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WO2001048203A1 - Nouvelle proteine et adn associe - Google Patents

Nouvelle proteine et adn associe Download PDF

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Publication number
WO2001048203A1
WO2001048203A1 PCT/JP2000/009236 JP0009236W WO0148203A1 WO 2001048203 A1 WO2001048203 A1 WO 2001048203A1 JP 0009236 W JP0009236 W JP 0009236W WO 0148203 A1 WO0148203 A1 WO 0148203A1
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WIPO (PCT)
Prior art keywords
protein
dna
present
salt
partial peptide
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Ceased
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PCT/JP2000/009236
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English (en)
Japanese (ja)
Inventor
Yasuaki Itoh
Kazunori Nishi
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Takeda Pharmaceutical Co Ltd
Original Assignee
Takeda Chemical Industries Ltd
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Filing date
Publication date
Application filed by Takeda Chemical Industries Ltd filed Critical Takeda Chemical Industries Ltd
Priority to AU22254/01A priority Critical patent/AU2225401A/en
Publication of WO2001048203A1 publication Critical patent/WO2001048203A1/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

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Classifications

    • 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
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P19/00—Drugs for skeletal disorders
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P19/00—Drugs for skeletal disorders
    • A61P19/02—Drugs for skeletal disorders for joint disorders, e.g. arthritis, arthrosis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • the present invention relates to a novel cell regulatory factor protein.
  • cytokines include lymphokines, monokinin, interferon, colony stimulating factor, tumor necrosis factor, etc.
  • ChM-1I chondromodulin-1 I
  • ChM-I is a glycoprotein of approximately 25 kDa purified from fetal cartilage in 1991 as a factor that modulates chondrocyte proliferation and differentiation.
  • Its precursor cDNA encodes a protein consisting of 335 amino acids, and has a transmembrane region near its N-terminus.
  • ChM_I precursor is first synthesized as a sugar chain-bound membrane protein.
  • ChM-I ChM-I encoded at the C-terminal end are produced [Biochem. Biophys. Res. Commun., Vol. 175, p. 971-977 (1991)].
  • Homologous genes of ChM-I have also been cloned from various mammals other than Escherichia coli, and human ChM-I has also been cloned [Eur. J. Biochem., Vol. 260, p.869- 878 (1999)].
  • ChM-I was originally obtained from soft fetal cartilage by using the synergistic DNA synthesis-promoting activity of basic fibroblast growth factor (bFGF) on chondrocytes.
  • bFGF basic fibroblast growth factor
  • One of the functional matrix proteins in bone it was later found to be an anti-angiogenic factor that inhibits the proliferation and lumen formation of vascular endothelial cells in cartilage [The Journal of Op. 'Biological' Chemistry (The Journal of Biological Chemistry) 272, 32419-32426 (1997)].
  • bFGF basic fibroblast growth factor
  • ChM-I has its gene discovered across species as described above, but it has a so-called so-called gene, which is found in other cytokines such as the Tumor Necrosis Factor (TNF) family.
  • TNF Tumor Necrosis Factor
  • the existence of other genes that form a structural family was not known.
  • other local factors, such as ChM-I which is expressed in a chondrocyte-specific manner and controls the proliferation and differentiation of the chondrocytes, exist in the living body is not always denied.
  • the unknown factors having the unique basic structure play a more important role in physiological functions and, in turn, regulate the development, differentiation, proliferation and maintenance of homeostasis.
  • endogenous factors are likely to provide completely new drugs themselves, which have a different mechanism of action from hitherto known, or target factors, and therefore fall into this category.
  • a novel cell regulator protein to be included was desired.
  • the present invention relates to a novel protein usable in biology, medicine, veterinary medicine, etc., a partial peptide thereof, or a salt thereof, a recombinant vector, a transformant, a method for producing the protein, the protein or a partial peptide.
  • An object of the present invention is to provide a drug containing the same, and an antibody against the protein or the like. Disclosure of the invention
  • the present inventors have conducted intensive studies to solve the above-mentioned problems, and as a result, Succeeded in discovering a cDNA having a novel nucleotide sequence that is frequently expressed in infants, etc., and discovered that the protein encoded by it was a new cell regulatory factor protein.
  • the protein according to (1) or the partial peptide according to (2) or a salt thereof which can be obtained using the screening method according to (12) or the screening kit according to (13).
  • a pharmaceutical comprising a compound or a salt thereof that promotes or inhibits the activity of
  • a pharmaceutical comprising the protein or a salt thereof according to the above (1) or the partial peptide or a salt thereof according to the above (2);
  • An amino acid sequence substantially identical to the amino acid sequence represented by SEQ ID NO: 1 is about 50% or more (preferably about 50% or more) of the amino acid sequence represented by SEQ ID NO: 1.
  • the amino acid sequence substantially identical to the amino acid sequence represented by SEQ ID NO: 1 is: (1) one or more amino acid sequences in the amino acid sequence represented by SEQ ID NO: 1 (preferably 1 to 30 Amino acid sequence in which about 1) amino acids have been deleted, 2) an amino acid sequence in which one or more (preferably about 1 to 30) amino acids have been added to the amino acid sequence represented by SEQ ID NO. An amino acid sequence in which one or more (preferably about 1 to 30) amino acids in the amino acid sequence represented by No. 1 have been substituted with another amino acid, or amino acids obtained by combining them.
  • the present invention can be used not only for basic research such as molecular weight markers, tissue markers, chromosome mapping, genetic disease identification, primers, probe design, etc., but also for cancer metastasis inhibition, cancer metastasis detection, For the treatment or prevention of various diseases in the fields of regulation of differentiation and proliferation of cells, induction of cytotoxicity, regulation of hematopoiesis, regulation of blood coagulation, infectious disease, metabolic regulation, wound burn healing, anti-inflammatory, fetal growth deficiency, gene therapy, etc.
  • FIG. 1 shows the nucleotide sequence of DNA encoding the protein of the present invention obtained in Example 1 and the amino acid sequence deduced from the nucleotide sequence.
  • the figure shows the insertion sequence side of the EcoRI recognition site of Vector-1 DNA.
  • FIG. 2 shows the homology between the human NCHM protein and various known chondromodulin I pre- and carcinoma proteins.
  • Nchm.pro indicates human NC HM protein, CHM1—HUMAN, pro produces human chondromodulin I precursor protein,
  • CHM1_B0VIN.pro contains ⁇ sicndromodulin I precursor protein
  • CHM1_RABBIT.pro indicates the Pseudochondromodulin I precursor protein
  • CHM1-RAT.pro indicates the rat chondromodulin I precursor protein (continued in FIG. 3).
  • FIG. 3 shows the homology between human NCHM protein and various known chondromodulin I precursor proteins (continuation of FIG. 2).
  • Proteins having the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 1 of the present invention include humans including fetuses and warm-blooded animals.
  • cells eg, moon-dried cells, spleen cells, nerve cells, glial cells, porcine 3 cells, bone marrow cells, mesangial cells
  • Cells Langerhans cells, epidermal cells, epithelial cells, endothelial cells, endothelial cells, fibroblasts, fiber cells, muscle cells, fat cells, immune cells (eg, macrophages, ⁇ cells, ⁇ cells, natural killer cells, mast cells, Neutrophils, basophils, eosinophils, monocytes, dendritic cells), megakaryocytes, synovial cells,
  • amino acid sequence substantially identical to the amino acid sequence represented by SEQ ID NO: 1 is at least about 50%, preferably at least about 60%, more preferably the amino acid sequence represented by SEQ ID NO: 1. About 70% or more, more preferably about 80% or more, particularly preferably about 90% or more, and most preferably about 95% or more homology. Amino acid sequence and the like.
  • Examples of the protein having an amino acid sequence substantially identical to the amino acid sequence represented by SEQ ID NO: 1 of the present invention include, for example, a protein having an amino acid sequence substantially identical to the amino acid sequence represented by the aforementioned SEQ ID NO: 1 Preferred is a protein having substantially the same properties as the protein having the amino acid sequence represented by SEQ ID NO: 1.
  • the properties of substantially the same quality include, for example, an angiogenesis inhibitory action.
  • substantially the same means that those properties are qualitatively the same. Therefore, properties such as angiogenesis inhibitory action are equivalent (eg, about 0.1 to 100 times, preferably about 0.5 to 10 times, more preferably 0.5 to 2 times).
  • properties such as angiogenesis inhibitory action are equivalent (eg, about 0.1 to 100 times, preferably about 0.5 to 10 times, more preferably 0.5 to 2 times).
  • quantitative factors such as the degree of these properties and the molecular weight of the protein may be different.
  • Examples of the protein of the present invention include: (1) one or two or more amino acids in the amino acid sequence represented by SEQ ID NO: 1 (preferably, about 1 to 30, preferably about 1 to 10, more preferably Is an amino acid sequence in which several (1 to 5) amino acids have been deleted, (2) 1 or more (preferably about 1 to 30 and preferably 1 to 1) amino acid sequences represented by SEQ ID NO: 1 An amino acid sequence to which about 0, more preferably a number (1 to 5) amino acids have been added; 3 1 or 2 or more amino acids (preferably 1 to 30 amino acids) to the amino acid sequence represented by SEQ ID NO: 1 The degree is preferably about 1 to 10, more preferably about 1 to 5 amino acids, or 1 or 2 in the amino acid sequence represented by SEQ ID NO: 1.
  • mutin such as a protein containing an amino acid sequence in which about 10 amino acids are substituted, and more preferably (1 to 5) amino acids, or an amino acid sequence combining them is also included.
  • the position of the insertion, deletion or substitution is not particularly limited.
  • the protein has a N-terminus at the left end (amino terminus) and a C-terminus at the right end (carboxyl terminus) according to the convention of peptide notation.
  • the proteins of the present invention including the protein containing the amino acid sequence represented by SEQ ID NO: 1,
  • the protein has a C-terminus, usually a hydroxyl group (one COOH) or a carboxylate (one C ⁇ —), but a C-terminus is an amide (—C ⁇ NH 2 ) or an ester (one COOR). You may.
  • R in the ester e.g., methyl, Echiru, n- propyl Le, isopropyl or n- heptyl etc.
  • C 3 _ 8 cycloalkyl such as cyclohexyl group, for example, Fe Le
  • C 6 1 2 Ariru group such ⁇ - naphthyl, for example, benzyl, phenethyl of which phenyl one -. 2 alkyl or ⁇ - naphthylmethyl etc.
  • ⁇ - naphthoquinone Chiru C i _ 2 alkyl groups In addition to C 7 -i 4 aralkyl groups, piva yloxymethyl group, which is widely used as an oral ester, is used.
  • the protein of the present invention When the protein of the present invention has a lipoxyl group (or carboxylate) other than the C-terminus, the protein of the present invention includes a lipoxyl group amidated or esterified.
  • the ester in this case, for example, the above-mentioned C-terminal ester and the like are used.
