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WO2002072816A1 - Proteine de recepteur murin de type kiss-1 et adn correspondant - Google Patents

Proteine de recepteur murin de type kiss-1 et adn correspondant Download PDF

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Publication number
WO2002072816A1
WO2002072816A1 PCT/JP2002/001656 JP0201656W WO02072816A1 WO 2002072816 A1 WO2002072816 A1 WO 2002072816A1 JP 0201656 W JP0201656 W JP 0201656W WO 02072816 A1 WO02072816 A1 WO 02072816A1
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WIPO (PCT)
Prior art keywords
protein
receptor protein
present
salt
coupled receptor
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English (en)
Japanese (ja)
Inventor
Yasuko Terao
Yasushi Shintani
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Takeda Pharmaceutical Co Ltd
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Takeda Chemical Industries Ltd
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Publication of WO2002072816A1 publication Critical patent/WO2002072816A1/fr
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/566—Immunoassay; Biospecific binding assay; Materials therefor using specific carrier or receptor proteins as ligand binding reagents where possible specific carrier or receptor proteins are classified with their target compounds
    • 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
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2500/00—Screening for compounds of potential therapeutic value

Definitions

  • the present invention relates to a novel G protein-coupled receptor protein or a salt thereof derived from mouse brain, DNA encoding the same, and the like.
  • G proteins conjugated guanine nucleotide-binding proteins
  • TMR seven transmembrane receptor proteins
  • G protein-coupled receptor One protein is present on the surface of each functional cell in living cells and organs, and molecules that regulate the function of those cells and organs, such as hormones, neurotransmitters, and physiologically active substances It plays a physiologically important role as a target for.
  • the receptor transmits a signal into the cell through binding to a physiologically active substance, and this signal causes various reactions such as suppression of activation and activation of the cell.
  • G protein-coupled receptor protein Elucidating the relationship between substances that regulate complex functions in cells and organs of various organisms and their specific receptor proteins, particularly G protein-coupled receptor protein, is important It will elucidate the functions of cells and organs and provide a very important means for drug development closely related to those functions.
  • physiological functions are regulated under the control of many hormones, hormone-like substances, neurotransmitters or bioactive substances.
  • physiologically active substances are present at various sites in the body, and regulate their physiological functions through their corresponding receptor proteins.
  • receptor proteins not in vivo
  • hormones, neurotransmitters, and other physiologically active substances and the structure of their receptor proteins has not yet been reported.
  • receptor proteins do not know whether subtypes exist.
  • the present inventors have found a G protein-coupled receptor protein derived from the rat brain stem periphery and human brain, and searched for a peptide having an intracellular Ca ion concentration increasing activity for the receptor.
  • the C-terminal peptide of the protein encoded by the cancer metastasis suppressor gene KiSS-1 (Genomics, Vol. 54, pp. 145-148, 1998) activates a G protein-coupled receptor derived from human brain.
  • KiSS-1 cancer metastasis suppressor gene
  • the present invention provides a mouse homolog receptor protein for a useful human G protein-coupled receptor protein. Disclosure of the invention
  • the present inventors have conducted intensive studies in order to solve the above-mentioned problems, and as a result, isolated cDNA encoding a sequence highly homologous to the human G protein-coupled receptor from mouse brain cDNA.
  • the entire nucleotide sequence was successfully analyzed.
  • this base sequence was translated into an amino acid sequence, it was confirmed that a protein having significantly higher homology to the amino acid sequence of the human G protein-coupled receptor protein was coded.
  • the present inventors have further studied based on these findings, and as a result, completed the present invention.
  • a G protein-type receptor protein or a salt thereof which comprises an amino acid sequence identical or substantially identical to the amino acid sequence represented by SEQ ID NO: 1;
  • the G protein-coupled receptor protein according to (1) which comprises the amino acid sequence represented by SEQ ID NO: 1;
  • the antibody according to (11) which is a neutralizing antibody that inactivates signal transmission of the G protein-coupled receptor-1 protein according to (1);
  • the G protein-coupled receptor protein according to (1) which can be obtained by using the G protein-coupled receptor protein according to (1) or the partial peptide according to (4) or a salt thereof. Or a ligand for its salt;
  • a ligand comprising the G protein-coupled receptor protein according to (1) or the partial peptide according to (4) or a salt thereof, and a G protein-coupled receptor protein according to (1).
  • a polysaccharide wherein the ligand has the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5 or SEQ ID NO: 6.
  • (21) a ligand obtainable by using the screening method according to (18) or the screening kit according to (19) and a G protein-coupled receptor protein or a salt thereof according to (1); A compound or a salt thereof that changes the binding property;
  • (22) a ligand obtainable using the screening method according to (18) or the screening kit according to (19), and a G protein-coupled receptor protein or a salt thereof according to (1);
  • a medicament comprising a compound that alters binding properties or a salt thereof;
  • a polynucleotide comprising a nucleotide sequence complementary to the polynucleotide of (5) or a part thereof;
  • (35) a method for preventing or treating cancer, which comprises administering to a mammal an effective amount of the compound according to (21), (30) or (31) or a salt thereof; Use of the compound or a salt thereof according to (21), (30) or (31) for the manufacture of a prophylactic or therapeutic agent for:
  • (41) a non-human mammalian embryonic stem cell in which DNA encoding the G protein-coupled receptor protein according to (1) is inactivated;
  • the present invention relates to a method for screening a compound that promotes or inhibits one activity or a salt thereof, and the like.
  • the protein is: (1) an amino acid sequence represented by SEQ ID NO: 1, or one or more in the amino acid sequence represented by SEQ ID NO: 1 (preferably about 1 to 10, more preferably several (1 to (5)) The amino acid sequence of which amino acid is deleted, (2) SEQ ID NO: 1 or 2 or more (preferably about 1 to 10, more preferably several (1 to 5) in the amino acid sequence represented by SEQ ID NO: 1) ) Of the amino acid sequence represented by SEQ ID NO: 1 or 2 or more (preferably about 1 to 10, more preferably several (1 to 5)) amino acids Contains an amino acid sequence substituted with another amino acid or an amino acid sequence combining them.
  • a compound that activates the G protein-coupled receptor protein or a salt thereof according to (1) is contacted with a cell containing the G protein-coupled receptor protein according to (1). And (ii) a compound that activates the G protein-coupled receptor protein or a salt thereof described in (1) above and a test compound containing the G protein-coupled receptor protein described in (1) above.
  • a method for screening a compound or a salt thereof that changes the binding property to a salt
  • the compound for activating the G protein-coupled receptor protein or the salt thereof described in (1) and a test compound are cultured with the transformant described in (9).
  • a pharmaceutical comprising a salt;
  • the screening kit according to (19) which comprises a cell containing the G protein-coupled receptor protein according to (1);
  • (62) A compound that can be obtained by using the screening kit according to (58) to (60) and that alters the binding between the ligand and the G protein-coupled receptor protein or salt thereof according to (1). Or a medicament characterized by containing a salt thereof,
  • the antibody according to (11), a test solution, and the labeled (1) The antibody according to (11), a test solution, and the labeled (1).
  • G protein-coupled receptor according to (1) which is competitively reacted with the G protein-coupled receptor protein or the partial peptide or the salt thereof according to (4), and is labeled with the antibody.
  • test solution and the antibody of (11) insolubilized on a carrier and the labeled antibody of (11) are allowed to react simultaneously or successively with each other.
  • FIG. 1 shows a hydrophobicity plot of the receptor protein of the present invention.
  • the G protein-coupled receptor protein of the present invention (hereinafter sometimes abbreviated as receptor protein) is a receptor protein having an amino acid sequence identical or substantially identical to the amino acid sequence represented by SEQ ID NO: 1. Evening quality.
  • the receptor protein of the present invention includes, for example, all cells (eg, spleen cells, nerve cells, and the like) of human non-human mammals (eg, guinea pigs, rats, mice, rabbits, puppies, sheep, birds, monkeys, etc.).
  • brain various parts of the brain (e.g., olfactory bulb, ⁇ nucleus, cerebral basal bulb, hippocampus, thalamus, hypothalamus, subthalamic Nucleus, cerebral cortex, medulla, cerebellum, cerebellum, occipital lober
  • amino acid sequence substantially identical to the amino acid sequence represented by SEQ ID NO: 1 include, for example, about 95% or more, preferably about 98% or more homology with the amino acid sequence represented by SEQ ID NO: 1. Amino acid sequence and the like.
  • Examples of the protein having an amino acid sequence substantially identical to the amino acid sequence represented by SEQ ID NO: 1 of the present invention include, for example, a protein having an amino acid sequence substantially identical to the amino acid sequence represented by SEQ ID NO: 1 Proteins having substantially the same activity as the amino acid sequence represented by SEQ ID NO: 1 are preferred.
  • substantially the same activity examples include a ligand binding activity and a signal transduction activity. Substantially the same means that their activities are the same in nature. Therefore, the activities such as ligand binding activity and signal transduction activity are equivalent (eg, about 0.01 to 100 times, preferably about 0.5 to 20 times, more preferably about 0.5 to 2 times). However, quantitative factors such as the degree of activity and the molecular weight of the protein may be different.
  • the measurement of the activity such as the ligand binding activity and signal transduction can be carried out according to a known method.
  • the activity can be measured according to a ligand determination method and a screening method described later.
  • the receptor protein of the present invention includes: (1) one or more (preferably about 1 to 10, more preferably several (1 to 5)) amino acids in the amino acid sequence represented by SEQ ID NO: 1; An amino acid sequence deleted from the amino acid sequence; (2) one or more (preferably about 1 to 10, more preferably several (1 to 5)) amino acids in the amino acid sequence represented by SEQ ID NO: 1 (3) One or more (preferably about 1 to 10, more preferably several (1 to 5)) amino acids in the amino acid sequence represented by SEQ ID NO: 1 Amino acids replaced by amino acids A protein containing an amino acid sequence or an amino acid sequence obtained by combining them is also used.
  • the amino acid sequence of the receptor protein is N-terminal (amino terminal) at the left end and C-terminal (carboxyl terminal) at the right end according to the convention of peptide labeling.
  • the receptor proteins of the present invention including the receptor protein containing the amino acid sequence represented by SEQ ID NO: 1, usually have a C-terminus of a carboxyl group (1-C00H) or a carboxylate (1-C00-).
  • the C-terminal may be an amide (—C0NH 2 ) or an ester (—C00R).
  • R in the ester e.g., methyl, Echiru, n- propyl, C HJ alkyl group such as isopropyl or n- butyl, Shikuropen chill, C 3, such as cyclohexyl - 8 cycloalkyl group, for example, phenyl, 6 _ 12 Ariru group Flip such over naphthyl, for example, benzyl, C 7 _ 14 Ararukiru groups such as flying one Nafuchiru C DOO 2 Aruki Le group such phenyl one CH group or flying one naphthylmethyl such phenethyl In addition, piva methoxymethyl group, which is widely used as an ester for oral use, is used.
  • the receptor protein of the present invention When the receptor protein of the present invention has a lipoxyl group (or carboxylate) other than the C-terminus, the receptor protein of the present invention includes a carboxyl group amidated or esterified.
  • the ester in this case, for example, the above-mentioned C-terminal ester and the like are used.
  • Amino group protecting groups Mechionin residues of N-terminal (e.g., formyl group, etc. ( ⁇ _ 6 Ashiru groups such as any Arukanoiru group of Asechiru) Protected by N-terminal, Daltamyl group generated by cleavage of N-terminal in vivo, pyroglutamine oxidation, Substituent on side chain of amino acid in molecule (for example, _ OH, --SH, amino group, imidazole group, indole group, Guanijino group, etc.) a suitable protecting group (e.g., those protected by formyl group, C M Ashiru group such as C 2 _ 6 Arukanoiru group such Asechiru), or a sugar chain Complex proteins, such as so-called glycoproteins, to which is bound.
  • N-terminal e.g., formyl group, etc.
  • ⁇ _ 6 Ashiru groups such as any Arukanoiru group of Ase
  • receptor protein of the present invention include, for example, SEQ ID NO: 1
  • a receptor protein containing the amino acid sequence described is used.
  • the partial peptide of the receptor protein of the present invention may be any partial peptide of the receptor protein of the present invention described above.
  • a partial peptide may be any partial peptide of the receptor protein of the present invention described above.
  • the receptor protein molecules of the present invention those that are exposed outside the cell membrane and have substantially the same activity are used.
  • substantially the same activity indicates, for example, ligand binding activity.
  • the measurement of the ligand binding activity can be performed in the same manner as described above.
  • the extracellular region (hydrophilic region) in the hydrophobicity plot analysis shown in FIG. Is the peptide containing the part analyzed to be.
  • a peptide partially containing a hydrophobic (Hydrophobic) site can also be used.
  • a peptide containing individual domains may be used, but a peptide containing a plurality of domains at the same time may be used.
  • the number of amino acids of the partial peptide of the present invention is at least 20 or more, preferably 50 or more, more preferably 100 or more of the amino acid sequences constituting the receptor protein of the present invention. Peptides and the like are preferred.
  • a substantially identical amino acid sequence refers to an amino acid sequence having about 90% or more, preferably about 95% or more, more preferably about 98% or more homology with these amino acid sequences.
  • the partial peptide of the present invention has the following features: (1) one or more (preferably about 1 to 10, more preferably several (1 to 5)) amino acids in the amino acid sequence are deleted; One or two or more (preferably about 1 to 10, more preferably several (1 to 5)) amino acids are added to the amino acid sequence, or 3 one or two or more ( Preferably, about 1 to 10, more preferably, about 1 to 5) amino acids may be substituted with another amino acid.
  • partial peptide C-terminal of the present invention are typically force Rupokishiru group (one C00H) or Karupokishireto (one C00-), as the protein of the present invention described above, C-terminal amide (- C0Nh 2) Or an ester (—C00R) The meaning is as defined above).
  • C-terminal amide - C0Nh 2
  • an ester —C00R
  • the partial peptide of the present invention has a lipoxyl group (or carboxylate) other than at the C-terminus, those in which the lipoxyl group is amidated or esterified are also included in the partial peptide of the present invention.
  • the ester in this case, for example, the above-mentioned C-terminal ester or the like is used.
  • the partial peptide of the present invention has an N-terminal methionine residue in which the amino group of the methionine residue is protected with a protecting group, and the N-terminal side is cleaved in vivo to produce G1n is pyroglutamine-oxidized, the substituent on the side chain of the amino acid in the molecule is protected by an appropriate protecting group, or a complex peptide such as a so-called glycopeptide to which a sugar chain is bound is also included. It is.
  • Examples of the salt of the receptor protein of the present invention or its partial peptide include a physiologically acceptable salt with an acid or a base, and a physiologically acceptable acid addition salt is particularly preferable.
  • Such salts include, for example, salts with inorganic acids (eg, hydrochloric acid, phosphoric acid, hydrobromic acid, sulfuric acid) or organic acids (eg, acetic acid, formic acid, propionic acid, fumaric acid, maleic acid) , Succinic acid, tartaric acid, citric acid, malic acid, oxalic acid, benzoic acid, methanesulfonic acid, benzenesulfonic acid) and the like.
  • inorganic acids eg, hydrochloric acid, phosphoric acid, hydrobromic acid, sulfuric acid
  • organic acids eg, acetic acid, formic acid, propionic acid, fumaric acid, maleic acid
  • Succinic acid tartaric acid, citric acid, malic acid, ox
  • the receptor protein of the present invention or a salt thereof can be produced from the above-mentioned human or non-human mammal cell or tissue by a known method for purifying a receptor protein, or the receptor of the present invention described later. It can also be produced by culturing a transformant containing DNA encoding the protein. Also, the protein can be produced by the protein synthesis method described later or according to the method.
  • the tissues or cells of a human / non-human mammal are homogenized and then extracted with an acid or the like, and the extract is subjected to reverse phase chromatography or ion exchange chromatography. Purification and isolation can be achieved by combining such chromatographies.
  • a commercially available resin for protein synthesis can be used.
  • resins include, for example, chloromethyl resin, hydroxymethyl resin, benzhydrylamine resin, aminomethyl resin, Alcohol resin, 4-methylbenzhydrylamine resin, PAM resin, 4-hydroxymethylmethylphenylacetamidomethyl resin, polyacrylamide resin, 4- (2 ', 4'-dimethoxyphenyl) Hydroxymethyl) phenoxy resin; and 4- (2 ′, 4′-dimethoxyphenyl-Fmoc aminoethyl) phenoxy resin.
