Screening for pharmacological compounds
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/158—Expression markers

Definitions

  • the present invention relates to a screening method for a novel protein, its DNA, a compound that regulates the expression or activity of the protein, a compound obtained by the screening method, a prophylactic or therapeutic agent for a bone or joint disease containing the compound, etc.
  • Background art for a novel protein, its DNA, a compound that regulates the expression or activity of the protein, a compound obtained by the screening method, a prophylactic or therapeutic agent for a bone or joint disease containing the compound, etc.
  • the olfactomedin domain (Conserved Domain Database pfam0219> OLF) is a protein structural unit consisting of about 250 amino acid residues and of unknown function. Proteins containing this domain are classified into two types: secreted proteins (including ER-localized proteins) and seven transmembrane receptors. Olfactomedin, a secreted protein, was first isolated as a component of the mucous extracellular matrix, and has been reported to be expressed in olfactory neuroepithelial cells of toad frogs (Yokoe H, Anolt RR, Proc Natl Acad Sci US A. 90: 4655-4659, 1993). Since then, proteins and genes containing this domain have been discovered in many species including humans.
  • Myocil in expressed in the ciliary body is well studied.
  • Myoci 1 in, also called TIGR is known to be one of the causative genes of glaucoma (Stone, EM. Et al. Science 275: 668-70, 1997).
  • GW112 (NP-006409) has been reported to be upregulated in epithelial cells of the mucosa of the colon in ulcerative colitis (Shinozaki, S. et al. Gut 48: 623-9, 2001).
  • Mouse Pancor tin Nagano, T. et al. Brain Res Mol Brain Res 53: 13-23 (1998).
  • Latrophilin is a receptor for the spider neurotoxin a-Latrotoxin, a G protein-coupled seven-transmembrane receptor, and is expressed in the brain in mice (Lelianova, VG, et al. J Biol. Chem 272, 21504-8 (1997).
  • T i ar in is a soluble receptor with angiogonist action of Latrophi 1 in (Wessely, 0 and De Robert is, EM Neuron, Vol 33, 489-491, 2002) .
  • HN0EL-iso GenBank Accession NP_064575
  • 0LFM1 NP_055094
  • 0LFM2 NP_477512
  • 0LFM3 AAK97473
  • Receptor-type olfactomedin domain-containing proteins include Latrophilin 1 (NP-036434, also called Lectomedin-1) in humans,
  • Lectomedin-2 (NP_055736.1) and Lectomedin-3 (NP-056051) are registered. Of these, the human Latrophilin 1 gene has been shown to be altered in breast cancer (White, G.R. Oncogene 17 (26), 3513-3519, 1998).
  • Osteoarthritis is a joint disease accompanied by chronic arthritis, and is a disease in which degeneration of cartilage causes destruction of cartilage and proliferative changes in bone and cartilage. Deformation of the joint such as formation of osteophytes at the epiphysis is also observed. Osteoarthritis increases with age, and it is said that over the age of 60, more than 80% of the knees, elbows, hips and spine exhibit symptoms of osteoarthritis. Treatment of osteoarthritis mainly focuses on coping therapies to control pain, including non-steroidal anti-inflammatory analgesics, hyaluronic acid injected directly into joints, and steroids. If advanced, arthroscopic surgery or severe pain or deformity, knee or hip joints are indicated for osteotomy or artificial joint replacement.
  • cDNA or oligonucleotide A standardized microarray method has been developed, and the technology for finding disease-specific changes in gene expression has become widespread, and its usefulness has been confirmed.
  • Affymetrix's GeneChip system is increasingly being used to diagnose diseases such as cancer and to discover drug discovery target genes. Attempts have been made to find genes and proteins that are specifically expressed in the joints of osteoarthritis patients and to use them to create therapeutics and discontinuations for osteoarthritis.
  • matrix meta-oral protease such as MMP-13 is increased in joints with osteoarthritis, and MMP inhibitors are being studied as joint destruction inhibitors.
  • DKFZP586L151 is an orthophedomedin domain-containing protein registered as GenBank Accession No. XP-034000, but its full-length sequence has not been reported.
  • a gene highly homologous to DKFZP586L151 has been cloned from mice (GenBank Accession No. XM_1 9545).
  • no relationship between olfactomedin domain-containing proteins and osteoarthritis has been reported. There is a need for safe and superior bone and joint disease diagnostics and prophylactic and therapeutic agents. Disclosure of the invention
  • the present inventors have conducted intensive studies in order to solve the above-mentioned problems, and as a result, have found an orfactomedin domain-containing gene whose expression is remarkably increased in osteoarthritis cartilage. As a result of repeated studies, the present invention has been completed.
  • a medicament comprising the protein of (1) or the partial peptide of (4) or a salt thereof,
  • a diagnostic agent comprising the polynucleotide of (5) above, (14) an antibody against the protein of (1) or the partial peptide or salt thereof of (4),
  • a polynucleotide comprising a nucleotide sequence complementary to or substantially complementary to the nucleotide sequence of the polynucleotide according to (5) or a part thereof,
  • a medicament comprising the compound according to (19a) or a salt thereof, (20) a drug comprising the protein according to (1) or a partial peptide thereof or a salt thereof, Compounds that regulate the activity of proteins of Is a kit for screening its salts,
  • (20b) a medicine comprising the compound or salt thereof according to (20a); (21) a protein gene according to (1), characterized by using the polynucleotide according to (5).
  • (21b) a medicine comprising the compound or salt thereof according to (21a); (22) expression of the protein gene according to (1), comprising the polynucleotide according to (5) Kit for screening a compound or a salt thereof that regulates
  • the bone or joint disease is a cartilage dysplasia, bone dysplasia, osteoporosis, osteoarthritis, rheumatoid arthritis, arthritis, synovitis, metabolic arthrosis or a joint disorder due to sports (above).
  • Bone and joint disease is caused by cartilage dysplasia, bone dysplasia, osteoporosis, osteoarthritis, rheumatoid arthritis, arthritis, synovitis, metabolic arthrosis or sports
  • the diagnostic agent according to the above (26) which is a joint disorder
  • a method for screening a compound or a salt thereof that modulates the transcription regulating activity of A (34a) a compound or a salt thereof obtained by using the screening method according to the above (34),
  • a compound or a salt thereof which inhibits the activity of a protein or a salt thereof having the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 1 in mammals, or A method for preventing or treating bone and joint diseases, which comprises administering an effective amount of a compound or a salt thereof that inhibits the expression of the protein gene.
  • FIG. 1 shows changes in the expression of the mouse chondromedin gene during the cartilage differentiation process of mouse C3H10T1 / 2 cells.
  • the vertical axis shows the value of the expression level of the mouse chondromedin gene at each time point examined relative to the expression level in DayO, and the horizontal axis shows the number of days after the start of differentiation culture.
  • FIG. 2 shows an increase in the anti-Trl antibody titer in the serum of a heron immunized with Trl.
  • the vertical axis shows the antibody titer
  • 'The horizontal axis shows the dilution ratio of peptide serum used as the primary antibody ⁇ : serum collected on Day 0, and' Hata shows the serum collected on Day 35, respectively. .
  • FIG. 3 shows an increase in the anti-Tr2 antibody titer in the serum of a heron immunized with Tr2.
  • the vertical axis indicates the antibody titer
  • the horizontal axis indicates the dilution ratio of the anti-peptide serum used as the primary antibody
  • ⁇ indicates the serum collected on Day 0
  • ⁇ indicates the serum collected on Day 35.
  • amino acid sequence substantially identical to the amino acid sequence represented by SEQ ID NO: 1 is 85% or more, preferably about 90% or more, and preferably about 95% or more of the amino acid sequence represented by SEQ ID NO: 1.
  • amino acid sequence having about 98% or more homology and the like are exemplified.
  • Examples of the protein having an amino acid sequence substantially the same as the amino acid sequence represented by SEQ ID NO: 1 include, for example, a protein having an amino acid sequence substantially the same as the amino acid sequence represented by SEQ ID NO: 1 described above. However, a protein having substantially the same activity as the protein containing the amino acid sequence represented by SEQ ID NO: 1 is preferable. - Examples of substantially the same activity include a chondrocyte differentiation regulating activity and a chondrocyte binding activity. Substantially identical indicates that the properties are biologically (eg, physiologically or pharmacologically) identical.
  • the measurement of chondrocyte differentiation regulation activity can be performed according to a known method, for example, the method described in J Cell Biochem 67, 498-513 (1997) or a method based thereon. can do.
  • the protein of the present invention is obtained from a culture supernatant or a cell extract of a cell capable of producing the protein of the present invention according to a known method (eg, an immunoaffinity column). Add this protein to cultured chondrocytes and culture at 37 ° C for 7-21 days.
  • the activity of regulating chondrocyte differentiation is measured by evaluating the proteodarin content or the phosphatase activity of chondrocytes according to the method described in the above-mentioned literature.
  • the binding activity to chondrocytes can be measured according to a known method.
  • the protein of the present invention is labeled with biotin according to a known method, for example, using!)-Piotinoyl- ⁇ -aminocaproic acid_ ⁇ -hydroxysuccinimide ester.
  • the labeled protein is added to cultured chondrocytes, and after culturing at 4 ° C for 1 hour, the cells are washed with PBS. Next, a complex of streptavidin and an enzyme (eg, alkaline phosphatase) is added. After culturing at 4 for 1 hour, the cells are washed with PBS.
  • an enzyme eg, alkaline phosphatase
  • the chemiluminescent substrate of the enzyme eg, Di sodium 2-chloro-5- (4-methoxyspiro (l,-dioxetane-3, 2 '-(5'-chloro) tricyc lo [3.3.1. 1. 3, 7] decan ⁇ -4-yl) -l-phenyl phosphate
  • the chemiluminescent substrate of the enzyme eg, Di sodium 2-chloro-5- (4-methoxyspiro (l,-dioxetane-3, 2 '-(5'-chloro) tricyc lo [3.3.1. 1. 3, 7] decan ⁇ -4-yl) -l-phenyl phosphate
  • Examples of the protein of the present invention include: (i) one or two or more amino acids in the amino acid sequence represented by SEQ ID NO: 1 (for example, about 1 to 50, preferably about 1 to 30; Is an amino acid sequence in which about 1 to 10 amino acids have been deleted, more preferably about 1 to 5 amino acids, and (ii) one or two or more amino acids (for example, 1 An amino acid sequence to which about 50, preferably about 1-30, preferably about 1-10, more preferably about (1-5) amino acids have been added; (iii) SEQ ID NO: 1 The amino acid sequence represented by 1 or 2 or more (for example, about 1 to 50, preferably about 1 to 30, An amino acid sequence in which about 1 to 10 amino acids have been inserted, more preferably about 1 to 5 amino acids, and (iv) one or two or more amino acids in the amino acid sequence represented by SEQ ID NO.
  • SEQ ID NO: 1 for example, about 1 to 50, preferably about 1 to 30
  • amino acid sequence in which about 1 to 50, preferably about 1 to 30 / preferably about 1 to 10, and more preferably a number (1 to 5) of amino acids have been replaced with another amino acid; Or (V) so-called mucins such as proteins containing an amino acid sequence obtained by combining them.
  • the position of the insertion, deletion or substitution is not particularly limited.
  • the left end is the N-terminus (amino terminus) and the right end is the C-terminus (potassium terminus) according to the convention of peptide notation.
  • the protein of the present invention including the protein containing the amino acid sequence represented by SEQ ID NO: 1, has a C-terminal lipoxyl group (-C00H), carboxylate (-COO-) ', amide ( —C0NH 2 ) or an ester (—C00R).
  • R ′ in the ester is, for example, an alkyl group such as methyl, ethyl, n-propyl, isopropyl, or n-butyl; for example, a C 3 cycloalkyl group such as cyclopentyl or cyclohexyl; for example, phenyl, C 6 _ 12 Ariru group such Hiichina Fuchiru, for example, benzyl, ⁇ 3 7 _ 14 such as alpha _ Nafuchiru C, _ 2 Al kill groups such as phenyl one C M alkyl or flying one naphthylmethyl such phenethyl An aralkyl group, a piperyloxymethyl group, or the like is used.
  • an alkyl group such as methyl, ethyl, n-propyl, isopropyl, or n-butyl
  • a C 3 cycloalkyl group such as cyclopentyl or cyclohe
  • the lipoxyl group is amidated or esterified.
  • the ester in this case, for example, the above-mentioned C-terminal ester and the like are used.
  • the amino group of the N-terminal amino acid residue (eg, methionine residue) is protected by a protecting group (eg, a formyl group, an acetyl group, etc., and a 6- alkanol group such as an acyl group).
  • a protecting group eg, a formyl group, an acetyl group, etc., and a 6- alkanol group such as an acyl group.
  • Protected, N-terminal glutamine residue generated by cleavage in vivo, pyroglutamine-oxidized, Substituent on side chain of amino acid in molecule are suitable protecting groups (for example, formyl groups, Those protected by such c Bok 6 Ashiru groups such as c Bok 6 Al force Noiru group such as methyl group), or include complex protein such Domo such as glycoproteins sugar chain is bound.
  • protein of the present invention include, for example, a protein containing the amino acid sequence represented by SEQ ID NO: 1.
  • Examples of the protein containing the amino acid sequence represented by SEQ ID NO: 1 include a protein containing the amino acid sequence represented by SEQ ID NO: 3.
  • the partial peptide of the protein of the present invention is a partial peptide of the protein of the present invention described above, and is preferably any peptide having the same properties as the protein of the present invention described above. For example, at least 20 or more, preferably 50 or more, more preferably 70 or more, more preferably 100 or more, most preferably 200 or more of the constituent amino acid sequences of the protein of the present invention.
  • Peptides having the above amino acid sequences are used.
  • a peptide having the amino acid sequence of the 1st to 290th amino acids of the amino acid sequence represented by SEQ ID NO: 1 is exemplified.
  • one or more (preferably about 1 to 10, more preferably a number (1 to 5)) amino acids in the amino acid sequence are deleted, Or 1 or 2 or more (preferably, about 1 to 20; more preferably, about 1 to 10; more preferably, about 1 to 5) amino acids are added to the amino acid sequence. Or one or more (preferably about 1 to 20, more preferably about 1 to 10, and more preferably a number (1 to 5)) amino acids in the amino acid sequence. Or one or more (preferably about 1 to 10, more preferably several, more preferably about 1 to 5) amino acids in the amino acid sequence are replaced with other amino acids. It may be.
  • the partial peptide used in the present invention the C-terminus force Rupokishiru group (- C00H) Karupokishireto (- C00-), amide (- C0NH 2) or may be anything Re is the ester (-C00R).
  • the partial peptide used in the present invention includes the protein of the present invention described above.
  • N-terminal amino acid residue eg, methionine residue
  • a glutamine residue generated by cleavage of the N-terminal side in vivo and oxidized with lipamine, a compound in which the substituent on the side chain of the amino acid in the molecule is protected with an appropriate protecting group, or a sugar chain Complex peptides, such as so-called glycopeptides, to which are bound.
  • the partial peptide used in the present invention can also be used as an antigen for producing an antibody.
  • salts with physiologically acceptable acids eg, inorganic acids, organic acids
  • bases eg, alkali metal salts
  • Preferred acid addition salts are:
  • Such salts include, for example, salts with inorganic acids (eg, hydrochloric acid, phosphoric acid, hydrobromic acid, sulfuric acid) or organic acids (eg, acetic acid, formic acid, propionic acid, fumaric acid, maleic acid) Succinic acid, tartaric acid, citric acid, malic acid, oxalic acid, benzoic acid, methanesulfonic acid, benzenesulfonic acid).
