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WO2004039407A1 - Inducteur d'apoptose - Google Patents

Inducteur d'apoptose Download PDF

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Publication number
WO2004039407A1
WO2004039407A1 PCT/JP2003/013920 JP0313920W WO2004039407A1 WO 2004039407 A1 WO2004039407 A1 WO 2004039407A1 JP 0313920 W JP0313920 W JP 0313920W WO 2004039407 A1 WO2004039407 A1 WO 2004039407A1
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WO
WIPO (PCT)
Prior art keywords
seq
amino acid
acid sequence
protein
salt
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Ceased
Application number
PCT/JP2003/013920
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English (en)
Japanese (ja)
Inventor
Kenichi Horikoshi
Osamu Kitahara
Takahiro Watanabe
Yoshio Taniyama
Satoru Nishizawa
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Takeda Pharmaceutical Co Ltd
Original Assignee
Takeda Pharmaceutical Co Ltd
Takeda Chemical Industries Ltd
Priority date (The priority date 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 date listed.)
Filing date
Publication date
Application filed by Takeda Pharmaceutical Co Ltd, Takeda Chemical Industries Ltd filed Critical Takeda Pharmaceutical Co Ltd
Priority to AU2003280642A priority Critical patent/AU2003280642A1/en
Publication of WO2004039407A1 publication Critical patent/WO2004039407A1/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

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Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
    • 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
    • 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
    • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
    • C07K14/4747—Apoptosis related proteins
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • C12Q1/6886—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/136—Screening for pharmacological compounds
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2510/00—Detection of programmed cell death, i.e. apoptosis

Definitions

  • the present invention relates to an apoptosis inducer and the like. More specifically, the present invention relates to an enzyme activity of HTRA3 and its substrate, an apoptosis-inducing agent based on inhibition of HTRA3 function, and a screening method thereof. The present invention also relates to an apoptosis inhibitor based on the promotion of the function of HTR A3 or an apoptosis inhibitor using a protein preparation and a screening method thereof.
  • DNA topoisomerase inhibitors can inhibit the recombination of DNA that has been cut during DNA replication. It is an agent that induces apoptosis by inhibiting it and exerts an antitumor effect. It utilizes the fact that DNA replication occurs more frequently in cancer cells than in normal cells.
  • Irinotecan hydrochloride which inhibits DNA topoisomerase II
  • etoposide 'teniposide which inhibits DNA topoisomerase II
  • these anticancer agents cause considerable damage to normal cells, and often have strong side effects especially on bone marrow where cell division is active. For this reason, chemotherapy is required to induce apoptosis in a tumor-specific manner, and various apoptosis-inducing anticancer drugs are currently being developed, but no anticancer drug that satisfies this demand is still available.
  • MMP matrix meta-oral protease
  • Perokinase plasminogen activator is a protease that acts on plasminogen in blood to release plasmin. Recent studies suggest that plasmaminogen activators and plasmin are the major players in MMP activation (EMBO J., (16), 2319-2332, 1997). . Furthermore, plasmin is known to act on latent TGF i3 and release active TGF; 3. (Activated TGF) 3 plays an important role in creating an environment in which cancer cells are prone to proliferate, invade, and metastasize because it promotes angiogenesis, accumulates extracellular matrix, and suppresses immunosuppression. It is believed that.
  • IGF Insulin-like growth factor
  • IGF-BP IGF-binding protein
  • HTRA3 Serine protease
  • GenBank: AY 040094 Serine protease
  • the present inventors have conducted intensive studies to solve the above problems, and as a result, have found a gene (Serine protease HTR A3 gene) whose expression is remarkably increased in cancer tissues, and further suppress the function of the gene. As a result, they found that apoptosis was induced. Further, by searching for the function of the gene, It has been found that it has the property. Based on this finding, as a result of further studies, they have found new experimental results on apoptosis inhibitory activity, a phenomenon of self-processing, and a substrate for protease activity, and completed the present invention.
  • a drug comprising the antisense polynucleotide according to (3); (6) a drug according to (5), which is an apoptosis inducer;
  • a medicine according to the above (5) or (6) which is an agent for preventing or treating cancer
  • a protein containing the amino acid sequence represented by SEQ ID NO: 5 or SEQ ID NO: 30 or a qualitatively identical amino acid sequence, a partial peptide thereof, or a salt thereof is used. Screening method for apoptosis inducer;
  • a kit for screening for apoptosis-inducing agents contains a protein having the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 5 or SEQ ID NO: 30, a partial peptide thereof, or a salt thereof.
  • a prophylactic / therapeutic agent for kidney cancer comprising a compound that inhibits or a salt thereof;
  • a prophylactic / therapeutic agent for Teng's carcinoma obtainable by using the screening method according to (33) or the screening kit according to (34); (36) a protein having a protease activity and containing an amino acid sequence identical or substantially identical to the amino acid sequence represented by SEQ ID NO: 5 or SEQ ID NO: 30;
  • a protease inhibitor comprising an antisense polynucleotide containing a partially complementary base sequence or a part thereof;
  • protease inhibitor comprising an antisense polynucleotide consisting of the base sequence represented by SEQ ID NO: 3, SEQ ID NO: 26 or SEQ ID NO: 28;
  • (41) comprising an antibody against a protein having the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 5 or SEQ ID NO: 30, or a partial peptide thereof or a salt thereof Protease inhibitors;
  • a polynucleotide having the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 5 or SEQ ID NO: 30 or a polynucleotide encoding a partial peptide thereof is used.
  • amino acid sequence represented by SEQ ID NO: 5 or SEQ ID NO: 30 obtainable by using the screening method described in (49) or the screening kit described in (50).
  • a polynucleotide coding for a protein having the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 5 or SEQ ID NO: 30 or a partial peptide thereof is used.
  • a protein professional A method for screening for a substrate for thease activity;
  • a kit for screening a substrate for the protease activity of the protein characterized by containing a polynucleotide encoding a protein or a partial peptide thereof having the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 5 or SEQ ID NO: 30.
  • a protein comprising the amino acid sequence represented by SEQ ID NO: 5 or SEQ ID NO: 30 obtainable by using the screening method according to (52) or the screening kit according to (53); A substrate for a protease activity of a protein having an amino acid sequence identical or substantially identical to the amino acid sequence represented by SEQ ID NO: 5 or SEQ ID NO: 30;
  • a method for preventing and treating Teng's carcinoma which comprises inhibiting the activity of a protein containing the same amino acid sequence or a partial peptide thereof or a salt thereof.
  • Apoptosis one cis induction method Ku amino acid sequence identical if represented, which comprises substantially inhibit expression of a gene of the same protein or parts thereof Bae flop comprising the amino acid sequence tides or salt thereof;
  • a method for preventing or treating hepatic cancer which comprises inhibiting the expression of a gene for a tide or a salt thereof;
  • (61) Inhibits the activity of a protein containing the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 5 or SEQ ID NO: 3.0, a partial peptide thereof, or a salt thereof Administering an effective amount of a compound or a salt thereof to a mammal.
  • (62) Inhibits the activity of a protein containing the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 5 or SEQ ID NO: 30 or a partial peptide thereof or a salt thereof
  • a method for inhibiting a protease which comprises administering an effective amount of a compound or a salt thereof to a mammal;
  • a method for preventing and treating kidney cancer which comprises administering an effective amount of a compound or a salt thereof to a mammal;
  • (70) a protein or a partial peptide thereof containing an amino acid sequence identical or substantially identical to the amino acid sequence represented by SEQ ID NO: 5 or SEQ ID NO: 30 for producing an apoptotic inducer; Use of a compound or a salt thereof that inhibits the expression of a gene of the salt;
  • a protein or a partial peptide thereof comprising an amino acid sequence identical or substantially identical to the amino acid sequence represented by SEQ ID NO: 5 or SEQ ID NO: 30 for production of a protease inhibitor Or use of a compound or a salt thereof that inhibits the expression of a gene for the salt thereof;
  • a protein containing an amino acid sequence identical or substantially identical to the amino acid sequence represented by SEQ ID NO: 5 or SEQ ID NO: 30 for the manufacture of a preventive or therapeutic agent for kidney cancer or a part thereof Use of a compound or a salt thereof that inhibits the expression of a peptide or a salt thereof gene;
  • the amino acid sequence substantially the same as the amino acid sequence represented by SEQ ID NO: 5 includes about 50% or more, preferably about 60% or more, and more preferably about 70% or more of the amino acid sequence represented by SEQ ID NO: 5. % Or more, more preferably about 80% or more, particularly preferably about 90% or more, and most preferably about 95% or more.
  • the amino acid sequence substantially the same as the amino acid sequence represented by SEQ ID NO: 30 includes about 50% or more, preferably about 60% or more, and more preferably about 70% or more of the amino acid sequence represented by SEQ ID NO: 30. % Or more, more preferably about 80% or more, particularly preferably about 90% or more, and most preferably about 95% or more.
  • Examples of the protein having an amino acid sequence substantially the same as the amino acid sequence represented by SEQ ID NO: 5 include, for example, a protein having an amino acid sequence substantially the same as the amino acid sequence represented by the aforementioned SEQ ID NO: 5 However, a protein having substantially the same activity as the protein containing the amino acid sequence represented by SEQ ID NO: 5 is preferred.
  • Examples of the protein having an amino acid sequence substantially the same as the amino acid sequence represented by SEQ ID NO: 30 include, for example, a protein containing the amino acid sequence substantially the same as the amino acid sequence represented by SEQ ID NO: 30 described above. However, a protein having substantially the same activity as the protein containing the amino acid sequence represented by SEQ ID NO: 30 is preferred.
  • substantially equivalent activities include, for example, apoptosis inhibitory activity and protease activity.
  • substantially identical indicates that the properties are qualitatively identical (eg, physiologically or pharmacologically). Therefore, it is preferable that the apoptosis inhibitory activity and the protease activity are equivalent (eg, about 0.01 to 100 times, preferably about 0.1 to 10 times, more preferably 0.5 to 2 times).
  • the quantitative factors such as the degree of these activities and the molecular weight of proteins may be different.
  • Apoptosis inhibitory activity can be measured by a method known per se, for example, a method using the enzymatic activity of caspases 3 and 7 that carry out apoptosis (Methods in Enzymology, (244), 615-31, 1994) or a method using the method. It can be measured by a combination of a method based on the method, a method for inhibiting the function of a specific gene using an antisense oligonucleotide, or a method based thereon.
  • the apoptosis-suppressing activity can be measured by using a substance in which a fluorescent substance is bound to a substrate specific to caspases 3 and 7, and a substance to which a quencher is bound simultaneously. That is, the antisense oligonucleotide of the protein of the present invention (eg, HTRA3) is introduced into cells expressing HTRA3 to inhibit the function of the HTRA3 gene, and then reacted with the above-mentioned substrate in an appropriate buffer.
  • the antisense oligonucleotide of the protein of the present invention eg, HTRA3
  • HTRA3 antisense oligonucleotide of the protein of the present invention
  • the degree of the apoptosis inhibitory activity of the protein of the present invention can be determined.
  • the protease activity can be measured by a method known per se, for example, a method utilizing the degradation of casein. More specifically, protease activity can be measured by preparing a casein fluorescently labeled product, allowing the protein of the present invention to act on the product, and measuring the increase in fluorescent signal derived from casein degradation products (Anal. Biochem. , (254), 144-147, 1997).
  • compounds that inhibit the enzymatic activity of the protein of the present invention and have apoptosis-inducing activity can be screened using the above-described protease activity measurement method.
  • the enzyme inhibitory activity of the protein of the present invention by the low molecular compound can be quantified by coexisting a low molecular compound in the above reaction system and measuring the decrease in the fluorescent signal derived from the casein degradation product.
  • the selected compound can be used as an apoptosis-inducing agent (in this specification, a compound having an apoptosis-inducing activity or a salt thereof may be referred to as an apoptosis-inducing agent) and used as a therapeutic agent for cancer such as Teng's carcinoma. . ⁇
  • the protein consisting of the amino acid sequence represented by SEQ ID NO: 30 corresponds to a product resulting from autolysis of the protein consisting of the amino acid sequence represented by SEQ ID NO: 5, as shown in Examples described later.
  • the amino acid sequence represented by 30 is a novel amino acid sequence.
  • the protein of the present invention it is possible to search for a protein that binds to the protein or a protein serving as a substrate.
  • the protein of the present invention is transiently expressed in cells using an expression vector, and the protein binding to the obtained protein of the present invention can be searched for by a method such as immunoprecipitation.
  • cytostatic factors such as' TGF] 3, their families (eg, BMP-4, GDF-5, Activin), and cell growth factors such as IGF are considered as proteins that bind to the protein of the present invention.
  • various proteins can be fluorescently labeled to search for proteins that serve as substrates for the protease activity of the protein of the present invention. It is possible.
  • proteins that are basement membrane components such as laminin-5, proteins that are components of extracellular matrix such as collagen, IGF activity regulators such as IGF-BP, and TGF i3 such as plasminogen Activity regulators, latent TGF3, and the like are considered as proteins that serve as substrates for the protease activity of the protein of the present invention.
  • Examples of the protein used in the present invention include: 1) 1 or 2 or more (for example, about 1 to 100, preferably 1) in the amino acid sequence represented by SEQ ID NO: 5 or SEQ ID NO: 30; About 30 amino acids, preferably about 1-10 amino acids, more preferably about 1 to 5 amino acids, 2) SEQ ID NO: 5 or SEQ ID NO: 30 One or two or more amino acids (for example, about 1 to 100, preferably about 1 to 30, preferably about 1 to 10, and more preferably about 1 to 5) Amino acid sequence, 3) 1 or 2 or more amino acid sequences represented by SEQ ID NO: 5 or SEQ ID NO: 30 (for example, about 1 to 100, preferably about 1 to 30, preferably about 1 to 10, More preferably, an amino acid having a number (1 to 5) of amino acids inserted therein Acid sequence, 4) 1 or 2 or more in the amino acid sequence represented by SEQ ID NO: 5 or SEQ ID NO: 30 (for example, about 1 to 100, preferably about 1 to 30, preferably 1 to 10 Also included are so-
  • the position of the insertion, deletion or substitution is not particularly limited.
  • the left end is the N-terminus (amino terminus) and the right end is the C-terminus (carboxyl terminus) in accordance with the convention of peptide notation.
  • the proteins used in the present invention include a carboxylate (-COOH), carboxylate (- COO—), amide (one CONH 2 ) or ester (one COOR).
  • esters as, for example, methyl, Echiru, n- propyl, isopropyl, alkyl groups such as n- butyl, cyclopentyl, C 3 _ 8 cycloalkyl group such as cyclohexyl, for example, phenyl, Facial C 6 _ 12 Ariru groups, such as single naphthyl le, for example, benzyl, phenylene Lou C such as phenethyl ⁇ - naphthyl such as an alkyl group or an ⁇ - naphthylmethyl - C 7 _ i 4 Ararukiru groups such as C i _ 2 alkyl group, such as pivaloyl Ruo carboxymethyl group is used.
  • alkyl groups such as n- butyl, cyclopentyl, C 3 _ 8 cycloalkyl group such as cyclohexyl, for example, phenyl, Facial C 6 _ 12
  • the protein used in the present invention has a lipoxyl group (or carboxylate) other than the C-terminal
  • the protein in which the lipoxyl group is amidated or esterified is also included in the protein used in the present invention.
  • the ester in this case for example, the above-mentioned C-terminal ester and the like are used.
  • an amino group at the N-terminal amino acid residue may have a protecting group (eg, a 6- alkyl group such as a C 6 alkanol such as a formyl group or an acetyl group).
  • a protecting group eg, a 6- alkyl group such as a C 6 alkanol such as a formyl group or an acetyl group.
  • protein used in the present invention include, for example, a protein containing the amino acid sequence represented by SEQ ID NO: 5, a protein containing the amino acid sequence represented by SEQ ID NO: 30 and the like.
