JP2004121245A - New protein and its dna - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a new protein having transporting activity to bile acid, steroid hormone, lipid soluble vitamin, neurotransmitter, etc., and a DNA encoding the protein, a method for screening compounds which promote or inhibit activity of the protein and a compound obtained by the screening. <P>SOLUTION: The protein or the polynucleotide is useful as a diagnostic marker, etc., for hyperlipidemia, digestive system disease, central nervous system disease, diabetes, pancreatic disease, respiratory disease, circulatory disease, cancer, etc., and the compounds etc., promoting or inhibiting the activities of the protein obtained by the screening method using the protein, can be used as a prophylactic/therapeutic agent, for example, above diseases. <P>COPYRIGHT: (C)2004,JPO

Description

The present invention provides a method for screening a novel sodium-dependent bile acid transporter protein, a polynucleotide encoding the protein, a compound that promotes or inhibits the activity of the protein, a compound that promotes or inhibits the expression of the protein gene, A compound obtained by the screening method is provided. Furthermore, the present invention relates to a prophylactic / therapeutic agent for central nervous system diseases (preferably Parkinson's syndrome and the like) and screening thereof.

Bile acids are synthesized in the liver and secreted into the small intestine, and play an important role in the small intestine in promoting absorption of lipids, fat-soluble vitamins, cholesterol, and the like. Bile acids are mainly efficiently reabsorbed from the small intestine (ileum), return to the liver via the portal vein, and are excreted again in bile (enterohepatic circulation). Since the body cholesterol pool size is feedback-controlled not only by dietary cholesterol but also by bile acids in the enterohepatic circulation, resorption of bile acids in the intestine using a bile acid adsorbent (anion exchange resin) is considered. Suppression has led to the treatment of hypercholesterolemia.

Parkinson's syndrome is a degenerative disease in which dopamine deficiency occurs in the striatum with nerve endings due to degeneration and loss of dopaminergic neurons in the substantia nigra, and extrapyramidal movement disorder appears. Muscle rigidity, slow motion, immobility and postural reflex disorder are the four main features. The prevalence is about 100 per 100,000. The etiology is unknown and the involvement of some poisoning substance is presumed.

Sodium-dependent bile acid transporters are thought to contribute to bile acid transport. In humans, two isoforms of the sodium-dependent bile acid transporter have been identified, and NTCP (Na + / taurocholate cotransporting polypeptide) is mainly expressed in the liver (Non-patent Document 1 J. Clin. Invest., Vol. 93). ISBT (Ileal sodium / bile salt cotransporter) is mainly expressed in the ileum and kidney (J. Biol. Chem., 270, 27228-27234, 1995). Regarding ISBT, it has been suggested that a gene mutation accompanied by amino acid substitution is directly associated with bile acid malabsorption (J. Clin. Invest., 99, 1880-1887, 1997).
The Journal of Clinical Investigation 93, 1326-1331, 1994 Genbank Accession No. BC019066

The sodium-dependent bile acid transporter is thought to play an important role in the transport of bile acids in the liver and small intestine, but its detailed mechanism and its relation to disease are not well understood. Elucidation of the role of sodium-dependent bile acid transporters in detailed substrate specificity and bile acid metabolism will lead to the development of therapeutics for diseases involving bile acid metabolism and the like.

(4) Following the determination of the human genome sequence, the genes responsible for central nervous system diseases such as Parkinson's syndrome have been dramatically discovered, and the pathogenesis of diseases for which the mechanism of onset was unknown until now has been elucidated. In the field of central nervous system diseases, drugs that target molecules that are specifically expressed in each disease, and that target the suppression of abnormal protein production and degradation, or the clearance or deposition of abnormal proteins that have been deposited, are required. Have been. At present, there are various Parkinson's therapeutic agents, but no excellent drug to cure the symptoms has yet to be developed.

The present inventors have conducted intensive studies in order to solve the above-mentioned problems, and as a result, have found a human sodium-dependent bile acid transporter protein (human TCH162) and its mouse ortholog (mouse TCH162). Human TCH162 shows 34% homology at the amino acid level to human NTCP (J. Clin. Invest., 93, 1326-1331, 1994), and functions as a sodium-dependent bile acid transporter. Can be done. Also, it is a part of the base sequence of Genbank Accession No. BC019066 (Non-Patent Document 2).

As a method for suppressing the above protein, for example, inhibiting the transport of bile acids, steroid hormones, fat-soluble vitamins, neurotransmitters (eg, dopamine, glutamic acid, enkephalin, etc.) or suppressing the transcription of the protein It is conceivable to reduce the expression level. Methods for activating the protein include, for example, promoting the transport of bile acids, steroid hormones, fat-soluble vitamins, neurotransmitters (eg, dopamine, glutamic acid, enkephalin, etc.), activating the promoter of the protein, and the like. It can be considered that the expression level is enhanced by stabilizing mRNA.

The present inventors have further studied based on these findings, and as a result, have completed the present invention.

That is, the present invention
(1) a protein containing an amino acid sequence identical or substantially identical to the amino acid sequence represented by SEQ ID NO: 1 or SEQ ID NO: 19, or a salt thereof;
(2) a protein comprising an amino acid sequence represented by SEQ ID NO: 1 or a salt thereof;
(3) a protein comprising an amino acid sequence represented by SEQ ID NO: 19 or a salt thereof;
(4) a partial peptide of the protein according to (1) or a salt thereof,
(5) a polynucleotide containing a polynucleotide encoding the protein of (1) or the partial peptide of (4),
(6) the polynucleotide according to (5), which is DNA;
(7) a DNA comprising the base sequence represented by SEQ ID NO: 20 or SEQ ID NO: 27,
(8) a recombinant vector containing the polynucleotide according to (5),
(9) a transformant transformed with the recombinant vector according to (8),
(10) culturing the transformant according to (9), producing and accumulating the protein according to (1) or the partial peptide according to (4), and collecting the protein; )) Or a method for producing the partial peptide according to (4) or a salt thereof,
(11) a medicine comprising the protein of (1) or the partial peptide of (4) or a salt thereof,
(12) a medicine comprising the polynucleotide according to the above (5),
(13) A diagnostic agent comprising the polynucleotide according to (5),
(14) an antibody against the protein according to (1) or the partial peptide according to (4) or a salt thereof;
(15) a diagnostic agent comprising the antibody according to (14),
(16) a medicine comprising the antibody according to the above (14),
(17) a polynucleotide having a base sequence complementary to or substantially complementary to the polynucleotide according to (5) or a part thereof;
(18) a medicine comprising the polynucleotide according to the above (17),
(19) The protein according to (1), the partial peptide according to (4) or a salt thereof, wherein the protein according to (1) or the partial peptide according to (4) or a salt thereof is used. A method for screening a compound or a salt thereof that promotes or inhibits the activity,
(20) The activity of the protein according to (1) or the partial peptide according to (4) or a salt thereof, comprising the protein according to (1) or the partial peptide according to (4) or a salt thereof. A kit for screening for a compound that promotes or inhibits or a salt thereof,
(21) Promotes the activity of the protein of (1), the partial peptide of (4) or a salt thereof obtained by using the screening method of (19) or the screening kit of (20). Or a compound that inhibits or a salt thereof,
(22) a medicine comprising the compound or a salt thereof according to (21),
(23) A method for screening a compound or a salt thereof that promotes or inhibits the expression of the protein gene according to (1), which comprises using the polynucleotide according to (5);
(24) A kit for screening a compound or a salt thereof that promotes or inhibits the expression of the protein gene according to (1), comprising the polynucleotide according to (5),
(25) a compound or a salt thereof that promotes or inhibits the expression of the protein gene according to (1), obtained by using the screening method according to (23) or the screening kit according to (24);
(26) a medicament comprising the compound according to (25) or a salt thereof,
(27) The method for quantifying a protein according to (1), comprising using the antibody according to (14),
(28) A method for diagnosing a disease associated with the function of the protein according to (1), which comprises using the quantification method according to (27);
(29) A method for screening a compound or a salt thereof that promotes or inhibits the expression of the protein according to (1), which comprises using the antibody according to (14).
(30) A kit for screening a compound or a salt thereof that promotes or inhibits the expression of the protein according to (1), comprising the antibody according to (14);
(31) A compound or a salt thereof that promotes or inhibits the expression of the protein according to (1), which is obtained by using the screening method according to (29) or the screening kit according to (30),
(32) a medicament comprising the compound according to (31) or a salt thereof,
(33) The medicament according to (11), (12), (16), (18), (22), (26) or (32), which is a prophylactic / therapeutic agent for a central nervous system disease.
(34) The diagnostic agent according to the above (13) or (15), which is a diagnostic agent for a central nervous system disease.
(35) a method for preventing or treating a central nervous system disease, which comprises administering to a mammal an effective amount of the compound according to (21), (25) or (31) or a salt thereof;
(36) Use of the compound or salt thereof according to (21), (25) or (31) for producing a prophylactic / therapeutic agent for a central nervous system disease is provided.

The protein, polynucleotide and antibody of the present invention include, for example, hyperlipidemia, digestive disorders (eg, irritable bowel syndrome, ulcerative colitis, Crohn's disease, ischemic colitis, gastritis, peptic ulcer, proctitis) ), Central nervous system disorders (eg, Alzheimer's disease, Parkinson's syndrome, depression, emotional disorders, schizophrenia, anxiety, etc.), diabetes, pancreatic disorders (eg, pancreatitis, cystic fibrosis, etc.) Insufficiency), respiratory disease (eg, chronic obstructive pulmonary disease (COPD), asthma, etc.), hypothyroidism, cardiovascular disease (eg, heart failure, arrhythmia, long QT syndrome, arteriosclerosis, etc.), cancer (eg, , Testicular tumor, ovarian cancer, breast cancer, esophageal cancer, lung cancer, kidney cancer, liver cancer, non-small cell lung cancer, prostate cancer, gastric cancer, bladder cancer, cervical cancer, colon cancer, rectal cancer, pancreatic cancer, thymoma, etc. Useful as a diagnostic marker A. It is preferably useful as a diagnostic marker for central nervous system diseases, particularly preferably for Parkinson's syndrome and the like. Compounds that promote or inhibit the activity of the protein obtained by a screening method using the protein, polynucleotide or antibody include compounds that promote or inhibit the expression of the protein gene, compounds that promote or inhibit the expression of the protein, and the like. For example, hyperlipidemia, gastrointestinal diseases (eg, irritable bowel syndrome, ulcerative colitis, Crohn's disease, ischemic colitis, gastritis, peptic ulcer, proctitis, etc.), central nervous system diseases (eg, , Alzheimer's disease, Parkinson's syndrome, depression, emotional disorders, schizophrenia, anxiety, etc.), diabetes, pancreatic disease (eg, pancreatic insufficiency such as pancreatitis, cystic fibrosis, etc.), respiratory disease ( Eg, chronic obstructive pulmonary disease (COPD), asthma, etc., hypothyroidism, cardiovascular disease (eg, heart failure, arrhythmia, long QT syndrome, arteriosclerosis, etc.) Cancer (eg, testicular, ovarian, breast, esophageal, lung, kidney, liver, non-small cell lung, prostate, gastric, bladder, cervical, colon, colon, rectal, pancreatic, Thymoma, etc.). Preferably, it is a prophylactic / therapeutic agent for central nervous system diseases and the like, particularly preferably a prophylactic / therapeutic agent for Parkinson's syndrome and the like.

A protein containing the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 1 or SEQ ID NO: 19 used in the present invention (hereinafter referred to as the protein of the present invention or the protein used in the present invention) May be) human or warm-blooded animal (eg, guinea pig, rat, mouse, chicken, rabbit, pig, sheep, cow, monkey, etc.) cells (eg, hepatocytes, splenocytes, neurons, glial cells, Pancreatic β cells, bone marrow cells, mesangial cells, Langerhans cells, epidermal cells, epithelial cells, goblet cells, endothelial cells, smooth muscle cells, fibroblasts, fiber cells, muscle cells, adipocytes, immune cells (eg, macrophages, T cells) Cells, B cells, natural killer cells, mast cells, neutrophils, basophils, eosinophils, monocytes), megakaryocytes, synovial cells, chondrocytes, bone fine cells , Osteoblasts, osteoclasts, mammary cells, hepatocytes or stromal cells, or precursors, stem cells or cancer cells of these cells) or any tissue in which these cells are present, for example, brain, each part of the brain (Eg, olfactory bulb, amygdala, basal sphere, hippocampus, thalamus, hypothalamus, cerebral cortex, medulla, cerebellum), spinal cord, pituitary, stomach, pancreas, kidney, liver, gonads, thyroid, gallbladder, bone marrow, adrenal Skin, muscle, lung, digestive tract (eg, large intestine, small intestine), blood vessels, heart, thymus, spleen, submandibular gland, peripheral blood, prostate, testicle, ovary, placenta, uterus, bone, joints, skeletal muscle, etc. Protein or a synthetic protein.

As the amino acid sequence substantially identical to the amino acid sequence represented by SEQ ID NO: 1, about 50% or more, preferably about 60% or more, preferably about 70% or more, More preferably, the amino acid sequence has about 80% or more, particularly preferably about 90% or more, and most preferably about 95% or more homology.

Examples of the protein containing an amino acid sequence substantially the same as the amino acid sequence represented by SEQ ID NO: 1 include, for example, a protein containing the same amino acid sequence as the above-mentioned amino acid sequence represented by SEQ ID NO: 1. And a protein having substantially the same activity as the protein having the amino acid sequence represented by SEQ ID NO: 1.

The amino acid sequence substantially the same as the amino acid sequence represented by SEQ ID NO: 19 includes about 50% or more, preferably about 60% or more, preferably about 70% or more as the amino acid sequence represented by SEQ ID NO: 19, More preferably, the amino acid sequence has about 80% or more, particularly preferably about 90% or more, and most preferably about 95% or more homology.

Examples of the protein having an amino acid sequence substantially identical to the amino acid sequence represented by SEQ ID NO: 19 include, for example, an amino acid sequence substantially identical to the amino acid sequence represented by SEQ ID NO: 19 described above. And a protein having substantially the same activity as the protein having the amino acid sequence represented by SEQ ID NO: 19.

Amino acid sequence homology was determined using the homology calculation algorithm NCBI BLAST (National Center for Biotechnology Information Basic Local Alignment Search Tool) under the following conditions (expected value = 10; gap allowed; matrix = BLOSUM62; filtering = OFF). Can be calculated.

Examples of substantially the same activity include transport of bile acids, steroid hormones, fat-soluble vitamins, neurotransmitters (eg, dopamine, glutamic acid, enkephalin, etc.) and the like. Substantially homogenous indicates that the properties are homogenous (eg, physiologically or pharmacologically). Therefore, transportation of bile acids, steroid hormones, fat-soluble vitamins, neurotransmitters (eg, dopamine, glutamic acid, enkephalin, etc.) is equivalent (eg, about 0.01 to 100 times, preferably about 0.1 to 10 times). , More preferably 0.5 to 2 times), but the quantitative factors such as the degree of these activities and the molecular weight of the protein may be different.

Activity such as transport of bile acids, steroid hormones, fat-soluble vitamins, neurotransmitters (eg, dopamine, glutamic acid, enkephalin, etc.) can be measured according to known methods. For example, Am. Physiol., Vol. 274, G157-169, 1998 or a method analogous thereto.

The protein of the present invention includes, for example, (1) (i) one or two or more amino acids in the amino acid sequence represented by SEQ ID NO: 1 (eg, about 1 to 200, preferably about 1 to 150, Is an amino acid sequence in which about 1 to 100, preferably about 1 to 50, preferably about 1 to 30, preferably about 1 to 10, and more preferably several (1 to 5) amino acids have been deleted. , (Ii) one or two or more amino acids in the amino acid sequence represented by SEQ ID NO: 1 (eg, about 1 to 200, preferably about 1 to 150, preferably about 1 to 100, preferably 1 to 50 (Preferably about 1 to 30, preferably about 1 to 10, more preferably about 1 to 5) amino acids, and (iii) the amino acid sequence represented by SEQ ID NO: 1. 1 or Or more (e.g., about 1 to 200, preferably about 1 to 150, preferably about 1 to 100, preferably about 1 to 50, preferably about 1 to 30, preferably about 1 to 10, More preferably, an amino acid sequence having a number (1 to 5) of amino acids inserted therein, and (iv) one or more (eg, about 1 to 200, preferably 1 to 200) amino acids in the amino acid sequence represented by SEQ ID NO: 1. About 1 to 150 pieces, preferably about 1 to 100 pieces, preferably about 1 to 50 pieces, preferably about 1 to 30 pieces, preferably about 1 to 10 pieces, and more preferably a number (1 to 5) pieces. A so-called mutein such as a protein containing an amino acid sequence in which an amino acid is substituted with another amino acid, or (v) an amino acid sequence obtained by combining them; (2) (i) an amino acid represented by SEQ ID NO: 19 1 or 2 or more in the row (for example, about 1 to 200 pieces, preferably about 1 to 150 pieces, preferably about 1 to 100 pieces, preferably about 1 to 50 pieces, preferably about 1 to 30 pieces, preferably An amino acid sequence in which about 1 to 10, more preferably a number (1 to 5) amino acids have been deleted, and (ii) one or more amino acids (for example, 1 to 200) in the amino acid sequence represented by SEQ ID NO: 19 Pieces, preferably about 1 to 150 pieces, preferably about 1 to 100 pieces, preferably about 1 to 50 pieces, preferably about 1 to 30 pieces, preferably about 1 to 10 pieces, and more preferably a number (1 to 1 (Iii) 1 or 2 or more amino acids (for example, about 1 to 200, preferably about 1 to 150, preferably 1) to the amino acid sequence represented by SEQ ID NO: 19; -10 An amino acid sequence in which about 0, preferably about 1 to 50, preferably about 1 to 30, preferably about 1 to 10, and more preferably a number (1 to 5) amino acids have been inserted, (iv 1) in the amino acid sequence represented by SEQ ID NO: 19 or more (eg, about 1 to 200, preferably about 1 to 150, preferably about 1 to 100, preferably about 1 to 50, Amino acid sequence in which about 1 to 30, preferably about 1 to 10, and more preferably number (1 to 5) amino acids have been substituted with other amino acids, or (v) an amino acid sequence combining them So-called muteins such as proteins containing

場合 When the amino acid sequence is inserted, deleted or substituted as described above, the position of the insertion, deletion or substitution is not particularly limited.

In the protein in the present specification, the left end is the N-terminus (amino terminus) and the right end is the C-terminus (carboxyl terminus) according to the convention of peptide labeling. Proteins of the present invention, C-terminal, carboxyl group (-COOH), a carboxylate (-COO ^-), may be any of an amide (-CONH ₂₎ or an ester (-COOR).

Here, as R in the ester, for example, a C _1-6 alkyl group such as methyl, ethyl, n-propyl, isopropyl and n-butyl, for example, a C _3-8 cycloalkyl group such as cyclopentyl and cyclohexyl, for example, phenyl , C _6-12 aryl groups such as α-naphthyl, for example, phenyl-C _1-2 alkyl groups such as benzyl and phenethyl or C _7- alkyl groups such as α-naphthyl-C _1-2 alkyl groups such as α-naphthylmethyl. _{14 An} aralkyl group, a pivaloyloxymethyl group and the like are used.

場合 When the protein of the present invention has a carboxyl group (or carboxylate) other than the C-terminus, a protein in which the carboxyl group is amidated or esterified is also included in the protein of the present invention. As the ester in this case, for example, the above-mentioned C-terminal ester and the like are used.

Furthermore, in the protein of the present invention, the amino group of the N-terminal amino acid residue (eg, a methionine residue) has a protecting group (eg, a C _1-6 acyl such as a C _1-6 alkanoyl such as a formyl group or an acetyl group). Group), N-terminal glutamine residue generated by cleavage in vivo, pyroglutamine-oxidized, substituent on the side chain of amino acid in the molecule (for example, -OH, -SH, Amino group, imidazole group, indole group, guanidino group, etc. protected with a suitable protecting group (eg, C _1-6 acyl group such as C _1-6 alkanoyl group such as formyl group and acetyl group) Or a complex protein such as a so-called glycoprotein to which a sugar chain is bound.

具体 Specific examples of the protein of the present invention include, for example, a protein containing the amino acid sequence represented by SEQ ID NO: 1, a protein containing the amino acid sequence represented by SEQ ID NO: 19, and the like.

部分 The partial peptide of the protein of the present invention is the partial peptide of the protein of the present invention described above, and preferably any peptide having the same properties as the protein of the present invention described above.

For example, at least 5 or more, preferably 10 or more, preferably 15 or more, preferably 20 or more, preferably 50 or more, more preferably 70 or more, more preferably 70 or more, of the constituent amino acid sequences of the protein of the present invention. Is a peptide having an amino acid sequence of 100 or more, most preferably 200 or more.

In the partial peptide used in the present invention, one or more (preferably about 1 to 10, more preferably a number (1 to 5)) amino acids in the amino acid sequence are deleted, or One or more (preferably about 1 to 20, more preferably about 1 to 10, and still more preferably, about 1 to 5) amino acids are added to the amino acid sequence, or the amino acid One or more (preferably about 1 to 20, more preferably about 1 to 10, and still more preferably, about 1 to 5) amino acids are inserted into the sequence, or One or more (preferably about 1 to 10, more preferably several, and still more preferably about 1 to 5) amino acids may be substituted with another amino acid.