  • an amino acid having an ⁇ -amino group and a side chain functional group appropriately protected is condensed on the resin in accordance with the known amino acid sequence of the target protein or peptide according to various known condensation methods.
  • proteins and peptides are 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 convert the target protein or partial peptide or its amide. get.
  • carbodiimides are particularly preferable.
  • the carpoimides DCC, N, N'-diisopropylcarpoimide, N-ethyl-N '-(3-dimethylaminoprolyl) carpoimide, and the like are used.
  • Activation by these involves adding the protected amino acid directly to the resin along with a racemization inhibitor additive (eg, HO Bt, HO OB t), or using a symmetrical acid anhydride or HO BT ester or HO OB t ester.
  • the protected amino acid can be added to the resin after activation of the protected amino acid in advance as a t-ester.
  • 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-methylpyrrolidone, 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; and esters such as methyl acetate and ethyl acetate. Used.
  • the reaction temperature is appropriately selected from the range known to be usable for the protein bond formation reaction, and is usually selected from the range of about 1 to 2 (TC to 50 ° C.
  • Activated amino acid Derivatives are usually used in a 1.5- to 4-fold excess.
  • unreacted amino acid can be acetylated using acetic anhydride or acetylimidazole.
  • Examples of the protecting group for the amino group of the starting material include Z, Boc, succinyl-pentyloxycarponyl, isopolnyloxycarponyl, 4-methoxybenzyloxycarponyl, C 1 _Z, Br—Z, 7 Damantyloxycarbonyl, trifluoroacetyl, phthaloyl, formyl, 2-nitrophenylsulfenyl, diphenylphosphinothioyl, Fmoc and the like are used.
  • the lipoxyl group can be, for example, alkyl-esterified (eg, methyl, ethyl, propyl, butyl, tert-butyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, 2-adamantyl, etc.) Or cyclic alkyl esterification), aralkyl esterification (for example, benzyl ester, 4-nitrobenzyl ester, 4-methoxybenzyl ester, 4-methoxybenzyl ester, benzhydryl esterification), fenasi It can be protected by esterification, benzyloxycarbonyl hydrazide, short-lived butoxycarbonyl hydrazide, trityl hydrazide, etc.
  • alkyl-esterified eg, methyl, ethyl, propyl, butyl, tert-butyl, cyclopen
  • the hydroxyl group of serine can be protected, for example, by esterification or etherification.
  • a group suitable for the esterification for example, a lower alkanol group such as an acetyl group, an aroyl group such as a benzoyl group, a group derived from carbonic acid such as a benzyloxycarbonyl group, an ethoxycarbonyl group and the like are used.
  • the group suitable for etherification include a benzyl group, a tetrahydropyranyl group, and a t-butyl group.
  • the protecting group of Fueno Ichiru hydroxyl group of tyrosine for example, B z I, C l 2 - B zl, 2- two Torobenjiru, B r- Z, protecting group of the imidazole of evening histidine such as single-tertiary butyl is used
  • Tos 4-methoxy-2,3,6-trimethylbenzenesulfonyl, DNP, benzyloxymethyl, Bum, Boc, Trt, Fmoc and the like are used.
  • Activated carbonyl groups of the raw materials include, for example, corresponding acid anhydrides, azides, active esters [alcohols (eg, pentachlorophenol, 2,4,5-trichlorophenol, 2,4 Dinitrophenol, cyanomethyl alcohol, paranitrophenol, HONB, N-hydroxysuccinimide, N-hydroxyphthalimide, and esters with HOBt).
  • active esters eg, pentachlorophenol, 2,4,5-trichlorophenol, 2,4 Dinitrophenol, cyanomethyl alcohol, paranitrophenol, HONB, N-hydroxysuccinimide, N-hydroxyphthalimide, and esters with HOBt.
  • 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 by acetic acid is also used.
  • the elimination reaction by the acid treatment is generally performed at a temperature of about ⁇ 20 ° C. to 40 ° C.
  • the protection of the functional group which should not be involved in the reaction of the raw materials, the protecting group, the elimination of the protective group, the activation of the functional group involved in the reaction, and the like can be appropriately selected from known groups or known means.
  • an amide form of a protein for example, first, after amidating and protecting the ⁇ -hydroxyl group of the amino acid at the terminal end of the amino acid, a peptide (protein) chain is added to the amino group side of a desired chain. After lengthening the protein, remove the Q-terminal amino-protecting group only from the ⁇ ⁇ -terminal of the peptide chain. A protein from which only the protein has been removed is produced, and both proteins are condensed in a mixed solvent as described above. Details of the condensation reaction are the same as described above. After purifying the protected protein obtained by the condensation, all the protecting groups are removed by the above-mentioned method to obtain a desired crude protein. The crude protein is purified by various known purification means, and the main fraction is freeze-dried to obtain an amide of the desired protein.
  • ester of a protein for example, after condensing the ⁇ -carboxyl group of the carboxy terminal amino acid with a desired alcohol to form an amino acid ester, the ester of the desired protein Can be obtained.
  • the partial peptide of the protein of the present invention or a salt thereof can be produced according to a known peptide synthesis method or by cleaving the protein of the present invention with an appropriate peptide.
  • a peptide synthesis method for example, any of a solid phase synthesis method and a liquid phase synthesis method may be used. That is, the target peptide can be produced by condensing a partial peptide or amino acid capable of constituting the protein of the present invention with the remaining portion, and if the product has a protecting group, removing the protecting group. .
  • Known condensation methods and elimination of protecting groups include, for example, the methods described in the following 1 to 5.
  • the partial peptide of the present invention can be purified and isolated by a combination of ordinary purification methods, for example, solvent extraction, distillation, column chromatography, liquid chromatography, and recrystallization.
  • the partial peptide obtained by the above method is a free form In some cases, it can be converted to an appropriate salt by a known method. Conversely, when it is obtained as a salt, it can be converted to a free form by a known method.
  • the polynucleotide encoding the receptor protein of the present invention may be any polynucleotide as long as it contains the nucleotide sequence (DNA or RNA, preferably DNA) encoding the receptor protein of the present invention.
  • the polynucleotide is RNA such as DNA or mRNA encoding the receptor protein of the present invention, and may be double-stranded or single-stranded. In the case of double-stranded, it may be double-stranded DNA, double-stranded RNA or DNA: RNA hybrid. If single stranded, it may be the sense strand (ie, the coding strand) or the antisense strand (ie, the non-coding strand).
  • the polynucleotide encoding the receptor protein of the present invention Using the polynucleotide encoding the receptor protein of the present invention, a method described in the publicly available experimental medicine special edition “New PCR and its Application” 15 (7), 1997 or a method analogous thereto, for example, a method such as TadMan PCR As a result, the mRNA of the receptor protein of the present invention can be quantified.
  • the DNA encoding the receptor protein of the present invention may be any of genomic DNA, genomic DNA library, cDNA derived from the above-described cells and tissues, cDNA library derived from the above-described cells and tissues, and synthetic DNA.
  • the vector used for the library may be any of pacteriophage, plasmid, cosmid, phagemid and the like. Alternatively, it can be directly amplified by Reverse Transcriptase Polymerase Chain Reaction (hereinafter abbreviated as RT-PCR method) using a preparation of a total RNA or mRNA fraction from the cells and tissues described above.
  • RT-PCR method Reverse Transcriptase Polymerase Chain Reaction
  • the DNA encoding the receptor protein of the present invention includes, for example, a DNA containing the nucleotide sequence represented by SEQ ID NO: 2 or a DNA having the nucleotide sequence represented by SEQ ID NO: 2
  • Examples of the DNA that hybridizes under the conditions include a nucleotide sequence having about 90% or more, preferably about 95% or more, and more preferably about 98% homology with the nucleotide sequence represented by SEQ ID NO: 2. DNA or the like is used.
  • Hybridization is performed by a known method or a method analogous thereto, such as the method described in Molecular Cloning 2nd (J. Sambrook et al., Cold Spring Harbor Lab. Press, 1989). It can be done according to. When a commercially available library is used, 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.
  • the high stringency conditions include, for example, conditions in which the sodium concentration is about 19 to 40 mM, preferably about 19 to 20 mM, and the temperature is about 50 to 70 ° C, preferably about 60 to 65 ° C. Show. In particular, a sodium concentration of about 19 mM and a temperature of about 65 ° C are most preferred.
  • DNA encoding a receptor protein containing an amino acid sequence represented by SEQ ID NO: 1 DNA containing a base sequence represented by SEQ ID NO: 2 or the like is used.
  • a polynucleotide comprising a part of the base sequence of DNA encoding the receptor protein of the present invention or a part of a base sequence complementary to the DNA is a partial peptide of the present invention described below. It is used to mean not only DNA to be loaded but also RNA.
  • an antisense polynucleotide capable of inhibiting the replication or expression of a G protein-coupled receptor protein gene is cloned or determined to have a G protein-coupled receptor. It can be designed and synthesized based on the nucleotide sequence information of the DNA encoding the protein.
  • a polynucleotide can hybridize to the RNA of the G protein-coupled receptor protein gene and inhibit the synthesis or function of the RNA, or it can be a G protein-coupled receptor protein. It can regulate and control the expression of G protein-coupled receptor protein protein through interaction with related RNA.
  • G protein-coupled receptor Yuichi protein-related RNA Polynucleotides that are complementary to selected sequences and those that can specifically hybridize to G protein-coupled receptor protein-related RNA are used to express G protein-coupled receptor protein genes in vivo and in vitro. It is useful for regulating and controlling blood pressure, and is also useful for treating or diagnosing diseases.
  • the 3'-end untranslated region, 3'-end palindrome region, and 3'-end hairpin loop can be selected as preferable target regions, but any region within the G protein-coupled receptor overnight protein gene is selected. Can.
  • the relationship between the target nucleic acid and the polynucleotide complementary to at least a part of the target region that is, the relationship between the target nucleic acid and the polynucleotide that can hybridize with the target can be said to be “antisense”.
  • Antisense polynucleotides are 2-deoxy D-report-containing polydeoxynucleotides, D-report-containing polydeoxynucleotides, N-daricosides of purine or pyrimidine bases
  • Other types of polynucleotides or other polymers with non-nucleotide backbones eg, commercially available protein nucleic acids and synthetic sequence-specific nucleic acid polymers
  • other polymers containing specialized linkages provided that the polymer Represents a pairing of bases as found in DNA or RNA, which contains a nucleotide having a configuration permitting base attachment).
  • 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) and those with known modifications, such as those with labels, caps, methylated, and one or more natural nucleotides known in the art.
  • an intramolecular nucleotide for example, having an uncharged bond (eg, methylphosphonate, phosphotriester, phosphoramidate, olebamate, etc.), having a charged bond or containing sulfur
  • Those having a bond for example, phosphorothioate, phosphomouth dithioate, etc.
  • protein for example, protein (nuclease, nuclease Those having side-chain groups such as zein inhibitors, toxins, antibodies, signal peptides, poly-L-lysine, etc., and sugars (eg, monosaccharides), and compounds with in-situ rate compounds (eg, , Acridine, psoralen (psora1en), etc., chelating compounds (eg, metals, radioactive metals, boron, oxidizing metals, etc.), those containing alkylating agents, It may have a modified bond (for example, ⁇ -anomeric bond (eg,
  • 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 nucleosides and modified nucleotides may also be modified at the sugar moiety, e.g., one or more hydroxyl groups are replaced with halogens, aliphatic groups, etc., or functional groups such as ethers, amines, etc. It may be converted to a group.
  • the antisense polynucleotide (nucleic acid) of the present invention is an RNA, a DNA, or a modified nucleic acid (RNA, DNA).
  • modified nucleic acid include, but are not limited to, sulfur derivatives of nucleic acids, thiophosphate derivatives, and polynucleoside amides which are resistant to degradation of polynucleonucleoside amides.
  • the antisense nucleic acid of the present invention can be preferably designed according to the following policy. That is, to make the antisense nucleic acid more stable in the cell, to increase the cell permeability of the antisense nucleic acid, to have a greater affinity for the target sense strand, and to be more toxic if it is toxic. Make sense nucleic acid less toxic.
  • the antisense nucleic acids of the present invention may contain altered or modified sugars, bases, or bonds, and may be provided in special forms such as liposomes and microspheres. Or be applied by gene therapy or given in an added form.
  • additional forms include polycations, such as polylysine, which act to neutralize the charge on the phosphate backbone, and lipids, which enhance interaction with cell membranes 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 arranged at the 3 'end or 5' end of a nucleic acid to 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 antisense nucleic acid inhibitory activity 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 a G protein-coupled receptor protein. it can.
  • the nucleic acid can be applied to cells by various known methods.
  • the antisense polynucleotide having a nucleotide sequence complementary to or substantially complementary to the nucleotide sequence of a polynucleotide (eg, DNA) encoding a protein or a partial peptide used in the present invention includes the present invention. It has a nucleotide sequence complementary to or substantially complementary to the nucleotide sequence of a polynucleotide (eg, DNA) and has an action capable of suppressing the expression of the polynucleotide (eg, DNA). As long as it is any antisense polynucleotide, it may be a chimeric strand of DNA and RNA, but antisense DNA is preferred.
  • the nucleotide sequence substantially complementary to the polynucleotide of the present invention is, for example, a nucleotide sequence complementary to the polynucleotide of the present invention (eg, DNA) (that is, the nucleotide sequence of the present invention).
  • a nucleotide sequence complementary to the polynucleotide of the present invention eg, DNA
  • the nucleotide sequence of the present invention is, for example, a nucleotide sequence complementary to the polynucleotide of the present invention (eg, DNA) (that is, the nucleotide sequence of the present invention).
  • the complement of the nucleotide sequence of the portion encoding the N-terminal portion of the protein of the present invention for example, the nucleotide sequence near the start codon.
  • Antisense polynucleotides having about 90% or more, preferably about 95% or more, more preferably about 98% or more homology with the strand are suitable.
  • an antisense polynucleotide having a nucleotide sequence complementary to or substantially complementary to the nucleotide sequence of the 5, untranslated region or 3 'untranslated region (preferably, the 5' untranslated region), or a portion thereof Is mentioned.
  • the antisense polynucleotide is generally composed of about 10 to 40 bases, preferably about 15 to 30 bases.
  • each nucleotide constituting the antisense polynucleotide is chemically modified, for example, with phosphorothioate, methylphosphonate, phosphorodithionate, etc. It may be substituted with a phosphate residue.
  • phosphate residue phosphate
  • These antisense polynucleotides can be produced using a known DNA synthesizer or the like.
  • the DNA encoding the partial peptide of the present invention may be any DNA containing the above-described nucleotide sequence encoding the partial peptide of the present invention. Any of a DNA library, the above-described cell / tissue-derived cDNA, the above-described cell / tissue-derived cDNA library, and synthetic DNA may be used.
  • the vector used for the library may be any of pacteriophage, plasmid, cosmid, phagemid and the like.
  • amplification can be performed directly by the RT-PCR method using an mRNA fraction prepared from the cells and tissues described above.
  • the DNA encoding the partial peptide of the present invention includes, for example, (1) a DNA having a partial nucleotide sequence of a DNA having a nucleotide sequence represented by SEQ ID NO: 2, or (2) a sequence No .: DNA represented by 2 and high stringency Partial nucleotide sequence of a DNA that encodes a protein that has a DNA that hybridizes under various conditions and has substantially the same activity as the protein peptide of the present invention, such as ligand binding activity and signal transduction activity.
  • a DNA having DNA is used.
  • Examples of the DNA that hybridizes with the DNA represented by SEQ ID NO: 2 under high stringent conditions include, for example, about 90% or more, preferably about 95% or more of the nucleotide sequence represented by SEQ ID NO: 2. More preferably, DNA containing a base sequence having about 98% or more homology is used.
  • DNA encoding the peptide of the present invention may be used.
  • Amplification by PCR using a synthetic DNA primer having a partial base sequence of the base sequence of SEQ ID NO: 1 or DNA encoding a part or the whole region of the receptor protein of the present invention is a DNA incorporated into an appropriate vector.
  • Selection can be performed by hybridization with a fragment or a fragment labeled with synthetic DNA.
  • the method of hybridization can be carried out, for example, according to the method described in Molecular 'Cloning (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.
  • the DNA base sequence can be replaced by the 0DA-LA PCR method using PCR or a known kit, for example, Mutan TM -super Express Km (Takara Shuzo), Mutan TM -K (Takara Shuzo) or the like.