  • the partial peptide of the protein used in the present invention is the partial peptide of the protein used in the present invention described above, and preferably has the same properties as the protein used in the present invention described above. Any one may be used. Specifically, for the purpose of preparing the antibody of the present invention described later, at least 20 or more, preferably 50 or more, of the amino acid sequence represented by SEQ ID NO: 5 or SEQ ID NO: 30 More preferably, peptides having an amino acid sequence of 70 or more, more preferably 100 or more, most preferably 200 or more are used.
  • one or more (preferably about 110, more preferably about (15)) amino acids in the amino acid sequence are deleted, Or 1 or 2 or more (preferably, about 120, more preferably, about 110, and still more preferably, a number (, 15)) Amino acids), or 1 or 2 or more (preferably about 1 to 20, more preferably about 1 to 10, more preferably about 1 to 5) to the amino acid sequence. Amino acids), 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. It may be substituted with another amino acid.
  • the C-terminus may be any one of a carboxylate group (-1 c ⁇ H), a carboxylate (_C ⁇ 0 _), an amide (-1 C ⁇ NH 2 ) and an ester (-COOR). It may be.
  • the partial peptide used in the present invention has a lipoxyl group (or lupoxylate) other than the C-terminus, and the N-terminal amino acid residue (eg, , A methionine residue) whose amino group is protected with a protecting group, a glutamine residue generated by cleavage of the N-terminal in vivo and pyroglutamine oxidation, a substituent on the side chain of an amino acid in the molecule Are protected by an appropriate protecting group, or a complex peptide such as a so-called sugar peptide to which a sugar chain is bound.
  • the partial peptide used in the present invention can also be used as an antigen for preparing an antibody.
  • salts with physiologically acceptable acids eg, inorganic acids, organic acids
  • bases eg, alkali metal salts
  • acid addition salts that are chemically acceptable 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, methanesulfonic
  • the protein, its partial peptide, or a salt thereof used in the present invention can be produced from the cells or tissues of humans or warm-blooded animals by the above-mentioned method for purifying a protein or contains a DNA encoding the protein. It can also be produced by culturing a transformant obtained. It can also be produced according to the peptide synthesis method described below. '
  • the cells When produced from human or mammalian tissues or cells, Alternatively, the cells can be homogenized, extracted with an acid or the like, and the extract can be purified and isolated by combining chromatography such as reverse phase chromatography and ion exchange chromatography.
  • chromatography such as reverse phase chromatography and ion exchange chromatography.
  • a commercially available resin for protein synthesis can be usually used.
  • resins 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'dimethoxyphenylhydroxymethyl) phenoxy resin, 4- (2,, 4, dimethoxyphenyl) -Fmoc Aminoethyl) phenoxy resin.
  • an amino acid having an ⁇ -amino group and a side chain functional group appropriately protected is condensed on the resin in accordance with the sequence of the target protein according to various known condensation methods.
  • a protein or partial peptide is cleaved from the resin, and at the same time, various protecting groups are removed.
  • an intramolecular disulfide bond formation reaction is carried out in a highly diluted solution to obtain a target protein or partial peptide or an amide thereof. get.
  • the condensation of the above protected amino acids various activating reagents that can be used for protein synthesis can be used, and carbodiimides are particularly preferable.
  • carpoimides DCC, N, N, diisopropyl carpoimide, N-ethyl N '-(3-dimethylaminoprolyl) carpoimide, and the like are used.
  • the protected amino acid may be added directly to the resin along with a racemization inhibitor additive (eg, HOB t, HOOB t), or the protected amino acid may be preformed as a symmetric anhydride or HOB t ester or HOOB t ester. It can be added to the resin after activation.
  • the solvent used for activating the protected amino acid or condensing with the resin can 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-methylpiperidone, and halogenated carbons such as methylene chloride and chloroform.
  • Hydrogens, alcohols such as trifluoroethanol, sulfoxides such as dimethyl sulfoxide, ethers such as pyridine, dioxane, and tetrahydrofuran; nitriles such as acetonitrile and propionitrile; methyl acetate and ethyl acetate; Esters or an appropriate mixture thereof are used.
  • 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, isopornyloxycarbonyl, 4-methoxybenzyloxycarponyl, C1Z, Br-Z, Damantyloxycarbonyl, trifluoroacetyl, phthaloyl, formyl, 212-trophenylsulfenyl, diphenylphosphinochioil, Fmoc and the like are used.
  • the carboxyl group may be, for example, a linear, branched or cyclic alkyl such as an alkyl esterified (eg, methyl, ethyl, propyl, butyl, t-butyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, 2-adamantyl).
  • alkyl esterified eg, methyl, ethyl, propyl, butyl, t-butyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, 2-adamantyl.
  • Esterification aralkyl esterification (e.g., benzyl ester, 4-nitrobenzyl ester, 4-methoxybenzyl ester, 4-methylbenzyl ester, benzhydryl esterification), phenacyl esterification, benzyloxycarponyl hydrazide , T-butoxycarbonyl hydrazide, trityl hydrazide, etc.
  • aralkyl esterification e.g., benzyl ester, 4-nitrobenzyl ester, 4-methoxybenzyl ester, 4-methylbenzyl ester, benzhydryl esterification
  • phenacyl esterification e.g., benzyloxycarponyl hydrazide , T-butoxycarbonyl hydrazide, trityl hydrazide, etc.
  • the hydroxyl group of serine can be protected, for example, by esterification or etherification.
  • Suitable groups for this esterification include, for example, lower (C ⁇ 6 ) alkanol groups such as acetyl, aroyl graves such as benzoyl, and groups derived from carbonic acid such as benzyloxycarbonyl and ethoxycarbonyl. Ira It is.
  • Examples of a group suitable for etherification include a benzyl group, a tetrahydrovinyl group, a t-butyl group, and the like.
  • the protecting group of the phenolic hydroxyl group of tyrosine for example, B z 1, C l 2 - B zl, 2_ nitrobenzyl, B r- Z, such as t- butyl are used.
  • imidazole protecting group of histidine examples include Tos, 4-methoxy-2,3,6-trimethylbenzenesulfonyl, DNP, benzyloxymethyl, Bum, Boc, Trt, and Fmoc. .
  • Activated carbonyl groups of the raw materials include, for example, corresponding acid anhydrides, azides, active esters [alcohols (eg, pentachlorophenol, 2,4,5-trichloromouth phenol, 2,4-dinitro Phenol, cyanomethyl alcohol, paranitrophenol, H ⁇ NB, N-hydroxysuccinimide, N-hydroxyphthalimide, and esters with HOB t)].
  • active esters eg, pentachlorophenol, 2,4,5-trichloromouth phenol, 2,4-dinitro Phenol, cyanomethyl alcohol, paranitrophenol, H ⁇ NB, N-hydroxysuccinimide, N-hydroxyphthalimide, and esters with HOB t
  • the activated amino group of the raw material for example, a corresponding phosphoric amide is used.
  • Methods for removing (eliminating) protecting groups include, for example, catalytic reduction in a hydrogen stream in the presence of a catalyst such as P—black or Pd—carbon, or hydrogen fluoride anhydride, methanesulfonic acid, Acid treatment with trifluoromethanesulfonic acid, trifluoroacetic acid or a mixture thereof, base treatment with diisopropylethylamine, triethylamine, piperidine, piperazine, etc., or with sodium in liquid ammonia Reduction is also used.
  • the elimination reaction by the acid treatment is generally performed at a temperature of about ⁇ 20 ° C. to 40 ° C.
  • anisol for example, anisol, phenol, thioanisole, methacrylol, paracresol
  • a force-thione scavenger such as dimethyl sulfide, 1,4-butanedithiol, 1,21-ethanedithiol and the like.
  • the 2,4-dinitrophenyl group used as an imidazole protecting group of histidine is removed by thiophenol treatment, and the formyl group used as an indole protecting group of tributofan is treated with 1,2-ethanedithiol, 1,4- In addition to deprotection by acid treatment in the presence of butanedithiol, etc., it is also removed by 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 protecting 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.
  • Another method for obtaining an amide form of a protein or partial peptide is, for example, first protecting the carboxy-terminal amino acid with amidation of the amino group, and then adding a peptide (protein) chain on the amino group side to the desired length. After extension, the protein or partial peptide from which only the protecting group for the N-terminal ⁇ -amino group of the peptide chain has been removed, and the protein or partial peptide from which only the protecting group for the C-terminal carbonyl group have been removed. It is produced and these proteins or peptides are condensed in a mixed solvent as described above. Details of the condensation reaction are the same as described above.
  • the crude protein or peptide can be purified by various known purification means, and the main fraction can be lyophilized to obtain the desired protein or peptide amide.
  • an ester of a protein or peptide for example, after condensing the ⁇ -hydroxyl group of the amino acid at the carboxy terminus with a desired alcohol to form an amino acid ester, An ester of a protein or a peptide can be obtained.
  • the partial peptide used in the present invention or a salt thereof can be produced according to a known peptide synthesis method, or by cleaving the protein used in the present invention with an appropriate peptide.
  • a method for synthesizing a peptide for example, any of a solid phase synthesis method and a liquid phase synthesis method may be used. That is, the target peptide is produced by condensing a partial peptide or amino acid that can constitute the partial peptide used in the present invention with the remaining portion, and if the product has a protecting group, removing the protecting group. Can be. Examples of the known condensation method and elimination of the protecting group include the methods described in the following 1) to 5).
  • the partial peptide used in the present invention can be purified and isolated by a combination of ordinary purification methods, for example, solvent extraction, distillation, column chromatography, liquid chromatography, recrystallization, etc. .
  • the partial peptide obtained by the above method is a free form, it can be converted into an appropriate salt by a known method or a method analogous thereto. Can be converted into a free form or another salt by a method according to the above.
  • the polynucleotide encoding the protein used in the present invention may be any polynucleotide as long as it contains the above-described nucleotide sequence encoding the protein used in the present invention.
  • it is DNA.
  • the DNA may be any of genomic DNA, genomic DNA library, cDNA derived from the above-described cells and tissues, cDNA library derived from the above-described cells and tissues, and synthetic DNA.
  • the vector used for the library may be any of pacteriophage, plasmid, cosmid, phagemid and the like.
  • a reverse RNA is directly prepared by using a total RNA or mRNA fraction prepared from the cells and tissues described above.
  • RT-PCR method Transcriptase Polymerase Chain Reaction
  • Examples of the DNA encoding the protein used in the present invention include, for example, a DNA containing the base sequence represented by SEQ ID NO: 6 or the base sequence represented by SEQ ID NO: 6 under highly stringent conditions. Any DNA can be used as long as it contains a nucleotide sequence that hybridizes with the above, and encodes a protein having substantially the same properties as the protein containing the amino acid sequence represented by SEQ ID NO: 5 described above. .
  • the DNA encoding the protein used in the present invention may be, for example, a DNA containing the nucleotide sequence represented by SEQ ID NO: 31 or a DNA containing the nucleotide sequence represented by SEQ ID NO: 31.
  • It contains a base sequence that hybridizes with the base sequence represented by SEQ ID NO: 31 under highly stringent conditions, and has substantially the same properties as the protein containing the amino acid sequence represented by SEQ ID NO: 30 described above. Any DNA may be used as long as it encodes a protein having the DNA.
  • DNA that can hybridize with the nucleotide sequence represented by SEQ ID NO: 6 under high stringency conditions include, for example, about 50% or more, preferably about 60% of the nucleotide sequence represented by SEQ ID NO: 6 Or more, more preferably about 70% or more, more preferably about 80% or more, particularly preferably about 90% or more, and most preferably about 95% or more.
  • a DNA that can hybridize with the nucleotide sequence represented by SEQ ID NO: 31 under high stringency conditions include, for example, about 50% or more of the nucleotide sequence represented by SEQ ID NO: 31, and preferably about 60%.
  • Hybridization is carried out by a method known per se or a method analogous thereto, for example, Molecular Cloning 2nd (J. Sambrook et al., Cold Spring Harbor Lab. Press, 1989).
  • the method can be performed according to the method described in, for example.
  • a commercially available library it can be performed according to the method described in the attached instruction manual. More preferably, it can be performed under high stringent conditions.
  • High stringency end conditions refer to, for example, a sodium concentration of about 19 to 40 mM, preferably about 19 to 2 OmM, and a temperature of about 50 to 70 T, preferably about 60 to 65 ° C. In particular, the case where the sodium concentration is about 19 mM and the temperature is about 65 is most preferable. More specifically, as a DNA encoding a protein containing the amino acid sequence represented by SEQ ID NO: 5, DNA containing the base sequence represented by SEQ ID NO: 6, or the like is used. As the DNA encoding the protein containing the amino acid sequence represented by SEQ ID NO: 30, DNA containing the base sequence represented by SEQ ID NO: 31 and the like are used.
  • the polynucleotide (eg, DNA) encoding the partial peptide used in the present invention may be any polynucleotide having the above-described nucleotide sequence encoding the partial peptide used in the present invention. Further, it may be any of genomic DNA, genomic DNA library, the above-described cell / tissue-derived cDNA, the above-mentioned cell / tissue-derived cDNA library, and synthetic DNA.
  • Examples of the DNA encoding the partial peptide used in the present invention include, for example, a DNA having a part of the DNA containing the base sequence represented by SEQ ID NO: 6 or SEQ ID NO: 31, or SEQ ID NO: 6 or SEQ ID NO: A DNA comprising a nucleotide sequence that hybridizes under high stringent conditions with the nucleotide sequence represented by 31, and a DNA containing a part of a DNA encoding a protein having substantially the same activity as the protein of the present invention. Used. DNA that can hybridize with the nucleotide sequence represented by SEQ ID NO: 6 or SEQ ID NO: 31 has the same significance as described above. The same method as described above is used for the method of the hybridization and the stringent conditions.
  • DNA amplified by PCR using a synthetic DNA primer having a part of the nucleotide sequence encoding the protein of the present invention, or DNA integrated into an appropriate vector is used as the DNA of the present invention.
  • the DNA base sequence can be converted using the ODA-LAPCR method using PCR, a known kit, for example, Mutan TM -super Express Km (Takara Shuzo Co., Ltd.), Mutan TM -K (Takara Shuzo Co., Ltd.), etc. It can be carried out according to a method known per se, such as the gapped duplex method and the Kunkel method, or a method analogous thereto. ,
  • the DNA encoding the cloned protein can be used as it is by itself or, if desired, digested with a restriction enzyme or added with a linker.
  • the DNA may have ATG as a translation initiation codon at its 5 'end, and may have TAA, TGA or TAG as a translation stop codon at its 3' end. These translation initiation codon and translation termination codon can also be added using an appropriate 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 of an appropriate expression vector. It can be manufactured by connecting downstream.
  • the vector examples include a plasmid derived from E. coli (eg, pBR322, pBR325, pUC12, pUC13), a plasmid derived from Bacillus subtilis (eg, pUB110, TP5, pC194), a plasmid derived from yeast (Eg, pSH19, pSH15), bacteriophage such as ⁇ phage, animal viruses such as retrovirus, vaccinia virus, baculovirus, etc., pAl-11, pXTl, pRc / CMV , PRc / RSV, pcDNAIZNeo, and the like.
  • E. coli eg, pBR322, pBR325, pUC12, pUC13
  • Bacillus subtilis eg, pUB110, TP5, pC194
  • yeast eg, pSH19, pSH15
  • bacteriophage such as ⁇
  • the promoter used in the present invention may be any promoter as long as it is an appropriate promoter corresponding to the host used for the expression of the gene.
  • SRa promoter when an animal cell is used as a host, SRa promoter, SV40 promoter, LTR promoter, CMV promoter, HSV-TK promoter and the like can be mentioned. Of these, it is preferable to use CMV (Site Megalovirus) promoter, SRo!
  • trp promoter one When the host is Eshierihia genus bacterium, trp promoter one, lac promoter one, re cA promoter, AP L promoter In the evening, the lpp promoter, T7 promoter, etc., when the host is a Bacillus genus, SP01 promoter, SPO2 promoter, penP promoter, etc., when the host is yeast, the PHO5 promoter, The PGK promoter, GAP promoter overnight, ADH promoter and the like are preferred. When the host is an insect cell, a polyhedrin promoter, a P10 promoter and the like are preferable.
