WO2003072780A1 - Nouvelles protéines, leurs adn et leur utilisation - Google Patents

Nouvelles protéines, leurs adn et leur utilisation Download PDF

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Publication number
WO2003072780A1
WO2003072780A1 PCT/JP2003/002133 JP0302133W WO03072780A1 WO 2003072780 A1 WO2003072780 A1 WO 2003072780A1 JP 0302133 W JP0302133 W JP 0302133W WO 03072780 A1 WO03072780 A1 WO 03072780A1
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Prior art keywords
protein
present
peptide
salt
seq
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PCT/JP2003/002133
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English (en)
Japanese (ja)
Inventor
Tsukasa Sugo
Masaaki Mori
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Takeda Chemical Industries, Ltd.
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Priority to AU2003211722A priority Critical patent/AU2003211722A1/en
Publication of WO2003072780A1 publication Critical patent/WO2003072780A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/72—Receptors; Cell surface antigens; Cell surface determinants for hormones
    • C07K14/723—G protein coupled receptor, e.g. TSHR-thyrotropin-receptor, LH/hCG receptor, FSH receptor
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A61P1/14—Prodigestives, e.g. acids, enzymes, appetite stimulants, antidyspeptics, tonics, antiflatulents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A61P1/16—Drugs for disorders of the alimentary tract or the digestive system for liver or gallbladder disorders, e.g. hepatoprotective agents, cholagogues, litholytics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P11/00—Drugs for disorders of the respiratory system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P15/00—Drugs for genital or sexual disorders; Contraceptives
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P15/00—Drugs for genital or sexual disorders; Contraceptives
    • A61P15/04—Drugs for genital or sexual disorders; Contraceptives for inducing labour or abortion; Uterotonics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P19/00—Drugs for skeletal disorders
    • A61P19/02—Drugs for skeletal disorders for joint disorders, e.g. arthritis, arthrosis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P19/00—Drugs for skeletal disorders
    • A61P19/06—Antigout agents, e.g. antihyperuricemic or uricosuric agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/04—Anorexiants; Antiobesity agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/06—Antihyperlipidemics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/08—Drugs for disorders of the metabolism for glucose homeostasis
    • A61P3/10—Drugs for disorders of the metabolism for glucose homeostasis for hyperglycaemia, e.g. antidiabetics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P7/00—Drugs for disorders of the blood or the extracellular fluid
    • A61P7/06—Antianaemics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A61P9/10—Drugs for disorders of the cardiovascular system for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A61P9/12—Antihypertensives
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • the present invention relates to a novel G protein-coupled receptor protein, a DNA encoding the same, and uses thereof.
  • anorexia anemia associated with anorexia
  • prevention and treatment of hypoproteinemia weak labor, laxative bleeding, uterine remodeling failure or milk stasis, or obesity
  • hyperphagia affective disorder, sex Dysfunction, labour, tonic contractions, preterm birth, Prader-Willi syndrome, diabetes and its complications, hypertension, hyperlipidemia, coronary atherosclerosis, gout, respiratory disease, fatty liver, infertility or
  • the present invention relates to screening for compounds having a prophylactic and therapeutic effect on osteoarthritis. Background art
  • Obesity is known to be a risk factor for the development of many lifestyle-related diseases such as diabetes and heart disease. With changes in eating habits and lifestyles, fertility is increasing, especially in developed countries, and it has become a very socially significant problem from the health economic perspective. Obesity is known to be caused by various factors.Recently, several peptides located in the hypothalamus, the feeding center, are involved in promoting or suppressing feeding behavior in mammals. Thus, the contribution of these peptides to obesity has received particular attention. This is because drugs that inhibit or activate the action of these peptides are considered to be potential central antiobesity drugs.
  • One such hypothalamic peptide, melanin-concentrating hormone (Melanin) melanin-concentrating hormone
  • MCH Concentrating Hormone
  • MCH is localized in the feeding center, the hypothalamic lateral area and the irregular zone. And were more likely to be involved in eating and drinking behavior, but Qu et al. First showed that MCH was involved in eating behavior as follows (Qu, D. et al., Nature, vol. 380). , 243-247, 1996). Expression of altered mRNA levels in the hypothalamus of ob / ob mice and wild-type mice, which are genetically obese models, was searched for by the di ferent al al di sp lay method. Was found to be significantly increased. Increased MCH gene expression was observed in both wild-type and ob / ob mice after a 24-hour fast.
  • MCH functioned as a food-stimulating peptide, but in fact, administration of MCH directly to the lateral ventricle of rats significantly increased food intake. In addition, it was reported that MCH knockout mice lost significant weight due to reduced food intake and increased metabolism.
  • mice deficient in the MCH gene have not been reported in animals knocked down by these peptides, and MCH functions as a particularly important regulator in eating behavior. it seems to do.
  • MCH gene-deficient mice did not show any prominent phenotype other than weight loss, it was suggested that suppressing the effect of MCH could lead to the development of anti-obesity drugs with few side effects.
  • SLC-1 (MCHK MCHR1) was previously known as a functional receptor for MCH (Nature, 400, 26-1265, 1999, Nature, 400, 265-269, 1999, Bi ochei. Bi ophys. Res. Commun., 261, 622-626, 1999, Nature Cell Biol., 1st, 267-271, 1999, FEBS Lett. 524, 1999, WO 00/40725, etc.), and recently, SLT (MCH2, CHR2) has been cloned as the second receptor subtype of MCH (J. Biol. Chem. , Volume 276, 20125-20129 Co., 2001, Biochem. Biophys. Res.Co, Vol. 283, 1013- 1018, 2001, Proc. Natl.
  • SLC-1 receptor is particularly strongly expressed in the hypothalamus region involved in feeding (arcus nucleus, ventral nucleus, dorsolateral nucleus, paraventricular nucleus) and in the nucleus accumbens which is thought to be involved in the reward system
  • SLT is expressed in the hippocampus and tonsils and is low in the hypothalamus, so SLC-1 is mainly involved in the inducing action of MCH, and SLT is It has been suggested that they may be involved in memory and emotion's effects.
  • SLT is distributed in the hypothalamus in areas different from SLC-1 (pronucleus, lateral nucleus, etc.), and is involved in feeding because it is located near the causative gene of childhood obesity.
  • the present inventors have conducted intensive studies in order to solve the above-mentioned problems, and as a result, have found that genes encoding monkey, canine, and feline SLTs showing homology to human SLT can be obtained from monkey, canine, and feline brain cDNA. More successfully found and cloned. In addition, these receptors bind to MCH. As a result of further study based on these findings, the present invention has been completed.
  • the present invention (1) a protein or a salt thereof containing an amino acid sequence identical or substantially identical to the amino acid sequence represented by SEQ ID NO: 4, SEQ ID NO: 14 or SEQ ID NO: 24;
  • a diagnostic agent comprising the polynucleotide according to (6),
  • a medical comprising the antisense polynucleotide according to (28).
  • a method for quantifying the mRNA of the protein according to (1) which comprises using the polynucleotide or a part thereof according to (6);
  • a method for screening a compound or a salt thereof that alters the expression level of the protein described in (1) above which comprises using the quantification method described in (30) above.
  • a medicament comprising the compound according to (35) or (36) or a salt thereof,
  • An obesity, hyperphagia, affective disorder, sexual dysfunction, or hyperactivity comprising administering to a mammal an effective amount of the compound according to (25) or a salt thereof.
  • Obesity, hyperphagia, affective disorders, sexual dysfunction, overwork labor, ankylosing uterine contractions, preterm birth, Prader-Willi syndrome, diabetes and its complications, hypertension, hyperlipidemia, coronary Provide the use of the compound described in (25) or a salt thereof for the manufacture of a prophylactic or therapeutic agent for arteriosclerosis, gout, respiratory disease, fatty liver, infertility or osteoarthritis. I do. BRIEF DESCRIPTION OF THE FIGURES
  • FIG. 1 is a graph showing the intracellular Ca ion elevating activity of various concentrations of MCH on cat SLT-expressing CH0 cells measured using FLIPR.
  • FIG. 2 is a graph of the expression level of the cat SLT gene in various parts of the cat brain.
  • the expression level was expressed as 1000 times the value obtained by dividing the expression level of the cat SLT gene for the same type II RNA level by the expression level of the cat GAPDH gene.
  • the protein containing the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 4, SEQ ID NO: 14 or SEQ ID NO: 24 (hereinafter, may be referred to as the protein of the present invention) is It is a G protein-coupled receptor protein and is used in human warm-blooded animals (eg, guinea pig, rat, mouse, chicken, Cells of egrets, bushes, sheep, sheep, monkeys, monkeys, dogs, cats, etc.
  • human warm-blooded animals eg, guinea pig, rat, mouse, chicken, Cells of egrets, bushes, sheep, sheep, monkeys, monkeys, dogs, cats, etc.
  • amino acid sequence substantially identical to the amino acid sequence represented by SEQ ID NO: 4 includes amino acids having 98% or more, preferably about 99% or more homology with the amino acid sequence represented by SEQ ID NO: 4. And the like.
  • Examples of the protein containing an amino acid sequence substantially identical to the amino acid sequence represented by SEQ ID NO: 4 include, for example, a protein containing an amino acid sequence substantially identical to the amino acid sequence represented by the aforementioned SEQ ID NO: 4 However, a protein having substantially the same activity as the protein having the amino acid sequence represented by SEQ ID NO: 4 is preferred.
  • amino acid sequence substantially the same as the amino acid sequence represented by SEQ ID NO: 14 includes 92% or more, preferably about 95% or more, more preferably about 95% or more of the amino acid sequence represented by SEQ ID NO: 14. Amino acid sequences having a homology of 97% or more, more preferably about 99% or more, may be mentioned.
  • proteins containing an amino acid sequence substantially the same as the amino acid sequence represented by SEQ ID NO: 14 include, for example, amino acids substantially the same as the amino acid sequence represented by the aforementioned SEQ ID NO: 14
  • a protein containing a sequence and having substantially the same activity as a protein having the amino acid sequence represented by SEQ ID NO: 14 is preferred.
  • the amino acid sequence substantially the same as the amino acid sequence represented by SEQ ID NO: 24 includes 92% or more, preferably about 95% or more, more preferably about 95% or more of the amino acid sequence represented by SEQ ID NO: 24. Amino acid sequences having a homology of 97% or more, more preferably about 99% or more, and the like can be mentioned.
  • proteins having an amino acid sequence substantially the same as the amino acid sequence represented by SEQ ID NO: 24 include, for example, amino acids substantially the same as the amino acid sequence represented by SEQ ID NO: 24 above.
  • a protein containing a sequence and having substantially the same activity as a protein having the amino acid sequence represented by SEQ ID NO: 24 is preferred.
  • Substantially the same activity includes, for example, a binding activity to MCH, a signal information transduction effect, and the like. Substantially the same means that their activities are the same in nature. Therefore, it is preferable that the activities such as the binding activity to MCH and the activity of signal transduction are equivalent (eg, about 0.5 to 2 times), but the quantitative factors such as the degree of these activities and the molecular weight of the protein are It may be different.
  • the activity such as the binding activity and the signal transduction action can be measured according to a method known per se or a method analogous thereto.
  • Examples of the protein of the present invention include (1) (i) one or two or more amino acids in the amino acid sequence represented by SEQ ID NO: 4 (eg, about 1 to 8, preferably about 1 to 5, (Ii) preferably one or two or more amino acids in the amino acid sequence represented by SEQ ID NO: 4 (for example, about 1 to 200, preferably 1 to 1); About 50, preferably about 1 to 100, preferably about;!
  • amino acids in the amino acid sequence represented by SEQ ID NO: 14 for example, about 1 to 200, preferably about 1 to 150, and preferably 1 to 1 About 100, preferably about 1 to 50, preferably about 1 to 30, preferably about 1 to 10, and more preferably the number (1 to 5) of amino acids is other amino acids.
  • a so-called mutin such as a protein containing a substituted amino acid sequence or (V) a protein containing an amino acid sequence combining them; 1 or 2 or more (for example, about 1 to 25, preferably about 1 to 10, and more preferably about 1 to 5) amino acids in the amino acid sequence represented by SEQ ID NO: 24; (Ii) one or two or more amino acid sequences represented by SEQ ID NO: 24 (for example, about 1 to 200, preferably about 1 to 150, and preferably About 100, preferably about 1 to 50, preferably about 1 to 30, preferably about 1 to 10, and more preferably about (1 to 5) amino acids (Iii) one or two or more amino acid sequences represented by SEQ ID NO: 24 (for example, about 1 to 200, preferably about 1 to 150, and preferably 1 to 1 About 100 pieces, preferably about 1 to 50 pieces, preferably about 1 to 30 pieces, preferably about 1 to 10 pieces, and more preferably Or (iv) one or two or more amino acids in the amino acid sequence represented by
  • a so-called mutin such as a protein containing an amino acid sequence in which a number (1 to 5) of amino acids are substituted with another amino acid, or (V) an amino acid sequence obtained by combining them is also included.
  • the position of the insertion, deletion or substitution is not particularly limited.
  • protein of the present invention examples include, for example, a protein containing the amino acid sequence represented by SEQ ID NO: 4, a protein containing the amino acid sequence represented by SEQ ID NO: 14, SEQ ID NO: 24 And proteins containing the amino acid sequence represented by
  • the partial peptide of the protein of the present invention (hereinafter sometimes abbreviated as the partial peptide of the present invention) is a partial peptide of the protein of the present invention described above, and is preferably the same as the above-described protein of the present invention. Any material having properties may be used.
  • the partial peptide of the present invention lacks one or more (preferably about 1 to 10, more preferably, about 1 to 5) amino acids in its amino acid sequence, Alternatively, one or more (preferably, about 1 to 20, more preferably, about 1 to 10, and more preferably, about 1 to 5) amino acids are added to the amino acid sequence. Or 1 or 2 or more amino acids in the amino acid sequence thereof (preferably, about 1 to 20; more preferably, about 1 to 10;
  • amino acids or one or more (preferably about 1 to 10, more preferably several, and more preferably 1 to (About 5 amino acids) may be substituted with another amino acid.
  • the partial peptide of the present invention for example, in the amino acid sequence represented by SEQ ID NO: 4, for example, the 1st to 41st amino acids, the 31st to 34th amino acids, SEQ ID NO: 1
  • the amino acid sequence represented by the amino acid sequence represented by SEQ ID NO: 24 the 1st to 41st amino acid sequence, the 31st amino acid sequence to the 34th amino acid sequence, SEQ ID NO: 24
  • the amino acid sequences of the 1st to 41st and the 31st to 330th amino acids are preferred.
  • a peptide containing the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 25 is a human Cells of warm-blooded animals (eg, guinea pigs, rats, mice, chicks, egrets, pigs, higgies, lions, monkeys, dogs, cats, etc.) (eg, hepatocytes, spleen cells, nerve cells, glial cells) 3 cells, bone marrow cells, mesangial cells, Langer's cells, epidermal cells, epithelial cells, goblet cells, endothelial cells, smooth muscle cells, fibroblasts, fiber cells, muscle cells, adipocytes, immune cells (Eg, macrophages, T cells, B cells, natural killer cells, mast cells, neutrophils, basophils, eosinophils, monocytes), megakaryocyte
  • Examples of the peptide having an amino acid sequence substantially identical to the amino acid sequence represented by SEQ ID NO: 25 include, for example, an amino acid sequence substantially identical to the amino acid sequence represented by SEQ ID NO: 25 Peptides having substantially the same activity as the peptide having the amino acid sequence represented by SEQ ID NO: 25 are preferred.
  • Substantially equivalent activities include, for example, SLT binding activity, etc. It is. Substantially the same indicates that the activity is the same in nature. Therefore, it is preferable that the binding activity to SLT is equivalent (eg, about 0.5 to 2 times), but quantitative factors such as the degree of this activity and the molecular weight of the protein may be different. No.
  • the binding activity can be measured according to a method known per se.
  • a peptide containing the amino acid sequence represented by SEQ ID NO: 25 is preferably used.
  • Examples of the peptide of the present invention include: (i) one or two or more amino acids in the amino acid sequence represented by SEQ ID NO: 25 (for example, about 1 to 30, preferably about 1 to 10; More preferably, an amino acid sequence in which a number (1 to 5) of amino acids are deleted, and (ii) one or two or more amino acids in the amino acid sequence represented by SEQ ID NO: 25 (for example, about 1 to 30 amino acids, Preferably about 1 to 10 amino acids, more preferably about 1 to 5 amino acids, and (iii) one or more amino acid sequences in the amino acid sequence represented by SEQ ID NO: 25. (For example, about 1 to 30 amino acids, preferably about 1 to 10 amino acids, and more preferably about 1 to 5 amino acids).
  • SEQ ID NO: 25 1 or 2 or more in the amino acid sequence for example, about 1 to 30, preferably about 1 to 10, more preferably
  • a peptide containing an amino acid sequence in which a number (1 to 5) of amino acids are substituted with another amino acid, or (V) an amino acid sequence obtained by combining them is also included.
  • the position of the insertion, deletion or substitution is not particularly limited.
  • Specific examples include a peptide containing the 5th to 19th partial sequences from the N-terminal of the amino acid sequence represented by SEQ ID NO: 25. More specifically, it has the amino acid sequence represented by SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30 or SEQ ID NO: 31. Peptides are examples. Preferably, a peptide having the amino acid sequence represented by SEQ ID NO: 28 is used.
  • Substantially identical substitutions of amino acids in the amino acid sequence may be selected, for example, from other amino acids of the class to which the amino acid belongs.
  • Non-polar (hydrophobic) amino acids include alanine, leucine, isoleucine, valin, proline, phenylalanine, tributofan, methionine, and the like.
  • Polar (neutral) amino acids include glycine, serine, threonine, cysteine, tyrosine, asparagine, and glutamine.
  • Positively charged (basic) amino acids include arginine, lysine and histidine.
  • Negatively charged (acidic) amino acids include aspartic acid and glutamic acid. '
  • the position at which the above-mentioned other amino acid is deleted or substituted is preferably a position other than Cys in the amino acids constituting the peptide of the present invention.
  • the peptide of the present invention may be labeled.
  • Specific examples include peptides labeled with an isotope by a method known per se, fluorescently labeled peptides (for example, fluorescently labeled with fluorescein), biotinylated peptides, enzyme-labeled peptides, and the like. .
  • Porton A labeled product of the peptide of the present invention prepared by a known method using one Hunter reagent may be used.
  • the left end of the protein and the peptide is the N-terminus (amino end) and the right end is the C-terminus (potassium terminal) according to the convention of peptide labeling.
  • This onset Ming proteins and peptides, C-terminal, carboxyl group (-C00H), carbo Kishireto (-C00-), amide (- C0M 2) or ester (- C00R) any derconnection may be of.
  • R in the ester e.g., methyl, Echiru, n- propyl, isopropyl, n - C, such as butyl, _ 6 alkyl groups, such as cyclopentyl, such as hexyl cyclo ⁇ 3 - 8 cycloalkyl group, for example, phenyl, alpha-naphthyl of which C 6 _ 12 7 aryl group, e.g., benzyl, phenyl, such as phenethyl - C, C 7 _ such as alpha-Nafuchiru alkyl groups such as _ 2 Al kill group or carry one naphthylmethyl
  • a 14 aralkyl group, a bivaloyloxymethyl group and the like are used.
  • the protein and peptide of the present invention have a lipoxyl group (or lipoxylate) other than at the C-terminus
  • those in which the carboxyl group is amidated or esterified are also included in the proteins and peptides of the present invention.
  • the ester in this case, for example, the above-mentioned C-terminal ester and the like are used.
  • amino acid residues at the N-terminus eg, Mechionin residues
  • Amino group protecting groups e.g., formyl group, etc. C M Ashiru group such Arukanoiru such Asechiru group
  • Substituent on the side chain of amino acid in the molecule for example, -OH, -SH, amino group, imidazo Ichiru group, indole group, etc. Guanijino group
  • a suitable protecting group e.g., formyl group, etc.
