JP2003000272A - New protein and its dna - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain a new esterase protein. SOLUTION: The new protein containing an amino acid sequence originated from a human being. The new protein containing the same or substantially same amino acid sequence as the above amino acid sequence. A partial polypeptide of the new protein or its salt. A DNA encoding the protein. A method for producing the protein. A medicine containing the protein or DNA. A method for screening a compound or its salt stimulating or inhibiting the esterase activity of the protein. A compound obtained by the screening method. A medicine containing the compound. Thereby, the protein, and the like, can be used as medicines for treating and preventing diseases such as arteriosclerosis, hyperlipemia, obesity, and diabetes. Further, the protein, and the like, are useful as materials for screening compounds or their salts having an activity to stimulate or inhibit the esterase activity of the protein.

Description

Detailed Description of the Invention

[0001]

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

[0002]

2. Description of the Related Art It is well known that lipases and esterases play important roles in digestion and absorption of dietary lipids, decomposition and accumulation of lipids in vivo, detoxification of harmful compounds and the like. Among them, hormone-sensitive lipase (Hormone-sensitive lipase, hereinafter abbreviated as HSL), which is widely expressed in tissues such as adipose tissue, testis, heart, skeletal muscle, pancreas, and aorta, is
It has been attracting attention as a regulator of fat accumulation and energy consumption in the body, glucose metabolism or arteriosclerotic lesion formation (Holm,
C and Osterlund, T. Methods in Molecular Biology,
Vol. 109, pp109-121). That is, HSL is a rate-limiting enzyme for degrading triglyceride (particularly triglyceride) in each tissue and produces energy in muscle tissue and brown adipocytes (Himms-Hagen, J.Prog. Lipid Res. 28, 67-115 (198
9)) and ligand production of nuclear receptors PPARs (Tontonoz, Pe
t al Cell 79, 1147-1156 (1994)) not only regulates the supply of fatty acids but also regulates insulin secretion in pancreatic β cells (Mulder, H et al Diabetes 48,
228-232 (1999)) has also been reported to be involved. In aorta and macrophages, neutral cholesteryl ester hydrolase (Neutral cholesterylester hydase) is responsible for the degradation of intracellular cholesteryl ester.
rolase (NCEH) has been suggested to be involved in the formation and regression of atherosclerotic plaques (Escarry, JL et al Arterioscler. Thromb. Vasc. B).
iol. 18, 991-998). However, in HSL-deficient mice produced by Osuga et al., Not only was there no difference in body weight from wild-type mice, but also hormone-sensitive triglyceride degrading activity in adipocytes and NCEH activity in macrophages remained significantly. In addition, there was no change in body heat production under cold conditions (Osuga, J. et al Pr.
oc. Natl. Acad. Sci. USA 97, 787-792 (2000)). Therefore, it is strongly suggested that other HSL-like lipases or esterases are involved in various lipolytic processes that have been conventionally associated with HSLs, and such lipases or esterases are By being involved in fatty acid production in cells and muscle tissues and foam formation of macrophages, arteriosclerosis and hyperlipidemia,
It is expected that it may play an important role in the development of diabetes and obesity. Bile salt-activated lipase (Li, F. a) has been reported as a neutral lipolytic enzyme that has been reported to be expressed in adipose tissue, aorta or macrophages.
nd Hui, DYJ Biol. Chem. 272, 28666-28671 (199
7)), lipoprotein lipase (Mattsson, L. et al J. Clin.
Invest. 92, 1759-1765 (1993)), monocyte / macrophage serine esterase-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. Biop
hys. Acta 424, 204-212 (1976)) are known, but these are localized in tissues and cells, substrate specificity, optimum pH of activity, presence of hormone sensitivity, macrophage foaming process. From the activity fluctuation pattern of
Since it is unlikely that the function of HSL can be replaced, it was desired to isolate a new lipase or esterase derived from adipocytes and macrophages.

[0003]

DISCLOSURE OF THE INVENTION The present invention provides a novel protein or partial peptide having esterase activity or the like or a salt thereof, a DNA encoding the protein or a partial peptide thereof, a recombinant vector, a transformant,
Method for producing the protein or its partial peptide or its salt, a drug containing the protein or its partial peptide or its salt or its DNA, an antibody against the protein or its partial peptide or its salt, the said protein or its partial peptide Also provided are a screening method / screening kit for a compound having an activity of promoting the esterase activity of a salt thereof, a compound obtained by the screening method, a medicine containing the compound, and the like.

[0004]

[Means for Solving the Problems] The GXSXG sequence (X represents an arbitrary amino acid) and the HG (or HGG) sequence located upstream thereof are known as amino acid sequences characteristic of HSL and its related lipases or esterases. (Har
ri, H et al Biochim. Biophys. Acta 1210, 249-253
(1994)). The present inventors have focused their attention on these sequences and have energetically searched for genes that are expressed in adipose tissue or macrophages, and as a result, 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 that replaces HSL. In addition, an animal with altered expression of the gene is useful as a novel model animal for arteriosclerosis, hyperlipidemia, obesity, and diabetes, and affects the expression of this gene and the activity of the protein encoded by this gene. The drug is useful as a therapeutic drug for arteriosclerosis, hyperlipidemia, obesity and diabetes based on a novel mechanism. The present inventors have completed the present invention as a result of further studies based on these findings.

That is, the present invention provides (1) SEQ ID NO: 1.
A protein or its salt containing the same or substantially the same amino acid sequence as the amino acid sequence represented by
(2) The protein or salt thereof described in (1) above, which contains the amino acid sequence represented by SEQ ID NO: 1, (3) a partial peptide or salt thereof of the protein described in (1) above, and (4) having esterase activity. It contains a protein or a salt thereof described in (1) above, a partial peptide or a salt thereof described in (3) above, and (5) a polynucleotide encoding the protein described in (1) above or a partial peptide described above in (2). Polynucleotide, (6)
The polynucleotide according to (5) above, which is DNA.
(7) The DNA according to (6) above, which contains the nucleotide sequence represented by SEQ ID NO: 2, (8) the recombinant vector containing the polynucleotide according to (5) above, (9) the above (8) above. (10) The transformant transformed with the recombinant vector, (10) the transformant described in (9) above is cultured to produce and accumulate the protein described in (1) above or the partial peptide described in (2) above, (1) A method for producing the protein according to (1) above, or the partial peptide according to (3) above or a salt thereof, which comprises:
1) A medicament comprising the protein according to (1) above, the partial peptide according to (3) above, or a salt thereof,
(12) A medicament comprising the polynucleotide according to (5) above, (13) a therapeutic or prophylactic agent for arteriosclerosis, hyperlipidemia, obesity or diabetes (11) or (1).
(2) The medicine, (14) the protein described in (1) above, the partial peptide described in (3) above, or a salt thereof, (15) a medicine comprising the antibody described in (14) above, (16) ) A diagnostic agent comprising the antibody according to (14) above, (17) a DNA encoding the protein according to (1) above or the partial peptide according to (3) above.
(18) A drug comprising the antisense DNA according to (17) above, (19) above (17)
A diagnostic agent containing the described antisense DNA, (2
0) The esterase activity of the protein according to (1) above, the partial peptide according to (2) above, or the partial peptide according to (2) above, wherein the protein according to (1) above, the partial peptide according to (3) above, or a salt thereof is used. (21) A method for screening a compound or a salt thereof having an activity of promoting or inhibiting, a protein according to (1) above, which comprises the protein according to (1) above or the partial peptide according to (3) above or a salt thereof. Alternatively, a screening kit for a compound or a salt thereof that has an activity of promoting or inhibiting the esterase activity of the partial peptide or salt thereof according to (3) above, (22) above (20)
Having the activity of promoting or inhibiting the esterase activity of the protein of (1) above, the partial peptide of (3) above, or a salt thereof obtained by using the screening method described above or the screening kit described in (21) above. Compound or salt thereof, (23) above (2
0) obtained by using the screening method described in 0) or the screening kit described in 21)
(24) A pharmaceutical comprising a compound having the activity of promoting the esterase activity of the protein according to (3) or the partial peptide according to (3) above or a salt thereof, (24)
The medicament according to (23) above, which is a therapeutic / preventive agent for arteriosclerosis, hyperlipidemia, obesity or diabetes, (25) above (2).
0) obtained by using the screening method described in 0) or the screening kit described in 21)
(26) A pharmaceutical comprising a compound having the activity of inhibiting the esterase activity of the protein according to (3) or the partial peptide according to (3) above or a salt thereof, (26)
The medicament according to (25) above, which is a therapeutic / preventive agent for obesity,
(27) The protein according to (1) or the partial peptide according to (3), which can be obtained from a mammal using the screening method according to (20) or the screening kit according to (21). Or a method for preventing and / or treating arteriosclerosis, hyperlipidemia, obesity or diabetes, which comprises administering an effective amount of a compound having an activity of promoting esterase activity of a salt thereof or a salt thereof (28) For a mammal, the protein according to (1) above, the partial peptide according to (3) above, or a protein thereof, which can be obtained using the screening method according to (20) above or the screening kit according to (21) above. Prevention of obesity, which comprises administering an effective amount of a compound having an activity of inhibiting esterase activity of salt or a salt thereof Treatment method, (29) Use of the screening method according to the above (20) or the screening kit according to the above (21) for producing a prophylactic / therapeutic agent for arteriosclerosis, hyperlipidemia, obesity or diabetes Use of a compound having the activity of promoting the esterase activity of the protein according to (1) above, the partial peptide according to (3) above, or a salt thereof that can be obtained, or (30) a prophylactic / therapeutic agent for obesity Or the screening method according to (2) above for producing
Use of a compound or a salt thereof having an activity of inhibiting the esterase activity of the protein according to (1) above, the partial peptide according to (3) above or a salt thereof, which can be obtained using the screening kit according to 1).
(31) The protein according to (1) above or (3) above
A non-human mammal having an exogenous DNA encoding the partial peptide described above or a mutant DNA thereof, (32) the animal of (31) above, wherein the non-human mammal is a rodent, (33) a rodent A non-human mammal containing the animal described in (32) above, which is a mouse or rat, (34) the foreign DNA encoding the protein described in (1) above or the partial peptide described in (3) above, or a mutant DNA thereof. A recombinant vector capable of expressing in (35)
Non-human mammalian embryonic stem cells in which the DNA encoding the protein described in (1) above or the partial peptide described in (3) above is inactivated, (36) the DNA is inactivated by introducing a reporter gene (35) Embryonic stem cells according to (35) above, (37) Neomycin resistant embryonic stem cells according to (35) above, (38) Embryonic stem cells according to (35) above, wherein the non-human mammal is a rodent. )
The embryonic stem cell according to the above (38), wherein the rodent is a mouse, (40) the protein encoding the protein according to the above (1) or the partial peptide according to the above (3), and the DNA expression deficient non-activated Human mammal, (41) The non-human mammal according to (40) above, 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. ) The non-human mammal described in (40) above, wherein the non-human mammal is a rodent, (43) the non-human mammal described in (42) above, wherein the rodent is a mouse,
And (44) a method for screening a compound or a salt thereof which promotes or inhibits the promoter activity to said DNA, which comprises administering a test compound to the animal according to (41) above and detecting the expression of a reporter gene To do.