  • an expression vector containing, if desired, an enhancer, a splicing signal, a polyA addition signal, a selection marker, an SV40 replication origin (hereinafter sometimes abbreviated as SV40 ori), and the like may be used.
  • the selection Ma one force one, for example, dihydrofolate reductase (hereinafter sometimes abbreviated as dh fr) gene [Mesotorekise Ichito (MTX) resistance], ampicillin phosphorus resistant gene (hereinafter sometimes abbreviated as Amp r Neomycin resistance gene (hereinafter sometimes abbreviated as Ne0r, G418 resistance), and the like.
  • dhfr gene when used as the primary candidate for selection using Chinese hamster cells deficient in the dhfr 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 of the protein of the present invention.
  • the host is a bacterium belonging to the genus Escherichia, PhoA signal sequence, OmpA signal sequence, etc.
  • the host is a bacterium belonging to the genus Bacillus, ⁇ -amylase signal sequence, subtilisin signal sequence, etc.
  • the host is yeast, MFa signal sequence, SUC2 signal sequence, etc.
  • the host is an animal cell, insulin signal sequence, ⁇ -interferon signal sequence, antibody molecule, signal sequence, etc. Are available respectively.
  • a transformant can be produced using the thus constructed vector containing DN, A encoding the protein of the present invention.
  • Escherichia bacteria for example, Escherichia bacteria, Bacillus bacteria, yeast, insect cells, insects, animal cells, and the like are used.
  • Escherichia examples include, for example, Escherichia coli K12 ⁇ DH1 (Pro Natl. Acad. Sci. USA), 60, 160 (1968)], J 103 [Nucleic Acids Research, Vol. 9, 309 (1 981)], JA 221 [Journal 'ob''Molekiyura' biology (Journal of Molecular Biology), 120, 517 (1978)], HB 101 (Journal of 'Molecular' Biology, 41, 459 (1969)), C 600 (Genetics
  • Bacillus bacteria examples include, for example, Bacillus subtilis MI114 [Gene, 24, 255 (1983)], 207-21 [Journal of Biochemistry, 95 Vol. 87 (1 984)].
  • yeast examples include, for example, Saccharomyces cerevisiae AH22, AH22 R-, NA87-11A, DKD-5D, 20B-12, Schizosaccharomyces pombe NC YC 1913, NCYC 2036, Pichia pastoris K ⁇ 71 and the like are used.
  • insect cells for example, when the virus is Ac AV, the cell line derived from the larvae of the night larvae (Spodoptera frugiperda cell; S f cell), the MGl cell derived from the midgut of Trichoplusia ni, and Trichoplusia ni Egg-derived High Five TM cells, Mamestra brass icae-derived cells, or cells from Est igmena acreafi are used.
  • the virus is BmNPV
  • a silkworm-derived cell line Boombyx mori N cell; BmN cell
  • Sf cells include Sf9 cells (ATCCCRL1711) and Sf21 cells (Vaughn, JL et al., In Vivo, 13, 213-217, (1977)). Used.
  • insects for example, silkworm larvae are used [Maeda et al., Nature, Vol. 315, 592 (1985)].
  • animal cells examples include monkey cell COS-7, Vero, Chinese hamster cell CH ⁇ (hereinafter abbreviated as CHO cell), and dhfr gene-deficient Chinese hamster cell CHO (hereinafter abbreviated as CHO (dhfr)) cell.
  • CHO cell Chinese hamster cell CH ⁇
  • dhfr dhfr gene-deficient Chinese hamster cell CHO
  • Transformation of a Bacillus bacterium can be performed, for example, according to the method described in Molecular & General Genetics, 168, 11 (1799).
  • Insect cells or insects can be transformed, for example, according to the method described in Bio / Technology, 6, 47-55 (1988). To transform animal cells, for example, see Cell Engineering Separate Volume 8 New Cell Engineering Experiment Protocol. 263—267 (1995) (published by Shujunsha), Virology, 52, 456 (1973). )).
  • a liquid medium is suitable as a medium used for the cultivation.
  • carbon sources include glucose, dextrin, soluble starch, and sucrose
  • nitrogen sources include ammonium salts, nitrates, corn chips, liquor, peptone, zein, meat extract, soybean meal, and potato extract.
  • examples of the inorganic or organic substance and the inorganic substance include calcium chloride, sodium dihydrogen phosphate, magnesium chloride, and the like.
  • yeast extract, vitamins, growth promoting factors and the like may be added.
  • the size of the medium is preferably about 5 to 8.
  • an M9 medium containing glucose and casamino acids As a medium for culturing the genus Escherichia, for example, an M9 medium containing glucose and casamino acids [Miller, Journal “Ob” Experimen-n 1 Journal of Experiments in Molecular Genetics), 431-433, Cold Spring Harbor Laboratory, New York 1972]. If necessary, a drug such as 33-indolylacrylic acid can be added in order to make the promoter 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 performed at about 30 to 40 ° C for about 6 to 24 hours, and aeration and stirring may be added as necessary.
  • the medium used is Grace's Insect Medium (Grace, TCC, Nature, 195, 788 (1962)). And the like to which additives such as the above are appropriately added are used.
  • the ⁇ of the culture medium is 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 / or agitation are added as necessary.
  • examples of the medium include a MEM medium containing about 5 to 20% fetal bovine serum [Science, 122, 501 (1952)], a DMEM medium [Virology, 8, 396 (1959)], RPM I 1640 medium [Journal of the American Medical Association] The Journal of the American Medical Association) 199, 519 (1967)], 199 medium [Proceding of the Society for the Biological Medicine], 73, 1 (1950) )] Is used.
  • the pH is between about 6 and 8. Culture is usually performed at about 30 ° C to 40 ° C for about 15 to 60 hours, and aeration and stirring are added as necessary.
  • the protein of the present invention can be produced in the cells, in the cell membrane, or outside the cells of the transformant.
  • the protein of the present invention can be separated and purified from the above culture by, for example, the following method.
  • cells or cells are collected by a known method, suspended in an appropriate buffer, and subjected to ultrasonic wave, lysozyme and / or freeze-thawing. After the cells or cells are destroyed by the method, a method of obtaining a crude extract of the protein 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.
  • Methods that mainly use differences in molecular weight such as methods, methods that use differences in charges, such as ion-exchange chromatography, methods that use specific affinity such as affinity chromatography, reverse-phase high-performance liquid chromatography, etc.
  • a method utilizing the difference in hydrophobicity, and a method utilizing the difference in isoelectric points such as isoelectric focusing.
  • the protein thus obtained when it is obtained as a free form, it can be converted to a salt by a method known per se or a method analogous thereto, and conversely, when the protein is obtained as a salt, a method known per se or The free form or other salt can be Can be converted.
  • the protein produced by the recombinant can be arbitrarily modified or the polypeptide can be partially removed by the action of an appropriate protein-modifying enzyme before or after purification.
  • an appropriate protein-modifying enzyme for example, trypsin, chymotrypsin, arginyl endopeptidase, protein kinase, glycosidase and the like are used. '
  • the presence of the thus-produced protein of the present invention can be measured by enzyme immunoassay or Western blotting using a specific antibody.
  • An antibody against the protein or partial peptide or a salt thereof used in the present invention may be any of a polyclonal antibody and a monoclonal antibody as long as it can recognize the protein or partial peptide or a salt thereof used in the present invention. It may be.
  • An antibody against the protein or partial peptide used in the present invention or a salt thereof (hereinafter sometimes simply referred to as the protein of the present invention in the description of the antibody) uses the protein of the present invention as an antigen, It can be produced according to a known antibody or antiserum 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 by itself or together with a carrier or diluent.
  • Complete Freund's adjuvant or incomplete Freund's adjuvant may be administered in order to enhance antibody production upon administration.
  • the administration is usually performed once every 2 to 6 weeks, for a total of about 2 to 10 times.
  • the warm-blooded animals to be used include, for example, monkeys, egrets, dogs, guinea pigs, mice, rats, sheep, goats, and chickens, and mice and rats are preferably used.
  • a warm-blooded animal immunized with the antigen for example, an individual with a mouse titer selected from mice is selected, and the spleen or lymph node is collected 2 to 5 days after the final immunization.
  • the antibody-producing cells contained in the above are fused with myeloma thorax of the same or xenogeneic animal to produce monoclonal antibody-producing hybrids.
  • 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 Keller and Milstein [Nature 256, 495 (1975)].
  • the fusion promoter include polyethylene glycol (PEG) and Sendai virus, but 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) and the number of myeloma cells used is about 1: 1 to 20: 1, and the concentration of PEG (preferably PEG 1000 to PEG6000) is about 10 to 80%.
  • PEG preferably PEG 1000 to PEG6000
  • 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, and then radioactive substances are added.
  • a solid phase eg, a microplate
  • Anti-globulin antibody labeled with an enzyme or an enzyme anti-mouse immunoglobulin antibody is used when the cells used for cell fusion are mice) or protein A, and a monoclonal antibody bound to a solid phase is detected.
  • 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 can be used as long as it can grow a hybridoma.
  • RPM-1640 medium containing 1-20%, preferably 10-20% fetal bovine serum, GIT medium containing 1-10% fetal bovine serum (Wako Pure Chemical Industries, Ltd.) or A serum-free medium for hybridoma culture (SFM-101, Nissui Pharmaceutical Co., Ltd.) can be used.
  • the culturing temperature is usually 20 to 40 T, preferably about 37: It is.
  • the culturing time is usually 5 days to 3 weeks, preferably 1 week to 2 weeks. Cultivation can usually be performed under 5% CO2.
  • 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 focusing, electrophoresis, ion exchangers (eg, DEAE ), Specific centrifugation method, gel filtration method, antigen-binding solid phase or specific purification method in which only antibody is collected using an active adsorbent such as protein A or protein G and the bond is dissociated to obtain the antibody. You can do it.
  • immunoglobulin separation and purification methods eg, salting out, alcohol precipitation, isoelectric focusing, electrophoresis, ion exchangers (eg, DEAE ), Specific centrifugation method, gel filtration method, antigen-binding solid phase or specific purification method in which only antibody is collected using an active adsorbent such as protein A or protein G and the bond is dissociated to obtain the antibody. You can do it.
  • the polyclonal antibody of the present invention can be produced according to a method known per se or a method analogous thereto.
  • an immunizing antigen protein Bohara
  • 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 monoclonal antibody production method.
  • the antibody can be produced by collecting the antibody-containing substance corresponding to the protein and separating and purifying the antibody.
  • the type of carrier protein and the mixing ratio of carrier and hapten are different from those of hapten immunized by cross-linking with carrier.
  • Any antibody may be cross-linked at any ratio as long as the antibody can be efficiently prepared.
  • serum serum albumin, thyroglobulin, hemocyanin, etc. may be used in a weight ratio of about 0 to 1 for hapten.
  • a method of coupling at a rate of 1 to 20 and preferably about 1 to 5 is used.
  • Various condensing agents can be used for force coupling between the hapten and the carrier.
  • glutaraldehyde, carbodiimide, a maleimide active ester, an active ester reagent containing a thiol group or a dithioviridyl group, or the like is used.
  • the condensation product is administered to a warm-blooded animal itself or together with a carrier or diluent at a site where antibody production is possible.
  • Complete Freund's adjuvant / incomplete Freund's adjuvant may be administered in order to enhance the antibody-producing ability upon administration. Throw The dose is usually given once every 2 to 6 weeks, about 3 to 10 times in total.
  • the polyclonal antibody can be collected from the blood, ascites, etc., preferably from the blood, of the warm-blooded animal immunized by the above method.
  • the measurement of the polyclonal antibody titer in the antiserum can be performed in the same manner as the measurement of the antibody titer in the antiserum described above. Separation and purification of the polyclonal antibody can be carried out according to the same immunoglobulin separation and purification method as the above-described separation and purification of the monoclonal antibody. '
  • the nucleotide sequence of a polynucleotide encoding a protein or a partial peptide used in the present invention eg, DNA (hereinafter, these DNAs may be abbreviated as the DNA of the present invention in the description of an antisense polynucleotide)
  • the antisense polynucleotide having a complementary or substantially complementary base sequence or a part thereof includes a complementary or substantially complementary base sequence of the polynucleotide (eg, DNA) of the present invention. Any antisense polynucleotide may be used as long as it has a basic sequence or a part thereof and has an action of suppressing the expression of the DNA, but antisense DNA is preferable.
  • the nucleotide sequence substantially complementary to the DNA of the present invention is, for example, about 70% of 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). % Or more, preferably about 80% or more, more preferably about 90% or more, and most preferably about 95% or more.
  • the combination of an antisense polynucleotide directed to translation inhibition is based on the nucleotide sequence of the portion coding for the N-terminal site of the protein of the present invention.
  • the antisense polynucleotide is (mouth) an antisense polynucleotide that directs RNA degradation by RNaseH, it is preferably at least about 70%, preferably about 70% or more, of the complementary strand of the entire nucleotide sequence of the DNA of the present invention including introns.
  • Antisense polynucleotides having about 80% or more, more preferably about 90% or more, and most preferably about 95% or more homology are each suitable.
  • An antisense polynucleotide having a base sequence complementary to the base sequence of the base sequence-containing DNA or a part thereof (more preferably, a base of the DNA containing the base sequence represented by SEQ ID NO: 6 or SEQ ID NO: 31) A base sequence complementary to the sequence, or an antisense polynucleotide having a part thereof).
  • an antisense polynucleotide consisting of the nucleotide sequence represented by SEQ ID NO: 3, SEQ ID NO: 26 or SEQ ID NO: 28 is preferably used.
  • An antisense polynucleotide is usually composed of about 10 to 40 bases, preferably about 1 to 5 to 30 bases.
  • Antisense DN to prevent degradation by hydrolases such as nuclease
  • the phosphate residue (phosphate) of each nucleotide constituting A may be substituted with a chemically modified phosphate residue such as phosphorothioate, methylphosphonate, or phosphorodithionate.
  • the sugar (deoxylipose) of each nucleotide may be substituted with a chemically modified sugar structure such as 2'-O-methylation, and the base (pyrimidine, purine) may also be chemically modified.
  • an antisense polynucleotide capable of inhibiting the replication or expression of the protein gene of the present invention is designed based on the nucleotide sequence information of the DNA encoding the cloned or determined protein. And can be synthesized.
  • a polynucleotide can hybridize with the RNA of the protein gene of the present invention, inhibit the synthesis or function of the RNA, or interact with the protein-related RNA of the present invention via an interaction with the protein-related RNA of the present invention. It can regulate and control the expression of the protein gene of the present invention.
  • a polynucleotide complementary to the selected sequence of the protein-related RNA of the present invention and a polynucleotide capable of specifically hybridizing with the protein-related RNA of the present invention.
  • Pide is useful for regulating and controlling the expression of the protein gene of the present invention in vivo and in vitro, and is also useful for treating or diagnosing diseases and the like.
  • the term "corresponding" means having homology or being complementary to a specific sequence of nucleotides, base sequences or nucleic acids including genes.
  • nucleotide, base sequence, or nucleic acid and a peptide (protein) refers to the amino acid of a peptide (protein) specified by the nucleotide (nucleic acid) sequence or its complement.
  • nucleotide (nucleic acid) sequence or its complement usually pointing. 5'-end hairpin loop of protein gene, 5'-end 6-base spare 'repeat, 5'-end untranslated region, polypeptide translation initiation codon, protein coding region, ORF translation stop codon, 3'-end untranslated region, 3
  • the palindrome region at the 'end and the hairpin loop at the 3' end may be selected as preferred regions of interest, but any region within the protein gene may be selected as a target.
