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WO2002050266A1 - Nouvelle proteine receptrice couplee a la proteine g et son adn - Google Patents

Nouvelle proteine receptrice couplee a la proteine g et son adn Download PDF

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Publication number
WO2002050266A1
WO2002050266A1 PCT/JP2001/011126 JP0111126W WO0250266A1 WO 2002050266 A1 WO2002050266 A1 WO 2002050266A1 JP 0111126 W JP0111126 W JP 0111126W WO 0250266 A1 WO0250266 A1 WO 0250266A1
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WO
WIPO (PCT)
Prior art keywords
protein
receptor protein
salt
present
coupled receptor
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Ceased
Application number
PCT/JP2001/011126
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English (en)
Japanese (ja)
Inventor
Natsuki Ikeda
Yasushi Shintani
Nobuyuki Miyajima
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Takeda Pharmaceutical Co Ltd
Original Assignee
Takeda Chemical Industries Ltd
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Filing date
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Priority to AU2002217455A priority Critical patent/AU2002217455A1/en
Publication of WO2002050266A1 publication Critical patent/WO2002050266A1/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • 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
    • 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
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/705—Assays involving receptors, cell surface antigens or cell surface determinants
    • G01N2333/72—Assays involving receptors, cell surface antigens or cell surface determinants for hormones
    • G01N2333/726—G protein coupled receptor, e.g. TSHR-thyrotropin-receptor, LH/hCG receptor, FSH
    • 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 mouse-derived G protein-coupled receptor protein or a salt thereof, and a DNA encoding the same.
  • G protein-coupled receptor protein 7-transmembrane receptor protein
  • G protein-coupled receptor protein is present on the surface of each functional cell in living cells and organs, and regulates the functions of those cells and organs, such as hormones, neurotransmitters, and bioactive substances. Plays a physiologically important role.
  • 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.
  • physiological functions are regulated under the control of many hormones, hormone-like substances, neurotransmitters or bioactive substances.
  • physiologically active substances are present in various parts of the body, and regulate their physiological functions through their corresponding receptor proteins.
  • hormones in the body neurotransmitters and other physiologically active substances.
  • Many of the quality structures have not yet been reported.
  • the G protein-coupled receptor searches for a new physiologically active substance (that is, a ligand) using its signal transduction action as an index. Useful for searching for strikes or angyo gonists. On the other hand, even if a physiological ligand is not found, by analyzing the physiological action of the receptor from an inactivation experiment (knockout animal) of the receptor, an agonist or an angist for the receptor can be identified. It is also possible to produce.
  • These ligands for these receptors eg, agonists and antagonists, can be expected to be used as preventive and therapeutic drugs for diseases associated with dysfunction of G protein-coupled receptors.
  • a decrease or increase in the function of the receptor in a living body based on a gene mutation of a G protein-coupled receptor may cause some disease.
  • the present invention can be applied not only to administration of angonist and agonist, but also to gene therapy by introducing the receptor gene into a living body (or a specific organ) or by introducing an antisense nucleic acid against the receptor gene.
  • the nucleotide sequence of the receptor is essential information for examining the presence or absence of a deletion or mutation in the gene, and the receptor gene is used for a disease associated with dysfunction of the receptor. It can also be applied to preventive Z treatments and diagnostics.
  • the present invention provides a novel G protein-coupled receptor protein useful as described above.
  • the G protein-coupled receptor protein or its partial peptide or a salt thereof contains a novel G protein-coupled receptor protein or its partial peptide or a salt thereof, and a polynucleotide (DNA, RNA or a derivative thereof) encoding the G protein-coupled receptor protein or its partial peptide.
  • the present inventors have isolated cDNA encoding a novel mouse-derived G protein-coupled receptor overnight protein and succeeded in analyzing the entire nucleotide sequence thereof.
  • this nucleotide sequence was translated into an amino acid sequence, the first to seventh transmembrane regions were confirmed on the hydrophobicity plot, and the protein encoded by these cDNAs was conjugated to seven transmembrane G-proteins. It was confirmed that it was a type receptor protein.
  • the present inventors have further studied based on these findings, and as a result, have completed the present invention. That is, the present invention
  • G protein-coupled receptor protein or a salt thereof which comprises the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 1;
  • the antibody according to (10) which is a neutralizing antibody that inactivates signal transduction of the G protein-coupled receptor 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 described in (1) or the partial peptide described in (3) or a salt thereof.
  • a pharmaceutical comprising the ligand of G protein-coupled receptor described in (14) above,
  • a ligand characterized by using the G protein-coupled receptor protein described in (1) above or the partial peptide described in (3) or a salt thereof, and a G protein-conjugated protein described in (1) above A method for screening a compound or a salt thereof that alters the binding to a receptor protein or a salt thereof,
  • a ligand comprising the G protein-coupled receptor protein described in (1) above or the partial peptide or salt thereof described in (3) above, and a G protein-coupled receptor protein described in (1) above Or a compound for screening a compound or a salt thereof that changes the binding property to a salt thereof,
  • a pharmaceutical comprising a compound that changes the binding property of
  • (22) a polynucleotide comprising a nucleotide sequence complementary to the polynucleotide of (4) or a part thereof,
  • the protein comprises: (1) an amino acid sequence represented by SEQ ID NO: 1; one or more amino acids in the amino acid sequence represented by SEQ ID NO: 1 (preferably about 1 to 30, more preferably 1 to 30); About 10 amino acids, more preferably several (1 to 5) amino acids are deleted, and 2 or more amino acids (preferably 1 to 5) in the amino acid sequence represented by SEQ ID NO: 1 An amino acid sequence to which about 30 amino acids are added, more preferably about 1 to 10 amino acids, and still more preferably several amino acids (1 to 5 amino acids); 3 one or more amino acids in the amino acid sequence represented by SEQ ID NO: 1 An amino acid sequence in which two or more (preferably about 1 to 30, more preferably about 1 to 10, and more preferably several (1 to 5)) amino acids have been substituted with another amino acid; or ⁇ ⁇ ⁇ ⁇ ⁇ Protein containing amino acid sequence combining them There above (1) G protein-coupled receptions evening one protein or salt thereof according,
  • the ligand is, for example, angiotensin, bombesin, canapinoid, cholecystokinin, glutamine, serotonin, melatonin, neuropeptide Y, opioid, purine, vasopressin, oxitosine, PACAP (e.g., PACA P 2 7, PACAP 3 8), secretin, glucagon, calcitonin, adrenomedullin, somatos, GHRH, CRF, ACTH, GRP, PTH, VIP (basoactive intestinal polypeptide), somatos, dopamine, Motilin, amylin, bradykinin, CGRP (calcitonin gene releated peptide), leukotriene, pancreastatin, prostaglandin, thromboxane, adenosine, adrenaline, chemokine superfamily (eg, IL-8, GROa, GRO ⁇ ) , GROA, NAP-2
  • the compound that activates the G protein-coupled receptor protein or a salt thereof described in (1) and a test compound are cultured in the transformant described in (8).
  • the transformant is brought into contact with the G protein-coupled receptor protein expressed in the cell membrane, the cell stimulating activity mediated by the G protein-coupled receptor protein is measured and compared.
  • the compound that activates the G protein-coupled receptor protein according to (1) is angiotensin, bombesin, canapinoid, cholecystokinin, glutamine, serotonin, melatonin, neuropeptide Y, obioid, Purine, vasopletsusin, oxytocin, PACAP (e.g., PACAP 27, PACAP 38), secretin, glucagon, calcitonin, adrenomejuulin, somatos quintin, GHRH, CRF, ACTH, GRP.PTH, VIP (vasoactive Intestinal polypeptide), somatostin, dopamine, motilin, amylin, bradykinin, CGRP (calcitonin gene-related peptide), leukotriene, pancreastatin, prostaglandin, troponpoxane, adenosine, adrenaline, chemokine sopain one Amiri (e.g.,
  • a compound or a salt thereof that alters the binding between the ligand obtainable by the screening method according to (38) to (45) and the G protein-coupled receptor protein or salt thereof according to (1) (48) a screening kit according to (18), which comprises a cell containing the G protein-coupled receptor protein according to (1); The screening kit according to (18), which comprises a membrane fraction of a cell containing the G protein-coupled receptor protein according to (1).
  • a pharmaceutical comprising a compound or a salt thereof that alters the binding property between the ligand and the G protein-coupled receptor protein or a salt thereof according to the above (1).
  • a recombinant vector which has a DNA encoding the G protein-coupled receptor protein described in (1) or a mutant DNA thereof and can be expressed in a non-human animal,
  • (61) a non-human mammal deficient in DNA expression, wherein the DNA encoding the G protein-coupled receptor protein of (1) is inactivated, and
  • the G protein-coupled receptor protein (hereinafter sometimes abbreviated as receptor protein) of the present invention is a receptor containing an amino acid sequence identical or substantially identical to the amino acid sequence represented by SEQ ID NO: 1. It is a protein.
  • the receptor protein of the present invention can be used, for example, in any cells (eg, spleen cells, nerve cells, glial cells) of human mammals (eg, guinea pigs, rats, mice, rabbits, bushes, sheep, birds, monkeys, etc.).
  • human mammals eg, guinea pigs, rats, mice, rabbits, bushes, sheep, birds, monkeys, etc.
  • the brain each part of the brain (e.g., olfactory bulb, nucleus punctiform nucleus, basal sphere, hippocampus, thalamus, hypothalamus, hypothalamus nucleus, cerebral cortex, medulla oblongata, cere
  • Examples of the amino acid sequence substantially the same as the amino acid sequence represented by SEQ ID NO: 1 include, for example, 85% or more, preferably about 90% or more, more preferably the amino acid sequence represented by SEQ ID NO: 1. Examples include amino acid sequences having about 95% or more homology. 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, an amino acid sequence substantially identical to the amino acid sequence represented by SEQ ID NO: 1 And a protein having substantially the same activity as the amino acid sequence represented by SEQ ID NO: 1.
  • substantially equivalent activities include, for example, ligand binding activity and signal transduction. Substantially the same means that their activities are the same in nature. Therefore, activities such as ligand binding activity and signal transduction are equivalent (e.g., about 0.01 to 100 times, preferably about 0.5 to 20 times, more preferably about 0.5 to 2 times). ), But the quantitative factors such as the degree of these activities and the molecular weight of the protein may be different.
  • the measurement of the activity such as the ligand binding activity and the signal information transmission activity can be performed according to a known method.
  • the activity can be measured according to the ligand determination method described later ⁇ screening method. .
  • the receptor protein of the present invention includes: (1) one or two or more amino acids in the amino acid sequence represented by SEQ ID NO: 1 (preferably, about 1 to 30, more preferably about 1 to 10) More preferably 1 to 2 amino acids (preferably 1 to 30 amino acids) in the amino acid sequence represented by SEQ ID NO: 1 in which several (1 to 5) amino acids have been deleted. Degree, more preferably about 1 to 10, more preferably several (1 to 5)) amino acid sequences, and 3 one or two amino acids in the amino acid sequence represented by SEQ ID NO: 1.
  • the receptor protein in this specification has the N-terminus at the left end (amino end) and the C-terminus at the right end (capillary end).
  • the receptor protein of the present invention including the receptor protein containing the amino acid sequence represented by SEQ ID NO: 1, has a C-terminal lipoxyl group (—COOH), a carboxyl group (—COO) —), Amide (one CO NH 2 ) or ester (one COOR).
  • R in the ester e.g., methyl, Echiru, n- propyl, ( ⁇ _ 6 alkyl groups such as isopropyl or n- butyl, cyclopentyl Le, C 3 _ 8 cycloalkyl group such as cyclohexyl, for example, phenyl, alpha-C 6, such as naphthyl -! i 2 ⁇ Li Ichiru group, e.g., benzyl, Hue second phenethyl Lou C _ 2 alkyl or a- naphthylmethyl etc. a- Nafuchiru C i _ 2 in addition to C 7 _ 1 4 Ararukiru group such as an alkyl group, such as Pibaroiru Okishimechiru group commonly used as an oral ester.
  • ⁇ _ 6 alkyl groups such as isopropyl or n- butyl, cyclopentyl Le, C 3 _ 8 cycloalky
  • the receptor protein of the present invention has a carbonyl group (or carboxylate) other than at the C-terminus
  • a protein in which the carboxyl group is amidated or esterified is also included in the receptor protein of the present invention.
  • the ester in this case, for example, the above-mentioned terminal ester and the like are used.
  • the receptions evening one protein of the present invention is the protein mentioned above, Amino group protecting groups Mechionin residues of N-terminal (e.g., formyl group, Flip 6 Ashiru group such as C 2 _ 6 Arukanoiru group such Asechiru ), N-terminal cleavage in vivo, and the darminyl mill group formed by oxidation of darminamine, substituents on the side chains of amino acids in the molecule (eg, _OH , protected in one SH, amino group, imidazole group, indole group, etc.
  • Amino group protecting groups Mechionin residues of N-terminal e.g., formyl group, Flip 6 Ashiru group such as C 2 _ 6 Arukanoiru group such Asechiru
  • N-terminal cleavage in vivo e.g., N-terminal cleavage in vivo
  • the darminyl mill group formed by oxidation of darminamine substituents on
  • Guanijino group appropriate protecting groups (e.g., formyl group, such as C E _ 6 Ashiru group such as C 2 _ 6 Arukanoiru group such as ⁇ cetyl) And complex proteins such as so-called glycoproteins to which sugar chains are bound.
