WO2002036765A1

WO2002036765A1 - Novel protein and dna thereof

Info

Publication number: WO2002036765A1
Application number: PCT/JP2001/009585
Authority: WO
Inventors: Ryuichi Tozawa; Eiji Sunahara; Yoshio Taniyama
Original assignee: Takeda Chemical Industries, Ltd.
Priority date: 2000-11-02
Filing date: 2001-11-01
Publication date: 2002-05-10
Also published as: AU2002215208A1

Abstract

An esterase protein having an amino acid sequence which is the same or substantially the same as the amino acid sequence represented by SEQ ID NO:1 or its salt is usable as a preventive or a remedy for diseases such as arteriosclerosis, hyperlipemia, obesity and diabetes. This protein or its salt is also useful as a material for screening a compound having an activity of accelerating or inhibiting the esterase activity of the protein or a salt of such a compound.

Description

Description New protein and its DNA technology

The present invention relates to a lipolytic enzyme protein derived from human adipose tissue or a salt thereof, and a DNA encoding the same. Background art

It is well known that lipases and esterases play an important role in digestion and absorption of dietary lipids, decomposition and accumulation of lipids in vivo, and detoxification of harmful compounds. In particular, hormone-sensitive lipase that is widely expressed in tissues such as adipose tissue, testis, heart, skeletal muscle, Teng, and aorta.

(Hormone-sensitive lipase, hereafter abbreviated as HSL) has been attracting attention as a regulator of fat accumulation and energy consumption in the body, glucose metabolism or atherosclerotic lesion formation

(Holm, C and Osterlund, T. Methods in Molecular Biology, Vol. 109, ppl09-12l).

In other words, HSL is the rate-limiting enzyme in the degradation of neutral fats (especially triglycerides) in various tissues, and energy production in muscle tissues and brown adipocytes (Himms-Hagen, J. Prog. Lipid Res. 28, 67-115 (1989)) Production of PPARs and nuclear receptors PPARs

(Tontonoz, P. et al Cell 79, 1147-1156 (1994)) Regulation of insulin secretion in cells that not only regulate the supply of fatty acids (Mulder, H et al Diabetes 48, 228-232 (1999)). In the aorta and macrophages, it is involved in the formation and regression of atherosclerotic lesions as neutral cholesteryl ester hydrase (neutral cholesterylester hydrolase), which is responsible for the degradation of intracellular cholesteryl ester. A possibility has been suggested (Escarry, JL et al Arterioscler. Thromb. Vase. Biol. 18, 991-998).

However, Osuga et al. Produced wild-type mice in BSL-deficient mice. Not only did not show a difference in body weight between the cells, but also hormone-sensitive triglyceride-degrading activity in adipocytes, NCEH activity in macrophages, and changes in body heat production under cold conditions. Not available (Osuga, J. et al Proc. Natl. Acad. Sci. USA 97, 787-792 (2000)). Therefore, it is strongly suggested that other HSL-like lipases or esterases are involved in the various lipolytic processes that were conventionally thought to involve HSL. Pases or esterases may play an important role in the development of arteriosclerosis and hyperlipidemia, diabetes and obesity by participating in fatty acid production and macrophage foaming in adipocytes and muscle tissue Is expected.

Neutral lipolytic enzymes that have been reported to be expressed in adipose tissue, aorta or macrophages include bile salt-activated lipase (Li, R and Hui, DYJ Biol. Chem. 272, 28666-28671 (1997) ), Lipoprotein lipase (Mattsson, L. et al J. Clin. Invest. 92, 1759-1765 (1993)), monocyte Z macrophage serine esterase-ze-1 (Zschunke, F. et al Blood 78, 506-) 512 (1991)), endothelium-derived lipase (Jaye, M. et al Nature Genet. 21, 424-428 (1999)), lysosomal acid lipase

(Sakurada, T. et al Biochim. Biophys. Acta 424, 204-212 (1976)), etc., which are localized in tissues and cells, substrate specificity, optimal activity pH, hormones, etc. It is unlikely that HSL can replace the function of HSL because of the sensitivity or the pattern of activity fluctuations during the foaming process of macrophages.Isolation of new lipase or esterase from adipocytes and macrophages is expected. Was rare. The present invention relates to a novel protein or a partial peptide having an esterase activity or the like, a salt thereof, a DNA encoding the protein or a partial peptide thereof, a recombinant vector, a transformant, the protein or a partial peptide thereof, A method for producing a salt, a medicament comprising the protein or its partial peptide or its salt or its DNA, an antibody against the protein or its partial peptide or its salt, and an antibody against the protein or its partial peptide or its salt Provided are a screening method for a compound having an activity of promoting esterase activity, a kit for screening, a compound obtained by the screening method, a medicament containing the compound, and the like. Disclosure of the invention

As amino acid sequences characteristic of HSL and its related lipase or esterase, the GXSXG sequence (X represents any amino acid) and the HG (or HGG) sequence located upstream thereof are known (Harri, H et al Biochim. Biophys. Acta 1210, 249-253 (1994)). The present inventors have paid attention to these sequences and have energetically searched for genes whose expression is observed in adipose tissue or macrophages. As a result, they have found a novel lipolytic enzyme gene as described in the present invention. Since this gene is expressed in adipose tissue, macrophages, and skeletal muscle, it is expected to function as a neutral lipolytic enzyme in place of HSL. In addition, animals whose gene expression has been modified are useful as novel model animals for arteriosclerosis, hyperlipidemia, obesity, and diabetes, as well as for the expression of this gene and the activity of the protein encoded by this gene. Influencing drugs are useful as therapeutic agents for arteriosclerosis, hyperlipidemia, obesity, and diabetes based on a novel mechanism.

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 a salt thereof,

(2) the protein or salt thereof according to the above (1), which comprises the amino acid sequence represented by SEQ ID NO: 1;

(3) a partial peptide of the protein according to (1) or a salt thereof,

(4) The protein or a salt thereof according to the above (1) having an esterase activity or the partial peptide or a salt thereof according to the above (3),

(5) a polynucleotide comprising a polynucleotide encoding the protein of (1) or the partial peptide of (2),

(6) the polynucleotide according to (5), which is a DNA,

(7) The DNA according to the above (6), comprising the base sequence represented by SEQ ID NO: 2.

(8) a recombinant vector containing the polynucleotide according to the above (5), (9) a transformant transformed with the recombinant vector according to (8) above,

(10) culturing the transformant described in (9) above, producing and accumulating the protein described in (1) or the partial peptide described in (2) above, and collecting the protein; 1) The method for producing the protein according to the above or the partial peptide or the salt thereof according to the above (3),

(1 1) a medicine comprising the protein of (1) or the partial peptide of (3) or a salt thereof,

(12) a medicine comprising the polynucleotide according to the above (5),

(13) The medicament according to the above (11) or (12), which is an agent for treating or preventing arteriosclerosis, hyperlipidemia, obesity or diabetes.

(14) an antibody against the protein according to (1) or the partial peptide according to (3) or a salt thereof,

(15) a medicament comprising the antibody according to the above (14),

(16) a diagnostic agent comprising the antibody according to (14),

(17) an antisense DNA comprising a nucleotide sequence complementary to DNA or a part thereof encoding the protein of (1) or the partial peptide of (3),

(18) a medicine comprising the antisense DNA according to (17),

(19) A diagnostic agent comprising the antisense DNA according to (17), (20) a protein according to (1), a partial peptide according to (3), or a salt thereof. A method for screening a compound or a salt thereof having an activity of promoting or inhibiting the esterase activity of the protein or the partial peptide or the salt thereof according to the above (1);

(21) The esterase activity of the protein of (1) or the partial peptide of (3) or a salt thereof comprising the protein of (1) or the partial peptide of (3) or a salt thereof described above. A kit for screening a compound having an activity of promoting or inhibiting or a salt thereof,

(22) The protein or the protein according to (1), which is obtained by using the screening method according to (20) or the screening kit according to (21). Is a compound having the activity of promoting or inhibiting the esterase activity of the partial peptide or a salt thereof according to the above (3), or a salt thereof;

(23) The esterase activity of the protein of (1) or the partial peptide of (3) or a salt thereof obtained by using the screening method of (20) or the screening kit of (21). A medicament comprising a compound having an accelerating activity or a salt thereof;

(24) The treatment of arteriosclerosis, hyperlipidemia, obesity or diabetes.

(25) The esterase activity of the protein of (1) or the partial peptide of (3) or a salt thereof obtained by using the screening method of (20) or the screening kit of (21). A medicament comprising a compound having an inhibitory activity or a salt thereof,

(26) the medicament according to the above (25), which is an agent for treating and preventing obesity;

(27) The protein according to (1) or the protein according to (3), which can be obtained from a mammal using the screening method according to (20) or the screening kit according to (21). Prevention or treatment of arteriosclerosis, hyperlipidemia, obesity or diabetes, which comprises administering an effective amount of a compound having a partial peptide or a salt thereof, which has an activity of promoting esterase activity, or a salt thereof. Treatment method,

(28) The protein according to (1) or the protein according to (3), which can be obtained from a mammal using the screening method according to (20) or the screening kit according to (21). A method for preventing and treating obesity, which comprises administering an effective amount of a compound having an activity of inhibiting the esterase activity of a partial peptide or a salt thereof, or a salt thereof.

(29) The screening method according to the above (20) or the screening kit according to the above (21) for producing an agent for preventing or treating arteriosclerosis, hyperlipidemia, obesity or diabetes. Use of a compound having an activity of promoting the esterase activity of the protein of (1) or the partial peptide of (3) or a salt thereof, or a salt thereof, (30) The protein according to (1) or the above, which can be obtained using the screening method according to (20) or the screening kit according to (21) for producing an agent for preventing or treating obesity. (3) Use of a compound or a salt thereof having an activity of inhibiting the esterase activity of the partial peptide or a salt thereof described above,

(31) a non-human mammal having exogenous DNA encoding the protein according to (1) or the partial peptide according to (3) or a mutant DNA thereof, (32) a non-human mammal is a rodent (33) The animal according to (32), wherein the rodent is a mouse or a rat, or (34) the protein according to (1) or the partial peptide according to (3) above, wherein the rodent is a mouse or a rat. A recombinant vector containing exogenous DNA or its mutant DNA, which can be expressed in a non-human mammal;

(35) a non-human mammalian embryonic stem cell in which DNA encoding the protein according to (1) or the partial peptide according to (3) is inactivated,

(36) The embryonic stem cell according to (35), wherein the DNA is inactivated by introducing a reporter gene,

(37) The embryonic stem cell according to (35), which is neomycin-resistant,

(38) The embryonic stem cell according to the above (35), wherein the non-human mammal is a rodent, the embryonic stem cell according to the above (38), wherein the rodent is a mouse,

(40) a non-human mammal deficient in expression of the DNA described above, wherein the DNA encoding the protein described in (1) or the partial peptide described in (3) is inactivated, (41) the DNA comprises a reporter gene. The non-human mammal according to the above (40), wherein the non-human mammal is inactivated by the introduction, and the reporter gene can be expressed under the control of a promoter for the DNA.

(42) the non-human mammal according to the above (40), wherein the non-human mammal is a rodent;

(43) the non-human mammal according to (42), wherein the rodent is a mouse; and

(44) The test compound is administered to the animal according to (41), And a method for screening for a compound or a salt thereof that promotes or inhibits the promoter activity of the DNA. BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows the results of expression distribution of LIP-15 in tissues. lane 1 is adipose tissue, lane 2 is aorta, lane 3 is bone marrow, lane 4 is adult brain, lane 5 is large intestine, lane 6 is menstrual brain, lane 7 is skeletal, lane 8 is kidney, lane 9 is leukocyte , Lane 10 is liver B, lane 11 is lung, lane 12 is breast, lane 13 is ovary, lane 13 is ovary, lane 14 is prostate, lane 15 is prostate, lane 16 is skeletal muscle, lane 17 ^: small, lane 18 Spleen, lane 19¾: testis, lane 20 indicates thymus, lan6 21 indicates uterus, lane 22 indicates adrenal gland, M indicates lOObp marker. BEST MODE FOR CARRYING OUT THE INVENTION

A protein containing the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 1 of the present invention (hereinafter sometimes referred to as the protein of the present invention) is a warm-blooded animal (eg, human) , Guinea pig, rat, mouse, chick, egret, porcupine, sheep, sword, monkey, etc.) cells (eg, hepatocytes, spleen cells, nerve cells, glial cells, kidney i3 cells, bone marrow cells, mesangial cells) , Langer's cell, epidermal cell, epithelial cell, endothelial cell, fibroblast, fiber cell, muscle cell, fat cell, immune cell (eg, macrophage, T cell, B cell, natural killer cell, mast cell, neutrophil Spheres, basophils, eosinophils, monocytes), megakaryocytes, synovial cells, chondrocytes, osteocytes, osteoblasts, osteoclasts, mammary cells, hepatocytes or stromal cells Vesicles, or their precursors, stem cells or cancer cells) or any tissue in which those cells are present, such as the brain, parts of the brain (eg, olfactory bulb, amygdala, basal sphere, hippocampus, Thalamus, hypothalamus, cerebral cortex, medulla, cerebellum), spinal cord, pituitary, stomach, kidney, kidney, liver, gonad, thyroid, gall bladder, bone marrow, adrenal gland, skin, muscle, lung, digestive tract (eg, colon, Small intestine), blood vessels (eg, aorta), heart, thymus, spleen, submandibular gland, peripheral blood, prostate, testicle, ovary, placenta, testis, uterus, bone, joint, skeletal muscle, mammary gland, aorta, etc. Hematopoietic cells or their cultured cells (eg, MEL, Ml, CTLL-2, HT-2, WE HI-3, HL-60, J OSK-1, K562, ML-1, MOLT-3, MOLT-4, MOLT-1, 10, CCRF-CEM, TALL-1, Jurkat, CCRT-HSB-1, 2, KE-37, SKW-3, HUT -78, HUT-102, H9, U937, THP-1, HEL, JK-1, CMK, KO-812, MEG-01, etc.), especially adipose tissue, aorta, large intestine, kidney, mammary gland, It may be a protein derived from the ovary, prostate, small intestine, spleen, testis, uterus and adrenal gland, or may be a synthetic protein.

The amino acid sequence substantially identical to SEQ ID NO: 1 is about 50% or more, preferably about 60% or more, more preferably about 70% or more, more preferably the amino acid sequence represented by SEQ ID NO: 1. Is an amino acid sequence having about 80% or more, particularly preferably about 90% or more, and most preferably about 95% or more homology.

Examples of a protein having an amino acid sequence substantially identical to the amino acid sequence represented by the amino acid sequence represented by SEQ ID NO: 1 of the present invention include, for example, the amino acid sequence substantially represented by the amino acid sequence represented by SEQ ID NO: 1 Preferred are proteins having the same amino acid sequence as the above, and having substantially the same activity as the protein containing the amino acid sequence represented by SEQ ID NO: 1.

Examples of substantially the same activity include esterase activity and the like. Substantially equivalent indicates that the activity is qualitatively (eg, physiologically or pharmacologically) equivalent. Therefore, it is preferable that the esterase activity is equivalent (eg, about 0.0 :! to 100 times, preferably, about 0.1 to 10 times, more preferably, 0.5 to 2 times). Quantitative factors such as the molecular weight of the protein may be different.

The esterase activity described in the present specification means an activity involved in the hydrolysis of carboxylate ester, phosphate ester, and sulfate ester, and is preferably involved in hydrolysis of carboxylate ester, particularly fatty acid ester. Means activity. Examples of the carboxylic acid ester used as a substrate include natural substrates such as neutral fats such as triacylglycerol, diacylglycerol, and monoacylglycerol, and sterol esters such as cholesteryl ester, and P-nitrofuran. Synthetic substrates such as phenyl butyrate may be used. The acyl group provided for the ester bond may have at least two carbon atoms, and may have fluorescence or emission activity. '

Esterase activity can be measured according to a method known per se, for example, according to a screening method described later.