  • inorganic acids eg, hydrochloric acid, phosphoric acid, hydrobromic acid, sulfuric acid
  • organic acids eg, acetic acid, formic acid, propionic acid, fumaric acid, maleic acid
  • Succinic acid tartaric acid, citric acid, malic acid, oxalic acid, benzoic acid, methane
  • the protein of the present invention can be produced from the above-mentioned human or warm-blooded animal cells or tissues by a known method for purifying a protein, or a protein containing a DNA encoding the protein. It can also be produced by culturing the transformant. It can also be produced according to the peptide synthesis method described below.
  • the human or mammalian tissues or cells are homogenized, then extracted with an acid or the like, and the resulting extract is subjected to reverse phase chromatography, ion exchange chromatography, etc. Purification and isolation can be performed by combining chromatography.
  • a commercially available resin for protein synthesis can be generally used.
  • a resin include chloromethyl resin, hydroxymethyl resin, benzylhydrylamine resin, aminomethyl resin, 4-benzyloxybenzyl alcohol resin, 4-methylbenzhydrylamine resin, P AM resin, 4-hide Mouth ximetermethylphenylacetamidomethyl resin, polyacrylamide resin, 4- (2 ', 4, -dimethoxyphenyl-1-hydroxymethyl) phenoxy resin, 4- (2,4,1-dimethoxyphenyl) F moc aminoethyl) phenoxy resin.
  • an amino acid in which the amino group and the side chain functional group are appropriately protected is condensed on the resin in accordance with the sequence of the target protein in accordance with various known condensation methods.
  • proteins or partial peptides are cut out from the resin, and at the same time, various protecting groups are removed.
  • an intramolecular disulfide bond formation reaction is carried out in a highly diluted solution to obtain the target protein or partial peptide. Or obtain their amide form.
  • the protected amino acid may be added directly to the resin along with a racemization inhibitor additive (eg, HOB t, HO OB t) or as a symmetrical anhydride or HOB t ester or HO ⁇ B t ester. It can be added to the resin after the protected amino acid has been activated in advance.
  • a racemization inhibitor additive eg, HOB t, HO OB t
  • the solvent used for activation of the protected amino acid or condensation with the resin can be appropriately selected from solvents known to be usable for protein condensation reactions.
  • acid amides such as N, N-dimethylformamide, N, N-dimethylacetoamide, N-methylpyrrolidone, halogenated hydrocarbons such as methylene chloride, chloroform, trifluoroethanol, etc.
  • Alcohols, sulfoxides such as dimethylsulfoxide, ethers such as pyridine, dioxane, and tetrahydrofuran; acetonitrile; Nitriles such as pionitrile in the mouth, esters such as methyl acetate and ethyl acetate, or an appropriate mixture thereof are used.
  • the reaction temperature is appropriately selected from a range known to be usable for the protein bond formation reaction, and is usually appropriately selected from a range of about ⁇ 20 ° C. to 50 ° C.
  • the activated amino acid derivative is usually used in a 1.5 to 4-fold excess.
  • Examples of the protecting group for the amino group of the starting material include: Z, Boc, t-pentyloxycarbonyl, isopolnyoxycarbonyl, 4-methoxybenzyloxy-caprolponyl, C1-Z, Br—Z Adamantyloxycarponyl, trifluoroacetyl, phthaloyl, formyl, 2-ditrophenylsulfenyl, diphenylphosphinothioyl, Fmoc and the like.
  • the lipoxyl group may be, for example, an alkyl esterified (eg, methyl, ethyl, propyl, butyl, t-butyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, 2-adamantyl, etc.) Cyclic alkyl esterification), aralkyl esterification (for example, benzyl ester, 4-nitrobenzyl ester, 4-methoxybenzyl ester, 4-chlorobenzyl ester, benzhydryl esterification), phenacyl esterification, benzyl ester It can be protected by oxycarbonylhydrazide, t-butoxycarbonylhydrazide, tritylhydrazide, or the like.
  • alkyl esterified eg, methyl, ethyl, propyl, butyl, t-butyl, cyclopentyl,
  • the hydroxyl group of serine can be protected, for example, by esterification or etherification.
  • a group suitable for the esterification for example, a group derived from carbonic acid such as a lower (C ⁇ 6 ) alkanoyl group such as an acetyl group, an aroyl group such as a benzoyl group, a benzyloxycarbonyl group, and an ethoxycarbonyl group is used.
  • groups suitable for etherification include a benzyl group, a tetrahydropyranyl group, and a t-butyl group.
  • groups for protecting the phenolic hydroxyl group of tyrosine for example, B z and C 1 2 one B zl, 2-nitrobenzyl, B r- Z, such as t- butyl are used.
  • protecting group for imidazole of histidine for example, Tos, 4-methoxy-2,3,6-trimethylbenzenesulfonyl, DNP, benzyloxymethyl, Bum, Boc, Trt, Fmoc and the like are used.
  • Activated carboxyl groups of the raw material include, for example, Hydrates, azides, active esters [alcohols (eg, pen phenol, 2,4,5-trichloro phenol, 2,4-dinitrophenol, cyanomethyl alcohol, paranitrophenol, HONB, N-hydroxysuccinimide) , N-hydroxyfurimide, esters with H ⁇ B t)] and the like.
  • active esters eg, pen phenol, 2,4,5-trichloro phenol, 2,4-dinitrophenol, cyanomethyl alcohol, paranitrophenol, HONB, N-hydroxysuccinimide) , N-hydroxyfurimide, esters with H ⁇ B t)
  • the activated amino group of the raw material for example, a corresponding phosphoramide is used.
  • -'Methods for removing (eliminating) protecting groups include, for example, catalytic reduction in a hydrogen stream in the presence of a catalyst such as Pd-black or Pd-carbon, or
  • Acid treatment with sulfonic acid, trifluoromethanesulfonic acid, trifluoroacetic acid or a mixture thereof, base treatment with diisopropylethylamine, triethylamine, piperidine, piperazine, etc., or in liquid ammonia Return by sodium is also used.
  • the elimination reaction by the above-mentioned acid treatment is generally carried out at a temperature of about 120 ° C to 40 ° C.
  • a cation scavenger such as ethyl, dimethyl sulfide, 1,4-butanedithiol or 1,2-ethanedithiol.
  • a cation scavenger such as ethyl, dimethyl sulfide, 1,4-butanedithiol or 1,2-ethanedithiol.
  • the 2,4-dinitrophenyl group used as an imidazole protecting group of histidine is removed by thiophenol treatment
  • the formyl group used as an indole protecting group of tributofan is 1,2-ethanedithiol, 1
  • alkali treatment with dilute sodium hydroxide solution, dilute ammonia and the like.
  • the protection of the functional group which should not be involved in the reaction of the raw material, the protection group, the elimination of the protective group, and the activation of the functional group involved in the reaction can be appropriately selected from known groups or known means.
  • an amide form of a protein or a partial peptide for example, first, the ⁇ -hydroxyl group of the amino acid at the carboxy terminal was protected by amidation to protect it.
  • an ester of a protein or peptide for example, after condensing a strong lipoxyl group of a terminal amino acid with a desired alcohol to form an amino acid ester, in the same manner as in the amide of a protein or peptide, It is possible to obtain a desired protein or an ester of a peptide.
  • the partial peptide or a salt thereof used in the present invention can be produced according to a peptide synthesis method known per se, or by cleaving the protein of the present invention with an appropriate peptidase.
  • a method for synthesizing a peptide for example, any of a solid phase synthesis method and a liquid phase synthesis method may be used. That is, the target peptide can be formed by condensing the remaining peptide or amino acid with the remaining peptide which can constitute the partial peptide used in the present invention for 5 minutes, and removing the protecting group when the product has a protecting group. Can be manufactured.
  • Known condensation methods and elimination of protecting groups include, for example, the methods described in the following (i) to (v).
  • the partial peptide obtained by the above method is a free form, it can be converted to an appropriate salt by a known method or a method analogous thereto, and conversely, when the partial peptide is obtained by a salt, a known method or long as the polynucleotide encoding the protein of t present invention can be converted to free form or another salt by methods analogous thereto, as it contains the base sequence encoding the protein used in the present onset bright described above Anything is acceptable.
  • it is DNA.
  • the DNA may be any of genomic DNA, genomic DNA library, cDNA derived from the above-mentioned cells and tissues, cDNA library derived from the above-mentioned cells and tissues, and synthetic DNA ; , Bacteriophage, plasmid, cosmid, phagemid and the like. Alternatively, it can be directly amplified by Reverse Transcriptase Polymerase Chain Reaction (hereinafter abbreviated as RT-PCR method) using a total RNA or mRNA fraction prepared from the above-mentioned cells and tissues.
  • RT-PCR method Reverse Transcriptase Polymerase Chain Reaction
  • Examples of the DNA encoding the protein of the present invention include: (i) a DNA containing the base sequence represented by SEQ ID NO: 2 or the base sequence represented by SEQ ID NO: 2 under conditions of high stringency end; A DNA encoding a protein containing a hybridizing base sequence and having substantially the same properties as the protein containing the amino acid sequence represented by SEQ ID NO: 1, (ii) the base sequence represented by SEQ ID NO: 4 Or a protein containing a nucleotide sequence that hybridizes under high stringent conditions with a nucleotide sequence represented by SEQ ID NO: 4 and a protein containing the amino acid sequence represented by SEQ ID NO: 3 Any DNA may be used as long as it encodes a protein having the same properties. ⁇
  • '' DN.A that can hybridize with the nucleotide sequence represented by SEQ ID NO: 2 or SEQ ID NO: 4 under high stringency conditions is, for example, represented by SEQ ID NO: 2 or SEQ ID NO: 4.
  • DNA containing a base sequence having about 90% or more, preferably about 95% or more, and preferably about 98% or more homology with the base sequence is used.
  • Hybridization is performed by a method known per se or a method analogous thereto, for example, For example, it can be carried out according to the method described in Molecular Cloning 2nd (J. Sambrook et al., Cold Spring Harbor Lab. Press, 1989). When a commercially available library is used, it can be performed according to the method described in the attached instruction manual. More preferably, it can be performed according to high stringency end conditions.
  • High stringency conditions include, for example, a sodium concentration of about 19 to 40 mM, preferably about 19 to 20 mM, and a temperature of about 50 to 70 ° C, preferably about 60 to 6 ° C. The conditions at 5 ° C are shown. In particular, the case where the sodium concentration is about 19 mM and the temperature is about 65 is most preferable. '
  • DNA encoding the protein containing the amino acid sequence represented by SEQ ID NO: 1 a DNA containing the base sequence represented by SEQ ID NO: 2, 2; SEQ ID NO: 3
  • DNA encoding the protein containing the amino acid sequence represented by SEQ ID NO: 4 a DNA containing the base sequence represented by SEQ ID NO: 4 or the like is used.
  • the DNA encoding the partial peptide of the present invention may be any as long as it contains the nucleotide sequence encoding the partial peptide used in the present invention described above. Further, it may be any one of a genome DNA, a genome DNA library, the above-mentioned cell and tissue-derived cDNA, the above-mentioned cell and tissue-derived cDNA library, and a synthetic DNA.
  • Examples of the DNA encoding the partial peptide used in the present invention include, for example, a DNA having a part of the DNA containing the nucleotide sequence represented by SEQ ID NO: 2 or SEQ ID NO: 4, or SEQ ID NO: A portion of DNA encoding a protein having a nucleotide sequence that hybridizes under high stringent conditions with the nucleotide sequence represented by SEQ ID NO: 2 or SEQ ID NO: 4, and having substantially the same activity as the protein of the present invention. And the like containing DNA.
  • the DNA hybridizable with the nucleotide sequence represented by SEQ ID NO: 2 or SEQ ID NO: 0.4 has the same significance as described above.
  • DNA that completely encodes the protein or partial peptide of the present invention (hereinafter, in the description of the cloning and expression of DNAs encoding them, these may be simply abbreviated as the protein of the present invention).
  • the DNA of the present invention is amplified by PCR using a synthetic DNA primer having a part of the nucleotide sequence encoding the protein of the present invention, or the DNA of the present invention is incorporated into an appropriate vector. Selection can be carried out by hybridization with a DNA fragment encoding a part or all of the protein or labeled with synthetic DNA. The method of hybridization is described in, for example, Molecular Cloning 2nd (J. Sambrook et al., Cold Spring Harbor Lab.
  • the DNA base sequence can be converted using PCR, a known kit, for example, Mutan TM -super Express Km (Takara Shuzo Co., Ltd.), Mutan TM -K (Takara Shuzo Co., Ltd.), etc., using the ODA-LA PCR method. It can be carried out according to a method known per se, such as the gapped duplex method or the Kunkel method, or a method analogous thereto. '
  • the DNA encoding the cloned protein can be used as it is, or it can be used after digestion with a restriction enzyme or adding a linker, if desired.
  • the DNA may have ATG as a translation initiation codon at its 5 'end, and may have TAA, TGA or TAG as a translation termination codon at its 3, terminal. These translation initiation codon and translation termination codon can be added using an appropriate synthetic DNA adapter.
  • the expression vector for the protein of the present invention may be prepared, for example, by (a) cutting out a DNA fragment of interest from DNA encoding the protein of the present invention, and (mouth) converting the DNA fragment downstream of a promoter in an appropriate expression vector. It can be manufactured by connecting to
  • the vector examples include a plasmid derived from E. coli (eg, pBR322, pBR325, pUC12, pUC13), a plasmid derived from Bacillus subtilis (eg, pUB110, pTP5, PC194), a plasmid derived from yeast (eg, SH19, p SH 15)
  • E. coli eg, pBR322, pBR325, pUC12, pUC13
  • Bacillus subtilis eg, pUB110, pTP5, PC194
  • yeast eg, SH19, p SH 15
  • animal viruses such as retrovirus, vaccinia virus, and baculovirus, pAl-11, pXT1 pRcZCMV, pRc / RSV, pcDNA I / Neo, etc. Used.
  • the promoter used in the present invention may be any promoter as long as it is appropriate for the host used for gene expression.
  • examples include SRa promoter, SV40 promoter, LTR promoter, CMV promoter, HSV-TK promoter, and the like.
  • CMV cytomegalovirus
  • SRo CMV (cytomegalovirus) promoter
  • the host is Eshierihia genus bacterium, trp promoter evening one, l ac promoter, re cA promoter, AP L flop Romo - evening -, promoter - and tau 7 promoter one, etc.
  • the host is a bacterium of the genus Bacillus
  • yeast PH05 promoter, PGK promoter, GAP promoter, ADH promoter, etc. are preferable.
  • a polyhedrin promoter, P10 pro overnight, etc. are preferred.
  • the expression vector may contain, in addition to the above, an enhancer, a splicing signal, a polyaddition signal, a selection marker, and an SV40 replication origin (hereinafter sometimes abbreviated as SV40 ori).
  • an enhancer e.g., a splicing signal
  • a polyaddition signal e.g., a selection marker
  • an SV40 replication origin e.g., SV40 ori
  • SV40 ori SV40 replication origin
  • the selection marker include a dihydrofolate reductase (hereinafter sometimes abbreviated as dhfr) gene [methotrexate (MTX) resistance] and an ampicillin resistance gene (hereinafter abbreviated as Amp 1 ). ), Neomycin resistance gene (hereinafter sometimes abbreviated as Ne of, G418 resistance), etc.