  • c, _ 6 Ashiru groups such Arukanoiru groups such as ⁇ Se butyl group), or Also included are complex proteins such as so-called glycoproteins to which sugar chains are bound.
  • the force Rupokishiru group (-C00H)
  • the force Lupo - Kishireto (-C00-)
  • amide (- C0Nh 2) or may be filed in any of ester (-C00R).
  • the partial peptide of the present invention includes, as in the case of the protein of the present invention, those having a carbonyl group (or carboxylate) other than the C-terminus, N-terminal amino acid residues (eg, methionine). Residue is protected with a protecting group, the glutamine residue formed by cleavage of the N-terminal in vivo is a pyroglutamate, and the substituent on the side chain of the amino acid in the molecule is appropriate. Also included are those protected with a protecting group or complex peptides such as so-called glycopeptides to which sugar chains are bonded.
  • Salts of the protein or partial peptide of the present invention and salts of the peptide of the present invention include salts with physiologically acceptable acids (eg, inorganic acids, organic acids) and bases (eg, alkali metal salts). Preference is given to the use of, especially the physiologically acceptable acid addition salts.
  • physiologically acceptable acids eg, inorganic acids, organic acids
  • bases eg, alkali metal salts
  • Such salts include, for example, salts with inorganic acids (eg, hydrochloric acid, phosphoric acid, hydrobromic acid, sulfuric acid), and organic acids (eg, acetic acid, formic acid, propionic acid, fumaric acid, maleic acid) Salts with formic acid, succinic acid, tartaric acid, citric acid, malic acid, oxalic acid, benzoic acid, methanesulfonic acid, 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
  • Salts with formic acid, succinic acid, tartaric acid, citric acid, malic acid, oxalic acid, benzoic acid, methanesulfonic acid, benzenesulfonic acid are used.
  • the protein of the present invention or a partial peptide thereof, the peptide of the present invention or a salt thereof can be produced from the above-mentioned human or warm-blooded animal cell or tissue by a known protein purification method. Can also be produced by culturing a transformant containing DNA encoding It can also be produced according to the peptide synthesis method described below.
  • the human or mammalian tissues or cells are homogenized and then extracted with an acid or the like, and the resulting extract is subjected to reverse phase chromatography, ion exchange chromatography, etc. Purification and isolation can be achieved by combining the above chromatography.
  • the protein of the present invention for the synthesis of the peptide of the present invention, its partial peptide, their amides or their salts, commercially available resins for protein / peptide synthesis can be used.
  • a resin for example, Methyl resin, hydroxymethyl resin, benzhydrylamine resin, aminomethyl resin, 4-benzyloxybenzyl alcohol resin, 4-methylbenzhydrylamine resin, PAM resin, 4-hydroxymethylmethylphenylacetamide Methyl resin, polyacrylamide resin, 4- (2 ', 4'-dimethoxyphenylhydroxymethyl) phenoxy resin, 41- (2', 4'-dimethoxyphenyl Fmocaminoethyl) phenoxy resin, etc. Can be.
  • an amino acid appropriately protected with an ⁇ -amino group and a side chain functional group is condensed on the resin in accordance with the sequence of the desired protein / peptide according to various known condensation methods. Let it. At the end of the reaction, proteins and peptides are cleaved from the resin, and at the same time, various protecting groups are removed. Further, an intramolecular disulfide bond formation reaction is performed in a highly diluted solution to obtain the desired protein, peptide or amide thereof.
  • the protected amino acid may be added directly to the resin along with a racemization inhibitor additive (eg, HOBt, HOOBt) or pre-protected as a symmetrical acid anhydride or HOBt ester or HOOBt ester. It can be added to the resin after activation of the amino acid.
  • a racemization inhibitor additive eg, HOBt, HOOBt
  • pre-protected as a symmetrical acid anhydride or HOBt ester or HOOBt ester it can be added to the resin after activation of the amino acid.
  • the solvent used for activating the protected amino acid or for condensing with the resin may be appropriately selected from solvents known to be usable for the protein-peptide condensation reaction.
  • acid amides such as N, N-dimethylformamide, N, N-dimethylacetamide, N-methylpyrrolidone, halogenated hydrocarbons such as methylene chloride and chloroform, alcohols such as trifluoroethanol , Sulfoxides such as dimethyl sulfoxide, ethers such as pyridine, dioxane, and tetrahydrofuran; nitriles such as acetonitrile and propionitrile; esters such as methyl acetate and ethyl acetate; or an appropriate mixture thereof.
  • the reaction temperature is appropriately selected from a range known to be usable for a protein bond formation reaction, and is usually appropriately selected from a range of about 120 ° (: to 5 Ot :).
  • the sensitized amino acid derivative is usually used in a 1.5 to 4-fold excess.
  • Examples of the protecting group for the amino group of the starting material include Z, Boc, Yuichi Sharipentyloxycarbonyl, Isoporonyloxycarbonyl, 4-methoxybenzyloxycarbonyl, C11Z, Br-Z , Adamantyloxycarbonyl, trifluoroacetyl, phthaloyl, formyl, 2-ditrophenylsulfenyl, diphenylphosphinothioyl, Fmoc and the like are used.
  • Lepoxyl groups can be, for example, alkyl esterified (e.g., methyl, ethyl, propyl, butyl, tert-butyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, 2-adamantyl, etc.).
  • alkyl esterified e.g., methyl, ethyl, propyl, butyl, tert-butyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, 2-adamantyl, etc.
  • aralkyl esterification e.g., benzyl ester, 412 trobenzyl ester, 4-methoxybenzyl ester, 4-methyl benzyl ester, benzhydryl esterification
  • phenacyl esterification Benzyloxycarbonyl hydrazide, tert-butoxycarbonyl hydrazide, trityl hydrazide and the like.
  • the hydroxyl group of serine can be protected, for example, by esterification or etherification.
  • a group suitable for this esterification for example, a lower alkanol group such as an acetyl group, an aroyl group such as a benzoyl group, a group derived from carbonic acid such as a benzyloxycarbonyl group, an ethoxycarponyl group, and the like are used.
  • Examples of a group suitable for etherification include a benzyl group, a tetrahydroviranyl group, and a t-butyl group.
  • the protecting group of the phenolic hydroxyl group of tyrosine for example, B zl, C 1 2 -
  • Bzl 2-nitrobenzyl, Br-Z, Yuichi Sharybutyl and the like are used.
  • protecting group for imidazole of histidine for example, Tos, 4-methoxy-12,3,6-trimethylbenzenesulfonyl, DNP, benzyloxymethyl, Bum, Boc, Trt, Fmoc and the like are used.
  • activated carboxylic acid groups of the raw material include, for example, corresponding acid anhydrides, azides, and activated esters [alcohols (for example, pentachlorophenol, 24,5-trichloromouth phenol, 2,4-dinitrophenol) , Cyanomethyl alcohol, paranitrophenol, HONB, N-hydroxysuccinimide, N-hydroxyphthalimide, and esters with HOBt).
  • activated amino group of the raw material for example, a corresponding phosphoric acid amide is used.
  • Methods for removing (eliminating) the protecting group include, for example, catalytic reduction in a hydrogen stream in the presence of a catalyst such as Pd-black or Pd-carbon, or hydrogen fluoride anhydride or methanesulfonic acid.
  • a catalyst such as Pd-black or Pd-carbon, or hydrogen fluoride anhydride or methanesulfonic acid.
  • the elimination reaction by the above acid treatment is generally carried out at a temperature of about 120 ° 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.
  • an amide of a protein or peptide for example, first, after amidating and protecting the ⁇ -hydroxyl group of the amino acid at the terminal end of the amino acid, a peptide chain is extended to a desired chain length on the amino side. After that, the protein / peptide obtained by removing only the protecting group for the ⁇ -amino group at the ⁇ -terminal of the peptide chain and the C-terminal lipoxyl group are removed. A protein and a peptide from which only the protecting group has been removed are produced, and the peptide and the protein are condensed in a mixed solvent as described above. Details of the condensation reaction are the same as described above.
  • the crude peptide / crude protein is purified by various known purification means, and the main fraction is freeze-dried to obtain an amide of the desired protein / peptide.
  • an ester of protein / peptide for example, after condensing the ⁇ -hydroxyloxyl group of a carboxy-terminal amino acid with a desired alcohol to form an amino acid ester, the same procedure as in the amide of protein / peptide is carried out. It is possible to obtain an ester of a protein and a peptide.
  • the peptide of the present invention and the protein of the present invention can be produced according to a peptide synthesis method known per se. Further, the partial peptide of the protein of the present invention or a salt thereof can be produced according to a peptide synthesis method known per se, or by cleaving the protein of the present invention with an appropriate peptidase.
  • any of a solid phase synthesis method and a liquid phase synthesis method may be used. That is, the peptide of the present invention is condensed with the peptide of the present invention or the partial peptide or amino acid capable of constituting the protein of the present invention and the remaining portion, and when the product has a protecting group, the protecting group is eliminated to remove the target peptide. Can be manufactured. Examples of the known condensation method and elimination of the protecting group include the methods described in the following (i) to (V).
  • the peptide of the present invention or the partial peptide of the present invention can be purified and isolated by a combination of ordinary purification methods, for example, solvent extraction, distillation, laffification, liquid chromatography, and recrystallization. .
  • solvent extraction for example, solvent extraction, distillation, laffification, liquid chromatography, and recrystallization.
  • the peptide or partial peptide obtained by the above method is in a free form, it can be converted to an appropriate salt by a known method.
  • the peptide or partial peptide is obtained in a salt form, it can be converted to a free form by a known method Can be converted.
  • the polynucleotide encoding the protein of the present invention may be any polynucleotide containing the above-described nucleotide sequence encoding the protein of the present invention.
  • it is DNA.
  • the DNA may be any of genomic DNA, genomic DNA library, cDNA derived from the cells and tissues described above, cDNA library derived from the cells and tissues described above, and synthetic DNA.
  • the vector used for the library may be any of bacteriophage, plasmid, cosmid, phagemid and the like.
  • a reverse RNA was directly prepared using the total RNA or mRNA fraction prepared from the cells and tissues described above.
  • RT-PCR method Transcriptase Polymerase Chain Reaction
  • the DI ⁇ A encoding the protein of the present invention includes, for example, (i) a DNA containing the nucleotide sequence represented by SEQ ID NO: 3 or a nucleotide sequence represented by SEQ ID NO: 3 under highly stringent conditions.
  • Examples of the DNA that can hybridize with the nucleotide sequence represented by SEQ ID NO: 3 under high stringency conditions include, for example, 98% or more, preferably about 99% or more of the nucleotide sequence represented by SEQ ID NO: 3 DNA containing a nucleotide sequence having homology of
  • Examples of the DNA that can hybridize with the nucleotide sequence represented by SEQ ID NO: 13 under high stringency conditions include, for example, a nucleotide sequence represented by SEQ ID NO: 13 and 92% or more, preferably about 9% or less.
  • a DNA containing a nucleotide sequence having a homology of 5% or more, more preferably about 97% or more, more preferably about 99% or more is used.
  • Examples of the DNA that can hybridize with the nucleotide sequence represented by SEQ ID NO: 23 under high stringent conditions include, for example, the nucleotide sequence represented by SEQ ID NO: 23 and 92% or more, preferably about 9% or less.
  • DNA containing a base sequence having a homology of 5% or more, more preferably about 97% or more, more preferably about 99% or more is used.
  • Hybridization is performed by a known method or a method equivalent thereto, such as the method described in Molecular Cloning (Molecular Cloning) 2nd (J. Sambrook et al., Cold Spring Harbor Lab. Press, 1989). It can be done according to. If a commercially available library is still 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.
  • High stringency conditions include, for example, a sodium concentration of about 19 to 40 mM, preferably about 19 to 20 mM, and a temperature of about 50 to 70, preferably about 60 to 65.
  • the conditions at ° C are shown. In particular, the case where the sodium concentration is about 19 mM and the temperature is about 65 is most preferable.
  • DNAs encoding the protein containing the amino acid sequence represented by SEQ ID NO: 4 include DNA containing the base sequence represented by SEQ ID NO: 3, and SEQ ID NO: 38.
  • the DNA encoding the protein having the amino acid sequence represented by SEQ ID NO: 14 has the nucleotide sequence represented by SEQ ID NO: 13 DNA, represented by SEQ ID NO: 39
  • Examples of the DNA encoding a protein having the amino acid sequence represented by SEQ ID NO: 24, such as a DNA having a nucleotide sequence represented by SEQ ID NO: 24, include a DNA having a base sequence represented by SEQ ID NO: 23, DNA containing the base sequence represented by 40 or the like is used.
  • the polynucleotide encoding the partial peptide of the present invention may be any polynucleotide as long as it contains a base sequence encoding the above-described partial peptide of the present invention.
  • DNA is preferred.
  • the DNA may be any of genomic DNA, genomic DNA library, cDNA derived from the above-described cells and tissues, cDNA library derived from the above-described cells and tissues, and synthetic DNA. '
  • Examples of the DNA encoding the partial peptide of the present invention include, for example, DNA having a part of the DNA having the base sequence represented by SEQ ID NO: 3, SEQ ID NO: 13 or SEQ ID NO: 23, or SEQ ID NO: 3 A DN having a nucleotide sequence that hybridizes under high stringent conditions to the nucleotide sequence represented by SEQ ID NO: 1.3 or SEQ ID NO: 23, and encoding a protein having substantially the same activity as the protein of the present invention. DNA containing a part of A is used.
  • the DNA that can be hybridized with the base sequence represented by SEQ ID NO: 3, SEQ ID NO: 13 or SEQ ID NO: 23 has the same significance as described above.
  • the DNA or DNA encoding the protein or partial peptide of the present invention (hereinafter, these may be simply referred to as the protein of the present invention in the description of the cloning and expression of the DNA encoding the same).
  • the DNA of the present invention is amplified by PCR using a synthetic DNA primer having a part of the nucleotide sequence encoding the protein of the present invention, or the DNA of the present invention is incorporated into an appropriate vector. Selection can be performed by hybridization with a DNA fragment encoding a part or all of the region or labeled with a synthetic DNA. The hybridization can be performed according to, for example, the method described in Molecular Cloning 2nd (J. Sabrook et al., Cold Spring Harbor Lab. Press, 1989). Ma 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 by PCR or a known kit such as Mutan TM -Super Express Km (Takara Shuzo) or Mutan TM -K (Takara Shuzo) using the 0DA-LA PCR method or the like. It can be performed according to a known method such as the gapped duplex method or the Kunkel method, or a method analogous thereto.
  • the DNA encoding the cloned protein can be used as it is depending on the purpose, or can be used after digestion with a restriction enzyme or addition of a linker, if desired.
  • the DNA may have ATG as a translation initiation codon on the 5, terminal side, and TAA, TGA or TAG as a translation termination codon on the 3, terminal side. These translation initiation codon and translation termination codon can also be added using a suitable synthetic DNA adapter.
  • the expression vector of the protein of the present invention can be prepared, for example, by (a) cutting out a DNA fragment of interest from DNA encoding the protein of the present invention, and (mouth) converting the DNA fragment into a promoter in an appropriate expression vector. It can be manufactured by connecting downstream of
  • the vector examples include a plasmid derived from E. coli (eg, pBR322, PBR32 5, pUC 12, pUC 13), a plasmid derived from Bacillus subtilis (eg, pUB 110, pTP 5, pC 194), a yeast-derived plasmid (eg, pSH19, pSH15)
  • pacteriophage such as ⁇ phage
  • animal viruses such as retrovirus, vaccinia virus, and baculovirus, pAl-11, pXTl, pR cZCMV, pRc / RSV, pc DNA I ZNe o and the like are used.
  • the promoter used in the present invention may be any suitable promoter corresponding to the host used for gene expression.
  • SRa promoter SV40 promoter, LTR promoter, CMV promoter, HSV-TK promoter, etc.
  • CMV (cytomegalovirus) promoter It is preferable to use SRo!
  • the host is Escherichia, the trp promoter, lac promoter, recA promoter, ⁇ PL promoter If the host is a Bacillus genus, SP01 promoter, SP02 promoter, penP promoter, etc.If the host is yeast, the host is yeast.
  • the expression vector may contain, in addition to the above, an enhancer, a splicing signal, a poly-A addition signal, a selection marker, and an SV40 replication origin (hereinafter sometimes abbreviated as SV400 ri), if desired. Anything can be used.
  • the selection marker one, for example, dihydrofolate reductase (hereinafter sometimes abbreviated as dh fr) gene [Mesotorekise Ichito (MTX) resistance], ampicillin phosphorus resistant gene (hereinafter sometimes abbreviated as Amp r ), Neomai, Shin-resistant gene (hereinafter sometimes abbreviated as Ne o r, include G418 resistance) and the like.
  • dh fr dihydrofolate reductase
  • Amp r ampicillin phosphorus resistant gene
  • Ne o r include G418 resistance
  • the target gene can be selected using a thymidine-free medium.
  • a signal sequence suitable for the host is added to the N-terminal side of the protein of the present invention. If the host is Escherichia, the Pho A signal sequence, Omp A signal sequence, etc., if the host is Bacillus, the amylase signal sequence, subtilisin signal sequence, etc. In the case of yeast, MFa signal sequence and SUC2 signal sequence can be used. When the host is an animal cell, insulin signal sequence, high interferon signal sequence, antibody molecule and signal sequence can be used, respectively. .
  • a transformant can be produced.
  • Examples of the host include Escherichia, Bacillus, yeast, insect cells, insects, animal cells, and the like.
  • Escherichia include, for example, Escherichia coli.
  • Bacillus bacteria examples include, for example, Bacillus subtilis (Bacillus).
  • subtilis MI114 (Gene, 24, 255 (1983)), 207-21 (Journal of Biochemistry, 95, 87 (1984)) and the like are used.
  • yeast include, for example, Saccharomyces cerevisiae AH22, AH22R—, ⁇ 87-11A, DKD-5D, 20B-12, Schizosaccharomyces porabe, NC YC 1913, NCYC 2036 Pastoris (Pichia pastoris) K # 71 or the like is used.
  • insect cells for example, when the virus is Ac NPV, a cell line derived from a larva of night roth moth (Spodoptera frugiperda cell; Sf 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, cells derived from Estigmena acrea, and the like are used.
  • Sf cells include Sf9 cells (ATCC
  • insects for example, silkworm larvae are used [Maeda et al., Nature, Vol. 315, 592 (1985)].
  • animal cells examples include monkey cell COS-7, Vero, Chinese hamster cell CHO (hereinafter abbreviated as CHO cell), dh fr gene-deficient chinini—zhamster cell CH ⁇ (hereinafter CHO (dh fr)). Cells), mouse L cells, mouse AtT-20, mouse myeoma cells, rat GH3, human FL cells, and the like.
  • Transformation of the genus Escherichia can be performed, for example, according to the method described in Proc. Natl; Acad. Sci. USA, 69, 2110 (1972) or Gene, 17, 107 (1982). it can. Transformation of Bacillus spp. Can be performed, for example, according to the method described in Molecular & General Genetics, Volume 168, 111 (1979).
  • the yeast is transformed according to the method described in, for example, Methods in Enzymology, Vol. 194, 182-187 (1991), Proc. Natl. Acad. Sci. USA, Vol. 75, 1929 (1978). be able to.
  • Insect cells or insects can be transformed, for example, according to the method described in Bio / Technology, 6, 47-55 (1988).
  • a liquid medium is suitable as a medium used for the cultivation, and a carbon source necessary for the growth of the transformant is contained therein.
  • Nitrogen sources inorganic substances and others.
  • carbon sources include glucose, dextrin, soluble starch, and sucrose.
  • nitrogen sources include ammonium salts, nitrates, corn chip lica, peptone, zein, meat extract, soybean meal, and potato extract.