As the partial peptide of the present invention, for example, a peptide having the 1st to 97th amino acids, the 169th to 191st amino acids, and the 254th to 277th amino acid sequences in the amino acid sequence represented by SEQ ID NO: 1; Peptides containing the 1st to 97th amino acids, the 169th to 191st amino acids, and the 254th to 277th amino acid sequences in the represented amino acid sequences are exemplified.

The partial peptide used in the present invention usually has a carboxyl group (—COOH) or a carboxylate (—COO ⁻ ) at the C-terminus, but has an amide (—CONH ₂ ) at the C-terminus as in the protein of the present invention described above. Or it may be an ester (-COOR).

Further, the partial peptides used in the present invention include those having a carboxyl group (or carboxylate) other than the C-terminus and N-terminal amino acid residues (eg, methionine), similarly to the aforementioned protein of the present invention. Residue), the amino group of which is protected by a protecting group, the glutamine residue formed by cleavage of the N-terminal side in vivo and pyroglutamine oxidation, 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.

部分 The partial peptide used in the present invention can also be used as an antigen for preparing an antibody. For example, in order to prepare the antibody of the present invention described below, for example, in the amino acid sequence represented by SEQ ID NO: 1, the 1st to 28th positions, the 99th to 129th positions, the 180th to 193rd positions, and the 246th to 286th positions And a peptide having the 1st to 97th amino acids, the 169th to 191st amino acids, and the 254th to 277th amino acids in the amino acid sequence represented by SEQ ID NO: 19.

As the salt of the protein or partial peptide of the present invention, salts with physiologically acceptable acids (eg, inorganic acids, organic acids) and bases (eg, alkali metal salts) are used. Preferred acid addition salts are: Such salts include, for example, salts with inorganic acids (eg, hydrochloric acid, phosphoric acid, hydrobromic acid, sulfuric acid) or organic acids (eg, acetic acid, formic acid, propionic acid, fumaric acid, maleic acid, succinic acid) Acids, tartaric acid, citric acid, malic acid, oxalic acid, benzoic acid, methanesulfonic acid, benzenesulfonic acid) and the like are used.

The protein of the present invention, a partial peptide thereof, or a salt thereof can be produced from the above-mentioned method for purifying a protein from human or warm-blooded animal cells or tissues, or a transformant containing a DNA encoding the protein. Can also be produced by culturing. Further, it can be produced according to the peptide synthesis method described later.

When producing from human or mammalian tissues or cells, the homogenized human or mammalian tissues or cells are extracted with an acid or the like, and the extract is subjected to chromatography such as reverse phase chromatography or ion exchange chromatography. Can be purified and isolated.

市 販 For synthesis of the protein or partial peptide of the present invention or a salt thereof, or an amide thereof, a commercially available resin for protein synthesis can be usually used. Examples of such a resin include chloromethyl resin, hydroxymethyl resin, benzhydrylamine resin, aminomethyl resin, 4-benzyloxybenzyl alcohol resin, 4-methylbenzhydrylamine resin, PAM resin, and 4-hydroxymethylmethyl. Phenylacetamidomethyl resin, polyacrylamide resin, 4- (2 ′, 4′-dimethoxyphenyl-hydroxymethyl) phenoxy resin, 4- (2 ′, 4′-dimethoxyphenyl-Fmocaminoethyl) phenoxy resin, and the like. it can. Using such a resin, an amino acid having an α-amino group and a side chain functional group appropriately protected is condensed on the resin in accordance with the sequence of the target protein according to various known condensation methods. At the end of the reaction, the protein or partial peptide is cleaved from the resin, and at the same time, various protecting groups are removed.In addition, an intramolecular disulfide bond formation reaction is performed in a highly diluted solution to obtain the target protein or partial peptide or an amide thereof. I do.

For the condensation of the above protected amino acids, various activating reagents that can be used for protein synthesis can be used, and carbodiimides are particularly preferable. As the carbodiimides, DCC, N, N′-diisopropylcarbodiimide, N-ethyl-N ′-(3-dimethylaminoprolyl) carbodiimide and the like are used. For activation by these, the protected amino acid was directly added to the resin together with a racemization inhibitor additive (for example, HOBt, HOOBt), or the protected amino acid was previously activated as a symmetric acid anhydride or HOBt ester or HOOBt ester. Later it can be added to the resin.

(4) The solvent used for activating the protected amino acid or condensing with the resin can be appropriately selected from solvents known to be usable for the protein condensation reaction. For example, acid amides such as N, N-dimethylformamide, N, N-dimethylacetamide, N-methylpyrrolidone, halogenated hydrocarbons such as methylene chloride and chloroform, alcohols such as trifluoroethanol, and dimethyl sulfoxide. Sulfoxides, ethers such as pyridine, dioxane, and tetrahydrofuran, nitriles such as acetonitrile and propionitrile, esters such as methyl acetate and ethyl acetate, and appropriate mixtures thereof are used. The reaction temperature is appropriately selected from a range known to be usable for a protein bond formation reaction, and is usually appropriately selected from a range of about -20 ° C to 50 ° C. The activated amino acid derivative is usually used in a 1.5 to 4-fold excess. As a result of the test using the ninhydrin reaction, when the condensation is insufficient, sufficient condensation can be performed by repeating the condensation reaction without removing the protecting group. When sufficient condensation cannot be obtained even by repeating the reaction, the unreacted amino acid can be acetylated using acetic anhydride or acetylimidazole, so that the subsequent reaction is not affected.

Examples of the protecting group for the amino group of the starting material include Z, Boc, t-pentyloxycarbonyl, isobornyloxycarbonyl, 4-methoxybenzyloxycarbonyl, Cl-Z, Br-Z, adamantyloxycarbonyl, trifluoroacetyl , Phthaloyl, formyl, 2-nitrophenylsulfenyl, diphenylphosphinothioyl, Fmoc and the like.

The carboxyl group may be, for example, a linear, branched or cyclic alkyl ester such as an alkyl esterified ester (eg, methyl, ethyl, propyl, butyl, t-butyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, 2-adamantyl). ), Aralkyl esterification (eg, benzyl ester, 4-nitrobenzyl ester, 4-methoxybenzyl ester, 4-chlorobenzyl ester, benzhydryl esterification), phenacyl esterification, benzyloxycarbonylhydrazide, t-butoxy It can be protected by carbonyl hydrazide, trityl hydrazide and the like.

The hydroxyl group of serine can be protected, for example, by esterification or etherification. Examples of groups suitable for the esterification include lower (C _1-6 ) alkanoyl groups such as an acetyl group, aroyl groups such as a benzoyl group, and groups derived from carbonic acid such as a benzyloxycarbonyl group and an ethoxycarbonyl group. Used. Examples of the group suitable for etherification include a benzyl group, a tetrahydropyranyl group, and a t-butyl group.

As the protecting group for the phenolic hydroxyl group of tyrosine, for example, Bzl, Cl ₂ -Bzl, 2-nitrobenzyl, Br-Z, t-butyl and the like are used.

@ As the imidazole protecting group for histidine, for example, Tos, 4-methoxy-2,3,6-trimethylbenzenesulfonyl, DNP, benzyloxymethyl, Bum, Boc, Trt, Fmoc and the like are used.

Examples of the activated carboxyl group of the raw material include, for example, a corresponding acid anhydride, azide, active ester [alcohol (eg, pentachlorophenol, 2,4,5-trichlorophenol, 2,4-dinitrophenol, Cyanomethyl alcohol, paranitrophenol, HONB, N-hydroxysuccinimide, N-hydroxyphthalimide, and an ester with HOBt). As the activated amino group of the raw material, for example, a corresponding phosphoric amide is used.

As a method for removing (eliminating) the protecting group, for example, catalytic reduction in a hydrogen stream in the presence of a catalyst such as Pd-black or Pd-carbon, or hydrogen fluoride anhydride, methanesulfonic acid, trifluoromethane, etc. Acid treatment with dichloromethane, trifluoroacetic acid or a mixture thereof, base treatment with diisopropylethylamine, triethylamine, piperidine, piperazine, etc., reduction with sodium in liquid ammonia, and the like are also used. The elimination reaction by the above acid treatment is generally performed at a temperature of about -20 ° C to 40 ° C. In the acid treatment, for example, anisole, phenol, thioanisole, metacresol, paracresol, dimethylsulfide, 1,4 -Addition of a ligand operable cation scavenger such as butanedithiol, 1,2-ethanedithiol, etc. is effective. Further, the 2,4-dinitrophenyl group used as an imidazole protecting group of histidine is removed by thiophenol treatment, and the formyl group used as an indole protecting group of tryptophan is the above 1,2-ethanedithiol, 1,4-butane. In addition to deprotection by acid treatment in the presence of dithiol or the like, it is also removed by alkali treatment with dilute sodium hydroxide solution, dilute ammonia or the like.

保護 The protection of the functional group which should not be involved in the reaction of the raw material, 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.

As another method for obtaining an amide form of a protein or a partial peptide, for example, after amidating and protecting the α-carboxyl group of the carboxy terminal amino acid, a peptide (protein) chain is added to the amino group side to a desired chain length. After the elongation, a protein or partial peptide from which only the protecting group for the N-terminal α-amino group of the peptide chain has been removed and a protein or partial peptide from which only the protecting group for the C-terminal carboxyl group has been removed, and these are produced. The protein or peptide is condensed in a mixed solvent as described above. Details of the condensation reaction are the same as described above. After purifying the protected protein or peptide obtained by the condensation, all the protecting groups are removed by the above-mentioned method to obtain a desired crude protein or peptide. This crude protein or peptide is purified by various known purification means, and the main fraction is lyophilized to obtain an amide of the desired protein or peptide.

In order to obtain an ester of a protein or peptide, for example, after condensing the α-carboxyl group of the carboxy terminal amino acid with a desired alcohol to form an amino acid ester, the desired protein or An ester of the peptide can be obtained.

The partial peptide or a salt thereof used in the present invention can be produced according to a known peptide synthesis method or by cleaving the protein of the present invention with an appropriate peptidase. As a method for synthesizing a peptide, for example, any of a solid phase synthesis method and a liquid phase synthesis method may be used. That is, the partial peptide or amino acid that can constitute the partial peptide used in the present invention is condensed with the remaining portion, and when the product has a protecting group, the protecting group is eliminated to produce the target peptide. it can. Known methods for condensation and elimination of protecting groups include, for example, the methods described in the following (a) to (e).
(A) M. Bodanszky and MA Ondetti, Peptide Synthesis, Interscience Publishers, New York (1966)
(B) Schroeder and Luebke, The Peptide, Academic Press, New York (1965)
(C) Nobuo Izumiya et al., Fundamentals and experiments of peptide synthesis, Maruzen Co., Ltd. (1975)
(D) Haruaki Yajima and Shunpei Sakakibara, Laboratory of Biochemistry 1, Protein Chemistry IV, 205, (1977)
(E) Supervised by Haruaki Yajima, Development of Continuing Pharmaceuticals, Vol. 14, Peptide Synthesis, Hirokawa Shoten After the reaction, conventional purification methods such as solvent extraction, distillation, column chromatography, liquid chromatography, recrystallization, etc. Can be used to purify and isolate the partial peptide used in the present invention. When the partial peptide obtained by the above method is a free form, it can be converted to an appropriate salt by a known method or a method analogous thereto, and conversely, when the partial peptide is obtained as a salt, a known method or analogous method It can be converted into a free form or another salt by a method.

ポ リ The polynucleotide encoding the protein of the present invention may be any polynucleotide as long as it contains the above-described nucleotide sequence encoding the protein of the present invention. Preferably it is DNA. The DNA may be any of genomic DNA, genomic DNA library, cDNA derived from the above-described cells / tissues, cDNA library derived from the aforementioned cells / tissues, and synthetic DNA.

ベ ク タ ー The vector used for the library may be any of bacteriophage, plasmid, cosmid, phagemid and the like. Alternatively, amplification can be carried out directly by Reverse Transcriptase Polymerase Chain Reaction (hereinafter abbreviated as RT-PCR method) using a total RNA or mRNA fraction prepared from the cells and tissues described above.

The DNA encoding the protein of the present invention includes, for example, (1) a DNA containing the base sequence represented by SEQ ID NO: 2 or a base sequence represented by SEQ ID NO: 2 hybridized under high stringent conditions. DNA encoding a protein having a soybean base sequence and having substantially the same properties as a protein containing the amino acid sequence represented by SEQ ID NO: 1, (2) a base sequence represented by SEQ ID NO: 15 Or a protein having a nucleotide sequence that hybridizes with the nucleotide sequence represented by SEQ ID NO: 15 under high stringent conditions, and substantially comprising the amino acid sequence represented by SEQ ID NO: 1 DNA encoding a protein having the same properties as described above, (3) DNA containing the nucleotide sequence represented by SEQ ID NO: 20, or SEQ ID NO: A protein having a nucleotide sequence that hybridizes under high stringent conditions to the nucleotide sequence represented by SEQ ID NO: 0 and having substantially the same properties as the protein containing the amino acid sequence represented by SEQ ID NO: 19 (4) a DNA containing the nucleotide sequence represented by SEQ ID NO: 27, or a nucleotide sequence that hybridizes under high stringent conditions to the nucleotide sequence represented by SEQ ID NO: 27; Any DNA may be used as long as it encodes a protein having substantially the same properties as the protein containing the amino acid sequence represented by No. 19.

Examples of the DNA that can hybridize with the nucleotide sequence represented by SEQ ID NO: 2, SEQ ID NO: 15, SEQ ID NO: 20, or SEQ ID NO: 27 under high stringent conditions include, for example, SEQ ID NO: 2, SEQ ID NO: 15, about 50% or more, preferably about 60% or more, more preferably about 70% or more, particularly preferably about 80% or more, and most preferably about 50% or more of the nucleotide sequence represented by SEQ ID NO: 20 or SEQ ID NO: 27. For example, DNA containing a base sequence having 90% or more homology is used.

The homology of the nucleotide sequences was determined using the homology calculation algorithm NCBI BLAST (National Center for Biotechnology Information Basic Local Alignment Search Tool) under the following conditions (expected value = 10; gap allowed; filtering = ON; match score = 1; It can be calculated by mismatch score = -3).

Hybridization can be carried out according to a known method or a method analogous thereto, for example, the method described in Molecular Cloning 2nd (J. Sambrook et al., Cold Spring Harbor Lab. Press, 1989). When a commercially available library is used, it can be performed according to the method described in the attached instruction manual. More preferably, it can be performed under high stringent conditions.

High stringency conditions refer to, for example, conditions of a sodium concentration of about 19 to 40 mM, preferably about 19 to 20 mM, and a temperature of about 50 to 70 ° C, preferably about 60 to 65 ° C. In particular, the case where the sodium concentration is about 19 mM and the temperature is about 65 ° C. is most preferable.

More specifically, as the DNA encoding the protein containing the amino acid sequence represented by SEQ ID NO: 1, the DNA containing the base sequence represented by SEQ ID NO: 2 or 15 is SEQ ID NO: As a DNA encoding a protein containing the amino acid sequence represented by SEQ ID NO: 19, a DNA containing the base sequence represented by SEQ ID NO: 20 or SEQ ID NO: 27 or the like is used.

ＤＮＡ The DNA encoding the partial peptide of the present invention may be any DNA containing the above-described nucleotide sequence encoding the partial peptide of the present invention. Further, any of genomic DNA, genomic DNA library, cDNA derived from the above-mentioned cells / tissues, cDNA library derived from the aforementioned cells / tissues, and synthetic DNA may be used.

Examples of the DNA encoding the partial peptide used in the present invention include a DNA having a part of a DNA having a base sequence represented by SEQ ID NO: 2, SEQ ID NO: 15, SEQ ID NO: 20 or SEQ ID NO: 27; Alternatively, it contains a nucleotide sequence that hybridizes with the nucleotide sequence represented by SEQ ID NO: 2, SEQ ID NO: 15, SEQ ID NO: 20 or SEQ ID NO: 27 under high stringent conditions, and substantially differs from the protein of the present invention. For example, a DNA containing a part of a DNA encoding a protein having the same activity can be used.

ＤＮＡ DNA capable of hybridizing with the nucleotide sequence represented by SEQ ID NO: 2, SEQ ID NO: 15, SEQ ID NO: 20 or SEQ ID NO: 27 has the same significance as described above.

は The same hybridization method and high stringency conditions as described above are used.

As a means for cloning a DNA that completely encodes the protein or partial peptide of the present invention (hereinafter, in the description of the cloning and expression of DNAs encoding them, these may be simply abbreviated to the protein of the present invention), Amplifying by a PCR method using a synthetic DNA primer having a part of the nucleotide sequence encoding the protein of the present invention, or incorporating a DNA incorporated in an appropriate vector into a DNA encoding a part or the whole region of the protein of the present invention. Selection can be performed by hybridization with fragments or those labeled with synthetic DNA. Hybridization can be performed, for example, according to the method described in Molecular Cloning 2nd (J. Sambrook et al., Cold Spring Harbor Lab. Press, 1989). When a commercially available library is used, it can be performed according to the method described in the attached instruction manual.

The DNA base sequence can be converted using PCR or a known kit such as Mutan ^™ -super Express Km (Takara Shuzo Co., Ltd.) or Mutan ^™ -K (Takara Shuzo Co., Ltd.) using the ODA-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.

ＤＮＡ The DNA encoding the cloned protein can be used as it is depending on the purpose, or can be digested with a restriction enzyme or added with a linker if desired. The DNA may have ATG as a translation initiation codon at the 5 'end and TAA, TGA or TAG as a translation termination codon at the 3' end. These translation initiation codon and translation termination codon can be added using an appropriate synthetic DNA adapter.

The expression vector for the protein of the present invention includes, for example, (a) cutting out a DNA fragment of interest from DNA encoding the protein of the present invention, and (b) ligating the DNA fragment downstream of a promoter in an appropriate expression vector. It can be manufactured by the following.

Examples of the vector include plasmids derived from Escherichia coli (eg, pBR322, pBR325, pUC12, pUC13), plasmids derived from Bacillus subtilis (eg, pUB110, pTP5, pC194), plasmids derived from yeast (eg, pSH19, pSH15), λ phage and the like. In addition to animal viruses such as bacteriophage, retrovirus, vaccinia virus, and baculovirus, pA1-11, pXT1, pRc / CMV, pRc / RSV, pcDNAI / Neo, and the like are used.

プ ロ モ ー タ ー The promoter used in the present invention may be any promoter that is appropriate for the host used for gene expression. For example, when an animal cell is used as a host, SRα promoter, SV40 promoter, LTR promoter, CMV promoter, HSV-TK promoter and the like can be mentioned.

の う ち Among them, it is preferable to use a CMV (cytomegalovirus) promoter, SRα promoter or the like. When the host is a bacterium belonging to the genus Escherichia, trp promoter, lac promoter, recA promoter, λPL promoter, lpp promoter, T7 promoter, etc., and when the host is a bacterium belonging to the genus Bacillus, SPO1 promoter, SPO2 promoter, penP promoter, etc. When the host is yeast, PHO5 promoter, PGK promoter, GAP promoter, ADH promoter and the like are preferable. When the host is an insect cell, a polyhedrin promoter, a P10 promoter and the like are preferable.

In addition to the above, an expression vector containing an enhancer, a splicing signal, a polyA addition signal, a selection marker, an SV40 replication origin (hereinafter sometimes abbreviated as SV40 ori), and the like may be used, if desired. it can. As the selection marker include dihydrofolate reductase (hereinafter sometimes abbreviated as dhfr) gene [methotrexate (MTX) resistance], ampicillin resistant gene (hereinafter sometimes abbreviated as Amp ^r), neomycin resistant gene (Hereinafter, may be abbreviated as Neo ^r , G418 resistance). In particular, when the dhfr gene is used as a selection marker using Chinese hamster cells deficient in dhfr gene, the target gene can be selected using a thymidine-free medium.

(4) If necessary, a signal sequence suitable for the host is added to the N-terminal side of the protein of the present invention. When the host is a bacterium belonging to the genus Escherichia, a PhoA signal sequence, an OmpA signal sequence, or the like is used. When the host is a bacterium belonging to the genus Bacillus, an α-amylase signal sequence, a subtilisin signal sequence, or the like is used. In some cases, MFα signal sequence, SUC2 signal sequence, etc., and when the host is an animal cell, insulin signal sequence, α-interferon signal sequence, antibody molecule, signal sequence, etc. can be used.

形 質 A transformant can be produced using the vector containing the DNA encoding the protein of the present invention thus constructed.

As the host, for example, Escherichia, Bacillus, yeast, insect cells, insects, animal cells and the like are used.

Specific examples of the genus Escherichia include, for example, Escherichia coli K12 DH1 [Processings of the National Academy of Sciences of the USA (Proc. Natl. Acad. Sci. USA), 60, 160 (1968)], JM103 [Nucleic Acids Research, 9, 309 (1981)], JA221 [Journal of Molecular Biology (Journal of Molecular Biology)]. Biology)], 120, 517 (1978)], HB101 [Journal of Molecular Biology, 41, 459 (1969)], C600 [Genetics, 39, 440 (1954)], etc. Is used.

Examples of Bacillus bacteria include, for example, Bacillus subtilis MI114 [Gene, 24, 255 (1983)], 207-21 [Journal of Biochemistry, 95, 87 (1984)]. )] Is used.