[0006]

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 warm blood. Animals (eg human, guinea pig, rat, mouse, chicken, rabbit, pig,
Sheep cells, bovine cells, monkey cells, etc.) (eg, hepatocytes, splenocytes, nerve cells, glial cells, pancreatic β cells, bone marrow cells,
Mesangial cells, Langerhans cells, epidermal cells, epithelial cells, endothelial cells, fibroblasts, fiber cells, muscle cells, adipocytes, immune cells (eg, macrophages, T cells, B cells, natural killer cells, mast cells, neutrophils) Sphere, basophil, eosinophil, monocyte), megakaryocyte, synovial cell, chondrocyte, osteocyte, osteoblast, osteoclast, mammary gland cell, hepatocyte or stromal cell, or precursor of these cells Cells, stem cells, cancer cells, etc.) or any tissue in which those cells exist, for example, the brain, various parts of the brain (eg, olfactory bulb, amygdala, basal sphere, hippocampus, thalamus, hypothalamus, cerebral cortex, medulla oblongata, Cerebellum), spinal cord, pituitary gland, stomach, pancreas, kidney, liver, gonad, thyroid, gallbladder, bone marrow, adrenal gland, skin, muscle, lung,
Digestive tract (eg, large intestine, small intestine), blood vessel (eg, aorta), heart, thymus, spleen, submandibular gland, peripheral blood, prostate, testis, ovary, placenta, testis, uterus, bone, joint, skeletal muscle, mammary gland , Aorta, etc., or blood cells or cultured cells thereof (eg, MEL, M1, CTLL-2, HT-2, WEH
I-3, HL-60, JOSK-1, K562, ML-
1, MOLT-3, MOLT-4, MOLT-10, C
CRF-CEM, TALL-1, Jurkat, CCR
T-HSB-2, KE-37, SKW-3, HUT-7
8, HUT-102, H9, U937, THP-1, H
EL, JK-1, CMK, KO-812, MEG-01
Etc.), in particular adipose tissue, aorta, large intestine, kidney, mammary gland, ovary, prostate, small intestine, spleen, testis, uterus and adrenal gland, or synthetic protein.

The amino acid sequence which is substantially the same as the amino acid sequence of SEQ ID NO: 1 is about 50% or more, preferably about 60% or more, with the amino acid sequence represented by SEQ ID NO: 1.
More preferably about 70% or more, more preferably about 80%.
%, Particularly preferably about 90% or more, most preferably about 95% or more homologous amino acid sequences. The protein having an amino acid sequence substantially the same as the amino acid sequence represented by the amino acid sequence represented by SEQ ID NO: 1 of the present invention is, for example, substantially the same as the amino acid sequence represented by the above SEQ ID NO: 1. Preferred is a protein containing the amino acid sequence of SEQ ID NO: 1 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. The term “substantially the same” means that the activity is qualitatively (eg, physiologically or pharmacologically) the same. Therefore, the esterase activity is equivalent (eg, about 0.01 to
It is preferably 100 times, preferably about 0.1 to 10 times, more preferably 0.5 to 2 times), but quantitative factors such as the degree of this activity and the molecular weight of the protein may be different. The esterase activity described in the present specification means
It means the activity involved in the hydrolysis of carboxylic acid ester, phosphoric acid ester, and sulfuric acid ester, and preferably the activity involved in the hydrolysis of carboxylic acid ester, especially fatty acid esters. Examples of the carboxylic acid ester compound as a substrate include neutral fats such as triacylglycerol, diacylglycerol and monoacylglycerol,
In addition to natural substrates such as sterol esters such as cholesteryl ester, synthetic substrates such as p-nitrophenyl butyrate may be used. Further, the acyl group provided for the ester bond may have two or more carbon atoms, and may have fluorescence or radioactivity. The esterase activity can be measured according to a method known per se, for example, the screening method described below.

The protein of the present invention is, for example, 1 or 2 or more (preferably about 1 to 30, preferably about 1 to 10 in the amino acid sequence represented by SEQ ID NO: 1, more preferably about 10). Is the number (1-5)
1 or 2 or more (preferably about 1 to 30, preferably about 1 to 10 and more preferably a number (1 to 5) in the amino acid sequence represented by SEQ ID NO: 1 in which the amino acid of 1) or 2 or more (preferably about 1 to 30, preferably about 1 to 10 and more preferably several (1) to the amino acid sequence represented by SEQ ID NO: 1). ~
5) the amino acid sequence in which the amino acid is inserted, or 1 or 2 or more (preferably about 1 to 30, preferably 1 to 10) in the amino acid sequence represented by SEQ ID NO: 1.
A so-called mutein such as a protein containing an amino acid sequence in which about one, more preferably several (1 to 5) amino acids are substituted with other amino acids, or an amino acid sequence combining them is also included.

The protein in the present specification has the N-terminal (amino terminal) at the left end and the C-terminal (carboxyl terminal) at the right end according to the convention of peptide notation. SEQ ID NO: including the protein containing the amino acid sequence represented by 1, the protein of the present invention, C-terminal, carboxyl group (-COOH), a carboxylate (-COO ^-),
It may be any of (-CONH ₂₎ or ester (-COOR). Here, as R in the ester,
For example, a C _1-6 alkyl group such as methyl, ethyl, n-propyl, isopropyl or n-butyl, for example,
C _3-8 cycloalkyl groups such as cyclopentyl and cyclohexyl, for example C such as phenyl and α-naphthyl
_6-12 aryl groups, for example, phenyl-C _1-2 alkyl groups such as benzyl and phenethyl, or C _7-14 such as α-naphthyl-C _1-2 alkyl groups such as α-naphthylmethyl.
In addition to the aralkyl group, a pivaloyloxymethyl group commonly used as an oral ester is used. When the protein of the present invention has a carboxyl group (or carboxylate) in addition to the C-terminal, those in which the carboxyl group is amidated or esterified are also included in the protein of the present invention. As the ester in this case, for example, the above-mentioned C-terminal ester or the like is used. Furthermore, in the protein of the present invention, the amino group of the N-terminal amino acid residue (eg, methionine residue) has a protecting group (eg,
Formyl group, C _1-6 alkanoyl and other C _1-6 acyl groups such as acetyl group), those in which the N-terminal glutamine residue produced by cleavage in vivo is pyroglutamine-oxidized, Substituents on the side chains of amino acids in the molecule (for example, -OH, -SH, amino group, imidazole group, indole group, guanidino group, etc.) are suitable protecting groups (for example, C ₁ -formyl group, acetyl group, etc.). ₍₆ ) _C1-6 acyl groups such as alkanoyl groups) and complex proteins such as so-called glycoproteins to which sugar chains are bound are also included. Specific examples of the protein of the present invention include, for example, a human-derived protein containing the amino acid sequence represented by SEQ ID NO: 1 (LIP
-5) etc. are mentioned.

The partial peptide of the protein of the present invention (hereinafter sometimes referred to as the partial peptide of the present invention) is the partial peptide of the above-mentioned protein of the present invention, preferably the above-mentioned partial protein of the present invention Any one may be used as long as it has a similar activity (eg, esterase activity). For example, it has at least 20 or more, preferably 50 or more, more preferably 70 or more, more preferably 100 or more, most preferably 200 or more amino acid sequences among the constituent amino acid sequences of the protein of the present invention, A peptide having esterase activity is used. The esterase activity can be measured in the same manner as above. In addition, the partial peptide of the present invention is 1 or 2 or more (preferably about 1 to 10 and more preferably several (1 to 5) in the above amino acid sequence.
1) or 2 or more (preferably about 1-20, more preferably about 1-10, more preferably several (1-5) amino acids in the above amino acid sequence.
1) or 2 or more (preferably about 1 to 10) amino acids in the above amino acid sequence,
(More preferably several, more preferably about 1 to 5)
The amino acid of may be substituted with another amino acid. In the partial peptide, the C-terminus carboxyl group of the present invention (-COOH), a carboxylate (-COO ^-) may be any of an amide (-CONH ₂₎ or an ester (-COOR). Further, in the partial peptide of the present invention, as in the above-mentioned receptor protein of the present invention, a peptide in which the amino group of the N-terminal methionine residue is protected by a protecting group, and the N-terminal side is cleaved in vivo to generate the partial peptide. Also included are those in which Gln is pyroglutamine-oxidized, those in which the substituents on the side chains of amino acids in the molecule are protected by appropriate protecting groups, or complex peptides such as so-called glycopeptides to which sugar chains are bound. Further, when the partial peptide of the present invention has a carboxyl group (or carboxylate) in addition to the C-terminal, those in which the carboxyl 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 or the like is used. Since the partial peptide of the present invention can be used as an antigen for producing an antibody, it does not necessarily have to have esterase activity.

