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WO2003091434A1 - Nouvelle proteine, et adn de cette proteine - Google Patents

Nouvelle proteine, et adn de cette proteine Download PDF

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Publication number
WO2003091434A1
WO2003091434A1 PCT/JP2003/005171 JP0305171W WO03091434A1 WO 2003091434 A1 WO2003091434 A1 WO 2003091434A1 JP 0305171 W JP0305171 W JP 0305171W WO 03091434 A1 WO03091434 A1 WO 03091434A1
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WIPO (PCT)
Prior art keywords
protein
present
seq
dna
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
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PCT/JP2003/005171
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English (en)
Japanese (ja)
Inventor
Atsushi Nakanishi
Yoji Sagiya
Hiroyuki Miya
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Takeda Pharmaceutical Co Ltd
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Takeda Chemical Industries Ltd
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Priority to AU2003235096A priority Critical patent/AU2003235096A1/en
Publication of WO2003091434A1 publication Critical patent/WO2003091434A1/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • the present invention provides a novel voltage-dependent Ca 2+ channel protein, a DNA encoding the protein, a method for screening a compound that regulates the activity of the protein, a compound obtained by the screening method, and the like.
  • sperm motility is activated by maturation, called capacitation, which enables an acrosome reaction.
  • Cyclic nucleotides, Ca 2+ ions, and intracellular pH are thought to be involved in the control of fertility.
  • sperm have voltage-dependent Ca 2+ channels ''
  • the conventional Cav has a 24 transmembrane structure with 4 units of 1 transit unit in the 6 transmembrane region.However, CatSper and Cat Sper2 have a homozygous or 6 transmembrane subunit similar to Kv. It is thought that it functions as a channel by forming a heterotetramer.
  • Catsper and CatSper2 are specifically expressed in testis, and in sperm, protein localization is found in the main part of the flagella.
  • CatSper knockout mice exhibit a sperm motility and male infertility phenotype, suggesting that CatSper families have important functions in sperm.
  • neither Catsper nor CatSper2 has been electrophysiologically proven to be a channel at this time, and it may be necessary to associate with an unknown subject.
  • Ca 2+ channels There are many molecular species in Ca 2+ channels, and their expression sites are also diverse. In addition, one cell often expresses multiple Ca 2+ channels. Changes in intracellular Ca 2+ concentration due to Ca M channel are physiologically important phenomena.In some cases, the relationship between abnormalities and diseases is known, but in other cases, detailed mechanism is not clear. Many. In spermatozoa, multiple Ca 2+ channels are expressed. Among them, the Cat Sper family is thought to play an important role, but the detailed mechanism of its true activity and involvement in sperm function is Obviously not.
  • Methods for regulating the protein include, for example, inhibiting or promoting the permeation of Ca 2+ ions, reducing the expression level by suppressing the transcription of the protein gene, activating the promoter of the protein gene, and stabilizing the mRNA. It can be considered that the expression level is enhanced by the conversion.
  • (6) a polynucleotide comprising a polynucleotide encoding the protein of (1) or the partial peptide of (5),
  • a diagnostic agent comprising the polynucleotide according to (6), (16) an antibody against the protein according to (1) or the partial peptide according to (5) or a salt thereof,
  • a polynucleotide comprising a nucleotide sequence complementary to or substantially complementary to the nucleotide sequence of the polynucleotide according to (6) or a part thereof.
  • (26) a screening kit for a compound or a salt thereof that promotes or inhibits the expression of the protein gene according to (1), which comprises the polynucleotide according to (6);
  • (31) A method for screening a compound or a salt thereof that promotes or inhibits expression of the protein according to (1), which comprises using the antibody according to (16).
  • (32) a kit for screening a compound or a salt thereof that promotes or inhibits the expression of the protein according to (1), which comprises the antibody according to (16);
  • (33) a compound or a salt thereof that promotes or inhibits the expression of the protein of (1), obtained by using the screening method of (31) or the screening kit of (32);
  • FIG. 1 is a diagram showing a comparison of amino acid sequences in transmembrane regions of human TCH207, human CatSper and human CatSper2.
  • TCH2Q7 is the sequence from the 92nd to the 306th of the amino acid sequence of human TCH207 (SEQ ID NO: 1)
  • CatSper is the human CatSper2 is the amino acid sequence of CatSper2a (Proc. Natl. Acad.Sci. USA, 98, 12527, 2001).
  • the sequence from the 104th to the 340th is shown.
  • S1 to S6 indicate transmembrane domains, and P indicates a pore region.
  • the mouth indicates an amino acid that matches in two or more of the three sequences.
  • the basic residues in S4 are indicated by ⁇ . Residues conserved in the voltage-dependent Ca 2+ channel family in the pore region are marked with *, and Asp important for Ca 2+ selectivity is marked with *.
  • FIG. 2 is a diagram showing the expression level of human TCH207 gene product in each tissue.
  • Results of human TCH207 expression in human tissue cDNAs [Human MTC panel I, Human MTC pane II (upper figure) and Human digivestive system MTC panel (lower figure): Clontech] measured by TaqMan PCR Is shown.
  • the vertical axis represents the expression level, and the number of copies per 1 n1 of cDNA solution of human TCH207 is divided by the number of copies of glyceral dehide-3-phosphate dehydrogenase (GAPDH) in an equal amount of each tissue cDNA. The value was multiplied by 100,000.
  • GPDH glyceral dehide-3-phosphate dehydrogenase
  • FIG. 3 is a diagram showing the expression level of human TCH207 gene product in each tissue.
  • the results of measuring the expression level of human TCH207 in human tissue cDNAs [Human fetal MTC pane l (upper figure) and Human tumor MTC panel (lower figure): Clontech] by TaqMan PCR are shown.
  • the vertical axis represents the expression level, and the copy number per liter of the human TCH207 cDM solution was divided by the copy number of glyceral dehide-3-phosphate dehydrogenase (GAPDH) in an equal amount of each tissue cDNA. Expressed as a value multiplied by 100,000.
  • GPDH glyceral dehide-3-phosphate dehydrogenase
  • FIG. 4 is a diagram showing a comparison of amino acid sequences between human TCH207 and mouse TCH207.
  • human indicates the amino acid sequence of human TCH207 (SEQ ID NO: 1)
  • mouse indicates the amino acid sequence of mouse TCH207 (SEQ ID NO: 42).
  • S1 to S6 indicate transmembrane domains
  • P indicates a pore region.
  • the mouth indicates the amino acids that are identical in the two sequences.
  • the basic residues in S4 are indicated by ⁇ , and residues conserved in the voltage-dependent Ca 2+ channel family in the pore region are marked with *, and Asp important for Ca 2+ selectivity is marked with *.
  • FIG. 5 is a diagram showing the expression level of mouse TCH207 gene product in each tissue.
  • the results of measuring the expression level of mouse TCH207 in mouse tissue cDNAs (Mouse MTC pane I and mouse MTC panel II: manufactured by Klontech) by TaqMan PCR are shown.
  • the vertical axis represents the expression level, and the number of copies per mouse cDNA solution of mouse TCH207 is divided by the number of copies of rodent glyceraldehide-3-phosphate dehydrogenase (GAPDH) in an equal amount of each tissue cDNA, and is 100,000 times. It was expressed by the value given.
  • a protein containing an amino acid sequence identical or substantially identical to the amino acid sequence represented by SEQ ID NO: 1, SEQ ID NO: 3 or SEQ ID NO: 42 (hereinafter referred to as the protein of the present invention or the protein used in the present invention; )
  • are cells of human warm-blooded animals eg, guinea pigs, rats, mice, chickens, egrets, bushes, sheep, whales, monkeys, etc.
  • hepatocytes, spleen cells, nerve cells eg, hepatocytes, spleen cells, nerve cells
  • Glial cells, gut iS cells, bone marrow cells mesangial cells, Langerhans cells, epidermal cells, epithelial cells, goblet cells, endothelial cells, smooth muscle cells, fibroblasts, fiber cells, muscle cells, fat cells, immune cells ( Eg, macrophages, T cells, B cells, natural killer cells, mast cells, neutrophils, basophils, e
  • amino acid sequence substantially the same as the amino acid sequence represented by SEQ ID NO: 1 is, for example, about 30% or more, preferably about 50% or more, and preferably about 30% or more of the amino acid sequence represented by SEQ ID NO: 1.
  • Examples of the protein containing an amino acid sequence substantially identical to the amino acid sequence represented by SEQ ID NO: 1 include, for example, the amino acid sequence represented by SEQ ID NO: 1
  • a protein containing substantially the same amino acid sequence and having substantially the same activity as the protein containing the amino acid sequence represented by SEQ ID NO: 1 is preferred.
  • the amino acid sequence substantially the same as the amino acid sequence represented by SEQ ID NO: 3 includes, for example, about 30% or more, preferably about 50% or more, and preferably about 60% or more of the amino acid sequence represented by SEQ ID NO: 3. % 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 containing an amino acid sequence substantially the same as the amino acid sequence represented by SEQ ID NO: 3 include, for example, a protein containing an amino acid sequence substantially the same as the amino acid sequence represented by the aforementioned SEQ ID NO: 3 However, a protein having substantially the same activity as the protein containing the amino acid sequence represented by SEQ ID NO: 3 is preferable.
  • the amino acid sequence substantially the same as the amino acid sequence represented by SEQ ID NO: 42 includes, for example, about 30% or more, preferably about 50% or more, and preferably about 60% An amino acid sequence having the above, more preferably about 80% or more, particularly preferably about 90% or more, and most preferably about 95% or more.
  • Examples of a protein having an amino acid sequence substantially the same as the amino acid sequence represented by SEQ ID NO: 42 include, for example, a protein having the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 42 described above. However, a protein having substantially the same activity as the protein having the amino acid sequence represented by SEQ ID NO: 42 is preferable.
  • substantially the same activity examples include cation (eg, Ca 2+ , K +, Na + , etc.) permeability activity and the like.
  • cation eg, Ca 2+ , K +, Na + , etc.
  • force thiones eg, Ca 2+ , K Na +, etc.
  • permeation activity eg, about 0.01 to: 100 times, preferably about 0.1 to 10 times, more preferably 0.5 times.
  • the quantitative factors such as the degree of these activities and the molecular weight of the protein may be different.
  • the protein of the present invention includes, for example, (i) one or more amino acids in the amino acid sequence represented by SEQ ID NO: 1, SEQ ID NO: 3 or SEQ ID NO: 42 (for example, 1 to 2 About 100 pieces, preferably about 1 to 150 pieces, preferably about 1 to 100 pieces, preferably about 1 to 50 pieces, preferably about 1 to 30 pieces, preferably 1 to 10 pieces.
  • the amino acid sequence represented by SEQ ID NO: 3 or SEQ ID NO: 42 has one or two or more amino acids (for example, about 1 to 200, preferably about 1 to 150, and preferably 1 to 100).
  • About 1 to 50, preferably about 1 to 50, preferably about 1 to 30, more preferably about 1 to 10 and more preferably about 1 to 5 amino acids have been inserted.
  • amino acids in the amino acid sequence represented by SEQ ID NO: 1, SEQ ID NO: 3 or SEQ ID NO: 42 for example, about 1 to 200, preferably 1 to 150
  • V a protein containing an amino acid sequence obtained by substituting an amino acid with another amino acid, or (V) an amino acid sequence obtained by combining them.
  • the amino acid sequence is inserted, deleted or substituted as described above, the position of the insertion, deletion or substitution is not particularly limited.
  • the protein has a N-terminus at the left end (amino terminus) and a C-terminus at the right end (capillary end) according to the convention of peptide labeling.
  • R in the ester e.g., methyl, Echiru, n- propyl, isopropyl
  • alkyl groups such as n- butyl, for example, C 3 _ 8 cycloalkyl group such as cyclohexyl cyclopentyl, to cyclo, for example, phenyl, ⁇ - naphthyl which any Ji 6 _ 12 Ariru group, e.g., benzyl, C 7 _ 14
  • Ararukiru groups such as flying one Nafuchiru CM alkyl group, such as phenylene Lou CM ⁇ alkyl group or a flying one naphthylmethyl such phenethyl, pivaloyl An oxymethyl group is used.
  • the protein used in the present invention has a lipoxyl group (or lipoxylate) at a position other than the C-terminus
  • the protein used in the present invention includes a protein in which the lipoxyl group is amidated or esterified. It is.
  • 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 (eg, methionine residue) has a protecting group (eg, an acyl group such as a Cw alkanol such as a formyl group or an acetyl group).
  • a protecting group eg, an acyl group such as a Cw alkanol such as a formyl group or an acetyl group.
  • N-terminal glutamine residue generated by cleavage in vivo, pyroglutamine oxidation, Substituent on the side chain of amino acid in the molecule (for example, - ⁇ H, one SH, amino group) , imidazo Ichiru group, India Ichiru group, etc.
  • suitable protecting group eg, C, such as formyl group, Asechiru group, - 6 etc. Ashiru groups such Arukanoiru groups
  • They are protected by shall Alternatively, It also includes complex proteins such as so-called sugar proteins to which sugar chains are bonded.
  • proteins containing the amino acid sequence represented by SEQ ID NO: 1 include, for example, a protein containing the amino acid sequence represented by SEQ ID NO: 1, a protein containing the amino acid sequence represented by SEQ ID NO: 3, and SEQ ID NO: 42. And proteins containing the amino acid sequence represented by
  • the partial peptide of the protein used in the present invention is the above-mentioned partial peptide of the protein of the present invention, and is preferably any one having the same properties as the above-mentioned protein of the present invention. Good.
  • one or more (preferably about 1 to 10, more preferably a number (1 to 5)) of amino acids in the amino acid sequence are deleted, Or 1 or 2 or more (preferably, about 1 to 20; more preferably, about 1 to 10; more preferably, about 1 to 5) amino acids are added to the amino acid sequence; or One or two or more (preferably about 1 to 20; more preferably about 1 to 10; and more preferably a number (1 to 5)) amino acids are inserted into the amino acid sequence; Alternatively, one or more (preferably, about 1 to 10, more preferably, several, and more preferably, about 1 to 5) amino acids in the amino acid sequence are substituted with another amino acid.
  • the partial peptide of the present invention includes SEQ ID NO: 1.
  • amino acid sequence represented by SEQ ID NO: 3 such as a peptide having the 1st to 91st amino acids and the 107th to 47th amino acids
  • a peptide having the amino acid sequence of the 307 to 428, having the amino acid sequence represented by SEQ ID NO: 42 for example, having the amino acid sequence of the 1st to 67th and the 283 to 442nd ⁇ Peptides and the like.
  • the partial peptide used in the present invention the C-terminus force Rupokishiru group (- C00H), carboxylate (- C00_), amide (-C0ffl 2) or ester - may be either (C00R).
  • the partial peptides used in the present invention include those having a carboxyl group (or carboxylate) other than the C-terminus, N-terminal amino acid residues (eg, Methionine residue) protected by a protecting group, glutamine residue generated by cleavage of N-terminal in vivo, and pyrroleamine oxidation, substitution of amino acid in the molecule on the side chain Also included are those in which the group is protected by an appropriate protecting group, and those in which a sugar chain is bonded, such as a complex peptide such as a so-called glycopeptide.