  • dfr gene when used as a selection marker using Chinese hamster cells lacking the dh fr gene, Genes can also be selected on
  • a signal sequence suitable for the host may be added to the protein of the present invention. Add to N terminal side.
  • a bacterium belonging to the genus Escherichia a ⁇ signal sequence, a 0A signal sequence, etc. is used. If the host is yeast, MFa signal sequence, SUC2 signal sequence, etc. If the host is an animal cell, the insulin 'signal sequence, the signal signal sequence, and the antibody Molecules and signal sequences can be used.
  • a transformant can be produced.
  • Escherichia bacteria for example, Escherichia bacteria, Bacillus bacteria, yeast, insect cells, insects, animal cells, and the like are used.
  • Escherichia include, for example, Escherichia coli.
  • Bacillus bacteria examples include, for example, Bacillus subtilis (Bacillus).
  • subtil is M I 14 [Gene, 24, 255 (1983)], 207-21 [Journal of Biochemistry, 95, 87 (1984)] and the like are used.
  • yeast examples include, for example, Saccharomyces cerevisiae AH22, AH22R—, NA87-11A, DKD—5D, 20B_12, Schizosaccharomyces pombe N CYC 1913, NCYC 2036, (Pichia pastoris) KM71 or the like is used.
  • Insect cells include, for example, when the virus is Ac NPV, a cell line derived from a larva of night moth (Spodoptera frugiperda cell; S f cell), MG1 cell derived from the midgut of Trichoplusia ni, High derived from egg of Trichoplusia ni Five TM cells,
  • the Sf cells include, for example, Sf9 cells (ATCC CRL1711), Sf21 cells (Vaughn, JL et al., 'In' vipo).
  • insects for example, silkworm larvae are used (Maeda et al.,
  • animal cells examples include monkey cell COS-7, Vero, Chinese hamster cell CHO (hereinafter abbreviated as CHO cell), dh fr gene-deficient Chinese hamster cell CHO (hereinafter CHO (dhfr_) cell). Abbreviations), mouse L cells, mouse AtT-20, mouse myeoma cells, rat GH3, human FL cells, etc. are used.
  • Natl. Acad. Sci. USA, 69, 2110 (1972) or Gene, 17, 107 (1982) can be used to transform Escherichia bacteria.
  • Insect cells or insects can be transformed, for example, according to the method described in Bio / Technology, 6, 47-55 (1988).
  • Transformation of animal cells is performed, for example, according to the method described in Cell Engineering Annex 8 New Cell Engineering Experimental Protocol. 263-267 (1995) (published by Shujunsha), Virology, 52, 456 (1973). be able to.
  • a liquid medium is suitable as a medium for cultivation, and a carbon source necessary for the growth of the transformant is contained therein.
  • carbon sources include nitrogen, such as glucose, dextrin, soluble starch, and sucrose.
  • Sources include, for example, ammonium salts, nitrates, inorganic or organic substances such as corn chipperica, peptone, casein, meat extract, soybean meal, potato extract, and inorganic substances such as calcium chloride, diphosphate Examples include sodium hydrogen and magnesium chloride.
  • yeast extract, vitamins, growth promoting factors and the like may be added.
  • the pH of the medium is preferably about 5-8.
  • Examples of a medium for culturing Escherichia species include, for example, M9 medium containing Darcos and casamino acids [Miller, Journal of Experiments in Molecular Genetics, 431-433, Cold Spring Harbor Laboratory, New York 19 72] is preferred.
  • an agent such as 3B_indolylacrylic acid can be added to make the promoter work efficiently if necessary.
  • the cultivation is usually performed at about 15 to 43 ° C for about 3 to 24 hours, and if necessary, aeration and stirring may be added.
  • the cultivation is usually performed at about 30 to 40 ° C for about 6 to 24 hours, and if necessary, aeration and stirring can be applied. .
  • the medium When culturing a transformant in which the host is yeast, for example, the medium may be Burkholder minimal medium [Bostin, KL et al., Proc. Natl. Acad. Sci. USA, 77, 4505 (1980) And SD medium containing 0.5% casamino acid [Bitter, GA et al., Proc. Natl. Acad. Sci. USA, 81, 5330 (1984)].
  • the pH of the medium is adjusted to about 5-8. Cultivation is usually performed at about 20 ° C to 35 for about 24 to 72 hours, with aeration and agitation as needed.
  • the medium When culturing a transformant in which the host is an animal cell, the medium may be, for example, a MEM medium containing about 5 to 20% fetal bovine serum [3 (: 161 ⁇ 6, 122, 501 (1952)], DMEM) Medium [Virology, 8, 396 (1959)], RPMI 1640 medium [The Journal of the American Medical Association, 199, 519 (1967)], 199 medium [Proceeding of the Society for the Biological Medicine, 73, 1 (1950)].
  • the pH is about 6-8.
  • the cultivation is usually performed at about 30 to 40 ° C for about 15 to 60 hours, and aeration and stirring are added as necessary.
  • the protein of the present invention can be produced in the cells of the transformant, in the cell membrane, or outside the cells.
  • the protein of the present invention can be separated and purified from the culture by, for example, the following method.
  • '' When extracting the protein of the present invention from cultured cells or cells, after culturing, collect the cells or cells by a known method, suspend this in an appropriate buffer, and sonicate, lysozyme and Z or freeze-thaw. After the cells or cells are destroyed by, for example, a method of obtaining a crude protein extract by centrifugation or filtration is appropriately used.
  • a protein modifier such as urea or hydrochloric guanidine in the buffers, may be included because surfactants such as preparative Li Bokun X- 1 0 0 TM.
  • the protein contained in the culture supernatant or the extract thus obtained can be purified by appropriately combining known separation and purification methods.
  • known separation and purification methods include methods that use solubility such as salting out and solvent precipitation, methods that mainly use differences in molecular weight such as dialysis, ultrafiltration, gel filtration, and SDS-polyacrylamide gel electrophoresis, and ion exchange.
  • Methods that use differences in charge such as chromatography, methods that use specific affinity, such as affinity chromatography, methods that use differences in hydrophobicity, such as reversed-phase high-performance liquid chromatography, and isoelectrics.
  • a method utilizing the difference between isoelectric points such as point electrophoresis is used.
  • the protein thus obtained when obtained in a free form, it can be converted to a salt by a method known per se or a method analogous thereto, and conversely, when a protein obtained in the form of a salt, a method known per se or analogous Depending on the method, it can be converted into a free form or other salts.
  • the protein produced by the recombinant was used before or after purification.
  • the modification enzyme can be arbitrarily modified or the polypeptide can be partially removed by the action of the modifying enzyme.
  • the protein modifying enzyme for example, trypsin, chymotrypsin, arginyl endopeptidase, petitinkinase, daricosidase and the like are used.
  • the presence of the protein of the present invention thus produced can be measured by enzyme immunoassay using a specific antibody, Western blotting, or the like.
  • the antibody against the protein or partial peptide of the present invention or a salt thereof may be any of a polyclonal antibody and a monoclonal antibody as long as it can recognize the protein or partial peptide of the present invention or a salt thereof.
  • an antibody against the protein or partial peptide of the present invention or a salt thereof uses the protein of the present invention as an antigen, and is known per se Can be produced according to the method for producing an antibody or antiserum. '' [Preparation of monoclonal antibody]
  • the protein of the present invention is administered to a warm-blooded animal itself or together with a carrier or diluent at a site where antibody production is possible by administration.
  • Complete Freund's adjuvant or incomplete Freund's adjuvant may be administered in order to enhance antibody production upon administration.
  • the administration is usually performed once every 2 to 6 weeks, for a total of about 2 to 10 times.
  • warm-blooded animals to be used include monkeys, rabbits, rabbits, dogs, guinea pigs, mice, rats, sheep, goats, and chickens, and mice and rats are preferably used. '
  • a warm-blooded animal immunized with the antigen for example, an individual with an antibody titer from a mouse is selected, and spleen or lymph nodes are collected 2 to 5 days after the final immunization.
  • a monoclonal antibody-producing hybridoma can be prepared by fusing the contained antibody-producing cells with myeloma cells of the same or different species.
  • the antibody titer in the antiserum can be measured, for example, by reacting the labeled protein described below with the antiserum and then reacting the activity of the labeling agent bound to the antibody. Can be measured.
  • the fusion operation can be performed according to a known method, for example, the method of Köhler and Milstein [Nature, 256, 49.5 (1975)].
  • the fusion promoter include polyethylene glycol (PEG) and Sendai virus.
  • PEG polyethylene glycol
  • Sendai virus Preferably, PEG is used.
  • ⁇ Myeloma cells include, for example, NS_1, P3U1, SP 2/0, AP — Myeloma cells of warm-blooded animals such as 1 are preferred, but P 3 U 1 is preferably used ⁇
  • the preferred ratio of the number of antibody-producing cells (spleen cells) to myeloma cells used is 1: 1 to 20 : About 1 and PEG (preferably PEG 1000 to PEG 6 000) is added at a concentration of about 10 to 80% and incubated at 20 to 40 ° C., preferably 30 to 37 ° C. for 1 to 10 minutes. Thus, cell fusion can be performed more efficiently. ⁇
  • a solid phase onto which a protein antigen is adsorbed directly or together with a carrier is added to the hybridoma culture supernatant.
  • an anti-immunoglobulin antibody (anti-mouse immunoglobulin antibody is used if the cell used for cell fusion is a mouse) or protein A labeled with a radioactive substance or an enzyme, and then bound to the solid phase
  • an anti-immunoglobulin antibody anti-mouse immunoglobulin antibody is used if the cell used for cell fusion is a mouse
  • protein A labeled with a radioactive substance or an enzyme and then bound to the solid phase
  • Monoclonal antibody detection method hybridoma culture supernatant was added to a solid phase to which anti-immune glopurin antibody or protein A was adsorbed, and proteins labeled with radioactive substances, enzymes, etc. were added and bound to the solid phase.
  • a method for detecting a monoclonal antibody is exemplified.
  • 'Selection of a monoclonal antibody can be performed according to a method known per se or a method analogous thereto. Usually, it can be performed in a medium for animal cells supplemented with HAT (hypoxanthine, aminopterin, thymidine).
  • HAT hyperxanthine, aminopterin, thymidine
  • any medium can be used as long as it can grow a hybridoma.
  • RP Ml 1640 medium containing 1-20%, preferably 10-20% fetal bovine serum, GIT medium containing 1-10% fetal bovine serum (Wako Pure Chemical Industries, Ltd.) or for hybridoma culture
  • a serum-free medium SFM-101, Nissui Pharmaceutical Co., Ltd.
  • the culture temperature is usually 20 to 40 ° C, preferably about 37T :.
  • Culture time is usually 5 days to 3 weeks, preferably 1 week to 2 weeks.
  • the culture can be usually performed under 5% carbon dioxide gas.
  • the antibody titer of the hybridoma culture supernatant can be measured in the same manner as the measurement of the antibody titer in the antiserum described above.
  • Monoclonal antibodies can be separated and purified by methods known per se, for example, immunoglobulin separation and purification methods (eg, salting out method, alcohol precipitation method, isoelectric point precipitation method, electrophoresis method, ion exchanger (eg, DEAE) Adsorption / desorption method, ultracentrifugation method, gel filtration method, antigen-binding solid phase or specific purification method of collecting antibodies only with an active adsorbent such as protein A or protein G and dissociating the bond to obtain antibodies) You can do it.
  • immunoglobulin separation and purification methods eg, salting out method, alcohol precipitation method, isoelectric point precipitation method, electrophoresis method, ion exchanger (eg, DEAE) Adsorption / desorption method, ultracentrifugation method, gel filtration method, antigen-binding solid phase or specific purification method of collecting antibodies only with an active adsorbent such as protein A or protein G and dissociating the bond to obtain antibodies
  • the polyclonal antibody of the present invention can be produced by a method known per se or a method analogous thereto. For example, an immunizing antigen (protein antigen) itself or a complex thereof with a carrier protein is formed, and immunization is performed on a warm-blooded animal in the same manner as in the above-described method for producing a monoclonal antibody.
  • the antibody can be produced by collecting the antibody-containing substance for the protein and separating and purifying the antibody.
  • the type of the carrier protein and the mixing ratio of the carrier and the hapten are different from those of the hapten immunized by cross-linking the carrier.
  • Any antibody may be cross-linked at any ratio as long as the antibody can be efficiently produced.
  • serum serum albumin, thyroglobulin, hemocyanin, etc. are used in a weight ratio of about 0.1 to 1 for hapten.
  • a method of coupling at a rate of 1 to 20, preferably about 1 to 5 is used.
  • various condensing agents can be used for force coupling between the hapten and the carrier.
  • daltaraldehyde carbodiimide, a maleimide active ester, an active ester reagent containing a thiol group or a dithioviridyl group, or the like is used.
  • the condensation product is administered to a warm-blooded animal at a site where antibody production is possible or together with a carrier or diluent.
  • Complete Freund's adjuvant or incomplete Freund's adjuvant may be administered to increase antibody production during administration.
  • the administration is usually performed once every about 2 to 6 weeks, about 3 to 10 times in total.
  • the polyclonal antibody can be collected from blood, ascites, etc. of a warm-blooded animal immunized by the above method. Preferably, it can be collected from the blood.
  • the measurement of the polyclonal antibody titer in the antiserum can be performed in the same manner as the measurement of the antibody titer in the antiserum described above. Separation and purification of the polyclonal antibody can be performed according to the same immunoglobulin separation and purification method as in the above-described separation and purification of the monoclonal antibody.
  • DNA encoding the protein or partial peptide of the present invention (hereinafter, these DNAs may be abbreviated as the DNA of the present invention in the description of the antisense polynucleotide), or substantially complementary to the base sequence of
  • An antisense polynucleotide containing a nucleotide sequence complementary to the DNA or a part thereof has a nucleotide sequence complementary to or substantially complementary to the nucleotide sequence of the DNA of the present invention, and Any antisense polynucleotide may be used as long as it has an action of suppressing expression, but antisense DNA is preferable.
  • the nucleotide sequence substantially complementary to the DNA of the present invention refers to, for example, the entire nucleotide sequence or a partial nucleotide sequence of the nucleotide sequence complementary to the DNA of the present invention (that is, the complementary strand of the DNA of the present invention).
  • Base sequences having about 70% or more, preferably about 80% or more, more preferably about 90% or more, and most preferably about 95% or more homology are exemplified.
  • the nucleotide sequence of the portion encoding the N-terminal site of the protein of the present invention for example, An antisense polynucleotide having a homology of about 70% or more, preferably about 80% or more, more preferably about 90% or more, and most preferably about 95% or more with a complementary strand of a base sequence near the start codon, etc.
  • an antisense polynucleotide that directs RNA degradation by RNaseH it is at least about 70%, preferably at least about 80%, more preferably at least about 70% of the complementary strand of the entire nucleotide sequence of the DNA of the present invention including introns.
  • Antisense polynucleotides having a homology of about 90% or more, and most preferably about 95% or more, are respectively suitable.
  • a base sequence complementary to or substantially complementary to the base sequence of DNA, or an antisense polynucleotide having a part thereof preferably, for example, the base represented by SEQ ID NO: 2 or SEQ ID NO: 4
  • An antisense polynucleotide having a complementary nucleotide sequence or a part thereof to the nucleotide sequence of the DNA containing the sequence (more preferably, a nucleotide sequence represented by SEQ ID NO: 2 or SEQ ID NO: 4;
  • An antisense polynucleotide having a base sequence complementary to the base sequence of DNA or a part thereof is usually composed of about 10 to 40 bases, preferably about 15 to 30 bases.