As the salt of the protein or partial peptide of the present invention, a salt with a physiologically acceptable acid (eg, inorganic acid, organic acid) or base (eg, alkali metal salt) is used. Acid-acceptable acid addition salts are preferred. Examples of such salts include 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). Acid, tartaric acid, citric acid, malic acid, oxalic acid, benzoic acid, methanesulfonic acid, benzenesulfonic acid) and salts thereof are used.

The protein of the present invention or a salt thereof can be produced from the cells or tissues of warm-blooded animals described above by a method known per se for protein purification, or a transformant containing a DNA encoding the protein described below. It can also be produced by culturing. It can also be produced according to the peptide synthesis method described below. When producing from tissues or cells of warm-blooded animals, homogenize tissues or cells of warm-blooded animals and then extract with acid etc., and combine the extract with chromatography such as reverse phase chromatography, ion exchange chromatography, etc. Thus, it can be purified and isolated.

For the synthesis of the protein or partial peptide of the present invention, its salt, or its amide, a commercially available resin for protein synthesis can be used. Examples of such resins include chloromethyl resin, hydroxymethyl resin, benzhydrylamine 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
Examples thereof include-(2 ', 4'-dimethoxyphenyl-Fmocaminoethyl) phenoxy resin. Using such a resin, an amino acid having an α-amino group and a side chain functional group appropriately protected is condensed on the resin according to the sequence of the desired protein or partial peptide according to various condensation methods known per se. At the end of the reaction, the protein or partial peptide is cleaved from the resin and at the same time various protective groups are removed,
Further, an intramolecular disulfide bond forming reaction is carried out in a highly diluted solution to obtain the target protein or partial peptide or its amide. Regarding the condensation of the above-mentioned protected amino acids, various activation reagents that can be used for protein synthesis can be used, but carbodiimides are particularly preferable. As the carbodiimides, DCC, N, N′-diisopropylcarbodiimide, N-ethyl-N ′-(3-dimethylaminoprolyl) carbodiimide and the like are used. For activation by these, additives for suppressing racemization (for example, HOB
t, HOOBt) and the protected amino acid is added directly to the resin, or the symmetrical acid anhydride or HOBt ester or HO
It can be added to the resin after previously performing activation of the protected amino acid as an OBt ester.

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

Examples of the protecting group for the amino group of the raw material include Z, Boc, t-pentyloxycarbonyl, isobornyloxycarbonyl, 4-methoxybenzyloxycarbonyl, Cl-Z, Br-Z, adamantyloxycarbonyl, Trifluoroacetyl, phthaloyl, formyl, 2
-Nitrophenylsulfenyl, diphenylphosphinothioyl oil, Fmoc and the like are used. The carboxyl group is
For example, alkyl esterification (eg, methyl, ethyl, propyl, butyl, t-butyl, cyclopentyl,
Cyclohexyl, cycloheptyl, cyclooctyl, 2
-Straight-chain, branched or cyclic alkyl esterification such as adamantyl), aralkyl esterification (eg benzyl ester, 4-nitrobenzyl ester, 4-methoxybenzyl ester, 4-chlorobenzyl ester, benzhydryl esterification) , Phenacyl esterification, benzyloxycarbonyl hydrazide formation, t-butoxycarbonyl hydrazide formation, trityl hydrazide formation and the like. The hydroxyl group of serine is
For example, it can be protected by esterification or etherification. Suitable groups for this esterification include, for example, lower (C _1-6 ) alkanoyl groups such as acetyl group, aroyl groups such as benzoyl group, groups derived from carbonic acid such as benzyloxycarbonyl group and ethoxycarbonyl group. Used. Examples of the group suitable for etherification include a benzyl group, a tetrahydropyranyl group, a t-butyl group and the like. Examples of the protective group for the phenolic hydroxyl group of tyrosine include Bzl, Cl ₂ -Bzl, 2-
Nitrobenzyl, Br-Z, t-butyl and the like are used.
Examples of the protecting group for imidazole of histidine include:
Tos, 4-methoxy-2,3,6-trimethylbenzenesulfonyl, DNP, benzyloxymethyl, Bum, Boc, Trt, Fmoc
Are used.

The activated carboxyl group of the raw material includes, for example, corresponding acid anhydrides, azides, active esters [alcohols (for example, pentachlorophenol, 2,4,5-trichlorophenol, 2,4- Ester with dinitrophenol, cyanomethyl alcohol, para-nitrophenol, HONB, N-hydroxysuccinimide, N-hydroxyphthalimide, HOBt), etc. are used. As the activated amino group of the raw material, for example, the corresponding phosphoric acid amide is used. Examples of the method for removing (eliminating) the protecting group include catalytic reduction in a hydrogen stream in the presence of a catalyst such as Pd-black or Pd-carbon,
Also, acid treatment with anhydrous hydrogen fluoride, methanesulfonic acid, trifluoromethanesulfonic acid, trifluoroacetic acid, or a mixture thereof, base treatment with diisopropylethylamine, triethylamine, piperidine, piperazine, or 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, anisole, phenol,
The addition of a cation trapping agent such as thioanisole, metacresol, paracresol, dimethyl sulfide, 1,4-butanedithiol, 1,2-ethanedithiol is effective. Also, the 2,4-dinitrophenyl group used as the imidazole protecting group of histidine is removed by thiophenol treatment, and the formyl group used as the indole protecting group of tryptophan is the above 1,2-ethanedithiol, 1,4-butane group. In addition to deprotection by acid treatment in the presence of dithiol or the like, it is also removed by alkali treatment with dilute sodium hydroxide solution, dilute ammonia or the like.

Protection of a functional group which should not be involved in the reaction of the starting material, removal of the protective group and removal of the protective group, activation of the functional group involved in the reaction and the like can be appropriately selected from known groups or known means. . As another method for obtaining an amide form of a protein or partial peptide, for example, first, the α-carboxyl group of the carboxy-terminal amino acid is amidated and protected, and then the peptide (protein) chain is added to the amino group side up to the desired chain length. After the extension, a protein or partial peptide in which only the N-terminal α-amino protecting group of the peptide chain is removed and a protein or partial peptide in which only the C-terminal carboxyl protecting group is removed are produced. The protein or partial peptide is condensed in a mixed solvent as described above. The details of the condensation reaction are the same as above. After the protected protein or partial peptide obtained by condensation is purified, all the protecting groups can be removed by the above method to obtain the desired crude protein or partial peptide. The crude protein or partial peptide can be purified using various known purification means, and the main fraction can be lyophilized to obtain the amide of the desired protein or partial peptide. To obtain an ester form of a protein or partial peptide, for example, the α-carboxyl group of the carboxy-terminal amino acid is condensed with a desired alcohol to form an amino acid ester, which is then treated in the same manner as the amide form of the protein or partial peptide. An ester form of a protein or partial peptide can be obtained.

The protein or partial peptide of the present invention or a salt thereof can be produced by publicly known methods for peptide synthesis, or by cleaving the protein of the present invention with an appropriate peptidase. The peptide synthesis method may be, for example, either a solid phase synthesis method or a liquid phase synthesis method. That is, the target peptide can be produced by condensing the partial peptide or amino acid that can form the partial peptide of the present invention with the residual portion and removing the protective group when the product has a protective group. Examples of known condensation methods and removal of protective groups include the methods described in the following (1) to (3). M. Bodanszky and MA Ondetti, Peptide Synthesis, Interscience Publisher
s, New York (1966) Schroeder and Luebke, The Peptid
e), Academic Press, New York (1965) Nobuo Izumiya et al., Fundamentals and experiments of peptide synthesis, Maruzen Co., Ltd.
(1975) Haruaki Yajima and Shunpei Sakakibara, Laboratory of Biochemistry 1, Protein Chemistry IV, 205, (1977) Supervised by Haruaki Yajima, Development of Pharmaceuticals, Volume 14, Peptide Synthesis, Hirokawa Shoten After that, the protein or partial peptide of the present invention can be purified and isolated by a combination of usual purification methods such as solvent extraction, distillation, column chromatography, liquid chromatography, and recrystallization. When the protein or partial peptide obtained by the above method is a free form, it can be converted into a suitable salt by a known method or a method analogous thereto, and conversely, when it is obtained by a salt, a known method or It can be converted into an educt or other salt by a method analogous thereto.

A polynucleotide encoding the protein of the present invention or a partial peptide thereof is a polynucleotide containing a base sequence (DNA or RNA, preferably DNA) encoding the above-mentioned protein of the present invention or a partial peptide thereof. It can be anything. Examples of the polynucleotide include R such as DNA and mRNA encoding the protein of the present invention or its partial peptide.
NA, which may be double-stranded or single-stranded. When it is double-stranded, it may be double-stranded DNA, double-stranded RNA or a hybrid of DNA: RNA. In the case of a single strand, it may be a sense strand (that is, a coding strand) or an antisense strand (that is, a non-coding strand). Using the polynucleotide encoding the protein of the present invention or a partial peptide thereof, for example, the publicly known special edition of Experimental Medicine, “New PCR and its Applications”, 15 (7), 1997.
The mRNA of the protein of the present invention or its partial peptide can be quantified by the method described or a method analogous thereto.

The DNA encoding the protein of the present invention may be any DNA as long as it contains the nucleotide sequence encoding the protein of the present invention. In addition, genomic DNA, genomic DNA library,
Any of the above-mentioned cell / tissue-derived cDNA, the above-mentioned cell / tissue-derived cDNA library, and synthetic DNA may be used. The vector used for the library may be any of bacteriophage, plasmid, cosmid, phagemid and the like. In addition, from the cells and tissues mentioned above
Reverse Transcriptase Polymerase Chain React directly using prepared total RNA or mRNA fraction
It can also be amplified by ion (hereinafter, abbreviated as RT-PCR method). DN encoding the protein of the present invention
As A, for example, a DNA containing the base sequence represented by SEQ ID NO: 2 or a DNA hybridizing with the DNA containing the base sequence represented by SEQ ID NO: 2 under high stringent conditions, SEQ ID NO: 1
Any DNA may be used as long as it encodes a protein having substantially the same activity (eg, esterase activity) as the protein containing the amino acid sequence represented by.