  • formyl group such as C E _ 6 Ashiru group such as C 2 _ 6 Arukanoiru group such as ⁇ cetyl
  • complex proteins such as so-called glycoproteins to which sugar chains are bound.
  • receptor protein of the present invention for example, a receptor protein containing the amino acid sequence represented by SEQ ID NO: 1 is used.
  • the partial peptide of the receptor protein of the present invention may be any peptide as long as it is the partial peptide of the receptor protein of the present invention described above.
  • the receptor protein molecules those that are exposed outside the cell membrane and have substantially the same receptor binding activity are used.
  • a partial peptide of the receptor protein having the amino acid sequence represented by SEQ ID NO: 1 the extracellular region (hydrophilicity) (Hydrophilic) site). Further, a peptide partially containing a hydrophobic site can also be used. A peptide containing individual domains may be used, but a partial peptide containing multiple 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 amino acids in the amino acid sequence of the receptor protein of the present invention. Peptides having a sequence are preferred.
  • 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 above amino acid sequence are deleted (2) One or more (preferably about 1 to 20, more preferably about 1 to 10, and more preferably several (1 to 5)) amino acids are added to the above amino acid sequence. Or 3 one or more (preferably about 1 to 10, more preferably several, more preferably about 1 to 5) amino acids in the above amino acid sequence are substituted with another amino acid. You may.
  • the partial peptide of the present invention may have any one of a lipoxyl group (—COOH), a propyloxylitol (—CO ⁇ _), an amide (—CONH 2 ), and an ester (—COO) at the C-terminus.
  • Good R is as defined above.
  • the partial peptide of the present invention has a carboxyl group (or carboxylate) other than the C-terminal, the partial peptide 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.
  • 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.
  • Py-glutamine-oxidized G 1 n formed, compounds in which the substituent on the side chain of amino acid in the molecule is protected with an appropriate protecting group, or complex such as a so-called glycopeptide with a sugar chain attached Peptides are also included.
  • the salt of the receptor protein or its partial peptide of the present invention may be an acid or Physiologically acceptable salts with bases are included, and physiologically acceptable acid addition salts are particularly preferred.
  • Such salts include, for example, salts with inorganic acids (eg, hydrochloric acid, phosphoric acid, hydrobromic acid, sulfuric acid) or organic acids (eg, acetic acid, formic acid, propionic acid, fumaric acid, maleic acid, succinic acid) Acids, tartaric acid, citric acid, malic acid, oxalic acid, benzoic acid, methanesulfonic acid, and benzenesulfonic acid) are used.
  • inorganic acids eg, hydrochloric acid, phosphoric acid, hydrobromic acid, sulfuric acid
  • organic acids eg, acetic acid, formic acid, propionic acid, fumaric acid, maleic acid, succinic acid
  • the receptor protein of the present invention or a salt thereof can be produced from the above-mentioned human or mammalian cell or tissue by a known method for purifying a receptor protein, or the present invention described later. It can also be produced by culturing a transformant containing DNA encoding the receptor protein. Also, the protein can be produced by the protein synthesis method described later or according to the method.
  • the human or mammalian tissues or cells are homogenized, extracted with an acid or the like, and the extract is subjected to reversed-phase chromatography or ion-exchange chromatography. Purification and isolation can be achieved by using a combination of chromatography methods.
  • a commercially available resin for protein synthesis can be usually used.
  • a resin include chloromethyl resin, hydroxymethyl resin, benzylhydrylamine resin, aminomethyl resin, 4-benzyloxybenzyl alcohol resin, 4-methylbenzhydrylamine resin, and P-methylbenzhydrylamine resin.
  • AM resin 4-hydroxymethylmethylphenylacetamidomethyl resin, polyacrylamide resin, 4 _ (2,4, dimethoxyphenylhydroxymethyl) phenoxy resin, 4-(2 ', 4, dimethoxyphenyl) L-Fmoc aminoethyl) phenoxy resin.
  • an amino acid in which the amino group and the side chain functional group are appropriately protected is condensed on the resin according to the known amino acid sequence of the target protein or peptide according to various known condensation methods.
  • a protein or peptide is cleaved from the resin, and at the same time, various protecting groups are removed.
  • an intramolecular disulfide bond formation reaction is carried out in a highly diluted solution to obtain a target protein or peptide or an amide thereof.
  • 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, HOBt, HOOBt), or symmetrical acid anhydride or HOBT or HOOBt ester.
  • a racemization inhibitor additive eg, HOBt, HOOBt
  • the solvent used for the activation of the protected amino acid and the condensation 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 acetate nitrile and propionitrile; esters such as methyl acetate and ethyl acetate; Used.
  • the reaction temperature is appropriately selected from a range known to be usable for a protein bond formation reaction, and is usually appropriately selected from a range of about ⁇ 20 ° C. to 50 ° C.
  • the activated amino acid derivative is usually used in a 1.5 to 4-fold excess.
  • Examples of the protecting group for the amino group of the starting material include Z, B oc, sulfur pentyl oxycarbonyl, isobornyloxycarbonyl, 4-methoxybenzyloxy carbonyl, C 11 Z, Br—Z, Adamantyloxycarbonyl, trifluoroacetyl, phthaloyl, formyl, 212-trophenylsulfenyl, diphenylphosphinothioyl, Fmoc and the like are used.
  • Carboxyl groups can be, for example, alkyl esterified (eg, methyl, ether, propyl, butyl, tertiary butyl, cyclopentyl, cyclohexyl, cyclo Linear, branched or cyclic alkyl esterification of heptyl, cyclooctyl, 2-adamantyl etc., aralkyl esterification (eg benzyl ester, 4-nitrobenzyl ester, 4-methoxybenzyl ester, 4-methoxybenzyl ester) Benzyl ester, benzhydryl esterification), phenacyl esterification, benzyloxy karponyl hydrazide, Yuichi Sharybutoxycarponyl hydrazide, trityl hydrazide and the like.
  • alkyl esterified eg, methyl, ether, propyl, butyl, tertiary butyl
  • the hydroxyl group of serine can be protected, for example, by esterification or etherification.
  • esterification for example, a lower alkanoyl 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.
  • groups suitable for etherification include, for example, a benzyl group, a tetrahydrobiranyl group, a t-butyl group and the like.
  • the protecting group of the phenolic hydroxyl group of tyrosine for example, B z 1, C 1 2 -B zl, 2- two Torobenjiru, B r- Z, such as evening one tertiary heptyl is used.
  • As the protecting group for imidazole of histidine for example, Tos, 4-methoxy-2,3,6-trimethylbenzenesulfonyl, DNP, benzyloxymethyl, Bum, Boc, Trt, Fmoc and the like are used.
  • activated carboxyl groups in the raw materials include, for example, corresponding acid anhydrides, azides, active esters [alcohols (eg, pentachlorophenol, 2,4,5-trichloromouth phenol, 2,4-dinitrophenol) , Cyanomethyl alcohol, paranitrophenol, HONB, N-hydroxysuccinimide, N-hydroxyfurimide, HOB tester) and the like.
  • active esters eg, pentachlorophenol, 2,4,5-trichloromouth phenol, 2,4-dinitrophenol
  • Cyanomethyl alcohol eg, paranitrophenol, HONB, N-hydroxysuccinimide, N-hydroxyfurimide, HOB tester
  • activated amino group of the raw material for example, a corresponding phosphoric amide is used.
  • Methods for removing (eliminating) protecting groups include, for example, catalytic reduction in a hydrogen stream in the presence of a catalyst such as Pd-black or Pd-carbon, or hydrogen fluoride anhydride, methanesulfonic acid, Acid treatment with dichloromethane, trifluoroacetic acid or a mixture thereof, base treatment with diisopropylethylamine, triethylamine, piperidine, piperazine, etc., reduction with sodium in liquid ammonia, etc. are also used. .
  • the elimination reaction by the above-mentioned acid treatment is generally 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 protective 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.
  • a peptide (protein) chain is extended to a desired chain length on the amino group side.
  • a protein was prepared by removing only the protecting group of the ⁇ -amino group at the ⁇ end of the peptide chain and a protein was obtained by removing only the protecting group of the lipoxyl group at the C-terminal of the peptide chain.
  • a mixed solvent Details of the condensation reaction are the same as described above.
  • all the protecting groups are removed by the above 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.
  • an ester form of the protein for example, after condensing the lpoxyl group of the carboxy terminal amino acid with a desired alcohol to form an amino acid ester, the desired amino acid ester is prepared in the same manner as in the case of the amide form of protein.
  • An ester form of the 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 peptidase.
  • a method for synthesizing a peptide for example, any of a solid phase synthesis method and a liquid phase synthesis method may be used.
  • the product when the partial peptide or amino acid that can constitute the protein of the present invention is condensed with the remaining portion, and the product has a protecting group.
  • the desired peptide can be produced by removing the protecting group.
  • Known methods for condensation and elimination of protecting groups include, for example, the methods described in (1) to (4) below.
  • 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 / recrystallization.
  • the partial peptide obtained by the above method is a free form, it can be converted to an appropriate salt by a known method, and conversely, when obtained as a salt, it can be converted to a free form by a known method. can do.
  • the polynucleotide encoding the receptor protein of the present invention may be any one containing the above-described nucleotide sequence (DNA or RNA, preferably DNA) encoding the receptor protein of the present invention. Any thing may be used.
  • the polynucleotide is RNA such as DNA or mRNA which encodes the receptor protein of the present invention, and may be double-stranded or single-stranded. In the case of a double strand, it may be a double-stranded DNA, a double-stranded RNA or a hybrid of DNA: RNA. In the case of a single strand, it may be a sense strand (that is, a coding strand) or an antisense strand (that is, a non-coding strand).
  • the polynucleotide encoding the receptor protein of the present invention for example, by the method described in the well-known experimental medicine special edition "New PCR and its application” 15 (7), 1997 or a method analogous thereto.
  • the mRNA of the receptor protein of the present invention can be quantified.
  • the DNA encoding the receptor protein of the present invention includes genomic DNA, Any of a nome DNA library, a cDNA derived from the above-described cells and tissues, a cDNA library derived from the above-described cells and tissues, and synthetic DNA may be used.
  • the vector used in the library may be any of bacteriophage, plasmid, cosmid, phagemid and the like. Alternatively, it can also be directly amplified by Reverse Transcriptase Polymerase Chain Reaction (hereinafter abbreviated as RT-PCR method) using a 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 for example, a DNA containing the base sequence represented by SEQ ID NO: 2 or SEQ ID NO: 3, or SEQ ID NO: 2 or the sequence It has a DNA that hybridizes under highly stringent conditions with DNA having the nucleotide sequence represented by No. 3 and has substantially the same activity as the receptor protein of the present invention (eg, ligand binding activity, signal Any DNA may be used as long as it encodes a receptor protein having an information transfer function.
  • DNA that hybridizes with DNA having the nucleotide sequence represented by SEQ ID NO: 2 or SEQ ID NO: 3 under high stringent conditions include, for example, 85% or more of the nucleotide sequence represented by SEQ ID NO: 2, Preferably, a DNA containing a base sequence having a homology of about 90% or more, more preferably about 95% or more is used.
  • Hybridization can be 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). Can be performed according to When a commercially available library is used, it can be performed according to the method described in the attached instruction manual. More preferably, it can be performed under high stringency conditions.
  • the high stringent conditions include, for example, conditions at a sodium concentration of about 19 to 4 OmM, preferably about 19 to 20 mM, and a temperature of about 50 to 70 ° C, preferably about 60 to 65 ° C. Show. In particular, the case where the sodium concentration is about 19 mM and the temperature is about 65 ° C. is most preferable.
  • a receptor containing the amino acid sequence represented by SEQ ID NO: 1 As the DNA encoding the protein, a DNA containing the base sequence represented by SEQ ID NO: 2 or SEQ ID NO: 3 or the like is used.
  • a part of the nucleotide sequence of the DNA encoding the receptor protein of the present invention or a polynucleotide containing a part of the nucleotide sequence complementary to the DNA refers to the following partial peptide of the present invention. It is used not only to include the coding DNA, but also to include the RNA.
  • a G protein-coupled receptor protein obtained by cloning or determining an antisense polynucleotide (nucleic acid) capable of inhibiting the replication or expression of a G protein-coupled receptor protein gene can be designed and synthesized based on the nucleotide sequence information of the DNA encoding Such a polynucleotide (nucleic acid) can hybridize to the RNA of a G protein-coupled receptor protein gene and can inhibit the synthesis or function of the RNA, or it can be a G protein-coupled receptor protein-related RNA. Can regulate and control the expression of G protein-coupled receptor protein gene through interaction with.
  • a polynucleotide complementary to the selected sequence of the G protein-coupled receptor protein-related RNA and a polynucleotide capable of specifically hybridizing to the G protein-coupled receptor and the overnight protein-related RNA are It is useful for regulating and controlling the expression of G protein-coupled receptor protein gene in vivo and in vitro, and is also useful for treating or diagnosing diseases and the like.
  • the term "corresponding" means having homology or being complementary to a specific sequence of nucleotides, base sequences or nucleic acids, including genes.
  • a “correspondence” between a nucleotide, base sequence or nucleic acid and a peptide (protein) is defined as the amino acid of the peptide (protein) normally specified by the nucleotide (nucleic acid) sequence or its complement.
  • the 3 'end untranslated region, the 3' end palindrome region, and the 3 'end hairpin loop can be selected as preferred target regions, but any region in the G protein-coupled receptor protein gene is selected. Can.
  • Antisense polynucleotides are polydeoxynucleotides containing 2-deoxy D_ribose, polynucleotides containing D-lipose, N-glycosides of purine or pyrimidine bases and other types.