  • the phosphate residues (phosphates) of each nucleotide constituting the antisense DNA are, for example, phosphorothioate, methylphosphonate, and phosphorodithionate. It may be substituted by an acid residue.
  • the sugar (doxy lipose) of each nucleotide may be substituted with a chemically modified sugar structure such as 2,1 O-dimethylation, or the base moiety (pyrimidine, purine) may be chemically modified. Any compound may be used as long as it hybridizes to DNA having the nucleotide sequence represented by SEQ ID NO: 2.
  • These antisense polynucleotides can be produced using a known DNA synthesizer or the like.
  • an antisense polynucleotide capable of inhibiting the replication or expression of the protein gene of the present invention is cloned or a nucleotide sequence of a DNA encoding the determined protein.
  • a polynucleotide can hybridize with the RNA of the protein gene of the present invention, inhibit the synthesis or function of the RNA, or interact with the protein-related RNA of the present invention.
  • the expression of the protein gene of the present invention can be regulated and controlled.
  • Polynucleotides that are complementary to the selected sequence of the protein-related RNA of the present invention, and that can specifically hybridize with the protein-related RNA of the present invention, can be used in vivo and in vitro. It is useful for regulating and controlling the expression of the protein gene of the invention, and is also useful for treating or diagnosing diseases. is there.
  • the term "corresponding" means having homology or being complementary to a specific sequence of nucleotides, base sequences or nucleic acids including genes.
  • nucleotide, nucleotide sequence or nucleic acid and a peptide (protein) usually refers to the amino acid of the peptide (protein) as directed by the nucleotide (nucleic acid) sequence or its complement. ing. 5 'end hairpin loop of protein gene, 5, end 6—base pair. Repeat, 5 terminal untranslated region, polypeptide translation initiation codon, protein coding region, ORF translation stop codon, 3 terminal untranslated region, 3 terminal palindrome region, and 3 terminal hairpin loop are preferred target regions But any region within the protein gene can be selected as the target.
  • the relationship between the target nucleic acid and the polynucleotide complementary to at least a part of the target region is as follows:
  • the relationship between the target nucleic acid and the polynucleotide that can hybridize with the target is:
  • Antisense polynucleotides include polynucleotides containing 2-dexoxy-D-ribose, polynucleotides containing D-lipose, and other types of polynucleotides that are N-glycosides of purine or pyrimidine bases.
  • Other polymers having a nucleotide or non-nucleotide backbone eg, commercially available protein nucleic acids and synthetic sequence-specific nucleic acid polymers
  • other polymers containing special linkages such as DNA or DNA
  • RNA hybrids can be double-stranded DNA, single-stranded DNA, double-stranded RNA, single-stranded RNA, and even DNA: RNA hybrids, and can also be unmodified polynucleotides (or unmodified polynucleotides).
  • Oligonucleotides and those with known modifications, for example, those with labels known in the art, those with caps, methylated ones, and one or more natural nucleotides as analogs Substituted with an intramolecular nucleotide, for example, having an uncharged bond (eg, methylphosphonate, phosphotriester, phosphoramidate, olebamate, etc.), a charged bond or a sulfur-containing bond (for example, those having phosphorothioate, phosphorodithioate, etc., for example, proteins (nucleases) ⁇ , Nucleic acid ⁇ f ⁇ Inhibitors, toxins, antibodies, signal peptides, poly-L-lysine, etc., and those with side chains such as sugars (eg, monosaccharides), etc.
  • an intramolecular nucleotide for example, having an uncharged bond (eg, methylphosphonate, phosphotriester, phosphoramidate, o
  • a calate compound eg, acridine, psoralen, etc.
  • a chelate compound eg, metal, radioactive metal, boron, oxidizable metal, etc.
  • an alkylating agent for example, a panomer-type nucleic acid.
  • nucleoside may include not only those containing purine and pyrimidine bases but also those containing other modified heterocyclic bases. . Such modifications may include methylated purines and pyrimidines, acylated purines and pyrimidines, or other heterocycles.
  • Modified nucleotides and modified nucleotides may also be modified at the sugar moiety, e.g., where one or more hydroxyl groups are replaced with halogens, aliphatic groups, etc., or functional groups such as ethers, amines, etc. May be converted to
  • the antisense polynucleotide of the present invention is RNA, DNA, or a modified nucleic acid (RNA, DNA).
  • modified nucleic acid include, but are not limited to, a sulfur derivative of a nucleic acid, a thiophosphoate derivative, and a polynucleoside amide which is resistant to decomposition of a polynucleonucleoside amide.
  • the antisense nucleic acid of the present invention can be preferably designed according to the following policy. That is, to make the antisense nucleic acid more stable in the cell, to make the antisense nucleic acid more cell-permeable, to make the target sense strand more compatible, and to be toxic. Minimize the toxicity of antisense nucleic acids.
  • the antisense nucleic acids of the present invention may contain altered or modified sugars, bases, or bonds, and may be in special forms such as ribosomes and microspheres. May be provided, applied from gene therapy, or given in an added form.
  • polycations such as polylysine, which acts to neutralize the charge of the phosphate skeleton
  • lipids which increase the interaction with the cell membrane or increase the uptake of nucleic acids (
  • hydrophobic substances such as phospholipid and cholesterol
  • Preferred lipids for addition include cholesterol and its derivatives (eg, cholesteryl chromate formate, cholic acid, etc.).
  • nucleic acids can be attached via a base, sugar, or intramolecular nucleoside bond.
  • Other groups include cap groups specifically arranged at the 3 'end or 5' end of nucleic acids for preventing degradation by nucleases such as exonuclease and RNase.
  • capping groups include, but are not limited to, hydroxyl-protecting groups known in the art, including glycols such as polyethylene glycol and tetraethylene glycol.
  • the inhibitory activity of the antisense nucleic acid can be examined using the transformant of the present invention, the in vivo or in vitro gene expression system of the present invention, or the in vivo or in vitro translation system of the protein of the present invention.
  • the nucleic acid can be applied to cells by various known methods.
  • the protein or partial peptide of the present invention or a salt thereof hereinafter, sometimes abbreviated as the protein of the present invention
  • the protein or partial peptide of the present invention will be described.
  • DNA of the present invention Coding DNA
  • antibody against the protein or partial peptide of the present invention or a salt thereof hereinafter sometimes abbreviated as the antibody of the present invention
  • DNA of the present invention an antisense polynucleotide as an antisense polynucleotide and the like will be described.
  • the protein of the present invention has increased expression in osteoarthritis cartilage and is secreted extracellularly, it can be used as a disease marker. That is, it is useful as a marker for early diagnosis of osteoarthritis cartilage and the like, determination of the severity of symptoms, prediction of disease progression, and determination of therapeutic effects.
  • the antisense of the present invention Spolynucleotides, compounds that regulate the activity of the protein of the present invention or salts thereof, and bodies of the present invention include, for example, cartilage dysplasia, bone dysplasia, osteoporosis, osteoarthritis, rheumatoid arthritis, arthritis, It can be used as a prophylactic and / or therapeutic agent for bone and joint diseases such as synovitis, metabolic arthrosis or joint disorders due to sports (eg, tennis elbow).
  • the protein of the present invention is increased in expression in osteoarthritis cartilage, and has a chondrocyte differentiation regulating activity, a chondrocyte binding activity, etc., thereby modulating (promoting or inhibiting) the activity of the protein of the present invention.
  • the protein of the present invention is useful as a reagent for screening a compound or a salt thereof that regulates the activity of the protein of the present invention.
  • the present invention provides a method for screening a compound or a salt thereof that regulates the activity of the protein of the present invention, which comprises using the protein of the present invention.
  • the present invention provides (1) promoting or inhibiting the activity of the protein of the present invention characterized by using the protein of the present invention (eg, chondrocyte differentiation regulating activity, chondrocyte binding activity, etc.)
  • a method for screening a compound or a salt thereof hereinafter may be abbreviated as an accelerator or an inhibitor, respectively.
  • activity of the protein of the present invention eg, chondrocyte differentiation regulating activity, binding activity to cartilage cells, etc.
  • activity of a mixture of the protein of the present invention and a test compound eg, And a salt or a salt thereof that regulates (promotes or inhibits) the activity of the protein of the present invention, which is characterized by comparing the activity of the protein of the present invention with the activity of regulating the differentiation of chondrocytes and the activity of binding to chondrocytes.
  • chondrocyte differentiation regulating activity can be performed according to a known method, for example, the method described in J Cell Biochem 67, 498-513 (1997) or a method based thereon. It can be measured according to the law.
  • the protein of the present invention is obtained from a culture supernatant or a cell extract of a cell capable of producing the protein of the present invention according to a known method (eg, an immunoaffinity column).
  • the protein and the test compound are added to the cultured chondrocytes and cultured at 37 ° C for 7 to 21 days.
  • the activity of regulating chondrocyte differentiation is measured by evaluating the proteodalican content or phosphatase activity of chondrocytes according to the method described in the above literature.
  • the test compound may be added to the cultured chondrocytes before, simultaneously with, or after the addition of the protein of the present invention, and preferably before the addition of the protein of the present invention.
  • the measurement of the binding activity to chondrocytes can be performed according to a known method.
  • a known method eg, immunoaffinity column
  • the chemiluminescent substrate of the enzyme e.g., Disodium 2-chloro-5- (4-methoxyspiro ⁇ l, 2-dioxetane-3,2 '_ (5'-chloro) tricyclo [3.3.1.1. 1. 3, 7] decan ⁇ -4-yl) -l-phenyl phosphate, etc.
  • the test compound may be added to the cultured chondrocytes before the addition of the protein of the present invention, simultaneously with or after the addition of the protein of the present invention, and preferably before the addition of the protein of the present invention.
  • a host transformed with a vector containing the above-described DNA encoding the protein of the present invention
  • a host for example, animal cells such as CHO cells are preferably used.
  • a transformant in which the protein of the present invention is expressed on a cell membrane by culturing by the method described above is preferably used.
  • the method for culturing cells capable of expressing the protein of the present invention comprises the steps of: It is the same as the culture method.
  • Test compounds include, for example, peptides, proteins, non-peptidic compounds, synthetic compounds, fermentation products, cell extracts, plant extracts, animal tissue extracts, and the like.
  • a test compound which increases the activity in the case of the above (ii) by about 20% or more, preferably 30% or more, more preferably about 50% or more as compared with the case of the above (i) As the compound that promotes the activity of the protein of the present invention, the activity in the case of the above (ii) is about 20% or more, preferably 30% or more, more preferably about 50% as compared with the case of the above (i).
  • a test compound that reduces the above can be selected as a compound that inhibits the activity of the protein of the present invention.
  • the compound having the activity of promoting the activity of the protein of the present invention is useful as a safe and low-toxic drug for enhancing the action of the protein of the present invention.
  • the compound having the activity of inhibiting the activity of the protein of the present invention is a safe and low toxic drug for suppressing the physiological activity of the protein of the present invention, for example, cartilage dysplasia, bone dysplasia, osteoporosis, osteoarthritis It is useful as a prophylactic / therapeutic agent for bone-joint diseases such as rheumatism, rheumatoid arthritis, arthritis, synovitis, metabolic arthropathy or joint disorders due to sports (eg, tennis elbow, etc.).
  • Compounds or salts thereof obtained using the screening method or the screening kit of the present invention include, for example, peptides, proteins, non-peptidic compounds, synthetic compounds, fermentation products, cell extracts, It is a compound selected from plant extracts, animal tissue extracts, and plasma.
  • the salt of the compound those similar to the aforementioned salts of the peptide of the present invention are used.
  • the expression of the gene encoding the protein of the present invention also increases in osteoarthritis cartilage, a compound that inhibits (inhibits or promotes, preferably inhibits) the expression of the gene encoding the protein of the present invention.
  • bones such as cartilage dysplasia, bone dysplasia, osteoporosis, osteoarthritis, rheumatoid arthritis, arthritis, synovitis, metabolic arthrosis or joint disorders due to sports (eg tennis elbow etc.) It can be used as a preventive and therapeutic agent for joint diseases. .
  • the DNA of the present invention corresponds to the gene encoding the protein of the present invention. It is useful as a reagent for screening a compound or a salt thereof that regulates expression.
  • the screening methods include (iii) culturing cells capable of producing the protein of the present invention, and (iv) culturing cells capable of producing the protein of the present invention in the presence of the test compound. And a screening method characterized by performing a comparison with the above.
  • the expression level of the gene (specifically, the amount of the protein of the present invention or the amount of mRNA encoding the protein) is measured and compared. .
  • test compound and cells having the ability to produce the protein of the present invention include the same cells as described above.
  • the amount of the protein is measured by a known method, for example, using an antibody recognizing the protein of the present invention, and analyzing the protein present in a cell extract or the like according to a method such as Western analysis, ELISA, or a method analogous thereto. Can be measured.
  • the amount of mRNA can be measured by a known method, for example, Northern hybridization using a nucleic acid containing the nucleotide sequence of SEQ ID NO: 2 or SEQ ID NO: 4 or a part thereof as a probe, or SEQ ID NO: 1 as a primer. : 2 or SEQ ID NO: 4 Using a nucleic acid containing the base sequence or a part thereof, it can be measured according to a PCR method or a method analogous thereto.
  • the gene expression level in the case (iv) is increased by about 20% or more, preferably 30% or more, more preferably about 50% or more as compared with the case of the above (iii).
  • a test compound that inhibits about 20% or more, preferably 30% or more, more preferably about 50% or more of the present invention It can be selected as a compound that suppresses the expression of a gene encoding a protein.
  • the present invention provides a transformant transformed with a recombinant DNA having a reporter gene linked downstream (under expression control) of the transcription regulatory region of the gene of the protein of the present invention in the presence and absence of a test compound.
  • a method for screening a compound that regulates (promotes or inhibits) the transcriptional regulatory activity which comprises measuring and comparing the reporter activities, and a screening kit for this method are also provided. .
  • Examples of the transcription regulatory region of the gene of the protein of the present invention include DNA having the same or substantially the same nucleotide sequence as the nucleotide sequence represented by SEQ ID NO: 8. '
  • DNA containing a nucleotide sequence substantially identical to the nucleotide sequence represented by SEQ ID NO: 8 has a nucleotide sequence that hybridizes under high stringency conditions, and is substantially the same as SEQ ID NO: 8. Any DNA may be used as long as it has transcription regulation activity (eg, promoter activity).
  • Examples of the DNA that can hybridize with the nucleotide sequence represented by SEQ ID NO: 8 under high stringent conditions include, for example, about 50% or more, preferably about 60%, of the nucleotide sequence represented by SEQ ID NO: 8. % Or more, more preferably about 70% or more, more preferably about 80% or more, particularly preferably about 90% or more, and most preferably about 95% or more. DNA or the like is used.
  • Hybridization can be performed by a method known per se or a method analogous thereto, for example, a method described in Molecular 'Cloning (Molecular Cloning) 2nd (J. Sambrook. Et al., Cold Spring Harbor Lab. Press, 1989). It can be done according to the method. When a commercially available library is used, it can be performed according to the method described in the attached instruction manual. More preferably, the reaction can be carried out in accordance with eight stringent conditions.
  • High stringency conditions include, for example, a sodium concentration of about 19 to 40 mM, preferably about 19 to 20 mM, and a temperature of about 50 to 70 ° C, preferably about 60 to 70 ° C.
  • the conditions at 65 ° C are shown. In particular, the case where the sodium concentration is about 19 mM and the temperature is about 65 ° C is most preferable.