  • amino acid residues e.g., Mechionin residues
  • N-terminal Amino group protecting groups e.g., formyl group, such as C Bok 6 ⁇ Rukanoiru such Asechiru group - 6 Ashiru group such as
  • substituents on the side chains of amino acids in the molecule eg, 1 OH, 1 SH
  • an appropriate protecting group for example, a C 6 acyl group such as an alkanoyl group such as a formyl group or an acetyl group
  • It also includes complex proteins such as so-called glycoproteins to which sugar chains are bonded.
  • protein of the present invention include, for example, a human-derived protein having an amino acid sequence represented by SEQ ID NO: 1.
  • the partial peptide of the protein of the present invention (hereinafter, sometimes referred to as the partial peptide of the present invention) is a partial peptide of the protein of the present invention described above, and preferably has the same properties as the protein of the present invention described above. Any material may be used as long as it has. For example, at least 5 or more, preferably 20 or more, more preferably 30 or more of the constituent amino acid sequences of the protein of the present invention, More preferably, a peptide having an amino acid sequence of 50 or more, most preferably 80 or more is used.
  • these peptides for example, they have the same or substantially the same amino acid sequence as the 49th to 31st amino acid sequence (SEQ ID NO: 2) of the amino acid sequence represented by SEQ ID NO: 1, and Peptides having substantially the same properties as the above proteins are preferred. More preferably, it has the same or substantially the same amino acid sequence as the amino acid sequence at positions 215 to 317 of the amino acid sequence represented by SEQ ID NO: 1 (SEQ ID NO: 3). And a peptide having substantially the same properties as the above-mentioned protein.
  • 1st to 28th, 2nd 6th to 95th, 3rd to 6th, 1st to 7th of the amino acid sequence represented by SEQ ID NO: 1 Has the same or substantially the same amino acid sequence as the amino acid sequence of the 1st, 5 1st to 744th, or 6 1st to 765th, and has substantially the same properties as the protein of the present invention.
  • Peptides are also examples.
  • substantially the same property has the same meaning as described above.
  • one or more (preferably about 1 to 10, more preferably a number of (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; Alternatively, one or more (preferably, about 1 to 20, more preferably, about 1 to 10, and more preferably, about 1 to 5) amino acids are inserted into 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 May be.
  • the C-terminus is usually a hydroxyl group (one CO OH) or a carpoxylate (one CO O_), but the C-terminus is an amide (—C ON H 2 ) or an ester (one COOR) It may be.
  • the partial peptide of the present invention has a lipoxyl group (or carboxylate) other than at the C-terminus, those in which the lipoxyl group is amidated or esterified are also included in the partial peptide of the present invention. It is.
  • the ester in this case, for example, C A terminal ester or the like is used.
  • the partial peptides of the present invention include those in which the amino group of the N-terminal amino acid residue (eg, methionine residue) is protected by a protecting group, and those formed by cleavage of the N-terminal side in vivo. Also included are those in which the amino acid residue has been oxidized by pyrodaltamine, those in which the substituents on the side chains of the amino acids in the molecule are protected with appropriate protecting groups, and those in which a sugar chain is bonded, such as a so-called glycopeptide. .
  • the partial peptide of the present invention can be used as an antigen for producing an antibody, it is not necessary to have the activity of the protein of the present invention.
  • 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, partial peptide or salt thereof of the present invention can be produced from the above-mentioned human warm-blooded animal cells or tissues by a known method for purifying a protein, or contains DNA encoding a protein described below. It can also be produced by culturing the transformant obtained. It can also be produced according to the peptide synthesis method described below.
  • the human or mammalian tissues or cells are homogenized, extracted with an acid or the like, and the extract is subjected to reversed-phase mouth chromatography, ion-exchange chromatography, etc. Purification and isolation can be performed by combining the above chromatography.
  • a commercially available resin for protein synthesis can be usually used.
  • a resin include chloromethyl resin, hydroxymethyl resin, benzhydrylamine resin, aminomethyl resin, 4-benzyloxybenzyl alcohol resin, 4-methylbenzhydrylamine resin, and PAM resin.
  • 4-hydroxymethylmethylphenylacetamidomethyl resin polyacrylamide Resin, 4- (2 ', 4'-dimethoxyphenyl-hydroxymethyl) phenoxy resin,-(2', 4'-dimethoxyphenyl-Fmocaminoethyl) phenoxy resin, and the like.
  • an amino acid having an ⁇ -amino group and a side chain functional group appropriately protected is condensed on the resin according to the various known condensation methods in accordance with the amino acid sequence of the target protein or peptide.
  • the protein is cleaved from the resin, and at the same time, various protecting groups are removed.
  • an intramolecular disulfide bond formation reaction is performed in a highly diluted solution to obtain the target protein or partial peptide or an amide thereof.
  • various activating reagents that can be used for protein synthesis can be used, and carbodiimides are particularly preferable.
  • the protected amino acid may be added directly to the resin along with a racemization inhibitor (eg, HOBt, HOOBt), or may be pre-formed as a symmetrical acid anhydride or HOBt ester or H ⁇ OBt ester. It can be added to the resin after activation of the protected amino acid.
  • a racemization inhibitor eg, HOBt, HOOBt
  • the solvent used for activating the protected amino acid or condensing with the resin may be appropriately selected from solvents known to be usable for the protein condensation reaction.
  • acid amides such as N, N-dimethylformamide, N, N-dimethylacetamide, N-methylpyrrolidone, halogenated hydrocarbons such as methylene chloride, chloroform, trifluoroethanol, etc.
  • Alcohols, sulfoxides such as dimethyl sulfoxide, ethers such as pyridine, dioxane, and tetrahydrofuran; nitriles such as acetonitrile and propionitrile; esters such as methyl acetate and ethyl acetate; or an appropriate mixture thereof.
  • Etc is an appropriate mixture thereof.
  • the reaction temperature is appropriately selected from a range known to be usable for a 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, C 1 _Z, Br—Z, and adaman Tyloxycarbonyl, trifluoroacetyl, phthaloyl, formyl, 2-ditrophenylsulfenyl, diphenylphosphinothioyl, Fmoc and the like are used.
  • the carboxyl group may be, for example, alkyl esterified (eg, methyl, ethyl, propyl, butyl, t-butyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, 2-adamantyl, etc.).
  • alkyl esterified eg, methyl, ethyl, propyl, butyl, t-butyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, 2-adamantyl, etc.
  • Alkyl esterification Alkyl esterification
  • aralkyl esterification for example, benzyl ester, 4-122 benzyl ester, 4-methoxybenzyl ester, 4-chloro benzyl ester, benzhydryl esterification
  • phenacyl esterification benzyl ester It can be protected by ziroxycarponyl hydrazide, t-butoxycarbonyl hydrazide, trityl hydrazide, or the like.
  • 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 ⁇ ) alkanol group such as an acetyl group, an aroyl group such as a benzoyl group, a benzyloxycarbonyl group, and an ethoxycarbonyl group can be used.
  • Examples of a group suitable for etherification include a benzyl group, a tetrahydropyranyl group, and a t-butyl group.
  • the protecting group of the phenolic hydroxyl group of tyrosine for example, B z and C l 2 - Bz l, 2- nitrobenzyl, B r- Z, such as t one-butyl is used.
  • As the protecting group for imidazole of histidine for example, Tos, 4-methoxy2,3,6-trimethylbenzenesulfonyl, DNP, benzyloxymethyl, Bum, Boc, Trt, Fmoc and the like are used.
  • Examples of the activated carbonyl group of the starting material include, for example, corresponding acid anhydrides, azides, active esters [alcohols (eg, phenol, 2,4,5-trichlorophenol, 2, 4-dinitrophenol, cyanomethyl alcohol, paranitrophenol, H ⁇ NB, N-hydroxysuccinimide , N-hydroxyfurimide, esters with H ⁇ B t)].
  • active esters eg, phenol, 2,4,5-trichlorophenol, 2, 4-dinitrophenol, cyanomethyl alcohol, paranitrophenol, H ⁇ NB, 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) the protecting group include, for example, catalytic reduction in a hydrogen stream in the presence of a catalyst such as Pd-black or Pd-carbon, or hydrogen fluoride anhydride or methanesulfonic acid.
  • a catalyst such as Pd-black or Pd-carbon, or hydrogen fluoride anhydride or methanesulfonic acid.
  • Acid treatment with trifluoromethanesulfonic acid, trifluoroacetic acid, or a mixture thereof; base treatment with diisopropylethylamine, triethylamine, piperidine, piperazine, etc .; or in liquid ammonia Reduction with sodium is also used.
  • the elimination reaction by the above acid treatment is generally carried out at a temperature of about ⁇ 20 ° C. to 40 ° C.
  • anisol for example, anisol, phenol, thioanisole, metacresol, paracresol
  • a cation scavenger such as dimethyl sulfide, 1,4-butanedithiol, 1,2-ethanedithiol and the like.
  • the 2,4-dinitrophenyl group used as an imidazole protecting group for histidine is removed by thiophenol treatment, and the formyl group used as an indole protecting group for tryptophan is replaced with the above 1, 2
  • the formyl group used as an indole protecting group for tryptophan is replaced with the above 1, 2
  • 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 materials, the protection group, the elimination of the protective group, the activation of the functional group involved in the reaction, and the like can be appropriately selected from known groups or known means.
  • an amide form of a protein for example, first, after amidating and protecting the 0! -Hydroxyl group of the carpoxy terminal amino acid, a peptide (protein) chain is extended to a desired chain length on the amino group side. After that, a protein in which only the protecting group of the 0: -amino group at the N-terminal of the peptide chain was removed and a protein in which only the protecting group of the C-terminal lipoxyl group was removed were produced. Condensation is performed in a mixed solvent as described above. Details of the condensation reaction are the same as described above. After purifying the protected protein obtained by the condensation, all the protecting groups are removed by the above method to obtain the crude protein of interest. This crude protein is purified using various known purification methods, and the main fraction is lyophilized to obtain the desired protein. An amide of the desired protein can be obtained.
  • an ester of a protein for example, after condensing a carboxylic acid group of a carboxy terminal amino acid with a desired alcohol to form an amino acid ester, an ester of a desired protein is obtained in the same manner as an amide of a protein be able to.
  • the partial peptide of the present invention or a salt thereof 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 desired peptide can be produced by condensing a partial peptide or amino acid that can constitute the partial peptide of the present invention with the remaining portion, and if the product has a protective group, removing the protective group to produce the desired peptide. it can.
  • Known condensation methods and elimination of protecting groups include, for example, the methods described in the following 1 to 5.
  • the partial peptide of the present invention can be purified and isolated by a combination of ordinary purification methods such as solvent extraction, distillation, column chromatography, liquid chromatography, and recrystallization.
  • the partial peptide obtained by the above method is an educt, it can be converted into an appropriate salt by a known method or a method analogous thereto.
  • the free form or another salt can be converted by a method analogous thereto.
  • the DNA encoding the protein of the present invention may be any DNA containing the above-described nucleotide sequence encoding the protein of the present invention.
  • genomic DNA, cDNA derived from the cells and tissues described above, and synthetic DNA Either may be used.
  • the vector used for the library may be any of pacteriophage, plasmid, cosmid, phagemid and the like. Alternatively, it can also 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 cells and tissues described above.