  • the inorganic or organic substance such as a liquid and the inorganic substance include calcium chloride, sodium dihydrogen phosphate, and magnesium chloride.
  • yeast extract, vitamins, growth promoting factors and the like may be added.
  • the pH of the medium is preferably about 5-8.
  • Examples of a medium for culturing Escherichia bacteria include, for example, an M9 medium containing glucose and casamino acids [Miller, Journal “Obexperiment”, “Molecular”, Journal of Experiments in
  • a drug such as 3; 6-indolylacrylic acid can be added to make the promoter work efficiently.
  • the host is a bacterium belonging to the genus Escherichia
  • the cultivation is usually carried out at about 15 to 43 ° C for about 3 to 24 hours, and if necessary, aeration and stirring can be applied.
  • cultivation is usually performed at about 30 to 40 ° C for about 6 to 24 hours.
  • the medium used is Grace's Insect Medium (Grace, TC, Nature, 195, 788 (1962)). Those to which additives such as serum are appropriately added are used.
  • the ⁇ of the culture medium is adjusted to about 6.2 to 6.4. Culture is usually performed at about 27 ° C for about 3 to 5 days, and aeration and agitation are added as necessary.
  • examples of the medium include a MEM medium containing about 5 to 20% fetal bovine serum (Science, 122, 501 (1952)), a DMEM medium [Virology, 8, 396 (1959)], RPMI 1640 medium [The Journal of the American Medical Association 199, 519 (1967)], 199 medium [Proceeding of the Society for the Biological Medicine, 73] , 1 (1950)].
  • the pH is about 6-8.
  • Cultivation is usually performed at about 30 ° C (: up to 40 ° C for about 15 to 60 hours, and aeration and agitation are added as necessary.
  • the protein of the present invention can be produced in the cells, in the cell membrane, or outside the cells of the transformant.
  • the protein of the present invention can be separated and purified from the culture by, for example, the following method.
  • the cells or cells are collected by a known method, suspended in an appropriate buffer, After lysing cells or cells by lysozyme and Z or freeze-thawing, a method of obtaining a crude protein extract by centrifugation or filtration is used as appropriate.
  • the buffer may contain a protein denaturant such as urea or guanidine hydrochloride, or a surfactant such as Triton X-1000 TM .
  • Purification of the protein contained in the culture supernatant or extract obtained in this manner can be performed by appropriately combining known separation and purification methods.
  • These known separation and purification methods mainly include methods using solubility such as salting out and solvent precipitation, dialysis, ultrafiltration, gel filtration, and SDS-polyacrylamide gel electrophoresis, mainly molecular weight.
  • Methods that utilize differences in charge methods that use differences in charge, such as ion-exchange chromatography, methods that use specific affinities, such as affinity chromatography, and hydrophobicity, such as reverse-phase high-performance liquid chromatography.
  • a method using the difference between the isoelectric points, such as an isoelectric focusing method, and the like, may be used.
  • the protein thus obtained when obtained in a free form, it can be converted to a salt by a known method or a method analogous thereto, and conversely, when the protein is obtained in the form of a salt, a known method or a method analogous thereto Can be converted into a free form or another salt.
  • the protein produced by the recombinant can be arbitrarily modified or the polypeptide can be partially removed by the action of an appropriate protein modifying enzyme before or after purification.
  • an appropriate protein modifying enzyme for example, trypsin, chymotrypsin, arginyl endopeptidase, protein kinase, glycosidase and the like are used.
  • the presence of the protein of the present invention thus produced can be measured by, for example, enzymatic immunoassay western blotting using a specific antibody.
  • the antibody against the protein or partial peptide of the present invention or a salt thereof may be any of a polyclonal antibody and a monoclonal antibody as long as it can recognize the protein or partial peptide of the present invention or a salt thereof.
  • An antibody against the protein or partial peptide of the present invention or a salt thereof (hereinafter, these may be simply abbreviated to the protein of the present invention in the description of the antibody) is obtained by using the protein of the present invention as an antigen.
  • the antibody or antiserum can be produced according to the following method.
  • the protein of the present invention is administered to a warm-blooded animal itself or together with a carrier or diluent at a site capable of producing an antibody upon administration.
  • 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 the warm-blooded animal to be used include monkeys, egrets, dogs, guinea pigs, mice, rats, sheep, goats, and chickens, and mice and rats are preferably used.
  • a warm-blooded animal immunized with an antigen for example, an individual with an antibody titer is selected from a mouse, and the spleen or lymph node is collected 2 to 5 days after the final immunization and contained in them.
  • an individual with an antibody titer is selected from a mouse, and the spleen or lymph node is collected 2 to 5 days after the final immunization and contained in them.
  • a monoclonal antibody-producing hybridoma can be prepared.
  • the antibody titer in the antiserum can be measured, for example, by reacting the labeled protein described below with the antiserum, and then measuring the activity of the labeling agent bound to the antibody.
  • the fusion operation can be performed according to a known method, for example, the method of Koehler and Milstein Nature, 256, 495 (1975)].
  • 'Fusion promoters include, for example, polyethylene glycol (PEG), Sendai virus and the like, and preferably PEG is used.
  • myeloma cells examples include myeloma cells of warm-blooded animals such as NS-1, P3U1, SP 2/0, and AP-1, but P3U1 is preferably used.
  • the preferred ratio between the number of antibody-producing cells (spleen cells) and the number of myeloma cells used is about 1: 1 to 20: 1, and PEG (preferably PEG1000 to PEG6000) is used at a concentration of about 10 to 80%.
  • Cell fusion can be carried out efficiently by adding the mixture and incubating at 20 to 40 ° C, preferably 30 to 37 ° C, for 1 to 10 minutes.
  • a hybridoma culture supernatant is added to a solid phase (eg, a microplate) on which a protein antigen is adsorbed directly or together with a carrier.
  • a solid phase eg, a microplate
  • an anti-immunoglobulin antibody (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 an enzyme, and a monoclonal antibody bound to the solid phase Antibody detection method, Add the hybridoma culture supernatant to a solid phase to which anti-immunoglobulin antibody or protein A is adsorbed, add proteins labeled with radioactive substances, enzymes, etc. Examples include a method for detecting a single antibody.
  • the selection of the monoclonal antibody can be performed according to a known method or a method analogous thereto. Usually, it can be performed in an animal cell culture medium supplemented with HAT (hypoxanthine, aminopterin, thymidine).
  • HAT hyperxanthine, aminopterin, thymidine
  • any medium can be used as long as it can grow a hybridoma.
  • RPMI 1640 medium containing 1 to 20%, preferably 10 to 20% fetal bovine serum, and 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 gas.
  • the antibody titer of the hybridoma culture supernatant can be measured in the same manner as the measurement of the antibody titer in the antiserum described above.
  • Monoclonal antibodies can be separated and purified by known methods, for example, immunoglobulin separation and purification methods (eg, salting out method, alcohol precipitation method, isoelectric point precipitation method, electrophoresis method, ion exchanger (eg, DEAE)).
  • immunoglobulin separation and purification methods eg, salting out method, alcohol precipitation method, isoelectric point precipitation method, electrophoresis method, ion exchanger (eg, DEAE)
  • an active adsorbent such as protein A or protein G and the bond is dissociated to obtain the antibody.
  • the polyclonal antibody of the present invention is produced according to a known method or a method analogous thereto. can do.
  • a immunizing antigen protein antigen
  • a complex thereof with a carrier protein is formed, and immunization is performed on a warm-blooded animal in the same manner as in the above-described method for producing a monoclonal antibody. It can be produced by collecting the contents and separating and purifying the antibody.
  • the type of the carrier protein and the mixing ratio between the carrier and the hapten are different from those of the hapten immunized by cross-linking the carrier.
  • Any antibody may be cross-linked at any ratio as long as it can be efficiently produced.
  • serum albumin, thyroglobulin, hemocyanin, etc. in a weight ratio of about 0.1 to hapten per hapten, may be used. -20, preferably about 1-5.
  • Various condensing agents can be used for force coupling between the hapten and the carrier, but glutaraldehyde, carbodiimide, a maleimide active ester, an active ester reagent containing a thiol group or a dithioviridyl group, or the like is used. .
  • the condensation product is administered to a warm-blooded animal itself or together with a carrier or diluent at a site where antibody production is possible.
  • Complete Freund's adjuvant / incomplete Freund's adjuvant may be administered in order to enhance the antibody-producing ability upon administration.
  • the administration is usually made once every about 2 to 6 weeks, for a total of about 3 to 10 times.
  • the polyclonal antibody can be collected from the blood, ascites, etc., preferably from the blood of a warm-blooded animal immunized by the above method.
  • the measurement of the polyclonal antibody titer in the antiserum can be performed in the same manner as the measurement of the antibody titer in the antiserum described above. Separation and purification of the polyclonal antibody can be performed according to the same method for separation and purification of immunoglobulin as in the above-described separation and purification of the monoclonal antibody.
  • an antisense polynucleotide having a substantially complementary base sequence or a part thereof includes a base sequence complementary to or substantially complementary to the base sequence of the DNA of the present invention or a part thereof. Any antisense having an action capable of suppressing the expression of the DNA. Although it may be a polynucleotide, antisense DNA is preferred.
  • the nucleotide sequence substantially complementary to the DNA of the present invention is, for example, the entire nucleotide sequence or a partial nucleotide sequence of the nucleotide sequence complementary to the DNA of the present invention (that is, the complementary strand of the DNA of the present invention). And about 70% or more, preferably about 80% or more, more preferably about 90% or more, and most preferably about 95% or more.
  • the complementary strand of the nucleotide sequence of the portion encoding the N-terminal portion of the protein of the present invention is about 70%. %, Preferably at least about 80%, more preferably at least about 90%, most preferably at least about 95%.
  • nucleotide sequence complementary or substantially complementary to the nucleotide sequence of DNA having the nucleotide sequence represented by SEQ ID NO: 3, SEQ ID NO: 13 or SEQ ID NO: 23 Or a part thereof, preferably, for example, a nucleotide sequence complementary to the nucleotide sequence of DNA having the nucleotide sequence represented by SEQ ID NO: 3, SEQ ID NO: 13 or SEQ ID NO: 23, or Antisense polynucleotides having a part thereof are exemplified.
  • An antisense polynucleotide is usually composed of about 10 to 40 bases, preferably about 15 to 30 bases.
  • the phosphate residues (phosphates) of each nucleotide constituting the antisense DNA are, for example, chemically modified phosphate residues such as phosphorothioate, methylphosphonate, and phosphorodithionate. May be substituted.
  • These antisense polynucleotides can be produced using a known DNA synthesizer or the like.
  • an antisense polynucleotide (nucleic acid) corresponding to the protein gene of the present invention, which can inhibit the replication or expression of the gene, is cloned or the determined protein is encoded. It can be designed and synthesized based on DNA base sequence information.
  • Such an antisense polynucleotide can hybridize to RNA of the protein gene of the present invention, inhibit the synthesis or function of the RNA, or bind to the protein-related RNA of the present invention.
  • Polynucleotides complementary to the selected sequence of the protein-related RNA of the present invention, and polynucleotides capable of specifically hybridizing with the protein-related RNA of the present invention include the protein of the present invention in vivo and in vitro. It is useful for regulating and controlling gene expression, and is also useful for treating or diagnosing diseases.
  • the term "corresponding" means having homology or being complementary to a specific sequence of nucleotides, base sequences or nucleic acids including genes.
  • nucleotide, nucleotide sequence or nucleic acid usually refers to the amino acids of the (directed) protein derived from the nucleotide (nucleic acid) sequence or its complement.
  • the 5 'end hairpin loop of the protein gene usually refers to the amino acids of the (directed) protein derived from the nucleotide (nucleic acid) sequence or its complement.
  • any region in a protein gene can be selected as a target.
  • a hairpin loop or the like can be selected as a preferred target region, any region in a protein gene can be selected as a target.
  • the nucleic acid of interest can hybridize to the target region, the nucleic acid of interest can be said to be "antisense" to the polynucleotide of the target region.
  • Antisense polynucleotides can be polydexoxy liponucleotides containing 2-deoxy D-reports, polyliponucleotides containing D-reports, N-glycosides of purine or pyrimidine bases and other types of polynucleotides.
  • Other polymers having nucleotide or non-nucleotide backbones eg, commercially available protein nucleic acids and synthetic sequence-specific nucleic acid polymers
  • other polymers containing special bonds provided that such polymers are found in DNA or RNA
  • nucleases nucleases, nuclease inhibitors, toxins, antibodies, signal peptides, poly-L-lysine, etc.
  • sugars eg, monosaccharides
  • containing acridine, psoralen, etc. containing chelating compounds (eg, metals, radioactive metals, boron, oxidizing metals, etc.), containing alkylating agents, modified bonds (Eg, a anomeric nucleic acid).
  • chelating compounds eg, metals, radioactive metals, boron, oxidizing metals, etc.
  • alkylating agents eg, a anomeric nucleic acid
  • nucleoside may include not only those containing purine and pyrimidine bases but also those having other modified heterocyclic bases.
  • Modified nucleotides and modified nucleotides may also be modified at the sugar moiety, e.g., where one or more hydroxyl groups have been replaced with a halogen, an aliphatic group, or the like, or a functional group, such as an ether or an amine. Conversion.
  • the antisense polynucleotide of the present invention is RNA, DNA or a modified nucleic acid (RNA, DNA).
  • modified nucleic acid include a sulfur derivative of a nucleic acid, a thiophosphate derivative, a polynucleoside amide / a nucleic acid that is resistant to degradation of oligonucleoside amide, and the like.
  • the antisense polynucleotide of the present invention can be designed, for example, as follows.
  • the antisense polynucleotide is more stable in the cell, the cell permeability of the antisense polynucleotide is increased, the affinity for the target sense strand is increased, and If so, reduce the toxicity of the antisense polynucleotide.
  • the antisense polynucleotides of the present invention may contain altered or modified sugars, bases, or bonds, may be provided in special forms such as ribosomes, microspheres, or may be applied by genetic therapy. Can be given in an added form.
  • the addition forms include polycations such as polylysine, which acts to neutralize the charge on the phosphate backbone, and lipids, which enhance the interaction with cell membranes and increase the uptake of nucleic acids ( For example, phosphoric acid, hydrophobic, cholesterol, etc.).
  • Preferred lipids for addition include cholesterol and its derivatives (eg, cholesteryl chromate formate, cholic acid, etc.). These can be attached to the 3 'or 5' end of the nucleic acid and can be attached via a base, sugar, or intramolecular nucleoside linkage.
  • Other groups are cap groups specifically located at the 3 'or 5' end of nucleic acids that prevent degradation by nucleases such as exonuclease and RNase. No. Examples of such a capping group include, but are not limited to, hydroxyl-protecting groups known in the art, such as diols such as polyethylene glycol and tetraethylene glycol.
  • the inhibitory activity of the antisense polynucleotide can be examined using the transformant of the present invention, the in vivo or in vitro gene expression system of the present invention, or the in vivo or in vitro translation system of the protein of the present invention. Can be.
  • the protein or partial peptide of the present invention or a salt thereof hereinafter sometimes abbreviated as the protein of the present invention
  • the DNA encoding the protein or partial peptide of the present invention hereinafter referred to as the D NA
  • an antibody against the protein or partial peptide of the present invention or a salt thereof hereinafter may be abbreviated as the antibody of the present invention
  • an antisense polynucleotide of the DNA of the present invention hereinafter may be referred to as an antibody.
  • an antibody which may be abbreviated as the antisense polynucleotide of the present invention.
  • the protein of the present invention is a G protein-coupled receptor protein, MC By binding to H, it plays an important role in promoting appetite (feeding) and promoting oxytocin secretion.
  • anorexia eg, nervous appetite
  • Anemia or hypoproteinia due to anorexia, weak labor, laxative bleeding, uterine remodeling failure, milk stasis, etc. develop various diseases.
  • the protein of the present invention and the DNA of the present invention include, for example, an appetite (feeding) enhancer, anorexia (eg, anorexia nervosa), anemia or hypoproteinemia associated with anorexia, weak labor pain It can be used as a medicament such as a preventive and remedy for flaccid hemorrhage, uterine remodeling failure, and milk stasis.
  • the protein of the present invention is reduced or deficient in the living body, and there is a patient in which the appetite (feeding) enhancement or oxytocin secretion promoting activity of MCH is not sufficiently or normally exerted
  • the DNA of the present invention When the DNA of the present invention is used as the above-described prophylactic or therapeutic agent, the DNA is used alone or in an appropriate vector such as a retrovirus vector, an adenovirus vector, or an adenovirus associated virus vector. Thereafter, it can be administered to humans or warm-blooded animals according to conventional means.
  • the DNA of the present invention can be administered as it is or in the form of a formulation together with a physiologically acceptable carrier such as an auxiliary agent for promoting uptake, and can be administered using a gene gun or a catheter such as a hide mouth gel catheter.
  • the protein of the present invention When the protein of the present invention is used as the prophylactic / therapeutic agent, the protein purified to at least 90%, preferably 95% or more, more preferably 98% or more, and still more preferably 99% or more. It is preferred to use.
  • the protein of the present invention can be used, for example, in the form of tablets, capsules, elixirs, microcapsules, and the like, which are sugar-coated as necessary, orally, or aseptically with water or other pharmaceutically acceptable liquids. It can be used parenterally in the form of injections, such as aqueous solutions or suspensions.
  • the protein of the present invention may be used together with physiologically acceptable carriers, flavoring agents, excipients, vehicles, preservatives, stabilizers, binders, and the like in a unit dosage form required for generally accepted pharmaceutical practice. It can be manufactured by mixing. 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.
  • binders such as gelatin, corn starch, tragacanth, gum arabic, excipients such as crystalline cellulose, corn starch, gelatin, Swelling agents such as alginic acid, lubricants such as magnesium stearate, sweeteners such as sucrose, lactose or saccharine, and flavoring agents such as peppermint, cocoa oil or cellulose.
  • Swelling agents such as alginic acid, lubricants such as magnesium stearate, sweeteners such as sucrose, lactose or saccharine, and flavoring agents such as peppermint, cocoa oil or cellulose.
  • sweeteners such as sucrose, lactose or saccharine
  • flavoring agents such as peppermint, cocoa oil or cellulose.
  • a liquid carrier such as oil and fat can be further contained in the above-mentioned type of material.
  • Sterile compositions for injection can be formulated according to standard pharmaceutical practice, such as dissolving or suspending the active substance in vehicles such as
  • Aqueous liquids for injection include, for example, physiological saline, isotonic solutions containing glucose and other adjuvants (eg, D-sorbitol, D-mannitol, sodium chloride, etc.).
  • Agents for example, alcohols (eg, alcohol), polyalcohols (eg, propylene glycol, polyethylene glycol, etc.), non-ionic surfactants (eg, Polysorbate 80 TM , HC ⁇ —50 etc.).
  • examples of the oily liquid include sesame oil, soybean, and oil, and may be used in combination with a solubilizing agent such as benzyl benzoate or benzyl alcohol.
  • buffers eg, phosphate buffer, sodium acetate, buffer, etc.
  • soothing agents eg, benzalkonium chloride, proforce hydrochloride, etc.
  • the prepared injection is usually filled in an appropriate ampoule.
  • Stabilizers eg, human serum albumin, polyethylene glycol, etc.
  • preservatives eg, benzyl alcohol, phenol, etc.
  • antioxidants etc. ' May be combined.
  • the prepared injection is usually filled in an appropriate ampoule.
  • the vector into which the DNA of the present invention has been inserted is also formulated in the same manner as described above, and is usually used parenterally.
  • the preparations obtained in this way are safe and have low toxicity, for example, in warm-blooded animals (e.g., humans, rats, mice, guinea pigs, egrets, birds, higgies, bush, pests, pests, cats, Dogs, monkeys, chimpanzees, etc.).