  • the transcription regulatory region eg, DNA containing a nucleotide sequence substantially identical to the nucleotide sequence represented by SEQ ID NO: 8
  • the transcription regulatory region is substantially the same as the nucleotide sequence represented by SEQ ID NO: 8.
  • a sequence obtained by adding an upstream genomic DNA sequence to a basic nucleotide sequence preferably, a promoter region.
  • the number of bases is, for example, about 10 Kb or less, preferably about 5 Kb or less, more preferably about 2 Kb or less, and most preferably about 1 Kb or less.
  • the c- repo overnight gene includes a sequence substantially the same as the base sequence represented by SEQ ID NO: 8 and further added with an intron sequence of the gene of the protein of the present invention.
  • E.g., 1 ac Z galactosidase gene), chloramphenic acid luciferase transferase (CAT), luciferase, growth factor, / 3-darctic nidase, alkaline phosphatase, Green f luorescent Protein (GFP), 3 _ lacquerase and the like are used.
  • a test compound that increases the amount of the repo overnight gene product (eg, mRNA, protein) by measuring the amount of the repo overnight gene product using a known method is used to regulate the transcription of the protein of the present invention. It can be selected as a compound having an activity of controlling (particularly promoting) its activity (preferably, promoter activity), that is, a compound having an activity of promoting the expression of the protein of the present invention. Conversely, a test compound that reduces the amount of the reporter gene product is a compound that has the activity of controlling (particularly inhibiting) the transcriptional regulatory activity (preferably, promoter activity) of the protein of the present invention, ie, the present invention. Can be selected as compounds having an activity of inhibiting the expression of the protein.
  • test compound is, for example, a compound selected from peptides, proteins, non-peptidic compounds, synthetic compounds, fermentation products, cell extracts, plant extracts, animal tissue extracts, and plasma.
  • the culture of the transformant can be carried out in the same manner as the above-mentioned transformant having the protein of the present invention.
  • the vector construction of the reporter gene and the Atsey method can be performed according to known techniques ( For example, Molecular Biotechnology 13, 29-43, 1999).
  • the screening kit of the present invention contains the protein or partial peptide of the present invention or a salt thereof, or a cell having the ability to produce the protein or partial peptide of the present invention.
  • the compound or a salt thereof obtained by using the screening method or the screening kit of the present invention may be a test compound as described above, for example, a peptide or a protein. Or a salt thereof, selected from the group consisting of protein, non-peptidic compound, synthetic compound, fermentation product, cell extract, plant extract, animal tissue extract, and plasma.
  • a compound or a salt thereof that regulates (preferably inhibits) the activity of the protein of the present invention a compound or a salt thereof that regulates the expression of a gene encoding the protein of the present invention
  • the compound having an action of controlling the promoter activity or a salt thereof is, for example, a cartilage abnormal condition, a bone abnormal condition, osteoporosis, osteoarthritis, rheumatoid arthritis, arthritis, synovitis, metabolic arthropathy or It is useful for preventing and treating bone and joint diseases such as joint disorders caused by sports (eg, tennis elbows).
  • a compound or a salt thereof obtained by using the screening method or the screening kit of the present invention is used as the above-mentioned prophylactic / therapeutic agent, it can be formulated into a preparation according to a conventional method.
  • compositions for oral administration include solid or liquid dosage forms, specifically tablets (including sugar-coated tablets and film-coated tablets), pills, granules, powders, capsules (soft capsules). ), Syrups, emulsions, suspensions and the like.
  • Such a composition is produced by a method known per se and contains a carrier, diluent or excipient commonly used in the field of pharmaceuticals.
  • a carrier for example, lactose, starch, sucrose, magnesium stearate and the like are used as carriers and excipients for tablets.
  • compositions for parenteral administration include injections, suppositories, and the like.
  • Injections include intravenous injections, subcutaneous injections, intradermal injections, intramuscular injections, intravenous injections, and joint injections. Includes dosage forms such as injections.
  • Such injections are prepared according to a method known per se, for example, by dissolving, suspending or emulsifying the antibody or a salt thereof in a sterile aqueous or oily liquid commonly used for injections.
  • aqueous liquid for injection for example, physiological saline, isotonic solution containing glucose and other auxiliary agents and the like are used, and a suitable solubilizing agent, for example, alcohol (eg, ethanol), polyalcohol ( Eg, propylene glycol, polyethylene glycol), non-y Surfactant [eg, Polysorbate 80, HCO-50
  • oily liquid for example, sesame oil, soybean oil, etc. are used, and benzyl benzoate, benzyl alcohol, etc. may be used in combination as a solubilizing agent.
  • the prepared injection is usually filled in an appropriate ampoule.
  • Suppositories used for rectal administration are prepared by mixing the above compound or a salt thereof with a conventional suppository base. .
  • the above-mentioned oral or parenteral pharmaceutical composition is conveniently prepared in the form of a dosage unit so as to conform to the dose of the active ingredient.
  • dosage unit forms include tablets, pills, capsules, injections (ampoules), and suppositories, and usually 5 to 500 mg per dosage unit form, and especially 5 to 500 mg for injections.
  • the above-mentioned antibody is contained in the following form: I 00 mg, and other dosage forms 10 to 250 mg. ,
  • compositions may contain other active ingredients as long as the compound and the above-mentioned compound do not cause an undesirable interaction.
  • the preparations obtained in this way are safe and low toxic, for example, in humans or in warm-blooded animals (eg, mice, rats, puppies, sheep, pigs, puppies, puppies, birds, cats, dogs, Monkeys, chimpanzees, etc.) orally or parenterally.
  • warm-blooded animals eg, mice, rats, puppies, sheep, pigs, puppies, puppies, birds, cats, dogs, Monkeys, chimpanzees, etc.
  • the dose of the compound or a salt thereof varies depending on its action, target disease, subject of administration, route of administration, and the like. For example, it inhibits the activity of the protein of the present invention for the purpose of treating osteoarthritis.
  • the compound or a salt thereof is administered orally, generally, for an adult (assuming a body weight of 60 kg), the compound or a salt thereof is administered in an amount of about 0 to 10 mg / day, preferably about 1.0 to 50 mg / day. More preferably, about 10 to 20 mg is administered.
  • the single dose of the compound or its salt may vary depending on the administration target, the target disease and the like.
  • the present invention is intended for the treatment of osteoarthritis.
  • the compound or a salt thereof which inhibits the activity of the protein of the present invention is administered to an adult (with a body weight of 60 kg) in the form of an injection
  • the compound or a salt thereof is preferably 0.01 to 3 Omg per day, preferably Is about 0. l 20m g, more preferably about 0.1-1 O mg, is administered by intra-articular injection.
  • the dose can be administered in terms of weight per 60 kg.
  • the protein of the present invention contributes to the differentiation and proliferation of chondrocytes and plays an important role in the development of osteoarthritis.
  • the DNA encoding the protein of the present invention is abnormal or defective, or when the expression level of the protein of the present invention is reduced, for example, abnormal cartilage formation, abnormal bone formation, osteoporosis, etc.
  • Various diseases such as bone and joint diseases such as osteoarthritis, rheumatoid arthritis, arthritis, synovitis, metabolic arthropathy or joint disorders due to sports (eg, tennis elbows) occur.
  • the protein of the present invention and DNA encoding the protein of the present invention include, for example, chondrodysplasia, bone dysplasia, osteoporosis, osteoarthritis, rheumatoid arthritis, arthritis, synovitis, metabolic lanchiasis It can also be used as a medicine for preventing or treating bone and joint diseases such as sports-related joint disorders (eg, tennis elbows). .
  • the protein encodes the protein.
  • administering the DNA to the patient and expressing the protein of the present invention in vivo inserting the above DNA into cells, expressing the protein of the present invention, and then transplanting the cells to the patient.
  • the role of the protein of the present invention in the patient can be sufficiently or normally exerted.
  • a of the present invention may be used alone or in the form of a retrovirus vector, adenovirus vector, adenovirus associated virus vector, or the like. After insertion into the appropriate vector, human or other warm blood Can be administered.
  • the polypeptide of the present invention (eg, DNA) may be formulated as it is or in combination with a physiologically acceptable carrier such as an auxiliary for promoting uptake, and a catheter such as a gene gun or a hydrogel catheter may be used. Can be administered.
  • the protein of the present invention is used as the above-mentioned prophylactic / therapeutic agent
  • the protein is purified to at least 90%, preferably at least 95%, more preferably at least 98%, and even more preferably at least 99%. It is preferable to use those that have been prepared.
  • the protein of the present invention can be used, for example, in the form of tablets, capsules, elixirs, microcapsules, etc., which are sugar-coated as required, orally, or with water or other pharmaceutically acceptable liquids. It can be used parenterally in the form of injections, such as sterile solutions or suspensions.
  • the protein or the like of the present invention may be used together with physiologically acceptable carriers, flavoring agents, excipients, vehicles, preservatives, stabilizers, binders, and the like in a unit dosage form generally required for the practice of preparations. It can be manufactured by mixing. The amount of the active ingredient in these preparations is such that a suitable dosage in the specified range can be obtained.
  • additives that can be incorporated into tablets, capsules, etc. include, but are not limited to, binders such as zelatin, corn starch, tragacanth, gum arabic, excipients such as crystalline cellulose, corn starch, gelatin, and alginic acid. Swelling agents such as sodium stearate, sweeteners such as sucrose, lactose or saccharin, and flavoring agents such as peppermint, cocoa oil or cellulose. When the unit dosage form is a capsule, a liquid carrier such as an oil or fat may be further contained in the above-mentioned dinner material.
  • Sterile compositions for injection should be formulated according to the normal formulation practice, for example, dissolving or suspending the active substance in vehicles such as water for injection, or naturally occurring vegetable oils such as sesame oil or coconut oil. Can be.
  • aqueous liquids for injection examples include physiological saline, isotonic solutions containing glucose and other adjuvants (eg, D-sorbitol, D-mannitol, sodium chloride, etc.).
  • Solubilizers eg, alcohols (eg, ethanol, etc.)-, polyalcohols (eg, propylene glycol, polye It may be used in combination with a nonionic surfactant (eg, polysorbate 80 TM , HCO-50, etc.).
  • examples of the oily liquid include sesame oil and soybean oil, and may be used in combination with solubilizers such as benzyl benzoate and benzyl alcohol.
  • buffers eg, phosphate buffer, sodium acetate buffer, etc.
  • soothing agents eg, benzalkonium chloride, hydrochloride proforce, etc.
  • stabilizers eg, human serum albumin, polyethylene glycol, etc.
  • Preservatives eg, benzyl alcohol, phenol, etc.
  • antioxidants eg, antioxidants and the like.
  • the prepared injection solution is usually filled in a suitable ampoule.
  • the vector into which the DNA of the present invention has been inserted is also formulated in the same manner as described above, and is usually used parenterally.
  • the preparations obtained in this way are safe and have low toxicity, for example, warm-blooded animals (eg, humans, rats, mice, guinea pigs, egrets, birds, higgies, bush, horses, cats, cats) , Dogs, monkeys, chimpanzees, etc.).
  • warm-blooded animals eg, humans, rats, mice, guinea pigs, egrets, birds, higgies, bush, horses, cats, cats
  • the dosage of the protein of the present invention varies depending on the target disease, the administration subject, the administration route, and the like.
  • the protein of the present invention when administered for the purpose of treating osteoarthritis, it is generally used.
  • the single dose of the protein when administered parenterally, the single dose of the protein varies depending on the administration subject, target disease, and the like.
  • the protein of the present invention is in the form of an injection for the treatment of osteoarthritis.
  • the protein When administered to an adult (assuming a body weight of 60 kg), the protein is injected into the affected area at a rate of about 0.01 to 30 mg, preferably about 0.1 to 2 Omg, more preferably about 0.1 to 10 mg per day. It is convenient to administer by doing so. In the case of other animals, the dose can be administered in terms of weight per 60 kg.
  • the antibody of the present invention can specifically recognize the protein of the present invention, It can be used for quantification of the protein of the present invention in a test wave, particularly for quantification by a sandwich immunoassay.
  • the antibody of the present invention and the test solution and the labeled protein of the present invention are competitively treated, and the ratio of the labeled protein of the present invention bound to the antibody is measured.
  • a method for quantifying the protein of the present invention in a test solution and
  • one antibody is an antibody that recognizes the N-terminal of the protein of the present invention and the other antibody is an antibody that reacts with the C-terminal of the protein of the present invention.
  • the protein of the present invention can be quantified using a monoclonal antibody against the protein of the present invention (hereinafter, sometimes referred to as the monoclonal antibody of the present invention), and can also be detected by tissue staining or the like.
  • the antibody molecule itself may be used, or the F (ab ') 2 , Fab', or Fab fraction of the body molecule may be used.
  • the method for quantifying the protein of the present invention using the antibody of the present invention is not particularly limited, and may be an antibody, an antigen, or an antibody-antigen complex corresponding to the amount of antigen (eg, the amount of protein) in the test solution. Any method may be used as long as it is a method for detecting the amount of ⁇ by chemical or physical means and calculating from a standard curve prepared using a standard solution containing a known amount of antigen. For example, nephrometry, a competitive method, an immunometric method, and a sandwich method are suitably used. From the viewpoint of sensitivity and specificity, it is particularly preferable to use a sandwich method described later.
  • a labeling agent used in a measurement method using a labeling substance for example, a radioisotope, an enzyme, a fluorescent substance, a luminescent substance and the like are used.
  • the radioisotope e.g., [125 1], [131 1], [3 ⁇ 4], and the like are used [14 c].
  • the enzyme a stable enzyme having a large specific activity is preferable.
  • i3_galactosidase, 0-Dalkosidase, alkaline phosphatase; ⁇ , peroxidase, malate dehydrogenase, etc. are used.
  • fluorescent substance for example, fluorescamine, fluorescein isothiocyanate, or the like is used.
  • luminescent substance for example, luminol, luminol derivative, luciferin, lucigenin and the like are used.
  • a biotin-avidin system can be used for binding the antibody or antigen to the labeling agent.
  • the carrier include insoluble polysaccharides such as agarose, dextran, and cellulose; synthetic resins such as polystyrene, polyacrylamide, and silicon; and glass.
  • the test solution is reacted with the insolubilized monoclonal antibody of the present invention (primary reaction), and further reacted with another labeled monoclonal antibody of the present invention (secondary reaction).
  • primary reaction the insolubilized monoclonal antibody of the present invention
  • secondary reaction another labeled monoclonal antibody of the present invention
  • the primary reaction and the secondary reaction may be performed in the reverse order, may be performed simultaneously, or may be performed at staggered times.
  • the labeling agent and the method of insolubilization can be based on those described above.
  • the antibody used for the solid phase antibody or the labeling antibody is not necessarily required to be one type, and two or more types of antibodies are used for the purpose of improving the measurement sensitivity and the like. Mixtures may be used.
  • the monoclonal antibody of the present invention used in the primary reaction and the secondary reaction is preferably an antibody having a different site to which the protein of the present invention binds.
  • the antibody used in the primary reaction and the secondary reaction are preferably, for example, when the antibody used in the secondary reaction recognizes the C-terminal of the protein of the present invention, the antibody used in the primary reaction is preferably Is an antibody that recognizes other than the C-terminal, for example, the N-terminal.
  • the monoclonal antibody of the present invention can be used in a measurement system other than the sandwich method, for example, a competitive method, an immunometric method, or a nephrometry.
  • a competitive method the antigen in the test solution and the labeled antigen are allowed to react competitively with the antibody, and then the unreacted labeled antigen (F) and the labeled antigen bound to the antibody (B) are separated.