  • the method can be performed according to known methods such as the gapped duplex method and the Kimkel method, or a method analogous thereto.
  • the DNA encoding the cloned receptor protein can be used as it is depending on the purpose, or can be digested with a restriction enzyme or added with a linker if desired.
  • the DNA may have ATG as a translation initiation codon at its 5 'end, and may have TAA, T08 or G8 at its 3' end as a translation stop codon. .
  • These translation initiation codon and translation termination codon can also be added using an appropriate synthetic DNA adapter.
  • the expression vector of the receptor protein of the present invention may be prepared, for example, by (a) cutting out a DNA fragment of interest from, for example, cDNA containing DNA encoding the receptor protein of the present invention; It can be produced by ligating downstream of a promoter in an expression vector.
  • vectors examples include Escherichia coli-derived plasmids (eg, pCR4, pCR2. PBR322, pBR325, pUC12, pUC13), Bacillus subtilis-derived plasmids (eg, ⁇ 110, pTP5, pC194), yeast-derived plasmids (eg, pSH19, pSH15)
  • pacteriophage such as phage
  • animal viruses such as retrovirus, vaccinia virus, and baculovirus, Al-Ik pXT pc / CMV, pRc / RSV, pcDNM / Neo and the like are used.
  • the promoter used in the present invention may be any promoter as long as it is appropriate for the host used for gene expression.
  • SRa promoter when animal cells are used as host, SRa promoter, SV40 promoter, LTR open motor, CMV promoter, HSV-TK promoter and the like can be mentioned.
  • the CMV promoter the SRa promoter and the like.
  • Escherichia such as trp promoter, lac promoter, recA promoter, ⁇ promoter, lpp promoter, etc.
  • yeast PH05 promoter, PGK promoter, GAP promoter, ADH promoter and the like are preferable.
  • a polyhedrin promoter, a P10 promoter and the like are preferable.
  • the expression vector may further include an enhancer, a splicing signal, a polyA addition signal, a selection marker, an SV40 replication origin (hereinafter sometimes abbreviated as SV4 Oori), and the like, if desired.
  • selectable markers include a dihydrofolate reductase (hereinafter sometimes abbreviated as dMr) gene (methotrexate (MTX) resistance) and an ampicillin resistance gene (hereinafter sometimes abbreviated as Amp 1 )
  • the neomycin resistance gene hereinafter sometimes abbreviated as Ne o r, include G418 resistance) and the like.
  • the dMr gene is used as a selection marker using CHO (dhfr) cells, the target gene can be selected using a thymidine-free medium.
  • a signal sequence suitable for the host is added to the N-terminal side of the receptor protein of the present invention.
  • the host is a bacterium belonging to the genus Escherichia, a PhoA signal sequence, an Omp A signal sequence, or the like. Signal sequence, SUC2 signal sequence, etc. If the host is an animal cell, insulin signal sequence, ' ⁇ _interferin signal sequence, antibody molecule, signal sequence, etc. it can.
  • a transformant can be produced using the vector containing the DNA encoding the receptor protein of the present invention thus constructed.
  • Escherichia bacteria for example, Escherichia bacteria, Bacillus bacteria, yeast, insect cells, insects, animal cells, and the like are used.
  • bacterium belonging to the genus Escherichia include Escherichia coli K12 ⁇ DH1 [Processings of the National Academy of Sciences, Op. Natl. Acad. Sci. USA), 60, 160 (1968)], JM103 [Nucleic Acids Research, 9, 309 (1981)], JA221 [Journal Op. Molecular] -Journal of Molecular Biology], 120, 517 (1978)], HB101 [Journal of Molecular Biology, 41, 459 (1969)], C600 [Genetics, 39, 440 (1954)], DH5a CInoue, H., Nojima, H.
  • Bacillus spp. include, for example, Bacillus subtilis MI114 [Gene, 24, 255 (1983)], 207-21 [Journal of Biochemistry, 95, 87] (1984)]
  • yeast include Saccharomyces cerevisiae AH22, A marauder, ONA87-11A, DKD-5D, 20B-12, Schizosaccharomyces pombe NCYC1913, NCYC2036, Pichia pastoris, etc. Is used.
  • insect cells for example, when the virus is AcNPV, a cell line derived from a larva of Spodoptera (Spodoptera frugiperda cell; S f cell), MG1 cell derived from the midgut of Trichoplusia ni, High Five TM derived from egg of Trichoplusia ni Cells,
  • Sf cells derived from Mamestra brassicae or cells derived from Estigmena acrea are used.
  • viruses When the virus is BmNPV, a silkworm-derived cell line (Bombyx mori N; BmN cell) or the like is used.
  • Sf cells for example, Sf9 cells (ATCC C L1711), Sf21 cells (Vaughn, JL et al., In Vivo, 13, 213-217 (1977)) and the like are used.
  • Sf9 cells ATCC C L1711
  • Sf21 cells Vaughn, JL et al., In Vivo, 13, 213-217 (1977)
  • insects for example, silkworm larvae are used [Maeda et al., Nature, 315, 592 (1985)].
  • animal cells examples include monkey cell COS-7, Vero, Chinese hamster cell CHO (hereinafter abbreviated as CHO cell), dhir gene-deficient Chinese hamster cell CHO (hereinafter abbreviated as dfD cell), mouse L cells, mouse AtT-20, mouse myeloma cells, rat GH3, human FL cells, etc. are used.
  • CHO cell Chinese hamster cell CHO
  • dfD cell dhir gene-deficient Chinese hamster cell CHO
  • mouse L cells mouse AtT-20, mouse myeloma cells, rat GH3, human FL cells, etc.
  • Transformation of Bacillus can be performed, for example, according to the method described in Molecular & General Genetics, Vol. 168, 111 (1979).
  • Transformation of insect cells or insects can be performed, for example, according to the method described in Bio / Technology, 6, 47-55 (1988).
  • a liquid medium is suitable as the medium used for the culturing, and a carbon source necessary for the growth of the transformant is contained therein.
  • the carbon source include glucose, dextrin, soluble starch, and sucrose.
  • the nitrogen source include ammonium salts, nitrates, corn chip liquor, peptone, casein, meat extract, soybean meal, and potato extract.
  • the inorganic or organic substance and the inorganic substance include calcium chloride, sodium dihydrogen phosphate, magnesium chloride, and the like.
  • yeast extract, vitamins, growth promoting factors and the like may be added.
  • the pH of the medium is preferably about 5-8.
  • an M9 medium containing glucose and casamino acids As a medium for cultivating a bacterium belonging to the genus Escherichia, for example, an M9 medium containing glucose and casamino acids [Miller, Journal of Expermentin, Molecular of Genetics] Genetics, 431-433, Cold Spring Harbor Laboratory, New York 1972] where it is necessary to add a drug such as 3) 3-indolylacrylic acid in order to make the promoter work efficiently. Can be.
  • culturing is usually performed at about 15 to 43 for about 3 to 24 hours, and if necessary, aeration and stirring can be added.
  • culturing is usually performed at about 30 to 40 for about 6 to 24 hours. If necessary, ventilation and stirring can be added.
  • a medium for example, Burkholder's minimum medium [Bostian, KL. Et al., "Processings of the National Academy” Pro Natl. Acad. Sci. USA, 77, 4505 (1980) J or SD medium containing 0.5% casamino acid GBitter, GA Proc. Natl. Acad. Sci. USA, 81, 5330 (1984), "Processings of the National Academy of Ops Sciences of the USA.” No. Medium! H) is preferably adjusted to about 5-8. Cultivation is usually performed at about 20 ° C to 35 ° C for about 24 to 72 hours, and aeration and stirring 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% of fetal bovine serum [Science, 122, 501 (1952)], a DMEM medium [Virology, Volume 8, 396 (1959)], RPMI 1640 medium [Journal 'The American of Medical Association', Volume 199, 519
  • the pH is about 6-8.
  • Culture is usually performed at about 30 ° C to 40 ° C for about 15 to 60 hours, and aeration and agitation are added as necessary.
  • the G protein-coupled receptor protein of the present invention can be produced in the transformant, in the cell membrane, or outside the cell.
  • the separation and purification of the receptor protein of the present invention from the culture can be performed, for example, by the following method.
  • the cells or cells are collected by a known method after culturing, suspended in an appropriate buffer, and subjected to ultrasound, lysozyme and Z Alternatively, a method in which cells or cells are ruptured by freeze-thawing or the like and then a crude extract of the receptor protein is obtained by centrifugation or filtration, etc., 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 receptor protein contained in the thus obtained culture supernatant or extract can be carried out 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.
  • Method using difference in charge method using charge difference such as ion exchange chromatography, method using specific affinity such as affinity mouth chromatography, hydrophobicity such as reverse phase high performance liquid chromatography, etc.
  • a method utilizing the difference between the isoelectric points such as a method utilizing the difference between the isoelectric points, and an isoelectric point electrophoresis method are used.
  • the receptor protein thus obtained When the receptor protein thus obtained is obtained in a free form, it can be converted into a salt by a known method or a method analogous thereto. It can be converted into a free form or another salt by an analogous method.
  • the receptor 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, daricosidase and the like are used.
  • the activity of the receptor protein or the salt thereof of the present invention thus produced can be measured by a binding experiment with a labeled ligand, an enzyme immunoassay using a specific antibody, or the like.
  • Antibodies against the receptor protein of the present invention or its partial peptide or its salt may be any of polyclonal antibodies and monoclonal antibodies as long as they can recognize the receptor protein or its partial peptide or its salt of the present invention. It may be.
  • An antibody against the receptor protein of the present invention or its partial peptide or a salt thereof may be a known antibody or a known antibody using the receptor protein of the present invention as an antigen. It can be produced according to the production method of antiserum. [Preparation of monoclonal antibody]
  • the receptor protein of the present invention or the like is administered to a mammal at a site capable of producing an antibody by administration itself or together with a carrier or a diluent.
  • Freund's complete adjuvant or incomplete Freund's adjuvant may be administered to enhance the antibody-producing ability.
  • the administration is usually performed once every 2 to 6 weeks, for a total of about 2 to 10 times. Examples of mammals to be used include monkeys, rabbits, dogs, guinea pigs, mice, rats, sheep, goats, and mice and rats are preferably used.
  • a warm-blooded animal immunized with the 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.
  • 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.
  • a monoclonal antibody-producing hybridoma can be prepared.
  • the antibody titer in the antiserum can be measured, for example, by reacting a labeled receptor protein or the like described below with the antiserum, and then measuring the activity of a labeling agent bound to the antibody.
  • the fusion operation can be carried out according to a known method, for example, the method of Koehler and Mills [Nature, 256, 495 (1975)].
  • the fusion promoter include polyethylene glycol (PEG) and Sendai virus, but PEG is preferably used.
  • Myeloma cells include, for example, NS-1, P3U1, SP2 / 0, etc. However, P 3 U 1 is preferably used.
  • the preferred ratio between the number of antibody-producing cells (spleen cells) and the number of myeloma cells used is about 1: 1 to 20: 1, and PEG (preferably PEG1000 to PEG6000) is added at a concentration of about 10 to 80%. Is about 20-40. C, preferably by incubating at about 30 to 37 ° C for about 1 to 10 minutes, efficient cell fusion can be performed.
  • hybridomas can be cultured on a solid phase (eg, microplate) on which an antigen such as receptor protein has been adsorbed directly or together with a carrier. And then add an anti-immunoglobulin antibody labeled with a radioactive substance or enzyme (if the cells used for cell fusion are mice, use an anti-mouse immunoglobulin antibody) or protein A and bind to the solid phase A hybridoma culture supernatant is added to a solid phase to which anti-immune glopurin antibody or protein A is adsorbed, and a receptor protein labeled with a radioactive substance or an enzyme is added.
  • a method for detecting a monoclonal antibody bound to a phase is exemplified.
  • the selection of the monoclonal antibody can be carried out according to a known method or a method analogous thereto. Usually, it can be carried out in a medium for animal cells to which HAT (hypoxanthine, aminopterin, thymidine) is added.
  • HAT hyperxanthine, aminopterin, thymidine
  • any medium can be used as long as hybridomas can grow.
  • RPMI 1640 medium containing 1-20%, preferably 10-20% fetal calf serum, GIT medium containing 1-10% fetal calf serum (Wako Pure Chemical Industries, Ltd.)
  • a serum-free medium SFM-101, Nissui Pharmaceutical Co., Ltd.
  • the culture temperature is usually 20 to 40 ° C, preferably about 37 ° C.
  • the culture time is usually 5 days to 3 weeks, preferably 1 week to 2 weeks.
  • the culture can be usually performed under 5% carbon dioxide gas.
  • the antibody titer of the hybridoma culture supernatant can be measured in the same manner as the measurement of the antibody titer in the antiserum described above.
  • Monoclonal antibodies can be separated and purified in the same manner as normal polyclonal antibodies, such as immunoglobulin separation and purification (e.g., salting out, alcohol precipitation, isoelectric focusing). Method, electrophoresis, adsorption / desorption using an ion exchanger (e.g., DEAE), ultracentrifugation, gel filtration, antigen-bound solid phase, or active antibody such as protein A or protein G. Specific purification method of dissociating the bond to obtain an antibody].
  • immunoglobulin separation and purification e.g., salting out, alcohol precipitation, isoelectric focusing.
  • electrophoresis e.g., electrophoresis, adsorption / desorption using an ion exchanger (e.g., DEAE), ultracentrifugation, gel filtration, antigen-bound solid phase, or active antibody such as protein A or protein G.
  • an ion exchanger e.g., DEAE
  • the polyclonal antibody of the present invention can be produced according to a known method or a method analogous thereto. For example, a complex of an immunizing antigen (antigen such as the protein of the present invention) and a carrier protein is formed, and a mammal is immunized in the same manner as in the above-described method for producing a monoclonal antibody. Yuichi It can be produced by collecting the antibody-containing substance against the protein etc. and separating and purifying the antibody.
  • the type of carrier protein and the mixing ratio of carrier to hapten are determined by the antibody against hapten immunized by cross-linking the carrier. Any efficiency can be achieved by cross-linking any material at any ratio.For example, serum serum albumin, thyroglobulin, keyhole limpet, hemocyanin, etc.
  • a method of coupling the hapten at a ratio of about 0.1 to 20, preferably about 1 to 5 with respect to 1 of the hapten is used.
  • various condensing agents can be used for force coupling between the hapten and the carrier.
  • an active ester reagent containing a daltaraldehyde, a carbodiimide, a maleimide active ester, a thiol group or a dithioviridyl group 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 can usually be performed once every about 2 to 6 weeks, for a total of about 3 to 10 times.
  • the polyclonal antibody can be collected from blood, ascites, or the like, preferably from blood, of the mammal immunized by the above method.
  • the measurement of the polyclonal antibody titer in the antiserum is the same as the measurement of the antibody titer in the serum described above. Can be measured in the following manner. 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.
  • the receptor protein of the present invention or its salt, its partial peptide or its salt, and the DNA encoding the receptor protein or its partial peptide are as follows: (1) a ligand for the G protein-coupled receptor protein of the present invention ( (2) prevention and / or therapeutic agent for diseases associated with dysfunction of the G protein-coupled receptor protein of the present invention, (3) genetic diagnostic agent, (4) G protein coupling of the present invention (5) Screening method for compounds (eg, agonists, antagonists, etc.) that change the binding property between the G protein-coupled receptor protein and the ligand of the present invention, (6) A compound that alters the binding between a G protein-coupled receptor protein and a ligand of the present invention (7) quantification of the receptor protein of the present invention or its partial peptide or a salt thereof, (8) the receptor protein of the present invention Or (9) creation of a non-human transgenic animal having a DNA encoding the G protein-coupled receptor protein of the present invention, (10) knockout An animal, (11) a method for
  • a ligand for a G protein-coupled receptor specific for human / non-human mammals can be obtained. Screening for compounds that alter the binding (eg, agonists, anthony gonists, etc.)
  • the agonist or angelic gonist can be used as an agent for preventing or treating various diseases.
  • the receptor protein of the present invention (sometimes abbreviated as DNA of the present invention) and the receptor protein of the present invention (hereinafter sometimes abbreviated as the antibody of the present invention) will be specifically described below.
  • the receptor protein of the present invention or its salt or the partial peptide or its salt of the present invention searches for or determines a ligand (agonist) for the receptor protein or its salt of the present invention.
  • the present invention provides a method for determining a ligand for the receptor protein of the present invention, which comprises contacting the receptor protein of the present invention or a salt thereof or the partial peptide of the present invention or a salt thereof with a test compound. provide.