  • polymers having a non-nucleotide backbone for example, commercially available protein nucleic acids and synthetic sequence-specific nucleic acid polymers
  • polymers containing special bonds provided that the polymer is DNA or RNA
  • Base pairing as found therein contains a nucleotide having a configuration permitting base attachment
  • They can be double-stranded DNA, single-stranded DNA, double-stranded RNA, single-stranded RNA, and even DNA: RNA hybrids, and can also be unmodified polynucleotides (or unmodified oligonucleotides).
  • nucleotide As well as those with known modifications, such as labeled, capped, methylated, and one or more natural nucleotides as analogs known in the art. Substituted, modified with an intramolecular nucleotide, for example, having an uncharged bond (eg, methylphosphonate, phosphotriester, phosphoramidate, olebamate, etc.), a charged or sulfur-containing bond (eg, Those having phosphorothioate, phosphorodithioate, etc., for example, proteins (nucleases, nucleases and inhibitors, Those having side chain groups such as syn, antibody, signal peptide, poly-L-lysine, etc.
  • an intramolecular nucleotide for example, having an uncharged bond (eg, methylphosphonate, phosphotriester, phosphoramidate, olebamate, etc.), a charged or sulfur-containing bond (eg, Those having phosphoroth
  • nucleic acid may include not only those containing purine and pyrimidine bases but also those having other modified heterocyclic bases. Such modifications may include methylated purines and pyrimidines, acylated purines and pyrimidines, or other heterocycles.
  • Modified nucleotides and modified nucleotides The moiety may be modified, for example, one or more hydroxyl groups may be substituted with a halogen, an aliphatic group, or the like, or may be converted to a functional group such as an ether or an amine.
  • the antisense polynucleotide (nucleic acid) of the present invention is an RNA, a DNA, or a modified nucleic acid (RNA, DNA).
  • modified nucleic acids include, but are not limited to, sulfur derivatives of nucleic acids, thiophosphate derivatives, and polynucleoside amides, which are resistant to degradation of oligonucleoside amides.
  • the antisense nucleic acid of the present invention can be preferably designed according to the following policy.
  • the antisense nucleic acid of the present invention may contain altered or modified sugars, bases, or bonds, and may be provided in a special form such as ribosome or microsphere, applied by gene therapy, or added. Can be given in a given form.
  • additional forms include polythiones, 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, phospholipid, cholesterol, etc.).
  • Preferred lipids for addition include cholesterol and its derivatives (eg, cholesteryl chromate formate, cholic acid, etc.).
  • nucleic acids can be attached via a base, sugar, or intramolecular nucleoside bond.
  • Other groups include cap groups specifically arranged at the 3 'end or 5' end of nucleic acids for preventing degradation by nucleases such as exonuclease and RNase.
  • the base for such caps is polyethylene Examples include, but are not limited to, hydroxyl-protecting groups known in the art, such as glycols such as recall and tetraethylene glycol.
  • the inhibitory activity of the antisense nucleic acid is examined using the transformant of the present invention, the in vivo or in vitro gene expression system of the present invention, or the in vivo or in vitro translation system of the G protein-coupled receptor protein. be able to.
  • the nucleic acid can be applied to cells by various known methods.
  • 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 genomic DNA, a genomic DNA library, the above-described cell / tissue-derived cDNA, the above-described cell / tissue-derived cDNA library, and a synthetic DNA may be used.
  • the vector used for the library may be any of pacteriophage, plasmid, cosmid, phagemid and the like. Alternatively, it can also be directly amplified by Reverse Transcriptase Polymerase Chain Reaction (hereinafter abbreviated as RT-PCR method) using an mRNA fraction prepared from the above-mentioned cell'tissue.
  • RT-PCR method Reverse Transcriptase Polymerase Chain Reaction
  • the DNA encoding the partial peptide of the present invention includes, for example, (1) a DNA having a partial base sequence of a DNA having a base sequence represented by SEQ ID NO: 2 or SEQ ID NO: 3. NA, or (2) having a DNA that hybridizes under high stringent conditions with the DNA represented by SEQ ID NO: 2 or SEQ ID NO: 3, and having substantially the same activity as the protein peptide of the present invention (eg, For example, a DNA having a partial base sequence of DNA encoding a protein having ligand binding activity, signal information transmitting action, etc.) may be used.
  • a DNA having a partial base sequence of DNA encoding a protein having ligand binding activity, signal information transmitting action, etc. may be used.
  • Examples of the DNA which hybridizes with the DNA represented by SEQ ID NO: 2 or SEQ ID NO: 3 under high stringent conditions include, for example, the nucleotide sequence represented by SEQ ID NO: 2 or SEQ ID NO: 3; A DNA containing a nucleotide sequence having a homology of 5% or more, preferably about 90% or more, more preferably about 95% or more is used.
  • DNA encoding the peptide of the present invention may be used.
  • Partial nucleotide sequence of the nucleotide sequence of Amplified by the PCR method using a synthetic DNA primer having the above, or labeled with a DNA fragment or a synthetic DNA encoding a part or the entire region of the receptor protein of the present invention, incorporating DNA into an appropriate vector It can be selected by hybridization with the one that has been done.
  • the hybridization method can be performed, for example, according to the method described in Molecular Cloning 2nd (J. Sambrook et al., Cold Spring Harbor Lab. Press, 1989). . When a commercially available library is used, it can be performed according to the method described in the attached instruction manual.
  • the DNA base sequence can be converted using PCR or a known kit, for example, Mutan TM -supper Express Ks (Takara Shuzo Co., Ltd.), Mutan TM -K (Takara Shuzo Co., Ltd.), etc. ODA—LAPCR method, Gapped plex method, Kunke method, and other known methods or methods similar thereto.
  • the DNA encoding the cloned receptor protein can be used as it is depending on the purpose, or can be used by digesting with a restriction enzyme or adding a linker if desired.
  • the DNA may have ATG as a translation initiation codon at the 5 'end and TAA, TGA or TAG as a translation termination codon at the 3' end. These translation initiation codon and translation termination codon can also be added using a suitable synthetic DNA adapter.
  • the expression vector of the receptor protein of the present invention includes, for example, (a) cutting out a DNA fragment of interest from DNA encoding the receptor protein of the present invention,
  • vectors include Escherichia coli-derived plasmids (eg, pCR4, pCR2.1, pBR322, pBR325, pUC12, pUC13), Bacillus subtilis-derived plasmids (eg, pUB110, pTP5, pC194), yeast
  • derived plasmids eg, pSH19, pSH15
  • bacteriophages such as phage, animal viruses such as retrovirus, vaccinia virus, baculovirus, etc., pA1-11, pXTl, pRc / CMV, pRc / RSV,; pc DNA I / Neo, etc. 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 hosts, SRa promoter, SV40 promoter, LTR promoter, CMV promoter, HSV-TK promoter and the like can be mentioned.
  • CMV Promo One Night SR Hi Promoter One, and the like.
  • the host is Eshierihia genus bacterium, trp promoter, 1 ac promoter - evening one, rec A promoter, AP L promoter of all, like 1 ro-ro promoter one is, when the host is Bacillus, S [rho Omicron 1
  • the host is a yeast, such as a promoter, S ⁇ 2 promoter overnight, pe ⁇ promoter, etc.
  • polyhedrin promoter overnight, P10 promoter and the like are preferable.
  • the expression vector may contain, in addition to the above, an enhancer, a splicing signal, a poly-A additional signal, a selection marker, and an SV40 replication origin (hereinafter sometimes abbreviated as SV40 ori), if desired.
  • SV40 ori an SV40 replication origin
  • the selection marker include dihydrofolate reductase (hereinafter, dh fr and sometimes you abbreviation) gene [Mesotorekise Ichito (MTX) resistance], ampicillin resistant gene (hereinafter sometimes abbreviated as Amp r there), the neomycin resistance gene (hereinafter sometimes abbreviated as Ne o r, include G418 resistance) and the like.
  • dhfr gene is used as a selection marker using CHO (dhfr_) cells, the target gene can be selected even on 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. If the host is Escherichia, Pho A signal sequence, Omp A signal sequence, etc., if the host is Bacillus, a; amylase signal sequence, subtilisin signal sequence, etc. If the host is yeast, MFa signal sequence, SUC2 signal sequence, etc.If the host is an animal cell, insulin signal sequence, single interferon signal sequence, antibody molecule, signal sequence Etc. can be used respectively.
  • a transformant can be produced using a vector containing
  • Escherichia bacteria for example, Escherichia bacteria, Bacillus bacteria, yeast, insect cells, insects, animal cells, and the like are used.
  • Escherichia * coli Escherichia col 0 Kl 2 ⁇ DH1 [Procedings. Ob 'The National' Academy 'Ob. Sciences' Ob. Natl. Acad. Sci. USA), 60, 160 (1968)], JM103 [Nucleic Acids Research], (Nucleic Acids Research), 9, 309 (1981)], J ⁇ 221 [Journal of Ob. Journal of Molecular Biology, 120, 517 (1978)], ⁇ 101 [Journal of the 'Molecular' Biology, 41, 459 (1969)], C 600 [Genetics] , 39, 440 (1954)], DH5a Clnoue, H., Nojima, H.
  • Bacillus bacteria examples include, for example, Bacillus subtilis MI114 [Gene, 24, 255 (1983)], 207-21 [Journal of Biochemistry, 95, 87 (1984)].
  • yeast examples include, for example, Saccharomyces cerevisiae AH22, AH22 R—, ⁇ 87-11 A, DKD—5D, 20 B-12, Scriizosaccliaromyces pombe NCYC1 913, NCYC 2036 Pichia (Pichia pastor is) is used.
  • Insect cells include, for example, when the virus is Ac NPV, a cell line derived from the larva of night moth (Spodoptera frugiperda cell; S f cell), MG1 cell derived from the midgut of Trichoplusia ni, and egg derived from Trichoplusia ni egg High Five TM cells, cells derived from Mamestra brassicae or cells derived from Estigmena acrea are used.
  • Sf cell a silkworm-derived cell line (Bombyx mori N; BmN cell) is used.
  • Sf cell include Sf9 cell (ATCC CRL1711) and Sf21 cell (Vaughn, J, et al., In Vivo, 13, 213-217, (1977)) Etc. are used.
  • 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), dh fr gene-deficient Chinese hamster cell CHO (hereinafter abbreviated as CHO (dh fr—) cell). ), Mouse L cells, mouse AtT-20, mouse myeloma cells, rat GH3, human FL cells, etc. are used.
  • 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, by the method described in Bio / Technology, 6, 47-55 (1988).
  • a liquid medium is suitable as a medium used for the cultivation.
  • Nitrogen sources, inorganic substances, etc. are included.
  • carbon sources include glucose, dextrin, soluble starch, and sucrose.
  • nitrogen sources include ammonium salts, nitrates, corn chip liquor, peptone, casein, meat extract, soybean meal, and potato extract.
  • Inorganic or organic substances such as liquids, and inorganic substances include, for example, 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.
  • a medium for cultivating a bacterium belonging to the genus Escherichia for example, an M9 medium containing glucose and casamino acids [Miller, Journal of Experiments, Molecular, Genetics (Journal of Experiments in Molecular Genetics), 431-433, Cold Spring Harbor Laboratory, New York 1972].
  • a drug such as 3j3-indolylacrylic acid can be added to make the promoter work efficiently.
  • cultivation is usually performed at about 15 to 43 ° C for about 3 to 24 hours, and if necessary, aeration and stirring can be applied.
  • the cultivation is usually performed at about 30 to 40 ° C for about 6 to 24 hours, and if necessary, aeration and stirring can be applied.
  • the medium When culturing a transformant in which the host is yeast, for example, the medium may be Burktiolder minimum medium [Bostian, KL et al., “Procedures of the National 'Academy' of Sciences”. Prob. The USA (Pro Natl. Acad. Sci. USA), 77, 4505 (1980)] and an SD medium containing 0.5% casamino acid [BiUer, GA et al. Proc. Natl. Acad. Sci. USA, 81, 5330 (1984)].
  • the pH of the culture medium is about 5
  • the culture is preferably adjusted to about 8 to 8.
  • the culture is usually performed at about 20 ° C to 35 ° C for about 24 to 72 hours.
  • the medium used is a 10% serum serum immobilized in Grace's Insect Medium (Grace, TCC, Nature, 195, 788 (1962)). And the like to which additives such as the above are appropriately added are used.
  • the ⁇ of the medium is adjusted to about 6.2 to 6.4. Culture is usually performed at about 27 ° C for about 3 to 5 days, and aeration and agitation are added as necessary.
  • examples of the medium include a MEM medium containing about 5 to 20% fetal bovine serum [Science, 122, 501 (1952)], DMEM Medium [Virology, 8, 396 (1959)], RPMI 1640 medium [Journal of the American Medical Association] 1999, 519 (1967)], 199 medium [Proceding of the Society for the Biological Medicine], 73, 1 (1950)]
  • the pH is about 6-8.
  • Culture is usually carried out at about 30 to 40 ° C for about 15 to 60 hours, and aeration and stirring are added as necessary.
  • the G protein-coupled receptor protein of the present invention can be produced inside the cell, in the cell membrane, or outside the cell of the transformant.
  • the receptor protein of the present invention can be separated and purified from the culture by, for example, the following method.
  • the cells or cells are collected by a known method, suspended in an appropriate buffer, and subjected to ultrasound, lysozyme and / or After the cells or cells are destroyed by freezing and thawing, a method of obtaining a crude extract of the receptor protein by centrifugation or filtration is appropriately used.