Examples of the protein of the present invention include: (1) one or two or more amino acids in the amino acid sequence represented by SEQ ID NO: 1 (preferably, about 1 to 30, preferably about 1 to 10, more preferably Is an amino acid sequence in which several (1 to 5) amino acids have been deleted, and 1 or 2 or more (preferably about 1 to 30 amino acids, preferably 1 amino acid amino acid sequence) in the amino acid sequence represented by SEQ ID NO: 1. An amino acid sequence to which about 10 amino acids have been added, more preferably a number (1 to 5) of amino acids, and 3) one or more amino acids (preferably 1 to 3) to the amino acid sequence represented by SEQ ID NO: 1 About 0, preferably about 1 to 10, more preferably about (1 to 5) amino acids, and 1 or 2 in the amino acid sequence represented by SEQ ID NO: 1. Is 2 or more (preferably about 1 to 30 pieces, preferably 1 to 1 A so-called mutin, such as a protein containing an amino acid sequence in which about 0, more preferably a number (1 to 5) amino acids are substituted with another amino acid, or an amino acid sequence obtained by combining them, is also included.

In the present specification, the protein has a N-terminus at the left end (amino terminus) and a C-terminus at the right end (capillary terminus) according to the convention of peptide labeling. The proteins of the present invention, including the protein containing the amino acid sequence represented by SEQ ID NO: 1, have a C-terminal lipoxyl group (—COOH), a carboxylate (1-C〇0—), It may be either C〇NH ₂ ) or ester (—COOR).

Here, as R in the ester, e.g., methyl, Echiru, n- propyl Le, C. ^ e alkyl group such as isopropyl or n- butyl, consequent opening pentyl, C ₃ _ ₈ cycloalkyl such as cyclohexyl group, for example, phenylene Le, 〇 ₆ _ _{1 2} Ariru groups, such as single-naphthyl, for example, benzyl, such as phenethyl phenylene Lou C E _ ₂ Arukiru group or α- naphthylmethyl etc. α- Naphthyl - Other ₄ Ararukiru groups such as C Bok ₂ alkyl groups, such as pivaloyl I Ruo carboxymethyl group which is generally used as an oral es ether is used.

When the protein of the present invention has a lipoxyl group (or carboxylate) at a position other than the C-terminus, the protein of the present invention includes a lipoxyl group amidated or esterified. For example, the above-mentioned C-terminal ester or the like is used as the ester of the banana.

Furthermore, in the protein of the present invention, the amino group at the N-terminal amino acid residue (eg, methionine residue) is protected by a protecting group (eg, an acyl group such as a formyl group, an acetyl group or the like, or a alkanoyl group). N-terminal daltamine residue generated by cleavage in vivo, which is oxidized with lipoamine, substituents on the side chains of amino acids in the molecule (e.g., 1H, 1SH, amino groups, those imidazole group, India Ichiru group, etc. Guanijino group) is protected with a suitable protecting group (e.g., formyl group, etc. (^ _ ₆ Ashiru groups such as E Arukanoiru groups such Asechiru group), Alternatively, a complex protein such as a so-called glycoprotein having a sugar chain bonded thereto is also included.

Specific examples of the protein of the present invention include, for example, a human-derived protein (LIP-5) containing the amino acid sequence represented by SEQ ID NO: 1. The partial peptide of the protein of the present invention (hereinafter, sometimes referred to as the partial peptide of the present invention) is a partial peptide of the protein of the present invention described above, and preferably has the same activity as the protein of the present invention described above. (Eg, esterase activity). For example, at least 20 or more, preferably 50 or more, more preferably 70 or more, more preferably 100 or more, and most preferably 200 or more of the constituent amino acid sequences of the protein of the present invention. Peptides having the above amino acid sequence and having esterase activity are used. Esterase activity can be measured in the same manner as described above. In addition, the partial peptide of the present invention may have the following deficiency: 1 or 2 or more (preferably about 1 to 10, more preferably several (1 to 5)) amino acids in the above amino acid sequence are deleted. (2) 1 or 2 or more amino acids in the above amino acid sequence (preferably about 1 to 20, more preferably about 1 to 10, and still more preferably several (1 to 5)) Or 3) one or more amino acids (preferably about 1 to 10, more preferably several, more preferably about 1 to 5) in the above amino acid sequence May be substituted.

The partial peptide of the present invention may have a carboxyl group (—CO OH), a carboxylate (—CO〇—) amide (—CO NH ₂ ) or an ester (—COOR) at the C-terminus.

Furthermore, the partial peptide of the present invention has a N-terminal methionine residue whose amino group is protected with a protecting group, and a N-terminal side is cleaved in vivo as in the case of the above-described receptor protein of the present invention. G1n is pyroglutamine-oxidized, and the substituent on the side chain of the amino acid in the molecule is protected with an appropriate protecting group, or a complex peptide such as a so-called glycopeptide to which a sugar chain is bound, etc. Is also included.

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

Since the partial peptide of the present invention can be used as an antigen for preparing an antibody, it does not necessarily have to have an esterase activity.

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. Acceptable acid addition salts are preferred. Such salts include, for example, salts with inorganic acids (eg, hydrochloric acid, phosphoric acid, hydrobromic acid, sulfuric acid) or organic acids (eg, acetic acid, formic acid, propionic acid, fumaric acid, maleic acid) Succinic acid, tartaric acid, citric acid, malic acid, oxalic acid, benzoic acid, methanesulfonic acid, benzenesulfonic acid).

The protein of the present invention or a salt thereof can be produced from a cell or tissue of a warm-blooded animal by a known method for purifying a protein, or a transformant containing a DNA encoding a protein described below can be produced. It can also be produced by culturing. Further, it can be produced according to the peptide synthesis method described later. In the case of production from tissues or cells of a warm-blooded animal, the tissues or cells of the warm-blooded animal are homogenized, extracted with an acid or the like, and the extract is subjected to chromatography such as reverse-phase chromatography or ion exchange chromatography. Purification and isolation can be performed by combining the above.

For the 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 used. Examples of such a resin include chloromethyl resin, hydroxymethyl resin, benzylhydrylamine resin, aminomethyl resin, 4-benzyloxybenzyl alcohol resin, 4-methylbenzhydrylamine resin, PAM resin, 4-hydroxymethylmethylphenylacetamidomethyl resin, polyacrylamide resin, 4- (2 ', 4'-dimethoxyphenyl-hydroxymethyl) phenoxy resin, 4-(2', 4 ' -Dimethoxyphenyl-Fmocaminoethyl) phenoxy resin and the like. Using such a resin, an amino acid having an appropriately protected amino group and side chain functional group is condensed on the resin in accordance with the sequence of the target protein or partial peptide according to various known condensation methods. Let it. At the end of the reaction, a 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 carried out in a highly diluted solution to convert the target protein or partial peptide or its amide. get.

For the condensation of the protected amino acids described above, various activating reagents that can be used for protein synthesis can be used, and carbodiimides are particularly preferable. As the carbodiimides, DCC, Ν, Ν′-diisopropylcarboimide, Ν-ethyl-Ν3-dimethylaminoprolyl) carbodiimide and the like are used. For activation by these, the protected amino acid is added directly to the resin together with a racemization inhibitor additive (eg, HOBt, HOOBt), or the protected amino acid is preliminarily converted to a symmetric acid anhydride or HOBt ester or HOOBt ester. It can be added to the resin after activation.

The solvent used for activating the protected amino acid or for condensing with the resin can be appropriately selected from solvents known to be usable for the protein condensation reaction. For example, N, N-dimethylformamide, N, N-dimethylacetamide, N-methyl Acid amides such as tylpyrrolidone, halogenated hydrocarbons such as methylene chloride and chloroform, alcohols such as trifluoroethanol, sulfoxides such as dimethyl sulfoxide, ethers such as pyridine, dioxane and tetrahydrofuran, and acetonitrile And nitriles such as propionitrile, esters such as methyl acetate and ethyl acetate, or an appropriate mixture thereof. The reaction temperature is appropriately selected from a range known to be usable for the protein bond formation reaction, and is usually appropriately selected from a range of about 120 ° 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 dihydrin reaction, if the condensation is insufficient, sufficient condensation can be performed by repeating the condensation reaction without removing the protecting group. If sufficient condensation is not obtained even after repeating the reaction, acetylation of unreacted amino acid with acetic anhydride or acetylimidazole is performed so that the subsequent reaction is not affected. be able to.

Examples of the protecting group for the amino group of the starting material include, for example, Z, Boc, t-pentyloxycapillonyl, isopolnyloxycarponyl, 4-methoxybenzyloxycarponyl, Cl-Z, Br-Z, adamantylo Xycarbonyl, trifluoroacetyl, phthaloyl, formyl, 2-nitrophenylsulfenyl, diphenylphosphinothioyl, Fmoc and the like are used.

The lipoxyl group may be, for example, a linear, branched or cyclic alkyl esterified (eg, methyl, ethyl, propyl, butyl, t-butyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, 2-adamantyl, etc.) Alkyl esterification), aralkyl esterification (eg, benzyl ester, 4-nitrobenzyl ester, 4-methoxybenzyl ester, 4-chlorobenzyl ester, benzhydryl esterification), phenacyl esterification, benzoyl ester It can be protected by xycarbonyl hydrazide, t-butoxycarbonyl hydrazide, trityl hydrazide and the like.

The hydroxyl group of serine can be protected, for example, by esterification or etherification. Suitable groups for this esterification include, for example, lower (di- ₆ ) alkanoyl groups such as acetyl group, aroyl groups such as benzoyl group, and benzyl group. Groups derived from carbonic acid such as an oxycarbonyl group and an ethoxycarbonyl group are used. Examples of a group suitable for etherification include a benzyl group, a tetrahydroviranyl group, and a t_butyl group.

As a 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, B Boc Trt Fmoc and the like are used.

Examples of the activated form of the carbonyl group of the raw material include, for example, corresponding acid anhydrides, azides, active esters (e.g., alcohols (e.g., pentachlorophenol, 2,4,5-trichloromouth phenol, 24-dinitro Esters with phenol, cyanomethyl alcohol, paranitrophenol, HONB N-hydroxysuccinimide, N-hydroxyphthalimide, HOBt). As the activated amino group of the raw material, for example, a corresponding phosphoric amide is used. Methods for removing (eliminating) protecting groups include, for example, catalytic reduction in a hydrogen stream in the presence of a catalyst such as Pd-black or Pd-carbon, or hydrogen fluoride anhydride or methanesulfonic acid. Acid treatment with trifluoromethanesulfonic acid, trifluoroacetic acid or a mixture thereof, base treatment with diisopropylethylamine, triethylamine, piperidine, piperazine, etc., and reduction with sodium in liquid ammonia Also used. The elimination reaction by the above acid treatment is generally carried out at a temperature of about −20 ° C. to 40 ° C. In the acid treatment, for example, anisol, phenol, thioanisole, methacrylol, paracresol, dimethyl Addition of a cation scavenger such as sulfide, 14-butanedithiol, 1,2-ethanedithiol, etc. is effective. Also, the 24_-dinitrophenyl group used as an imidazole protecting group of histidine is removed by thiophenol treatment, and the formyl group used as an indole protecting group of tributofan is 1,2-ethanedithiol, 1,4-butanedithiol, etc. In addition to deprotection by acid treatment in the presence of sulfur, it is also removed by alkaline treatment with dilute sodium hydroxide solution, dilute ammonia and the like. The protection of the functional group which should not be involved in the reaction of the raw materials, the protection group, the elimination of the protective group, and the activation of the functional group involved in the reaction 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, first, a carboxy-terminal amino acid is protected by amidation of a lipoxyl group, and then a peptide (protein) chain having a desired chain length is attached to the amino group side. After that, the protein or partial peptide from which only the protecting group for the N-terminal amino group of the peptide chain is removed and the protein or partial peptide from which only the protecting group for the C-terminal lipoxyl group is removed. It is prepared and the proteins or partial peptides are condensed in a mixed solvent as described above. The details of the condensation reaction are the same as described above. After purifying the protected protein or partial peptide obtained by the condensation, all the protecting groups are removed by the above-mentioned method to obtain a desired crude protein or partial peptide. The crude protein or partial peptide is purified by various known purification means, and the main fraction is freeze-dried to obtain an amide of the desired protein or partial peptide.

In order to obtain an ester of a protein or a partial peptide, for example, after condensing the thiol-oxyl group of the amino acid at the terminal end of the carboxyl with a desired alcohol to form an amino acid ester, the same procedure as in the amide of the protein or the partial peptide is carried out. An ester of the desired protein or partial peptide can be obtained.

The protein or partial peptide of the present invention or a salt thereof can be produced according to a peptide synthesis method known per se, or by cleaving the protein of the present invention with an appropriate peptide. 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 of 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 desired peptide. be able to. Examples of the known condensation method and elimination of the protecting group include the methods described in the following ① to ⑤.

®M. Bodanszky and MA Ondetti, Peptide Synthesis, Interscience Publishers, New York (1966 ② Schroeder and Luebke, The Peptide, Academic Press, New York (1965)

(3) Nobuo Izumiya et al. Basics and experiments on peptide synthesis, Maruzen Co., Ltd. (1975)

治 Haruaki Yajima and Shunpei Sakakibara, Laboratory for Biochemical Experiments 1, Protein Chemistry IV, 205, (1977)

治 Supervised by Haruaki Yajima, Development of Continuing Drugs, Vol. 14, Peptide Synthesis, Hirokawa Shoten

After the reaction, it is possible to purify and isolate the protein or partial peptide of the present invention by a combination of ordinary purification methods such as solvent extraction, distillation, column chromatography, liquid chromatography, and recrystallization. it can. When the protein or 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 protein or a partial peptide is obtained as a salt, It can be converted to a free form or another salt by a method or a method analogous thereto.

The polynucleotide encoding the protein of the present invention or its partial peptide is not limited as long as it contains the nucleotide sequence (DNA or RNA, preferably DNA) encoding the above-described protein of the present invention or its partial peptide. Anything may be used. The polynucleotide is RNA such as DNA or mRNA encoding the protein of the present invention or its partial peptide, and may be double-stranded or single-stranded. In the case of double-stranded, it may be double-stranded DNA, double-stranded RNA or DNA: RNA hybrid. In the case of a single strand, it may be a sense strand (ie, a coding strand) or an antisense strand (ie, a non-coding strand).

Using a polynucleotide encoding the protein or its partial peptide of the present invention, for example, by the method described in the known experimental medicine special edition `` New PCR and its application '' 15 (7), 1997 or a method analogous thereto, The mRNA of the protein of the present invention or its partial peptide can be quantified.

The DNA encoding the protein of the present invention may be any DNA as long as it contains the above-described nucleotide sequence encoding the protein of the present invention. Also, genomic DNA, genomic DNA library, CDNA, a cDNA library derived from the above-mentioned cells and tissues, or a synthetic DNA.

The vector used for the library may be any of pacteriophage, plasmid, cosmid, phagemid and the like. In addition, a reverse RNA was directly prepared by using a total RNA or mRNA fraction prepared from the above-mentioned cells.

It can also be amplified by Transcriptase Polymerase Chain Reaction (hereinafter abbreviated as RT-PCR method).

The DNA encoding the protein of the present invention includes, for example, DNA containing the nucleotide sequence represented by SEQ ID NO: 2 or DNA containing the nucleotide sequence represented by SEQ ID NO: 2 under high stringent conditions. Any DNA that encodes a protein having a hybridizing DNA and having substantially the same activity (eg, esterase activity, etc.) as the protein containing the amino acid sequence represented by SEQ ID NO: 1 It may be something.