  • the partial peptide used in the present invention can also be used as an antigen for producing an antibody.
  • an antibody for example, for the purpose of preparing the antibody of the present invention described below, for example, in the amino acid sequence represented by SEQ ID NO: 1, the 19th to 46th, the 49th to 77th, In the amino acid sequence represented by SEQ ID NO: 3, such as a peptide having an amino acid sequence at positions 268 to 283, 313 to 341 and 410 to 440, for example, having an amino acid sequence at positions 410 to 440
  • SEQ ID NO: 42 for example, positions 19 to 52, positions 244 to 259, positions 288 to 316, positions 322 to 352, and positions 32 to 352.
  • the protein of the present invention may form a multimer (eg, homotetramer, heterotetramer, etc.).
  • the heteromultimer include a heteromultimer with CatSper, CatSper2 and the like.
  • salts with physiologically acceptable acids eg, inorganic acids, organic acids
  • bases eg, alkali metal salts
  • Preferred acid addition salts are:
  • Such salts include, for example, salts with inorganic acids (eg, hydrochloric acid, phosphoric acid, hydrobromic acid, sulfuric acid) or organic acids (eg, acetic acid, formic acid, propionic acid, fumaric acid, maleic acid, succinic acid) Acids, tartaric acid, citric acid, malic acid, oxalic acid, benzoic acid, salts with methanes) sulfonic acid, benzenesulphonic acid) are used.
  • 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
  • the protein of the present invention or a partial peptide thereof or a salt thereof can be produced by the above-mentioned method for purifying a protein from human or warm-blooded animal cells or tissues, or can be a trait containing a DNA encoding the protein. It can also be produced by culturing the transformant. It can also be produced according to the peptide synthesis method described below.
  • a commercially available resin for protein synthesis can be usually used.
  • resins include, for example, chloromethyl resin, hydroxymethyl resin, benzhydrylamine resin, aminomethyl resin, 4-benzyloxybenzyl alcohol Resin, 4-methylbenzhydrylamine resin, PAM resin, 4-hydroxymethylmethylphenylacetamidomethyl resin, polyacrylamide resin, 4- (2,, 4, dimethoxyphenylhydroxymethyl) phenoxy resin, 4 — (2 ', 4'-dimethoxyphenyl-Fmocaminoethyl) 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 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 pre-activated as a symmetric anhydride or HOB t ester or HOOB t ester. Can be added to the resin after the conversion.
  • a racemization inhibitor additive eg, HOB t, HOOB t
  • the protected amino acid may be pre-activated as a symmetric anhydride or HOB t ester or HOOB t ester. Can be added to the resin after the conversion.
  • 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-methylvinylidone, halogenated hydrocarbons such as methylene chloride and chloroform, alcohols such as trifluoroethanol , Sulfoxides such as dimethyl sulfoxide, ethers such as pyridine, dioxane, and tetrahydrofuran; nitriles such as acetonitrile and propionitrile; esters such as methyl acetate and ethyl acetate; or an appropriate mixture thereof. .
  • 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, isopolnyloxycarbonyl, 4-methoxybenzyloxycarbonyl, C1_Z, Br-Z, and 7damant.
  • Tyloxycarbonyl, trifluoroacetyl, phthaloyl, formyl, 2-ditrophenylsulfenyl, diphenylphosphinothioyl, Fmoc and the like are used.
  • the lipoxyl group can be, for example, alkyl esterified (eg, methyl, ethyl, propyl, butyl, t-butyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, 2-adamantyl, etc.).
  • alkyl esterified eg, methyl, ethyl, propyl, butyl, t-butyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, 2-adamantyl, etc.
  • aralkyl esterification e.g., benzyl ester, 4-nitrobenzyl ester, 4-methoxybenzyl ester, 4-chlorobenzyl ester, benzhydryl esterification
  • phenacyl esterification benzyloxy It can be protected by carbonylation, t-butoxycarbonylhydrazide, tritylhydrazide and the like.
  • the hydroxyl group of serine can be protected, for example, by esterification or etherification.
  • a group suitable for the esterification for example, a group derived from carbonic acid such as a lower (Cw) alkanol group such as an acetyl group, an aroyl group such as a benzoyl group, a benzyloxycarbonyl group, and an ethoxycarbonyl group is used.
  • groups suitable for etherification include a benzyl group, a tetrahydropyranyl group, and a t-butyl group.
  • the protecting group of the phenolic hydroxyl group of tyrosine for example, Bz 1, C l 2 - Bz l, 2- two Torobenjiru, B r- Z, such as t- butyl are used.
  • the protecting group for imidazole of histidine for example, Tos, 4-methoxy-1,2,3,6-trimethylbenzenesulfonyl, DNP, benzyloxymethyl, Bum, Boc, Trt, Fmoc and the like are used.
  • the activated carboxyl groups of the raw materials include, for example, corresponding acid anhydrides, azides, and activated esters [alcohols (eg, phenol, 2,4,5—trichlorophenol, 2,4— Dinitrophenol, cyanomethyl alcohol, paranitrophenol, HONB, N-hydroxysuccinimide, N-hydroxyfurimide, HO Bt) and the like).
  • the activated amino group of the raw material for example, a corresponding phosphoric amide is used.
  • Methods for removing (eliminating) the protecting group include, for example, catalytic reduction in a hydrogen stream in the presence of a catalyst such as Pd-black or Pd-carbon, or hydrogen fluoride anhydride or methanesulfonic acid.
  • a catalyst such as Pd-black or Pd-carbon, or hydrogen fluoride anhydride or methanesulfonic acid.
  • Acid treatment with trifluoromethanesulfonic acid, trifluoroacetic acid or a mixture thereof, base treatment with diisopropylethylamine, triethylamine, piperidine, piperazine, etc., and sodium in liquid ammonia Reduction is also used.
  • the elimination reaction by the above acid treatment is generally carried out at a temperature of about ⁇ 20 ° C. to 40 ° C.
  • a ligand-operating cation scavenger such as dimethyl sulfide, 1,4-butanedithiol, 1,2-ethanedithiol, etc.
  • a ligand-operating cation scavenger such as dimethyl sulfide, 1,4-butanedithiol, 1,2-ethanedithiol, etc.
  • 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 substituted with 1,2-ethanedithiol, 1,4 described above.
  • alkali treatment with dilute sodium hydroxide solution, dilute ammonia and the like.
  • the protection of the functional group that should not be involved in the reaction of the raw materials, the removal of the protective group and the protective group ′, the activation of the functional group involved in the reaction, and the like can be appropriately selected from known groups or known means.
  • an amide of a protein or partial peptide for example, first, amidation of the ⁇ -hydroxyl group of the amino acid at the carboxy terminal is protected by amidation, and a peptide (protein) chain is added to the amino group side of the desired chain. After lengthening the protein, the protein or partial peptide is obtained by removing only the protecting group for the amino group at the ⁇ end of the peptide chain. And a protein or partial peptide from which only the protecting group for the carboxyl group at the C-terminus is removed, and these proteins or peptides are condensed in the above-mentioned mixed solvent. Details of the condensation reaction are the same as described above.
  • the crude protein or peptide can be purified using various known purification means, and the main fraction can be freeze-dried to obtain the desired protein or peptide amide.
  • an ester of a protein or peptide for example, after condensing an ⁇ -carboxyl group of a carboxy-terminal amino acid with a desired alcohol to form an amino acid ester, a desired protein is prepared in the same manner as in the amide of a protein or peptide Alternatively, an ester of the 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 partial peptide or amino acid capable of constituting the partial peptide used in the present invention is condensed with the remaining portion, and when the product has a protecting group, the protecting group is eliminated to thereby remove the desired compound.
  • Peptides can be manufactured. Examples of known condensation methods and elimination of protecting groups include the methods described in the following (a) to (e).
  • the partial peptide used in the present invention can be purified and isolated by a combination of fibrous liquid chromatography and recrystallization.
  • the partial peptide obtained by the above method is a free form, it can be converted to an appropriate salt by a known method or a method analogous thereto, and conversely, when the partial peptide is obtained by a salt, the known method or Can be converted into a free form or another salt by a method according to the above.
  • the polynucleotide encoding the protein of the present invention may be any polynucleotide containing the above-described nucleotide sequence encoding the protein of the present invention.
  • it is DNA.
  • the DNA may be any of a genomic DNA, a genomic DNA library, the above-described cells and tissues-derived cDNA, the above-described cells and tissues-derived cDNA library, and a synthetic DNA.
  • the vector used for the library may be any of pacteriophage, plasmid, cosmid, phagemid and the like.
  • using a preparation of the total RNA or mRNA fraction from the above-described cell may be any of pacteriophage, plasmid, cosmid, phagemid and the like.
  • Amplification can also be carried out by the Transcriptase Polymerase Chain Reaction (hereinafter abbreviated as RT-PCR method).
  • RT-PCR method Transcriptase Polymerase Chain Reaction
  • Examples of the DNA encoding the protein of the present invention include: (i) a DNA containing the nucleotide sequence represented by SEQ ID NO: 2 or SEQ ID NO: 22, or SEQ ID NO: 2 or SEQ ID NO: 2 Encodes a protein having a nucleotide sequence that hybridizes under high stringent conditions to the nucleotide sequence represented by 2 and having substantially the same properties as the protein containing the amino acid sequence represented by SEQ ID NO: 1 DNA, (ii) a DNA containing the nucleotide sequence represented by SEQ ID NO: 4 or SEQ ID NO: 23, or highly stringent with the nucleotide sequence represented by SEQ ID NO: 4 or SEQ ID NO: 23 DNA encoding a protein having a nucleotide sequence that hybridizes under the conditions and having substantially the same properties as a protein containing the amino acid sequence represented by SEQ ID NO: 3, (iii) SEQ ID NO: 4 3, a DNA containing the base sequence represented by SEQ ID NO: 35 or S
  • DNA that can hybridize with the nucleotide sequence represented by SEQ ID NO: 2 or SEQ ID NO: 22 under high stringency conditions include, for example, the nucleotide sequence represented by SEQ ID NO: 2 or SEQ ID NO: 22.
  • DNA having a base sequence having the same is used.
  • DNAs that can hybridize with SEQ ID NO: 43, SEQ ID NO: 35 or SEQ ID NO: 41 under high stringent conditions include, for example, bases represented by SEQ ID NO: 43, SEQ ID NO: 35 or SEQ ID NO: 41 Has about 40% or more homology with the sequence, preferably about 50% or more, preferably about 60% or more, more preferably about 80% or more, particularly preferably about 90% or more, and most preferably about 95% or more.
  • a DNA containing a base sequence or the like is used.
  • Hybridization can be performed according to a known method or a method analogous thereto, for example, the method described in Molecular Cloning 2nd (J. Sambrook et al., Cold Spring Harbor Lab. Press, 1989). .
  • the procedure 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 conditions refer to, for example, a sodium concentration of about 19 to 40 mM, preferably about 19 to 20 mM, and a temperature of about 50 to 70 ° C, preferably about 60 to 65 ° C. In particular, the sodium concentration is about 19 mM and the temperature is about 65. Is most preferred.
  • the DNA encoding the protein containing the amino acid sequence represented by SEQ ID NO: 1 is a DNA containing the base sequence represented by SEQ ID NO: 2; represented by SEQ ID NO: 22
  • Examples of DNA that encodes a protein containing the amino acid sequence represented by SEQ ID NO: 3, such as DNA containing the nucleotide sequence include DNA and sequence containing the nucleotide sequence represented by SEQ ID NO: 4.
  • the DNA containing the base sequence represented by SEQ ID NO: 23, etc. is a DNA encoding the protein containing the amino acid sequence represented by SEQ ID NO: 42.
  • DNA containing the base sequence represented by SEQ ID NO: 35, DNA containing the base sequence represented by SEQ ID NO: 41, or the like is used.
  • the DNA encoding the partial peptide used in the present invention may be any DNA containing a base sequence encoding the partial peptide used in the present invention described above. Further, it may be any of genomic DNA, genomic DNA library, cDNA derived from cells and tissues described above, cDNA library derived from cells and tissues described above, and synthetic DNA.
  • Examples of the DNA ′ encoding the partial peptide used in the present invention include: SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 43, SEQ ID NO: 35 or SEQ ID NO: DNA having a part of the DNA having the nucleotide sequence represented by 41, or SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 43, SEQ ID NO: 35 or sequence It contains a nucleotide sequence that hybridizes under high stringent conditions to the nucleotide sequence represented by No. 41, and contains a part of DNA encoding a protein having substantially the same activity as the protein of the present invention. DNA or the like is used.
  • SEQ ID NO: 2 SEQ ID NO: 4, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 43, SEQ ID NO: 35 or SEQ ID NO: 41
  • the DNA that can hybridize with the base sequence represented by SEQ ID NO: Show the same significance.
  • Hybridization methods and high stringency conditions are the same as described above.
  • the protein and partial peptide of the present invention (hereinafter referred to as the protein of the present invention in the description of the cloning and expression of DNAs encoding them)
  • a means for cloning the DNA that completely encodes the DNA sequence there may be used a method of amplifying by PCR using a synthetic DNA primer having a part of the nucleotide sequence encoding the protein of the present invention, or an appropriate method.
  • the DNA incorporated in the vector can be selected by hybridization with a DNA fragment encoding a part or the entire region of the protein of the present invention or a DNA labeled with a synthetic DNA. Hybridization can be performed according to, for example, the method described in Molecular Cloning 2nd (J. Sambrook et al., Cold Spring Harbor Lab. Press, 1989). When a commercially available library is used, it can be performed according to the method described in the attached instruction manual.
  • Conversion of the nucleotide sequence of DNA is performed using PCR or a known kit, for example, Mutan TM -supe.r Express Km (Takara Shuzo), Mutan TM -K (Takara Shuzo), or the like. It can be carried out according to a known method such as the PCR method, the Gapped dup 1 ex method, the Kunke 1 method, or a method analogous thereto.
  • the DNA encoding the cloned protein can be used as it is depending on the purpose, or can be used after digestion with a restriction enzyme or addition of a linker, if desired.
  • the DNA may have ATG as a translation initiation codon at the 5 'end and TAA, TGA or TAG as a translation stop codon at the 3' end. These translation initiation codon and translation termination codon can also be added using an appropriate synthetic DNA adapter.
  • the expression vector for the protein of the present invention includes, for example, (a) cutting out a target DA fragment from DNA encoding the protein of the present invention, and (mouth) downstream of the promoter in a suitable expression vector. It can be manufactured by connecting to.
  • the vector examples include a plasmid derived from Escherichia coli (eg, pBR322, pBR325, pUC12, pUC13), a plasmid derived from Bacillus subtilis (eg, pUB110, TP5, pC194), a plasmid derived from yeast (eg, Bacteriophages such as pSHl9, pSHl5) and ⁇ phage; animal viruses such as retrovirus, vaccinia virus, and baculovirus; pAl-11, pXTl, pRc / CMV, pRc / RSV, pcDNA I / Ne o and the like are used.
  • Escherichia coli eg, pBR322, pBR325, pUC12, pUC13
  • Bacillus subtilis eg, pUB110, TP5, pC194
  • yeast eg, Bacteriophages such as pSH
  • the promoter used in the present invention may be any promoter as long as it is appropriate for the host used for gene expression.