  • RT-PCR method reverse transcriptase polymerase chain reaction
  • DNA encoding the protein of the present invention for example, a DNA containing the nucleotide sequence represented by SEQ ID NO: 4 or a DNA sequence comprising the nucleotide sequence represented by SEQ ID NO: 4 is hybridized under high stringent conditions. Any DNA may be used as long as it has a nucleotide sequence and encodes a protein having substantially the same properties as the protein of the present invention (eg, angiogenesis inhibitory action).
  • Examples of a DNA that can hybridize with the base sequence represented by SEQ ID NO: 4 under high stringency conditions include, for example, about 60% or more, preferably about 70% or more of the base sequence represented by SEQ ID NO: 4. More preferably, DNA containing a base sequence having about 80% or more homology 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. 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 carried out under high 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 6 ° C. The conditions at 5 ° C are shown.
  • DNA having the base sequence represented by SEQ ID NO: 4 and the like are used.
  • the DNA encoding the partial peptide of the present invention may be any DNA containing the above-described nucleotide sequence encoding the partial peptide of the present invention.
  • genomic DNA, the above-mentioned cell and tissue-derived cDNA, and synthetic D Any of NA may be used.
  • Examples of the DNA encoding the partial peptide of the present invention include: (1) DNA having the nucleotide sequence represented by SEQ ID NO: 5 or the base sequence represented by SEQ ID NO: 5 and highly stringent conditions. DNA having a partial base sequence of DNA having a base sequence that hybridizes below and encoding a protein having substantially the same properties as the protein of the present invention; (2) a base sequence represented by SEQ ID NO: 6 Has a nucleotide sequence that hybridizes under high stringent conditions with DNA having a sequence or the nucleotide sequence represented by SEQ ID NO: 6, and encodes a protein having substantially the same properties as the protein of the present invention. DNA having a partial base sequence of DNA or the like is used.
  • the DNA hybridizable with the nucleotide sequence represented by SEQ ID NO: 5 or SEQ ID NO: 6 has the same significance as described above. .
  • the DNA encoding the partial peptide having the amino acid sequence represented by SEQ ID NO: 2 includes, for example, the DNA having the base sequence represented by SEQ ID NO: 5, and the amino acid represented by SEQ ID NO: 3.
  • DNA having the base sequence represented by SEQ ID NO: 6 and the like are used as the DNA encoding the partial peptide having the sequence.
  • the DNA or the partial peptide of the present invention may be used as the DNA or the partial peptide.
  • amplification is performed by PCR using a synthetic DNA primer having a partial base sequence of the protein of the present invention, or the DNA incorporated into an appropriate vector is used for cloning of the protein of the present invention.
  • Selection can be performed by hybridization with a DNA fragment coding for a part or the entire region or a DNA fragment labeled with a synthetic DNA. Hybridization can be performed according to, for example, the method described in Molecular Cloning 2nd (J. Sambrook et al., Cold Spring Harbor Lab. Press, 1989). it can. When using a commercially available library, follow the method described in the attached instruction manual. So you can do it.
  • Conversion of the DNA base sequence can be performed using PCR or a known kit, for example, Mutan TM -Supper Express (Takara Shuzo Co., Ltd.), Mutan TM — K (Takara Shuzo Co., Ltd.), etc.
  • the method can be carried out according to a method known per se, such as the LA PCR method, the Gapped duplicate method, the Kunke 1 method, or a method analogous thereto.
  • the DNA encoding the cloned protein can be used as it is depending on the purpose, or can be used by digesting with a restriction enzyme or adding a linker if desired.
  • the DNA may have ATG as a translation initiation codon at the 5 'end, and TAA, TGA or TAG as a translation stop codon at the 3' end. These translation initiation codon and translation termination codon can also be added using a suitable synthetic DNA adapter.
  • the expression vector of 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 into a promoter in an appropriate expression vector. It can be manufactured by connecting it every night.
  • Escherichia coli-derived plasmids eg, pBR322, pBR325, pUC12, pUC13
  • Bacillus subtilis-derived plasmids eg, pUB110, pTP5, pC194
  • yeast-derived plasmids eg, , PSH19, pSH15
  • bacteriophage such as phage ⁇
  • animal viruses such as retrovirus, vaccinia virus, baculovirus, etc., pAl-11, pXT1, pRc / CMV, pRc / RSV, pc DNA I ZNeo or the like is 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.
  • SRa promoter when animal cells are used as hosts, SRa promoter, SV40 promoter, HIV / LTR promoter, CMV promoter, HSV-TK promoter and the like can be mentioned.
  • CMV cytomegalovirus
  • SRo CMV (cytomegalovirus) promoter, SRo!
  • the host is a bacterium belonging to the genus Escherichia, If the host is a Bacillus genus, such as trp promoter, lac promoter, recA promoter, ⁇ PL promoter, promoter, and so on. If the host is Bacillus, SP01 promoter, SP02 promoter, etc.
  • yeast such as a penP promoter, a PH5 promoter, a PGK promoter, a GAP promoter, an ADH promoter, and the like are preferable.
  • a polyhedrin promoter, a P10 promoter and the like are preferable.
  • the expression vector may contain, in addition to the above, an enhancer, a splicing signal, a polyA addition signal, a selection marker, and an SV40 replication origin (hereinafter sometimes abbreviated as SV40 ori), if desired.
  • the selection Ma one car, for example, dihydrofolate reductase (hereinafter sometimes abbreviated as dhfr) gene [methotrexate (MTX) resistance], ampicillin resistant gene (hereinafter sometimes abbreviated as Amp r), Neomai Shin-resistant gene (hereinafter sometimes abbreviated as Ne o r, G418 resistance).
  • dh fr gene is used as a selection marker using Chinese hamster cells deficient in the dh fr gene
  • the target gene can be selected using a thymidine-free medium.
  • a signal sequence suitable for the host is added to the N-terminal side of the protein of the present invention.
  • the PhoA * signal sequence and the Omp ⁇ signal sequence are used.
  • the human amylase signal sequence and the subtilisin signal sequence are used.
  • the host is a Bacillus genus
  • the human amylase signal sequence and the subtilisin signal sequence are used.
  • the insulin signal sequence, ⁇ -inulin ferron ⁇ signal sequence, antibody molecule ⁇ signal sequence Etc. can be used respectively.
  • a transformant can be produced using the vector containing the DNA encoding the protein of the present invention thus constructed.
  • 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 spp. include, for example, Bacillus subtilis MI 114 [Gene, 24, 255 (1983)], 207-21 [Journal of Biochemistry, Vol. 95. , 87 (1 984)].
  • yeast examples include, for example, Saccharomyces cerevisiae AH22, AH22R—, ⁇ 87-11A, DKD-5D, 20B—12, Schizosaccharomyces pombe NCYC 1913, NCYC2036, (Pichia pastor is) KM 71 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 roth moth (Spodoptera frugiperda cell; S f cell), MG 1 cell derived from the midgut of Tric hop 1 usiani, Tric hop High Five TM cells derived from lusiani eggs, cells derived from Mame strabrassicae, or cells derived from Estigme naacrea are used.
  • the virus is BmNPV
  • a silkworm-derived cell line Boombyx mori N cell; Bm N cell
  • Sf cells include, for example, Sf9 cells (ATCC CRL1711), Sf21 cells [Vaughn, J.L et al., In Vivo, 13, 213-217, (1977) 3 Are used.
  • insects for example, silkworm larvae are used [Maeda et al., Nature, Vol. 315, 592 (1985)].
  • animal cells examples include monkey cells COS-7 (COS 7), Vero, Chinese hamster cells CHO (hereinafter abbreviated as CHO cells), and dh fr Gene-deficient Chinese hamster cells CHO (hereinafter abbreviated as CH ⁇ (dhfr_) cells), mouse L cells, mouse AtT-20, mouse myeloid cells, rat GH3, human FL cells, etc. Used.
  • Bacillus spp. Can be transformed, for example, according to the method described in Molecular & General Genetics, Vol. 168, 111 (1979).
  • Transformation of insect cells or insects can be performed, for example, according to the method described in Bio / Technology, 6, 4755 (1988).
  • a liquid medium is suitable as a medium used for culturing, and a carbon source necessary for the growth of the transformant is contained therein.
  • the carbon source include glucose, dextrin, soluble starch, and sucrose.
  • examples of the nitrogen source include amptonium salts, nitrates, corn chip lica, peptone, casein, meat extract, and soybean meal.
  • the organic substance and the inorganic substance include calcium chloride, sodium dihydrogen phosphate, 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.
  • a culture medium for culturing the genus Escherichia for example, an M9 medium containing glucose and casamino acid [Miller, Journal, Journal of Molecular Gelatins] of Experiments in Molecular Genetics), 431-433, Cold Spring Harbor Laboratory, New York 1972].
  • an agent such as, for example, 3] 3-indolylacrylic acid can be added to make the promotion work efficiently.
  • 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 carried out at about 30 to 40 for about 6 to 24 hours, and if necessary, aeration and stirring can be applied.
  • the medium used is Grace's Insect Medium (Grace, TCC, Nature, 195, 788 (1962)).
  • a solution to which an additive such as a serum is appropriately added 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.
  • 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 [Science, 122 Volume, 501 (1952)], DMEM medium [Virology, 8,
  • the pH is about 6-8.
  • Culture is usually about 30 ° (: ⁇
  • the protein of the present invention can be produced in cells, cell membranes or extracellular cells of the transformant.
  • the protein of the present invention can be separated and purified from the culture by, for example, the following method.
  • the cells or cells When extracting the protein of the present invention from cultured cells or cells, after culturing, the cells or cells are collected by a known method, suspended in an appropriate buffer, and then subjected to ultrasound, lysozyme and Z or freezing. After the cells or cells are disrupted by thawing or the like, a method of obtaining a crude protein extract by centrifugation or filtration is appropriately used.
  • the buffer may contain a protein denaturant such as urea or guanidine hydrochloride, or a surfactant such as Triton X-100 TM.
  • the protein contained in the culture supernatant or extract obtained in this manner can be purified by appropriately combining known separation and purification methods.
  • known separation and purification methods include methods using solubility such as salting out and solvent precipitation, dialysis, ultrafiltration, gel filtration, and SDS-polyacrylamide gel electrophoresis, mainly for molecular weight analysis.
  • Method using difference method using charge difference such as ion exchange chromatography, method using specific affinity such as affinity chromatography, and hydrophobicity such as reversed phase high performance liquid chromatography.
  • a method using a difference, a method using an isoelectric point difference such as an isoelectric focusing method, and the like are used.
  • the protein thus obtained is obtained in a free form, a method known per se Alternatively, it can be converted to a salt by a method analogous thereto, and conversely, when it is obtained as a salt, it can be converted to a free form or another salt by a method known per se or a method analogous thereto.
  • the protein produced by the recombinant can be arbitrarily modified or the polypeptide can be partially removed by applying an appropriate protein modifying enzyme before or after purification.
  • an appropriate protein modifying enzyme for example, trypsin, chymotrypsin, arginyl endopeptidase, proteinase, glycosidase and the like are used.
  • the presence of the thus-produced protein of the present invention or a salt thereof can be measured by enzyme blotting using a specific antibody or the like.