  • F labeled antigen
  • B labeled antigen bound to the antibody
  • a soluble phase is used as the antibody
  • B / F separation is performed using polyethylene glycol
  • a liquid phase method using a second antibody against the antibody or a solid phased antibody is used as the first antibody.
  • An immobilization method using a soluble antibody as the first antibody and an immobilized antibody as the second antibody is used.
  • the antigen in the test solution and the immobilized antigen are subjected to a competitive reaction with a certain amount of the labeled antibody, and then the solid phase and the liquid phase are separated.
  • the antigen is allowed to react with an excessive amount of the labeled antibody, then the immobilized antigen is added, and the unreacted labeled antibody is bound to the solid phase, and then the solid phase and the liquid phase are separated.
  • the amount of label in either phase is measured to determine the amount of antigen in the test solution.
  • nephrometry the amount of insoluble sediment generated as a result of an antigen-antibody reaction in a gel or in a solution is measured. Even when the amount of antigen in the test solution is small and only a small amount of sediment is obtained, laser nephrometry utilizing scattering by a laser is preferably used.
  • the protein of the present invention can be quantified with high sensitivity by using the antibody of the present invention.
  • abnormal cartilage formation, abnormal bone formation, or osteoporosis A bone or joint disease, such as osteoarthritis, rheumatoid arthritis, arthritis, synovitis, metabolic arthropathy or sports-related joint disorders (eg, Tenis elbow, etc.) or may be affected in the future It can be diagnosed as high.
  • the antibody of the present invention can be used for detecting the protein of the present invention present in a subject such as a body fluid or a tissue.
  • preparation of an antibody column used for purifying the protein of the present invention, detection of the protein of the present invention in each fraction at the time of purification, analysis of the behavior of the protein of the present invention in test cells, etc. can be used for (4) Gene diagnostics
  • the DNA of the present invention can be used, for example, in humans or warm-blooded animals (for example, rats, mice, guinea pigs, egrets, birds, higgies, bush, horses, horses, cats, dogs) by using them as probes. , A monkey, a chimpanzee, etc.), the abnormality (gene abnormality) of DNA or mRNA encoding the protein of the present invention or a partial peptide thereof can be detected.
  • ⁇ whose expression is decreased or the DNA or mRNA is increased is useful as a diagnostic agent for genes such as overexpression.
  • the above-described genetic diagnosis using the DNA of the present invention can be performed, for example, by the known Northern hybridization or PCR-SSCP method (Genomics, Vol. 5, pp. 874-879). (1989), Proceed ings of the National Academy of Sciences of the
  • the antisense polynucleotide of the present invention which can complementarily bind to the DNA of the present invention and suppresses the expression of the DNA, has low toxicity, and inhibits the function of the protein of the present invention or the function of the DNA of the present invention in vivo.
  • Prevention of joint diseases ⁇ Can be used as a therapeutic agent.
  • the antisense polynucleotide is used as the above-mentioned prophylactic / therapeutic agent, it can be formulated and administered according to a method known per se.
  • a human or It can be administered orally or parenterally to mammals (eg, rats, egrets, sheep, sheep, bush, birds, cats, dogs, monkeys, etc.).
  • the antisense polynucleotide can be administered as it is or in the form of a formulation together with a physiologically acceptable carrier such as an adjuvant to promote ingestion, and can be administered by a gene gun or a catheter such as a hydrogel. Alternatively, they can be aerosolized and administered topically into the trachea as an inhalant.
  • antisense polynucleotides are used alone or in the form of ribosomes for the purpose of improving pharmacokinetics, prolonging the half-life and improving the efficiency of cellular uptake.
  • Formulation (injection) with the body may be administered intravenously, subcutaneously, or into the M node.
  • the dosage of the antisense polynucleotide varies depending on the target disease, the administration subject, the administration route, and the like.For example, when the antisense polynucleotide of the present invention is administered for the purpose of treating osteoarthritis, Generally, for an adult (body weight of 6 O kg), about 0.1 to 10 O mg of the antisense polynucleotide is administered per day.
  • antisense polynucleotide is used in a tissue or a cell of the present invention.
  • It can also be used as a diagnostic oligonucleotide probe for examining the presence of DNA and its expression status.
  • the present invention also includes a double-stranded RNA containing a part of the RNA encoding the protein of the present invention, a lipozyme containing a part of the RNA encoding the protein of the present invention, and the like. Gene expression, and the function of the protein of the present invention or the DNA used in the present invention in vivo can be suppressed.
  • cartilage dysplasia for example, cartilage dysplasia, bone dysplasia, osteoporosis, osteoarthritis It can be used as a prophylactic and therapeutic agent for bone and joint diseases such as osteoarthritis, rheumatoid arthritis, arthritis, synovitis, metabolic arthropathy, and joint disorders due to sports (eg, tennis elbow).
  • bone and joint diseases such as osteoarthritis, rheumatoid arthritis, arthritis, synovitis, metabolic arthropathy, and joint disorders due to sports (eg, tennis elbow).
  • the double-stranded RNA can be designed and manufactured based on the sequence of the polynucleotide of the present invention according to a known method (eg, Nature, 411, 494, 2001).
  • the lipozyme can be designed and manufactured based on the sequence of the polynucleotide of the present invention according to a known method (eg, TRENDS in Molecular Medicine, Vol. 7, pp. 221, 2001). For example, it can be produced by linking a known lipozyme to a part of the RNA encoding the protein of the present invention.
  • 'A part of the RNA encoding the protein of the present invention includes a portion (RNA fragment) adjacent to the cleavage site on the RNA of the present invention which can be cleaved by a known lipozyme.
  • RNA or lipozyme When the above double-stranded RNA or lipozyme is used as the prophylactic or therapeutic agent, it can be formulated and administered in the same manner as an antisense polynucleotide.
  • Monkey When the above double-stranded RNA or lipozyme is used as the prophylactic or therapeutic agent, it can be formulated and administered in the same manner as an antisense polynucleotide.
  • the bovine of the present invention which has the action of neutralizing the activity of the protein of the present invention, includes cartilage dysplasia, bone dysplasia, osteoporosis, osteoarthritis, rheumatoid arthritis, arthritis, synovitis, metabolic arthrosis Or it can be used as a prophylactic or therapeutic agent for bone and joint diseases such as sports-related joint disorders (eg, tennis elbows).
  • the prophylactic / therapeutic agent for the above-mentioned diseases containing the antibody of the present invention has low toxicity, and is used as it is as a liquid or as a pharmaceutical composition of an appropriate dosage form, in humans or mammals (eg, rat, porcupine, sheep, pig, It can be administered orally or parenterally (eg, intra-articular) to mice, cats, dogs, monkeys, etc.). Dosage should be
  • the antibody of the present invention is usually used as a single dose.
  • the antibody also good c present invention increased depending on the symptoms, itself still as possible out be administered as an appropriate pharmaceutical composition.
  • composition used for the above administration contains the above antibody or a salt thereof and a pharmacologically acceptable carrier, diluent or vehicle.
  • a pharmacologically acceptable carrier e.g., intra-articular administration. It is preferably provided as an inhalant.
  • compositions may contain another active ingredient as long as the composition does not cause an undesirable interaction with the above-mentioned antibody.
  • the present invention relates to a DNA encoding the exogenous protein of the present invention (hereinafter abbreviated as the exogenous DNA of the present invention) or a mutant DNA thereof (the exogenous mutant DN of the present invention).
  • A may be abbreviated as A).
  • Non-human mammals having the exogenous DNA of the present invention or the mutant DNA thereof include unfertilized eggs, fertilized eggs, germ cells including spermatozoa and their progenitor cells, and the like.
  • the calcium phosphate method, the electric pulse method, and the ribofection method It can be produced by transferring the desired DNA by a coagulation method, microinjection method, particle gun method, DEAE-dextran method or the like.
  • the exogenous DNA of the present invention can be transferred to somatic cells, organs of living organisms, tissue cells, and the like, and used for cell culture, tissue culture, and the like. Can be fused with the above-mentioned germ cells by a cell fusion method known per se to produce the DNA transgenic animal of the present invention.
  • mice for example, porcupines, pigs, higgins, goats, magpies, dogs, cats, guinea pigs, hamsters, mice, rats and the like are used.
  • mice for example, C57BL / 6 strains and DBA2 strains as pure strains
  • rat eg, Wistar, SD, etc.
  • mammal in the recombinant vector that can be expressed in mammals, human and the like can be mentioned in addition to the above-mentioned non-human mammals.
  • the exogenous DNA of the present invention refers to the DN of the present invention that a non-human mammal originally has. Rather than A, it refers to the DNA of the present invention once isolated and extracted from mammals.
  • mutant DNA of the present invention those in which a mutation (for example, mutation) has occurred in the base sequence of the original DNA of the present invention, specifically, addition of base, deletion, substitution with another base, etc.
  • the DNA in which the occurrence has occurred is used, and the abnormal DNA is also included.
  • the abnormal DNA refers to a DNA that expresses an abnormal protein of the present invention.
  • a DNA that expresses a protein that suppresses the function of the normal protein of the present invention is used. .
  • the exogenous DNA of the present invention may be derived from a mammal that is the same or different from the animal of interest.
  • the human DNA of the present invention when transferred, it is derived from various mammals (eg, egrets, dogs, cats, guinea pigs, hamsters, rats, mice, etc.) having the DNA of the present invention having high homology thereto.
  • Microinjection of the DNA construct of the present invention into a fertilized egg of a target mammal, for example, a mouse fertilized egg, downstream of various promoters capable of expressing the DNA of the present invention.
  • a DNA-transferred mammal that highly expresses the DNA of the present invention can be produced.
  • Examples of the expression vector of the protein of the present invention include a plasmid derived from Escherichia coli, a plasmid derived from Bacillus subtilis, a plasmid derived from yeast, a bacteriophage such as ⁇ phage, a retrovirus such as Moroni leukemia virus, vaccinia virus or baculovirus. Animal viruses such as viruses are used. Among them, a plasmid derived from Escherichia coli, a plasmid derived from Bacillus subtilis or a plasmid derived from yeast are preferably used.
  • Examples of the promoter that regulates the expression of DN ⁇ include, for example, DNA derived from (i) virus (eg, simian virus, cytomegalovirus, Moroni leukemia virus, JC virus, breast cancer virus, poliovirus, etc.).
  • virus eg, simian virus, cytomegalovirus, Moroni leukemia virus, JC virus, breast cancer virus, poliovirus, etc.
  • Promoters (ii) promoters derived from various mammals (human, egret, dog, cat, guinea pig, nomster, rat, mouse, etc.), for example, albumin, Insulin II, perobrakin II, elastase, erythropoietin, endocerin, muscle creatine kinase, glial fibrillary acidic protein, daluzin S-transferase, platelet-derived growth factor; 6, keratin Kl, K10 and ⁇ 14, Collagen types I and II, cyclic AMP-dependent protein kinase / 3 I-subunit, dystrophin, tartrate-resistant alkaline phosphatase, atrial natriuretic factor, endothelial receptor thymic synkinase (generally Abbreviated as T ie 2), sodium potassium adenosine 3 kinase
  • a promoter or the like is used.
  • the cytomegalovirus promoter capable of high expression throughout the whole body the promoter of human peptide chain elongation factor 1 (EF-1), the human and chicken / 3-actin promoters, etc. are preferable.
  • the above vector preferably has a sequence that terminates transcription of the messenger RNA of interest in a DNA-transferred mammal (generally referred to as Yuichi Minei Yuichi).
  • a DNA-transferred mammal generally referred to as Yuichi Minei Yuichi.
  • it is derived from viruses and various mammals.
  • a sequence of each DNA can be used, and preferably, SV40 terminator of simian virus or the like is used.
  • the splicing signal of each DNA, the enhancer region, a part of the intron of eukaryotic DNA, etc. are promoted 5 'upstream of the promoter region and the promoter region for the purpose of further expressing the target exogenous DNA. It is also possible to link between the Yuichi region and the translation region, or at 3, downstream of the translation region.
  • the normal translation region of the protein of the present invention is derived from humans or various mammals (eg, egrets, dogs, cats, guinea pigs, hamsters, rats, mice, etc.).
  • Liver, kidney, thyroid cells, fibroblast-derived DNA, and commercially available Prepared as all or part of genomic DNA from various genomic DN'A libraries, or by known methods from liver, kidney, thyroid cells, or fibrocyte-derived RNA
  • the obtained complementary DNA can be obtained as a raw material.
  • the foreign abnormal DNA can produce a translation region obtained by mutating the translation region of a normal protein obtained from the above-described cells or tissues by a point mutation induction method.
  • the translation region can be prepared as a DNA construct that can be expressed in a transgenic animal by a conventional DNA engineering technique in which it is ligated downstream of the aforementioned promoter and, if desired, upstream of the transcription termination site.
  • Transfer of the exogenous DNA of the present invention at the fertilized egg cell stage is ensured to be present in all germ cells and somatic cells of the target mammal.
  • the presence of the exogenous DNA of the present invention in the germinal cells of the produced animal after DNA transfer means that all the progeny of the produced animal retain the exogenous DNA of the present invention in all of the germ cells and somatic cells Means to do.
  • the progeny of such animals that inherit the exogenous DNA of the present invention have the exogenous DNA of the present invention in all of their germinal and somatic cells.
  • the non-human mammal to which the exogenous normal DNA of the present invention has been transferred is confirmed to stably maintain the exogenous DNA by mating, and is subcultured as an animal having the DNA in a normal breeding environment. I can do it.
  • Transfer of the exogenous DNA of the present invention at the fertilized egg cell stage is ensured to be present in excess in all germ cells and somatic cells of the target mammal.
  • Excessive presence of the exogenous DNA of the present invention in the germinal cells of the produced animal after DNA transfer indicates that all of the offspring of the produced animal contain the exogenous DNA of the present invention in all of its germ cells and somatic cells. Means to have.
  • the progeny of this type of animal that has inherited the exogenous DNA of the present invention has an excess of the exogenous DNA of the present invention in all its germ cells and somatic cells.
  • the non-human mammal having the normal DNA of the present invention expresses the normal DNA of the present invention at a high level. It may develop hyperfunction and can be used as a disease model animal. For example, using the normal DNA-transferred animal of the present invention to elucidate the pathological mechanism of the hyperactivity of the protein of the present invention and diseases associated with the protein of the present invention. It is possible to do.
  • a prophylactic / therapeutic agent for a disease associated with the protein of the present invention for example, cartilage Bone and joint disorders such as dysplasia, bone dysplasia, osteoporosis, osteoarthritis, rheumatoid arthritis, arthritis, synovitis, metabolic arthropathy or joint disorders due to sports (eg tennis elbow) It can also be used for screening tests for prophylactic 'therapeutic agents.
  • the non-human mammal having the foreign abnormal DNA of the present invention should be subcultured in a normal breeding environment as an animal having the DNA after confirming that the foreign DNA is stably retained by the crossing. Can be done. Furthermore, the desired foreign DNA can be incorporated into the aforementioned plasmid and used as a raw material.
  • the DNA construct with the promoter can be prepared by a usual DNA engineering technique. The transfer of the abnormal DNA of the present invention at the fertilized egg cell stage is ensured to be present in all germ cells and somatic cells of the target mammal.
  • the presence of the abnormal DNA of the present invention in the germinal cells of the animal produced after transfer of the DNA means that the offspring of the animal produced have the abnormal DNA of the present invention in all of the germinal and somatic cells.
  • the progeny of this type of animal that has inherited the exogenous DNA of the present invention has the abnormal DNA of the present invention in all of its germinal and somatic cells.