  • SRo when animal cells are used as host, SRo; promoter, SV40 promoter, LTR promoter, CMV promoter, HSV-TK promoter and the like can be mentioned. Of these, it is preferable to use the CMV (cytomegalovirus) promoter, SRo; Promo-Yuichi, and the like.
  • the host is Escherichia
  • the host is Bacillus, such as tr ⁇ promoter, lac promoter, recA promoter, ⁇ PL promoter, lpp promoter, T7 promoter, etc.
  • the expression vector may contain, in addition to the above, an enhancer, a splicing signal, a poly-A addition signal, a selection primer, and an SV40 replication origin (hereinafter sometimes abbreviated as SV400 ri), if desired. Can be used.
  • Selection methods include, for example, dihydrofolate reductase (hereinafter sometimes abbreviated as dh fr) gene (methotrexate (MTX) resistance), ampicillin resistance gene (hereinafter sometimes abbreviated as Amp ⁇ ) And neomycin resistance gene (hereinafter sometimes abbreviated as Neor, G418 resistance) and the like.
  • dh fr dihydrofolate reductase
  • MTX metalhotrexate
  • Amp ⁇ ampicillin resistance gene
  • Neor neomycin resistance gene
  • a signal sequence suitable for the host is added to the N-terminal side of the protein of the present invention.
  • the host is Escherichia, Pho A signal sequence, OmpA signal sequence, etc.
  • the host is amylase signal sequence, subtilisin, signal sequence, etc.
  • yeast use MFo; signal sequence, SUC2 signal sequence, etc.
  • the host is an animal cell, use insulin 'signal sequence, one interferon' signal sequence, antibody molecule, signal sequence, etc. it can.
  • a transformant can be produced.
  • Escherichia bacteria for example, Escherichia bacteria, Bacillus bacteria, yeast, insect cells, insects, animal cells, and the like are used.
  • Escherichia include, for example, Escherichia coli.
  • Bacillus genus for example, Bacillus subtilis MI114 [Gene, 24, 255 (1983)], 207-21 [Journal of 'Biochemistry, 95, 87 (1984)] and the like are used. .
  • yeast examples include, for example, Saccharomyces cerevisiae AH22, AH22R ", NA87-11A, DKD-5D, 20B-12, Schizosaccharomyces pombe NC YC 1913, NCYC2036, Pichia Pastoris (Pichia pastoris) K # 71 or the like is used.
  • insect cells for example, when the virus is AcNPV, a cell line derived from a larva of night moth (Spodoptera frugiperda cell; Sf cell), a MG1 cell derived from the midgut of Trichoplusia ni, and an egg derived from Trichoplusia ni egg High Five TM cells, cells derived from Mamestra brassicae or cells derived from Estigmena acrea are used.
  • Sf cells include Sf9 cells (ATCC CRL1711), Sf21 cells (Vaughn, J.L., et al., In Vivo).
  • insects for example, silkworm larvae are used [Maeda et al., Nature, Vol. 315, 592 (1985)].
  • animal cells for example, monkey cells COS-7, Vero, Chinese Muster cell CHO (hereinafter abbreviated as CH ⁇ cell), dh fr gene-deficient chinini — Zhamster cell CHO (hereinafter abbreviated as CHO (dh fr ”) cell), mouse L cell, mouse At T-20, mouse myeloma cell Rat GH3, human FL cells, etc. are used.
  • Transformation of insect cells or insects can be performed, for example, according to the method described in Bio / Technology, 6, 47-55 (1988).
  • Transformation of animal cells can be performed, for example, by the method described in Cell Engineering Annex 8 New Cell Engineering Experimental Protocol. 263-267 (1995) (published by Shujunsha), Virology, 52, 456 (1973). It can be performed according to the law. '
  • a liquid medium is suitable as a medium used for the cultivation, and a carbon source necessary for the growth of the transformant is contained therein.
  • the carbon source include glucose, dextrin, soluble starch, and sucrose.
  • the nitrogen source include ammonium salts, nitrates, corn chipperica, peptone, power zein, meat extract, soybean meal, and potato extract.
  • the inorganic or organic substance such as a liquid and the inorganic substance include calcium chloride, sodium dihydrogen phosphate, and magnesium chloride.
  • yeast extract, vitamins, growth promoting factors and the like may be added.
  • the pH of the medium is preferably about 5-8.
  • Examples of a culture medium for culturing Escherichia bacteria include glucose, M9 medium containing noric acid (Miller, Journal of Experiments in
  • a drug such as 3 ⁇ _indolylacrylic acid can be added in order to make the promoter work efficiently as needed.
  • the cultivation is usually carried out at about 15 to 43 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.
  • the host is yeast
  • Burkholder's minimal medium Bostian, KL et al., Proc. Natl. Acad. Sci. (1980)
  • an SD medium containing 0.5% casamino acid Bitter, GA et al., Proc. Natl. Acad. Sci. USA, 81, 5330 (1984)
  • the pH of the medium is adjusted to about 5-8.
  • Culture is usually performed at about 20 to 35 for about 24 to 72 hours, and aeration and agitation are added as necessary.
  • Grace's Insect Medium (Grace, T.C.C, Nature, 195, 788 (1962)) to which an additive such as immobilized 10% serum is appropriately added is used.
  • the pH of the medium is preferably adjusted to about 6.2 to 6.4.
  • Culture is usually performed at about 27 ° C for about 3 to 5 days, and aeration and agitation are added as necessary.
  • the medium When culturing a transformant in which the host is an animal cell, the medium may be, for example, a MEM medium containing about 5 to 20% fetal bovine serum [Science, 122, 501 (1952)], a DMEM medium [Virology , 8, 396 (1959)], RPMI 1640 medium [The Journal of the American Medical Association 199, 519 (1967)], 199 medium
  • the pH is about 6-8.
  • Cultivation is usually carried out at about 30 to 40 ° C for about 15 to 60 hours, and aeration and stirring are added as necessary.
  • the protein of the present invention can be produced in the cells, in the cell membrane, or outside the cells of the transformant.
  • the following method Can be performed.
  • the cells or cells When extracting the protein of the present invention from cultured cells or cells, after culturing, the cells or cells are collected by a known method, suspended in an appropriate buffer, and subjected to sonication, lysozyme and Z or freeze-thawing. After the cells or cells are destroyed by the method described above, a method of obtaining a crude protein extract by centrifugation or filtration is used as appropriate.
  • the buffer may contain a protein denaturing agent such as urea or guanidine hydrochloride, or a surfactant such as Triton X-100 TM.
  • Purification of the protein contained in the culture supernatant or extract obtained in this manner can be performed by appropriately combining known separation and purification methods.
  • These known separation and purification methods mainly include methods using solubility such as salting out and solvent precipitation, dialysis, ultrafiltration, gel filtration, and SDS-polyacrylamide gel electrophoresis, mainly molecular weight.
  • Methods that use differences in charge methods that use differences in charge, such as ion-exchange chromatography, methods that use specific affinities, such as affinity chromatography, and differences in hydrophobicity, such as reverse-phase high-performance liquid chromatography.
  • a method using the difference between isoelectric points, such as isoelectric focusing, is used.
  • the protein thus obtained when obtained in a free form, it can be converted to a salt by a known method or a method analogous thereto, and conversely, when the protein is obtained in the form of a salt, a known method or a method analogous thereto Can be converted into a free form or another salt.
  • 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 protein of the present invention thus produced is determined by using a specific antibody.
  • the antibody against the protein or partial peptide or a salt thereof used in the present invention may be a polyclonal antibody or a monoclonal antibody as long as it can recognize the protein or partial peptide or a salt thereof used in the present invention. You may.
  • An antibody against the protein or partial peptide used in the present invention or a salt thereof (hereinafter, these may be simply abbreviated to the protein of the present invention in the description of the antibody) uses the protein of the present invention as an antigen, Can be produced according to the antibody or antisera production method described above.
  • the protein of the present invention is administered to a warm-blooded animal at a site where the antibody can be produced by administration to itself or together with a carrier or a diluent.
  • Complete Freund's adjuvant / incomplete Freund's adjuvant may be administered in order to enhance antibody production upon administration. Administration is usually performed once every 2 to 6 weeks, for a total of about 2 to 10 times.
  • Examples of the warm-blooded animal to be used include monkeys, egrets, dogs, guinea pigs, mice, rats, sheep, goats, and chickens, and mice and rats are preferably used.
  • a warm-blooded animal immunized with an antigen for example, an individual with an antibody titer is selected from a mouse, and the spleen or lymph node is collected 2 to 5 days after the final immunization and contained in them.
  • an individual with an antibody titer is selected from a mouse, and the spleen or lymph node is collected 2 to 5 days after the final immunization and contained in them.
  • a monoclonal antibody-producing hybridoma can be prepared.
  • the antibody titer in the antiserum can be measured, for example, by reacting the 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 carried out according to a known method, for example, the method of Kohler and Milstein [Nature, 256, 495 (1975)].
  • the fusion promoter include polyethylene glycol (PEG) and Sendai virus, but PEG is preferably used.
  • PEG polyethylene glycol
  • the myeloma cells include myeloma cells of warm-blooded animals such as NS-1, P3U1, SP2 / 0, and AP-1, and P3U1 is preferably used.
  • the preferred ratio between the number of antibody-producing cells (spleen cells) and the number of myeloma cells is about 1: 1 to 20: 1, and PEG (preferably PEG1000 to PEG6000) is added at a concentration of about 10 to 80%.
  • Cell fusion can be performed efficiently by incubating at 20 to 40 ° C (preferably at 30 to 37 ° C for 1 to 10 minutes.
  • Various methods can be used to screen monoclonal antibody-producing hybridomas. For example, 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 an anti-immunoglobulin antibody (labeled with a radioactive substance or an enzyme) is added.
  • a solid phase eg, a microplate
  • an anti-immunoglobulin antibody labeled with a radioactive substance or an enzyme
  • mouse used for cell fusion is mouse, anti-mouse immunoglobulin antibody is used) or protein A is added and monoclonal antibody bound to solid phase is detected Monoclonal antibody bound to solid phase by adding hybridoma culture supernatant to solid phase to which anti-immunoglobulin antibody or protein A is adsorbed, adding proteins labeled with radioactive substances, enzymes, etc. And the like.
  • the selection of the monoclonal antibody can be performed according to a known method or a method analogous thereto. Usually, it can be performed in an animal cell culture medium supplemented with HAT (hypoxanthine, aminopterin, thymidine).
  • HAT hyperxanthine, aminopterin, thymidine
  • any medium can be used as long as it can grow a hybridoma.
  • RPMI 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 hybridoma culture Serum-free medium (SFM-101, Nissui Pharmaceutical Co., Ltd.) can be used.
  • the culture temperature is usually 20 to 40 ° C, preferably about 37 ° C.
  • the culture time is generally 5 days to 3 weeks, preferably 1 week to 2 weeks.
  • the culture can be usually performed under 5% carbon dioxide gas.
  • the antibody titer of the hybridoma culture supernatant can be measured in the same manner as the measurement of the antibody titer in the antiserum described above.
  • Monoclonal antibodies can be separated and purified by known methods, for example, immunoglobulin separation and purification methods (eg, salting out method, alcohol precipitation method, isoelectric point precipitation method, electrophoresis method, ion exchanger (eg, DEAE)). Absorption / desorption method, ultracentrifugation method, gel filtration method, antigen-binding solid phase or antibody only using active adsorbent such as protein A or protein G Specific Purification Method for Obtaining Antibodies by Collecting and Dissociating the Bonds]. , immunoglobulin separation and purification methods (eg, salting out method, alcohol precipitation method, isoelectric point precipitation method, electrophoresis method, ion exchanger (eg, DEAE)). Absorption / desorption method, ultracentrifugation method, gel filtration method, antigen-binding solid phase or antibody only using active adsorbent such as protein A or protein G Specific Purification Method for Obtaining Antibodies by Collecting and Dissociating
  • the polyclonal antibody of the present invention can be produced according to a known method or a method analogous thereto.
  • a immunizing antigen protein antigen
  • a warm-blooded animal is immunized in the same manner as in the above-described monoclonal antibody production method.
  • the antibody can be produced by collecting an antibody-containing substance and separating and purifying the antibody.
  • the type of carrier-protein and the mixing ratio of the carrier and the hapten are determined by the antibody against the hapten immunized by cross-linking the carrier.
  • any kind may be cross-linked at any ratio.
  • a serum albumin, a thyroglobulin, a hemocyanin, etc. in a weight ratio of about 0.1 to hapten per 1 hapten.
  • a method of coupling at a ratio of 20 and preferably about 1 to 5 is used.
  • various condensing agents can be used for force coupling between the hapten and the carrier.
  • daltaraldehyde, carbodiimide, a maleimide active ester, an active ester reagent containing a thiol group or a dithioviridyl group, or the like is used.
  • the condensation product is administered to a warm-blooded animal itself or together with a carrier or diluent at a site where antibody production is possible.
  • Complete Freund's adjuvant or incomplete Freund's adjuvant may be administered in order to enhance the antibody-producing ability upon administration. The administration is usually made once every about 2 to 6 weeks, for a total of about 3 to 10 times.
  • the polyclonal antibody can be collected from the blood, ascites, etc., preferably from the blood of a 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 performed according to the same method for separation and purification of immunoglobulin as in the above-described separation and purification of the monoclonal antibody.
  • DNAs encoding the protein or partial peptide of the present invention are abbreviated as DNAs of the present invention.
  • an antisense polynucleotide having a nucleotide sequence complementary to or substantially complementary to the nucleotide sequence of the present invention or a nucleotide sequence complementary to the nucleotide sequence of the DNA of the present invention.
  • any antisense polynucleotide may be used as long as it has a substantially complementary base sequence or a part thereof and has an action capable of suppressing the expression of the DNA, and may be any antisense polynucleotide. Is preferred.
  • 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 homology. In particular, about 70% of the total nucleotide sequence of the complementary strand of the DNA of the present invention and the complementary strand of the nucleotide sequence encoding the N-terminal portion of the protein of the present invention (for example, the nucleotide sequence near the start codon). As described above, antisense polynucleotides having a homology of preferably about 80% or more, more preferably about 90% or more, and most preferably about 95% or more are suitable.
  • An antisense polynucleotide having a base sequence complementary to or substantially complementary to a base sequence, or a part thereof preferably, for example, SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 43, SEQ ID NO: 35 or SEQ ID NO: 41, a nucleotide sequence complementary to the nucleotide sequence of the DNA having the nucleotide sequence represented by SEQ ID NO: 41, or an antisense polynucleotide having a part thereof, and the like.
  • An antisense polynucleotide is usually composed of about 10 to 40, preferably about 15 to 30 bases.
  • the phosphate residue (phosphate) of each nucleotide constituting the antisense DNA is, for example, a chemically modified phosphorus such as phosphorothioate, methylphosphonate, or phosphorodithionate. It may be substituted by an acid residue.
  • These antisense polynucleotides can be produced using a known DNA synthesizer or the like. According to the present invention, an antisense polynucleotide (nucleic acid) capable of inhibiting the replication or expression of the protein gene of the present invention has been cloned or determined. It can be designed based on and synthesized.
  • Such a polynucleotide can hybridize with RNA of the protein gene of the present invention, inhibit the synthesis or function of the RNA, or interact with the protein-related RNA of the present invention.