  • Antisense polyliponucleotides can be polydeoxyliponucleotides containing 2-deoxy D-reports, polynucleotides containing D-reports, N-glycosides of purine or pyrimidine bases or other types of polynucleotides. Nucleotides or other polymers with non-nucleotide backbones
  • RNA hybrids can be double-stranded DNA, single-stranded DNA, double-stranded RNA, single-stranded RNA, and even DNA: RNA hybrids, as well as unmodified polynucleotides (or unmodified oligonucleotides), and Include those with known modifications, e.g., those with labels known in the art, capped, methylated, one or more natural nucleotides replaced by analogs, Intramolecularly modified nucleotides, such as those with uncharged bonds (eg, methylphosphonates, phosphotriesters, phosphoramidates, olebamates, etc.), charged or sulfur-containing bonds (eg, phosphoro Those having a thioate, a phosphorodithioate, etc., for example, a protein (nu Nucleases, nucleases / inhibitors, toxins, antibodies, signal peptides, poly-L-lysine, etc.) or sugars (for
  • nucleoside may include not only those containing purine and pyrimidine bases but also those having other modified heterocyclic bases. Such modifications may include methylated purines and pyrimidines, acylated purines and pyrimidines, or other heterocycles. Modified nucleotides and modified nucleotides may also be modified at the sugar moiety, e.g., where one or more hydroxyl groups are replaced with halogens, aliphatic groups, etc., or functional groups such as ethers, amines, etc. It may be converted to
  • the antisense polynucleotide (nucleic acid) of the present invention is RNA, DNA, or a modified nucleic acid (RNA, DNA).
  • modified nucleic acid include, but are not limited to, sulfur derivatives of nucleic acids, thiophosphate derivatives, and those resistant to degradation of polynucleoside amides and oligonucleoside amides.
  • the antisense nucleic acid of the present invention can be preferably designed according to the following policy. That is, to make the antisense nucleic acid more stable in the cell, to make the antisense nucleic acid more cell permeable, to have a greater affinity for the target sense strand, and to be more toxic if it is toxic. Make sense nucleic acids less toxic.
  • the antisense nucleic acids of the present invention may contain altered or modified sugars, bases, or bonds, and may be provided in special forms such as ribosomes and microspheres. Or be applied by gene therapy or given in an added form.
  • additional forms include polycations, such as polylysine, which act to neutralize the charge on the phosphate backbone, and lipids, which enhance interaction with cell membranes or increase nucleic acid uptake.
  • polycations such as polylysine, which act to neutralize the charge on the phosphate backbone
  • lipids which enhance interaction with cell membranes or increase nucleic acid uptake.
  • hydrophobic substances such as phospholipid and cholesterol
  • Preferred lipids for addition include cholesterol and its derivatives (eg, cholesteryl chromate formate, cholic acid, etc.).
  • Such a substance can be attached to the 3 'end or 5' end of a nucleic acid, and can be attached via a base, sugar, or intramolecular nucleoside bond.
  • Other groups include cap groups specifically arranged at the 3 'end or 5' end of a nucleic acid, which prevent degradation by nucleases such as exonuclease and RNase.
  • capping groups include, but are not limited to, hydroxyl-protecting groups known in the art, including glycols such as polyethylene glycol and tetraethylene glycol.
  • the inhibitory activity of the antisense nucleic acid can be examined using the transformant of the present invention, the in vivo or in vitro gene expression system of the present invention, or the in vivo or in vitro translation system of the protein of the present invention.
  • the nucleic acid can be applied to cells by various methods known per se.
  • the protein or partial peptide of the present invention or a salt thereof hereinafter, sometimes abbreviated as the protein of the present invention
  • the protein or partial peptide of the present invention may be used.
  • DNA of the present invention DNA encoding the peptide
  • an antibody against the protein or partial peptide of the present invention or a salt thereof hereinafter sometimes abbreviated as the antibody of the present invention
  • antisense polynucleotide of the DNA of the present invention hereinafter sometimes abbreviated as the antisense polynucleotide of the present invention
  • the expression of the protein of the present invention increases in cancer tissues, it can be used as a tumor marker. In other words, it is useful as a tool for early diagnosis of cancer tissue and estimation of the extent of lesion development. Therefore, a drug containing the antisense polynucleotide of the gene encoding the protein of the present invention, a compound that inhibits the activity of the protein of the present invention or a salt thereof, or an antibody against the protein of the present invention.
  • Is for example, colon cancer, breast cancer, lung cancer, prostate cancer, esophageal cancer, stomach cancer, liver cancer, biliary tract cancer, spleen cancer, renal cancer, bladder cancer, uterine cancer, testis cancer, thyroid cancer, kidney cancer, brain tumor or blood
  • cancers such as tumors (preferably Teng's carcinoma), apoptosis inducing (or promoting) agents, protease inhibitors (in the present specification, compounds having protease inhibitory activity or salts thereof) It may be referred to as a protease inhibitor.)
  • the gene of the protein of the present invention has low expression in normal tissues and high expression in cancer tissues, particularly in kidney cancer tissues, a drug based on inhibition of the physiological function of apoptosis suppression is assumed. In this case, it is possible to develop a prophylactic / therapeutic agent for cancer that has a low pharmacological action on normal tissues, that is, a drug with few side effects.
  • the expression of the protein of the present invention is increased in cancer tissues. Further, when the activity of the protein of the present invention is inhibited, the protease activity is inhibited. In addition, when the activity of the protein of the present invention is inhibited, cancer cells induce apoptosis. Accordingly, the compounds or salts thereof that inhibit the activity of the protein of the present invention include, for example, colon cancer, breast cancer, lung cancer, prostate cancer, esophagus cancer, stomach cancer, liver cancer, biliary tract cancer, spleen cancer, kidney cancer, bladder cancer, It can be used as a therapeutic or prophylactic agent for cancers such as uterine cancer, testis cancer, thyroid cancer, thyroid cancer, brain tumor or blood tumor (preferably, kidney cancer, etc.).
  • the compound or its salt that inhibits the activity of the protein of the present invention can also be used as, for example, a protease inhibitor, an apoptosis inducing (or promoting) agent and the like.
  • a protease inhibitor an apoptosis inducing (or promoting) agent and the like.
  • it can be used as a therapeutic / preventive agent for rheumatoid arthritis, osteoarthritis, myocardial infarction, arteriosclerosis, etc.
  • By inhibiting the protease activity of the protein it can also be used as a therapeutic agent for treating neurodegenerative diseases (such as Alzheimer's disease).
  • the protein of the present invention is useful as a reagent for screening a compound or a salt thereof that inhibits the activity of the protein of the present invention (eg, protease activity, apoptosis-suppressing activity, etc.). provide.
  • a host transformed with a vector containing DNA encoding the protein of the present invention described above is used.
  • the host for example, animal cells such as COS 7 cells, CHI cells, and HEK293 cells are preferably used.
  • a transformant in which the protein of the present invention is expressed intracellularly or extracellularly by culturing by the method described above is preferably used.
  • the method for culturing cells capable of expressing the protein of the present invention is the same as the above-described method for culturing the transformant of the present invention.
  • Test compounds include, for example, peptides, proteins, non-peptidic compounds, synthetic compounds, fermentation products, cell extracts, plant extracts, animal tissue extracts, and the like.
  • test compound can be selected as a compound that inhibits the activity of the protein of the present invention.
  • the protease activity in the case (iii) is inhibited by about 20% or more, preferably 30% or more, more preferably about 50% or more, as compared with the case of the above (V).
  • a test compound can be selected as a compound that inhibits the activity of the protein of the present invention.
  • the compound having the activity of inhibiting the activity of the protein of the present invention is useful as a low-toxicity drug for suppressing the physiological activity of the protein of the present invention.
  • the compound or a salt thereof is, for example, a compound selected from peptides, proteins, non-peptidic compounds, synthetic compounds, fermentation products, cell extracts, plant extracts, animal tissue extracts, plasma and the like.
  • the salt of the compound those similar to the aforementioned salts of the peptide of the present invention are used.
  • compounds that inhibit the expression of the gene of the protein of the present invention or salts thereof include, for example, breast cancer, colon cancer, breast cancer, lung cancer, prostate cancer,
  • cancers such as esophageal cancer, stomach cancer, liver cancer, biliary tract cancer, spleen cancer, kidney cancer, bladder cancer, uterine cancer, testicular cancer, thyroid cancer, kidney cancer, brain tumor or blood tumor (preferably, liver cancer)
  • liver cancer preferably, liver cancer
  • the compound that inhibits the expression of the gene encoding the protein of the present invention or a salt thereof can also be used, for example, as a protease inhibitor, an apoptosis induction (or promotion) agent, and the like. ' ⁇
  • the polynucleotide (eg, DNA) of the present invention is useful as a reagent for screening a compound that inhibits the expression of the protein gene of the present invention or a salt thereof.
  • the screening methods include (a method of culturing cells capable of producing the protein of the present invention, and a method of (vi) culturing cells capable of producing the protein used in the present invention in the presence of a test compound.
  • a screening method that specializes in making a comparison with the case in which it is performed is mentioned.
  • the expression level of the gene (specifically, the amount of the protein of the present invention or the amount of mRNA encoding the protein) in the cases (V) and (vi) is measured and compared. I do. ,
  • test compound and cells having the ability to produce the protein of the present invention include the same cells as described above.
  • the amount of the protein is measured by a known method, for example, using an antibody that recognizes the protein of the present invention, and analyzing the protein present in a cell extract or the like according to a method such as Western analysis, ELISA, or a method analogous thereto. Can be measured.
  • the amount of mRNA is measured by a known method, for example, Northern hybridization using a nucleic acid containing SEQ ID NO: 6, SEQ ID NO: 31 or a part thereof as a probe. It can be measured according to a PCR method using a nucleic acid containing SEQ ID NO: 6, SEQ ID NO: 31 or a part thereof as a primer or primer, or a method analogous thereto.
  • the compound can be selected as a compound that inhibits the expression of the gene of the protein of the present invention.
  • the screening kit of the present invention contains the protein or partial peptide used in the present invention or a salt thereof, or a cell capable of producing the protein or partial peptide used in the present invention.
  • the compound obtained by using the screening method or the screening kit of the present invention or a salt thereof may be a test compound described above, for example, a peptide, a protein, a non-peptidic compound, a synthetic compound, a fermentation product, a cell extract, A compound or a salt thereof selected from a plant extract, an animal tissue extract, plasma, or the like, and a compound or a salt thereof that inhibits the activity of the protein of the present invention (eg, apoptosis inhibitory activity, etc.); Or a salt thereof, which inhibits the expression of the gene for the protein of
  • salt of the compound those similar to the aforementioned salts of the protein of the present invention are used.
  • the compound that inhibits the activity of the protein of the present invention or a salt thereof, and the compound or a salt thereof that inhibits the expression of the protein gene of the present invention are, for example, colon cancer, breast cancer, lung cancer, prostate cancer, esophageal cancer, and gastric cancer, respectively.
  • Treatment of cancers such as liver, biliary tract, spleen, kidney, bladder, uterus, testis, thyroid, kidney, brain or blood tumors (preferably, kidney cancer) ⁇ It is useful as a prophylactic agent or as a protease inhibitor, apoptosis inducing (or promoting) agent, etc.
  • the compound or a salt thereof obtained by using the screening method or the screening kit of the present invention is used as the above-mentioned therapeutic or prophylactic agent, it can be formulated into a preparation according to a conventional method.
  • compositions for oral administration include solid or liquid dosage forms such as tablets (including sugar-coated tablets and film-coated tablets), pills, granules, powders, turnips and the like.
  • Cell preparations including soft capsules), syrups, emulsions, suspensions and the like can be mentioned.
  • Such a composition is produced by a method known per se and contains a carrier, diluent or excipient commonly used in the field of formulation. 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, etc. are used.
  • Injections are intravenous, subcutaneous, intradermal, intramuscular, intravenous, intraarticular. Dosage forms such as agents.
  • Such injections are prepared according to a method known per se, for example, by dissolving, suspending or emulsifying the above compound or a salt thereof in a sterile aqueous or oily liquid usually used for injections.
  • aqueous solution for injection for example, physiological saline, isotonic solution containing glucose and other adjuvants is used, and a suitable solubilizing agent, for example, alcohol (eg, ethanol), polyalcohol (eg, , Propylene glycol, polyethylene glycol), nonionic surfactants (eg, polysorbate 80, HCO- 50 (polyoxyeihylene (5 Omol) adduci of hydrogenated castor oil)), etc. Is also good.
  • 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 an appropriate sample.
  • a suppository for rectal administration is prepared by mixing the above compound or a salt thereof with a usual suppository base.
  • the above-mentioned oral or parenteral pharmaceutical composition is conveniently prepared in a unit dosage form so as to be compatible with the dosage of the active ingredient.
  • dosage unit dosage forms include tablets, pills, capsules, injections (ampoules), suppositories, etc., and usually 5 to 500 mg, especially It is preferable that the compound contains 5 to 100 mg for an injection and 10 to 25 Omg for the other dosage forms.
  • compositions may contain another active ingredient as long as the composition does not cause an undesirable interaction with the above compound.
  • the preparations obtained in this way are safe and low toxic, for example, in humans or warm-blooded animals (eg, mice, rats, puppies, sheep, pigs, puppies, puppies, birds, It can be administered orally or parenterally to cats, dogs, monkeys, chimpanzees, etc.).
  • warm-blooded animals eg, mice, rats, puppies, sheep, pigs, puppies, puppies, birds, It can be administered orally or parenterally to 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, route of administration, and the like.
  • a compound or a compound that inhibits the activity of the protein of the present invention for the purpose of treating kidney cancer is
  • the salt is orally administered, generally, in an adult (with a body weight of 60 kg), the compound or its salt is administered in an amount of about 0.1 to ⁇ 0 Omg per day, preferably about 1.0 to 5 Omg, per day. More preferably, about 1.0 to 20 mg is administered.
  • the single dose of the compound or a salt thereof varies depending on the subject to be administered, the target disease, and the like.
  • the activity of the protein of the present invention for the treatment of kidney cancer may be reduced.
  • the inhibitory compound or a salt thereof is usually administered to an adult (with a body weight of 60 kg) in the form of an injection, the compound or a salt thereof is used in an amount of about 0.01 to 3 Omg per day, preferably about 0 to 3 Omg. It is convenient to administer about 1-2 Omg, more preferably about 0.1-10 mg, by injection to the cancerous lesion. In the case of other animals, the dose can be administered per 60 kg body weight.
  • 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. In particular, it can be used for quantification by a 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 the 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, an antigen, or an antibody-Bohara corresponding to the amount of antigen (eg, the amount of protein) in the test solution. Any method may be used as long as the method detects the amount of the complex by chemical or physical means and calculates this from a standard curve prepared using a standard solution containing a known amount of antigen. .
  • nephrometry, a competition method, an immunometric method and a sandwich method are suitably used, but it is particularly preferable to use a sandwich method described later in terms of sensitivity and specificity.
  • 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. Radioisotopes, if example embodiment, [1 2 5 I], [1 3 1 I], [3 H], and [1 4 C] used.
  • the above-mentioned enzymes those which are stable and have a large specific activity are preferable. For example, 3-galactosidase,
  • fluorescent substance for example, fluorescamine, fluorescein isothiosinate and the like are used.
  • luminescent substance for example, luminol, luminol derivative, reluciferin, lucigenin and the like are used.
  • a biotin-avidin system can be used for binding the antibody or antigen to the labeling agent.
  • the carrier include insoluble polysaccharides such as agarose, dextran, and cellulose; synthetic resins such as polystyrene, polyacrylamide, and silicon; and glass.
  • the test solution is reacted with the insolubilized monoclonal antibody of the present invention (primary reaction), and another labeled monoclonal antibody of the present invention is further reacted.
  • secondary reaction the amount of the protein of the present invention in the test solution can be quantified by measuring the activity of the labeling agent on the insolubilized carrier.
  • 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 the measurement sensitivity and the like. May be used.
  • the monoclonal antibody of the present invention used in the primary reaction and the secondary reaction has different binding sites for the protein of the present invention.