Examples of yeast include Saccharomyces cerevisiae ^{(Saccharomyces cerevisiae) AH22, AH22R -} , NA87-11A, DKD-5D, 20B-12, Schizosaccharomyces pombe (Schizosaccharomyces pombe) NCYC1913, NCYC2036, such as Pichia pastoris (Pichia pastoris) KM71 is Used.

Insect cells include, for example, when the virus is AcNPV, 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, High Five ™ cell derived from egg of Trichoplusia ni And cells derived from Mamestra brassicae or cells derived from Estigmena acrea. When the virus is BmNPV, a silkworm-derived cell line (Bombyx mori N cell; BmN cell) or the like is used. As the Sf cells, for example, Sf9 cells (ATCC CRL 1711), Sf21 cells (Vaughn, J.L., et al., In Vivo, 13, 213-217, (1977)) and the like are used.

As the insect, for example, silkworm larvae are used [Maeda et al., Nature, 315, 592 (1985)].

Examples of animal cells include monkey cell COS-7, Vero, Chinese hamster cell CHO (hereinafter, CHO cells), dhfr gene deficient Chinese hamster cell CHO (hereinafter, CHO (dhfr ^-) cell), mouse L Cells, mouse AtT-20, mouse myeloma cells, rat GH3, human FL cells and the like are used.

In order to transform Escherichia, for example, Procagings of the National Academy of Sciences of the USA (Proc. Natl. Acad. Sci. USA), 69, 2110 ( 1972) and Gene, Vol. 17, 107 (1982).

Transformation of Bacillus sp. Can be performed, for example, according to the method described in Molecular & General Genetics, Vol. 168, 111 (1979).

For transforming yeast, for example, Methods in Enzymology, 194, 182-187 (1991), Proceedings of the National Academy of Sciences of Science -The USA (Proc. Natl. Acad. Sci. USA), 75, 1929 (1978).

Insect cells or insects can be transformed, for example, according to the method described in Bio / Technology, 6, 47-55 (1988).

{To transform animal cells, see, eg, Cell Engineering Separate Volume 8} New Cell Engineering Experiment Protocol. 263-267 (1995) (published by Shujunsha) and Virology, Vol. 52, 456 (1973).

Thus, a transformant transformed with the expression vector containing the DNA encoding the protein can be obtained.

When culturing a transformant whose host is a genus Escherichia or Bacillus, a liquid medium is suitable as a medium used for the culturing, and a carbon source necessary for growth of the transformant is used therein. Nitrogen sources, inorganic substances, etc. are included. As a carbon source, for example, glucose, dextrin, soluble starch, sucrose, etc., as a nitrogen source, for example, ammonium salts, nitrates, corn steep liquor, peptone, casein, meat extract, soybean meal, potato extract and the like Examples of the inorganic or organic substance and the inorganic substance include calcium chloride, sodium dihydrogen phosphate, and magnesium chloride. In addition, yeast extract, vitamins, growth promoting factors and the like may be added. The pH of the medium is preferably about 5 to 8.

As a medium for culturing the genus Escherichia, for example, an M9 medium containing glucose and casamino acid [Miller, Journal of Experiments in Molecular Genetics] 431-433, Cold Spring Harbor Laboratory, New York 1972]. If necessary, an agent such as 3β-indolylacrylic acid can be added in order to make the promoter work efficiently.

場合 When the host is a bacterium belonging to the genus Escherichia, the cultivation is usually performed at about 15 to 43 ° C. for about 3 to 24 hours, and if necessary, aeration and stirring can be added.

場合 When the host is a bacterium belonging to the genus Bacillus, the cultivation is usually carried out at about 30 to 40 ° C. for about 6 to 24 hours, and if necessary, aeration and stirring can be added.

When culturing a transformant in which the host is yeast, as a medium, for example, Burkholder's minimum medium [Bostian, KL et al., Processings of the National Academy of Sciences of Science] Natl. Acad. Sci. USA, 77, 4505 (1980)] or an SD medium containing 0.5% casamino acid [Bitter, GA et al., Processings of the National. Academy of Sciences of the USA (Proc. Natl. Acad. Sci. USA), 81, 5330 (1984)]. Preferably, the pH of the medium is adjusted to about 5-8. The cultivation is usually performed at about 20 ° C. to 35 ° C. for about 24 to 72 hours, and if necessary, aeration and stirring are added.

When culturing an insect cell or a transformant whose host is an insect, 10% bovine serum immobilized in Grace's Insect Medium (Grace, TCC, Nature, 195, 788 (1962)) is added as a medium. What added the thing suitably is used. The pH of the medium is preferably adjusted to about 6.2 to 6.4. The cultivation is usually performed at about 27 ° C. for about 3 to 5 days, and if necessary, aeration or stirring is added.

When culturing a transformant in which the host is an animal cell, examples of the medium include a MEM medium containing about 5 to 20% fetal bovine serum [Science, Vol. 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, Vol. 73, 1 (1950)]. Preferably, the pH is between about 6 and 8. The cultivation is usually performed at about 30 ° C. to 40 ° C. for about 15 to 60 hours, and if necessary, aeration and stirring are added.

As described above, the protein of the present invention can be produced in the cells of the transformant, in the cell membrane, or outside the cells.

分離 The protein of the present invention can be separated and purified from the culture by, for example, the following method.

When extracting the protein of the present invention from cultured cells or cells, the cells or cells are collected by a known method after culturing, suspended in an appropriate buffer, and subjected to sonication, lysozyme and / or freeze-thawing. After the cells or cells are destroyed by the method, a method of obtaining a crude extract of the protein by centrifugation or filtration is used as appropriate. The buffer may contain a protein denaturant such as urea or guanidine hydrochloride, or a surfactant such as Triton X-100 ^™ . When the protein is secreted into the culture solution, after the culture, the supernatant is separated from the cells or cells by a known method, and the supernatant is collected.

培養 The protein contained in the thus obtained culture supernatant or extract can be purified by appropriately combining known separation and purification methods. These known separation and purification methods include methods utilizing solubility such as salting out and solvent precipitation, dialysis, ultrafiltration, gel filtration, and SDS-polyacrylamide gel electrophoresis, mainly molecular weight. Using differences in charge, methods using charge differences such as ion exchange chromatography, methods using specific affinity such as affinity chromatography, and using differences in hydrophobicity such as reversed-phase high-performance liquid chromatography A method utilizing a difference between isoelectric points, such as an isoelectric focusing method, is used.

When the thus obtained protein is 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 as a salt, by a known method or a method analogous thereto, It can be converted to a free form or other salts.

The protein produced by the recombinant can be arbitrarily modified or the polypeptide can be partially removed before or after purification by the action of an appropriate protein modifying enzyme. As the 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 an enzyme immunoassay using a specific antibody, Western blotting, or the like.

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 as the protein of the present invention) using the protein of the present invention as an antigen, is a known antibody or It can be produced according to the method for producing antiserum.
[Preparation of monoclonal antibody]
(A) Preparation of Monoclonal Antibody-Producing Cell The protein of the present invention is administered to a warm-blooded animal by itself or together with a carrier and a diluent at a site where antibody production is possible by administration. Complete Freund's adjuvant or incomplete Freund's adjuvant may be administered in order to enhance the antibody-producing ability upon administration. The administration is usually performed once every 2 to 6 weeks, for a total of about 2 to 10 times. Examples of the warm-blooded animal used include monkeys, rabbits, dogs, guinea pigs, mice, rats, sheep, goats, and chickens, and mice and rats are preferably used.

When preparing monoclonal antibody-producing cells, a warm-blooded animal immunized with an antigen, for example, an individual having 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. By fusing the antibody-producing cells thus obtained with myeloma cells of the same or different species, a monoclonal antibody-producing hybridoma can be prepared. The antibody titer in the antiserum can be measured, for example, by reacting a labeled protein described below with the antiserum, and then measuring the activity of a labeling agent bound to the antibody. The fusion operation can be performed according to a known method, for example, the method of Kohler and Milstein [Nature, 256, 495 (1975)]. Examples of the fusion promoter include polyethylene glycol (PEG) and Sendai virus, but PEG is preferably used.

Examples of myeloma cells include myeloma cells of warm-blooded animals such as NS-1, P3U1, SP2 / 0, and AP-1, but P3U1 is preferably used. The preferred ratio between the number of antibody-producing cells (spleen cells) and the number of myeloma cells used is about 1: 1 to 20: 1, and PEG (preferably PEG1000 to PEG6000) is added at a concentration of about 10 to 80%. By incubating at 20 to 40 ° C, preferably 30 to 37 ° C for 1 to 10 minutes, cell fusion can be carried out efficiently.

Various methods can be used to screen for monoclonal antibody-producing hybridomas. For example, a hybridoma culture supernatant is added to a solid phase (eg, a microplate) on which a protein antigen is directly or adsorbed together with a carrier, and then radioactive substances or A method for detecting a monoclonal antibody bound to a solid phase by adding an anti-immunoglobulin antibody labeled with an enzyme or the like (an anti-mouse immunoglobulin antibody is used when cells used for cell fusion are mice) or protein A; A method in which a hybridoma culture supernatant is added to a solid phase to which globulin antibodies or protein A is adsorbed, a protein labeled with a radioactive substance, an enzyme, or the like is added, and a monoclonal antibody bound to the solid phase is detected.

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 a medium for animal cells supplemented with HAT (hypoxanthine, aminopterin, thymidine). As a selection and breeding medium, any medium can be used as long as a hybridoma can grow. For example, RPMI 1640 medium containing 1 to 20%, preferably 10 to 20% fetal bovine serum, GIT medium containing 1 to 10% fetal bovine serum (Wako Pure Chemical Industries, Ltd.) or serum-free for hybridoma culture A medium (SFM-101, Nissui Pharmaceutical Co., Ltd.) or the like can be used. The culturing temperature is usually 20 to 40 ° C, preferably about 37 ° C. The culture time is usually 5 days to 3 weeks, preferably 1 week to 2 weeks. The culture can be usually performed under 5% carbon dioxide gas. The antibody titer of the hybridoma culture supernatant can be measured in the same manner as the measurement of the antibody titer in the antiserum described above.
(B) Purification of monoclonal antibody 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) adsorption / desorption method, ultracentrifugation method, gel filtration method, specific method of collecting only antibody by antigen-binding solid phase or active adsorbent such as protein A or protein G and dissociating the bond to obtain antibody Purification Method].
(Preparation of polyclonal antibody)
The polyclonal antibody of the present invention can be produced according to a known method or a method analogous thereto. For example, a immunizing antigen (protein antigen) itself or 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 a substance and separating and purifying the antibody.

Regarding the complex of an immunizing antigen and a carrier protein used to immunize a warm-blooded animal, the type of the carrier protein and the mixing ratio between the carrier and the hapten are such that the antibody is efficiently cross-linked to the hapten immunized by crosslinking the carrier. If possible, any material may be cross-linked at any ratio. For example, bovine serum albumin, bovine thyroglobulin, hemocyanin and the like are used in a weight ratio of about 0.1 to 20, preferably about 1 to 20, relative to hapten 1. A method of coupling at a rate of ~ 5 is used.

Various condensing agents can be used for coupling the hapten and the carrier. For example, glutaraldehyde, carbodiimide, a maleimide active ester, an active ester reagent containing a thiol group or a dithioviridyl group, or the like is used.

(4) The condensation product is administered to a warm-blooded animal at a site capable of producing an antibody itself or together with a carrier and a diluent. Complete Freund's adjuvant or incomplete Freund's adjuvant may be administered in order to enhance the antibody-producing ability upon administration. The administration is usually performed once every about 2 to 6 weeks, about 3 to 10 times in total.

The polyclonal antibody can be collected from the blood, ascites, etc., preferably from the blood of a warm-blooded animal immunized by the above method.

ポ リ Polyclonal antibody titer in antiserum can be measured in the same manner as in the above-described measurement of antibody titer in antiserum. The separation and purification of the polyclonal antibody can be performed according to the same immunoglobulin separation and purification method as the above-mentioned separation and purification of the monoclonal antibody.

Complementary or substantially complementary to the base sequence of DNA encoding the protein or partial peptide of the present invention (hereinafter, these DNAs may be abbreviated as the DNA of the present invention in the description of the antisense polynucleotide). The antisense polynucleotide having a complementary base sequence or a part thereof has a base sequence complementary to or substantially complementary to the base sequence of the DNA of the present invention or a part thereof, and Any antisense polynucleotide may be used as long as it has an action of suppressing expression, but antisense DNA is preferable.

The nucleotide sequence substantially complementary to the DNA of the present invention is, for example, about 70% of the total nucleotide sequence or a partial nucleotide sequence of the nucleotide sequence complementary to the DNA of the present invention (that is, the complementary strand of the DNA of the present invention). % Or more, preferably about 80% or more, more preferably about 90% or more, most preferably about 95% or more. In particular, of the entire base sequence of the complementary strand of the DNA of the present invention, the base sequence of the portion encoding the N-terminal site of the protein of the present invention (for example, a base sequence near the start codon, etc.) is at least about 70% or more complementary to Antisense polynucleotides having preferably at least about 80%, more preferably at least about 90%, most preferably at least about 95% homology are suitable.

Specifically, a nucleotide sequence complementary to or substantially complementary to the nucleotide sequence of the DNA having the nucleotide sequence represented by SEQ ID NO: 2, SEQ ID NO: 15, SEQ ID NO: 20, or SEQ ID NO: 27, Or a base sequence complementary to a base sequence of a DNA having a base sequence represented by SEQ ID NO: 2, SEQ ID NO: 15, SEQ ID NO: 20 or SEQ ID NO: 27, preferably an antisense polynucleotide having a part thereof, Or an antisense polynucleotide having a part thereof.

Antisense polynucleotide is usually composed of about 10 to 40 bases, preferably about 15 to 30 bases.

In order to prevent degradation by a hydrolase such as nuclease, the phosphate residue (phosphate) of each nucleotide constituting the antisense DNA is replaced with, for example, a chemically modified phosphate residue such as phosphorothioate, methylphosphonate or phosphorodithionate. It may be substituted. These antisense polynucleotides can be produced using a known DNA synthesizer or the like.

According to the present invention, an antisense polynucleotide (nucleic acid) capable of inhibiting the replication or expression of the protein gene of the present invention is designed based on the cloned or determined nucleotide sequence information of the DNA encoding the protein. And can be synthesized. Such a polynucleotide (nucleic acid) can hybridize with the RNA of the protein gene of the present invention and inhibit the synthesis or function of the RNA, or through the interaction with the protein-related RNA of the present invention. Thus, the expression of the protein gene of the present invention can be regulated and controlled. Polynucleotides that are complementary to a selected sequence of the protein-associated RNA of the present invention, and that can specifically hybridize with the protein-associated RNA of the present invention, can be used in vivo and in vitro to produce the protein gene of the present invention. It is useful for regulating and controlling the expression of, and also useful for treating or diagnosing diseases and the like. The term “corresponding” means having homology or being complementary to a specific sequence of nucleotides, base sequences or nucleic acids including genes. "Corresponding" between a nucleotide, base sequence or nucleic acid and a peptide (protein) usually refers to the amino acid of the peptide (protein) as directed by the nucleotide (nucleic acid) sequence or its complement. . 5 'end hairpin loop of protein gene, 5' end 6-base pair repeat, 5 'end untranslated region, polypeptide translation start codon, protein coding region, ORF translation stop codon, 3' end untranslated region, 3 ' The terminal palindrome region and the 3'-end hairpin loop may be selected as preferred regions of interest, but any region within the protein gene may be selected as a target.

関係 The relationship between the target nucleic acid and the polynucleotide complementary to at least a part of the target region can be said to be that the relationship between the target nucleic acid and the polynucleotide that can hybridize with the target is “antisense”. Antisense polynucleotides include polynucleotides containing 2-deoxy-D-ribose, polynucleotides containing D-ribose, other types of polynucleotides that are N-glycosides of purine or pyrimidine bases, or Other polymers having non-nucleotide backbones (eg, commercially available protein nucleic acids and synthetic sequence-specific nucleic acid polymers) or other polymers containing special bonds, provided that the polymer is a base such as found in DNA or RNA And nucleotides having a configuration that allows base attachment). They can be double-stranded DNA, single-stranded DNA, double-stranded RNA, single-stranded RNA, and also DNA: RNA hybrids, and can also be unmodified polynucleotides (or unmodified oligonucleotides), or even known. Such as labeled, capped, methylated, one or more naturally-occurring nucleotides replaced by analogs, intramolecular nucleotides Modified, eg, those having an uncharged bond (eg, methylphosphonate, phosphotriester, phosphoramidate, carbamate, etc.), charged or sulfur-containing bonds (eg, phosphorothioate, phosphorodithioate, etc.) ), Such as proteins (nucleases, nuclease inhibitors, Those having side-chain groups such as syn, antibody, signal peptide, poly-L-lysine, etc. and sugars (eg, monosaccharide), those having intercalating compounds (eg, acridine, psoralen) Containing chelating compounds (eg, metals, radioactive metals, boron, oxidizing metals, etc.), those containing alkylating agents, those having modified bonds (eg, α-anomeric nucleic acids) Etc.). Here, "nucleoside", "nucleotide", and "nucleic acid" may include not only those containing purine and pyrimidine bases but also those having other modified heterocyclic bases. Such modifications may include methylated purines and pyrimidines, acylated purines and pyrimidines, or other heterocycles. Modified nucleotides and modified nucleotides may also be modified at the sugar moiety, e.g., one or more hydroxyl groups are replaced by halogens, aliphatic groups, etc., or by functional groups such as ethers, amines, etc. It may have been converted.

ア ン チ The antisense polynucleotide (nucleic acid) of the present invention is RNA, DNA, or a modified nucleic acid (RNA, DNA). Specific examples of the modified nucleic acid include, but are not limited to, sulfur derivatives and thiophosphate derivatives of nucleic acids, and those that are resistant to degradation of polynucleoside amides and oligonucleoside amides. The antisense nucleic acid of the present invention can be preferably designed according to the following policy. That is, to make the antisense nucleic acid more stable in the cell, to make the antisense nucleic acid more cell permeable, to have a greater affinity for the target sense strand, and to be more toxic if it is toxic. Minimize the toxicity of sense nucleic acids.

Many such modifications are known in the art, for example, J. Kawakami et al., Pharm Tech Japan, Vol. 8, pp. 247, 1992; Vol. 8, pp. 395, 1992; ST Crooke et al. Ed. , Antisense Research and Applications, CRC Press, 1993.

The antisense nucleic acid of the present invention may contain altered or modified sugars, bases or bonds, may be provided in a special form such as liposomes or microspheres, may be applied by gene therapy, It could be provided in an added form. Thus, in the form of addition, polycations such as polylysine which acts to neutralize the charge of the phosphate skeleton, lipids which enhance the interaction with the cell membrane or increase the uptake of nucleic acids ( For example, hydrophobic substances such as phospholipid and cholesterol can be mentioned. Preferred lipids for addition include cholesterol and its derivatives (eg, cholesteryl chloroformate, cholic acid, etc.). These can be attached to the 3 'end or 5' end of the nucleic acid, and can be attached via a base, sugar, or intramolecular nucleoside bond. Other groups include cap groups specifically arranged at the 3 ′ end or 5 ′ end of a nucleic acid for preventing degradation by nucleases such as exonuclease and RNase. Such capping groups include, but are not limited to, hydroxyl-protecting groups known in the art, including glycols such as polyethylene glycol and tetraethylene glycol.

(4) The antisense nucleic acid inhibitory activity can be examined using the transformant of the present invention, the in vivo or in vitro gene expression system of the present invention, or the in vivo or in vitro translation system of the protein of the present invention. The nucleic acid can be applied to cells by various known methods.

Hereinafter, 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 DNA of the present invention) ), 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), and an antisense polynucleotide of the DNA of the present invention (hereinafter, the antisense polynucleotide of the present invention). (May be abbreviated as nucleotide).

Pharmaceuticals containing a compound that inhibits the activity of the protein of the present invention or a salt thereof, for example, inhibit the transport of bile acids, steroid hormones, fat-soluble vitamins, neurotransmitters (eg, dopamine, glutamic acid, enkephalin, etc.). Thus, for example, hyperlipidemia, gastrointestinal diseases (eg, irritable bowel syndrome, ulcerative colitis, Crohn's disease, ischemic colitis, gastritis, peptic ulcer, proctitis, etc.), central nervous system diseases ( Eg, Alzheimer's disease, Parkinson's syndrome, depression, emotional disorders, schizophrenia, anxiety, etc.), diabetes, pancreatic disorders (eg, pancreatic insufficiency such as pancreatitis, cystic fibrosis), respiratory diseases (Eg, chronic obstructive pulmonary disease (COPD), asthma, etc.), hypothyroidism, cardiovascular disease (eg, heart failure, arrhythmia, long QT syndrome, arteriosclerosis, etc.), cancer (eg, testicular tumor) , Ovarian, breast, esophageal, lung, kidney, liver, non-small cell lung, prostate, gastric, bladder, cervical, colon, rectal, pancreatic, thymoma, etc.) It can be used as a prophylactic / therapeutic agent.