  • the expression of the protein gene of the present invention can be regulated and controlled.
  • Polynucleotides that are complementary to the selected sequence of the protein-related RNA of the present invention and polynucleotides that can specifically hybridize with the protein-related RNA of the present invention can be used in vivo and in vitro. Thus, they are useful for regulating and controlling the expression of the protein gene of the present invention, and are also useful for treating or diagnosing diseases and the like.
  • corresponding means having homology or being complementary to a nucleotide, base sequence or a specific sequence of a nucleic acid including a gene.
  • a “correspondence” between a nucleotide, base sequence or nucleic acid and a peptide (protein) usually refers to the amino acids of the peptide (protein) as directed by the nucleotide (nucleic acid) sequence or its complement. pointing.
  • the 3 'end palindrome region and the 3' end hairpin loop may be selected as preferred regions of interest, but any region within the protein gene may be selected as a target.
  • the relationship between the target nucleic acid and a polynucleotide complementary to at least a part of the target region can be said to be "antisense" if the relationship between the target nucleic acid and a polynucleotide that can hybridize with the target is.
  • Antisense polynucleotides may be polynucleotides containing 2-deoxy D-reports, polynucleotides containing DU portions, other types of polynucleotides that are N-glycosides of purine or pyrimidine bases, or non-polynucleotides.
  • Other polymers having a nucleotide backbone eg, commercially available nucleic acid polymers specific to protein nucleic acids and synthetic sequences
  • other polymers containing special bonds provided that the polymer is found in DNA or RNA Allow base pairing and base attachment Containing nucleotides having different arrangements).
  • RNA hybrids can be double-stranded DNA, single-stranded DNA, double-stranded RNA, single-stranded RNA, and even DNA: RNA hybrids, and can be unmodified polynucleotides (or unmodified oligonucleotides).
  • Nucleotides as well as those with known modifications, e.g., those with a label, capped, methylated, or one or more naturally occurring nucleotides with analogs, as known in the art , Modified with an intramolecular nucleotide, for example, having an uncharged bond (eg, methylphosphonate, phosphotriester, phosphoramidate, olebamate, etc.), a charged bond or a sulfur-containing bond (eg, Those having phosphorothioate, phosphorodithioate, etc., such as proteins (nucleases, nucleases and inhibitors) , Toxin, antibodies, signal peptides, poly-L-lysine, etc.) or sugars (for example, monosaccharides), etc., or those having side chain groups, and inter-rate compounds (for example, acridine, psoralen, etc.) Having chelating compounds (eg, metals, radioactive metals,
  • 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 have been replaced with halogens, aliphatic groups, etc., or ethers, It may be converted to a functional group such as amine.
  • the antisense polynucleotide of the present invention is RNA, DNA, or a modified nucleic acid (RNA, DNA).
  • modified nucleic acids include, but are not limited to, sulfuric acid derivatives of nucleic acids, thiophosphate derivatives, and polynucleoside amides, which are resistant to degradation of oligonucleoside amides.
  • the antisense nucleic acid of the present invention can be preferably designed according to the following policy. In other words, the antisense nucleic acid that is more stable in cells Increase the nucleic acid's cell permeability, increase its affinity for the target sense strand, and, if toxic, reduce the toxicity of the antisense nucleic acid.
  • the antisense nucleic acids of the present invention may contain altered or modified sugars, bases, or bonds, may be provided in special forms such as ribosomes or microspheres, may be applied by gene therapy, It could be given in additional 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 and increase nucleic acid uptake (eg, , Phospholipids, cholesterol, etc.).
  • Preferred lipids for addition include cholesterol and its derivatives (eg, cholesteryl chromate formate, cholic acid, etc.).
  • These can be attached to the 3 'end or 5' end of the nucleic acid and can be attached via a base, sugar, or intramolecular nucleoside bond.
  • Other groups include capping groups specifically located at the 3 'or 5' end of nucleic acids that prevent degradation by nucleases such as exonuclease and RNase.
  • capping groups include, but are not limited to, hydroxyl-protecting groups known in the art, such as glycols such as polyethylene glycol and tetraethylene glycol.
  • the inhibitory activity of the antisense nucleic acid can be examined using the transformant of the present invention, the in vivo or in vitro gene expression system of the present invention, or the in vivo or in vitro translation system of the protein of the present invention.
  • the nucleic acid can be applied to cells by various known methods.
  • the protein or partial peptide of the present invention or a salt thereof hereinafter, may be abbreviated as the protein of the present invention
  • the protein or partial peptide of the present invention (Hereinafter sometimes abbreviated as the DNA of the present invention), an antibody against the protein or partial peptide of the present invention or a salt thereof (hereinafter sometimes abbreviated as the antibody of the present invention), and the present invention.
  • the use of the DNA antisense polynucleotide (hereinafter sometimes abbreviated as the antisense polynucleotide of the present invention) will be described.
  • Pharmaceuticals containing a compound that inhibits the activity of the protein of the present invention or a salt thereof include, for example, cations (eg, Ca 2+ , KNa +, etc.) Irregular menstruation, dysmenorrhea, amenorrhea, weight loss amenorrhea, secondary amenorrhea, azoospermia, hypospermia, sperm annihilation, asthenia, spermatogenesis disorder, testicular dysfunction, etc.) , Cancer (e.g., testicular tumor, ovarian cancer, breast cancer, esophageal cancer, lung cancer, kidney cancer, liver cancer, non-small cell lung cancer, prostate cancer, gastric cancer, bladder cancer, cervical cancer, colon cancer, rectal cancer, ⁇ It can be used as a prophylactic / therapeutic agent for organ cancer, thymoma, etc., and as a contraceptive drug. It is preferably used as a contraceptive or the like.
  • a medicament containing a compound or a salt thereof that promotes the activity of the protein of the present invention can be used, for example, by promoting cation (eg, Ca 2+ , KNa +, etc.) permeation activity to obtain, for example, infertility ( E.g., irregular menstruation, dysmenorrhea, amenorrhea, weight-loss amenorrhea, secondary amenorrhea, azoospermia, hypospermia, sperm annihilation, asthenia, spermatogenesis, testicular dysfunction (Eg, testicular tumor, ovarian cancer, breast cancer, esophageal cancer, lung cancer, kidney cancer, liver cancer, non-small cell lung cancer, prostate cancer, gastric cancer, bladder cancer, cervical cancer, colon cancer, rectal cancer) • Prophylactic / therapeutic agents such as thyroid cancer, thymoma, etc., and contraceptives and other medicines. It is preferably used as an infertility
  • a preventive / therapeutic agent for various diseases related to the protein of the present invention [1] A preventive / therapeutic agent for various diseases related to the protein of the present invention.
  • the protein of the present invention has a cation (eg, Ca 2+ , K +, Na +, etc.) permeability activity, and contributes to the permeation thereof, thereby affecting cell functions such as sperm motility and fertilization ability. Plays an important role.
  • a cation eg, Ca 2+ , K +, Na +, etc.
  • cancer eg, testicular tumor, ovarian cancer, breast cancer, esophageal cancer, lung cancer, kidney cancer, liver cancer, non-small cell lung cancer, prostate cancer, gastric cancer, bladder cancer, cervical cancer, colon cancer, rectal cancer, Teng Teng Organ cancer, thymoma, etc.
  • infertility eg, irregular menstruation, dysmenorrhea, amenorrhea, weight loss amenorrhea, secondary amenorrhea, azoospermia, hypospermia, sperm annihilation,
  • diseases such as asthenozoospermia, spermatogenesis disorder, and testicular dysfunction develop.
  • the protein of the present invention and the DNA of the present invention may be used, for example, for infertility (eg, irregular menstruation, dysmenorrhea, amenorrhea, weight-loss amenorrhea, secondary amenorrhea, azoospermia, sperm Hypoplasia, aspermia, asthenozoospermia, spermatogenesis disorder, testicular dysfunction, etc., cancer (eg, testicular tumor, ovarian cancer, breast cancer, esophageal cancer, lung cancer, kidney cancer, liver cancer, non-small cell lung cancer, prostate It can be used as a prophylactic / therapeutic agent for cancer, stomach cancer, bladder cancer, cervical cancer, colon cancer, rectal cancer, kidney cancer, thymoma, etc., and as a medicament for contraceptives. It is preferably used as an agent for preventing or treating infertility.
  • infertility eg, irregular menstruation, dysmenorrhea,
  • the DNA of the present invention When the DNA of the present invention is used as the above-mentioned prophylactic or therapeutic agent, the DNA is used alone or inserted into an appropriate vector such as a retrovirus vector, an adenovirus vector, or an adenovirus associated virus vector. Thereafter, it can be administered to humans or warm-blooded animals according to conventional means.
  • the DNA of the present invention can be administered as it is or in the form of a formulation with a physiologically acceptable carrier such as an adjuvant for promoting uptake, using a gene gun or a catheter such as a hydrogel catheter.
  • the protein of the present invention is used as the above-mentioned prophylactic / therapeutic agent, at least 90%, preferably 95% or more, more preferably 98% or more, still more preferably It is preferable to use one purified to 99% or more.
  • the protein of the present invention can be used, for example, in the form of tablets, capsules, elixirs, microcapsules, etc., which are sugar-coated as required, orally, or in water or other pharmaceutically acceptable liquids. It can be used parenterally in the form of injections, such as aqueous solutions or suspensions.
  • the protein of the present invention may be used together with physiologically acceptable carriers, flavors, excipients, vehicles, preservatives, stabilizers, binders, and the like in a unit dosage form generally required for the practice of pharmaceutical preparations. It can be manufactured by mixing. The amount of active ingredient in these preparations is such that a suitable dosage in the specified range can be obtained.
  • Additives that can be incorporated into tablets, capsules, etc. include, for example, binders such as gelatin, corn starch, tragacanth, gum arabic, excipients such as crystalline cellulose, corn starch, gelatin, alginic acid, etc. Swelling agents such as magnesium stearate, sweeteners such as sucrose, lactose or saccharin, and flavoring agents such as peppermint, cocoa oil or cellulose.
  • a liquid carrier such as oil and fat can be further contained in the above-mentioned type of material.
  • Sterile compositions for injection can be formulated according to standard pharmaceutical practice, such as dissolving or suspending the active substance in vehicles such as water for injection, and naturally occurring vegetable oils such as sesame oil and coconut oil. it can.
  • aqueous solutions for injection examples include physiological saline, isotonic solutions containing glucose and other adjuvants (eg, D-sorbyl, D-mannitol, sodium chloride, etc.), and the like.
  • Suitable solubilizers such as alcohols (eg, ethanol), polyalcohols (eg, propylene glycol, polyethylene glycol), nonionic surfactants (eg, Polysorbate 80 TM, HCO— 50 etc.).
  • the oily liquid examples include sesame oil and soybean oil, and may be used in combination with a solubilizing agent such as benzyl benzoate or benzyl alcohol.
  • buffers eg, phosphate buffer, sodium acetate buffer, etc.
  • soothing agents eg, benzalkonium chloride, proactive hydrochloride, etc.
  • stabilizers eg, human serum albumin, polyethylene glycol, etc.
  • preservatives eg, benzyl alcohol, phenol, etc.
  • antioxidants eg, antioxidants and the like.
  • the prepared injection is usually filled in an appropriate ampoule.
  • the vector into which the DNA of the present invention is inserted is also formulated in the same manner as described above, and is usually used parenterally.
  • the preparations obtained in this way are safe and have low toxicity, for example, in warm-blooded animals (e.g., humans, rats, mice, guinea pigs, egrets, birds, higgies, bush, pests, pests, cats, Dogs, monkeys, chimpanzees, etc.).
  • warm-blooded animals e.g., humans, rats, mice, guinea pigs, egrets, birds, higgies, bush, pests, pests, cats, Dogs, monkeys, chimpanzees, etc.
  • the dosage of the protein of the present invention varies depending on the target disease, the subject of administration, the administration route, and the like.
  • the protein of the present invention when administered for the purpose of treating infertility, it is generally required for an adult (body weight 60). ), the protein is administered from about 0.1 to: L0 Omg, preferably from about 1.0 to 50 mg, more preferably from about 1.0 to 20 mg per day.
  • the single dose of the protein varies depending on the administration subject, target disease, and the like.
  • the protein of the present invention may be administered in the form of an injection to an adult (body weight) for the purpose of treating infertility.
  • the protein of the present invention is useful as a reagent for screening a compound that promotes or inhibits the activity of the protein of the present invention or a salt thereof.
  • the present invention relates to (1) a compound or a salt thereof that promotes or inhibits the activity (for example, cation permeation activity, etc.) of the protein of the present invention characterized by using the protein of the present invention (hereinafter referred to as “promoter”).
  • promotes or inhibits the activity for example, cation permeation activity, etc.
  • agents and inhibitors may be abbreviated). More specifically, for example,
  • the present invention provides a method for screening for an accelerator or an inhibitor, comprising comparing a cation (eg, Ca2 + , K +, Na +, etc.) permeation activity of a mixture of cells having the following and a test compound.
  • a cation eg, Ca2 + , K +, Na +, etc.
  • the change in membrane potential accompanying the permeation of cations is It is characterized in that it is measured and compared as an index of cation permeation.
  • Test compounds include, for example, peptides, proteins, non-peptidic compounds, synthetic compounds, fermentation products, cell extracts, plant extracts, animal tissue extracts, and the like. Or a known compound.
  • cells having the ability to produce the protein of the present invention are prepared by suspending them in a buffer suitable for screening.
  • Buffers include cations (eg, Ca 2+ , K +, ⁇ a) of the protein of the present invention, such as a phosphate buffer and a borate buffer having a pH of about 4 to 10 (preferably, a pH of about 6 to 8). Any buffer may be used as long as it does not inhibit the permeation activity.
  • a host transformed with a vector containing the above-described DNA encoding the protein of the present invention is used.
  • a host for example, animal cells such as CHO cells are preferably used.
  • a transformant in which the protein of the present invention is expressed on a cell membrane by culturing by the method described above is preferably used.
  • the cation (eg, C a M , K + , Na +, etc.) permeation activity of the protein of the present invention can be determined by a known method, for example, the method described in J. Biol. Chem., 275, 12237, 2000. Alternatively, it can be measured according to a method analogous thereto.
  • the cation (eg, Ca 2+ , K Na +, etc.) permeation activity in the case (ii) above is about 20% or more, preferably 30% or more, more preferably Can be selected as a compound or salt thereof that promotes the activity of the protein of the present invention.
  • the permeation activity of the cation (eg, Ca 2+ , KNa +, etc.) in the case (ii) above is about 20% or more, preferably 30%, as compared with the case (i).
  • a test compound that inhibits (or suppresses) more preferably about 50% or more can be selected as a compound that inhibits the activity of the protein of the present invention or a salt thereof.
  • a gene such as secreted alkaline phosphatase or luciferase was inserted into the Byone stream of the promoter of the protein gene of the present invention, expressed in the various cells described above, and the test compound was brought into contact with the cells.
  • a compound or a salt thereof that promotes or suppresses the expression of the protein of the present invention ie, promotes or inhibits the activity of the protein of the present invention
  • the salt can be screened.
