WO2004039974A1 - Novel protein and dna thereof - Google Patents

Novel protein and dna thereof 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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Prior art keywords
protein
dna
present
salt
seq
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PCT/JP2003/013919
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French (fr)
Japanese (ja)
Inventor
Tatsuya Watanabe
Masakazu Inazuka
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Takeda Pharmaceutical Company Limited
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Priority to AU2003280641A priority Critical patent/AU2003280641A1/en
Publication of WO2004039974A1 publication Critical patent/WO2004039974A1/en

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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

A protein which is useful as, for example, a diagnostic marker for osteitis, arthritis, etc. This protein and compounds etc. promoting or inhibiting (preferably inhibiting) the activity of the protein or the expression of a gene of the protein, which are obtained by a screening method with the use of the above protein, are usable as highly safe drugs such as preventives/remedies for, e.g., the above-described diseases.

Description

明 細書 新規タンパク質およびその DN A ' '技術分野 '  Description New protein and its DNA '' 'Technical field'
本発明は、 新規タンパク質、 その DNA、 該タンパク質の発現または活性を 調節する化合物等のスクリーニング方法、 該スクリーニング方法で得られる化 合物、 該化合物を含有する骨 ·関節疾患の予防 ·治療剤などに関する。 背景技術  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. About. Background art
オルファクトメジンドメイン （Conserved Domain Database pfam0219> OLF) は、 約 250アミノ酸残基からなる、 それ自体は機能不明のタンパク質構造単位で ある。 このドメインを含むタンパク質は、 分泌型タンパク質 （ER局在型も含 む） と 7回膜貫通型受容体型の二つに分類されている。 分泌型タンパクとしては Olfactomedinが粘液性の細胞外マトリックスの構成成分として最初に単離され、 ゥシガエルの嗅覚神経上皮細胞で発現していることが報告されている （Yokoe H, An olt RR、 Proc Natl Acad Sci U S A. 90 : 4655-4659、 1993) 。 その後、 ヒ トを含む多くの生物種でこのドメインを含むタンパク質や遺伝子が発見されて いる。 ヒト由来の遺伝子としては毛様体で発現している Myocil in (Kubota, R. ら Genomics 41 :.360- 369、 1997) がよく研究されている。 Myoci 1 inは TIGRとも 呼ばれ、 緑内障の原因遺伝子の 1つであることが知られている （Stone, EM. ら Science 275： 668-70, 1997) 。 GW112 (NP一 006409) は潰瘍性大腸炎の大腸粘 膜の上皮細胞で発現が上昇していることが報告されている （Shinozaki, S.ら Gut 48： 623-9、 2001) 。 マウスの Pancor tin (Nagano, T.ら Brain Res Mol Brain Res 53： 13-23 (1998). Nagano, T.ら， J Neurochem 75, 卜 8、 2000) は大脳皮質で発現しており、 神経分化に必須な夕ンパク質であると考えられて いる。 ニヮトリの Noelin (Barembaura M, ら Nat Cell Biol 2000 219-25) は神 経管による神経提 （neural crest) 細胞の生産を調^ ϊしていることが報告され ている。 また、 アフリカッメガエルの Tiarin (Hiroshi Tsuda ら、 Neuron, Vol 33， 515-528, 2002) は神経管の組織形成に関与する分泌因子であると考えられ ている。 このように、 これらのオルファクトメジンドメインを有するタンパク 質は細胞外に分泌され、 神経の組織形成を制御する新しい範疇の生理活性物質 と考えられる。 一方、 Latrophilinはクモの神経毒素である a- Latrotoxinの受 容体であり、 Gタンパク質共役型 7回膜貫通型受容体であり、 マウスでは脳で発 現している （Lelianova, V. G. , ら. J Biol Chem 272, 21504-8 (1997)。 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. As a human-derived gene, Myocil in expressed in the ciliary body (Kubota, R. et al. Genomics 41: 360-369, 1997) 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). Nagano, T. et al., J Neurochem 75, U8, 2000) is expressed in the cerebral cortex, It is considered to be an essential evening protein. It has been reported that the chicken Noelin (Barembaura M, et al. Nat Cell Biol 2000 219-25) regulates the production of neural crest cells by neural tubes. Also, African Megafrog Tiarin (Hiroshi Tsuda et al., Neuron, Vol. 33, 515-528, 2002) are thought to be secreted factors involved in neural tube tissue formation. Thus, proteins having these olfactmedin domains are secreted extracellularly and are considered to be a new category of physiologically active substances that control neural tissue formation. Latrophilin, on the other hand, 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 i nは L a t r oph i 1 i nのアン夕ゴニスト作用を有する可溶性受容体とする仮説も 提唱されている （Wessely, 0 and De Robert is, E.M. Neuron, Vol 33, 489- 491, 2002) 。 上記の他にもヒト由来の分泌型オルファクトメジンドメイン含有 タンパク質として、 GenBankには HN0EL- iso (GenBank Accession NP_064575) 、 0LFM1 (NP_055094) 、 0LFM2 (NP_477512) 、 0LFM3 (AAK97473) などが登録され ている。 また受容体型オルファクトメジンドメイン含有タンパク質としては、 ヒトでも Latrophilin 1 (NP— 036434、 Lectomedin- 1とも呼ばれる） 、 A hypothesis has also been proposed that 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) . In addition to the above, HN0EL-iso (GenBank Accession NP_064575), 0LFM1 (NP_055094), 0LFM2 (NP_477512), 0LFM3 (AAK97473), etc. are registered in GenBank as human-derived secretory olfactomedin domain-containing proteins. . Receptor-type olfactomedin domain-containing proteins include Latrophilin 1 (NP-036434, also called Lectomedin-1) in humans,
Lectomedin-2 (NP_055736.1) , Lectomedin- 3 (NP— 056051) などが登録されて いる。 この内、 ヒト Latrophilin 1遺伝子は乳がんでは変化していることが示さ れている (White,G.R¾ Oncogene 17 (26)， 3513-3519、 1998) 。 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).
変形性関節症は慢性の関節炎を伴う関節疾患で、 軟骨の退行変性により、 軟 骨の破壊と骨や軟骨の増殖性変化を来たす病気である。 骨端に骨棘形成するな どの関節の変形も認められる。 変形性関節症は年令とともに増加し、 60才以上 になると膝、 肘、 股関節、 脊椎では、 80%以上が変形性関節症の症状を呈する と言われている。 変形性関節症の治療は痛みを抑制する対処療法が中心であり、 非ステロイド性消炎鎮痛剤や関節に直接注射するヒアルロン酸やステロイド剤 ' などが使用される。 進行した場合、 関節鏡視下手術や痛みや変形が強い場合、 膝や股関節では骨切り術や人工関節置換などの手術の適応となる。 しかし、 人 ェ関節には寿命があるため 55才迄はこの手術を避けることが好ましいと考えら ' れ、 関節の退行性変化を抑制する治療法の開発が望まれている。 さらに、 変形 性関節症は X腺撮影などの装置による診断法しかなく、 より簡便な診断法の開発 も望まれている。  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. However, since human joints have a long life, it is considered preferable to avoid this operation until the age of 55, and the development of a treatment to suppress degenerative changes in joints is desired. Furthermore, osteoarthritis can only be diagnosed using a device such as an X-ray gland, and the development of a simpler diagnostic method is also desired.
遺伝子発現を網羅的に解析するために、 cDNAまたはオリゴヌクレオチドを固 定化したマイクロアレイ法が開発され、 疾患特異的な遺伝子発現の変化を見出 'す技術が普及し、 その有用性が確認されている。 例えば、 Affymetrix社の GeneChipシステムはがんなどの疾患の診断や創薬標的遺伝子の発見に多用され つつある。 変形性関節症患者の関節で特異的に発現している遺伝子やタンパク 質を見出し、 そこから変形性関節症の治療薬や 断薬を創出する試みが行われ ている。 例えば、 MMP- 13などのマトリックスメタ口プロテアーゼは変形性関節 症の関節で発現が上昇していることが知られており、 MMP阻害剤を関節破壊抑制 薬として検討されている。 For comprehensive analysis of gene expression, 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. For example, 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. For example, it is known that the expression of 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は、 GenBank Accession No. XP— 034000として登録されているォ ルファクトメジンドメイン含有タンパク質であるが、 全長配列は報告されてい ない。 また DKFZP586L151と相同性の高い遺伝子がマウスからクロ一エングされ ている （GenBank Accession No. XM_1 9545) 。 しかし、 オルファクトメジンド メイン含有タンパク質と変形性関節症との関係はこれまで報告さていない。 安全で優れた骨 ·関節疾患の診断薬及び予防 ·治療剤が求められている。 発明の開示  DKFZP586L151 is an orthophedomedin domain-containing protein registered as GenBank Accession No. XP-034000, but its full-length sequence has not been reported. In addition, a gene highly homologous to DKFZP586L151 has been cloned from mice (GenBank Accession No. XM_1 9545). However, 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
本発明者らは、 上記の課題を解決するために鋭意研究を重ねた結果、 変形性 関節症軟骨に発現が顕著に増加するオルファクトメジンドメイン含有遺伝子を. 見出し、 この知見に基づいて、 さらに検討を重ねた結果、 本発明を完成するに 至った。  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.
すなわち、 本発明は、  That is, the present invention
(1) 配列番号： 1で表されるアミノ酸配列と同一もしくは実質的に同一の ァミノ酸配列を含有するタンパク質またはその塩、  (1) a protein or a salt thereof containing an amino acid sequence identical or substantially identical to the amino acid sequence represented by SEQ ID NO: 1,
(2) 配列番号： 1で表されるアミノ酸配列からなるタンパク質またはその 塩、  (2) a protein consisting of the amino acid sequence represented by SEQ ID NO: 1 or a salt thereof;
(3) 配列番号： 3で表されるアミノ酸配列からなるタンパク質またはその 塩、  (3) a protein consisting of the amino acid sequence represented by SEQ ID NO: 3 or a salt thereof;
(4) 上記 （1) 記載のタンパク質の部分ペプチドまたはその塩、  (4) a partial peptide of the protein according to (1) or a salt thereof,
(5) 上記 （1) 記載のタンパク質または上記 （4) 記載の部分ペプチドを コードするポリヌクレオチドを含有するポリヌクレオチド、(5) The protein of (1) or the partial peptide of (4) A polynucleotide containing the encoding polynucleotide,
(6) DNAである上記 （5) 記載のポリヌクレオチド、 (6) the polynucleotide according to the above (5), which is a DNA;
(7) 配列番号： 2または配列番号： 4で表される塩基配列からなるポリヌ クレオチド、  (7) a polynucleotide having a base sequence represented by SEQ ID NO: 2 or SEQ ID NO: 4,
(8) 上記 （5) 記載のポリヌクレオチドを含有する組換えべクタ一、 (8) a recombinant vector containing the polynucleotide according to (5),
(9) 上記 （8) 記載の組換えべクタ一で形質転換された形質転換体、(9) a transformant transformed with the recombinant vector according to (8),
(10) 上記 （9) 記載の形質転換体を培養し、 上記 （1) 記載のタンパク 質または上記 （4) 記載の部分ペプチドを生成、 蓄積せしめ、 これを採取する ことを特徴とする上記 （1) 記載のタンパク質もしくは上記 （4) 記載の部分 ぺプチドまたはその塩の製造法、 (10) culturing the transformant according to (9) above, producing and accumulating the protein according to (1) or the partial peptide according to (4), and collecting the protein; 1) The method for producing the protein described in the above or the partial peptide described in the above (4) or a salt thereof,
(11) 上記 （1) 記載のタンパク質もしくは上記 （4) 記載の部分べプチ ドまたはその塩を含有してなる医薬、  (11) a medicament comprising the protein of (1) or the partial peptide of (4) or a salt thereof,
(12) 上記 （5) 記載のポリヌクレオチドを含有してなる医薬、  (12) a medicine comprising the polynucleotide according to the above (5),
(13) 上記 （5) 記載のポリヌクレオチドを含有してなる診断薬、 (14) 上記 （1) 記載のタンパク質もしくは上記 （4) 記載の部分べプチ ドまたはその塩に対する抗体、  (13) 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),
(15) 上記 （14) 記載の抗体を含有してなる診断薬、  (15) a diagnostic agent comprising the antibody according to (14) above,
(16) 上記 （14) 記載の抗体を含有してなる医薬、  (16) a medicament comprising the antibody according to (14) above,
(17) 上記 （5) 記載のポリヌクレオキドの塩基配列に相補的もしくは実 質的に相補的な塩基配列またはその一部を含有するポリヌクレオチド、  (17) 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,
(18) 上記 （17) 記載のポリヌクレオチドを含有してなる医薬、 (18) a medicament comprising the polynucleotide according to (17),
(19) 上記 （1) 記載のタンパク質もしくはその部分ペプチドまたはその 塩を用いることを特徴とする、 上記 （1) 記載のタンパク質の活性を調節する 化合物またはその塩のスクリ一ニング方法、 (19) A method for screening a compound or a salt thereof that regulates the activity of the protein according to (1), which comprises using the protein or partial peptide thereof or a salt thereof according to (1) above.
(19 a) 上記 （19) 記載のスクリーニング方法を用いて得られる化合物 またはその塩、  (19a) a compound obtained by using the screening method according to (19) or a salt thereof,
(19 b) 上記 （19 a) 記載の化合物またはその塩を含有してなる医薬、 (20) 上記 (1) 記載のタンパク質もしくはその部分ペプチドまたはその 塩を含有してなる、 上記 （1) 記載のタンパク質の活性を調節する化合物また はその塩のスクリーニング用キット、 (19b) 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,
(20 a) 上記 （20) 記載のスクリーニング用キットを用いて得られる化 合物またはその塩、  (20a) a compound or a salt thereof obtained using the screening kit according to (20),
(20 b) 上記 （20 a) 記載の化合物またはその塩を含有してなる医薬、 (21) 上記 （5) 記載のポリヌクレオチドを用いることを特徴とする、 上 記 （1) 記載のタンパク質遺伝子の発現を調節する化合物またはその塩の ク リ一ニング方法、  (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). A method for cleaning a compound or a salt thereof that regulates the expression of
(21 a) 上記 （21) 記載のスクリーニング方法を用いて得られる化合物 またはその塩、  (21a) a compound obtained by using the screening method according to (21) or a salt thereof,
(21 b) 上記 （21 a) 記載の化合物またはその塩を含有してなる医薬、 (22) 上記 （5) 記載のポリヌクレオチドを含有してなる、 上記 （1) 記 載のタンパク質遺伝子の発現を調節する化合物またはその塩のスクリーニング 用キット、  (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
(22 a) 上記 （22) 記載のスクリーニング用キットを用いて得られる化 合物またはその塩、  (22a) a compound or a salt thereof obtained using the screening kit according to (22),
(22 b) 上記 （22 a) 記載の化合物またはその塩を含有してなる医薬、 (22b) a medicament comprising the compound according to (22a) or a salt thereof,
(23) 上記 （14) 記載の抗体を用いることを特徴とする上記 （1) 記載 のタンパク質の定量方法、 (23) The method for quantifying a protein according to (1), wherein the antibody according to (14) is used.
(24) 骨 ·関節疾患の予防'治療剤である上記 （12) または （16) 記 載の医薬、 '  (24) The medicament according to (12) or (16) above, which is a therapeutic agent for preventing bone and joint diseases.
(24 a) 骨 ·関節疾患の予防 ·治療剤である上記 （19 b) 、 （20 b) (24a) The above (19b) and (20b) which are preventive and therapeutic agents for bone and joint diseases
(21 b) または （22 b) 記載の医薬、 (21b) or the medicament according to (22b),
(25) 骨，関節疾患が、 軟骨形成異常、 骨形成異常、 骨粗鬆症、 変形性関 節症、 慢性関節リウマチ、 関節炎、 滑膜炎、 代謝性関節症またはスポーツによ る関節障害である上記 （24) 記載の医薬、  (25) 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). 24) The medicament according to the above,
(26) 骨 ·関節疾患の診断薬である上記 （13) または （15) 記載の診' 断薬、  (26) The diagnostic drug according to (13) or (15) above, which is a diagnostic drug for bone and joint diseases.
(27) 骨'関節疾患が、 軟骨形成異常、 骨形成異常、 骨粗鬆症、 変形性関 節症、 慢性関節リウマチ、 関節炎、 滑膜炎、 代謝性関節症またはスポーツによ る関節障害である上記 （26) 記載の診断薬、 (27) 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
(28) 配列番号： 8で表わされる塩基配列と同一または実質的に同一の塩 基配列を含有する DNA、  (28) a DNA comprising a base sequence identical or substantially identical to the base sequence represented by SEQ ID NO: 8,
(29) E列番号： 8で表わされる塩基配列と同一または実質的に同一の塩 基配列を含有する DNAの転写調節領域である上記 （28) 記載の DNA、  (29) E The DNA according to the above (28), which is a transcription regulatory region of a DNA containing the same or substantially the same base sequence as the base sequence represented by E column number: 8,
(30) 転写調節領域がプロモーター領域である上記 （29) 記載の DNA、 (30) the DNA according to the above (29), wherein the transcription control region is a promoter region;
(31) 上記 （.28) 記載の DNAを含有する組換えべクタ一、 (31) A recombinant vector containing the DNA described in (.28) above,
(32) 配列番号： 8で表わされる塩基配列と同一または実質的に同一の塩 基配列を含有する DN Aの転写調節領域の下流にレポ一夕一遺伝子を有する D N Aを含有する上記 （31) 記載の組換えべクタ一、  (32) The above-mentioned (31), which contains a DNA having a repo allele downstream of a transcriptional regulatory region of DNA containing a nucleotide sequence identical or substantially identical to the nucleotide sequence represented by SEQ ID NO: 8. Described recombination vector,
(33) 上記 （32) 記載の組換えべクタ一を含有する形質転換体、  (33) a transformant containing the recombinant vector according to (32),
(34) 上記 （28) 記載の DNAを用いることを特徴とする、 配列番号： (34) SEQ ID NO: characterized by using the DNA of (28) above.
2で表わされる塩基配列と同一または実質的に同一の塩基配列を含有する DNDN containing the same or substantially the same nucleotide sequence as the nucleotide sequence represented by 2
Aの転写調節活性を調節する化合物またはその塩のスクリーニング方法、 (34 a) 上記 （34) 記載のスクリーニング方法を用いて得られる化合物 またはその塩、 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),
(34 b) 上記 （34 a) 記載の化合物またはその塩を含有してなる医薬、， (34 c) 骨 ·関節疾患の予防 ·治療剤である上記 （34b) 記載の医薬、 (35) 上記 （33) 記載の形質転換体を用いる上記 （34) 記載のスクリ 一二ング方法、  (34b) a medicament comprising the compound or salt thereof according to (34a), (34c) a medicament according to (34b), which is a preventive or therapeutic agent for bone / joint disease, (33) The screening method according to (34) using the transformant according to (34),
(36) 哺乳動物に対して、 配列番号： 1で表されるアミノ酸配列と同一も しくは実質的に同一のァミノ酸配列を含有するタンパク質またはその塩の活性 を阻害する化合物もしくはその塩、 または該タンパク質遺伝子の発現を阻害す る化合物もしくはその塩の有効量を投与することを特徴とする、 骨 ·関節疾患 の予防 ·治療法、  (36) 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.
(37) 配列番号： 1で表されるアミノ酸配列と同一もしくは実質的に同一 のアミノ酸配列を含有するタンパク質またはその塩の活性を阻害する、 または 該タンパク質遺伝子の発現を阻害することを特徴とする、 骨 ·関節疾患の予 防 ·治療法、 ( 3 8 ) 骨 ·関節疾患の予防 ·治療剤を製造するための、 配列番号： 1で表 されるァミノ酸配列と同一もしくは実質的に同一のァミノ酸配列を含有する夕 ンパク質またはその塩の活性を阻害する化合物もしくはその塩、 または該夕ン パク質遺伝子の発現を阻害する化合物もしくはその塩の使用などに関する。 図面の簡単な説明 (37) inhibiting the activity of a protein or a salt thereof containing the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 1, or inhibiting the expression of the protein gene The prevention and treatment of bone and joint diseases, (38) A protein or a salt thereof containing the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 1 for producing a preventive or therapeutic agent for bone or joint disease. The use of a compound or a salt thereof that inhibits the activity of the compound, or a compound or a salt thereof that inhibits the expression of the protein gene. BRIEF DESCRIPTION OF THE FIGURES
図 1は、 マウス C3H10T1/2細胞の軟骨分化過程におけるマウス chondromedin 遺伝子の発現変動を示す。 図中、 縦軸は、 調べた各時点におけるマウス chondromedin遺伝子の発現量を DayOにおける発現量で相対化した値を、 横軸は 分化培養開始後の日数を示す。  FIG. 1 shows changes in the expression of the mouse chondromedin gene during the cartilage differentiation process of mouse C3H10T1 / 2 cells. In the figure, 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.
図 2は、 Trlを免疫感作したゥサギの血清における抗 Trl抗体価の上昇を示す 。 図中、 縦軸は抗体価を、'横軸は一次抗体として用いた^:ペプチド血清の希釈 倍率を、 〇ば Day 0に採取した血清、 '秦は Day 35 に採取した血清をそれぞれ示 す。  FIG. 2 shows an increase in the anti-Trl antibody titer in the serum of a heron immunized with Trl. In the figure, 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. .
図 3は、 Tr2を免疫感作したゥサギの血清における抗 Tr2抗体価の上昇を示す 。 図中、 縦軸は抗体価を、 横軸は一次抗体として用いた抗ペプチド血清の希釈 倍率を、 〇は Day 0に採取した血清、 參は Day 35 に採取した血清をそれぞれ示 す。 発明を実施するための最良の形態  FIG. 3 shows an increase in the anti-Tr2 antibody titer in the serum of a heron immunized with Tr2. In the figure, the vertical axis indicates the antibody titer, the horizontal axis indicates the dilution ratio of the anti-peptide serum used as the primary antibody, 〇 indicates the serum collected on Day 0, and 參 indicates the serum collected on Day 35. BEST MODE FOR CARRYING OUT THE INVENTION
配列番号： 1で表されるアミノ酸配列と実質的に同一のアミノ酸配列として は、 配列番号： 1で表わされるアミノ酸配列と 8 5 %以上、 好ましくは約 9 0 %以上、 好ましくは約 9 5 %以上、 好ましくは約 9 8 %以上の相同性を有す るァミノ酸配列などが挙げられる。  The 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. As described above, preferably, an amino acid sequence having about 98% or more homology and the like are exemplified.
配列番号： 1で表されるアミノ酸配列と実質的に同一のアミノ酸配列を含有 するタンパク質としては、 例えば、 前記の配列番号： 1で表されるアミノ酸配 列と実質的に同一のアミノ酸配列を含有し、 配列番号： 1で表されるアミノ酸 配列を含有するタンパク質と実質的に同質の活性を有するタンパク質などが好 ましい。 - 実質的に同質の活性としては、 例えば、 軟骨細胞の分化調節活性、 軟骨細胞 への結合活性などがあげられる。 実質的に同質とは、 それらの性質が生物学的 に （例、 生理学的に、 または薬理学的に;） 同質であることを示す。 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.
軟骨細胞の分化調節活生の測定は、 公知の方法に準じて行うことができ、 例 えば、 J Cel l Biochem 67, 498-513 (1997)に記載の方法またはそれに準じた方 法などに従って測定することができる。 例えば、 本発明のタンパク質を産生す る能力を有する細胞の培養上清または細胞抽出物から、 公知方法 （例、 免疫ァ フィニティーカラム） に従い、 本発明のタンパク質を得る。 このタンパク質を 培養軟骨細胞に添加し、 37°Cで 7日〜 21日間培養する。 次に上記文献に挙げられ た方法に従い軟骨細胞のプロテオダリ力ン含量またはアル力リ性ホスファタ一 ゼ活性を評価することにより、 軟骨細胞の分化調節活性を測定する。  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. For example, 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. Next, 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.
軟骨細胞への結合活性の測定は、 公知の方法に準じて行うことができる。 例 えば、 本発明のタンパク質を、 公知の方法に準じ、 例えば!) -ピオチノィル - ε - アミノカプロン酸 _Ν-ハイ.ドロキシサクシィミドエステルを用いてピオチン標識 する。 該標識タンパク質を培養軟骨細胞に添加し、 4°Cで一時間培養後、 細胞を PBSで洗浄する。 次に、 ストレプトアビジンおよび酵素 （例、 アルカリ性ホスフ ァタ一ゼ） の複合体を添加し、 4 で一時間培養後、 細胞を PBSで洗浄する。 最 後に該酵素の化学発光基質 （例、 Di sodium 2-chloro-5- (4-methoxyspi ro {l , - dioxetane-3, 2' - (5' -chloro) tr icyc lo [3. 3. 1. 1. 3， 7] decan}-4-yl) -l-phenyl phosphateなど） を添加レ、 発生する光を測定することにより、 本発明のタンパ ク質の軟骨細胞への結合活性を測定する。  The binding activity to chondrocytes can be measured according to a known method. For example, the protein of the present invention is labeled with biotin according to a known method, for example, using!)-Piotinoyl-ε-aminocaproic acid_Ν-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. Finally, 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), and measure the light generated to determine the binding activity of the protein of the present invention to chondrocytes. .
また、 本発明のタンパク質としては、 例えば、 （i) 配列番号： 1で表される アミノ酸配列中の 1または 2個以上 （例えば 1〜 5 0個程度、 好ましくは 1〜 3 0個程度、 好ましくは 1〜1 0個程度、 さらに好ましくは数 （1〜5 ) 個） のアミノ酸が欠失したアミノ酸配列、 （i i) 配列番号： 1で表されるアミノ酸 配列に 1または 2個以上 （例えば 1〜 5 0個程度、 好ましくは 1〜3 0個程度、 好ましくは 1〜1 0個程度、 さらに好ましくは数 （1〜5 ) 個) のアミノ酸が 付加したアミノ酸配列、 （i i i) 配列番号： 1で表されるアミノ酸配列に 1また は 2個以上 （例えば 1〜 5 0個程度、 好ましくは 1〜3 0個程度、 好ましくは 1〜 1 0個程度、 さらに好ましくは数 （1〜 5 ) 滅） のアミノ酸が挿入された アミノ酸配列、 （iv) 配列番号：. Γで表されるアミノ酸配列中の 1または 2個 以上 （例えば 1〜 5 0個程度、 好ましくは 1〜 3 0個程度/好ましくは 1〜 1 0個程度、 さらに好ましくは数 （1〜 5 ) 個） のアミノ酸が他のアミノ酸で置 換されたアミノ酸配列、 または （V) それらを組み合わせたアミノ酸配列を含有 するタンパク質などのいわゆるムティンも含まれる。 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. An 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.
上記のようにアミノ酸配列が挿入、 欠失または置換されている場合、 その揷 入、 欠失または置換の位置としては、 とくに限定されない。  When the amino acid sequence is inserted, deleted or substituted as described above, the position of the insertion, deletion or substitution is not particularly limited.
本明細書におけるタンパク質は、 ペプチド標記の慣例に従って左端が N末端 (ァミノ末端） 、 右端が C末端 （力ルポキシル末端） である。 配列番号： 1で 表わされるアミノ酸配列を含有するタンパク質をはじめとする、 本発明のタン パク質は、 C末端が力ルポキシル基 (-C00H) 、 カルポキシレート（-COO—)' 、 ァ ミド （-C0NH2) またはエステル （- C00R) の何れであってもよい。 In the protein in the present specification, 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).
ここでエステルにおける R'としては、 例えば、 メチル、 ェチル、 n—プロピ ル、 イソプロピル、 n—ブチルなどの アルキル基、 例えば、 シクロペンチ ル、 シクロへキシルなどの C3 ンクロアルキル基、 例えば、 フエニル、 ひ一ナ フチルなどの C 6_12ァリール基、 例えば、 ベンジル、 フェネチルなどのフエニル 一 C Mアルキル基もしくはひ一ナフチルメチルなどの α _ナフチルー C ,_2アル キル基などの <37_14ァラルキル基、 ピパロィルォキシメチル基などが用いられる 本発明のタンパク質が C末端以外に力ルポキシル基 （またはカルポキシレー ト) を有している場合、 力ルポキシル基がアミド化またはエステル化されてい るものも本発明のタンパク質に含まれる。 この場合のエステルとしては、 例え ば上記した C末端のエステルなどが用いられる。 Here, 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. When the protein of the present invention has a lipoxyl group (or carboxylate) other than the C-terminal, the lipoxyl group is amidated or esterified. Are also included in the protein of the present invention. As the ester in this case, for example, the above-mentioned C-terminal ester and the like are used.
さらに、 本発明のタンパク質には、 N末端のアミノ酸残基 （例、 メチォニン 残基） のァミノ基が保護基 （例えば、 ホルミル基、 ァセチル基などの〇卜6アル カノィルなどの ァシル基など） で保護されているもの、 生体内で切断され て生成する N末端のグルタミン残基がピログルタミン酸化したもの、 分子内の アミノ酸の側鎖上の置換基 （例えば- 0H、 - SH、 アミノ基、 イミダゾール基、 ィ ンドール基、 グァニジノ基など） が適当な保護基 （例えば、 ホルミル基、 ァセ チル基などの c卜6アル力ノィル基などの c卜 6ァシル基など） で保護されている もの、 あるいは糖鎖が結合したいわゆる糖タンパク質などの複合タンパク質な ども含まれる。 Furthermore, in the protein of the present invention, 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). Protected, N-terminal glutamine residue generated by cleavage in vivo, pyroglutamine-oxidized, Substituent on side chain of amino acid in molecule (eg, -OH, -SH, amino group, imidazole Groups, indole groups, guanidino groups, etc.) 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.
本発明のタンパク質の具体例としては、 例えば、 配列番号： 1で表されるァ ミノ酸配列を含有するタンパク質などがあげられる。  Specific examples of the protein of the present invention include, for example, a protein containing the amino acid sequence represented by SEQ ID NO: 1.
配列番号： 1で表されるアミノ酸配列を含有するタンパク質としては、 配列 番号： 3で表されるアミノ酸配列を含有するタンパク質などが挙げられる。 ' 本発明のタンパク質の部分べプチドとしては、 前記した本発明のタンパク質 の部分ペプチドであって、 好ましくは、 前記した本発明のタンパク質と同様の 性質を有するものであればいずれのものでもよい。 例えば、 本発明のタンパク 質の構成アミノ酸配列のうち少なくとも 2 0個以上、 好ましくは 5 0個以上、 さらに好ましくは 7 0個以上、 より好ましくは 1 0 0個以上、 最も好ましくは 2 0 0個以上のアミノ酸配列を有するペプチドなどが用いられる。 例えば、 配 列番号： 1で表されるアミノ酸配列の第 1〜2 9 0番目のアミノ酸配列を有す るべプチドなどが挙げられる。  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. For example, 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.