  • Test compounds include known ligands (e.g., angiotensin, bombesin, canapinoid, cholecystokinin, glutamine, serotonin, melatonin, neuropeptide Y, o "oid, 'purine, vasoprescin, oxytocin, PACAP ( E.g., PACAP 27, PACAP 38), secretin, glucagon, calcitonin, adrenomedullin, somatos-tin, GHRH, CRF, ACTH, GRP, PTH, VIP (Vasoactive Intestinal and Related polypeptide) ), Somatostatin, dopamine, motilin, amylin, bradykinin, CGRP (calcitonin gene releated peptide), leukotriene, pancreastatin, prostaglandin, thromboxane, adenosine, adrenaline, chemokine superfamily (Example: CXC
  • the ligand determination method of the present invention uses the receptor protein of the present invention or a partial peptide thereof or a salt thereof, or constructs an expression system of a recombinant receptor protein, and comprises the expression system.
  • the receptor binding system used in the present invention it binds to the receptor binding protein of the present invention and has a cell stimulating activity (for example, arachidonic acid release, acetylcholine release, intracellular Ca 2+ release, intracellular c).
  • Activity to promote or suppress AMP generation, intracellular CAMP suppression, intracellular cGMP generation, inositol phosphate production, cell membrane potential fluctuation, intracellular protein phosphorylation, c-fos activation, pH decrease, etc. (Eg, peptides, proteins, non-peptidic compounds, synthetic compounds, fermentation products, etc.) or a salt thereof.
  • a receptor compound of the present invention or a partial peptide thereof is contacted with a test compound, for example, binding of a test compound to the receptor protein or the partial peptide It is characterized by measuring the amount and cell stimulating activity.
  • the present invention provides
  • the labeled test compound is transferred to the receptor protein of the present invention or its salt or Is a method for measuring the amount of a labeled test compound bound to the protein or a salt thereof or to the partial peptide or a salt thereof when the test compound is brought into contact with the partial peptide of the present invention or a salt thereof.
  • a method for determining a ligand for a receptor protein or a salt thereof
  • the labeled test compound When the labeled test compound is brought into contact with the receptor protein expressed on the cell membrane by culturing a transformant containing the DNA encoding the receptor protein of the present invention, the labeled test compound A method for determining a ligand for a receptor protein of the present invention, which comprises measuring the amount of binding to the receptor protein or a salt thereof;
  • ⁇ Cell stimulating activity via receptor protein when a test compound is brought into contact with cells containing the receptor protein of the present invention for example, arachidonic acid release, acetylcholine release, intracellular Ca 2+ release, Intracellular cAMP production, Intracellular cAMP production, Intracellular cGMP production, Inositol phosphate production, Cell membrane potential fluctuation, Intracellular protein phosphorylation, Activation of c-fos, Decrease in pH, etc.
  • a method for determining a ligand for the receptor protein or a salt thereof of the present invention characterized by measuring
  • a transformant containing DNA encoding the receptor protein of the present invention for example, arachidonic acid release, acetylcholine release, intracellular Ca2 + release, intracellular cAMP production, intracellular cAMP suppression, intracellular cGMP production, inositol phosphate production, cell membrane potential fluctuation, Phosphorylation of c-protein, activation of fos, activation or suppression of pH reduction, etc.
  • a method for determining a ligand for the receptor protein or a salt thereof of the present invention will provide a.
  • the receptor protein used in the ligand determination method may be any receptor protein containing the above-described receptor protein of the present invention or the partial peptide of the present invention. Reception night and evening The protein is suitable.
  • the above-described expression method is used, but it is preferable to produce the DNA encoding the receptor protein in mammalian cells or insect cells.
  • a complementary DNA is usually used as the DNA fragment encoding the protein portion of interest, but is not necessarily limited to this.
  • a gene fragment or a synthetic DNA may be used.
  • the DNA fragment should be a nuclear polysaccharide belonging to baculovirus using an insect as a host.
  • Nuclear polyhedrosis virus (NPV) polyhedrin promoter SV40-derived promoter, retrovirus promoter overnight, meta-oral thionine promoter, human heat shock promoter, cytomegalovirus promoter It is preferable to incorporate it downstream such as the SR promoter.
  • Examination of the amount and quality of the expressed receptor can be performed by a known method. For example, the method is carried out according to the method described in the document [Nambi, P. et al., The Journal of Biological Chemistry (J. Biol. Chem.), 267, 19555-19559, 1992]. Can be.
  • the receptor protein of the present invention or a partial peptide thereof or a salt thereof includes a receptor protein or a partial peptide thereof or a salt thereof purified according to a known method.
  • a cell containing the receptor protein or a cell membrane fraction thereof may be used.
  • the cells When cells containing the receptor protein of the present invention are used in the ligand determination method of the present invention, the cells may be immobilized with daltaraldehyde, formalin, or the like.
  • the immobilization method can be performed according to a known method.
  • the cell containing the receptor protein of the present invention refers to a host cell expressing the receptor protein of the present invention.
  • Escherichia coli, Bacillus subtilis, yeast, insect cells, animal cells and the like are used as the host cell.
  • the cell membrane fraction refers to a fraction containing a large amount of cell membrane obtained by a known method after cell disruption.
  • Cells can be crushed by crushing the cells with a Potter-Elvehjem homogenizer, ⁇ ⁇ ⁇ one ring blender ⁇ ⁇ ⁇ crushing with a polytron (Kinema tica), crushing with ultrasonic waves, pressing with a French press, etc. Crushing by ejecting cells from a thin nozzle can be mentioned.
  • a fractionation method by centrifugal force such as a fractionation centrifugation method or a density gradient centrifugation method is mainly used.
  • the cell lysate is centrifuged at a low speed (500 rpm to 300 rpm) for a short time (usually about 1 minute to 10 minutes), and the supernatant is further spun at a high speed (150 rpm to 300 rpm).
  • the mixture is centrifuged usually at 0,000 rpm for 30 minutes to 2 hours, and the resulting precipitate is used as a membrane fraction.
  • the membrane fraction is rich in the expressed receptor protein and membrane components such as cell-derived phospholipids and membrane proteins.
  • the amount of the receptor protein of the cells or during the membrane fraction containing the receptor protein is preferably from 1 0 3 to 1 0 8 molecules per cell, that 1 is 0 5-1 0 7 molecules It is suitable.
  • receptor protein fraction it is desirable to use a natural receptor protein fraction or a recombinant receptor protein fraction having an activity equivalent to that of the natural receptor protein fraction.
  • equivalent activity refers to equivalent ligand binding activity, signal transduction activity and the like.
  • a cell or a membrane fraction of the cell containing the receptor protein of the present invention is subjected to a buffer suitable for the determination method.
  • a buffer suitable for the determination method Prepare a standard preparation by resuspending the suspension. Any buffer may be used as long as it does not inhibit the binding between the ligand and the receptor protein, such as a phosphate buffer having a pH of 4 to 10 (preferably pH 6 to 8) and a tris-hydrochloride buffer.
  • various proteins such as detergents such as CHAPS, Tween-80 TM (Kao-Ichi Atlas), digitonin, dexcholate, serum albumin, and gelatin are added to the buffer to reduce non-specific binding. You can also.
  • professional Protease inhibitors such as PMS F, Leptin, E-64 (manufactured by Peptide Research Laboratories), and Peptide may also be added for the purpose of suppressing receptor degradation and ligand degradation by the protease.
  • PMS F, Leptin, E-64 manufactured by Peptide Research Laboratories
  • Peptide may also be added for the purpose of suppressing receptor degradation and ligand degradation by the protease.
  • 0.0 lm 1 to 1 Om 1 of the receptor solution was labeled with a fixed amount (5000 cpm to 500,000 cpm) of [ 3 H], [ 125 I], [ 14 C], [ 35 S], etc. The test compound is allowed to coexist.
  • the reaction is carried out at about 0 ° C to 50 ° (: preferably at about 4 ° C to 37 ° C for about 20 minutes to 24 hours, preferably for about 30 minutes to 3 hours.
  • the radioactivity remaining on the glass fiber filter paper is measured with a liquid scintillation counter or a counter ..
  • a test compound having (B-NSB) exceeding O cpm can be selected as a ligand (agonist) for the receptor protein of the present invention or a salt thereof.
  • the cell stimulating activity mediated by the receptor protein for example, arachidonic acid release, acetylcholine release, intracellular Ca 2+ release, Intracellular cAMP generation, Intracellular cAMP suppression, Intracellular cGMP generation, Inositol phosphate production, Cell membrane potential fluctuation, Intracellular protein phosphorylation, Activation of c-fos, Decrease in pH, etc.
  • Activity or inhibitory activity can be measured using a known method or a commercially available measurement kit. Specifically, first, cells containing the receptor protein are cultured in a multiwell plate or the like.
  • the assay Before determining the ligand, replace with a fresh medium or an appropriate buffer that is not toxic to cells, add test compounds, etc., incubate for a certain period of time, and then extract cells or collect supernatant. Quantify the product produced according to each method. If the production of a substance (for example, arachidonic acid) as an indicator of cell stimulating activity is difficult due to the presence of a degrading enzyme contained in the cells, the assay may be performed by adding an inhibitor against the degrading enzyme. . In addition, the activity such as cAMP production suppression can be detected as a production suppression effect on cells whose basal production has been increased by forskolin or the like.
  • a substance for example, arachidonic acid
  • the activity such as cAMP production suppression can be detected as a production suppression effect on cells whose basal production has been increased by forskolin or the like.
  • the kit for determining a ligand that binds to the receptor protein of the present invention or a salt thereof is a receptor protein of the present invention or a salt thereof, a partial peptide or a salt thereof of the present invention, a cell containing the receptor protein of the present invention, or It contains the membrane fraction of the cells containing the receptor protein of the present invention.
  • Examples of the kit for determining a ligand of the present invention include the following.
  • CHO cells expressing the receptor protein of the present invention were subcultured on a 12-well plate at 5 ⁇ 10 5 cells / well, and cultured for 2 days at 37, 5% C 2 , and 95% air.
  • Test compounds that are poorly soluble in water should be dissolved in dimethylformamide, DMSO, methanol, etc.
  • the same as the labeled compound is prepared at a concentration 100 to 1000 times higher.
  • Examples of the ligand capable of binding to the receptor protein or a salt thereof of the present invention include, for example, substances specifically present in the hypothalamus, cerebral cortex, colon cancer, lung cancer, and the like.
  • the receptor protein of the present invention or the DNA encoding the receptor protein can be used according to the action of the ligand.
  • Can be used as a medicament such as an agent for preventing and / or treating a disease associated with dysfunction of the receptor protein of the present invention.
  • the receptor protein of the present invention is reduced in vivo, the physiological effect of receptor protein cannot be expected (the receptor protein deficiency of the present invention).
  • the receptor protein deficiency of the present invention By administering one protein to the patient to replenish the amount of the receptor protein of the present invention, or (2) administering the DNA encoding the receptor protein of the present invention to the patient for expression. Or (mouth) Inserting and expressing the DNA encoding the receptor protein of the present invention into target cells, and then transplanting the cells into the patient, for example, to obtain the present invention in the patient's body.
  • the receptor protein of the present invention and the DNA encoding the receptor protein of the present invention can be used as a safe and low toxic agent for preventing and / or treating diseases associated with dysfunction of the receptor protein of the present invention. It is useful as a medicament.
  • the DNA which is a receptor for the ligand is used.
  • the receptor protein of the present invention is useful as a preventive or therapeutic drug for any cancer (for example, lung cancer, stomach cancer, liver cancer, knee cancer, colon cancer, rectum cancer, colon cancer, prostate cancer, ovarian cancer, cervical cancer, breast cancer, etc.). is there.
  • the receptor protein of the present invention is used as the prophylactic / therapeutic agent, it can be formulated according to a conventional method.
  • the DNA of the present invention when a DNA encoding the receptor protein of the present invention (hereinafter sometimes abbreviated as the DNA of the present invention) is used as the prophylactic or therapeutic agent, the DNA of the present invention may be used alone or retrograde. After insertion into a suitable vector such as a virus vector, an adenovirus vector, or an adenovirus associated virus vector, it can be carried out according to a conventional method.
  • the DNA of the present invention can be administered as it is or together with an adjuvant for promoting uptake, using a gene gun or a catheter such as a hydrogel catheter.
  • the receptor protein of the present invention or (2) DNA encoding the receptor protein is orally provided as a sugar-coated tablet, capsule, elixir, microcapsule or the like, if necessary. It can be used parenterally in the form of injectable solutions, such as sterile solutions with water or other pharmaceutically acceptable liquids, or suspensions.
  • injectable solutions such as sterile solutions with water or other pharmaceutically acceptable liquids, or suspensions.
  • Excipients 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 And bulking agents such as magnesium stearate, sweeteners such as sucrose, lactose or saccharin, and flavoring agents such as peppermint, cocoa oil or cellulose.
  • binders such as gelatin, corn starch, tragacanth, gum arabic
  • excipients such as crystalline cellulose, corn starch, gelatin, alginic acid And bulking 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.
  • Sterile compositions for injection can be formulated according to normal 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, coconut oil and the like.
  • Aqueous liquids for injection include, for example, saline, dextrose and other Isotonic solutions containing adjuvants (eg, D-sorbitol, D-mannitol, sodium chloride, etc.) are used, and suitable solubilizing agents, such as alcohols (eg, ethanol), polyalcohols (eg, It may be used in combination with propylene glycol, polyethylene glycol), nonionic surfactants (eg, polysorbate 80 (TM), HCO-50).
  • suitable solubilizing agents such as alcohols (eg, ethanol), polyalcohols (eg, It may be used in combination with propylene glycol, polyethylene glycol), nonionic surfactants (eg, polysorbate 80 (TM), HCO-50).
  • prophylactic / therapeutic agent examples include a buffer (for example, phosphate buffer and sodium acetate buffer), a soothing agent (for example, benzalkonium chloride, procaine hydrochloride, etc.), a stabilizer (for example, human serum It may be blended with albumin, polyethylene glycol, etc.), preservatives (eg, benzyl alcohol, phenol, etc.), antioxidants, etc.
  • a buffer for example, phosphate buffer and sodium acetate buffer
  • a soothing agent for example, benzalkonium chloride, procaine hydrochloride, etc.
  • a stabilizer for example, human serum It may be blended with albumin, polyethylene glycol, etc.
  • preservatives eg, benzyl alcohol, phenol, etc.
  • antioxidants etc.
  • the prepared injection solution is usually filled into a suitable ampoule.
  • the preparations obtained in this way are safe and low toxic, so they can be used, for example, in mammals (eg, humans, rats,
  • the dosage of the receptor protein of the present invention varies depending on the administration subject, target organ, symptoms, administration method, and the like.
  • oral administration for example, in a cancer patient (as 60 kg), it is generally required to be administered per day. It is about 0.1 mg to 100 mg, preferably about 1.0 to 50 mg, more preferably about 1.0 to 2 Omg.
  • parenteral administration the single dose varies depending on the administration target, target organ, symptoms, administration method, etc.
  • the dose can be administered in terms of 6 O kg.
  • the dose of the DNA encoding the receptor protein of the present invention varies depending on the administration subject, target organ, symptoms, administration method, and the like.
  • oral administration in general, cancer patients (6O about 0.1 to 100 mg per day, preferably about 1.0 to 50 mg, more preferably about 1.0 to 20 mg per day.
  • parenteral administration the single dose varies depending on the administration target, target organ, symptoms, administration method, etc.For example, in the case of injection, it is usually used, for example, in cancer patients (as 60 kg), It is convenient to administer about 0.01 to 30 mg, preferably about 0.1 to 2 Omg, more preferably about 0.1 to 10 mg per day by intravenous injection. In the case of other animals, the dose can be administered in terms of 60 kg.
  • the DNA encoding the receptor protein of the present invention can be used as a probe to produce human or human mammals (eg, rat, mouse, rabbit, sheep, pig, pig, cat, dog, monkey, etc.). ) Can detect abnormalities (genetic abnormalities) in the DNA or mRNA encoding the receptor protein of the present invention, for example, damage, mutation, or reduced expression of the DNA or mRNA; It is useful as a diagnostic agent for genes such as an increase in expression or overexpression.
  • the above-mentioned genetic diagnosis using DNA encoding the receptor protein of the present invention can be carried out, for example, by the known Northern hybridization or PCR-SSCP method (Genomics, Vol. 5, pp. 874-879). 1989), Proceedings of the National Academy of Sciences of the United States of America, Vol. 86, pp. 2766-2770, Procedings of the National Academy of Sciences of the United States of America (1989)).