  • the antibody against the protein, partial peptide or salt thereof of the present invention may be any of a polyclonal antibody and a monoclonal antibody as long as it can recognize the protein, partial peptide or salt thereof of the present invention.
  • An antibody against the protein, partial peptide or a salt thereof of the present invention uses the protein of the present invention as an antigen, and is a known antibody or antibody. It can be produced according to the serum production method.
  • the protein of the present invention is administered to a warm-blooded animal at a site capable of producing an antibody upon administration, itself or together with a carrier or diluent.
  • complete Freund's adjuvant ⁇ incomplete broth adjuvant may be administered.
  • Administration is usually performed once every 2 to 6 weeks, for a total of about 2 to 10 times.
  • warm-blooded animal used include monkeys, rabbits, rabbits, dogs, guinea pigs, mice, rats, sheep, goats, and chickens, and mice and rats are preferably used.
  • monoclonal antibody-producing cells When producing monoclonal antibody-producing cells, select a warm-blooded animal immunized with the antigen, for example, an individual with an antibody titer from a mouse, and collect the spleen or lymph nodes 2 to 5 days after the final immunization and include them in them.
  • Antibody-producing cells By fusing with a myeloma cell of a seed animal, a hybridoma producing a monoclonal antibody can be prepared.
  • the antibody titer in the antiserum can be measured, for example, by reacting a labeled protein described below with the antiserum, and then measuring the activity of the labeling agent bound to the antibody.
  • the fusion operation can be performed according to a known method, for example, the method of Koehler and Milstein (Nature, 256, 495 (1975)).
  • the fusion promoter include polyethylene glycol (PEG) and Sendai virus, and PEG is preferably used.
  • myeloma cells examples include myeloma cells of warm-blooded animals such as NS-1, P3U1, SP2 / 0, and AP-1, but P3U1 is preferably used.
  • the preferred ratio between the number of antibody-producing cells (spleen cells) used and the number of myeloma cells used is about 1: 1-20: 1, and PEG (preferably PEG1000-PEG6000) is used at a concentration of about 10-80%.
  • Cell fusion can be performed efficiently by adding the mixture and incubating at 20 to 40 ° (preferably 30 to 37 ° C) for 1 to 10 minutes.
  • a hybridoma culture supernatant is added to a solid phase (eg, a microplate) on which a protein antigen is adsorbed directly or together with a carrier.
  • a solid phase eg, a microplate
  • an anti-immunoglobulin antibody labeled with a radioactive substance or enzyme is used if the cells used for cell fusion are mice
  • peptide A is added, and the monoclonal antibody bound to the solid phase is added.
  • Antibody detection method Add the hybridoma culture supernatant to a solid phase to which anti-immune globulin antibody or protein A is adsorbed, add proteins labeled with radioactive substances, enzymes, etc. And a method for detecting a null antibody.
  • the selection of the 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 may 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.)
  • a serum-free culture medium for hybridoma culture SFM-101, Nissui Pharmaceutical Co., Ltd.
  • the cultivation temperature is usually 20 to 40 ° C, preferably about 37 ° C.
  • the culturing 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, alcohol precipitation, isoelectric precipitation, electrophoresis, ion exchangers (eg, DEAE) Adsorption / desorption method, ultracentrifugation method, gel filtration method, specific purification method in which only antibodies are collected using an antigen-binding solid phase or an active adsorbent such as protein A or protein G, and the bonds are dissociated to obtain antibodies.) It can be done according to.
  • immunoglobulin separation and purification methods eg, salting out, alcohol precipitation, isoelectric precipitation, electrophoresis, ion exchangers (eg, DEAE)
  • Adsorption / desorption method eg, ultracentrifugation method, gel filtration method, specific purification method in which only antibodies are collected using an antigen-binding solid phase or an active adsorbent such as protein A or protein G, and the bonds are
  • the polyclonal antibody of the present invention can be produced according to 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 carrier protein and the mixing ratio of carrier-1 to hapten are determined by the antibody against hapten immunized by cross-linking with carrier. If it is possible to efficiently crosslink hapten, any substance may be cross-linked at any ratio. A method of pulling force 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.
  • an active ester reagent containing a daltaraldehyde ⁇ carbodiimide, a maleimide active ester, a thiol group, or a dithioviridyl group 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 in order to enhance the antibody-producing ability upon administration.
  • the administration is usually performed once every about 2 to 6 weeks, for a total of about 3 to 10 ⁇ .
  • the polyclonal antibody can be collected from the blood, ascites, or the like of a warm-blooded animal immunized by the above method, preferably from the blood.
  • the polyclonal antibody titer in the antiserum can be measured in the same manner as the measurement of the antibody titer in the antiserum described above.
  • the separation and purification of the polyclonal antibody can be carried out according to the same method for separating and purifying immunoglobulins as in the above-described separation and purification of the monoclonal antibody.
  • DNA of the present invention is a DNA of the present invention in the description of antisense DNA.
  • Any antisense DNA having a base sequence can be used as long as it has a base sequence complementary to or substantially complementary to the DNA of the present invention and has an action capable of suppressing the expression of the DNA.
  • Antisense DNA may be used.
  • the nucleotide sequence substantially complementary to the DNA of the present invention is, 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). And about 70% or more, preferably about 80% or more, more preferably about 90% or more, and most preferably about 95% or more.
  • the complementary sequence of the nucleotide sequence of the portion encoding the N-terminal portion of the protein of the present invention is approximately 70%.
  • An antisense DNA having a homology of at least about 80%, preferably at least about 80%, more preferably at least about 90%, most preferably at least about 95% is suitable. These antisense DNAs can be produced using a known DNA synthesizer or the like.
  • a protein, a partial peptide or a salt thereof of the present invention hereinafter sometimes abbreviated as a protein of the present invention
  • a DNA encoding the protein or a partial peptide of the present invention hereinafter abbreviated as a DNA of the present invention
  • An antibody against the protein, partial peptide or a salt thereof of the present invention hereinafter, the antibody of the present invention
  • the protein of the present invention is expressed in a tissue-specific manner, it can be used as a tissue marker. That is, it is useful as a marker for detecting tissue differentiation, disease state, metastasis of cancer, and the like. It can also be used for fractionation of corresponding receptors and binding proteins. Furthermore, it can be used as a panel for high-throughput screening known per se to examine biological activity. It can also be used for genetic disease research by performing chromosome mapping.
  • the protein of the present invention exists as a cell regulatory factor in a living body, the protein or the like of the present invention or the DNA of the present invention has an abnormality or is defective, or the expression level is abnormally decreased or If elevated, for example, bone-cartilage ⁇ joint disease, cancer (malignant tumor), other pathological angiogenesis, fetal growth failure, organ failure, gastrointestinal disorders (eg, ulcerative colitis, irritability) Various diseases such as bowel syndrome, Crohn's disease and exocrine disorders (eg, Siedalen syndrome, Teng cystic fibrosis) develop.
  • cancer malignant tumor
  • other pathological angiogenesis fetal growth failure
  • organ failure eg, ulcerative colitis, irritability
  • Various diseases such as bowel syndrome, Crohn's disease and exocrine disorders (eg, Siedalen syndrome, Teng cystic fibrosis) develop.
  • the protein of the present invention and the DNA of the present invention can be used, for example, in bone 'cartilage / joint disease, cancer (malignant tumor), pathological angiogenesis other than the above, fetal growth failure, organ failure, gastrointestinal disorder (eg, , Ulcerative colitis, irritable bowel syndrome, Crohn's disease), exocrine disorders (eg, Siedalen's syndrome, cystic fibrosis), etc. It can be used as a medicine for treating or preventing various diseases.
  • cancer malignant tumor
  • pathological angiogenesis other than the above fetal growth failure
  • organ failure eg, , Ulcerative colitis, irritable bowel syndrome, Crohn's disease
  • exocrine disorders eg, Siedalen's syndrome, cystic fibrosis
  • the DNA of the present invention is And then expressing the protein of the present invention in vivo.
  • the DNA of the present invention is And then expressing the protein of the present invention in vivo.
  • the role of the protein of the present invention in the patient can be sufficiently or normally exerted.
  • the DNA of the present invention When the DNA of the present invention is used as the above-mentioned therapeutic / prophylactic agent, the DNA may be used alone or in a retrovirus vector, adenovirus vector, adenovirus. After insertion into a suitable vector, such as a related virus vector, it can be administered to humans or warm-blooded animals according to conventional means.
  • a suitable vector such as a related virus vector
  • the DNA of the present invention can be administered as it is or in the form of a formulation together with a bio-recognized carrier such as an adjuvant for promoting uptake, and administered with a gene gun or a catheter such as a hydrogel catheter.
  • the protein of the present invention is used as a therapeutic or prophylactic 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 and the like of the present invention can be used as an auxiliary agent and a protective agent for the differentiation of various cells and organ regeneration in the field of regenerative medicine.
  • 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 of the present invention is mixed with a physiologically acceptable carrier, flavoring agent, excipient, vehicle, preservative, stabilizer, binder, etc. in a unit dosage form generally required for the practice of a pharmaceutical preparation. It can be manufactured by this.
  • the amount of active ingredient in these preparations is such that a suitable dosage in the specified range can be obtained.
  • binders such as gelatin, corn starch, tragacanth, gum arabic
  • excipients such as crystalline cellulose, corn starch, gelatin, and alginic acid.
  • a bulking agent such as magnesium stearate, a sweetener such as sucrose, lactose or saccharin, a flavoring agent such as peppermint, cocoa oil or cherry, and the like are used.
  • the unit dosage form is a capsule
  • the above-mentioned dinner material may further contain a liquid carrier such as oil and fat.
  • Sterile compositions for injection can be formulated according to standard pharmaceutical practice, such as dissolving or suspending the active substance in vehicles such as water for injection, or naturally occurring vegetable oils such as sesame oil or coconut oil. it
  • Aqueous injection solutions include, for example, saline, isotonic solutions containing dextrose and other adjuvants (eg, D-Sorbi! ⁇ L, D-mannitol, sodium chloride)
  • Suitable solubilizers for example, alcohols (eg, ethanol), polyalcohols (eg, propylene glycol, polyethylene glycol, etc.), nonionic surfactants (eg, polysorbate 80) TM , HC ⁇ -50, etc.).
  • examples of the oily liquid include sesame oil and soybean oil, and may be used in combination with a solubilizing agent such as benzyl benzoate or benzyl alcohol.
  • buffers for example, phosphate buffer, sodium acetate buffer, etc.
  • soothing agents for example, benzalkonium chloride, proprochlorinate, etc.
  • stabilizers for example, human serum albumin, polyethylene glycol, etc.
  • Preservatives eg, benzyl alcohol, phenol, etc.
  • antioxidants eg, antioxidants and the like.
  • 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, in humans or warm-blooded animals (eg, rats, mice, guinea pigs, egrets, birds, higgies, pigs, pigs, dogs, cats). , Dogs, monkeys, chimpanzees, etc.).
  • the dosage of the protein of the present invention varies depending on the target disease, the subject of administration, the administration route, and the like.