また、 本発明で用いられる部分ペプチドは、 そのアミノ酸配列中の 1または 2個以上 （好ましくは、 1〜1 0個程度、 さらに好ましくは数 （1〜5 ) 個） のアミノ酸が欠失し、 または、.そのアミノ酸配列に 1または 2個以上 （好まし くは、 1〜2 0個程度、 より好ましくは 1〜1 0個程度、 さらに好ましくは数 ( 1〜5 ) 個） のアミノ酸が付加し、 または、 そのアミノ酸配列に 1または 2 個以上 （好ましくは、 1〜2 0個程度、 より好ましくは 1〜1 0個程度、 さら に好ましくは数 （1〜5 ) 個） のアミノ酸が揷入され、 または、 そのアミノ酸 配列中の 1または 2個以上 （好ましくは、 1〜1 0個程度、 より好ましくは数 個、 さらに好ましくは 1〜5個程度） のアミノ酸が他のアミノ酸で置換されて いてもよい。  In the partial peptide used in the present invention, 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.
また、 本発明で用いられる部分ペプチドは C末端が力ルポキシル基 （- C00H) カルポキシレート （- C00-) 、 アミド （- C0NH2) またはエステル （-C00R) の何 れであってもよい。 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).
さらに、 本発明で用いられる部分ペプチドには、 前記した本発明のタンパク 質と同様に、 C末端以外に力ルポキシル基 （またはカルポキシレート） を有し ているもの、 N末端のアミノ酸残基 （例、 メチォニン残基） のァミノ基が保護 基で保護されているもの、 N端側が生体内で切断され生成し グルタミン残基 がピ口ダル夕ミン酸化したもの、 分子内のアミノ酸の側鎖上の置換基が適当な 保護基で保護されているもの、 あるいは糖鎖が結合したいわゆる糖ペプチドな どの複合ペプチドなども含まれる。 Further, the partial peptide used in the present invention includes the protein of the present invention described above. As well as those having a C-terminal amino acid residue (or carboxylate) at the C-terminus, N-terminal amino acid residue (eg, methionine residue) whose amino group is protected by a protecting group , 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.
本発明で用いられる部分ペプチドは抗体作成のための抗原としても用いるこ とができる。  The partial peptide used in the present invention can also be used as an antigen for producing an antibody.
本発明のタンパク質または部分ペプチドの塩としては、 生理学的に許容され る酸 （例、 無機酸、 有機酸） や塩基 （例、 アルカリ金属塩） などとの塩が用い られ、 とりわけ生理学的に許容される酸付加塩が好ましい。 この様な塩として は、 例えば、 無機酸 （例えば、 塩酸、 リン酸、 臭化水素酸、 硫酸） との塩、 あ るいは有機酸 （例えば、 酢酸、 ギ酸、 プロピオン酸、 フマル酸、 マレイン酸、 コハク酸、 酒石酸、 クェン酸、 リンゴ酸、 蓚酸、 安息香酸、 メタンスルホン酸、 ベンゼンスルホン酸） との塩などが用いられる。  As the salt of the protein or partial peptide of the present invention, salts with physiologically acceptable acids (eg, inorganic acids, organic acids) and bases (eg, alkali metal salts) are used. 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).
本発明のタンパク質もしくはその部分ペプチドまたはその塩は、 前述したヒ トや温血動物の細胞または組織から自体公知のタンパク質の精製方法によって 製造することもできるし、 タンパク質をコードする D N Aを含有する形質転換 体を培養することによつても製造することができる。 また、 後述のペプチド合 成法に準じて製造することもできる。  The protein of the present invention, its partial peptide, or a salt thereof 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.
ヒトゃ哺乳動物の組織または細胞から製造する場合、 ヒトゃ哺乳動物の組織 または細胞をホモジナイズした後、 酸などで抽出を行ない、 該抽出液を逆相ク 口マトグラフィ一、 イオン交換クロマトグラフィ一などのクロマトグラフィー を組み合わせることにより精製単離することができる。  When producing from human or mammalian tissues or cells, 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.
本発明のタンパク質もしくは部分ペプチドまたはその塩、 またはそのアミド 体の合成には、 通常市販のタンパク質合成用樹脂を用いることができる。 その ような樹脂としては、 例えば、 クロロメチル樹脂、 ヒドロキシメチル樹脂、 ベ ンズヒドリルァミン樹脂、 アミノメ'チル樹脂、 4一べンジルォキシベンジルァ ルコール樹脂、 4一メチルベンズヒドリルァミン樹脂、 P AM樹脂、 4—ヒド 口キシメテルメチルフエニルァセトアミドメチル樹脂、 ポリアクリルアミド樹 脂、 4— ( 2 ', 4，—ジメトキシフエ二ル一ヒドロキシメチル） フエノキシ樹脂、 4— ( 2，， 4，一ジメトキシフエニル— F m o cアミノエチル） フエノキシ樹脂 などを挙げることができる。 このような樹脂を用い、 ひーァミノ基と側鎖官能 基を適当に保護したアミノ酸を、 目的とするタンパク質の配列通りに、 自体公 知の各種縮合方法に従い、 樹脂上で縮合させる。 反応の最後に榭脂からタンパ ク質または部分べプチドを切り出すと同時に各種保護基を除去し、 さらに高希 釈溶液中で分子内ジスルフィド結合形成反応を実施し、 目的のタンパク質もし くは部分ペプチドまたはそれらのアミド体を取得する。 For the synthesis of the protein or partial peptide of the present invention, a salt thereof, or an amide thereof, a commercially available resin for protein synthesis can be generally used. Examples of such 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. Using such a 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. At the end of the reaction, proteins or partial peptides are cut out from the resin, and at the same time, various protecting groups are removed.In addition, 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.
上記した保護アミノ酸の縮合に関しては、 タンパク質合成に使用できる各種 活性化試薬を用いることができるが、 特に、 カルポジイミド類がよい。 力ルポ ジイミド類としては、 D C C、 N, N，ージイソプロピルカルポジイミド、 N— ェチル— N，— ( 3—ジメチルァミノプロリル) カルポジイミドなどが用いられ る。 これらによる活性化にはラセミ化抑制添加剤 （例えば、 H O B t , HO O B t ) とともに保護アミノ酸を直接樹脂に添加するかまたは、 対称酸無水物ま たは H O B tエステルあるいは HO〇B tエステルとしてあらかじめ保護アミ ノ酸の活性化を行なった後に樹脂に添加することができる。  For the condensation of the protected amino acids described above, various activating reagents that can be used for protein synthesis can be used, and carbodiimides are particularly preferable. As the disulfide diimides, DCC, N, N, diisopropylcarbodiimide, N-ethyl-N,-(3-dimethylaminoprolyl) carbodiimide and the like are used. For these activations, 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〇B t ester. It can be added to the resin after the protected amino acid has been activated in advance.
保護アミノ酸の活性化や樹脂との縮合に用いられる溶媒としては、 タンパク 質縮合反応に使用しうることが知られている溶媒から適宜選択されうる。 例え ば、 N, N—ジメチルホルムアミド， N, N—ジメチルァセトアミド， N—メ チルピロリドンなどの酸アミド類、 塩化メチレン， クロ口ホルムなどのハロゲ ン化炭化水素類、 トリフルォロエタノールなどのアルコール類、 ジメチルスル ホキシドなどのスルホキシド類、 ピリジン, ジォキサン， テトラヒドロフラン などのエーテル類、 ァセトニトリル， フ。口ピオ二トリルなどの二トリル類、 酢 酸メチル， 酢酸ェチルなどのエステル類あるいはこれらの適宜の混合物などが 用いられる。 反応温度は夕ンパク質結合形成反応に使用され得ることが知られ ている範囲から適宜選択され、 通常約— 2 0 °C〜5 0 °Cの範囲から適宜選択さ れる。 活性化されたアミノ酸誘導体は通常 1 . 5〜4倍過剰で用いられる。 ニン ヒドリン反応を用いたテストの結果、 縮合が不十分な場合には保護基の脱離を 行なうことなく縮合反応を繰り返すことにより十分な縮合を行なうことができ る。 反応を繰り返しても十分な縮合が得られないときには、 無水酢酸またはァ セチルイミダゾ一ルを用いて未反応ァミノ酸をァセチル化することによって、 後の反応に影響を与えないようにすることができる。 · 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. For example, 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. As a result of the test using the ninhydrin reaction, if the condensation is insufficient, Sufficient condensation can be performed by repeating the condensation reaction without performing. If sufficient condensation is not obtained even after repeating the reaction, unreacted amino acid can be acetylated with acetic anhydride or acetylimidazole so that the subsequent reaction is not affected. . ·
原料のァミノ基の保護基としては、 例えば、 Z、、： B o c、 t一ペンチルォキ シカルボニル、 イソポルニルォキシカルポニル、 4—メトキシベンジルォキシ 力ルポニル、 C 1 一 Z、 B r— Z、 ァダマンチルォキシカルポニル、 トリフル ォロアセチル、 フタロイル、 ホルミル、 2—二トロフエニルスルフエ二ル、 ジ フエニルホスフイノチオイル、 Fm o cなどが用いられる。  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.
力ルポキシル基は、 例えば、 アルキルエステル化 （例えば、 メチル、 ェチル、 プロピル、 ブチル、 t一ブチル、 シクロペンチル、 シクロへキシル、 シクロへ プチル、 シクロォクチル、 2—ァダマンチルなどの直鎖状、 分枝状もしくは環 状アルキルエステル化) 、 ァラルキルエステル化 (例えば、 ベンジルエステル、 4—ニトロべンジルエステル、 4ーメトキシベンジルエステル、 4—クロ口べ ンジルエステル、 ベンズヒドリルエステル化） 、 フエナシルエステル化、 ベン ジルォキシカルボニルヒドラジド化、 t—ブトキシカルポニルヒドラジド化、 トリチルヒドラジド化などによつて保護することができる。  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.
セリンの水酸基は、 例えば、 エステル化またはエーテル化によって保護する ことができる。 このエステル化に適する基としては、 例えば、 ァセチル基など の低級 （C ^ 6) アルカノィル基、 ベンゾィル基などのァロイル基、 ベンジル ォキシカルポニル基、 エトキシカルポニル基などの炭酸から誘導される基など が用いられる。 また、 ェ一テル化に適する基としては、 例えば、 ベンジル基、 テトラヒドロピラニル基、 t一ブチル基などである。 . チロシンのフエノール性水酸基の保護基としては、 例えば、 B z し C 1 2 一 B z l、 2—ニトロベンジル、 B r— Z、 t—ブチルなどが用いられる。 The hydroxyl group of serine can be protected, for example, by esterification or etherification. As 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. . Examples of groups suitable for etherification include a benzyl group, a tetrahydropyranyl group, and a t-butyl group. . Examples of 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.
ヒスチジンのイミダゾールの保護基としては、 例えば、 T o s、 4ーメトキ シ— 2， 3， 6—トリメチルベンゼンスルホニル、 D N P、 ベンジルォキシメ チル、 B u m、 B o c、 T r t、 F m o cなどが用いられる。  As the 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.
原料のカルボキシル基の活性化されたものとしては、 例えば、 対応する酸無 水物、 アジド、 活性エステル 〔アルコール （例えば、 ペン夕クロ口フエノール、 2 , 4， 5—トリクロ口フエノール、 2， 4—ジニトロフエノール、 シァノメ チルアルコール、 パラニトロフエノ一ル、 H O N B、 N—ヒドロキシスクシミ ド、 N—ヒドロキシフ夕ルイミド、 H〇B t ) とのエステル〕 などが用いられ る。 原料のァミノ基の活性化されたものとしては、 例えば、 対応するリン酸ァ ミドが用いられる。 - ' 保護基の除去 （脱離） 方法としては、 例えば、 P d—黒あるいは P d—炭素 などの触媒の存在下での水素気流中での接触還元や、 また、 無水フッ化水素、 メタンスルホン酸、 トリフルォロメタンスルホン酸、 トリフルォロ酢酸あるい はこれらの混合液などによる酸処理や、 ジイソプロピルェチルァミン、 トリエ チルァミン、 ピぺリジン、 ピぺラジンなどによる塩基処理、 また液体アンモニ ァ中ナトリウムによる還 なども用いられる。 上記酸処理による脱離反応は、 一般に約一 2 0 °C〜4 0 °Cの温度で行なわれるが、 酸処理においては、 !Jえば、 ァニソ一ル、 フエノール、 チオアニソール、 メタクレゾール、 パラクレゾ一ル、 ジメチルスルフィド、 1 , 4一ブタンジチオール、 1， 2—エタンジチオール などのようなカチオン捕捉剤の添加が有効である。 また、 ヒスチジンのイミダ ゾ一ル保護基として用いられる 2， 4—ジニトロフエニル基はチオフエノ一ル 処理により除去され、 トリブトファンのインドール保護基として用いられるホ ルミル基は上記の 1， 2—エタンジチオール、 1 , 4一ブタンジチオールなど の存在下の酸処理による脱保護以外に、 希水酸化ナトリウム溶液、 希アンモニ ァなどによるアルカリ処理によっても除去される。 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〇B t)] and the like. As 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 anhydrous hydrogen fluoride or methane. 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. In the case of the acid treatment, anisol, phenol, thioanisole, methacresol, paracresol, etc. It is effective to add a cation scavenger such as ethyl, dimethyl sulfide, 1,4-butanedithiol or 1,2-ethanedithiol. In addition, the 2,4-dinitrophenyl group used as an imidazole protecting group of histidine is removed by thiophenol treatment, and the formyl group used as an indole protecting group of tributofan is 1,2-ethanedithiol, 1 In addition to deprotection by acid treatment in the presence of 1,4-butanedithiol, etc., it is also removed by alkali treatment with dilute sodium hydroxide solution, dilute ammonia and the like.
原料の反応に関与すべきでない官能基の保護ならびに保護基、 およびその保 護基の脱離、 反応に関与する官能基の活性化などは公知の基または公知の手段 から適宜選択しうる。  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.
タンパク質または部分べプチドのアミ)ド体を得る別の方法としては、 例えば、 まず、 カルポキシ末端アミノ酸の α—力ルポキシル基をアミド化して保護した  As another method for obtaining an amide) form of a protein or a partial peptide, for example, first, the α-hydroxyl group of the amino acid at the carboxy terminal was protected by amidation to protect it.
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- 後、 アミノ基側にペプチド （タンパク質） 鎖を所望の鎖長まで延ばした後、 該 ぺプチド鎖の Ν末端の α—ァミノ基の保護基のみを除いた夕ンパク質または部 分ペプチドと C末端の力ルポキシル基の保護基のみを除去したタンパク質また は部分ペプチドとを製造し、 これらのタンパク質またはペプチドを上記したよ うな混合溶媒中で縮合させる。 縮合反応の詳細については上記と同様である。 縮合により得られた保護タンパク質またはペプチドを精製した後、 上記方法に よりすべての保護基を除去し、 所望の粗夕ンパク質またはべプチドを得ること ができる。 この粗タンパク質またはペプチドは既知の各種精製手段を駆使して 精製し、 主要画分を凍結乾燥することで所望のタンパク質またはペプチドのァ ミド体を得ることができる。 -After that, after extending the peptide (protein) chain to the desired chain length on the amino group side, the protein or the partial peptide and C, which are obtained by removing only the protecting group for the α-amino group at the Ν end of the peptide chain, A protein or protein in which only the protecting group of the terminal lipoxyl group has been Produces a partial peptide, and condenses these proteins or peptides in a mixed solvent as described above. Details of the condensation reaction are the same as described above. After purifying the protected protein or peptide obtained by the condensation, all the protecting groups are removed by the above-mentioned method, and a desired crude protein or peptide can be obtained. The crude protein or peptide is purified by using various known purification means, and the main fraction is freeze-dried to obtain an amide of the desired protein or peptide.
タンパク質またはペプチドのエステル体を得るには、 例えば、 力ルポキシ末 端アミノ酸のひ—力ルポキシル基を所望のアルコール類と縮合しアミノ酸エス テルとした後、 タンパク質またはペプチドのアミド体と同様にして、 所望の夕 ンパク質またはべプチドのエステル体を得ることができる。  In order to obtain 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.
本発明で用いられる部分ペプチドまたはそれらの塩は、 自体公知のペプチド の合成法に従って、 あるいは本発明のタンパク質を適当なぺプチダ一ゼで切断 することによって製造することができる。 ペプチドの合成法としては、 例えば、 固相合成法、 液相合成法のいずれによっても良い。 すなわち、 本発明で用いら れる部分べプチドを構成し得る ¾5分べプチドもしくはアミノ酸と残余部分とを 縮合させ、 生成物が保護基を有する場合は保護基を脱離することにより目的の ぺプチドを製造することができる。 公知の縮合方法や保護基の脱離としては、 例えば、 以下の （i) 〜 (v) に記載された方法が挙げられる。  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. As 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).
(i) M. Bodanszkyおよび M丄 Ondet t K Pept ide Synthes is, Intersc ience Publ ishers, New York (1966年）  (i) M. Bodanszky and M 丄 Ondet t K Peptide Synthes is, Interscience Publ ishers, New York (1966)
(i i) Schroederおよび Luebke、 The Pept ide, Academic Press, New York (1965年）  (ii) Schroeder and Luebke, The Peptide, Academic Press, New York (1965)
(i i i) 泉屋信夫他、 ペプチド合成の基礎と実験、 丸善 (株） （1975年）  (iii) Nobuo Izumiya et al., Peptide Synthesis Basics and Experiments, Maruzen Co., Ltd. (1975)
(iv) 矢島治明 および榊原俊平、 生化学実験講座 1、 タンパク質の化学 IV、 - 205、 （1977年）  (iv) Haruaki Yajima and Shunpei Sakakibara, Laboratory of Biochemistry 1, Protein Chemistry IV, -205, (1977)
(v) 矢島治明監修、 続医薬品の開発、 第 14巻、 ペプチド合成、 広川書店 また、 反応後は通常の精製法、 例えば、 溶媒抽出 '蒸留 'カラムクロマトグ ラフィ一'液体クロマトグラフィー '再結晶などを組み合わせて本発明で用い られる部分べプチドを精製単離することができる。 上記方法で得られる部分べ プチドが遊離体である場合は、 公知の方法あるいはそれに準じる方法によって 適当な塩に変換することができるし、 逆に塩で得られた場合は、 公知の方法あ るいはそれに準じる方法によって遊離体または他の塩に変換することができる t 本発明のタンパク質をコードするポリヌクレオチドとしては、 前述した本発 明で用いられるタンパク質をコードする塩基配列を含有するものであればいか なるもの あってもよい。 好ましくは DNAである。 DNAとしては、 ゲノム DNA、 ゲノム DNAライブラリ一、 前記した細胞 ·組織由来の c DNA、 前 記した細胞 '組織由来の cDNAライブラリ一、 合成 DNAのいずれでもよい ; ライブラリ一に使用するべクタ一は、 バクテリオファージ、 .プラスミド、 コ スミド、 ファージミドなどいずれであってもよい。 また、 前記した細胞 '組織 より totalRNAまたは mRNA画分を調製したものを用いて直接 Reverse Transcriptase Polymerase Chain Reaction (以下、 RT-PCR法と略称する) によ つて増幅することもできる。 (v) Supervision of Haruaki Yajima, Development of Continuing Pharmaceuticals, Vol. 14, Peptide Synthesis, Hirokawa Shoten After the reaction, the usual purification method, for example, solvent extraction 'distillation' column chromatography 'liquid chromatography' liquid chromatography 'recrystallization Used in the present invention The resulting partial peptide can be purified and isolated. When the partial peptide obtained by the above method is a free form, it can be converted to an appropriate salt by a known method or a method 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. Preferably 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.
本発明のタンパク質をコードする DNAとしては、 例えば、 （i) 配列番号： 2で表される塩基配列を含有する DNA、 または配列番号： 2で表される塩基 配列とハイストリンジエンドな条件下でハイブリダィズする塩基配列を含有し- 配列番号： 1で表されるアミノ酸配列を含有するタンパク質と実質的に同質の 性質を有するタンパク質をコードする DNA、 (ii) 配列番号： 4で表される 塩基配列を含有する DNA、 または配列番号： 4で表される塩基配列とハイス トリンジェントな条件下でハイブリダィズする塩基配列を含有し、 配列番号： 3で表されるアミノ酸配列を含有するタンパク質と実質的に同質の性質を有す るタンパク質をコードする DNAであれば何れのものでもよい。 ·  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. ·
' 配列番号： 2または配列番号： 4で表される塩基配列とハイストリンジェン トな条件下でハイブリダィズできる DN.Aとしては、 例えば、 配列番号： 2ま たは配列番号： 4で表される塩基配列と約 90%以上、 好ましくは約 95%以 上、 好ましくは約 98 %以上の相同性を有する塩基配列を含有する DNAなど が用いられる。 '' 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.
ハイブリダィゼ一シヨンは、 自体公知の方法あるいはそれに準じる方法、 例 えば、 Mol ecular Cloning 2nd (J. Sambrook et al . , Cold Spring Harbor Lab. Press, 1989) に記載の方法などに従って行なうことができる。 また、 市販のラ イブラリーを使用する場合、 添付の使用説明書に記載の方法に従って行なうこ とができる。 より好ましくは、 ハイストリンジエンドな条件に従って行なうこ とができる。 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.
ハイストリシジェントな条件とは、 例えば、 ナトリウム濃度が約 1 9〜4 0 mM、 好ましくは約 1 9〜2 O mMで、 温度が約 5 0〜7 0 °C、 好ましくは約 6 0〜6 5 °Cの条件を示す。 特に、 ナトリウム濃度が約 1 9 mMで温度が約 6 5 の場合が最も好ましい。'  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. '
より具体的には、 配列番号： 1で表されるアミノ酸配列を含有するタンパク 質をコードする D NAとしては、 配列番号：，2で表される塩基配列を含有する D NA、 配列番号： 3で表されるアミノ酸配列を含有するタンパク質をコード する D NAとしては、 配列番号： 4で表される塩基配列を含有する D NAなど が用いられる。  More specifically, as the 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 As the 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.
本発明の部分ペプチドをコードする D NAとしては、 前述した本発明で用い られる部分べプチドをコードする塩基配列を含有するものであればいかなるも のであってもよい。 また、 ゲノム D NA、 ゲノム' D NAライブラリ一、 前記し た細胞 ·組織由来の c D NA、 前記した細胞 ·組織由来の c D NAライブラリ ―、 合成 D N Aのいずれでもよい。  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.
本発明で用いられる部分ペプチドをコ一ドする D NAとしては、 例えば、 配 列番号： 2または配列番号： 4で表される塩基配列を含有する D NAの一部分 を有する D NA、 または配列番号： 2または配列番号： 4で表される塩基配列 とハイストリンジェントな条件下でハイプリダイズする塩基配列を含有し、 本 発明のタンパク質と実質的に同質の活性を有するタンパク質をコードする D N Aの一部分を含有する D NAなどが用いられる。  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.
配列番号： 2または配列番号：.4で表される塩基配列とハイブリダィズでき る D NAは、 前記と同意義を示す。  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.
ハイブリダィゼ一シヨンの方法およびハイストリンジェントな条件は前記と 同様のものが用いられる。 本発明のタンパク質、 部分ペプチド （以下、 これらをコードする DNAのク ローニングおよび発現の説明においては、 これらを単に本発明のタン八°ク質と 略記する場合がある） を完全にコ ドする DNAのクローニングの手段として は、 本発明のタンパク質をコードする塩基配列の一部分を有する合成 DN Aプ ライマーを用いて PC R法によって増幅するか、 または適当なベクタ一に組み 込んだ D N Aを本発明の夕ンパク質の一部あるいは全領域をコードする DNA 断片もしくは合成 D N Aを用いて標識したものとのハイプリダイゼ一シヨンに よって選別することができる。 ハイブリダィゼーシヨンの方法は、 例えば、 ， Molecular Cloning 2nd (J. Sambrook et al. , Cold Spring Harbor Lab. The same hybridization method and high stringency conditions as described above are used. 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). As a means for cloning, 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.
Press, 1989) に記載の方法などに従って行なうことができる。 また、 市販のラ イブラリーを使用する場合、 添付の使用説明書に記載の方法に従って行なうこ とができる。' Press, 1989). When a commercially available library is used, it can be performed according to the method described in the attached instruction manual. '
DNAの塩基配列の変換は、 PCR、 公知のキット、 例えば、 Mutan™- super Express Km (宝酒造 （株） ） 、 Mutan™-K (宝酒造 （株） ） 等を用いて、 ODA- LA PCR法、 Gapped duplex法、 Kunkel法等の自体公知の方法あるいはそれらに準じ る方法に従って行なうことができる。 '  The DNA base sequence can be converted using PCR, a known kit, for example, Mutan ™ -super Express Km (Takara Shuzo Co., Ltd.), Mutan ™ -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. '
クローン化されたタンパク質をコ一ドする DN Aは目的によりそのまま、 ま たは所望により制限酵素で消化したり、 リンカーを付加したりして使用するこ とができる。 該 DNAはその 5'末端側に翻訳開始コドンとしての ATGを有し， また 3，末端側には翻訳終止コドンとしての TAA、 TGAまたは TAGを有レ ていてもよい。 これらの翻訳開始コドンや翻訳終止コドンは、 適当な合成 DN Aアダプタ一を用いて付加することもできる。  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.
本発明のタンパク質の発現べクタ一は、 例えば、 （ィ） 本発明のタンパク質 をコードする DNAから目的とする DNA断片を切り出し、 （口） 該 DNA断 片を適当な発現ベクター中のプロモーターの下流に連結することにより製造す ることができる。  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
ベクターとしては、 大腸菌由来のプラスミド （例、 pBR 322， pBR3 25, pUC 12, pUC 13) 、 枯草菌由来のプラスミド （例、 pUB 11 0, pTP 5, P C 194) 、 酵母由来プラスミド （例、 p SH19， p SH 15) 、 λファ一ジなどのバクテリオファージ、 レトロウイルス， ワクシニア ウィルス， バキュロウィルスなどの動物ウィルスなどの他、 pAl— 11、 p XT 1 pRcZCMV、 pRc/RSV, p c D NA I /N e oなどが用い られる。 Examples of the vector 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) In addition to bacteriophage such as λ phage, animal viruses such as retrovirus, vaccinia virus, and baculovirus, pAl-11, pXT1 pRcZCMV, pRc / RSV, pcDNA I / Neo, etc. Used.
本発明で用いられるプロモー夕一としては、 遺伝子の発現に用いる宿主に対 応して適切なプロモータ一であればいかなるものでもよい。 例えば、 動物細胞 を宿主として用いる場合は、 SRaプロモ一夕一、 SV40プロモ一夕一、 L TRプロモータ一、 CMVプロモータ一、 HS V- TKプロモータ一などが挙げ ' られる。  The promoter used in the present invention may be any promoter as long as it is appropriate for the host used for gene expression. For example, when an animal cell is used as a host, examples include SRa promoter, SV40 promoter, LTR promoter, CMV promoter, HSV-TK promoter, and the like.
これらのうち、 CMV (サイトメガロウィルス） プロモータ一、 SRo;プロ モ一夕一などを用いるのが好ましい。 宿主がェシエリヒア属菌である場合は、 t r pプロモー夕一、 l acプロモーター、 r e cAプロモーター、 APLプ ロモ—夕—、 プロモータ—、 τ 7プロモータ一などが、 宿主がバチルス 属菌である場合は、 SPOlプロモーター、 SP02プロモ一夕一、 p enP プロモーターなど、，宿主が酵母である場合は、 PH05プロモ一夕一、 PGK プロモーター、 GAPプロモーター、 ADHプロモーターなどが好ましい。 宿 , 主が昆虫細胞である場合は、 ポリヘドリンプロモーター、 P 10プロ 一夕一 などが好ましい。 - 発現べクタ一には、 以上の他に、 所望によりェンハンサー、 スプライシング シグナル、 ポリ Α付加シグナル、 選択マーカ一、 SV40複製オリジン （以下、 S V40 o r iと略称する場合がある） などを含有しているものを用いること ができる。 選択マーカ一としては、 例えば、 ジヒドロ葉酸還元酵素 （以下、 d h f rと略称する場合がある） 遺伝子 〔メソトレキセ一ト （MTX) 耐性〕 、 アンピシリン耐性遺伝子 （以下、 Amp1"と略称する塲合がある） 、 ネオマイ シン耐性遺伝子 （以下、 Ne ofと略称する場合がある、 G418耐性） 等が 挙げられる。 特に、 dh f r遺伝子欠損チャイニーズハムスター細胞を用いて d f r遺伝子を選択マーカ一として使用する場合、 目的遺伝子をチミジンを 含まない培地によっても選択できる。 Of these, it is preferable to use the CMV (cytomegalovirus) promoter, SRo; When 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. When the host is a bacterium of the genus Bacillus When the host is yeast, PH05 promoter, PGK promoter, GAP promoter, ADH promoter, etc. are preferable. When the host is mainly insect cells, 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). Can be used. Examples of 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. In particular, when the dfr gene is used as a selection marker using Chinese hamster cells lacking the dh fr gene, Genes can also be selected on thymidine-free media.
また、 必要に応じて、 宿主に合ったシグナル配列を、 本発明のタンパク質の N端末側に付加する。 宿主がェシエリヒア属菌である場合は、 ΡΙιοΑ ·シグナル 配列、 0即 A ·シグナル配列などが、 宿主がバチルス属菌である場合は、 ーァ ミラ一ゼ*シグナル配列、 サブチリシン ·シグナル配列などが、 宿主が酵母で ある場合は、 MF a ·シグナル配列、 SUC 2 ·シグナル配列など、 宿主が動 物細胞である場合には、 インシュリン 'シグナル配列、 ひ一イン夕一フエ口 ン ·シグナル配列、 抗体分子 ·シグナル配列などがそれぞれ利用できる。 Also, if necessary, a signal sequence suitable for the host may be added to the protein of the present invention. Add to N terminal side. When the host is a bacterium belonging to the genus Escherichia, a ΡΙιοΑ 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.
このようにして構築された本発明のタンパク質をコードする DN Aを含有す るべクタ一を用いて、 形質転換体を製造することができる。  Using the vector containing the DNA encoding the protein of the present invention thus constructed, a transformant can be produced.
宿主としては、 例えば、 ェシエリヒア属菌、 バチルス属菌、 酵母、 昆虫細胞、 昆虫、 動物細胞などが用いられる。  As the host, for example, Escherichia bacteria, Bacillus bacteria, yeast, insect cells, insects, animal cells, and the like are used.
ェシエリヒア属菌の具体例としては、 例えば、 ェシエリヒア 'コリ  Specific examples of the genus Escherichia include, for example, Escherichia coli.
(Escherichia coli) K 12 - TDH 1 [Proc. Natl. Acad. Sci. USA, 60 巻， 160(1968)〕 ， JM103 [Nucleic Acids Research, 9巻， 309(1981)〕 ， J A221 〔JournaL of Molecular Bi'ology, 120卷， 517(1978)〕 , HB 101 [Journal of Molecular Biology, 41卷， 459(1969)〕 ， C 600 [Genetics, 39 巻， 440(1954)〕 などが用いられる。  (Escherichia coli) K 12-TDH 1 [Proc. Natl. Acad. Sci. USA, 60, 160 (1968)], JM103 [Nucleic Acids Research, 9, 309 (1981)], JA221 [JournaL of Molecular Bi'ology, 120 volumes, 517 (1978)], HB101 [Journal of Molecular Biology, 41 volumes, 459 (1969)], C600 [Genetics, 39 volumes, 440 (1954)] and the like are used.