Examples of the DNA that hybridizes with the DNA containing the nucleotide sequence represented by SEQ ID NO: 2 under high stringency conditions include, for example, about 70% or more, preferably about 70% or more of the nucleotide sequence represented by SEQ ID NO: 2. A DNA having a nucleotide sequence having a homology of 80% or more, more preferably about 90% or more, and most preferably about 95% or more is used.

Hybridization can be carried out by a method known per se or a method analogous thereto, for example, as described in Molecular Cloning 2nd (J. Sambrook et al., Cold Spring Harbor Lab. Press, 1989). It can be done according to the method. 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 end conditions include, for example, a sodium concentration of about 19 to 40 mM, preferably about 19 to 20 mM, and a temperature of about 50 to 70 ° C, preferably about 60 to 65 ° C. Is shown. In particular, the case where the sodium concentration is about 19 mM and the temperature is about 65 ° C is most preferable.

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

The DNA encoding the partial peptide of the present invention may be any as long as it contains the above-described nucleotide sequence encoding the partial peptide of the present invention. Further, it may be any of genomic DNA, genomic DNA library, cDNA derived from the cells and tissues described above, cDNA library derived from the cells and tissues described above, and synthetic DNA.

Examples of the DNA encoding the partial peptide of the present invention include, for example, a DNA having a partial base sequence of DNA containing the base sequence represented by SEQ ID NO: 2, or a DNA containing the base sequence represented by SEQ ID NO: 2 And a DNA having a DNA that hybridizes under high stringent conditions and having substantially the same activity (eg, esterase activity) as the protein containing the amino acid sequence represented by SEQ ID NO: 1. For example, DNA having a partial base sequence of the encoding DNA is used.

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

Examples of the DNA that hybridizes with the DNA containing the nucleotide sequence represented by SEQ ID NO: 2 under high stringent conditions include, for example, about 70% or more, preferably about 80%, of the nucleotide sequence represented by SEQ ID NO: 2. % Or more, more preferably about 90% or more, and still more preferably about 95% or more.

A polynucleotide comprising a part of the nucleotide sequence of DNA encoding the protein of the present invention or a partial peptide thereof, or a polynucleotide containing a part of a nucleotide sequence complementary to the DNA is a fragment encoding the partial peptide of the present invention. It is used to mean not only DNA but also RNA.

'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 cloned or encodes the determined protein of the present invention. It can be designed and synthesized based on the DNA sequence information of DNA. Such polynucleotides (nucleic acids) It can hybridize with RNA of the protein gene and inhibit the synthesis or function of the RNA, or regulate the expression of the protein gene of the present invention through the interaction with the protein-related RNA of the present invention. Adjustment 'can be controlled. Polynucleotides that are complementary to a selected sequence of the protein-related RNA of the present invention, and that can specifically hybridize with the protein-related RNA of the present invention, are those of the present invention in vivo and in vitro. It is useful for regulating and controlling the expression of protein genes, and is also useful for treating or diagnosing diseases. The term "corresponding" means having homology or being complementary to a specific sequence of nucleotides, base sequences or nucleic acids including genes. The “correspondence” between a nucleotide, base sequence or nucleic acid and a peptide (protein) usually refers to the amino acid of a peptide (protein) in the direction derived from the nucleotide (nucleic acid) sequence or its complement. I have. 5'-end hairpin loop, 5'-end 6-base spare'-lipet, 5'-end untranslated region, peptide translation start codon, protein coding region, ORF translation stop codon, 3'-end The translation region, the 3 'end palindrome region, and the 3' end hairpin loop may be selected as preferred regions of interest, but any region within the protein gene of the present invention may be selected as the region of interest.

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 "antisense" when the relationship between the target nucleic acid and the polynucleotide that can hybridize with the target is. Antisense polynucleotides include polynucleotides containing 2-dexoxy-D-reports, polynucleotides containing D-reports, and other types of polynucleotides that are N-glycosides of purine or pyrimidine bases. Nucleotides or other polymers with non-nucleotide backbones (eg, commercially available protein nucleic acids and synthetic sequence-specific nucleic acid polymers) or other polymers containing special linkages (such as those found in DNA or RNA) (Including nucleotides having a configuration permitting the attachment of a base), and the like. They can be double-stranded DNA, single-stranded DNA, double-stranded RNA, single-stranded RNA, and even DNA: RNA hybrids, as well as unmodified polynucleotides. (Or unmodified oligonucleotides), as well as those with known modifications, such as those with labels, caps, methylated, Natural nucleotides substituted with analogs, modified intranucleotides, such as those with uncharged bonds (eg, methylphosphonates, phosphotriesters, phosphoramidates, olebamates, etc.) Having a bond or a sulfur-containing bond (eg, phosphorothioate, phosphorodithioate, etc.), such as proteins (nucleases, nuclease inhibitors, toxins, antibodies, signal peptides, poly-L-lysine, etc.) and sugars ( (E.g., monosaccharides), etc. Yuichi Currents containing current compounds (eg, acridine, psoralen, etc.), those containing chelating compounds (eg, metals, radioactive metals, boron, oxidizing metals, etc.), those containing alkylating agents, It may have a modified bond (for example, a nucleic acid of an anoma type 1). 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 complexes. Modified nucleotides and modified nucleotides may also be modified at the sugar moiety, e.g., where one or more hydroxyl groups are replaced with halogens, aliphatic groups, etc., or functionalities such as ethers, amines, etc. It may be converted to a group.

The antisense polynucleotide (nucleic acid) of the present invention is RNA, DNA, or a modified nucleic acid (RNA, DNA). Specific examples of modified nucleic acids include, but are not limited to, sulfur derivatives of nucleic acids ゃ thiophosphoate derivatives, and polynucleoside amides のもの oligonucleoside amides that are resistant to degradation. . 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.

Thus, many 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 acids of the present invention may contain altered or modified sugars, bases, or bonds, are provided in special forms such as ribosomes, microspheres, are applied or added by gene therapy. Can be given in a written form. Thus, the addition forms include polycations such as polylysine, which acts to neutralize the charge of the phosphate skeleton, and lipids, which enhance the interaction with cell membranes or increase the uptake of nucleic acids ( For example, phospholipids, cholesterol, etc.). Preferred lipids for addition include cholesterol and its derivatives (eg, cholesteryl chromate formate, cholic acid, etc.). These can be attached to the 3 'end or 5' end of the nucleic acid, and can be attached via a base, sugar, or intramolecular nucleoside bond. Other groups include cap groups specifically located at the 3 'or 5' end of the nucleic acid to prevent degradation by nucleases such as exonuclease and RNase. It is. 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.

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

The DNA encoding the protein of the present invention or a partial peptide thereof may be labeled by a method known per se, and specifically, isotope-labeled DNA, fluorescently-labeled DNA (for example, Fluorescent labeling with fluorescein), biotinylated one or enzyme-labeled one. It completely encodes the protein or partial peptide of the present invention (hereinafter, these proteins and the like are described. In the description of cloning and expression of A, these proteins and the like may be simply abbreviated as the protein of the present invention). DNA can be cloned by PCR using the synthetic DNA primer having the partial nucleotide sequence of the protein of the present invention, or by PCR using the DNA incorporated into an appropriate vector. Selection can be carried out by hybridization with a DNA fragment encoding a part or the entire region of the protein of the invention or labeled with a synthetic DNA. Hybridization can be performed according to, for example, the method described in Molecular Cloning (Molecular Cloning) 2nd (J. Sambrook et al., Cold Spring Harbor Lab. Press, 1989). When a commercially available library is used, it can be performed according to the method described in the attached instruction manual.

DNA base sequence conversion can be performed using PCR or a known kit, for example, MutanTM-super Express Km (Takara Shuzo Co., Ltd.) or MutanTM-K (Takara Shuzo Co., Ltd.), using ODA-LA PCR or Gapped duplex. The method can be performed according to a method known per se, such as the method or the Kunkel method, or a method analogous thereto.

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

The expression vector for the protein of the present invention may be prepared, for example, by (i) cutting out a DNA fragment of interest from DNA encoding the protein of the present invention, and (mouth) converting the DNA fragment downstream of a promoter in an appropriate expression vector. It can be manufactured by connecting to

Examples of the vector include a plasmid derived from Escherichia coli (eg, pBR322, pBR325, pUC12, pUC13), a plasmid derived from Bacillus subtilis (eg, pUB110, pTP5, pC194), a plasmid derived from yeast ( Example, p SH 19, p SH 15), pacteriophage such as λ phage, animal viruses such as retrovirus, vaccinia virus, and baculovirus, as well as pAl-11, pXT1, pRcZCMV, pRc / RSV, pcDNAIZNeo, etc. Used.

The promoter used in the present invention may be any promoter as long as it is appropriate for the host used for gene expression. For example, when an animal cell is used as a host, examples include the SRCK promoter, SV40 promoter, LTR promoter, CMV promoter, and HSV-TK promoter.

Of these, it is preferable to use the CMV (cytomegalovirus) promoter, SRa promoter and the like. When the host is Eshierihia genus bacterium, trp promoter, lac promoter mono-, re cA promoter, lambda PL flop Romo - evening -, iota _{[rho [rho} promoter - coater, and tau 7 promoter one host is a Bacillus subtilis If the host is yeast, PH05 promoter, PGK promoter, GAP promoter, ADH promoter, etc. are preferable. When the host is an insect cell, polyhedrin promoter and P10 promoter are preferred.

The expression vector may contain, in addition to the above, an enhancer, a splicing signal, a poly-A addition signal, a selection marker, and an SV40 replication origin (hereinafter sometimes abbreviated as SV40 ori), if desired. Can be used. Examples of the selectable marker include a dihydrofolate reductase (hereinafter sometimes abbreviated as dhfr) gene [methotrexate (MTX) resistance], an ampicillin resistance gene (hereinafter sometimes abbreviated as Amp ¹ ), and neomycin. Syn-resistance gene (hereinafter sometimes abbreviated as Ne ο ¹ · G418 resistance), etc. Particularly, when the dh fr gene is used as a selection marker in Chinese hamster cells lacking the dh fr gene, the target gene Can also be selected with a thymidine-free medium.

Also, if necessary, a signal sequence suitable for the host may be added to the protein of the present invention. Add to N terminal side. If the host is a genus Escherichia, PhoA signal sequence, OmpA signal sequence, etc.If the host is a Bacillus genus, amylase signal sequence, subtilisin signal sequence, etc. In some cases, MF · signal sequence, SUC2 · signal sequence, etc. When the host is an animal cell, insulin signal sequence, α-interferon · signal sequence, antibody molecule · signal sequence, etc. Available. Using the vector containing the DNA encoding the protein of the present invention thus constructed, a transformant can be produced.

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

Specific examples of the genus Escherichia include, for example, Escherichia coli

(Escherichia coli) K 12 · DH 1 [Proc. Natl. Acad. Sci. USA], 6 0, 160 (1968)], JM 103 [Nucleic Acids Research], (Nucleic Acids Research), 9, 309 (1981)], JA 221 [Journal of 'Molecular'] Biology (Journal of Molecular Biology), 120, 5 17 (19778)], HB101 [Journal 'ob' Molecular Biology, 41, 459 (1969)] , C600 [Genetics, 39, 440 (1954)], etc. are used.

Examples of the bacterium belonging to the genus Bacillus include, for example, Bacillus subtilis MI114 (Gene, 24, 255 (1983)), 207—21 [Journal of Biochemistry (Journal of Biochemistry). ), 95, 87 (1 984)].

Examples of yeast include Saccharomyces cerevisiae AH22, AH22R-, NA87-11A, DKD-5D, 20B-12 and Schizosaccharomyces pombe NCYC1 913, NCYC 2036, Pichia pastoris KM71 and the like are used. As insect cells, for example, when the virus is Ac NPV, a cell line derived from a larva of night rob moth (Spodoptera frugiperda cell; Sί cell), MG1 cell derived from the midgut of Trichoplusia, and High derived from an egg of Trichoplusia ni Five ™ cells,

Cells derived from Mamestra brassicae or cells derived from Estigmena acrea are used. When the virus is BmNP V, a silkworm-derived cell line (Bombyx mori N cell; BmN cell) or the like is used. Examples of the Sf cell include Sf9 cell (ATCC CRL1711) and Sf21 cell (Vaughn, JL et al., In Vivo, 13, 213-217, (1977)) Are used.

As insects, for example, silkworm larvae and the like are used [Maeda et al., Neichia- (Nature), 315, 592 (1998)].

Animal cells include, for example, monkey cells COS-7, Vero, Chinese Eight Muster cells CHO (hereinafter abbreviated as CHO cells), dhfr gene-deficient Chinese Hams Yuichi cells CHO (hereinafter abbreviated as CHO (dh fr-) cells) ), Mouse L cells, mouse AtT-20, mouse myeloma cells, rat GH3, and human FL cells.

In order to transform Escherichia sp., For example, Prosessing's Ob-The National Academie. ' , 69, 211 (1972) and Gene (17), 107 (1982).

Transformation of Bacillus spp. Can be performed, for example, according to the method described in Molecular & General Genetics, Volume 168, 11 (1979). it can.

To transform yeast, see, for example, Methods in Enzymology, Vol. 194, 182—187 (1991), Proceedings of the National Academy. Natl. Acad. Sci. USA, _{75, 1929} (1978), and the like.

To transform insect cells or insects, e.g., bio / technology (Bio / Technology), 6, 47-55 (1988).

To transform animal cells, for example, see Cell Engineering Separate Volume 8 New Cell Engineering Experimental Protocol. 263-26 7 (19995) (published by Shujunsha), Virology, Volume 52 , 4556 (1973).

Thus, a transformant transformed with the expression vector containing 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 culturing, and a carbon source necessary for the growth of the transformant is contained therein. , Nitrogen sources, inorganic substances and others. Examples of the carbon source include glucose, dextrin, soluble starch, and sucrose.Examples of the nitrogen source include ammonium salts, nitrates, corn steep liquor, peptone, casein, meat extract, soybean meal, and potato. Examples of the inorganic or organic substance and the inorganic substance such as the extract include calcium chloride, sodium dihydrogen phosphate, and magnesium chloride. In addition, yeast extract, vitamins, growth promoting factors and the like may be added. The medium: H is preferably about 5-8.

Examples of a medium for culturing Escherichia bacteria include, for example, an M9 medium containing glucose and casamino acids [Miller, Journal of Journal of Experiments in Journal. Molecular Genetics), 4d1-4dd, Cold Spring Harbor Laboratory, New York 1972]. If necessary, an agent such as, for example, 3 / 3-indolylacrylic acid can be added in order to make the promotion work efficiently.

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

When the host is a bacterium belonging to the genus Bacillus, 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 applied.

When culturing a transformant whose host is yeast, for example, Burk BurMiolder Minimal Medium [; Bostian, KL et al., Proc. Natl. Acad. Sci. USA, Proc. Of the National Academy of Sciences, Obsc. ), 77, 4505 (1980)] and SD medium containing 0.5% casamino acid DBitter, G. Α: et al., Processings of the National Academy of Sciences. The USA (Proc. Natl. Acad. Sci. USA), 81, 5330 (1 984)]. The pH of the medium is preferably adjusted to about 5-8. Culture is usually about

Performed in 20 ° C~3 5 ° C to about 24 to 72 hours, when _{the c} host aerated or agitated as necessary, the transformant is cultivated insect cells or insects, the culture medium, Grace's Insect Medium ( Grace, TCC, Nature, 195, 788 (1962)) to which immobilized 10% serum serum or the like is appropriately added. The pH of the medium is preferably adjusted to about 6.2 to 6.4. Culture is usually performed at about 27 ° C for about 3 to 5 days, and aeration and agitation are added as necessary.