On the other hand, a medicament containing a compound or a salt thereof that promotes the activity of the protein of the present invention can transport bile acids, steroid hormones, fat-soluble vitamins, neurotransmitters (eg, dopamine, glutamic acid, enkephalin, etc.). By promoting, for example, hyperlipidemia, digestive disorders (eg, irritable bowel syndrome, ulcerative colitis, Crohn's disease, ischemic colitis, gastritis, peptic ulcer, proctitis, etc.), central nervous system Diseases (eg, Alzheimer's disease, Parkinson's syndrome, depression, emotional disorders, schizophrenia, anxiety neurosis, etc.), diabetes, pancreatic diseases (eg, pancreatic insufficiency such as pancreatitis, cystic fibrosis, etc.), respiratory System diseases (eg, chronic obstructive pulmonary disease (COPD), asthma, etc.), hypothyroidism, cardiovascular diseases (eg, heart failure, arrhythmia, long QT syndrome, arteriosclerosis, etc.), cancer (eg, Tumor, ovarian cancer, breast cancer, esophageal cancer, lung cancer, kidney cancer, liver cancer, non-small cell lung cancer, prostate cancer, stomach cancer, bladder cancer, cervical cancer, colon cancer, rectal cancer, pancreatic cancer, thymoma, etc.) Can be used as a prophylactic / therapeutic agent.
[1] Agent for Prevention / Treatment of Various Diseases Related to the Protein of the Present Invention The protein of the present invention has a transport activity of bile acids, steroid hormones, fat-soluble vitamins, neurotransmitters (eg, dopamine, glutamic acid, enkephalin, etc.). It contributes to the transport of bile acids, steroid hormones, fat-soluble vitamins, neurotransmitters (eg, dopamine, glutamic acid, enkephalin, etc.) and plays an important role in bile acid metabolism.

Therefore, when the DNA encoding the protein of the present invention is abnormal or defective, or when the expression level of the protein of the present invention is reduced, for example, hyperlipidemia, digestive diseases (eg, Irritable bowel syndrome, ulcerative colitis, Crohn's disease, ischemic colitis, gastritis, peptic ulcer, proctitis, etc., central nervous system disorders (eg, Alzheimer's disease, Parkinson's syndrome, depression, emotional disorders, integration Ataxia, anxiety, etc.), diabetes, pancreatic disease (eg, pancreatitis, pancreatic insufficiency such as cystic fibrosis), respiratory disease (eg, chronic obstructive pulmonary disease (COPD), asthma, etc.) , Hypothyroidism, cardiovascular disease (eg, heart failure, arrhythmia, long QT syndrome, arteriosclerosis, etc.), cancer (eg, testicular tumor, ovarian cancer, breast cancer, esophageal cancer, lung cancer, kidney cancer, liver cancer, non-small cell) Lung cancer, prostate cancer, gastric cancer Bladder cancer, cervical cancer, colon cancer, rectal cancer, pancreatic cancer, various diseases such as thymoma, etc.) to develop.

Therefore, the protein of the present invention and the DNA of the present invention include, for example, hyperlipidemia, digestive diseases (eg, irritable bowel syndrome, ulcerative colitis, Crohn's disease, ischemic colitis, gastritis, peptic ulcer, Proctitis), central nervous system disorders (eg, Alzheimer's disease, Parkinson's syndrome, depression, emotional disorders, schizophrenia, anxiety, etc.), diabetes, pancreatic disorders (eg, pancreatitis, cystic fibrosis, etc.) Pancreatic insufficiency, etc.), respiratory diseases (eg, chronic obstructive pulmonary disease (COPD), asthma, etc.), hypothyroidism, cardiovascular diseases (eg, heart failure, arrhythmia, long QT syndrome, arteriosclerosis, etc.), cancer (Eg, testicular tumor, ovarian cancer, breast cancer, esophageal cancer, lung cancer, kidney cancer, liver cancer, non-small cell lung cancer, prostate cancer, gastric cancer, bladder cancer, cervical cancer, colon cancer, rectum cancer, pancreatic cancer, breast cancer Medicines such as prophylactic / therapeutic agents such as adenomas) To be able to use. Preferably, it is a prophylactic / therapeutic agent for central nervous system diseases and the like, particularly preferably a prophylactic / therapeutic agent for Parkinson's syndrome and the like.

For example, since the protein of the present invention is reduced or deficient in the living body, the transport activity of bile acids, steroid hormones, fat-soluble vitamins, neurotransmitters (eg, dopamine, glutamic acid, enkephalin, etc.) is sufficient, Alternatively, when there is a patient who does not normally exert, (a) administering the DNA of the present invention to the patient and expressing the protein of the present invention in vivo, (b) inserting the DNA of the present invention into cells, By expressing the protein of the present invention and then transplanting the cells into a patient, or (c) administering the protein of the present invention to the patient, the role of the protein of the present invention in the patient can be sufficiently improved. Or normal.

When the DNA of the present invention is used as the above-described prophylactic / therapeutic agent, the DNA 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 to humans or warm-blooded animals. 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 adjuvant for promoting uptake, using a gene gun or a catheter such as a hydrogel catheter.

When the protein of the present invention is used as the above-mentioned prophylactic / therapeutic agent, it is preferable to use a protein purified to at least 90%, preferably 95% or more, more preferably 98% or more, and still more preferably 99% or more. preferable.

The protein or the like of the present invention can be, for example, sterilized with water or other pharmaceutically acceptable liquids orally as tablets, capsules, elixirs, microcapsules and the like, if necessary coated with sugar. It can be used parenterally in the form of injections, such as aqueous solutions or suspensions. For example, the protein or the like of the present invention is mixed with physiologically acceptable carriers, flavors, excipients, vehicles, preservatives, stabilizers, binders, and the like in a unit dosage form generally required for the practice of preparations. Can be manufactured. The amount of the active ingredient in these preparations is such that a proper dosage in the specified range can be obtained.

Examples of additives that can be incorporated into tablets, capsules, and the like include binders such as gelatin, corn starch, tragacanth, and acacia, excipients such as crystalline cellulose, corn starch, gelatin, and alginic acid. Useful bulking agents, lubricants such as magnesium stearate, sweeteners such as sucrose, lactose or saccharin, flavoring agents such as peppermint, reddish oil or cherry and the like are used. When the preparation unit form is a capsule, 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 normal pharmaceutical practice such as dissolving or suspending the active substance in vehicles such as water for injection, and naturally occurring vegetable oils such as sesame oil, coconut oil and the like.

Aqueous liquids for injection include, for example, physiological saline, isotonic solutions containing glucose and other adjuvants (eg, D-sorbitol, D-mannitol, sodium chloride, etc.), and suitable solubilizing agents. For example, it may be used in combination with an alcohol (eg, ethanol or the like), a polyalcohol (eg, propylene glycol, polyethylene glycol or the like), a nonionic surfactant (eg, polysorbate 80 ^™ , HCO-50, or the like). Examples of the oily liquid include sesame oil and soybean oil, and may be used in combination with solubilizers such as benzyl benzoate and benzyl alcohol. Also, buffers (eg, phosphate buffer, sodium acetate buffer, etc.), soothing agents (eg, benzalkonium chloride, procaine hydrochloride, etc.), stabilizers (eg, human serum albumin, polyethylene glycol, etc.), You may mix | blend with a preservative (for example, benzyl alcohol, phenol etc.), an antioxidant, etc. The prepared injection is usually filled in an appropriate ampoule.

ベ ク タ ー The vector into which the DNA of the present invention is inserted is also formulated in the same manner as described above, and is usually used parenterally.

The preparation obtained in this way is safe and low toxic, and thus, for example, is useful for warm-blooded animals (eg, human, rat, mouse, guinea pig, rabbit, bird, sheep, pig, cow, horse, cat, dog, monkey). , Chimpanzees, etc.).

The dose of the protein or the like of the present invention varies depending on the target disease, the administration subject, the administration route, and the like. For example, when the protein or the like of the present invention is orally administered for the purpose of treating Parkinson's syndrome, it is generally adult (body weight). 60 kg), the protein or the like is administered in an amount of about 0.1 mg to 100 mg, preferably about 1.0 to 50 mg, more preferably about 1.0 to 20 mg per day. When administered parenterally, the single dose of the protein or the like varies depending on the administration subject, target disease, and the like. For example, for the purpose of treating Parkinson's syndrome, the protein or the like of the present invention may be used in the form of an injection ( (As a body weight of 60 kg), the protein or the like is administered by intravenous injection of about 0.01 to 30 mg, preferably about 0.1 to 20 mg, more preferably about 0.1 to 10 mg per day. It is convenient. In the case of other animals, the amount can be administered in terms of the weight per 60 kg.
[2] Screening of drug candidate compounds for disease The protein of the present invention is useful as a reagent for screening a compound or a salt thereof that promotes or inhibits the activity of the protein of the present invention.

The present invention provides (1) the activity of the protein of the present invention characterized by using the protein of the present invention (eg, bile acids, steroid hormones, fat-soluble vitamins, neurotransmitters (eg, dopamine, glutamic acid, enkephalin, etc.)) The present invention provides a method for screening a compound or a salt thereof (hereinafter, may be abbreviated as an accelerator or an inhibitor, respectively) that promotes or inhibits transport or the like. More specifically, for example,
(2) (i) transport activity of cells capable of producing the protein of the present invention such as bile acids, steroid hormones, fat-soluble vitamins, and neurotransmitters (eg, dopamine, glutamic acid, enkephalin, etc.) and (ii) the present invention It is characterized by comparing the transport activity of a mixture of cells having the ability to produce the protein of the invention with a test compound, such as bile acids, steroid hormones, fat-soluble vitamins, and neurotransmitters (eg, dopamine, glutamic acid, enkephalin, etc.). And a method for screening for an accelerator or an inhibitor.

Specifically, in the above screening method, for example, in cases (i) and (ii), transport of bile acids, steroid hormones, fat-soluble vitamins, neurotransmitters (eg, dopamine, glutamic acid, enkephalin, etc.) Is measured with a radiolabeled substrate or a fluorescent dye, and compared using the transport of bile acids, steroid hormones, fat-soluble vitamins, neurotransmitters (eg, dopamine, glutamic acid, enkephalin, etc.) as an index. It is.

Test compounds include, for example, peptides, proteins, non-peptidic compounds, synthetic compounds, fermentation products, cell extracts, plant extracts, animal tissue extracts, and the like. Or a known compound.

実 施 In order to carry out the above screening method, cells having the ability to produce the protein of the present invention are prepared by suspending them in a buffer suitable for screening. The buffer includes a bile acid, a steroid hormone, a fat-soluble vitamin, a neurotransmitter (eg, a bile acid) of the protein of the present invention, such as a phosphate buffer or a borate buffer having a pH of about 4 to 10 (preferably, a pH of about 6 to 8). Any buffer that does not inhibit the transport activity, such as dopamine, glutamic acid, enkephalin and the like, may be used.

細胞 As a cell having the ability to produce the protein of the present invention, for example, a host (transformant) transformed with a vector containing the DNA encoding the protein of the present invention described above is used. As the host, for example, animal cells such as CHO cells are preferably used. For the screening, for example, a transformant in which the protein of the present invention is expressed on a cell membrane by culturing by the method described above is preferably used.

The transport activity of the protein of the present invention such as bile acids, steroid hormones, fat-soluble vitamins, and neurotransmitters (eg, dopamine, glutamic acid, enkephalin, etc.) can be determined by known methods, for example, Am. J. Physiol. It can be measured according to the method described on page G157-169, 1998 or a method analogous thereto.

For example, the activity of transporting bile acids, steroid hormones, fat-soluble vitamins, neurotransmitters (eg, dopamine, glutamic acid, enkephalin, etc.) in the case of the above (ii) is about 20 times that of the above (i). A test compound that promotes the activity of the protein of the present invention or a salt thereof can be selected as a test compound that promotes the activity of the protein of the present invention by at least 30%, preferably at least 30%, more preferably at least about 50%.

Also, for example, the transport activity of bile acids, steroid hormones, fat-soluble vitamins, neurotransmitters (eg, dopamine, glutamic acid, enkephalin, etc.) in the case of the above (ii) is compared with the case of the above (i). A test compound that inhibits (or suppresses) about 20% or more, preferably 30% or more, more preferably about 50% or more can be selected as a compound that inhibits the activity of the protein of the present invention or a salt thereof.

In addition, a gene such as secretory alkaline phosphatase or luciferase is inserted downstream of the promoter of the protein gene of the present invention, expressed in the above-described various cells, and activated to activate the enzyme activity when the test compound is brought into contact with the cells. By searching for an inhibitory compound or a salt thereof, a compound or a salt thereof that promotes or suppresses the expression of the protein of the present invention (that is, promotes or inhibits the activity of the protein of the present invention) can be screened.

(4) The polynucleotide encoding the protein of the present invention is useful as a reagent for screening a compound or its salt that promotes or inhibits the expression of the protein gene of the present invention.

The present invention relates to (3) a compound or a salt thereof which promotes or inhibits the expression of the protein gene of the present invention, which is characterized by using a polynucleotide encoding the protein of the present invention (hereinafter referred to as a promoter and an inhibitor, respectively). May be provided), more specifically, for example,
(4) A comparison is made between (iii) culturing cells capable of producing the protein of the present invention and (iv) culturing a mixture of cells capable of producing the protein of the present invention and a test compound. A method for screening for an accelerator or an inhibitor is provided.

In the above-mentioned screening method, for example, in the cases (iii) and (iv), the expression level of the protein gene of the present invention (specifically, the amount of the protein of the present invention or the amount of mRNA encoding the protein) is measured. And compare.

Test compounds include, for example, peptides, proteins, non-peptidic compounds, synthetic compounds, fermentation products, cell extracts, plant extracts, animal tissue extracts, and the like. Or a known compound.

実 施 In order to carry out the above screening method, cells having the ability to produce the protein of the present invention are prepared by suspending them in a buffer suitable for screening. The buffer may be any buffer that does not inhibit the expression of the gene of the protein of the present invention, such as a phosphate buffer or a borate buffer having a pH of about 4 to 10 (preferably, a pH of about 6 to 8).

細胞 As a cell having the ability to produce the protein of the present invention, for example, a host (transformant) transformed with a vector containing the DNA encoding the protein of the present invention described above is used. As the host, for example, animal cells such as CHO cells are preferably used. For the screening, for example, a transformant in which the protein of the present invention is expressed on a cell membrane by culturing by the method described above is preferably used.

The amount of the protein of the present invention can be measured by a known method, for example, using an antibody recognizing the protein of the present invention, and analyzing the protein present in a cell extract or the like by a method such as Western analysis, ELISA, or the like. It can be measured according to the method.

The expression level of the protein gene of the present invention can be determined by a known method, for example, a method such as Northern blotting, reverse transcription-polymerase chain reaction (RT-PCR), a real-time PCR analysis system (manufactured by ABI, TaqMan polymerase chain reaction) or the like. It can be measured according to a corresponding method.

For example, a test that promotes the expression level of the protein gene of the present invention in the case of the above (iv) by about 20% or more, preferably 30% or more, more preferably about 50% or more as compared with the case of the above (iii). The compound can be selected as a compound that promotes the expression of the protein gene of the present invention or a salt thereof.

For example, a test that inhibits the expression level of the protein gene of the present invention in the case of the above (iv) by about 20% or more, preferably 30% or more, more preferably about 50% or more as compared with the case of the above (iii). The compound can be selected as a compound that inhibits the expression of the protein gene of the present invention or a salt thereof.

Furthermore, the antibody of the present invention is useful as a reagent for screening a compound or a salt thereof that promotes or inhibits the expression of the protein of the present invention.

The present invention relates to (5) a compound or a salt thereof which promotes or inhibits the expression of the protein of the present invention, which is characterized by using the antibody of the present invention (hereinafter sometimes abbreviated as a promoter or an inhibitor, respectively). Provide a screening method, more specifically, for example,
(6) A comparison is made between (v) culturing cells capable of producing the protein of the present invention and (vi) culturing a mixture of cells capable of producing the protein of the present invention and a test compound. A method for screening for an accelerator or an inhibitor is provided.

In the above screening method, for example, the expression level of the protein of the present invention (specifically, the amount of the protein of the present invention) in the cases (v) and (vi) is measured using the antibody of the present invention (for example, The expression of the protein of the present invention is detected, the expression level of the protein of the present invention is quantified, and the like, and compared.

Test compounds include, for example, peptides, proteins, non-peptidic compounds, synthetic compounds, fermentation products, cell extracts, plant extracts, animal tissue extracts, and the like. Or a known compound.

実 施 In order to carry out the above screening method, cells having the ability to produce the protein of the present invention are prepared by suspending them in a buffer suitable for screening. The buffer may be any buffer that does not inhibit the expression of the protein of the present invention, such as a phosphate buffer or a borate buffer having a pH of about 4 to 10 (preferably, a pH of about 6 to 8).

細胞 As a cell having the ability to produce the protein of the present invention, for example, a host (transformant) transformed with a vector containing the DNA encoding the protein of the present invention described above is used. As the host, for example, animal cells such as CHO cells are preferably used. For the screening, for example, a transformant in which the protein of the present invention is expressed on a cell membrane by culturing by the method described above is preferably used.

The amount of the protein of the present invention can be measured by a known method, for example, using an antibody recognizing the protein of the present invention, and analyzing the protein present in a cell extract or the like by a method such as Western analysis, ELISA, or the like. It can be measured according to the method.

For example, a test compound which promotes the expression level of the protein of the present invention in the case of the above (vi) by about 20% or more, preferably 30% or more, more preferably about 50% or more as compared with the case of the above (v) Can be selected as a compound or its salt that promotes the expression of the protein of the present invention.

For example, a test compound that inhibits the expression level of the protein of the present invention in the case of the above (vi) by about 20% or more, preferably 30% or more, more preferably about 50% or more as compared with the case of the above (v) Can be selected as a compound that inhibits the expression of the protein of the present invention or a salt thereof.

ス ク リ ー ニ ン グ The screening kit of the present invention contains a protein or a partial peptide of the present invention or a salt thereof, or a cell capable of producing the protein or partial peptide of the present invention.

Compounds or salts thereof obtained by using the screening method or the screening kit of the present invention are test compounds described above, for example, peptides, proteins, non-peptide compounds, synthetic compounds, fermentation products, cell extracts, plant extracts. , A compound selected from animal tissue extract, plasma and the like, or a salt thereof, and the activity of the protein of the present invention (eg, bile acids, steroid hormones, fat-soluble vitamins, neurotransmitters (eg, dopamine, glutamic acid, enkephalin, etc.) Or a salt thereof which promotes or inhibits the transport activity such as

塩 As the salt of the compound, those similar to the aforementioned salts of the protein of the present invention are used.

Compounds or salts thereof which promote the activity of the protein of the present invention include, for example, hyperlipidemia, digestive disorders (eg, irritable bowel syndrome, ulcerative colitis, Crohn's disease, ischemic colitis, gastritis, peptic Ulcers, proctitis, etc.), central nervous system disorders (eg, Alzheimer's disease, Parkinson's syndrome, depression, emotional disorders, schizophrenia, anxiety, etc.), diabetes, pancreatic disorders (eg, pancreatitis, cystic fibrosis) Pancreatic insufficiency, etc.), respiratory diseases (eg, chronic obstructive pulmonary disease (COPD), asthma, etc.), hypothyroidism, cardiovascular diseases (eg, heart failure, arrhythmia, long QT syndrome, arteriosclerosis, etc.) , Cancer (eg, testicular tumor, ovarian cancer, breast cancer, esophageal cancer, lung cancer, kidney cancer, liver cancer, non-small cell lung cancer, prostate cancer, gastric cancer, bladder cancer, cervical cancer, colon cancer, rectal cancer, pancreatic cancer , Thymoma, etc.) As useful. Preferably, it is a prophylactic / therapeutic agent for central nervous system diseases and the like, particularly preferably a prophylactic / therapeutic agent for Parkinson's syndrome and the like.

Further, the compound or a salt thereof that inhibits the activity of the protein of the present invention includes, for example, hyperlipidemia, digestive disorders (eg, irritable bowel syndrome, ulcerative colitis, Crohn's disease, ischemic colitis, gastritis, Peptic ulcer, proctitis, etc.), central nervous system disorders (eg, Alzheimer's disease, Parkinson's syndrome, depression, emotional disorders, schizophrenia, anxiety, etc.), diabetes, pancreatic disorders (eg, pancreatitis, pancreatic cystic) Pancreatic insufficiency such as fibrosis, respiratory disease (eg, chronic obstructive pulmonary disease (COPD), asthma, etc.), hypothyroidism, cardiovascular disease (eg, heart failure, arrhythmia, long QT syndrome, arteriosclerosis) ), Cancer (eg, testicular tumor, ovarian cancer, breast cancer, esophageal cancer, lung cancer, kidney cancer, liver cancer, non-small cell lung cancer, prostate cancer, gastric cancer, bladder cancer, cervical cancer, colon cancer, rectal cancer, Pancreatic cancer, thymoma, etc.) It is useful as a pharmaceutical. Preferably, it is a prophylactic / therapeutic agent for central nervous system diseases and the like, particularly preferably a prophylactic / therapeutic agent for Parkinson's syndrome and the like.