  • the receptor protein of the present invention has a binding property to the ligand protein, it is possible to quantify the receptor concentration in the living body with high sensitivity.
  • the quantification method of the present invention can be used, for example, in combination with a competition method. That is, the ligand concentration in the test sample can be measured by bringing the test sample into contact with the receptor protein of the present invention. Specifically, for example, It can be used according to the method described in (1) or (2) below or a method analogous thereto.
  • a method for screening a compound that changes the binding property between the receptor protein or its salt of the present invention and a ligand eg, agonist, antagonist, etc.
  • the present invention provides Efficient screening of compounds (eg, peptides, proteins, non-peptidic compounds, synthetic compounds, fermentation products, etc.) or salts thereof that alters the binding between receptor proteins and ligand proteins it can.
  • the ligand used here is a ligand whose properties are well known, for example, the same as or substantially the same as the amino acid sequence represented by SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5 or SEQ ID NO: 6.
  • Polypeptides or salts thereof characterized by containing the same amino acid sequence are preferred.
  • Such compounds for example, a cell stimulating activity via a G protein-coupled receptors (e.g., Arakidon acid release, acetylcholine release, intracellular C a 2 + release, intracellular c AM P generation, intracellular c GMP production, inositol phosphate production, cell membrane potential fluctuations, phosphorylation of intracellular proteins, activation of c-fos, activity to promote or suppress pH reduction, etc.
  • a G protein-coupled receptors e.g., Arakidon acid release, acetylcholine release, intracellular C a 2 + release, intracellular c AM P generation, intracellular c GMP production, inositol phosphate production, cell membrane potential fluctuations, phosphorylation of intracellular proteins, activation of c-fos, activity to promote or suppress pH reduction, etc.
  • Agonist for receptor protein (mouth) a compound not having the cell stimulating activity (so-called receptor agonist for receptor protein), or
  • the present invention relates to (i) the case where the ligand is brought into contact with the receptor protein of the present invention, and (ii) the case where the ligand is brought into contact with the receptor protein and the test compound of the present invention.
  • the present invention is characterized by performing a comparison.
  • the screening method of the present invention is characterized by measuring, for example, the amount of ligand binding to the receptor protein of the present invention, cell stimulating activity, and the like in the cases (i) and (ii), and comparing them.
  • the present invention provides
  • the receptor may be labeled instead of the ligand.
  • the labeled ligand protein and the test compound contain the receptor protein of the present invention. Measuring the amount of binding of the labeled ligand protein to the cell or the membrane fraction when the cell is contacted with the cell or the membrane fraction of the cell, and comparing the ligand protein with the receptor of the present invention. A method of screening for a compound or a salt thereof that changes the binding property to one protein,
  • ⁇ Contact the ligand protein with cells containing the receptor protein of the present invention.
  • Cell stimulating activity via the receptor protein of the present invention eg, arachidonic acid release
  • a cell containing the receptor protein of the present invention Acetylcholine release, intracellular Ca 2+ release, intracellular cAMP generation, intracellular cGMP generation, inositol phosphoric acid production, cell membrane potential fluctuation, intracellular protein phosphorylation, c_fos activity And the activity of promoting or suppressing the decrease of pH, etc.
  • Screening method and
  • a compound that inhibits the binding between the ligand and the receptor protein of the present invention can be efficiently screened. Furthermore, it is possible to easily evaluate whether the screened compound is an agonist or an engonist.
  • the receptor protein of the present invention used in the screening method of the present invention may be any one as long as it contains the above-described receptor protein of the present invention. Mammalian viscera cell membrane fractions containing the receptor protein are preferred. However, in order to obtain a large amount of the receptor protein of the present invention used for screening, it is necessary to express a large amount of the receptor protein of the present invention using a recombinant. It is suitable for night and night.
  • the above-mentioned method is used to produce the receptor protein of the present invention, but it is preferable to express the DNA in mammalian cells or insect cells.
  • Complementary DNA is used as the DNA fragment encoding the protein portion of interest, but is not necessarily limited thereto.
  • a gene fragment or a synthetic DNA may be used.
  • the DNA fragment should be prepared by transferring the DNA fragment to a nuclear polyhedrosis virus belonging to the baculo-mouth virus using an insect as a host. nuclear
  • polyhedros is virus (NPV) polyhedrin promoter, SV40-derived promoter, retroviral promoter, meta-mouth thionine promoter, human heat shock promoter, cytomegalovirus promoter, SR CK promoter, etc. It is preferably incorporated downstream of.
  • the amount and quality of the expressed receptor can be detected by a known method. For example, it can be carried out according to the method described in the literature [Nambi, P. et al., The Journal of Biological Chemistry (J. Biol. Chem.), 267, 19555-19559, 1992]. it can.
  • the receptor protein-containing protein may be a receptor protein purified according to a known method, or a cell containing the receptor protein may be used. Alternatively, a membrane fraction of a cell containing the receptor protein may be used.
  • the cells When cells containing the receptor protein of the present invention are used in the screening method of the present invention, the cells may be immobilized with daltaraldehyde, formalin, or the like.
  • the immobilization method can be performed according to a known method.
  • Cells containing the receptor protein of the present invention include host cells that express the receptor protein.
  • Examples of the host cells include Escherichia coli, Bacillus subtilis, yeast, insect cells, animal cells, and the like. preferable.
  • the cell membrane fraction refers to a fraction containing a large amount of cell membrane obtained by a known method after cell disruption.
  • Cell crushing methods include PoUer-Elvehj em-type homogenizer, crushing cells, and Warlin Blender ⁇ Polytron.
  • centrifugal fractionation methods such as differential centrifugation and density gradient centrifugation are mainly used.
  • the cell lysate is centrifuged at a low speed (500 rpm to 3000 rpm) for a short time (typically about 1 minute to 10 minutes), and the supernatant is further centrifuged at a higher speed (1500 rpm to 30000 rpm), usually 30 min. Centrifuge for 1 minute to 2 hours, and use the resulting precipitate as the membrane fraction.
  • the membrane fraction is rich in the expressed receptor protein of the present invention and membrane components such as cell-derived phospholipids and membrane proteins.
  • the amount of the receptor protein in the cells and membrane fractions containing the receptor protein of the present invention is preferably 10 3 to 10 8 molecules per cell, and 10 5 to 10 7 molecules per cell. Preferably it is a molecule.
  • the receptor protein fraction of the present invention is preferably a natural receptor protein fraction of the present invention or a recombinant receptor protein fraction having an activity equivalent thereto.
  • equivalent activity refers to equivalent ligand binding activity, signal transduction action, and the like.
  • labeled ligand a labeled ligand, a labeled ligand analog compound and the like are used.
  • ligands labeled with [ 3 H], [ 125 I], [ 14 C], [ 35 S] eg, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5 or SEQ ID NO: 6)
  • a polypeptide characterized by containing the same or substantially the same amino acid sequence as the amino acid sequence represented by or a salt thereof, etc. A polypeptide characterized by containing the same or substantially the same amino acid sequence as the amino acid sequence represented by or a salt thereof, etc.
  • a cell containing the receptor protein of the present invention or a membrane fraction of the cell is subjected to screening.
  • a receptor protein preparation is prepared by suspending in a buffer suitable for screening.
  • Any buffer may be used as long as it does not inhibit the binding between the ligand and the receptor protein, such as a phosphate buffer having a pH of 4 to 10 (preferably pH 6 to 8) or a tris-hydrochloride buffer.
  • a surfactant such as CHAPS, Tween-80 TM (Kaoichi Atlas), digitonin, or dexcholate can be added to the buffer.
  • a protease inhibitor such as PMSF, leptin, E-64 (manufactured by Peptide Research Institute), or peptide sutin is added for the purpose of suppressing the degradation of the receptor by the protease and the degradation of the ligand protein of the present invention.
  • a certain amount 5000 c pm ⁇ 500000 c pm
  • the reaction is carried out at about 0 ° C to 50 ° C, preferably about 4 ° C to 37 ° C, for about 20 minutes to 24 hours, preferably for about 30 minutes to 3 hours.
  • the reaction solution is filtered through a glass fiber filter paper and the like, washed with an appropriate amount of the same buffer, and the radioactivity remaining on the glass fiber filter paper is measured using a liquid scintillation counter or a glass counter.
  • the specific binding amount (B-NS B) becomes For example, a test compound having 50% or less can be selected as a candidate substance having a competitive inhibitory ability.
  • cell stimulating activity mediated by the receptor protein of the present invention for example, Promotes acetylcholine release, intracellular Ca release, intracellular cAMP generation, intracellular cGMP generation, inositol phosphate production, cell membrane potential fluctuation, intracellular protein phosphorylation, c-fos activation, pH reduction, etc.
  • Activity or inhibitory activity can be measured using a known method or a commercially available measurement kit.
  • cells containing the receptor protein of the present invention are cultured on a multiwell plate or the like. Before performing screening, replace the medium with fresh medium or an appropriate buffer that is not toxic to cells. After incubating for a certain period of time, extract cells or collect the supernatant, and quantitate the resulting product according to each method. If the production of a substance (for example, arachidonic acid) as an indicator of cell stimulating activity is difficult due to a degrading enzyme contained in a cell, the assay may be performed by adding an inhibitor against the degrading enzyme. Good. In addition, activities such as inhibition of cAMP production can be detected as production inhibitory effects on cells whose basic production has been increased with forskolin or the like.
  • a substance for example, arachidonic acid
  • cells expressing the appropriate receptor protein of the present invention are required.
  • the cells expressing the receptor protein of the present invention a cell line having a natural receptor protein, a cell line expressing the above-mentioned recombinant receptor protein and the like are preferable.
  • test compounds for example, peptides, proteins, non-peptidic compounds, synthetic compounds, fermentation products, cell extracts, plant extracts, animal tissue extracts, etc. are used, and these compounds are novel compounds. Or a known compound.
  • a screening kit for a compound or a salt thereof that changes the binding property between a ligand protein and the receptor protein of the present invention comprises a receptor protein of the present invention, a cell containing the receptor protein of the present invention, or a receptor protein of the present invention. And those containing the membrane fraction of the containing cells.
  • screening kit of the present invention examples include the following. 1. Screening reagent
  • the C HO cells expressing the receptor protein of the present invention 1 2-well plates and passaged 5 X 1 0 5 or Z holes, 3 7 ° C, 5% C 0 2, 9 5% air for 2 days Culture Things.
  • the ligand protein is dissolved in PBS containing 0.1% ⁇ serum albumin (manufactured by Sigma) to become ImM, and stored at -20 ° C.
  • the CHO cells expressing the receptor protein of the present invention cultured on a 12-well tissue culture plate were washed twice with 1 ml of measurement buffer f at night, and then 490 1 measurement buffer was added to each well. Add.
  • the compound or a salt thereof obtained by using the screening method or the screening kit of the present invention comprises a ligand protein and a receptor protein of the present invention. And a compound having an action of changing the binding to a compound.
  • cell stimulating activity for example, arachidonic acid release, acetylcholine release, intracellular Ca 2
  • + Release intracellular cAMP production, intracellular cGMP production, inositol phosphate production, cell membrane potential fluctuation, intracellular protein phosphorylation, activation of c-fos, decrease of pH, etc.
  • a compound having an inhibitory activity, etc. (a so-called agonist against the receptor protein of the present invention)
  • Examples of the compound include a peptide, a protein, a non-peptidic compound, a synthetic compound, a fermentation product, and the like, and these compounds may be novel compounds or known compounds.
  • the agonist against the receptor protein of the present invention has the same activity as the physiological activity of the ligand protein for the receptor protein of the present invention, it is safe and low toxic according to the ligand activity.
  • any cancer for example, lung cancer, stomach cancer, liver cancer, ⁇ cancer, colorectal cancer, rectum cancer, colon cancer, prostate cancer, It is useful for the prevention or treatment of ovarian cancer, uterine face cancer, breast cancer, etc.).
  • the agonist against the receptor protein of the present invention since the agonist against the receptor protein of the present invention has a placental function regulating action, it prevents choriocarcinoma, hydatidiform mole, invasive mole, miscarriage, fetal growth deficiency, abnormal glucose metabolism, abnormal lipid metabolism, or induced labor. Or useful for therapeutic drugs.
  • the antagonist against the receptor protein of the present invention can suppress the physiological activity of the ligand protein for the receptor protein of the present invention.
  • Useful as The compound that decreases the binding force between the ligand protein and the receptor protein of the present invention is useful as a safe and low-toxic drug for reducing the physiological activity of the ligand protein for the receptor protein of the present invention. is there.
  • the compound or a salt thereof can be used as the above-mentioned pharmaceutical composition, it can be carried out according to a conventional method.
  • tablets, capsules, elixirs, microcapsules, sterile solutions, suspensions, and the like can be prepared in the same manner as the above-mentioned drug containing the DNA of the present invention.
  • the preparations obtained in this way are safe and have low toxicity, so they can be administered to mammals (eg, humans, rats, mice, puppies, sheep, pigs, puppies, cats, dogs, monkeys, etc.). can do.
  • mammals eg, humans, rats, mice, puppies, sheep, pigs, puppies, cats, dogs, monkeys, etc.
  • the dose of the compound or a salt thereof (in the case of agonist) varies depending on the administration subject, target organ, symptom, administration method, and the like.
  • oral administration in general, for example, cancer patients (6O (as kg) from about 0.1 to: L0 Omg, preferably about 1.0 to 5 Omg, more preferably about 1.0 to 20 mg per day.
  • parenteral administration the single dose varies depending on the administration target, target organ, symptoms, administration method, etc.
  • an injection it is usually, for example, a cancer patient (as 6 Okg)
  • a cancer patient as 6 Okg
  • the dose can be administered in terms of 60 kg.
  • a preventive and / or therapeutic agent for various diseases containing a compound (agonist, antagonist) that alters the binding between the ligand protein and the receptor protein of the present invention.
  • the receptor protein of the present invention has a cancer metastasis inhibitory activity, it can be used for any cancer (for example, lung cancer, stomach cancer, liver cancer, knee cancer, colon cancer, rectal cancer, colon cancer, prostate cancer, ovarian cancer, It is useful as a prophylactic or therapeutic drug for child cancer (face cancer, breast cancer, etc.).
  • the receptor protein of the present invention since the receptor protein of the present invention has a placental function regulating action, it prevents or treats choriocarcinoma, hydatidiform mole, invasive mole, miscarriage, fetal growth failure, glucose metabolism abnormality, lipid metabolism abnormality or induction of parturition. Useful for medicine. Therefore, compounds that alter the binding between the ligand protein and the receptor protein of the present invention (agonists, angelic gonists) are dysfunctional or insufficient or excessive in the ligand protein. It can be used as a prophylactic and / or therapeutic agent for related diseases.
  • the compound When used as a preventive and / or therapeutic agent for a disease associated with dysfunction or deficiency or excess of a ligand protein, it can be formulated according to a conventional method.
  • the compound can be used as a sugar-coated tablet, capsule, elixir, microcapsule or the like as needed, orally, or aseptic solution with water or another pharmaceutically acceptable liquid. It can be used parenterally or in the form of injections such as suspensions.
  • the compound is mixed with known physiologically acceptable carriers, flavoring agents, 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 The amount of the active ingredient in these preparations is such that an appropriate dose in the specified range can be obtained.
  • Excipients 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 And bulking agents such as magnesium stearate, sweeteners such as sucrose, lactose or saccharin, and flavoring agents such as peppermint, cocoa oil or cellulose.
  • binders such as gelatin, corn starch, tragacanth, gum arabic
  • excipients such as crystalline cellulose, corn starch, gelatin, alginic acid And bulking 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.
  • Sterile compositions for injection can be formulated according to conventional methods, such as by 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. Wear.
  • vehicles such as water for injection, and naturally-occurring vegetable oils such as sesame oil and coconut oil.
  • aqueous solution for injection for example, physiological saline, isotonic solution containing glucose and other adjuvants (for example, D-sorbitol, D-mannitol, sodium chloride, etc.) and the like are used.
  • Agents for example, alcohols (eg, ethanol), polyalcohols (eg, propylene glycol, polyethylene glycol), nonionic surfactants (eg, polysorbate 80 (TM), HCO-50 ) May be used together.
  • oily liquid for example, sesame oil, soybean oil and the like are used, and may be used in combination with solubilizers such as benzyl benzoate and benzyl alcohol.