バチルス属菌としては、 例えば、 バチルス ·サブチルス （Bacillus  Examples of Bacillus bacteria include, for example, Bacillus subtilis (Bacillus
subtil is) M I 1 14 〔Gene， 24巻， 255 (1983)〕 ， 207 -21 [Journal of Biochemistry, 95巻， 87(1984)〕 などが用いられる。 subtil is) M I 14 [Gene, 24, 255 (1983)], 207-21 [Journal of Biochemistry, 95, 87 (1984)] and the like are used.
酵母としては、 例えば、 サッカロマイセス セレピシェ （Saccharomyces cerevisiae) AH22, AH22R—， NA87 - 1 1 A, DKD—5D, 2 0B_ 12、 シゾサッカロマイセス ボンべ (Schizosaccharomyces pombe) N CYC 191 3, NCYC 2036、 ピキア パストリス （Pichia pastoris) KM 7 1などが用いられる。  Examples of yeast 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.
昆虫細胞としては、 例えば、 ウィルスが Ac NPVの場合は、 夜盗蛾の幼虫 由来株化細胞 (Spodoptera frugiperda cell； S f細胞) 、 Trichoplusia niの 中腸由来の MG1細胞、 Trichoplusia niの卵由来の High Five™細胞、  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 ™ cells,
Mamestra brassicae由来の細胞または Estigmena acrea由来の細胞などが用いら れる。 ウィルスが BmNP Vの場合は、 蚕由来株化細胞 （Bombyx mori 細 胞； BmN細胞） などが用いられる。 該 S f細胞としては、 例えば、 S f 9細 胞 （ATCC CRL1711) 、 S f 2 1細胞 （以上、 Vaughn, J.L.ら、 'イン 'ヴィポCells derived from Mamestra brassicae or cells derived from Estigmena acrea are used. When the virus is BmNP V, a silkworm-derived cell line (Bombyx mori cells) Vesicles; BmN cells). The Sf cells include, for example, Sf9 cells (ATCC CRL1711), Sf21 cells (Vaughn, JL et al., 'In' vipo).
(In Vivo) ，13， 213-217, (1977)) などが用いられる。 (In Vivo), 13, 213-217, (1977)).
昆虫としては、 例えば、 カイコの幼虫などが用いられる 〔前田ら、  As insects, for example, silkworm larvae are used (Maeda et al.,
Nature, 315巻， 592 (1985)〕 。 Nature, Vol. 315, 592 (1985)].
動物細胞としては、 例えば、 サル細胞 COS— 7, Ve r o, チャイニーズ ハムスター細胞 CHO (以下、 CHO細胞と略記） ， dh f r遺伝子欠損チヤ ィニーズハムスター細胞 CHO (以下、 CHO (dh f r_) 細胞と略記） , マウス L細胞， マウス A t T— 20， マウスミエ口一マ細胞， ラット GH3， ヒト FL細胞などが用いられる。  Examples of animal cells 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.
ェシエリヒア属菌を形質転換するには、 例えば、 Proc. Natl. Acad. Sci. USA, 69巻， 2110 (1972)や Gene, 17巻, 107 (1982)などに記載の方法に従って行なう ことができる。  Natl. Acad. Sci. USA, 69, 2110 (1972) or Gene, 17, 107 (1982) can be used to transform Escherichia bacteria.
バチルス属菌を形質転換するには、 例えば、 Molecular & General  To transform Bacillus, for example, use Molecular & General
Gene ti c s , 168巻， 111 (1979)などに記載の方法に従つて行なうことができる。 酵母を形質転換するには、 例えば、 Methods in Enzymology, 194巻， 182 - 187(1991)、 Proc. Natl. Acad. Sci. USA, 75巻， 1929(1978)などに記載の方法に 従って行なうことができる。 ， Genetics, 168, 111 (1979) and the like. To transform yeast, for example, the method described in Methods in Enzymology, Vol. 194, 182-187 (1991), Proc. Natl. Acad. Sci. USA, Vol. 75, 1929 (1978) can be used. Can be. ,
昆虫細胞または昆虫を形質転換するには、 例えば、 Bio/Technology, 6, 47- 55 (1988)などに記載の方法に従って行なうことができる。  Insect cells or insects can be transformed, for example, according to the method described in Bio / Technology, 6, 47-55 (1988).
動物細胞を形質転換するには、 例えば、 細胞工学別冊 8 新細胞工学実験プ ロトコール. 263-267 (1995) (秀潤社発行) 、 Virology, 52巻, 456(1973)に記載 の方法に従って行なうことができる。  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.
このようにして、 タンパク質をコードする DN Aを含有する発現ベクターで 形質転換された形質転換体を得ることができる。  Thus, a transformant transformed with the expression vector containing the DNA encoding the protein can be obtained.
宿主がェシエリヒア属菌、 バチルス属菌である形質転換体を培養する際、 培 養に使用される培地としては液体培地が適当であり、 その中には該形質転換体 の生育に必要な炭素源、 窒素源、 無機物その他が含有せしめられる。 炭素源と しては、 例えば、 グルコース、 デキストリン、 可溶性澱粉、 ショ糖など、 窒素 源としては、 例えば、 アンモニゥム塩類、 硝酸塩類、 コーンスチープ' リカ一、 ペプトン、 カゼイン、 肉エキス、 大豆粕、 バレイショ抽出液などの無機または 有機物質、 無機物としては、 例えば、 塩化カルシウム、 リン酸二水素ナトリウ ム、 塩化マグネシウムなどが挙げられる。 また、 酵母エキス、 ビタミン類、 生 長促進因子などを添加してもよい。 培地の pHは約 5〜8が望ましい。 When culturing a transformant whose host is a bacterium belonging to the genus Escherichia or Bacillus, a liquid medium is suitable as a medium for cultivation, and a carbon source necessary for the growth of the transformant is contained therein. , Nitrogen sources, inorganic substances and others. Examples of 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. In addition, yeast extract, vitamins, growth promoting factors and the like may be added. The pH of the medium is preferably about 5-8.
ェシエリヒア属菌を培養する際の培地としては、 例えば、 ダルコ一ス、 カザ ミノ酸を含む M9培地 〔ミラー （Miller) ， Journal of Experiments in Molecular Genetics, 431-433, Cold Spring Harbor Laboratory, New York 19 72〕 が好ましい。 ここに必要によりプロモータ一を効率よく働かせるために、 例えば、 3B_インドリルアクリル酸のような薬剤を加えることができる。  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. Here, for example, an agent such as 3B_indolylacrylic acid can be added to make the promoter work efficiently if necessary.
宿主がェシエリヒア属菌の場合、 培養は通常約 15〜43°Cで約 3〜24時 間行ない、 必要により、 通気や撹拌を加えることもできる。  When the host is a bacterium belonging to the genus Escherichia, 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.
宿主がバチルス属菌の場合、 培養は通常約 30〜 40 °Cで約 6〜 24時間行 ない、 必要により通気や撹拌を加えることもできる。 .  When the host is a bacterium belonging to the genus Bacillus, 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. .
宿主が酵母である形質転換体を培養する際、 培地としては、 例えば、 バーク ホールダー (Burkholder) 最小培地 [Bosti n, K. L. ら、 Proc. Natl. Acad. Sci. USA, 77巻， 4505 (1980)〕 や 0.5 %カザミノ酸を含有する S D培地 [Bitter, G. A. ら、 Proc. Natl. Acad. Sci. USA, 81巻， 5330(1984)〕 が挙げられる。 培 地の p Hは約 5〜 8に調整ずるのが好ましい。 培養は通常約 20 °C〜 35 で '約 24〜72時間行ない、 必要に応じて通気や撹拌を加える。  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)]. Preferably, 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.
宿主が昆虫細胞または昆虫である形質転換体を培養する際、 培地としては、 Grace's Insect Medium (Grace, T. C. C. , Nature, 195, 788 (1962)) に非動化し た 10%ゥシ血清等の添加物を適宜加えたものなどが用いられ 。 培地の pH は約 6. 2〜 6. 4に調整するのが好ましい。 培養は通常約 27 °Cで約 3〜 5 日間行ない、 必要に応じて通気や撹拌を加える。  When culturing an insect cell or a transformant whose host is an insect, add 10% serum inactivated to Grace's Insect Medium (Grace, TCC, Nature, 195, 788 (1962)). What added the thing suitably is used. The pH of the medium is preferably adjusted to about 6.2 to 6.4. Culture is usually performed at about 27 ° C for about 3 to 5 days, and aeration and agitation are added as necessary.
宿主が動物細胞である形質転換体を培養する際、 培地としては、 例えば、 約 5〜20 %の胎児牛血清を含む MEM培地 〔3(：161^6,122巻，501(1952)〕 , DM EM培地 [Virology, 8巻， 396 (1959)〕 ， RPM I 1640培地 〔The Journal of the American Medical Association 199巻， 519 (1967)〕 ， 199培地 [Proceeding of the Society for the Biological Medicine, 73巻， 1 (1950)〕 などが用いられる。 p Hは約 6〜8であるのが好ましい。 培養は通常約 3 0〜 4 0 °Cで約 1 5〜6 0時間行ない、 必要に応じて通気や撹拌を加える。 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)]. Preferably, 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.
以上のようにして、 形質転換体の細胞内、 細胞膜または細胞外に本発明の夕 ンパク質を生成せしめることができる。  As described above, the protein of the present invention can be produced in the cells of the transformant, in the cell membrane, or outside the cells.
上記培養物から本発明のタンパク質を分離精製するには、 例えば、 下記の方 法により行なうことができる。 ' 本発明のタンパク質を培養菌体あるいは細胞から抽出するに際しては、 培養 後、 公知の方法で菌体あるいは細胞を集め、 これを適当な緩衝液に懸濁し、 超 音波、 リゾチームおよび Zまたは凍結融解などによって菌体あるいは細胞を破 壊したのち、 遠心分離やろ過によりタンパ^質の粗抽出液を得る方法などが適 宜用いられる。 緩衝液の中に尿素や塩酸グァニジンなどの蛋白質変性剤や、 ト リ卜ン X— 1 0 0 TMなどめ界面活性剤が含まれていてもよい。 培養液中にタン パク質が分泌される場合には、 培養終了後、 それ自体公知の方法で菌体あるい は細胞と上清とを分離し、 上清を集める。 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. When the protein is secreted into the culture solution, after the culture is completed, the cells or cells are separated from the supernatant by a method known per se, and the supernatant is collected.
このようにして得られた培養上清、 あるいは抽出液中に含まれる夕ンパク質 の精製は、 自体公知の分離 ·精製法を適切に組み合わせて行なうことができる, これらの公知の分離、 精製法としては、 塩析ゃ溶媒沈澱法などの溶解度を利用 する方法、 透析法、 限外ろ過法、 ゲルろ過法、 および S D S—ポリアクリルァ ミドゲル電気泳動法などの主として分子量の差を利用する方法、 イオン交換ク 口マトグラフィ一などの荷電の差を利用する方法、 ァフィ二ティークロマトグ ラフィ一などの特異的親和性を利用する方法、 逆相高速液体クロマトグラフィ —などの疎水性の差を利用する方法、 等電点電気泳動法などの等電点の差を利 用する方法などが用いられる。  The protein contained in the culture supernatant or the extract thus obtained can be purified by appropriately combining known separation and purification methods. These known separation and purification methods Examples 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.
かくして得られるタンパク質が遊離体で得られた場合には、 自体公知の方法 あるいはそれに準じる方法によって塩に変換することができ、.逆に塩で得られ た場合には自体公知の方法あるいはそれに準じる方法により、 遊離体または他 の塩に変換することができる。  When the protein thus obtained is 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.
なお、 組換え体が産生するタンパク質を、'精製前または精製後に適当な蛋白 修飾酵素を作用させることにより、 任意に修飾を加えたり、 ポリペプチドを部 分的に除去することもできる。 蛋白修飾酵素としては、 例えば、 トリプシン、 キモトリブシン、 アルギニルエンドべプチダ一ゼ、 プ tiティンキナ一ゼ、 ダリ コシダ一ゼなどが用いられる。 In addition, 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. As the protein modifying enzyme, for example, trypsin, chymotrypsin, arginyl endopeptidase, petitinkinase, daricosidase and the like are used.
かくして生成する本発明のタンパク質の存在は、 特異抗体を用いたェンザィ ムィムノアッセィやウェスタンプロッティングなどにより測定することができ る。  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.
本発明の夕ンパク質もしくは部分べプチドまたはその塩に対する抗体は、 本 発明のタンパク質もしくは部分ペプチドまたはその塩を認識し得る抗体であれ ば、 ポリクローナル抗体、 モノクローナル抗体の何れであってもよい。  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.
本発明のタンパク質もしくは部分ペプチドまたはその塩 （以下、 抗体の説明 においては、 これらを単に本発明のタンパク質と略記する場合がある） に対す る抗体は、 本発明のタンパク質を抗原として用い、 自体公知の抗体または抗血 清の製造法に従って製造することができる。 ' 〔モノクローナル抗体の作製〕  An antibody against the protein or partial peptide of the present invention or a salt thereof (hereinafter sometimes simply referred to as the protein of the present invention in the description of the antibody) 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]
( a ) モノクローナル抗体産生細胞の作製  (a) Preparation of monoclonal antibody-producing cells
本発明の夕ンパク質は、 温血動物に対して投与により抗体産生が可能な部位 にそれ自体あるいは担体、 希釈剤とともに投与される。 投与に際して抗体産生 能を高めるため、 完全フロイントアジュバントや不完全フロイントアジュバン トを投与してもよい。 投与は通常 2〜 6週毎に 1回ずつ、 計 2〜1 0回程度行 われる。 用いられる温血動物としては、 例えば、 サル、 ゥサギ、 ィヌ、 モルモ ット、 マウス、 ラット、 ヒッジ、 ャギ、 ニヮトリが挙げられるが、 マウスおよ びラットが好ましく用いられる。 '  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. Examples of 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. '
モノクローナル抗体産生細胞の作製に際しては、 抗原で免疫された温血動物、 例えばマウスから抗体価の認められた個体を選択し最終免疫の 2〜 5日後に脾 臓またはリンパ節を採取し、 それらに含まれる抗体産生細胞を同種または異種 動物の 髄腫細胞と融合させることにより、 モノクローナル抗体産生ハイプリ ドーマを調製することができる。 抗血清中の抗体価の測定は、 例えば、 後記の 標識化タンパク質と抗血清とを反応させたのち、 抗体に結合した標識剤の活性 を測定することにより行なうことができる。 融合操作は既知の方法、 例えば、 ケ一ラーとミルスタインの方法 〔Nature、 256、 49.5 (1975)] に従い実施するこ とができる。 融合促進剤としては、 例えば、 ポリエチレングリコール （PE G) やセンダイウィルスなどが挙げられるが、 好ましくは PEGが用いられる < 骨髄腫細胞としては、 例えば、 NS_ 1、 P 3U1、 S P 2/0, AP— 1 などの温血動物の骨髄腫細胞が挙げられるが、 P 3 U 1が好ましく用いられる < 用いられる抗体産生細胞 （脾臓細胞） 数と骨髄腫細胞数との好ましい比率は 1 ： 1〜20 ： 1程度であり、 PEG (好ましくは PEG 1000〜PEG6 000) が 10〜 80 %程度の濃度で添加され、 20〜 40 °C、 好ましくは 3 0〜37°Cで 1〜10分間インキュベートすることにより効率よく細胞融合を 実施できる。 · When preparing monoclonal antibody-producing cells, 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)]. Examples of the fusion promoter include polyethylene glycol (PEG) and 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. ·
モノク口一ナル抗体産生ハイプリドーマのスクリ一ニングには種々の方法が 使用できるが、 例えば、 タンパク質抗原を直接あるいは担体とともに吸着させ た固相 （例、 マイクロプレート） ハイプリドーマ培養上清を添加し、 次に放 射性物質や酵素などで標識した抗免疫グロブリン抗体 （細胞融合に用いられる 細胞がマウスの場合、 抗マウス免疫グロブリン抗体が用いられる） またはプロ ティン Aを加え、 固相に結合したモノクロ一ナル抗体を検出する方法、 抗免疫 グロプリン抗体またはプロテイン Aを吸着させた固相にハイプリド一マ培養上 清を添加し、 放射性物質や酵素などで標識したタンパク質を加え、 固相に結合 したモノクローナル抗体を検出する方法などが挙げられる。  Various methods can be used to screen the monoclonal antibody-producing hybridoma. For example, a solid phase (eg, microplate) onto which a protein antigen is adsorbed directly or together with a carrier is added to the hybridoma culture supernatant. Then, 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 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.
'モノクロ一ナル抗体の選別は、 自体公知あるいはそれに準じる方法に従って 行なうことができる。 通常 HAT (ヒポキサンチン、 アミノプテリン、 チミジ ン） を添加した動物細胞用培地で行なうことができる。 選別および育種用培地 としては、 ハイプリドーマが生育できるものならばどのような培地を用いても 良い。 例えば、 1〜20%、 好ましくは 10〜 20 %の牛胎児血清を含む R P Ml 1640培地、 1〜 10 %の牛胎児血清を含む G I T培地 （和光純薬工業 (株） ） あるいはハイプリドーマ培養用無血清培地 （SFM— 101、 日水製 薬 （株） ) などを用いることができる。 培養温度は、 通常 20〜40°C、 好ま しくは約 37T:である。 培養時間は、 通常 5日〜 3週間、 好ましくは 1週間〜 2週間である。 培養は、 通常 5 %炭酸ガス下で行なうことができる。 ハイプリ ドーマ培養上清の抗体価は、 上記の抗血清中の抗体価の測定と同様にして測定 できる。 '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). As a selection and breeding medium, any medium can be used as long as it can grow a hybridoma. For example, 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.) can be used. 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.
( b ) モノクローナル抗体の精製  (b) Purification of monoclonal antibodies
モノクローナル抗体の分離精製は、 自体公知の方法、 例えば、 免疫グロプリ ンの分離精製法 〔例、 塩析法、 アルコール沈殿法、 等電点沈殿法、 電気泳動法、 イオン交換体 （例、 D E A E) による吸脱着法、 超遠心法、 ゲルろ過法、 抗原 結合固相あるいはプロティン Aあるいはプロティン Gなどの活性吸着剤により 抗体のみを採取し、 結合を解離させて抗体を得る特異的精製法〕 に従って行な うことができる。  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.
〔ポリクローナル抗体の作製〕  (Preparation of polyclonal antibody)
本発明のポリクローナル抗体は、 それ自体公知あるいはそれに準じる方法に 従って製造することができる。 例えば、 免疫抗原 （タンパク質抗原） 自体、 あ るいはそれとキャリアー蛋白質との複合体をつくり、 上記のモノクローナル抗 体の製造法と同様に温血動物に免疫を行ない、 該免疫動物から本発明のタンパ ク質に対する抗体含有物を採取して、 抗体の分離精製を行なうことにより製造 することができる。  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.
温血動物を免疫するために用いられる免疫抗原とキャリア一蛋白質との複合 体に関し、 キヤリァ一蛋白質の種類およびキヤリァ一とハプテンとの混合比は、 キヤリァ一に架橋させて免疫したハプテンに対して抗体が効率良くできれば、 どの様なものをどの様な比率で架橋させてもよいが、 例えば、 ゥシ血清アルブ ミンゃゥシサイログロブリン、 へモシァニン等を重量比でハプテン 1に対し、 約 0 . 1〜2 0、 好ましくは約 1〜 5の割合でカプルさせる方法が用いられる。 また、 ハプテンとキャリアーの力プリングには、 種々の縮合剤を用いること ができるが、 ダルタルアルデヒドやカルポジイミド、 マレイミド活性エステル、 チオール基、 ジチオビリジル基を含有する活性エステル試薬等が用いられる。 縮合生成物は、 温血動物に対して、 抗体産生が可能な部位にそれ自体あるい は担体、 希釈剤とともに投与される。 投与に際して抗体産生能を高めるため、 完全フロイントアジュバントゃ不完全フロイントアジュバントを投与してもよ レ^ 投与は、 通常約 2〜 6週毎に 1回ずつ、 計約 3〜10回程度行なわれる。 ポリクローナル抗体は、 上記の方法で免疫された温血動物の血液、 腹水など. 好ましくは血液から採取することができる。 Regarding the complex of the immunizing antigen and the carrier protein used for immunizing a warm-blooded animal, 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.For example, 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. In addition, various condensing agents can be used for force coupling between the hapten and the carrier. For example, 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.
抗血清中のポリクローナル抗体価の測定は、 上記の抗血清中の抗体価の測定 と同様にして測定できる。 ポリクロ一ナル抗体の分離精製は、 上記のモノクロ ーナル抗体の分離精製と同様の免疫グロブリンの分離精製法に従って行なうこ とができる。  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.
本発明のタンパク質または部分ペプチドをコードする DNA , (以下、 アンチ センスポリヌクレオチドの説明においては、 これらの DNAを本発明の DNA と略記する場合がある） の塩基配列に相補的な、 または実質的に相補的な塩基 配列またはその一部を含有するアンチセンスポリヌクレオチドとしては、 本発 明の DN Aの塩基配列に相補的な、 または実質的に相補的な塩基配列を有し、 該 D N Aの発現を抑制し得る作用を有するものであれば、 いずれのアンチセン スポリヌクレオチドであってもよいが、 アンチセンス DN Aが好ましい。  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.
本発明の DNAに実質的に相補的な塩基配列とは、 例えば、 本発明の DNA に相補的な塩基配列 （すなわち、 本発明の DNAの相補鎖） の全塩基配列ある いは部分塩基配列と約 70 %以上、 好ましくは約 80 %以上、 より好ましくは 約 90%以上、 最も好ましくは約 95%以上の相同性を有する塩基配列などが 挙げられる。 特に、 本発明の DNAの相補鎖の全塩基配列うち、 （ィ） 翻訳阻 害を指向したアンチセンスポリヌクレオチドの場合は、 本発明のタンパク質の N末端部位をコードする部分の塩基配列 （例えば、 開始コドン付近の塩基配列 など） の相補鎖と約 70%以上、 好ましくは約 80%以上、 より好ましくは約 90%以上、 最も好ましくは約 95%以上の相同性を有するアンチセンスポリ ヌクレオチドが、 （口） RNa s eHによる RNA分解を指向するアンチセン スポリヌクレオチドの場合は、 イントロンを含む本発明の DN Aの全塩基配列 の相補鎖と約 70%以上、 好ましくは約 80%以上、 より好ましくは約 90% 以上、 最も好ましくは約 95%以上の相同性を有するアンチセンスポリヌクレ ォチドがそれぞれ好適である。  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. In particular, of the entire nucleotide sequence of the complementary strand of the DNA of the present invention, (a) in the case of an antisense polynucleotide directed to translation inhibition, 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. (Mouth) In the case of 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.
具体的には、 配列番号： 2または配列番号： 4で表わされる塩基配列を含有 する D NAの塩基配列に相補的な、 もしくは実質的に相補的な塩基配列、 また はその一部分を有するアンチセンスポリヌクレオチド、 好ましくは例えば、 配 列番号： 2または配列番号： 4で表わされる塩基配列を含有する D NAの塩基 配列に.相補な塩基配列、 またはその一部分を有するアンチセンスポリヌクレオ' チド （より好ましくは、 配列番号： 2または配列番号： 4で表わされる塩基配 列を含有する D N Aの塩基配列に相補な塩基配列、 またはその一部分を有する アンチセンスポリヌクレオチドは通常、 1 0〜4 0個程度、 好ましくは 1 5 〜3 0個程度の塩基から構成される。 Specifically, it contains the nucleotide sequence represented by SEQ ID NO: 2 or SEQ ID NO: 4. 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.
ヌクレアーゼなどの加水分解酵素による分解を防ぐために、 アンチセンス D N Aを構成する各ヌクレオチドのりん酸残基 （ホスフェート） は、 例えば、 ホ スホロチォエート、 メチルホスホネ一卜、 ホスホロジチォネートなどの化.学修 飾りん酸残基に置換されていてもよい。 また、 各ヌクレオチドの糖 (デォキシ リポース） は、 2，一 O二メチル化などの化学修飾糖構造に置換されていてもよ いし、 塩基部分 （ピリミジン、 プリン） も化学修飾を受けたものであってもよ く、 配列番号： 2で表わされる塩基配列を有する D NAにハイブリダィズする ものであればいずれのものでもよい。 これらのアンチセンスポリヌクレオチド は、 公知の D NA合成装置などを用いて製造することができる。  To prevent degradation by hydrolytic enzymes such as nucleases, 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. In addition, 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.
本発明に従えば、 本発明のタンパク質遺伝子の複製または発現を阻害するこ とのできるアンチセンスポリヌクレオチド （核酸） を、 クローン化した、 ある いは決定されたタンパク質をコードする D NAの塩基配列情報に基づき設計し, 合成しうる。 かかるポリヌクレオチド （核酸） は、 本発明のタンパク質遺伝子 の R NAとハイブリダィズすることができ、 該 R NAの合成または機能を阻害 することができるか、 あるいは本発明のタンパク質関連 R N A .との相互作用を 介して本発明のタンパク質遺伝子の発現を調節 ·制御することができる。 本発 明のタンパク質関連 R NAの選択された配列に相補的なポリヌクレオチド、 お よび本発明のタンパク質関連 R N Aと特異的にハイプリダイズすることができ るポリヌクレオチドは、 生体内および生体外で本発明のタンパク質遺伝子の発 現を調節 ·制御するのに有用であり、 また病気などの治療または診断に有用で ある。 用語 「対応する」 とは、 遺伝子を含めたヌクレオチド、 塩基配列または 核酸の特定の配列に相同性を有するあるいは相補的であることを意味する。 ヌ クレオチド、 塩基配列または核酸とペプチド （タンパク質） との間で 「対応す る」 とは、 ヌクレオチド （核酸） の配列またはその相補体から誘導される指令 にあるペプチド （タンパク質） のアミノ酸を通常指している。 タンパク質遺伝 子の 5 '端ヘアピンループ、 5，端 6—ベースペア。 リピート、 5，端非翻訳領域、 ポリペプチド翻訳開始コドン、 タンパク質コード領域、 O R F翻訳終止コドン、 3，端非翻訳領域、 3，端パリンドローム領域、 および 3，端ヘアピンループは好 ましい対象領域として選択しうるが、 タンパク質遺伝子内の如何なる領域も対 象として選択しう.る。 According to the present invention, an antisense polynucleotide (nucleic acid) 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. Can be designed and synthesized based on information. Such a polynucleotide (nucleic acid) 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. Thus, 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. The “correspondence” between a 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.
目的核酸と、 対象領域の少なくとも一部に相補的なポリヌクレオチドとの関 係は、 対象物とハイプリダイズすることができるポリヌクレオチドとの関係は、 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:
「アンチセンス」 であるということができる。 アンチセンスポリヌクレオチド は、 2—デォキシ— D—リボースを含有しているポリヌクレオチド、 D—リポ —スを含有しているポリヌクレオチド、 プリンまたはピリミジン塩基の N—グ リコシドであるその他のタイプのポリヌクレオチド、 あるいは非ヌクレオチド 骨格を有するその他のポリマ一 （例えば、 市販のタンパク質核酸および合成配 列特異的な核酸ポリマー） または特殊な結合を含有するその他のポリマー （伹 し、 該ポリマ一は D NAや R NA中に見出されるような塩基のペアリングや塩' 基の付着を許容する配置をもつヌクレオチドを含有する） などが挙げられる。 それらは、 二本鎖 D NA、 一本鎖 D NA、 二本鎖 R NA、 一本鎖 R NA、 さら に D NA : R NAハイブリッドであることができ、 さらに非修飾ポリヌクレオ チド （または非修飾オリゴヌクレオチド） 、 さらには公知の修飾の付加された もの、 例えば当該分野で知られた標識のあるもの、 キャップの付いたもの、 メ チル化されたもの、 1個以上の天然のヌクレオチドを類縁物で置換したもの、 分子内ヌクレオチド修飾のされたもの、 例えば非荷電結合 （例えば、 メチルホ スホネート、 ホスホトリエステル、 ホスホルアミデート、 力ルバメートなど） を持つもの、 電荷を有する結合または硫黄含有結合 （例えば、 ホスホロチォェ —ト、 ホスホロジチォェ一トなど） を持つもの、 例えばタンパク質 （ヌクレア —ゼ、 ヌクレア一 ^ f ·インヒビ夕一、 トキシン、 抗体、 シグナルペプチド、 ポ リー L—リジンなど） や糖 （例えば、 モノサッカライドなど） などの側鎖基を 有しているもの、 イン夕一カレ一ト化合物 （例えば、 ァクリジン、 ソラレンな ど） を持つもの、 キレート化合物 （例えば、 金属、 放射活性をもつ金属、 ホウ 素、 酸化性の金属など） を含有するもの、 アルキル化剤を含有するもの、 修飾 された結合を持つもの （例えば、 έァノマー型の核酸など） であってもよい。 こ こで 「ヌクレオシド」 、 「ヌクレオチド」 および 「核酸」 とは、 プリンおよび ピリミジ'ン塩基を含有するのみでなく、 修飾されたその他の複素環型塩基をも つようなものを含んでいて良い。 こうした修飾物は、 メチル化されたプリンお よびピリミジン、 ァシル化されたプリンおよびピリミジン、 あるいはその他の 複素環を含むものであってよい。 修飾されたヌクレオチドおよび修飾されたヌ クレオチドはまた糖部分が修飾されていてよく、 例えば、 1個以上の水酸基が ハロゲンとか、 脂肪族基などで置換されていたり、 あるいはエーテル、 ァミン などの官能基に変換されていてよい。 It can be said that it is "antisense". 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) or other polymers containing special linkages (such as DNA or DNA) (Including nucleotides having a configuration that allows base pairing and attachment of a salt group as found in RNA). They 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) ゼ, Nucleic acid ^ f · Inhibitors, toxins, antibodies, signal peptides, poly-L-lysine, etc., and those with side chains such as sugars (eg, monosaccharides), etc. Contains a calate compound (eg, acridine, psoralen, etc.), contains a chelate compound (eg, metal, radioactive metal, boron, oxidizable metal, etc.), contains an alkylating agent Or a substance having a modified bond (for example, a panomer-type nucleic acid). Here, “nucleoside”, “nucleotide” and “nucleic acid” 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
本発明のアンチセンスポリヌクレオチドは、 RNA、 D NA、 あるいは修飾 された核酸 （R NA、 D N A) である。 修飾された核酸の具体例としては核酸 の硫黄誘導体ゃチォホスフエ一ト誘導体、 そしてポリヌクレオシドアミドゃォ リゴヌクレオシドアミドの分解に抵抗性のものが挙げられるが、 それに限定さ れるものではない。 本発明のアンチセンス核酸は次のような方針で好ましく設 計されうる。 すなわち、 細胞内でのアンチセンス核酸をより安定なものにする， アンチセンス核酸の細胞透過性をより高める、 目標とするセンス鎖に対する親 和性をより大きなものにする、 そしてもし毒性があるならァンチセンス核酸の 毒性をより小さなものにする。  The antisense polynucleotide of the present invention is RNA, DNA, or a modified nucleic acid (RNA, DNA). Specific examples of the 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.