  • warm-blooded animals e.g., humans, rats, mice, guinea pigs, egrets, birds, higgies, bush, pests, pests, cats, Dogs, monkeys, chimpanzees, etc.
  • the dosage of the protein of the present invention varies depending on the target disease, the administration subject, the administration route, and the like.
  • the adult as 60 kg
  • the single dose varies depending on the administration subject, the target disease, and the like.
  • the protein of the present invention when administered to an adult (with a body weight of 60 kg) in the form of an injection for the purpose of treating anorexia nervosa, It is convenient to administer the protein by injecting about 0.01 to 30 mg, preferably about 0.1 to 20 mg, more preferably about 0.1 to 10 mg of the protein per day into the affected area. 60 kg for other animals Amount converted to have enough can be administered.
  • the protein of the present invention is useful as a reagent for screening a compound or a salt thereof that changes the binding between the peptide of the present invention (eg, MCH) and the protein of the present invention.
  • a compound or a salt thereof that changes the binding between the peptide of the present invention (eg, MCH) and the protein of the present invention.
  • a method for screening a salt thereof, or a kit for screening a compound or a salt thereof, which changes the binding property between the peptide of the present invention and the protein of the present invention, which comprises using the peptide of the present invention and the protein of the present invention (
  • the screening method of the present invention and the kit for screening of the present invention are abbreviated in detail below.
  • a compound that alters the binding between the peptide of the present invention and the protein of the present invention a compound that alters the binding between the peptide of the present invention and the protein of the present invention (for example, peptide, protein, non-peptide) Compounds, synthetic compounds, fermentation products, etc.) or salts thereof.
  • Such compounds cell stimulating activity via protein of the present invention (e.g., Arakidon acid release, acetylcholine release, intracellular C a 2 + release, intracellular c AM P producing formation, intracellular c AM P production inhibition , Intracellular cGMP production, Inositol phosphate production, Cell membrane potential fluctuation, Intracellular protein phosphorylation, c-fos activation, pH decrease, cAMP-dependent protein kinase activation, cGMP Activation of protein-dependent protein kinase, activation of phospholipid-dependent protein kinase, activity of promoting or inhibiting the activation of microtubule-associated protein kinase (MAP kinase), etc.
  • MAP kinase microtubule-associated protein kinase
  • the present invention relates to (i) the case where the protein of the present invention is brought into contact with the peptide of the present invention, and (ii) the case where the above-mentioned protein of the present invention is brought into contact with the peptide of the present invention and a test compound. And a method for screening a compound or a salt thereof, which changes the binding property between the peptide of the present invention and the protein of the present invention, characterized in that the comparison is performed.
  • the screening method of the present invention (i) the case where the above-mentioned protein of the present invention is brought into contact with the peptide of the present invention and (ii) the above-mentioned protein of the present invention which comes in contact with the peptide of the present invention and a test compound.
  • the amount of the peptide of the present invention bound to the protein of the present invention, the cell stimulating activity and the like are measured and compared.
  • screening method of the present invention include, for example, (a) the peptide of the present invention relative to the protein of the present invention when the peptide of the present invention is brought into contact with the protein of the present invention, and when the peptide and the test compound of the present invention are brought into contact with the protein of the present invention; A method for screening a compound or a salt thereof that changes the binding property between the peptide of the present invention and the protein of the present invention, which comprises measuring and comparing the amount of binding;
  • the peptide of the present invention and the peptide of the present invention are characterized by measuring and comparing the amount of binding of the peptide of the present invention to the cell or the membrane fraction when brought into contact with the membrane fraction of the cell.
  • the peptide of the present invention is a labeled peptide, such as the screening method according to any one of (a) to (c) above,
  • the protein of the present invention used in the screening method of the present invention may be any protein containing the above-described protein of the present invention.
  • the protein of the present invention which is expressed in a large amount using a recombinant is suitable.
  • the aforementioned methods and the like are used.
  • the preparation method described later when cells containing the protein of the present invention or the cell membrane fraction are used, the preparation method described later may be followed.
  • the cell When a cell containing the protein of the present invention is used, the cell may be immobilized with datalaldehyde, formalin, or the like.
  • the immobilization method can be performed according to a known method.
  • the cell containing the protein of the present invention refers to a host cell expressing the protein of the present invention. Examples of the host cell include the aforementioned Escherichia coli, Bacillus subtilis, yeast, insect cells, animal cells and the like. .
  • the membrane fraction refers to a fraction containing a large amount of cell membrane obtained by a known method after cell disruption.
  • the cells can be crushed by crushing the cells with a Potter-Elvehj em homogenizer, crushing with a Warlinda blender-Polytron (Kinematica), crushing with ultrasonic waves, or pressing with a French press. Crushing by ejecting cells from thin nozzles.
  • centrifugal fractionation methods such as differential centrifugation and density gradient centrifugation are mainly used.
  • the cell lysate is centrifuged at a low speed (500-3000 rpm) for a short time (usually about 1-10 minutes), and the supernatant is further centrifuged at a high speed (15000-30,000 rpm) for 30 minutes to 2 hours to obtain a precipitate.
  • a low speed 500-3000 rpm
  • a high speed 15000-30,000 rpm
  • the membrane fraction is rich in the expressed protein of the present invention and membrane components such as cell-derived phospholipids and membrane proteins.
  • the amount of the protein of the present invention in the cells or membrane fraction containing the protein of the present invention is preferably 10 3 to 10 8 molecules per cell, more preferably 10 5 to 10 7 molecules per cell. is there.
  • the protein fraction of the present invention and the peptide of the present invention are used.
  • the protein fraction of the present invention is preferably a natural protein fraction of the present invention or a recombinant protein fraction of the present invention having an activity equivalent thereto.
  • the equivalent activity means equivalent ligand binding activity and the like.
  • the labeled peptide such as a radioactive isotope (e.g., [125 1], [131 1], [3 ⁇ 4], [ "C], [32 P], [33 P], etc.
  • Fluorescent substances eg, cyanine fluorescent dyes (eg, Cy2, Cy3, Cy5, Cy5.5, Cy7 (manufactured by Amersham Bioscience), etc.), fluorescamine, fluorescein sothiosinate, etc.
  • enzymes eg, —galactosidase, j6—labeled with darcosidase, alkaline phosphatase, oxidase, malate dehydrogenase, etc.
  • luminescent substances eg, luminol, luminol derivatives, J-reciferin, lucigenin, etc.
  • piotin lanthanide, etc.
  • the peptide of the present invention prepared by a known method using a Porton-Hunter reagent can be used. It is also possible to use the body.
  • a compound that alters the binding between the peptide of the present invention and the protein of the present invention first, cells containing the protein of the present invention or the membrane fraction of the cells are screened.
  • a buffer suitable for tanning Any buffer may be used as long as it does not inhibit the binding between the ligand and the receptor, such as a phosphate buffer having a pH of 4 to 10 (preferably, a pH of 6 to 8), such as Tris-HCl buffer.
  • a surfactant such as CHAPS, Tween-80 TM (Kao-Atlas), digitonin, and dexcholate can be added to the buffer.
  • a protease inhibitor such as PMSF, leptin, E-64 (manufactured by Peptide Research Laboratories), and peptide sutin is added to suppress the degradation of the protein of the present invention and the peptide of the present invention by protease. You can also.
  • a certain amount 5,000 to 500,000 cpm
  • NBS non-specific binding
  • the reaction is carried out at 0 to 50 ° C, preferably 4 to 37 ° C, for 20 minutes to 24 hours, preferably 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 a counter.
  • the specific binding amount (B-NSB) is For example, a test compound having 50% or less can be selected as a candidate substance having a competitive inhibitory ability.
  • BIAcore manufactured by Amersham Pharmacia Biotech
  • the peptide of the present invention is immobilized on a sensor chip by an amino coupling method according to the protocol attached to the device, and contains a cell containing the protein of the present invention or a DNA encoding the protein of the present invention.
  • the protein of the present invention is immobilized on a sensor chip, and a buffer solution such as a phosphate buffer or a Tris buffer containing the peptide of the present invention or the peptide of the present invention and a test compound is passed over the sensor chip.
  • a buffer solution such as a phosphate buffer or a Tris buffer containing the peptide of the present invention or the peptide of the present invention and a test compound is passed over the sensor chip.
  • Test compounds include those similar to the above.
  • Atsusi system mediated cell stimulating activities proteins of the present invention e.g., Arakidon acid release, Asechirukori emissions release, intracellular C a 2 + release, intracellular c AM P generated , Inhibition of intracellular cAMP production, Intracellular cGMP production, Wild boar!
  • ⁇ -Monophosphate production Cell membrane potential fluctuation, Intracellular protein Phosphorylation, activation of c-fos, decrease of pH, activation of cAMP-dependent protein kinase, activation of cGMP-dependent protein kinase, activation of phospholipid-dependent protein kinase,
  • the activity of promoting or suppressing the activation of microtubule-associated protein kinase (MAP kinase), etc.) can be measured using a known method or a commercially available measurement kit. Specifically, first, cells containing the protein of the present invention are cultured on a multiwell plate or the like.
  • cells expressing an appropriate protein of the present invention are used.
  • the cells expressing the protein of the present invention the above-mentioned recombinant cell lines expressing the protein of the present invention and the like are desirable.
  • the transformant expressing the protein of the present invention may be a stable expression strain or a transient expression strain.
  • the same kind of animal cells as described above are used.
  • Test compounds include, for example, peptides, proteins, non-peptidic compounds, synthetic compounds, fermentation products, cell extracts, plant extracts, animal tissue extracts, and the like.
  • GTPaS When a receptor-expressing cell is stimulated by a receptor agonist, an intracellular G protein is activated and GTP is bound. This phenomenon is also observed in the membrane fraction of receptor-expressing cells. Normally, GTP is hydrolyzed to GDP, but if GTPaS is added to the reaction solution at this time, GTP7S binds to G protein like GTP, but is hydrolyzed. Without G cells The state of being bonded to the membrane is maintained. If labeled GTPaS is used, the activity of stimulating receptor agonist receptor-expressing cells can be measured by measuring the labeled GTPaS remaining on the cell membrane.
  • the stimulatory activity of the peptide of the present invention on the protein-expressing cell of the present invention is measured, whereby a compound that changes the binding property between the peptide of the present invention and the protein of the present invention is obtained. You can screen.
  • This method is performed using a membrane fraction containing the protein of the present invention.
  • the substance exhibiting the activity of promoting the GTP-A binding to the protein membrane fraction of the present invention is agonist.
  • the case where the peptide of the present invention is brought into contact with the protein cell membrane fraction of the present invention in the presence of labeled GTP TS and the case where the peptide and test compound of the present invention are brought into contact with the protein cell membrane fraction of the present invention.
  • a compound that alters the binding property between the peptide of the present invention and the protein of the present invention is determined. Screen.
  • the peptide GTP T S binding promoting activity test compound showing activity to suppress to proteins the cell membrane fraction of the present invention according to the present invention can be selected as Ah Ru candidates competitive inhibition.
  • screening of agonist is carried out by bringing only the test compound into contact with the protein cell membrane fraction of the present invention and measuring the GTP-AS binding promoting activity to the protein cell membrane fraction of the present invention. You can also.
  • the cell membrane fraction containing the protein of the present invention prepared according to known methods, membrane dilution buffer (50 mM T ris, 5 mM M g C 1 2, 150 mM N a C 1, 1 M GDP, 0. Dilute with 1% BSA; pH 7.4).
  • the dilution ratio depends on the expression level of the receptor. This was dispensed by 0. 2 ml min F a 1 c 0 n 2053, a peptide or peptide and a test compound of the present invention of the present invention was added, further to a final concentration of 200 pM added [35 S] GTPrS.
  • wash ice-cold buffer 50 mM Tr is, 5mM MgC 1 2, 150 mM NaCl, 0.1% BSA, 0.05% CHAP S; pH 7.4
  • Add 15 ml and filter through glass fiber filter paper GFZF. After incubating for 65 and 30 minutes and drying, it was bound to the membrane fraction remaining on the filter paper with a liquid scintillation counter.
  • the radioactivity of [ 35 S] GTPrS is measured.
  • the radioactivity of the experimental group to which only the peptide of the present invention was added was 100%, and the radioactivity of the experimental group to which the peptide of the present invention was not added was 100%.
  • test compound having a GTPaS binding promoting activity of, for example, 50% or less can be selected as a candidate substance having competitive inhibitory ability.
  • the production of intracellular cAMP is suppressed by stimulation of the peptide of the present invention.
  • the binding between the peptide of the present invention and the protein of the present invention is changed.
  • the compound can be screened.
  • a compound that alters the binding between the peptide of the present invention and the protein of the present invention is determined by measuring the activity of inhibiting the production of intracellular cAMP in the cell when the cell is brought into contact with the cell and comparing the results. I do.
  • a substance that increases the amount of intracellular cAMP for example, forskolin, calcitonin and the like are used.
  • the amount of cAMP produced in the protein-expressing cells of the present invention is determined by comparing the amount of anti-cAMP antibody obtained by immunizing mice, rats, rabbits, goats, rabbits, etc. with [ 125 1] -labeled cAMP (both commercially available products). Can be measured using an RIA system by using EIA or an EIA system combining an anti-cAMP antibody and labeled cAMP. Moreover, the anti-cAMP antibody, using a protein A or beads containing scintillant which is secured using such antibodies to etc.
  • c A of the protein-expressing cell of the present invention by the peptide of the present invention A test compound exhibiting an activity of inhibiting MP production inhibitory activity can be selected as a candidate substance having a competitive inhibition ability.
  • a compound showing an agonist activity can be screened by contacting only the test compound with the protein-expressing cell of the present invention and examining the cAMP production inhibitory activity.
  • Cells expressing the protein of the present invention are seeded on a 24-well plate at 5 ⁇ 10 4 ce 1 LZwe 11 and cultured for 48 hours. Wash the cells with Hank's buffer (pH 7.4) containing 0.2 ⁇ 3-isobutyl-methylxanthine, 0.05% BSA and 2 OmM HEPES (hereinafter abbreviated as reaction buffer 1). Then add 0.5 ml of reaction buffer and incubate in the incubator for 30 minutes.
  • Hank's buffer pH 7.4
  • reaction buffer 1 containing 0.2 ⁇ 3-isobutyl-methylxanthine, 0.05% BSA and 2 OmM HEPES
  • reaction buffer Excluding the reaction buffer, add 0.25 ml of the reaction buffer to the cells, and then add 1 M of the peptide of the present invention or 1 M of the peptide of the present invention and 2 M forskolin to which the test compound is added. Add 0.25 ml of reaction buffer to the cells and incubate at 37 ° C for 24 minutes. Stop the reaction by adding 100 1 of 20% perchloric acid, and then leave it on ice for 1 hour to extract intracellular cAMP. The amount of cAMP in the extract is measured using a cAMP EIA kit (Amersham Pharmacia Biotech).
  • the amount of cAMP produced by the stimulation of forskolin was defined as 100%, and the amount of cAMP suppressed by the addition of 1 / M of the peptide of the present invention was defined as 0%.
  • the effect of the test compound on is calculated.
  • a test compound that inhibits the activity of the peptide of the present invention and has a cAMP production activity of, for example, 50% or more can be selected as a candidate substance capable of competitive inhibition.
  • the peptide of the present invention when using the protein-expressing cell of the present invention, which exhibits the property of increasing the amount of intracellular CAMP by stimulation of the peptide of the present invention, the peptide of the present invention was brought into contact with the protein-expressing cell of the present invention. And a test compound obtained by contacting the peptide of the present invention and the test compound with the protein-expressing cell of the present invention, by measuring and comparing the intracellular cAMP production promoting activity of the cell. Compounds that alter the binding to the proteins of the invention can be screened. In this method, a test compound showing an activity of inhibiting the cAMP production promoting activity of the protein-expressing cells of the present invention by the peptide of the present invention can be selected as a candidate substance having an antagonistic inhibitory ability. ''
  • a compound exhibiting agonist activity can be screened by contacting the test compound alone with the protein-expressing cell of the present invention and examining cAMP production promoting activity.
  • the AMP production promoting activity can be measured by adding the peptide of the present invention or the peptide of the present invention and the test compound to the protein-expressing cells of the present invention (eg, animal cells such as CHO cells) without adding forskolin in the above screening method.
  • the amount of cAMP produced by the addition of is determined and measured by the method described above.
  • the peptide of the present invention and the protein of the present invention can be measured.
  • Compounds that alter binding can be screened.
  • DNA containing CRE (cAMP response element) is inserted into the vector upstream of the reporter gene to obtain the CRE—repo overnight gene vector.
  • CRE cAMP response element
  • stimulation accompanied by an increase in cAMP is accompanied by CRE-mediated expression of the reporter gene, and subsequent stimulation of the gene product (protein) of the repo overnight gene. Induce production.
  • the reporter gene protein by measuring the enzymatic activity of the reporter gene protein, it is possible to detect a change in the amount of CAMP in cells into which the CRE-repo overnight gene vector has been introduced.
  • the peptide of the present invention when the peptide of the present invention is brought into contact with the protein-expressing cell of the present invention into which the peptide of the present invention has been introduced in the presence of a substance that increases the amount of intracellular cAMP, the peptide of the present invention And the test compound was brought into contact with the protein-expressing cell of the present invention when the CRE-repo overnight gene vector was introduced. The enzyme activity of the reporter gene protein was measured and compared. Screening for a compound that alters the binding between the peptide of the present invention and the protein of the present invention.
  • a substance that increases the amount of intracellular CAMP for example, forskolin, calcitonin and the like are used.
  • the vector for example, Pitka Gene Basic Vector, Pitka Gene Enhancer Vector (Toyo Ink Manufacturing Co., Ltd.) and the like are used.
  • the CRE-containing DNA is inserted into the above-mentioned vector in the repo overnight gene, for example, the multicloning site upstream of the luciferase gene, to obtain the CRE-reporter gene vector.
  • a test compound that restores the inhibition of the enzyme activity of the reporter gene protein by the peptide of the present invention can be selected as a candidate substance capable of competitive inhibition.
  • agonists can be screened by bringing only the test compound into contact with the protein-expressing cells of the present invention and measuring the same inhibition of the amount of luminescence increased by forskolin stimulation as with the peptides of the present invention.
  • Cells expressing the protein of the present invention into which the CRE-repo overnight gene (luciferase) has been introduced are seeded on a 24-well plate at 5 ⁇ 10 3 ce 1 lZwe 11 and cultured for 48 hours.
  • the cells are washed with Hank's buffer (pH 7.4) containing 0.2 mM 3_isobutyl-methylxanthine, 0.05% BSA and 20 mM HE PES (hereinafter abbreviated as reaction buffer). Then add 0.5 ml of application buffer and incubate for 30 minutes in the incubator.
  • the peptides of the present invention provide for luciferase-induced luminescence upon forskolin stimulation. Suppress increase in volume. A compound that restores the inhibition can be selected as a candidate substance having competitive inhibitory ability.
  • reporter genes for example, alkaline phosphatase, chloramphene
  • Acetyl trans f erase or a gene such as iS-galactosidase may be used.
  • the enzyme activity of these reporter gene proteins is measured according to a known method or using a commercially available measurement kit.
  • Alkaline phosphatase activity can be measured using, for example, Lumi-Phos 530 manufactured by Wako Pure Chemical, and chloramphenicol acetyltransferase can be measured using, for example, FAST CAT chrol amphenicol manufactured by Wako Pure Chemical.
  • the i3-galactosidase activity is measured using, for example, Acetyl trans erase erase Assay KiT using, for example, Aurora Ga1-XE manufactured by Wako Pure Chemical Industries.
  • the protein-expressing cells of the present invention release arachidonic acid metabolites extracellularly by stimulation of the peptides of the present invention.
  • This reaction to measure the stimulating activity of the peptide of the present invention on the cells expressing the protein of the present invention, it is possible to screen for a compound that alters the binding property between the peptide of the present invention and the protein of the present invention. .