The DNA that hybridizes with the DNA containing the base sequence represented by SEQ ID NO: 2 under high stringent conditions is, for example, about 70% or more, preferably about 70% or more, of the base sequence represented by SEQ ID NO: 2. About 80%
DN containing a base sequence having a homology of not less than 90%, more preferably not less than about 90%, and most preferably not less than about 95%.
A or the like is used. Hybridization is a method known per se or a method analogous thereto, for example, Molecular Cloning 2nd (J.
Sambrook et al., Cold Spring Harbor Lab. Press, 198
It can be performed according to the method described in 9). 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 carried out under high stringent conditions. The high stringent condition means, for example, a condition that the sodium concentration is about 19 to 40 mM, preferably about 19 to 20 mM, and the temperature is about 50 to 70 ° C, preferably about 60 to 65 ° C. Particularly, the case where the sodium concentration is about 19 mM and the temperature is about 65 ° C. is most preferable. More specifically, as the DNA encoding the protein containing the amino acid sequence represented by SEQ ID NO: 1, DNA containing the base sequence represented by SEQ ID NO: 2 or the like is used.

DNA encoding the partial peptide of the present invention
Any may be used as long as it contains the 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 / tissues described above, cDNA library derived from the cells / tissues described above, and synthetic DNA. DNA encoding the partial peptide of the present invention
Is, for example, a DNA having a partial base sequence of a DNA containing the base sequence represented by SEQ ID NO: 2, or a DNA containing the base sequence represented by SEQ ID NO: 2.
D which encodes a protein having DNA that hybridizes under high stringency conditions with and having substantially the same activity (eg, esterase activity) as the protein containing the amino acid sequence represented by SEQ ID NO: 1.
DNA having a partial base sequence of NA is used.
The same hybridization method and high stringent conditions as described above are used. The DNA that hybridizes with the DNA containing the base sequence represented by SEQ ID NO: 2 under high stringent conditions is, for example, about 70% or more, preferably about 80% or more with the base sequence represented by SEQ ID NO: 2. , More preferably about 90% or more, and even more preferably about 95% or more homologous DNA containing a nucleotide sequence.

Part of the nucleotide sequence of DNA encoding the protein of the present invention or its partial peptide, or the DN
A polynucleotide containing a part of a base sequence complementary to A is a DNA encoding the partial peptide of the present invention.
Is used to mean not only RNA 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 a cloned or determined DNA encoding the protein of the present invention.
Can be designed and synthesized based on the nucleotide sequence information of Such a polynucleotide (nucleic acid) can hybridize with RNA of the protein gene of the present invention.
Or the expression of the protein gene of the present invention can be regulated / controlled via the interaction with the protein-related RNA of the present invention. The polynucleotide complementary to the selected sequence of the protein-related RNA of the present invention and the polynucleotide capable of specifically hybridizing with the protein-related RNA of the present invention can be used as a protein gene of the present invention in vivo and in vitro. It is useful for regulating / controlling the expression of erythrocyte, and for treating or diagnosing diseases and the like. The term "corresponding" means having homology or being complementary to a specific sequence of nucleotides, base sequences or nucleic acids including genes. "Corresponding" between a nucleotide (base sequence or nucleic acid) and a peptide (protein) usually refers to an amino acid of a peptide (protein) in a command derived from the nucleotide (nucleic acid) sequence or its complement. . 5′-end hairpin loop of the protein gene of the present invention, 5′-end 6-base pair repeat, 5′-end untranslated region, peptide translation initiation codon, protein coding region, ORF translation stop codon, 3′-end untranslated region, The 3'-end palindromic region and the 3'-end hairpin loop can be selected as preferred regions of interest, but any region within the protein gene of the present invention can be selected as a region of interest.

The relationship between the target nucleic acid and the polynucleotide complementary to at least a part of the target region, and the relationship between the polynucleotide capable of hybridizing with the target region can be said to be "antisense". . Antisense polynucleotides include polynucleotides containing 2-deoxy-D-ribose, polynucleotides containing D-ribose, other types of polynucleotides that are N-glycosides of purine or pyrimidine bases, Alternatively, other polymers having a non-nucleotide skeleton (for example, commercially available protein nucleic acids and synthetic sequence-specific nucleic acid polymers) or other polymers containing a special bond (provided that the polymer is DNA or RN).
Containing a nucleotide having a configuration that allows base pairing and base attachment as found in A). They are double-stranded DNA, single-stranded DNA,
It can be double-stranded RNA, single-stranded RNA, or even a DNA: RNA hybrid, and also unmodified polynucleotides (or unmodified oligonucleotides), as well as those with known modifications, such as are known in the art. Labeled, capped, methylated, substituted with one or more natural nucleotides by analogs, modified intramolecularly with nucleotides such as uncharged bonds (eg, With methylphosphonates, phosphotriesters, phosphoramidates, carbamates, etc., with charged or sulfur-containing bonds (eg phosphorothioates, phosphorodithioates, etc.), eg proteins (nucleases, nuclease inhibitors) , Toxin, antibody, signal peptide, poly-L- Having a side chain group such as gin) or sugar (eg, monosaccharide), having an intercurrent compound (eg, acridine, psoralen, etc.), chelate compound (eg, metal, radioactive) Metal, boron, an oxidizing metal, etc.), an alkylating agent, or a modified bond (for example, α-anomeric nucleic acid). As used herein, the term "nucleoside", "nucleotide" and "nucleic acid" may include those having not only purine and pyrimidine bases but also other modified heterocyclic bases. Such modifications may include methylated purines and pyrimidines, acylated purines and pyrimidines, or other heterocycles. Modified nucleotides and modified nucleotides may also have sugar moieties modified, such as one or more hydroxyl groups being halogen,
Substituted with aliphatic groups, or ether,
It may be converted into a functional group such as an amine.

The antisense polynucleotide (nucleic acid) of the present invention is RNA, DNA or a modified nucleic acid (RNA, DNA). Specific examples of the modified nucleic acid include, but are not limited to, sulfur derivatives and thiophosphate derivatives of nucleic acids, and those resistant to degradation of polynucleoside amides and oligonucleoside amides. The antisense nucleic acid of the present invention can be preferably designed according to the following policy. That is, it makes the antisense nucleic acid more stable in the cell, increases the cell permeability of the antisense nucleic acid, increases the affinity for the target sense strand, and Make sense nucleic acid less toxic. Thus many modifications are known in the art, for example J. Kawakami et al., Pharm Tech Japan, Vo.
l. 8, pp. 247, 1992; Vol. 8, pp. 395, 1992; ST Cr
ooke et al. ed., Antisense Research and Applicatio
ns, CRC Press, 1993 etc. The antisense nucleic acid of the present invention has an altered or modified sugar,
It may contain a base or a bond, and may be provided in a special form such as liposome, microsphere, applied by gene therapy, or provided in an added form. Thus, as an additive form, a polycationic substance such as polylysine that acts to neutralize the charge of the phosphate skeleton, a lipid that enhances the interaction with the cell membrane or the uptake of nucleic acid ( Examples thereof include crude aqueous substances such as phospholipid and cholesterol). Preferred lipids to add include cholesterol and its derivatives (eg cholesteryl chloroformate, cholic acid, etc.). These can be attached to the 3'-end or 5'-end of the nucleic acid, and can be attached via a base, a sugar, or an intramolecular nucleoside bond. Other groups include
A capping group specifically arranged at the 3'-end or the 5'-end of a nucleic acid, which includes a group for preventing decomposition by nucleases such as exonuclease and RNase. Examples of such a capping group include, but are not limited to, hydroxyl group-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 its partial peptide may be labeled by a method known per se, specifically, isotope-labeled one, fluorescent-labeled one (for example, fluorescence by fluorescein, etc.). Label), biotinylated one, enzyme labeled one and the like.

Proteins or partial peptides of the present invention (hereinafter, in the description of cloning and expression of DNAs encoding these proteins, these proteins may be simply referred to as the proteins of the present invention)
As a means for cloning the DNA encoding the complete DNA, a synthetic D having a partial base sequence of the protein of the present invention is used.
Amplification by PCR method using NA primer,
Alternatively, the DNA incorporated into an appropriate vector can be selected by hybridization with a DNA fragment that encodes a part or the entire region of the protein of the present invention or that labeled with a synthetic DNA. The hybridization method is, for example, molecular cloning (Molecular Cloning) 2nd (J. Sambrook e
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. Conversion of the nucleotide sequence of DNA can be performed by PCR or a known kit such as Mutan ^™ -su.
Using perExpress Km (Takara Shuzo Co., Ltd.), Mutan ^TM -K (Takara Shuzo Co., Ltd.), etc., ODA-LAPCR method, Gapped duplex method,
It can be carried out according to a method known per se such as Kunkel method or a method analogous thereto. The DNA encoding the cloned protein can be used as it is, or if desired after digestion with a restriction enzyme or addition of a linker, depending on the purpose. The DNA has ATG as a translation initiation codon at the 5'-terminal side, and 3 '
The terminal side may have TAA, TGA, or TAG as a translation termination codon. These translation initiation codon and translation termination codon can be added using an appropriate synthetic DNA adaptor. The expression vector of the protein of the present invention includes, for example, (a) cutting out a target DNA fragment from DNA encoding the protein of the present invention,
(B) It can be produced by ligating the DNA fragment downstream of the promoter in an appropriate expression vector.

As a vector, a plasmid derived from Escherichia coli (eg, pBR322, pBR325, pUC12, pBR322
UC13), a Bacillus subtilis-derived plasmid (eg, pUB11)
0, pTP5, pC194), yeast-derived plasmids (eg, pSH19, pSH15), bacteriophages such as λ phage, animal viruses such as retrovirus, vaccinia virus, baculovirus, etc.
A1-11, pXT1, pRc / CMV, pRc / RS
V, pcDNAI / Neo, etc. are used. The promoter used in the present invention may be any promoter as long as it is suitable for the host used for gene expression. For example, when an animal cell is used as a host, SRα promoter, SV40 promoter, L promoter
TR promoter, CMV promoter, HSV-TK
Examples include promoters. Of these, CMV
It is preferable to use (cytomegalovirus) promoter, SRα promoter and the like. When the host is a bacterium belonging to the genus Escherichia, trp promoter, lac promoter, recA promoter, λPL promoter,
lpp promoter, T7 promoter, etc., when the host is a Bacillus genus, SPO1 promoter, S
When the host is yeast such as PO2 promoter and penP promoter, PHO5 promoter, PGK promoter, GAP promoter, ADH promoter and the like are preferable. When the host is an insect cell, polyhedrin promoter, P10 promoter and the like are preferable.