  • the non-human mammal having the abnormal DNA of the present invention expresses the abnormal DNA of the present invention at a high level, and finally inhibits the function of the endogenous normal DNA to thereby finally produce the protein of the present invention.
  • Function inactive type refractory disease Can be used as an animal.
  • using the abnormal DNA-transferred animal of the present invention it is possible to elucidate the pathological mechanism of the function-inactive refractory of the protein of the present invention and to examine a method for treating this disease.
  • the abnormal DNA highly expressing animal of the present invention can be used to inhibit the function of a normal protein by the abnormal protein of the present invention in the function-inactive refractory disease of the protein of the present invention. Action).
  • a preventive / therapeutic agent for the protein of the present invention or a functionally inactive type refractory disease for example, Bone and joints such as cartilage dysplasia, bone dysplasia, osteoporosis, osteoarthritis, rheumatoid arthritis, arthritis, synovitis, metabolic arthrosis or joint disorders due to sports (eg tennis elbow) It can also be used in screening tests for disease prevention-therapeutic agents.
  • the DNA-transferred animal of the present invention in order to develop a therapeutic agent for a disease associated with the protein of the present invention, including the inactive type refractory type of the protein of the present invention, using the DNA-transferred animal of the present invention, It is possible to provide an effective and rapid screening method for a therapeutic agent for the disease by using the method and the quantitative method. Also, using the DNA transfer product of the present invention or the exogenous DNA expression vector of the present invention, it is possible to examine and develop a method for treating a DNA associated with the protein of the present invention.
  • the present invention provides a non-human mammalian embryonic stem cell in which the DNA of the present invention has been inactivated, and a non-human mammal deficient in expression of the DNA of the present invention.
  • neomycin an embryonic stem cell according to (1), which is resistant to neomycin
  • the embryonic stem cell according to (1) which is a non-human mammal rodent
  • the embryonic stem cell according to (4) wherein the rodent is a mouse
  • the DNA is inactivated by introducing a reporter gene (eg, a ⁇ _galactosidase gene derived from Escherichia coli), and the reporter gene is regulated by the promoter of D ⁇ of the present invention.
  • a reporter gene eg, a ⁇ _galactosidase gene derived from Escherichia coli
  • a compound or a salt thereof that promotes or inhibits the promoter activity of the DNA of the present invention which comprises administering a test compound to the animal described in (7) and detecting the expression of a repo overnight gene.
  • the non-human mammalian embryonic stem cells in which the DNA of the present invention has been inactivated are defined as artificially mutating the DNA of the present non-human mammal, which suppresses the expression ability of the DNA.
  • the DNA substantially does not have the ability to express the protein of the present invention by substantially eliminating the activity of the protein of the present invention encoded by the DNA (hereinafter referred to as the knockout DNA of the present invention). This may be referred to as "embryonic stem cells" of non-human mammals (hereinafter abbreviated as ES cells).
  • non-human mammal the same one as described above is used.
  • the method of artificially mutating the DNA of the present invention can be performed, for example, by deleting a part or all of the DNA sequence and inserting or substituting another DNA by a genetic engineering technique.
  • the knockout DNA of the present invention may be prepared by, for example, shifting the reading frame of the codon or disrupting the function of the promoter or exon by these mutations.
  • non-human mammalian embryonic stem cells in which the DNA of the present invention is inactivated include, for example, The DNA of the present invention possessed by a non-human mammal to be isolated is isolated and its exon part is a drug resistance gene represented by a neomycin resistance gene, a hygromycin resistance gene, or lacZ ( ⁇ -galactosidase gene), cat Disruption of exon function by inserting a repo overnight gene represented by (chloramphenicol acetyltransferase gene) or termination of gene transcription in the intron between exons
  • a DNA sequence for example, the po1yA additional signal
  • a DNA strand having a DNA sequence constructed so as to disrupt the gene ) Is introduced into the chromosome of the animal by, for example,
  • ES cells from which the DNA of the present invention is inactivated by the homologous recombination method or the like for example, those already established as described above may be used, and the method of the known Evans and Kaufma It may be newly established according to.
  • mouse ES cells currently, 129 ES cells are generally used, but since the immunological background is not clear, a pure line that substitutes them and immunologically inherited is used.
  • the number of eggs collected from C57BLZ6 mice or C57BL / 6 was improved by crossing with DBA / 2 (C57BLZ6 and DBA / 2 It is also possible to use a mouse established using F and F.
  • the BD mouse has the advantage of a large number of eggs collected and the eggs are robust, and has the background of C57BL / 6 mice.
  • ES cells obtained using this can be advantageously used when pathological model mice are created, because the genetic background can be replaced by C57BLZ6 mice by backcrossing with C57BLZ6 mice. .
  • ES cells when ES cells are established, blastocysts 3.5 days after fertilization are generally used. Early embryos can be obtained. Either male or female ES cells may be used, but male ES cells are generally more convenient for producing breeding line chimeras. It is also desirable to discriminate between males and females as soon as possible in order to reduce the complexity of culturing.
  • An example of a method for determining the sex of ES cells is a method of amplifying and detecting a gene in the sex-determining region on the Y chromosome by PCR.
  • the for example G-banding method requires about 1.0 6 cells for karyotype analysis, requires one colony of about ES cell number (about 50)
  • the primary selection of ES cells in the early stage of culture can be performed by discriminating between males and females, and the early stages of culture can be greatly reduced by enabling the selection of male cells at an early stage.
  • Embryonic stem cell lines obtained in this way usually have very good proliferative potential, but must be carefully subcultured because they tend to lose their ontogenetic potential.
  • a suitable feeder cell such as STO fibroblasts
  • a carbon dioxide incubator preferably 5% carbon dioxide, 95% air or 5% oxygen
  • LIF 1-10000 U / ml
  • 5% carbon dioxide, 90% air at about 37, etc.
  • trypsin ZEDTA solution usually 0.001 to 0.5% trypsin / 0.1 to 5 mM EDTA, preferably about Single cells are treated by 0.1% trypsin (ZlmMEDTA) treatment and seeded on a freshly prepared feeder cell.
  • Such subculture is usually performed every 1 to 3 days. At this time, it is desirable to observe the cells, and if morphologically abnormal cells are found, discard the cultured cells.
  • ES cells are differentiated into various types of cells, such as parietal, visceral, and cardiac muscle, by monolayer culture up to high density or suspension culture until cell clumps are formed under appropriate conditions.
  • CM. J. Evans and MH Kaufman Nature 292, 154, 1981; GR Martin Proc. Natl. Acad. Sci. USA 78, 7634, 1981; Doetschman et al., Journal 'ob. Embryology 'and experimental' morphology, Vol. 87, p. 27, 1985
  • the DNA-deficient cells of the present invention obtained by differentiating the ES cells of the present invention are Useful in cell biology studies of proteins.
  • the DNA expression-deficient non-human mammal of the present invention can be obtained by determining the mRNA level of the animal by a known method.
  • the expression can be distinguished from normal animals by measuring indirectly and comparing the expression levels indirectly.
  • -As the non-human mammal the same ones as described above are used. ,
  • the non-human mammal deficient in DNA expression of the present invention can be obtained, for example, by introducing the targeting vector prepared as described above into a mouse embryonic stem cell or mouse egg cell, and introducing the DNA of the present invention into the targeting vector.
  • the DNA of the present invention can be knocked out by homologous recombination of the inactivated DNA sequence to replace the DNA of the present invention on the chromosome of mouse embryonic stem cells or mouse egg cells by gene homologous recombination. it can.
  • Cells in which the DNA of the present invention has been knocked out are subjected to Southern hybridization analysis using a DNA sequence on or near the DNA of the present invention as a probe or a DNA sequence on a targeting vector, and evening gettering.
  • the determination can be made by PCR analysis using the DNA sequence of the neighboring region other than the DNA of the present invention derived from the mouse used as the vector as a primer.
  • the cell line in which the DNA of the present invention has been inactivated by gene homologous recombination is cloned, and the cell is cultured at an appropriate time, for example, at the 8-cell stage.
  • the chimeric embryo is injected into a human mammalian embryo or blastocyst, and the resulting chimeric embryo is transplanted into the uterus of the pseudopregnant non-human mammal.
  • the produced animal is a chimeric animal composed of both a normal cell having the DNA locus of the present invention and an artificially mutated cell having the DNA locus of the present invention.
  • all tissues are artificially obtained from the population obtained by crossing such a chimeric individual with a normal individual. It can be obtained by selecting an individual composed of cells having the DNA locus of the present invention having a mutation, for example, by judging coat color.
  • the individual obtained in this manner is usually an individual having a heterozygous expression of the protein of the present invention, which is crossed with an individual having a heterozygous expression of the protein of the present invention. A homozygous deficient individual can be obtained.
  • the targeting vector can be inserted into the chromosome by injecting a DNA solution into the nucleus of the egg cell by microinjection.
  • the introduced transgenic non-human mammal can be obtained, and can be obtained by selecting a transgenic non-human mammal having a mutation in the DNA locus of the present invention by homologous recombination as compared to these transgenic non-human mammals.
  • the animal individual obtained by the crossing should be confirmed that the DNA has been knocked out and subjected to rearing in a normal rearing environment. Can be.
  • the germline can be obtained and maintained according to a conventional method. That is, by crossing male and female animals having the inactivated DNA, a homozygous animal having the inactivated DNA on both homologous chromosomes can be obtained.
  • the obtained homozygous animal can be efficiently obtained by rearing the mother animal in such a manner that one normal individual and a plurality of homozygous animals are obtained.
  • homozygous and heterozygous animals having the inactivated DNA are bred and passaged.
  • the non-human mammalian embryonic stem cells in which the DNA of the present invention has been inactivated are extremely useful for producing the non-human mammal deficient in expression of the DNA of the present invention.
  • the non-human mammal deficient in expression of the DNA of the present invention lacks various biological activities that can be induced by the protein of the present invention, it may cause inactivation of the biological activity of the protein of the present invention. It is useful for investigating the causes of these diseases and examining treatment methods.
  • the non-human mammal deficient in DNA expression of the present invention can be used for screening for a compound having a therapeutic / preventive effect against diseases caused by DNA deficiency or damage of the present invention.
  • the present invention comprises administering a test compound to a non-human mammal deficient in expression of the DNA of the present invention, and observing and measuring changes in the animal.
  • a method for screening a compound or a salt thereof having a therapeutic or preventive effect on a disease caused by the disease is provided.
  • the non-human female deficient in expression of the DNA of the present invention used in the screening method examples of the dairy animal include those described above.
  • Test compounds include, for example, peptides, proteins, non-peptidic compounds, synthetic compounds, fermentation products, cell extracts, plant extracts, animal tissue extracts, and plasma, and these compounds are novel compounds. Or a known compound '.
  • a non-human mammal deficient in expression of the DNA of the present invention is treated with a test compound and compared with a non-treated control animal, and the change in each organ, tissue, symptom of disease or the like of the animal is used as an index.
  • the therapeutic and prophylactic effects of the test compound can be tested.
  • test compound for example, oral administration, intravenous injection, or the like is used, and it can be appropriately selected according to the symptoms of the test animal, properties of the test compound, and the like.
  • the dose of the test compound can be appropriately selected according to the administration method, the properties of the test compound, and the like.
  • bone and joint diseases such as cartilage dysplasia, bone dysplasia, osteoporosis, osteoarthritis, rheumatoid arthritis, arthritis, synovitis, metabolic arthropathy, or joint disorders due to sports (eg, tennis elbow, etc.)
  • a test compound is administered to a non-human mammal deficient in expression of the DNA of the present invention, and the joints, cartilage, etc. of the animal are observed over time, and Observe the symptoms of. .
  • the test compound when the test compound is administered to a test animal, the above-mentioned disease symptoms of the test animal are improved by about 10% or more, preferably about 30% or more, more preferably about 50% or more.
  • the test compound can be selected as a compound having a therapeutic-prophylactic effect against the above-mentioned diseases.
  • the compound obtained by using the screening method is a compound selected from the test compounds described above, and has a therapeutic / preventive effect against a disease caused by deficiency or damage of the protein of the present invention. It can be used as a medicament such as a safe and low toxic prophylactic / therapeutic agent for the disease. Furthermore, a compound derived from the compound obtained by the above-mentioned screening can be used in the same manner.
  • the compound obtained by the screening method may form a salt.
  • salts of the products salts with physiologically acceptable acids (eg, inorganic acids, organic acids, etc.) and bases (eg, alkali metals, etc.) are used, and especially physiologically acceptable acid addition salts Is preferred.
  • physiologically acceptable acids eg, inorganic acids, organic acids, etc.
  • bases eg, alkali metals, etc.
  • physiologically acceptable acid addition salts Is preferred.
  • Such salts include, for example, salts with inorganic acids (eg, salts of acids, phosphoric acid, hydrobromic acid, sulfuric acid, etc.) or organic acids (eg, acetic acid, formic acid, propionic acid, fumaric acid, etc.).
  • Acid maleic acid, succinic acid, tartaric acid, citric acid, and salts with malic acid, oxalic acid, benzoic acid, methanesulfonic acid, benzenesulfonic acid, etc
  • a drug containing the compound or a salt thereof obtained by the screening method can be produced in the same manner as the above-mentioned drug containing the protein of the present invention.
  • the preparations obtained in this way are safe and of low toxicity and are, for example, suitable for humans or mammals (for example, rats, mice, guinea pigs, egrets, sheep, pigs, horses, cats, cats). , Dogs, monkeys, etc.).
  • the dose of the compound or a salt thereof varies depending on the target disease, the administration subject, the administration route, and the like.
  • the deformability of an adult is generally
  • the compound is administered from about 0.1 to: L0 Omg, preferably from about 1.0 to 5 Omg, more preferably from about 1.0 to 20 mg per day.
  • the single dose of the compound varies depending on the administration target, the target disease, etc., but, for example, the compound is usually in the form of an injection in an adult (assuming a body weight of 6 Okg).
  • the compound When administered to a patient with arthropathy, the compound is administered by intravenous injection at a dose of about 0.01 to 3 Omg, preferably about 0.1 to 20 mg, more preferably about 0.1 to 1 Omg per day. It is convenient. In the case of other animals, the dose can be administered in terms of weight per 60 kg.
  • the present invention relates to a compound which promotes or inhibits the activity of a promoter of the DNA of the present invention, which comprises administering a test compound to a non-human mammal deficient in expressing the DNA of the present invention and detecting the expression of a reporter gene. Or provide a method of screening for its salts.
  • the non-bit mammal deficient in DNA expression of the present invention is inactivated by introducing a repo overnight gene, and the repo overnight gene is a DNA of the present invention. Those that can be expressed under the control of a promoter for are used.
  • test compound examples include the same compounds as described above.
  • Zea gene (1 acZ), soluble alkaline phosphatase gene or luciferase gene are preferred.
  • the reporter gene is under the control of the promoter of the DNA of the present invention, so that Trace the expression of the substance encoded by the gene
  • the activity of the promoter can be detected.
  • a tissue expressing the protein of the present invention originally 6-galactosidase is expressed instead of the protein of the invention. Therefore, for example, by staining with a reagent that serves as a substrate for ⁇ -galactosidase such as 5-bromo-4-monocloth-3-indolyl- ⁇ -galactopyranoside (X-gal), In addition, the state of expression of the protein of the present invention in animal organisms can be observed.