  • the arachidonic acid metabolite-releasing activity can be measured by measuring the labeled arachidonic acid metabolite released outside the cell by incorporating the labeled arachidonic acid into the cells expressing the protein of the present invention in advance. can do. Specifically, when the peptide of the present invention is brought into contact with a cell expressing the protein of the present invention containing labeled arachidonic acid, the peptide of the present invention and the test compound are brought into contact with the cell of the present invention containing labeled arachidonic acid. By measuring and comparing the arachidonic acid metabolite release activity when brought into contact with a protein-expressing cell, a compound that changes the binding property between the peptide of the present invention and the protein of the present invention is screened. .
  • a test compound that inhibits the arachidonic acid metabolite release activity of the peptide of the present invention can be selected as a candidate substance having a competitive inhibition ability.
  • the test compound alone is brought into contact with the protein-expressing cell of the present invention, and the arachidonic acid metabolite releasing activity of the protein-expressing cell of the present invention is examined by a known method.
  • a compound exhibiting agonist activity can also be screened.
  • the protein-expressing cells of the present invention are seeded on a 24-well plate at 5 ⁇ 10 4 ce 11 / we 11, and cultured for 24 hours, and [ 3 H] arachidonic acid is added to 0.25 Ci / well. After 16 hours, wash the cells with Hanks buffer (pH 7.4) containing 0.05% BSA and 20 mM HE PES (hereinafter abbreviated as reaction buffer 1). A reaction buffer 500 ⁇ 1 containing a final concentration of 10 M of the peptide of the present invention or a final concentration of 10 / M of the peptide of the present invention and a test compound is added to each we11. After incubating at 37 ° C for 60 minutes, the reaction solution 4001 is added to the scintillator overnight, and the amount of [ 3 H] arachidonic acid metabolite + released in the reaction solution is measured by scintillation counting.
  • reaction buffer containing peptide free [3 H] Arakidon the amount of acid metabolites as 100%, free [3 H] Arakidon acid when the test compound added 'to addition of (test compound non-added additive) Calculate the amount of metabolite.
  • test compound having an arachidonic acid metabolite releasing activity of, for example, 50% or less can be selected as a candidate substance having an antagonistic ability.
  • the intracellular Ca concentration increases by stimulation with the peptide of the present invention.
  • the stimulating activity of the peptide of the present invention on the protein-expressing cell of the present invention is measured to screen for a compound that changes the binding property between the peptide of the present invention and the protein of the present invention. be able to. '
  • the intracellular calcium concentration in the case where the peptide of the present invention is brought into contact with the protein-expressing cell of the present invention and in the case where the peptide of the present invention and the test compound are brought into contact with the protein-expressing cell of the present invention By measuring and comparing the increasing activity, a compound that changes the binding property between the peptide of the present invention and the protein of the present invention is screened. The measurement is performed according to a known method.
  • an increase in intracellular calcium concentration by the peptide of the present invention is suppressed.
  • the test compound to be tested can be selected as a candidate substance capable of competitive inhibition.
  • an agonist can be screened by measuring an increase in fluorescence intensity due to the addition of the test compound alone.
  • the protein-expressing cells of the present invention are seeded on a sterilized cover glass for a microscope. Two days later, the culture solution is replaced with HBSS suspended in 4 mM Fura-2AM (Dojindo Laboratories), and incubated at room temperature. Leave for 30 minutes. After washing with HBSS, place a coverslip on the cuvette, add the peptide of the present invention or the peptide of the present invention and a test compound, and emit fluorescence at 505 nm at excitation wavelengths of 300 nm and 380 nm. Measure the increase in intensity ratio with a fluorimeter and compare.
  • FLIPR manufactured by Molecular Devices
  • F1uo-3AM manufactured by Dojindo Laboratories
  • F1uo-3AM manufactured by Dojindo Laboratories
  • a protein gene (eg, aequorin, etc.) that emits light due to an increase in intracellular Ca ion is co-expressed in the protein-expressing cell of the present invention, and the intracellular Ca ion concentration is increased.
  • the gene protein eg, aequorin or the like
  • a gene for a protein that emits light when the intracellular Ca ion increases is co-expressed, and the protein-expressing cell of the present invention is seeded on a 96-well plate, and the peptide of the present invention or the Add the peptide and test compound, measure the increase in the ratio of fluorescence intensity with a fluorimeter, and compare.
  • a test compound that suppresses an increase in fluorescence intensity due to the peptide of the present invention can be selected as a candidate substance having a competitive inhibition ability.
  • clone 24 of ETA (endothelin A receptor) -expressing CH0 cell Abbreviated as ETA24 cells. Journal of Pharmacology and Experimental
  • Therapeutics Vol. 279, pp. 675-685, 1996) was used to measure the activity of increasing the intracellular Ca ion concentration of the protein-expressing cells of the present invention and ETA24 cells using FLIPR (manufactured by Molecular Devices). This is performed using The cells expressing the protein of the present invention and ETA24 cells should be subcultured in DMEM supplemented with 10% dialyzed fetal calf serum (hereinafter referred to as dFBS). Protein expressing cells of the present invention, suspended in medium (10% d FBS-DMEM) such that ETA24 cells each 15xl0 4 cells / ml, in 96 well plate for FLIPR (Black plate clear bottom.
  • medium 10% d FBS-DMEM
  • H / HBSS Nisy Hanks 2 (Nissui Pharmaceutical Co., Ltd.) 9.8 g, sodium hydrogen carbonate 0.35 g, HEPES 4.77 g, adjusted to pH 7.4 with sodium hydroxide solution, filter sterilized) 20 ml, 250 mM Probenecid 200 ⁇ Mix 200 fetal serum (FBS).
  • Fluo 3-AM Diojindo Research Laboratories 2 piles (50 xg) was converted to dimethyl sulfoxide 401, 20% Pluronic acid
  • H / HBSS-Probenecid-FBS Molecular Probes 40, and added to the above H / HBSS-Probenecid-FBS. After mixing, pipette 100 l of each well into the cell plate from which the culture solution has been removed using an 8-tube pipette. % C0 2 incubator base 1 hour at 37 ° C for at one coater in incubate (dye the loading). H / HBSS 1501 containing 2.5 mM Probenecid and 0.1% CHAPS is added to the sample for each assay (each fraction), diluted, and transferred to a 96-well plate for FLIPR (V-Bottom plate, Coster) (hereafter, sample plate) ).
  • FLIPR V-Bottom plate, Coster
  • a peptide of the present invention was brought into contact with a protein-expressing cell of the present invention in the presence of labeled inositol, and a peptide and a test compound of the present invention were brought into contact with a protein-expressing cell of the present invention.
  • the inositol triphosphate-producing activity is measured and compared to screen for a compound that alters the binding property between the peptide of the present invention and the protein of the present invention. The measurement is performed according to a known method. .
  • a test compound that suppresses inositol triphosphate-producing activity can be selected as a candidate substance capable of competitive inhibition.
  • agonists can be screened by bringing only the test compound into contact with the protein-expressing cells of the present invention and measuring the increase in inositol triphosphate production.
  • the cells expressing the protein of the present invention are seeded on a 24-well plate and cultured for one day. Thereafter, the cells are cultured for 1 day in a medium supplemented with myo- [2-3 ⁇ 4] inositol (2.5 Ci / well), and the cells are thoroughly washed with a medium without radioactive inositol. After adding the peptide of the present invention or the peptide and the test compound of the present invention, 10% perchloric acid is added to stop the reaction. 1. neutralized with 5M potassium hydroxide and 60 mM HEPES solution and then passed through a column packed with AGlx8 tree fat (Bi o-Rad) in 0.
  • the radioactivity eluted with 1 M ammonium formate and 0.1 M formic acid is measured with a liquid scintillation counter.
  • the radioactivity when the peptide of the present invention is not added is 0%, and the radioactivity when the peptide of the present invention is added is 100%.
  • test compound having an inositol triphosphate-producing activity of, for example, 50% or less can be selected as a candidate substance having an antagonistic ability.
  • DNA containing a TRE is inserted upstream of the reporter gene of the vector to obtain a TRE-repo overnight gene vector.
  • TPA response element DNA containing a TRE (TPA response element) is inserted upstream of the reporter gene of the vector to obtain a TRE-repo overnight gene vector.
  • stimulation accompanied by an increase in the intracellular calcium concentration is caused by the TRE-mediated repo overnight gene expression and the subsequent reporter Induces the production of the gene product (protein) of the gene.
  • the TRE-reporter gene vector by measuring the enzymatic activity of the reporter gene protein, it is possible to detect fluctuations in the amount of calcium in the cells into which the TRE-reporter gene vector has been introduced.
  • the peptide of the present invention when the peptide of the present invention is brought into contact with a TRE-reporter gene vector-introduced protein-expressing cell of the present invention, the peptide of the present invention and the test compound are expressed in the TRE-reporter gene.
  • the vector examples include Pitska Gene Basic Vector and Pitska Gene Enhancer Vector (Toyo Ink Mfg. Co., Ltd.).
  • the TRE-containing DNA is inserted into a reporter gene of the above vector, for example, a multi-cloning site upstream of the luciferase gene, to obtain a TRE-reporter gene vector.
  • a test compound that suppresses the enzymatic activity of the repo overnight gene protein by the peptide of the present invention can be selected as a candidate substance having a competitive inhibition ability.
  • agonist screening is performed by contacting only the test compound with the TRE-repo allele-introduced gene-introduced protein-expressing cells of the present invention and measuring the increase in luminescence in the same manner as the peptide of the present invention. You can do it.
  • luciferase As a reporter gene.
  • Cells expressing the protein of the present invention into which the TRE-reporter gene (luciferase) has been introduced are seeded on a 24-well plate at 510 3 6 1 176 11 and cultured for 48 hours. After washing the cells with Hanks buffer (pH 7.4) containing 0.05% BSA and 20 mM HEPES, 10 nM of the peptide of the present invention or 10 nM of the peptide of the present invention and a test compound are added thereto, and the mixture is added at 37 ° C. And react for 60 minutes.
  • Hanks buffer pH 7.4
  • BSA Hanks buffer
  • 10 nM of the peptide of the present invention or 10 nM of the peptide of the present invention and a test compound are added thereto, and the mixture is added at 37 ° C. And react for 60 minutes.
  • Lyse the cells with a cell lysing agent for Pitka Gene (Toyo Ink Mfg. Co., Ltd.) and add a luminescent substrate (Toyo Ink Mfg. Co., Ltd.) to the lysate.
  • Luminescence from luciferase is measured using a luminometer, liquid scintillation counter or top counter. The amount of luminescence by luciferase when the peptide of the present invention is added and when ⁇ ⁇ the peptide of the present invention and a test compound are added are measured and compared.
  • the increase in intracellular calcium by the peptide of the present invention increases the amount of luminescence by luciferase.
  • a compound that suppresses this increase can be selected as a candidate substance capable of competitive inhibition.
  • Reporter genes include, for example, alkaline phosphatase, chloramphenic acid, acetylfurantransferrase ⁇ chloramphenicol
  • Genes such as acetyltransferase) and _galactosidase may be used.
  • the enzyme activities of these reporter gene proteins are measured according to a known method or using a commercially available measurement kit.
  • '' Alkali phosphatase activity was measured using, for example, Lumi-Phos 530 manufactured by Wako Pure Chemical, and chloramphenicol acetyltransferase was evaluated using, for example, FAST CAT chrola immediately manufactured by Wako Pure Chemical.
  • S-galactosidase activity is measured using, for example, Aurora Gat XE manufactured by Wako Pure Chemical Industries.
  • MAP kinase is activated and proliferates by stimulation of the peptide of the present invention.
  • this reaction to measure the stimulating activity of the peptide of the present invention on the cells expressing the protein of the present invention, it is possible to screen for a compound that alters the binding property between the peptide of the present invention and the protein of the present invention. .
  • the peptide of the present invention was brought into contact with a cell expressing the protein of the present invention.
  • the binding between the peptide of the present invention and the protein of the present invention can be determined. Screen for compounds that alter the.
  • Proliferation of the protein-expressing cells of the present invention may be measured, for example, by measuring MAP kinase activity, thymidine uptake activity, cell number, and the like.
  • MAP kinase activity after adding the peptide of the present invention or the peptide of the present invention and a test compound to the cells expressing the protein of the present invention, an anti-VIAP kinase antibody was used from a cell lysate. After obtaining the MAP kinase fraction by immunoprecipitation, the MAP kinase activity is measured using a known method, for example, MAP Kinase Assay II- [ 32 P] -ATP manufactured by Wako Pure Chemical Industries, and compared.
  • the protein-expressing cells of the present invention were seeded on a 24-well plate, cultured, added with the peptide of the present invention or the peptide of the present invention and a test compound, and then labeled with radioactivity.
  • thymidine e.g., [methy Bok 3 H] - thymidine down, etc.
  • the cells were lysed and the radioactivity of the thymidine incorporated into the cells, by counting in a liquid scintillator one Chillon counter, thymidylate ' Measure gin uptake activity and compare.
  • the protein-expressing cells of the present invention were seeded on a 24-well plate, cultured, and after adding the peptide of the present invention or the peptide of the present invention and a test compound, MTT (3- ( Add 4,5-dimethyl-2-thiazolyl) -2,5-diphenyl-2H-tetrazolium bromide).
  • MTT (3- ( Add 4,5-dimethyl-2-thiazolyl) -2,5-diphenyl-2H-tetrazolium bromide).
  • MTT formazan in which MTT has been changed by being taken into cells is dissolved in an aqueous solution of isopropanol acidified with hydrochloric acid, and then measured by absorption at 570 nm for comparison.
  • a test compound that suppresses the growth of the protein-expressing cell of the present invention can be selected as a candidate substance having a competitive inhibition ability.
  • agonists can be screened by bringing only the test compound into contact with the protein-expressing cell of the present invention and measuring the cell proliferation activity similar to that of the peptide of the present invention.
  • the evening protein-expressing cells of the present invention are seeded at 5,000 cells / well in a 24-well plate and cultured for one day. The cells are then starved in a serum-free medium for 2 days.
  • the peptide or peptide and a test compound of the present invention of the present invention after incubation was added to the cells 24 hours, [methy Bok 3 H] - thymidine was added Ueru per 0. 015MBq, incubation 6 hours. After washing the cells with PBS, add methanol and let stand for 10 minutes. Next, add 5% trichloroacetic acid and let stand for 15 minutes. Wash the fixed cells four times with distilled water. Lyse the cells with 0.3N sodium hydroxide solution and measure the radioactivity in the lysate with a liquid scintillation counter.
  • a test compound that suppresses an increase in radioactivity when the peptide of the present invention is added can be selected as a candidate substance capable of competitive inhibition.
  • the stimulation of the peptide of the present invention activates the potassium channel, and intracellular K ions flow out of the cell.
  • the stimulating activity of the peptide of the present invention on cells expressing the protein of the present invention is measured to screen for compounds that alter the binding between the peptide of the present invention and the protein of the present invention. be able to.
  • Rb ions rubberidium ions
  • Rb ions which are homologous to K ions, flow out of the cell through potassium channels without distinction from K ions.
  • the protein-expressing cells of the present invention a radioactive isotope R b After allowed incorporation of ([8 6 R b]) , the 8 6 R b flowing out by stimulation of the peptides of the present invention
  • the stimulating activity of the peptide of the present invention on the cells expressing the protein of the present invention is measured.
  • the peptide of the present invention when the peptide of the present invention is brought into contact with the protein-expressing cell of the present invention in the presence of 86 Rb, the peptide of the present invention and the test compound are brought into contact with the protein-expressing cell of the present invention. in the case of it is to measure the efflux activity of 8 6 R b, by comparing, for screening the compound that changes the binding property between the protein of the peptide and the invention of the present invention.
  • the 8 6 R b test compound to suppress an increase in efflux activity of by peptide stimulation of the present invention can be selected as a candidate substance capable of competitive inhibition.
  • only the test compound is brought into contact with the protein-expressing cell of the present invention, It is also possible to perform the disk re-learning of Agonisu Bok by measuring the increase in peptide similar 8 6 R b of efflux activity.
  • the protein-expressing cells of the present invention are seeded on a 24-well plate and cultured for 2 days. Thereafter, the cells are incubated for 2 hours in a medium containing 1 mCiZm1 and 86 RbC1. Wash cells thoroughly with medium to completely remove 86 RbC1 in the external solution.
  • the peptide of the present invention or the peptide of the present invention and a test compound are added to the cells, and after 30 minutes, the external solution is collected, and the radioactivity is measured and compared at a local time.
  • a test compound that suppresses an increase in the 86 Rb efflux activity due to peptide stimulation of the present invention can be selected as a candidate substance having a competitive inhibitory ability.
  • the extracellular pH change is measured using, for example, a Cytosensor device (Molecular Devices). ,
  • a test compound that suppresses the extracellular pH change caused by the peptide of the present invention can be selected as a candidate substance capable of competitive inhibition.
  • agonist screening can be performed by bringing only the test compound into contact with the protein-expressing cell of the present invention and measuring the extracellular pH change similar to that of the peptide of the present invention.
  • the protein-expressing cells of the present invention are cultured overnight in a capsule for a Cytosensor device, set in the chamber of the device, and set to 0.1% for about 2 hours until the extracellular pH is stabilized.
  • Perfuse 1 to 1640 medium (Molecular Devices) containing 83-8. After the pH is stabilized, a medium containing the peptide of the present invention or the peptide of the present invention and the test compound is perfused on the cells. Measure and compare the pH change of the medium caused by perfusion.
  • a compound that suppresses the extracellular pH change caused by the peptide of the present invention can be selected as a candidate substance having a competitive inhibition ability.
  • the sex pheromone receptor, ST e2, of the haploida-mating type (MAT) of yeast (Sacc aromyces cerevisiae) is conjugated to the G protein, Gpa1, and responds to the sex pheromone mating factor.
  • MAT haploida-mating type
  • Gpa1 G protein
  • Stel 2 induces the expression of various proteins (eg, FUS 1 involved in conjugation).
  • the control factor S st 2 functions in the above process in a suppressive manner.
  • a yeast into which a receptor gene has been introduced is produced, a signal transduction system in the yeast cell is activated by stimulation of the receptor gene, and the resulting proliferation is used as an index.
  • Attempts have been made to measure the reaction between a protein and a receptor (Trends in Biotechnology, 15, 487-494, 1997).
  • a compound capable of changing the binding property between the peptide of the present invention and the protein of the present invention can be screened.
  • the genes encoding Ste2 and Gpa1 of the MAT ⁇ yeast are removed, and instead, the protein gene of the present invention and a gene encoding the Gpa1-Gai2 fusion protein are introduced.
  • the gene encoding Far is removed to prevent ceU-cycle arrest from occurring, and the gene encoding Sst is removed to increase the sensitivity of the response to the peptide of the present invention.
  • the FUS1—HIS3 gene in which the histidine biosynthesis gene HIS3 is linked to FUS1 is introduced. This genetic recombination operation can be performed, for example, by replacing the somatostin-tin receptor-type-1 (SSTR2) gene with the protein of the present invention in the method described in Molecular and Cellular Biology, vol.
  • SSTR2 somatostin-tin receptor-type-1
  • the transformed yeast thus constructed reacts with high sensitivity to the peptide of the present invention, resulting in the activation of MAP kinase, the synthesis of histidine biosynthesis enzyme, and the histidine-deficient medium. It is possible to grow in.
  • the above-described protein-expressing yeast of the present invention (the Ste2 gene and the Gpa1 gene were removed, the protein gene of the present invention and the Gpa1-Gai2 fusion protein-encoding gene were introduced, and the Far gene was introduced.
  • S ⁇ yeast with the FUS1-1HIS3 gene removed and the Sst gene removed is cultured in a histidine-deficient medium, and the peptide of the present invention or the peptide of the present invention and a test compound are contacted with each other; By measuring and comparing the growth of the yeast, it is possible to screen for compounds that alter the binding between the peptide of the present invention and the protein of the present invention. In this method, a test compound that suppresses the growth of the yeast can be selected as a candidate substance having a competitive inhibition ability.