  • the protein of the present invention when administered for the purpose of treating malignant tumors, generally, the adult ( (As 60 kg), the protein is administered in an amount of about lmg to 100 mg, preferably about 10 to 500 mg, more preferably about 10 to 20 mg per day.
  • the single dose of the protein varies depending on the administration subject, target disease, and the like.
  • the protein of the present invention is injected into an adult (body weight) in the form of an injection.
  • the amount converted per 60 kg can be administered.
  • the protein of the present invention regulates cells in vivo (especially human fetal skeletal muscle, intestinal tract, etc.) Since it exists as a factor, the compound that promotes the function of the protein of the present invention or a salt thereof includes, for example, bone, cartilage, and joint diseases, cancer (malignant tumor), pathological angiogenesis other than those described above, fetal growth dysfunction, and organs. It can be used as a medicine for treating and preventing insufficiency and gastrointestinal disorders.
  • the compound or its salt that inhibits the function of the protein of the present invention can be used as a medicament such as an agent for treating or preventing a disease caused by excessive production 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 promotes or inhibits the function of the protein of the present invention.
  • the present invention relates to a compound or a salt thereof which promotes the function of the protein of the present invention, a partial peptide thereof or a salt thereof (hereinafter referred to as a promoter). Or a compound that inhibits the function of the protein of the present invention, a partial peptide thereof or a salt thereof (hereinafter may be abbreviated as an inhibitor).
  • the screening kit of the present invention contains the protein, partial peptide or salt thereof of the present invention.
  • Compounds or salts thereof obtained using the screening method or screening kit of the present invention include, for example, peptides, proteins, non-peptide compounds, synthetic compounds, fermentation products, cell extracts, plant extracts, animal tissues It is a compound selected from extracts, plasma, and the like, and is a compound that promotes or inhibits the function of the protein of the present invention.
  • salt of the compound those similar to the aforementioned salts of the protein of the present invention are used.
  • a compound obtained by using the screening method or the screening kit of the present invention is used as the above-mentioned therapeutic / prophylactic agent, it can be carried out according to conventional means.
  • the above-mentioned drug containing the protein of the present invention for example, tablets, capsules, elixirs, microcapsules, etc., which are sugar-coated as needed, orally, or water or other of It can be used parenterally in the form of injections, such as sterile solutions with pharmaceutically acceptable liquids or suspensions.
  • the preparations obtained in this way are safe and of low toxicity and are, for example, used in humans or warm-blooded animals (for example, mice, rats, puppies, sheep, pigs, puppies, pumas, birds, cats, dogs). , Monkeys, chimpanzees, etc.).
  • the dose of the compound or a salt thereof varies depending on its action, target disease, subject to be administered, administration route, and the like.
  • compounds that promote the function of the protein of the present invention for the purpose of treating malignant tumors When administered orally, generally in adults (assuming a body weight of 60 kg), the compound is used in an amount of about 0.1 to 100 mg, preferably about 1.0 to 5 Omg, more preferably about 1.
  • the single dose of the compound varies depending on the administration subject, target disease, and the like.
  • a compound that promotes the function of the protein of the present invention for the purpose of treating a malignant tumor may be used.
  • the compound When administered to an adult (as 6 O kg) usually in the form of an injection, the compound is administered in an amount of about 0.01 to 3 Omg per day, preferably about 0.1 to 20 mg, more preferably about 0.1 to 20 mg per day. It is convenient to administer about 1 to 1 Omg by intravenous injection. For other animals, the dose can be administered in terms of 60 kg.
  • a compound that inhibits the function of the protein of the present invention when a compound that inhibits the function of the protein of the present invention is orally administered, generally, in an adult (assuming a body weight of 6 O kg), the compound is used in an amount of about 0.1 to: L 0 Omg per day, preferably Is administered at about 1.0 to 5 Omg, more preferably about 1.0 to 2 Omg.
  • the single dose of the compound varies depending on the administration subject, target disease, and the like.
  • the compound is administered by intravenous injection at a rate 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 to administer. In the case of other animals, the dose can be administered in terms of 60 kg.
  • An antibody against the protein of the present invention (hereinafter sometimes abbreviated as the antibody of the present invention) can specifically recognize the protein of the present invention.
  • the present invention can be used for quantification of the protein of the present invention, particularly quantification by sandwich immunoassay.
  • 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 F (ab ') 2 , Fab', or Fab fraction of the antibody 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, antigen, or antibody-antigen corresponding to the amount of antigen (eg, the amount of protein) in the test solution. Any method may be used as long as the amount of the complex is detected by chemical or physical means and calculated 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 for example, [1 2 5 I], [1 3 1 I], [3 H], and [1 4 C] used.
  • the above enzyme a stable enzyme having a large specific activity is preferable.
  • i3-galactosidase,] 3-dalcosidase, alkaline phosphatase, oxidase, malate dehydrogenase and the like are used.
  • the fluorescent substance for example, fluorescamine, fluorescein isothiosinate and the like are used.
  • Luminescent substances include, for example, luminol, luminol derivatives, luciferin, lucige Nin and the like are used. Further, 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 in accordance with those described above.
  • the antibody used for the solid phase antibody or the labeling antibody does not necessarily need to be one kind, and a mixture of two or more kinds of antibodies is used for the purpose of improving measurement sensitivity and the like. 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 is, for example, 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 An antibody that recognizes other than the C-terminal, for example, the N-terminal, is used.
  • the monoclonal antibody of the present invention can be used in a measurement system other than the sandwich method, for example, a competition method, an immunometric method, or a nephelometry.
  • 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 (B) bound to the antibody are separated.
  • B / F separation The labeling amount of either B or F is measured, and the amount of antigen in the test solution is determined.
  • a soluble antibody is used as an antibody
  • BZF separation is performed by polyethylene render recall, a liquid phase method using a second antibody against the antibody, and the like.
  • An immobilized antibody is used as the first antibody, or an immobilized method using a soluble 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 quantify the amount of antigen in the test solution.
  • nephelometry 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 laser-scattering is preferably used.
  • the protein measurement system of the present invention may be constructed by adding ordinary technical considerations of those skilled in the art to ordinary conditions and operations in each method. For details of these general technical means, reference can be made to reviews and documents.
  • the protein of the present invention can be quantified with high sensitivity.
  • the concentration of the protein of the present invention is reduced by quantifying the concentration of the protein of the present invention using the antibody of the present invention, for example, bone, cartilage, joint disease, cancer (malignant) Tumor) can be diagnosed as a disease other than the above, such as pathological angiogenesis, organ failure, gastrointestinal tract disorder, or exocrine disorder, or is likely to be affected in the future.
  • a disease other than the above such as pathological angiogenesis, organ failure, gastrointestinal tract disorder, or exocrine disorder, or is likely to be affected in the future.
  • 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
  • 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. , Monkeys, chimpanzees, etc.), it is possible to detect DNA (mRNA) abnormality (gene abnormality) encoding the protein of the present invention or a partial peptide thereof, for example, damage or mutation of the DNA or mRNA.
  • mRNA DNA
  • mRNA abnormality gene abnormality
  • a gene with a decreased expression or an increase in the DNA or mRNA is useful as a diagnostic agent for a gene 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), Proceedings of the National Academy of Sciences of the United States of America, Vol. 86, pp. 2766-2770 (1989) ), DNA microarray, etc.
  • DNA microarray when expression reduction is detected by Northern hybridization, DNA microarray, etc., or when DNA mutation is detected by PCR-SSCP method, DNA microarray, etc. ⁇ Can be diagnosed as having a high possibility of cartilage, joint disease, cancer (malignant tumor), other pathological angiogenesis, fetal growth failure, organ failure, gastrointestinal tract disorders, exocrine disorders, etc. You.
  • Antisense DNA which complementarily binds to the DNA of the present invention and can suppress the expression of the DNA, can suppress the function of the protein of the present invention or the DNA of the present invention in a living body. However, it can be safely used as an agent for treating and preventing diseases caused by excessive expression of the protein of the present invention.
  • the above-mentioned antisense DNA is used as the above-mentioned therapeutic / prophylactic agent, it can be carried out in the same manner as the aforementioned therapeutic / prophylactic agent for various diseases containing the DNA of the present invention.
  • the antisense DNA when used, the antisense DNA is used alone or inserted into an appropriate vector such as a retrovirus vector, an adenovirus vector, an adenovirus associated virus vector, and the like, followed by a conventional method. It can be administered orally or parenterally to humans or mammals (eg, rats, puppies, sheep, sheep, bush, puppies, cats, dogs, monkeys, etc.).
  • the antisense DNA can be administered as it is or in the form of a formulation together with a physiologically acceptable carrier such as an auxiliary agent for promoting uptake, and administered with a gene gun or a catheter such as a hydrogel catheter. Alternatively, they can be aerosolized and administered intratracheally as an inhalant.
  • the dosage of the antisense DNA varies depending on the target disease, the administration subject, the administration route, and the like.
  • the antisense DNA of the present invention when it is locally administered into the trachea as an inhalant, it is generally used in adults ( At a body weight of 60 kg), about 0.1 to 100 mg of the antisense DNA is administered daily.
  • the antisense DNA can also be used as a diagnostic oligonucleotide probe for examining the presence of the DNA of the present invention in tissues and cells and the state of expression thereof.
  • the antibody of the present invention which has the activity of neutralizing the activity of the protein of the present invention, can be used, for example, as a medicament such as an agent for treating or preventing a disease caused by overexpression of the protein of the present invention.
  • the therapeutic and prophylactic agent for the above-mentioned diseases containing the antibody of the present invention is safe and low toxic, Oral or parenteral to humans or mammals (eg, rat, puppies, higgs, bush, puppies, cats, dogs, monkeys, etc.) as a liquid solution or as a pharmaceutical composition in an appropriate dosage form Can be administered in a controlled manner.
  • the dose varies depending on the administration subject, target disease, symptoms, administration route and the like.
  • the dose of the antibody of the present invention as a single dose is usually about 0.01 to 2 OmgZ kg, preferably about 0.1 mg / kg.
  • the antibodies of the present invention can be administered by themselves or as a suitable pharmaceutical composition.
  • the pharmaceutical composition used for the above administration contains the antibody of the present invention and a pharmacologically acceptable carrier, diluent or excipient.
  • Such compositions are provided in dosage forms suitable for oral or parenteral administration.
  • 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 and the like). ), Syrups, emulsions, suspensions and the like.
  • Such a composition is produced by a method known per se and contains a carrier, diluent or excipient usually used in the field of formulation.
  • a carrier for example, lactose, starch, sucrose, magnesium stearate and the like are used as carriers and excipients for tablets.
  • compositions for parenteral administration for example, injections, suppositories and the like are used.
  • Injections are in the form of intravenous injections, subcutaneous injections, intradermal injections, intramuscular injections, drip injections and the like. Is included.
  • 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 liquids for injection examples include ⁇ saline, isotonic solution containing glucose and other adjuvants, and suitable solubilizing agents such as alcohol (eg, ethanol) and polyalcohol ( For example, propylene glycol, polyethylene glycol), nonionic surfactant [for example, polysorbate 80, HCO- 50 (polyoxyethylene (5 O mol) adduct) of hydrogenated castor oil)]].