  • the polynucleotide encoding the protein of the present invention is useful as a reagent for screening a compound or a salt thereof that promotes or inhibits the expression of the protein gene of the present invention.
  • the present invention relates to (3) a compound or a salt thereof that promotes or inhibits the expression of the protein gene of the present invention, which comprises using a polynucleotide encoding the protein of the present invention (hereinafter, abbreviated as a promoter and an inhibitor, respectively) May be provided), and more specifically, for example,
  • the present invention provides a method of screening for an accelerator or an inhibitor, characterized in that the method is carried out.
  • the expression level of the protein gene of the present invention (specifically, the amount of the protein of the present invention or mRNA encoding the protein) Measure) and compare.
  • Test compounds include, for example, peptides, proteins, non-peptidic compounds, synthetic compounds, fermentation products, cell extracts, plant extracts, animal tissue extracts, etc., and these compounds may be novel compounds. However, a known compound may be used.
  • cells having the ability to produce the protein of the present invention are prepared by suspending them in a buffer suitable for screening.
  • the buffer may be any buffer that does not inhibit the expression of the protein gene of the present invention, such as a phosphate buffer or a borate buffer having a pH of about 4 to 10 (preferably, a pH of about 6 to 8). Either may be used.
  • a host transformed with a vector containing DNA encoding the protein of the present invention described above is used.
  • a host for example, animal cells such as CHO cells are preferably used.
  • a transformant in which the protein of the present invention is expressed on a cell membrane by culturing by the method described above is preferably used.
  • the amount of the protein of the present invention can be measured by a known method, for example, using an antibody recognizing the protein of the present invention to detect the protein present in a cell extract or the like, using a method such as Western analysis, ELISA, or the like. It can be measured in accordance with the corresponding method.
  • the expression level of the protein gene of the present invention can be determined by a known method, for example, Northern blotting, Reverse transcription-polymerase chain react ion (RT-PCR), Real-time PCR analysis system (manufactured by ABI, TaqMan polymerase chain reactant). ion) or a method analogous thereto.
  • RT-PCR Reverse transcription-polymerase chain react ion
  • Real-time PCR analysis system manufactured by ABI, TaqMan polymerase chain reactant. ion
  • a method analogous thereto for example, Northern blotting, Reverse transcription-polymerase chain react ion (RT-PCR), Real-time PCR analysis system (manufactured by ABI, TaqMan polymerase chain reactant). ion) or a method analogous thereto.
  • the expression level of the protein gene of the present invention in the case of the above (iv) is about 20% or more, preferably 30% or more, more preferably about 5% or more as compared with the case of the above (iii).
  • a test compound that promotes 0% or more can be selected as a compound that promotes expression of the protein gene of the present invention or a salt thereof.
  • the expression level of the protein gene of the present invention in the case of the above (iv) is about 20% or more, preferably 30% or more, more preferably about 5% or more as compared with the case of the above (iii).
  • a test compound that inhibits 0% or more can be selected as a compound that inhibits expression of the protein gene of the present invention or a salt thereof.
  • the antibody of the present invention is useful as a reagent for screening a compound or its salt that promotes or inhibits the expression of the protein of the present invention.
  • the present invention provides (5) a compound or a salt thereof that promotes or inhibits the expression of the protein of the present invention, which is characterized by using the antibody of the present invention (hereinafter, may be abbreviated as a promoter or an inhibitor, respectively).
  • a promoter or an inhibitor may be abbreviated as a promoter or an inhibitor, respectively.
  • the expression level (specifically, the protein mass of the present invention) of the protein of the present invention in the cases (V) and (vi) is measured using the antibody of the present invention (example). Then, the expression of the protein of the present invention is detected, and the expression level of the protein of the present invention is quantified.
  • Test compounds include, for example, peptides, proteins, non-peptidic compounds, synthetic compounds, fermentation products, cell extracts, plant extracts, animal tissue extracts, and the like. Or a known compound. '
  • cells having the ability to produce the protein of the present invention are prepared by suspending them in a buffer suitable for screening.
  • Buffers include 13 ⁇ 1 about 4 to 10 (preferably about 6 to 8) phosphate buffer, borate buffer, and other cations of the protein of the present invention (eg, Ca 2+ , K ⁇ a + ) Any buffer may be used as long as it does not inhibit the permeation activity.
  • a host transformed with a vector containing the DNA encoding the protein of the present invention described above is used.
  • a host for example, animal cells such as CHO cells are preferably used.
  • a transformant in which the protein of the present invention is expressed on a cell membrane by culturing by the method described above is preferably used.
  • the amount of the protein of the present invention can be measured by a known method, for example, using an antibody that recognizes the protein of the present invention to detect the protein present in a cell extract or the like. It can be measured according to a method such as protein analysis, ELISA, or a method analogous thereto.
  • the expression level of the protein of the present invention in the case of the above (vi) is promoted by about 20% or more, preferably 30% or more, more preferably about 50% or more as compared with the case of the above (v).
  • the test compound to be tested can be selected as a compound that promotes the expression of the protein of the present invention or a salt thereof.
  • the expression level of the protein of the present invention in the case of the above (vi) 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).
  • the test compound to be tested can be selected as a compound that inhibits the expression of the protein of the present invention or a salt thereof.
  • the screening kit of the present invention contains cells capable of producing the protein or partial peptide used in the present invention or a salt thereof, or the protein or partial peptide used in the present invention.
  • Compounds or salts thereof obtained by using the screening method or screening kit of the present invention can be prepared from the test compounds described above, for example, peptides, proteins, non-peptidic compounds, synthetic compounds, fermentation products, cell extracts, and plant extracts. Liquid, animal tissue extract, plasma or the like, and a compound or a salt thereof that promotes or inhibits the activity (eg, cation permeation activity, etc.) of the protein of the present invention.
  • salt of the compound those similar to the aforementioned salts of the protein of the present invention are used.
  • 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 protein gene of the present invention, compounds or salts thereof that promote the expression of the protein of the present invention include, for example, infertility (E.g., irregular menstruation, dysmenorrhea, amenorrhea, weight loss amenorrhea, secondary amenorrhea, azoospermia, hypospermia, sperm death, asthenia, spermatogenesis disorder, testicular function Insufficiency), cancer (eg, testicular tumor, ovarian cancer, breast cancer, esophageal cancer, lung cancer, kidney cancer, liver cancer, non-small cell lung cancer, prostate cancer, stomach cancer, bladder jesus, cervical cancer, colon cancer, rectum It is useful as a preventive agent for cancer, Teng's carcinoma, thymoma, etc., and as a medicine such as a contraceptive.
  • the compound or its salt that inhibits the activity of the protein of the present invention, the compound or its salt that inhibits the expression of the protein gene of the present invention, and the compound or its salt that inhibits the expression of the protein of the present invention include, for example, Symptoms (e.g., irregular menstruation, dysmenorrhea, amenorrhea, weight-loss amenorrhea, secondary amenorrhea, azoospermia, hypospermia, sperm death, asthenia asthenia, spermatogenesis, testicular function Failure), cancer (eg, testicular tumor, ovarian cancer, breast cancer, esophageal cancer, lung cancer, kidney cancer, liver cancer, non-small cell lung cancer, prostate cancer, gastric cancer, bladder cancer, cervical pole, colon cancer, rectal cancer , Tengen ⁇ thymoma, etc.) are useful as preventive and therapeutic agents, and as contraceptives and other medicines.
  • Symptoms e.g., irregular men
  • a contraceptive or the like it is preferably used as a contraceptive or the like.
  • a compound or a salt thereof obtained by using the screening method or the screening kit of the present invention can be formulated according to a conventional method.
  • tablets, capsules, elixirs, microcapsules, sterile solutions, suspensions and the like can be used.
  • the preparations obtained in this way are safe and low toxic and can be used, for example, in humans or in warm-blooded animals (eg mice, rats, puppies, higgs, pigs, puppies, puppies, birds, cats, dogs, monkeys). , Chimpanzees, etc.) can be administered orally or parenterally.
  • warm-blooded animals eg mice, rats, puppies, higgs, pigs, puppies, puppies, birds, cats, dogs, monkeys.
  • Chimpanzees, etc. can be administered orally or parenterally.
  • 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 its compound that promotes the activity of the protein of the present invention for the purpose of treating infertility can be used.
  • the salt is administered orally, generally, in an adult (assuming a body weight of 60 kg), the compound or a salt thereof is used in an amount of about 0.1 to 10 Omg, preferably about 1.0 to 5 Omg per day. More preferably about 1.0 to 20 mg.
  • the single dose of the compound or a salt thereof varies depending on the administration subject, target disease, etc., for example, to promote the activity of the protein of the present invention for the purpose of treating infertility.
  • the compound or a salt thereof When a compound or a salt thereof is administered to an adult (with a body weight of 6 O kg) usually in the form of an injection, the compound or a salt thereof is administered in an amount of about 0.01 to 3 O mg per day, preferably about 0. It is convenient to administer! -2 O mg, more preferably about 0.1-1 O mg, by intravenous injection. In the case of other animals, * can be administered in terms of the body weight per 60 kg.
  • Quantification of the protein of the present invention, its partial peptide or its salt Since the antibody of the present invention can specifically recognize the protein of the present invention, the quantification of the protein of the present invention in a test solution, particularly It can be used for quantification by sandwich immunoassay.
  • the protein of the present invention can be quantified using a monoclonal antibody against the protein of the present invention (hereinafter sometimes referred to as the monoclonal antibody of the present invention), and can also be detected by tissue staining or the like.
  • the antibody molecule itself may be used, or 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-antigen complex corresponding to the amount of antigen (eg, the amount of protein) in the test solution. Any measurement method may be used as long as the amount of the body is detected by chemical or physical means, and this is calculated from a standard curve prepared using a standard solution containing a known amount of antigen. . For example, nephelometry, competition method, immunometric method and sandwich method are preferably used, but it is particularly preferable to use the 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 include Eg to [l25 i] c I3i n, [3 H.], and [i4 c] used.
  • the above-mentioned enzyme those which are stable and have a large specific activity are preferable. For example, / 3-galactosidase, j3-darcosidase, alkaline phosphatase, peroxidase, malate dehydrogenase and the like are used. .
  • fluorescent substance for example, fluorescamine, fluorescein isothiosinate and the like are used.
  • luminescent substance for example, luminol, luminol derivative, luciferin, lucigenin and the like are used.
  • a biotin-avidin system can be used for binding the antibody or antigen to the labeling agent.
  • the carrier include insoluble polysaccharides such as agarose, dextran, and cellulose; synthetic resins such as polystyrene, polyacrylamide, and silicon; and glass.
  • the test solution is reacted with the insolubilized monoclonal antibody of the present invention (primary reaction), and further reacted with another labeled monoclonal antibody of the present invention (secondary reaction).
  • primary reaction the insolubilized monoclonal antibody of the present invention
  • secondary reaction another labeled monoclonal antibody of the present invention
  • the primary reaction and the secondary reaction may be performed in the reverse order, may be performed simultaneously, or may be performed at staggered times.
  • the labeling agent and the method of insolubilization can be in accordance with those described above.
  • the antibody used for the solid phase antibody or the labeling antibody does not necessarily need to be one kind, and a mixture of two or more kinds of antibodies is used for the purpose of improving measurement sensitivity and the like. May be used.
  • the monoclonal antibody of the present invention used in the primary reaction and the secondary reaction is an antibody having a different site to which the protein of the present invention binds. It is preferably used. That is, the antibody used in the primary reaction and the secondary reaction is, for example, when the antibody used in the secondary reaction recognizes the C-terminal of the protein of the present invention, the antibody used in the primary reaction is Preferably, an antibody that recognizes other than the C-terminal, such as the N-terminal, is used.
  • Measuring system other than the sandwich method using the monoclonal antibody of the present invention for example, in the competition method, which can be used in the competition method, the immunometric method or the nephrometry, the antigen in the test solution and the labeled antigen are allowed to react competitively with the antibody, and then the unreacted labeled antigen (F ) And the labeled antigen (B) bound to the antibody are separated (BZF separation), the labeling amount of either B or F is measured, and the amount of antigen in the test solution is quantified.
  • the competition method which can be used in the competition method, the immunometric method or the nephrometry
  • the antigen in the test solution and the labeled antigen are allowed to react competitively with the antibody, and then the unreacted labeled antigen (F ) And the labeled antigen (B) bound to the antibody are separated (BZF separation), the labeling amount of either B or F is measured, and the amount of antigen in the test solution is quantified.
  • a soluble antibody is used as the 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. After reacting the antigen with an excess amount of the labeled antibody, 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. Next, the amount of label in either phase is measured to determine the amount of antigen in the test solution.
  • nephrometry the amount of insoluble sediment resulting from an antigen-antibody reaction in a gel or in a solution is measured. Even when the amount of antigen in the test solution is small and only a small amount of sediment is obtained, laser nephrometry utilizing laser scattering is preferably used.
  • the protein measuring system of the present invention may be constructed by adding ordinary technical considerations of those skilled in the art to ordinary conditions and operation methods in each method. For details of these general technical means, reference can be made to reviews, documents, etc.
  • the protein of the present invention can be quantified with high sensitivity by using the antibody of the present invention.
  • the concentration of the protein of the present invention is reduced by quantifying the concentration of the protein of the present invention using the antibody of the present invention, for example, cancer (eg, testicular tumor, ovarian cancer, breast cancer) , Esophageal cancer, lung cancer, kidney cancer, liver cancer, non-small cell lung cancer, prostate cancer, stomach cancer, '' 'bladder cancer, cervical cancer, colon cancer, rectum cancer, kidney cancer, thymoma, etc.)
  • Pregnancy e.g., irregular menstruation, dysmenorrhea, amenorrhea, weight loss amenorrhea, secondary amenorrhea, azoospermia, hypospermia, sperm annihilation, asthenia asthenia, impaired spermatogenesis, Testis dysfunction, etc.
  • pregnancy e.g., irregular menstruation, dysmenorrhea, amenorrhea, weight loss amenorrhea, secondary amenorrhea,
  • cancer eg, testicular tumor, ovarian cancer, breast cancer, esophageal cancer, lung cancer, kidney cancer, liver cancer, non-small cell lung cancer, Prostate cancer, gastric cancer, bladder cancer, cervical cancer, colon cancer, rectal cancer, renal cancer, thymoma, etc.
  • infertility eg, irregular menstruation, dysmenorrhea, amenorrhea, no weight loss
  • Menstruation secondary amenorrhea, azoospermia, hypospermia, spermatozoa, asthenozoospermia, spermatogenesis disorder, testicular dysfunction, etc.
  • 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.
  • a subject such as a body fluid or a tissue.
  • the detection of the protein of the present invention in each fraction during purification, and the analysis of the behavior of the protein of the present invention in test cells, etc. Can be used.
  • the DNA of the present invention can be used, for example, by using it as a probe in humans or warm-blooded animals (eg, rats, mice, guinea pigs, egrets, birds, higgies, bush, Abnormalities (genetic abnormalities) of DNA or mRNA encoding the protein of the present invention or a partial peptide thereof in a mouse, a cat, a cat, a dog, a monkey, a chimpanzee, etc.
  • the DNA or mRNA is useful as a diagnostic agent for a gene such as damage, sudden mutation or reduced expression, and increased or overexpressed DNA or mRNA.