  • agonist screening can also be performed by bringing only the test compound into contact with the above-described yeast expressing the protein of the present invention and measuring the growth of yeast similar to the peptide of the present invention.
  • the above-described protein-expressing yeast of the present invention is cultured overnight in a liquid medium of a complete synthetic medium, and then added to a dissolved agar medium from which histidine has been removed to a concentration of 2 ⁇ 10 4 ce 11 / m 1. Then sow in a 9 x 9 cm square petri dish. After the agar solidifies, a sterile filter paper impregnated with the peptide of the present invention or the peptide of the present invention and the test compound is placed on the agar surface, and cultured at 30 ° C for 3 days. The effect of the test compound is to compare the growth of yeast around the filter paper with the use of sterile filter paper impregnated with only the peptide of the present invention. In addition, the peptide of the present invention is added to the agar medium from which histidine has been removed in advance, and the test compound is soaked in sterile filter paper to culture the yeast. You may observe that they are affected.
  • a compound that suppresses the growth of yeast can be selected as a candidate substance having a competitive inhibition ability.
  • RNA-introduced African Xenopus oocytes RNA-introduced African Xenopus oocytes
  • test compound of the present invention By measuring and comparing changes in cell membrane potential when brought into contact with the introduced Xenopus laevis oocytes, a compound that changes the binding property between the peptide of the present invention and the protein of the present invention is screened. I do.
  • a test compound that suppresses a change in cell membrane potential can be selected as a candidate substance having a competitive inhibition ability.
  • agonists can be screened by bringing only test compounds into contact with the protein gene of the present invention, RNA-introduced African oocytes, and measuring changes in cell membrane potential similar to those of the peptides of the present invention.
  • the protein gene mRN ⁇ of the present invention may be prepared from a tissue or a cell, or may be transcribed from a plasmid in vitro.
  • the protein gene mRNA of the present invention was cultured in MBS solution at 20 ° C for 3 days, and this was cultured in a voltage clamp equipped with Ringer solution. Insert the potential fixing glass microelectrode and the potential measurement glass microelectrode into the cell, and place the electrode outside the cell. When the potential is stabilized, a Ringer solution containing the peptide of the present invention or the peptide of the present invention and a test compound is passed, and the change in potential is recorded.
  • the effect of the test compound can be measured by comparing the change in the cell membrane potential of the oocytes of the present invention into which the protein gene of the present invention has been transfected with RNA, compared to the case where a Ringer solution containing only the peptide of the present invention is applied. .
  • a compound that suppresses cell membrane electrical changes can be selected as a candidate substance having a competitive inhibition ability.
  • the increase in the amount of change in the potential facilitates the measurement, and thus, the pO1A-added RNA of each G protein gene may be introduced.
  • polyA-added RNA of a gene of a protein eg, aequorin, etc.
  • a screening kit for a compound or a salt thereof that alters the binding property between the peptide of the present invention and the protein of the present invention includes a protein of the present invention, a cell containing the protein of the present invention, or a cell containing the protein of the present invention.
  • a membrane fraction, and a peptide of the present invention includes a protein of the present invention, a cell containing the protein of the present invention, or a cell containing the protein of the present invention.
  • screening kit of the present invention examples include the following. 1. Screening reagent
  • CH0 cells expressing the protein of the present invention were subcultured in 12-well plates at 5 ⁇ 10 5 Z-wells and cultured at 37 ° C., 5% CO 2 , 95% air for 2 days. .
  • the peptide of the present invention is dissolved to a concentration of lmM in PBS containing 0.1% ⁇ serum albumin (Sigma) and stored at -20 ° C.
  • test compound solution (ii) 10- 3 ⁇ 1 ( ) M 5 1 added, the peptide of the present invention labeled 5 1 was added to react at room temperature for one hour.
  • 5/1 of the peptide of the present invention is added instead of the test compound.
  • NSB Non-specific Binding ''
  • the compound or a salt thereof obtained by using the screening method or the screening kit of the present invention is a compound that changes the binding (inhibits or promotes the binding) between the peptide of the present invention and the protein of the present invention.
  • a compound having a cell stimulating activity via the protein of the present invention or a salt thereof a so-called agonist of the protein of the present invention
  • a compound not having the stimulating activity a so-called antagonist of the protein of the present invention.
  • Such compounds include peptides, proteins, non-peptidic compounds, synthetic compounds, fermentation products, and the like. It may be a novel compound or a known compound.
  • the specific method for evaluating whether the above-mentioned protein of the present invention is an agonist or an antagonist may be according to the following ( ⁇ ) or ( ⁇ ).
  • the binding assay shown in the above screening method is performed to obtain a compound that changes the binding property (particularly, inhibits the binding) between the peptide of the present invention and the protein of the present invention. Is determined whether it has the above-described cell stimulating activity via the protein of the present invention.
  • a compound having a cell stimulating activity or a salt thereof is an agonist of the protein of the present invention, and a compound or a salt thereof having no such activity is an agonist of the protein of the present invention.
  • test compound is brought into contact with a cell containing the protein of the present invention, and the cell stimulating activity mediated by the protein of the present invention is measured.
  • the compound having a cell stimulating activity or a salt thereof is an agonist of the protein of the present invention.
  • a compound that activates the protein of the present invention for example, a peptide of the present invention or an agonist of the protein of the present invention
  • a cell containing the protein of the present invention The cell stimulating activity mediated by the protein of the present invention when the compound to be converted and the test compound are brought into contact with the cells containing the protein of the present invention is measured and compared.
  • the compound capable of decreasing the cell stimulating activity of the compound activating the protein of the present invention or a salt thereof is an antagonist of the protein of the present invention.
  • the agonist of the protein of the present invention has the same activity as the physiological activity of the peptide of the present invention on the protein of the present invention, it is useful as a safe and low-toxic drug like the peptide of the present invention. It is.
  • the protein antagonist of the present invention can suppress the physiological activity of the peptide of the present invention on the protein of the present invention, and is therefore useful as a safe and low-toxic drug for suppressing the receptor activity. is there.
  • the protein of the present invention is involved in an appetite (feeding) promoting action and an oxytocin secretion promoting action. Therefore, among the compounds obtained using the above-mentioned screening method or screening kit, agonists of the protein of the present invention include, for example, an appetite (feeding) enhancer, anorexia (eg, anorexia nervosa), Appetite It can be used as a medicament for the prevention and treatment of anemia or hypoproteinemia due to tremor, weak labor, laxative bleeding, uterine remodeling failure, and milk stasis.
  • the protein antagonists of the present invention include, for example, obesity (eg, malignant mastocytosis), exogenous obesity, hyperinsulin inar obesity, hyperplasma Hyperplasmic obesity ⁇ hypophyseal adiposity, hypoplasmic obesity> hypothyroid obesity, hypothalamic obesity, hypothalamic obesity, symptoms Obesity (sy immediate tomatic obesity), childhood obesity (infantile obesity), upper body obesity, alimentary obesity, hypogonadal obesity, systemic mastocytosis
  • central obesity central obesity
  • hyperphagia affective disorders, sexual dysfunction, overwork, tonic contractions, fetal asphyxia, uterine rupture, tubal lacerations, preterm birth, Prader-Willi syndrome, glucoseuria and Its complications (eg, diabetic nephropathy, diabetic retinopathy, diabetic neuropathy, etc.), hypertension, hyperlipidemia, coronary atherosclerosis, gout, respiratory disease (Pickwick syndrome, sleep apnea syndrome) It can be used as a medicine for prophylactic and therapeutic agents such as fatty liver, infertility, osteoarthritis, etc. (especially antiobesity agents, appetite (feeding) regulators, etc.).
  • a pharmaceutically acceptable salt or the like is used as a salt of the compound obtained by using the above-described screening method or screening kit.
  • a pharmaceutically acceptable salt or the like examples thereof include salts with inorganic bases, salts with organic bases, salts with inorganic acids, salts with organic acids, and salts with basic or acidic amino acids.
  • the salt with an inorganic base include an alkali metal salt such as a sodium salt and a potassium salt, an alkaline earth metal salt such as a calcium salt and a magnesium salt, and an aluminum salt and an ammonium salt.
  • Preferred examples of the salt with an organic base include, for example, trimethylamine, triethylamine, pyridine, picoline, 2,6-alutidine, ethanolamine, genoaluminamine, triethanolamine, cyclohexylamine, dicyclohexane.
  • Examples include salts with xylamine, N, N, and dibenzylethylenediamine.
  • Preferred examples of the salt with an inorganic acid include salts with hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric acid, and the like.
  • Suitable examples of salts with organic acids include, for example, formic acid, acetic acid, propionic acid, fumaric acid, oxalic acid, tartaric acid, maleic acid, citric acid, succinic acid, malic acid, methanesulfonic acid, benzenesulfonic acid, benzoic acid And the like.
  • salts with a basic amino acid include salts with, for example, arginine, lysine, orcinus
  • salts with an acidic amino acid include, for example, salts with aspartic acid, glutamic acid, etc. Is raised.
  • a compound or a salt thereof obtained by using the screening method or the screening kit of the present invention can be carried out according to a conventional method.
  • It can be used parenterally in the form of injections, such as aqueous solutions or suspensions.
  • a physiologically acceptable carrier, flavoring agent, excipient, vehicle, preservative, stabilizer, binder and the like in a generally accepted unit dosage form.
  • 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, gum arabic, excipients such as crystalline cellulose, corn starch, gelatin, alginic acid, etc.
  • binders such as gelatin, corn starch, tragacanth gum, gum arabic
  • excipients such as crystalline cellulose, corn starch, gelatin, alginic acid, etc.
  • a leavening agent such as magnesium stearate
  • a sweetening agent such as sucrose, lactose or saccharin
  • a flavoring agent such as peppermint, cocoa oil or cellulose
  • a liquid carrier such as oils and fats can be further contained in the above-mentioned type of material.
  • Sterile compositions for injection include active substances in vehicles such as water for injection, natural substances such as sesame oil, coconut oil and the like. It can be formulated according to the usual formulation practice such as dissolv
  • Aqueous liquids for injection include, for example, physiological saline, isotonic solutions containing glucose and other adjuvants (eg, D-sorbitol, D-mannitol, sodium chloride, etc.).
  • Agents such as alcohols (eg, ethanol), polyalcohols (eg, propylene glycol, polyethylene glycol), nonionic surfactants (eg, Polysorbate 80 TM, HCO-50) and the like may be used in combination.
  • examples of the oily liquid include sesame oil and soybean oil, which may be used in combination with a dissolution aid such as benzyl benzoate or benzyl alcohol.
  • buffers eg, phosphate buffer, sodium acetate buffer
  • the prepared injection solution is usually filled in a suitable ampoule.
  • the preparations obtained in this way are safe and low toxic, and can be used, for example, in mammals (eg, humans, mice, rats, guinea pigs, egrets, sheep, sheep, bushus, dogs, cats, dogs, monkeys, chimpanzees, etc. ) Can be administered.
  • mammals eg, humans, mice, rats, guinea pigs, egrets, sheep, sheep, bushus, dogs, cats, dogs, monkeys, chimpanzees, etc.
  • the dose of a compound or a salt thereof obtained by using the screening method or the screening kit of the present invention varies depending on symptoms and the like.
  • the single dose varies depending on the administration subject, target organ, symptoms, administration method, etc.
  • injection into adult obese patients with a body weight of 60 kg
  • the dose can be administered in terms of 60 kg.
  • the antibody of the present invention can specifically recognize the protein of the present invention, It can be used for quantification of the protein of the present invention in a test solution, particularly for quantification by sandwich immunoassay.
  • the present invention provides a method for quantifying the protein of the present invention in a test solution, characterized in that in the quantification method of the above (ii), one of the antibodies is an antibody that recognizes the N-terminal of the protein of the present invention, and the other is It is desirable that the above-mentioned antibody reacts with the C-terminal of the protein of the present invention.
  • the protein of the present invention can be quantified using a monoclonal antibody against the protein of the present invention (hereinafter sometimes referred to as the monoclonal antibody of the present invention), and can also be detected by tissue staining or the like.
  • the anti-body molecule itself may be used, or the F (ab,) 2 , Fab ′ or Fab fraction of the antibody molecule may be used.
  • the method for quantifying the protein of the present invention using the antibody of the present invention is not particularly limited, and may be an antibody, an antigen, or an antibody-antigen complex corresponding to the amount of antigen (eg, the amount of protein) in the test solution. Any measurement method may be used as long as the amount of the body is detected by chemical or physical means, and this is calculated from a standard curve prepared using a standard solution containing a known amount of antigen. . For example, nephrometry, competition method, immunometric method and sandwich method are preferably used, but it is particularly preferable to use the sandwich method described later in terms of sensitivity and specificity.
  • radioactive isotopes elemental eg, [125 1], [131 1], [3 ⁇ 4], [ '4 C], [32 P], [33 P], [ 35s ], etc.
  • fluorescent substances eg, cyanine fluorescent dyes (eg, Cy2, Cy3, Cy5, Cy5.5, Cy7 (manufactured by Amersham Biosciences), etc.), fluorescamine, fluorescenisochi Etc.
  • enzymes eg, -galactosidase,) 3-darcosidase, Lucariphosphatase, passoxidase, malate dehydrogenase, etc.
  • luminescent substances eg, luminol, luminol derivatives, luciferin, lucigenin, etc.
  • a biotin-avidin system can be used for binding the antibody or antigen to the labeling agent.
  • the carrier include insoluble polysaccharides such as agarose, dextran, and cellulose; synthetic resins such as polystyrene, polyacrylamide, and silicon; and glass.
  • a test solution is reacted with the insolubilized monoclonal antibody of the present invention (primary reaction), and further reacted with another labeled monoclonal antibody of the present invention (secondary reaction).
  • primary reaction the insolubilized monoclonal antibody of the present invention
  • secondary reaction another labeled monoclonal antibody of the present invention
  • the primary reaction and the secondary reaction may be performed in the reverse order, may be performed simultaneously, or may be performed at staggered times.
  • the labeling agent and the method of insolubilization can be in accordance with those described above.
  • the antibody used for the solid phase antibody or the labeling antibody does not necessarily need to be one kind, and a mixture of two or more kinds of antibodies is used for the purpose of improving measurement sensitivity and the like. May be used.
  • the monoclonal antibody of the present invention used in the primary reaction and the secondary reaction is preferably an antibody having a different site to which the protein of the present invention binds.
  • the antibody used in the primary reaction and the secondary reaction is, for example, when the antibody used in the secondary reaction recognizes the C-terminal of the protein of the present invention, the antibody used in the primary reaction is Preferably, an antibody that recognizes other than the C-terminal, for example, the N-terminal, is used.
  • the monoclonal antibody of the present invention can be used in a measurement system other than the sandwich method, for example, a competition method, an immunometric method or a nephelometry.
  • a competition method the antigen in the test solution and the labeled antigen are used for the antibody.
  • the unreacted labeled antigen (F) and the labeled antigen (B) bound to the antibody are separated (BZF separation), and the amount of labeling of either B or F is measured. Quantify the amount of antigen in the test solution You.
  • a soluble antibody was used as the antibody
  • B / F separation was performed using polyethylene glycol
  • a liquid phase method using a second antibody against the antibody was used as the first antibody.
  • a solid phase antibody was used as the first antibody.
  • an immobilization method using a soluble first antibody and an immobilized antibody as the second antibody is used.
  • the antigen in the test solution and the immobilized antigen are subjected to a competitive reaction with a certain amount of labeled antibody, and then the solid phase and the liquid phase are separated.
  • the antigen is allowed to react with an excess amount of the labeled antibody, then the immobilized antigen is added, and the unreacted labeled antibody is bound to the solid phase, and then the solid phase and the liquid phase are separated.
  • the amount of label in either phase is measured to determine the amount of antigen in the test solution.
  • nephrometry the amount of insoluble sediment resulting from an antigen-antibody reaction in a gel or in a solution is measured. Even when the amount of antigen in the test solution is small and only a small amount of sediment is obtained, laser-nephrometry using laser-dispersion is preferably used.
  • the protein measuring system of the present invention may be constructed by adding ordinary technical considerations of those skilled in the art to ordinary conditions and operation methods in each method. For details of these general technical means, reference can be made to reviews, documents, etc.
  • the protein of the present invention can be quantified with high sensitivity by using the antibody of the present invention.
  • anorexia eg, anorexia nervosa, etc.
  • Anemia or hypoproteinemia due to anorexia, weak labor, lax hemorrhage, uterine remodeling failure, milk stasis, etc. can be diagnosed as having a high possibility of developing.
  • an increase in the concentration of the protein of the present invention is detected, for example, obesity (eg, vein mastocytosis, exogenous obesity, hyperinsulinic obesity, hyperplasmic obesity, pituitary) Obesity, reduced plasma obesity, hypothyroid obesity, hypothalamic obesity, symptomatic obesity, pediatric obesity, upper body obesity, dietary obesity, hypogonadism, systemic mastocytosis, simple obesity , Central obesity, etc.), hyperphagia, affective disorders, sexual dysfunction, excessive labor, tonic contractions, fetal asphyxia, uterine rupture, facial lacerations, preterm birth, Prader-Willi syndrome, diabetes and its complications Disease (eg, diabetic nephropathy, diabetic retinopathy, diabetic neuropathy, etc.), hypertension, hyperlipidemia, coronary atherosclerosis, gout, respiratory disease (Pickck syndrome, sleep apnea syndrome), Fatty liver, infertility It can be diagnosed that the possibility of osteoarthritis and the
  • the antibody of the present invention can be used for detecting the protein of the present invention present in a subject such as a body fluid or a tissue.
  • a subject such as a body fluid or a tissue.
  • the detection of the protein of the present invention in each fraction during purification, and the analysis of the behavior of the protein of the present invention in test cells, etc. Can be used.
  • the DNA of the present invention can be used, for example, in humans or warm-blooded animals (e.g., rats, mice, guinea pigs, egrets, birds, higgies, bushes, horses, cats, cats, dogs, Abnormalities (genetic abnormalities) in the DNA or mRNA encoding the protein of the present invention or a partial peptide thereof in monkeys, chimpanzees, etc.). It is useful as a gene diagnostic agent for mutation or decrease in expression, and increase or excessive expression of the DNA or mRNA.
  • warm-blooded animals e.g., rats, mice, guinea pigs, egrets, birds, higgies, bushes, horses, cats, cats, dogs, Abnormalities (genetic abnormalities) in the DNA or mRNA encoding the protein of the present invention or a partial peptide thereof in monkeys, chimpanzees, etc.
  • the above-described genetic diagnosis using the DNA of the present invention can be performed, for example, by the well-known Northern Hybridization ⁇ PCR-SSCP method (Genomics, Vol. 5, pp. 874-879 (1989)) Proceedings of the National Academy of Sciences of The United States of America, Vol. 86, pp. 2766-2770 (1989)).
  • obesity eg, malignant mastocytosis, exogenous obesity, hyperinsulinic obesity, hyperplasmic obesity, pituitary obesity, hypoplasma
  • Obesity hypothyroid obesity, hypothalamic obesity, symptomatic obesity, childhood obesity, upper body obesity, dietary obesity, hypogonadism obesity, systemic mastocytosis, simple obesity, centrality Obesity, etc.
  • hyperphagia affective disorders, sexual dysfunction, excessive labor, tonic contractions, fetal asphyxia, uterine rupture, tubal lacerations, preterm birth, Prader-Willi syndrome, diabetes and its complications ( Eg, diabetic nephropathy, diabetic retinopathy, diabetic neuropathy), hypertension, hyperlipidemia, coronary atherosclerosis, gout, respiratory disease (Pickwick syndrome, sleep apnea syndrome), fat Liver, It can be diagnosed that the possibility of infertility or osteoarthritis is high.