こうした修飾は当該分野で数多く知られており、 例えば J . Kawakami e t al .， Pharm Tech Japan, Vo l . 8, pp. 247, 1992 ; Vol . 8, pp. 395, 1992 ; S. T.  Many such modifications are known in the art, for example, J. Kawakami et al., Pharm Tech Japan, Vol. 8, pp. 247, 1992; Vol. 8, pp. 395, 1992; S. T.
Crooke et al . ed. , Ant i sense Research and Appl i cat i ons, CRC Press, 1993 などに開示がある。 '  Crooke et al. Ed., Ant isense Research and Appli cations, CRC Press, 1993. '
本発明のアンチセンス核酸は、 変化せしめられたり、 修飾された糖、 塩基、 結合を含有していて良く、 リボゾーム、 ミクロスフエアのような特殊な形態で 供与されたり、 遺伝子治療 より適用されたり、 付加された形態で与えられる ことができうる。 こうして付加形態で用いられるものとしては、 リン酸基骨格 の電荷を中和するように働くポリリジンのようなポリカチオン体、 細胞膜との 相互作用を高めたり、 核酸の取込みを増大せしめるような脂質 （例えば、 ホス ホリピド、 コレステロールなど） といった疎水性のものが挙げられる。 付加す るに好ましい脂質としては、 コレステロールやその誘導体 （例えば、 コレステ リルクロ口ホルメート、 コール酸など） が挙げられる。 こうしたものは、.核酸 の 3 '端あるいは 5 '端に付着させることができ、 塩基、 糖、 分子内ヌクレオシ ド結合を介して付着させることができうる。 その他の基としては、 核酸の 3 '端 あるいは 5 '端に特異的に配置されたキャップ用の基で、 ェキソヌクレア一ゼ、 R N a s eなどのヌクレアーゼによる分解を阻止するためのものが挙げられる。 こうしたキャップ用の基としては、 ポリエチレングリコール、 テトラエチレン グリコールなどのグリコールをはじめとした当該分野で知られた水酸基の保護 基が挙げられるが、 それに限定されるものではない。 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. Thus, in the form of addition, polycations such as polylysine, which acts to neutralize the charge of the phosphate skeleton, and lipids, which increase the interaction with the cell membrane or increase the uptake of nucleic acids ( For example, hydrophobic substances such as phospholipid and cholesterol) can be mentioned. Preferred lipids for addition include cholesterol and its derivatives (eg, cholesteryl chromate formate, cholic acid, etc.). These can be attached to the 3 'end or 5' end of a nucleic acid, and 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. Such capping groups include, but are not limited to, hydroxyl-protecting groups known in the art, including glycols such as polyethylene glycol and tetraethylene glycol.
アンチセンス核酸の阻害活性は、 本発明の形質転換体、 本発明の生体内や生 体外の遺伝子発現系、 あるいは本発明のタンパク質の生体内や生体外の翻訳系 を用いて調べることができる。 該核酸は公知の各種の方法で細胞に適用できる 以下に、 本発明のタンパク質もしくは部分ペプチドまたはその塩 （以下、 本 発明のタンパク質と略記する場合がある） 、 本発明のタンパク質または部分べ プチドをコ一ドする D NA (以下、 本発明の D NAと略記する場合がある） 、 本発明のタンパク質もしくは部分ペプチドまたはその塩に対する抗体 （以下、 本発明の抗体と略記する場合がある） 、 および本発明の D N Aのアンチセンス ポリヌクレオチド （以下、 本発明のアンチセンスポリヌクレオチドと略記する 場合がある） 等の用途を説明する。  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. Hereinafter, 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. Coding DNA (hereinafter sometimes abbreviated as DNA of the present invention), an 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), and The use of the DNA of the present invention as an antisense polynucleotide (hereinafter, sometimes abbreviated as the antisense polynucleotide of the present invention) and the like will be described.
本発明のタンパク質は、 変形性関節症軟骨で発現が増加し、 細胞外へ分泌さ れるので、 疾患マーカ一として利用することが出来る。 すなわち、 変形性関節 症軟骨などにおける早期診断、 症状の重症度の判定、 疾患進行の予測および? 療効果判定のためのマ一カーとして有用である。 よって、 本発明のアンチセン スポリヌクレオチド、 本発明のタンパク質の活性を調節する化合物またはその 塩、 本発明のお体などは、 例えば、 軟骨形成異常、 骨形成異常、 骨粗鬆症、 変 形性関節症、 慢性関節リウマチ、 関節炎、 滑膜炎、 代謝性関節症またはスポー ッによる関節障害 （例、 テニス肘など） などの骨 ·関節疾患の予防 ·治療剤と して使用することができる。 Since 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. Therefore, 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).
( 1 ) 疾病に対する医薬候補化合物のスクリーニング (1) Screening of drug candidate compounds for diseases
本発明の夕ンパク質は変形性関節症軟骨で発現'が増加し、 軟骨細胞の分化調 節活性、 軟骨細胞への結合活性などを有するので、 本発明のタンパク質の活性 を調節 （促進または阻害、 好ましくは阻害） する化合物またはその塩は、 軟骨 形成異常、 骨形成異常、 骨粗鬆症、 変形性関節症、 慢性関節リウマチ、 関節炎、 滑膜炎、 代謝性関節 またはスポーツによる関節障害 （例、 テニス肘など） な どの骨 ·関節疾患の予防 ·治療剤として使用することができる。  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 compound or a salt thereof that inhibits, preferably inhibits cartilage dysplasia, bone dysplasia, osteoporosis, osteoarthritis, rheumatoid arthritis, arthritis, synovitis, metabolic joints or sports-related joint disorders (eg, tennis elbows) It can be used as a preventive and therapeutic agent for bones and joint diseases.
したがって、 本発明のタンパク質は、 本発明のタンパク質の活性を調節する 化合物またはその塩のスクリーニングのための試薬として有用である。  Therefore, 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.
すなわち、 本発明は、 本発明のタンパク質を用いることを特徴とする本発明 のタンパク質の活性を調節する化合物またはその塩のスクリーニング方法を提 供する。  That is, 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.
, 本発明は、 （1 ) 本発明のタンパク質を用いることを特徴とする本発明の夕 ンパク質の活性 （例、 軟骨細胞の分化調節活性、 軟骨細胞への結合活性など） を促進または阻害する化合物またはその塩 （以下、 それぞれ促進剤、 阻害剤と 略記する場合がある） のスクリーニング方法を提供する。 具体的には、 例えば、 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.) Provided is a method for screening a compound or a salt thereof (hereinafter may be abbreviated as an accelerator or an inhibitor, respectively). Specifically, for example,
( 2 ) (i) 本発明のタンパク質の活性 （例、 軟骨細胞の分化調節活性、 軟骨細 胞への結合活性など） と、 （i i) 本発明のタンパク寳と試験化合物の混合物の 活性 （例、 軟骨細胞の分化調節活性、 軟骨細胞への結合活性など） を比較する ことを特徴する本発明のタンパク質の活性を調節 （促進または阻害） する化合 物またはその塩のスクリーニング方法が用いられる。 - 軟骨細胞の分化調節活性の測定は、 公知の方法に準じて行うことができ、 例 えば、 J Cel l Biochem 67, 498-513 (1997)に記載の方法またはそれに準じた方 法などに従つて測定することができる。 (2) (i) activity of the protein of the present invention (eg, chondrocyte differentiation regulating activity, binding activity to cartilage cells, etc.); and (ii) 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. -The measurement of 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.
例えば、 本発明のタンパク質を産生する能力を有する細胞の培養上清または 細胞抽出物から、 公知方法 （例、 免疫ァフィ二ティーカラム） に従い、 本発明 のタンパク質を得る。 このタンパク質および試験化合物を培養軟骨細胞に添加 し、 37°Cで 7日〜 21日間培養する。 次に上記文献に挙げられた方法に従い軟骨細 胞のプロテオダリカン含量またはアル力リ性ホスファタ一ゼ活性を評価するこ とにより、 軟骨細胞の分化調節活性を測定する。 試験化合物の培養軟骨細胞へ の添加は、 本発明のタンパク質の添加前、 同時またはタンパク質添加後のいず れに行ってもよく、 本発明のタンパク質の添加前が好ましい。  For example, 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. Next, 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.
.軟骨細胞への結合活性の測定は、 公知の方法に準じて行うことができる。 例えば、 本発明のタンパク質を産生する能力を有する細胞の培養上清または 細胞抽出物から、 公知方法 （例、 免疫ァフィ二ティ一カラム） に従い、 本発明 The measurement of the binding activity to chondrocytes can be performed according to a known method. For example, according to a known method (eg, immunoaffinity column) from a culture supernatant or a cell extract of a cell capable of producing the protein of the present invention,
'のタンパク質を得、 例えば D-ピオチノィル- ε -アミノカプロン酸- N -ハイドロキ シサクシイミドエステルを用いてピオチン標識する。 該標識タンパク質および 試験化合物を培養軟骨細胞に添加し、 4°Cで一時間培養後、 細胞を PBSで洗浄す る。 次に、 ストレプトアビジンおよび酵素 （例、 アルカリ性ホスファターゼ） の複合体を添 ¾1し、 4°Cで一時間培養後、 細胞を PBSで洗浄する。 最後に該酵素 の化学発光基質 （例、 Disodium 2-chloro-5- (4-methoxyspiro {l , 2-dioxetane- 3， 2' _ (5' - chloro) tr icyc lo [3. 3. 1. 1. 3, 7] decan}-4-yl) -l-phenyl phosphateな ど） を添加し、 発生する光を測定することにより、 本発明のタンパク質の軟骨 細胞への結合活性を測定する。 試験化合物の培養軟骨細胞への添加は、 本発明 の夕ンパク質添加前、' 同時または夕ンパク質添加後のいずれに行ってもよく、 本発明のタンパク質添加前が好ましい。 本発明のタンパク質を産生する能力 を有する細胞としては、 例えば、 前述した本発明のタンパク質をコードする D NAを含有するべクタ一で形質転換された宿主 （形質転換体） が用いられる。 宿主としては、 例えば、 C HO細胞などの動物細胞が好ましく用いられる。 該 スクリーニングには、 例えば、 前述の方法で培養することによって、 本発明の タンパク質を細胞膜上に発現させた形質転換体が好ましく用いられる。 本発明 のタンパク質を発現し得る細胞の培養方法は、 前記した本発明の形質変換体の 培養法と同様である。 ', And labeled with biotin using, for example, D-piotinoyl-ε-aminocaproic acid-N-hydroxysuccinimide ester. The labeled protein and the test compound are added to the cultured chondrocytes, and cultured at 4 ° C for 1 hour, and the cells are washed with PBS. Next, a complex of streptavidin and an enzyme (eg, alkaline phosphatase) is added, and after culturing at 4 ° C for 1 hour, the cells are washed with PBS. Finally, 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.), and the generated light is measured to determine the binding activity of the protein of the present invention to chondrocytes. 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. As the cell having the ability to produce the protein of the present invention, for example, a host (transformant) transformed with a vector containing the above-described DNA encoding the protein of the present invention is used. As a host, for example, animal cells such as CHO cells are preferably used. For the screening, for example, 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.
試験化合物としては、 例えばペプチド、 タンパク質、 非ペプチド性化合物、 合成化合物、 発酵生産物、 細胞抽出液、 植物抽出液、 動物組織抽出液などがあ け'られる。  Test compounds include, for example, peptides, proteins, non-peptidic compounds, synthetic compounds, fermentation products, cell extracts, plant extracts, animal tissue extracts, and the like.
例えば、 上記 （i i) の場合における活性が上記 （i) の場合に比べて、 約 2 0 %以上、 好ましくは 3 0 %以上、 より好ましくは約 5 0 %以上上昇させる試 験化合物を、 本発明のタンパク質の活性を促進する化合物として、 上記 （i i) の場合における活性が上記 （i) の場合に比べて、 約 2 0 %以上、 好ましくは 3 0 %以上、 より好ましくは約 5 0 %以上減少させる試験化合物を本 明のタン パク質の活性を阻害する化合物として選択することができる。  For example, 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.
本発明のタンパク質の活性を促進する活性を有する化合物は、 本発明のタン パク質の作用を増強するための安全で低毒性な医薬として有用である。  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.
本発明のタンパク質の活性を阻害する活性を有する化合物は、 本発明のタン パク質の生理活性を抑制するための安全で低毒性な医薬、 例えば軟骨形成異常、 骨形成異常、 骨粗鬆症、 変形性関節症、 慢性関節リウマチ、 関節炎、 滑膜炎、 代謝性関節症またはスポーツによる関節障害 （例、 テニス肘など） などの骨 - 関節疾患の予防 ·治療剤として有用である。  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.).
本発明のスクリ一二ング方法またはスクリ一二ング用キットを用いて得られ る化合物またはその塩は、 例えば、 ペプチド、 タンパク質、 非ペプチド性化合 物、 合成化合物、 発酵生産物、 細胞抽出液、 植物抽出液、 動物組織抽出液、 血 漿などから選ばれた化合物である。 該化合物の塩としては、 前記した本発明の ペプチドの塩と同様のものが用いられる。  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. As the salt of the compound, those similar to the aforementioned salts of the peptide of the present invention are used.
さらに、 本発明のタンパク質をコードする遺伝子も、 変形性関節症軟骨にお いて発現が増加するので、 本発明のタンパク質をコードする遺伝子の発現を詾 節 （阻害または促進、 好ましくは阻害） する化合物またはその塩も、 軟骨形成 異常、 骨形成異常、 骨粗鬆症、 変形性関節症、 慢性関節リウマチ、 関節炎、 滑 膜炎、 代謝性関節症またはスポーツによる関節障害 （例、 テニス肘など） など の骨 ·関節疾患の予防 ·治療剤として使用することができる。 .  Furthermore, since 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. Or its salts, 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. .
したがって、 本発明の D N Aは、 本発明のタンパク質をコードする遺伝子の 発現を調節する化合物またはその塩のスクリーニングのための試薬として有用 である。 Therefore, 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.
スクリーニング方法としては、 （i i i) 本発明のタンパク質を産生する能力を 有する細胞を培養した場合と、 （iv) 試験化合物の存在下、 本発明のタンパク 質を産生する能力を有する細胞を培養した場合との比較を行うことを特徴とす るスクリーニング方法が挙げられる。  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.
' 上記方法において、 （i i i) と （iv) の場合における、 前記遺伝子の発現量 (具体的には、 本発明のタンパク質量または前記タンパク質をコードする mR ' NA量） を測定して、 比較する。 'In the above method, in the cases (iii) and (iv), 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. .
試験化合物および本発明のタンパク質を産生する能力を有する細胞としては、 上記と同様のものが挙げられる。  Examples of the test compound and cells having the ability to produce the protein of the present invention include the same cells as described above.
タンパク質量の測定は、 公知の方法、 例えば、 本発明のタンパク質を認識す る抗体を用いて、 細胞抽出液中などに存在する前記タンパク質を、 ウエスタン 解析、 E L I S A法などの方法またはそれに準じる方法に従い測定することが できる。  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.
mR NA量の測定は、 公知の方法、 例えば、 プローブとして配列番号： 2も しくは配列番号： 4の塩基配列またはその一部を含有する核酸を用いるノーザ ンハイブリダィゼ一シヨン、 あるいはプライマ一として配列番号： 2もしくは 配列番号： 4の塩基配列またはその一部を含有する核酸を用いる' P C R法また はそれに準じる方法に従い測定することができる。  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.
例えば、 上記 （iv) の場合における遺伝子発現量を、 上記 （i i i) の場合に比. ベて、 約 2 0 %以上、 好ましくは 3 0 %以上、 より好ましくは約 5 0 %以上上 昇させる試験化合物を、 本発明のタンパク質をコードする遺伝子の発現を促進 する化合物として、 約 2 0 %以上、 好ましくは 3 0 %以上、 より好ましくは約 5 0 %以上阻害する試験化合物を、 本発明のタンパク質をコードする遺伝子の 発現を抑制する化合物として選択することができる。  For example, 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). As a compound that promotes the expression of the gene encoding the protein of the present invention, 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.
さらに、 本発明は、 本発明のタンパク質の遺伝子の転写調節領域の下流 （発 現制御下） にレポーター遺伝子を連結した組換え D NAで形質転換した形質転 換体を、 試験化合物の存在下および非存在下で培養した場合における、 それぞ れのレポ一ター活性を測定し、 比較することを特徴とする該転写調節活性を調 節 （促進または阻害） する化合物のスクリ一 ング方法、 およびこの方法のた めのスクリーニング用キットも提供する。 Furthermore, 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. When cultured in the presence 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. .
本発明のタンパク質の遺伝子の転写調節領域としては、 配列番号： 8で表さ れる塩基配列と同一または実質的に同一な塩基配列を含有する D N Aなどが挙 げられる。 '  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. '
配列番号： 8で表される塩基配列と実質的に同一な塩基配列を含有する D N Aとしては、 ハイストリンジェントな条件下でハイプリダイズする塩基配列を 有し、 配列番号： 8と実質的に同質の転写調節活性 （例、 プロモータ一活性な ど） を有する D N Aであれば何れのものでもよい。  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).
配列番号： 8で表される塩基配列とハイス卜リンジェン卜な条件下でハイブ リダィズできる D N Aとしては、 例えば、 配列番号： 8で表される塩基配列と 約 5 0 %以上， 好ましくは約 6 0 %以上、 さらに ましくは約 7 0 %以上、 よ り好ましくは約 8 0 %以上、 特に好ましくは約 9 0 %以上、 最も好ましくは約 9 5 %以上の相同性を有する塩基配列を含有する D NAなどが用いられる。 ハイブリダィゼーシヨンは、 自体公知の方法あるいはそれに準じる方法、 例 えば、 モレキュラー 'クローニング （Molecul ar Cloning) 2nd (J. Sambrook . et al .， Cold Spr ing Harbor Lab. Press, 1989) に記載の方法などに従って行 なうことができる。 また、 市販のライブラリ一を使用する場合、.添付の使用説 明書に記載の方法に従って行なうことができる。 より好ましくは、 八イストリ ンジェントな条件に従って行なうことができる。  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.
ハイストリンジェントな条件とは、 例えば、 ナトリウム濃度が約 1 9〜4 0 mM、 好ましくは約 1 9〜 2 0 mMで、 温度が約 5 0〜 7 0 °C、.好ましくは約 6 0〜6 5 °Cの条件を示す。 特に、 ナトリウム濃度が約 1 9 mMで温度が約 6 5 °Cの場合が最も好ましい。  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.
また、 転写調節領域 （例、 配列番号： 8で表される塩基配列と実質的に同一 な塩基配列を含有する D N A等） としては、 配列番号： 8で表される塩基配列 と実質的に同一な塩基配列に、 さらに 5，上流のゲノム D N A配列が付加された 配列なども挙げられる （好ましくはプロモーター領域） 。 付加する塩基配列の 塩基数は、 例えば約 1 0 K b以下、 好ましくは約 5 K b以下、 さらに好ましく は約 2 K b以下、 最も好ましくは約 1 K b以下の配列数が挙げられる。 さらに、 配列番号： 8で表される塩基配列と実質的に同一な塩基配列に、 さらに本発明 のタンパク質の遺伝子のイントロンの配列が付加された配列なども挙げられる c レポ一夕一遺伝子としては、 例えば、 1 a c Z ガラクトシダ一ゼ遺伝 子） 、 クロラムフエニコ一ルァセチルトランスフェラ一ゼ （C AT) 、 ルシフ エラ一ゼ、 成長因子、 /3—ダルク口ニダーゼ、 アルカリホスファタ一ゼ、 Green f luorescent protein (GFP) , 3 _ラク夕マ一ゼなどが用いられる。 In addition, the transcription regulatory region (eg, DNA containing a nucleotide sequence substantially identical to the nucleotide sequence represented by SEQ ID NO: 8) is substantially the same as the nucleotide sequence represented by SEQ ID NO: 8. And a sequence obtained by adding an upstream genomic DNA sequence to a basic nucleotide sequence (preferably, a promoter region). Of the base sequence to be added 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. Further, 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.
レポ一夕一遺伝子産物 （例、 mR NA、 タンパク質） の量を公知の方法を用 いて測定することによって、 レポ一夕一遺伝子産物の量を増加させる試験化合 物を本発明のタンパク質の転写調節活性 （好ましくはプロモーター活性） を制 御 （特に促進） する作用を有する化合物、 すなわち本発明のタンパク質の発現 を促進する活性を有する化合物として選択できる。 逆に、 レポーター遺伝子産 物の量を減少させる試験化合物を本発明のタンパク質の転写調節活性 （好まし くはプロモー夕一活性） を制御 （特に阻害） する作用を有する化合物、 すなわ ち本発明のタンパク質の発現を阻害する活性を有する化合物として選択するこ とができる。  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.
試験化合物としては、 例えば、 ペプチド、 タンパク質、 非ペプチド性化合物、 合成化合物、 発酵生産物、 細胞抽出液、 植物抽出液、 動物組織抽出液、 血漿な どから選ばれた化合物である。  The 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.
形質転換体の培養は、 前記の本発明のタンパク質を^ "有する形質転換体と同 様にして行うことができる。 レポ一ター遺伝子のベクタ一構築やアツセィ法は 公知の技術に従うことができる （例、 Molecular Biotechnology 13, 29-43, 1999) 。  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).
本発明のスクリーニング用キットは、 本発明のタンパク質もしくは部分ぺプ チドまたはその塩、 または本発明のタンパク質もしくは部分べプチドを産生す る能力を有する細胞を含有するものである。 ，  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. ,
本発明のスクリ一ニング方法またはスクリーニング用キットを用いて得られ る化合物またはその塩は、 上記した試験化合物、 例えば、 ペプチド、 タンパク 質、 非ペプチド性化合物、 合成化合物、 発酵生産物、 細胞抽出液、 植物抽出液、 動物組織抽出液、 血漿などから選ばれた化合物またはその塩である。 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.
• 該化合物の塩としては、 前記した本発明のタンパク質の塩と同様のものが用 いられる。  • As the salt of the compound, those similar to the salts of the protein of the present invention described above are used.
本発明のタンパク質の活性を調節 （好ましくは阻害） する化合物またはその 塩、 本発明のタンパク質をコードする遺伝子の発現を調節する化合物またはそ' の塩、 本発明のタンパク質の転写調節活性 （好ましくはプロモーター活性） を 制御する作用を有する化合物またはその塩は、 それぞれ、 例えば、 軟骨形成異 常、 骨形成異常、 骨粗鬆症、 変形性関節症、 慢性関節リウマチ、 関節炎、 滑膜 炎、 代謝性関節症またはスポーツによる関節障害 （例、 テニス肘など） などの 骨 ·関節疾患の予防 ·治療刳として有用である。  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).
本発明のスクリーニング方法またはスクリーニング用キットを用いて得られ る化合物またはその塩を上述の予防 ·治療剤として使用する場合、 常套手段に 従って製剤化することができる。  When 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.
例えば、 経口投与のための組成物としては、 固体または液体の剤形、 具体的 には錠剤 （糖衣錠、 フィルムコ一ティング錠を含む) 、 丸剤、 顆粒剤、 散剤、 ' カプセル剤 （ソフトカプセル剤を含む） 、 シロップ剤、 乳剤、 懸濁剤などがあ げられる。 かかる組成物は自体公知の方法によって製造され、 製剤分野におい て通常用いられる担体、 希釈剤もしくは賦形剤を含有するものである。 例えば、 錠剤用の担体、 賦形剤としては、 乳糖、 でんぷん、 蔗糖、ステアリン酸マグネ シゥムなどが用いられる。  For example, 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. For example, lactose, starch, sucrose, magnesium stearate and the like are used as carriers and excipients for tablets.
非経口投与のための組成物としては、 例えば、 注射剤、 坐剤などが用いられ、 注射剤は静脈注射剤、 皮下注射剤、 皮内注射剤、 筋肉注射剤、 点滴注射剤、 関 節内注射剤などの剤形を包含する。 かかる注射剤は、 自体公知の方法に従って、 例えば、 上記抗体またはその塩を通常注射剤に用いられる無菌の水性もしくは 油性液に溶解、 懸濁または乳化することによって調製する。 注射用の水性液と しては、 例えば、 生理食塩水、 ブドウ糖やその他の補助薬を含む等張液などが 用いられ、 適当な溶解補助剤、 例えば、 アルコール （例、 エタノール） 、 ポリ アルコール （例、 プロピレングリコール、 ポリエチレングリコール） 、 非ィォ ン界面活性剤 〔例、 ポリソルべ一ト 80、 H CO— 50 Examples of 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. As an 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
(.polyoxyethylene (50mol) adduct of hydrogenated castor oil) j なこと併 用してもよい。 油性液としては、 例えば、 ゴマ油、 大豆油などが用いられ、 溶 解補助剤として安息香酸ベンジル、 ベンジルアルコールなどを併用してもよい 調製された注射液は、 通常、 適当なアンプルに充填される。 直腸投与に用いら れる坐剤は、 上記化合物またはその塩を通常の坐薬用基剤に混合することによ つて調製される。 .  (.polyoxyethylene (50mol) adduct of hydrogenated castor oil) As the 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. .
上記の経口用または非経口用医薬組成物は、 活性成分の投与量に適合するよ うな投薬単位の剤形に調製されることが好都合である。 かかる投薬単位の剤形 としては、 錠剤、 丸剤、 カプセル剤、 注射剤 （アンプル） 、 坐剤などが例示さ れ、 それぞれの投薬単位剤形当たり通常 5〜500mg、 とりわけ ¾射剤では 5〜： I 00mg、 その他の剤形では 10〜250 mgの上記抗体が含有されて いることが好ましい。 ，  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. Examples of such 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. It is preferable that the above-mentioned antibody is contained in the following form: I 00 mg, and other dosage forms 10 to 250 mg. ,
なお前記した各組成物は、 上記化合物との配合により好ましくない相互作用 を生じない限り他の活性成分を含有してもよい。  Each of the above-mentioned compositions may contain other active ingredients as long as the compound and the above-mentioned compound do not cause an undesirable interaction.
このようにして得られる製剤は安全で低毒性であるので、 例えば、 ヒトまた は温血動物 （例えば、 マウス、 ラット、 ゥサギ、 ヒッジ、 ブタ、 ゥシ、 ゥマ、 トリ、 ネコ、 ィヌ、 サル、 チンパンジーなど) に対して経口的にまたは非経口 的に投与することができる。  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.
該化合物またはその塩の投与量は、 その作用、 対象疾患、 投与対象、 投与ル —トなどにより差異はあるが、 例えば、 変形性関節症の治療の目的で本発明の タンパク質の活性を阻害する化合物またはその塩を経口投与する場合、 一般的 に成人 （体重 60 kgとして） においては、 一日につき該化合物またはその塩 を約 0.；!〜 10 Omg、 好ましくは約 1. 0〜50mg、 より好ましくは約 1 0〜20mg投与する。 非経,口的に投与する場合は、 該ィ匕合物またはその塩の 1回投与量は投与対象、 対象疾患などによっても異なるが、 例えば、 変形性関 節症の治療の目的で本発明のタンパク質の活性を阻害する化合物またはその塩 を注射剤の形で通常成人 （体重 60 kgとして） に投与する場合、 一日につき 該化合物またはその塩を約 0. 01〜3 Omg、 好ま Lくは約 0。 l 20m g、 より好ましくは約 0 . 1〜1 O m gを関節内注射により投与するのが好都 合である。 他の動物の場合も、 体重 6 0 k g当たりに換算した量を投与するこ とができる。 ' ( 2 ) 本発明のタンパク質が関与する各種疾病の予防 ·治療剤 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. When 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. In the case of non-oral or oral administration, the single dose of the compound or its salt may vary depending on the administration target, the target disease and the like. For example, the present invention is intended for the treatment of osteoarthritis. When a 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. In the case of other animals, the dose can be administered in terms of weight per 60 kg. '' (2) Prevention and treatment of various diseases related to the protein of the present invention
本発明のタンパク質は、 軟骨細胞の分化 ·増殖に寄与するとともに、 変形性 関節症発症に重要な役割を果 している。  The protein of the present invention contributes to the differentiation and proliferation of chondrocytes and plays an important role in the development of osteoarthritis.
本発明のタンパク質をコードする D NAに異常があったり、 欠損している場 合あるいは本発明のタンパク質の発現量が減少している場合には、 例えば、 軟 骨形成異常、 骨形成異常、 骨粗鬆症、 変形性関節症、 慢性関節リウマチ、 関節 炎、 滑膜炎、 代謝性関節症またはスポーツによる関節障害 （例、 テニス肘な ど） などの骨 ·関節疾患などの種々の疾患が発症する。  When 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.
したがって、 本発明のタンパク質および本発明のタンパク質をコードする D NAは、 例えば、 軟骨形成異常、 骨形成異常、 骨粗鬆症、 変形性関節症、 慢性 関節リウマチ、 関節炎、 滑膜炎、 代謝性蘭節症またはスポーツによる関節障害 (例、 テニス肘など） などの骨 ·関節疾患の予防 ·治療剤などの医薬として使 用することができる。.  Therefore, 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). .
例えば、 生体内において本発明のタンパク質が減少あるいは欠損しているた めに、 軟骨細胞の分化 ·増殖が十分に、.あるいは正常に発揮されない患者がい る場合に、 （ィ） 該タンパク質をコードする D N Aをその患者に投与し、 生体 内で本発明のタンパク質を発現させることによって、 （口） 細胞に上記 D NA を挿入し、 本発明のタンパク質を発現させた後に、 その細胞を患者に移植する ことによって、 または （八） 本発明のタンパク質をその患者に投与することな どによって、 その患者における,本発明のタンパク質の役割を十分に、 あるいは 正常に発揮させることができる。 ,  For example, if there is a patient in which chondrocyte differentiation and proliferation are not sufficiently or properly performed due to a decrease or deletion of the protein of the present invention in a living body, or (i) the protein encodes the protein. By administering the DNA to the patient and expressing the protein of the present invention in vivo, (oral) inserting the above DNA into cells, expressing the protein of the present invention, and then transplanting the cells to the patient. Thus, or (8) by administering the protein of the present invention to the patient, the role of the protein of the present invention in the patient can be sufficiently or normally exerted. ,
本発明のポリヌクレオチド （例、 D NA) を上記の予防 ·治療剤として使用 する場合は、 本発明の Aを単独あるいはレトロウイルスベクター、 アデノ ウィルスベクター、 アデノウイルスァソシエーテツドウィルスベクタ一などの 適当なベクターに挿入した後、 常套手段に従って、 ヒトまたはその他の温血動 物に投与することができる。 本発明のポリペプチド （例、 D NA) は、 そのま まで、 あるいは摂取促進のための補助剤などの生理学的に認められる担体とと もに製剤化し、 遺伝子銃やハイドロゲルカテーテルのようなカテーテルによつ て投与できる。 When the polynucleotide (eg, DNA) of the present invention is used as the above-mentioned prophylactic / therapeutic agent, 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.