  • alcohol eg, ethanol
  • polyalcohol for example, propylene glycol, polyethylene glycol
  • nonionic surfactant for example, polysorbate 80, HCO- 50 (polyoxyethylene (5 O mol) adduct) of hydrogenated castor oil)
  • the oily liquid for example, sesame oil, soybean oil, and the like are used, and benzyl benzoate, benzyl alcohol, and the like may be used in combination as a solubilizing agent.
  • the prepared injection solution is usually filled in a suitable ampoule.
  • compositions are conveniently prepared in dosage unit forms to be compatible with the dosage of the active ingredient.
  • dosage unit dosage forms include tablets, pills, capsules, injections (ampoules), and suppositories, and usually 5 to 500 mg per dosage unit dosage form, and in particular, 5 to: It is preferred that the above antibody be contained in L 00 mg and other dosage forms in an amount of 10 to 250 mg.
  • compositions may contain other active ingredients as long as the composition does not cause an undesirable interaction with the above-mentioned antibody.
  • the present invention relates to a DNA encoding an exogenous protein of the present invention (hereinafter abbreviated as the exogenous DNA of the present invention) or a mutant DNA thereof (sometimes abbreviated as the exogenous mutant DNA of the present invention).
  • 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 Transfer of the desired DNA by coagulation, microinjection, particle injection, DEAE-dextran, etc. Can be produced.
  • the exogenous DNA of the present invention can be transferred to somatic cells, organs of living organisms, tissue cells, and the like, and can be used for cell culture, tissue culture, and the like. Can be fused with the above-mentioned germinal cells by a cell fusion method known per se to produce the DNA transgenic animal of the present invention.
  • non-human mammal for example, red sea lions, bushes, higgins, goats, night egrets, dogs, cats, guinea pigs, hamsters, mice, rats, and the like are used.
  • BDFi strains B6D2Fi strains, BALB / c strains, ICR strains, etc.
  • rats eg, Wistar, SD, etc.
  • Examples of the “mammal” in the recombinant vector that can be expressed in mammals include humans and the like in addition to the above-mentioned non-human mammals.
  • the exogenous DNA of the present invention refers to the DNA of the present invention once isolated and extracted from a mammal, not the DNA of the present invention originally possessed by a non-human mammal.
  • mutant DNA of the present invention those having a mutation (for example, mutation) in the base sequence of the original DNA of the present invention, specifically, addition or deletion of bases, DNA with substitution or the like is used, and also includes abnormal DNA.
  • the abnormal DNA means 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.
  • the exogenous DNA of the present invention may be derived from a mammal of the same or different species as 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.
  • a DNA construct eg, vector, etc.
  • 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, a vaccinia virus or a paculovirus.
  • animal viruses such as E. coli are used.
  • a plasmid derived from Escherichia coli, a plasmid derived from Bacillus subtilis or a plasmid derived from yeast are preferably used.
  • promoters that regulate the above DNA expression include: (1) virus
  • Expression can be re-Bok Megarou Virus promoters, human polypeptide chain elongation factor 1a (EF-1) promoters, human and chicken / 3-actin promoters and the like are preferred.
  • EF-1 human polypeptide chain elongation factor 1a
  • human and chicken / 3-actin promoters and the like are preferred.
  • the vector described above is the target messenger R
  • It preferably has a sequence that terminates the transcription of NA (generally called terminator).
  • terminator a sequence of each DNA derived from a virus and various mammals can be used.
  • the virus SV40 and Minute1 are used.
  • the splicing signal of each DNA, the enhancer region, a part of the intron of eukaryotic DNA, etc. are placed 5 'upstream of the promoter region, the promoter region, and the translation for the purpose of further expressing the target exogenous DNA. It is also possible to connect between the regions or 3 'downstream of the translation region depending on the purpose.
  • the translation region can be produced as a DNA construct that can be expressed in a transposed animal by a conventional DNA engineering technique in which the above-mentioned promoter is ligated to the downstream of the promoter and optionally to the 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 of the progeny of the produced animal and the exogenous DN of the present invention
  • the progeny of such an animal that inherits the exogenous DNA of the present invention has the exogenous DNA of the present invention in all of its germ cells 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. You 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 germ cells of the produced animal after DNA transfer indicates that all the offspring of the produced animal have the exogenous DNA of the present invention in all of their germ cells and somatic cells.
  • the offspring of an animal of this species that has inherited the exogenous DNA of the present invention have the exogenous DN of the present invention in all of its germinal and somatic cells. It has A 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, and finally promotes the function of endogenous normal DNA to thereby finally obtain the protein of the present invention.
  • hyperfunction may develop, and it can be used as a model animal for the disease.
  • the mammal into which the exogenous normal DNA of the present invention has been transferred has an increased symptom of the released protein of the present invention, it can be used for a screening test for a therapeutic drug for a disease associated with the protein of the present invention. It is.
  • a non-human mammal having the foreign abnormal DNA of the present invention can be subcultured in a normal breeding environment as an animal having the DNA after confirming that the foreign DNA is stably retained by mating. I can do it.
  • the desired exogenous DNA can be incorporated into the above-mentioned plasmid and used as a source substance.
  • a DNA construct with a promoter can be prepared by ordinary DNA engineering techniques. 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 after the transfer of the DNA means that the progeny of the animal has the abnormal DNA of the present invention in all of its 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 germ cells and somatic cells.
  • Non-human mammals having the abnormal DNA of the present invention in which the abnormal DNA of the present invention is highly expressed, ultimately inhibit the function of endogenous normal DNA.
  • the protein of the present invention may become functionally inactive refractory, and can be used as a disease model 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.
  • the abnormal DNA highly expressing animal of the present invention is useful for inhibiting the function of the normal protein by the abnormal protein of the present invention (dominant negative activity) in the inactive refractory disease of the protein of the present invention.
  • the mammal into which the foreign abnormal DNA of the present invention has been transferred has an increased symptom of the released protein of the present invention, it is also used in a screening test for a therapeutic drug for a functionally inactive refractory disease of the protein of the present invention. Available.
  • a protein specifically expressed or activated by the protein of the present invention by directly analyzing the DNA or RNA in the tissue of the DNA-transferred animal of the present invention or analyzing the protein tissue expressed by the DNA. Analysis of the relationship with
  • ⁇ ⁇ Isolation and purification of the mutant protein of the present invention and production of its antibody can be considered. Further, using the DNA-transferred animal of the present invention, it is possible to examine the clinical symptoms of diseases related to the protein of the present invention, including the inactive type refractory type of the protein of the present invention, and the like. Obtain more detailed pathological findings in each organ of the disease model related to the protein of the present invention, develop new treatment methods, and contribute to the research and treatment of secondary diseases caused by the disease. Can be.
  • each organ is removed from the DNA-transferred animal of the present invention, minced, and then obtained by using a protease such as trypsin to obtain free DNA-transferred cells and culturing them.
  • a protease such as trypsin
  • the present inventors can identify the protein-producing cells of the present invention, examine their relationship to apoptosis, differentiation or proliferation, or examine their signal transduction mechanisms, and investigate their abnormalities. It is an effective research material for elucidating proteins and their actions.
  • the above-mentioned test was performed. 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 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.
  • the DNA is inactivated by introducing a repo overnight gene (eg, an i3-galactosidase gene derived from Escherichia coli), and the repo overnight gene is expressed under the control of a promoter for the DNA of the present invention.
  • a repo overnight gene eg, an i3-galactosidase gene derived from Escherichia coli
  • a promoter overnight activity against DNA of the present invention which comprises administering a test compound to the animal according to (7) and detecting the expression of a repo overnight gene.
  • a method for screening a compound or a salt thereof that promotes or inhibits A non-human mammalian embryonic stem cell in which the DNA of the present invention has been inactivated is defined as a DNA that is capable of suppressing DNA expression ability by artificially mutating the DNA of the present invention in the non-human mammal.
  • the DNA does not substantially have the ability to express the protein of the present invention (hereinafter referred to as the present invention).
  • Non-human mammalian embryonic stem cells 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 carried out, for example, by deleting part or all of the DNA sequence by 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 codons or destroying the function of promoters or exons.
  • 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 portion is a drug resistance gene typified by a neomycin resistance gene, a hygromycin resistance gene, or lacZ (one galactosidase gene), cat (clo Inserting a reporter gene 1 ⁇ 2 represented by the ramphenicol acetyltransferase gene) disrupts exon function or terminates transcription of the gene in the intron between exons.
  • a drug resistance gene typified by a neomycin resistance gene, a hygromycin resistance gene, or lacZ (one galactosidase gene), cat (clo Inserting a reporter gene 1 ⁇ 2 represented by the ramphenicol acetyltransferase gene) disrupts exon function or terminates transcription of the gene in the intron between exons.
  • a DNA strand having a DNA sequence constructed so as to disrupt the gene (hereinafter abbreviated as targeting vector 1) is introduced into the chromosome of the animal by, for example, homologous recombination, and the obtained ES cells are used in the present invention.
  • ES cells for inactivating the DNA of the present invention by the homologous recombination method or the like for example, those already established as described above may be used. It may be newly established according to the method of Ka uf ma.
  • Ka uf ma for example, in the case of mouse ES cells, currently, 129 ES cells are generally used, but since the immunological background is not clear, an alternative pure immunological and genetically
  • BDFi mice C57BLZ6 that have improved the number of eggs collected from C57BL / 6 mice or C57BL / 6 by crossing with DBA / 2 It is also possible to use a mouse established using the F of the mouse and DBA / 2, etc.
  • the BDFi mouse has the advantage of a large number of eggs collected and the robustness of the egg, as well as the advantage of the C57BL / 6 mouse.
  • the ES cells obtained by using this can be used to backcross C57BLZ6 mice to generate C57BL / 6 mice by cross-crossing C57BLZ6 mice. Can be used advantageously.
  • blastocysts 3.5 days after fertilization are generally used. Many 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.
  • a method for determining the sex of ES cells a method of amplifying and detecting a gene in the sex-determining region on the Y chromosome by PCR is mentioned.
  • this method conventionally, for example G-banding method, requires about 10 6 cells for karyotype analysis, than suffices ES cell number of about 1 colony (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 early selection of male cells.
  • the secondary selection can be performed, for example, by confirming the number of chromosomes by the G-banding method.
  • Embryonic stem cell lines obtained in this way usually have very good proliferative properties, 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 1-1 000 OU / ml
  • 5% CO 2, 90% air at about 37 ° C.
  • trypsin / EDTA solution usually 0.001-0.5% trypsin 0.1
  • mM EDTA preferably about 0.1% tribcine / ImM EDTA
  • 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 muscles, by monolayer culture up to high density or suspension culture until cell clumps are formed under appropriate conditions.
  • MJ Evans and MH Kaufman Nature, Vol. 292, pp. 154, 1981; GR Martin Proc. Of National Academy of Sciences. (Proc. Natl. Acad. Sci. USA) Vol. 78, p. 7634, 1981; TC Doe ts chinan, et al., Journal of Ob. , 1985]
  • the DNA-deficient cell of the present invention obtained by differentiating the ES cell of the present invention is useful in the cell biological examination of the protein of the present invention in the in vitro mouth.