  • the buffer may contain a protein denaturant such as urea or guanidine hydrochloride, or a surfactant such as Triton X-100 TM . If the receptor protein is secreted into the culture solution, after the culture is completed, the supernatant is separated from the cells or cells by a known method, and the supernatant is collected.
  • 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 include methods utilizing solubility such as salting out and solvent precipitation, dialysis, ultrafiltration, gel filtration, and SDS-polyacrylamide gel electrophoresis, and other molecular weight methods.
  • Methods that utilize differences in charge methods that use differences in charge, such as ion-exchange chromatography, methods that use specific affinities, such as affinity chromatography, methods that use reverse-phase high-performance liquid chromatography, etc.
  • a method utilizing a difference in hydrophobicity, a method utilizing a difference in isoelectric point such as isoelectric focusing, and the like are used.
  • the receptor protein obtained in this manner When the receptor protein obtained in this manner is obtained in a free form, it can be converted to a salt by a known method or a method analogous thereto.
  • the compound can be converted into a free form or another salt by a method or a method analogous thereto.
  • Recombinant receptor protein is treated with an appropriate protein-modifying enzyme before or after purification, so that it can be modified arbitrarily or the polypeptide can be partially removed.
  • an appropriate protein-modifying enzyme for example, trypsin, chymotrypsin, arginyl endopeptidase, protein kinase, glycosidase and the like are used.
  • the activity of the receptor protein of the present invention or a salt thereof thus produced can be measured by a binding experiment with a labeled ligand, an enzyme immunoassay using a specific antibody, or the like.
  • the antibody against the receptor protein of the present invention or its partial peptide or its salt may be any of a polyclonal antibody and a monoclonal antibody, as long as it 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 prepared by using the receptor protein of the present invention as an antigen and a known antibody. It can be produced according to a method for producing an antibody or antiserum.
  • the receptor protein of the present invention or the like is administered to a mammal at a site where the antibody can be produced by administration to itself or together with a carrier or a diluent.
  • Complete Freund's adjuvant / incomplete Freund's adjuvant may be administered in order to enhance antibody production upon administration. Administration is usually performed once every 2 to 6 weeks, for a total of 2 to 10 times. Examples of mammals to be used include monkeys, rabbits, dogs, guinea pigs, mice, rats, sheep, and goats, and mice and rats are preferably used.
  • a warm-blooded animal immunized with the antigen for example, a mouse 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 measurement of the antibody titer in the antiserum can be performed, for example, by reacting a labeled receptor protein or the like described below with the antiserum, and then measuring the activity of the labeling agent bound to the antibody.
  • the fusion operation can be performed according to a known method, for example, the method of Keller and Milstein [Nature, 256, 495 (1975)].
  • the fusion promoter include polyethylene glycol (PEG) and Sendai virus, and preferably PEG is used.
  • myeloma cells examples include NS_1, P3U1, and SP2Z0, and P3U1 is preferably used.
  • the preferred ratio between the number of antibody-producing cells (spleen cells) and the number of myeloma cells to be used is about 1: 1 to 20: 1, and PEG (preferably PEG1000 to PEG6000) is added at a concentration of about 10 to 80%.
  • PEG preferably PEG1000 to PEG6000
  • hybridomas can be cultured on a solid phase (eg, microplate) on which an antigen such as a receptor protein is adsorbed directly or together with a carrier. Then, an anti-immunoglobulin antibody (anti-mouse immunoglobulin antibody is used if the cell used for cell fusion is a mouse) or protein A, labeled with a radioactive substance or enzyme, is added to the solid phase.
  • a solid phase eg, microplate
  • an antigen such as a receptor protein
  • an anti-immunoglobulin antibody anti-mouse immunoglobulin antibody is used if the cell used for cell fusion is a mouse
  • protein A labeled with a radioactive substance or enzyme
  • Method for detecting bound monoclonal antibody, anti-immunoglobulin is to add the hybridoma culture supernatant to the solid phase to which the antibody or protein A has been adsorbed, add a receptor protein labeled with a radioactive substance or an enzyme, etc. No.
  • 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 it can grow hybridomas.
  • RPMI 1640 medium containing 1 to 20%, preferably 10 to 20% fetal bovine serum, GIT medium containing 1 to 10% fetal bovine serum (Wako Pure Chemical Industries, Ltd.)
  • serum-free medium for hybridoma culture SFM-101, Nissui Pharmaceutical Co., Ltd.
  • the culture temperature is usually 20 to 40, preferably about 37 ° C.
  • the culture time is generally 5 days to 3 weeks, preferably 1 week to 2 weeks.
  • the culture can be usually performed under 5% carbon dioxide.
  • 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.
  • immunoglobulin separation and purification methods eg, salting out, alcohol precipitation, isoelectric precipitation, electrophoresis, ion exchangers
  • DEAE adsorption / desorption method
  • ultracentrifugation method ultracentrifugation method
  • gel filtration method antigen-bound solid phase or active adsorbent such as protein A or protein G to collect only antibody and dissociate to obtain antibody Specific purification method.
  • 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 (an 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. It can be produced by collecting antibody-containing substances for overnight proteins and the like and separating and purifying the antibodies.
  • any antibody can be cross-linked at any ratio as long as antibodies can be efficiently produced against the hapten immunized by cross-linking with the carrier.
  • a method is used in which whole, limpet, and hemocyanin are coupled in a weight ratio of about 0.1 to 20, preferably about 1 to 5, with respect to hapten 1.
  • various condensing agents can be used for force coupling between the hapten and the carrier, but a dartalaldehyde, a carbodiimide, a maleimide active ester, an active ester reagent containing a thiol group or a dithioviridyl group, or the like is used. .
  • the condensation product is administered to a warm-blooded animal at a site where antibody production is possible, by itself or together with a carrier and a diluent.
  • Complete Freund's adjuvant / 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 preferably collected from blood, such as blood or ascites, of a mammal immunized by the above method.
  • the measurement of the polyclonal antibody titer in the antiserum can be performed in the same manner as the measurement of the antibody titer in the serum described above. Separation and purification of the polyclonal antibody can be carried out according to the same immunoglobulin separation and purification method as in the above-mentioned separation and purification of the monoclonal antibody.
  • the DNA encoding the receptor protein of the present invention or its salt, its partial peptide or its salt, and the receptor protein or its partial peptide is:
  • the binding of ligand to a G protein-coupled receptor specific to humans and mammals can be improved.
  • a compound to be changed eg, agonist, angstromist etc.
  • the agonist or angstromist can be used as an agent for preventing or treating various diseases.
  • a receptor protein or a partial peptide of the present invention or a salt thereof hereinafter sometimes abbreviated as the receptor protein of the present invention
  • a DNA encoding the receptor protein of the present invention or a partial peptide thereof hereinafter, referred to as The use of the antibody of the present invention (which may be 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 of the present invention or its salt. It is useful as a reagent for
  • the present invention is characterized in that the test compound is brought into contact with the receptor protein of the present invention or a salt thereof or the partial peptide of the present invention or a salt thereof.
  • the present invention provides a method for determining a ligand for the receptor protein of the present invention.
  • known ligands e.g., angiotensin, bombesin, canapinoid, cholecystokinin, glutamine, serotonin, melatonin, new oral peptide Y, opioid, purine, vasoprescin, oxotocin, PACAP (e.g., PACAP 27, PACAP 38), secretin, glucagon, calcitonin, adrenomedullin, somatos, GHRH, CRF, ACTH, GRP, PTH, VIP (Vasoactive Intestinal and Rerated Polypeptide), somatostin, dopamine , Motilin, amylin, bradykinin, CGRP (calcitonin gene-related peptide), leukotriene, pan creatinine, prostaglandin, tropoxane, adenosine, adrenaline, chemokine parper family
  • GRO GRO
  • Tissue extracts such as cell culture supernatant
  • the tissue extract, cell culture supernatant, and the like are added to the receptor protein of the present invention, and fractionated while measuring cell stimulating activity, etc., to finally obtain a single ligand. .
  • the ligand determination method of the present invention uses the receptor protein of the present invention or its partial peptide or a salt thereof, or constructs an expression system for a recombinant receptor protein, and Use the receptor binding assay system used And by binding to cell stimulating activity to the receptor protein of the present invention (e.g., ⁇ Rakidon acid release, Asechirukorin release, intracellular C a 2 + release, intracellular c AM P production, intracellular c GM P product, phosphatidylinositol A compound having an activity of promoting or inhibiting acid production, cell membrane potential fluctuation, intracellular protein phosphorylation, c-fos activation, pH reduction, etc. (eg, peptide, protein, non-peptidic compound) , Synthetic compounds, fermentation products, etc.) or salts thereof.
  • cell stimulating activity e.g., ⁇ Rakidon acid release, Asechirukorin release, intracellular C a 2 + release, intracellular c AM P production, intracellular c
  • a receptor compound of the present invention or a partial peptide thereof is brought into contact 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
  • a method for determining a ligand for a receptor protein of the present invention which comprises measuring the amount of binding of a labeled test compound to the receptor protein or a salt thereof;
  • a test compound When a test compound is brought into contact with a cell containing the receptor protein of the present invention, cell stimulating activity via receptor protein (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 phosphate
  • receptor protein 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 phosphate
  • a method for determining the ligand for the receptor protein or a salt thereof of the present invention which comprises measuring the activity of promoting or inhibiting c-fos activation, pH reduction, etc. , and
  • ⁇ ⁇ Via the receptor protein when the test compound is brought into contact with the receptor protein expressed on the cell membrane by culturing a transformant containing DNA encoding the receptor protein of the present invention.
  • cell stimulating activities e.g., Arakidon acid release, Asechirukorin release, intracellular C a 2 + release, intracellular c AM P production, intracellular c GM P, production of inositol phosphate, changes in cell membrane potential, phosphorus intracellular proteins
  • the activity of promoting or suppressing oxidation, activation of c-fos, reduction of pH, and the like) of the receptor of the present invention or a salt thereof. provide.
  • the receptor protein used in the ligand determination method may be any of the receptor proteins of the present invention described above or those containing the partial peptide of the present invention. Receptor proteins that are expressed in large amounts are suitable.
  • the expression method described above is used to produce the receptor protein of the present invention, but it is preferable to express the DNA encoding the receptor protein in mammalian cells or insect cells.
  • Complementary DNA is usually used as the DNA fragment encoding the protein portion of interest, but is not necessarily limited to this.
  • gene fragments or synthetic DNA may be used.
  • the DNA fragment In order to introduce a DNA fragment encoding the receptor protein of the present invention into host animal cells and express them efficiently, the DNA fragment must be transformed into a nucleopolyhedron belonging to a baculovirus using an insect as a host.
  • Nuclear polyliedros is virus (NPV) polyhedrin promoter, SV40-derived promoter, retrovirus promoter, metallothionein promoter, human heat shock promoter, cytomegalovirus promoter, SRa promoter Preferably, it is incorporated downstream, such as overnight.
  • NMV virus
  • SV40-derived promoter SV40-derived promoter
  • retrovirus promoter metallothionein promoter
  • human heat shock promoter cytomegalovirus promoter
  • SRa promoter a promoter
  • SRa promoter incorporated downstream, such as overnight.
  • Examination of the quantity and quality of the expressed receptor can be performed by a known method. For example, in the literature [Nambi, P. et al., The Journal of Biological Chemistry (J. Biol. Chem.), 267, 19555-19559, 1992].
  • the receptor protein of the present invention or a partial peptide thereof or a salt thereof includes the receptor protein or a partial peptide thereof or a salt thereof purified according to a known method. Or 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 method for determining a ligand of the present invention, the cells may be immobilized with dartalaldehyde, 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.
  • the host cell include Escherichia coli, Bacillus subtilis, yeast, insect cells, animal cells, and the like. Is used.
  • 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 crushed by crushing the cells with a Potter-Elvejem-type homogenizer, crushing with a Warlinda blender or a polytron (Kinematica), crushing with ultrasonic waves, or pressing with a French press. Crushing by ejecting cells from a thin nozzle may be mentioned.
  • 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 spun at a higher speed (15000 rpm to 30000 rpm) for usually 30 minutes. Centrifuge for ⁇ 2 hours, and use the resulting precipitate as the 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 in the cells containing the receptor protein and the membrane fraction thereof is preferably 10 3 to 10 8 molecules per cell, and more preferably 10 5 to 10 7 molecules per cell. It is suitable.
  • receptor protein fraction a natural receptor protein fraction or a recombinant receptor protein fraction having an activity equivalent thereto is desirable.
  • “equivalent activity” refers to equivalent ligand binding activity, signal transduction activity and the like.
  • CXC chemokine subfamily MCAF / MCP-1, MCP-2, MCP-3, MCP-4, eotaxin, RANTES, MIPla, MIP-1j3, HCC_1, MIP_3a / LARC, MIP-3 / ELC, 1—309, TARC, MI PF—1, MI PF-2 / eot ax in-2, MDC, DC-CK1 / PARC, SLC, etc.
  • CC chemokine subfamily 1 ymp hotactin, etc.
  • Chemokinein subfamily such as fracta 1 kine), endothelin, enterogastrin, histamine, neurotensin, TRH, pancreatic polypeptide, galanin, lysophosphatidic acid (LPA), Sphingosine monophosphate is preferred.
  • a receptor preparation is prepared by suspending the membrane fraction of cells or cells containing the receptor protein of the present invention in a buffer suitable for the determination method.