Compounds or salts thereof that promote the expression of the protein gene of the present invention include, for example, hyperlipidemia, digestive disorders (eg, irritable bowel syndrome, ulcerative colitis, Crohn's disease, ischemic colitis, gastritis, digestion, Ulcers, proctitis, etc.), central nervous system disorders (eg, Alzheimer's disease, Parkinson's syndrome, depression, emotional disorders, schizophrenia, anxiety, etc.), diabetes, pancreatic disorders (eg, pancreatitis, cystic fibrosis) Pancreatic insufficiency such as dysfunction), respiratory disease (eg, chronic obstructive pulmonary disease (COPD), asthma, etc.), hypothyroidism, circulatory disease (eg, heart failure, arrhythmia, long QT syndrome, arteriosclerosis, etc.) ), Cancer (eg, testicular tumor, ovarian cancer, breast cancer, esophageal cancer, lung cancer, kidney cancer, liver cancer, non-small cell lung cancer, prostate cancer, gastric cancer, bladder cancer, cervical cancer, colon cancer, rectal cancer, pancreas) Cancer, thymoma, etc.) It is useful as a pharmaceutical. Preferably, it is a prophylactic / therapeutic agent for central nervous system diseases and the like, particularly preferably a prophylactic / therapeutic agent for Parkinson's syndrome and the like.

Further, the compound or a salt thereof that inhibits the expression of the protein gene of the present invention includes, for example, hyperlipidemia, gastrointestinal diseases (eg, irritable bowel syndrome, ulcerative colitis, Crohn's disease, ischemic colitis, gastritis) , Peptic ulcer, proctitis, etc.), central nervous system diseases (eg, Alzheimer's disease, Parkinson's syndrome, depression, emotional disorders, schizophrenia, anxiety neurosis, etc.), diabetes, pancreatic diseases (eg, pancreatitis, pancreatic cyst) Pancreatic insufficiency such as cystic fibrosis, respiratory disease (eg, chronic obstructive pulmonary disease (COPD), asthma, etc.), hypothyroidism, cardiovascular disease (eg, heart failure, arrhythmia, long QT syndrome, artery Sclerosis, etc.), cancer (eg, testicular tumor, ovarian cancer, breast cancer, esophageal cancer, lung cancer, kidney cancer, liver cancer, non-small cell lung cancer, prostate cancer, gastric cancer, bladder cancer, cervical cancer, colon cancer, rectal cancer) , Pancreatic cancer, thymoma, etc.) Agents are useful as pharmaceuticals such as. Preferably, it is a prophylactic / therapeutic agent for central nervous system diseases and the like, particularly preferably a prophylactic / therapeutic agent for Parkinson's syndrome and the like.

Compounds or salts thereof that promote the expression of the protein of the present invention include, for example, hyperlipidemia, digestive disorders (eg, irritable bowel syndrome, ulcerative colitis, Crohn's disease, ischemic colitis, gastritis, peptic Ulcers, proctitis, etc.), central nervous system disorders (eg, Alzheimer's disease, Parkinson's syndrome, depression, emotional disorders, schizophrenia, anxiety, etc.), diabetes, pancreatic disorders (eg, pancreatitis, cystic fibrosis) Pancreatic insufficiency, etc.), respiratory diseases (eg, chronic obstructive pulmonary disease (COPD), asthma, etc.), hypothyroidism, cardiovascular diseases (eg, heart failure, arrhythmia, long QT syndrome, arteriosclerosis, etc.) , Cancer (eg, testicular tumor, ovarian cancer, breast cancer, esophageal cancer, lung cancer, kidney cancer, liver cancer, non-small cell lung cancer, prostate cancer, gastric cancer, bladder cancer, cervical cancer, colon cancer, rectal cancer, pancreatic cancer , Thymoma, etc.) As useful. Preferably, it is a prophylactic / therapeutic agent for central nervous system diseases and the like, particularly preferably a prophylactic / therapeutic agent for Parkinson's syndrome and the like.

Further, the compound or a salt thereof that inhibits the expression of the protein of the present invention includes, for example, hyperlipidemia, digestive diseases (eg, irritable bowel syndrome, ulcerative colitis, Crohn's disease, ischemic colitis, gastritis, Peptic ulcer, proctitis, etc.), central nervous system disorders (eg, Alzheimer's disease, Parkinson's syndrome, depression, emotional disorders, schizophrenia, anxiety, etc.), diabetes, pancreatic disorders (eg, pancreatitis, pancreatic cystic) Pancreatic insufficiency such as fibrosis, respiratory disease (eg, chronic obstructive pulmonary disease (COPD), asthma, etc.), hypothyroidism, cardiovascular disease (eg, heart failure, arrhythmia, long QT syndrome, arteriosclerosis) ), Cancer (eg, testicular tumor, ovarian cancer, breast cancer, esophageal cancer, lung cancer, kidney cancer, liver cancer, non-small cell lung cancer, prostate cancer, gastric cancer, bladder cancer, cervical cancer, colon cancer, rectal cancer, Pancreatic cancer, thymoma, etc.) It is useful as a pharmaceutical. Preferably, it is a prophylactic / therapeutic agent for central nervous system diseases and the like, particularly preferably a prophylactic / therapeutic agent for Parkinson's syndrome and the like.

化合物 When the compound or its salt obtained by using the screening method or the screening kit of the present invention is used as the above-mentioned prophylactic / therapeutic agent, it can be formulated according to a conventional method. For example, tablets, capsules, elixirs, microcapsules, sterile solutions, suspensions and the like can be used.

The preparations obtained in this way are safe and low toxic, for example, human or warm-blooded animals (eg, mouse, rat, rabbit, sheep, pig, cow, horse, bird, cat, dog, monkey, chimpanzee, etc.) ) Can be administered orally or parenterally.

The dose of the compound or a salt thereof varies depending on its action, target disease, administration subject, administration route, and the like.For example, a compound or a salt thereof that promotes the activity of the protein of the present invention for the purpose of treating Parkinson's syndrome is used. In the case of oral administration, generally, in an adult (with a body weight of 60 kg), the compound or a salt thereof is used in an amount of about 0.1 to 100 mg, preferably about 1.0 to 50 mg, more preferably about 1.0 to 50 mg per day. Administer 20 mg. When administered parenterally, the single dose of the compound or a salt thereof varies depending on the administration subject, target disease, and the like. For example, a compound or a compound that promotes the activity of the protein of the present invention for the purpose of treating Parkinson's syndrome or When the salt is administered to an adult (with a body weight of 60 kg) usually in the form of an injection, the compound or a salt thereof is used in an amount of about 0.01 to 30 mg, preferably about 0.1 to 20 mg, more preferably about 0.1 to 20 mg per day. Advantageously, 1 to 10 mg is administered by intravenous injection. In the case of other animals, the amount can be administered in terms of the weight per 60 kg.
[3] Quantification of the protein of the present invention, its partial peptide or its salt Since the antibody of the present invention can specifically recognize the protein of the present invention, the quantification of the protein of the present invention in a test solution, particularly It can be used for quantification by sandwich immunoassay and the like.

That is, the present invention
(I) reacting the antibody of the present invention with a test solution and a labeled protein of the present invention competitively, and measuring the ratio of the labeled protein of the present invention bound to the antibody; And (ii) simultaneously or sequentially reacting the test solution with the antibody of the present invention insolubilized on a carrier and another antibody of the present invention labeled on a carrier. The present invention provides a method for quantifying the protein of the present invention in a test solution, which comprises measuring the activity of a labeling agent on an insolubilized carrier after the reaction.

In the quantification method (ii), it is desirable that one antibody is an antibody that recognizes the N-terminal of the protein of the present invention and the other antibody is an antibody that reacts with the C-terminal of the protein of the present invention.

In addition, 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. For these purposes, the antibody molecule itself may be used, or 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 the antibody, antigen, or antibody-antigen complex corresponding to the amount of the antigen (eg, the amount of the protein) in the test solution is measured. Any measurement method may be used as long as the amount is detected by chemical or physical means, and the amount is calculated from a standard curve prepared using a standard solution containing a known amount of antigen. For example, nephelometry, competition method, immunometric method and sandwich method are suitably used, but it is particularly preferable to use the sandwich method described later in terms of sensitivity and specificity.

As a labeling agent used in a measuring method using a labeling substance, for example, a radioisotope, an enzyme, a fluorescent substance, a luminescent substance and the like are used. As the radioisotope, for example, [ ¹²⁵ I], [ ¹³¹ I], [ ³ H], [ ¹⁴ C] and the like are used. As the above-mentioned enzyme, a stable enzyme having a large specific activity is preferable. For example, β-galactosidase, β-glucosidase, alkaline phosphatase, peroxidase, malate dehydrogenase and the like are used. As the fluorescent substance, for example, fluorescamine, fluorescein isothiocyanate and the like are used. As the luminescent substance, for example, luminol, a luminol derivative, luciferin, lucigenin and the like are used. Further, a biotin-avidin system can be used for binding the antibody or antigen to the labeling agent.

(4) For the insolubilization of the antigen or antibody, physical adsorption may be used, or a method using a chemical bond usually used for insolubilizing and immobilizing proteins or enzymes may be used. Examples of the carrier include insoluble polysaccharides such as agarose, dextran, and cellulose; synthetic resins such as polystyrene, polyacrylamide, and silicon; and glass.

In the sandwich method, the test solution is reacted with the insolubilized monoclonal antibody of the present invention (primary reaction), and further reacted with another labeled monoclonal antibody of the present invention (secondary reaction). By measuring the activity of the labeling agent, the amount of the protein of the present invention in the test solution can be determined. 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. In addition, in the immunoassay by the sandwich method, the antibody used for the solid phase antibody or the labeling antibody is not necessarily one kind, and a mixture of two or more kinds of antibodies is used for the purpose of improving measurement sensitivity and the like. You may.

は In the method for measuring the protein of the present invention by the sandwich method of the present invention, as the monoclonal antibody of the present invention used in the primary reaction and the secondary reaction, an antibody having a different site to which the protein of the present invention binds is preferably used. That is, the antibody used in the primary reaction and the secondary reaction is preferably, 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, for example, the N-terminal other than the C-terminal is used.

モ ノ ク ロ ー ナ ル The monoclonal antibody of the present invention can be used in a measurement system other than the sandwich method, for example, a competition method, an immunometric method, or a nephrometry.

In the competition method, after the antigen in the test solution and the labeled antigen are allowed to react competitively with the antibody, the unreacted labeled antigen (F) and the labeled antigen (B) bound to the antibody are separated. (B / F separation), the amount of any of B and F is measured, and the amount of antigen in the test solution is quantified. In this reaction method, a soluble antibody is used as the antibody, B / F separation is performed using polyethylene glycol, a liquid phase method using a second antibody against the antibody, or a solid phase antibody is used as the first antibody. A solid-phase method using a soluble antibody as the first antibody and using a solid-phased antibody as the second antibody is used.
In the immunometric method, the antigen in the test solution and the immobilized antigen are subjected to a competitive reaction with a fixed amount of the labeled antibody, and then the solid phase and the liquid phase are separated. And an excess amount of the labeled antibody, and then immobilized antigen is added to bind unreacted labeled antibody to the solid phase, and then the solid phase and the liquid phase are separated. Next, the amount of label in any phase is measured to determine the amount of antigen in the test solution.

ネ In nephelometry, the amount of insoluble sediment resulting from the antigen-antibody reaction in a gel or in a solution is measured. Even when the amount of antigen in the test solution is small and only a small amount of sediment is obtained, laser nephrometry utilizing laser scattering is preferably used.

適用 In applying each of these immunoassays to the quantification method of the present invention, no special conditions, operations and the like need to be set. The protein measurement 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, books, and the like.

For example, "Radioimmunoassay" edited by Hiroshi Irie (Kodansha, published in 1974), "Radioimmunoassay" continued (edited by Kodansha, published in 1979), and "Enzyme immunoassay" edited by Eiji Ishikawa (Medical Shoin, Showa) Ed. Ishikawa et al., “Enzyme Immunoassay” (2nd edition) (Issue Shoin, 1982), Eiji Ishikawa et al. “Enzyme Immunoassay” (Third Edition), 3rd edition (Medical Shoin, Showa) Vol. 70 (Immunochemical Techniques (Part A)), Vol. 73 (Immunochemical Techniques (Part B)), Vol. 74 (Immunochemical Techniques (Part C)), Vol. 74 (Immunochemical Techniques (Part C)) 84 (Immunochemical Techniques (Part D: Selected Immunoassays)), ibid.Vol. 92 (Immunochemical Techniques (Part E: Monoclonal Antibodies and General Immunoassay Methods)), ibid.Vol. 121 (Immunochemical Techniques (Part I: Hybridoma Technology and Monoclonal Antibodies). Academic Press Company)).

As described above, the protein of the present invention can be quantified with high sensitivity by using the antibody of the present invention.

Furthermore, when a decrease in the concentration of the protein of the present invention is detected by quantifying the concentration of the protein of the present invention using the antibody of the present invention, for example, hyperlipidemia, gastrointestinal diseases (eg, hypersensitivity) Inflammatory bowel syndrome, ulcerative colitis, Crohn's disease, ischemic colitis, gastritis, peptic ulcer, proctitis, etc., central nervous system disorders (eg, Alzheimer's disease, Parkinson's syndrome, depression, emotional disorders, schizophrenia) ), Diabetes, pancreatic disease (eg, pancreatitis, pancreatic insufficiency such as cystic fibrosis), respiratory disease (eg, chronic obstructive pulmonary disease (COPD), asthma, etc.), thyroid gland Functional decline, cardiovascular disease (eg, heart failure, arrhythmia, long QT syndrome, arteriosclerosis, etc.), cancer (eg, testicular tumor, ovarian cancer, breast cancer, esophageal cancer, lung cancer, kidney cancer, liver cancer, non-small cell lung cancer, Prostate cancer, stomach cancer, bladder cancer, uterus Part cancer, colon cancer, rectal cancer, pancreatic cancer, thymoma, etc.) can be diagnosed as likely to be developed. Conversely, for example, when an increase in the concentration of the protein of the present invention is detected, for example, hyperlipidemia, gastrointestinal diseases (eg, irritable bowel syndrome, ulcerative colitis, Crohn's disease, ischemic colitis, Gastritis, peptic ulcer, proctitis, etc.), central nervous system disorders (eg, Alzheimer's disease, Parkinson's syndrome, depression, emotional disorders, schizophrenia, anxiety, etc.), diabetes, pancreatic disorders (eg, pancreatitis, pancreas) Pancreatic insufficiency such as cystic fibrosis, respiratory disease (eg, chronic obstructive pulmonary disease (COPD), asthma, etc.), hypothyroidism, cardiovascular disease (eg, heart failure, arrhythmia, long QT syndrome, Arteriosclerosis, etc.), cancer (eg, testicular tumor, ovarian cancer, breast cancer, esophageal cancer, lung cancer, kidney cancer, liver cancer, non-small cell lung cancer, prostate cancer, gastric cancer, bladder cancer, cervical cancer, colon cancer, rectum Cancer, pancreatic cancer, thymoma, etc.) It can be diagnosed with a high sex.

Further, 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. Further, for preparation of an antibody column used for purifying the protein of the present invention, detection of the protein of the present invention in each fraction at the time of purification, analysis of the behavior of the protein of the present invention in test cells, etc. Can be used.
[4] Gene diagnostic agent The DNA of the present invention can be used, for example, as a probe to produce a human or warm-blooded animal (eg, rat, mouse, guinea pig, rabbit, bird, sheep, pig, cow, horse, cat, dog) , Monkeys, chimpanzees, etc.) can detect abnormalities (genetic abnormalities) in DNA or mRNA encoding the protein of the present invention or partial peptides thereof, for example, damage, mutation or reduced expression of the DNA or mRNA, It is useful as an agent for diagnosing a gene such as an increase or excessive expression of the DNA or mRNA.

The above-described genetic diagnosis using the DNA of the present invention can be performed, for example, by the known Northern hybridization or PCR-SSCP method (Genomics, Vol. 5, pp. 874-879 (1989), Processings of the -Proceedings of the National Academy of Sciences of the United States of America, Vol. 86, pp. 2766-2770 (1989)).

For example, when increased expression is detected by Northern hybridization, for example, hyperlipidemia, gastrointestinal diseases (eg, irritable bowel syndrome, ulcerative colitis, Crohn's disease, ischemic colitis, gastritis, peptic ulcer, Proctitis), central nervous system disorders (eg, Alzheimer's disease, Parkinson's syndrome, depression, emotional disorders, schizophrenia, anxiety, etc.), diabetes, pancreatic disorders (eg, pancreatitis, cystic fibrosis, etc.) Pancreatic insufficiency, etc.), respiratory diseases (eg, chronic obstructive pulmonary disease (COPD), asthma, etc.), hypothyroidism, cardiovascular diseases (eg, heart failure, arrhythmia, long QT syndrome, arteriosclerosis, etc.), cancer (Eg, testicular, ovarian, breast, esophageal, lung, kidney, liver, non-small cell lung, prostate, gastric, bladder, cervical, colon, rectal, pancreatic, breast Adenoma) It can be diagnosed as high. Conversely, when a decrease in the expression is detected or a mutation in the DNA is detected by the PCR-SSCP method, for example, hyperlipidemia, gastrointestinal diseases (eg, irritable bowel syndrome, ulcerative colitis, Crohn's disease, ischemic colitis, gastritis, peptic ulcer, proctitis, etc.), central nervous system disorders (eg, Alzheimer's disease, Parkinson's syndrome, depression, emotional disorders, schizophrenia, anxiety, etc.), diabetes, Pancreatic disease (eg, pancreatitis, pancreatic insufficiency such as cystic fibrosis), respiratory disease (eg, chronic obstructive pulmonary disease (COPD), asthma, etc.), hypothyroidism, cardiovascular disease (eg, Heart failure, arrhythmia, long QT syndrome, arteriosclerosis, etc.), cancer (eg, testicular tumor, ovarian cancer, breast cancer, esophageal cancer, lung cancer, kidney cancer, liver cancer, non-small cell lung cancer, prostate cancer, gastric cancer, bladder cancer, uterus) Cervical, colon, rectal, pancreatic, breast It can be diagnosed that it is highly likely that in such tumors, etc.).
[5] Pharmaceuticals Containing Antisense Polynucleotide The antisense polynucleotide of the present invention, which can complementarily bind to the DNA of the present invention and suppress the expression of the DNA, has low toxicity and exhibits the present invention in vivo. Activity or function (eg, bile acids, steroid hormones, fat-soluble vitamins, transport activities of neurotransmitters (eg, dopamine, glutamic acid, enkephalin, etc.), etc.) can be suppressed, For example, hyperlipidemia, gastrointestinal diseases (eg, irritable bowel syndrome, ulcerative colitis, Crohn's disease, ischemic colitis, gastritis, peptic ulcer, proctitis, etc.), central nervous system diseases (eg, Alzheimer's disease) Disease, Parkinson's syndrome, depression, emotional disorders, schizophrenia, anxiety, etc.), diabetes, pancreatic disease (eg, pancreatitis, cystic fibrosis, etc.) Pancreatic insufficiency, etc.), respiratory diseases (eg, chronic obstructive pulmonary disease (COPD), asthma, etc.), hypothyroidism, cardiovascular diseases (eg, heart failure, arrhythmia, long QT syndrome, arteriosclerosis, etc.), cancer (Eg, testicular, ovarian, breast, esophageal, lung, kidney, liver, non-small cell lung, prostate, gastric, bladder, cervical, colon, rectal, pancreatic, breast Adenoma, etc.). Preferably, it is a prophylactic / therapeutic agent for central nervous system diseases and the like, particularly preferably a prophylactic / therapeutic agent for Parkinson's syndrome and the like.

When the antisense polynucleotide is used as the prophylactic / therapeutic agent, it can be formulated and administered according to a known method.

For example, when the antisense polynucleotide is 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, or the like, and then inserted into a human or mammal according to a conventional method. It can be administered orally or parenterally to animals (eg, rats, rabbits, sheep, pigs, cows, 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 using a gene gun or a catheter such as a hydrogel catheter.

The dose 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 administered to an affected part for the purpose of treating Parkinson's syndrome, a general method is used. In adults (weight 60 kg), about 0.1 to 100 mg of the antisense polynucleotide is administered per day.

Furthermore, the antisense polynucleotide can also be used as a diagnostic oligonucleotide probe for examining the presence of the DNA of the present invention in tissues or cells and its expression status.

Further, the present invention provides
(I) a double-stranded RNA containing a part of RNA encoding the protein of the present invention and RNA complementary thereto,
(Ii) a medicine comprising the double-stranded RNA,
(Iii) a ribozyme containing a part of RNA encoding the protein of the present invention,
(Iv) a medicament comprising the ribozyme,
(V) An expression vector or the like containing a gene (DNA) encoding the ribozyme is also provided.

Like the above-mentioned antisense polynucleotide, double-stranded RNA, ribozyme and the like can also destroy RNA transcribed from the DNA of the present invention or suppress the function thereof, and can be used in vivo for the protein of the present invention or the ribozyme. Since the function of the DNA used can be suppressed, for example, hyperlipidemia, digestive disorders (eg, irritable bowel syndrome, ulcerative colitis, Crohn's disease, ischemic colitis, gastritis, peptic ulcer, Proctitis), central nervous system disorders (eg, Alzheimer's disease, Parkinson's syndrome, depression, emotional disorders, schizophrenia, anxiety, etc.), diabetes, pancreatic disorders (eg, pancreatitis, cystic fibrosis, etc.) Pancreatic insufficiency, etc., respiratory diseases (eg, chronic obstructive pulmonary disease (COPD), asthma, etc.), hypothyroidism, cardiovascular diseases (eg, heart failure, arrhythmia, long QT syndrome, Pulse sclerosis), cancer (eg, testicular tumor, ovarian cancer, breast cancer, esophageal cancer, lung cancer, kidney cancer, liver cancer, non-small cell lung cancer, prostate cancer, gastric cancer, bladder cancer, cervical cancer, colon cancer, rectum Cancer, pancreatic cancer, thymoma, etc.). Preferably, it is a prophylactic / therapeutic agent for central nervous system diseases and the like, particularly preferably a prophylactic / therapeutic agent for Parkinson's syndrome and the like.