  • a reagent that serves as a substrate for ⁇ -galactosidase such as 5-bromo-4-monocloth-3-indolyl- ⁇ -galactopyranoside (X-gal)
  • the protein-deficient mouse of the present invention or a tissue section thereof is fixed with dartalaldehyde or the like, washed with phosphate buffered saline (PBS), and then stained with X-ga1 at room temperature.
  • PBS phosphate buffered saline
  • the 8-galactosidase reaction may be stopped by washing the tissue specimen with ImMED TAZPBS solution, and the coloration may be observed.
  • mRNA encoding 1acZ may be detected according to a conventional method. .
  • the compound or a salt thereof obtained by using the above-mentioned screening method is a compound selected from the above-mentioned test compounds, and is a compound that promotes or inhibits the activity of promoter of DNA of the present invention.
  • the compound obtained by the screening method may form a salt,
  • a salt with a physiologically acceptable acid eg, an inorganic acid, etc.
  • a base eg, an organic acid, etc.
  • a physiologically acceptable acid addition salt is particularly preferable.
  • Such salts include, for example, salts with inorganic acids (eg, hydrochloric acid, phosphoric acid, hydrobromic acid, sulfuric acid, etc.) and organic acids (eg, acetic acid, formic acid, propionic acid, fumaric acid, maleic acid, Salts with acids, succinic acid, tartaric acid, citric acid, malic acid, oxalic acid, benzoic acid, methanesulfonic acid, benzenesulfonic acid, etc. are used.
  • inorganic acids eg, hydrochloric acid, phosphoric acid, hydrobromic acid, sulfuric acid, etc.
  • organic acids eg, acetic acid, formic acid, propionic acid, fumaric acid, maleic acid
  • Salts with acids succinic acid, tartaric acid, citric acid, malic acid, oxalic acid, benzoic acid, methanesulfonic acid, benzenesulfonic acid, etc.
  • the compound or its salt that promotes or inhibits the activity of the promoter of the DNA of the present invention or the salt thereof can regulate the expression of the protein of the present invention and regulate the function of the protein.
  • Useful as a prophylactic / therapeutic agent for bone and joint diseases such as abnormalities, osteoporosis, osteoarthritis, rheumatoid arthritis, arthritis, synovitis, metabolic arthrosis, or joint disorders due to sports (eg, tennis elbow, etc.) is there.
  • a drug containing the compound or a salt thereof obtained by the screening method can be produced in the same manner as the above-mentioned drug containing the protein of the present invention or a salt thereof.
  • the preparations obtained in this way are safe and of low toxicity and can be used, for example, in humans or mammals (for example, rats, mice, guinea pigs, egrets, sheep, pigs, pigs, dogs, cats, dogs). , Monkeys, etc.).
  • the dose of the compound or a salt thereof varies depending on the target disease, the subject of the administration, the administration route, and the like.
  • the compound of the present invention that inhibits the promoter activity for DNA when administered parenterally, generally the adult In a patient with osteoarthritis weighing 60 kg (as weight as 60 kg), the compound may be used in an amount of about 0.1 to 100 mg per day, preferably about 1.0 to 50 mg, more preferably about 1 to 50 mg. Administer 0 to 20 mg.
  • the single dose of the compound varies depending on the administration subject, target disease, and the like.
  • a compound that inhibits the promoter activity of DNA of the present invention may be used as an injection.
  • the non-human mammal deficient in DNA expression of the present invention is extremely useful for screening a compound or a salt thereof that promotes or inhibits the activity of the promoter for DNA of the present invention, The present invention can greatly contribute to investigating the causes of various diseases caused by insufficient DNA expression or to develop preventive and therapeutic agents.
  • transgenic animal In addition, using a DNA containing the promoter region of the protein of the present invention, genes encoding various proteins are ligated downstream thereof and injected into egg cells of an animal to produce a so-called transgenic animal (transgenic animal). ) Makes it possible to specifically synthesize the protein and examine its effects on living organisms. Further, by binding an appropriate repo overnight gene to a portion of the above promoter and establishing a cell line that expresses the gene, specific promotion or suppression of the in vivo production ability of the protein of the present invention itself is achieved. It can be used as a search system for low-molecular compounds that have an effect.
  • bases, amino acids, and the like are indicated by abbreviations based on the abbreviations by the IUPAC- IUB Commission on Biochemical Nomenclature or commonly used abbreviations in the art, and examples thereof are described below.
  • amino acids can have optical isomers, the L-form is indicated unless otherwise specified.
  • FIG. 9 shows the amino acid sequence of mature chondromedin protein obtained in Example 2, [SEQ ID NO: 2]
  • Example 3 shows the base sequence obtained in Example 2.
  • Example 3 shows the nucleotide sequence of a primer used in Example 2.
  • Example 3 shows the nucleotide sequence of a primer used in Example 2.
  • FIG. 6 shows the nucleotide sequence of a primer used in Example 4.
  • Example 7 shows the nucleotide sequence of a primer used in Example 4.
  • Example 7 shows the nucleotide sequence of a primer used in Example 4.
  • Example 7 shows the nucleotide sequence of a primer used in Example 4.
  • Example 6 shows the nucleotide sequence of a probe used in Example 4.
  • [SEQ ID NO: 15] 2 shows the amino acid sequence of a synthetic peptide Tr2.
  • Example 14 shows the base sequence of an oligonucleotide constituting the adapter 1 used in Example 6. 5 'is phosphorylated. '' Escherichia coli DH5 ⁇ / ⁇ 2300 obtained in Example 2 described below has been used since November 20, 2002, 1-1 1-1 Higashi, Tsukuba City, Ibaraki Prefecture 1 Chuo No. 6 (Zip code 305-
  • OA cartilage (1 knee joint, 5 hip joints), normal articular cartilage (1 knee joint, 1 hip joint) to clarify genes that are upregulated in osteoarthritis cartilage (0A cartilage)
  • total RNA (Table 1) extracted from 22 other normal tissues (1 case each) as a material
  • gene expression analysis was performed using an oligonucleotide microarray (Human Genome intestine A; Affymetrix).
  • DKFZP586L151 (GenBank Accession No. XP-034000) A gene-based search was performed based on the gene sequence.
  • the expected PCR product of approximately 2.3 kbp was observed in the reaction solution obtained from cDNAs of placenta, lung, skeletal muscle, small intestine, testis, uterus, fetal kidney and fetal liver. These were mixed and the PCR amplification product was purified using a Strataprep PCR purification kit (Stratagene). The obtained DNA fragment of about 2.3 kb was cloned into E. coli DH5 using pCR-Script cloning kit (Stratagene), and the plasmid was
  • Plasmid DNA was isolated from the obtained Escherichia coli DH5 ⁇ / ⁇ 2300, and the nucleotide sequence of a DNA fragment of about 2.3 kb inserted into PTB2300 was determined by a fluorescent Dye-Terminator method.
  • SEQ ID NO: 6 and SEQ ID NO: 7 SEQ ID NO: 5 and the known nucleotide sequence of the DKFZP586L151 gene (GenBank Accession No.
  • XP_034000 The nucleotide sequence of the open reading frame is shown in SEQ ID NO: 4.
  • the amino acid sequence predicted from the nucleotide sequence represented by SEQ ID NO: 4 is shown in SEQ ID NO: 3.
  • amino acid sequence represented by SEQ ID NO: 3 is DKFZP586L151 (GenBank Accession It is an amino acid sequence that extends 314 residues to the N-terminal side of the amino acid sequence of No. XP-034000) and has 82% homology with mouse 1110018N05Rik (GenBank Accession No. XM_129545). '
  • the nucleotide sequence represented by SEQ ID NO: 4 is a nucleotide sequence obtained by extending 826 base on the 5th side of the nucleotide sequence of DKFZP586L151, and has 86% homology with 'mouse 1110018N05Rik'.
  • the protein having the amino acid sequence (SEQ ID NO: 1) obtained by removing the signal peptide from the amino acid sequence represented by SEQ ID NO: 3 was named as mature chondromedin protein.
  • the nucleotide sequence encoding the amino acid sequence represented by SEQ ID NO: 1 is shown in SEQ ID NO: 2.
  • SEQ ID NO: 8 is the promoter sequence of chondromedin present in the 5 ′ upstream region of the nucleotide sequence represented by SEQ ID NO: 2.
  • the binding site of the basic transcription factor TFII-I and its cooperating USF was issued. It is considered to be a type of promoter that operates through an initiator element (Inr).
  • binding sites for transcription factors such as SMAD-3 and SMAD-4, which are involved in signaling of Sox9 and TGF-i3 receptor superfamily involved in cartilage differentiation, were found. It was expected that chondromedin would be promoted in chondrocyte differentiation by stimulation of BMP or increased expression of Sox9.
  • a sequence containing the nucleotide sequence represented by SEQ ID NO: 8 is linked upstream of the repo overnight gene 0 luciferase, alkaline phosphatase, etc.), and is linked to various cell lines (ATDC5, C3H10T1 / 2, etc.). By introducing the gene, it can be used for searching for an expression regulator of this gene.
  • SEQ ID NO: 8 A sequence containing the nucleotide sequence represented by SEQ ID NO: 8 is linked upstream of the repo overnight gene 0 luciferase, alkaline phosphatase, etc.), and is linked to various cell lines (ATDC5, C3H10T1 / 2, etc.).
  • 1110018N05Rik (GenBank Accession No. XM_129545) is abbreviated as mouse chondromedin gene.
  • C3H10T1 / 2 cells which are mouse-derived mesenchymal stem cells, are stimulated with BMP-2 or BMP-4 to induce cartilage differentiation in vitro (Ahrens M, et al., DNA Cell Biol.
  • C3H10T1 / 2 cells (Cancer Res., 33: 3239-3249, 1973) purchased from Dainippon Pharmaceutical were cultured in the presence of BMP-4 for 3, 6 and 13 days. Cells were lysed with RLT buffer (Qiagen) before (Day 0), 3 days (Day 3), 6 days (Day 6) and 13 days (Day 13) after BMP-4 addition. , QIAshredder (Qiagen), -8 (saved in TC.) The sample was thawed, and cell-derived total RNA was prepared using the RNeasy mini kit (Qiagen). To remove DNA, we followed the optional protocol of Dnase treatment on the column.
  • Quantitative RT-PCR was performed using the obtained total RM as type III, and the expression level of mouse chondromedin gene was examined.
  • a primer (SEQ ID NO: 9 and SEQ ID NO: 10) for mouse chondromedin mRNA was designed and used based on the nucleotide sequence XM-129545 registered in Genbank.
  • RT-PCR and signal detection were performed on a 7700 sequence detection system (Applied Biosystems). First incubate at 50 ° C for 30 minutes After performing the heating and then heating at 95 ° C for 15 minutes, 40 cycles of amplification at 15 ° C for 15 seconds and 60 ° C for 60 seconds were performed.
  • cDNA was synthesized from the above-described total RNA of C3H10T1 / 2 cells using SensiScript reverse transpciptrase (Qiagen).
  • this cDNA was converted into type III, and PCR and signal detection were performed on a 7700 sequence detection system (Applied Biosystems) using TaqMan Universal PCR Master Mix (Applied Biosystems) as a reagent. After first heating at 95 ° C for 10 minutes, 40 cycles of amplification at 94 ° C for 15 seconds and 60 ° C for 60 seconds were performed.
  • the fluorescent signal value obtained by quantitative PCR of mouse chondromedin was normalized by the fluorescent signal value obtained for mouse j3-actin. Furthermore, the standardized expression level of mouse chondromedin on Day 3, Day 6 and Day 13 was divided by the standardized expression level on Day 0 and relativized.
  • Trl SEQ ID NO: 14
  • Tr2 SEQ ID NO: 15
  • Partial blood collection was performed immediately before the first sensitization (Day 0) and on the 35th day (Day 35). Get The antibody titers of the obtained sera were measured by ELISA using Fanglia peptide as a solid phase, and the results are shown in FIG. 2 (anti-Trl antiserum) and FIG. 3 (anti-Tr2 antiserum). An increase in antibody titer was confirmed for all sera. Finally, on day 56 after sensitization, whole blood was collected to obtain anti-Trl antiserum and anti-Tr2 antiserum.
  • the plasmid PTB2300 obtained in Example 2 was digested with the restriction enzyme Spel, the resulting non-blunt ends were blunt-ended using Pfu Turbo DNA polymerase (Stratagene), and then Xbal linker was used using DNA ligase. (Takara Shuzo) was inserted.
  • the resulting plasmid was digested with EcoRI and Ncol, and the two adapters C5'-pAATTCACCGC-3 '(SEQ ID NO: 16) and 5'-pCATGGCGGTG-3' (SEQ ID NO: 17) were obtained between the obtained non-sticky ends. ) (5′-p indicates 5 ′ phosphorylation)] was inserted by the reaction of DNA ligase.
  • the resulting plasmid was digested with Hindlll and Xbal, and d the fragment excised the cDNA fragment of about 2.3 kb containing the open reading frame of human chondromedin gene between the Hindi II and Xbal sites of pcDNA5 / T0 (Invitrogen Corp.) Insertion yielded the human chondromedin expression plasmid pcDNA5 / TO-hCdm.
  • COS-7 cells were seeded on a culture plate, and after about 24 hours, pcDNA5 / T0 (Invitrogen) or pcDNA5 / T (HiCdm obtained above) was added to Lipofectamine2000 reagent.
  • the anti-Trl antiserum or immuno-IgG derived therefrom can be used for diagnosis / prevention / treatment of diseases associated with elevated human chondromedin, for example, osteoarthritis.
  • the human chondromedin expression vector is human
  • the protein or DNA of the present invention can be used as a diagnostic tool for osteoarthritis.
  • Polynucleotides include, for example, cartilage dysplasia, bone dysplasia, osteoporosis, osteoarthritis, rheumatoid arthritis, arthritis, synovitis, metabolic arthrosis or joint disorders due to sports (eg tennis elbow, etc.) Bone of. It can be used as an agent for preventing or treating arthritis.

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Abstract

Cette invention concerne une protéine qui sert, notamment, de marqueur diagnostique de l'ostéite, de l'arthrite, etc. Cette protéine et des composés, qui servent à promouvoir ou à inhiber (de préférence à inhiber) l'activité de la protéine ou l'expression d'un gène de la protéine, et qui sont obtenus par une méthode de criblage à l'aide de la protéine susmentionnée, peuvent être utilisés comme médicaments sans danger, par exemple comme préventifs/remèdes, notamment pour les maladies susmentionnées.
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Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2000018922A2 (fr) * 1998-10-01 2000-04-06 Incyte Genomics, Inc. Proteines humaines associees a des glucides
WO2001053312A1 (fr) * 1999-12-23 2001-07-26 Hyseq, Inc. Nouveaux acides nucleiques et polypeptides

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2000018922A2 (fr) * 1998-10-01 2000-04-06 Incyte Genomics, Inc. Proteines humaines associees a des glucides
WO2001053312A1 (fr) * 1999-12-23 2001-07-26 Hyseq, Inc. Nouveaux acides nucleiques et polypeptides

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
DATABASE GENBANK [online] KAWAI, J ET AL: "Functional annotation of a full-length mouse cDNA collection.", XP002974972, accession no. NCBI Database accession no. ak042424 *
DATABASE GENBANK [online] STRAUSBERG, RL ET AL: "Generation and intial analysis of more than 15,000 full-length human and mouse cDNA sequences.", XP002974973, accession no. NCBI Database accession no. BC047472 *
NATURE, vol. 409, 8 February 2001 (2001-02-08), pages 685 - 690 *
PROC NATL ACAD SCI USA, vol. 99, no. 26, 24 December 2001 (2001-12-24), pages 16899 - 16903 *

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