  • the buffer may be any buffer that does not inhibit the binding of the ligand to the receptor protein, such as a phosphate buffer of pH 4 to 10 (preferably pH 6 to 8) or a buffer of Tris-monohydrochloride. Good.
  • various proteins such as surfactants such as CHAPS, Tween-80 TM (Kao-Ichi Atlas), digitonin and dexcholate, serum albumin, and gelatin are used to reduce non-specific binding.
  • protease inhibitors such as PMS F, leptin, E-64 (manufactured by Peptide Research Institute), and peptidyltin can be added to suppress the degradation of receptors and ligands by proteases.
  • PMS F protein-specific silicate
  • leptin leptin
  • E-64 manufactured by Peptide Research Institute
  • peptidyltin can be added to suppress the degradation of receptors and ligands by proteases.
  • 0. to 0 1m l ⁇ 1 0m l of the receptions evening first solution a certain amount (50 0 0 c pm ⁇ 50 0 0 0 0 c pm) of [3 H], [125 I], [14 C] Co-exist with a test compound labeled with [ 35 S].
  • the reaction is carried out at about 0 to 50 ° C, preferably about 4 ° C to 37, 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 or the like, washed with an appropriate amount of the same buffer, and the radioactivity remaining on the glass fiber filter is measured using a liquid scintillation counter or an arc-counter.
  • a test compound having a count (B-NSB) exceeding 0 cpm obtained by subtracting the non-specific binding amount (NS B) from the total binding amount (B) is used as a ligand (agon) for the receptor protein of the present invention or a salt thereof.
  • Second strike can be selected.
  • the cell stimulating activity via the receptor protein for example, arachidonic acid release, acetylcholine release, intracellular release
  • Activity that promotes Ca 2+ release, intracellular c AMP generation, intracellular c GMP generation, inositol phosphate production, cell membrane potential fluctuation, intracellular protein phosphorylation, activation of c-fos, lowering of pH, etc. Or its inhibitory activity can be measured using a known method or a commercially available measurement kit. Specifically, first, cells containing the receptor protein are cultured on a multiwell plate or the like.
  • the medium Before determining the ligand, replace the medium with a fresh medium or an appropriate buffer that is not toxic to cells. After incubating for a certain period of time after adding the cells, extract the cells or collect the supernatant, and quantitate the produced product according to each method.
  • a substance for example, arachidonic acid
  • an inhibitor for the degrading enzyme may be added to perform the assay.
  • 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 includes a receptor protein of the present invention or a salt thereof, a partial peptide or a salt thereof of the present invention, and a cell containing the receptor protein of the present invention. Or a membrane fraction of cells containing the receptor protein of the present invention.
  • kits 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 ° C., 5% C 2 , and 95% air.
  • Ligands capable of binding to the receptor protein or its salts of the present invention include, for example, brain, large intestine, small intestine, knee, ovary, stomach, heart, liver, testis, placenta, lung, spinal cord, spleen , Thymus, kidney, duodenum, adrenal gland, prostate, pituitary gland, uterus, etc.
  • Specific examples include angiotensin, bombesin, canapinoid, cholecystokinin, glutamine, serotonin, and mela.
  • the receptor protein of the present invention or (2) the receptor protein of the present invention can be converted according to the action of the ligand.
  • the encoding DNA 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 physiological function of the ligand cannot be expected because the receptor protein of the present invention is reduced in a living body (the deficiency of the receptor protein protein).
  • the amount of receptor protein in the patient's body is increased by transplanting the cells into the patient, etc. Can be fully exhibited. That is, DNA encoding the receptor protein of the present invention is useful as a safe and low-toxic agent for preventing and / or treating diseases associated with dysfunction of the receptor protein of the present invention.
  • the receptor protein of the present invention is a G protein-coupled receptor protein, mouse GIR (Glucocorticoid-Induced Receptor) [Molecular 'Endocrinology (Mo 1. Endocrinol.), Vol. 5, pp. 1331-1338, 1991].
  • GIR Glucocorticoid-Induced Receptor
  • the DNA encoding the receptor protein or the receptor protein of the present invention includes, for example, central diseases (eg, Alzheimer's disease, dementia, eating disorders, etc.), endocrine diseases (eg, hypertension, gonad dysfunction, Thyroid dysfunction, pituitary dysfunction, etc.), metabolic disorders (eg, diabetes, lipid metabolism disorders, hyperlipidemia, etc.), cancers (eg, non-small cell lung cancer, ovarian cancer, prostate cancer, gastric cancer, bladder cancer, breast cancer) It is useful for the prevention and / or treatment of cervical cancer, colon cancer, rectal cancer, etc., and heart diseases (eg, angina pectoris, myocardial infarction, etc.).
  • central diseases eg, Alzheimer's disease, dementia, eating disorders, etc.
  • endocrine diseases eg, hypertension, gonad dysfunction, Thyroid dysfunction, pituitary dysfunction, etc.
  • metabolic disorders eg, diabetes, lipid metabolism disorders, hyperlipidemia, etc.
  • cancers eg, non-
  • the receptor protein of the present invention When used as the above-mentioned prophylactic / therapeutic agent, it can be formulated according to a conventional method.
  • DNA of the present invention when DNA encoding the receptor protein of the present invention (hereinafter sometimes abbreviated as DNA of the present invention) is used as the above-mentioned prophylactic or therapeutic agent, the DNA of the present invention may be used alone or in a retrovirus vector or adenovirus. After insertion into a suitable vector such as a viral vector or an adenovirus associated virus vector, the method 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, by a power gun such as a gene gun or a hydrogel catheter.
  • a power gun such as a gene gun or a hydrogel catheter.
  • the receptor protein of the present invention or (2) DNA encoding the receptor protein may be orally administered as sugar-coated tablets, capsules, elixirs, microcapsules, etc., if necessary. It can be used parenterally in the form of injections, such as sterile solutions with water or other pharmaceutically acceptable liquids, or suspensions.
  • Swelling agent such as Lubricants such as magnesium stearate, sweetening agents such as sucrose, lactose or saccharin, flavoring agents such as peppermint, cocoa oil or cellulose are used.
  • 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 by dissolving or suspending the active substance in a vehicle such as water for injection, or naturally occurring vegetable oils such as sesame oil or 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.
  • an alcohol e.g., ethanol
  • Poriaruko Lumpur eg, propylene glycol, polyethylene glycol
  • nonionic surface active agents e.g., polysorbate one DOO 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 / therapeutic agents examples include, for example, buffers (for example, phosphate buffer and sodium acetate buffer), soothing agents (for example, benzalkonium chloride, pro-active acid 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, pro-active acid hydrochloride, etc.
  • stabilizers for example, 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, human and mammals (for example, rats, mice, rabbits, sheep, bush, birds, cats, dogs, monkeys, etc.) Can be administered.
  • human and mammals for example, rats, mice, rabbits, sheep, bush, birds, cats, dogs, monkeys, etc.
  • 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 in general, for example, in a patient with cancer (assuming 60 kg), one day From about 0.1 mg to about 100 mg, preferably from about 1.0 to 5 Omg, more preferably from about 1.0 to 2 Omg.
  • parenteral administration 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). 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 Okg.
  • the dosage of the DNA of the present invention 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 6 Okg), one day About 0.1 mg to 100 mg, preferably about 1.0 to 50 mg, and 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 usually, for example, a cancer patient (60 kg)
  • the amount converted per 60 kg can be administered.
  • the DNA of the present invention can be used as a probe to produce the receptor of the present invention in a human or non-human mammal (eg, rat, mouse, mouse, egret, sheep, bush, horse, cat, dog, monkey, etc.).
  • An abnormality (gene abnormality) in DNA or mRNA encoding one protein or a partial peptide thereof can be detected, for example, damage, mutation or reduced expression of the DNA or mRNA, or DNA or mRNA. It is useful as an agent for genetic diagnosis of increased RNA or overexpression.
  • the above-described genetic diagnosis using the DNA of the present invention includes, for example, known Northern hybridization and PCR-SSCP method (Genomics, Vol. 5, pp. 874-879 (1989), Procedings Procedings of the National Academy of Sciences of the United States of America, Vol. 86, pp. 2766-2770 (1989)) Can be implemented.
  • the DNA of the present invention can be used as a probe to screen for a compound that changes the expression level of the receptor protein of the present invention or a partial peptide thereof. Can be used.
  • the present invention relates to, for example, (i) non-human mammal blood, (2) specific organ, (3) tissue or cells isolated from the organ, or (ii) the receptor of the present invention contained in a transformant or the like.
  • the present invention provides a method for screening a compound that changes the expression level of the receptor protein or its partial peptide of the present invention by measuring the mRNA amount of the protein or its partial peptide.
  • the measurement of the amount of mRNA of the receptor protein of the present invention or its partial peptide is specifically performed as follows.
  • non-human mammals e.g., mice, rats, rabbits, sheep, sheep, bush, horses, cats, dogs, monkeys, etc., more specifically, dementia rats, obese mice, 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.) ), And after a certain period of time, obtain blood or a specific organ (eg, brain, lung, colon, etc.), or a tissue or cell isolated from the organ.
  • a specific organ eg, brain, lung, colon, etc.
  • mRNA of the receptor protein of the present invention or its partial peptide contained in the obtained cells for example, mRNA is extracted from cells or the like by a conventional method, and for example, a technique such as TaQMan PCR is used. Thus, it can be analyzed by performing Northern blot by a known method.
  • 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. Can be quantified and analyzed in the same manner.
  • 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 drug or physical 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 ) Quantifying and analyzing the mRNA amount of the receptor protein of the present invention or its partial peptide contained in the cells,
  • test compound is mixed with the medium, and after culturing for a certain period of time (from 1 day to 7 days, preferably from 1 day to 3 days, more preferably after 2 days) 3 days later) by quantifying and analyzing the mRNA amount of the receptor protein of the present invention or a partial peptide thereof 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 or its partial peptide of the present invention.
  • the cell stimulating activity via G protein-coupled receptor protein for example, arachidonic acid release, acetylcholine release, intracellular Ca 2+ Promotes release, intracellular cAMP generation, intracellular cGMP generation, inositol phosphate production, fluctuations in cell membrane potential, phosphorylation of intracellular proteins, activation of c-fos, decrease in pH, etc.
  • G protein-coupled receptor protein for example, arachidonic acid release, acetylcholine release, intracellular Ca 2+ Promotes release, intracellular cAMP generation, intracellular cGMP generation, inositol phosphate production, fluctuations in cell membrane potential, phosphorylation of intracellular proteins, activation of c-fos, decrease in pH, etc.
  • Examples of the compound include a peptide, a protein, a non-peptidic compound, a synthetic compound, a fermentation product, and the like. 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 of the present invention or the like.
  • 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 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 have low toxicity, so they can be used, for example, in humans and mammals (for example, rats, mice, rabbits, sheep, pigs, pigs, 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.
  • oral administration in general, for example, in a cancer patient (as 60 kg), It is about 0.1-100 mg per day, preferably about 1.0-50 mg, more preferably about 1.0-20 mg.
  • parenteral administration the single dose varies depending on the administration target, target organ, symptoms, administration method, etc.
  • injection it is usually used, for example, in cancer patients (60 kg).
  • the dose can be administered in terms of 60 kg.
  • the receptor protein of the present invention can be used in various tissues (eg, brain, large intestine, small intestine, knee, ovary, stomach, heart, liver, testis, placenta, lung, spinal cord, spleen, thymus, kidney, duodenum) , Adrenal gland, prostate, pituitary gland, uterus, etc.). Therefore, the compound of the present invention that alters the expression level of the receptor protein or its partial peptide can be used as a prophylactic and / or therapeutic agent for diseases 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 tablet, capsule, elixir, microcapsule, or the like, if necessary, orally or aseptic solution with water or other pharmaceutically acceptable liquid, Alternatively, it can be used parenterally in the form of injections such as suspensions.
  • known carriers, flavoring agents, and excipients which are physiologically acceptable for the compound It can be manufactured by admixing it with agents, vehicles, preservatives, stabilizers, binders and the like in the unit dosage form generally required for the practice of formulations. 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 by dissolving or suspending the active substance in a vehicle such as water for injection, or naturally occurring vegetable oils such as sesame oil or coconut oil. it can.
  • a vehicle such as water for injection, or naturally occurring vegetable oils such as sesame oil or coconut oil.
  • a 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.
  • an alcohol e.g., ethanol
  • Poriaruko - Le eg, propylene glycol, polyethylene glycol
  • nonionic surface active agents e.g., 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 benzo
  • prophylactic / therapeutic agent examples include a buffer (for example, a phosphate buffer and a sodium acetate buffer), a soothing agent (for example, benzalkonium chloride, proactive hydrochloride, etc.), a stabilizer (for example, , Human serum albumin, polyethylene glycol, etc.), preservatives (eg, benzyl alcohol, phenol, etc.), antioxidants and the like.
  • a buffer for example, a phosphate buffer and a sodium acetate buffer
  • a soothing agent for example, benzalkonium chloride, proactive hydrochloride, etc.
  • a stabilizer for example, 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 have low toxicity, so they can be used, for example, in humans and mammals (for example, rats, mice, rabbits, sheep, pigs, pigs, cats, dogs, monkeys, etc.). Can be administered.
  • the dose of the compound or its salt depends on the administration target, target organ, symptoms, administration method, etc.
  • administration target in general, for example, in cancer patients (as 60 kg), about 0.1 to 100 mg, preferably about 1.0 to 50 mg, more preferably about 1 to 50 mg per day 0 to 20 mg.