  • the non-human mammal deficient in DNA expression of the present invention can be distinguished from a normal animal by measuring the mRNA level of the animal using a known method and indirectly comparing the expression level.
  • non-human mammal those similar to the aforementioned can be used.
  • the non-human mammal deficient in DNA expression of the present invention may be obtained, for example, by introducing the evening-getting vector prepared as described above into a mouse embryonic stem cell or a mouse egg cell, and introducing the evening-getting vector into the present invention.
  • DNA inactivated DNA sequence of the mouse embryonic stem cells or mouse egg cells The DNA of the present invention can be knocked out by homologous recombination replacing the DNA of the present invention on the chromosome.
  • Cells in which the DNA of the present invention has been knocked out can be analyzed by Southern hybridization analysis using DNA sequences on or near the DNA of the present invention as a probe or DNA sequences on a getter vector. The determination can be carried out by PCR analysis using the DNA sequence of a neighboring region other than the DNA of the present invention derived from the mouse used as one getting vector as a primer.
  • the cell line in which the DNA of the present invention has been inactivated is cloned by gene homologous recombination, and the cells are collected at an appropriate time, for example, at the 8-cell stage.
  • the chimeric embryo is injected into a non-human mammal embryo or blastocyst, and is transplanted into the uterus of the pseudo-pregnant 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 the tissues are more artificial than 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 obtained by adding a mutation to, for example, by judging a color.
  • the individuals obtained in this manner are usually individuals with heterozygous expression of the protein of the present invention, which are crossed with individuals without heteroexpression of the protein of the present invention, and homozygous expression of the protein of the present invention from their offspring. Defective individuals can be obtained.
  • a transgenic non-human mammal having a chromosome into which a gettering vector has been introduced can be obtained by injecting a DNA solution into the egg cell nucleus by a microinjection method.
  • transgenic non-human mammals they can be obtained by selecting those having a mutation in the DNA locus of the present invention by homologous recombination.
  • the animal individual obtained by mating is confirmed to be knocked out of the DNA, and is reared in a normal rearing environment. be able to.
  • the acquisition and maintenance of the germ line may be performed according to a conventional method. That is, by crossing male and female animals having the inactivated DNA, the inactivated DNA is obtained. To obtain homozygous animals having both homologous chromosomes. 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.
  • the non-human mammalian embryonic stem cells in which the DNA of the present invention is inactivated are very useful for producing the non-human mammal deficient in the 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 be caused by 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, because they can serve as a model for such diseases.
  • the non-human mammal deficient in expression of the DNA of the present invention includes diseases (eg, bone, cartilage, joint disease, cancer (malignant tumor), and diseased blood vessels other than those described above) resulting from the deficiency or damage of the DNA of the present invention. It can be used to screen compounds that have therapeutic and / or preventive effects on newborns, fetal growth failure, organ failure, gastrointestinal disorders, exocrine disorders, etc.). 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.
  • the present invention provides a method for screening a compound or a salt thereof having a therapeutic / preventive effect on a disease caused by the disease.
  • Examples of the non-human mammal deficient in DNA expression of the present invention used in the screening method include the same as 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 human mammal deficient in expression of the DNA of the present invention is treated with a test compound and compared with an untreated control animal, and changes in organs, tissues, disease symptoms, etc. of the animal are indicated as an index.
  • Methods for treating a test animal with a test compound include, for example, oral administration, intravenous injection, etc., which are 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.
  • the compound obtained by using the screening method of the present invention is a compound selected from the test compounds described above, and is a disease caused by protein deficiency or damage of the present invention (eg, bone, cartilage, joint disease, cancer) (Malignant tumors), pathological vascular neoplasia, organ failure, gastrointestinal tract disorders, exocrine disorders, etc.). Can be used as Furthermore, compounds derived from the compounds obtained by the above screening can be used in the same manner.
  • the compound obtained by the screening method may form a salt.
  • the salt of the compound include physiologically acceptable acids (eg, inorganic acids, organic acids) and bases (eg, alkali metals). And particularly preferably a physiologically acceptable acid addition salt.
  • 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, Salts with succinic acid, tartaric acid, citric acid, malic acid, oxalic acid, benzoic acid, methanesulfonic acid, benzenesulfonic acid) are used.
  • 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 low toxic and can be used, for example, in humans or mammals (eg, 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 administration subject, the administration route, and the like.
  • the compound when administered for the purpose of treating malignant tumors, it is generally used for adults (body weight 6). 0 kg), the compound is administered at about 0.1 to 10 Omg, preferably about 1.0 to 5 Omg, more preferably about 1.0 to 20 mg per day.
  • a single dose of the compound may be
  • the compound when administered to a normal adult (as 60 kg) in the form of an injection for the purpose of treating a malignant tumor, the compound may be administered in an amount of about 0.01 per day. It is convenient to administer by intravenous injection about -3 Omg, preferably about 0.1-20 mg, more preferably about 0.1-1 Omg.
  • the dose can be administered in terms of 60 kg.
  • the present invention provides a test compound administered to a non-human mammal deficient in expression of a DNA of the present invention, and detects or promotes the activity of a promoter against the DNA of the present invention, which is characterized by detecting the expression of a reporter gene.
  • the non-human mammal deficient in expression of DNA of the present invention includes, among the above-described non-human mammals deficient in expression of DNA of the present invention, the DNA of the present invention in which a repo allelic gene is introduced.
  • a gene capable of being expressed under the control of a promoter for the DNA of the present invention is used.
  • test compound examples include the same compounds as described above.
  • the same one as described above is used, and a 0-galactosidase gene (1 ac Z), a soluble alkaline phosphatase gene or a luciferase gene is suitable.
  • the reporter gene is under the control of a promoter for the DNA of the present invention, the substance encoded by the reporter gene By tracing the expression of the promoter, the activity of the promoter can be detected.
  • a tissue that expresses the protein of the present invention originally 3-galactosidase is expressed instead of the protein of the invention.
  • i3-galactosidase such as 5-bromo-4-chloro-1-indolyl-3-galactopyranoside (X-gal)
  • the protein-deficient mouse of the present invention or a tissue section thereof is fixed with daltaraldehyde or the like, washed with phosphate buffered saline (PBS), and then stained with X-ga1 at room temperature or at room temperature. After reacting at about 7 ° C for about 30 minutes to 1 hour, the jS-galactosidase reaction is stopped by washing the tissue sample with 1 mM EDTA / PBS solution, and the color is observed. Good.
  • mRNA encoding lacZ 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 promoter activity of the DNA of the present invention.
  • the compound obtained by the screening method may form a salt.
  • the salt of the compound include salts with physiologically acceptable acids (eg, inorganic acids) and bases (eg, organic acids). Salts are used, and physiologically acceptable acid addition salts are preferred.
  • Such salts include, for example, salts with inorganic acids (eg, hydrochloric acid, phosphoric acid, hydrobromic acid, sulfuric acid), Alternatively, salts with 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) and the like are used. .
  • the compound of the present invention or a salt thereof that promotes the activity of a promoter against DNA can promote the expression of the protein of the present invention and promote the function of the protein, for example, bone, cartilage, joint disease, cancer ( Malignant tumors), and other useful drugs such as safe and low-toxic therapeutic and prophylactic agents for diseases such as pathological angiogenesis, organ failure, gastrointestinal disorders, and exocrine disorders.
  • a drug containing the compound or its salt 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 its salt.
  • the preparations obtained in this way are safe and have low toxicity, for example, in humans or mammals (for example, rats, mice, guinea pigs, egrets, sheep, pigs, Pests, pests, cats, dogs, monkeys, etc.).
  • the dose of the compound or salt thereof varies depending on the target disease, the subject of administration, the route of administration, and the like.
  • the single dose of the compound varies depending on the administration subject, target disease, and the like.
  • a compound that promotes the promoter activity of the DNA of the present invention for the purpose of treating a malignant tumor may be injected.
  • an adult usually in the form of about 0.01 to 30 mg of the compound per day, preferably about 0.1 to 2 Omg, more preferably about 0.1 to 1 mg / day, It is convenient to administer about 10 mg by intravenous injection. In the case of other animals, the dose can be administered in terms of 60 kg.
  • a compound of the present invention that inhibits promoter activity against DNA when orally administered, generally, in an adult (assuming a body weight of 6 O kg), the compound is used in an amount of about 0.1 to 10 O mg per day. Preferably, about 1.0 to 50 mg, more preferably about 1.0 to 20 mg is administered.
  • the single dose of the compound varies depending on the administration subject, target disease, etc., but the compound of the present invention which inhibits promoter activity on DNA is usually in the form of an injection.
  • about 0.01 to 30 mg, preferably about 0.1 to 2 Omg, more preferably about 0.1 to 10 mg of the compound per day is administered intravenously. It is convenient to administer by injection. In the case of other animals, the amount converted per 60 kg can be administered.
  • the non-human mammal deficient in expression of the DNA 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 the DNA of the present invention. It can greatly contribute to the investigation of the causes of various diseases caused by insufficient DNA expression or the development of therapeutic drugs.
  • bases and amino acids are indicated by abbreviations, the abbreviations by IUPAC-I UB Commission on Biochemical Nomenclature Alternatively, it is based on a common abbreviation in the art, examples of which are described below.
  • amino acids may have optical isomers, the L-form is indicated unless otherwise specified.
  • FIG 1 shows the amino acid sequence of the protein of the present invention (human NCHM).
  • amino acid sequence at positions 215 to 317 of the amino acid sequence represented by SEQ ID NO: 1 is shown.
  • FIG. 1 shows the nucleotide sequence of DNA encoding the protein of the present invention (human NCHM) having the amino acid sequence represented by SEQ ID NO: 1.
  • Example 1 shows the nucleotide sequence of oligo DNA used in Example 1 described later.
  • Example 1 shows the nucleotide sequence of oligo DNA used in Example 1 described later.
  • the transformant Es che richiacol obtained in Example 1 described later DH5 a / pTBN3 has been deposited with the Fermentation Research Institute (I FO) at 17-85, 2-10-Honcho, Yodogawa-ku, Osaka-shi, Osaka since January 18, 2000 as accession number I FO 163 54. They have been deposited with the National Institute of Advanced Industrial Science and Technology (NI BH) at the Ministry of International Trade and Industry, National Institute of Industrial Science and Technology (NI BH) at 1-11, Higashi, Tsukuba, Ibaraki Prefecture, Japan, as deposit number FER M BP-7017.
  • NI BH National Institute of Advanced Industrial Science and Technology
  • NI BH National Institute of Advanced Industrial Science and Technology
  • NI BH National Institute of Industrial Science and Technology
  • the cDNA encoding the human NCHM protein of the present invention is as follows: Obtained by CR method.