A 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).

A ribozyme can be designed and produced based on the sequence of the polynucleotide of the present invention according to a known method (eg, TRENDS in Molecular Medicine, Vol. 7, pp. 221, 2001). For example, it can be produced by substituting a part of a known ribozyme sequence with a part of RNA encoding the protein of the present invention. As a part of the RNA encoding the protein of the present invention, a sequence in the vicinity of a consensus sequence NUX (where N represents all bases and X represents a base other than G) which can be cleaved by a known ribozyme, and the like are included. No.

When the above-mentioned double-stranded RNA or ribozyme is used as the above-mentioned prophylactic / therapeutic agent, it can be formulated and administered in the same manner as an antisense polynucleotide. The expression vector (v) is used in the same manner as known gene therapy methods and the like, and is used as the above-mentioned prophylactic / therapeutic agent.
[6] Production of Animal Having DNA of the Present Invention The present invention relates to a DNA encoding the foreign protein of the present invention (hereinafter abbreviated as the foreign DNA of the present invention) or a mutant DNA thereof (the foreign DNA of the present invention). (May be abbreviated as mutant DNA).

That is, the present invention
(1) a non-human mammal having the exogenous DNA of the present invention or a mutant DNA thereof,
(2) The animal according to (1) above, wherein the non-human mammal is a rodent.
(3) The animal according to the above (2), wherein the rodent is a mouse or a rat, and (4) a recombinant vector containing the exogenous DNA of the present invention or a mutant DNA thereof and capable of being expressed in a mammal. I do.

Non-human mammals having the exogenous DNA of the present invention or the mutant DNA thereof (hereinafter abbreviated as the DNA-transferred animal of the present invention) can be used for non-fertilized eggs, fertilized eggs, germ cells including spermatozoa and their progenitor cells, And preferably at the stage of embryonic development in non-human mammal development (more preferably at the stage of single cells or fertilized eggs and generally before the 8-cell stage), the calcium phosphate method, the electric pulse method, the lipofection method, the agglutination method, It can be produced by introducing a target DNA by a microinjection method, a particle gun method, a DEAE-dextran method, or the like. Further, by the DNA introduction method, the exogenous DNA of the present invention can be introduced into somatic cells, organs of living organisms, tissue cells, and the like, and used for cell culture, tissue culture, and the like. The DNA-transfected animal of the present invention can also be produced by fusing the above-mentioned germ cells with a known cell fusion method.

As the non-human mammal, for example, cow, pig, sheep, goat, rabbit, dog, cat, guinea pig, hamster, mouse, rat and the like are used. Among them, rodents, which are relatively short in ontogeny and biological cycle in terms of preparation of disease animal model systems, and are easy to breed, especially mice (for example, hybrids such as C57BL / 6 strain and DBA2 strain as pure strains) As a system, _one B6C3F system, _one BDF system, _one B6D2F system, a BALB / c system, an ICR system, or a rat (for example, Wistar, SD, etc.) is preferable.

「As the“ mammal ”in the recombinant vector that can be expressed in mammals, humans and the like can be mentioned in addition to the above-mentioned non-human mammals.

外 The exogenous DNA of the present invention refers to the DNA of the present invention once isolated and extracted from a mammal, not the DNA of the present invention originally possessed by a non-human mammal.

As the mutant DNA of the present invention, those having a mutation (for example, mutation) in the base sequence of the original DNA of the present invention, specifically, base addition, deletion, substitution with another base, etc. The resulting DNA or the like is used, and also includes abnormal DNA.

異常 The abnormal DNA means a DNA that expresses an abnormal protein of the present invention, and for example, a DNA that expresses a protein that suppresses the function of a normal protein of the present invention is used.

(4) The exogenous DNA of the present invention may be derived from mammals of the same species or different species from the target animal. When introducing the DNA of the present invention into a target animal, it is generally advantageous to use the DNA as a DNA construct linked downstream of a promoter capable of being expressed in animal cells. For example, when the human DNA of the present invention is introduced, DNAs derived from various mammals (eg, rabbits, dogs, cats, guinea pigs, hamsters, rats, mice, etc.) having the DNA of the present invention having high homology thereto are expressed. Highly expressing the DNA of the present invention by microinjecting a DNA construct (eg, vector, etc.) in which the human DNA of the present invention is bound downstream of various promoters that can be made into a fertilized egg of a target mammal, for example, a mouse fertilized egg. DNA-introduced mammals can be created.

Examples of the expression vector of the protein of the present invention include plasmids derived from Escherichia coli, plasmids derived from Bacillus subtilis, plasmids derived from yeast, bacteriophages such as λ phage, retroviruses such as Moloney leukemia virus, animal viruses such as vaccinia virus or baculovirus. Are used. Among them, a plasmid derived from Escherichia coli, a plasmid derived from Bacillus subtilis or a plasmid derived from yeast is preferably used.

Examples of the promoter for controlling the DNA expression include, for example, (i) a promoter of a DNA derived from a virus (eg, simian virus, cytomegalovirus, Moloney leukemia virus, JC virus, breast cancer virus, poliovirus, etc.); ) Promoters derived from various mammals (human, rabbit, dog, cat, guinea pig, hamster, rat, mouse, etc.), for example, albumin, insulin II, uroplakin II, elastase, erythropoietin, endothelin, muscle creatine kinase, glial fibrillary acid Protein, glutathione S-transferase, platelet-derived growth factor β, keratins K1, K10 and K14, collagen type I and type II, cyclic AMP-dependent protein kinase βI subunit, dystrophy Fin, tartrate-resistant alkaline phosphatase, atrial natriuretic factor, endothelial receptor tyrosine kinase (commonly abbreviated as Tie2), sodium potassium adenosine 3 kinase (Na, K-ATPase), neurofilament light chain, metallothionein I and IIA, metalloproteinase 1 tissue inhibitor, MHC class I antigen (H-2L), H-ras, renin, dopamine β-hydroxylase, thyroid peroxidase (TPO), polypeptide chain elongation factor 1α (EF-1α), β Actin, α and β myosin heavy chain, myosin light chain 1 and 2, myelin basic protein, thyroglobulin, Thy-1, immunoglobulin, heavy chain variable region (VNP), serum amyloid P component, myoglobin, troponin C, smooth muscle Actin, pre-pro-enkephalin A, such as a promoter, such as vasopressin is used. Among them, a cytomegalovirus promoter capable of high expression throughout the body, a promoter of human polypeptide chain elongation factor 1α (EF-1α), human and chicken β-actin promoters, and the like are preferable.

The above vector preferably has a sequence that terminates transcription of the target mRNA in a DNA-transfected mammal (generally called a terminator). For example, a sequence of each DNA derived from a virus and from various mammals is used. Preferably, a simian virus SV40 terminator or the like is used.

In addition, a splicing signal of each DNA, an enhancer region, a part of an intron of a eukaryotic DNA, and the like are placed 5 ′ upstream of the promoter region, between the promoter region and the translation region, or between the translation region for the purpose of further expressing the desired foreign DNA. It is also possible depending on the purpose to connect to the 3 ′ downstream の.

The normal translation region of the protein of the present invention can be obtained from liver, kidney, thyroid cells, fibroblast-derived DNA derived from humans or various mammals (eg, rabbits, dogs, cats, guinea pigs, hamsters, rats, mice, etc.) and commercially available DNAs. From a variety of genomic DNA libraries, or as a starting material, a complementary DNA prepared by known methods from RNA derived from liver, kidney, thyroid cells, and fibroblasts. In addition, a translation region obtained by mutating a translation region of a normal polypeptide obtained from the above-mentioned cells or tissues by a point mutagenesis method can be used for the exogenous abnormal DNA.

翻 訳 The translation region can be prepared as a DNA construct that can be expressed in an introduced animal by a conventional DNA engineering technique in which it is ligated to the downstream of the aforementioned promoter and, if desired, to the upstream of the transcription termination site.

導入 Introduction of the exogenous DNA of the present invention at the fertilized egg cell stage is ensured to be present in all germ cells and somatic cells of the target mammal. The presence of the exogenous DNA of the present invention in the germinal cells of the produced animal after the introduction of the DNA means that all progeny of the produced animal will retain the exogenous DNA of the present invention in all of the germ cells and somatic cells. means. The offspring of such animals that have inherited the exogenous DNA of the present invention have the exogenous DNA of the present invention in all of their germ cells and somatic cells.

非 The 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 maintained by mating.

導入 The introduction of the exogenous DNA of the present invention at the fertilized egg cell stage is ensured to be present in excess in all germ cells and somatic cells of the target mammal. Excessive presence of the exogenous DNA of the present invention in the germinal cells of the produced animal after DNA introduction means that all the offspring of the produced animal have the exogenous DNA of the present invention in all of their germinal and somatic cells. Means Progeny of such animals that have inherited the exogenous DNA of the present invention have an excess of the exogenous DNA of the present invention in all of their germinal and somatic cells.

(4) A homozygous animal having the introduced DNA on both homologous chromosomes is obtained, and by crossing these male and female animals, all the offspring can be bred to have the DNA in excess.

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 promotes the function of the endogenous normal DNA to eventually cause hyperactivity of the protein of the present invention. Occasionally, it can be used as a disease model animal. For example, using the normal DNA-transfected animal of the present invention, it is possible to elucidate the pathological mechanism of the hyperactivity of the protein of the present invention and diseases associated with the protein of the present invention, and to examine a method for treating these diseases. It is possible.

Further, since the mammal into which the exogenous normal DNA of the present invention has been introduced has an increased symptom of the released protein of the present invention, it can be used for a screening test for a therapeutic drug for a disease associated with the protein of the present invention. .

On the other hand, 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. Furthermore, the desired foreign DNA can be used as a raw material by incorporating it into the aforementioned plasmid. The DNA construct with the promoter can be prepared by a usual DNA engineering technique. 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 produced animal after DNA transfer means that all the offspring of the produced animal have the abnormal DNA of the present invention in all of its germ cells and somatic cells. The progeny of such animals that have inherited the exogenous DNA of the present invention have the abnormal DNA of the present invention in all of their germinal 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 breed the cells so that all offspring have the DNA.

The non-human mammal having the abnormal DNA of the present invention, in which the abnormal DNA of the present invention is highly expressed, inhibits the function of endogenous normal DNA, and finally, the functionally inactive form of the protein of the present invention. It can be refractory and can be used as a model animal for the disease. For example, by 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.

Also, as a specific possibility, the abnormal DNA-highly expressing animal of the present invention can inhibit the abnormal protein of the present invention (dominant negative action) by the abnormal protein of the present invention in the inactive refractory disease of the protein of the present invention. A model to elucidate.

In addition, since 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 for a therapeutic drug screening test for the protein of the present invention or a functionally inactive refractory disease thereof. It is.

In addition, other possible uses of the two kinds of the DNA-introduced animals of the present invention include, for example,
(I) use as a cell source for tissue culture,
(Ii) a protein specifically expressed or activated by the protein of the present invention by directly analyzing DNA or RNA in the tissue of the DNA-transfected animal of the present invention, or analyzing a polypeptide tissue expressed by the DNA; Analysis of the relationship with
(Iii) culturing cells of DNA-bearing tissue by standard tissue culture techniques and using them to study the function of cells from tissues that are generally difficult to culture;
(Iv) Screening of a drug that enhances the function of the cell by using the cell described in (iii) above, and (v) Isolation and purification of the mutant protein of the present invention and production of an antibody thereof are conceivable.

Furthermore, using the DNA-introduced animal of the present invention, it is possible to examine the clinical symptoms of diseases related to the protein of the present invention, including the inactive refractory disease of the protein of the present invention, etc. More detailed pathological findings in each organ of the disease model related to the disease can be obtained, which can contribute to the development of a new treatment method, and further to the research and treatment of a secondary disease caused by the disease.

In addition, it is possible to take out each organ from the DNA-introduced animal of the present invention, cut it into small pieces, and then use a proteolytic enzyme such as trypsin to obtain the released DNA-introduced cells, culture them, or systematize the cultured cells. . Furthermore, it is possible to examine the specificity of the protein-producing cell of the present invention, its relationship with apoptosis, differentiation or proliferation, or its signal transduction mechanism, and its abnormalities. It is an effective research material for

Further, in order to develop 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, It is possible to provide an effective and rapid screening method for a therapeutic agent for the disease by using a method or the like. In addition, using the DNA-introduced animal of the present invention or the exogenous DNA expression vector of the present invention, it is possible to examine and develop a DNA treatment method for a disease associated with the protein of the present invention.
[7] Knockout Animal The present invention provides a non-human mammal embryonic stem cell in which the DNA of the present invention is inactivated and a non-human mammal deficient in expression of the DNA of the present invention.

That is, the present invention
(1) a non-human mammalian embryonic stem cell in which the DNA of the present invention has been inactivated,
(2) The embryonic stem cell according to (1), wherein the DNA is inactivated by introducing a reporter gene (eg, a β-galactosidase gene derived from Escherichia coli).
(3) The embryonic stem cell according to (1), which is neomycin-resistant,
(4) the embryonic stem cell according to (1), wherein the non-human mammal is a rodent;
(5) The embryonic stem cell according to (4), wherein the rodent is a mouse,
(6) a non-human mammal deficient in expression of the DNA of the present invention, wherein the DNA is inactivated;
(7) The above (6), wherein the DNA is inactivated by introducing a reporter gene (eg, a β-galactosidase gene derived from Escherichia coli), and the reporter gene can be expressed under the control of a promoter for the DNA of the present invention. The non-human mammal of the description,
(8) the non-human mammal according to the above (6), wherein the non-human mammal is a rodent;
(9) A test compound is administered to the non-human mammal according to (8), wherein the rodent is a mouse, and (10) the animal according to (7), and the expression of the reporter gene is detected. And a method for screening a compound or a salt thereof that promotes or inhibits the promoter activity of the DNA of the present invention.

A non-human mammalian embryonic stem cell in which the DNA of the present invention has been inactivated is an artificially mutated DNA of the present invention possessed by the non-human mammal, which suppresses the expression ability of the DNA, or By substantially losing the activity of the protein of the present invention encoded by the DNA, the DNA does not substantially have the ability to express the protein of the present invention (hereinafter, may be referred to as the knockout DNA of the present invention). ) Non-human mammalian embryonic stem cells (hereinafter abbreviated as ES cells).

As the non-human mammal, the same one as described above is used.

方法 The method of artificially mutating the DNA of the present invention can be performed, for example, by deleting part or all of the DNA sequence and inserting or substituting another DNA by genetic engineering techniques. With these mutations, the knockout DNA of the present invention may be prepared, for example, by shifting the codon reading frame or disrupting the function of the promoter or exon.

Specific examples of the non-human mammalian embryonic stem cells in which the DNA of the present invention has been inactivated (hereinafter abbreviated as the DNA-inactivated ES cells of the present invention or the knockout ES cells of the present invention) include, for example, The DNA of the present invention possessed by a non-human mammal is isolated and its exon portion is a drug resistance gene typified by a neomycin resistance gene or a hygromycin resistance gene, or lacZ (β-galactosidase gene), cat (chloramphenicol acetyl). A reporter gene or the like typified by a transferase gene) to disrupt the exon function, or insert a DNA sequence that terminates gene transcription into an intron between exons (for example, a polyA addition signal); By making it impossible to synthesize complete mRNA As a result, a DNA strand having a DNA sequence constructed so as to disrupt the gene (hereinafter abbreviated as a targeting vector) is introduced into the chromosome of the animal by, for example, homologous recombination, and the resulting ES cells are used in the present invention. Analysis by Southern hybridization analysis using a DNA sequence on or in the vicinity of the DNA as a probe, or PCR using a DNA sequence on a targeting vector and a DNA sequence of a neighboring region other than the DNA of the present invention used for the preparation of the targeting vector as primers However, it can be obtained by selecting the knockout ES cells of the present invention.

As the ES cells from which the DNA of the present invention is inactivated by the homologous recombination method or the like, for example, those already established as described above may be used, or according to the known Evans and Kaufman method. May be newly established. For example, in the case of mouse ES cells, currently, 129-line ES cells are generally used. for example the purpose of acquiring the obvious ES cells, the BDF ₁ mice (C57BL / 6 and DBA / 2 for the egg collection number of lack of C57BL / 6 mice and C57BL / 6 were improved by crossing with DBA / 2 Those established using F ₁ ) can also be used favorably. BDF ₁ mice are often egg number, and, in addition to the advantage that the egg is tough, since with C57BL / 6 mice in the background, when the ES cells obtained therefrom, which were generated pathological model mice , C57BL / 6 mice can be advantageously used in that their genetic background can be replaced with C57BL / 6 mice by backcrossing.

In addition, when ES cells are established, blastocysts 3.5 days after fertilization are generally used. However, a large number of blastocysts can be efficiently obtained by collecting embryos at the 8-cell stage and culturing them up to blastocysts. Early embryos can be obtained.

Either male or female ES cells may be used, but male ES cells are generally more convenient for producing a germline chimera. It is also desirable to discriminate between males and females as soon as possible in order to reduce cumbersome culture labor.

As a method for determining the sex of ES cells, for example, a method of amplifying and detecting a gene in a sex-determining region on the Y chromosome by a PCR method can be mentioned. By using this method, the number of ES cells of about 1 colony (about 50 cells) is sufficient, whereas the conventional method required about 10 ⁶ cells for karyotype analysis. The primary selection of ES cells in the early stage can be performed by discriminating between male and female, and the early stage of culture can be greatly reduced by enabling the selection of male cells at an early stage.

{Circle around (2)} The secondary selection can be performed, for example, by confirming the number of chromosomes by the G-banding method. The number of chromosomes in the obtained ES cells is desirably 100% of the normal number. However, when it is difficult due to physical operations at the time of establishment, after knocking out the gene of the ES cells, the normal cells (for example, chromosomes in mice) are knocked out. It is desirable to clone again into cells (number 2n = 40).

胚 The embryonic stem cell line obtained in this way usually has very good growth properties, but must be subcultured carefully since it tends to lose its ontogenic ability. For example, on a suitable feeder cell such as STO fibroblast, in the presence of LIF (1-10000 U / ml) in a carbon dioxide incubator (preferably 5% carbon dioxide, 95% air or 5% oxygen, 5% The cells are cultured by a method such as culturing at about 37 ° C. in carbon dioxide gas and 90% air. At the time of subculture, for example, trypsin / EDTA solution (usually 0.001 to 0.5% trypsin / 0.1 to 5 mM EDTA, Preferably, a method is used in which cells are made into single cells by treatment with about 0.1% trypsin / 1 mM EDTA and seeded on newly prepared feeder cells. Such subculture is usually performed every 1 to 3 days. At this time, it is desirable to observe the cells and, if any morphologically abnormal cells are found, discard the cultured cells.

ES cells are differentiated into various types of cells, such as parietal muscle, visceral muscle, and cardiac muscle, by monolayer culture up to high density or suspension culture until cell clumps are formed under appropriate conditions. [MJ Evans and MH Kaufman, Nature 292, 154, 1981; GR Martin} Proceedings of National Academy of Sciences USA (Proc. Natl. Acad. Sci. USA) 78, 7634, 1981; TC Doetschman et al., Journal of Embryology and Experimental Morphology, 87, 27, 1985]. The DNA-deficient cells of the present invention obtained by differentiation are useful in in vitro cell biology studies of the proteins of the present invention.

The non-human mammal deficient in DNA expression of the present invention can be distinguished from a normal animal by measuring the mRNA level of the animal using a known method and indirectly comparing the expression level.

非 As the non-human mammal, the same one as described above is used.

The non-human mammal deficient in expression of the DNA of the present invention is, for example, a DNA in which the targeting vector prepared as described above is introduced into mouse embryonic stem cells or mouse egg cells, and the DNA of the targeting vector is inactivated by the introduction. The DNA of the present invention can be knocked out by homologous recombination in which the sequence replaces the DNA of the present invention on the chromosome of mouse embryonic stem cells or mouse egg cells by homologous recombination.

Cells in which the DNA of the present invention has been knocked out can be used as a DNA sequence on a Southern hybridization analysis or targeting vector using the DNA sequence on or near the DNA of the present invention as a probe, and the DNA of the present invention derived from the mouse used for the targeting vector. It can be determined by analysis by PCR using a DNA sequence of a nearby region other than DNA as a primer. When non-human mammalian embryonic stem cells are used, a cell line in which the DNA of the present invention has been inactivated by gene homologous recombination is cloned, and the cells are cultured at an appropriate time, for example, at the 8-cell stage of non-human mammalian cells. The chimeric embryo is injected into an animal embryo or blastocyst, and the resulting chimeric embryo is transplanted into the uterus of the pseudopregnant non-human mammal. The produced animal is a chimeric animal comprising both cells having the normal DNA locus of the present invention and cells having the artificially mutated DNA locus of the present invention.