In addition to the above, the expression vector may optionally contain an enhancer, a splicing signal, a poly A addition signal, a selection marker, an SV40 replication origin (hereinafter sometimes abbreviated as SV40 ori) and the like. Can be used. Examples of selection markers include dihydrofolate reductase (hereinafter, dhfr).
The gene [methotrexate (M
TX) resistance], ampicillin resistance gene (hereinafter referred to as Amp
^r ), neomycin resistance gene (hereinafter sometimes abbreviated as Neo ^r , G418 resistance) and the like. In particular, when the dhfr gene is used as a selection marker using Chinese hamster cells lacking the dhfr gene, the target gene can also be selected by a thymidine-free medium. In addition, a signal sequence suitable for the host is added to the N-terminal side of the protein of the present invention, if necessary. When the host is a bacterium of the genus Escherichia, the PhoA signal sequence, OmpA signal sequence, etc., and when the host is a bacterium of the genus Bacillus, the α-amylase signal sequence, the subtilisin signal sequence, etc., the host is yeast. In some cases, the MFα signal sequence,
When the host is an animal cell such as SUC2 / signal sequence, an insulin signal sequence, an α-interferon signal sequence, an antibody molecule / signal sequence, etc. can be used respectively. Using the vector containing the DNA encoding the protein of the present invention thus constructed, transformants can be produced.

As the host, for example, Escherichia, Bacillus, yeast, insect cells, insects, animal cells and the like are used. Specific examples of Escherichia bacteria include:
For example, Escherichia coli K1
2. DH1 [Proc. Natl. Acad. Sci. USA], 60
Vol. 160 (1968)], JM103 [Nukuirekku.
Acids Research, (Nucleic Acids Research), 9
Vol., 309 (1981)], JA221 [Journal of Molecular Biology (Journal of Molecul
ar Biology), 120, 517 (1978)], HB1
01 [Journal of Molecular Biology, 41, 459 (1969)], C600 [Genetics, 39, 440 (1954)] and the like are used. Examples of the genus Bacillus include, for example, Bacillus subtilis MI114.
[Gene, 24, 255 (1983)], 207-21
[Journal of Biochemistry
Biochemistry), Vol. 95, 87 (1984)] and the like. Examples of yeast include Saccharomyces
Saccharomyces cerevisiae AH22, A
^{H22R -, NA87-11A, DKD-5D} , 20B
-12, Schizosaccharomyces pombe (Schizosaccha
romyces pombe) NCYC1913, NCYC203
6. Pichia pastoris KM71
Are used.

Examples of insect cells include virus A
In the case of cNPV, larvae of the night-thief moth (Spodop
tera frugiperda cell; Sf cell), Trichoplusia ni
MG1 cells from the midgut of the mouse, H from the eggs of Trichoplusia ni
igh Five ^TM cells, cells from Mamestra brassicae or
Cells derived from Estigmena acrea are used. When the virus is BmNPV, the silkworm-derived cell line (Bombyx mor
i N cells; BmN cells) and the like are used. Examples of the Sf cells include Sf9 cells (ATCC CRL1711), Sf
21 cells (above Vaughn, JL et al., In Vivo
ivo), 13, 213-217, (1977)) is used. Examples of insects include silkworm larvae [Maeda et al., Nature, 315, 592 (198).
5)]. Animal cells include, for example, monkey cells COS-
7, Vero, Chinese hamster cell CHO (hereinafter abbreviated as CHO cell), dhfr gene-deficient Chinese hamster cell CHO (hereinafter CHO (dhf
r ^-) cell), mouse L cells, mouse AtT-2
0, mouse myeloma cells, rat GH3, human FL cells and the like are used. For transforming Escherichia, for example, Proc. Natl. Acad. Sci. USA, 69.
Volume, 2110 (1972) and Gene, Volume 17, 1
07 (1982) and the like.

For transforming Bacillus, for example, Molecular & General Genetics, Vol. 168,
111 (1979) and the like. For transforming yeast, for example, Methods in Enzymolog
y), 194, 182-187 (1991), Procedures of the National Academy of Sciences of the USA (Proc. Na).
tl. Acad. Sci. USA), 75, 1929 (197).
It can be performed according to the method described in 8). Transformation of insect cells or insects can be performed, for example, according to the method described in Bio / Technology, 6, 47-55 (1988) and the like. To transform animal cells, see, eg, Cell Engineering Supplement 8, New Cell Engineering Experimental Protocol. 263-267 (1995)
(Published by Shujunsha), Virology, Volume 52,
456 (1973). In this way, the DNA encoding the protein
It is possible to obtain a transformant transformed with an expression vector containing When the host is culturing a transformant that is a bacterium of the genus Escherichia, a bacterium of the genus Bacillus, a liquid medium is suitable as a medium used for the culture, and among them, a carbon source necessary for the growth of the transformant, A nitrogen source, an inorganic substance, etc. are contained. Examples of the carbon source include glucose, dextrin, soluble starch and sucrose, and examples of the nitrogen source include ammonium salts, nitrates, corn steep liquor, peptone, casein, meat extract, soybean meal and potato extract. Examples of the inorganic or organic substance and the inorganic substance include calcium chloride, sodium dihydrogen phosphate, magnesium chloride and the like. In addition, yeast extract, vitamins, growth promoting factor and the like may be added. The pH of the medium is preferably about 5-8.

As a medium for culturing Escherichia, for example, M9 medium containing glucose and casamino acid [Miller, Journal of Experiments in Molecular Genetics (Journa
l of Experiments in Molecular Genetics), 431-
433, Cold Spring Harbor Laboratory, New York1
972] is preferable. If necessary, a drug such as 3β-indolylacrylic acid can be added to the promoter so as to work efficiently. When the host is a bacterium belonging to the genus Escherichia, the culture is usually carried out at about 15 to 43 ° C. for about 3 to 24 hours, and if necessary, aeration and stirring can be added. When the host is a bacterium of the genus Bacillus, the culture is usually carried out at about 30 to 40 ° C. for about 6 to 24 hours, and aeration and stirring can be added if necessary. When culturing a transformant whose host is yeast, examples of the medium include Burkholder's minimum medium [Bostian, KL
Et al., Proc. Natl. Acad. Sci. USA, 77, 450.
5 (1980)] or SD medium containing 0.5% casamino acid [Bitter, GA et al., Procedures of the
National Academy of Sciences of the USA (Proc. Natl. Acad. Sci. US
A), 81, 5330 (1984)].
The pH of the medium is preferably adjusted to about 5-8. Culturing is usually carried out at about 20 ° C to 35 ° C for about 24 to 72 hours, and aeration and agitation are added if necessary. When culturing a transformant whose host is an insect cell or an insect, the medium is Grac
e's Insect Medium (Grace, TCC, Nature (Natur
e), 195, 788 (1962)) to which appropriate additives such as immobilized 10% bovine serum have been added. PH of medium
Is preferably adjusted to about 6.2-6.4. Culturing is usually performed at about 27 ° C. for about 3 to 5 days, and aeration and agitation are added if necessary. When culturing a transformant whose host is an animal cell, the medium is, for example, MEM medium containing about 5 to 20% fetal bovine serum [Science, 12
Volume 2, 501 (1952)], DMEM medium [Virology, Volume 8, 396 (1959)], RPMI
1640 medium [Journal of the American
Medical Association (The Journal of the A
merican Medical Association) 199, 519 (19
67)], 199 medium [Proceeding of the Society for the Biological Medi
cine), 73, 1 (1950)] and the like. p
H is preferably about 6-8. Culture is usually about 30 ℃
Perform at about -40 ° C for about 15-60 hours, adding aeration and agitation as needed. As described above, the protein of the present invention can be produced inside the transformant, inside the cell membrane or outside the cell.

The protein of the present invention can be separated and purified from the above culture by the following method, for example. When the protein of the present invention is extracted from cultured bacterial cells or cells, after the culture, the bacterial cells or cells are collected by a known method, suspended in an appropriate buffer solution, and then subjected to ultrasonic waves, lysozyme and / or freeze-thawing. After destroying the bacterial cells or cells by the method, a method of obtaining a crude protein extract by centrifugation or filtration is appropriately used. The buffer solution may contain a protein denaturing agent such as urea or guanidine hydrochloride, or a surfactant such as Triton X-100 ^™ . When the protein is secreted into the culture medium, after the culture is completed, the cells or cells are separated from the supernatant by a method known per se, and the supernatant is collected. The protein contained in the thus obtained culture supernatant or the extract can be purified by appropriately combining separation / purification methods known per se. Known separation and purification methods for these include methods utilizing solubility such as salting out and solvent precipitation, dialysis, ultrafiltration, gel filtration, and SD.
Methods utilizing mainly differences in molecular weight such as S-polyacrylamide gel electrophoresis, methods utilizing differences in charge such as ion exchange chromatography, methods utilizing specific affinity such as affinity chromatography,
A method utilizing a difference in hydrophobicity such as reversed-phase high performance liquid chromatography and a method utilizing a difference in isoelectric point such as isoelectric focusing are used.

When the thus obtained protein is obtained as a free form, it can be converted into a salt by a method known per se or a method analogous thereto, and conversely when it is obtained as a salt, a method known per se or It can be converted into a free form or another salt by a method similar to that. In addition, the protein produced by the recombinant can be optionally modified or the polypeptide can be partially removed by acting an appropriate protein-modifying enzyme before or after the purification. Examples of the protein-modifying enzyme include trypsin, chymotrypsin, arginyl endopeptidase, protein kinase, glycosidase and the like. The presence or activity of the thus-produced protein of the present invention or a salt thereof can be measured by an enzyme immunoassay using a specific antibody, a fatty acid release amount (enzyme activity amount) due to the decomposition of neutral lipids, and the like.