  • parenteral administration the single dose varies depending on the administration target, target organ, symptoms, administration method, etc., for example, usually in the form of injection, for example, in cancer patients (60 kg) It is convenient to administer about 0.01 to 3 Omg per day, preferably about 0.1 to 2 Omg, more preferably about 0.1 to 1 Omg per day by intravenous injection. For other animals, the dose can be administered in terms of 60 kg.
  • the receptor protein of the present invention has a binding property to a ligand, the ligand concentration in a living body can be quantified 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 in accordance with the method described in (1) or (2) below or a method analogous thereto.
  • a method for screening a compound (eg, agonist, angonist, etc.) that changes the binding property between a G protein-coupled receptor protein and a ligand of the present invention By constructing an expression system for an evening protein or the like and using a receptor evening binding system using the expression system, a compound that changes the binding between a ligand and the receptor protein of the present invention (for example, Peptides, proteins, non-peptidic compounds, synthetic compounds, fermentation products, etc.) or salts thereof can be efficiently screened.
  • a compound that changes the binding between a ligand and the receptor protein of the present invention for example, Peptides, proteins, non-peptidic compounds, synthetic compounds, fermentation products, etc.
  • Such compounds include (a) cell stimulation via G protein-coupled receptor Activity (e.g., Arakidon acid release, acetylcholine release, intracellular C a 2 + release, intracellular c AM P production, intracellular c GMP production, inositol phosphate production, change in cell membrane potential, phosphorylation of intracellular proteins, c one : a compound having an activity of promoting or suppressing the activation of fos, a decrease in pH, etc.
  • G protein-coupled receptor Activity e.g., Arakidon acid release, acetylcholine release, intracellular C a 2 + release, intracellular c AM P production, intracellular c GMP production, inositol phosphate production, change in cell membrane potential, phosphorylation of intracellular proteins
  • c one a compound having an activity of promoting or suppressing the activation of fos, a decrease in pH, etc.
  • agonist against the receptor protein of the present invention A compound that does not have (a so-called angonist for the receptor protein of the present invention), (8) a compound that enhances the binding force between the ligand and the G protein-coupled receptor protein of the present invention, or (2) a ligand And the G protein-coupled receptor protein of the present invention, and the like. Screening is preferably performed by the ligand determination method described above).
  • the present invention relates to (i) the case where the receptor protein of the present invention or its partial peptide or a salt thereof is brought into contact with a ligand; and (ii) the protein or its partial peptide of the present invention or its partial peptide.
  • a method for screening the salt is provided.
  • the screening method of the present invention is characterized in that, in the cases (i) and (ii), for example, the amount of a ligand bound to the receptor protein or the like, the cell stimulating activity, etc. are measured and compared. . More specifically, the present invention provides
  • a compound that activates the receptor protein of the present invention is contacted with a cell containing the receptor protein of the present invention.
  • Receptor-mediated cell stimulating activity eg, arachidonic acid release, acetylcholine
  • a compound or test compound that activates the receptor protein or the like of the present invention is brought into contact with cells containing the receptor protein or the like of the present invention.
  • a method of screening a compound or its salt that alters the binding and
  • a compound that activates the receptor protein of the present invention (eg, a ligand for the receptor protein of the present invention) is cultured on a cell membrane by culturing a transformant containing the DNA of the present invention.
  • a cell membrane is obtained by culturing a transformant containing the DNA of the present invention with a compound which activates the receptor protein of the present invention or the like and a test compound when activating the receptor protein or the like of the present invention.
  • the cell stimulating activity mediated by the receptacle Puta e.g., Arakidon acid release, acetylcholine release, intracellular C a 2 + release, intracellular c AM P product, cells C GMP production, inositol phosphate production, cell membrane potential fluctuation, intracellular protein phosphorylation, activation of c-fos, p
  • a method for screening a compound or a salt thereof that changes the binding property between the ligand and the receptor protein of the present invention or the like. e.g., Arakidon acid release, acetylcholine release, intracellular C a 2 + release, intracellular c AM P product, cells C GMP production, inositol phosphate production, cell membrane potential fluctuation, intracellular protein phosphorylation, activation of c-fos, p
  • a method for screening a compound or a salt thereof that changes the binding property between the ligand and the receptor protein of the present invention or the like. e.
  • a cell, tissue or cell membrane containing a G protein-coupled receptor protein such as a rat is screened.
  • a test to confirm whether the candidate compound actually inhibits the binding of the human G protein-coupled receptor protein to the ligand was required. If the cell, tissue, or cell membrane fraction is used as it is, other receptor proteins are also mixed, so it was difficult to actually screen for an agonist or an antagonist for the target receptor protein.
  • the receptor protein of the present invention by using the receptor protein of the present invention, primary screening is not required, and a compound that inhibits binding between a ligand and a G protein-coupled receptor protein can be efficiently screened. Furthermore, it is possible to easily evaluate whether the screened compound is an agonist or an engonist.
  • the receptor protein or the like of the present invention used in the screening method of the present invention may be any as long as it contains the above-mentioned receptor protein of the present invention.
  • Cell membrane fractions of mammalian organs containing the receptor protein of the invention and the like are preferred.
  • human-derived receptor proteins expressed in large amounts using recombinants and the like are suitable for screening.
  • the method described above is used to produce the receptor protein and the like of the present invention. It is preferable to carry out the method by expressing the DNA of the present invention in mammalian cells and insect cells.
  • Complementary DNA is used as the DNA fragment to be encoded, but is not necessarily limited thereto.
  • gene fragments or synthetic DNA may be used.
  • a DNA fragment encoding the receptor protein of the present invention is transferred to a host. In order to introduce the DNA fragment into the target cells and to express them efficiently, the DNA fragment must be expressed by the polyhedrin promoter of nuclear polyhedrosis virus (NPV) belonging to the baculovirus, which is home to insects.
  • NPV nuclear polyhedrosis virus
  • the promoter into the downstream of a promoter derived from SV40, a retrovirus promoter, a metamouth thionein promoter, a human heat shock promoter, a cytomegalovirus promoter, and an SR promoter. Inspection of the amount and quality of the expressed receptor can be performed 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, pp. 19555-19559, 1992]. .
  • the protein containing the receptor protein of the present invention may be a receptor protein or the like purified according to a known method, or may be a receptor protein or the like. May be used, or a membrane fraction of cells containing the receptor protein or the like may be used.
  • the cells When cells containing the receptor protein or the like 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.
  • the cells containing the receptor protein and the like of the present invention include host cells that express the receptor protein and the like, and the host cells include Escherichia coli, Bacillus subtilis, yeast, and insect cells. Animal cells and the like are preferred.
  • 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 disrupted by crushing the cells with a Potter-E lvehjem homogenizer, crushing with a single ring blender or polytron (Kinematica), crushing with ultrasonic waves, pressing the cells while pressing with a French press, etc. And crushing by jetting from a thin nozzle.
  • 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 300 rpm) for a short time (usually about 1 to 10 minutes), and the supernatant is further centrifuged at a high speed (1500 to 300 rpm). (000 rpm) for 30 minutes to 2 hours, and the resulting precipitate is used as a membrane fraction.
  • the membrane fraction the expressed receptor protein and the like and cell-derived phospholipids and membrane proteins were contained. It contains a lot of membrane components such as protein.
  • the amount of receptor protein in a cell or membrane fraction containing the receptor protein or the like is preferably 10 3 to 10 8 molecules per cell, and more preferably 10 5 to 10 7 molecules per cell. .
  • an appropriate receptor protein fraction and a labeled ligand are required. It is.
  • receptor protein fraction a natural receptor protein fraction or a recombinant receptor protein fraction having the same activity as that of the receptor is desirable. Indicates equivalent ligand binding activity, signal transduction action, etc.
  • labeled ligand a labeled ligand, a labeled ligand analog compound, or the like is used.
  • a labeled ligand for example [3 H], [125 I], [14 C], etc.
  • a cell containing the receptor protein of the present invention or a membrane fraction of the cell is used.
  • the buffer may be any buffer such as a phosphate buffer having a pH of 4 to 10 (preferably pH 6 to 8) or a buffer such as a tris-monohydrochloride buffer which does not inhibit the binding between the ligand and the receptor protein.
  • Surfactants such as CHAPS, Tween-80 TM (Kao-Atlas), digitonin, and dexcholate can also be added to the buffer to reduce non-specific binding.
  • a protease inhibitor such as PMSF, leptin, E_64 (manufactured by Peptide Research Laboratories), and pepstatin can be added for the purpose of suppressing the degradation of receptor and ligand by protease.
  • a protease inhibitor such as PMSF, leptin, E_64 (manufactured by Peptide Research Laboratories), and pepstatin can be added for the purpose of suppressing the degradation of receptor and ligand by protease.
  • the reaction is carried out at about O to 50 ° (: desirably at about 4 ° C to 37 ° C, for about 20 minutes to 24 hours, desirably for about 30 minutes to 3 hours.
  • filter with a glass fiber filter paper, etc. after washing with the same buffer and foremost, the radioactivity you remain on the glass fiber filter paper is then measured by a liquid scintillation counter evening one or ⁇ over the counter. count in the absence of substances that antagonize nonspecific binding amount from the (B Q) (NSB ) Is subtracted from the count (B.-NSB) as 100%.
  • a test compound with a specific binding amount (B-NSB) of 50% or less is selected as a candidate substance with competitive inhibition ability. be able to.
  • a cell stimulating activity via receptor protein 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, Activity that promotes or suppresses a decrease in pH, etc.
  • a cell stimulating activity via receptor protein 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, Activity that promotes or suppresses a decrease in pH, etc.
  • ⁇ Measurement can be performed using a known method or a commercially available measurement kit.
  • cells containing the receptor protein or the like of the present invention are cultured on a multiwell plate or the like.
  • replace the cells with a fresh medium or an appropriate buffer that is not toxic to the cells, add the test compound, etc., incubate for a certain period of time, extract the cells, or collect the supernatant.
  • the products produced are quantified according to the respective method. If the production of a substance (for example, arachidonic acid) as an indicator of the cell stimulating activity is difficult to be assayed by a degrading enzyme contained in a cell, an inhibitor for the degrading enzyme may be added to perform the assay.
  • activities such as cAMP production suppression can be detected as a production suppression effect on cells whose basic production has been increased by forskolin or the like.
  • Cells expressing an appropriate receptor protein are required.
  • Cells expressing the receptor protein or the like of the present invention include a cell line having the natural receptor of the present invention or the like, Cell lines expressing the recombinant receptor protein and the like are desirable.
  • test compounds for example, peptides, proteins, non-peptidic compounds, synthetic compounds, fermentation products, cell extracts, plant extracts, animal tissue extracts, etc. are used. Or a known compound.
  • a screening kit for a compound or a salt thereof that alters the binding between the ligand and the receptor protein of the present invention or the like includes cells containing the receptor protein of the present invention, the receptor protein of the present invention, etc. Or those containing the membrane fraction of cells containing the receptor protein of the present invention.
  • Examples of the screening kit 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 ° C., 5% CO 2 and 95% air.
  • the compound or a salt thereof obtained by using the screening method or the screening kit of the present invention is a compound having an action of changing the binding property between the ligand and the receptor protein of the present invention.
  • Cell stimulating activity eg, arachidonic acid release, acetylcholine release, intracellular Ca 2+ release, intracellular cAMP production, intracellular cGMP production, inositol phosphate production, (A so-called agonist against the receptor protein of the present invention) having an activity of promoting or suppressing cell membrane potential fluctuation, intracellular protein phosphorylation, activation of c-fos, reduction of pH, etc.
  • a compound not having the cell stimulating activity (so-called an antagonist to the receptor protein of the present invention) (8) a compound that enhances the binding force between the ligand and the G protein-coupled receptor protein of the present invention, or (2) reduces the binding force between the ligand and the G protein-coupled 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. These compounds may be novel compounds or known compounds.
  • the agonist against the receptor protein of the present invention etc. has the same activity as the physiological activity of the ligand for the receptor protein etc. of the present invention. Useful as a medicine.
  • Angonist against the receptor protein etc. of the present invention can suppress the physiological activity of the ligand for the receptor protein etc. of the present invention, and thus is useful as a safe and low toxic drug for suppressing the ligand activity. .
  • a compound that enhances the binding force between the ligand and the G protein-coupled receptor of the present invention is a safe and low-toxic compound for enhancing the physiological activity of the ligand for the receptor protein or the like of the present invention. Useful as a medicine.
  • a compound that reduces the binding force between the ligand and the G protein-coupled receptor protein of the present invention is a safe and low-toxic drug for reducing the physiological activity of the ligand for the receptor protein of the present invention and the like.
  • a compound or a salt thereof obtained by using the screening method or screening kit of the present invention is used as the above-mentioned pharmaceutical composition, it can be carried out according to a conventional method.
  • disintegrants, capsules, elixirs, microcapsules, sterile solutions, suspensions, and the like can be prepared in the same manner as in the above-described medicine containing the receptor protein of the present invention.
  • the preparations obtained in this way are safe and low toxic, for example, against human mammals (for example, rats, puppies, higgs, bushes, cats, 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.
  • oral administration for example, in a cancer patient (as 6 O kg)
  • one dose is generally used.
  • parenteral administration the single dose varies depending on the administration target, target organ, symptoms, administration method, etc.
  • injection it is usually used, for example, in cancer patients (as 60 kg). It is convenient to administer about 0.01 to 3 Omg per day, preferably about 0.1 to 2 Omg, more preferably about 0.1 to 1 Omg per day by intravenous injection. is there.
  • the dose can be administered in terms of 60 kg.