When culturing a transformant in which the host is an animal cell, the medium may be, for example, a MEM medium containing about 5 to 20% fetal bovine serum [Science, 122, 501, (1952)] ], DMEM medium [Virology, Vol. 8,

396 (1 959)], RPMI 1640 medium [The Journal of the American Medical Association] 199, 5 19 (1967)] 199 medium [Proceeding of the Society for the Biological Medicine, 73, 1, 1950)]. Preferably, the pH is about 6-8. The cultivation is usually performed at about 30 ° C to 40 for about 15 to 60 hours, and aeration and agitation are added as necessary.

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

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

When extracting the protein of the present invention from cultured cells or cells, Then, the cells or cells are collected by a known method, suspended in an appropriate buffer, and disrupted by ultrasonication, lysozyme, Z or freeze-thawing, etc., and then the protein is centrifuged or filtered. A method for obtaining a crude extract of the above is appropriately used. The buffer may contain a protein denaturing agent such as urea or guanidine hydrochloride, or a surfactant such as Triton X-10 ° ^TM . When protein is secreted into the culture solution, after culturing, the supernatant is separated from the cells or cells by a method known per se, and the supernatant is collected.

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

When the protein thus obtained is obtained in a free form, it can be converted to a salt by a method known per se or a method analogous thereto, and conversely, when the protein obtained is a salt, a method known per se or analogous thereto Can be converted to a free form or another salt.

The protein produced by the recombinant can be arbitrarily modified or the polypeptide can be partially removed by the action of an appropriate protein-modifying enzyme before or after purification. As the protein modifying enzyme, for example, trypsin, chymotrypsin, arginyl endopeptidase, protein kinase, glycosidase and the like are used.

The presence or activity of the thus-produced protein of the present invention or a salt thereof can be measured by enzymimnoassay using a specific antibody, the amount of released fatty acids (enzyme activity) due to the decomposition of neutral lipids, and the like. it can.

Antibodies against the protein, partial peptide or salt thereof of the present invention Polyclonal antibodies and monoclonal antibodies may be used as long as they can recognize the light proteins, partial peptides or salts thereof.

An antibody against the protein, the partial peptide or a salt thereof of the present invention (hereinafter, these proteins and the like may be simply abbreviated to the protein of the present invention in the description of the antibody) is obtained by using the protein of the present invention as an antigen. The antibody or antiserum can be produced by a known method for producing an antibody or antiserum.

[Preparation of monoclonal antibody]

(a) Preparation of monoclonal antibody-producing cells

The protein of the present invention is administered to a warm-blooded animal itself or together with a carrier or a diluent at a site capable of producing an antibody upon administration. Complete Freund's adjuvant / incomplete Freund's adjuvant may be administered in order to enhance antibody production upon administration. 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, 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 the antigen, for example, a mouse with an antibody titer is selected from the 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 the labeling agent bound to the antibody. The fusion operation can be performed according to a known method, for example, the method of Köhler 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 1: 1 to 20: 1; PEG (preferably PEG 1000 to PEG 6 000) S is added at a concentration of about 10 to 80%; 20 to 4 Ot: preferably 1 to 30 to 37 ° C. By incubating for up to 10 minutes, cell fusion can be carried out efficiently.

Various methods can be used to screen monoclonal antibody-producing hybridomas. For example, a hybridoma culture supernatant is added to a solid phase (eg, a microplate) on which a protein antigen is adsorbed directly or together with a carrier. Anti-immunoglobulin antibody (anti-mouse immunoglobulin antibody is used if the cells used for cell fusion are mice) or porcine tin A added to the solid phase Monoclonal antibody detection method, monoclonal antibody bound to solid phase by adding hybridoma culture supernatant to solid phase adsorbing anti-immune glopurin antibody or protein A, adding protein labeled with radioactive substance, enzyme, etc. Examples include a method of detecting an antibody.

Selection of the monoclonal antibody can be performed according to a method known per se 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 it can grow a hybridoma. For example, RPMI 1640 medium containing 1-20%, preferably 10-20% fetal bovine serum, GIT medium containing 1-10% fetal bovine serum (Wako Pure Chemical Industries, Ltd.) or Hachibidooma A serum-free culture medium (SFM-101, Nissui Pharmaceutical Co., Ltd.) or the like can be used. Culture temperature is usually 20 to 40 ° C, preferably about 37 ° C. The culturing time is usually 5 days to 3 weeks, preferably 1 week to 2 weeks. The culture can be usually performed under 5% carbon dioxide. The antibody titer of the hybridoma culture supernatant can be measured in the same manner as the measurement of the antibody titer in the antiserum described above.

(b) Purification of monoclonal antibodies

Monoclonal antibodies can be separated and purified by methods known per se, for example, immunoglobulin separation and purification methods (eg, salting out, alcohol precipitation, isoelectric precipitation, electrophoresis, ion exchangers (eg, DEAE) Adsorption / desorption method, ultracentrifugation, gel filtration, antigen Specific antibody purification method in which only the antibody is collected using a binding solid phase or an active adsorbent such as protein A or protein G, and the binding is dissociated to obtain the antibody.

(Preparation of polyclonal antibody)

The polyclonal antibody of the present invention can be produced according to a method known per se or a method analogous thereto. For example, a immunizing antigen (the protein antigen of the present invention) 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 monoclonal antibody production method described above. The antibody can be produced by collecting a substance containing the antibody against the protein of the present invention and separating and purifying the antibody.

Regarding the complex of the immunizing antigen and the carrier protein used for immunizing warm-blooded animals, the type of carrier protein and the mixture ratio of carrier-1 and hapten are different from those of hapten immunized by cross-linking with carrier-1. Any antibody may be cross-linked at any ratio as long as the antibody can be efficiently produced.For example, serum albumin, psilogloproline, and hemocyanin, etc. are used in a weight ratio of hapten 1 to hapten 1. About 0.1 to 20, preferably about 1 to 5 is used.

In addition, various condensing agents can be used for force coupling between the hapten and the carrier. For example, an active ester reagent containing a daltaraldehyde ゃ carpoimide, a maleimide active ester, a thiol group, or a dithioviridyl group is used. The condensation product is administered to a warm-blooded animal at a site where antibody production is possible, or together with a carrier or 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, for a total of about 3 to 10 times. The polyclonal antibody can be collected from the blood, ascites, etc., preferably from the blood of a warm-blooded animal immunized by the above method.

The measurement of the polyclonal antibody titer in the antiserum can be performed in the same manner as the measurement of the antibody titer in the antiserum described above. Separation and purification of polyclonal antibodies are performed according to the same immunoglobulin separation and purification method as for monoclonal antibodies described above. be able to.

Hereinafter, a protein or partial peptide of the present invention or a salt thereof (hereinafter, sometimes abbreviated as the protein of the present invention), a DNA encoding the protein or partial peptide of the present invention (hereinafter, referred to as a DNA of the present invention) The use of antibodies against the protein, partial peptide, or a salt thereof of the present invention (hereinafter sometimes abbreviated as the antibody of the present invention) will be described.

[1] An agent for treating or preventing various diseases related to the protein of the present invention

The protein of the present invention contributes to the regulation of the degradation of neutral lipids (eg, neutral fat and cholesteryl ester), and specifically has triglyceride-degrading activity, and thus encodes the protein of the present invention. When the DNA is abnormal or deficient, or when the expression level of the protein of the present invention is reduced, various diseases such as arteriosclerosis, hyperlipidemia, obesity, and diabetes are considered. I get sick.

Therefore, the protein and the like of the present invention and the DNA of the present invention can be used as a medicament such as an agent for treating or preventing various diseases such as arteriosclerosis, hyperlipidemia, obesity, and diabetes.

For example, when there is a patient in whom the regulation of neutral lipid degradation in cells is not sufficiently or normally exerted because the protein of the present invention is reduced or deleted in vivo, After administering the DNA of the present invention to the patient and expressing the protein or the like of the present invention in vivo, the (mouth) cell is introduced with the DNA of the present invention and the protein or the like of the present invention is expressed. By transplanting the cells into a patient, or (c) by administering the protein or the like of the present invention to the patient, the role of the protein or the like of the present invention in the patient can be sufficiently or normally exerted. it can.

When the DNA of the present invention is used as the above-mentioned therapeutic or prophylactic agent, the DNA is inserted alone or into an appropriate vector such as a retrovirus vector, an adenovirus vector, or an adenovirus associated virus vector. Thereafter, it can be administered to humans or warm-blooded animals according to conventional means. The DNA of the present invention may be used as it is or may be physiologically recognized as an auxiliary agent for promoting uptake. And a carrier such as a gene gun or a hydrogel catheter.

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

The protein and the like of the present invention can be used, for example, as tablets or capsules, capsules, elixirs, microcapsules, and the like, if necessary, orally, or water or other pharmaceutically acceptable liquids. It can be used parenterally in the form of an injection such as a sterile solution or suspension. For example, the protein of the present invention may be used together with physiologically acceptable carriers, flavoring agents, excipients, vehicles, preservatives, stabilizers, binders, and the like in a unit dosage form generally required for the practice of preparations. It can be manufactured by mixing. The amount of the active ingredient in these preparations is such that a suitable dosage in the specified range can be obtained.

Examples of additives that can be mixed with tablets and capsules include binders such as gelatin, corn starch, tragacanth, gum arabic, excipients such as crystalline cellulose, corn starch, gelatin, alginic acid, and the like. Such leavening agents, lubricants such as magnesium stearate, sweeteners such as sucrose, lactose or saccharin, and flavoring agents such as peppermint, cocoa oil or cherry are used. When the unit dosage form is a capsule, the above-mentioned dinner material may further contain a liquid carrier such as oil and fat. Sterile compositions for injection can be formulated according to standard pharmaceutical practice such as dissolving or suspending the active substance in vehicles such as water for injection, and naturally occurring vegetable oils such as sesame oil and coconut oil. it can.

Aqueous liquids for injection include, for example, physiological saline, isotonic solutions containing glucose and other adjuvants (eg, D-sorbitol, D-mannitol, sodium chloride, etc.). Agents, for example, alcohols (eg, ethanol, etc.), polyalcohols (eg, propylene glycol, polyethylene glycol, etc.), nonionic surfactants (eg, Polysorbate 8 OTM, HCO-50, etc.) You may use together. As an oily liquid, for example, Sesame oil, soybean oil and the like may be mentioned, and may be used in combination with benzyl benzoate, benzyl alcohol and the like as a solubilizing agent. In addition, buffers (for example, phosphate buffer, sodium acetate buffer, etc.), soothing agents (for example, benzalkonium chloride, prochloride hydrochloride, etc.), stabilizers (for example, human serum albumin, polyethylene glycol, etc.) ), Preservatives (eg, benzyl alcohol, phenol, etc.), antioxidants and the like. The prepared injection is usually filled in an appropriate ampoule.

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

The preparations obtained in this way are safe and have low toxicity, for example, warm-blooded animals (eg, humans, rats, mice, guinea pigs, egrets, birds, higgies, dogs, cats, cats, cats) , Dogs, monkeys, chimpanzees, etc.).

The dose of the protein or the like of the present invention varies depending on the target disease, the subject of administration, 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 arteriosclerosis, generally the adult In (as 6 O k), about 0.1 mg to 10 O mg, preferably about 1.0 to 5 O mg, more preferably about 1.0 to 20 mg of the protein or the like is administered per day. When administered parenterally, the single dose of the protein or the like varies depending on the administration target, target disease, and the like.For example, the protein or the like of the present invention is in the form of an injection for the treatment of arteriosclerosis. When administered to an adult (assuming a body weight of 60 kg), the protein or the like per day is about 0.01 to 3 Omg, preferably about 0.1 to 20 mg, more preferably about 0.1 to 10 mg per day. It is convenient to administer the degree by injecting it into the affected area. In the case of other animals, the dose can be administered in terms of 60 kg. [2] Screening of drug candidate compounds for disease

Since the protein or the like of the present invention has an esterase activity, especially a triglyceride-decomposing activity, a compound or a salt thereof which promotes the function of the protein or the like of the present invention (eg, esterase activity, etc.) includes, for example, arteriosclerosis and hyperlipidemia. It can be used as a medicine for treating and preventing obesity and diabetes. In addition, the inheritance of the protein of the present invention Since a child is specifically expressed in adipose tissue, a compound or a salt thereof that inhibits a function of a protein or the like (eg, esterase activity) of the present invention suppresses accumulation of free fatty acids due to triglyceride decomposition. It can be used, for example, as a medicament for preventing and treating obesity.

Therefore, the protein or the like of the present invention is useful as a probe for screening a compound or a salt thereof that promotes or inhibits the function of the protein or the like of the present invention.

That is, the present invention uses the protein or partial peptide of the present invention or a salt thereof (hereinafter, sometimes abbreviated as the protein of the present invention). The present invention provides a method for screening a compound having an activity of promoting or inhibiting

(1) Cells having the ability to express the gene of the protein of the present invention are cultured in the presence of the test compound, and the DNA of the present invention or its complementary DNA or a partial DNA thereof is used to transform the protein of the present invention. A method for screening a compound having an activity of promoting or inhibiting the esterase activity of the protein of the present invention or a salt thereof, which comprises measuring the amount of mRNA to be encoded, more specifically,

② (i) the expression level of mRNA of the protein of the present invention when cells having the ability to express the gene of the protein of the present invention are cultured, and (ii) the ability to express the gene of the protein of the present invention. A compound having the activity of promoting or inhibiting the esterase activity of the protein of the present invention, which is compared with the amount of mRNA of the protein of the present invention when the cells are cultured in the presence of the test compound. And a method for screening a substance or a salt thereof.

Examples of the cells having the ability to express the gene of the protein of the present invention include the above-mentioned known warm-blooded animal cells (preferably, fat cells, macrophages, and skeletal muscle cells) and the genes of the protein of the present invention. Examples include transformed animal cells. The animal cell transformed by introducing the gene of the protein of the present invention can be produced by the above-described method. Culture of cells having the ability to express the gene of the protein of the present invention is performed in the same manner as in a known animal cell culture method. For example, as a medium, MEM medium containing about 5 to 20% of fetal bovine serum [Science, 122, 501 (1952)], DMEM medium [Virology, 8 volumes, 3 96 (1959)], RPMI 1640 medium [Journal of the American Medical Association No. 199, 5 19 (1 967 Proceding of the Society for the Biological Medicine, Vol. 73, 1 (1950)] The pH is preferably about 6 to 8. Culture is usually performed at about 30 to 40 ° C. for about 15 to 60 hours, and if necessary, aeration and stirring may be added.

In order to compare the expression level of mRNA by the hybridization method, a method known per se or a method similar thereto, for example, Molecular Cloning 2nd (J. Sambrook et al "Cold Spring Harbor Lab. Press, 1989).

Specifically, the amount of mRNA encoding the protein of the present invention is determined by measuring the amount of mRNA extracted from cells according to a method known per se and DNA encoding the gene of the protein of the present invention or its complement. It is carried out by contacting DNA or its partial DNA and measuring the amount of mRNA bound to the gene DNA of the protein of the present invention or its complementary DNA. The amount of mRNA bound to the complementary DNA of the gene DNA of the protein of the present invention by labeling the complementary DNA of the DNA gene of the protein of the present invention or its partial DNA with, for example, a radioisotope or a dye. Can be easily measured. As the radioisotope, for example, ³² P, ³ H or the like is used. As the dye, for example, fluorescein FAM (manufactured by PE Biosystems), JE (manufactured by PE Biosystems), TAMRA (manufactured by PE Biosystems) , R 色素 X (manufactured by PE Biosystems), Cy5 (manufactured by Amersham), Cy3 (manufactured by Amersham) and the like are used.

In addition, the amount of mRNA of the protein of the present invention is determined by converting RNA extracted from cells into cDNA by reverse transcriptase and then coding for the gene of the protein of the present invention. This can be carried out by measuring the amount of cDNA amplified by PCR using DNA to be loaded or its complementary DNA or its partial DNA as a primer.