The antibody against the protein, partial peptide or salt thereof of the present invention is an antibody capable of recognizing the protein, partial peptide or salt thereof of the present invention,
It may be either a polyclonal antibody or a monoclonal antibody. An antibody against the protein, partial peptide or a salt thereof of the present invention (hereinafter, these proteins and the like may be simply referred to as the protein of the present invention in the description of antibodies) uses the protein of the present invention as an antigen, and It can be produced according to a known antibody or antiserum production method. [Preparation of Monoclonal Antibody] (a) Preparation of Monoclonal Antibody-Producing Cell The protein of the present invention is administered to warm-blooded animals at a site where antibody production is possible by itself or together with a carrier or diluent. Complete Freund's adjuvant or incomplete Freund's adjuvant may be administered to enhance the antibody-producing ability upon administration. The administration is usually performed once every 2 to 6 weeks, about 2 to 10 times in total. Examples of warm-blooded animals used include monkeys, rabbits, dogs, guinea pigs, mice, rats, sheep, goats, and chickens, and mice and rats are preferably used. When preparing monoclonal antibody-producing cells, warm-blooded animals immunized with the antigen, for example, individuals with antibody titers are selected from mice, and spleens or lymph nodes are collected 2 to 5 days after the final immunization. A monoclonal antibody-producing hybridoma can be prepared by fusing the resulting antibody-producing cells with myeloma cells of the same or different species. The antibody titer in the antiserum can be measured, for example, by reacting the labeled protein described below with the antiserum and then measuring the activity of the labeling agent bound to the antibody. The fusion operation is a known method, for example, the method of Kohler and Milstein [Nature, 256, 495 (1
975)]. Examples of the fusion accelerator include polyethylene glycol (PEG) and Sendai virus, and PEG is preferably used.

Examples of myeloma cells include NS-1,
Examples include myeloma cells of warm-blooded animals such as P3U1, SP2 / 0 and AP-1, but P3U1 is preferably used. The preferred ratio of the number of antibody-producing cells (spleen cells) to the number of myeloma cells used is about 1: 1 to 20: 1,
PEG (preferably PEG1000-PEG6000)
Is added at a concentration of about 10 to 80%, and 20 to 40 ° C.,
Cell fusion can be efficiently performed by preferably incubating at 30 to 37 ° C. for 1 to 10 minutes. Although various methods can be used for screening a monoclonal antibody-producing hybridoma, for example, a hybridoma culture supernatant is added to a solid phase (eg, a microplate) on which a protein antigen is directly or adsorbed with a carrier, and then a radioactive substance or Anti-immunoglobulin antibody labeled with an enzyme (when the cells used for cell fusion are mice, anti-mouse immunoglobulin antibody is used) or protein A
And detecting the monoclonal antibody bound to the solid phase, adding the hybridoma culture supernatant to the solid phase adsorbing the anti-immunoglobulin antibody or protein A, adding the protein labeled with a radioactive substance or enzyme, Examples include a method of detecting a monoclonal antibody bound to a phase. The monoclonal antibody can be selected according to a method known per se or a method analogous thereto. Normal H
It can be performed in a medium for animal cells supplemented with AT (hypoxanthine, aminopterin, thymidine). As the medium for selection and breeding, any medium may be used as long as the hybridoma can grow. For example, 1
RPMI 1640 medium containing -20%, preferably 10-20% fetal bovine serum, GIT medium containing 1-10% fetal bovine serum (Wako Pure Chemical Industries, Ltd.) or serum-free medium for hybridoma culture (SFM) -101, Nissui Pharmaceutical Co., Ltd., etc. can be used. The culture temperature is usually 20 to 40 ° C, preferably about 37 ° C. Cultivation time is usually 5 days to 3 weeks, preferably 1 week to 2 weeks. Culturing can usually be performed under 5% carbon dioxide gas. The antibody titer of the hybridoma culture supernatant can be measured in the same manner as the above antibody titer measurement in antiserum.

(B) Purification of monoclonal antibody Monoclonal antibody can be separated and purified by a method known per se, for example, a method for separating and purifying immunoglobulin [eg salting out method, alcohol precipitation method, isoelectric focusing method, electrophoresis. Method, adsorption / desorption method using ion exchanger (eg, DEAE), ultracentrifugation method, gel filtration method, antigen-binding solid phase or active adsorbent such as protein A or protein G to collect only antibody to dissociate the bond. Specific Purification Method for Obtaining Antibody].

[Preparation of Polyclonal Antibody] The polyclonal antibody of the present invention can be manufactured by a method known per se or a method analogous thereto. For example, an immune antigen (protein antigen of the present invention) itself or a complex thereof with a carrier protein is formed, and a warm-blooded animal is immunized in the same manner as in the above-described method for producing a monoclonal antibody. It can be produced by collecting the antibody-containing product against and isolating 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 mixing ratio of carrier and hapten are such that the antibody is effective against the hapten immunized by crosslinking with the carrier. If possible, any kind may be cross-linked in any ratio, but, for example, bovine serum albumin, bovine thyroglobulin, hemocyanin, etc. in a weight ratio of about 0.1 to 20, preferably about 1 to 1 hapten. A method of coupling at a rate of 5 is used. Although various condensing agents can be used for coupling the hapten and carrier, glutaraldehyde, carbodiimide, maleimide active ester, active ester reagent containing thiol group, dithiopyridyl group, etc. are used. The condensation product is administered to a warm-blooded animal at a site where antibodies can be produced, by itself or together with a carrier and a diluent.
Complete Freund's adjuvant or incomplete Freund's adjuvant may be administered to enhance the antibody-producing ability upon administration. The administration is usually performed once every about 2 to 6 weeks, about 3 to 10 times in total. The polyclonal antibody can be collected from the blood, ascites, etc., of the warm-blooded animal immunized by the above method, preferably from the blood. The polyclonal antibody titer in the antiserum can be measured in the same manner as the above-mentioned antibody titer in the antiserum. Separation and purification of the polyclonal antibody can be performed according to the same immunoglobulin separation and purification method as the above-described separation and purification of the monoclonal antibody.

Below, the protein or partial peptide of the present invention or a salt thereof (hereinafter sometimes abbreviated as the protein of the present invention, etc.), DNA encoding the protein or partial peptide of the present invention (hereinafter referred to as the DNA of the present invention) And an antibody against the protein, partial peptide or a salt thereof of the present invention (hereinafter sometimes abbreviated as the antibody of the present invention).

[1] Remedies for Preventing and Treating Various Diseases Involved by the Protein of the Present Invention The protein of the present invention contributes to the regulation of the degradation of neutral lipids (for example, neutral fat and cholesteryl ester). Has a triglyceride degrading activity, etc., and therefore, when the DNA encoding the protein of the present invention is abnormal or defective, or when the expression level of the protein of the present invention is reduced, for example, Various diseases such as arteriosclerosis, hyperlipidemia, obesity and diabetes develop. Therefore, the protein and the like of the present invention and the DNA of the present invention can be used as a medicine such as a therapeutic / preventive agent for various diseases such as arteriosclerosis, hyperlipidemia, obesity, diabetes and the like. For example, when there is a patient in which the regulation of the degradation of neutral lipids in cells is not sufficiently or normally exerted due to the decrease or deficiency of the protein of the present invention in vivo, (a) the DNA of the present invention (B) Inserting the DNA of the present invention into a cell by expressing the protein or the like of the present invention in vivo and expressing the protein or the like of the present invention, and then transplanting the cell into the 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.
When the DNA of the present invention is used as the above-mentioned therapeutic / prophylactic agent, the DNA is inserted alone or in an appropriate vector such as a retrovirus vector, an adenovirus vector, an adenovirus associated virus vector, and then, according to a conventional method, It can be administered to humans or warm-blooded animals. The DNA of the present invention can be administered as it is, or can be formulated with a physiologically acceptable carrier such as an auxiliary agent for promoting intake, and can be administered by a gene gun or a catheter such as a hydrogel catheter. When the protein or the like of the present invention is used as the above-mentioned therapeutic / prophylactic agent, at least 90%, preferably 95% or more, more preferably 98% or more, still more preferably 99% or more, is used. Is preferred.

The protein and the like of the present invention are, for example, tablets, capsules, elixirs, sugar-coated as necessary,
It can be used orally as a microcapsule or the like, or parenterally in the form of an injectable solution such as a sterile solution or suspension with water or another pharmaceutically acceptable liquid. For example, the protein or the like of the present invention is admixed with a physiologically acceptable carrier, flavoring agent, excipient, vehicle, preservative, stabilizer, binder, etc. in a unit dosage form generally required for drug formulation. It can be manufactured by The amount of active ingredient in these preparations is such that a suitable dose within the indicated range can be obtained. Additives that can be mixed with tablets, capsules, etc. include, for example, binders such as gelatin, corn starch, tragacanth, acacia, excipients such as crystalline cellulose, corn starch, gelatin, alginic acid, etc. Puffing agents, lubricants such as magnesium stearate, sweeteners such as sucrose, lactose or saccharin, flavoring agents such as peppermint, red oil or cherry and the like are used. When the unit dosage form is a capsule, a liquid carrier such as fat may be included in addition to materials of the above type. Sterile compositions for injection can be formulated according to conventional pharmaceutical practices such as dissolving or suspending the active substance in a vehicle such as water for injection, naturally occurring vegetable oils such as sesame oil, coconut oil and the like. Examples of the aqueous solution for injection include physiological saline, isotonic solution containing glucose and other adjuvants (for example, D-sorbitol, D-mannitol, sodium chloride, etc.), and a suitable solubilizing agent. For example, it may be used in combination with alcohol (eg, ethanol), polyalcohol (eg, propylene glycol, polyethylene glycol, etc.), nonionic surfactant (eg, Polysorbate 80 ^™ , HCO-50, etc.). Examples of the oily liquid include sesame oil, soybean oil and the like, which may be used in combination with solubilizing agents such as benzyl benzoate and benzyl alcohol. In addition, buffers (eg, phosphate buffer, sodium acetate buffer, etc.), soothing agents (eg, benzalkonium chloride, procaine hydrochloride, etc.), stabilizers (eg, human serum albumin, polyethylene glycol, etc.), Preservatives (eg,
(Benzyl alcohol, phenol, etc.), antioxidant, etc. The prepared injection solution is usually filled in a suitable ampoule. The vector into which the DNA of the present invention has been inserted is also formulated as in the above, and is usually used parenterally.