  • the body is preferably used. That is, for example, when the antibody used in the primary reaction and 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 nephrometry.
  • a competition method after the antigen in the test solution and the labeled antigen are allowed to react competitively with the antibody, the unreacted labeled antigen (F) and the labeled antigen (B) bound to the antibody are separated.
  • B / F separation Measure the amount of labeling for either B or F, and quantify the amount of antigen in the test solution.
  • a soluble antibody is used as an antibody
  • BZF separation is performed using polyethylene glycol
  • a liquid phase method using a second antibody against the antibody
  • a solid phase antibody is used as the first antibody.
  • An immobilization 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 labeled antibody, and then the solid phase and the liquid phase are separated.
  • the antigen in the solution is allowed to react with an excessive amount of the labeled antibody, and then the immobilized antigen is added to bind the unreacted labeled antibody to the solid phase, and then the solid phase and the liquid phase are separated.
  • the amount of the label in either phase is measured to determine the amount of the antigen in the test solution.
  • nephrometry the amount of insoluble sediment resulting from the antigen-antibody reaction in a gel or in a solution is measured. Small amount of antigen in test solution, small amount of sediment Even when only an object can be obtained, laser nephrometry utilizing scattering by laser is preferably used.
  • the protein measuring system of the present invention may be constructed by adding ordinary technical considerations of those skilled in the art to ordinary conditions and operating methods in each method. For details of these general technical means, reference can be made to reviews and written documents.
  • the protein of the present invention can be quantified with high sensitivity by using the antibody of the present invention.
  • an increase in the concentration of the protein of the present invention is detected by quantifying the concentration of the protein of the present invention using the antibody of the present invention, for example, colon cancer, breast cancer, lung cancer, prostate cancer, esophagus Cancer, such as cancer, stomach cancer, liver cancer, biliary tract cancer, spleen cancer, renal cancer, bladder cancer, uterine cancer, testicular cancer, thyroid cancer, kidney cancer, brain tumor or blood tumor, or may be affected in the future Can be diagnosed as high.
  • the antibody of the present invention can be used for detecting the protein of the present invention present in a subject such as a body fluid or a tissue.
  • preparation of an antibody column used for purifying the protein of the present invention, and protein of the present invention in each fraction at the time of purification Of the present invention analysis of the behavior of the protein of the present invention in test cells, and the like.
  • the DNA of the present invention can be used, for example, in humans or warm-blooded animals (e.g., rats, mice, guinea pigs, egrets, birds, higgies, pigs, pigs, dogs, cats, dogs, monkeys, DNA or mRNA encoding the protein of the present invention or a partial peptide thereof (gene abnormality) in a chimpanzee or the like can be detected.
  • DNA or mRNA damage, mutation, or mutation of the DNA or mRNA can be detected. It is useful as a diagnostic agent for genes such as decreased expression, increased or overexpressed DNA or mRNA.
  • the above-described genetic diagnosis using the DNA of the present invention includes, for example, the known Northern Eighth hybridization and PCR-SSCP method (Genomics, Vol. 5, pp. 874-879 (1989), ⁇ Ob ⁇ The National Academy of Sciences ⁇ Ob ⁇ Sciences ⁇ Ob ⁇ U.S.A. (Proceedings of the National Academy of Sciences of the United States of America), 86, 2766-2770 (1 989 )).
  • colon cancer for example, colon cancer, breast cancer, lung cancer, prostate cancer, esophageal cancer, gastric cancer
  • a cancer such as liver cancer, biliary tract cancer, spleen cancer, renal cancer, bladder cancer, uterine cancer, testicular cancer, thyroid cancer, kidney cancer, brain tumor or blood tumor.
  • the antisense polynucleotide of the present invention which can complementarily bind to the DNA of the present invention and suppresses the expression of the DNA, has low toxicity, and functions of the protein of the present invention or the DNA of the present invention in vivo.
  • apoptosis inhibitory activity can be suppressed, for example, colon cancer, breast cancer, lung cancer, prostate cancer, esophagus cancer, stomach cancer, liver cancer, biliary tract cancer, spleen cancer, kidney cancer, bladder cancer, uterine cancer It can be used as a therapeutic or prophylactic agent for cancer such as testicular cancer, thyroid cancer, thyroid cancer, brain tumor or blood tumor (preferably, cancer of the kidney).
  • the antisense polynucleotide of the present invention is, for example, For example, it can be used as an apoptosis inducing (or promoting) agent, a protease inhibitor and the like. '
  • antisense polynucleotide When used as the above-mentioned therapeutic / prophylactic agent, inducer (or enhancer), inhibitor or the like, it can be formulated and administered according to a method known per se.
  • the antisense polynucleotide after inserting the antisense polynucleotide alone or into an appropriate vector such as a retrovirus vector, an adenovirus vector, an adenovirus vector, an adenovirus vector, etc., according to a conventional method, It can be administered orally or parenterally to humans or mammals (eg, rats, puppies, sheep, sheep, bush, foxes, cats, dogs, monkeys, etc.).
  • the antisense polynucleotide can be administered as it is or in the form of a formulation together with a physiologically acceptable carrier such as an adjuvant for promoting uptake, and administered by a gene gun or a catheter such as a hydrogel catheter. Alternatively, they can be made into an aerosol and administered locally into the trachea as an inhalant.
  • the antisense polynucleotide is formulated alone or together with a carrier such as liposome (injection), and intravenously or subcutaneously. Etc. may be administered.
  • the dose of the antisense polynucleotide varies depending on the target disease, the subject of administration, the route of administration, and the like.For example, when the antisense polynucleotide of the present invention is administered for the purpose of treating kidney cancer, a general method is used. For adults (weight 60 kg), the antisense polynucleotide is administered in an amount of about 0.1 to 10 O mg per day. Furthermore, the antisense polynucleotide can also be used as a diagnostic oligonucleotide probe for examining the presence of DNA of the present invention in tissues and cells and the state of expression thereof.
  • RNA encoding the protein of the present invention Can also suppress the expression of the gene of the present invention, and suppress the function of the protein used in the present invention or ': ⁇ : D ⁇ ⁇ ⁇ ⁇ ⁇ used in the present invention in vivo.
  • colorectal cancer breast cancer, lung cancer, prostate cancer, esophageal cancer, stomach cancer, liver cancer, biliary tract cancer, spleen cancer, kidney cancer, bladder cancer, uterine cancer, testicular cancer, thyroid cancer, tiger cancer
  • cancers such as brain tumors or hematological tumors (preferably, for example, Teng's carcinoma), apoptosis-inducing (or promoting) agents, and protease inhibitors.
  • the double-stranded RNA can be designed and produced based on the sequence of the polynucleotide of the present invention according to a known method (eg, Nature, 411, 494, 2001).
  • the ribozyme can be designed and manufactured based on the sequence of the polynucleotide of the present invention according to a known method (eg, TRENDS in Molecular Medicine, Vol. 7, pp. 221, 2001). .
  • it can be produced by linking a known lipozyme to a part of RNA encoding the protein of the present invention.
  • a part of the RNA encoding the protein of the present invention includes a portion (RNA fragment) adjacent to the cleavage site on the RNA of the present invention which can be cleaved by a known lipozyme.
  • RNA or lipozyme When the above-described double-stranded RNA or lipozyme is used as the above-mentioned prophylactic / therapeutic agent, inducer (or enhancer) or inhibitor, it can be prepared and administered in the same manner as an antisense polynucleotide.
  • the antibodies of the present invention include, for example, colon cancer, breast cancer, lung cancer, prostate cancer, esophageal cancer, stomach cancer, liver cancer, biliary tract cancer, spleen cancer, renal cancer, bladder cancer, uterine cancer, testicular cancer, thyroid cancer, kidney cancer, It can be used as a prophylactic / therapeutic agent (eg, pectin etc.) for cancer such as brain tumor or hematological tumor (preferably, Teng's carcinoma etc.).
  • the antibody of the present invention can also be used, for example, as an apoptosis inducing (or promoting) agent or a protease inhibitor.
  • the therapeutic / prophylactic agent, inducer (or promoter), or inhibitor for the above-mentioned diseases containing the antibody of the present invention has low toxicity and can be used as a liquid or as a pharmaceutical composition in an appropriate dosage form.
  • animals eg, rats, egrets, sheep, sheep, bush, pests, cats, dogs, monkeys, etc.
  • it can be administered as a vaccine according to a standard method.
  • the antibody of the present invention may be administered as it is or as an appropriate pharmaceutical composition. May be administered.
  • the pharmaceutical composition used for administration may contain the antibody of the present invention or a salt thereof and a pharmacologically acceptable carrier, diluent or excipient.
  • Such a pharmaceutical composition is provided as a dosage form suitable for oral or parenteral administration.
  • compositions for parenteral administration for example, injections, suppositories, vaccines, etc. are used.
  • Injections are intravenous, subcutaneous, intradermal, intramuscular, intravenous, etc. May be included.
  • Such an injection can be prepared according to a known method.
  • Injection preparations can be prepared, for example, by dissolving, suspending, or emulsifying the antibody of the present invention or a salt thereof in a sterile aqueous liquid or oily liquid used for usual injections.
  • aqueous liquid for injection for example, physiological saline, isotonic solution containing budose sugar and other adjuvants, and the like
  • suitable solubilizing agents for example, alcohol (eg, ethanol), polyalcohol ( For example, propylene glycol, polyethylene glycol), nonionic surfactant [eg, polysorbate 80, HCO-50 (olyoxyethylene (50 mol) adduct of hydrogenated castor oil)] and the like may be used in combination.
  • alcohol eg, ethanol
  • polyalcohol for example, propylene glycol, polyethylene glycol
  • nonionic surfactant eg, polysorbate 80, HCO-50 (olyoxyethylene (50 mol) adduct of hydrogenated castor oil)
  • 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
  • compositions for oral administration include solid or liquid dosage forms, specifically tablets (including dragees and film-coated tablets), pills, granules, powders, capsules (soft capsules) Syrups, emulsions, suspensions and the like.
  • Such compositions are prepared by known methods and may contain carriers, diluents or excipients commonly used in the field of formulation.
  • carriers and excipients for tablets for example, lactose, starch, sucrose, and magnesium stearate are used.
  • parenteral or oral pharmaceutical composition is conveniently prepared in the form of a dosage unit so as to match the dosage of the active ingredient.
  • dosage unit forms include, for example, tablets, pills, capsules, injections (ampoules), and suppositories.
  • the antibody content is usually about 5 to 50 mg per dosage unit dosage form, 03013920
  • the dose of the above-mentioned prophylactic, therapeutic, inducer, or inhibitor containing the antibody of the present invention varies depending on the administration target, target disease, symptoms, administration route, and the like.
  • the antibody of the present invention is used as a single dose, usually about 0.01 to 2 Omg / kg body weight, preferably about 0.1 to 1 Omg / kg body weight, and more preferably about 0.1 lmg / kg body weight. It is convenient to administer the SmgZkg body child by intravenous injection about 1 to 5 times a day, preferably about 1 to 3 times a day. In the case of other parenteral administration and oral administration, an equivalent dose can be administered. If the symptoms are particularly severe, the dose may be increased accordingly.
  • the antibody of the present invention can be administered by itself or as a suitable pharmaceutical composition.
  • the pharmaceutical composition used for the administration contains the antibody or a salt thereof and a pharmacologically acceptable carrier, diluent or excipient.
  • a composition is provided as a dosage form suitable for oral or parenteral administration (eg, intravascular injection, subcutaneous injection, etc.).
  • compositions may contain other active ingredients as long as the composition does not cause an undesirable interaction with the above-mentioned antibody. .
  • An apoptosis inducing agent comprising a compound having an angle of inhibition of activity of HTRA3 or a salt thereof of the present invention and a compound or a salt thereof having an activity of inhibiting expression of HTRA3 are contained.
  • An apoptosis-inducing agent comprising; a "protease inhibitor comprising a compound having an HTRA3 activity inhibitory action or a salt thereof”; and "a compound having a HTRA3 expression inhibitory action or a salt thereof.
  • the “compound having an activity of inhibiting the activity of HTR A3” may be any compound having an activity of inhibiting the activity of HTR A3, such as colorectal cancer, breast cancer, lung cancer, prostate cancer, esophageal cancer, gastric cancer, Prevention and treatment of cancers such as liver cancer, biliary tract cancer, spleen cancer, renal cancer, bladder cancer, uterine cancer, testicular cancer, thyroid cancer, kidney cancer, brain tumor or blood tumor (preferably, kidney cancer) Used as
  • HTRA 3 expression inhibitory compound Any compound may be used as long as it has the following compounds, for example, colon cancer, breast cancer, lung cancer, prostate cancer, esophageal cancer, stomach cancer, liver cancer, biliary tract cancer, spleen cancer, kidney cancer, bladder cancer, uterine cancer, testicular cancer, It is used as a prophylactic / therapeutic agent for cancer such as thyroid cancer, thyroid cancer, brain tumor or blood tumor (preferably thyroid cancer).
  • the prophylactic / therapeutic agent is produced in the same manner as described above.
  • the present invention relates to a DNA encoding the exogenous protein of the present invention (hereinafter abbreviated as the exogenous DNA of the present invention) or a mutant DNA thereof (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, the Lipofection, • DNA can be produced by transferring the target DNA by a method such as a coagulation method, a coagulation method, a microinjection method, a particle gun method, or a DEAE-dextran method.
  • the exogenous DNA of the present invention can be transferred to somatic cells, organs of living organisms, tissue cells, and the like, and used for cell culture, tissue culture, and the like. Can be fused with the above-mentioned germinal cells by a cell fusion method known per se to produce the DNA-transferred animal of the present invention.
  • non-human mammal for example, red sea lions, bushes, hidge, goats, blue egrets, dogs, cats, guinea pigs, eight musters, mice, rats and the like are used.
  • ontogeny and biological cycle are relatively short in terms of the creation of disease animal model systems, JP2003 / 013920
  • mice for example, pure strains such as C57B LZ6 strains and DBA2 strains, and hybrid strains such as B6C3 strains, BDFi strains, BGDSFi strains, BALB / c strain, ICR strain, etc.
  • rat eg, Wistar, SD, etc.
  • mammals humans and the like can be mentioned in addition to the above-mentioned non-human mammals.
  • the exogenous DNA of the present invention refers to the 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 a DNA having a mutation (for example, mutation) in the base sequence of the original DNA of the present invention, specifically, addition or deletion of a base, substitution with another base, etc. DNA that has been used is used, and abnormal DNA is also included.
  • a mutation for example, mutation
  • the abnormal DNA refers to a DNA that expresses an abnormal protein of the present invention, and for example, a DNA that expresses a protein that suppresses the function of the normal protein of the present invention is used.
  • the exogenous DNA of the present invention may be derived from mammals of the same or different species as the target animal.
  • the DNA construct eg, vector-1 to which the human DNA of the present invention is bound is fertilized into a fertilized egg of a target mammal, for example, a mouse fertilized egg.
  • a DNA-transferring mammal of the present invention that highly expresses DNA 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 baculovirus.
  • animal viruses such as Among them, plasmids derived from Escherichia coli, plasmids derived from Bacillus subtilis or plasmids derived from yeast are preferred. It is used well.
  • Examples of the promoter for controlling the DNA expression include: 1) a promoter derived from a virus (eg, Simian virus, cytomegalovirus, Moroni monoleukemia virus, JC virus, breast cancer virus, polio virus, etc.); ) Promoters derived from various mammals (human, egret, dog, cat, guinea pig, hamus, rat, mouse, etc.), for example, albumin, insulin II, peroplaskin II, erasose, erythropoietin, endothelin , Muscle creatine kinase, glial fibrillary acidic protein, daltathione S_transferase, platelet-derived growth factor) 3, keratin Kl, 1 ⁇ 10 ⁇ and 1 ⁇ 14, collagen types I and II, Cyclic AMP-dependent protein kinase / 3 I subunit, dist fin , Tartrate-resistant alkaline phosphatase, atrial natri
  • the vector preferably has a sequence that terminates the transcription of the messenger RNA of interest in a DNA-transferred mammal (generally called a terminator).