本発明のタンパク質を上記の予防 ·治療剤として使用する場合は、 少なくと も 9 0 %、 好ましくは 9 5 %以上、 より好ましくは 9 8 %以上、 さらに好まし くは 9 9 %以上に精製されたものを使用するのが好ましい。  When 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.
本発明のタンパク質は、 例えば、 必要に応じて糖衣を施した錠剤、 カプセル 剤、 エリキシル剤、 マイクロカプセル剤などとして経口的に、 あるいは水もし くはそれ以外の薬学的に許容し得る液との無菌性溶液、 または懸濁液剤などの 注射剤の形で非経口的に使用できる。 例えば、 本発明のタンパク質等を生理学 的に認められる担体、 香味剤、 賦形剤、 べヒクル、 防腐剤、 安定剤、 結合剤な どとともに一般に認められた製剤実施に要求される単位用量形態で混和するこ とによって製造することができる。 これら製剤における有効成分量は指示され た範囲の適当な用量が得られるようにするものである。  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. For example, 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.
錠剤、 カプセル剤などに混和することができる添加剤としては、'例えば、 ゼ ■ ラチン、 コーンスターチ、 トラガント、 アラビアゴムのような結合剤、 結晶性 セルロースのような賦形剤、 コーンスターチ、 ゼラチン、 アルギン酸などのよ うな膨化剤、 ステアリン酸マ^ネシウムのような潤滑剤、 ショ糖、 乳糖または サッカリンのような甘味剤、 ペパーミント、 ァカモノ油またはチェリ一のよう な香味剤などが用.いられる。 調剤単位形態がカプセルである場合には、 前記夕 ィプの材料にさらに油脂のような液状担体を含有することができる。 注射のた めの無菌組成物は注射用水のようなべヒクル中の活性物質、 胡麻油、 椰子油な どのような天然産出植物油などを溶解または懸濁させるなどの通常の製剤実施 に従つて処方することができる。  Examples of 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.
注射用の水性液としては、 例えば、 生理食塩水、 ブドウ糖やその他の補助薬 を含む等張液 （例えば、 D—ソルビトール、 D—マンニト一ル、 塩化ナトリウ ムなど） などが挙げられ、 適当な溶解補助剤、 例えば、 アルコール （例えば、 エタノールなど） -、 ポリアルコール (例えば、 プロピレングリコール、 ポリエ チレングリコールなど） 、 非イオン性界面活性剤 （例えば、 ポリソルベート 8 0TM、 HCO— 50など） などと併用してもよい。 油性液としては、 例えば、 ゴマ油、 大豆油などが挙げられ、 溶解補助剤として安息香酸ベンジル、 ベンジ ルアルコールなどと併用してもよい。 また、 緩衝剤 （例えば、 リン酸塩緩衝液、 酢酸ナトリウム緩衝液など） 、 無痛化剤 （例えば、 塩化ベンザルコニゥム、 塩 酸プロ力インなど） 、 安定剤 （例えば、 ヒト血清アルブミン、 ポリエチレング リコールなど） 、 保存剤 （例えば、 ベンジルアルコール、 フエノールなど） 、 酸化防止剤などと配合してもよい。 調製された注射液は、 通常、 適当なアンプ ルに充填される。 Examples of aqueous liquids for injection 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 ™ , 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. In addition, 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 and the like. The prepared injection solution is usually filled in a suitable ampoule.
本発明の DNAが挿入されたべクタ一も上記と同様に製剤化され、 通常、 非 経口的に使用される。  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.
このようにして得られる製剤は、 安全で低毒性であるので、 例えば、 温血動 物 （例えば、 ヒト、 ラット、 マウス、 モルモット、 ゥサギ、 トリ、 ヒッジ、 ブ 夕、 ゥシ、 ゥマ、 ネコ、 ィヌ、 サル、 チンパンジーなど) に対して投与するこ とができる。  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.).
本発明のタンパク質の投与量は、 対象疾患、 投与対象、 投与ルートなどによ り差異はあるが、 例えば、 変形性関節症の治療目的で本発明のタンパク質を経 口投与する場合、 一般的に成人 （体重 60 kgとして） においては、 一日につ き該タンパク質を約 0. 1〜10 Omg、 好ましくは約 1. 0〜50mg、 よ り好ましくは約 1. 0〜2 Omg投与する。 非経口的に投与する場合は、 該タ ンパク質の 1回投与量は投与対象、 対象疾患などによっても異なるが、 例えば、 変形性関節症の治療目的で本発明のタンパク質を注射剤の形で成人 （体重 60 kgとして） に投与する場合、 一日につき該タンパク質を約ひ. 01〜30m g、 好ましくは約 0. 1〜2 Omg、 より好ましくは約 0. l〜10mgを患 部に注射することにより投与するのが好都合である。 他の動物の場合も、 体重 60 k g当たりに換算した量を投与することができる。  The dosage of the protein of the present invention varies depending on the target disease, the administration subject, the administration route, and the like.For example, when the protein of the present invention is orally administered for the purpose of treating osteoarthritis, it is generally used. For an adult (assuming a body weight of 60 kg), about 0.1 to 10 Omg, preferably about 1.0 to 50 mg, more preferably about 1.0 to 2 Omg of the protein is administered daily. When administered parenterally, the single dose of the protein varies depending on the administration subject, target disease, and the like.For example, the protein of the present invention is in the form of an injection for the treatment of osteoarthritis. 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.
(3) 本発明のタンパク質、 その部分ペプチドまたはその塩の定量 (3) Quantification of the protein of the present invention, its partial peptide or its salt
本発明の抗体は、 本発明のタンパク質を特異的に認識することができるので、 被検波中の本発明のタンパク質の定量、 特にサンドイッチ免疫測定法による定 量などに使用することができる。 Since 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.
すなわち、 本発明は、  That is, the present invention
(0 本発明の抗体と、 被検液および標識化された本発明のタンパク質とを競合 的に皮応させ、 該抗体に結合した標識化された本発明のタンパク質の割合を測 定することを特徴とする被検液中の本発明のタンパク質の定量法、 および  (0) 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
00 被検液と担体上に不溶化した本発明の抗体および標識化された本発明の 別の抗体とを同時あるいは連続的に反応させたのち、 不溶化担体上の標識剤の 活性を測定することを特徴とする被検液中の本発明のタンパク質の定量法を提 供する。  After reacting the test solution with the antibody of the present invention insolubilized on the carrier and another labeled antibody of the present invention simultaneously or continuously, it is necessary to measure the activity of the labeling agent on the insolubilized carrier. Provided is a method for quantifying the protein of the present invention in a test solution, which is a feature of the present invention.
上記 （i i) の定量法においては、 一方の抗体が本発明のタンパク質の N端部 を認識する抗体で、 他方の抗体が本発明のタンパク質の C端部に反応する抗体 であることが望ましい。  In the quantification method (ii), it is desirable that 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.
また、 本発明のタンパク質に対するモノクローナル抗体 （以下、 本発明のモ ノクローナル钪体と称する場合がある） を用いて本発明のタンパク質の定量を 行なえるほか、 組織染色等による検出を行なうこともできる。 これらの目的に は、 抗体分子そのものを用いてもよく、 また、 お体分子の F (ab' ) 2 、 Fab'、 あ るいは Fab画分を用いてもよい。 In addition, 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. For these purposes, the antibody molecule itself may be used, or the F (ab ') 2 , Fab', or Fab fraction of the body molecule may be used.
, 本発明の抗体を用いる本発明のタンパク質の定量法は、 特に制限されるべき ものではなく、 被測定液中の抗原量 （例えば、 タンパク質量） に対応した抗体、 抗原もしくは抗体一抗原複合体の量を化学的または物理的手段により検出し、 これを既知量の抗原を含む標準液を用いて作製した標準曲線より算出する測定 法であれば、 いずれの測定法を用いてもよい。 例えば、 ネフロメトリ一、 競合 法、 ィムノメトリック法およびサンドイッチ法が好適に用いられるが、 感度、 特異性の点で、 後述するサンドイッチ法を用いるのが特に好ましい。  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 γ 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.
標識物質を用いる測定法に用いられる標識剤としては、 例えば、 放射性同位 元素、 酵素、 蛍光物質、 発光物質などが用いられる。 放射性同位元素としては、 例えば、 〔1251〕 、 〔1311〕 、 〔¾〕 、 〔14c〕 などが用いられる。 上記酵素とし ては、 安定で比活性の大きなものが好ましく、 例えば、 i3 _ガラクトシダーゼ、 0—ダルコシダ一ゼ、 アルカリホスファタ一;^、 パーォキシダ一ゼ、 リンゴ酸 脱水素酵素などが用いられる。 蛍光物質としては、 例えば、 フルォレスカミン、 フルォレツセンイソチオシァネ一卜などが用いられる。 発光物質としては、 例 えば、 ルミノール、 ルミノール誘導体、 ルシフェリン、 ルシゲニンなどが用い . られる。 さらに、 抗体あるいは抗原と標識剤との結合にピオチン一アビジン系 を用いることもできる。 As 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. As the radioisotope, e.g., [125 1], [131 1], [¾], and the like are used [14 c]. As the above-mentioned enzyme, a stable enzyme having a large specific activity is preferable. For example, i3_galactosidase, 0-Dalkosidase, alkaline phosphatase; ^, peroxidase, malate dehydrogenase, etc. are used. As the fluorescent substance, for example, fluorescamine, fluorescein isothiocyanate, or the like is used. As the luminescent substance, for example, luminol, luminol derivative, luciferin, lucigenin and the like are used. Further, a biotin-avidin system can be used for binding the antibody or antigen to the labeling agent.
抗原あるいは抗体の不溶化に当っては、 物理吸着を用いてもよく、 また通常 タンパク質あるいは酵素等を不溶化、 固定化するのに用いられる化学結合を用 いる方法でもよい。 担体としては、 ァガロース、 デキストラン、 セルロースな どの不溶性多糖類、 ポリスチレン、 ポリアクリルアミド、 シリコン等の合成樹 脂、 あるいはガラス等が挙げられる。  For the insolubilization of an antigen or an antibody, physical adsorption may be used, or a method using a chemical bond usually used for insolubilizing and immobilizing a protein or an enzyme may be used. Examples of the carrier include insoluble polysaccharides such as agarose, dextran, and cellulose; synthetic resins such as polystyrene, polyacrylamide, and silicon; and glass.
サンドィツチ法においては不溶化した本発明のモノクローナル抗体に被検液 を反応させ （1次反応） 、 さらに標識化した別の本発明のモノクローナル抗体 を反応させ （2次反応） たのち、 不溶化担体上の標識剤の活性を測定すること により被検液中の本発明のタンパク質量を定量することができる。 1次反応と 2次反応は逆の順序に行っても、 また、 同時に行なってもよいし時間をずらし て行なってもよい。 標識化剤および不溶化の方法は前記のそれらに準じる と ができる。 また、 サンドイッチ法による免疫測定法において、 固相用抗体ある いは標識用抗体に用いられる抗体は必ずしも 1種類である必要はなく、 測定感 度を向上させる等の目的で 2種類以上の抗体の混合物を用いてもよい。  In the sandwich method, 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). By measuring the activity of the labeling agent, the amount of the protein of the present invention in the test solution can be determined. 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. In addition, in the immunoassay by the sandwich method, 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.
本発明のサンドイッチ法による本発明のタンパク質の測定法においては、 1 次反応と 2次反応に用いられる本発明の.モノクローナル抗体は、 本発明のタン パク質の結合する部位が相異なる抗体が好ましく用いられる。 すなわち、 1次 反応および 2次反応に用いられる钪体は、 例えば、 2次反応で用いられる抗体 が、 本発明のタンパク質の C端部を認識する場合、 1次反応で用いられる抗体 は、 好ましくは C端部以外、 例えば N端部を認識する抗体が用いられる。  In the method for measuring the protein of the present invention by the sandwich method of the present invention, 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. Used. That is, 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.
本発明のモノク口一ナル抗体をサンドィツチ法以外の測定システム、 例えば、 競合法、 ィムノメトリック法あるいはネフロメトリーなどに用いることができ る。 競合法では、 被検液中の抗原と標識抗原とを抗体に対して競合的に反応させ たのち、 未反応の標識抗原（F ) と、 抗体と結合した標識抗原 (B ) とを分離し (B /F分離） 、 B， Fいずれかの標識量を測定し、 被検液中の抗原量を定量 する。 本反応法には、 抗体として可溶性抗体を用い、 B / F分離をポリエチレ ングリコール、 前記抗体に対する第 2抗体などを用いる液相法、 および、 第 1 抗体として固相化抗体を用いるか、 あるいは、 第 1抗体は可溶性のものを用い 第 2抗体として固相化抗体を用いる固相化法とが用いられる。 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. In the 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. (B / F separation) Measure the amount of labeling for either B or F, and quantify the amount of antigen in the test solution. In this reaction method, 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.
ィムノメトリック法では、 被検液中の抗原と固相化抗原とを一定量の標識化 抗体に対して競合反応させた後固相と液相を分離するか、 あるいは、 被検液中 の抗原と過剰量の標識化抗体とを反応させ、 次に固相化抗原を加え未反応の標 識化抗体を固相に結合させたのち、 固相と液相を分離する。 次に、 いずれかの 相の標識量を測定し被検液中の抗原量を定量する。  In the immunometric method, 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. Next, the amount of label in either phase is measured to determine the amount of antigen in the test solution.
また、 ネフロメトリ一では、 ゲル内あるいは溶液中で抗原抗体反応の結果生 じた不溶性の沈降物の量を測定する。 被検液中の抗原量が僅かであり、 少量の 沈降物しか得られない場合にもレーザ一の散乱を利用するレーザーネフロメト リーなどが好適に用いられる。  In 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.
これら個々の免疫学的測定法を本発明の定量方法に適用するにあたっては、 特別の条件、 操作等の設定は必要とされない。 それぞれの方法における通常の 条件、 操作法に当業者の通常の技術的配慮を加えて本発明の夕ンパク質の測定 系を構築すればよい。 これらの一般的な技術手段の詳細については、 総説、 成 書などを参照することができる。 ·  In applying these individual immunoassays to the quantification method of the present invention, no special conditions, operations, and the like need to be set. What is necessary is just to construct the protein measurement system of the present invention by adding ordinary technical considerations of those skilled in the art to ordinary conditions and operation methods in each method. For details of these general technical means, reference can be made to reviews and documents. ·
例えば、 入江 寛編 「ラジオィムノアツセィ」 （講談社、 昭和 4 9年発行） ， 入江 寛編 「続ラジオィムノアツセィ」 （講談社、 昭和 5 4年発行） 、 石川栄 治ら編 「酵素免疫測定法」 （医学書院、 昭和 5 3年発行） 、 石川栄治ら編 「酵 素免疫測定法」 （第 2版） （医学書院、 昭和 5 7年発行） 、 石川栄治ら編 「酵 素免疫測定法」 （第 3版） （医学書院、 昭和 6 2年発行） 、 「Methods in ENZYMOLOGYJ Vol . 70 (Immunochemical Techniques (Part A) )、 同書 Vol .  For example, edited by Hiro Irie “Radio Nonotsusei” (Kodansha, published in Showa 49), edited by Hiroshi Irie “Radio Imunoatsusei” (Kodansha, published in 1954), edited by Eiji Ishikawa et al. "Measurement Method" (Medical Publishing, published in 1978), Eiji Ishikawa et al., "Enzyme Immunoassay" (Second Edition) (Medical Publishing, published in 1977), Eiji Ishikawa, et al., "Enzyme Immunoassay" (3rd edition) (Medical Shoin, published in 1962), “Methods in ENZYMOLOGYJ Vol. 70 (Immunochemical Techniques (Part A))”, Vol.
73 (Immunochemical Techniques (Part B) )、 同書 Vol . 74 (Immunochemical Techniques (Part C))、 同書 Vol. 84 (Immunochemical Techniques (Part fi:Selected Immunoassays)) > 同書 Vol. 92 (Immunochemical Techniques (Part E:Monoclonal Antibodies and General Immunoassay Methods))、'同書 Vol. 121 (Immunochemical Techniques (Part I： Hybrido a Technology and 73 (Immunochemical Techniques (Part B)), ibid.Vol. 74 (Immunochemical Techniques (Part C)), ibid.Vol. 84 (Immunochemical Techniques (Part B) fi: Selected Immunoassays))> Ibid.Vol. 92 (Immunochemical Techniques (Part E: Monoclonal Antibodies and General Immunoassay Methods)), 'Ibid.Vol. 121 (Immunochemical Techniques (Part I: Hybrido a Technology and
Monoclonal Ant ibodies)) (以上、 アカデミックプレス社発行）などを参照するこ とができる。 > Monoclonal Ant ibodies)) (above, published by Academic Press). >
以上のようにして、 本発明の抗 を用いることによって、 本発明のタンパク 質を感度良く定量することができる。  As described above, the protein of the present invention can be quantified with high sensitivity by using the antibody of the present invention.
さらには、 本発明の抗体を用いて本発明のタンパク質の濃度を定量すること によって、 本発明のタンパク質の濃度の増加または減少が検出された場合、 例 えば、 軟骨形成異常、 骨形成異常、 骨粗鬆症、 変形性関節症、 慢性関節リウマ チ、 関節炎、 滑膜炎、 代謝性関節症またはスポーツによる関節障害 （例、 テニ ス肘など） などの骨 ·関節疾患である、 または将来罹患する可能性が高いと診 断することができる。  Furthermore, when an increase or decrease in the concentration of the protein of the present invention is detected by quantifying the concentration of the protein of the present invention using the antibody of the present invention, for example, 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.
また、 本発明の抗体は、 体液や組織などの被検体中に存在する本発明のタン パク質を検出するために使用することができる。 また、 本発明のタンパク質を 精製するために使用する抗体カラムの作製、 精製時の各分画中の本発明のタン パク質の検出、 被検細胞内における本発明のタンパク質の挙動の分析などのた めに使用することができる。 (4) 遺伝子診断薬  Further, 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. In addition, 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
本発明の DN Aは、 例えば、 プローブとして使用することにより、 ヒトまた は温血動物 （例えば、 ラット、 マウス、 モルモット、 ゥサギ、 トリ、 ヒッジ、 ブ夕、 ゥシ、 ゥマ、 ネコ、 ィヌ、 サル、 チンパンジ一など） における本発明の タンパク質またはその部分ペプチドをコードする DN Aまたは mRN Aの異常 (遺伝子異常） を検出することができるので、 例えば、 該 DNAまたは mRN Aの損傷、 突然変異あるいは発現低下や、 該 DNAまたは mRNAの増加ある ^は発現過多などの遺伝子診断薬として有用である。  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. Alternatively, ^ whose expression is decreased or the DNA or mRNA is increased is useful as a diagnostic agent for genes such as overexpression.
本発明の DN Aを用いる上記の遺伝子診断は、 例えば、 自.体公知のノーザン ハイブリダィゼーシヨンや P CR— S S CP法 （Genomics，第 5巻， 874〜879頁 (1989年) 、 Proceed ings of the Nat i onal Academy of Sc i ences o f the 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
Uni ted S t ates of Amer ica，第 86巻， 2766〜2770頁（1989年） ) などにより実施す ることができる。 ' 例えば、 ノーザンハイブリダィゼ一シヨンにより発現過多または減少が検出 された場合や P C R— S S C P法により D N Aの突然変異が検出された場合は、 例えば、 軟骨形成異常、 骨形成異常、 骨粗鬆症、 変形性関節症、 慢性関節リウ マチ、 関節炎、 滑膜炎、 代謝性関節症またはスポーツによる関節障害 （例、 'テ ニス肘など） などの骨 ·関節疾患である可能性が高いと診断することができる。 ( 5 ) アンチセンスポリヌクレオチドを含有する医薬 . United States of America, Vol. 86, pp. 2766-2770 (1989)). '' For example, if overexpression or decrease is detected by Northern hybridization or DNA mutation is detected by PCR-SSCP method, for example, chondrodysplasia, bone dysplasia, osteoporosis, deformity Diagnosis of a high likelihood of bone and joint disease, such as osteoarthritis, rheumatoid arthritis, arthritis, synovitis, metabolic arthropathy, or joint disorders due to sports (eg, 'tennis elbow') it can. (5) A drug containing an antisense polynucleotide.
本発明の D N Aに相補的に結合し、 該 D N Aの発現を抑制することができる 本発明のアンチセンスポリヌクレオチドは低毒性であり、 生体内における本発 明のタンパク質または本発明の D N Aの機能を抑制することができるので、 例 えば、 軟骨形成異常、 骨形成異常、 骨粗鬆症、 変形性関節症、 慢性関節リウマ チ、 関節炎、 滑膜炎、 代謝性関節症またはスポーツによる関節障害 （例、 テニ ス肘など） などの骨 ·関節疾患の予防 ·治療剤として使用することができる。 上記ァンチセンスポリヌクレオチドを上記の予防 ·治療剤として使用する場 合、 自体公知の方法に従って製剤化し、 投与することができる。  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. For example, cartilage dysplasia, bone dysplasia, osteoporosis, osteoarthritis, rheumatoid arthritis, arthritis, synovitis, metabolic arthropathy or joint disorders due to sports (eg, tennis) Bone such as elbow) · Prevention of joint diseases · Can be used as a therapeutic agent. When 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.
また、 例えば、 前記のアンチセンスポリヌクレオチドを単独あるいはレトロ ウィルスベクタ一、 アデノウイルスベクター、 アデノウイルスァソシェ一テツ ドウィルスベクタ一などの適当なベクタ一に挿入した後、 常套手段に従って、 ヒトまたは哺乳動物 （例、 ラット、 ゥサギ、 ヒッジ、 ブ夕、 ゥシ、 ネコ、 ィヌ、 サルなど） に対して経口的または非経口的に投与することができる。 該アンチ センスポリヌクレオチドは、 そのままで、 あるいは摂取促進のために補助剤な どの生理学的に認められる担体とともに製剤化し、 遺伝子銃やハイド口ゲル力 テーテルのようなカテーテルによって投与できる。 あるいは、 エアロゾル化し て吸入剤として気管内に局所投与することもできる。  Further, for example, after inserting the above-mentioned antisense polynucleotide alone or into a suitable vector such as a retrovirus vector, an adenovirus vector, an adenovirus associated virus vector, or the like, 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.
さらに、 体内動態の改良、 半減期の長期化、 細胞内取り込み効率の改善を目 的に、 前記のアンチセンスポリヌクレオチドを単独またはリボゾームなどの担 体とともに製剤 （注射剤） 化し、 静脈、 皮下または M節腔内等に投与してもよ い。 Furthermore, the above-mentioned 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.
該アンチセンスポリヌクレオチドの投与量は、 対象疾患、 投与対象、 投与ル ートなどにより差異はあるが、 例えば、 変形性関節症の治療の目的で本発明の アンチセンスポリヌクレオチドを投与する場合、 一般的に成人 （体重 6 O k g ) においては、 一日につき該アンチセンスポリヌクレオチドを約 0 . 1〜1 0 O m g投与する。  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.
さらに、 該アンチセンスボリヌクレオチドは、 組織や細胞における本発明の Further, the antisense polynucleotide is used in a tissue or a cell of the present invention.
D N Aの存在やその発現状況を調べるための診断用オリゴヌクレオチドプロ一 ブとして使用することもできる。 It can also be used as a diagnostic oligonucleotide probe for examining the presence of DNA and its expression status.
上記アンチセンスポリヌクレオチドと同様に、 本発明のタンパク質をコード する R NAの一部を含有する二重鎖 R NA、 本発明のタンパク質をコードする R N Aの一部を含有するリポザィムなども、 本発明の遺伝子の発現を抑制する ことができ、 生体内における本発明のタンパク質または本発明で用いられる D N Aの機能を抑制することができるので、 例えば、 軟骨形成異常、 骨形成異常、 骨粗鬆症、 変形性関節症、 慢性関節リウマチ、 関節炎、 滑膜炎、 代謝性関節症 またはスポ一ッによる関節障害 （例、 テニス肘など） などの骨 ·関節疾患の予 防 ·治療剤などとして使用することができる。  Similarly to the above-mentioned antisense polynucleotide, 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. 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).
二重鎖 R NAは、 公知の方法 （例、 Nature, 411巻， 494頁， 2001年） に準じ て、 本発明のポリヌクレオチドの配列を基に設計して製造することができる。 リポザィムは、 公知の方法 （例、 TRENDS in Molecular Medic ine, 7巻， 221 頁， 2001年） に準じて、 本発明のポリヌクレオチドの配列を基に設計して製造 することができる。 例えば、 本発明のタンパク質をコードする R NAの一部に 公知のリポザィムを連結することによって製造することができる。'本発明の夕 ンパク質をコードする R NAの一部としては、 公知のリポザィムによって切断 され得る本発明の R NA上の切断部位に近接した部分 （R NA断片） が挙げら れる。  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.
上記の二重鎖 R NAまたはリポザィムを上記予防 ·治療剤として使用する場 合、 アンチセンスポリヌクレオチドと同様にして製剤化し、 投与することがで さる。 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
(6) 本発明の抗体.を含有する医薬 (6) a medicine containing the antibody of the present invention.
本発明のタンパク質の活性を中和する作用を有する本発明の坊体は、 軟骨形 成異常、 骨形成異常、 骨粗鬆症、 変形性関節症、 慢性関節リウマチ、 関節炎、 滑膜炎、 代謝性関節症またはスポーツによる関節障害 （例、 テニス肘など） な どの骨 ·関節疾患の予防 ·治療剤として使用することができる。  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).
本発明の抗体を含有する上記疾患の予防 ·治療剤は低毒性であり、 そのまま 液剤として、 または適当な剤型の医薬組成物として、 ヒトまたは哺乳動物 （例、 ラット、 ゥサギ、 ヒッジ、 ブタ、 ゥシ、 ネコ、 ィヌ、 サルなど） に対して経口 的または非経口的 （例、 関節内投与） に投与することができる。 投与量は、 投  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
/ 一 与対象、 対象疾患、 症状、 投与ルートなどによっても異なるが、 例えば、 成人 の変形性関節症の治療 ·予防のために使用する場合には、 本発明の抗体を 1回 量として、 通常 0.01〜20mg/k g体重程度、 好ましくは 0.1〜1 Om gZkg体重程度、 さらに好ましくは 0. l〜5mg/kg体重程度を、 1日 1 〜5回程度、 好ましくは 1日 1〜3回程^、 粉末吸入剤により投与するのが好 都合である。 他の非経口投与および経口投与の場合もこれに準ずる量を投与す ることができる。 症状が特に重い場合には、 その症状に応じて増量してもよい c 本発明の抗体は、 それ自体まだは適当な医薬組成物として投与することがで きる。 上記投与に用いられる医薬組成物は、 上記抗体またはその塩と薬理学的 に許容され得る担体、 希釈剤もしくは陚形剤とを含むものである。 かかる組成 物は、 経口または非経口投与 （例、 関節内投与） に適する剤形として提供され る。 好ましくは吸入剤として提供される。 / Depending on the target, target disease, symptoms, administration route, etc., for example, when used for the treatment or prevention of osteoarthritis in adults, the antibody of the present invention is usually used as a single dose. About 0.01 to 20 mg / kg body weight, preferably about 0.1 to 1 Om gZ kg body weight, more preferably about 0.1 to 5 mg / kg body weight, about 1 to 5 times a day, preferably about 1 to 3 times a day ^, It is conveniently administered by powder inhalation. In the case of other parenteral administration and oral administration, an equivalent amount can be administered. When the condition is especially severe, the antibody also good c present invention increased depending on the symptoms, itself still as possible out be administered as an appropriate pharmaceutical composition. The pharmaceutical composition used for the above administration contains the above antibody or a salt thereof and a pharmacologically acceptable carrier, diluent or vehicle. Such compositions are provided in dosage forms suitable for oral or parenteral administration (eg, intra-articular administration). It is preferably provided as an inhalant.
なお前記した各組成物は、 上記抗体との配合により好ましくない相互作用を 生じない限り他の活性成分を含有してもよい。  Each of the above-mentioned compositions may contain another active ingredient as long as the composition does not cause an undesirable interaction with the above-mentioned antibody.
(7) DNA転移動物 . (7) DNA transfer animal.
本発明は、 外来性の本発明のタンパク質をコードする DNA (以下、 本発明 の外来性 D.N Aと略記する） またはその変異 DN A (本発明の外来性変異 DN Aと略記する場合がある） を有する非ヒ卜哺乳動物を提供する。 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).
すなわち、 本発明は、  That is, the present invention
(1) 本発明の外来性 DNAまたはその変異 DNAを有する非ヒト哺乳動物、 (1) a non-human mammal having the exogenous DNA of the present invention or a mutant DNA thereof,
(2) 非ヒト哺乳動物がゲッ歯動物である第 （1)記載の動物、 (2) the animal according to (1), wherein the non-human mammal is a rodent;
(3) ゲッ歯動物がマウスまたはラットである第 （2) 記載の動物、 および (4) 本発明の外来性 DNAまたはその変異 DNAを含有し、 哺乳動物におい て発現しうる組換えベクターを提供するものである。  (3) The animal according to (2), wherein the rodent is a mouse or a rat; and (4) a recombinant vector containing the exogenous DNA of the present invention or a mutant DNA thereof and capable of being expressed in a mammal. Is what you do.
本発明の外来性 DNAまたはその変異 DNAを有する非ヒ卜哺乳動物 （以下、 本発明の DNA転移動物と略記する） は、 未受精卵、 受精卵、 精子およびその 始原細胞を含む胚芽細胞などに対して、 好ましくは、 非ヒト哺乳動物の発生に おける胚発生の段階 （さらに好ましくは、 単細胞または受精卵細胞の段階でか つ一般に 8細胞期以前） に、 リン酸カルシウム法、 電気パルス法、 リボフェク シヨン法、 凝集法、 マイクロインジェクション法、 パーティクルガン法、 DE AE—デキストラン法などにより目的とする DN Aを転移することによって作 出することができる。 また、 該 DNA転移方法により、 体細胞、 生体の臓器、 組織細胞などに目的とする本発明の外来性 DNAを転移し、 細胞培養、 組織培 養などに利用することもでき、 さらに、 これら細胞を上述の胚芽細胞と自体公 知の細胞融合法により融合させることこより本発明の DN A転移動物を作出す ることもできる。  Non-human mammals having the exogenous DNA of the present invention or the mutant DNA thereof (hereinafter abbreviated as the DNA-transferred animal of the present invention) include unfertilized eggs, fertilized eggs, germ cells including spermatozoa and their progenitor cells, and the like. In contrast, preferably, during the stage of embryonic development in non-human mammal development (more preferably, at the stage of a single cell or a fertilized egg and generally before the 8-cell stage), 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. Further, by the DNA transfer method, 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.