When a part of the germ cells of the chimeric animal has a mutated DNA locus of the present invention, all tissues are artificially mutated from a population obtained by crossing such a chimeric individual with a normal individual. It can be obtained by selecting individuals composed of cells having the added DNA locus of the present invention, for example, by judging coat color or the like. The individual thus obtained is usually an individual with a heterozygous expression of the protein of the present invention, which is crossed with an individual with a heterozygous expression of the protein of the present invention, and homozygous expression of the protein of the present invention from their offspring. Defective individuals can be obtained.

In the case of using an egg cell, for example, a transgenic non-human mammal having a targeting vector introduced into a chromosome can be obtained by injecting a DNA solution into a nucleus of an egg by a microinjection method, and these transgenic non-human mammals can be obtained. Compared to mammals, they can be obtained by selecting those having a mutation in the DNA locus of the present invention by homologous recombination.

(4) In the individual knocked out of the DNA of the present invention in this way, animal individuals obtained by mating can confirm that the DNA has been knocked out, and can be reared in a normal rearing environment.

生殖 Furthermore, the acquisition and maintenance of the germ line may be performed in accordance with ordinary methods. That is, by crossing male and female animals having the inactivated DNA, a homozygous animal having the inactivated DNA on both homologous chromosomes can be obtained. The obtained homozygous animal can be efficiently obtained by rearing the mother animal in such a manner that there are one normal animal and one homozygote. By mating male and female heterozygous animals, homozygous and heterozygous animals having the inactivated DNA are bred and subcultured.

非 The non-human mammalian embryonic stem cells in which the DNA of the present invention is inactivated are extremely useful for producing a non-human mammal deficient in expressing the DNA of the present invention.

In addition, since the non-human mammal deficient in expression of the DNA of the present invention lacks various biological activities that can be induced by the protein of the present invention, a model of a disease caused by inactivation of the biological activity of the protein of the present invention. Therefore, it is useful for investigating the causes of these diseases and examining treatment methods.
[7a] A method for screening for a compound having a preventive / therapeutic effect on diseases caused by DNA deficiency or damage of the present invention The non-human mammal deficient in DNA expression of the present invention may be deficient or damaged by the DNA of the present invention. Can be used for screening for compounds that have a prophylactic / therapeutic effect on diseases caused by.

That is, the present invention is characterized by administering a test compound to a non-human mammal deficient in expressing the DNA of the present invention, and observing and measuring a change in the animal. Provided is a method for screening a compound having a prophylactic or therapeutic effect on a disease or a salt thereof.

ＤＮＡ The non-human mammal deficient in expressing DNA of the present invention used in the screening method includes the same ones as described above.

Test compounds include, for example, peptides, proteins, non-peptidic compounds, synthetic compounds, fermentation products, cell extracts, plant extracts, animal tissue extracts, plasma, and the like. These compounds are novel compounds. Or a known compound.

Specifically, a non-human mammal deficient in expression of the DNA of the present invention is treated with a test compound, compared with an untreated control animal, and tested using changes in each organ, tissue, disease symptoms, etc. of the animal as an index. The prophylactic and therapeutic effects of the compounds can be tested.

(4) As a method of treating a test animal with a test compound, for example, oral administration, intravenous injection, or the like is used, and it can be appropriately selected according to the symptoms of the test animal, the properties of the test compound, and the like. The dose of the test compound can be appropriately selected according to the administration method, the properties of the test compound, and the like.

For example, when screening for a compound having a therapeutic effect on Parkinson's syndrome, a test compound is administered to a non-human mammal deficient in expression of DNA encoding the protein of the present invention, and the amount of dopamine and its metabolite in the brain of the animal is administered. Of the animal, observation of the number or degeneration of nerve cells in the substantia nigra, measurement of the amount of movement of the animal, or analysis of the behavior pattern.

The compound obtained by using the screening method is a compound selected from the test compounds described above, and has a prophylactic / therapeutic effect against a disease caused by deficiency or damage of the protein of the present invention. It can be used as a safe and low toxic drug such as a prophylactic / therapeutic agent. Furthermore, compounds derived from the compounds obtained by the above screening can be used in the same manner.

The compound obtained by the screening method may form a salt. Examples of the salt of the compound include physiologically acceptable acids (eg, inorganic acids, organic acids, etc.) and bases (eg, alkali metals, etc.). ) Is used, and a physiologically acceptable acid addition salt is particularly preferable. Such salts include, for example, salts with inorganic acids (eg, hydrochloric acid, phosphoric acid, hydrobromic acid, sulfuric acid, etc.) or organic acids (eg, acetic acid, formic acid, propionic acid, fumaric acid, maleic acid, Salts with succinic acid, tartaric acid, citric acid, malic acid, oxalic acid, benzoic acid, methanesulfonic acid, benzenesulfonic acid, and the like are used.

医 薬 A drug containing the compound or a salt thereof obtained by the screening method can be produced in the same manner as the above-mentioned drug containing the protein of the present invention.

The preparations obtained in this way are safe and have low toxicity, for example, human or other mammals (eg, rat, mouse, guinea pig, rabbit, sheep, pig, cow, horse, cat, dog, monkey, etc.) ) Can be administered.

The dose of the compound or a salt thereof varies depending on the target disease, the administration subject, the administration route, and the like. For example, when the compound is orally administered, it is generally used in an adult (assuming a body weight of 60 kg) patient with Parkinson's syndrome. Administers about 0.1 to 100 mg, preferably about 1.0 to 50 mg, more preferably about 1.0 to 20 mg of the compound per day. When administered parenterally, the single dose of the compound varies depending on the administration subject, target disease, and the like. For example, the compound is usually administered in the form of an injection to an adult (with a body weight of 60 kg) of a patient with Parkinson's syndrome. It is convenient to administer about 0.01 to 30 mg, preferably about 0.1 to 20 mg, more preferably about 0.1 to 10 mg of the compound by intravenous injection per day. In the case of other animals, the amount can be administered in terms of the weight per 60 kg.
[7b] A method for screening a compound that promotes or inhibits the activity of a promoter for the DNA of the present invention The present invention provides a method for administering a test compound to a non-human mammal deficient in expressing the DNA of the present invention, and detecting the expression of a reporter gene. The present invention provides a method for screening a compound or a salt thereof that promotes or inhibits the activity of a promoter for DNA of the present invention.

In the above-mentioned screening method, the non-human mammal deficient in expression of DNA of the present invention may be a non-human mammal deficient in expression of DNA of the present invention in which the DNA of the present invention is inactivated by introducing a reporter gene, A reporter gene that can be expressed under the control of a promoter for the DNA of the present invention is used.

(4) Examples of the test compound include the same compounds as described above.

As the reporter gene, the same one as described above is used, and a β-galactosidase gene (lacZ), a soluble alkaline phosphatase gene, a luciferase gene and the like are preferable.

In a non-human mammal deficient in expression of the DNA of the present invention in which the DNA of the present invention is replaced with a reporter gene, since the reporter gene is under the control of the promoter for the DNA of the present invention, the expression of the substance encoded by the reporter gene is traced. By doing so, the activity of the promoter can be detected.

For example, when a part of the DNA region encoding the protein of the present invention is replaced with a β-galactosidase gene (lacZ) derived from Escherichia coli, the tissue that expresses the protein of the present invention should be used instead of the protein of the present invention. Β-galactosidase is expressed. Therefore, for example, the present invention can be easily performed by staining with a reagent serving as a substrate for β-galactosidase such as 5-bromo-4-chloro-3-indolyl-β-galactopyranoside (X-gal). Of the protein in an animal body can be observed. Specifically, the protein-deficient mouse of the present invention or a tissue section thereof is fixed with glutaraldehyde or the like, washed with phosphate buffered saline (PBS), and then stained with X-gal at room temperature or around 37 ° C. After reacting for about 30 minutes to 1 hour, the β-galactosidase reaction may be stopped by washing the tissue specimen with a 1 mM @ EDTA / PBS solution, and coloration may be observed. Further, mRNA encoding lacZ may be detected according to a conventional method.

The compound or its salt obtained by using the above-mentioned screening method is a compound selected from the above-mentioned test compounds, and is a compound that promotes or inhibits the promoter activity of the DNA of the present invention.

The compound obtained by the screening method may form a salt, and the salt of the compound may include a physiologically acceptable acid (eg, an inorganic acid) and a base (eg, an organic acid). And particularly preferably a physiologically acceptable acid addition salt. Such salts include, for example, salts with inorganic acids (eg, hydrochloric acid, phosphoric acid, hydrobromic acid, sulfuric acid, etc.) or organic acids (eg, acetic acid, formic acid, propionic acid, fumaric acid, maleic acid, Salts with succinic acid, tartaric acid, citric acid, malic acid, oxalic acid, benzoic acid, methanesulfonic acid, benzenesulfonic acid, and the like are used.

Since the compound or a salt thereof that promotes the promoter activity of the DNA of the present invention can promote the expression of the protein of the present invention and promote the function of the protein, for example, hyperlipidemia, gastrointestinal diseases (eg, Irritable bowel syndrome, ulcerative colitis, Crohn's disease, ischemic colitis, gastritis, peptic ulcer, proctitis, etc., central nervous system disorders (eg, Alzheimer's disease, Parkinson's syndrome, depression, emotional disorders, integration Ataxia, anxiety, etc.), diabetes, pancreatic diseases (eg, pancreatitis, pancreatic insufficiency such as cystic fibrosis), respiratory diseases (eg, chronic obstructive pulmonary disease (COPD), asthma) , Hypothyroidism, cardiovascular disease (eg, heart failure, arrhythmia, long QT syndrome, arteriosclerosis, etc.), cancer (eg, testicular tumor, ovarian cancer, breast cancer, esophageal cancer, lung cancer, kidney cancer, liver cancer, non-small cell) Lung cancer, prostate Cancer, stomach cancer, bladder cancer, cervical cancer, colon cancer, rectal cancer, pancreatic cancer, thymoma, etc.) are useful as pharmaceuticals such as prophylactic and therapeutic agents such as. Preferably, it is a prophylactic / therapeutic agent for central nervous system diseases and the like, particularly preferably a prophylactic / therapeutic agent for Parkinson's syndrome and the like.

Further, the compound or its salt that inhibits the promoter activity of the DNA of the present invention can inhibit the expression of the protein of the present invention and inhibit the function of the protein. E.g., irritable bowel syndrome, ulcerative colitis, Crohn's disease, ischemic colitis, gastritis, peptic ulcer, proctitis, etc., central nervous system disorders (e.g., Alzheimer's disease, Parkinson's syndrome, depression, emotional disorders, Schizophrenia, anxiety, etc.), diabetes, pancreatic disease (eg, pancreatitis, pancreatic insufficiency such as cystic fibrosis), respiratory disease (eg, chronic obstructive pulmonary disease (COPD), asthma, etc.) ), Hypothyroidism, cardiovascular disease (eg, heart failure, arrhythmia, long QT syndrome, arteriosclerosis, etc.), cancer (eg, testicular tumor, ovarian cancer, breast cancer, esophageal cancer, lung cancer, kidney cancer, liver cancer, non-small) Cell lung cancer, Tatsusengan, stomach cancer, bladder cancer, cervical cancer, colon cancer, rectal cancer, pancreatic cancer, thymoma, etc.) are useful as pharmaceuticals such as prophylactic and therapeutic agents such as. Preferably, it is a prophylactic / therapeutic agent for central nervous system diseases and the like, particularly preferably a prophylactic / therapeutic agent for Parkinson's syndrome and the like.

Furthermore, compounds derived from the compounds obtained by the above screening can be used in the same manner.

医 薬 A drug containing the compound or a salt thereof obtained by the screening method can be produced in the same manner as the above-mentioned drug containing the protein of the present invention or a salt thereof.

The preparations obtained in this way are safe and have low toxicity, for example, human or other mammals (eg, rat, mouse, guinea pig, rabbit, sheep, pig, cow, horse, cat, dog, monkey, etc.) ) Can be administered.

The dose of the compound or a salt thereof varies depending on the disease to be treated, the subject of administration, the administration route, and the like. For example, when the compound of the present invention that promotes the promoter activity for DNA is orally administered, generally the adult (body weight) is used. In a patient with Parkinson's syndrome (as 60 kg), about 0.1 to 100 mg, preferably about 1.0 to 50 mg, more preferably about 1.0 to 20 mg of the compound is administered per day. When administered parenterally, the single dose of the compound varies depending on the administration subject, target disease, and the like. For example, the compound of the present invention that promotes the promoter activity for DNA is usually administered in the form of an injection to an adult ( When administered to a Parkinson's syndrome patient (with a body weight of 60 kg), about 0.01 to 30 mg, preferably about 0.1 to 20 mg, more preferably about 0.1 to 10 mg of the compound per day is injected intravenously. It is convenient to administer by In the case of other animals, the amount can be administered in terms of the weight per 60 kg.

On the other hand, for example, when a compound of the present invention that inhibits promoter activity for DNA is orally administered, generally, in an adult (assuming a body weight of 60 kg) Parkinson's syndrome patient, about 0.1 to 100 mg of the compound per day is used. Preferably about 1.0 to 50 mg, more preferably about 1.0 to 20 mg is administered. When administered parenterally, the single dose of the compound varies depending on the administration subject, target disease, and the like. For example, a compound that inhibits the promoter activity of the DNA of the present invention is usually administered in the form of an injection to an adult ( When administered to a Parkinson's syndrome patient (with a body weight of 60 kg), about 0.01 to 30 mg, preferably about 0.1 to 20 mg, more preferably about 0.1 to 10 mg of the compound per day is injected intravenously. It is convenient to administer by In the case of other animals, the amount can be administered in terms of the weight per 60 kg.

As described above, the non-human mammal deficient in expression of the DNA of the present invention is extremely useful for screening for a compound or a salt thereof that promotes or inhibits the activity of the promoter for the DNA of the present invention, It can greatly contribute to investigating the cause of various diseases caused by it or to develop preventive and therapeutic drugs.

Further, using a DNA containing the promoter region of the protein of the present invention, genes encoding various proteins are ligated downstream thereof and injected into egg cells of an animal to produce a so-called transgenic animal (transgenic animal). Once produced, the polypeptide can be specifically synthesized and its action in the living body can be examined. Furthermore, if a suitable reporter gene is bound to the above promoter portion, and a cell line that expresses the reporter gene is established, a low-molecular compound having an action of specifically promoting or suppressing the production ability of the protein itself of the present invention in the body. Can be used as a search system.

塩 基 In the present specification and the drawings, the abbreviations for bases, amino acids and the like are based on the abbreviations by the IUPAC-IUB Commission on Biochemical Nomenclature or commonly used abbreviations in this field, and examples thereof are described below. When an amino acid can have an optical isomer, the L-form is indicated unless otherwise specified.

DNA: deoxyribonucleic acid cDNA: complementary deoxyribonucleic acid A: adenine T: thymine G: guanine C: cytosine RNA: ribonucleic acid mRNA: messenger ribonucleic acid dATP: deoxyadenosine triphosphate dTTP: deoxythymidine triphosphate dGTP: deoxyguanosine triphosphate Phosphate dCTP: Deoxycytidine triphosphate ATP: Adenosine triphosphate EDTA: Ethylenediaminetetraacetic acid SDS: Sodium dodecyl sulfate Gly: Glycine Ala: Alanine Val: Valine Leu: Leucine Ile: Isoleucine Ser: Serine Thr: Threin Crythine : Methionine Glu: Glutamate Asp: Aspartic acid Lys: Lysine Arg : Arginine His: Histidine Phe: Phenylalanine Tyr: Tyrosine Trp: Tryptophan Pro: Proline Asn: Asparagine Gln: Glutamine pGlu: Pyroglutamic acid The substituents, protecting groups and reagents frequently used in the present specification are represented by the following symbols. write.

Me: methyl group Et: ethyl group Bu: butyl group Ph: phenyl group TC: thiazolidine -4 (R) - carboxamide group Tos: p-toluenesulfonyl CHO: formyl Bzl: benzyl Cl ₂ -Bzl: 2,6-dichlorobenzyl Bom: benzyloxymethyl Z: benzyloxycarbonyl Cl-Z: 2-chlorobenzyloxycarbonyl Br-Z: 2-bromobenzyloxycarbonyl Boc: t-butoxycarbonyl DNP: dinitrophenyl Trt: trityl Bum: t-butoxymethyl Fmoc : N-9-fluorenylmethoxycarbonyl HOBt: 1-hydroxybenztriazole HOOBT: 3,4-dihydro-3-hydroxy-4-oxo-
1,2,3-benzotriazine HONB: 1-hydroxy-5-norbornene-2,3-dicarboximide DCC: N, N'-dicyclohexylcarbodiimide The sequence numbers in the sequence listing in the present specification indicate the following sequences. .
[SEQ ID NO: 1]
1 shows the amino acid sequence of human TCH162 protein obtained in Example 1.
[SEQ ID NO: 2]
1 shows the nucleotide sequence of DNA encoding human TCH162 protein having the amino acid sequence represented by SEQ ID NO: 1.
[SEQ ID NO: 3]
2 shows the base sequence of primer A3 used in Example 1.
[SEQ ID NO: 4]
2 shows the base sequence of primer B3 used in Example 1.
[SEQ ID NO: 5]
2 shows the nucleotide sequence of primer SP6 used in Example 1.
[SEQ ID NO: 6]
2 shows the nucleotide sequence of primer T7 used in Example 1.
[SEQ ID NO: 7]
2 shows the nucleotide sequence of primer F1 used in Example 1.
[SEQ ID NO: 8]
2 shows the base sequence of primer F2 used in Example 1.
[SEQ ID NO: 9]
2 shows the base sequence of primer F3 used in Example 1.
[SEQ ID NO: 10]
2 shows the base sequence of primer R1 used in Example 1.
[SEQ ID NO: 11]
3 shows the base sequence of primer R2 used in Example 1.
[SEQ ID NO: 12]
2 shows the base sequence of primer R3 used in Example 1.
[SEQ ID NO: 13]
2 shows the base sequence of primer A2 used in Example 1.
[SEQ ID NO: 14]
2 shows the base sequence of primer B2 used in Example 1.
[SEQ ID NO: 15]
1 shows the nucleotide sequence of cDNA containing the full-length human TCH162 gene obtained in Example 1.
[SEQ ID NO: 16]
3 shows the base sequence of primer TF2 used in Example 2.
[SEQ ID NO: 17]
3 shows the base sequence of primer TR2 used in Example 2.
[SEQ ID NO: 18]
4 shows the base sequence of TaqMan probe T2 used in Example 2.
[SEQ ID NO: 19]
4 shows the amino acid sequence of mouse TCH162 protein obtained in Example 3.
[SEQ ID NO: 20]
This shows the nucleotide sequence of DNA encoding the mouse TCH162 protein having the amino acid sequence represented by SEQ ID NO: 19.
[SEQ ID NO: 21]
3 shows the nucleotide sequence of primer mA3 used in Example 3.
[SEQ ID NO: 22]
3 shows the nucleotide sequence of primer mB3 used in Example 3.
[SEQ ID NO: 23]
3 shows the nucleotide sequence of primer mA1 used in Example 3.
[SEQ ID NO: 24]
3 shows the base sequence of primer mB1 used in Example 3.
[SEQ ID NO: 25]
3 shows the nucleotide sequence of primer mTF used in Example 3.
[SEQ ID NO: 26]
3 shows the nucleotide sequence of primer mTR used in Example 3.
[SEQ ID NO: 27]
3 shows the nucleotide sequence of cDNA containing the full-length mouse TCH162 gene obtained in Example 3.
[SEQ ID NO: 28]
7 shows the base sequence of TaqMan probe mT1 used in Example 4.
[SEQ ID NO: 29]
14 shows the base sequence of primer OF used in Example 6.
[SEQ ID NO: 30]
14 shows the base sequence of primer OR used in Example 6.
[SEQ ID NO: 31]
14 shows the base sequence of primer BGH RV used in Example 6.
[SEQ ID NO: 32]
14 shows the nucleotide sequence of primer TF used in Example 8.
[SEQ ID NO: 33]
14 shows the base sequence of primer TR used in Example 8.
[SEQ ID NO: 34]
14 shows the base sequence of TaqMan probe T1 used in Example 8.

The transformant Escherichia coli TOP10 / pCR-BluntII-TCH162 obtained in Example 1 described below was obtained from July 24, 2003 at 1-1-1, Higashi 1-chome, Chuo No. 6 (Zip code 305-8566), Tsukuba City, Ibaraki Prefecture, Japan. Deposited with the National Institute of Advanced Industrial Science and Technology, Patent Organism Depositary under the deposit number FERM BP-8434.

The transformant Escherichia coli TOP10 / pCR-BluntII-mTCH162 obtained in Example 3 described below was obtained from July 24, 2003 at 1-1-1, Higashi 1-chome, Chuo No. 6 (zip code 305-8566), Tsukuba City, Ibaraki Prefecture, Japan. Deposited with the National Institute of Advanced Industrial Science and Technology (AIST) at the Patent Organism Depositary under the deposit number FERM BP-8435.

Hereinafter, the present invention will be described more specifically with reference to Examples, but the present invention is not limited thereto. The gene manipulation using Escherichia coli, Mori Cloning ^{(molecular cloning, 2 nd, J.Sambrook} et al., Cold Spring Harbor Lab. Press, 1989 years) according to the method described in.
Example 1
Cloning of human TCH162 gene cDNA Using two types of primer DNAs, primer A3 (SEQ ID NO: 3) and primer B3 (SEQ ID NO: 4), a human brain cDNA library (Takara Shuzo) and a human small intestinal cDNA library (Takara Shuzo) Was performed using Pyrobest DNA Polymerase (Takara Shuzo) under the following conditions (1) to (3).