As the complementary DNA of the gene DNA of the protein of the present invention used for measuring the amount of mRNA of the protein of the present invention, a DNA having a sequence complementary to the gene DNA (upper chain) of the protein of the present invention (lower chain) ). As the partial DNA of the gene DNA of the protein of the present invention, for example, in the nucleotide sequence of the DNA gene of the protein of the present invention, about 10 to 2200 contiguous, preferably about 10 to 300, more preferably about 10 to 300 A base sequence composed of about 10 to 30 bases is exemplified. Examples of the partial DNA of the DNA complementary to the DNA gene of the protein of the present invention include DNA having a sequence complementary to the partial DNA of the DNA encoding the protein of the present invention. That is, for example, in the base sequence of the gene DNA of the protein of the present invention, a base sequence composed of about 10 to 2200 bases, preferably about 10 to 300 bases, and more preferably about 10 to 30 bases. A DNA having a complementary sequence is exemplified.

Examples of the primers used for PCR include DNA having a base sequence represented by SEQ ID NO: 5, DNA having a base sequence represented by SEQ ID NO: 6, and the like.

A test compound that increases the amount of mRNA of the protein of the present invention can be selected as a compound having the activity of promoting the expression of the protein gene of the present invention, and the amount of mRNA of the protein of the present invention can be reduced. The test compound to be reduced can be selected as a compound having the activity of inhibiting the expression of the gene of the protein of the present invention.

In addition, the present invention

(3) Known promoter of the protein of the present invention (2) Cells transformed with DNA obtained by cloning the enhancer region from genomic DNA and ligating upstream of an appropriate repo overnight gene (eg, fat cells, macrophages, skeletal muscle cells, etc.) ) Is cultured in the presence of a test compound, and the expression of a reporter gene is detected in place of the expression of the protein of the present invention. The present invention provides a method for screening a compound having an activity of promoting or inhibiting the activity or a salt thereof.

As the reporter gene, for example, a staining marker gene such as lacZ (; 3-galactosidase gene) and the like are used.

A test compound that increases the amount of a reporter gene product is measured by measuring the amount of a reporter gene product (eg, mRNA, protein) using a method known per se to promote the expression of the gene of the protein of the present invention. A test compound that reduces the amount of a reporter gene product can be selected as a compound that has an activity to inhibit the expression of the gene of the protein of the present invention.

The cells can be cultured in the same manner as in the known animal cell culture described above. Furthermore, the present invention

④ (i) An RI-labeled metabolic precursor (hereinafter sometimes referred to as a precursor. Specifically, a ³ H-labeled or i ⁴ C-labeled olein is added to a cell capable of producing the protein of the present invention. (Ii) fatty acids such as acids, glycerol, etc.) and (ii) culturing by contacting RI-labeled precursors and test compounds with cells capable of producing the protein of the present invention. put that neutral lipids (e.g. triglycerides, cholesteryl esters, etc.) in the case of the production of biochemical analysis (Yuki British Tsuyoshihen, p _P 235-272 Nankodo, 1984) the method according to, for example other It has an activity of promoting or inhibiting the esterase activity of the protein of the present invention, which is measured and compared by thin layer chromatography or column chromatography. Compound or to provide a subscription one Nin grayed method of a salt thereof.

The cells can be cultured in the same manner as in the known animal cell culture described above. A test compound that suppresses the production of neutral lipids or increases the production of degradation products of neutral lipids can be selected as a compound having the activity of promoting the gene expression of the protein of the present invention. A test compound that promotes the production of neutral lipids or decreases the production of neutral lipid digests can be selected as a compound having the activity of inhibiting the gene expression of the protein of the present invention. The esterase activity of the protein or the like of the present invention can be determined by a method known per se, for example, Holm C. and Osterlund T. Methods m Molecular Biology, edited by

It can be measured according to the method described in M.H. Doolittle and K. Reue, Vol. 109, ppl09_121 or a method analogous thereto.

For example, a test compound which increases the esterase activity in the case of the above (ii) by about 20% or more, preferably 30% or more, more preferably about 50% or more as compared with the case of the above (i), according to the present invention Can be selected as compounds that promote esterase activity, such as proteins.

On the other hand, a test compound in which the esterase activity in the case of (ii) is reduced by about 20% or more, preferably 30% or more, more preferably about 50% or more as compared with the case of (i). Can be selected as a compound that inhibits esterase activity such as the protein of the present invention.

Further, the present invention provides

本 A cell characterized by culturing cells capable of expressing the gene of the protein of the present invention in the presence of a test compound, and measuring the expression level of the protein of the present invention using an antibody against the protein of the present invention. A method for screening a compound having an activity of promoting or inhibiting the esterase activity of the protein of the present invention or a salt thereof, specifically,

⑥ (i) the expression level of the protein of the present invention when cells having the ability to express the gene of the protein of the present invention are cultured, and (ii) the cells having the ability to express the gene of the protein of the present invention as test compounds. Is characterized by measuring and comparing the expression level of the protein of the present invention when cultivated in the presence of an antibody of the protein of the present invention, and promoting or inhibiting the esterase activity of the protein of the present invention. A method for screening a compound having activity or a salt thereof is provided. The antibody of the protein of the present invention can be produced by the method described above. The cells can be cultured in the same manner as in the known animal cell culture described above. The expression level of the protein of the present invention can be quantified according to the method for quantifying the protein of the present invention shown in the following [3].

That is, more specifically, ⑦ (i) Cells having the ability to express the gene of the protein of the present invention are cultured, and the antibody of the protein of the present invention competes with the culture solution (test solution), the labeled protein of the present invention, and the like. (Ii) culturing cells having the ability to express the gene of the protein of the present invention in the presence of a test compound, and adding an antibody of the protein of the present invention to the culture solution (test solution) And comparing the ratio of the labeled protein of the present invention bound to the antibody with the case where the labeled protein of the present invention is competitively reacted. A method for screening a compound having an activity of promoting or inhibiting the esterase activity of the protein of the present invention or a salt thereof,

⑧ (i) culturing cells having the ability to express the gene of the protein of the present invention, the culture solution (test solution) and the antibody of the protein of the present invention insolubilized on a carrier and the labeled antibody of the present invention; When reacting simultaneously or sequentially with another antibody,

(ii) culturing cells having the ability to express the gene of the protein of the present invention in the presence of a test compound, and incubating the culture solution (test solution) with the antibody of the protein of the present invention insolubilized on a carrier; The esterase activity of the protein of the present invention is characterized by measuring the activity of the labeling agent on the insolubilized carrier when the labeled antibody is reacted simultaneously or successively with another antibody of the present invention. Provided is a method for screening a compound having a promoting or inhibiting activity or a salt thereof.

In the above method (2), it is preferable 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 and the like of the present invention.

In the screening method described above, examples of test compounds include peptides, proteins, non-peptide compounds, synthetic compounds, fermentation products, cell extracts, plant extracts, animal tissue extracts, and the like. May be a novel compound or a known compound.

The screening kit of the present invention contains a cell capable of expressing the gene of the protein of the present invention, a labeled protein of the present invention, an antibody against the protein of the present invention, and the like.

Obtained using the screening method or the screening kit of the present invention. The compounds or salts thereof are selected from the test compounds described above, for example, peptides, proteins, non-peptide compounds, synthetic compounds, fermentation products, cell extracts, plant extracts, animal tissue extracts, plasma, etc. It is a compound having an activity of promoting the function of the protein or the like of the present invention (eg, esterase activity and the like). As the salt of the compound, those similar to the aforementioned salts of the protein of the present invention are used.

Compounds having an activity of promoting the function of the protein or the like (eg, esterase activity) of the present invention include, for example, pharmaceuticals such as agents for treating and preventing diseases such as arteriosclerosis, hyperlipidemia, obesity, and diabetes. Can be used as

On the other hand, a compound having one activity that inhibits the function of the protein or the like (eg, esterase activity and the like) of the present invention can be used as a medicament such as a therapeutic / prophylactic agent for diseases such as obesity.

When a compound obtained by using the screening method or the screening kit of the present invention is used as the above-mentioned therapeutic / prophylactic agent, it can be carried out according to conventional means. For example, in the same manner as the above-mentioned drug containing the protein of the present invention, orally or parenterally as tablets, capsules, elixirs, microcapsules, sterile solutions, suspensions, etc. Can be. The preparations obtained in this way are safe and have low toxicity, for example, in warm-blooded animals (e.g. humans, mice, rats, puppies, sheep, bush, puppies, puppies, birds, cats, dogs, Monkeys, chimpanzees, etc.). The dose of the compound or a salt thereof varies depending on its action, target disease, subject to be administered, route of administration, and the like.For example, the function of the protein or the like of the present invention for the purpose of treating hyperlipidemia is considered. When the enhancing compound is administered orally, generally in adults (assuming a body weight of 60 kg), the compound is present in an amount of about 0.1 to 10 Omg, preferably about 1.0 to 5 Omg per day. More preferably, about 1.0 to 20 mg is administered. In the case of parenteral administration, the single dose of the compound varies depending on the administration subject, target disease, etc., for example, it promotes the function of the protein of the present invention for the purpose of treating hyperlipidemia. When the compound is administered to an adult (as 60 kg), usually in the form of an injection, the compound is administered in an amount of about 0.01 to 3 Omg per day, preferably about 0.1 to 3 Omg. It is convenient to administer about 2 O mg, more preferably about 0.1 to 1 O mg, by intravenous injection. For other animals, the equivalent dose per 60 kg can be administered.

[3] Quantification of the protein of the present invention, its partial peptide or a salt thereof An antibody against the protein or the like of the present invention (hereinafter sometimes abbreviated as the antibody of the present invention) specifically recognizes the protein or the like of the present invention. Therefore, it can be used for quantification of the protein of the present invention in a test solution, particularly for quantification by a sandwich immunoassay.

That is, the present invention

(i) The antibody of the present invention is competitively reacted with a test solution and a labeled protein of the present invention, and the percentage of the labeled protein of the present invention bound to the antibody is measured. A method for quantifying the protein of the present invention in a test solution, characterized in that:

(ii) Simultaneously or continuously reacting the test solution with the antibody of the present invention immobilized on the carrier and another labeled antibody of the present invention, and then measuring the activity of the labeling agent on the insoluble carrier. A method for quantifying the protein or the like of the present invention in a test solution,

(iii) A method for diagnosing diseases such as arteriosclerosis, hyperlipidemia, obesity, and diabetes using the quantitative method described in (i) or (ii) above.

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

In addition, the protein and the like of the present invention can be quantified using a monoclonal antibody against the protein and the like of the present invention (hereinafter sometimes referred to as the monoclonal antibody of the present invention), and can also be detected by tissue staining and 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 or the like of the present invention using the antibody of the present invention is not particularly limited, and may be an antibody, antigen or antibody-antigen complex corresponding to the amount of antigen (eg, the amount of protein) in the test solution. Detects body mass by chemical or physical means However, any measurement method may be used as long as it is calculated from a standard curve prepared using a standard solution containing a known amount of antigen. For example, nephelometry, a competition method, an immunometric method and a sandwich method are preferably used, but it is particularly preferable to use a sandwich method described later in terms of sensitivity and specificity. As a labeling agent used in a measurement method using a labeling substance, for example, a radioisotope, an enzyme, a fluorescent substance, a luminescent substance and the like are used. As the radioisotope, for example, ^{[1 2 5} I], ^{[1 3 1} I], ^[3 H], and ^{[1 4} C] used. As the above enzyme, a stable enzyme having a large specific activity is preferable. For example,) 3-galactosidase, / 3-dalcosidase, alkaline phosphatase, peroxidase, malate dehydrogenase and the like are used. As the fluorescent substance, for example, fluorescamine, fluorescein isothiosinate and the like are used. As the luminescent substance, for example, luminol, luminol derivative, luciferin, lucigenin and the like are used. Further, a biotin-avidin system may be used for binding the antibody or antigen to the labeling agent.

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, a test solution is reacted with the insolubilized monoclonal antibody of the present invention (primary reaction), and further reacted with another labeled monoclonal antibody of the present invention (secondary reaction). 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. Also, in the immunoassay by the sandwich method, the antibody used for the solid phase antibody or the labeling antibody is not necessarily required to be one type, and a mixture of two or more types of antibodies is used for the purpose of improving measurement sensitivity and the like. May be used. In the method for measuring the protein or the like of the present invention by the sandwich method of the present invention, As the monoclonal antibody of the present invention used for the primary reaction and the secondary reaction, an antibody having a different binding site such as the protein of the present invention is preferably used. That is, the antibody used in the primary reaction and the secondary reaction is, for example, when the antibody used in the secondary reaction recognizes the C-terminal of the protein or the like of the present invention, the antibody used in the primary reaction is Preferably, an antibody that recognizes other than the C-terminal, for example, the N-terminal, is used. The monoclonal antibody of the present invention can be used in a measurement system other than the sandwich method, for example, a competitive 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. (BZF separation) The labeling amount of either B or F is measured, and the amount of antigen in the test solution is determined. In this reaction method, a soluble antibody is used as an antibody, BZF separation is performed using polyethylene glycol, a liquid phase method using a second antibody against the antibody, a solid phase antibody is used as the first antibody, or An immobilization method using an immobilized antibody as the second antibody and a soluble antibody is used for the first antibody. In the immunometric method, the antigen in the test solution and the immobilized antigen are subjected to a competitive reaction with a certain amount of labeled antibody, and then the solid phase and the liquid phase are separated. Then, an antigen is reacted with an excessive amount of the labeled antibody, then the immobilized antigen is added to bind the unreacted labeled antibody to the solid phase, and then the solid phase and the liquid phase are separated. Next, the amount of label in either phase is measured to quantify the amount of antigen in the test solution.

In addition, in nephelometry, the amount of insoluble precipitate generated as a result of an antigen-antibody reaction in a gel or in a solution is measured. Even when the amount of antigen in the test solution is small and only a small amount of sediment is obtained, laser-nephrometry utilizing laser scattering is preferably used.

In applying these individual immunological measurement methods to the quantification method of the present invention, no special conditions, operations, and the like need to be set. A measurement system for the protein 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 and textbooks. For example, edited by Hiro Irie, "Radio Imuno Atsushi" (Kodansha, published in 1949), edited by Hiro Irie, "Radio Imno Atssey" (Kodansha, published in 1954), edited by Eiji Ishikawa et al. "Measurement Method" (Medical Shoin, published in 1958), "Immunoenzyme Assay" edited by Eiji Ishikawa et al. (Second Edition) (Medical Shoin, published in 1977), "Enzyme Immunoassay" edited by Eiji Ishikawa et al. (3rd edition) (Medical Shoin, published in 1962), "Methods in ENZYMOLOGYJ Vol. 70 (Immunochemical Techniques (Part A)), Ibid. Vol. 73 (Immunochemical Techniques (Part B)), Ibid. Vol.

74 (Immunochemical Techniques (Part C)), ibid.Vol. 84 (Immunochemical Techniques (Part D -'Selected Immunoassays))% ii Vol.

92 (Immunochemical Techniques (Part E: Monoclonal Antibodies and

General Immunoassay Methods)) ^ See the same book Vol. 12l (Immunochemical, published by Academic Press).

As described above, the protein and the like 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, arteriosclerosis, hyperlipidemia, It can be diagnosed as a disease such as obesity or diabetes, or is likely to be affected in the future.

On the other hand, when the concentration of the protein or the like of the present invention is determined by quantifying the concentration of the protein or the like of the present invention using the antibody of the present invention, for example, a disease such as obesity is detected. Or it can be diagnosed as likely to be affected in the future.