非ヒト哺乳動物としては、 例えば、 ゥシ、 ブタ、 ヒッジ、 ャギ、 ゥサギ、 ィ ヌ、 ネコ、 モルモット、 ハムスター、 マウス、 ラットなどが用いられる。 なか でも、 病体動物モデル系の作成の面から個体発生および生物サイクルが比較的 短く、 また、 繁殖が容易なゲッ歯動物、 とりわけマウス （例えば、 純系として、 C 57BL/6系統， DBA 2系統など、 交雑系として、 B 6 C 3 F ^系統, ' BDFt系統， BGDSFi系統， BALBZc系統， I CR系統など） または ラット （例えば、 Wi s t a r， SDなど） などが好ましい。  As non-human mammals, for example, porcupines, pigs, higgins, goats, magpies, dogs, cats, guinea pigs, hamsters, mice, rats and the like are used. Above all, rodents with relatively short ontogeny and biological cycle in terms of the creation of disease animal model systems, and easy to breed rodents, especially mice (for example, C57BL / 6 strains and DBA2 strains as pure strains) As the crossing strain, B 6 C 3 F ^ strain, 'BDFt strain, BGDSFi strain, BALBZc strain, ICR strain, etc.) or rat (eg, Wistar, SD, etc.) are preferable.
哺乳動物において発現しうる組換えベクターにおける 「哺乳動物」 としては、 上記の非ヒト哺乳動物の他にヒトなどがあげられる。  As the “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.
■ 本発明の外来性 DNAとは、 非ヒト哺乳動物が本来有している本発明の DN Aではなく、 いったん哺乳動物から単離 ·抽出された本発明の D N Aをいう。 本発明の変異 D N Aとしては、 元の本発明の D N Aの塩基配列に変異 （例え ば、 突然変異など） が生じたもの、 具体的には、 塩基の付加、 欠損、 他の塩基 への置換などが生じた D NAなどが用いられ、 また、 異常 D NAも含まれる。 該異常 D NAとしては、 異常な本発明のタンパク質を発現させる D NA.を意 味し、 例えば、 正常な本発明のタンパク質の機能を抑制するタンパク質を発現 させる D NAなどが用いられる。 . ■ 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. As the 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. For example, a DNA that expresses a protein that suppresses the function of the normal protein of the present invention is used. .
本発明の外来性 D NAは、 対象とする動物と同種あるいは異種のどちらの哺 乳動物由来のものであってもよい。 本発明の D NAを対象動物に転移させるに あたっては、 該 D NAを動物細胞で発現させ るプロモータ一の下流に結合し た D NAコンストラクトとして用いるのが一般に有利である。 例えば、 本発明 のヒト D NAを転移させる場合、 これと相同性が高い本発明の D NAを有する 各種哺乳動物 （例えば、 ゥサギ、 ィヌ、 ネコ、 モルモット、 ハムスター、 ラッ 卜、 マウスなど） 由来の D N Aを発現させうる各種プロモーターの下流に、 本 発明のヒト D NAを結合した D NAコンストラクト （例、 ベクタ一など） を対 象哺乳動物の受精卵、 例えば、 マウス受精卵へマイクロインジェクションする ことによつて本発明の D N Aを高発現する D N A転移哺乳動物を作出すること ができる。  The exogenous DNA of the present invention may be derived from a mammal that is the same or different from the animal of interest. In transferring the DNA of the present invention to a target animal, it is generally advantageous to use the DNA as a DNA construct linked downstream of a promoter that is expressed in animal cells. For example, when the human DNA of the present invention is 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 (eg, a vector) 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. Thus, a DNA-transferred mammal that highly expresses the DNA of the present invention can be produced.
本発明のタンパク質の発現べクタ一としては、 大腸菌由来のプラスミド、 枯 草菌由来のプラスミド、 酵母由来のプラスミド、 λファージなどのバクテリオ ファージ、 モロニ一白血病ウィルスなどのレトロウイルス、 ワクシニアウィル スまたはバキュロウィルスなどの動物ウイルスなどが用いられる。 なかでも、 大腸菌由来のプラスミド、 枯草菌由来のプラスミドまたは酵母由来のプラスミ ドなどが好ましく用いられる。  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 λ 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.
上記の D N Α発現調節を行なうプロモーターとしては、 例えば、 （i) ウィル ス （例、 シミアンウィルス、 サイトメガロウィルス、 モロニ一白血病ウィルス、 J Cウィルス、 乳癌ウィルス、 ポリオウイルスなど） に由来する D NAのプロ モーター、 （i i) 各種哺乳動物 （ヒト、 ゥサギ、 ィヌ、 ネコ、 モルモット、 ノ ムスター、 ラット、 マウスなど） 由来のプロモータ一、 例えば、 アルブミン、 インスリン I I、 ゥロブラキン I I、 エラスターゼ、 エリスロポエチン、 ェン ドセリン、 筋クレアチンキナーゼ、 グリア線維性酸性タンパク質、 ダル夕チォ ン S—トランスフェラ一ゼ、 血小板由来成長因子 ;6、 ケラチン K.l， K10お よび Κ14、 コラーゲン I型および I I型、 サイクリック AMP依存タンパク 質キナ一ゼ /3 Iサブュニット、 ジストロフィン、 酒石酸抵抗性アル力リホスフ ァ夕一ゼ、 心房ナトリウム利尿性因子、 内皮レセプターチ口シンキナーゼ (一 般に T i e 2と略される） 、 ナトリウムカリウムアデノシン 3リン酸化酵素 Examples of the promoter that regulates the expression of DNΑ include, for example, DNA derived from (i) 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 Κ14, 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
(Na, K一 ATP a s e) 、 ニューロフィラメント軽鎖、 メタ口チォネイン Iおよび I IA、 メタ口プロティナーゼ 1'組織インヒビ夕一、 MHCクラス I 坊原 （H— 2L) 、 H— r a s、 レニン、 ド一パミン β— 7_Κ酸化酵素、 甲状腺べ ルォキシダーゼ （ΤΡΟ) 、 ペプチド鎖延^因子 1ひ （EF— 1ひ） 、 /3ァク チン、 ひおよび /3ミオシン重鎖、 ミオシン軽鎖 1および 2、 ミエリ 基礎タン パク質、 チログロブリン、 Thy— 1、 免疫グロブリン、 H鎖可変部 （VN P) 、 血清アミロイド Pコンポ一ネン 、 ミオグロビン、 トロポニン C、 平滑 筋 ァクチン、 プレプロエンケフアリン A、 バソプレシンなどのプロモーター などが用いられる。 なかでも、 全身で高発現することが可能なサイトメガロウ ィルスプロモーター、 ヒトペプチド鎖延長因子 1ひ （EF— 1ひ） のプロモー 夕一、 ヒトおよびニヮトリ /3ァクチンプロモーターなどが好適である。  (Na, K-ATPase), neurofilament light chain, meta-oral thionein I and I IA, meta-oral proteinase 1 'tissue inhibitor Yuichi, MHC class I Bobara (H-2L), H-ras, renin, de Monopamine β-7_Κ oxidase, thyroid gland oxidase (ΤΡΟ), peptide chain elongation factor 1 (EF-1), / 3 actin, spider and / 3 myosin heavy chain, myosin light chain 1 and 2, Myeri basic protein, thyroglobulin, Thy-1, immunoglobulin, heavy chain variable region (VNP), serum amyloid P component, myoglobin, troponin C, smooth muscle actin, preproenkephalin A, vasopressin, etc. A promoter or the like is used. Among them, 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.
上記ベクターは、 DNA転移哺乳動物において目的とするメッセンジャー R N Aの転写を終結する配列 （一般に夕一ミネ夕一と呼ばれる） を有しているこ とが好ましく、 例えば、 ウィルス由来および各種哺乳動物由来の各 DNAの配 列を用いることができ、 好ましくは、 シミアンウィルスの SV40ターミネタ —などが用いられる。  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). For example, 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.
その他、 目的とする外来性 DNAをさらに高発現させる目的で各 DNAのス プライシングシグナル、 ェンハンサ一領域、.真核 DN Aのイントロンの一部な どをプロモー夕一領域の 5'上流、 プロモ一夕一領域と翻訳領域間あるいは翻訳 領域の 3，下流 に連結することも目的により可能である。  In addition, 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.
正常な本発明のタンパク質の翻訳領域は、 ヒトまたは各種哺乳動物 （例えば、 ゥサギ、 ィヌ、 ネコ、 モルモット、 ハムスター、 ラット、 マウスなど） 由来の 肝臓、 腎臓、 甲状腺細胞、 線維芽細胞由来 DNAおよび市販 各種ゲノム DN' Aライブラリーよりゲノム DNAの全てあるいは一部として、 または肝臓、 腎 臓、 甲状腺細胞、 線維 細胞由来 RNAより公知の方法により調製された相補 DNAを原料として取得することが出来る。 また、 外来性の異常 DNAは、 上 記の細胞または組織より得られた正常なタンパク質の翻訳領域を点突然変異誘 発法により変異した翻訳領域を作製することができる。 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. In addition, 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.
該翻訳領域は転移動物において発現しうる DNAコンストラクトとして、 前 記のプロモーターの下流および所望により転写終結部位の上流に連結させる通 常の D N A工学的手法により作製することができる。  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.
受精卵細胞段階における本発明の外来性 DNAの転移は、 対象哺乳動物の胚 芽細胞および体細胞のすべてに存在するように確保される。 DNA転移後の作 出動物の胚芽細胞において、 本発明の外来性 DNAが存在することは、 作出動 物の後代がすべて、 その胚芽細胞および体細胞のすべてに本発明の外来性 DN Aを保持することを意味する。 本発明の外来性 D N Aを受け継いだこの種の動 物の子孫はその胚芽細胞および体細胞のすべてに本発明の外来性 DN Aを有す る。  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.
本発明の外来性正常 DN Aを転移させた非ヒト哺乳動物は、 交配により外来 性 DNAを安定に保持することを確認して、 該 DN A保有動物として通常の飼 育環境で継代飼育することが出来る。  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.
受精卵細胞段階における本発明の外来性 DNAの転移は、 対象哺乳動物の胚 芽細胞および体細胞の全てに過剰に存在するように確保される。 DNA転移後 の作出動物の胚芽細胞において本発明の外来性 DN Aが過剰に存在することは、 作出動物の子孫が全てその胚芽細胞および体細胞の全てに本発明の外来性 DN Aを過剰に有することを意味する。 本発明の外来性 DN Aを受け継いだこの種 の動物の子孫はそO胚芽細胞および体細胞の全てに本発明の外来性 DN Aを過 剰に有する。  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.
導入 DNAを相同染色体の両方に持つホモザィゴート動物を取得し、 この雌 雄の動物を交配することによりすべての子孫が該 DN Aを過剰に有するように 繁殖継代することができる。' 本発明の正常 D NAを有する非ヒト哺乳動物は、 本発明の正常 D NAが高発 現させられており、 内在性の正常 D NAの機能を促進することにより最終的に 本発明のタンパク質の機能亢進症を発症することがあり、 その病態モデル動物 として利用することができる。 例えば、 本発明の正常 D N A転移動物を用いて、 本発明のタンパク質の機能亢進症や、 本発明のタンパク質が関連する疾患の病 . '態機序の解明およびこれらの疾患の治療方法の検討を行なうことが可能である。 By obtaining a homozygous animal having the introduced DNA on both homologous chromosomes and mating the male and female animals, it is possible to breed the cells so that all offspring have the DNA in excess. ' 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.
また、 本発明の外来性正常 D NAを転移させた哺乳動物は、 遊離した本発明 のタンパク質の増加症状を有することから、 本発明のタンパク質に関連する疾 患に対する予防 ·治療剤、 例えば、 軟骨形成異常、 骨形成異常、 骨粗鬆症、 変 形性関節症、 慢性関節リウマチ、 関節炎、 滑膜炎、 代謝性関節症またはスポ一 ッによる関節障害 （例、 テニス肘等） などの骨 ·関節疾患の予防'治療剤のス クリーニング試験にも利用可能である。  In addition, since the mammal to which the exogenous normal DNA of the present invention has been transferred has an increased symptom of the free protein of the present invention, 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.
一方、 本発明の外来性異常 D NAを有する非ヒト哺乳動物は、 交配により外 来性 D N Aを安定に保持することを確認して該 D N A保有動物として通常の飼 育環境で継代飼育することが出来る。 さらに、 目的とする外来 D NAを前述の プラスミドに組み込んで原料として用いることが きる。 プロモ一ターとの D NAコンストラク卜は、 通常の D NA工学的手法によって作製することができ る。 受精卵細胞段階における本発明の異常 D N Aの転移は、 対象哺乳動物の胚 芽細胞および体細胞の全てに存在するように確保される。 D N A転移後の作出 動物の胚芽細胞において本発明の異常 D N Aが存在することは、 作出動物の子 孫が全てその胚芽細胞および体細胞の全てに本発明の異常 D N Aを有すること を意味する。 本発明の外来性 D NAを受け継いだこの種の動物の子孫は、 その 胚芽細胞および体細胞の全てに本発明の異常 D N Aを有する。 導入 D NAを相 同染色体の両方に持つホモザィゴート動物を取得し、 この雌雄の動物を交配す ることによりすべての子孫が該 D N Aを有するように繁殖継代することができ る。  On the other hand, 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. By obtaining a homozygous animal having the introduced DNA on both homologous chromosomes, and crossing the male and female animals, it is possible to breed subculture so that all offspring have the DNA.
本発明の異常 D N Aを有する非ヒト哺乳動物は、 本発明の異常 D N Aが高発 現させられており、 内在性の正常 D NAの機能を阻害することにより最終的に 本発明の夕ンパク質の機能不活性型不応症となることがあり、 その病態モデル 動物として利用することができる。 例えば、 本発明の異常 D N A転移動物を用 いて、 本発明のタンパク質の機能不活性型不応症の病態機序の解明およびこの 疾患を治療方法の検討を行なうことが可能である。 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. For example, 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.
また、 具体的な利用可能性としては、 本発明の異常 D N A高発現動物は、 本 発明のタンパク質の機能不活性型不応症における本発明の異常タンパク質によ る正常タンパク質の機能阻害 （dominant negat ive作用） を解明するモデルとな る。  In addition, as a specific possibility, 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).
また、 本発明の外来異常 D N Aを転移させた哺乳動物は、 遊離した本発明の タンパク質の増加症状を有することから、 本発明のタンパク質または機能不活 性型不応症に対する予防 ·治療剤、 例えば、 軟骨形成異常、 骨形成異常、 骨粗 鬆症、 変形性関節症、 慢性関節リウマチ、 関節炎、 滑膜炎、 代謝性関節症また はスポーツによる関節障害 （例、 テニス肘等） などの骨 ·関節疾患の予防-治 療剤のスクリ一ニング試験にも利用可能である。  Further, since the mammal to which the foreign abnormal DNA of the present invention has been transferred has an increased symptom of the free protein of the present invention, 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.
また、 上記 2種類の本発明の D N A転移動物のその他の利用可能性として、 例えば、  In addition, other possible uses of the above two types of DNA transgenic animals of the present invention include, for example,
(i) 組織培養のための細胞源としての使用、 ' (i) use as a cell source for tissue culture;
(i i) 本発明の D NA転移動物の組織中の D NAもしくは R N Aを直接分析す るか、 または D NAにより発現されたペプチド組織を分析することによる、 本 発明のタンパク質により特異的に発現あるいは活性化するペプチドとの関連性 についての解析、 (ii) Direct analysis of DNA or RNA in the tissue of the DNA-transferred animal of the present invention, or analysis of peptide tissue expressed by the DNA to specifically express or express the protein of the present invention. Analysis of the relationship with the activating peptide,
(i i i) ' D NAを有する組織の細胞を標準組織培養技術により培養し、 これらを 使用して、 一般に培養困難な組織からの細胞の機能の研究、  (iii) '' cells of a tissue having DNA are cultured by standard tissue culture techniques, and these are used to study the function of cells from tissues that are generally difficult to culture,
(iv) 上記 （i i i) 記載の細胞を用いることによる細胞の機能を高めるような薬 剤のスクリーニング、 および  (iv) screening for a drug that enhances cell function by using the cell described in (iii) above, and
(V) 本発明の変異タンパク質を単離精製およびその抗体作製などが考えられる。 さらに、 本発明の D NA転移動物を用いて、 本発明のタンパク質の機能不活 性型不応症などを含む、 本発明のタンパク質に関連する疾患の臨床症状を調べ ,ることができ、 また、 本発明のタンパク質に関連する疾患モデルの各臓器にお けるより詳細な病理学的所見が得られ、 新しい治療方法の開発、 さらには、 該 疾患による二次的疾患の研究および治療に貢献することができる。 (V) Isolation and purification of the mutant protein of the present invention and production of its antibody can be considered. Furthermore, using the DNA-transferred animal of the present invention, it is possible to examine the clinical symptoms of a disease associated with the protein of the present invention, including a functionally inactive type refractory disease of the protein of the present invention, etc. More detailed pathological findings in each organ of the disease model related to the protein of the present invention can be obtained, and a new therapeutic method can be developed. It can contribute to the research and treatment of secondary diseases caused by diseases.
また、 本発明の DNA転移動物から各臓器を取り出し、 細切後、 トリプシン などのタンパク質分解酵素により、 遊離した DN A転移細胞の取得、 その培養 またはその培養細胞の系統化を行なうことが可能である。 さらに、 本発明の夕 ンパク質産生細胞の特定化、 アポトーシス、 分化あるいは増殖との関連性、 ま たはそれらにおけるシグナル伝達機構を調べ、 それらの異常を調べることなど ができ、 本発明のタンパク質およびその作用解明のための有効な研究材料とな る。  In addition, it is possible to take out each organ from the DNA-transferred animal of the present invention, cut it into small pieces, and then use a proteolytic enzyme such as trypsin to obtain released DNA-transferred cells, culture them, or systematize the cultured cells. is there. Furthermore, it is possible to identify the protein-producing cells of the present invention, examine their relationship with apoptosis, differentiation or proliferation, or investigate their signal transduction mechanisms, and investigate their abnormalities. It is an effective research material for elucidating its action.
さらに、 本発明の DNA転移動物を用いて、 本発明のタンパク質の機能不活 性型不応症を含む、 本発明のタンパク質に関連する疾患の治療薬の開発を行な うために、 上述の検査法および定量法などを用いて、 有効で迅速な該疾患治療 薬のスクリーニング法を提供することが可能となる。 また、 本発明の DN A転 移動物または本発明の外来性 DN A発現ベクターを用いて、 本発明のタンパク 質が関連する疾患の DN A治療法を検討、 開発ずることが可能である。  Further, 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.
(8) ノックアウト動物 . (8) Knockout animals.
本発明は、 本発明の DNAが不活性化された非ヒト哺乳動物胚幹細胞および 本発明の DNA発現不全非ヒト哺乳動物を提供する。  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.
すなわち、 本発明は、  That is, the present invention
( 1 ) 本発明の DN Aが不活性化された非ヒト哺乳動物胚幹細胞、  (1) a non-human mammalian embryonic stem cell in which the DNA of the present invention has been inactivated,
(2) 該 DNAがレポ一夕一遺伝子 （例、 大腸菌由来の β_ガラクトシダ一ゼ遺 伝子） を導入することにより不活性化された第 （1) 項記載の胚幹細胞、  (2) The embryonic stem cell according to (1), wherein the DNA is inactivated by introducing a repo overnight gene (eg, a β_galactosidase enzyme gene derived from Escherichia coli).
(3) ネオマイシン ,ノ耐性である第 （1) 項記載の胚幹細胞、  (3) neomycin, an embryonic stem cell according to (1), which is resistant to neomycin,
(4) 非ヒト哺乳動物 ゲッ歯動物である第 （1) 項記載の胚幹細胞、 (5) ゲッ歯動物がマウスである第 （4) 項記載の胚幹細胞、  (4) the embryonic stem cell according to (1), which is a non-human mammal rodent; (5) the embryonic stem cell according to (4), wherein the rodent is a mouse;
(6) 本発明の DNAが不活性化された該 DNA発現不全非ヒト哺乳動物、 (6) a DNA-deficient non-human mammal in which the DNA of the present invention has been inactivated,
(7) 該 DNAがレポーター遺伝子 （例、 大腸菌由来の β_ガラクトシダーゼ遺 伝子） を導入することにより不活性化され、 該レポ一夕一遺伝子が本発明の D ΝΑに対するプロモ一ターの制御下で発現しうる第 （6) 項記載の非ヒト哺乳 動物、 (7) The DNA is inactivated by introducing a reporter gene (eg, a β_galactosidase gene derived from Escherichia coli), and the reporter gene is regulated by the promoter of DΝΑ of the present invention. Non-human mammal according to item (6), which can be expressed in animal,
(8) 非ヒト哺乳動 がゲッ歯動物である第 （6) 項記載の非ヒト哺乳動物、 (8) The non-human mammal according to (6), wherein the non-human mammal is a rodent.
(9) ゲッ歯動物がマウスである第 （8) 項記載の非ヒト哺乳動物、 および(9) the non-human mammal according to (8), wherein the rodent is a mouse; and
(10) 第 （7) 項記載の動物に、 試験化合物を投与し、 レポ一夕一遺伝子の 発現を検出することを特徴とする本発明の DNAに対するプロモーター活性を 促進または阻害する化合物またはその塩のスクリ一ニング方法を提供する。 本発明の DNAが不活性化された非ヒト哺乳動物胚幹細胞とは、 該非ヒト哺 乳動物が有する本堯明の DN Aに人為的に変異を加えることにより、 DN Aの 発現能を抑制するか、 もしくは該 DNAがコードしている本発明のタンパク質 の活性を実質的に喪失させることにより、 DNAが実質的に本発明のタンパク 質の発現能を有さない （以下、 本発明のノックアウト DNAと称することがあ る） 非ヒト哺乳動物の胚幹細胞 （以下、 ES細胞と略記する） をいう。 (10) 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. To provide a screening method. 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. Alternatively, 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).
非ヒト哺乳動物としては、 前記と同様のものが用いられる。  As the non-human mammal, the same one as described above is used.
本発明の DNAに人為的に変異を加える方法としては、 例えば、 遺伝子工学 的手法により該 DN A配列の一部又は全部の削除、 他 DNAを揷入または置換 させることによって行なうことができる。 これらの変異により、 例えば、 コド ンの読み取り枠をずらしたり、 プロモータ一あるいはェキソンの機能を破壊す ることにより本発明のノックァゥト DNAを作製すればよい。  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.
本発明の DN Aが不活性化された非ヒト哺乳動物胚幹細胞 （以下、 本発明の DNA不活性化 ES細胞または本発明のノックアウト ES細胞と略記する） の 具体例としては、 例えば、 目的とする非ヒト哺乳動物が有する本発明の DNA を単離し、 そのェキソン部分にネオマイシン耐性遺伝子、 ハイグロマイシン耐 性遺伝子を代表とする薬剤耐性遺伝子、 あるいは l ac Z (β—ガラクトシダー ゼ遺伝子） 、 c a t (クロラムフエニコールァセチルトランスフェラ一ゼ遺伝 子） を代表とするレポ一夕 遺伝子等を挿入することによりェキソンの機能を 破壊するか、 あるいはェキソン間のイントロン部分に遺伝子の転写を終結させ る DNA配列 （例えば、 p o 1 yA付加シグナルなど） を挿入し、 完全なメッ センジャ一 RNAを合成できなくすることによって、 結果的に遺伝子を破壊す るように構築した DNA配列を有する DNA鎖 （以下、 ター ターと略記する） を、 例えば相同組換え法により該動物の染色体に導入し、 得 られた ES細胞について本発明の DN A上あるいはその近傍の DN A配列をプ ローブとしたサザンハイブリダィゼ一ション解析あるいは夕一ゲッティングべ クタ一上の DN A配列と夕ーゲッティングベクター作製に使用した本発明の D N A以外の近傍領域の D N A配列をプライマーとした P C R法により解析し、 本発明のノックアウト ES細胞を選別することにより得ることができる。 Specific examples of the non-human mammalian embryonic stem cells in which the DNA of the present invention is inactivated (hereinafter, abbreviated as the DNA-inactivated ES cells of the present invention or the knockout ES cells of the present invention) 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 (β-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 By inserting a DNA sequence (for example, the po1yA additional signal) and preventing the synthesis of complete messenger RNA, 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, homologous recombination, and the obtained ES cells are subjected to Southern hybridization using the DNA sequence on or near the DNA of the present invention as a probe. Analysis by PCR using the DNA sequence of the DNA fragment on the DNA analysis sequence or the DNA sequence of the neighboring region other than the DNA of the present invention used in the preparation of the evening getter vector as primers. Knockout can be obtained by selecting ES cells.
また、 相同組換え法等により'本発明の DNAを不活化させる元の ES細胞と しては、 例えば、 前述のような既に樹立されたものを用いてもよく、 また公知 Evansと Kaufmaの方法に準じて新しく樹立したものでもよい。 例えば、 マウスの ES細胞の場合、 現在、 一般的には 129系の ES細胞が使用されているが、 免疫学的背景がはつきりしていないので、 これに代わる純系で免疫学的に遺伝 的背景が明らかな ES細胞を取得するなどの目的で例えば、 C57BLZ6マ ウスや C 57 BL/ 6の採卵数の少なさを DBA/ 2との交雑により改善した マウス （C 57 BLZ6と DBA/2との F を用いて樹立したもの なども良好に用いうる。 BD マウスは、 採卵数が多く、 かつ、 卵が丈夫で あるという利点に加えて、 C 57 BL/6マウスを背景に持つので、 これを用 いて得られた ES細胞は病態モデルマウスを作出したとき、 C 57BLZ6マ ウスとバッククロスすることでその遺伝的背景を C 57BLZ6マウスに代え ることが可能である点で有利に用い得る。  Further, as the 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. For example, in the case of 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. For example, for the purpose of obtaining ES cells with a clear background, 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. .
また、 ES細胞を樹立する場合、 一般には受精後 3.5日目の胚盤胞を使用す るが、 これ以外に 8細胞期胚を採卵し胚盤胞まで培養して用いることにより効 率よく多数の初期胚を取得することができる。 ' また、 雌雄いずれの ES細胞を用いてもよいが、 通常雄の ES細胞の方が生 殖系列キメラを作出するのに都合が良い。 また、 煩雑な培養の手間を削減する ためにもできるだけ早く雌雄の判別を行なうことが望ましい。  In addition, 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.
ES細胞の雌雄の判定方法としては、 例えば、 PCR法により Y染色体上の 性決定領域の遺伝子を増幅、 検出する方法が、 その 1例としてあげることがで きる。 この方法を使用すれば、 従来、 核型分析をするのに約 1.06個の細胞数 を要していたのに対して、 1コロニー程度の ES細胞数 （約 50個） で済むの で、 培養初期における E S細胞の第一次セレクシヨンを雌雄の判別で行なうこ とが可能であり、 早期に雄細胞の選定を可能にしたことにより培養初期の手間 は大幅に削減できる。 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. Using this method, conventionally, 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) Thus, 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.
また、 第二次セレクションとしては、 例えば、 G—バンディ ：グ法による染 色体数の確認等により行うことができる。 得られる ES細胞の染色体数は正常 数の 10'0%が望ましいが、 樹立の際の物理的操作等の関係上 ¾難な場合は、 ES細胞の遺伝子をノックアウトした後、 正常細胞 （例えば、 マウスでは染色 体数が 2 n = 40である細胞） に再びクロ一ニングすることが望ましい。  The secondary selection can be performed by, for example, confirming the number of chromosomes by the G-bandy method. It is desirable that the number of chromosomes in the obtained ES cells is 10'0% of the normal number. However, if it is difficult due to physical operations at the time of establishment, after knocking out the gene of the ES cells, the normal cells (for example, It is desirable to clone again to cells with 2 n = 40 chromosomes in mice).
このようにして得られた胚幹細胞株は、 通常その増殖性は大変良いが、 個体 発生できる能力を失いやすいので、 注意深く継代培養することが必要である。 例えば、 S TO繊維芽細胞のような適当なフィーダ一細胞上で L I F (1〜 10000U/ml) 存在下に炭酸ガス培養器内 （好ましくは、 5%炭酸ガス、 95 %空 気または 5%酸素、 5%炭酸ガス、 90%空気） で約 37 で培養するなどの 方法で培養し、 継代時には、 例えば、 トリプシン ZEDTA溶液 （通常 0.001〜 0.5%トリプシン /0. l〜5mM EDTA、 好ましくは約 0.1 %トリプシン ZlmM E DTA) 処理により単細胞化し、 新たに用意したフィーダ一細胞上に播種する 方法などがとられる。 このような継代は、 通常 1〜3日毎に行なうが、 この際 に細胞の観察を行い、 形態的に異常な細胞が見受けられた場合はその培養細胞 は放棄することが望まれる。  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. For example, on a suitable feeder cell, such as STO fibroblasts, in a carbon dioxide incubator (preferably 5% carbon dioxide, 95% air or 5% oxygen) in the presence of LIF (1-10000 U / ml) , 5% carbon dioxide, 90% air) at about 37, etc., and at the time of subculture, for example, 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細胞は、 適当な条件により、 高密度に至るまで単層培養するか、 または 細胞集塊を形成するまで浮遊培養することにより、 頭頂筋、 内臓筋、 心筋など の種々のタイプの細胞に分化させることが可能であり CM. J. Evans及び M. H. Kaufman, Nature第 292卷、 154頁、 1981年； G. R. Martin Proc. Natl. Acad. Sci. U.S.A. 第 78巻、 7634頁、 1981年； Τ· C. Doetschman ら、 ジャーナル'ォ ブ。ェンブリオロジ一'アンド ·ェクスペリメンタル 'モルフォロジ一、 第 87 巻、 27頁、 1985年〕 、 本発明の ES細胞を分化させて得られる本発明の DN A 発現不全細胞は、 インビトロにおける本発明のタンパク質の細胞生物学的検討 において有用である。  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.
本発明の DNA発現不全非ヒト哺乳動物は、 該動物の mRNA量を公知方法 を用いて測定して間接的にその発現量を比較することにより、 正常動物と区別 することが可能である。 - 該非ヒト哺乳動物としては、 前記と同様のものが用い.られる。， 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. ,
本発明の D NA発現不全非ヒト哺乳動物は、 例えば、 前述のようにして作製 したターゲッティングベクタ一をマウス胚幹細胞またはマウス卵細胞に導入し、 導入により夕一ゲッティングベクタ一の本発明の D N Aが不活性化された D N A配列が遺伝子相同組換えにより、 マウス胚幹細胞またはマウス卵細胞の染色 体上の本発明の D N Aと入れ換わる相同組換えをさせることにより、 本発明の D NAをノックアウトさせることができる。  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.
本発明の D NAがノックアウトされた細胞は、 本発明の D NA上またはその 近傍の D N A配列をプローブとしたサザンハイブリダイゼ一ション解析または ターゲッティングベクタ一上の D NA配列と、 夕一ゲッティングベクターに使 用したマウス由来の本発明の D N A以外の近傍領域の D N A配列とをプライマ 一とした P C R法による解析で判定することができる。 非ヒト哺乳動物胚幹細 胞を用いた場合は、 遺伝子相同組換えにより、 本発明の D N Aが不活性化され た細胞株をクローエングし、 その細胞を適当な時期、 例えば、 8細胞期の非ヒ ト哺乳動物胚または胚盤胞に注入し、 作製したキメラ胚を偽妊娠させた該非ヒ ト哺乳動物の子宮に移植する。 作出された動物は正常な本発明の D NA座をも つ細胞と人為的に変異した本発明の D N A座をもつ細胞との両者から構成され るキメラ動物である。  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. When a non-human mammalian embryonic stem cell is used, 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.