The preparation thus obtained is safe and has low toxicity. Dog, monkey, chimpanzee, etc.). The dose of the protein or the like of the present invention varies depending on the target disease, the administration subject, the administration route, etc. (As 60 kg), the protein etc. is about 0.1 mg to 100 mg per day, preferably about 1.
The dosage is 0 to 50 mg, more preferably about 1.0 to 20 mg. When administered parenterally, 1 of the protein, etc.
Although the dose may vary depending on the administration subject, target disease, etc. About 0.01 to 3 for proteins
It is convenient to administer about 0 mg, preferably about 0.1 to 20 mg, and more preferably about 0.1 to 10 mg by injection into the affected area. In the case of other animals, the dose converted per 60 kg can be administered.

[2] Screening for Drug Candidate Compounds against Diseases Since the protein etc. of the present invention have esterase activity, especially triglyceride degrading activity, a compound or its compound which promotes the function (eg, esterase activity etc.) of the protein etc. of the present invention The salt can be used as a medicine such as a therapeutic / preventive agent for arteriosclerosis, hyperlipidemia, obesity and diabetes. Further, since the gene of the protein of the present invention is specifically expressed in adipose tissue, the compound or its salt that inhibits the function of the protein of the present invention (eg, esterase activity etc.) is caused by triglyceride decomposition. Since it can suppress the accumulation of free fatty acids, it can be used as a medicine such as a prophylactic / therapeutic agent for obesity. Therefore, the protein or the like of the present invention is useful as a probe for screening a compound or its salt 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, may be abbreviated as the protein of the present invention), and the function of the protein of the present invention (for example, esterase activity and the like). The present invention provides a method for screening a compound having the activity of promoting or inhibiting the activity of, specifically, culturing cells having the ability to express the gene of the protein of the present invention in the presence of a test compound, A compound having the activity of promoting or inhibiting the esterase activity of the protein of the present invention, which comprises measuring the amount of mRNA encoding the protein of the present invention using DNA encoding the protein or its complementary DNA or partial DNA thereof. Or a method for screening a salt thereof, more specifically, (i) the method of the present invention. mR of the protein of the present invention when culturing cells capable of expressing the gene of Park protein
The expression amount of NA is compared with (ii) the amount 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 in the presence of a test compound. The present invention also provides a method for screening a compound or salt thereof having an activity of promoting or inhibiting the esterase activity of the protein of the present invention.

The cells having the ability to express the gene of the protein of the present invention include, for example, the above-mentioned known warm-blooded animal cells (preferably adipocytes, macrophages, skeletal muscle cells) and the gene of the protein of the present invention. Examples of the transformed animal cells include: Animal cells transformed by introducing the gene of the protein of the present invention can be produced by the above-mentioned method. Culturing of cells having the ability to express the gene of the protein of the present invention is carried out in the same manner as known animal cell culture methods. For example, the medium is about 5
MEM medium containing 20% fetal bovine serum [Science, Volume 122, 501 (1952)], DME
M medium [Virology, 8 volumes, 396 (1
959)], RPMI 1640 medium [Journal of the American Medical Association (Th
e Journal of the American Medical Association) 1
99, 519 (1967)], 199 medium [Proceeding of the Society fort]
he Biological Medicine), 73, 1 (1950)]. The pH is preferably about 6-8. Culturing is usually performed at about 30 to 40 ° C. for about 15 to 60 hours, and aeration and stirring may be added if necessary.

To compare the expression levels of mRNAs by the hybridization method, a method known per se or a method analogous thereto, for example, Molecular Cloning 2nd (J. Sambrook et al., C
Old Spring Harbor Lab. Press, 1989) and the like. Specifically, the amount of mRNA encoding the protein of the present invention is measured by RNA 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 D
It is carried out by contacting NA 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. By labeling the complementary DNA of the gene DNA of the protein of the present invention or its partial DNA with, for example, a radioisotope or a dye, the amount of mRNA bound to the complementary DNA of the gene DNA of the protein of the present invention can be easily measured. . As the radioisotope, for example, ³² P, ³ H or the like is used, and as the dye, for example, fluorescein or FAM.
(PE Biosystems), JOE (PE Biosystems), TAMRA (PE Biosystems), ROX (PE
Biosystems), Cy5 (Amersham), Cy3
A fluorescent dye such as (Amersham) is used.

The amount of mRNA of the protein of the present invention can be determined by measuring the RNA extracted from cells with a reverse transcriptase cD.
After conversion into NA, the DNA encoding the gene of the protein of the present invention or its complementary DNA or its part D
This can be done by measuring the amount of cDNA amplified by PCR using NA as a primer. Complementary DNA of gene DNA of the protein of the present invention used for measuring the amount of mRNA of the protein of the present invention
Is the gene DNA of the protein of the present invention (upper chain)
DNA having a sequence complementary to (lower strand) can be mentioned.
The partial DNA of the gene DNA of the protein of the present invention is, for example, about 10 to 2,200 continuous, preferably 10 in the base sequence of the gene DNA of the protein of the present invention.
The base sequence includes about 300 to about 300 bases, more preferably about 10 to 30 bases. Examples of the partial DNA of the complementary DNA of the gene DNA 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, about 10 to 2200 consecutive bases,
Preferably about 10 to 300, more preferably 10
An example of the DNA is a DNA having a sequence complementary to a base sequence composed of about 30 bases. Examples of the primer used in PCR include DNA having the base sequence represented by SEQ ID NO: 5 and DNA having the base sequence represented by SEQ ID NO: 6. A test compound that increases the amount of mRNA of the protein of the present invention can be selected as a compound having an activity of promoting expression of the gene of the protein of the present invention, and a test compound that decreases the amount of mRNA of the protein of the present invention Can be selected as a compound having an activity of inhibiting the gene expression of the protein of the present invention.

The present invention also relates to cells (for example, adipocytes, macrophages, etc.) transformed with DNA in which a known promoter or enhancer region of the protein of the present invention is cloned from genomic DNA and ligated upstream of an appropriate reporter gene. Skeletal muscle cells, etc.) are cultured in the presence of a test compound, and the expression of the reporter gene is detected instead of the expression of the protein of the present invention, thereby promoting or inhibiting the esterase activity of the protein of the present invention. A method for screening a compound having: or a salt thereof is provided. As the reporter gene, for example, a staining marker gene such as lacZ (β-galactosidase gene) and the like are used. By measuring the amount of the reporter gene product (eg, mRNA, protein) using a method known per se, a test compound which increases the amount of the reporter gene product has an activity of promoting the gene expression of the protein of the present invention. A compound that can be selected as a compound, and a test compound that reduces the amount of the reporter gene product can be selected as a compound having an activity of inhibiting the gene expression of the protein of the present invention. Culturing of cells can be performed in the same manner as the above-described known animal cell culture.

[0048] Further, the present invention is (i) RI labeled metabolic precursor protein into a cell capable of producing the present invention (hereinafter sometimes referred to as precursor. Specifically the ³ H-labeled or ⁽¹⁴⁾ C-labeled fatty acids such as oleic acid, glycerol, etc.) and (ii) RI-labeled precursors and test compounds for cells capable of producing the protein of the present invention The amount of neutral lipids (eg triglyceride, cholesteryl ester, etc.) produced when contacted with cultivated was determined by biochemical analysis (edited by Hideki Yuki, pp.
235-272, Nankodo, 1984), which promotes or inhibits the esterase activity of the protein of the present invention, which is characterized by being measured and compared by, for example, a thin layer chromatography method or a column chromatography method. Provided is a method for screening a compound having activity or a salt thereof. Culturing of cells can be performed in the same manner as the above-described known animal cell culture. 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 expression of the gene of the protein of the present invention. A test compound that promotes the production amount or reduces the production amount of the degradation product of neutral lipid can be selected as a compound having an activity of inhibiting the expression of the gene 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 Osterlu.
nd T. Methods in Molecular Biology, edited by MH
It can be measured according to the method described in Doolittle and K. Reue, Vol. 109, pp109-121 or a method similar thereto. For example, the esterase activity in the case of (ii) above is about 20% or more as compared with the case of (i) above,
A test compound that increases preferably by 30% or more, more preferably by about 50% or more can be selected as a compound that promotes esterase activity of the protein or the like of the present invention. On the other hand, the test compound that reduces the esterase activity in the case of (ii) by about 20% or more, preferably by 30% or more, more preferably by about 50% or more as compared with the case of (i) above is used for the protein of the present invention and the like. Can be selected as a compound that inhibits the esterase activity of

Furthermore, in the present invention, cells having the ability to express the gene of the protein of the present invention are cultured in the presence of a test compound, and the expression level of the protein of the present invention is measured using an antibody of the protein of the present invention. A method for screening a compound or a salt thereof having an activity of promoting or inhibiting the esterase activity of the protein of the present invention, specifically, (i) a cell having the ability to express the gene of the protein of the present invention And (ii) the expression level of the protein of the present invention when the cells having the ability to express the gene of the protein of the present invention are cultured in the presence of the test compound. Promotion or inhibition of esterase activity of the protein of the present invention, characterized by being measured and compared using an antibody of the protein of the present invention Compound or to provide a screening method for a salt having that activity. The antibody of the protein of the present invention can be produced by the method described above. Culturing of cells can be performed in the same manner as the above-described known animal cell culture. 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 [3] below.