  • a DNA sequence can be used, and preferably, an SV40 terminator of simian virus or the like is used.
  • the splicing signal of each DNA, the enhancer region, a part of the intron of eukaryotic DNA, etc. are used to further express the target foreign DNA. • 5 'upstream of the promoter region, between the promoter region and the translation region Alternatively, it can be linked to the 3 'downstream of the translation region depending on the purpose. .
  • the normal translation region of the protein of the present invention is derived from human or various mammals (eg, egrets, dogs, cats, guinea pigs, hamsters, rats, mice, etc.), kidney, thyroid cells, fibroblasts It is possible to obtain all or part of genomic DNA from DNA and various commercially available genomic DNA libraries, or complementary DNA prepared by known methods from liver, kidney, thyroid cells, and fibroblast-derived RNA as raw materials. I can do it.
  • the foreign abnormal DNA can produce a translation region obtained by mutating the translation region of a normal protein obtained from the above cells or tissues by point mutagenesis.
  • the translation region can be prepared as a DNA construct that can be expressed in a transgenic animal by a conventional DNA engineering technique in which it is ligated downstream of the above promoter and, if desired, upstream of the transcription termination site.
  • Transfer of the exogenous DNA of the present invention at the fertilized egg cell stage is ensured to be present in all germ cells and somatic cells of the target mammal.
  • the presence of the exogenous DNA of the present invention in the germinal cells of the transgenic animal after transfer of the DNA indicates that the exogenous DNA of the present invention is present in all the progeny of the transgenic animal and in all of its germ cells and somatic cells. Means to keep.
  • the progeny of this type of animal that has inherited 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 exogenous normal DNA of the present invention was transferred
  • the non-human mammal was confirmed to stably maintain the exogenous DNA by mating, and was subcultured as a DNA-bearing animal in a normal breeding environment. I can do it. '
  • Transfer of the exogenous DNA of the present invention at the fertilized egg cell stage is ensured to be present in excess in all germ cells and somatic cells of the target mammal.
  • Excessive presence of the exogenous DNA of the present invention in the germinal cells of the animal after the transfer of the DNA indicates that the offspring of the animal of the present invention is present in all the germinal and somatic cells of the animal. It means having in excess.
  • This kind of animal inherited the exogenous DNA of the present invention Progeny have excessive amounts of the exogenous DNA of the present invention in all of their germ cells and somatic cells.
  • the normal DNA of the present invention is highly expressed, and the function of the protein of the present invention is finally enhanced by promoting the function of endogenous normal DNA.
  • the disease may develop and can be used as a model animal for the disease.
  • using the normal DNA-transferred animal of the present invention it is possible to elucidate the pathological mechanism of the hyperactivity of the protein of the present invention and diseases associated with the protein of the present invention, and to examine a method for treating these diseases. It is possible.
  • a preventive / therapeutic agent for a disease associated with the protein of the present invention such as colon cancer, Cancers such as breast, lung, prostate, esophagus, stomach, liver, biliary, spleen, kidney, bladder, uterus, testes, thyroid, kidney, brain, or blood tumors
  • a preventive / therapeutic agent for a disease associated with the protein of the present invention such as colon cancer, Cancers such as breast, lung, prostate, esophagus, stomach, liver, biliary, spleen, kidney, bladder, uterus, testes, thyroid, kidney, brain, or blood tumors
  • prophylactic and therapeutic agents such as kidney cancer
  • a non-human mammal having the exogenous abnormal DNA of the present invention can be subcultured in a normal breeding environment as an animal having the DNA after confirming that the exogenous DNA is stably maintained by the cross. I can do it. Furthermore, the desired foreign DNA can be incorporated into the above-mentioned plasmid and used as a raw material.
  • the DNA construct with the 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 germ cells of the produced animal after DNA transfer means that all the offspring of the produced animal have the abnormal DNA of the present invention in all of the germinal and somatic cells. .
  • Progeny of this type of animal that has inherited the exogenous DNA of the present invention have the abnormal DNA of the present invention in all of its germinal and somatic cells.
  • a homozygous animal having the introduced DNA on both homologous chromosomes is obtained, and all the offspring are obtained by mating the male and female animals.
  • the offspring can be bred to have the DNA.
  • the abnormal DNA of the present invention is highly expressed, and the function of the protein of the present invention is finally impaired by inhibiting the function of endogenous normal DNA. It may be active refractory and can be used as a model animal for the disease. 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 characterized in that the abnormal protein of the present invention inhibits the function of a normal protein (dominant negative action) in the function-inactive refractory disease of the protein of the present invention. It becomes a model to elucidate.
  • the agent for preventing or treating the protein of the present invention or a functionally inactive type refractory disease for example, Cancers such as colorectal, breast, lung, prostate, esophageal, stomach, liver, biliary, spleen, kidney, bladder, uterus, testes, thyroid, kidney, brain or blood tumors It can also be used for screening tests for prophylactic or therapeutic agents (preferably, Teng's carcinoma).
  • the protein of the present invention is functionally inactive. It is possible to examine clinical symptoms of diseases related to the protein of the present invention, including type refractory disease, etc., and obtain more detailed pathological findings in each organ of the disease model related to the protein of the present invention. Thus, it can contribute to the development of new treatment methods, and the research and treatment of secondary diseases caused by the diseases.
  • the protein of the present invention can be identified, its relationship with apoptosis, differentiation or proliferation, or its signal transduction mechanism can be examined, and its abnormality can be examined. It is an effective research material for elucidation.
  • a therapeutic agent for a disease associated with the protein of the present invention including a functionally inactive refractory type of the protein of the present invention
  • using the DNA-transferred animal of the present invention Using a quantitative method or the like, it is possible to provide an effective and rapid screening method for the therapeutic agent for the disease.
  • using the DNA transgenic animal of the present invention or the exogenous DNA expression vector of the present invention it is possible to study and develop a method for treating a DNA associated with the protein of the present invention. is there.
  • the present invention provides a non-human mammalian embryonic stem cell in which the DNA of the present invention is inactivated and a non-human mammal deficient in expression of the DNA of the present invention.
  • the DNA is inactivated by introducing a reporter gene (eg, a 3-galactosidase gene derived from Escherichia coli), and the reporter gene is transformed into the DN of the present invention.
  • a reporter gene eg, a 3-galactosidase gene derived from Escherichia coli
  • the non-human mammal according to (6) which can be expressed under the control of a promoter for A,
  • a compound that promotes or inhibits the promoter overnight activity against DNA of the present invention which comprises administering a test compound to the animal described in (7) and detecting the expression of a reporter gene. Or a method for screening a salt thereof.
  • the non-human mammalian embryonic stem cells in which the DNA of the present invention has been inactivated are artificially mutated DNA of the present invention possessed by the non-human mammal to suppress the expression of the DNA, or By substantially losing the activity of the protein of the present invention encoded by the DNA, the DNA does not substantially have the ability to express the protein of the present invention (hereinafter sometimes referred to as the knockout DNA of 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 for artificially mutating the DNA of the present invention can be performed, for example, by deleting a part or all of the DNA sequence by a genetic engineering technique, and inserting or substituting another DNA.
  • the knockout DNA of the present invention may be prepared by, for example, shifting the codon reading frame or disrupting the function of the promoter or exon by these mutations.
  • Non-human mammalian embryonic stem cells in which the DNA of the present invention has been inactivated include, for example, The DNA of the present invention possessed by a non-human mammal is isolated and its exon portion is a drug resistance gene typified by a neomycin resistance gene, a hygromycin resistance gene, or 1 ac Z (/ 3-galactosidase gene), cat (Chloramphenicol acetyltransferase gene) to destroy the exon function by introducing a reporter gene or the like, or to terminate transcription of the gene in the intron portion between exons.
  • a drug resistance gene typified by a neomycin resistance gene, a hygromycin resistance gene, or 1 ac Z (/ 3-galactosidase gene), cat (Chloramphenicol acetyltransferase gene) to destroy the exon function by introducing a reporter gene or the like, or to terminate transcription of the gene in the intron portion between
  • DNA strand having the DN A sequence was constructed so as to destroy the gene (hereinafter, abbreviated as targeting computing vector) to.
  • a sequence for example, a po 1 yA additional signal
  • DNA strand having the DN A sequence was constructed so as to destroy the gene (hereinafter, abbreviated as targeting computing vector) to.
  • DNA is introduced into the chromosome of the animal by homologous recombination, and the obtained ES cells are subjected to Southern hybridization analysis using the DNA sequence on or near the DNA of the present invention as a probe, or DNA on the targeting vector. It can be obtained by analyzing the sequence and the DNA sequence of the neighboring region other than the DNA of the present invention used for preparing the targeting vector by PCR using the primers and selecting the knockout ES cells of 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, or according to the known method of Evans and Kaufma. May be newly established.
  • mouse ES cells currently, 129 ES cells are generally used, but since the immunological background is not clear, an alternative pure immunological and genetically
  • C57BL / 6 mice or BDFi mice C57BLZ6 Those established using F 1 ) with DBA / 2 can also be used favorably.
  • BDFi mice have the advantage of high number of eggs collected and robust eggs, and also have C57BLZ6 mice as a background, so the ES cells obtained using them produced pathological model mice. It can be advantageously used because the genetic background can be replaced by C57BL / 6 mice by backcrossing with C57BLZ6 mice.
  • blastocysts 3.5 days after fertilization are generally used, but in addition to this, a large number of cells can be efficiently collected by collecting 8-cell embryos and culturing them up to blastocysts. Early embryos can be obtained.
  • Either male or female ES cells may be used, but male ES cells are generally more convenient for producing a germline chimera. It is also desirable to discriminate between males and females as soon as possible in order to reduce the complexity of culturing.
  • An example of a method for determining the sex of ES cells is a method of amplifying and detecting a gene in the sex-determining region on the Y chromosome by PCR.
  • this method conventionally, for example G-banding method, it requires about 1 0 6 cells for karyotype analysis, since suffices ES cell number of about 1 colony (about 50), Culture first
  • the primary selection of ES cells in the stage can be performed by gender discrimination, and the early selection of male cells can greatly reduce the labor required in the initial culture.
  • the embryonic stem cell line obtained in this way usually has very good growth potential, but it must be carefully subcultured because it tends to lose its ability to generate individuals.
  • a suitable feeder cell such as STO fibroblasts
  • a CO2 incubator preferably 5% CO2, 95% air
  • LIF 1-1 000 OU / ml
  • trypsin / EDTA solution usually 0.001-0.5
  • % Trypsin Z0.1-1.5 mM EDTA, preferably about 0.1% trypsin / ImM EDTA
  • Such subculture is usually performed every 11 to 13 days. At this time, cells should be observed, and if morphologically abnormal cells are found, it is desirable to discard the cultured cells. .
  • ES cells can be transformed into various types of cells, such as parietal, visceral, and cardiac muscle, by monolayer culture up to high density or suspension culture until cell clumps are formed under appropriate conditions.
  • MJ Evans and MH Kaufman Nature 292, 154, 1981; GR Martin Proceedings of the National Academy of Sciences Proc. Natl. Acad. Sci. USA) 78, 7634, 1981; T. Doetschman et al., Journal of Embryology and Experimental Morphology, 87, 27, 1985.
  • the DNA deficient cells of the present invention obtained by differentiating the ES cells of the present invention are useful in the cell biology of the protein of the present invention in the mouth of in vivo.
  • the non-human mammal deficient in expression of the DNA of the present invention can be distinguished from a normal animal by measuring the mRNA amount of the animal using a known method and indirectly comparing the expression level. .
  • non-human mammal those similar to the above can be used.
  • the non-human mammal deficient in DNA expression of the present invention may be obtained, for example, by introducing the evening getter vector prepared as described above into a mouse embryonic stem cell or a mouse egg cell and introducing the same into the present invention.
  • DNA of the present invention is inactivated by homologous recombination to replace the DNA sequence of the present invention on the chromosome of mouse embryonic stem cells or mouse egg cells by gene homologous recombination, thereby knocking out the DNA of the present invention. be able to.
  • the cells in which the DNA of the present invention has been knocked out are the DNA sequence on the Southern hybridization analysis or evening getter vector using the DNA sequence on or near the DNA of the present invention as a probe and the evening getter vector.
  • the DNA can be determined by PCR analysis using the DNA sequence of the neighboring region other than the DNA of the present invention derived from the mouse used as a primer.
  • the cell line in which the DNA of the present invention has been inactivated is cloned by homologous gene recombination, and the cells are cultured at an appropriate time, for example, at the 8-cell stage of non-human cells.
  • the chimeric embryo is injected into a mammalian embryo or blastocyst, and the produced chimeric embryo is transplanted into the uterus of the pseudopregnant non-human mammal.
  • the produced animal is a chimeric animal comprising both cells having the normal DNA locus of the present invention and cells having the artificially mutated DNA locus of the present invention.
  • a bamase having the DNA locus of the present invention in which a part of the germ cells of the chimeric animal is mutated From the population obtained by crossing such a chimeric individual with a normal individual, all tissues are artificially obtained. It can be obtained by selecting an individual composed of cells having the DNA locus of the present invention to which the mutation has been added, for example, by judging coat color.
  • the individuals obtained in this manner are usually individuals deficient in the hetero-expression of the protein of the present invention, mated with individuals deficient in the hetero-expression of the protein of the present invention, and obtained from their offspring to obtain the protein of the present invention. It is possible to obtain an individual lacking homo-expression.
  • a transgenic non-human mammal in which the targeting vector has been introduced into the chromosome can be obtained by injecting a DNA solution into the transgenic non-human mammal. It can be obtained by selecting those with a mutation in the DNA locus.
  • the animal individual obtained by mating can confirm that the DNA has been knocked out, and can be reared in a normal rearing environment.
  • the germline can be obtained and maintained according to a conventional method. That is, by mating male and female animals having the inactivated DNA, a homozygous animal having the inactivated DNA on both homologous chromosomes can be obtained.
  • the obtained homozygous animal can be efficiently obtained by breeding the mother animal in such a manner as to have one normal animal and one homozygote.
  • male and female heterozygous animals homozygous and heterozygous animals having the inactivated DNA are bred and subcultured.
  • the non-human mammal embryonic stem cells in which the DNA of the present invention is inactivated are very useful for producing the non-human mammal deficient in expression of the DNA of the present invention.
  • the non-human mammal deficient in expression of the DNA of the present invention lacks various biological activities that can be induced by the protein of the present invention, a disease caused by inactivation of the biological activity of the protein of the present invention. Since it can be a model, it is useful for investigating the causes of these diseases and studying treatment methods.
  • the non-human mammal deficient in expression of the DNA of the present invention can be used for screening for a compound having a therapeutic: preventive effect against a disease caused by deficiency or damage of the DNA of the present invention.
  • the present invention is characterized in that a test compound is administered to a non-human mammal deficient in expression of the DNA of the present invention, and changes in the animal are observed and measured.
  • the present invention provides a method for screening a compound or a salt thereof, which has a therapeutic / preventive effect on a disease such as cancer (preferably, Teng pancreatic cancer).
  • a disease such as cancer (preferably, Teng pancreatic cancer).
  • Examples of the non-human mammal deficient in expressing DNA of the present invention used in the screening method include the same ones 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.These compounds are novel compounds. Or a known compound.
  • a non-human mammal deficient in expression of the DNA of the present invention is treated with a test compound, compared with a non-treated control animal, and changes in organs, tissues, disease symptoms, etc. of the animal are used as indicators. Therapeutic and prophylactic effects of test compounds can be tested.
  • test compound for example, oral administration, intravenous injection and the like are used, and it can be appropriately selected according to the symptoms of the test animal, the 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.
  • test compound is administered to a non-human mammal deficient in expressing DNA of the present invention, and the test compound non-administered group and cancer
  • the difference in the degree of onset of cancer and the difference in the degree of healing of cancer are observed over time in the above tissues.