(1) 94 ° C for 2 minutes (2) 98 ° C for 10 seconds -68 ° C for 30 seconds -72 ° C for 3 minutes for 30 cycles (3) 72 ° C for 10 minutes After gel electrophoresis of the obtained amplification product, a 1.5 kbp band was The DNA was excised and cloned using Zero Blunt TOPO PCR cloning kit (manufactured by Invitrogen). Clones derived from cDNA libraries of the brain and small intestine were combined one by one to obtain two clones. This was used as a primer DNA [primer SP6 (SEQ ID NO: 5), primer T7 (SEQ ID NO: 6), primer F1 (SEQ ID NO: 7), primer F2 (SEQ ID NO: 8), primer F3 (SEQ ID NO: 9), Primer R1 (SEQ ID NO: 10), Primer R2 (SEQ ID NO: 11), Primer R3 (SEQ ID NO: 12), Primer A2 (SEQ ID NO: 13), Primer B2 (SEQ ID NO: 14)] and BigDye Terminator Cycle Sequencing The reaction was carried out using Kit (Applied Biosystems), and the nucleotide sequence of the inserted cDNA fragment was determined using a DNA sequencer ABI PRISM 3100 DNA Analyzer (Applied Biosystems). As a result, the obtained two clones contained the same DNA fragment and had a base sequence of 1538 (SEQ ID NO: 15). This cDNA fragment encodes a 437 amino acid sequence (SEQ ID NO: 1) (SEQ ID NO: 2), and a protein containing the amino acid sequence represented by SEQ ID NO: 1 was named human TCH162 protein.

形 質 A transformant having the plasmid containing the above-mentioned cDNA fragment was named Escherichia coli TOP10 / pCR-BluntII-TCH162.

When homology search was performed on owl using Blast P (Nucleic Acids Res., Vol. 25, p. 3389, 1997), human TCH162 belongs to the sodium-dependent bile acid transporter family. It was found to be a gene (FIG. 1). Human TCH162 is at the base level with sodium taurocholate co-transporting polypeptide (NTCP), a sodium-dependent bile acid transporter reported in humans (J. Clin. Invest., 93, 1326, 1994). Showed homology of 39% and 34% at the amino acid level. Human TCH162 had the same amino acid sequence as Genbank Accession No. BC019066.
Example 2
Analysis of tissue distribution of human TCH162 gene product Two kinds of primer DNAs [primer TF2 (SEQ ID NO: 16) and primer TR2 (SEQ ID NO: 17)] and TaqMan probe T2 (sequence) were designed from the nucleotide sequence encoding human TCH162. No. 18) in the cDNA of each human tissue (Human MTC panel I and II, Human digestive system MTC panel; Clontech, Human Adult Digestive System Normal Tissue cDNA Panel I and V; BioChain) The expression level of human TCH162 was measured by TaqMan PCR. The reaction was first performed at 50 ° C. for 2 minutes and then at 95 ° C. for 10 minutes using the ABI PRISM 7900 sequence detection system (Applied Biosystems) using TaqMan Universal PCR Master Mix (Applied Biosystems). Forty cycles were repeated at 95 ° C. for 15 seconds and 60 ° C. for one minute as one reaction cycle, and detection was performed simultaneously. The cDNA of each human tissue used for the measurement is shown in [Table 1].

[Table 1]
cDNA organization
Human MTC panel I Heart, brain, placenta, lung, liver, skeletal muscle, kidney, pancreas
Human MTC panel II spleen, thymus, prostate, testis, ovaries, small intestine, colon,
Peripheral blood leukocytes
Human digestive liver, esophagus, stomach, duodenum, jejunum, ileum, ileocecum,
system MTC panel Cecum, ascending colon, transverse colon, descending colon, rectum
Human Adult Digestive I; esophagus, stomach, small intestine, colon
System Normal Tissue V; Duodenum, jejunum, ileum, cecum
cDNA Panel I and V

The results are shown in FIGS. 2, 3 and 4. The TCH162 gene product (mRNA) is slightly expressed in Human MTC panel I and MTC panel II in heart, placenta, liver, skeletal muscle, kidney, spleen, thymus, ovary, peripheral leukocytes, prostate, testis, Some expression was observed in the colon, strong expression was observed in the pancreas, lung and brain, and strongest expression was observed in the small intestine.

(4) In the Human digestive system MTC panel, strong expression was observed in all regions from the stomach to the rectum, and particularly strong expression was observed in the ileum and jejunum.

In the Human Adult Digestive System Normal Tissue cDNA Panels I and V, strong expression was observed in the small intestine (jejunum, ileum), cecum and colon, and the strongest expression was observed particularly in the ileum.
Example 3
Cloning of cDNA encoding mouse TCH162 protein Using two kinds of primer DNAs [primer mA3 (SEQ ID NO: 21) and primer mB3 (SEQ ID NO: 22)], mouse brain Marathon-Ready cDNA (Clontech) was used. PCR was performed using Pyrobest DNA Polymerase (Takara Shuzo) under the following conditions (1) to (3).

(1) 94 ° C for 2 minutes (2) 98 ° C for 10 seconds -65 ° C for 30 seconds -72 ° C for 3.5 minutes for 30 cycles (3) 72 ° C for 10 minutes The amplified product obtained was subjected to Zero Blunt TOPO PCR Cloning kit (Invitrogen). To obtain the plasmid pCR-BluntII-mTCH162.

This was used as a primer DNA [primer SP6 (SEQ ID NO: 5), primer T7 (SEQ ID NO: 6), primer mA1 (SEQ ID NO: 23), primer mB1 (SEQ ID NO: 24), primer mTF (SEQ ID NO: 25), Primer mTR (SEQ ID NO: 26)] and BigDye Terminator Cycle Sequencing Kit (manufactured by Applied Biosystems), and the base sequence of the inserted cDNA fragment was analyzed using a DNA sequencer ABI PRISM 3100 DNA Analyzer (Applied Biosystems). (Manufactured by the company). As a result, the clone had 1398 base sequences (SEQ ID NO: 27). This cDNA fragment encodes a 437 amino acid sequence (SEQ ID NO: 19) (SEQ ID NO: 20), and the protein having the amino acid sequence represented by SEQ ID NO: 19 was named mouse TCH162 protein.

形 質 A transformant having a plasmid containing the above-mentioned cDNA fragment was named Escherichia coli TOP10 / pCR-BluntII-mTCH162.

When a homology search was performed on owl using Blast P [Nucleic Acids Res., Vol. 25, p. 3389, 1997], mouse TCH162 was found to be NTCP (J. Clin. Invest. 93, p. 1326, 1994), showing 41% homology at the base level and 35% homology at the amino acid level. Further, it showed a homology of 40% at the base level and 33% at the amino acid level with mouse NTCP (Genbank: NP # 035517), which is a liver sodium-dependent bile acid transporter reported in mice.

Mouse TCH162 showed homology of 81% at the base level and 86% at the amino acid level with human TCH162, indicating that mouse TCH162 is a mouse ortholog of human TCH162 (FIG. 5).
Example 4
Analysis of tissue distribution of mouse TCH162 gene product Two types of primer DNAs [primer mTF (SEQ ID NO: 25) and primer mTR (SEQ ID NO: 26)] and TaqMan probe mT1 (sequence) were designed from the nucleotide sequence encoding mouse TCH162. No. 28), mouse tissues (bone marrow, eyes, lymph nodes, smooth muscle, prostate, thymus, stomach, uterus, heart, brain, spleen, lung, liver, skeletal muscle, kidney, testis, embryo ( 7 days), embryo (11 days), embryo (15 days), embryo (17 days)) cDNA (Mouse MTC panel I and Mouse MTC panel II: Clontech) expression level (copy number) of mouse TCH162 It was measured by TaqMan PCR. For the Human Adult Digestive System Normal Tissue cDNA Panels I and V, the expression level (copy number) of GAPDH was also measured using TaqMan GAPDH control reagents (manufactured by Applied Biosystems). The reaction was first performed at 50 ° C. for 2 minutes and then at 95 ° C. for 10 minutes using the ABI PRISM 7900 sequence detection system (Applied Biosystems) using TaqMan Universal PCR Master Mix (Applied Biosystems). Forty cycles were repeated at 95 ° C. for 15 seconds and 60 ° C. for one minute as one reaction cycle, and detection was performed simultaneously.

The results are shown in FIG.

Mouse TCH162 gene product (mRNA) is available in Mouse MTC panel I and MTC panel II.
Expression was found in a wide range of tissues, but relatively strong expression was found in eyes, smooth muscle, and prostate, and even stronger in embryos (15 days) and embryos (17 days). Day) showed the strongest expression.
Example 5
Analysis of tissue distribution of mouse TCH162 gene product in 7-week-old BALB / c mice Tissues of 7-week-old BALB / c mice (cerebrum, medulla, hippocampus, cerebellum, spinal cord, eyeball, forestomach, posterior stomach, duodenum, jejunum , Colon, rectum, cecum, lung, trachea, skeletal muscle, heart, kidney, pancreas, liver, spleen, thymus, testis, ovary, uterus, prostate, skin; 1-10, male except ovary and uterus) Total RNA was prepared using the RNeasy Mini Kit (Qiagen). Reverse transcription was performed on the prepared total RNA using TaqMan Revease Transcription Reagents (manufactured by Applied Biosystems) to prepare cDNA. For this, the expression level (copy number) of mouse TCH162 was determined using the primer mTF (SEQ ID NO: 25), the primer mTR (SEQ ID NO: 26), and the TaqMan probe mT1 (SEQ ID NO: 28) used in Example 4. It was measured by TaqMan PCR. For the same cDNA, the expression level (copy number) of rodent GAPDH was also measured using TaqMan Rodent GAPDH Control Reagents VIC Probe (manufactured by Applied Biosystems). The reaction was first performed at 50 ° C. for 2 minutes and then at 95 ° C. for 10 minutes using the ABI PRISM 7900 sequence detection system (Applied Biosystems) using TaqMan Universal PCR Master Mix (Applied Biosystems). Forty cycles were repeated at 95 ° C. for 15 seconds and 60 ° C. for one minute as one reaction cycle, and detection was performed simultaneously.

The results are shown in FIG.

The mouse TCH162 gene product (mRNA) is highly expressed in the digestive tract tissues (fore stomach, hind stomach, duodenum, jejunum, colon, rectum, cecum) and cerebrum of each tissue of 7-week-old BALB / c mice, The highest expression was found in the medulla and spinal cord.
Example 6
Construction of Human TCH162 Expression Vector Using 10 ng of the plasmid obtained in Example 1 as a template, Pyrobest DNA Polymerase (manufactured by Takara Shuzo) using primer OF (SEQ ID NO: 29) and primer OR (SEQ ID NO: 30), PCR was performed under the conditions (1) to (3). The 5′-end primer OF and the 3′-end primer OR were designed to add a Hind III site and an Eco RI site, respectively, to the 5 ′ end for cloning into a vector.

(1) 94 ° C for 2 minutes (2) 30 cycles of 98 ° C for 10 seconds -65 ° C for 30 seconds -72 ° C for 3 minutes (3) 72 ° C for 10 minutes After gel electrophoresis of the PCR reaction solution, the main band was purified. The PCR fragment thus obtained was digested with the restriction enzymes HindIII and EcoRI by incubating at 37 ° C. for 1 hour, and the reaction solution was purified by gel electrophoresis. This was ligated to Hind III site and Eco RI site of pcDNA3.1 (+) (Invitrogen), which is an animal cell expression vector, using Takara ligation kit ver.2 (Takara Shuzo). This ligation reaction solution was subjected to ethanol precipitation treatment, and then transformed into competent cells, Escherichia coli TOP10 (manufactured by Invitrogen). A plasmid was prepared from a plurality of colonies thus obtained, and this base sequence was used as a primer DNA [primer BGH RV (SEQ ID NO: 31), primer T7 (SEQ ID NO: 6), primer F2 (SEQ ID NO: 8), R2 (SEQ ID NO: 11), primer A2 (SEQ ID NO: 13), primer B2 (SEQ ID NO: 14)] and BigDye Terminator Cycle Sequencing Kit (manufactured by Applied Biosystems). It was confirmed using an ABI PRISM 3100 DNA analyzer (manufactured by Applied Biosystems). A transformant having this plasmid was named Escherichia coli TOP10 / pCDNA3.1 (+)-TCH162.
Example 7
Preparation of Human TCH162-Expressing CHO Cell Line Escherichia coli TOP10 / pCDNA3.1 (+)-TCH162 was cultured, and plasmid DNA was prepared from the Escherichia coli body using the EndoFree Plasmid Maxi Kit (Qiagen). This plasmid DNA was introduced into CHO dhfr ⁻ cells using FuGENE 6 Transfection Reagent (Roche) according to the attached protocol. A mixture of 2 μg of the plasmid DNA and the transfection reagent was added to a 6 cm diameter petri dish seeded with 3 × 10 ⁶ CHO dhfr ⁻ cells 24 hours before. After culturing for 1 day in MEMα medium containing 10% fetal bovine serum, the cells were detached by trypsin treatment, and the collected cells were appropriately diluted and seeded on a 10 cm Petri dish. After an additional 24 hours, 0.5 mg / ml G418 was added to the medium, and then human TCH162-expressing cells were selected in the medium containing 0.5-1.0 mg / ml G418 for 10 days. 96 colonies of monoclonal human TCH162-expressing cells growing in the G418-containing selection medium were selected.
Example 8
Measurement of transgene expression level in human TCH162-expressing CHO cell line using TaqMan PCR method The human TCH162-expressing CHO cell line prepared in Example 7 was cultured in a 48-well plate or 24-well plate, and the cells proliferated were subjected to RNeasy 96 Kit (Qiagen). Was used to prepare total RNA. Reverse transcription was performed on the prepared total RNA using TaqMan Revease Transcription Reagents (manufactured by Applied Biosystems) to prepare cDNA. For this, the expression level of human TCH162 was measured by TaqMan PCR using primer TF (SEQ ID NO: 32) and primer TR (SEQ ID NO: 33) and TaqMan probe T1 (SEQ ID NO: 34). The reaction was first performed at 50 ° C. for 2 minutes and then at 95 ° C. for 10 minutes using the ABI PRISM 7900 sequence detection system (Applied Biosystems) using TaqMan Universal PCR Master Mix (Applied Biosystems). Forty cycles were repeated at 95 ° C. for 15 seconds and 60 ° C. for one minute as one reaction cycle, and detection was performed simultaneously. Clone No. 35 was selected as a human TCH162 gene high expression cell line.
Example 9
Expression analysis of human TCH162 gene database The expression of human TCH162 in each major normal brain region (cerebral cortex, temporal lobe, cerebellar hemisphere, hippocampus, hypothalamus, medulla oblongata, spinal cord and substantia nigra) using GeneLogic database Was.

The results are shown in FIG.

This indicates that the human TCH162 gene is specifically expressed in the substantia nigra.

FIG. 4 shows a comparison of amino acid sequences of human TCH162 and sodium-dependent bile acid transporter (NTCP). In the figure, TCH162 indicates the amino acid sequence of human TCH162, and NTCP indicates the amino acid sequence of sodium-dependent bile acid transporter (NTCP). □ indicates amino acids that are identical in the two sequences. BRIEF DESCRIPTION OF DRAWINGS FIG. 1 is a view showing the expression level of a human TCH162 gene product in each tissue. The results of measuring the expression level of human TCH162 in human human tissue cDNAs (Human MTC panel I and MTC panel II by Clontech) by TaqMan PCR are shown. The expression level was represented by the number of copies per μl of the cDNA solution. BRIEF DESCRIPTION OF DRAWINGS FIG. 1 is a view showing the expression level of a human TCH162 gene product in each tissue. The results of measuring the expression level of human TCH162 in human tissue cDNAs (Human digestive system MTC panel: Clontech) by TaqMan PCR are shown. The expression level was represented by the number of copies per μl of the cDNA solution. BRIEF DESCRIPTION OF DRAWINGS FIG. 1 is a view showing the expression level of a human TCH162 gene product in each tissue. The results of measuring the expression level of human TCH162 in human tissue cDNAs (Human Adult Digestive System Normal Tissue cDNA Panels I and V: manufactured by BioChain) using TaqMan PCR are shown. The expression level was represented by a value obtained by dividing the copy number per 1 μl of the TCH162 cDNA solution by the copy number of glyceraldehide-3-phosphate dehydrogenase (GAPDH) in an equal amount of each tissue cDNA. FIG. 4 is a diagram showing a comparison of amino acid sequences of human TCH162 (hTCH162) and mouse TCH162 (mTCH162). In the figure, hTCH162 indicates the amino acid sequence of human TCH162, and mTCH162 indicates the amino acid sequence of mouse TCH162. □ indicates amino acids that are identical in the two sequences. It is a figure showing the expression level in mouse | mouth TCH162 gene product in each tissue cDNA (Mouse MTC panel I and MTC panel II: Clontech). BRIEF DESCRIPTION OF THE DRAWINGS It is a figure showing the expression level in 7-week-old BALB / c mouse each tissue of the mouse TCH162 gene product. The expression level was expressed as a value obtained by dividing the copy number per 1 μl of the mouse TCH162 cDNA solution by the copy number of rodent glyceraldehide-3-phosphate dehydrogenase (rGAPDH) in an equal amount of each tissue cDNA. The results of the expression level of human TCH162 using Gene Logic Database are shown.

Claims (36)

A protein containing the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 1 or SEQ ID NO: 19, or a salt thereof. A protein comprising an amino acid sequence represented by SEQ ID NO: 1 or a salt thereof. A protein comprising an amino acid sequence represented by SEQ ID NO: 19 or a salt thereof. A partial peptide of the protein according to claim 1 or a salt thereof. A polynucleotide comprising a polynucleotide encoding the protein according to claim 1 or the partial peptide according to claim 4. The polynucleotide according to claim 5, which is DNA. A DNA consisting of the nucleotide sequence represented by SEQ ID NO: 20 or SEQ ID NO: 27. A recombinant vector containing the polynucleotide according to claim 5. A transformant transformed with the recombinant vector according to claim 8. The protein according to claim 1, wherein the transformant according to claim 9 is cultured to produce and accumulate the protein according to claim 1 or the partial peptide according to claim 4, and the collected protein is collected. 4. The method for producing the partial peptide or a salt thereof according to 4. A medicament comprising the protein according to claim 1, the partial peptide according to claim 4, or a salt thereof. A pharmaceutical comprising the polynucleotide according to claim 5. A diagnostic agent comprising the polynucleotide according to claim 5. An antibody against the protein according to claim 1, the partial peptide according to claim 4, or a salt thereof. A diagnostic agent comprising the antibody according to claim 14. A medicament comprising the antibody according to claim 14. A polynucleotide having a base sequence complementary to or substantially complementary to the polynucleotide according to claim 5, or a part thereof. A medicament comprising the polynucleotide according to claim 17. A compound that promotes or inhibits the activity of the protein according to claim 1, the partial peptide according to claim 4, or a salt thereof, wherein the protein according to claim 1 or the partial peptide according to claim 4 or a salt thereof is used. Or a method of screening for a salt thereof. A compound that promotes or inhibits the activity of the protein according to claim 1, the partial peptide according to claim 4, or a salt thereof, or a compound thereof, comprising the protein according to claim 1, the partial peptide according to claim 4, or a salt thereof. Salt screening kit. A compound or salt thereof that promotes or inhibits the activity of the protein of claim 1 or the partial peptide of claim 4 or a salt thereof, which is obtained by using the screening method of claim 19 or the screening kit of claim 20. . A medicament comprising the compound according to claim 21 or a salt thereof. A method for screening a compound or a salt thereof that promotes or inhibits expression of the protein gene according to claim 1, wherein the polynucleotide according to claim 5 is used. A kit for screening a compound or a salt thereof that promotes or inhibits the expression of the protein gene according to claim 1, comprising the polynucleotide according to claim 5. A compound or its salt that promotes or inhibits the expression of the protein gene according to claim 1, which is obtained by using the screening method according to claim 23 or the screening kit according to claim 24. A medicament comprising the compound according to claim 25 or a salt thereof. A method for quantifying a protein according to claim 1, wherein the antibody according to claim 14 is used. A method for diagnosing a disease associated with the function of the protein according to claim 1, wherein the method according to claim 27 is used. A method for screening a compound or a salt thereof that promotes or inhibits expression of the protein according to claim 1, characterized by using the antibody according to claim 14. A kit for screening a compound or a salt thereof, which promotes or inhibits expression of the protein according to claim 1, comprising the antibody according to claim 14. A compound or a salt thereof that promotes or inhibits the expression of the protein according to claim 1, which is obtained by using the screening method according to claim 29 or the screening kit according to claim 30. A medicament comprising the compound according to claim 31 or a salt thereof. The medicament according to claim 11, 12, 16, 18, 18, 22, 26 or 32, which is an agent for preventing or treating central nervous system diseases. The diagnostic agent according to claim 13 or 15, which is a diagnostic agent for a central nervous system disease. A method for preventing or treating central nervous system diseases, which comprises administering an effective amount of the compound according to claim 21, 25 or 31 or a salt thereof to a mammal. Use of the compound according to claim 21, 25 or 31 or a salt thereof for the manufacture of a prophylactic / therapeutic agent for central nervous system diseases.

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