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. In addition, 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 for

[4] A drug containing the antibody of the present invention The antibody of the present invention, which has the activity of neutralizing the activity of the protein of the present invention or its partial peptide, includes, for example, a disease (eg, obesity) caused by overexpression of the protein of the present invention or its partial peptide. It can be used as a medicament such as a preventive or remedy for the disease.

The therapeutic or prophylactic agent for the above-mentioned diseases containing the antibody of the present invention can be used as it is as a liquid preparation or as a pharmaceutical composition in an appropriate dosage form, in humans or mammals (eg, rat, egret, sheep, hidge, bush, Can be administered orally or parenterally to mice, cats, dogs, monkeys, etc.). The dosage varies depending on the administration subject, target disease, symptoms, administration route, and the like.For example, for use in adults, the antibody of the present invention is usually administered in a dose of 0.01 to 2 Omg per day. kg body weight, preferably about 0.1 to 1 O mg / kg body weight, more preferably 0.1 to 5 mg Z kg body weight, about 1 to 5 times a day, preferably 1 to 3 times a day To the extent it is convenient to administer by intravenous injection. In the case of other parenteral administration and oral administration, an equivalent amount can be administered. If the symptoms are particularly severe, the dose may be increased accordingly.

The antibody of the present invention can be administered by itself or as a suitable pharmaceutical composition. The pharmaceutical composition used for the above administration contains the above or a salt thereof and a pharmacologically acceptable carrier, diluent or excipient. Such compositions are provided in dosage forms suitable for oral or parenteral administration.

That is, for example, compositions for oral administration include solid or liquid dosage forms, specifically tablets (including sugar-coated tablets and film-coated tablets), pills, granules, powders, capsules (soft capsules) ), Syrups, emulsions, suspensions and the like. Such a composition is produced by a method known per se and contains a carrier, diluent or excipient commonly used in the field of pharmaceuticals. For example, lactose, starch, sucrose, magnesium stearate and the like are used as carriers and excipients for tablets.

As compositions for parenteral administration, for example, injections, suppositories and the like are used. Injections are in the form of intravenous injections, subcutaneous injections, intradermal injections, intramuscular injections, drip injections and the like. Is included. Such an injection is prepared according to a method known per se, for example, It is prepared by dissolving, suspending or emulsifying the antibody or a salt thereof in a sterile aqueous or oily liquid commonly used for injections. Examples of aqueous solutions for injection include physiological saline, isotonic solutions containing glucose and other adjuvants, and suitable solubilizing agents, for example, alcohols (eg, ethanol), polyalcohols (eg, , Propylene glycol, polyethylene glycol), nonionic surfactants [eg, polysorbate 80, HCO-50 (polyoxyethylene (50 mol) adduct of hydrogenated castor oil)] and the like. As the oily liquid, for example, sesame oil, soybean oil, and the like are used, and benzyl benzoate, benzyl alcohol, and the like may be used in combination as a solubilizing agent. The prepared injection solution is usually filled in a suitable ampoule. A suppository for rectal administration is prepared by mixing the above antibody or a salt thereof with a usual suppository base.

The above-mentioned oral or parenteral pharmaceutical composition is conveniently prepared in a unit dosage form so as to be compatible with the dosage of the active ingredient. Examples of such dosage unit dosage forms include tablets, pills, capsules, injections (ampoules), suppositories and the like. Usually, each dosage unit dosage form is 5 to 500 mg, especially 5 to 1 mg for injections. It is preferable that the above-mentioned antibody is contained in 0 Omg, and 10 to 250 mg in other dosage forms.

Each of the above-mentioned compositions may contain other active ingredients as long as the composition does not cause an undesirable interaction with the above-mentioned antibody.

(5) Gene diagnostic agent

The DNA or antisense DNA of the present invention can be used, for example, by using it as a probe to produce a warm-blooded animal (eg, human, rat, mouse, guinea pig, egret, bird, higgie, bush, horse, cat, cat). , Dogs, monkeys, chimpanzees, etc.), it is possible to detect abnormalities (genetic abnormalities) in DNA or mRNA encoding the protein of the present invention or partial peptides thereof, such as damage to the DNA or mRNA, sudden It is useful as a gene diagnostic agent for mutation or decreased expression, increase of the DNA or mRNA or overexpression. The above-mentioned genetic diagnosis using the DNA of the present invention includes, for example, known Northern hybridization and PCR-SSCP method (Genomics, Vol. 5, pp. 874-879 (1989), Proceedings of the National Academy of Sciences' ob ''Sciences' ob '

Vol. 86, pp. 2766-2770 (1989)).

For example, when expression reduction is detected by Northern hybridization or when DNA mutation is detected by PCR-SSCP method, for example, arteriosclerosis, hyperlipidemia, obesity, diabetes Can be diagnosed as having a high possibility of being a disease such as

[6] a drug containing an antisense polynucleotide

The antisense polynucleotide of the present invention, which can complementarily bind to the polynucleotide (eg, DNA) of the present invention and suppresses the expression of the polynucleotide (eg, DNA), has low toxicity, Since the function of the protein of the present invention or its partial peptide or its salt, or the polynucleotide of the present invention (eg, DNA) can be suppressed, for example, it may be caused by overexpression of the protein of the present invention or its partial peptide. It can be used as a preventive or therapeutic agent for diseases (eg, obesity).

When the above antisense polynucleotide is used as the above therapeutic or prophylactic agent, the antisense polynucleotide can be formulated in the same manner as in the case of the above-described polynucleotide of the present invention.

The preparations obtained in this way have low toxicity and are orally or parenterally administered to humans or mammals (eg rats, puppies, higgs, bush, puppies, cats, dogs, monkeys, etc.). Can be administered in a controlled manner.

The antisense polynucleotide can be administered as it is or together with a physiologically acceptable carrier such as an auxiliary for promoting uptake by a gene gun or a catheter such as a hydrogel catheter.

The dose of the antisense polynucleotide varies depending on the target disease, the subject to be administered, the route of administration, and the like. For example, when the antisense nucleotide of the present invention is locally administered to an organ for the purpose of treating obesity, it can be administered to an adult ( Weight 60 kg) About 0.1 ~ per day: LOO mg.

Further, the antisense polynucleotide can also be used as a diagnostic oligonucleotide probe for examining the presence of the DNA of the present invention in a tissue or a cell and the state of its expression.

The present invention further provides

(1) A double-stranded RNA containing a part of the RNA encoding the protein of the present invention or a partial peptide thereof and an RNA complementary thereto,

(2) a medicament comprising the double-chain RNA,

(3) a ribozyme containing a part of RNA encoding the protein of the present invention or a partial peptide thereof,

(4) To provide a medicine containing the lipozyme.

These double-stranded RNAs (RNAi; RNA interference method), lipozymes, and the like can suppress the expression of the polynucleotide (eg, DNA) of the present invention in the same manner as the antisense polynucleotide described above. Since the function of the protein of the present invention or its partial peptide or a salt thereof or the polynucleotide (eg, DNA) of the present invention can be suppressed, for example, overexpression of the protein of the present invention or its partial peptide It can be used as a prophylactic or therapeutic agent for diseases caused by the disease (eg, obesity).

The double-stranded RNA can be designed and manufactured based on the sequence of the polynucleotide of the present invention according to a known method (eg, Nature, 411, 494, 2001). The lipozyme can be designed and manufactured based on the sequence of the polynucleotide of the present invention according to a known method (eg, TRENDS in Molecular Medicine, Vol. 7, pp. 221, 2001). For example, it can be produced by linking a known lipozyme to a part of RNA encoding the protein of the present invention or a partial peptide thereof. A part of the RNA encoding the protein of the present invention or a partial peptide thereof includes a portion (RNA fragment) adjacent to the cleavage site on the RNA of the present invention which can be cleaved by a known lipozyme.

When the above double-stranded RNA or lipozyme is used as the above-mentioned prophylactic / therapeutic agent, it can be formulated and administered in the same manner as an antisense polynucleotide. Monkey

[7] DNA transfer animal

The present invention relates to a DNA encoding an exogenous protein of the present invention (hereinafter abbreviated as the exogenous DNA of the present invention) or a mutant DNA thereof (the abbreviated as the exogenous mutant DNA of the present invention). A non-human mammal having the formula

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), wherein the non-human mammal is a rodent;

(3) The animal according to (2), wherein the rodent is a mouse or 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. Is what you do.

Non-human mammals having the exogenous DNA of the present invention or the mutant DNA thereof (hereinafter abbreviated as the DNA transgenic animal of the present invention) include unfertilized eggs, fertilized eggs, germ cells including spermatozoa and their progenitor cells, and the like. Preferably, during 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, It can be produced by transferring the desired DNA by a method such as a coagulation method, a coagulation method, a microinjection method, a particle gun method, or a DEAE-dextran method. Further, by the DNA transfer method, the exogenous DNA of the present invention can be transferred to somatic cells, organs of living organisms, tissue cells, and the like, and used for cell culture, tissue culture, and the like. The DNA transgenic animal of the present invention can also be produced by fusing the cells with the above-mentioned germ cells by a cell fusion method known per se.

As non-human mammals, for example, porcupines, pigs, sheep, goats, goats, egrets, dogs, cats, guinea pigs, eighty-one mussels, mice, rats and the like are used. Above all, rodents with a relatively short ontogeny and biological cycle in terms of the preparation of disease animal model systems, and easily breedable rodents, especially mice (for example, C57BL / 6 strains and DBA2 strains as pure strains) As a crossing system, BSCS Ft system, BDFi system, BeDSFi system, BALB / c system, ICR system, etc.) Is preferably a rat (eg, Wistar, SD, etc.).

"Mammals" in a recombinant vector that can be expressed in mammals include humans in addition to the above-mentioned non-human mammals.

The exogenous DNA of the present invention refers not to the DNA of the present invention originally possessed by non-human mammals, but to the DNA of the present invention once isolated and extracted from the mammal. As the mutant DNA of the present invention, a mutation (for example, mutation) in the base sequence of the original DNA of the present invention, specifically, addition or deletion of a base, DNA with substitution or the like is used, and also includes abnormal DNA. The abnormal DNA means a DNA that expresses an abnormal protein of the present invention, for example, a DNA that expresses a protein that suppresses the function of a normal protein of the present invention, and the like.

The exogenous DNA of the present invention may be derived from a mammal that is the same or different from the animal of interest. In transferring the DNA of the present invention to 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 transferred, it may be derived from various mammals (eg, egrets, dogs, cats, guinea pigs, hamsters, rats, mice, etc.) having the DNA of the present invention having high homology thereto. The present invention is achieved by microinjecting a DNA construct (eg, a vector) or the like to which the human DNA of the present invention is bound downstream of various promoters capable of expressing DNA into a fertilized egg of a target mammal, for example, a mouse fertilized egg. Thus, it is possible to create a DNA transgenic mammal that highly expresses DNA.

Examples of the expression vector of the protein of the present invention include a plasmid derived from Escherichia coli, a plasmid derived from Bacillus subtilis, a plasmid derived from yeast, a bacterium phage such as λ phage, a retrovirus such as Moroni leukemia virus, vaccinia virus or baculovirus. Animal viruses such as viruses are used. Among them, a plasmid derived from Escherichia coli, a plasmid derived from Bacillus subtilis, or a plasmid derived from yeast are preferably used.

Examples of the promoter that regulates DNA expression include, for example, (Eg, Simian virus, cytomegalovirus, Moroni leukemia virus, JC virus, breast cancer virus, poliovirus, etc.), promoters of various mammals (humans, egrets, canines, cats, guinea pigs, hams) Evening, rat, mouse, etc.), for example, albumin, insulin II, peroplacin II, elastase, erythropoietin, endothelin, muscle creatine kinase, glial fibrillary acidic protein, dalyuthione S-trans Ferrase, platelet-derived growth factor] 3, keratins Kl, K10 and K14, collagen type I and type II, cyclic AMP-dependent protein kinase / 3I subunit, dystrophin, tartrate-resistant alkaline phosphate Fase, atrial natriuretic factor, Endothelial receptor osteosynkinase (generally abbreviated as Tie 2), sodium potassium adenosine 3 kinase (Na, K-ATPase), neurofilament light chain, metamouth thionein I and IIA, meta Oral proteinase 1 tissue inhibitor, MHC class I antigen (H-2L), H-ras, renin, dopamine] 3-hydroxylase, thyroid peroxidase (TPO), polypeptide chain elongation factor 1 ( EF-1α), β-actin, a; and 3 myosin heavy chains, myosin light chains 1 and 2, myelin basic protein, thyroglobulin, Thy-1, immunoglobulin, heavy chain variable region (VNP), Serum amyloid P component, myoglobin, troponin C, smooth muscle α-actin, preproenkephalin Α, promoters such as vasopressin, etc. are used. Among them, a cytomegalovirus promoter capable of high expression throughout the body, a promoter of human polypeptide chain elongation factor 1a; (EF-1), a human and a chicken / 3-actin promoter, and the like are preferable. The vector preferably has a sequence that terminates transcription of a messenger RNA of interest in a DNA-transferred mammal (generally referred to as "taminator"). The DNA sequence of each of the above can be used, and preferably, Simian virus SV40 or the like is used.

In addition, in order to further express the desired exogenous DNA, the splicing signal of each DNA, the enhancer region, a part of the intron of eukaryotic DNA, etc. It is also possible to connect a promoter 5 ′ upstream of the promoter region, between the promoter region and the translation region, or 3 ′ downstream of the translation region.

The normal translation region of the protein of the present invention is derived from liver, kidney, thyroid cells, fibroblasts derived from various mammals (eg, humans, rabbits, dogs, cats, guinea pigs, hamsters, rats, mice, etc.). All or part of genomic DNA from DNA and various commercially available genomic DNA libraries, or complementary DNA prepared by known methods from liver, kidney, thyroid cell, and fibroblast-derived RNA as raw materials Can be done. In addition, a translation region obtained by mutating a translation region of a normal protein obtained from the above-described cells or tissue by a point mutagenesis method can be used for the exogenous abnormal DNA.

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

Transfer 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 animal after the transfer of the DNA indicates that all of the progeny of the animal and the foreign DN of the present invention will be present in all of the germinal and somatic cells. It means holding A. The progeny 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 germinal and somatic cells.

The non-human mammal to which the exogenous normal DNA of the present invention has been transferred is confirmed to stably maintain exogenous DNA by mating, and is subcultured as an animal having the DNA in a normal breeding environment. I can do it.

Transfer of the exogenous DNA of the present invention at the fertilized egg cell stage is ensured to be present in excess in all germinal 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 transfer indicates that all of the offspring of the produced animal contain the exogenous DNA of the present invention in all of its germinal and somatic cells. Means to have. The offspring of an animal of this species that has inherited the exogenous DNA of the present invention, It has A in excess.

By obtaining homozygous animals having the introduced DNA on both homologous chromosomes and mating the male and female animals, all offspring can be subcultured so as to have the DNA in excess.

In the non-human mammal having the normal DNA of the present invention, the normal DNA of the present invention is highly expressed, and the function of the protein of the present invention is ultimately enhanced by promoting the function of endogenous normal DNA. The disease may develop and can be used as a model animal for the disease. For example, using the normal DNA-transferred animal of the present invention 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 study treatment methods for these diseases. Is possible.

In addition, since the mammal into which the exogenous normal DNA of the present invention has been transferred has an increased symptom of the free protein of the present invention, it can be used for screening tests for therapeutic drugs against diseases related to the protein of the present invention. It is.

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 incorporated into the above-mentioned plasmid and used as a source substance. A DNA construct with a promoter can be prepared by ordinary DNA engineering techniques. The transfer 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 germ 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 the germ cells and somatic cells. . The offspring 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. By obtaining a homozygous animal having the introduced DNA on both homologous chromosomes, and breeding the male and female animals, it is possible to breed so that all offspring have the DNA.