  • the above-described genetic diagnosis using the DNA of the present invention includes, for example, the well-known Northern Hybridization and the PCR-SSCP method (Genomics, Vol. 5, pp. 874-879 (1989), Proc. Proceedings of the National Academy of Sciences of the United States of America; Vol. 86, pp. 2766-2770 (1989)) Can be implemented.
  • cancer eg, testicular tumor, ovarian cancer, breast cancer, esophagus cancer, lung cancer, kidney cancer, liver cancer, non-small cell lung cancer, prostate cancer
  • Stomach cancer bladder cancer, cervical cancer, colon cancer, rectal cancer
  • Teng's cancer, thymoma, etc. infertility (eg, irregular menstruation, dysmenorrhea, amenorrhea, weight loss amenorrhea, Secondary amenorrhea, azoospermia, azoospermia, sperm killing, asthenozoospermia, spermatogenesis disorder, testicular dysfunction, etc.).
  • infertility eg, irregular menstruation, dysmenorrhea, amenorrhea, weight loss amenorrhea, Secondary amenorrhea, azoospermia, azoospermia, sperm killing, asthenozoospermia, spermatogenesis disorder, test
  • a decrease in expression is detected or a sudden mutation of DNA is detected by the PCR-SS CP method, it may include, for example, cancer (eg, testicular tumor, ovarian cancer, breast cancer, esophageal cancer, lung cancer) , Kidney cancer, liver cancer, non-small cell lung cancer, prostate cancer, stomach cancer, bladder cancer, cervical cancer, colon cancer, rectal cancer, Tengler cancer, thymoma, etc.), infertility (eg, irregular menstruation, menstruation) Dysmenorrhea, amenorrhea, weight-loss amenorrhea, secondary amenorrhea, azoospermia, hypospermia, sperm annihilation, asthenia asperity, spermatogenesis disorder, testicular dysfunction, etc.) Can be diagnosed as highly likely.
  • cancer eg, testicular tumor, ovarian cancer, breast cancer, esophageal cancer, lung cancer
  • Kidney cancer liver cancer, non
  • the antisense polynucleotide of the present invention which complementarily binds to the DNA of the present invention and can suppress the expression of the DNA, has low toxicity, and the function of the protein of the present invention or the DNA of the present invention in vivo (Example) , Cations (eg, Ca 2+ , K Na + Etc.) can be controlled, for example, infertility (eg, irregular menstruation, dysmenorrhea, amenorrhea, weight loss amenorrhea, secondary amenorrhea, azoospermia, Spermia, sperm annihilation, asthenozoospermia, spermatogenesis disorder, testicular dysfunction, etc., cancer (eg, testicular tumor, ovarian cancer, breast cancer, esophageal cancer, lung cancer, kidney cancer, liver cancer, non-small cell lung cancer, Prostate, gastric, bladder, cervical, colon, rectal, renal, thymoma,
  • the above antisense polynucleotide When used as the above medicine, it can be formulated and administered according to a known method.
  • the antisense polynucleotide when used, the antisense polynucleotide is used, the antisense polynucleotide is used alone or after being inserted into an appropriate vector such as a retrovirus vector, an adenovirus vector, an adenovirus associated virus vector, or the like. It can be administered orally or parenterally to humans or mammals (eg, rats, rabbits, sheep, sheep, pigeons, horses, cats, dogs, monkeys, etc.) according to conventional means.
  • 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 auxiliary agent for promoting uptake, and can be administered by a gene gun or a catheter such as a hydrogel catheter.
  • the dosage of the antisense polynucleotide varies depending on the target disease, the administration subject, the administration route, and the like.For example, when the antisense polynucleotide of the present invention is locally administered to testis for the purpose of treating infertility, Generally, in an adult (body weight of 6 O kg), about 0.1 to 100 mg of the antisense polynucleotide is administered per day.
  • antisense polynucleotide can also be used as a diagnostic oligonucleotide probe for examining the presence or expression of the DNA of the present invention in tissues or cells.
  • RNA double-stranded RNA
  • protein of the present invention A lipozyme containing a portion of the RNA
  • V An expression vector containing a gene (DNA) encoding the lipozyme is provided.
  • double-stranded RNA, lipozyme, etc. can also destroy RNA or transcribe RNA from the DNA of the present invention, and can be used in the present invention in vivo.
  • infertility eg, irregular menstruation, dysmenorrhea, amenorrhea, weight-loss amenorrhea, secondary amenorrhea, Azoospermia, hypospermia, sperm annihilation, asthenozoospermia, spermatogenesis disorder, testicular dysfunction, etc.
  • cancer eg, testicular tumor, ovarian cancer, breast cancer, esophageal cancer, lung cancer, kidney cancer, liver cancer, non It can be used as a prophylactic or therapeutic agent for small cell lung cancer, prostate cancer, stomach cancer, bladder cancer, cervical cancer, colon cancer, rectal cancer, kidney cancer, thymoma, etc. it can. It is preferably used as a contraceptive.
  • the double-stranded RNA can be designed and manufactured based on the sequence of the polynucleotide of the present invention according to a known method (eg, Nature, 411, 494, 2001).
  • the lipozyme can be designed and produced based on the sequence of the polynucleotide of the present invention according to a known method (eg, TRENDS in Molecular Medicine, Vol. 7, pp. 221, 2001). For example, it can be produced by substituting a part of a known lipozyme sequence with a part of an RNA encoding the protein of the present invention.
  • a sequence in the vicinity of a consensus sequence NUX (where N represents all bases and X represents a base other than G) which can be cleaved by a known lipozyme. And the like.
  • RNA or lipozyme When the above double-stranded RNA or lipozyme is used as the above-mentioned prophylactic / therapeutic agent, it can be formulated and administered in the same manner as an antisense polynucleotide.
  • the expression vector (V) is used in the same manner as known gene therapy methods and the like, and is used as the above-mentioned prophylactic / therapeutic agent.
  • the DNA encoding the exogenous protein of the present invention (hereinafter abbreviated as the exogenous DNA of the present invention) or its mutant DNA (the exogenous mutant DNA of the present invention may be abbreviated).
  • the present invention provides: (1) a non-human mammal having the exogenous DNA of the present invention or a mutant DNA thereof,
  • 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, electric pulse It can be produced by introducing the target DNA by the lipofection method, the coagulation method, the microinjection method, the particle gun method, or the DEAE-dextran method.
  • exogenous DNA of the present invention which is intended for somatic cells, organs of living organisms, tissue cells, and the like, can be introduced by the DNA introduction method and used for cell culture, tissue culture, and the like. Is fused with the above-mentioned germinal cells by a known cell fusion method to produce the DNA-introduced animal of the present invention.
  • mice for example, red sea lions, bushes, higgins, goats, gray egrets, dogs, cats, guinea pigs, hamsters, mice, rats, and the like are used.
  • mice for example, pure strains such as C57B LZ6 and DBA2
  • rat eg, Wistar, SD, etc.
  • Examples of the “mammal” in the recombinant vector that can be expressed in mammals include humans and the like in addition to the above-mentioned non-human mammals.
  • the exogenous DNA of the present invention refers to the DNA of the present invention once isolated and extracted from a mammal, not the DNA of the present invention originally possessed by a non-human mammal.
  • mutant DNA of the present invention those in which a mutation (for example, mutation) has occurred in the base sequence of the original DNA of the present invention, specifically, addition or deletion of a base, substitution with another base, or the like. Such DNAs are used, and abnormal DNAs are also included.
  • the abnormal DNA means 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 a mammal that is the same or different from the animal of interest.
  • a promoter capable of expressing in animal cells For example, when introducing the human DNA of the present invention, various mammals having the DNA of the present invention having a high homology with the DNA are introduced.
  • a vector into which a DNA of the present invention is highly expressed can be produced by microinjecting a vector (such as a vector) into a fertilized egg of a target mammal, for example, a mouse fertilized egg.
  • Examples of the expression vector of the protein of the present invention include plasmids derived from Escherichia coli, plasmids derived from Bacillus subtilis, plasmids derived from yeast, pateriophage such as ⁇ phage, retroviruses such as Moroni leukemia virus, vaccinia virus and the like.
  • animal viruses such as baculovirus are used.
  • a plasmid derived from Escherichia coli, a plasmid derived from Bacillus subtilis or a plasmid derived from yeast are preferably used.
  • Examples of the promoter that regulates the expression of DNA include, for example, DNA derived from (i) virus (eg, Simian virus, cytomegalovirus, Moroni leukemia virus, JC virus, breast cancer virus, poliovirus, etc.).
  • virus eg, Simian virus, cytomegalovirus, Moroni leukemia virus, JC virus, breast cancer virus, poliovirus, etc.
  • Promoters derived from various mammals for example, albumin, insulin Phosphorus II, perobrakin II, elastase, erythropoietin, endothelin, muscle creatine kinase, glial fibrillary acidic protein, daltathione S-transferase, platelet-derived growth factor] 3, keratins Kl, K10 and K14, cola Type I and type II, cyclic AMP-dependent protein kinase i3 I subunit, dystrophin, tartrate-resistant alkaline phosphatase, atrial sodium diuretic factor, endothelial receptor yuichi tyrosine kinase (commonly abbreviated as Tie 2), Sodium potassium adenosine 3 kinase (Na, K-ATPase), neurofila
  • Immunoglobulin, heavy chain variable region (VNP), serum amyloid P component, myoglobin, troponin (:, smooth muscle exactin, prebub enkephalin A, promoter such as vasopressin, etc. are used.
  • Sactin promoter, and the like are suitable.
  • the vector preferably has a sequence that terminates transcription of the target mRNA in a DNA-introduced mammal (generally referred to as terminator), for example, from a virus and from various mammals.
  • terminator a DNA-introduced mammal
  • the sequence of each DNA can be used, and preferably, Simian virus SV40 or the like is used.
  • the splicing signal of each DNA, the enhancer region, a part of the intron of the eukaryotic DNA, etc. are used to further express the target exogenous DNA at the 5 'upstream of the promoter region and the promoter region. It is also possible to link between translation regions or 3 ′ downstream of the translation region depending on the purpose.
  • the normal translation region of the protein of the present invention is DNA derived from liver, kidney, thyroid cells, fibroblasts derived from humans or various mammals (eg, egrets, dogs, cats, guinea pigs, hamsters, rats, mice, etc.).
  • various commercially available genomic DNA libraries Complementary DNA prepared by a known method from RNA derived from liver, kidney, thyroid cells, and fibroblasts can be obtained as a raw material as all or part of genomic DNA from the library.
  • an exogenous abnormal DNA can produce a translation region obtained by mutating a normal polypeptide translation region 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 an introduced animal by a conventional DNA engineering technique in which it is linked to the downstream of the promoter and, if desired, to the upstream of the transcription termination site.
  • the presence of the exogenous DNA of the present invention in the germinal cells of the transgenic animal after the introduction of DNA indicates that the progeny of the transgenic animal retains the exogenous DNA of the present invention in all of the germ cells and somatic cells Means to do.
  • 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 germinal and somatic cells.
  • a non-human mammal into which the exogenous normal DNA of the present invention has been introduced can be subcultured in a normal breeding environment as an animal having the DNA after confirming that the exogenous DNA is stably retained by the crossing. .
  • exogenous DNA of the present invention is provided at the fertilized egg cell stage to be present in excess in all germ cells and somatic cells of the target mammal.
  • Excessive presence of the exogenous DNA of the present invention in the germinal cells of the produced animal after the introduction of the DNA indicates that all the offspring of the produced animal have the exogenous DNA of the present invention in all of the germinal and somatic cells.
  • the progeny of this type of animal that has inherited the exogenous DNA of the present invention has an excess of the exogenous DNA of the present invention in all of its germ cells and somatic cells.
  • the non-human mammal having the normal DNA of the present invention expresses the normal DNA of the present invention at a high level, and finally promotes the function of endogenous normal DNA, thereby finally obtaining the protein of the present invention. May develop hyperactivity in the animal Can be used.
  • the normal DNA-transfected 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.
  • the non-human mammal having the exogenous abnormal DNA of the present invention is subcultured in a normal breeding environment as an animal having the DNA after confirming that the exogenous DNA is stably maintained by mating. I can do it. Furthermore, the desired foreign DNA can be incorporated into the above-mentioned plasmid and used as a raw material.
  • a DNA construct with the Promo One Night can be created by ordinary DNA engineering techniques. Introduction of the abnormal DNA of the present invention at the fertilized egg cell stage is ensured to be present in all germ cells and somatic cells of the target mammal.
  • the presence of the abnormal DNA of the present invention in the germinal cells of the animal after the transfer of DNA means that all the offspring of the animal have the abnormal DNA of the present invention in all of the germinal and somatic cells.
  • the progeny of this type of animal that has inherited the exogenous DNA of the present invention has the abnormal DNA of the present invention in all of its germ cells and somatic cells.
  • a homozygous animal having the introduced DNA on both homologous chromosomes is obtained, and by crossing these male and female animals, all 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 ultimately achieved by inhibiting the function of endogenous normal DNA.
  • Inactive refractory disease may occur and can be used as a model animal for the disease.
  • using the abnormal DNA-introduced animal of the present invention it is possible to elucidate the pathological mechanism of the function-inactive refractory of the protein of the present invention and to examine a method for treating this disease.
  • the abnormal DNA highly expressing animal of the present invention can be used to inhibit the function of a normal protein by the abnormal protein of the present invention in the inactive refractory disease of the protein of the present invention (dominant negative activity). Action).
  • the mammal into which the foreign abnormal DNA of the present invention has been introduced is free from the protein of the present invention. Since it has an increased protein symptom, it can also be used for a therapeutic drug screening test for the protein of the present invention or its functionally inactive refractory disease.
  • cells of tissues having DNA are cultured by standard tissue culture techniques, and these are used to study the function of cells from generally difficult tissues,
  • each organ from the DNA-introduced animal of the present invention cut it into small pieces, and then use a proteolytic enzyme such as trypsin to obtain the released DNA-introduced cells, culture them, or systematize the cultured cells. It is.
  • the protein producing cell of the present invention can be identified, examined for its relationship with apoptosis, differentiation or proliferation, or its signal transduction mechanism, and its abnormalities can be examined. It is an effective research material for elucidating its action.
  • the DNA-introduced animal of the present invention in order to use the DNA-introduced animal of the present invention to develop a therapeutic agent for a disease associated with the protein of the present invention, including a refractory inactive type of the protein of the present invention, It is possible to provide an effective and rapid screening method of the therapeutic agent for the disease by using a quantitative method and the like.
  • the DNA-introduced animal of the present invention Alternatively, using the exogenous DNA expression vector of the present invention, it is possible to study and develop a method for treating DNA associated with the protein of the present invention. .
  • the present invention provides a non-human mammalian embryonic stem cell in which the DNA of the present invention has been inactivated, and a non-human mammal deficient in expression of the DNA of the present invention.
  • a non-human mammal deficient in expression of the DNA in which the DNA of the present invention has been inactivated comprises a repo overnight gene (eg, a / 3-galactosidase gene derived from Escherichia coli);
  • a repo overnight gene eg, a / 3-galactosidase gene derived from Escherichia coli.
  • a test compound is administered to the non-human mammal according to (8), wherein the rodent is a mouse, and (10) the animal according to (7), and the expression of the reporter gene is detected.