  • solubilizers such as benzyl benzoate and benzyl alcohol.
  • the prophylactic / therapeutic agent include a buffer (for example, a phosphate buffer and a sodium acetate buffer), a soothing agent (for example, benzalkonium chloride, procaine hydrochloride, etc.), a stabilizer (for example, human serum It may be blended with albumin, polyethylene glycol, etc.), preservatives (eg, benzyl alcohol, phenol, etc.), antioxidants, etc.
  • the prepared injection solution is usually filled in a suitable ampoule.
  • the preparations obtained in this way are safe and have low toxicity, so they can be administered to mammals (eg, humans, rats, mice, puppies, sheep, stags, puppies, cats, dogs, monkeys, etc.) can do
  • the dose of the compound or a salt thereof varies depending on the administration subject, target organ, symptom, administration method, and the like.
  • the daily dose is generally one day.
  • the single dose varies depending on the administration target, target organ, symptoms, administration method, and the like. It is convenient to administer about 0.01 to 3 Omg per day, preferably about 0.1 to 20 mg, more preferably about 0.1 to 1 Omg by intravenous injection.
  • the dose can be administered in terms of 6 O kg.
  • the present invention provides, for example, U) a method wherein a test solution and a labeled receptor protein are allowed to competitively react with an antibody against the receptor protein of the present invention, and the labeled receptor protein bound to the antibody is reacted.
  • a method for quantifying the receptor protein of the present invention in a test solution which comprises measuring a protein ratio;
  • one antibody is an antibody that recognizes the N-terminal of the receptor protein of the present invention and the other antibody is an antibody that reacts with the C-terminal of the receptor protein of the present invention.
  • the receptor protein of the present invention can be measured using a monoclonal antibody against the receptor protein of the present invention (hereinafter sometimes referred to as a monoclonal antibody of the present invention), and detection by tissue staining or the like can be performed. You can do it.
  • the antibody molecule itself may be used, or F (ab ') 2 , Fab', or Fab fraction of the antibody molecule may be used.
  • the measurement method using an antibody against the receptor protein 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 an antigen (for example, the amount of a receptor protein) in a test solution.
  • any measurement method may be used as long as the amount of the body is detected by chemical or physical means and calculated from a standard curve prepared using a standard solution containing a known amount of the antigen.
  • nephrometry, a competition method, an immunometric method, and a sandwich method are suitably used, but in terms of sensitivity and specificity, it is particularly preferable to use a sandwich method described later.
  • a labeling agent used in a measuring method using a labeling substance for example, a radioisotope, an enzyme, a fluorescent substance, a luminescent substance and the like are used. Radioisotopes, if example embodiment, [1 2 5 I], [1 3 1 I], [3 H], and [1 4 C] used.
  • the enzyme is preferably a stable enzyme having a large specific activity. For example, / 3-galactosidase; 3-dalcosidase, alkaline phosphatase, peroxidase, and lignoic acid dehydrogenase are used.
  • the fluorescent substance for example, fluorescein, fluorescein isothiosinate and the like are used.
  • the 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.
  • insolubilization of the antigen or antibody physical adsorption may be used, and the chemical bond usually used to insolubilize and immobilize proteins or enzymes is used. May be used.
  • carrier for example, insoluble polysaccharides such as agarose, dextran, and cell mouth, synthetic resins such as polystyrene, polyacrylamide, and silicon, and glass are used.
  • the test solution is reacted with the insolubilized monoclonal antibody of the present invention (primary reaction), and further reacted with the labeled monoclonal antibody of the present invention (secondary reaction).
  • primary reaction the insolubilized monoclonal antibody of the present invention
  • secondary reaction the 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 is not necessarily one kind, and a mixture of two or more kinds of antibodies is used for the purpose of improving measurement sensitivity and the like. May be used.
  • the monoclonal antibody of the present invention used in the primary reaction and the secondary reaction is preferably an antibody having a different binding site to the receptor protein. 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 receptor protein, the antibody used in the primary reaction is Preferably, an antibody that recognizes other than the C-terminal, for example, the N-terminal, is used.
  • the monoclonal antibody against the receptor protein of the present invention can be used in a measurement system other than the San Deutsch method, for example, a competition method, an immunometric method, or a nephrometry.
  • a competition method after the antigen in the test solution and the labeled antigen are allowed to react competitively with the antibody, the unreacted labeled antigen, (F), and the labeled antigen (B) bound to the antibody are compared with each other. After separation (B / F separation), the amount of B or F label is measured, and the amount of antigen in the test solution is quantified.
  • a soluble antibody is used as an antibody
  • B / F 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
  • a solid-phase method using a soluble antibody as the first antibody and using a solid-phased antibody as the second antibody is used.
  • a fixed amount of labeled antigen is After a competitive reaction with the body, the solid phase and the liquid phase are separated, or the antigen in the test solution is allowed to react with an excessive amount of the labeled antibody, and then the immobilized antigen is added and unreacted After binding the labeled antibody to the solid phase, 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.
  • nephelometry the amount of insoluble sediment generated as a result of an antigen-antibody reaction in a gel or in a solution is measured. Even when the amount of antigen in the test solution is small and only a small amount of precipitate is obtained, laser nephrometry utilizing laser scattering is preferably used.
  • the receptor protein of the present invention can be quantified with high sensitivity by using the antibody of the present invention.
  • each of the proteins associated with dysfunction of the receptor protein of the present invention can be obtained. Diagnosis of seed diseases can be made.
  • the antibody against the receptor protein of the present invention can be used for specifically detecting the receptor protein of the present invention present in a subject such as a body fluid or a tissue.
  • preparation of an antibody column used for purifying the receptor protein of the present invention, detection of the receptor protein of the present invention in each fraction at the time of purification, and receptor protein of the present invention in test cells It can be used for analyzing the behavior of the robot.
  • the neutralizing activity of the antibody against the receptor protein of the present invention against the receptor protein means, in other words, an activity of inactivating the signaling function involved in the receptor protein. Therefore, when the antibody has a neutralizing activity, signal transduction involving the receptor protein, for example, cell stimulating activity via the receptor protein (eg, arachidonic acid release, acetylcholine release, intracellular Ca) Promotes 2+ release, intracellular cAMP production, intracellular cGMP production, inositol phosphate production, cell membrane potential fluctuation, intracellular protein phosphorylation, activation of C_fOS, decrease in pH, etc. Activity or inhibitory activity) can be inactivated. Therefore, it can be used for prevention and / or treatment of diseases caused by overexpression of the receptor protein and the like.
  • cell stimulating activity via the receptor protein eg, arachidonic acid release, acetylcholine release, intracellular Ca
  • promotes 2+ release intracellular cAMP production, intracellular cGMP production, inosito
  • a transgenic animal expressing the receptor protein of the present invention can be created.
  • animals include mammals (for example, rats, mice, rats, sheep, bush, sea lions, cats, dogs, monkeys, and the like), with mice being particularly preferred.
  • the DNA of the present invention In transferring the DNA of the present invention to a target animal, it is generally advantageous to use the DNA as a gene construct linked downstream of a promoter capable of being expressed in animal cells.
  • a gene construct linked to downstream of various promoters capable of expressing the DNA of the present invention derived from an animal having high homology to animal cells in an animal cell may be used, for example, To mouse fertilized egg By microinjection, a DNA-transferred animal that highly produces the receptor protein of the present invention can be produced.
  • a ubiquitous expression promoter such as a virus derived from virus or meta-mouth thionein may be used, and preferably, an NGF gene promoter specifically expressed in the brain, a genolase gene promoter, etc. Used.
  • the presence of the receptor protein of the present invention in the germinal cells of the animal produced after the transfer of DNA means that the offspring of the animal produced have the receptor protein of the present invention in all of the germ cells and somatic cells.
  • the offspring of such animals that have inherited the gene will have the receptor protein of the invention in all of their germinal and somatic cells.
  • the DNA-transferred animal of the present invention After confirming that the DNA-transferred animal of the present invention stably retains the gene by mating, it can be reared in a normal breeding environment as the DNA-bearing animal. Furthermore, by crossing male and female animals having the target DNA, homozygous animals having the transgene on both homologous chromosomes are obtained, and by crossing the male and female animals, all the offspring will have the DNA Breeding to have The animal to which the DNA of the present invention has been transferred has high expression of the receptor protein of the present invention, and thus is useful as an animal for screening an agonist or angonist against the receptor protein of the present invention.
  • the transgenic animals of the present invention can also be used as a source of cells for tissue culture.
  • the present invention can be performed by directly analyzing DNA or RNA in the tissue of the DNA-transferred mouse of the present invention, or by analyzing the tissue in which the receptor protein of the present invention expressed by a gene is present. Can be analyzed for receptor proteins.
  • the cells of a tissue having the receptor protein of the present invention are cultured by standard tissue culture techniques and used to study the function of cells from generally difficult-to-cultivate tissues such as those derived from brain and peripheral tissues. can do.
  • a drug that enhances the function of various tissues can be selected.
  • the receptor protein of the present invention can be isolated and purified therefrom. (10) Knockout animal
  • the present invention provides a non-human mammalian embryonic stem cell in which the DNA of the present invention has been inactivated, and a non-human mammal deficient in expression of the DNA of the present invention.
  • the DNA is inactivated by introducing a reporter gene (eg, a jS-galactosidase gene derived from Escherichia coli), and the reporter gene can be expressed under the control of the DNA promoter of the present invention.
  • a reporter gene eg, a jS-galactosidase gene derived from Escherichia coli
  • a non-human mammalian embryonic stem cell in which the DNA of the present invention has been inactivated is an artificially mutated DNA of the present invention possessed by the non-human mammal, which suppresses the expression of DNA, or By substantially losing the activity of the polypeptide of the present invention encoded by the DNA, the DNA has substantially no ability to express the polypeptide of the present invention (hereinafter referred to as the knockout DNA of the present invention).
  • This may be referred to as a "human embryonic stem cell” (hereinafter abbreviated as ES cell).
  • non-human mammal the same one as described above is used.
  • Methods for artificially mutating the DNA of the present invention include, for example, genetic engineering
  • the method can be carried out by partially or entirely deleting the DNA sequence, or inserting or substituting another DNA.
  • the knockout DNA of the present invention may be prepared by, for example, shifting the codon reading frame or disrupting the function of the promoter or exon by these mutations.
  • non-human mammalian embryonic stem cells in which the DNA of the present invention 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 lacZ (jQ-galactosidase gene), cat ( DNA that disrupts the function of exons by inserting a repo allele gene (e.g., oral ramphenico-l-acetyltransferase gene) or terminates gene transcription in the intron between exons.
  • a drug resistance gene represented by a neomycin resistance gene, a hygromycin resistance gene, or lacZ (jQ-galactosidase gene), cat ( DNA that disrupts the function of exons by inserting a repo allele gene (e.g., oral ramphenico-l-acetyltransferase gene) or terminates gene transcription in
  • a DNA chain having a DNA sequence constructed so as to disrupt the gene (hereinafter abbreviated as a targeting vector) is introduced into the chromosome of the animal by, for example, a homologous recombination method, and the obtained ES cells are used in the present invention.
  • the primers were used for Southern hybridization analysis using the DNA sequence on or near the DNA as a probe, or for the DNA sequence on the evening-getting vector and the DNA sequence in the neighboring region other than the DNA of the present invention used for producing the evening-getting vector.
  • the knockout ES cells of the present invention can be obtained by analyzing by the PCR method described above.
  • ES cells from which the DNA of the present invention is inactivated by the homologous recombination method or the like for example, those already established as described above may be used, or the ES cells may be obtained by the known Evans and Kaufman method. A newly established one may be used.
  • mouse ES cells currently, 129 ES cells are generally used, but the immunological background is not clear, so a pure line of immunogens that can be used instead
  • BDFi mice C 57 BLZ6 Established using F with DBAZ2 It can be used favorably.
  • BDF 1 mice have the advantage of high number of eggs collected and robust eggs, and also have C57BL / 6 mice as their background. Then, it can be advantageously used in that the genetic background can be replaced by C57BL / 6 mice by backcrossing with C57BL / 6 mice.
  • 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 early stage can be performed by discriminating between males and females, and the early stages of culture can be greatly reduced by enabling the selection of male cells at an early stage.
  • Embryonic stem cell lines obtained in this way usually have very good proliferative properties, but they must be carefully subcultured because they tend to lose their ability to generate individuals.
  • a suitable feeder cell such as STO fibroblasts
  • a carbon dioxide incubator preferably 5% carbon dioxide, 95% air or Incubate at about 37 ° C in 5% oxygen, 5% carbon dioxide, 90% air, etc. at the time of subculture.
  • trypsin / EDTA solution usually 0.001-0.
  • a single cell is obtained by treatment with 5% trypsin / 0.1-5 mM EDTA (preferably about 0.1% trypsin / ImM EDTA), and the cells are seeded on a freshly prepared feeder cell. Such subculture is usually performed every 1 to 3 days. At this time, it is desirable to observe the cells, and if morphologically abnormal cells are found, discard the cultured cells.
  • ES cells can be cultured in monolayers at 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
  • the non-human mammal deficient in DNA expression of the present invention can be distinguished from a normal animal by measuring the mRNA amount of the animal using a known method and indirectly comparing the expression levels. It is.
  • non-human mammal those similar to the aforementioned can be used.
  • the non-human mammal deficient in expression of the DNA of the present invention can be obtained, for example, by introducing the targeting vector prepared as described above into a mouse embryonic stem cell or a mouse egg cell, and introducing the targeting vector into the DNA of the present invention.
  • Knockout of the DNA of the present invention by homologous recombination in which the inactivated DNA sequence replaces the DNA of the present invention on the chromosome of mouse embryonic stem cells or mouse egg cells by gene homologous recombination. Can be.
  • the cells in which the DNA of the present invention has been knocked out are used for Southern hybridization analysis or evening using a DNA sequence on or near the DNA of the present invention as a probe, and a DNA sequence on a targeting vector. And the DNA sequence of a neighboring region other than the DNA of the present invention derived from the mouse as a primer. It can be determined by analysis by the PCR method.
  • the cell line in which the DNA of the present invention has been inactivated is cloned by homologous gene recombination, and the cell line is cloned at an appropriate time, for example, at the 8-cell stage.
  • the chimeric embryo is injected into a human mammalian embryo or blastocyst, and the resulting chimeric embryo is transplanted into the uterus of the pseudo-pregnant non-human mammal.
  • the produced animal 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. Can be obtained by, for example, selecting an individual composed of cells having the DNA locus of the present invention to which the DNA has been added by judging coat color or the like.
  • the individual thus obtained is usually an individual having a heterozygous expression of the polypeptide of the present invention, which is crossed with an individual having a heterozygous expression of the polypeptide of the present invention.
  • An individual having a deficiency in homoexpression of the peptide can be obtained.
  • a transgenic non-human mammal having a chromosome into which the evening-getting vector has been introduced can be obtained by injecting a DNA solution into the nucleus of an egg cell by a microinjection method.
  • transgenic non-human mammals they can be obtained by selecting those having a mutation in the DNA locus of the present invention by homologous recombination of the gene.
  • the individual in which the DNA of the present invention has been knocked out can be bred in an ordinary breeding environment after confirming that the DNA has been knocked out in the animal individual obtained by mating. .
  • 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 cell in which the DNA of the present invention has been inactivated is the DNA of the present invention. It is very useful for creating non-expressed non-human mammals.
  • non-human mammal deficient in expression of the DNA of the present invention lacks various biological activities that can be induced by the polypeptide of the present invention. It can be a model for the causal disease, and is useful for investigating the cause of these diseases and studying treatment methods.
  • the non-human mammal deficient in expression of the DNA of the present invention can be used for screening for a compound having a therapeutic / preventive effect against diseases caused by the deficiency or damage of the DNA of the present invention.
  • the present invention is characterized in that a test compound is administered to a non-human mammal deficient in expression of the DNA of the present invention, and changes in the animal are observed and measured.
  • a method for screening a compound or a salt thereof, which has a therapeutic or preventive effect on a disease caused by the disease is provided.
  • Examples of the non-human mammal deficient in DNA expression of the present invention used in the screening method include the same as described above.
  • Test compounds include, for example, peptides, proteins, non-peptidic compounds, synthetic compounds, fermentation products, cell extracts, plant extracts, animal tissue extracts, and plasma.These compounds are novel compounds. Or a known compound.