Non-human mammals having the abnormal DNA of the present invention have a high incidence of the abnormal DNA of the present invention. Inhibition of the function of endogenous normal DNA may eventually result in a functionally inactive refractory of the protein of the present invention, which can be used as a disease model animal. For example, using the abnormal DNA-transferred 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.

In addition, as a specific possibility, the abnormal DNA highly expressing animal of the present invention is useful for inhibiting the function of the normal protein by the abnormal protein of the present invention (dominant negative action) in the inactive refractory disease of the protein of the present invention. It becomes a model to elucidate.

In addition, since the mammal into which the foreign abnormal DNA of the present invention has been transferred has an increased symptom of the released protein of the present invention, it can be used for a therapeutic drug screening test for a functionally inactive refractory disease of the protein of the present invention. It is.

In addition, other possible uses of the above two types of DNA transgenic animals of the present invention include, for example,

① Use as a cell source for tissue culture,

(2) By directly analyzing the DNA or RNA in the tissue of the DNA-transferred animal of the present invention or by analyzing the protein tissue expressed by the DNA, a protein specifically expressed or activated by the protein of the present invention can be identified. Analysis of the relevance of

(3) Cells from tissues having DNA are cultured by standard tissue culture techniques, and these are used to study the function of cells from tissues that are generally difficult to culture.

薬剤 Screening of drugs that enhance cell function by using the cells described in ③ above, and.

単離 Isolation and purification of the mutant protein of the present invention and production of its antibody are considered. Furthermore, using the DNA transgenic animal of the present invention, it is possible to examine clinical symptoms of diseases related to the protein of the present invention, including refractory inactive type of the protein of the present invention, etc. More detailed pathological findings in each organ of the disease model related to this protein can be obtained, which can contribute to the development of new treatment methods and the research and treatment of secondary diseases caused by the disease. . Further, it is possible to take out each organ from the DNA-transferred animal of the present invention, shred it, and then use a proteolytic enzyme such as tryp to obtain free DNA-transferred cells, culture them, or systematize the cultured cells. . Furthermore, the protein of the present invention and its effects can be examined by examining the relationship between the protein-producing cells of the present invention, its relation to differentiation or proliferation, or its signal transduction mechanism, and examining their abnormalities. It is an effective research material for elucidation.

Furthermore, in order to develop a therapeutic agent for a disease associated with the protein of the present invention, including the inactive type refractory type of the protein of the present invention, using the DNA-transferred animal of the present invention, the above-mentioned test was conducted. It is possible to provide an effective and rapid screening method for a therapeutic agent for the disease by using the method and the quantification method. Further, using the DNA transfer product of the present invention or the exogenous DNA expression vector of the present invention, it is possible to study and develop a method for treating a DNA associated with the protein of the present invention.

[8] Knockout animal

The present invention provides a non-human mammalian 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 | 3-galactosidase gene derived from Escherichia coli); (1) the embryonic stem cell according to the above,

(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 DNA-deficient non-human mammal in which the DNA of the present invention has been inactivated,

(7) The DNA is inactivated by introducing a reporter overnight gene (eg, a / 3-galactosidase gene derived from Escherichia coli), and the reporter gene is used in the present invention.

The non-human mammal according to (6), which can be expressed under the control of promoters for DNA overnight.

(8) the non-human mammal according to (6), wherein the non-human mammal is a rodent;

(9) the non-human mammal according to (8), wherein the rodent is a mouse; and (10) A test compound is administered to the animal described in (7), and the expression of a reporter gene is detected. A screening method is provided. A non-human mammalian embryonic stem cell in which the DNA of the present invention has been inactivated refers to a DNA of the present invention possessed by the non-human mammal, wherein the DNA of the present invention is artificially mutated to suppress the expression ability of the DNA, or By substantially eliminating the activity of the protein of the present invention encoded by the DNA, the DNA has substantially no ability to express the protein of the present invention (hereinafter referred to as the knockout DNA of the present invention). This may be referred to as a non-human mammalian embryonic stem cell (hereinafter abbreviated as ES cell).

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

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

Specific examples of the non-human mammalian embryonic stem cells in which the DNA of the present invention is 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 a neomycin resistance gene, a drug resistance gene typified by a hygromycin resistance gene, a 1 ac Z (one galactosidase enzyme), a cat (clo The exon function is destroyed by introducing a reporter gene or the like represented by the ramphenicol acetyltransferase gene, or a DNA sequence that terminates gene transcription in the intron between exons (for example, polyA addition signal) to prevent synthesis of the complete messenger RNA, resulting in gene DNA A DNA strand having a DNA sequence constructed so as to be disrupted (hereinafter abbreviated as a targeting vector) is introduced into the chromosome of the animal by, for example, a homologous recombination method, and the obtained ES cell is subjected to the DN of the present invention. Southern hybridization analysis or evening gettering using the DNA sequence on or near A The DNA sequence on the vector and the DNA sequence of the neighboring region other than the DNA sequence of the present invention used for the preparation of the getter vector were used as primers for PCR. it can be obtained by selecting _£ Further, as the phase parent ES cells to inactivate the DNA of the present invention by homologous recombination methods and the like, for example, may be used after a strain already established as described above It may be newly established according to the known method of Evans and Kaufma. For example, in the case of mouse ES cells, currently, 129 ES cells are generally used, but since the immunological background is not clear, an alternative pure immunological and genetically For the purpose of obtaining ES cells with a clear background, for example, BDFi mice (C57BLZ6 and DBAZ2 with C57BLZ6 and DBAZ2) BDFi mice can be used satisfactorily because they have a high number of eggs collected and their eggs are robust, and they have C57BLZ6 mice as their background. The ES cells obtained by using the cells can be advantageously used when a pathological model mouse is produced, because the genetic background can be replaced with C57BLZ6 mouse by backcrossing with C57B LZ6 mouse.

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

Either male or female ES cells may be used, but male ES cells are generally more convenient for producing breeding line chimeras. It is also desirable to discriminate between males and females as soon as possible in order to reduce the complexity of culturing.

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 preferably 100% of the normal number. However, if it is difficult due to physical operations at the time of establishment, knock out the gene of the ES cells, and It is desirable to clone again into cells with 2 n = 40).

Embryonic stem cell lines obtained in this way usually have very good proliferative properties, but must be carefully subcultured because they tend to lose their ontogenetic potential. For example, on a suitable feeder cell, such as STO fibroblasts, in a carbon dioxide incubator (preferably 5% carbon dioxide, 95% air or 5%) in the presence of LIF (1-10000 U / ml). Incubate at about 37 ° C in 5% oxygen, 5% carbon dioxide, 90% air) .At the time of subculture, for example, trypsin / EDTA solution (usually 0.001-0.5% trypsin 0.1-5mM EDTA, preferably Can be converted into single cells by treatment with about 0.1% trypsin / ImM EDTA) and seeded on a freshly prepared feeder cell. Such subculture is usually performed every 1 to 3 days. At this time, it is desirable to observe the cells, and if any morphologically abnormal cells are found, discard the cultured cells.

ES cells are differentiated into various types of cells, such as parietal, visceral, and cardiac muscles, by monolayer culture up to high density or suspension culture until cell clumps are formed under appropriate conditions. [MJ Evans and MH Kaufman, Nature, Vol. 292, p. 154, 1981; GR Martin Proceedings of the National Academy of Sciences. Proc. Natl. Acad. Sci. USA) 78, 7634, 1981; TC Doetschman et al., Journal of Ob. However, the DNA-deficient cells of the present invention obtained by differentiating the ES cells of the present invention are useful in examining the cell biology of the protein of the present invention in the mouth of in vivo.

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. As the non-human mammal, those similar to the aforementioned can be used.

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

The cells in which the DNA of the present invention was knocked out were used for Southern hybridization analysis or a DNA sequence on a targeting vector using the DNA sequence on or near the DNA of the present invention as a probe, and a targeting vector. It can be determined by analysis by PCR using a mouse as a primer and a DNA sequence in a neighboring region other than the DNA of the present invention derived from a mouse. When a non-human mammalian embryonic stem cell is used, a cell line in which the DNA of the present invention has been inactivated by gene homologous recombination is cloned, and the cell is cultured at an appropriate time, for example, at the 8-cell stage. 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 composed of both a normal cell having the DNA locus of the present invention and an artificially mutated cell having the 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 obtained from the population obtained by crossing such a chimeric individual with a normal individual. It can be obtained by selecting individuals composed of cells having the DNA locus of the present invention with mutation added thereto, for example, by judging coat color or the like. The individual thus obtained is usually an individual having a heterozygous expression of the protein of the present invention, which is crossed with an individual having a heterozygous expression of the protein of the present invention, and homozygous expression of the protein of the present invention is obtained 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 getter vector introduced into a chromosome can be obtained by injecting a DNA solution into the egg cell nucleus by a microinjection method. Of the present invention by homologous recombination compared to non-human mammals Obtained by selecting those with mutations in the DNA locus.

When the DNA of the present invention is knocked out in this manner, the animal obtained by mating is also confirmed to have knocked out the DNA, and is reared in a normal rearing environment. be able to.

Furthermore, the acquisition and maintenance of the germ line may be performed according to a conventional method. That is, by mating male and female animals having the inactivated DNA, a homozygote animal having the inactivated DNA on both homologous chromosomes can be obtained. The obtained homozygous animal can be efficiently obtained by rearing the mother animal in such a manner that one normal individual and one homozygote are obtained. The homozygous and heterozygous animals having the inactivated DNA are bred by crossing male and female heterozygous animals.

The 'non-human DNA-inactivated non-human' mammalian embryonic stem cells are very useful for producing the 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, it may be caused by inactivation of the biological activity of the protein of the present invention. It is useful for investigating the causes of these diseases and examining treatment methods, because they can serve as a model for such diseases.

[8a] A method for screening a compound having a therapeutic / preventive effect against diseases caused by DNA deficiency or damage according to the present invention

The non-human mammal deficient in expression of the DNA of the present invention is treated for diseases (eg, arteriosclerosis, hyperlipidemia, obesity, diabetes, etc.) caused by the DNA deficiency or damage of the present invention. It can be used for screening for compounds having a prophylactic effect. That is, the present invention is characterized by administering a test compound to a non-human mammal deficient in expressing DNA of the present invention and observing and measuring changes in the animal. The present invention provides a method for screening a compound or a salt thereof having a therapeutic or preventive effect on a disease to be treated.

Examples of the non-human mammal deficient in DNA expression of the present invention used in the screening method include the same ones as described above.

Test compounds include, for example, peptides, proteins, non-peptidic compounds, Examples include synthetic compounds, fermentation products, cell extracts, plant extracts, animal tissue extracts, and plasma. These compounds may be novel compounds or known compounds.

Specifically, a non-human mammal deficient in expression of the DNA of the present invention is treated with a test compound, compared with a non-treated control animal, and changes in organs, tissues, disease symptoms, etc. of the animal are used as indices. The test compound can be tested for its therapeutic and prophylactic effects.

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

For example, when screening a compound having a therapeutic / prophylactic effect against arteriosclerosis, a glucose-loading treatment is administered to a non-human mammal deficient in expression of the DNA of the present invention, and the test compound is administered before or after the glucose-loading treatment. Is administered, and the blood glucose level and weight change of the animal are measured over time.

In the screening method, when a test compound is administered to a test animal, if the blood glucose level of the test animal decreases by about 10% or more, preferably about 30% or more, more preferably about 50% or more, the test The compound can be selected as a compound having therapeutic and preventive effects on arteriosclerosis.

The compound obtained by using the screening method of the present invention is a compound selected from the test compounds described above, and is a disease caused by deficiency or damage of the protein or the like of the present invention (eg, arteriosclerosis, etc.). ) Has therapeutic and prophylactic effects, and thus can be used as a safe and low-toxic therapeutic and prophylactic agent for the disease. 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) and bases (eg, alkali metals). 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) or organic acids (eg, acetic acid, formic acid, propio Acid, fumaric acid, maleic acid, succinic acid, tartaric acid, citric acid, malic acid, oxalic acid, benzoic acid, methanesulfonic acid, benzenesulfonic acid). 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, mammals (eg, humans, rats, mice, guinea pigs, egrets, higgs, bushus, dogs, dogs, cats, dogs) , Monkeys, etc.).

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 for the purpose of treating arteriosclerosis, it is generally required for an adult (body weight 6 (As O kg), the compound is administered from about 0.1 to about 100 mg L per day, preferably from about 1.0 to 50 mg, more preferably from about 1.0 to 2 Omg. In the case of parenteral administration, the single dose of the compound varies depending on the administration subject, the target disease, and the like. For example, the compound is usually administered in the form of a propellant for the treatment of arteriosclerosis in an adult ( (As 6 O kg), the compound is intravenously injected at a rate of about 0.01 to 3 Omg, preferably about 0.1 to 20 mg, more preferably about 0.1 to 1 Omg per day. It is convenient to administer by the following. For other animals, the dose can be administered in terms of 6 O kg.

[8b] A method for screening a compound that promotes the activity of a promoter for DNA of the present invention

The present invention provides a test compound administered to a non-human mammal deficient in expression of the DNA of the present invention to detect the expression of a reporter gene, thereby promoting the activity of the promoter for the DNA of the present invention. Or a method for screening a compound or a salt thereof to be inhibited.

In the above-described screening method, the non-human mammal deficient in expression of the DNA of the present invention includes, among the non-human mammals deficient in expressing the DNA of the present invention, the DNA of the present invention inactivated by introducing a reporter gene. Wherein the gene can be expressed under the control of a promoter for the DNA of the present invention. Is used.

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 j8-galactosidase gene (lacZ), a soluble alkaline phosphatase gene or a luciferase gene is 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 repo overnight gene, since the reporter gene is under the control of a promoter for the DNA of the present invention, the reporter gene By tracing the expression of the substance encoded by the promoter, one can detect the activity of the promoter overnight.

For example, when a part of the DNA region encoding the protein of the present invention is replaced by a β-galactosidase gene (1 ac 由来) derived from Escherichia coli, the tissue that originally expresses the protein of the present invention Thus, jS_galactosidase is expressed instead of the protein of the present invention. Therefore, for example, staining is carried out using a reagent which is a substrate of / 3-galactosidase, such as 5-bromo-4-chloro-3, -indolyl-] 3-galactopyranoside (X-ga1). This makes it possible to easily observe the expression state of the protein of the present invention in an animal body. Specifically, the protein-deficient mouse of the present invention or a tissue section thereof is fixed with dartalaldehyde or the like, washed with phosphate buffered saline (PBS), and then stained with X-ga1 at room temperature or at 37 ° C. After reacting at about 30 ° C. for about 30 minutes to 1 hour, the j3-galactosidase reaction is stopped by washing the tissue specimen with a 1 mM EDTAZPBS solution, and the coloration may be observed. Further, mRNA encoding 1acZ may be detected according to a conventional method.

The compound or a salt thereof 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 DNA promoter activity of the present invention.

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

The compound or salt thereof that promotes the promoter activity of DNA of the present invention over the entire DNA can promote the expression of the protein of the present invention and promote the function of the protein. For example, arteriosclerosis, hyperlipidemia, etc. It is useful as a safe and low toxic therapeutic and prophylactic agent for diseases such as diabetes, obesity, and diabetes.

On the other hand, the compound of the present invention which inhibits the activity of the promoter against DNA, or a salt thereof, can inhibit the expression of the protein of the present invention and inhibit the function of the protein. It is useful as a drug such as a safe and low-toxic treatment and prophylactic agent for the drug.

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

The preparations obtained in this way are safe and have low toxicity and can be used, for example, in mammals (for example, humans, rats, mice, guinea pigs, egrets, sheep, pigs, pigs, dogs, cats, dogs, Monkeys).