  • the disease symptom of the test animal is improved by about 10% or more, preferably about 3'0% or more, more preferably about 50% or more.
  • the test compound can be selected as a compound having a therapeutic / preventive effect on the above-mentioned diseases.
  • the compound obtained by using the screening method is a compound selected from the test compounds described above, and has a therapeutic / preventive effect against a disease caused by a deficiency or damage of the protein of the present invention. It can be used as a safe and low toxic prophylaxis drug as a therapeutic agent. Further, a compound derived from the compound obtained by the above screening can be used in the same manner.
  • Diseases caused by protein deficiency or damage of the present invention include, for example, Neurodegenerative diseases such as Alzheimer's disease, Huntington's disease, amyotrophic lateral sclerosis; ischemic heart diseases such as angina pectoris and myocardial infarction; and female infertility.
  • compounds or salts thereof that promote the activity of the protein of the present invention compounds or salts thereof that promote the expression of the gene of the protein of the present invention, the protein itself of the present invention, the gene of the protein of the present invention, etc.
  • 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, etc.) and bases (eg, alkali metals, etc.). And the like, and particularly preferred are physiologically acceptable acid addition salts.
  • Such salts include, for example, salts with inorganic acids' (eg, hydrochloric acid, phosphoric acid, hydrobromic acid, sulfuric acid, etc.) and organic acids (eg, acetic acid, formic acid, propionic acid, fumaric acid, maleic acid, Acid, succinic acid, tartaric acid, citric acid, malic acid, oxalic acid, benzoic acid, methanesulfonic acid, benzenesulfonic acid, etc.).
  • a drug containing the compound or a salt thereof obtained by the screening method can be produced in the same manner as the above-mentioned drug containing the protein of the present invention.
  • the preparations obtained in this way are safe and low toxic and can be used, for example, in humans or mammals (eg, rats, mice, guinea pigs, egrets, sheep, sheep, bushus, dogs, dogs, cats, dogs). , Monkeys, etc.).
  • the dosage of the compound or a salt thereof varies depending on the target disease, the administration subject, the administration route, and the like.
  • the adult with a body weight of 6 O kg
  • About 0.1 to 100 111, preferably about 1.0 to 50 mg, and more preferably about 1.0 to 20 mg of the compound per day are administered to the patient with Teng's cancer.
  • the single dose of the compound varies depending on the administration subject, the target disease and the like.
  • about 0.1 to 3 Omg of the compound per day preferably about 0.1 to 2 Omg, more preferably about 0.1 to 1 Omg per day. It is convenient to administer about Omg by intravenous injection. In the case of other animals, the dose can be administered in terms of 60 kg.
  • the present invention provides a non-human mammal deficient in expression of the DNA of the present invention, which comprises administering a test compound to detect the expression of a repo overnight gene.
  • a method for screening a compound to be inhibited or a salt thereof is provided.
  • the non-human mammal deficient in expressing DNA of the present invention may be a non-human mammal deficient in expressing DNA of the present invention, wherein the DNA of the present invention is obtained by introducing a repo allele gene. Those inactivated and capable of expressing the repo overnight gene under the control of the promoter for the DNA of the present invention are used.
  • test compound examples include the same compounds as described above.
  • reporter gene the same one as described above is used, and a j3_galactosidase gene (1 ac Z), a soluble alkaline phosphatase gene or a luciferase gene is suitable.
  • the tissue originally expressing the protein of the present invention may Instead of the protein of the present invention,) 3-galactosidase is expressed. Therefore, for example, by staining with a reagent serving as a substrate for 3-galactosidase (such as 5-promo-4-chloro-3-indolyl-3) -galactopyranoside (X-gal),
  • a reagent serving as a substrate for 3-galactosidase such as 5-promo-4-chloro-3-indolyl-3) -galactopyranoside (X-gal)
  • the expression state of the protein of the present invention in an animal body can be observed.
  • the protein-deficient mouse of the present invention or a tissue section thereof is fixed with glutaraldehyde or the like, and a phosphate-buffered physiological diet is used.
  • RNA encoding 1acZ may be detected according to a conventional method.
  • the compound or a salt thereof obtained by the above screening method is a compound selected from the test compounds described above, and is a compound that promotes or inhibits the promoter activity for DNA of the present invention.
  • the compound obtained by the screening method may form a salt
  • the salt of the compound may be a physiologically acceptable acid (eg, an inorganic acid) or a base (eg, an alkali metal).
  • physiologically acceptable acid addition salts include salts with inorganic acids (eg, hydrochloric acid, phosphoric acid, hydrobromic acid, sulfuric acid, etc.), and organic acids (eg, acetic acid, formic acid, propionic acid, fumaric acid, maleic acid, Salts with succinic acid, tartaric acid, citric acid, malic acid, oxalic acid, benzoic acid, methanesulfonic acid, benzenesulfonic acid, etc.
  • inorganic acids eg, hydrochloric acid, phosphoric acid, hydrobromic acid, sulfuric acid, etc.
  • organic acids eg, acetic acid, formic acid, propionic acid, fumaric acid, maleic acid, Salts with succinic acid, tartaric acid
  • the compound of the present invention or a salt thereof that inhibits promoter activity against DNA can inhibit the expression of the protein of the present invention and inhibit the function of the protein, for example, colon cancer, breast cancer, lung cancer, prostate cancer, esophagus Cancer, such as cancer, stomach cancer, liver cancer, biliary tract cancer, spleen cancer, kidney cancer, bladder cancer, uterine cancer, testicular cancer, thyroid cancer, kidney cancer, brain tumor or blood tumor (preferably knee cancer etc.)
  • Prophylaxis is useful as a therapeutic agent.
  • compounds derived from the compounds obtained by the above self-screening can be used in the same manner.
  • a drug containing the compound or a salt thereof obtained by the screening method can be produced in the same manner as the above-mentioned drug containing the protein of the present invention or a salt thereof.
  • the preparations obtained in this way are safe and low toxic and can be used, for example, in humans or mammals (eg, rats, mice, guinea pigs, egrets, sheep, sheep, bushus, dogs, dogs, cats, dogs). , Monkeys, etc.).
  • the dose of the compound or a salt thereof varies depending on the disease to be treated, the subject to be administered, the administration route, and the like.For example, it inhibits the promoter activity of the DNA of the present invention.
  • the compound When the compound is orally administered, generally, in an adult (assuming a body weight of 60 kg) a patient with renal cancer, about 0.1 to 100 mg, preferably about 1.0 to 50 mg, more preferably about 0.1 to 100 mg of the compound per day is used. Is administered at about 1.0-20 mg.
  • the single dose of the compound varies depending on the administration subject, target disease, and the like.
  • a compound that inhibits the promoter activity of DNA of the present invention is usually administered in the form of an injection to an adult.
  • the compound When administered to a patient with Teng's carcinoma (as 60 kg), 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. It is convenient to administer about 1 to 1 Omg by intravenous injection. In the case of other animals, the dose can be administered in terms of 60 kg.
  • the non-human mammal deficient in expression of the DNA of the present invention is extremely useful for screening a conjugate or a salt thereof that promotes or inhibits the activity of the promoter for the DNA of the present invention,
  • the present invention can greatly contribute to the investigation of the cause of various diseases caused by insufficient DNA expression or the development of preventive and therapeutic agents.
  • genes encoding various proteins are ligated downstream thereof and injected into an egg cell of an animal to produce a so-called transgenic animal (gene transfer). Animal), it is possible to specifically synthesize the protein and study its effects in the living body.
  • an appropriate reporter gene is linked to a part of the promoter, and cells that express it Once a strain is established, it can be used as a search system for low-molecular compounds that have the action of specifically promoting or suppressing the ability of the protein itself of the present invention to produce in the body.
  • bases, amino acids, and the like are indicated by abbreviations based on the abbreviations by the IUPAC-IUB Commission on Biochemical Nomenclature or conventional abbreviations in the field, and examples thereof are described below.
  • optical isomer of an amino acid the L-form is indicated unless otherwise specified.
  • HONB 1-hydroxy-5-norpolene-2,3-dicarpoxide
  • SEQ ID NOs in the sequence listing in the present specification show the following sequences. [SEQ ID NO: 1]
  • Example 1 shows the nucleotide sequence of a primer used in Example 1.
  • Example 1 shows the nucleotide sequence of a primer used in Example 1.
  • Example 3 shows the base sequence of the antisense oligonucleotide used in Example 2.
  • 1 shows the nucleotide sequence of DNA containing the full length gene encoding HTRA3.
  • Example 3 shows the nucleotide sequence of a primer used in Example 3.
  • Example 3 shows the nucleotide sequence of a primer used in Example 3.
  • Example 3 shows the nucleotide sequence of a primer used in Example 3.
  • Example 3 shows the nucleotide sequence of a primer used in Example 3.
  • Example 3 shows the nucleotide sequence of a primer used in Example 3.
  • (SEQ ID NO: r 14) 3 shows the nucleotide sequence of a DNA containing the full-length gene encoding HTRA3 used in Example 3.
  • Example 3 shows the nucleotide sequence of DNA containing a part of the full-length gene encoding HTR A3 used in Example 3.
  • FIG. 5 shows the nucleotide sequence of a DNA containing a part of the full-length gene encoding HTRA3 used in Example 3.
  • Example 3 shows the nucleotide sequence of a DNA containing a part of the full-length gene encoding HTRA3 used in Example 3.
  • Example 7 shows the nucleotide sequence of a primer used in Example 6.
  • Example 7 shows the nucleotide sequence containing the DNA encoding the HTRA3S305A mutant in the HTRA3S305A mutant expression vector 6) obtained in Example 6.
  • Example 7 shows the nucleotide sequence containing DNA encoding the HTRA 3 S305A mutant in the HTRA3S305A mutant expression vector 7) obtained in Example 6.
  • Example 7 shows the nucleotide sequence of a primer used in Example 7.
  • Example 7 shows the nucleotide sequence of a primer used in Example 7.
  • Example 7 shows the nucleotide sequence of a probe used in Example 7.
  • HTRA 3 S 305 A mutant (The serine residue at position 305 is replaced with an alanine residue. 2 shows the amino acid sequence of the present variant.
  • 26 shows the base sequence (reverse sequence of the base sequence represented by SEQ ID NO: 26) of the control oligonucleotide used in Example 9.
  • 26 shows the base sequence (reverse sequence of the base sequence represented by SEQ ID NO: 28) of the control oligonucleotide used in Example 9.
  • FIG. 2 shows the amino acid sequence of a 37 kDa protein obtained by autolysis of human HTRA3 obtained in Example 10.
  • RNA derived from human hepatocellular carcinoma tissue
  • RNA Direct Clinical Access
  • RNA derived from normal tissue around Teng carcinoma tissue
  • the expression level of the HTR A3 gene was quantified by a PCR method using two primers, Primer 1 (SEQ ID NO: 1) and Primer 1 (SEQ ID NO: 2).
  • the composition of the reaction solution was prepared by using the above cDNA 0.51 as type II, 101 volumes of HotStarTaq Master Mix (QIAGEN), 10 M of primer 1 (SEQ ID NO: 1) and 10 M of primer 1 (SEQ ID NO: 2) was added to each of 0.51, and distilled water was added to 8.51 to give a liquid volume of 201.
  • the PCR reaction is repeated 95 times at 95 ° C for 15 minutes, followed by 30 cycles of 94 ° C for 30 seconds, 60 ° C for 30 seconds, and 72 ° C for 30 seconds. Was done.
  • PCR product derived from the HTRA3 gene was obtained from 5 cases of total RNA derived from a kidney cancer tissue. However, no PCR product was detected from 4 cases of total RNA derived from normal tissues. Therefore, it was revealed that the expression of the HTRA3 gene in human hepatic cancer tissues was significantly enhanced as compared with normal tissues.
  • the internal standard of total RNA used for type I was the expression level of the GAPDH gene quantified using TaqMan Rodent GAPDH Control Reagent VIC Probe (Applied Biosystems).
  • the GAPDH gene PCR reaction conditions were the same as those for the HTR A3 gene.As a result, PCR products derived from the GAPDH gene were detected from the total RNA in all 9 cases, and the HTR A3 gene in normal tissues was detected. It was proved that undetection of the derived PCR product was not due to RNA degradation. '
  • HTRA3 antisense oligonucleotide introduction experiment was performed.
  • an antisense SEQ ID NO: 3,
  • a phosphorothioated oligonucleotide was synthesized, purified by HPLC, and used for an introduction experiment (Amersham Pharmacia Biotech).
  • the reverse sequence SEQ ID NO: 4 of the base sequence represented by SEQ ID NO: 3 was similarly used as a control oligonucleotide, phosphorothioated, and purified by HPLC (Amersham Pharmacia Biotech).
  • oligonucleotide 37. C, 5% C_ ⁇ 2 days after culture in 2 conditions, was introduced oligonucleotide. Oligonucleotides were introduced using ⁇ -50 Reagent (Promega) according to the standard experimental protocol. Introduce oligonucleotide, 37. C, 5% C_ ⁇ 2 days after culture in 2 conditions were investigated apoptosis inhibitory activity. The apoptosis activity of the cells was quantified by measuring the enzyme activities of caspases 3 and 7. The enzyme activities of caspases 3 and 7 were quantified using the Apo-ONE Homogeneous Caspase-3 / 7 Assay (Promega), using a substrate solution of 501 per 1 ⁇ l according to a standard experimental protocol.
  • HTR A3 was not only significantly increased in the hepatic cancer tissue, but also involved in apoptosis of cancer cells.
  • HTR A3 expression vector 1 in which the double-stranded DNA shown in SEQ ID NO: 8 was inserted into pc DNA 3.1 (+) (Invitrogen) was prepared.
  • Double-stranded DNA was synthesized by PCR using oligonucleotides (SEQ ID NO: 9 and SEQ ID NO: 11) as primers.
  • the composition of the reaction in this reaction was as follows: MTC panel ovary cDNA (Clontech) 0.3 K ExT aq 10 X buffer 2.01, Ex Taq 0.10 M primers 0.5, 1 2.5 mM dNTP1.6 ⁇ and distilled water 151 were added to adjust the liquid volume to 201.
  • PCR reaction is 98.
  • PCR product was diluted 100 times with distilled water, and the target sequence was amplified by PCR reaction.
  • the composition of the reaction solution in the reaction was such that PCR product diluent 31 was used as type ⁇ , and ExTaQ10X buffer 20 ⁇ 1, ExTaq1 ⁇ , 2.5 mM dNTP (16 l) and distilled water (1501) were added to adjust the liquid volume to 200 I.
  • a cycle of 98 ° C ⁇ 30 seconds, 65 ° C ⁇ 30 seconds, 72 ° C ⁇ 90 seconds is repeated 10 times after 98 ° C ⁇ 1 minute, and then A cycle of 98 ° C for 30 seconds, 60 ° 30 seconds, and 72 ° C for 90 seconds was repeated 20 times, and finally an extension reaction was performed at 72 ° C for 7 minutes.
  • purification was carried out using a PCR Purification Kit (Qiagen).
  • the purified PCR product was digested with restriction enzymes XbaI and KpnI, and the reaction solution was subjected to agarose gel electrophoresis, and then purified using a gel extraction kit (Qiagen).
  • pcDNA3.1 (+) was digested with restriction enzymes XbaI and KpnI, followed by agarose gel electrophoresis, and then purified with a gel extraction kit (Qiagen). 75 ng of the PCR product, 60 ng of the vector fragment, and DNA ligation kit Ver2 solution I51 (Yukara) were mixed, and the ligated reaction was carried out at 16 ° C for 30 minutes. After the ligation reaction, Escherichia coli DH5Q! was transformed, and eight transformants obtained were selected.
  • DNA sequencer (370 I).
  • DNA sequencer 370 I
  • the nucleotide sequence was determined, and a peptide that matched SEQ ID NO: 8 (containing all ORFs) was selected.