該キメラ動物の生殖細胞の一部が変異した本発明の D N A座をもつ場合、 こ のようなキメラ個体と正常個体を交配することにより得られた個体群より、 全 ての組織が人為的に変異を加えた本発明の D N A座をもつ細胞で構成された個 体を、 例えば、 コートカラ一の判定等により選別することにより得られる。 こ のようにして得られた個体は、 通常、 本発明のタンパク質のヘテロ発現不全個 体であり、 本発明のタンパク質のヘテロ発現不全個体同志を交配し、 それらの 産仔から本発明のタンパク質のホモ発現不全個体を得ることができる。  When a part of the germ cells of the chimeric animal has a mutated 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.
卵細胞を使用する場合は、 例えば、 卵細胞核内にマイクロインジェクション 法で D N A溶液を注入することによりタ一ゲッティングベクタ一を染色体内に 導入したトランスジエニック非ヒト哺乳動物を得ることができ、 これらのトラ ンスジエニック非ヒト哺乳動物に比べて、 遺伝子相同組換え より本発明の D N A座に変異のあるものを選択することにより得られる。 When using an egg cell, for example, 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.
このようにして本発明の D NAがノックアウトされている個体は、 交配によ り得られた動物個体も詨 D N Aがノックアウトされていることを確認して通常 の飼育環境で飼育継代を行なうことができる。  In the case of the individual knocked out of the DNA of the present invention in this manner, 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.
さらに、 生殖系列の取得および保持についても常法に従えばよい。 すなわち- 該不活化 D N Aの保有する雌雄の動物を交配することにより、 該不活化 D N A を相同染色体の両方に持つホモザィゴート動物を取得しうる。 得られたホモザ ィゴ一ト動物は、 母親動物に対して、 正常個体 1， ホモザィゴ一ト複数になる ような状態で飼育することにより効率的に得ることができる。 ヘテロザィゴー ト動物の雌雄を交配することにより、 該不活化 D N Aを有するホモザィゴ一ト およびへテロザィゴ一ト動物を繁殖継代する。  Furthermore, 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. By mating male and female heterozygous animals, homozygous and heterozygous animals having the inactivated DNA are bred and passaged.
本発明の D NAが不活性化された非ヒト哺乳動物胚幹細胞は、 本発明の D N A発現不全非ヒト哺乳動物を作出する上で、 非常に有用である。  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.
' また、 本発明の D NA発現不全非ヒト哺乳動物は、 本発明のタンパク質によ り誘導され得る種々の生物活性を欠失するため、 本発明のタンパク質の生物活 性の不活性化を原因とする疾病のモデルとなり得るので、 これらの疾病の原因 究明及び治療法の検討に有用である。  '' In addition, since 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.
( 8 a ) 本発明の D NAの欠損や損傷などに起因する疾病に対して治療 ·予防 効果を有する化合物のスクリーニング方法  (8a) A method for screening a compound having a therapeutic / preventive effect against diseases caused by DNA deficiency or damage, etc. of the present invention
本発明の D NA発現不全非ヒト哺乳動物は、 本発明の D N Aの欠損や損傷な どに起因する疾病に対して治療 ·予防効果を有する化合物のスクリーニングに 用いることができる。  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.
すなわち、 本発明は、 本発明の D NA発現不全非ヒト哺乳動物に試験化合物 を投与し、 該動物の変化を観察 ·測定することを特徴とする、 本発明の D NA の欠損や損傷などに起因する疾病に対して治療 ·予防効果を有する化合物また はその塩のスクリーニング方法を提供する。  That is, 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. Provided is a method for screening a compound or a salt thereof having a therapeutic or preventive effect on a disease caused by the disease.
該スクリ一ニング方法において用いられる本発明の D N A発現不全非ヒト哺 乳動物としては、 前記と同様のものがあげられる。 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.
試験化合物としては、 例えば、 ペプチド、 タンパク質、 非ペプチド性化合物、 合成化合物、 発酵生産物、 細胞抽出液、 植物抽出液、 動物組織抽出液、 血漿な どがあげられ、 これら化合物は新規な化合物であってもよいし、 公知の化合物 ' であってもよい。  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 '.
具体的には、 本発明の D NA発現不全非ヒト哺乳動物を、 試験化合物で処理 し、 無処理の対照動物と比較し、 該動物の各器官、 組織、 疾病の症状などの変 化を指標として試験化合物の治療 ·予防効果を試験することができる。  Specifically, 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. As a test, the therapeutic and prophylactic effects of the test compound can be tested.
試験動物を試験化合物で処理する方法としては、 例えば、 経口投与、 静脈注 射などが用いられ、 試験動物の症状、 試験化合物の性質などにあわせて適宜選 択することができる。 また、 試験化合物の投与量は、 投与方法、 試験化合物の 性質などにあわせて適宜選択することができる。  As a method of treating a test animal with a 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.
例えば、 軟骨形成異常、 骨形成異常、 骨粗鬆症、 変形性関節症、 慢性関節リ ゥマチ、 関節炎、 滑膜炎、 代謝性関節症またはスポーツによる関節障害 （例、 テニス肘等） などの骨 ·関節疾患に対して治療 '予防効果を有する化合物をス クリーニングする場合、 本発明の D NA発現不全非ヒト哺乳動物に試験化合物 を投与し、 該動物の関節や軟骨などを経時的に観察し、 上記疾患の症状を観察 する。 .  For example, 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.) When screening for a compound having a prophylactic effect, 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. .
該スクリー,ニング方法において、 試験動物に試験化合物を投与した場合、 該 試験動物の上記疾患症状が約 1 0 %以上、 好ましくは約 3 0 %以上、 より好ま しくは約 5 0 %以上改善した場合、 該試験化合物を上記の疾患に対して治療- 予防効果を有する化合物として選択することができる。  In the screening method, 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. In this case, the test compound can be selected as a compound having a therapeutic-prophylactic effect against the above-mentioned diseases.
該スクリーニング方法を用いて得られる化合物は、 上記した試験化合物から 選ばれた化合物であり、 本発明のタンパク質の欠損や損傷などによって引き起 こされる疾患に対して治療 ·予防効果を有するので、 該疾患に対する安全で低 毒性な予防 ·治療剤などの医薬として使用することができる。 さらに、 上記ス クリーニングで得られた化合物から誘導される化合物も同様に用いることがで さる。  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.
該スクリーニング方法で得られた化合物は塩を形成していてもよく、 該化合 物の塩としては、 生理学的に許容される酸 （例、 無機酸、 有機酸など） や塩基 (例、 アルカリ金属など） などとの塩が用いられ、 とりわけ生理学的に許容さ れる酸付加塩が好ましい。 この様な塩としては、 例えば、 無機酸 （例えば、 塩 '酸、，リン酸、 臭化水素酸、 硫酸など） との塩、 あるいは有機酸 ： (例えば、 酢酸、 ' ギ酸、 プロピオン酸、 フマル酸、 マレイン酸、 コハク酸、 酒石酸、 クェン酸、 . リンゴ酸、 蓚酸、 安息香酸、 メタンスルホン酸、 ベンゼンスルホン酸など） と の塩などが用いちれる。 The compound obtained by the screening method may form a salt. As 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. 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.).
該スクリーニング方法で得られた化合物またはその塩を含有する医薬は、 前 記した本発明のタンパク質を含有する医薬と同様にして製造することができる。 このようにして得られる製剤は、 安全で低毒性であるので、 例えば、 ヒトま たは哺乳動物 （例えば、 ラット、 マウス、 モルモット、 ゥサギ、 ヒ.ッジ、 ブタ、 ゥシ、 ゥマ、 ネコ、 ィヌ、 サルなど） に対して投与することができる。  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.).
該化合物またはその塩の投与量は、 対象疾患、 投与対象、 投与ルートなどに より差異はあるが、 例えば、 該化合物を経口投与する場合、 一般的に成人 （体 重 60 kgとして） の変形性関節症患者においては、 一日につき該化合物を約 0.1〜： L 0 Omg、 好ましくは約 1. 0〜5 Omg、 より好ましくは約 1. 0 〜20mg投与する。 非経口的に投与する場合は、 該化合物の 1回投与量は投 与対象、 対象疾患などによっても異なるが、 例えば、 該化合物を注射剤の形で 通常成人 （体重 6 Okgとして） の変形性関節症患者に投与する場合、 一日に つき該化合物を約 0. 01〜3 Omg、 好ましくは約 0. l〜20mg、 より 好ましくは約 0. 1〜1 Omgを静脈注射により投与するのが好都合である。 他の動物の場合も、 体重 60 kg当たりに換算した量を投与することができる。  The dose of the compound or a salt thereof varies depending on the target disease, the administration subject, the administration route, and the like. For example, when the compound is orally administered, the deformability of an adult (assuming a body weight of 60 kg) is generally In patients with arthrosis, 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. In the case of parenteral administration, 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). 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.
(8 b) 本発明の DN Aに対するプロモーターの活性を促進または阻害する化 合物をスクリーニング方法  (8b) Method for screening for a compound that promotes or inhibits the activity of a promoter for DNA of the present invention
本発明は、 本発明の DN A発現不全非ヒト哺乳動物に、 試験化合物を投与し、 レポーター遺伝子の発現を検出することを特徴とする本発明の DNAに対する プロモータ一の活性を促進または阻害する化合物またはその塩のスクリーニン グ方法を提供する。  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.
上記スクリーニング方法において、 本発明の DN A発現不全非ビ卜哺乳動物 としては、 前記した本発明の DNA発現不全非ヒト哺乳動物の中でも、 本発明 の DN Aがレポ一夕一遺伝子を導入することにより不活性化され、 該レポ一夕 一遺伝子が本発明の D N Aに対するプロモータ一の制御下で発現しうるものが 用いられる。 In the above-mentioned screening method, the non-bit mammal deficient in DNA expression of the present invention Among the non-human mammals deficient in expression of the DNA of the present invention, the DNA 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.
試験化合物としては、 前記 同様のものがあげられる。  Examples of the test compound include the same compounds as described above.
レポ一夕一遺伝子としては、 前記と同様のものが用いられ、 β—ガラクトシダ As the repo overnight gene, the same gene as described above is used, and β-galactosida
—ゼ遺伝子 (1 a c Z) 、 可溶性アルカリホスファタ一ゼ遺伝子またはルシフ エラ—ゼ遺伝子などが好適である。 ' 本発明の DN Aをレポーター遺伝子で置換された本発明の DN A発現不全非 ヒト哺乳動物では、 レポーター遺伝子が本発明の DNAに対するプロモ一夕一 の支配下に存在するので、 レポ一夕一遺伝子がコードする物質の発現をトレーZea gene (1 acZ), soluble alkaline phosphatase gene or luciferase gene are preferred. '' In a non-human mammal deficient in expression of the DNA of the present invention in which the DNA of the present invention has been replaced with a reporter gene, 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
！ スすることにより、 プロモーターの活性を検出す.ることができる。 ! Thus, the activity of the promoter can be detected.
例えば、 本発明のタンパク質をコードする DN A領域の一部を大腸菌由来の β 一ガラクトシダ一ゼ遺伝子 ( 1 a c Ζ) で置換している場合、 本来、 本発明の タンパク質の発現する組織で、 本発明のタンパク質の代わりに 6—ガラクトシダ —ゼが発現する。 従って、 例えば、 5—ブロモ—4一クロ口— 3—インドリル —β—ガラクトピラノシド （X— g a l) のような β—ガラクトシダ一ゼの基質 となる試薬を用いて染色することにより、 簡便に本発明のタンパク質の動物生 体内における発現状態を観察することができる。 具体的には、 本発明のタンパ ク質欠損マウスまたはその組織切片をダルタルアルデヒドなどで固定し、 リン 酸緩衝生理食塩液 (PBS) で洗浄後、 X— g a 1を含む染色液で、 室温また は 37刼付近で、 約 30分ないし 1時間反応させた後、 組織標本を ImM ED TAZPBS溶液で洗浄することによって、 8—ガラクトシダ一ゼ反応を停止さ せ、 呈色を観察すればよい。 また、 常法に従い、 1 a c Zをコードする mRN Aを検出してもよい。 .  For example, when a part of the DNA region encoding the protein of the present invention is replaced with a β-galactosidase gene (1 ac 由来) derived from Escherichia coli, 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 β-galactosidase such as 5-bromo-4-monocloth-3-indolyl-β-galactopyranoside (X-gal), In addition, the state of expression of the protein of the present invention in animal organisms can be observed. Specifically, 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. Alternatively, after reacting at about 37 刼 for about 30 minutes to 1 hour, the 8-galactosidase reaction may be stopped by washing the tissue specimen with ImMED TAZPBS solution, and the coloration may be observed. In addition, mRNA encoding 1acZ may be detected according to a conventional method. .
上記スクリ一ニング方法を用いて得られる化合物またはその塩は、 上記した 試験化合物から選ばれた化合物であり、 本発明の DN Aに対するプロモータ一 活性を促進または阻害する化合物である。  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.
該スクリ一エング方法で得られた化合物は塩を形成していてもよく、 該化合 物の塩としては、 生理学的に許容される酸 （例、 無機酸など） や塩基 （例、 有 機酸など） などとの塩が用いられ、 とりわけ生理学的に許容される酸付加塩が 好ましい。 この様な塩とじては、 例えば、 無機酸 （例えば、 塩酸、 リン酸、 臭 化水素酸、 硫酸など) との塩、 あるいは有機酸 （例えば、 酢酸、 ギ酸、 プロピ オン酸、 フマル酸、 マレイン酸、 コハク酸、 酒石酸、 クェン酸、 リンゴ酸、 蓚 酸、 安息香酸、 メタンスルホン酸、 ベンゼンスルホン酸など） との塩などが用 いられる。 The compound obtained by the screening method may form a salt, As the salt of the product, a salt with a physiologically acceptable acid (eg, an inorganic acid, etc.) or a base (eg, an organic acid, etc.) is used, and 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.
本発明の D N Aに対するプロモータ一活性を促進または阻害する化合物また はその塩は、 本発明のタンパク質の発現の調節、 該タンパク質の機能を調節す ることができるので、 例えば、 軟骨形成異常、 骨形成異常、 骨粗鬆症、 変形性 関節症、 慢性関節リウマチ、 関節炎、 滑膜炎、 代謝性関節症またはスポーツに よる関節障害 （例、 テニス肘など） などの骨 ·関節疾患の予防 ·治療剤として 有用である。  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.
さらに、 上記スク.リーニングで得られた化合物から誘導される化合物も同様 に用いることができる。  Further, compounds derived from the compounds obtained by the above screening can be used in the same manner.
該スクリーニング方法で得られた化合物またはその塩を含有する医薬は、 前 記した本発明のタンパク質またはその塩を含有する医薬と同様にして製造する ことができる。  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.
このようにして得られる製剤は、 安全で低毒性であるので、 例えば、 ヒトま たは哺乳動物 （例えば、 ラット、 マウス、 モルモット、 ゥサギ、 ヒッジ、 ブタ. ゥシ、 ゥマ、 ネコ、 ィヌ、 サルなど） に対して投与することができる。  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.).
該化合物またはその塩の投与量は、 対象疾患、 投与対象、 投与ルートなどに より差異はあるが、 例えば、 本発明の D N Aに対するプロモーター活性を阻害 する化合物を経口投与する場^、 一般的に成人 （体重 6 0 k gとして） の変形 性関節症患者においては、 一日につき該化合物を約 0 . 1〜1 0 0 m g、 好まし くは約 1 . 0〜5 0 m g、 より好ましくは約 1 . 0〜2 0 m g投与する。 非経 口的に投与する場合は、 該化合物の 1回投与量は投与対象、 対象疾患などによ つても異なるが、 例えば、 本発明の D NAに対するプロモーター活性を阻害す る化合物を注射剤の形で通常成人 （体重 6 0 k gとして） の変形性関節症患者 に投与する場合、 一日につき該化合物を約 0. 01〜30mg、 好ましくは約 0. 1〜2 Omg、 より好ましくは約 0. 1〜1 Omgを静脈注射により投与 するのが好都合である。 他の動物の場合も、 体重 60 kg当たりに換算した量 を投与することができる。 · このように、 本発明の DNA発現不全非ヒ卜哺乳動物は、 本発明の DN Aに 対するプロモーターの活性を促進または阻害する化合物またはその塩をスクリ 一二ングする上で極めて有用であり、 本発明の DN A発現不全に起因する各種 疾患の原因究明または予防 ·治療剤の開発に大きく貢献することができる。 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. For example, when the compound of the present invention that inhibits the promoter activity for DNA is orally administered, 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. When administered parenterally, the single dose of the compound varies depending on the administration subject, target disease, and the like.For example, a compound that inhibits the promoter activity of DNA of the present invention may be used as an injection. Adult patients with osteoarthritis in normal form (as weight as 60 kg) It is convenient to administer about 0.01 to 30 mg, preferably about 0.1 to 2 Omg, more preferably about 0.1 to 1 Omg of the compound per day by intravenous injection. In the case of other animals, the dose can be administered in terms of weight per 60 kg. Thus, 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.
また、 本発明のタンパク質のプロモータ一領域を含有する DNAを使って、 その下流に種々のタンパクをコードする遺伝子を連結し、 これを動物の卵細胞 に注入していわゆるトランスジエニック動物 （遺伝子移入動物） を作成すれば、 特異的にその夕ンパク質を合成させ、 その生体での作用を検討することも可能 となる。 さらに上記プロモ一夕一部分に適当なレポ一夕一遺伝子を結合させ、 これが発現するような細胞株を樹立すれば、 本発明のタンパク質そのものの体 内での産生能力を特異的に促進もしくは抑制する作用を持つ低分子化合物の探 率系として使用できる。 本明細書において、 塩基やアミノ酸などを略号で表示する場合、 IUPAC- IUB Commission on Biochemical Nomenclature による略号あるいは当該分野におけ る慣用略号に基づくものであり、 その例を下記する。 またアミノ酸に関し光学 異性体があり得る場合は、 特に明示しなければ L体を示すものとする。  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. In the present specification, 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. When amino acids can have optical isomers, the L-form is indicated unless otherwise specified.
DNA デォキシリポ核酸  DNA Deoxylipo nucleic acid
c DNA 相補的デォキシリポ核酸  c DNA complementary deoxylipo nucleic acid
A アデニン  A adenine
T チミン  T thymine
G グァニン  G Guanin
C C
RNA リポ核酸 RNA liponucleic acid
mRNA 一リポ核酸 dATP '三リン酸 mRNA monoliponucleic acid dATP 'Triphosphate
dTTP '三リン酸 dTTP 'Triphosphate
dGTP デォキシグァノシン三リン酸 dGTP Deoxyguanosine triphosphate
dCTP デォキシシチジン三リン酸 dCTP Deoxycytidine triphosphate
ATP アデノシン三リン酸 ATP adenosine triphosphate
EDTA エチレンジァミン四齚酸 EDTA Ethylenediaminetetraacid
SDS ドデシル硫酸ナトリウム SDS sodium dodecyl sulfate
G 1 y G 1 y
A 1 a ァラニン A 1 a Alanin
Va 1 パリン Va 1 Palin
Le u Le u
I 1 e I 1 e
S e r セリン S e r serine
Th r スレオニン Th r threonine
Cy s Cy s
Me t メチォニン Me t Methionin
G 1 u グルタミン酸 G 1 u Glutamic acid
As p ァスパラギン酸 As p Aspartic acid
L y s リジン Lys lysine
A r g アルギニン A r g Arginine
H i s ヒスチジン H is histidine
P h e フエ二ルァラニン P h e feniralanin
Ty r チロシン Ty r tyrosine
T r p トリブトファン T r p Tribute fan
P r o プロリン Pro proline
A s n ァスパラギン A s n asparagine
G 1 n グルタミン G 1 n Glutamine
pG 1 u ピログルタミン酸 pG 1 u pyroglutamic acid
S e c (selenocysteine) また、 本明細 ：中で繁用される置換基、 保護基および試薬を下記の記号で表 記する。 S ec (selenocysteine) In addition, substituents, protecting groups and reagents frequently used in the present specification are represented by the following symbols.
Me メチル基  Me methyl group
E t ェチル基  Etethyl group
B u ブチル基  B u butyl group
Ph フエニル基  Ph phenyl group
TC チアゾリジン— 4 (R) 一力ルポキサミ 'ド基  TC thiazolidine—4 (R) lipoxamide
T o s p—トルエンスルフォニル  T os p—toluenesulfonyl
CHO ホルミル  CHO Holmill
B z 1 ベンジル  B z 1 benzyl
Cl Bzl 2, 6—ジクロ口べンジル  Cl Bzl 2, 6—Ventil
Bom ベンジルォキシメチル  Bom benzyloxymethyl
Z ベンジルォキシカルポニル  Z benzyloxycarponyl
C 1 -z 2—クロ口べンジルォキシカルポニル ' B r - Z 2一ブロモベンジルォキシカルポニル  C 1 -z 2 -Chlorobenzoyloxycarbonyl 'Br-Z 2 -Bromobenzyloxycarbonyl
B o c t一ブトキシカルポニル  B oc t-butoxycarponyl
DNP ジニトロフエニル  DNP dinitrophenyl
T r t トリチル  T r t Trityl
Bum t—ブトキシメチル  Bum t—butoxymethyl
Fmo c N— 9一フルォレニルメトキシカルポニル  Fmo c N— 9-Fluorenylmethoxycarbonyl
HOB t 1ーヒドロキシべンズトリアゾール  HOB t 1-hydroxybenztriazole
HOOB t 3， 4ージヒドロー 3—ヒドロキシー 4一ォキソ一  HOOB t 3,4 dihydro-3-hydroxy-4oxo
1, 2, 3一べンゾ卜リアジン  1, 2, 3 Benzotriazine
HONB 1 -ヒドロキシ- 5 -ノルポルネン- 2, 3-ジカルボキシイミド DCC 本願明細書の配列表の配列番号は、 以下の配列を示す。  HONB 1-Hydroxy-5-norporene-2,3-dicarboximide DCC The sequence numbers in the sequence listing in the present specification show the following sequences.
〔配列番号： 1〕 '  [SEQ ID NO: 1] '
実施例 2で得られた成熟型 chondromedin夕ンパク質のアミノ酸配列を示す, 〔配列番号： 2〕 FIG. 9 shows the amino acid sequence of mature chondromedin protein obtained in Example 2, [SEQ ID NO: 2]
配列番号： 1で表されるアミノ酸配列を有する成熟型 chondromedinタンパク質 をコードする DNAの塩基配列を示す。 This shows the base sequence of DNA encoding the mature chondromedin protein having the amino acid sequence represented by SEQ ID NO: 1.
〔配列番号： 3〕 '  [SEQ ID NO: 3] '
実施例 2で得られた chondr omed in前駆体夕ンパク質のァミノ酸配列を示す。 〔配列番号： 4〕 2 shows the amino acid sequence of chondr omed in precursor protein obtained in Example 2. [SEQ ID NO: 4]
配列番号： 3で表されるアミノ酸配列を有するタンパク質をコードする DNA の塩基配列を示す。 This shows the base sequence of DNA encoding the protein having the amino acid sequence represented by SEQ ID NO: 3.
〔配列番号： 5〕  [SEQ ID NO: 5]
実施例 2で得られた塩基配列を示す。 3 shows the base sequence obtained in Example 2.
〔配列番号： 6〕 ·  [SEQ ID NO: 6]
実施例 2で用いられたプライマーの塩基配列を示す。 3 shows the nucleotide sequence of a primer used in Example 2.
〔配列番号： 7〕  [SEQ ID NO: 7]
実施例 2で用いられたプライマーの塩基配列を示す。 3 shows the nucleotide sequence of a primer used in Example 2.
〔配列番号： 8〕  [SEQ ID NO: 8]
実施例 3で用いられた塩基配列を示す。 3 shows the base sequence used in Example 3.
〔配列番号： ' 9〕  [SEQ ID NO: 9]
実施例 4で用いられたプライマーの塩基配列を示す。 6 shows the nucleotide sequence of a primer used in Example 4.
〔配列番号： 10〕  [SEQ ID NO: 10]
実施例 4で用いられたプライマ一の塩基配列を示す。 7 shows the nucleotide sequence of a primer used in Example 4.
〔配列番号： 11〕  [SEQ ID NO: 11]
実施例 4で用いられたプライマ一の塩基配列を示す。 7 shows the nucleotide sequence of a primer used in Example 4.
〔配列番号： 12〕  [SEQ ID NO: 12]
実施例 4で用いられたプライマ一の塩基配列を示す。 7 shows the nucleotide sequence of a primer used in Example 4.
〔配列番号： 13〕  [SEQ ID NO: 13]
実施例 4で用いられたプローブの塩基配列を示す。 6 shows the nucleotide sequence of a probe used in Example 4.
〔配列番号： 14〕  [SEQ ID NO: 14]
合成ペプチド Tr 1のアミノ酸配列を示す。 1 shows the amino acid sequence of synthetic peptide Tr1.
〔配列番号： 15〕 合成べプチド Tr2のアミノ酸配列を示す。 [SEQ ID NO: 15] 2 shows the amino acid sequence of a synthetic peptide Tr2.
〔配列番号： 16〕  [SEQ ID NO: 16]
実施例 6で用いられたアダプタ一を構成するオリゴヌクレオチドの塩基配列を 示す。 5'はリン酸化されている。 14 shows the base sequence of an oligonucleotide constituting the adapter 1 used in Example 6. 5 'is phosphorylated.
〔配列番号： 17〕  [SEQ ID NO: 17]
実施例 6で用いられたアダプタ一を構成するオリゴヌクレオチドの塩基配列を 示す。 5'はリン酸化されている。 ' 後述の実施例 2で得られた大腸菌 （Escherichia coli) DH5 α/ρΤΒ2300は、 2002年 11月 20日から茨城県つくば市東 1丁目 1番地 1 中央第 6 (郵便番号 305 -14 shows the base sequence of an oligonucleotide constituting the adapter 1 used in Example 6. 5 'is phosphorylated. '' Escherichia coli DH5α / ρΤΒ2300 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-
8566) の独立行政法人鸾業技術総合研究所 特許生物寄託センタ一に受託番号 FERM BP- 8240として寄託されている。 以下に実施例を挙げて本発明を更に具体的に説明するが、 本発明はそれに限 定されるものではない。 なお、 大腸菌を用いての遺伝子操作法は、 Molecular cloning, 2nd, J. Sambrook et al. , Cold Spring Harbor Lab. Press, 1989年に記 載されている方法に従った。 ， 8566) at the National Institute of Advanced Industrial Science and Technology (AIST) under the deposit number FERM BP-8240. Hereinafter, the present invention will be described more specifically with reference to Examples, but the present invention is not limited thereto. The gene manipulation using Escherichia coli, Molecular cloning, 2 nd, J. Sambrook et al., According to the methods that are placing serial in Cold Spring Harbor Lab. Press, 1989 years. ,
実施例 1 Example 1
変形性関節症軟骨 （0A軟骨） で発現亢進している遺伝子群を明らかにするた め、 OA軟骨 （膝関節 1例、 股関節 5例） 、 正常関節軟骨 （膝関節 1例、 股関節 1 例） 、 その他 22種類の正常組織 （各 1例） から抽出された total RNA (表 1) を 材料とし、 oligonucleotide microarray (Human Genome腸 A; Af fymetrix社) を用いて遺伝子発現解析を行った。  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) Using 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).
実験方法は、 Affymetrix社の実験手引き書 （Expression analysis technical manual) に従った。 OA軟骨と正常関節軟骨の遺伝子発現プロファイルを比較し . た結果、 DKFZP586L151 (GenBank Accession No. XP一 034000) 遺伝子の発現量は、 解析した全ての組織中、 0A軟骨において最大であった（表 2)。 . 〔表 1〕 The experimental method followed Affymetrix's experiment guide (Expression analysis technical manual). As a result of comparing the gene expression profiles of OA cartilage and normal articular cartilage, the expression level of DKFZP586L151 (GenBank Accession No. XP-1 034000) gene was the highest in 0A cartilage among all tissues analyzed (Table 2). ). . 〔table 1〕
RNAを抽出した組織 販: π:兀  RNA extracted tissue Sales: π: vat
変形性膝関節症軟骨 Di rect CI i ni cal Access社 変形性股関節症軟骨 Di rect CI inical Access社 正常膝関節軟骨 Di rect CI inical Access社 正常股関節 Di rect CI i ni cal Access社 肋軟骨 BioChain Insti tute ¾ 脂肪 BioChain Institute社 骨格筋 CI on tech社  Osteoarthritis cartilage Di rect CI ini cal Access Co. osteoarthritis cartilage Di rect CI inical Access Normal knee cartilage Di rect CI inical Access Co.脂肪 Fat BioChain Institute Skeletal muscle CI on tech
心臓 Clontech社  Heart Clontech
Clontech社  Clontech
副腎 Clontech社  Adrenal gland Clontech
肝臓 Clontech社'  Liver Clontech '
薛臓 Clontech社  Xuele Clontech
脾臓 Clontech社  Spleen Clontech
Clontech社  Clontech
肺 Clontech社  Lung Clontech
全 Clontech社  All Clontech
小 flea Clontech社  Small flea Clontech
甲状腺 Clontech社  Thyroid Clontech
胸腺 Clontech社  Thymus Clontech
乳腺 Clontech社  Mammary gland Clontech
唾液腺 Clontech社  Salivary gland Clontech
Clontech社  Clontech
BioChain Institute社 大腸 BioChain Institute社 . 子宮 Clontech.社  BioChain Institute Inc. Large Intestine BioChain Institute Inc. Uterus Clontech. Inc.
子宮頸 BioChain Institute社 〔表 2〕 Cervical BioChain Institute (Table 2)
組織一 遺伝子発現量  Tissue-gene expression level
変形性膝関節症軟骨 8.9  Osteoarthritis cartilage 8.9
b  b
変形性股関節症軟骨 5.9  Osteoarthritis cartilage 5.9
正常膝関節軟骨 ND  Normal knee cartilage ND
正常股関節 ND  Normal hip ND
肋軟骨 0.6  Costal cartilage 0.6
脂肪 ND  Fat ND
骨格筋 ND  Skeletal muscle ND
心臓 ND  Heart ND
ND  ND
J腎 ND  J kidney ND
肝臓 ND  Liver ND
脬臓 ND  Gut ND
脾 te ND  Spleen te ND
ND  ND
肺 ND  Lung ND
全脳 ND  Whole brain ND
小 flea ND  Small flea ND
甲状腺 ND  Thyroid ND
胸腺 ND  Thymus ND
乳腺 0.6  Mammary gland 0.6
唾液腺 ND  Salivary gland ND
ND ND
ND ND
大腸 ND  Large intestine ND
子宮 0.5  Uterus 0.5
子宮頸 ND  Cervical ND
a遺伝子発現量は、 oligonucleotide microarrayで発現が検出された 全遺伝子の発現量の中央値を〗として標準化した。 aThe gene expression level was normalized using the median of the expression levels of all genes whose expression was detected by the oligonucleotide microarray as〗.
b  b
平均値を示した （n=5)  Average value was shown (n = 5)
ND; not detected 実施例 2 ND; not detected Example 2
DKFZP586L151 (GenBank Accession No. XP一 034000) 遺伝子配列を基にデ一夕 ベース検索を行ったところ、 マウスオルソログと考えられる遺伝子  DKFZP586L151 (GenBank Accession No. XP-034000) A gene-based search was performed based on the gene sequence.