The dose of the compound or a salt thereof varies depending on the target disease, the administration subject, the administration route, and the like. When administered, generally, for an adult (assuming a body weight of 60 kg), the compound is administered at about 0.1 to 100 mg, preferably about 1.0 to 5 Omg, more preferably about 1.0 to 2 Omg per day. When administered parenterally, a single dose of the compound varies depending on the administration subject, target disease, and the like, but, for example, it promotes the promoter activity of the DNA of the present invention for the purpose of treating arteriosclerosis. When the compound to be administered is usually administered to an adult (as 6 Okg) in the form of an injection, the compound is administered in an amount of about 0.01 to 3 Omg per day, preferably about 0.1 to 2 Omg, more preferably about 0.1 to 2 Omg. It is convenient to administer about 0.1 to about 10 mg by intravenous injection. In the case of other animals, the dose can be administered in terms of 60 kg. As described above, the non-human mammal deficient in expression of the DNA of the present invention is extremely useful for screening a compound or its salt that promotes or inhibits the activity of the promoter of the DNA of the present invention, and the DNA of the present invention. It can greatly contribute to investigating the cause of various diseases caused by expression deficiency or to develop therapeutic drugs. In addition, using a DNA containing the promoter overnight 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 (gene). Creating an introduced animal) will allow the protein to be specifically synthesized and its effects on the organism to be studied. Furthermore, by binding an appropriate reporter gene to the above promoter portion and establishing a cell line that expresses it, the action of specifically promoting or suppressing the in vivo production ability of the protein of the present invention itself can be achieved. It can be used as a search system for low molecular weight compounds having By analyzing the promoter portion, it is also possible to find a new cis element and a transcription factor binding thereto. In the present specification and drawings, bases, amino acids, and the like are indicated by abbreviations based on the fflg issued by the IUBA Commission on Biochemical iSiomenclature or the commonly used abbreviations in the art, and examples thereof are described below. When amino acids may have optical isomers, the L-form is indicated unless otherwise specified.

DNA: Deoxyribonucleic acid

cDNA: Complementary deoxylipo nucleic acid

A: Adenine

T: Thymine

G: Guanin

C: Shitoshin

RNA: Liponucleic acid

mRNA: messenger liponucleic acid

dATP: Deoxyadenosine triphosphate

dTTP: Deoxythymidine triphosphate

dGTP: Deoxyguanosine diphosphate d CTP Deoxycytidine triphosphate

ATP Adenosine triphosphate

EDTA ethylenediaminetetraacetic acid

SD S Sodium dodecyl sulfate

G 1 y Glycine

A la alanine

V a 1 Valine

Leu leucine

I 1 e isoleucine

S e r serine

Th r threonine

Cy s,

Me t Methionin

G 1 u Glutamic acid

As aspartic acid

Lys lysine

A r g Arginine

H is histidine

P h e feniralanin

T y r tyrosine

T r p tryptophan

Pro proline

A s n asparagine

G 1 n Glutamine

p G 1 u pyroglutamic acid

The substituents, protecting groups and reagents frequently used in the present specification are represented by the following symbols.

Me: methyl group

E t: ethyl group B u: butyl group

P: phenyl group

TC: thiazolidine_4 (R) —carpoxamide group

To s: p-toluenesulfonyl

CHO: Formyl

B z 1: benzyl

Cl ₂ Bzl: 2,6-dicyclo benzene

Bom: benzyloxymethyl

Z: benzyloxycarponyl

C 1-Z: 2-clozen benzyloxycarponyl

B r— Z.: 2-bromobenzyloxycarponyl

Bo c: t-butoxycarponyl

DNP: dinitrophenyl

Trt: Trityl

Bum: t-butoxymethyl

Fmo c: N-9-fluorenylmethoxycarbonyl

HOB t: 1-hydroxybenztriazole

HOOB t: 3,4-dihydro-3-hydroxy-4-oxo-1

1,2,3-benzotriazine

HONB: 1-hydroxy-5-norpolene-2,3-dicarpoxyimide The sequence numbers in the sequence listing in the present specification show the following sequences.

[SEQ ID NO: 1]

1 shows the amino acid sequence of the human adipocyte-derived protein (LIP-5) of the present invention. [SEQ ID NO: 2]

1 shows the nucleotide sequence of DNA encoding the human adipocyte-derived protein (LIP-5) of the present invention having the amino acid sequence represented by SEQ ID NO: 1.

[SEQ ID NO: 3]

In Example 1, the DNA encoding the human-derived protein of the present invention was cloned. The base sequence of the synthetic primer used for the pairing is shown.

[SEQ ID NO: 4]

In Example 1, the nucleotide sequence of a synthetic primer used for cloning DNA encoding the human-derived protein of the present invention is shown.

[SEQ ID NO: 5]

3 shows the nucleotide sequence of a synthetic primer used in the analysis of the expression distribution in human tissues in Example 2.

[SEQ ID NO: 6]

1 shows the nucleotide sequence of a synthetic primer used in the analysis of the expression distribution in human tissues in Example 2.

Escherichiacoli DH5cu / pTB2177 carrying the plasmid ρΤΒ2177 obtained in Example 1 described below has been used since January 13, 2000, Tsukuba East 1-chome, 1-1, Ibaraki Pref. 305-8 566) at the National Institute of Advanced Industrial Science and Technology (AIST) Patent Depositary Depositary Center (formerly NIBH, National Institute of Advanced Industrial Science and Technology (NI BH)) under the deposit number FERM B P-7364 in 2000. It has been deposited with the Fermentation Research Institute (IFO) at 2-1 '7-85, Yodogawa-cho, Yodogawa-ku, Osaka, Japan since October 31 under the deposit number IF 0 16495.

Example

Hereinafter, the present invention will be described more specifically with reference to Examples, but the present invention is not limited thereto. In addition, the genetic engineering method using Escherichia coli followed the method described in Molecular Cloning. Example 1 Cloning of cDNA encoding human adipocyte-derived lipolytic enzyme protein and determination of nucleotide sequence

Using a human adipocyte Marathon-Ready cDNA (CLONTECH) as a cyclized form, a PCR reaction was carried out using two primers, primer 1 (SEQ ID NO: 3) and primer 1 2 (SEQ ID NO: 4). The composition of the reaction solution used in the reaction was as follows: cDNA 2 ^ 1 was used as a type II, Pfu Turbo DNA polymerase (STRATAGENE) 0.2 ^ 1 amount, primer 1 (SEQ ID NO: 3) and primer 2 (SEQ ID NO: 4) Is 0.5 iM each, 200 M of each dNTPs and 21 of the buffer attached to the enzyme were added to make a volume of 201. The PCR reaction is repeated at 94 ° C for 2 minutes, followed by a cycle of 94 ° C for 1 minute, 65 ° C for 1 minute, and 72 ° C for 2 minutes 35 times. Was done. After protruding adenine at the 3 'end using ExTaq (Takara Shuzo), the PCR reaction product was used, and the plasmid vector pCR2, l was used according to the recipe of DNA Ligation Kit Ver.2 (Takara Shuzo).

(Invitrogen). This was introduced into Escherichia coli DH5, and a clone having a cDNA (insert) was selected in an LB agar medium containing ampicillin. As a result of analyzing the nucleotide sequence of each clone, a clone containing a DNA having the nucleotide sequence represented by SEQ ID NO: 2 and a cDNA (insert) encoding a novel neutral lipolytic enzyme protein was obtained. . This clone (transformant) is

(Escherichia coli) DH5α / ρΤΒ2177.

In addition, a novel lipolytic enzyme protein containing an amino acid sequence (SEQ ID NO: 1) derived from the nucleotide sequence represented by SEQ ID NO: 2 was named LIΡ-5.

Example 2 Examination of expression distribution in human tissues

22 types (1. adipose tissue, 2. aorta, 3. bone marrow, 4. adult brain, 5. large intestine, 6. fetal brain, 7. heart, 8. kidney, 9. leukocyte, 10. liver, 11. lung, 12. Mammary gland, 13. Ovary, 14. Spleen, 15. Prostate, 16. Skeletal muscle, 17. Small intestine, 18. Spleen, 19. Testis, 20. Thymus, 21. Uterus, 22. Adrenal gland) A Marathon-Ready cDNA (CLONTECH) was used as type II, and a PCR reaction was performed using two primers, Primer 1 (SEQ ID NO: 5) and Primer 2 (SEQ ID NO: 6). The composition of the reaction solution used in this reaction was as follows: 0.5 l of the above cDNA was used as type II, and 0.11 amount of ExTaq (Takara Shuzo), Primer-1 (SEQ ID NO: 5) and Primer-2 (SEQ ID NO: 6) were 0.5 200 M of each of M and dNTPs and 21 of the buffer attached to the enzyme were added to make a liquid volume of 201. The PCR reaction was repeated at 94 ° C for 1 minute, followed by a cycle of 94 ° C for 30 seconds, 6030 seconds, and 72 minutes for 35 minutes, followed by extension at 72 ° C for 5 minutes. . The obtained reaction solution 5 ^ 1 was analyzed by 1.5% agarose gel electrophoresis. The length of the PCR product was 324 bp.

As a result, LIP-5 was expressed in adipose tissue, aorta, large intestine, kidney, mammary gland, ovary, prostate, small intestine, spleen, testis, uterus and adrenal gland.

Figure 1 shows the results of LIP-5 expression distribution. Industrial applicability

The protein of the present invention and DNA encoding the same can be used as a therapeutic / prophylactic agent for diseases such as arteriosclerosis, hyperlipidemia, obesity, and diabetes. In addition, a cell capable of expressing the protein of the present invention or the gene of the protein of the present invention is useful as a reagent for screening a compound or a salt thereof that promotes or inhibits the esterase activity of the protein of the present invention. . Furthermore, since an antibody against the protein of the present invention can specifically recognize the protein of the present invention, it can be used for quantification of the protein of the present invention in a test solution.

Claims

The scope of the claims

1. A protein containing an amino acid sequence identical or substantially identical to the amino acid sequence represented by SEQ ID NO: 1, or a salt thereof.

2. The protein or salt thereof according to claim 1, which comprises the amino acid sequence represented by SEQ ID NO: 1.

3. A partial peptide of the protein according to claim 1, or a salt thereof.

4. The protein according to claim 1, which has an esterase activity, or a salt thereof, or the partial peptide according to claim 3, or a salt thereof.

5. A polynucleotide comprising the polynucleotide encoding the protein according to claim 1 or the partial peptide according to claim 2.

6. The polynucleotide according to claim 5, which is a DNA.

7. The DNA according to claim 6, comprising the nucleotide sequence represented by SEQ ID NO: 2.

8. A recombinant vector containing the polynucleotide according to claim 5.

9. A transformant transformed with the recombinant vector according to claim 8.

10. 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 2, and collect the protein. A method for producing the partial peptide or a salt thereof according to claim 3.

1 1. A medicament comprising the protein according to claim 1 or the partial peptide according to claim 3 or a salt thereof.

12. A medicament comprising the polynucleotide according to claim 5.

13. The medicament according to claim 11 or 12, which is an agent for treating or preventing arteriosclerosis, hyperlipidemia, obesity or diabetes.

14. An antibody against the protein according to claim 1, the partial peptide according to claim 3, or a salt thereof.

15. A medicament comprising the antibody according to claim 14.

16. A diagnostic agent comprising the antibody according to claim 14.

17.Encodes the protein of claim 1 or the partial peptide of claim 3 Antisense DNA comprising a base sequence complementary to the DNA to be formed or a part thereof.

18. A drug comprising the antisense DNA according to claim 17.

19. A diagnostic agent comprising the antisense DNA according to claim 17. .

20. The protein according to claim 1 or the partial peptide according to claim 3 or a salt thereof, wherein the proteinase according to claim 1 or the partial peptide according to claim 2 or a salt thereof promotes esterase activity or A method for screening a compound having an inhibitory activity or a salt thereof.

21. The esterase activity of the protein of claim 1 or the partial peptide of claim 3 or a salt thereof comprising the protein of claim 1 or the partial peptide of claim 3 or a salt thereof. A kit for screening a compound having a promoting or inhibiting activity or a salt thereof.

22. Promote the esterase activity of the protein of claim 1, or the partial peptide of claim 3, or a salt thereof obtained by using the screening method of claim 20 or the screening kit of claim 21. Or a compound having an inhibitory activity or a salt thereof.

23. Promote the esterase activity of the protein according to claim 1 or the partial peptide according to claim 3 or a salt thereof obtained by using the screening method according to claim 20 or the screening kit according to claim 21. A medicament comprising a compound having an activity of causing or a salt thereof.

24. The medicament according to claim 23, which is an agent for treating or preventing arteriosclerosis, hyperlipidemia, obesity or diabetes.

25. Inhibits the esterase activity of the protein of claim 1 or the partial peptide of claim 3 or a salt thereof obtained by using the screening method of claim 20 or the screening kit of claim 21. A pharmaceutical comprising an active compound or a salt thereof.

26. The medicament according to claim 25, which is an agent for treating and preventing obesity.

27. The tannin according to claim 1, which can be obtained from a mammal using the screening method according to claim 20 or the screening kit according to claim 21. Atherosclerosis, hyperlipidemia, obesity, which comprises administering an effective amount of a compound having an activity of promoting the esterase activity of the protein or the partial peptide of claim 3 or a salt thereof, or a salt thereof. How to prevent or treat diabetes or diabetes.

28. The protein according to claim 1 or the partial peptide according to claim 3, which can be obtained from a mammal using the screening method according to claim 20 or the screening kit according to claim 21. Or a method for preventing and treating obesity, which comprises administering an effective amount of a compound having an activity of inhibiting the esterase activity of a salt thereof or an effective amount of a salt thereof.

29. The screening method according to claim 20 or the screening kit according to claim 21 for producing a prophylactic / therapeutic agent for arteriosclerosis, hyperlipidemia, obesity or diabetes. Use of a compound having an activity of promoting esterase activity of the protein according to claim 1 or the partial peptide according to claim 3, or a salt thereof, or a salt thereof, which can be obtained.

30. The protein or claim according to claim 20, which can be obtained by using the screening method according to claim 20 or the screening kit according to claim 21 for producing an agent for preventing or treating obesity. Use of a compound having an activity of inhibiting the esterase activity of the partial peptide or the salt thereof according to Item 3, or a salt thereof.

31. A non-human mammal having an exogenous DNA encoding the protein according to claim 1 or the partial peptide according to claim 3, or a mutant DNA thereof.

32. The animal of claim 31, wherein the non-human mammal is a rodent.

33. The animal of claim 32, wherein the rodent is a mouse or a rat.

34. A recombinant vector containing an exogenous DNA encoding the protein according to claim 1 or the partial peptide according to claim 3 or a mutant DNA thereof and capable of being expressed in a non-human mammal.

35. A non-human mammalian embryonic stem cell in which DNA encoding the protein according to claim 1 or the partial peptide according to claim 3 has been inactivated.

36. The embryonic stem cell according to claim 35, wherein said DNA has been inactivated by introducing a reporter gene.

37. The embryonic stem cell of claim 35, which is neomycin resistant.

38. The embryonic stem cell of claim 35, wherein the non-human mammal is a rodent.

39. The embryonic stem cell according to claim 38, wherein the rodent is a mouse.

40. A non-human mammal deficient in DNA expression, wherein the DNA encoding the protein according to claim 1 or the partial peptide according to claim 3 is inactivated.

41. The non-human mammal according to claim 40, wherein the DNA is inactivated by introducing a reporter gene, and the reporter gene can be expressed under the control of a promoter for the DNA.

42. The non-human mammal of claim 40, wherein the non-human mammal is a rodent.

43. The non-human mammal of claim 42, wherein the rodent is a mouse.

44. A method for screening a compound or a salt thereof that promotes or inhibits the activity of a promoter for DNA, comprising administering a test compound to the animal according to claim 41 and detecting expression of a reporter gene.