  • the present invention also provides a method for screening a compound or a salt thereof which promotes or inhibits the activity of a promoter for DNA of the present invention.
  • a non-human mammalian embryonic stem cell in which the DNA of the present invention has been inactivated refers to a DNA which is artificially mutated to the DNA of the present invention possessed by the non-human mammal, thereby suppressing the expression ability 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). )
  • ES cells Non-human mammalian embryonic stem cells
  • the same one as described above is used.
  • the method of artificially mutating the DNA of the present invention can be performed, for example, by deleting a part or all of the DNA sequence and inserting or substituting another DNA sequence by a genetic engineering technique.
  • the knockout DNA of the present invention may be prepared, for example, by shifting the codon reading frame or disrupting the function of the promoter or exon.
  • non-human mammalian embryonic stem cells in which the DNA of the present invention is inactivated include, for example, The DNA of the present invention possessed by a non-human mammal is isolated, and its exon portion is a drug resistance gene represented by a neomycin resistance gene, a hygromycin resistance gene, or 1 ac Z ( ⁇ -galactosidase gene), cat ( A DNA sequence that disrupts the function of exons by inserting a reporter gene or the like typified by the chloramphenicylacetyltransferase gene or terminates gene transcription in the intron between exons (eg, , Po1yA additional signal, etc.) and the inability to synthesize complete mRNA
  • a DNA strand having a DNA sequence constructed so as to be disrupted hereinafter abbreviated as “gettering vector 1”)
  • Southern hybridization analysis using the DNA sequence on or near the DNA as a probe, or the DNA sequence on the evening-getting vector and the neighborhood other than the DNA of the present invention used in the production of the evening-getting vector It can be obtained by analyzing by the PCR method using the DNA sequence of the region as a primer and selecting the knockout ES cells of the present invention.
  • ES cells from which the DNA of the present invention is inactivated by the homologous recombination method or the like for example, those already established may be used as described above, or the method described in the known method of Evans and Kaufman may be used. A newly established one may be used.
  • mouse ES cells currently, 129 ES cells are generally used, but since their immunological background has not been fixed, a pure line that substitutes them has been used for immunological inheritance.
  • the number of eggs collected from C57BLZ6 mice and C57BL / 6 was improved by crossing with DBAZ2.
  • Mouse (C57BLZ6 and DBAZ2 established using F can also be used favorably.
  • BDFi mice have the advantage of large number of eggs collected and robust eggs. Therefore, when ES cells obtained using this method are used to create a disease model mouse, the genetic background can be replaced with C57BLZ6 mice by backcrossing with C57BL / 6 mice. It can be used advantageously where possible.
  • blastocysts 3.5 days after fertilization are generally used. Early embryos can be obtained.
  • 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, requires about 10 6 cells for karyotype analysis, since suffices ES cell number of about 1 colony (about 50), culture
  • the primary selection of ES cells in the initial stage can be performed by gender discrimination, and the selection of male cells at an early stage greatly reduces the labor required in the initial culture.
  • Embryonic stem cell lines obtained in this way usually have very good proliferative properties, but they must be carefully subcultured because they tend to lose their ability to generate individuals.
  • a suitable feeder cell such as ST0 fibroblasts
  • a carbon dioxide incubator preferably 5% carbon dioxide, 95% air or 5% oxygen, in the presence of LIF (1 to 10,000 U / ml)). 5% CO2, 90% air
  • trypsin / EDTA solution usually 0.001 to 0.5% trypsin / 0.1 to 5 mM EDTA, preferably about 0.1% trypsin / ImM EDTA
  • Such subculture is usually performed every 1 to 3 days. At this time, it is desirable to observe the cells, and if morphologically abnormal cells are found, discard the cultured cells.
  • '' ES cells can be cultured in monolayers up to high densities or in suspension cultures to form cell clumps under appropriate conditions to produce various types of cells such as parietal, visceral, and cardiac muscles. (MJ Evans and MH
  • DNA-deficient cells of the present invention obtained by differentiating the ES cells of the present invention are cells of the protein of the present invention in the mouth of the in vivo. Useful in biological studies.
  • the non-human mammal deficient in DNA expression of the present invention can be distinguished from a normal animal by measuring the mRNA level of the animal using a known method and indirectly comparing the expression level.
  • non-human mammal those similar to the aforementioned can be used.
  • the non-human mammal deficient in DNA expression of the present invention may be prepared by, for example, introducing the evening-getting vector prepared as described above into a mouse embryonic stem cell or a mouse egg cell, and introducing the evening-getting vector into the present invention.
  • the DNA sequence obtained by inactivating the DNA of the present invention is subjected to homologous recombination to replace the DNA of the present invention on the chromosome of mouse embryonic stem cells or mouse egg cells by gene homologous recombination, thereby obtaining the DNA of the present invention. Can be knocked out.
  • the cells in which the DNA of the present invention was knocked out were subjected to Southern hybridization analysis using the DNA sequence on or near the DNA of the present invention as a probe, or the DNA sequence on the targeting vector, and the mouse used as the targeting vector. It can be determined by PCR analysis using the DNA sequence of the vicinity region other than the DNA of the present invention derived from the primer as the primer.
  • the cell line in which the DNA of the present invention has been inactivated is cloned by homologous recombination, and the cells are cloned at an appropriate time, for example, at the 8-cell stage of a non-human mammal embryo or scutellum.
  • the chimeric embryo thus prepared is transplanted into the uterus of the non-human mammal that has been pseudopregnant.
  • the animal produced is a chimeric animal composed of both cells having the normal DNA locus of the present invention and cells having the artificially altered DNA locus of the present invention.
  • all tissues are artificially mutated from a population obtained by crossing such a chimeric individual with a normal individual. It can be obtained by selecting individuals composed of cells having the added DNA locus of the present invention, for example, by judging coat color or the like.
  • the individuals obtained in this manner are usually individuals deficient in the hetero-expression of the protein of the present invention, and mated with individuals deficient in the hetero-expression of the protein of the present invention. A homozygous deficient individual can be obtained.
  • a transgenic non-human mammal having a getter vector introduced into a chromosome can be obtained by injecting a DNA solution into the nucleus of the egg cell by microinjection.
  • a transgenic non-human mammal of the present invention by selecting a gene having a mutation at the DNA locus of the present invention by homologous recombination.
  • the animal individual obtained by mating is confirmed to have knocked out the DNA, and breeding is carried out in a normal breeding environment. be able to.
  • the germline can be obtained and maintained according to a standard 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 rearing the mother animal in such a manner that one normal individual and plural homozygotes are obtained. By mating male and female heterozygous animals, homozygous and heterozygous animals having the inactivated DNA are bred and subcultured.
  • the non-human mammalian embryonic stem cells in which the DNA of the present invention has been inactivated are extremely useful for producing the non-human mammal deficient in expression of the DNA of the present invention.
  • the non-human mammal deficient in expression of the DNA of the present invention lacks various biological activities that can be induced by the protein of the present invention, 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 examining 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 diseases caused by deficiency or damage of the DNA of the present invention.
  • the present invention comprises administering a test compound to a non-human mammal deficient in expression of the DNA of the present invention, and observing and measuring changes in the animal.
  • the non-human mammal deficient in DNA expression of the present invention used in the screening method includes 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, and these compounds are novel compounds. Or a known compound.
  • a non-human mammal deficient in expression of the DNA of the present invention is treated with a test compound and compared with a non-treated control animal, and changes in organs, tissues, disease symptoms, etc. of the animal are indicated.
  • the therapeutic and prophylactic effects of the test compound 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.
  • a test compound when screening for a compound having a therapeutic effect on infertility, a test compound is administered to a male non-human mammal deficient in expression of DNA of the present invention, and the same amount as that of a normal female control animal is used. After breeding for a period, determine the proportion of pregnant females.
  • the compound obtained by using the screening method is a compound selected from the test compounds described above, and has a prophylactic / therapeutic effect against a disease caused by deficiency or damage of the protein of the present invention. It can be used as a medicament such as a safe and low toxic prophylactic or therapeutic agent. Further, a compound derived from the compound obtained by the above screening can also be used.
  • 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 physiologically acceptable acid addition salts are particularly preferable.
  • 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) , Succinic acid, tartaric acid, quinic 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
  • Succinic acid tartaric acid, quinic 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 drug containing the protein of the present invention described above.
  • the preparations obtained in this way are safe and of low toxicity, for example in humans or other mammals (for example, rats, mice, guinea pigs, egrets, sheep, pigs, pigs, dogs, cats, animals And monkeys and monkeys).
  • the dose of the compound or a salt thereof varies depending on the target disease, the administration subject, the administration route, and the like. For example, when the compound is orally administered, generally, the adult (assuming a body weight of 60 kg) is infertile.
  • 0.1 to 10 Omg preferably about 1.0 to 50 mg, more preferably about 1.0 to 20 mg of the compound is administered per day.
  • the single dose of the compound varies depending on the administration subject, target disease, and the like.
  • [7b] a compound that promotes or inhibits the activity of the promoter for the DNA of the present invention. Screening method
  • the present invention provides a test compound administered to a non-human mammal deficient in expression of a DNA of the present invention, and detects or enhances the expression of a reporter gene.
  • the non-human mammal deficient in expression of the DNA of the present invention may be one of the above-mentioned non-human mammals deficient in expression of the DNA of the present invention in which the DNA of the present invention is not introduced by introducing a reporter gene. Those which are activated and which can express the repo overnight gene under the control of the promoter over the DNA of the present invention are used.
  • test compound examples include the same compounds as described above.
  • the same one as described above is used, and a
  • the reporter gene is present under the control of the promoter for the DNA of the present invention since the repo overnight gene is present.
  • the protein-deficient mouse of the present invention or a tissue section thereof is fixed with datalaldehyde or the like, washed with phosphate buffered saline (PBS), and then stained with X-ga1 at room temperature or at room temperature. After reacting for about 30 minutes to 1 hour in the vicinity at 37, ⁇ -galactosidase reaction was stopped by washing the tissue specimen with ImM EDTAZPBS solution, and the color was observed. I just think.
  • mRNA encoding 1 ac Z 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 include physiologically acceptable acids (eg, inorganic acids) and bases (eg, organic acids).
  • physiologically acceptable acid addition salts are preferred.
  • Such salts include, for example, inorganic acids (eg, hydrochloric acid, phosphoric acid, hydrobromic acid, sulfuric acid, etc.) ' With organic acids (eg, acetic acid, formic acid, propionic acid, fumaric acid, maleic acid, succinic acid, tartaric acid, cunic acid, malic acid, oxalic acid, benzoic acid, methanesulfonic acid, benzenesulfonic acid, etc.) Salts and the like are used.
  • the compound or a salt thereof that promotes the promoter activity of the DNA of the present invention can promote the expression of the protein of the present invention and promote the function of the protein.
  • infertility eg, irregular menstruation, menstruation
  • cancer eg , Testicular tumor, ovarian cancer, breast cancer, esophageal cancer, lung cancer, kidney cancer, liver cancer, non-small cell lung cancer, prostate cancer, stomach cancer, bladder cancer, cervical cancer, colon cancer, rectal cancer, kidney cancer, thymoma It is useful as a prophylactic / therapeutic agent, and as a contraceptive drug. It is preferably used as an agent for preventing or treating infertility.
  • the compound of the present invention or a salt thereof that inhibits the promoter activity on DNA can inhibit the expression of the protein of the present invention and inhibit the function of the protein.
  • infertility eg, irregular menstruation, Dysmenorrhea, amenorrhea, weight loss amenorrhea, secondary amenorrhea, azoospermia, hypospermia, sperm annihilation, asthenia, spermatogenesis, testicular dysfunction, etc.
  • cancer E.g., testicular tumor, ovarian cancer, breast cancer, esophageal cancer, lung cancer, kidney cancer, liver cancer, non-small cell lung cancer, prostate cancer, stomach cancer, bladder cancer, cervical cancer, colon cancer, rectal cancer, renal cancer, It is useful as a prophylactic / therapeutic agent for thymoma, etc., and as a medicine for contraceptives. It is preferably used as a contraceptive or the like.
  • the compounds derived from the compounds obtained in the above screening can be similarly used. Can be used.
  • a drug containing the compound or a salt thereof obtained by the screening method can be produced in the same manner as the above-mentioned drug containing the protein of the present invention or a salt thereof.
  • the preparations obtained in this way are safe and of low toxicity, for example in humans or other mammals (for example, rats, mice, guinea pigs, egrets, sheep, pigs, pigs, dogs, cats, animals And monkeys and monkeys).
  • the dose of the compound or a salt thereof varies depending on the target disease, the administration subject, the administration route, and the like.
  • the compound of the present invention that promotes the promoter activity for DNA is administered orally, generally, For an adult (assuming a body weight of 60 kg) patient with infertility, the compound is administered in an amount of about 0.1 to 100 mg, preferably about 1.0 to 50 mg, more preferably about 1.0 to 20 mg per day.
  • the single dose of the compound varies depending on the administration subject, target disease, and the like.
  • the compound of the present invention which promotes the activity of the promoter for DNA is usually in the form of an injection.
  • the compound When administered to an adult (assuming a body weight of 60 kg) infertile patient, 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 1 Omg per day.
  • 1 Omg is administered by static J31 injection.
  • the dose can be administered in terms of weight per 60 kg.
  • the compound of the present invention that inhibits the promoter activity for DNA is orally administered, generally, in an adult (assuming a body weight of 6 O kg) infertility patient, the compound is administered in an amount of about 0.1 to 10 mg / day.
  • Administer 10 Omg preferably about 1.0-50 mg, more preferably about 1.0-20 mg.
  • the single dose of the compound varies depending on the administration subject, target disease, and the like.
  • the compound of the present invention that inhibits promoter activity on DNA is usually administered in the form of an injection to an adult ( When administered to an infertile patient (with a body weight of 6 O kg), the compound is administered in an amount of about 0.01 to 3 Omg per day, preferably about 0.1 to 2 Omg, more preferably about 0.1 to 1 Omg per day.
  • Omg is conveniently administered by intravenous injection. In the case of other animals, the dose can be administered in terms of weight per 60 kg.
  • the non-human mammal deficient in expression of the DNA of the present invention is not compatible with the DNA of the present invention. It is extremely useful for screening compounds or salts thereof that promote or inhibit the activity of the promoter that promotes the activity of the promoter, and greatly contribute to the investigation of the causes of various diseases caused by DNA expression deficiency of the present invention or the development of therapeutic drugs. be able to.
  • transgenic animals using a DNA containing the entire promoter region of the protein of the present invention, genes encoding various proteins are ligated downstream thereof and injected into egg cells of an animal to produce a so-called transgenic animal.
  • Transgenic animals it is possible to specifically synthesize the polypeptide and study its effects in the living body.