  • a preventive and / or therapeutic agent for various diseases containing a compound (agonist, angonist) that changes the binding property between a G protein-coupled receptor protein and a ligand of the present invention.
  • the receptor protein of the present invention is considered to play some important roles in vivo, such as central function, circulatory function, digestive function, and cardiac function. Therefore, compounds (agonists, antagonists) that alter the binding property between the receptor protein of the present invention and the ligand and ligands for the receptor protein of the present invention may be diseases associated with dysfunction of the receptor protein of the present invention. Can be used as a prophylactic and / or therapeutic agent for Z.
  • central diseases for example, Alzheimer's disease, dementia, eating disorders, etc.
  • endocrine diseases for example, hypertension, gonad dysfunction, thyroid dysfunction, pituitary dysfunction, etc.
  • metabolic diseases for example, diabetes
  • cancer for example, non-small cell lung cancer, ovarian cancer, prostate cancer, stomach cancer, bladder cancer, breast cancer, cervical cancer, colon cancer, rectal cancer, etc.
  • heart disease for example, it can be used as a prophylactic and / or therapeutic agent for angina pectoris, myocardial infarction, etc.
  • the compound or ligand when used as a prophylactic and / or therapeutic agent for a disease associated with dysfunction of the receptor protein of the present invention, it can be formulated according to conventional means.
  • the compound or ligand can be sterilized with tablets or capsules, elixirs, microcapsules, or the like, which are sugar-coated as necessary, or with water or other pharmaceutically acceptable liquids. It can be used parenterally in the form of injections, such as solutions or suspensions.
  • the compound is mixed with known physiologically acceptable carriers, flavoring agents, excipients, vehicles, preservatives, stabilizers, binders, and the like in the unit dosage form generally required for the practice of pharmaceutical preparations. Can be manufactured. The amount of the active ingredient in these preparations is such that a suitable dosage in the specified range can be obtained.
  • Swelling agent such as Lubricants such as magnesium stearate, sweeteners such as sucrose, lactose or saccharin, flavoring agents such as peppermint, cocoa oil or cellulose are used.
  • 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 by dissolving or suspending the active substance in a vehicle such as water for injection, or naturally occurring vegetable oils such as sesame oil or coconut oil. it can.
  • aqueous solution for injection for example, physiological saline, isotonic solution containing glucose and other auxiliary agents (eg, D-sorbitol, D-mannitol, sodium salt, etc.) and the like are used.
  • dissolution aid such as an alcohol (e.g., ethanol), Poriaruko Lumpur (eg, propylene glycol, polyethylene glycol), nonionic surface active agents (e.g., polysorbate 8 0 TM, HCO - 5 0 ) , such as a combination You may.
  • 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 (for example, phosphate buffer and sodium acetate buffer), soothing agents (for example, benzalkonium chloride, pro-active acid 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, pro-active acid hydrochloride, etc.
  • stabilizers for example, Human serum albumin, polyethylene glycol, etc.
  • preservatives eg, benzyl alcohol, phenol, etc.
  • antioxidants antioxidants and the like.
  • the above-mentioned prophylactic / therapeutic agent can be used in combination with an appropriate drug, for example, as a DDS product in which an organ or tissue in which the receptor protein of the present invention is highly expressed is specifically obtained.
  • the preparations obtained in this way are safe and low toxic, for example, in humans (eg, rats, mice, egrets, sheep, pigs, pests, 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 for example, in cancer patients (as 60 kg)
  • the It is about 0.1 to 100 mg per day, preferably about 1.0 to 50 mg, more preferably about 1.0 to 20 mg.
  • parenteral administration the single dose varies depending on the administration target, target organ, symptoms, administration method, etc., for example,
  • injections for example, in cancer patients (as 60 kg), about 0.01 to 3 Omg per day, preferably about 0.1 to 2 Omg, more preferably about 0.1 to 1 Omg per day It is convenient to administer about Omg by intravenous injection.
  • the dose can be administered in terms of 60 kg.
  • the antibody of the present invention can specifically recognize the receptor protein or the like of the present invention, it is used for quantification of the receptor protein of the present invention in a test solution, particularly for quantification by sandwich immunoassay. can do. That is, the present invention, for example,
  • test solution characterized by reacting the antibody of the present invention with a test solution and a labeled receptor protein, etc., and measuring the ratio of the labeled receptor protein bound to the antibody;
  • the present invention provides a method for quantifying the receptor protein of the present invention in a test solution, characterized by the following features.
  • one of the antibodies is an antibody that recognizes the N-terminal of the receptor protein of the present invention or the like, and the other antibody is an antibody that reacts with the C-terminal of the receptor protein or the like of the present invention. Is preferred.
  • the monoclonal antibody of the present invention In addition to measuring the receptor protein of the present invention using a monoclonal antibody against the receptor protein of the present invention (hereinafter sometimes referred to as the monoclonal antibody of the present invention), detection by tissue staining or the like is also possible. it can.
  • the antibody molecule itself may be used, or F (ab ') 2 , Fab', or Fab fraction of the antibody molecule may be used.
  • the assay method using an antibody against the receptor protein or the like of the present invention is not particularly limited, and may be an antibody, antigen or antibody corresponding to the antigen amount (for example, receptor protein amount) in the test solution.
  • Any method that detects the amount of the antigen complex by chemical or physical means and calculates this from a standard curve prepared using a standard solution containing a known amount of antigen can be used. May be used.
  • nephrometry, competition method, immunometric method, and sandwich method are suitably used, but from the viewpoint of sensitivity and specificity, it is particularly preferable to use the San German 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.
  • the radioisotope for example, [1 2 5 I], [1 3 1 I], [3 H], and [1 4 C] used.
  • the enzyme a stable enzyme having a large specific activity is preferable.
  • the fluorescent substance for example, fluorescamine, fluorescein isothiosinate and the like are used.
  • the luminescent substance for example, luminol, a luminol derivative, reluciferin, lucigenin and the like are used.
  • a biotin-avidin system can be used for binding the antibody or antigen to the labeling agent.
  • physical adsorption may be used, or a method using a chemical bond usually used for insolubilizing and immobilizing proteins, enzymes, and the like may be used.
  • the carrier for example, insoluble polysaccharides such as agarose, dextran, and cellulose; synthetic resins such as polystyrene, polyacrylamide, and silicone; and glass are used.
  • a 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 does not necessarily need to be one kind, and a mixture of two or more kinds of antibodies is used for the purpose of improving measurement sensitivity and the like. May be used.
  • the monoclonal antibody of the present invention used in the primary reaction and the secondary reaction has different binding sites for the receptor protein and the like. Used. That is, primary reaction And the antibody used in the secondary reaction is, for example, if the antibody used in the secondary reaction recognizes the C-terminal of the receptor protein, the antibody used in the primary reaction is preferably the C-terminal For example, an antibody that recognizes an N-terminal other than the N-terminal is used.
  • the monoclonal antibody of the present invention can be used in a measurement system other than the sandwich method, for example, a competitive method, an immunometric method, or a nephrometry method.
  • a competitive method an antigen in a test solution and a labeled antigen are allowed to react competitively with an antibody, and then the unreacted labeled antigen, (F) and the labeled antigen (B) bound to the antibody are separated.
  • BZF separation The amount of any of B and F is measured, and the amount of antigen in the test solution is quantified.
  • a soluble antibody is used as the antibody
  • B / F separation is performed using polyethylene glycol
  • a liquid phase method using a second antibody to the above antibody or a solid phase antibody is used as the first antibody.
  • An immobilization method using a soluble first antibody and an immobilized antibody as the second antibody is used.
  • the antigen in the test solution and the immobilized antigen are subjected to a competitive reaction with a fixed amount of the labeled antibody, and then the solid phase and the liquid phase are separated.
  • the antigen is allowed to react with an excessive amount of the labeled antibody, then the immobilized antigen is added, and the unreacted labeled antibody is bound to the solid phase, and then the solid phase and the liquid phase are separated.
  • the amount of label in either phase is measured, and the amount of antigen in the test solution is quantified.
  • nephrometry the amount of insoluble sediment generated as a result of an antigen-antibody reaction in a gel or in a solution is measured. Even when the amount of antigen in the test solution is small and only a small amount of sediment is obtained, laser-nephrometry utilizing laser scattering is suitably used.
  • the receptor protein of the present invention or its salt can be quantified with high sensitivity by using the antibody of the present invention.
  • the antibody of the present invention can be used for specifically detecting the receptor protein of the present invention and the like present in a subject such as a body fluid or a tissue.
  • preparation of an antibody column used for purifying the receptor protein and the like of the present invention, detection of the receptor of the present invention in each fraction at the time of purification, and detection of the receptor of the present invention in test cells It can be used for analysis of the behavior of one protein.
  • the antibody of the present invention can specifically recognize the receptor protein of the present invention or its partial peptide or a salt thereof, the amount of the receptor protein of the present invention in the cell membrane or the amount of its partial peptide can be recognized. Can be used for screening for compounds that change
  • the cell membrane fraction is isolated, and the receptor protein of the present invention contained in the cell membrane fraction or a portion thereof 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 quantifying the peptide,
  • 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 a cell membrane by confirming the protein on the cell membrane by quantifying the degree of staining.
  • 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 a cell membrane by confirming the protein on the cell membrane.
  • the quantification 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 for example, mice, rats, puppies, higgs, bush, puppies, cats, dogs, monkeys, etc .; more specifically, dementia rats, obese mice
  • 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
  • blood or specific organs eg, brain, lung, colon, etc.
  • tissues or cells isolated from the organs are obtained.
  • the obtained organs, tissues or cells are suspended in, for example, an appropriate buffer (for example, Tris-HCl buffer, phosphate buffer, Hess buffer, etc.) and the organs, tissues or cells are suspended.
  • an appropriate buffer for example, Tris-HCl buffer, phosphate buffer, Hess buffer, etc.
  • Rupture, detergents eg, Triton XI 00 TM , Chromatography such as down 2 0 TM
  • the cell membrane fraction have use a technique such as column fractionation.
  • 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, crushing with a Warlinda blender or a polytron (Kinematica), crushing with ultrasonic waves, or pressing with a French press. While crushing by ejecting cells from a fine nozzle.
  • 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 300 rpm) for a short time (usually about 1 to 10 minutes), and the supernatant is further spun at a high speed (150 rpm to 150 rpm). The mixture is centrifuged at 300 rpm for 30 minutes to 2 hours, and the resulting precipitate is used as a membrane fraction.
  • the membrane fraction is rich in 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 ⁇ stan blot can be performed by known means.
  • a transformant expressing the receptor protein of the present invention or its partial peptide is prepared according to the above method, and the receptor protein or its partial peptide of the present invention contained in the cell membrane fraction is prepared. It can be quantified.
  • 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:
  • a given time before drug or physical stress is given to a normal or disease model non-human mammal (30 minutes to 24 hours before, preferably 30 minutes to 12 hours before, More preferably 1 hour to 6 hours before) or after a certain time (30 minutes to 3 days, preferably 1 hour to 2 days, more preferably 1 hour to 24 hours), or a drug or physical
  • the test compound is administered at the same time as the target stress, and after a lapse of a certain time after administration (30 minutes to 3 days, preferably 1 hour to 2 days, more preferably 1 hour After 24 hours) can be carried out by quantifying the amount of the receptor protein of the present invention or its partial peptide in the cell membrane,
  • test compound is mixed with the medium, and after culturing for a certain period of time (from 1 day to 7 days, preferably from 1 day to 3 days, more preferably after 2 days) 3 days later) by quantifying the amount of the receptor protein of the present invention or its partial peptide in the cell membrane.
  • non-human mammals for example, mice, rats, rabbits, higgs, bushus, horses, cats, dogs, monkeys, etc., more specifically, dementia rats, obese mice, atherosclerosis Drugs (eg, anti-dementia drugs, anti-hypertensive drugs, anti-cancer drugs, anti-obesity drugs, etc.) or physical stress (eg, flooding stress, electric shock, light, low temperature, etc.)
  • drugs eg, anti-dementia drugs, anti-hypertensive drugs, anti-cancer drugs, anti-obesity drugs, etc.
  • physical stress eg, flooding stress, electric shock, light, low temperature, etc.
  • blood, or specific organs e.g., brain, large intestine, small intestine, england, ovary, stomach, heart, liver, testis, placenta, lung, spinal cord, spleen, thymus, kidney, Obtain tissues or cells isolated from the duodenum, adrenal gland, prostate, pit
  • the obtained organs, tissues or cells are cut into tissue sections according to a conventional method, and immunostained using the antibody of the present invention.
  • the receptor protein of the present invention in the cell membrane or The amount of the partial peptide can be confirmed.
  • 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 the cell membrane.
  • the cell stimulating activity via G protein-coupled receptor receptor for example, arachidonic acid release, acetylcholine release, intracellular Ca 2+ release, Intracellular cAMP generation, Intracellular cGMP generation, Compounds that enhance or inhibit toll phosphate production, fluctuations in cell membrane potential, phosphorylation of intracellular proteins, activation of C-fOS, reduction of pH, etc.
  • the compound include a peptide, a protein, a non-peptidic compound, a synthetic compound, a fermentation product, and the like. 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 of the present invention.
  • 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 have low toxicity, for example, in humans and non-human mammals (eg, rats, mice, rabbits, sheep, sheep, bush, birds, cats, dogs, monkeys, etc.) Can be administered.
  • non-human mammals eg, rats, mice, rabbits, sheep, sheep, bush, birds, cats, dogs, monkeys, etc.