  • anorexia eg, anorexia nervosa
  • anorexia associated with anorexia It can be diagnosed as likely to be low protein, weak labor, lax hemorrhage, uterine remodeling failure, and milk stasis.
  • the antisense polynucleotide of the present invention which complementarily binds to the DNA of the present invention and can suppress the expression of the DNA, has low toxicity, and functions of the protein of the present invention or the DNA of the present invention in vivo (eg, Since it can suppress eating (appetite) promoting activity, for example, obesity (eg, malignant mastocytosis, exogenous obesity, hyperinsulinic obesity, hyperplasmic obesity, pituitary obesity) , Hypoplasmic obesity, hypothyroid obesity, hypothalamic obesity, symptomatic obesity, childhood obesity, upper body obesity, dietary obesity, hypogonadism, systemic mastocytosis, simple obesity , Central obesity), Hyperphagia, affective disorders, sexual dysfunction, overwork labor, tonic contractions, fetal asphyxia, ruptured pupils, facial tears, premature birth, Prader-Willi syndrome, diabetes and its complications (eg, diabetic Nephropathy, diabetic retinopathy, diabetic neuropathy, etc.), hypertension,
  • the above-mentioned antisense polynucleotide When used as the above-mentioned prophylactic / therapeutic agent, it can be formulated and administered according to a known method.
  • the antisense polynucleotide when used, the antisense polynucleotide is inserted alone or into a suitable vector such as a retrovirus vector, an adenovirus vector, an adenovirus associated virus vector, and the like. It can be administered orally or parenterally to humans or mammals (eg, rats, puppies, sheep, pigs, puppies, cats, dogs, monkeys, etc.).
  • the antisense polynucleotide can be administered as it is or in the form of a formulation together with a physiologically acceptable carrier such as an auxiliary agent for promoting uptake, and can be administered by a gene gun or a catheter such as a hydrogel catheter.
  • the dosage of the antisense polynucleotide varies depending on the target disease, the administration subject, the administration route, and the like.For example, when the antisense polynucleotide of the present invention is intravenously administered for the purpose of treating obesity, Generally, in an adult (body weight 60 kg), about 0.1 to 100 mg of the antisense polynucleotide is administered per day.
  • antisense polynucleotide can also be used as a diagnostic oligonucleotide probe for examining the presence or expression of the DNA of the present invention in tissues or cells.
  • a medicament comprising the lipozyme
  • an expression vector or the like containing the gene (DNA) encoding the lipozyme is provided.
  • double-stranded RNA, lipozyme and the like can disrupt RNA transcribed from the DNA of the present invention or suppress the function thereof, and the protein or the same of the present invention in vivo.
  • Obstructions for example, obesity (eg, malignant mastocytosis, exogenous obesity, hyperinsulinic obesity, hyperplasmic obesity, pituitary obesity, Plasma obesity, hypothyroid obesity, hypothalamic obesity, symptomatic obesity, pediatric obesity, upper body obesity, dietary obesity, hypogonadism, systemic mastocytosis, simple obesity, centrality Obesity, etc.), hyperphagia, affective disorders, sexual dysfunction, overwork labor, tonic contractions, fetal asphyxia, ruptured uterus, tubal lacerations, preterm birth, Prader-Willi syndrome, diabetes and its complications (Eg, diabetic nephropathy, diabetic retinopathy, diabetic neuropathy, etc.), hypertension, hyperlipidemia, coronary arteriosclerosis, gout, respiratory disease (Pickwick syndrome, sleep apnea syndrome group) ), Fatty liver, infertility, osteoarthritis, etc. (especially anti-obesity agents, appetite (especially anti-obe
  • the double-stranded RNA can be designed and produced based on the sequence of the polynucleotide of the present invention according to a known method (eg, Nature, 411, 494, 2001).
  • the lipozyme can be designed and manufactured based on the sequence of the polynucleotide of the present invention according to a known method (eg, TRENDS in Molecular Medicine, Vol. 7, pp. 221, 2001). For example, it can be produced by substituting a part of a known lipozyme sequence with a part of RNA encoding the protein of the present invention.
  • a consensus sequence NU X (where N represents all bases, X represents a base other than G) which can be cleaved by a known lipozyme, And the like.
  • RNA or lipozyme When the above-mentioned double-stranded RNA or lipozyme is used as the above-mentioned prophylactic / therapeutic agent, it can be formulated and administered in the same manner as the antisense polynucleotide.
  • the expression vector (V) is used in the same manner as known gene therapy methods and the like, and is used as the above-mentioned prophylactic / therapeutic agent.
  • the antibody of the present invention having the action of neutralizing the activity of the protein of the present invention can suppress the function of the protein of the present invention (eg, inactivate signal transduction), for example, obesity (eg, obesity) Malignant mastocytosis, exogenous obesity, hyperinsulinic obesity, hyperplasmic obesity, pituitary obesity, hypoplasmic obesity, hypothyroid obesity, hypothalamic obesity, symptomatic obesity, childhood obesity , Upper body obesity, dietary obesity, hypogonadism, obesity, systemic mastocytosis, simple obesity, central obesity, etc.), hyperphagia, affective disorder, sexual dysfunction, labour, tonicity Uterine contractions, fetal asphyxia, uterine rupture, ruptured tubules, preterm birth, Prader-Willi syndrome, diabetes and its complications (eg, diabetic nephropathy, diabetic retinopathy, diabetic neuropathy, etc.), hypertension, Hyperlipidemia, coronary Pulse sclerosis, gout, respiratory disease (Pickwick syndrome
  • the antibody of the present invention having an activity of activating the activity of the protein of the present invention can promote the function of the protein of the present invention (eg, activate signal transduction), for example, increase appetite (feeding) Drugs, anorexia (eg, anorexia nervosa), anemia or hypoproteinemia associated with anorexia, weak labor, lax bleeding, uterine remodeling failure, milk stasis, etc. it can.
  • anorexia eg, anorexia nervosa
  • anemia or hypoproteinemia associated with anorexia weak labor, lax bleeding, uterine remodeling failure, milk stasis, etc. it can.
  • the antibodies of the present invention can be administered by themselves or as a suitable pharmaceutical composition.
  • the pharmaceutical composition used for the administration contains the antibody or a salt thereof and a pharmacologically acceptable carrier, diluent or excipient.
  • Such compositions are provided in dosage forms suitable for oral or parenteral administration.
  • compositions for oral administration include solid or liquid dosage forms, specifically tablets (including sugar-coated tablets and film-coated tablets), pills, granules, powders, capsules ( Soft capsules), syrups, emulsions, suspensions and the like.
  • Such a composition is produced by a known method and contains a carrier, diluent or excipient usually used in the pharmaceutical field.
  • a carrier for example, lactose, starch, sucrose, magnesium stearate and the like are used as carriers and excipients for tablets.
  • compositions for parenteral administration for example, injections, suppositories, etc. are used.
  • Injections are in the form of intravenous injections, subcutaneous injections, intradermal injections, intramuscular injections, infusions, etc. Is included. Such injections are prepared according to known methods, for example, by dissolving, suspending or emulsifying the antibody or a salt thereof in a sterile aqueous or oily liquid commonly used for injections.
  • Aqueous liquids for injection include, for example, saline, isotonic solutions containing glucose and other adjuvants, and suitable solubilizing agents, such as alcohol (eg, ethanol) and polyalcohol (eg, , Propylene glycol, polyethylene glycol), nonionic surfactants (eg, polysorbate 80, HCO-50 (polyoxythylene (50 mol) adduc tof hydrogenated cas tor oil)), etc. Good.
  • alcohol eg, ethanol
  • polyalcohol eg, Propylene glycol, polyethylene glycol
  • nonionic surfactants eg, polysorbate 80, HCO-50 (polyoxythylene (50 mol) adduc tof hydrogenated cas tor oil)
  • oily liquid for example, sesame oil, soybean oil, and the like are used, and benzyl benzoate, benzyl alcohol, and the like may be used as a solubilizing agent.
  • the prepared injection is usually filled
  • each dosage unit dosage form is 5 to 500 mg, especially for injections.
  • 5 to 100 mg, and other dosage forms contain 10 to 25 Omg of the antibody.
  • compositions may contain other active ingredients as long as no undesirable interaction occurs due to the combination with the above antibody.
  • the prophylactic / therapeutic agent for the above-mentioned diseases containing the antibody of the present invention has low toxicity and can be used as it is as a liquid or as a pharmaceutical composition of an appropriate dosage form, in humans or mammals (eg, rat, egret, sheep, etc.). Can be administered orally or parenterally (eg, intravenously) to mice, dogs, cats, cats, dogs, monkeys, etc.). The dosage varies depending on the administration subject, target disease, symptoms, administration route, and the like.
  • 0.01 to 20 mg / kg body weight preferably 0.1 to 10 mg / kg body weight, more preferably 0.1 to 5 mg / kg body weight, about 1 to 5 times a day, preferably 1 to 5 times a day About 3 times as injection It is convenient to give. In the case of other parenteral administration and oral administration, an equivalent amount can be administered. If the symptoms are particularly severe, the dose may be increased accordingly.
  • the antibody of the present invention is also useful, for example, as a diagnostic agent for the above-mentioned diseases and the like.
  • the present invention has a DNA encoding the exogenous protein of the present invention (hereinafter abbreviated as the exogenous DNA of the present invention) or a mutant DNA thereof (sometimes abbreviated as the exogenous mutant DNA of the present invention).
  • the exogenous DNA of the present invention or a mutant DNA thereof (sometimes abbreviated as the exogenous mutant DNA of the present invention).
  • a non-human mammal is provided.
  • Non-human mammals having the exogenous DNA of the present invention or the mutant DNA thereof include unfertilized eggs, fertilized eggs, germ cells including spermatozoa and their progenitor cells, and the like.
  • the calcium phosphate method, the electric pulse method, the Lipofection It can be produced by introducing the target DNA by a method such as a coagulation method, a coagulation method, a microinjection method, a particle gun method, or a DEAE-dextran method.
  • the exogenous DNA of the present invention intended for somatic cells, organs of living organisms, tissue cells, and the like can be introduced and used for cell culture, tissue culture, and the like.
  • the DNA-introduced animal of the present invention can also be produced by fusing the cells with the above-mentioned germ cells by a known cell fusion method.
  • mice for example, pure strains such as C57B LZ6 strain and DBA2 strain, and hybrid strains such as BSC SFi strain, BDFi strain, B6D2 Ft strain, BALBZc strain, ICR strain, etc.
  • light eg, Wistar, SD, etc.
  • Examples of the “mammal” in the recombinant vector that can be expressed in mammals include humans and the like in addition to the above-mentioned non-human mammals.
  • exogenous DNA of the present invention does not mean the DNA of the present invention originally possessed by a non-human mammal but refers to the DNA of the present invention once isolated and extracted from the mammal.
  • mutant DNA of the present invention DNA having a mutation (for example, mutation) in the base sequence of the original DNA of the present invention, specifically, addition, deletion, or substitution of another base to another base DNA that has been used is used, and also includes abnormal DNA.
  • mutation for example, mutation
  • the abnormal DNA means a DNA that expresses an abnormal protein of the present invention.
  • a DNA that expresses a protein that suppresses the function of the normal protein of the present invention is used.
  • the exogenous DNA of the present invention may be derived from a mammal of the same species or a different species as the target animal.
  • a promoter capable of being expressed in animal cells e.g, egret, dog, cat, guinea pig, hamster, rat, mouse, etc.
  • the DNA construct of the present invention eg, a vector
  • a target mammal eg, a mouse fertilized egg
  • various promoters capable of expressing the DNA of the present invention downstream of various promoters capable of expressing the DNA of the present invention.
  • a DNA-introduced mammal that highly expresses the DNA of the present invention can be produced.
  • Examples of the expression vector of the protein of the present invention include a plasmid derived from Escherichia coli, a plasmid derived from Bacillus subtilis, a plasmid derived from yeast, a bacteriophage such as ⁇ phage, a retrovirus such as Moroni leukemia virus, a vaccinia virus and the like.
  • animal viruses such as baculovirus are used.
  • plasmids derived from Escherichia coli, plasmids derived from Bacillus subtilis or plasmids derived from yeast are preferred. It is used well. ',
  • promoters that regulate the DNA expression include, for example, (i) DNA promoters derived from viruses (eg, Simian virus, cytomegalovirus, Moroni leukemia virus, JC virus, breast cancer virus, poliovirus, etc.) Yuichi, (ii) Promoters derived from various mammals (humans, egrets, dogs, cats, guinea pigs, mussels, rats, mice, etc.), such as albumin, insulin II, ⁇ roplakin II, Erasu evening Protease, erythropoietin, endothelin, muscle creatine kinase, glial fibrillary acidic protein, dalyuthione S-transferase, platelet-derived growth factor 13, keratin Kl, 10 and 14, collagen I And II, cyclic AMP-dependent protein kinase / 3I subunit, dystrophy Int, tartrate-resistant alkaline phosphatase, atrial sodium diuretic factor, end
  • the vector preferably has a sequence that terminates the transcription of the target mRNA in the DNA-transfected mammal (generally referred to as "Yuichi Mineta-1").
  • Yuichi Mineta-1 the DNA-transfected mammal
  • the sequence of each DNA can be used.
  • Simian virus SV40 or the like is used.
  • the splicing of each DNA for the purpose of further expressing the desired foreign DNA It is also possible to link a licensing signal, an enhancer region, a part of an intron of a eukaryotic DNA, etc., at 5, upstream of the promoter region, between the promoter region and the translation region, or 3 'downstream of the translation region.
  • the normal translation region of the protein of the present invention is DNA derived from liver, kidney, thyroid cells, fibroblasts derived from humans or various mammals (eg, egrets, dogs, cats, guinea pigs, hamsters, rats, mice, etc.). And all or part of genomic DNA from various commercially available genomic DNA libraries, or complementary DNA prepared by known methods from liver, kidney, thyroid cells, and fibroblast-derived RNA as raw materials I can do it.
  • an exogenous abnormal DNA can produce a translation region obtained by mutating a normal polypeptide translation region obtained from the above cells or tissues by a point mutagenesis method.
  • the translation region is a DNA construct that can be expressed in an introduced animal, which is usually ligated to the downstream of the promoter and optionally the upstream of a transcription termination site.
  • the exogenous DNA of the present invention at the fertilized egg cell stage is ensured to be present in all germ cells and somatic cells of the target mammal.
  • the presence of the exogenous DNA of the present invention in the germinal cells of the transgenic animal after the DNA transfer means that all the progeny of the transgenic animal retain the exogenous DNA of the present invention in all of the germ cells and somatic cells Means that.
  • the offspring of this type of animal that has inherited the exogenous DNA of the present invention have the exogenous DNA of the present invention in all of its germ cells and somatic cells.
  • a non-human mammal into which the exogenous normal DNA of the present invention has been introduced can be subcultured in a normal breeding environment as an animal having the DNA after confirming that the exogenous DNA is stably retained by the crossing. .
  • exogenous DNA of the present invention is provided to be present in excess in all germ cells and somatic cells of the target mammal.
  • Excessive presence of the exogenous DNA of the present invention in the germinal cells of the produced animal after the introduction of the DNA indicates that all the offspring of the produced animal carry the exogenous DNA of the present invention in all of its germ cells and somatic cells. It means every time you have it.
  • the progeny of this type of animal that has inherited the exogenous DNA of the present invention has an excess of the exogenous DNA of the present invention in all of its germinal and somatic cells.
  • the non-human mammal having the normal DNA of the present invention expresses the normal DNA of the present invention at a high level, and finally promotes the function of the endogenous normal DNA, thereby finally obtaining the protein of the present invention. May develop functional hyperplasia, and can be used as a disease model animal. For example, using the normal DNA-introduced animal of the present invention to elucidate the pathological mechanism of hyperactivity of the protein of the present invention and diseases associated with the protein of the present invention, and to examine a method for treating these diseases. Is possible.
  • a non-human mammal having the exogenous abnormal DNA of the present invention can be subcultured in a normal breeding environment as an animal having the DNA after confirming that the exogenous DNA is stably maintained by mating. I can do it.
  • the desired exogenous DNA can be incorporated into the above-mentioned plasmid and used as a raw material.
  • the DNA construct with the promoter can be prepared by ordinary DNA engineering techniques. Introduction of the abnormal DNA of the present invention at the fertilized egg cell stage is ensured to be present in all germ cells and somatic cells of the target mammal.
  • the presence of the abnormal DNA of the present invention in the germinal cells of the animal produced after the transfer of DNA means that all the offspring of the animal produced have the abnormal DNA of the present invention in all of the germ cells and somatic cells.
  • the progeny of this type of animal that has inherited the exogenous DNA of the present invention has the abnormal DNA of the present invention in all of its germ cells and somatic cells. 'A homozygous animal having the introduced DNA on both homologous chromosomes is obtained, and by crossing these male and female animals, it is possible to propagate the offspring so that all offspring have the DNA.
  • the non-human mammal having the abnormal DNA of the present invention expresses the abnormal DNA of the present invention at a high level, and finally inhibits the function of endogenous normal DNA, thereby ultimately obtaining the protein of the present invention. In some cases, it becomes a functional inactive refractory disease and can be used as a model animal for the disease. For example, using the abnormal DNA-introduced animal of the present invention, It is possible to elucidate the pathological mechanism of the function-inactive refractory of the protein of the present invention and to examine a method for treating this disease.
  • the abnormal DNA-highly expressing animal of the present invention can be used to inhibit the function of a normal protein by the abnormal protein of the present invention in function-inactive refractory disease of the protein of the present invention (dominant negatase). ive effect).
  • the mammal into which the foreign abnormal DNA of the present invention has been introduced has an increased symptom of the released protein of the present invention, it can be used in a therapeutic drug screening test for the protein of the present invention or its functionally inactive refractory disease. Is also available.
  • cells of tissues having DNA are cultured by standard tissue culture techniques, and these are used to study the function of cells from generally difficult tissues,
  • a therapeutic agent for a disease associated with the protein of the present invention including a functionally inactive refractory type of the protein of the present invention
  • using the DNA-transfected animal of the present invention Using a quantitative method or the like, it is possible to provide an effective and rapid screening method for the therapeutic agent for the disease.
  • using the animal into which the DNA of the present invention has been introduced or the exogenous DNA expression vector of the present invention it is possible to study and develop a method for treating DNA associated with the protein of the present invention.
  • bases, amino acids, and the like are represented by abbreviations based on the respective abbreviations by the I UPAC- IUB Commission on Biochemical Nomenclature or commonly used abbreviations in the art, and examples thereof are described below.
  • the L-form is indicated unless otherwise specified.
  • a 1 a Alanine
  • Th r Threonine
  • 1 shows the nucleotide sequence of cDNA encoding the cynomolgus SLT receptor.
  • Fig. 3 shows the amino acid sequence of the cynomolgus monkey SLT receptor.
  • [SEQ ID NO: 5] 2 shows the nucleotide sequence of primer 11 used in the PCR reaction in Example 2 below.
  • FIG. 3 shows the nucleotide sequence of cDNA encoding the C-terminal region of the feline SLT receptor obtained in Example 2 below.
  • Example 3 shows the nucleotide sequence of cDNA encoding the N-terminal region of the feline SLT receptor obtained in Example 3 below.
  • 1 shows the nucleotide sequence of a cDNA encoding a cat SLT receptor.
  • Example 5 shows the nucleotide sequence of cDNA encoding the C-terminal region of canine SLT receptor obtained in Example 5 below.
  • [SEQ ID NO: 18] 7 shows the nucleotide sequence of primer 11 used in the PCR reaction in Example 6 below. [SEQ ID NO: 19]
  • Example 7 shows the nucleotide sequence of cDNA encoding the N-terminal region of canine SLT receptor obtained in Example 6 below.
  • 1 shows the nucleotide sequence of cDNA encoding canine SLT receptor.