  • an appropriate repo overnight gene to a part of the above-mentioned promoter and establishing a cell line from which the gene is derived, the action of specifically promoting or suppressing the ability of the protein itself of the present invention to produce in the body can be achieved. It can be used as a search system for low molecular weight compounds having
  • the antibody of the present invention having the activity of neutralizing the activity of the protein of the present invention includes, for example, cancer (eg, testicular tumor, ovarian cancer, breast cancer, esophagus cancer, lung cancer, kidney cancer, liver cancer, non-small cell lung cancer, Prostate cancer, gastric cancer, bladder cancer, cervical cancer, colon cancer, rectal cancer, renal cancer, thymoma, etc., infertility (eg, irregular menstruation, dysmenorrhea, amenorrhea, weight loss amenorrhea) Prophylaxis, secondary amenorrhea, azoospermia, hypospermia, spermatozoa, asthenozoospermia, spermatogenesis disorder, testicular dysfunction, etc.) Can be. It is preferably used as a contraceptive or the like.
  • cancer eg, testicular tumor, ovarian cancer, breast cancer, esophagus cancer, lung cancer, kidney cancer, liver cancer, non
  • the prophylactic / therapeutic agent for the above-mentioned diseases containing the antibody of the present invention has low toxicity and is used as it is as a liquid or as a pharmaceutical composition of an appropriate dosage form, in humans or mammals (eg, rats, egrets, sheep, etc.). Can be administered orally or parenterally (eg, intravenously) to pigs, pigs, cats, cats, dogs, monkeys, etc.).
  • the dosage varies depending on the administration subject, target disease, symptoms, administration route and the like.For example, when used for the treatment of adult infertility, the antibody of the present invention is usually administered in a dose of 0.1 mg.
  • O m gZ kg body weight preferably 0.1-: LO mg Z kg body weight, more preferably 0.1-5 mg Z kg body weight, about 1-5 times a day, It is convenient to administer by intravenous injection or intra-articular injection preferably about 1 to 3 times a day. In the case of other parenteral administration and oral administration, an equivalent amount can be administered. If the symptoms are particularly severe, the dose may be increased accordingly.
  • the antibodies of the present invention can be administered by themselves 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.
  • Such compositions are provided in dosage forms suitable for oral or parenteral administration (eg, intravenous injection).
  • compositions may contain another active ingredient as long as the composition does not cause an undesirable interaction with the above-mentioned antibody.
  • bases, amino acids, and the like are indicated by abbreviations based on the abbreviations by the IUPAC-IUB Commission on Biochemical Nomenclature or commonly used abbreviations in the relevant field, and examples thereof are described below.
  • an amino acid can have optical isomers, the L-form is indicated unless otherwise specified.
  • sequence numbers in the sequence listing in the present specification indicate the following sequences.
  • Example 3 shows the amino acid sequence of the human TCH 207 protein obtained in Example 2.
  • Example 2 shows the amino acid sequence of human TCH207D436 N protein obtained in Example 2.
  • Example 1 shows the nucleotide sequence of primer A AP1 used in Example 1.
  • Example 1 shows the nucleotide sequence of primer A AP2 used in Example 1.
  • Example 5 shows the 5-terminal sequence of human TCH207 gene cDNA identified by the RACE reaction in Example 1.
  • [SEQ ID NO: 18] 3 shows the nucleotide sequence of primer T7 used in Example 2.
  • Example 1 shows the nucleotide sequence of cDNII containing the full-length human TCH207 gene obtained in Example 2.
  • Example 3 shows the nucleotide sequence of cDNA containing the human TCH 207D436 N full-length gene obtained in Example 2.
  • FIG. 5 shows the full-length sequence of mouse TCH207 gene cDNA identified by the RACE reaction in Example 4. .
  • FIG. 9 shows the nucleotide sequence of primer mA6 used in Example 5.
  • FIG. 5 shows the nucleotide sequence of cDNA containing the full-length mouse TCH207 gene obtained in Example 5.
  • FIG. 9 shows the nucleotide sequence of primer mA8 used in Example 6.
  • Example 7 shows the nucleotide sequence of TaqMan probe mT1 used in Example 6.
  • Escherichia coli TOP10 / pCRII-TCH207 obtained in Example 2 described below was obtained from 1-1-1 Tsukuba-Higashi, Ibaraki, Japan 1-Central 6 (Postal code 305 -8566), the National Institute of Advanced Industrial Science and Technology, Patent Organism Depositary, with accession number FERM BP-8055 from May 14, 2002, 2-17-85 Jusanhoncho, Yodogawa-ku, Osaka-shi (postal service) No. 532-8686) and deposited at the Fermentation Research Institute under the accession number IF0 16796.
  • R2 is Primer R2 (SEQ ID NO: 5)
  • F2R is Primer F2R (SEQ ID NO: 6)
  • F3R is the primer F3R (SEQ ID NO: 7)
  • R4R is the primer R4R (SEQ ID NO: 8)
  • R5R represents primer R5R (SEQ ID NO: 9)
  • F5 represents primer F5 (SEQ ID NO: 10).
  • the 5 ′ terminal sequence of human TCH207 gene cDNA (SEQ ID NO: 13) was determined from the sequences obtained by setl, set2, and set3 from the sequences obtained by seU, set5, and set6.
  • the 3 ′ terminal sequence of the TCH207 gene cDNA (SEQ ID NO: 14) was identified.
  • a 1.5 kbp band was cut out, treated with ExTaq DNA polymerase (manufactured by Takara Shuzo) at 72 ° C for 20 minutes, and then cloned using a TOPO TA Cloning kit dual promoter (manufactured by Invitrogen).
  • ExTaq DNA polymerase manufactured by Takara Shuzo
  • TOPO TA Cloning kit dual promoter manufactured by Invitrogen
  • This plasmid was used for primer DNA [primer SP6 (SEQ ID NO: 17), primer T7 (SEQ ID NO: 18), primer F1 (SEQ ID NO: 19), primer F2R (SEQ ID NO: 5), primer R1 (SEQ ID NO: 20), primer R5 (SEQ ID NO: 21), primer R5R (SEQ ID NO: 9)], and BigDye Terminator Cycle Sequencing Kit (manufactured by Applied Biosystems).
  • the base sequence of the inserted cDNA fragment was determined using a DNA sequencer ABI PRISM 3100 DNA Analyzer (manufactured by Applied Biosystems).
  • the cDNA fragment encodes 472 amino acid sequences (SEQ ID NO: 1) (SEQ ID NO: 2), and the protein containing the amino acid sequence was named human TCH207 protein.
  • a transformant having a plasmid containing the cDNA fragment was named Escherichia coli TOP 10 / pCRII-TCH207.
  • the expression level (copy number) of glyceraldehide-3-phosphate dehydrogenase (GAPDH) was also measured using TaqMan GAPDH control reagents (Applied Biosystems).
  • the reaction was carried out using TaqMan Universal PCR Master Mix (manufactured by Applied Biosystems) at 50 ° C for 2 minutes at ABI PRISM 7900 sequence detection system (manufactured by Applied Biosystems), then at 95 ° C for 10 minutes, and then The reaction was repeated 40 times, with one reaction cycle consisting of 15 seconds at 95 ° C and 1 minute at 60 ° C, and detection was performed simultaneously.
  • the human TCH207 gene product (mRNA) showed very strong selective expression in the testis, with some expression in the stomach, placenta, fetal kidney, breast, lung, colon and kidney cancers. Expression was not detected in the Human blood fractions MTC panel and the immunen immune system MTC panel.
  • primer DNA for 5'-RACE [primer niBl (SEQ ID NO: 27), primer mB3 (SEQ ID NO: 28), primer mB4 (SEQ ID NO: 29), the primer mB5 (SEQ ID NO: 30) and the primer mB6 (SEQ ID NO: 31)]
  • primer DNA for 3'-RACE [primer mA3 (SEQ ID NO: .32), primer niA4 (sequence No. 33) or primer mA5 (SEQ ID NO: 34)] and a primer API (SEQ ID NO: 11), and the following conditions (1) using Advantage 2 DNA Polymerase (Clontech).
  • the RACE reaction was performed in () to (5).
  • mouse testis Marathon-Ready cDNA (Clontech) was used.
  • PCR was performed using Pyrobes t DNA polymerase (Takara Shuzo) under the following conditions (1) to (5). (1) 94 ° C for 3 minutes
  • primer mB4 SEQ ID NO: 29
  • primer mB8 SEQ ID NO: 40
  • BigDye Terminator Cycle Sequencing Kit Applied Biosystems.
  • the nucleotide sequence of the cDNA fragment used was determined using the MA Sequencer ABI PRISM 3100 DNA Analyzer (manufactured by Applied Biosystems). .
  • the obtained clone had 1536 base sequences (SEQ ID NO:
  • This sequence completely corresponded to a part (14th to 1549th) of the full-length cDNA represented by SEQ ID NO: 35.
  • This cDNA fragment contains 442 amino acid sequences (SEQ ID NO:
  • mouse TCH207 protein is encoded (SEQ ID NO: 43), and the protein containing this amino acid sequence was designated as mouse TCH207 protein.
  • a transformant having the plasmid containing the above-mentioned cDNA fragment was designated as Escherichia coli TOP10 / pCRI I-mTCH207.
  • mouse TCH207 has the same structural characteristics as human TCH207, it is highly likely that mouse TCH207 functions as a subunit of voltage-dependent Ca 2+ channel.
  • primer-mA8 SEQ ID NO: 44
  • primer-mB9 SEQ ID NO: 45
  • TaqMan probe mTl SEQ ID NO: 46
  • Tissues bone marrow, eyes, lymph nodes, smooth muscle, prostate, thymus, stomach, uterus, heart, brain, spleen, lung, liver, skeletal muscle, kidney, testis, embryo (7 days), embryo (11 Day), embryo (15 days), embryo (17 days))
  • cDNA Mae MTC panel I and Mouse MTC panel II: Clontech expression level (copy number) of mouse TCH207 was measured by TaqMan PCR. .
  • the expression level (copy number) of rodent glyceral dehi de-3-phosphate dehydrogenase (GAPDH) was also measured using TaqMan rodent GAPDH control reagents (manufactured by Applied Biosystems). The reaction was first performed at 50 ° C for 2 minutes at ABI PRISM 7900 sequence detection system (Applied Biosystems) using TaqMan Universal PCR Master Mix (Applied Biosystems) and then at 95 ° C for 10 minutes. After the incubation, the reaction was repeated 40 cycles, each cycle consisting of 15 seconds at 95 ° C and 1 minute at 60, and the detection was performed simultaneously. ,
  • Fig. 5 shows the results.
  • the mouse TCH207 gene product (mRNA) showed selective and very strong expression in testis.
  • the protein of the present invention may be used, for example, for infertility (eg, irregular menstruation, dysmenorrhea, amenorrhea, weight-loss amenorrhea, secondary amenorrhea, azoospermia, hypospermia, sperm killing, sperm Asthenia, spermatogenesis disorder, testicular dysfunction, etc.), cancer (eg, testicular tumor, ovarian cancer, breast cancer, esophageal cancer, lung cancer, kidney cancer, liver cancer, non-small cell lung cancer, prostate cancer, gastric cancer, bladder cancer, Diagnostic markers for cervical cancer, colon cancer, rectal cancer, kidney cancer, thymoma, etc.)
  • infertility eg, irregular menstruation, dysmenorrhea, amenorrhea
  • azoospermia eg., hypospermia
  • sperm killing sperm Asthenia
  • spermatogenesis disorder spermatogenesis disorder,
  • Amenorrhea weight loss amenorrhea, secondary amenorrhea, azoospermia, spermia, sperm killing, asthenozoospermia, spermatogenesis disorder, testicular dysfunction, etc.
  • cancer eg, testicular tumor, ovarian cancer
  • breast cancer esophageal cancer
  • lung cancer kidney cancer, liver cancer, non-small cell lung cancer, prostate cancer, stomach cancer, bladder cancer, cervical cancer, colon cancer, rectal cancer, kidney cancer, thymoma, etc. It can be used as a medicament such as a therapeutic agent or contraceptive.
  • the compound that promotes the activity of the protein or a salt thereof is preferably used as an agent for preventing or treating infertility.
  • the compound or its salt that inhibits the activity of the protein is preferably used as a contraceptive or the like.

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Abstract

La présente invention concerne une protéine du canal Ca2+ dépendante du potentiel, ainsi que notamment un ADN codant cette protéine, ADN et protéine convenant comme marqueurs de diagnostic du cancer, de la stérilité, etc. L'invention concerne également des composés favorisant ou inhibant l'activité de cette protéine. Ces composés, qui s'obtiennent par un procédé de recherche systématique utilisant la protéine, conviennent notamment pour la thérapie ou la prophylaxie de ces maladies.
PCT/JP2003/005171 2002-04-24 2003-04-23 Nouvelle proteine, et adn de cette proteine Ceased WO2003091434A1 (fr)

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Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1430125A4 (fr) * 2001-05-03 2005-01-19 Childrens Medical Center Canal cationique specifique du sperme, et utilisations correspondantes
US7339029B2 (en) 2001-10-22 2008-03-04 Children's Medical Center Corporation Sperm-specific cation channel, CatSper2, and uses therefor
US8110668B2 (en) 2002-08-07 2012-02-07 Children's Medical Center Corporation Sperm-specific cation channel, catsper-3 and uses therefor
US8137918B2 (en) 2002-08-07 2012-03-20 Children's Medical Center Corporation Sperm-specific cation channel, CatSper4, and uses therefor
US8729248B2 (en) 2001-10-22 2014-05-20 Children's Medical Center Corporation Sperm-specific cation channel, CATSPER2, and uses therefor

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
OKAZAKI Y. ET AL.: "Analysis of the mouse transcriptome based on functional annotation of 60, 770 full-length cDNAs", NATURE, vol. 420, December 2002 (2002-12-01), pages 563 - 573, XP002252038 *
QUILL T.A. ET AL.: "A voltage-gated ion channel expressed specifically in spermatozoa", PROC. NATL. ACAD. SCI. USA, vol. 98, no. 22, 2001, pages 12527 - 12531, XP002253116 *

Cited By (10)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1430125A4 (fr) * 2001-05-03 2005-01-19 Childrens Medical Center Canal cationique specifique du sperme, et utilisations correspondantes
US8211666B2 (en) 2001-05-03 2012-07-03 Children's Medical Center Corporation Sperm-specific cation channel, CATSPER1, and uses therefor
US8852860B2 (en) 2001-05-03 2014-10-07 Children's Medical Center Corporation Sperm-specific cation channel, CatSper1 and uses therefor
US7339029B2 (en) 2001-10-22 2008-03-04 Children's Medical Center Corporation Sperm-specific cation channel, CatSper2, and uses therefor
US8729248B2 (en) 2001-10-22 2014-05-20 Children's Medical Center Corporation Sperm-specific cation channel, CATSPER2, and uses therefor
US9618521B2 (en) 2001-10-22 2017-04-11 Children's Medical Center Corporation Sperm-specific cation channel, Catsper2, and uses therefor
US8110668B2 (en) 2002-08-07 2012-02-07 Children's Medical Center Corporation Sperm-specific cation channel, catsper-3 and uses therefor
US8137918B2 (en) 2002-08-07 2012-03-20 Children's Medical Center Corporation Sperm-specific cation channel, CatSper4, and uses therefor
US8835105B2 (en) 2002-08-07 2014-09-16 Children's Medical Center Corporation Sperm-specific cation channel, CatSper4, and uses therefor
US8859197B2 (en) 2002-08-07 2014-10-14 Children's Medical Center Corporation Sperm-specific cation channel, CATSPER3, and uses therefor

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