  • the dose of the compound or a salt thereof varies depending on the administration subject, target organ, symptoms, administration method, and the like. However, in the case of oral administration, for example, in a patient with cancer (as 60 kg), one dose is required. From about 0.1 to about L: 0 mg, preferably from about 1.0 to 50 mg, more preferably from about 1.0 to 20 mg per day.
  • the single dose varies depending on the subject of administration, target organ, symptoms, administration method, etc.
  • it is usually used, for example, in cancer patients (as 6 O kg). It is convenient to administer about 0.01 to 3 Omg per day, preferably about 0.1 to 2 Omg, more preferably about 0.1 to 1 Omg per day by intravenous injection. is there.
  • the dose can be administered in terms of 60 kg.
  • the receptor protein of the present invention can be used in various tissues (eg, brain, large intestine, small intestine, knee, ovary, stomach, heart, liver, testis, placenta, lung, spinal cord, spleen, thymus, kidney, It may play some important role in the duodenum, adrenal gland, prostate, pituitary, uterus, etc. Therefore, a compound that alters the amount of the receptor protein of the present invention or its partial peptide in the cell membrane can be used as an agent for preventing and / or treating a disease associated with dysfunction of the receptor protein of the present invention. When the compound is used as a prophylactic and / or therapeutic agent for a disease associated with dysfunction of the receptor protein of the present invention, it can be formulated according to conventional methods.
  • 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 other pharmaceutically acceptable liquid, Alternatively, they can be used parenterally in the form of injections such as suspensions.
  • the compound is mixed with known physiologically acceptable carriers, flavoring agents, excipients, vehicles, preservatives, stabilizers, binders, etc. in a unit dosage form generally required for the practice of pharmaceutical preparations. Can be manufactured. 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 cell mouth, corn starch, gelatin, A swelling agent such as alginic acid, a lubricant such as magnesium stearate, a sweetening agent such as sucrose, lactose or saccharin, a flavoring agent such as peppermint, cocoa oil or cherry are used.
  • 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 by dissolving or suspending the active substance in a vehicle such as water for injection, or naturally occurring vegetable oils such as sesame oil or coconut oil. it can.
  • Aqueous liquids for injection include, for example, saline, isotonic solutions containing glucose and other adjuvants (Eg, D-sorbitol, D-mannitol, sodium chloride, etc.), and suitable solubilizing agents, for example, alcohols (eg, ethanol), polyalcohols (eg, propylene glycol, polyethylene glycol), non- It may be used in combination with an ionic surfactant (eg, polysorbate 80 TM , HCO-50).
  • 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 agent examples include a buffer (for example, a phosphate buffer and a sodium acetate buffer), a soothing agent (for example, benzalkonium chloride, proactive hydrochloride, etc.), a stabilizer (for example, , Human serum albumin, polyethylene glycol, etc.), preservatives (eg, benzyl alcohol, phenol, etc.), antioxidants and the like.
  • a buffer for example, a phosphate buffer and a sodium acetate buffer
  • a soothing agent for example, benzalkonium chloride, proactive hydrochloride, etc.
  • a stabilizer for example, 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, so they can be used, for example, in humans and mammals (for example, rats, mice, puppies, sheep, bush, puppies, cats, dogs, monkeys, etc.). Can be administered.
  • the dose of the compound or a salt thereof varies depending on the subject of administration, target organ, symptoms, administration method, and the like.
  • oral administration for example, in a patient with cancer (as 60 kg), the daily About 0.1 to: L 00 mg, preferably about 1.0 to 50 mg, more preferably about 1.0 to 2 Omg.
  • the single dose varies depending on the subject of administration, target organ, symptoms, administration method, etc.
  • it is usually used, for example, in cancer patients (as 6 O kg).
  • the dose can be administered in terms of 60 kg.
  • the neutralizing activity of an antibody against the receptor protein of the present invention, its partial peptide, or a salt thereof against the receptor protein or the like means an activity of inactivating a signal transduction function involving the receptor protein.
  • signal transduction involving the receptor protein for example, cell stimulating activity via the receptor protein (eg, arachidonic acid release, acetylcholine release, intracellular C Promotes a2 + release, intracellular cAMP production, intracellular cGMP production, inositol phosphate production, fluctuations in cell membrane potential, phosphorylation of intracellular proteins, activation of c-fos, decrease in pH, etc. Activity or activity to suppress). Therefore, it can be used for prevention and Z or treatment of diseases caused by overexpression of the receptor protein.
  • transgenic animals that express the receptor protein and the like of the present invention can be produced.
  • animals include human or non-human mammals (eg, rat, mouse, egret, sheep, sheep, bush, oyster, cat, dog, monkey, etc.) (hereinafter sometimes abbreviated as animal).
  • human or non-human mammals eg, rat, mouse, egret, sheep, sheep, bush, oyster, cat, dog, monkey, etc.
  • animal hereinafter sometimes abbreviated as animal.
  • mouse, magpie, rat and the like are particularly preferable.
  • the DNA of the present invention When introducing the DNA of the present invention into a subject 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 the downstream of one of various promoters capable of expressing the DNA of the present invention derived from an animal having high homology to animal cells is used.
  • a DNA-introduced animal that produces high levels of the receptor protein of the present invention or the like can be produced by microinjection into a fertilized egret egg.
  • a ubiquitous expression promoter such as a virus-derived promoter or meta-mouth thionein can be used.
  • a promoter of a gene that is specifically expressed in brain, lung, stomach, heart, kidney and the like is used. Is used.
  • the presence of the receptor protein or the like of the present invention in the germ cells of the produced animal after the DNA transfer means that all the offspring of the produced animal have the receptor protein or the like of the present invention in all of the germ cells and somatic cells thing Means
  • the progeny of this type of animal that has inherited the gene has the receptor protein of the present invention in all of its germ cells and somatic cells.
  • the DNA-introduced animal of the present invention After confirming that the DNA-introduced animal of the present invention stably retains the gene by crossing, it can be reared in an ordinary breeding environment as the DNA-bearing animal. Furthermore, by crossing male and female animals having the desired DNA, homozygous animals having the transgene on both homologous chromosomes are obtained, and by crossing the male and female animals, all progeny can obtain the DNA. Breeding can be subcultured to have.
  • the animal into which the DNA of the present invention has been introduced expresses the receptor protein of the present invention at a high level, it can be used as an animal for screening an agonist or an gonist against the receptor protein of the present invention. Useful.
  • the DNA-introduced animal of the present invention can also be used as a cell source for tissue culture.
  • tissue culture For example, by directly praying DNA or RNA in the tissue of the DNA-introduced mouse of the present invention, or by analyzing the tissue in which the receptor protein of the present invention expressed by the gene is present, the receptor of the present invention can be obtained.
  • One protein can be analyzed.
  • the cells of a tissue having the receptor protein of the present invention and the like are cultured by standard tissue culture techniques, and the functions of cells from tissues that are generally difficult to culture, such as those derived from brain and peripheral tissues, are used. Can be studied.
  • 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.
  • bases, amino acids, and the like are indicated by abbreviations based on the abbreviations by the IUPAC-IUB Commission on Biochemical Nomenclature or commonly used abbreviations in the art, and examples thereof are described below.
  • amino acids can have optical isomers, the L-form is indicated unless otherwise specified.
  • RNA Messenger ribonucleic acid dATP Deoxyadenosine triphosphate dTTP Deoxythymidine triphosphate dGTP Deoxyguanosine triphosphate d CTP Deoxycytidine triphosphate ATP Adenosine triphosphate
  • sequence numbers in the sequence listing in the present specification indicate the following sequences.
  • FIG. 1 shows an amino acid sequence of a mouse-derived novel G protein-coupled receptor protein TGR 33 of the present invention.
  • FIG. 1 shows the nucleotide sequence of cDNA encoding the novel mouse-derived G protein-coupled receptor protein TGR 33 of the present invention.
  • FIG. 1 shows the nucleotide sequence of cDNA encoding the novel mouse-derived G protein-coupled receptor protein TGR 33 of the present invention.
  • Example 1 shows the nucleotide sequence of a primer used in Example 1.
  • Example 1 shows the nucleotide sequence of a primer used in Example 1.
  • the transformant Escherichia coli DH5 ⁇ / ⁇ CR2.1—TOPO—mTGR33 obtained in Example 1 described below has been used since February 8, 2001 in Ibaraki Prefecture, Japan. 1-1, Higashi, Tsukuba 1 1 Chuo No.
  • CDNA encoding the TGR33 protein of the present invention was obtained by the following PCR method.
  • the oligo DNA having the nucleotide sequence represented by SEQ ID NO: 4 is used as a sense strand primer
  • the oligo DNA having the nucleotide sequence represented by SEQ ID NO: 5 is used as an antisense strand primer
  • 5 M Advant age 2Po 1 ymerase Mix (manufactured by CLONTECH) 0.241, DMSO (4%), and Marathon—Ready Mo usec DNA 1 ibrary
  • a mixed solution 1 21 containing fetal cDNA solution 1 ⁇ 1 of CLONTECH Co., Ltd. was prepared, and the mixture was first prepared using a thermal cycler (Gene Amp PCR system mo del 9700 (PerkinElmer)).
  • TA cloning was performed using PCR 2.1-TOPO (manufactured by NVI TROGEN) to determine the nucleotide sequence, and the plasmid was introduced into a competent cell of Escherichia coli DH5 ⁇ strain. From the colonies of the ampicillin-resistant transformants appearing on the L ⁇ agar medium containing ampicillin, clones containing the plasmid in which the foreign D ⁇ fragment had been inserted were selected by Co10 ny PCR, and the introduced DNA was selected.
  • nucleotide sequence of cDNA (SEQ ID NO: 2), which has higher homology to GIR (Glucocorticoid-Induced Receptor) than the PCR product and encodes a novel amino acid sequence (SEQ ID NO: 1) I got
  • TGR33 The novel G protein-coupled receptor protein containing the amino acid sequence represented by SEQ ID NO: 1 was named TGR33.
  • TGR33 showed 35% homology at the amino acid level with mouse GIR.
  • nucleotide sequence of cDNA encoding TGR33 (SEQ ID NO: 3) was obtained by the same operation.
  • Escherichia coli harboring a plasmid having the nucleotide sequence represented by SEQ ID NO: 3 was designated as Escherichia coli (Escherichiacocoi) DH5a / pCR2.1-I TOPO_m TGR33.
  • the polynucleotide (for example, DNA, RNA and their derivatives) encoding the G protein-coupled receptor protein or its partial peptide or its salt, the receptor protein or its partial peptide of the present invention may be a ⁇ ligand (agonist) ⁇ . 2) Acquisition of antibodies and antiserum, 3) Construction of a recombinant receptor expression protein expression system, 2) Development of a receptor binding assay system using the same expression system and screening of drug candidate compounds, 4) Structural analysis Similar ligands ⁇ Drug design based on comparison with receptor Yuichi, ⁇ ⁇ Reagents for the preparation of probes and PCR primers in gene diagnosis, 7Transgenic animals or ⁇ ⁇ Gene therapy drugs, etc. Etc. can be used.

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Abstract

L'invention vise à mettre au point une nouvelle protéine s'utilisant pour cribler un agoniste, un antagoniste, etc. L'invention concerne plus spécifiquement une nouvelle protéine réceptrice couplée à la protéine G présentant une séquence d'acide aminé identique ou sensiblement identique à la séquence d'acide aminé représentée par SEQ ID n°1 ou son sel, un polynucléotide la codant ; l'identification d'un ligand (un agoniste) ; l'obtention d'un anticorps et d'un antisérum et la construction d'un système d'expression de protéine réceptrice recombinée à l'aide de ceux-ci ; la mise au point d'un système de dosage lié au récepteur et criblage d'un composé admissible de médicament à l'aide dudit système d'expression ; la conception de médicaments sur la base de la comparaison avec un récepteur de ligand de structure similaire ; et son utilisation par exemple dans la préparation d'une sonde en thérapie génique et d'une amorce de PCR.
PCT/JP2001/011126 2000-12-20 2001-12-19 Nouvelle proteine receptrice couplee a la proteine g et son adn Ceased WO2002050266A1 (fr)

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Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0789076A2 (fr) * 1996-02-07 1997-08-13 Takeda Chemical Industries, Ltd. Protéines récepteurs couplés à la protéine G, leur production et utilisation
EP0877083A1 (fr) * 1997-05-07 1998-11-11 Smithkline Beecham Corporation Récepteur humain (HCEPT09), connecté à la protéine G
WO2000008133A1 (fr) * 1998-08-06 2000-02-17 Merck & Co., Inc. NOUVELLE SEQUENCE D'ADNc D'UN NOUVEAU RECEPTEUR COUPLE A LA PROTEINE G

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0789076A2 (fr) * 1996-02-07 1997-08-13 Takeda Chemical Industries, Ltd. Protéines récepteurs couplés à la protéine G, leur production et utilisation
EP0877083A1 (fr) * 1997-05-07 1998-11-11 Smithkline Beecham Corporation Récepteur humain (HCEPT09), connecté à la protéine G
WO2000008133A1 (fr) * 1998-08-06 2000-02-17 Merck & Co., Inc. NOUVELLE SEQUENCE D'ADNc D'UN NOUVEAU RECEPTEUR COUPLE A LA PROTEINE G

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
CARNINCI P. ET AL.: "Normalization and subtraction of cap-trapper-selected cDNAs to prepare full-lenght cDNA libraries for rapid discovery of new genes", GENOME RESEARCH, vol. 10, August 2000 (2000-08-01), pages 1317 - 1630, XP002944079 *

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