  • a non-human mammal deficient in expression of the DNA of the present invention is treated with a test compound, compared with a non-treated control animal, and changes in organs, tissues, disease symptoms, etc. of the animal are used as indices.
  • the test compound can be tested for its therapeutic and prophylactic effects.
  • 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.
  • central diseases eg, Alzheimer's disease, dementia, eating disorders, etc.
  • endocrine Diseases eg, hypertension, gonad dysfunction, thyroid dysfunction, pituitary dysfunction, etc.
  • metabolic diseases eg, diabetes, lipid metabolism, hyperlipidemia, etc.
  • cancer eg, non-small cell lung cancer, ovary Cancer
  • prostate cancer stomach cancer, bladder cancer, breast cancer, cervical cancer, colon cancer, rectal cancer, etc.
  • the animals are subjected to glucose tolerance treatment, the test compound is administered before or after the glucose tolerance treatment, and the blood glucose level and weight change of the animals are measured over time.
  • the cancer metastasis of the test animal is reduced by about 10% or more, preferably about 30% or more, more preferably about 50% or more.
  • the test compound can be selected as a compound having a preventive / therapeutic effect on the above-mentioned diseases.
  • the compound obtained using the screening method is a compound selected from the test compounds described above, and has a preventive / therapeutic effect against a disease caused by deficiency or damage of the polypeptide of the present invention. It can be used as a medicament such as a safe and low-toxic preventive / therapeutic agent for the disease. Further, a compound derived from the compound obtained by the above screening can be used in the same manner.
  • the compound obtained by the screening method may form a salt.
  • the salt of the compound include physiologically acceptable acids (eg, inorganic acids, organic acids, etc.) and bases (eg, alkali metals, etc.). And the like, and a physiologically acceptable acid addition salt is 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, citric acid, malic acid, oxalic acid, benzoic acid, methanesulfonic acid, benzenesulfonic acid, etc.).
  • inorganic acids eg, hydrochloric acid, phosphoric acid, hydrobromic acid, sulfuric acid, etc.
  • organic acids eg, acetic acid, formic acid, propionic acid, fumaric acid, maleic acid
  • Succinic acid tartaric acid, citric acid, malic acid, oxalic acid, benzoic acid, methanesulfonic acid, benzenesulfonic acid, etc.
  • a drug containing the compound or a salt thereof obtained by the screening method can be produced in the same manner as the drug containing the polypeptide of the present invention described above.
  • the preparations obtained in this way are safe and low toxic, and thus can be used, for example, in humans or non-human mammals (eg, rats, mice, guinea pigs, egrets, sheep, sheep, horses, horses, cats, Dogs, monkeys, etc.).
  • the dose of the compound or a salt thereof varies depending on the target disease, the subject of administration, the administration route, and the like. About 0.1 to 100 mg, preferably about 1.0 to 0.50 mg, more preferably about 1.0 to 20 mg of the compound per day.
  • the single dose of the compound will vary depending on the administration target, target disease, etc. In this case, about 0.01 to 30 mg, preferably about 0.1 to 2 Omg, more preferably about 0.1 to 10 mg of the compound per day is administered by intravenous injection. is there. In the case of other animals, the dose can be administered in terms of 60 kg.
  • the present invention provides a test compound administered to a non-human mammal deficient in expression of the DNA of the present invention, and detects or enhances the expression of a reporter gene.
  • the non-human mammal deficient in expressing DNA of the present invention may be a non-human mammal deficient in expressing DNA of the present invention in which the DNA of the present invention introduces a repo overnight gene. And those in which the reporter gene can be expressed under the control of a promoter for the DNA of the present invention.
  • test compound examples include the same compounds as described above.
  • the reporter gene the same one as described above is used, and a / 3-galactosidase gene (1 acZ), a soluble alkaline phosphatase gene or a luciferase gene is preferable.
  • the reporter gene is Trace expression of encoded material
  • the activity of the promoter can be detected.
  • a tissue that originally expresses the polypeptide of the present invention may J8-galactosidase is expressed instead of the polypeptide of the present invention. Therefore, for example, by staining with a reagent that is a base of / 3-galactosidase, such as 5-bromo-4-chloro-3-indolyl-j3-galactopyranoside (X-gal), The state of expression of the polypeptide of the present invention in an animal body can be easily observed.
  • the polypeptide-deficient mouse of the present invention or a tissue section thereof is fixed with dartalaldehyde or the like, washed with phosphate buffered saline (PBS), and then stained with X-ga1 at room temperature.
  • PBS phosphate buffered saline
  • the tissue sample is washed with an ImM EDTA / PBS solution to stop the] -galactosidase reaction, and color development may be observed.
  • mRNA encoding 1acZ may be detected according to a conventional method.
  • the compound or a salt thereof obtained by the above screening method is a compound selected from the test compounds described above, and is a compound that promotes or inhibits the promoter activity of the 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). Salts with, for example, are preferred, and physiologically acceptable acid addition salts are particularly preferred.
  • Such salts include, for example, 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, citric acid, malic acid, oxalic acid, benzoic acid, methanesulfonic acid, benzenesulfonic acid, etc.) And the like.
  • 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
  • the compound or its salt that promotes the promoter activity for the DNA of the present invention can promote the expression of the polypeptide of the present invention and promote the function of the polypeptide, and thus, for example, central diseases (for example, Alzheimer's disease) Diseases, dementia, eating disorders, etc.), endocrine disorders (eg, hypertension, gonad dysfunction, thyroid dysfunction, pituitary dysfunction, etc.), metabolic disorders (eg, diabetes, dyslipidemia, hyperlipidemia) Disease), cancer (for example, it is useful as a medicine for non-small cell lung cancer, ovarian cancer, prostate cancer, stomach cancer, bladder cancer, breast cancer, cervical cancer, colon cancer, rectal cancer, etc.).
  • central diseases for example, Alzheimer's disease
  • endocrine disorders eg, hypertension, gonad dysfunction, thyroid dysfunction, pituitary dysfunction, etc.
  • metabolic disorders eg, diabetes, dyslipidemia, hyperlipidemia
  • cancer for example, it is useful as a medicine for non-small cell lung cancer, ovarian cancer, prostate
  • the compound of the present invention or a salt thereof that inhibits the promoter activity against DNA can inhibit the expression of the polypeptide of the present invention and inhibit the function of the polypeptide.
  • a central disease eg, Alzheimer's disease
  • endocrine disorders eg, hypertension, gonadal dysfunction, thyroid dysfunction, pituitary dysfunction, etc.
  • metabolic disorders eg, diabetes, lipid metabolism, hyperlipidemia, etc.
  • cancer eg, non-small cell lung cancer, ovarian cancer, prostate cancer, gastric cancer, bladder cancer, breast cancer, cervical cancer, colon cancer, rectal cancer, etc.
  • compounds derived from the compounds obtained by the above screening can be used in the same manner.
  • a medicament containing the compound or a salt thereof obtained by the screening method can be produced in the same manner as the above-mentioned medicament containing the polypeptide of the present invention or a salt thereof.
  • the preparations obtained in this way are safe and low toxic and can be used, for example, in humans or non-human mammals (e.g., rats, mice, guinea pigs, egrets, sheep, sheep, butter, horsetails, horses, cats, Dogs, monkeys, etc.).
  • the dose of the compound or a salt thereof varies depending on the target disease, the subject of administration, the administration route, and the like.
  • the compound of the present invention that promotes the promoter activity for DNA is orally administered, In adult (as a body weight 6 O kg) cancer patients, about 0.1 to 10 O mg, preferably about 1.0 to 50 mg, more preferably about 1.0 to about 10 mg of the compound 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 that promotes the promoter overnight activity against DNA may be used as an injection.
  • the compound When administered to an adult (as 60 kg) cancer patient in the form of the compound, the compound is administered in an amount of about 0.01 to 30 mg, preferably about 0.1 to 20 mg, more preferably about 0.1 to 30 mg per day. It is convenient to administer about 0.1 to 1 Omg by intravenous injection. In the case of other animals, the amount converted per 60 kg can be administered.
  • a compound that inhibits the promoter When administered orally, generally in adult (with a body weight of 60 kg) cancer patients, about 0.1 to 100 mg, preferably about 1.0 to 50 mg, and more preferably about 1. Administer 0-20 mg.
  • the single dose of the compound varies depending on the administration subject, target disease, and the like.
  • the compound When administered to a human (as 60 kg) cancer patient, the compound is administered in an amount of about 0.01 to 30 mg, preferably about 0.1 to 20 mg, more preferably about 0.1 to 1 Omg per day. Is conveniently administered by intravenous injection. For other animals, the dose can be administered in terms of 6 O kg.
  • the non-human mammal deficient in expression of the DNA of the present invention is extremely useful for screening a compound or a salt thereof that promotes or inhibits the activity of the promoter for the DNA of the present invention.
  • Investigating or preventing the causes of various diseases caused by insufficient expression of DNA can greatly contribute to the development of therapeutic drugs.
  • transgenic animal In addition, using a DNA containing the promoter region of the polypeptide of the present invention, genes encoding various proteins are ligated downstream thereof and injected into egg cells of an animal to produce a so-called transgenic animal (transgenic animal). ) Makes it possible to specifically synthesize the polypeptide and study its effects on living organisms. Furthermore, by binding an appropriate repo overnight gene to the above promoter portion and establishing a cell line in which this is expressed, the polypeptide of the present invention has an action of specifically promoting or suppressing the production ability of the polypeptide itself in the body. It can be used as a search system for low molecular compounds.
  • the DNA of the present invention can be used for screening a compound that changes the expression level of the receptor protein or its partial peptide of the present invention.
  • the present invention includes, for example, (i) non-human mammal's (2) blood, (2) a specific organ, (3) a tissue or cell isolated from the organ, or (ii) a transformant.
  • a method for screening for a compound that changes the expression level of the receptor protein of the present invention or its partial peptide by measuring the mRNA amount of the receptor protein of the present invention or its partial peptide is provided.
  • the measurement of the amount of mRNA of the receptor protein or its partial peptide of the present invention is specifically carried out as follows.
  • non-human mammals for example, mice, rats, rabbits, sheep, sheep, bush, horses, cats, dogs, monkeys, etc., more specifically, dementia rats, obese mice, arteriosclerosis ⁇ Drugs (eg, anti-dementia drugs, antihypertensive drugs, anti-cancer drugs, anti-obesity drugs, etc.) or physical stress (eg, flooding stress, electric shock, light / dark, low temperature, etc.)
  • Drugs eg, anti-dementia drugs, antihypertensive drugs, anti-cancer drugs, anti-obesity drugs, etc.
  • physical stress eg, flooding stress, electric shock, light / dark, low temperature, etc.
  • the mRNA of the receptor protein of the present invention or its partial peptide contained in the obtained cells can be obtained, for example, by extracting mRNA from cells or the like by a usual method, for example, by a method such as TaqMan PCR. It can be quantified by using it, and can also be analyzed by performing Northern blot by known means.
  • a transformant expressing the receptor protein of the present invention or a partial peptide thereof is prepared according to the above method, and the mRNA of the receptor protein of the present invention or the partial peptide thereof contained in the transformant is prepared. It can be quantified and analyzed in the same way.
  • Screening for a compound that changes the expression level of the receptor protein or its partial peptide of the present invention comprises:
  • test compound is administered at the same time as the target stress, and after a certain period of time after administration (30 minutes to 3 days, preferably 1 hour to 2 days, more preferably 1 hour to 24 hours), the cells Receptor protein of the present invention contained in Alternatively, it can be performed by quantifying and analyzing the mRNA amount of the partial peptide,
  • the test compound is mixed in the medium, and after culturing for a certain period of time (1 day to 7 days, preferably 1 day to 3 days, more preferably 2 days to 3 days) A day later), the amount can be determined by quantifying and analyzing the mRNA amount of the receptor protein of the present invention or its partial peptide contained in the transformant.
  • the compound or a salt thereof obtained by using the screening method of the present invention is a compound having an action of changing the expression level of the receptor protein of the present invention or a partial peptide thereof.
  • the cell stimulating activity via G protein-coupled receptor eg, arachidonic acid release, acetylcholine release, intracellular Ca 2+ release
  • the Rukoto, Ru compound der to decrease the cell-stimulating activity.
  • Examples of the compound include a peptide, a protein, a non-peptidic compound, a synthetic compound, a fermentation product, and the like, and these compounds may be novel compounds or known compounds.
  • the compound that enhances the cell stimulating activity is useful as a safe and low toxic drug for enhancing the physiological activity of the receptor protein or the like of the present invention.
  • the compound that reduces the cell stimulating activity is useful as a safe and low-toxic drug for reducing the physiological activity of the receptor protein of the present invention or the like.
  • a compound or a salt thereof obtained by using the screening method of the present invention is used as a pharmaceutical composition, it can be performed according to a conventional method.
  • tablets, capsules, elixirs, microcapsules, sterile solutions, suspensions, and the like can be prepared in the same manner as the above-mentioned pharmaceuticals containing the receptor protein of the present invention.
  • the preparations obtained in this way are safe and low toxic, for example, in humans and other mammals (eg, rats, mice, puppies, higgs, bush, puppies, cats, dogs, monkeys, etc.). Can be administered.
  • the dose of the compound or a salt thereof varies depending on the administration subject, target organ, symptoms, administration method, and the like.
  • oral administration in general, for example, in a cancer patient (as 60 kg), About 0.1 to 100 mg, preferably about 1.0 to 50 mg, more preferably about 1.0 to 2 Omg per day.
  • parenteral administration the single dose varies depending on the administration target, target organ, symptoms, administration method, etc.
  • an injection it is usually used, for example, in a cancer patient (as 6 O kg). It is convenient to administer about 0.01 to 3 Omg / day, preferably about 0.1 to 20 mg / day, more preferably about 0.1 to 1 Omg / day, by intravenous injection.
  • the dose can be administered in terms of 60 kg
  • a preventive and / or therapeutic agent for various diseases containing a compound that changes the expression level of the receptor protein or its partial peptide of the present invention
  • the receptor protein of the present invention is considered to play some important role in vivo such as central function. Therefore, the compound of the present invention that alters the expression level of the receptor protein or a partial peptide thereof can be used as an agent for preventing and / or treating a disease associated with dysfunction of the receptor protein of the present invention. .
  • the compound when used as a prophylactic and / or therapeutic agent for diseases associated with dysfunction of the receptor protein of the present invention, it can be formulated according to conventional means.
  • the compound can be used as a sugar-coated tablet, capsule, elixir, microcapsule, etc., orally, or aseptic solution with water or another pharmaceutically acceptable liquid, if necessary. It can be used parenterally or in the form of injections, such as suspensions.
  • the compound can be a known carrier physiologically recognized, It can be manufactured by mixing the compound with excipients, excipients, vehicles, preservatives, stabilizers, binders and the like in the unit dosage form generally required for the practice of the formulation. The amount of the active ingredient in these preparations is such that a suitable dosage in the specified range can be obtained.
  • Excipients 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 Swelling agents such as sucrose, lubricating agents such as magnesium stearate, sweetening agents such as sucrose, lactose or saccharine, and flavoring agents such as peppermint, cocoa oil or cherry.
  • the unit dosage form is a capsule, the above type of material can further contain a liquid carrier such as an oil or fat.
  • Sterile compositions for injection can be formulated according to standard pharmaceutical practice, such as dissolving or suspending the active substance in vehicles such as water for injection, and naturally occurring vegetable oils such as sesame oil and coconut oil. it can.
  • aqueous solution for injection for example, physiological saline, isotonic solution containing glucose and other adjuvants (eg, D-sorbitol, D-mannitol, sodium chloride, etc.) and the like are used.
  • Agents such as alcohol (eg, ethanol), polyalcohol (eg, propylene glycol, polyethylene daricol), nonionic surfactants (eg, polysorbate 80 TM, HCO-50) .
  • oily liquid for example, sesame oil, soybean oil and the like are used, and may be used in combination with solubilizers such as benzyl benzoate and benzyl alcohol.
  • prophylactic and therapeutic agents include, for example, buffers (for example, phosphate buffer and sodium acetate buffer), soothing agents (for example, benzalkonium chloride, procaine hydrochloride, etc.), stabilizers (for example, human serum Albumin, polyethylene glycol, etc.), preservatives (eg, benzyl alcohol, phenol, etc.), antioxidants and the like.
  • buffers for example, phosphate buffer and sodium acetate buffer
  • soothing agents for example, benzalkonium chloride, procaine hydrochloride, etc.
  • stabilizers for example, human serum Albumin, polyethylene glycol, etc.