1110018N05Rik (GenBank Accession No. XM_1 9545) を見出した。 この塩基配 列を基に、 Ensemblのヒト cDNA (Genscan) データベース 1110018N05Rik (GenBank Accession No. XM_1 9545) was found. Based on this base sequence, Ensembl's human cDNA (Genscan) database
(http：〃 www. ensembl.org/homo_sapiens/) を検索して、 ヒト DKFZP586L151の 翻訳開始コドンを含むと考えられる塩基配列 （配列番号： 5) を見出した。 こ の配列および DKFZP586L151遺伝子の塩基配列 （GenBank Accession No.  By searching (http: .www.ensembl.org / homo_sapiens /), a nucleotide sequence (SEQ ID NO: 5) thought to contain the translation initiation codon of human DKFZP586L151 was found. This sequence and the nucleotide sequence of the DKFZP586L151 gene (GenBank Accession No.
XP— 0.34000) に基づき、 2種の合成プライマ一 （配列番号： 6および配列番号： 7) を設計した。 このプライマ一ペアを用い、 ヒト各種組織由来 cDNA (クロン テック社） を铸型として PCRを行った。 耐熱性 DNAポリメラ一ゼには Stratagene 社から購入した uTurboを用いた。 反応溶液の組成は PfuTurboの使用説明書に 従った。 始めに 95°C2分の加熱を行い、 次に 98C10秒— 65 30秒— 72 3分 30秒 からなる増幅過程を 35サイクル実行、 最後に 72°C10分のインキュベーションを 行った。 胎盤、 肺、 骨格筋、 小腸、 精巣、 子宮、 胎児腎臓および胎児肝臓の cDNAから得られた反応液で、 予想される約 2.3kbpの PCR産物が認められた。 これ らを混合し、 Strataprep PCR purification kit (Stratagene社) を用いて PCR 増幅産物を精製した。 得られた約 2.3kbの DNA断片を pCR-Script cloning kit (Stratagene社） を用いて大腸菌 DH5ひにクローン化し、 そのプラスミドをBased on XP—0.34000), two synthetic primers (SEQ ID NO: 6 and SEQ ID NO: 7) were designed. Using this primer pair, PCR was performed using cDNA from various human tissues (Clontech) as type III. UTurbo purchased from Stratagene was used for the thermostable DNA polymerase. The composition of the reaction solution was in accordance with the instruction manual for PfuTurbo. First, heating was performed at 95 ° C for 2 minutes, followed by 35 cycles of an amplification process consisting of 98C for 10 seconds—65 30 seconds—723.3 minutes and 30 seconds, followed by incubation at 72 ° C for 10 minutes. 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
PTB2300と命名した。 得られた大腸菌 (Escherichia coli) DH5 α/ρΤΒ2300から プラスミド DNAを単離し、 PTB2300に挿入された約 2.3kb の DNA断片を、 蛍光 Dye - Terminator法によりその塩基配列を決定した。 得られた塩基配列において、 配 列番号： 6および配列番号： 7で示した 2種のプライマ一配列間に、 配列番号： 5および DKFZP586L151遺伝子の既知塩基配列 （GenBank Accession No. Named PTB2300. Plasmid DNA was isolated from the obtained Escherichia coli DH5α / ρΤΒ2300, and the nucleotide sequence of a DNA fragment of about 2.3 kb inserted into PTB2300 was determined by a fluorescent Dye-Terminator method. In the obtained nucleotide sequence, between the two primer sequences shown in 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) を含有するオープンリーディングフレームが見出された。 該ォ一プ ンリーディングフレームの塩基配列を配列番号： 4に示す。 配列番号： 4で表 される塩基配列から予想されるアミノ酸配列を配列番号： 3に示す。  XP_034000) was found. 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.
配列番号： 3で示されるアミノ酸配列は、 DKFZP586L151 (GenBank Accession No. XP一 034000) のアミノ酸配列の N末側に 314残基伸長したアミノ酸配列であ り、 マウス 1110018N05Rik (GenBank Accession No. XM_129545) とは 82%の相 同性を有する。 ' The 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). '
配列番号： 4で示される塩基配列は、 DKFZP586L151の塩基配列の 5，側に 826塩 基を伸長した塩基配列であり、'マウス 1110018N05Rikとは 86%の,相同性を有する。 分泌シグナル予測プログラムである SignalP (Protein Engineering 12巻， 3- 9頁、 1999年） で配列番号： 3で表されるアミノ酸配列を解析したところ、 N末 端の 22残基からなる AKPRLLVLYFALIVVPAWVSSは、 分泌シグナルぺプチドと予測さ れた。 配列番号： 3で表されるアミノ酸配列からシグナルペプチドを除いたァ ミノ酸配列 （配列番号： 1) を有するタンパク質を、 成熟型 chondromedinタン パク質と命名した。 配列番号： 1で表されるアミノ酸配列をコードする塩基配 列を配列番号： 2に示す。 実施例 3  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'. Analysis of the amino acid sequence represented by SEQ ID NO: 3 using SignalP (Protein Engineering Vol. 12, pp. 3-9, 1999), a secretion signal prediction program, revealed that AKPRLLVLYFALIVVPAWVSS consisting of the N-terminal 22 residues was secreted. Expected to be a signal peptide. 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. Example 3
実施例 2で得られだ chondromedinの cDNA配列を墓に、 ENSEMBLヒトゲノムデ一 夕ベース (http://www. ensembl. org/homo— sapiens/) を BLASTサーチした。 その 結果、 cDNAの 5，上流を含むゲノム配列を見出した。 この配列を第一ェキソンと プロモーター領域予測プログラムである FirstEF (First Exon Finder, Ramana V. ら Nature Genetics 29, 412—417, 2001) で解析し、 転写制御領域を含む と考えられるゲノム配列 （配列番号： 8) を見出した。 配列番号： 8で.表され る塩基配列は、 配列番号： 2で表される塩基 列の 5'上流域に存在する chondromedinのプロモーター配列である。  Using the cDNA sequence of chondromedin obtained in Example 2 as a grave, a BLAST search was performed on the ENSEMBL human genome database (http://www.ensembl.org/homo-sapiens/). As a result, a genomic sequence containing 5, upstream of cDNA was found. This sequence was analyzed with the first exon and promoter region prediction program FirstEF (First Exon Finder, Ramana V. et al. Nature Genetics 29, 412-417, 2001), and the genomic sequence (SEQ ID NO: : 8) was found. The nucleotide sequence represented by 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.
配列番号： 8で表される塩基配列を、 転写因子の結合サイトデ一夕ベースで ある TRANSFAC (Wingender, Eら Nucleic Acids Res. 29, 281-283. 2001) に対 し、 転写因子結合サイト予測ソフトである Match version 1.5 (Goessling, E. ら、 Proceedings of the German Conference on Bioinformat ics (GCB ' 01), GBF Braunschweig E. Wingender, R. Hofestaedt, I. Liebich (eds.), 158- 161 (2001)) で解析した結果を表 3に示す。 なお、 TRANSFACおよび Matchは h t tp：〃 www, b i obase. de/で最新版が公開されている。
Figure imgf000076_0001
基本転写因子である TFII-Iおよびこれと協同的に作用する USFの結合部位が見 出された。 Initiator element (Inr) を介して作動するタイプのプロモータ一 と考えられる。 また、 軟骨の分化に関係する Sox9、 TGF-i3受容体スーパ一ファ ミリーのシグナル伝達に関与する SMAD-3や SMAD- 4などの転写因子の結合サイト が見出された。 chondromedinは軟骨細胞分化において BMP刺激や Sox9の発現上昇 により産生促進されることが予想された。 配列番号： 8で表される塩基配列を 含む配列をレポ一夕一遺伝子 0レシフェラーゼ、 アルカリホスファタ一ゼな ど） の上流に結合し、 各種の細胞株 （ATDC5、 C3H10T1/2など） に導入すること により、 本遺伝子の発現調節薬の探索に用いることができる。 実施例 4 Based on the nucleotide sequence represented by SEQ ID NO: 8, a transcription factor binding site prediction software for TRANSFAC (Wingender, E et al., Nucleic Acids Res. 29, 281-283. 2001) Match version 1.5 (Goessling, E. et al., Proceedings of the German Conference on Bioinformatics (GCB '01), GBF Braunschweig E. Wingender, R. Hofestaedt, I. Liebich (eds.), 158-161 (2001) Table 3 shows the results of the analysis in ()). The latest versions of TRANSFAC and Match are published at ht tp: 〃www, bi obase. De /.
Figure imgf000076_0001
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). In addition, 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. Example 4
以下、 1110018N05Rik (GenBank Accession No. XM_129545) をマウス chondromedin遺伝子と略記する。  Hereinafter, 1110018N05Rik (GenBank Accession No. XM_129545) is abbreviated as mouse chondromedin gene.
マウス由来の間葉系幹細胞である C3H10T1/2細胞を BMP-2または BMP- 4で刺激し て、 in vitroで軟骨分化を誘導させ (Ahrens M, et al., DNA Cell Biol.  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.
12:871-80., 1993) 、 この過程におけるマウス chondromedin遺伝子の発現変動 を以下のように解析した。 12: 871-80., 1993), and the change in the expression of the mouse chondromedin gene in this process was analyzed as follows.
大日本製薬から購入した C3H10T1/2細胞 (Cancer Res., 33: 3239-3249, 1973) を BMP- 4存在下、 3、 6および 13日間培養した。 BMP- 4の添加前 （Day 0) 、 添加後 3日目 （Day 3) 、 6日目 （Day 6) および 13日目 (Day 13) に、 細胞を RLT 緩衝液 (Qiagen社） で溶解し、 QIAshredder (Qiagen社） 処理した後、 - 8(TC で保存した。 このサンプルを解凍し、 RNeasy mini kit (Qiagen社） を用いて 細胞由来 total RNAを調製した。 この際、 混在する微量のゲノム DNAを除去する ために、 カラム上で Dnase処理を行うオプションプロトコ一ルに従った。  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.
得られた total RMを铸型として定量的 RT-PCRを行い、 マウス chondromedin遺 伝子の発現量を調べた。 Genbank登録の塩基配列 XM— 129545を基に、 マウス chondromedin mRNAに対するプライマ一 （配列番号： 9および配列番号： 1 0) を設計して用いた。 試寒こは、 Quant i-Tect SYBR green RT-PCR kit (Qiagen社)を使用し、 7700 sequence detection system (Applied Biosystems 社） 上で RT- PCRおよびシグナル検出を行った。 始めに 50°C、 30分のインキュべ —シヨンを行い、 次いで 95°Cで 15分間の加熱を行った後、 °C- 15秒、 60°C-60 秒の増幅過程を 40サイクル行った。 " 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. Using a Quanti-Tect SYBR green RT-PCR kit (Qiagen), 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. "
次に、 マウス ]3- actin遺伝子の発現量を調べた。 Genbank登録の塩基配列を基 に、 マウス /3- aciin mRNAに対するプライマー （配列番号： 1 1および配列番 号： 12) および TaqManプローブ （配列番号： 1 3) を設計して用いた。 第一 段階では、 SensiScript reverse transpciptrase (Qiagen社) を用いて、 刖述 の C3H10T1/2細胞の total RNAから cDNAを合成した。 第二段階ではこの cDNAを铸 型とし、 試薬に TaqMan Universal PCR Master Mix (Applied Biosystems社），を 使用し、 7700 sequence detection system (Applied Biosystems社） 上で PCR およびシグナル検出を行った。 始めに 95°Cで 10分間の加熱を行った後、 94°C - 15 秒、 60°C-60秒の増幅過程を 40サイクル行った。  Next, the expression level of the mouse] 3-actin gene was examined. Based on the nucleotide sequence registered in Genbank, primers (SEQ ID NO: 11 and SEQ ID NO: 12) for mouse / 3-aciin mRNA and a TaqMan probe (SEQ ID NO: 13) were designed and used. In the first step, cDNA was synthesized from the above-described total RNA of C3H10T1 / 2 cells using SensiScript reverse transpciptrase (Qiagen). In the second step, 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.
Day 0、 Day 3、 Day 6 および Day 13のサンプルのそれぞれにっき、 マウス chondromedin の定量的 PCRで得られた蛍光シグナル値を マウス j3 -act inについ て得られた蛍光シグナル値で標準化した。 さらに Day 3、 Day 6 および Day 13に おけるマウス chondromedinの標準化発現量を Day 0における標準化発現量で割り 込んで相対化した。  For each of the samples on Day 0, Day 3, Day 6 and Day 13, 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.
結果を図 1に示す。  The results are shown in Figure 1.
これより、 マウス chondromedin遺伝子は、 マウス C3H10T1/2細胞の軟骨分化に 伴い発現上昇することが示された。 ' 実施例 5  This indicates that the expression of the mouse chondromedin gene increases with cartilage differentiation of mouse C3H10T1 / 2 cells. '' Example 5
ヒト chondromedinの部分ペプチドに対するゥサギ抗血清の作製  Production of anti-Egret antiserum against partial peptide of human chondromedin
Antibodies A LABORATORY MANUAL (E. Harlow and D. Lane. , Cold Spring Harbor Lab. Press, 1988) に記載の方法などに従い、 2種の合成ペプチド Trl (配列番号： 14) および Tr2 (配列番号： 15) を Fmoc固相合成法を用いて合 成した。 これらのペプチドにキーホールリンペットへモシァニン （KLH) をキヤ リア一タンパク質として結合させた複合体を抗原として、 それぞれ別個にゥサ ギを免疫感作した。  According to the method described in Antibodies A LABORATORY MANUAL (E. Harlow and D. Lane., Cold Spring Harbor Lab. Press, 1988), two synthetic peptides Trl (SEQ ID NO: 14) and Tr2 (SEQ ID NO: 15) Was synthesized using the Fmoc solid phase synthesis method. The rabbits were individually immunized with a complex of these peptides and mosquisin (KLH) bound to a keyhole limpet as a carrier protein.
初回感作の直前 （Day 0) および 35日目 （Day 35) に部分採血を行った。 得ら れた血清の抗体価を、 枋原ペプチドを固相に用いた ELISAで測定した結果を図 2 (抗 Trl抗血清） および図 3 (抗 Tr2抗與清） に示す。 いずれの血清についても 抗体価の上昇が確認された。 最終的に感作後 56日目に全採血を行い、 抗 Trl抗血 清および抗 Tr 2抗血清を取得した。 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.
'  '
実施例 6 Example 6
COS- 7細胞を用いたヒト chondromedinタンパク質の一過性発現  Transient expression of human chondromedin protein using COS-7 cells
(1) 発現ベクターの構築  (1) Construction of expression vector
実施例 2で得られたプラスミド PTB2300を制限酵素 Spelで切断し、 得られた非 平滑末端を Pfu Turbo DNAポリメラ一ゼ （Stratagene社） を用いて平滑化した 後、 DNAリガ一 を用いて Xbalリンカ一 く宝酒造） を挿入した。  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.
得られたプラスミドを EcoRIおよび Ncolで切断し、 得られた非粘着末端間に 2 種アダプター C5' -pAATTCACCGC-3' (配列番号： 16) および 5' -pCATGGCGGTG- 3' (配列番号： 1 7) (5'- pは、 5'リン酸化を表す） 〕 を DNAリガーゼの反応に より挿入した。  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.
得られたプラスミドを Hindlllおよび Xbalで消化し、 ヒト chondromedin遺伝子 のオープンリーディングフレームを含む約 2.3 kb の cDNA断片を切り出した d この断片を PCDNA5/T0 (Invitrogen社） の Hindi IIおよび Xbal 部位の間に挿入 してヒト chondromedin発現プラスミド pcDNA5/ TO-hCdmを得た。 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.
(2) COS- 7 細胞へのトランスフエクシヨン  (2) Transfection to COS-7 cells
COS- 7細胞を培養プレートに播種し、 約 24時間後に、 pcDNA5/T0 (Invitrogen 社） または上記で得られた pcDNA5/T(HiCdm を、 Lipofectamine2000試薬  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.
(Invitrogen) を用いてトランスフエクシヨンした。 3日後に細胞を PBSで洗浄 し、 スクレイパーを用いて培養プレートから剥がし取った後、 - 遠心回収し- 80°C に保存した。  (Invitrogen). Three days later, the cells were washed with PBS, peeled off from the culture plate using a scraper,-collected by centrifugation, and stored at -80 ° C.
(3) 抗ペプチド抗体によるヒト chondromedinタンパク質の検出  (3) Detection of human chondromedin protein by anti-peptide antibody
上記保存した細胞を、 氷上で解凍した後、 Laemliのサンプルバッファー Thaw the stored cells on ice, then use Laemli sample buffer
(Laemrali, U. K. (1970) Nature 227,680-685) を添加、 95°Cで 5分加熱して、 細胞総タンパク質を可溶化した。 このサンプルを SDS- PAGEで.展開した後、 ニト ロセルロースフィルタ一 （バイオ。ラド社） にタンパク質を転写、 実施例 5で '得 έ>れた抗 Trl抗血清を一次抗体に、 抗'ゥサギ IgG-アルカリ性ホスファターゼ複 合体（Invi trogen社） を二次抗体に用いて、 ウエスタンプロットを行った。 その 結果、 ヒト chondromedinを導入した C0S-7細胞でのみバンドが検出された。 (Laemrali, UK (1970) Nature 227,680-685) and heated at 95 ° C. for 5 minutes to solubilize total cellular proteins. After developing this sample by SDS-PAGE, The protein was transferred to a rocellulose filter (Bio-Rad), and the anti-Trl antiserum obtained in Example 5 was used as the primary antibody, and the anti-Egret IgG-alkaline phosphatase conjugate (Invitrogen) was used as the primary antibody. Western plot was performed using the secondary antibody. As a result, a band was detected only in C0S-7 cells into which human chondromedin was introduced.
以上の操作は、 Ant ibodies A LABORATORY MANUAL (E. Har low and D. Lane. , Cold Spr ing Harbor Lab. Press, 1988) に記載の方法、 用いた試薬や材料に添 付のプロトコールに従った。  The above operation was performed according to the method described in Antibodies A LABORATORY MANUAL (E. Harlow and D. Lane., Cold Spring Harbor Lab. Press, 1988), and the protocol attached to the reagents and materials used.
これより、 钪 Trl抗血清またはそれに由来する免疫 IgGは、 ヒト chondromedin の上昇を伴う疾患、、 例えば変形性関節症などの診断、 予防 '治療に用いること が /ti来ることがわかる。 またヒト chondromedin発現ベクターは、 ヒト  From this, it can be seen that 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
chondromedinタンパク質の生産に用いることができる。 産業上の利用可能性  It can be used to produce chondromedin protein. Industrial applicability
本発明のタンパク質または D NAは、 骨'関節炎の診断マ一力一として使用 できる。  The protein or DNA of the present invention can be used as a diagnostic tool for osteoarthritis.
本発明のタンパク質の活性を調節 （好ましくは阻害） する化合物またはその 塩、 該タンパク質の遺伝子の発現を調節 （好ましくは阻害） する化合物または その塩、 該タンパク質に対する中和抗体、 本発明のアンチセンスポリヌクレオ チドなどは、 例えば、 軟骨形成異常、 骨形成異常、 骨粗鬆症、 変形性関節症、 慢性関節リウマチ、 関節炎、 滑膜炎、 代謝性関節症またはスポーツによる関節 障害 （例、 テニス肘等） などの骨。関節炎の予防'治療剤として使用すること ができる。  Compounds or salts thereof that regulate (preferably inhibit) the activity of the protein of the present invention, compounds or salts thereof that regulate (preferably inhibit) the expression of the gene of the protein, neutralizing antibodies against the protein, antisense of the present invention 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.

Claims

請求 の 範囲  The scope of the claims
I . 配列番号： 1で表されるアミノ酸配列と同一もしくは実質的に同一のァ ミノ酸配列を含有するタンパク質またはその塩。 I. A protein containing an amino acid sequence identical or substantially identical to the amino acid sequence represented by SEQ ID NO: 1, or a salt thereof.
2 . 配列番号： 1で表されるアミノ酸配列からなるタンパク質またはその塩。2. A protein comprising an amino acid sequence represented by SEQ ID NO: 1 or a salt thereof.
3 . 配列番号： 3で表されるアミノ酸配列からなるタンパク質またはその塩。3. A protein comprising an amino acid sequence represented by SEQ ID NO: 3 or a salt thereof.
4. 請求項 1記載のタンパク質の部分べプチドまたはその塩。 4. A partial peptide of the protein according to claim 1, or a salt thereof.
5 . 請求項 1記載のタンパク質または請求項 4記載の部分べプチドをコード するポリヌクレオチドを含有するポリヌクレオチド。  5. A polynucleotide comprising a polynucleotide encoding the protein according to claim 1 or the partial peptide according to claim 4.
6 . D NAである請求項 5記載のポリヌクレオチド。 6. The polynucleotide according to claim 5, which is DNA.
7。 配列番号： 2または配列番号： で表される塩基配列からなるポリヌク レオチド。 1 7. A polynucleotide consisting of the nucleotide sequence represented by SEQ ID NO: 2 or SEQ ID NO: One
8 . 請求項 5記載のポリヌクレオチドを含有する組換えベクター。  8. A recombinant vector containing the polynucleotide according to claim 5.
9 . 請求項 8記載の組換えベクターで形質転換された形質転換体。  9. A transformant transformed with the recombinant vector according to claim 8.
1 0 . 請求項 9記載の形質転換体を培養し、 請求項 1記載のタンパク質また は請求項 4記載の部分ペプチドを生成、 蓄積せしめ、 これを採取することを特 徴とする請求項 1記載の夕ンパク質もしくは請求項 4記載の部分べプチドまた はその塩の製造法。 10. The method according to claim 1, wherein the transformant according to claim 9 is cultured to produce and accumulate the protein according to claim 1 or the partial peptide according to claim 4, and collect this. A method for producing the protein of claim 1 or the partial peptide or the salt thereof according to claim 4.
I I . 請求項 1記載のタンパク質もしくは請求項 4記載の部分ペプチドまた はその塩を含有してなる医薬。  II. A medicament comprising the protein according to claim 1 or the partial peptide according to claim 4 or a salt thereof.
1 2 . 請求項 5記載のポリヌクレオチドを含有してなる医薬。 ' 12. A pharmaceutical comprising the polynucleotide according to claim 5. '
1 3 . 請求項 5記載のポリヌクレオチドを含有してなる診断薬。 13. A diagnostic agent comprising the polynucleotide according to claim 5.
1 4. 請求項 1記載の夕ンパク質もしくは請求項 4記載の部分べプチドまた はその塩に対する抗体。  1 4. An antibody against the protein according to claim 1 or the partial peptide or a salt thereof according to claim 4.
1 5 . 請求項 1 4記載の抗体を含有してなる診断薬。 15. A diagnostic agent comprising the antibody according to claim 14.
1 6。 請求項 1 4記載の抗体を含有してなる医薬。  1 6. A medicament comprising the antibody according to claim 14.
1 7。 請求項 5記載のポリヌクレオチドの塩基配列に相補的もしくは実質的 に相補的な塩基配列またはその一部を含有するポリヌクレオチド。  1 7. A polynucleotide comprising a base sequence complementary to or substantially complementary to the base sequence of the polynucleotide according to claim 5, or a part thereof.
1 8。 請求項 1 7記載のポリヌクレオチドを含有してなる医薬。 1 8. A medicament comprising the polynucleotide according to claim 17.
1 9 . 請求項 1記載のタンパク質もしくはその部分ペプチドまたはその塩を 用いることを特徴とする、 請求項 1記載の夕ンパク質の活性を調節する化合物 またはその塩のスクリーニング方法。 19. The method for screening a compound or a salt thereof that regulates the activity of protein according to claim 1, wherein the protein or a partial peptide thereof or a salt thereof according to claim 1 is used.
2 0 . 請求項 1記載のタンパク質もしくはその部分ペプチドまたはその塩を 含有してなる、 請求項 1記載のタンパク質の活性を調節する化合物またはその 塩のスクリーニング用キット。  20. A screening kit for a compound or a salt thereof that regulates the activity of the protein according to claim 1, comprising the protein according to claim 1 or a partial peptide thereof or a salt thereof.
2 1 . 請求項 5記載のポリヌクレオチド ¾用いることを特徴とする、 請求項 1記載のタンパク質遺伝子の発現を調節する化合物またはその塩のスクリ一二 ング方法。  21. A method for screening a compound or a salt thereof that regulates expression of the protein gene according to claim 1, wherein the polynucleotide according to claim 5 is used.
2 2 . 請求項 5記載のポリヌクレオチドを含有してなる、 請求項 1記載の夕 ンパク質遺伝子の発現を調節する化合物またはその塩のスクリ ニング用キッ 卜。  22. A screening kit for a compound or a salt thereof, which regulates expression of the protein gene according to claim 1, comprising the polynucleotide according to claim 5.
2 3 . 請求項 1 4記載の抗体を用いることを特徴とする請求項 1記載のタン パク質の定量方法。  23. The method for quantifying a protein according to claim 1, wherein the antibody according to claim 14 is used.
2 4. 骨 ·関節疾患の予防 ·治療剤である請求項 1 2または請求項 1 6記載 の医薬。  24. The medicament according to claim 12 or claim 16, which is a preventive or therapeutic agent for bone / joint disease.
2 5 . 骨 ·関節疾患が、 軟骨形成異常、 骨形成異常、 骨粗鬆症、 変形性関節 症、 慢性関節リウマチ、 関節炎、 滑膜炎、 代謝性関節症またはスポーツによる 関節障害である請求項 2 4記載の医薬。  25. The method according to claim 24, wherein the bone and joint diseases are cartilage dysplasia, bone dysplasia, osteoporosis, osteoarthritis, rheumatoid arthritis, arthritis, synovitis, metabolic arthropathy, or joint disorders due to sports. Medicine.
2 6 . 骨 ·関節疾患の診断薬である請求項 1 3または請求項 1 5記載の診断  26. The diagnosis according to claim 13 or claim 15, which is a diagnostic agent for bone and joint diseases.
2 7 . 骨 ·関節疾患が、 軟骨形成異常、 骨形成異常、 骨粗鬆症、 変形性関節 症、 慢性関節リウマチ、 関節炎、 滑膜炎、 代謝性関節症またはスポーツによる 関節障害である請求項 2 6記載の診断薬。 27. The method according to claim 26, wherein the bone and joint diseases are cartilage dysplasia, bone dysplasia, osteoporosis, osteoarthritis, rheumatoid arthritis, arthritis, synovitis, metabolic arthrosis, or joint disorders due to sports. Diagnostics.
2 8 . 配列番号： 8で表わされる塩基配列と同一または実質的に同一の塩基 配列を含有する D NA。  28. A DNA containing the same or substantially the same nucleotide sequence as the nucleotide sequence represented by SEQ ID NO: 8.
2 9 . 配列番号： 8で表わされる塩基配列と同一または実質的に同一の塩基 配列を含有する D N Aの転写調節領域である請求項 2 8記載の D N A。  29. The DNA of claim 28, which is a DNA transcriptional regulatory region containing the same or substantially the same nucleotide sequence as the nucleotide sequence represented by SEQ ID NO: 8.
3 0 . 転写調節領域がプロモータ一領域である請求項 2 9記載の D NA。 30. The DNA of claim 29, wherein the transcription regulatory region is a promoter region.
3 1 . 請求項 2 8記載の D NAを含有する組換えベクター。 31. A recombinant vector containing the DNA according to claim 28.
3 2 . 配列番号： 8で表わされる塩基配列と同一または実質的に同一の塩基 配列を含有する D NAの転写調節領域の下流にレポ一夕一遺伝子を有する D N Aを含有する請求項 3 1記載の組換えべクタ一。  32. The method according to claim 31, which comprises a DNA having a repo allele downstream of a transcriptional regulatory region of a DNA containing the same or substantially the same nucleotide sequence as the nucleotide sequence represented by SEQ ID NO: 8. Recombination vector.
5 3 3 . 請求項 3 2記載の組換えベクターを含有する形質転換体。  53 3. A transformant containing the recombinant vector according to claim 32.
3 4. 請求項 2 8記載の D NAを用いることを特徴とする、 配列番号： 2で 表わされる塩基配列と同一または実質的に同一の塩基配列を含有する D N Aの 転写調節活性を調節する化合物またはその塩のスクリーニング方法。  3 4. A compound that regulates the transcriptional regulatory activity of a DNA containing the same or substantially the same nucleotide sequence as the nucleotide sequence represented by SEQ ID NO: 2, wherein the DNA according to claim 28 is used. Or a method of screening for a salt thereof.
' 3 5 . 請求項 3 3記載の形質転換体を用いる請求項 3 4記載のスクリ一ニン 35. The scrinin according to claim 34, wherein the transformant according to claim 33 is used.
10 グ方法。 10 way.
3 6 . 哺乳動物に対して、 配列番号： 1で表されるアミノ酸配列と同一もし くは実質的に同一のアミノ酸配列を含有するタンパク質またはその塩の活性を 阻害する化合物もしくはその塩、 または該タンパク質遺伝子の発現を阻害する 化合物もしくはその塩の有効量を投与することを特徴とする、 骨 ·関節疾患の 15 予防 ·治療法。  36. A compound or a salt thereof that inhibits the activity of a protein or a salt thereof containing the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 1, 15. A method for preventing and treating bone and joint diseases, comprising administering an effective amount of a compound or a salt thereof that inhibits expression of a protein gene.
3 7 . 配列番号： 1で表されるアミノ酸配列と同一もしくは実質的に同一の ァミノ酸配列を含有するタンパク質またはその塩の活性を阻害する、 または該 . タンパク質遺伝子の発現を阻害することを特徴とする、 骨 ·関節疾患の予防 · 治療法。  37. It inhibits the activity of a protein containing an amino acid sequence identical or substantially identical to the amino acid sequence represented by SEQ ID NO: 1 or a salt thereof, or inhibits the expression of the protein gene. To prevent and treat bone and joint diseases.
20 3 8 . 骨 ·関節疾患の予防 ·治療剤を製造するための、 配列番号： 1で表さ れるァミノ酸配列と同一もしくは実質的に同一のァミノ酸配列を含有する夕ン パク質またはその塩の活性を阻害する化合物もしくはその塩、 または該タンパ ク質遺伝子の発現を阻害する化合物もしくはその塩の使用。  20 3 8. A protein containing an amino acid sequence identical or substantially identical to the amino acid sequence represented by SEQ ID NO: 1 or a protein thereof for producing a preventive or therapeutic agent for bone or joint disease Use of a compound or a salt thereof that inhibits the activity of a salt, or a compound or a salt thereof that inhibits the expression of the protein gene.
PCT/JP2003/013919 2002-10-30 2003-10-30 Novel protein and dna thereof WO2004039974A1 (en)

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Citations (2)

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