  • the oligo DNA represented by SEQ ID NO: 7 (TCAGCAGTGGTCTCTC AGTCCTCTCA) as the sense strand primer, represented by SEQ ID NO: 8
  • A) as antisense strand primers 5 Opmol each, Premix Taq TM (Ex Taq TM Version) (Takara Shuzo Co., Ltd.) 25/1, ⁇ type 0A as human fetal skeletal muscle cDNA solution (Clontech)
  • Premix Taq TM Ex Taq TM Version
  • ⁇ type 0A human fetal skeletal muscle cDNA solution
  • Clontech Prepare a mixed solution 501 containing 0.5 l, and first use a thermal cycler (Gen eAmp R PCR system mo del 9700 (Pakinkin Elma)) at 94 ° C. After 1 minute, repeat 35 cycles of 1 cycle at 94 ° C for 20 seconds, 50 ° C for 30 seconds, 72 ° C for 3 minutes, and extend the PCR reaction at 72 ° C for 5 minutes. went.
  • a thermal cycler Gene eAmp R PCR system mo del 9700 (Pakinkin Elma)
  • plasmid DNA and pTBN3 were prepared.
  • pTBN3 was used as a type I DNA, and AB I PR I SM R Big Dy e Terminator Cycle Se quenc inng FS Ready React on Kit (Perkin According to the conditions of the attached document, the sequencing reaction using Samma cycler was performed using a thermocycler [Gene Amp R PGR system model 9700 (PerkinElmer)].
  • the encoded NCHM protein contains a highly hydrophobic region corresponding to the transmembrane region of the cell membrane-localizing protein from the amino acid residue at serine (Ser) 28 to glycine (Gly) at position 49.
  • Rat chondromodulin I precursor protein Rat chondromodulin I precursor protein, human chondromodulin I precursor protein, ⁇ sicndromodulin I precursor protein, and ⁇ The homology with the heron chondromodulin I precursor protein is 39% at the amino acid level and 3% at the amino acid level.
  • Example (E c oR I- E c oR I 1. 1 kb fragment of PTBN3) ⁇ DNA fragment according to 1 20 ng and Non one 32 P] dCTP (Ame rsh am : 60 Using 00 CiZmmo 1) 51, a DNA probe was prepared by the method of Multitip DNA DNA label 1 ingsystem (Amersham: RPN. 1601 Y). Using this probe, hybridization was performed on a human multi-tissue northern plot (CLONTECH: # 7759-1, # 7760-1) and a human RNA master blot (CLONTECH: # 7770-1). Hybridization and washing conditions were performed according to the attached manual, and detection was performed using BAS-2000 (Fujifilm). As a result, it was clarified that the protein was expressed in specific tissues and organs including at least the large intestine, small intestine, and exocrine glands, in addition to the human fetal skeletal muscle described in Example 1.
  • Example 3 Example 3
  • primer-DNA is chemically synthesized based on the nucleotide sequence of cDNA which encodes the NCHM protein obtained in Example 1.
  • One is 5'-CGAATTC AC-3 '(SEQ ID NO: 9), which has an anchor sequence containing a restriction element EcoRI recognition site at the 5' end, a start codon and +145 to It is an oligo DNA containing a sense sequence up to +172 (the translation start site is set to +1).
  • GGCCACCCA-3 (SEQ ID NO: 10), which is an oligo having a sequence in which an antisense sequence from +925 to 10,951 is connected to the 3rd side of the anchor sequence containing the restriction enzyme Sa1I recognition site. DNA.
  • pCAN 618 FLAG is derived from the plasmid vector pCAN618, has a neomycin resistance gene as a selection marker, and clones the DNA fragment encoding the target protein into its cloning sites EcoRI and SalI.
  • the target protein is FLAG It can also be expressed as a fusion protein.
  • the above PCR cloning DNA fragment was also inserted into pCAN618 FLAG for the purpose of expressing a fusion protein of NC.HM protein extracellular region and FLAG epitope (with one Va1 residue inserted between them). Obtain the expression vector plasmid pNCHMtra-F.
  • COS 7 cells (1.2 x 10 5 cells) were cultured in Dulbecco's modified minimal medium (DMEM) containing 10% fetal bovine serum (FBS) for 24 hours using a 6-well plate.
  • DMEM Dulbecco's modified minimal medium
  • FBS fetal bovine serum
  • the expression plasmid pNCHMtra-F (0.4 g / well) is introduced using lipofectamine (GI bco BRL). 24 hours after introduction, replace with the above new medium, and 5 hours later, replace with FBS-free Op t I-MEM (GI bco BRL), culture for 36 hours, and obtain the culture supernatant and cell extract .
  • the cell extract was washed twice with phosphate buffered saline (PBS) containing physiological saline and dissolved and extracted with Tris-SDS sample buffer.
  • PBS phosphate buffered saline
  • the culture supernatant was appropriately concentrated by ultrafiltration (3,000 molecular weight cut). Then mix with an equal volume of Tris SDS sample buffer. After heat treatment, these samples are electrophoresed on a 15% -25% SDS-polyacrylamide gel, and then transferred from the gel onto a PVDF membrane (Amersham parmac iabiotech). Next, remove the PVDF membrane with Block Ace (Snow Brand Milk Products) for 1 hour. Block and react with anti-FLAG monoclonal antibody (10 Mg / m 1; Kodak) in PBS containing 0.05% Tween 20 (PBS-T) for 2 hours.
  • the fusion protein was purified by scaling up the above-described gene transfer experiment into COS 7 cells and preparing a large amount of the culture supernatant or RIPA buffer (150 m, M NaCl, 1% Nonidet P-40 ( Anti-FLAG monoclonal antibody M2 (Sigma) from crude cell extract using NP-40), 0.1% SDS, 0.5% sodium deoxycholate, 40 mM Tris-HCl (pH 7.5) Is performed by affinity chromatography coupled to a column.
  • RIPA buffer 150 m, M NaCl, 1% Nonidet P-40 ( Anti-FLAG monoclonal antibody M2 (Sigma) from crude cell extract using NP-40), 0.1% SDS, 0.5% sodium deoxycholate, 40 mM Tris-HCl (pH 7.5) Is performed by affinity chromatography coupled to a column.
  • the target protein thus obtained is used for assaying various biological activities.
  • an assay is performed using an angiogenesis kit (Kurabo Industries). Industrial applicability
  • the protein of the present invention and DNA encoding the same include, for example, bone, cartilage, and joint diseases, cancer (malignant tumors), pathological angiogenesis other than those described above, fetal growth failure, organ failure, digestive tract disorders, exocrine disorders, and the like. It can be used as a treatment / prevention agent for diseases. Further, the protein of the present invention is useful as a reagent for screening a compound or a salt thereof that promotes or inhibits the activity of the protein of the present invention. Furthermore, since the antibody against the protein 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 solution.

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Abstract

L'invention concerne une nouvelle protéine et un ADN codant ladite protéine, qu'il est possible d'utiliser comme remèdes/prophylactiques dans le cadre de maladies telles que les maladies des os, des cartilages ou des articulations, le cancer (tumeur maligne), l'angiogenèse pathologique autre que les affections précitées, l'insuffisance organique, les troubles du tube digestif et l'insuffisance de la sécrétion exocrine. Ladite protéine est également utilisée comme réactif pour le criblage d'un composé, ou de son sel, favorisant ou inhibant l'activité de cette protéine. En outre, il est possible d'utiliser un anticorps dirigé contre cette protéine, ledit anticorps pouvant reconnaître ladite protéine de manière spécifique, pour quantifier la protéine dans un échantillon liquide par exemple.
PCT/JP2000/009236 1999-12-27 2000-12-26 Nouvelle proteine et adn associe Ceased WO2001048203A1 (fr)

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Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1191099A4 (fr) * 1999-06-30 2002-08-07 Takeda Chemical Industries Ltd Nouveau polypeptide et adn de ce polypeptide
JP2003520590A (ja) * 2000-01-19 2003-07-08 アムジェン インコーポレイテッド コンドロモジュリンｉ関連ペプチド
EP1219710A4 (fr) * 1999-09-29 2004-07-14 Teijin Ltd Polypeptides et genes les codant
EP1946765A4 (fr) * 2005-09-22 2009-10-21 Keiichi Fukuda Médicament pour maladie en rapport avec l angiogenèse comportant la chondromoduline-i comme principe actif

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EP0473080A2 (fr) * 1990-08-23 1992-03-04 Mitsubishi Chemical Corporation Protéine chondromodulin-I
WO2000012708A2 (fr) * 1998-09-01 2000-03-09 Genentech, Inc. Nouveaux pro-polypeptides et sequences correspondantes
WO2000029579A1 (fr) * 1998-11-13 2000-05-25 Zymogenetics, Inc. Proteine de type chondromoduline de mammmifere
WO2000043495A2 (fr) * 1999-01-19 2000-07-27 Human Genome Sciences, Inc. Proteines humaines secretees (33)

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Publication number Priority date Publication date Assignee Title
EP0473080A2 (fr) * 1990-08-23 1992-03-04 Mitsubishi Chemical Corporation Protéine chondromodulin-I
WO2000012708A2 (fr) * 1998-09-01 2000-03-09 Genentech, Inc. Nouveaux pro-polypeptides et sequences correspondantes
WO2000029579A1 (fr) * 1998-11-13 2000-05-25 Zymogenetics, Inc. Proteine de type chondromoduline de mammmifere
WO2000043495A2 (fr) * 1999-01-19 2000-07-27 Human Genome Sciences, Inc. Proteines humaines secretees (33)

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Title
HIRAKI Y. ET AL.: "Molecular cloning of human chondromodulin-1 a cartilage-derived growth modulating factor and its expression in Chinese hamuster ovary cells", EUR. J. BIOCHEM., vol. 260, no. 3, March 1999 (1999-03-01), pages 869 - 878, XP002944164 *
NEAME P.J. ET AL.: "An 18-kDa glycoprotein from bovine nasal cartilage. Isolation and primary structure of small, cartilage-derived glycoprotein", J. BIOL. CHEM., vol. 265, no. 17, 1990, pages 9628 - 9633, XP002944156 *
SHUKUNAMI C. ET AL.: "Expression of cartilage-specific functional matrix chondromodulin-1 mRNA in rabbit growth plate chondrocytes and its responsiveness to growth stimuli in vitro", BIOCHEM. BIOPHYS. RES. COMMUN., vol. 249, 1998, pages 885 - 890, XP002944163 *

Cited By (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1191099A4 (fr) * 1999-06-30 2002-08-07 Takeda Chemical Industries Ltd Nouveau polypeptide et adn de ce polypeptide
US6797483B1 (en) 1999-06-30 2004-09-28 Takeda Chemical Industries, Ltd. Polypeptide and DNA thereof
EP1219710A4 (fr) * 1999-09-29 2004-07-14 Teijin Ltd Polypeptides et genes les codant
US7575922B2 (en) 1999-09-29 2009-08-18 Teijin Limited Polypeptide and gene encoding the same
US8030277B2 (en) 1999-09-29 2011-10-04 Teijin Limited Polypeptide and gene encoding the same
JP2003520590A (ja) * 2000-01-19 2003-07-08 アムジェン インコーポレイテッド コンドロモジュリンｉ関連ペプチド
EP1946765A4 (fr) * 2005-09-22 2009-10-21 Keiichi Fukuda Médicament pour maladie en rapport avec l angiogenèse comportant la chondromoduline-i comme principe actif

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