  • preservatives eg, benzyl alcohol, phenol, etc.
  • antioxidants eg, benzyl alcohol, phenol, etc.
  • the prepared injection solution is usually filled in a suitable ampoule.
  • the preparations obtained in this way are safe and low toxic, for example, in humans and other mammals (eg, rats, mice, puppies, higgs
  • the dose of the compound or a salt thereof varies depending on the administration subject, target organ, symptoms, administration method, and the like. It is about 0.1-100 mg per day, preferably about 1.0-50 mg, more preferably about 1.0-20 mg. In the case of parenteral administration, the single dose varies depending on the administration target, target organ, symptoms, administration method and the like. It is convenient to administer about 0.01 to 3 Omg per day, preferably about 0.1 to 20 mg, more preferably about 0.1 to 1 Omg by intravenous injection. For other animals, the dose can be administered in terms of 60 kg
  • the antibody of the present invention can specifically recognize the receptor protein of the present invention or its partial peptide or its salt, the amount of the receptor protein of the present invention or its partial peptide in the cell membrane can be reduced. It can be used for screening compounds to be changed.
  • Non-human mammal 1) Blood, 2) Specific organs, 3) Tissues or cells isolated from the organs are destroyed, the cell membrane fraction is isolated, and the receptor of the present invention contained in the cell membrane fraction
  • the cell membrane fraction is isolated, and the receptor protein of the present invention or its partial peptide contained in the cell membrane fraction is isolated.
  • Non-human mammal blood, specific organs, and tissues isolated from organs Alternatively, after slicing the cells, etc., the immunostaining method is used to quantify the degree of staining of the receptor protein on the cell surface, thereby confirming the protein on the cell membrane.
  • the immunostaining method is used to quantify the degree of staining of the receptor protein on the cell surface, thereby confirming the protein on the cell membrane.
  • Transformants expressing the receptor protein of the present invention or a partial peptide thereof are sectioned, and then immunostaining is used to quantify the degree of staining of the receptor protein on the cell surface.
  • the present invention provides a method for screening a compound that changes the amount of the receptor protein of the present invention or its partial peptide in the cell membrane by confirming the protein on the cell membrane.
  • the quantitative determination of the receptor protein of the present invention or its partial peptide contained in the cell membrane fraction is specifically performed as follows.
  • non-human mammals eg, mice, rats, rabbits, sheep, pigs, pigs, cats, dogs, monkeys, etc., more specifically, dementia rats, obese mice, arteriosclerotic rabbits
  • Drugs eg, anti-dementia drugs, antihypertensive drugs, anti-cancer drugs, anti-obesity drugs, etc.
  • physical stress eg, waterlogging stress, electric shock, light / dark, low temperature, etc.
  • blood or a specific organ eg, brain, lung, colon, etc.
  • tissue or cells isolated from the organ is obtained.
  • the obtained organ, tissue or cell is suspended in, for example, an appropriate buffer (for example, Tris-HCl buffer, phosphate buffer, Hess buffer, etc.) to destroy the organ, tissue or cell.
  • an appropriate buffer for example, Tris-HCl buffer, phosphate buffer, Hess buffer, etc.
  • a cell membrane fraction is obtained by using a surfactant (for example, Triton XI 00 TM, Tween 20 TM, etc.), and further using techniques such as centrifugation, filtration, and column fractionation.
  • a surfactant for example, Triton XI 00 TM, Tween 20 TM, etc.
  • the cell membrane fraction refers to a cell membrane-rich fraction obtained by disrupting cells and then obtained by a known method.
  • Cells can be disrupted by crushing the cells with a Poer-Elvehjem homogenizer, crushing with a Warlinda blender Polytron (manufactured by Kinematica), crushing by ultrasonic waves, or pressing the cells while applying pressure with a French press. Crushing by ejecting from a thin nozzle is exemplified.
  • Cell membrane fractionation is performed by centrifugal separation, such as differential centrifugation or density gradient centrifugation. Is mainly used.
  • the cell lysate is centrifuged at a low speed (500 rpm to 300 rpm) for a short time (usually about 1 to 10 minutes), and the supernatant is further spun at a higher speed (150 rpm to 300 rpm).
  • the mixture is centrifuged usually at 0,000 rpm for 30 minutes to 2 hours, and the resulting precipitate is used as a membrane fraction.
  • the membrane fraction contains a large amount of expressed receptor proteins and membrane components such as cell-derived phospholipids and membrane proteins.
  • the receptor protein of the present invention or its partial peptide contained in the cell membrane fraction can be quantified by, for example, a sandwich immunoassay using the antibody of the present invention, Western blot analysis, or the like.
  • Such a sandwich immunoassay can be performed in the same manner as described above, and the Western blot can be performed by known means.
  • Screening for a compound that alters the amount of the receptor protein of the present invention or its partial peptide in the cell membrane is performed by:
  • test compound is administered at the same time as the target stress, and after a certain period of time after the administration (30 minutes to 3 days, preferably 1 hour to 2 days, more preferably 1 hour to 24 hours), the cell membrane
  • test compound is mixed into the medium, and after a certain period of culture (after 1 day to 7 days, preferably after 1 day to 3 days, more preferably
  • the obtained organ, tissue or cell is cut into a tissue section according to a conventional method, and immunostaining is performed using the antibody of the present invention.
  • immunostaining is performed using the antibody of the present invention.
  • the compound or a salt thereof obtained by using the screening method of the present invention is a compound having an action of changing the amount of the receptor protein of the present invention or a partial peptide thereof in a cell membrane.
  • the cell stimulating activity via G protein-coupled receptor eg, arachidonic acid release, acetylcholine release, intracellular Ca 2 + Release, intracellular cAMP production, intracellular cAMP suppression, intracellular cGMP production, inositol phosphate production, cell membrane potential fluctuation, intracellular protein phosphorylation, c-fos activation, a compound that enhances the activity of promoting or suppressing pH reduction, etc.
  • the receptor protein of the present invention in the cell membrane or By reducing the amount of a partial peptide thereof, a compound that decrease the cell-stimulating activity.
  • Examples of the compound include a peptide, a protein, a non-peptidic compound, a synthetic compound, and a fermentation product. These compounds may be a novel compound or a known compound.
  • the compound that enhances the cell stimulating activity may be a compound such as the receptor protein of the present invention. It is useful as a safe and low toxic drug for enhancing physical activity.
  • the compound that attenuates the cell stimulating activity is useful as a safe and low toxic drug for decreasing the physiological activity of the receptor protein of the present invention.
  • a compound or a salt thereof obtained by using the screening method of the present invention is used as a pharmaceutical composition, it can be carried out according to a conventional method.
  • tablets, capsules, elixirs, microcapsules, sterile solutions, suspensions, and the like can be prepared in the same manner as the above-mentioned drug containing the receptor protein of the present invention.
  • the preparations obtained in this way are safe and low toxic, for example, in humans and other mammals (eg, rats, mice, puppies, higgs, bush, puppies, cats, dogs, monkeys, etc.). Can be administered.
  • the dose of the compound or a salt thereof varies depending on the administration subject, target organ, symptom, administration method, and the like.
  • the daily dose is generally one day.
  • the single dose varies depending on the administration target, target organ, symptoms, administration method, etc.
  • it is usually used, for example, in cancer patients (as 6 O kg).
  • the amount converted per 60 kg can be administered.
  • a preventive and / or therapeutic agent for various diseases containing a compound that alters the amount of the receptor protein or its partial peptide of the present invention in the cell membrane The receptor protein of the present invention is, as described above, for example, It may play some important role in the body. Therefore, a compound that changes the amount of the receptor protein of the present invention or its partial peptide in the cell membrane can be used as a preventive and / or therapeutic agent for a disease associated with dysfunction of the receptor protein of the present invention.
  • the compound is used to prevent or prevent diseases associated with dysfunction of the receptor protein of the present invention.
  • it can be formulated according to conventional means.
  • the compound can be used as a sugar-coated tablet, capsule, elixir, microcapsule or the like as needed, orally, or aseptic solution with water or another pharmaceutically acceptable liquid. It can be used parenterally or in the form of injections such as suspensions.
  • the compound is mixed with known physiologically acceptable carriers, flavoring agents, 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 The amount of the active ingredient in these preparations is such that a suitable dosage in the specified range can be obtained.
  • Additives that can be incorporated into tablets, capsules, etc. include, 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 cherry.
  • the unit dosage form is a capsule, the above type of material can further contain a liquid carrier such as an oil or fat.
  • Sterile compositions for injection can be formulated according to standard pharmaceutical practice, such as dissolving or suspending the active substance in vehicles such as water for injection, and naturally occurring vegetable oils such as sesame oil and coconut oil. it can.
  • aqueous liquid for injection include physiological saline, isotonic solution containing glucose and other adjuvants (eg, D-sorbitol, D-mannitol, sodium chloride, etc.) and the like.
  • an alcohol e.g., ethanol
  • polyalcohol e.g., propylene glycol, polyethylene Dali call
  • a nonionic surfactant eg, polysorbate 8 0 TM, HCO- 5 0
  • the oily liquid for example, sesame oil, soybean oil and the like are used, and may be used in combination with solubilizers such as benzyl benzoate and benzyl alcohol.
  • prophylactic / therapeutic agents examples include, for example, buffers (eg, phosphate buffer, sodium acetate buffer), soothing agents (eg, benzalkonium chloride, procaine hydrochloride). ), Stabilizers (eg, human serum albumin, polyethylene glycol, etc.), preservatives (eg, benzyl alcohol, phenol, etc.), antioxidants and the like.
  • buffers eg, phosphate buffer, sodium acetate buffer
  • soothing agents eg, benzalkonium chloride, procaine hydrochloride
  • Stabilizers eg, human serum albumin, polyethylene glycol, etc.
  • preservatives eg, benzyl alcohol, phenol, etc.
  • antioxidants antioxidants and the like.
  • the prepared injection solution is usually filled in a suitable ampoule.
  • the preparations obtained in this way are safe and low toxic, for example, in humans and other mammals (eg, rats, mice, puppies, higgs, bush, puppies, cats
  • the dose of the compound or a salt thereof varies depending on the administration subject, target organ, symptoms, administration method, and the like.
  • About 0.1 to 10 Omg preferably about 1.0 to 50 mg, more preferably about 1.0 to 20 mg.
  • the single dose varies depending on the administration target, target organ, symptoms, administration method, etc. It is convenient to administer about 0.01 to 30 mg, preferably about 0.1 to 20 mg, more preferably about 0.1 to 1 Omg per day by intravenous injection. In the case of other animals, the dose can be administered in terms of 6 O kg.
  • TC thiazolidine-4 (R)--carboxamide group
  • Substituents, protecting groups and reagents frequently used in the present specification are represented by the following symbols.
  • FIG 2 shows the amino acid sequence of the receptor protein of the present invention.
  • FIG. 1 shows the nucleotide sequence of DNA encoding the receptor protein of the present invention.
  • 1 shows the amino acid sequence of the human ligand protein of the receptor protein of the present invention.
  • FIG. 1 shows the amino acid sequence of mouse type 1 ligand protein of the receptor protein of the present invention.
  • 1 shows the amino acid sequence of the mouse type 2 ligand protein of the receptor protein of the present invention.
  • Example 2 shows the nucleotide sequence of primer 2 used in Example 1 described later.
  • Escherichia coli DH5aI pCR-Bluntll-mOT7T175 obtained in Example 1 described below has been used since January 11, 2001, 1-1 1-1 Tsukuba-Higashi, Ibaraki Pref. 8566) was deposited with the National Institute of Advanced Industrial Science and Technology (AIST) at the Patent Organism Depositary (formerly Ministry of Economy, Trade and Industry, National Institute of Advanced Industrial Science and Technology (NIBH)) under the accession number FERM BP-7428. No. 17-85, Jusanhoncho 2-chome, Yodogawa-ku, Osaka-shi, Osaka since December 22, 2012
  • Example 1 Cloning of cDNA encoding novel G protein-coupled receptor protein from mouse whole brain and determination of nucleotide sequence
  • a mouse whole brain cDNA (CL0NTECH) was used as type I, and a PCR reaction was carried out using two primers, primer 1 (SEQ ID NO: 7) and primer 2 (SEQ ID NO: 8).
  • the composition of the reaction solution used in the reaction was as follows: the cDNA was used as a 1/10 type II, 1/50 volume of Pfu Turbo DNA Polymerase (STRATAGENE), 0.2 ⁇ M each of Primer-1 and Primer-2, dNTPs 200 M and the buffer added to the enzyme were added to make a volume of 25 II 1.
  • the PCR reaction is: 1 After 94 ° C for 2 minutes, 2 3 cycles of 94 seconds for 20 seconds, 72 ° C for 2 minutes, 3 3 cycles of 94 ° O for 20 seconds, 68 ° C for 2 minutes A cycle of 94 ° C. for 20 seconds, 62 ° C. for 20 seconds, and 68 ° C. for 1 minute and 30 seconds was repeated 38 times, and finally, an extension reaction was performed for 68.7 minutes.
  • the reaction product after the PCR reaction is subjected to Zero-blunt TOPO TA Cloning
  • the plasmid vector was subcloned into pCR—Blunt II-TOPO (Invitrogen) according to the kit (Invitrogen).
  • the transformant having the mOT7T175 sequence was designated as Escherichia coli thigh / pCR-Blunt II-mOT7T175. '' Industrial applicability
  • the G protein-coupled receptor protein of the present invention or its partial peptide or its salt, the polynucleotide encoding the receptor protein or its partial peptide are as follows: Acquisition of antibodies and antisera, 2Construction of a recombinant receptor protein expression system, 3Development of a receptor binding assay system using the same expression system and screening of drug candidate compounds, 4Structurally similar ligands ⁇ It can be used for drug design based on comparison with receptors, ⁇ ⁇ reagents for preparing probes and PCR primers in gene diagnosis, 6transgenic animals or 7gene preventive and therapeutic drugs, etc. .

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Abstract

L'invention concerne une protéine utile dans le criblage notamment d'agonistes et d'antagonistes. Elle concerne, plus spécifiquement, une protéine de récepteur d'origine murine, ou un de ses sels, un ADN codant pour cette protéine, un procédé de criblage et un nécessaire de criblage pour un composé capable d'altérer les propriétés de liaison de cette protéine à un ligand, ainsi qu'un composé, ou un sel de ce composé, obtenu par le criblage.
PCT/JP2002/001656 2001-02-26 2002-02-25 Proteine de recepteur murin de type kiss-1 et adn correspondant Ceased WO2002072816A1 (fr)

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1363655A4 (fr) * 2000-12-18 2005-04-06 Merck & Co Inc Molecules d'acides nucleiques isolees codant un recepteur humain et murin couple a la proteine g - gpr54 -, proteines codees, cellules transformees par ces molecules et utilisations correspondantes

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1997013778A1 (fr) * 1995-10-11 1997-04-17 The Penn State Research Foundation Gene suppresseur de metastases
WO2000024890A1 (fr) * 1998-10-27 2000-05-04 Takeda Chemical Industries, Ltd. Nouvellesroteines receptrices couplees aux proteines g, leurs adn et leursigands
WO2000050563A2 (fr) * 1999-02-24 2000-08-31 Merck & Co., Inc. Recepteur couple a une proteine g ressemblant aux recepteurs de galanine
WO2001075104A1 (fr) * 2000-03-30 2001-10-11 Takeda Chemical Industries, Ltd. Nouvelle proteine, adn codant pour celle-ci, et son procede de production

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1997013778A1 (fr) * 1995-10-11 1997-04-17 The Penn State Research Foundation Gene suppresseur de metastases
WO2000024890A1 (fr) * 1998-10-27 2000-05-04 Takeda Chemical Industries, Ltd. Nouvellesroteines receptrices couplees aux proteines g, leurs adn et leursigands
WO2000050563A2 (fr) * 1999-02-24 2000-08-31 Merck & Co., Inc. Recepteur couple a une proteine g ressemblant aux recepteurs de galanine
WO2001075104A1 (fr) * 2000-03-30 2001-10-11 Takeda Chemical Industries, Ltd. Nouvelle proteine, adn codant pour celle-ci, et son procede de production

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1363655A4 (fr) * 2000-12-18 2005-04-06 Merck & Co Inc Molecules d'acides nucleiques isolees codant un recepteur humain et murin couple a la proteine g - gpr54 -, proteines codees, cellules transformees par ces molecules et utilisations correspondantes

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