  • the expression vector 1) obtained earlier was designated as type I, and the primers were oligonucleotides (SEQ ID NO: 9 and SEQ ID NO: 12) as SEQ ID NO: 14 and SEQ ID NO: 15 as SEQ ID NO: 15 (SEQ ID NO: 9 and SEQ ID NO: 13),
  • SEQ ID NO: 16 SEQ ID NO: 10 and SEQ ID NO: 12
  • SEQ ID NO: 17 SEQ ID NO: 10 and SEQ ID NO: 11
  • the composition of the reaction solution in this reaction was as follows: 4.7 n1 expression vector 1) 21, lO Xpfu buffer 15 / xl, pfuturbo 0.251, 10 M primers (SEQ ID NO: (SEQ ID NO: 9 and SEQ ID NO: 13) or (SEQ ID NO: 10 and SEQ ID NO: 12) or (SEQ ID NO: 10 and SEQ ID NO: 11) 1, 2.5 mM dNTP 41 and distilled water 36.251 were added to adjust the liquid volume to 501.
  • the PCR reaction is repeated 98 times at 98 ° C for 1 minute, then at 98 ° C for 30 seconds, at 65 ° 30 seconds, at 72 ° C for 90 seconds, and then repeated at 98 ° C for 30 seconds at 60 ° C.
  • a cycle of 30 seconds and 72 ° C for 90 seconds was repeated 15 times, and finally an extension reaction was performed at 72 ° C for 7 minutes.
  • Each of the synthesized PCR products was subjected to agarose gel electrophoresis, purified by a gel extraction kit (Qiagen), and digested with restriction enzymes XbaI and KpnI. The obtained fragment was purified using a PCR Purification Kit (Qiagen).
  • the expression vector: 3 XFLAG-CMV9, 13 and ⁇ 4 were digested with restriction enzymes XbaI and KpnI, subjected to agarose gel electrophoresis, and then purified using a gel extraction kit (Qiagen).
  • the ligation reaction was carried out at 16 ° C for 30 minutes using the PCR product and the vector fragment with 5 Ong and 3 Ong, respectively, using the DNA Ligation Kit Ver2 solution.
  • Escherichia coli DH5 was transformed and eight of the resulting transformants were selected.
  • DNA is extracted, a sequence reaction is performed using Big Dye Miner Cycler Sequencing Ver.
  • SEQ ID NO: 14 all containing ORF
  • SEQ ID NO: 15 C-terminal PDZ domain deletion
  • SEQ ID NO: 16 N-terminal signal sequence deletion
  • SEQ ID NO: 17 N-terminal A clone matching (signal sequence deletion) was selected.
  • COS-7 cells were cultured in serum-containing DMEM medium, and 9 ⁇ 10 5 cells were seeded on a 10 cm dish (Falcon) the day before gene transfer. After washing with serum-free DMEM medium, 12 g of expression vector 2), 3), 4), 5), empty vector P 3 XFLA G—CMV 14, p 3 XFL AG—CMV 13 or p 3 XFL AG—Add 6 ml of serum-free medium containing CMV 9 and 36 1 TRANSF AST (Promega), and transfer genes into two 10 cm dishes with each expression vector and empty vector.
  • serum-free DMEM medium 12 g of expression vector 2
  • p 3 XFL AG CMV 13
  • TRANSF AST TransF AST
  • Enriched culture supernatant obtained in Example 4 25 to 75_ ⁇ 1 EnzChek Protease Assay Kit green fluorescence Digestion Buffer (Molecular Probes), 100 1 of 10 ig / ml EnzChek Protease Assay Kit green fluorescence BOD I PY-FL A labeled casein substrate solution (Molecular Probes) was mixed and reacted at 37 for 4 hours. After the reaction, serum and phenol red-free D, MEM medium 251, 75 ⁇ 1 Using a mixture of the above as a background, an excitation wavelength of 485 nm and a fluorescence wavelength of 538 nm were measured with a fluoroscan fluorescence plate reader (Lab Systems).
  • the double stranded sequence shown in SEQ ID NO: 20 was obtained using the Quicchage Site-Directed Mutagenesis Kit (Stratagene).
  • HTRA3 S305A mutant (SEQ ID NO: 21) in which the DNA was inserted into pcDNA3.1 (+) (Invitrogen) and the double-stranded DNA shown in SEQ ID NO: 21 was inserted into ⁇ 3XFLAG-CMV14 No .: 25)
  • Expression vectors 6) (SEQ ID NO: 20) and 7) (SEQ ID NO: 21) were prepared.
  • the composition of the reaction mixture in the reaction was 10 ng of the expression vectors 1) and 2) obtained in Example 3, 10 X reaction buffer 5.0 n 1, futurbo DNA polymerase 1.01, 2.7 M
  • the primers were added to each other at 5 // 2.5 mM dNTP 11 and distilled water 331 were added to adjust the liquid volume to 51 l.
  • the PCR reaction was performed by repeating a cycle of 95 ° C for 30 seconds, 95 ° C for 30 seconds, 55 ° C for 1 minute, and 68 ° C for 8 minutes 12 times. After the reaction, the PCR reaction solution was digested with 11 restriction enzymes Dpnl. After digestion, E. coli XL—Blue was transformed, the selected E.
  • HTRA3 In order to further verify the apoptosis-inhibiting activity of HTRA3 described in Example 2, as a test cell, a cell line PANC-1 derived from Teng's carcinoma (Int. J. Cancer, Vol. 5, p. 741-747, 1975) , Purchased from ATCC).
  • the HTRA3 antisense oligonucleotide (SEQ ID NO: 3) and the control oligonucleotide (SEQ ID NO: 4) were prepared using the phosphorothioated oligonucleotide described in Example 2. Was used.
  • Apoptosis was induced as in 153 cells, indicating that HTRA3 has apoptosis inhibitory activity.
  • a reverse transcription reaction was performed from the total RA using a TaqMan Gold RT-PCR Kit (Applied Biosystems) to obtain a cDNA solution.
  • the composition of the reaction solution and the reaction conditions in the reaction were in accordance with the attached standard experimental protocol.
  • the expression level of the HTRA3 gene was quantified using the TaqMan PCR method.
  • the oligonucleotides represented by SEQ ID NO: 22 and SEQ ID NO: 23 were used as primers. Professional
  • the FAM and TAMRA-labeled oligonucleotides represented by SEQ ID NO: 24 were used as 25 nucleotides.
  • TaqMan PCR was performed using TaqMan Universal Mixture (Applied Biosystems) according to its standard cold protocol. The final concentration of each primer was 250 nM, and the final concentration of the probe was 100 nM. The PCR reaction was performed at 95 ° C: 15 seconds and 65: 60 seconds for 40 cycles.
  • the internal standard for HTRA3 gene expression was GAPDH gene expression.
  • the quantification of the expression level of the GAPDH gene was performed using TaqMan GAPDH Control Reagents (Applied Biosystems) according to the standard experimental protocol.
  • the amount of HTRA3 remarkable in the antisense oligonucleotide-transfected cells was higher than that in the control (reverse) oligonucleotide-transfected cells (100%).
  • Decreased gene mRNA was observed (49%).
  • the introduction of antisense oligonucleotides showed a decrease in the mRNA of the HTRA3 gene, suggesting that it may be caused by the decrease in the mRNA of the HTRA3 gene, which induces apoptosis in cells.
  • an experiment was performed to introduce an HTRA3 expression vector.
  • an HTRA3 expression vector an expression vector 2) containing all ORFs represented by SEQ ID NO: 14 obtained in Example 3) was used.
  • the expression vector of the HTRA3 gene represented by SEQ ID NO: 2.06) (serine residue 305 as an alanine residue in the protein sequence)
  • the substituted mutant (SEQ ID NO: 25) was used.
  • a pancreatic cancer-derived cell line PAN C-1 (Int. J. Cancer, 15, 741-747, 1975, purchased from ATCC, 1975) was used as a test cell. 10,000 cells / well of PANCl-1 were seeded on a 96-well clear bottom black plate (Falcon). After 2 days of culture at 3 7 ° C, 5% C0 2 conditions, each of the empty base Kuta one, expression vectors 2), it was introduced 6). TransfasT Reagent (Promega) was used for the introduction, following the standard experimental protocol. The final concentration of each empty vector and expression vector was InM. Was introduced, at 37, after two days of culture in the 5% C0 2 condition were examined apoptosis inhibitory activity.
  • the apoptotic activity of the cells was quantified by measuring the enzymatic activity of caspases 3 and 7.
  • Enzyme activity of caspases 3 and 7 The sex was determined using Apo-ONE Homogeneous Caspase-3 / 7 Assay (Promega), using 50 L of substrate solution per gel according to a standard experimental protocol.
  • the caspases 3 and 7 were remarkably higher in the cells transduced with HTRA3 expression 2). Inhibition of the enzyme activity was observed (75%).
  • apoptosis inhibitory activity was quantified by measuring the enzyme activities of caspases 3 and 7.
  • the enzymatic activities of caspases 3 and 7 were determined using Apo-ONE Homogeneous Caspase-3 / 7 Assay (Promega), using a substrate solution per 1 ⁇ l, according to standard experimental protocols.
  • the caspases 3 and 7 were significantly higher in the HTRA3-expressing vector 2) transfected cells than in the control cell (empty vector: P 3 XFLAG—CMV 14) transfected cells (100%). Inhibition of the enzyme activity was observed (49%). Furthermore, this inhibitory effect was not observed in HTRA3 expression vector 6) transfected cells in which the 3rd serine residue was substituted with an alanine residue (104%). In other words, similar apoptosis-inhibitory activity in PANC-11 cells was also observed in SW480 cells, indicating that seric protease activity was important.
  • a pancreatic cancer-derived cell line PANC-1 (Int. J. Cancer, 15, 741-747, 1975, purchased from ATCC, 1975) was used as a test cell. 100 000 cells / well of PANCl-1 were seeded on 96-well clear bottom black plates (Falcon). 3 7 ° C, 2 days after culture in 5% C0 2 of Article ⁇ cattle were introduced oligonucleotide. Oligonucleotides were introduced using Transfasr Reagent (Promega) according to the standard experimental protocol. After introducing the oligonucleotide and culturing at 37 ° C. and 5% CO 2 for 2 days, the apoptosis inhibitory activity was examined. The apoptotic activity of the cells was quantified by measuring the enzymatic activity of caspases 3 and 7. The enzyme activities of caspases 3 and 7 are determined by Apo-ONE Homogeneous
  • the FLAG-labeled HTRA3 expression vector 2) (SEQ ID NO: 14) was transfected into the HEK293 cell line, selected with 750 g / m 1 G418, and a FLAG-labeled HTRA3 stable expression strain was isolated.
  • the FLAG-labeled HTRA3 stable expression strain was cultured in DMEM containing 10% FBS for 6 days, and 450 ml of the culture supernatant was obtained. The obtained culture supernatant was centrifuged at 2500 rpm for 10 minutes, and 10-fold concentrated TBS was added.
  • the FLAG-labeled HTR A3 stable expression strain obtained in Example 10 was cultured in OPTI-MEM (Invitrogen) for 3 days, and 400 ml of a culture supernatant was obtained. The obtained culture supernatant was centrifuged at 2000 rpm for 10 minutes, and 10-fold concentration of TBS was added. The supernatant was added to a 5 ml FLAG antibody bead (Sigma) column, washed with 200 ml of TBS, and eluted 4 times with 5 ml of 100 g Zm1 3XFLAG peptide (Sigma) (fraction 1-4).
  • the resulting flux Yeons 2 and 3 were dialyzed against TBS three times with a Slide ALizer (Pierce) to obtain 1.4 mgZm1 of FLAG-labeled HTRA3.
  • crudely purified HTRA3 0.94 was added to 125 nM IGFBP-5 (Austral Biologicals), 25 mM Hepes (pH 7.4), 25 mM CHES, 50 mM NaCl, 0.02 The reaction was carried out at 37 ° C. for 14.5 hr in a 20 l reaction solution containing 5% Tween 20 and 1.5 mM DTT.
  • HTRA3 1.48 was replaced with 125'11 I GFBP-2 (Austral Biologics), 25 mM Hepes (pH 7.4), 25 mM CHES, 50 mM NaCl, The reaction was carried out at 37 ° C for 12 hr in a 20 ⁇ l reaction solution containing 1.025% Tween 20 and 1.5 mM DTT. After the reaction, add 2 1 2 X 1 aemm 1 i buffer (5%) 3 mercaptomethanol and treat at 98 ° C for 5 minutes to obtain a 15-25% gradient gel (Daiichi Kagaku). SDS-PAGE was carried out with a (drug) and transferred to a PVDF membrane (Amersham).
  • the PV DF membrane was blocked for 1 hour with Block Ac e (Dainippon Pharmaceutical Co., Ltd.), and then washed 3 times (5 minutes each) with TBS containing 0.1% Tween 20.
  • the reaction was carried out for 2 hours with 2 g / m1 of IGFBP_2 antibody (R & D Systems) or 1 gZm1 of IGFBP-5 ⁇ ⁇ ⁇ (Austral Biologicals). Thereafter, the cells were washed three times with TBS containing 0.1% Tween 20 (for 5 minutes each), and then diluted 100 fold with TBS containing 0.1% Tween 20 to give A1 ka1ine Phosphatase-labeled antibody.
  • the reaction was carried out for 1 hour with a mouse or anti-mouse IgG antibody (Sigma). After the reaction, the color was developed with Western Blue S tabilized S ubstrate for Alkaline Phosphatase (Promega). As a result, the band of IGFB P-2 was reduced by HTRA3, and a band considered to be a degradation product of 30 kDa was observed. In addition, the band of IGFBP-5 was lost by HTR A3. These results indicate that HTRA3 degrades I &? 8? -2 and 1 GFBP-5. Industrial applicability
  • the protein used in the present invention is specifically expressed in cancer cells, and is a diagnostic marker for cancer. Therefore, compounds that inhibit the activity of the protein or salts thereof, compounds that inhibit the expression of the protein gene or salts thereof include, for example, colon cancer, breast cancer, lung cancer, prostate cancer, esophageal cancer, gastric cancer, Prevention of cancers such as liver cancer, biliary tract cancer, spleen cancer, renal cancer, bladder cancer, uterine cancer, testicular cancer, thyroid cancer, Tengler cancer, brain tumor or blood tumor (preferably, kidney cancer) Can be used safely.
  • the compound or its salt that inhibits the activity of the protein or the compound or its salt that inhibits the expression of the protein gene may be safely used as, for example, an apoptosis inducing (or promoting) agent, a protease inhibitor or the like. You can also.
  • the antisense polynucleotide or antibody of the present invention can inhibit the expression of the protein used in the present invention, and includes, for example, colorectal cancer, breast cancer, lung cancer, prostate cancer, esophageal cancer, stomach cancer, liver cancer, biliary tract cancer,
  • a prophylactic / therapeutic agent for cancer such as spleen cancer, kidney cancer, bladder cancer, uterine cancer, testicular cancer, thyroid cancer, thyroid cancer, brain tumor or hematological tumor (preferably, cancer of the kidney), or apoptotic cancer It can be used safely as a cis-inducing (or facilitating) agent, protease inhibitor, etc.

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Abstract

Cette invention concerne un inducteur d'apoptose ou analogue contenant un composé ou son sel inhibant l'activité d'une protéine renfermant un acide aminé, qui est identique ou sensiblement identique à la séquence d'acides aminés de HTRA3, son fragment peptidique ou un sel de celui-ci ou l'expression du gène de celui-ci.
PCT/JP2003/013920 2002-11-01 2003-10-30 Inducteur d'apoptose Ceased WO2004039407A1 (fr)

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN104007263A (zh) * 2014-05-22 2014-08-27 复旦大学附属中山医院 HtrA3免疫组化试剂在制备预测肺癌术后复发转移试剂中的应用
CN104007263B (zh) * 2014-05-22 2015-09-30 复旦大学附属中山医院 HtrA3免疫组化试剂在制备预测肺癌术后复发转移试剂中的应用

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