  • 1 shows the amino acid sequence of MCH. .
  • Example 1 shows the nucleotide sequence of cDNA which encodes the entire length of the cynomolgus monkey SLT receptor obtained in Example 1 below.
  • Example 7 shows the nucleotide sequence of cDNA encoding the full length of cat SLT receptor obtained in Example 4 below.
  • Example 7 shows the nucleotide sequence of cDNA encoding the entire length of the canine SLT receptor obtained in Example 7 below.
  • Escherichia col i T0P10 / pCR4_monSLT obtained in Example 1 described below is an independent administrative corporation of Chuo No. 6 (Zip code 305-8566) 1-1 Toto, Tsukuba-shi, Ibaraki, Japan from June 4, 2002. Accession No. FERM BP-8066 to National Institute of Advanced Industrial Science and Technology Patent Organism Depositary Has been deposited as
  • Escherichia coli TOPlO / pcDNA-catSLT obtained in Example 4 described below has been administered by Independent Administration of 1-1 Chukoku No. 6 Tsukuba Totoro, Ibaraki, Japan (Postal Code 305-8566) since April 25, 2002.
  • the Patent Organism Depositary the deposit number FERM BP-8030, from April 16, 2002, 2-17-85, Jusanhoncho, Yodogawa-ku, Osaka-shi, Osaka (postal code 532-8686) They have been deposited with the Fermentation Research Institute (IF0) under the accession number IF0 16789.
  • Escherichia coli TOPlO / pAKKO-dogSLT obtained in Example 11 described below has been established on April 25, 2002, as an independent company of 1-1 Chukoku No. 1-1, Tsukuba Totoro, Ibaraki, Japan (zip code 305-8566). Deposited number at Patent Organism Depositary, National Institute of Advanced Industrial Science and Technology
  • RNA was prepared from the hypothalamus of the cynomolgus monkey using Isogen (Futtsubon Gene), a poly (A) + RNA fraction was prepared using Oligotex (dT) 30 (Takara Shuzo). ⁇ oly (A) T RNA 2. Power, et al. Superscript reversetranscr iptase
  • a cynomolgus monkey hypothalamic cDNA A PCR reaction was performed using the ⁇ type. The reaction was carried out using a cDNA equivalent to 20 ng of mRNA as a type II in a volume of 201.
  • the composition of the reaction solution was a primer concentration of 0.5 M, a dNTP mixture solution of 0.2 mM, Z-Taq (Takara Shuzo).
  • the cycle for amplification is After keeping it at 94 ° C for 120 seconds, a cycle of 98 ° C for 1 second and 72 ° C for 30 seconds was repeated 40 times.
  • the resulting reaction solution 21 was subcloned into a plasmid vector pcr4 using a 1 "0-0 TA cloning kit (Invitrogen), and then introduced into E. coli T0P10.
  • a poly (A) + RNA fraction was prepared using Oligotex (dT) 30 (Takara Shuzo).
  • Type III for 3'-RACE was prepared based on feline whole brain poly (A) + RNA using SMART RACE cDNA Amplification kit.
  • the primer set used for the RACE PCR reaction was as follows: the first PCR reaction included the Universal Primer Mix and primer 1 (SEQ ID NO: 5) attached to the kit; the second PCR reaction included the Nested Universal Primer and primer 2 (sequence number) attached to the kit. : 6) was used. The reaction was carried out in the form of ⁇ with a volume of 201 liquid, ie, 11 * 6 equivalents of the reversely transcribed 00 ⁇ .
  • the composition of the reaction solution was a primer concentration of 0.5 zM, a dNTP mixed solution of 0.2 mM, Advantage? Polymerase Mix (Clontech) 1/50 volume ⁇ 10-fold concentrated Buffer 1/10 volume.
  • the cycle for amplification is 94X ⁇ 5 seconds for both PCRs,
  • Type I for 5'-RACE was prepared from feline whole brain poly (A) + RNA using Marathon ready cDNA Amplification'kit.
  • the set of primers used for the RACE PCR reaction included adapter primer 1 for the Marathon ready cDNA Amplification kit in the first PCR reaction.
  • Primer 2 (SEQ ID NO: 9) and primer 2 (SEQ ID NO: 9) for Marathon ready cDNA Amplification kit were used in the second PCR reaction. The reaction was carried out in the amount of 20 ng of the reverse transcribed cDNA corresponding to 2 ng of mRNA at a volume of 20 / i1.
  • the composition of the reaction solution was primer concentration 0.5 M, dNTP mixture 0.2 mM, Advantage 2 Polymerase Mix (Clontech) 1/50 volume, and 10-fold concentrated Buffer 1/10 volume.
  • the cycle for amplification is 10 cycles of 94 ° C for 5 seconds, 66 ° C for 120 seconds for both PCRs, 94 ° C for 5 seconds, 60 ° C for 20 seconds, 72 ° C for After repeating the cycle of 120 seconds 25 times, it was kept at 72 ° C for 10 minutes.
  • the reaction product was electrophoresed on a 1.2% Seakem GTG Agarose (Takara Shuzo), and a band around 500 bp, which was visible when stained with ethidium amide, was extracted with the GeneClean Spin kit (Bio 101). After subcloning into plasmid vector pcr4-T0P0 using E. coli, Escherichia coli E0 was introduced. Plasmid DNA was purified from the resulting transformant using QIA prep8 mini prep (Qiagen). The reaction for base sequence determination was performed using BigDye Terminator Cycle Sequence Ready Reaction Kit (Perkin Elmer). As a result of decoding using a fluorescent automatic sequencer, a base sequence represented by SEQ ID NO: 10 including the N-terminal region of cat SLT was obtained.
  • the cycle for amplification is as follows: after holding at 94 ° C for 15 seconds, 98t: 1 second, 64 ° C, 15 seconds, 72 ° C Keep warm for a minute.
  • the reaction product was electrophoresed on 1.2% Seakein GTG Agarose (Takara Shuzo), and a band around 1200 bp, which was visible when stained with ethidium bromide, was extracted using the GeneClean Spin kit (Bio 101) and the Eukaryotic T0P0 TA cloning kit (Invitrogen) After subcloning into plasmid vector pcDNA3.1 / V5 / His-T0P0 using Escherichia coli, E. coli TOP10 was introduced.
  • Plasmid DM was purified from the resulting transformant using QIA prep8 mini prep (Qiagen). The reaction for base sequence determination was performed using a BigDye Terminator Cycle Sequence Ready Reaction Kit (Pakkin Elma). As a result of decoding using a fluorescent automatic sequencer, the nucleotide sequence represented by SEQ ID NO: 39 was obtained. Since this sequence contained the nucleotide sequence (SEQ ID NO: 13) encoding the entire amino acid sequence of cat SLT (SEQ ID NO: 14), Escherichia coli TOP10 was transformed with this plasmid to transform Escherichia coli TOP10. / pcDNA-catSLT was obtained.
  • SEQ ID NO: 13 the nucleotide sequence represented by SEQ ID NO: 13
  • SEQ ID NO: 14 the nucleotide sequence represented by SEQ ID NO: 14
  • a poly (A) + RNA fraction was prepared using Oligotex (dT) 30 (Takara Shuzo).
  • a type for 3'_RACE was prepared using SMART RACE cDNA Amplification kit based on canine frontal lobe poly (A) + RNA.
  • the primer set used for the RACE PCR reaction was the Universal Primer Mix and primer 1 (SEQ ID NO: 15) attached to the kit in the fourth PCR reaction, and the Nested Universal Primer and primer 1 in the kit for the second PCR reaction. (SEQ ID NO: 16) was used. The reaction was performed by converting 20 ng of reverse-transcribed cDNA into Performed with 1 liquid volume.
  • the composition of the reaction solution was primer concentration 0.5 / M, dNTP mixture 0.2 mM, Advantage2 Polymerase Mix (Clontech) 1/50 volume ⁇ 10-fold concentrated Buffer 1/10 volume.
  • the cycles for amplification were as follows: 94 ° C for 5 seconds, 72 ° C for 90 seconds, three cycles for each PCR, 94 ° C for 5 seconds, 70 ° C for 90 seconds, three cycles. After repeating the cycle of ° C for 5 seconds and 68 ° C for 90 seconds 40 times, it was kept at 72 ° C for 10 minutes.
  • reaction product was electrophoresed on 1.2% Seakem GTG Agarose (Takara Shuzo), and a band around 900 bp, which was visible when stained with ethidium bromide, was extracted with the GeneClean Spin kit (Bio 101). ) was used for subcloning into a plasmid vector pc-TOPO, and then introduced into E. coli T0P10. Plasmid DNA was purified from the resulting transformant using QIA prep8 mini prep (Qiagen). The reaction for base sequence determination is BigDye Terminator Cycle Sequence Ready
  • -Type II for RACE was prepared from canine frontal lobe poly (A) + RNA using Marathon ready cDNA Amplification kit.
  • the primer set used for the RACE PCR reaction was the adapter primer 1 and primer 1 (SEQ ID NO: 18) for the Marathon ready cDNA Amplification kit in the first PCR reaction, and the Marathon ready cDNA Amplification kit in the second PCR reaction.
  • the adaptor primer 2 and the primer 1 (SEQ ID NO: 19) were used.
  • the reaction was carried out in the form of type II with a volume of 201, corresponding to 2 ng of mRNA of the reverse-transcribed cDNA.
  • the composition of the reaction solution was primer concentration 0.5 M, dNTP mixture 0.2 mM,
  • a cycle of 66 ° C for 120 seconds was repeated 10 times, a cycle of 94 for 5 seconds, a cycle of 60 ° C for 20 seconds, and a cycle of 72 for 120 seconds was repeated 25 times, followed by incubation at 72 ° C for 10 minutes.
  • Reactants to 1.2% Seakem GTG 1.2% Seakem GTG
  • primer 1 SEQ ID NO: 21
  • 3 'PCR was performed by setting Primer 2 (SEQ ID NO: 22) to the untranslated region.
  • the reaction was carried out in the form of type II with a volume of 201, corresponding to 2 ng of mRNA of the reverse-transcribed cDNA.
  • the composition of the reaction solution was a primer concentration of 0.5 M, a dNTP mixed solution of 0.2 mL, a Z-Taq (TaKaRa) 1/50 volume, and a 10-fold concentrated Buffer 1/10 volume.
  • the cycle for amplification is as follows: after holding at 94 ° C for 15 seconds, repeat the cycle of 98 ° C for 1 second, 64 ° C for 15 seconds, 72 ° C for 20 seconds 40 times, and then for 72 ° C. For 2 minutes.
  • the reaction product was electrophoresed on a 1.2% Seakem GTG Agarose (Takara Shuzo), and a band around 1200 bp, which was visible when stained with ethidium umide, was extracted with the GeneClean Spin kit (Bio 101).
  • Eukaryotic T0P0 TA cloning kit (Invitrogen) After subcloning into the plasmid vector PCDNA3.1 / V5 / His-TOP0 using Escherichia coli, Escherichia coli T0P10 was introduced. Plasmid DNA was purified from the resulting transformant using QIA prep8 mini prep (Qiagen). The reaction for base sequence determination was carried out using BigDye Terminator Cycle Sequence Ready Reaction Kit (Pakkin Elma). As a result of decoding using a fluorescent automatic sequencer, the base sequence represented by SEQ ID NO: 40 was obtained.
  • This sequence contained the nucleotide sequence (SEQ ID NO: 23) encoding the entire amino acid sequence of canine SLT (SEQ ID NO: 24).
  • Bacterium TOP10 was transformed to obtain Escherichia coli TOPlO / pcDNA-dogSLT.
  • Plasmids were prepared from the cloned Escherichia coli using the Plasmid Midi Kit (Qiagen) and cut with restriction enzymes Sal I and Spe I to cut out the insert. After the electrophoresis, the insert DNA was cut out from an agarose gel with a force razor, and then recovered by performing fragmentation, phenol extraction, phenol-chloroform extraction, and ethanol precipitation.
  • Plasmid Midi Kit Plasmid Midi Kit (Qiagen). This was added to the CHO dhfr cell 500,000 cells / 150 1 O tiMEM (Gibco) cell suspension at 50 g / ml, and 240 V 960 F pulse was applied to the cells by Genepulsa (Bio-Rad).
  • CHO / catSLT cells are suspended in DMEM containing 10% dialyzed fetal serum to a concentration of 15 x 10 4 cells / ml and dispensed into a 96-well plate for FLIPR (Black plate clear bottom, Costar). 200 1 by implantation into the Ueru using (3.0X10 4 cells / 200 l / ⁇ El), 5% C0 2 was 37 ° C De ⁇ cultured at incubator base one coater in was used to Atsusi (hereinafter This plate is called a cell plate).
  • HANKS '/ HBSS Nesy Hanks 2
  • a sample plate was prepared by adding MCH at a concentration of 1. The color of the cell plate After finishing the washing, wash the cell plate 4 times using a plate washer (Molecular Devices) with HANKS '/ HBSS and 2.5 mM Probenecid using a washing buffer. The cell plate and the sample plate were set on the FLIPR and assembled (the sample from the sample plate was transferred to the cell plate by the FLIPR).
  • MCH increased the intracellular Ca ion concentration of feline SLT-expressing CH0 cells in a concentration-dependent manner (Fig. 1).
  • the increase in intracellular Ca ion concentration is indicated by the increase in fluorescence of the dye loaded on the cell caused by Ca.
  • RNA fraction was prepared using ISOGEN (Futtsubon Gene). The obtained RNA was digested with RNase by digestion with proteinase K (Invitrogen), DNase I digested genomic DNA using the Message Clean Kit (GenHunter), and total RNA lxg at each site was extracted.
  • Reverse transcription was carried out using Superscript II reversetranscriptase (Invitrogen) as a template and random primers according to the attached manual to produce cDNA.
  • the standard cDNA was prepared by PCR using the primers 1 and 2 (SEQ ID NO: 32 and SEQ ID NO: 33) in the form of 200 xl using primers 1 and 2 (SEQ ID NO: 32 and SEQ ID NO: 33) with 50 pg of the plasmid pcDNA-catSLT obtained in Example 4. Done and created.
  • the composition was performed using primers 1 and 2, 0.5 M, 2.5 mM MgCl 2 , dNTP 0.2 niM, AmliTaq Gold (Pakinkin Elmer) 1/100 volume, and 10-fold concentrated Ampl iTaq Gold Buffer 1/10 volume.
  • the reaction was incubated at 95 ° C for 10 minutes, and then repeated at 95 ° C for 15 seconds, 60 at 15 seconds, and 72 ° C for 10 seconds 40 times.
  • the amplification product is purified from the reaction mixture using the PCR purification kit (Qiagen), the absorbance at 260 nm is measured, the concentration is calculated, the exact copy number is calculated, and the copy is diluted with distilled water.
  • a standard cDNA solution of 7 copies of lxlO was prepared. Also,
  • TaqMan PCR probes and primers were designed using Primer Express (Version I.O) (PE Biosystems).
  • the expression level was calculated using ABI PRISM 7700 SDS software. The number of cycles at the moment when the fluorescence intensity of the reporter reaches the set value is plotted on the vertical axis. A standard curve was created using the logarithmic value of the initial concentration as the horizontal axis. The initial concentration of each reverse transcription product was calculated from the standard curve, and the expression amount of the cat SLT gene per 25 ng of total RNA at each site was determined. Further, the expression level of the feline GAP DH gene in each sample was determined using the primers # 3 (SEQ ID NO: 35), primer 4 (SEQ ID NO: 36) and the probe (Fam-ccaggagcgagatcccgcca-Tamura) shown in SEQ ID NO: 37. The expression of the feline SLT gene was corrected by the TaqMan: PCR method in the same manner as in the analysis of the SLT gene expression level.
  • the expression level of the feline SLT gene relative to the feline GAPDH gene was 0.084% in the frontal lobe, 0.32% in the temporal lobe, 0.046% in the occipital lobe, 0.056% in the parietal lobe, 0.086% in the pons, and 0.012% in the cerebellum.
  • a plasmid into which a gene encoding the full-length amino acid sequence of canine SLT to which the sequence was confirmed in Example 7 and a Sal I recognition sequence was added on the 5 'side and a Spe I recognition sequence was added on the 3' side was added.
  • E. coli was transformed with pcDNA-dogSLT. Plasmids were prepared from the E. coli clones using Plasmid Midi Kit (Qiagen) and cut with restriction enzymes Sal I and Spe I to cut out the insert.
  • proteins, polynucleotides, antisense polynucleotides and antibodies of the present invention include, for example, anorexia (eg, anorexia nervosa), anemia or hypoproteinemia associated with anorexia, weak labor, lax hemorrhage, uterus It is useful as a marker for diagnosis of retro failure, milk stasis, etc.
  • anorexia eg, anorexia nervosa
  • anemia or hypoproteinemia associated with anorexia weak labor
  • lax hemorrhage uterus It is useful as a marker for diagnosis of retro failure, milk stasis, etc.
  • An agonist of the protein obtained by a screening method using the protein and the peptide of the present invention includes an appetite (feeding) enhancer, anorexia (eg, anorexia nervosa), anemia associated with anorexia, As a prophylactic / therapeutic agent for hypoproteinemia, weak labor, laxative bleeding, uterine remodeling failure, milk stasis, etc.
  • anorexia eg, anorexia nervosa
  • anemia associated with anorexia As a prophylactic / therapeutic agent for hypoproteinemia, weak labor, laxative bleeding, uterine remodeling failure, milk stasis, etc.
  • Obesity hyperplasmic obesity, pituitary obesity, hypoplasmic obesity, hypothyroid obesity, hypothalamic obesity, symptomatic obesity, childhood obesity, upper body obesity, dietary obesity, hypogonadism Obesity, systemic mastocytosis, simple obesity, central obesity, etc.), hyperphagia, affective disorders, sexual dysfunction, excessive labor, tonic contractions, fetal distress, uterine rupture , Vascular laceration, premature birth, Prader-Willi syndrome, diabetes and its complications (eg, diabetic nephropathy, diabetic retinopathy, diabetic neuropathy, etc.), hypertension, hyperlipidemia, coronary atherosclerosis, Prevention and treatment of gout, respiratory diseases (Pickwick syndrome, sleep apnea syndrome), fatty liver, infertility, osteoarthritis (especially antiobesity agents, appetite (feeding) regulators, etc.) It can be used as an agent.
  • respiratory diseases Pickwick syndrome, sleep apnea syndrome

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Abstract

L'invention porte sur de nouvelles protéines, d'origine simiesque, féline et canine et leurs sels, sur des polynucléotides codant pour elles, sur leurs anticorps, etc. pouvant par exemple servir de marqueurs de diagnostic d'inappétence, d'inertie utérine etc., sur des composés obtenus par criblage à l'aide desdites protéines et de MCH pouvant servir de stimulateur d'appétit et d'agent de prévention ou de traitement de l'inappétence
PCT/JP2003/002133 2002-02-27 2003-02-26 Nouvelles protéines, leurs adn et leur utilisation WO2003072780A1 (fr)

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2004023870A1 (fr) * 2002-09-10 2004-03-25 Takeda Pharmaceutical Company Limited Animal transgénique à slt humain

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2001070975A1 (fr) * 2000-03-24 2001-09-27 Yamanouchi Pharmaceutical Co., Ltd. Nouveau recepteur d'hormone concentrant la melanine

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2001070975A1 (fr) * 2000-03-24 2001-09-27 Yamanouchi Pharmaceutical Co., Ltd. Nouveau recepteur d'hormone concentrant la melanine

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
HILL J. ET AL.: "Molecular cloning and functional characterization of MCH2, a novel human MCH receptor", J. BIOL. CHEM., vol. 276, no. 23, June 2001 (2001-06-01), pages 20125 - 20129, XP002947038 *

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2004023870A1 (fr) * 2002-09-10 2004-03-25 Takeda Pharmaceutical Company Limited Animal transgénique à slt humain

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