More specifically, (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, the culture solution (test solution) and labeled In the case of competitively reacting with the protein of the present invention and (ii) culturing cells having the ability to express the gene of the protein of the present invention in the presence of a test compound, , The ratio of the labeled protein of the present invention bound to the antibody is compared with the case where the culture solution (test liquid) and the labeled protein of the present invention are 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, (i) having the ability to express the gene of the protein of the present invention When culturing cells, the culture solution (test solution) and the antibody of the protein of the present invention insolubilized on a carrier and another antibody of the present invention labeled are reacted simultaneously or successively, (Ii) A cell having the ability to express the gene of the protein of the present invention is cultured in the presence of a test compound, and the antibody of the protein of the present invention which has been insolubilized on the culture medium (test liquid) and a carrier and labeled The activity of promoting or inhibiting the esterase activity of the protein of the present invention, characterized in that the activity of the labeling agent on the insolubilized carrier is measured when it is reacted with another antibody of the present invention simultaneously or continuously. A method for screening a compound having: or a salt thereof is provided. In the above method, it is desirable that one antibody recognizes the N-terminal portion of the protein of the present invention and the other antibody reacts with the C-terminal portion of the protein of the present invention. In the screening method described above, examples of the test compound include peptides, proteins, non-peptidic compounds, synthetic compounds, fermentation products, cell extracts, plant extracts, animal tissue extracts, and the like. The compound may be a known compound or a known compound.

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

The compound or its salt obtained by using the screening method or the screening kit of the present invention is a test compound described above, for example, a peptide, protein, non-peptidic compound, synthetic compound, fermentation product, cell extract, It is a compound selected from a plant extract, an animal tissue extract, plasma and the like, and is a compound having an activity of promoting the function (eg, esterase activity) of the protein of the present invention. As the salt of the compound, those similar to the salts of the protein of the present invention described above are used. The compound of the present invention having an activity of promoting functions such as proteinase activity (eg, esterase activity) is used as a drug such as a therapeutic / prophylactic agent for diseases such as arteriosclerosis, hyperlipidemia, obesity and diabetes. Can be used. On the other hand, the compound having an activity of inhibiting the function of the protein or the like (eg, esterase activity) of the present invention can be used as a medicine such as a therapeutic / prophylactic agent for diseases such as obesity.

When the 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 in a conventional manner. For example, in the same manner as the above-mentioned pharmaceutical containing the protein of the present invention, orally or parenterally administered as tablets, capsules, elixirs, microcapsules, sterile solutions, suspensions and the like. You can Since the preparation thus obtained is safe and has low toxicity, for example, warm-blooded animals (for example, humans, mice,
Rat, rabbit, sheep, pig, cow, horse, bird, cat, dog, monkey, chimpanzee, etc.). The dose of the compound or its salt varies depending on its action, target disease, administration subject, administration route, etc. When administered orally,
Generally, in an adult human (as a body weight of 60 kg), the compound is about 0.1 to 100 mg, preferably about 1.0 to 50 mg, more preferably about 1.0 to 20 mg per day.
Administer. When administered parenterally, the single dose of the compound varies depending on the administration subject, target disease, etc.
For example, for the purpose of treating hyperlipidemia, a compound that promotes the function of the protein of the present invention or the like in the form of an injection is usually used for adults (60k).
g)), the amount of the compound is about 0.01 to 30 mg per day, preferably about 0.1 to 20 m
It is convenient to administer about g, more preferably about 0.1 to 10 mg by intravenous injection. In the case of other animals, the dose converted per 60 kg can be administered.

[3] Quantification of Protein of the Present Invention, Partial Peptides or Salts Thereof The antibody against the protein of the present invention (hereinafter sometimes abbreviated as the antibody of the present invention) is specific for the protein of the present invention. Since it can be recognized specifically, it can be used for quantification of the protein or the like of the present invention in a test solution, particularly for quantification by sandwich immunoassay. That is, the present invention provides (i) the antibody of the present invention, the test liquid, the labeled protein of the present invention, and the like, which are competitively reacted to bind to the labeled protein of the present invention. A method for quantifying the protein or the like of the present invention in a test solution, which comprises measuring the ratio of (ii) the test solution and the antibody of the present invention insolubilized on a carrier, and another of the labeled present invention A method for quantifying the protein or the like of the present invention in a test solution, which comprises reacting with an antibody simultaneously or continuously and then measuring the activity of the labeling agent on the insolubilized carrier, and (iii) above (i) Or a method for diagnosing diseases such as arteriosclerosis, hyperlipidemia, obesity and diabetes using the quantitative method described in (ii).
In the quantification method of (ii) above, one antibody is an antibody that recognizes the N-terminal portion of the protein of the present invention and the other antibody is an antibody that reacts with the C-terminal portion of the protein of the present invention. desirable.

Further, a monoclonal antibody against the protein or the like of the present invention (hereinafter sometimes referred to as the monoclonal antibody of the present invention) can be used to quantify the protein or the like of the present invention, and detection by tissue staining or the like can be performed. You can also 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, an antigen or an antibody-antigen complex corresponding to the amount of antigen (eg, amount of protein) in the liquid to be measured. Any measuring method may be used as long as it is a method of detecting the amount of the compound by chemical or physical means and calculating it from a standard curve prepared using a standard solution containing a known amount of the antigen. For example, nephrometry, competitive method, immunometric method and sandwich method are preferably used,
From the viewpoint of sensitivity and specificity, it is particularly preferable to use the sandwich method described later. As the labeling agent used in the measuring method using the labeling substance, for example, a radioisotope, an enzyme, a fluorescent substance, a luminescent substance, etc. are used. Examples of radioactive isotopes include [ ¹²⁵ I], [ ¹³¹ I], [ ³ H], [
¹⁴ C] or the like is used. As the above-mentioned enzyme, those having a stable and large specific activity are preferable, and for example, β-galactosidase, β-glucosidase, alkaline phosphatase, peroxidase, malate dehydrogenase and the like are used. As the fluorescent substance, for example, fluorescamine, fluorescein isothiocyanate, etc. are used. Luminol, a luminol derivative, luciferin, lucigenin, etc. are used as a luminescent substance, for example. Furthermore, the biotin-avidin system can be used for binding the antibody or the antigen to the labeling agent.

For the insolubilization of an antigen or an antibody, physical adsorption may be used, or a method using a chemical bond which is usually used for insolubilizing or immobilizing proteins or enzymes. 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 (first reaction), and further reacted with another labeled monoclonal antibody of the present invention (secondary reaction), and then on the insolubilized carrier. The amount of the protein of the present invention in the test liquid can be quantified by measuring the activity of the labeling agent. The primary reaction and the secondary reaction may be carried out in the reverse order, may be carried out simultaneously, or may be carried out at different times. The labeling agent and the method of insolubilization can be the same as those described above. In addition, in the immunoassay method by the sandwich method,
The antibody used for the solid phase antibody or the labeling antibody is not necessarily one type, and a mixture of two or more types of antibodies may be used for the purpose of improving the measurement sensitivity. In the method for measuring the protein of the present invention by the sandwich method of the present invention, the monoclonal antibody of the present invention used in the primary reaction and the secondary reaction is preferably an antibody having different binding sites for the protein of the present invention. Be done. That is, the antibody used in the primary reaction and the secondary reaction is preferably, for example, when the antibody used in the secondary reaction recognizes the C-terminal portion of the protein of the present invention, the antibody used in the primary reaction is preferably An antibody that recognizes an N-terminal other than the C-terminal is used.

The monoclonal antibody of the present invention can be used in a measuring system other than the sandwich method, for example, a competitive method, an immunometric method or nephrometry. In the competitive method, the antigen in the test solution and the labeled antigen are reacted competitively with the antibody, and then the unreacted labeled antigen is reacted.
(F) and the labeled antigen (B) bound to the antibody are separated (B / F separation), the labeled amount of either B or F is measured, and the amount of antigen in the test liquid is quantified. In this reaction method, a soluble antibody is used as the antibody, a liquid phase method using polyethylene glycol, a second antibody against the antibody is used for B / F separation, and a solid phase antibody is used as the first antibody, or The first antibody is soluble and the second antibody is a solid-phased antibody. In the immunometric method, the antigen in the test liquid and the solid-phased antigen are competitively reacted with a fixed amount of the labeled antibody and then the solid phase and the liquid phase are separated, or the antigen in the test liquid is separated. After reacting with an excess amount of the labeled antibody and then adding the immobilized antigen to bind the unreacted labeled antibody to the solid phase, the solid phase and the liquid phase are separated.
Next, the labeled amount of either phase is measured to quantify the amount of antigen in the test liquid. In nephrometry, the amount of insoluble precipitate produced as a result of the antigen-antibody reaction in the gel or in the solution is measured. Even when the amount of antigen in the test liquid is small and only a small amount of precipitate can be obtained, laser nephrometry utilizing laser scattering is preferably used.

When applying these individual immunological measurement methods to the quantification method of the present invention, it is not necessary to set special conditions, operations and the like. The measurement system for the protein or the like of the present invention may be constructed by adding ordinary technical consideration to those skilled in the art to the usual conditions and operating methods in each method. For details of these general technical means, reference can be made to reviews, books, and the like. For example, Hiroshi Irie "Radioimmunoassay" (Kodansha, published in 1974), Hiroshi Irie "Radioimmunoassay" (Kodansha, published in 1979), Eiji Ishikawa et al. "Enzyme Immunoassay" (Medical Shoin, Showa Showa) 1993), Eiji Ishikawa et al., "Enzyme Immunoassay" (second edition) (Medical Shoin, published in 1982), Eiji Ishikawa, et al. "Enzyme Immunoassay" (3rd edition) (Medicine Shoin, Showa) 1987), "Methods in ENZYMOLOGY" Vol. 70 (Immunochem
ical Techniques (Part A)), ibid Vol. 73 (Immunochem
ical Techniques (Part B)), ibid Vol. 74 (Immunochem
ical Techniques (Part C)), ibid Vol. 84 (Immunochem
ical Techniques (Part D: Selected Immunoassays)),
Ibid Vol. 92 (Immunochemical Techniques (Part E: Mono
clonal Antibodies and General Immunoassay Method
s)), ibid Vol. 121 (Immunochemical Techniques (Part
I: Hybridoma Technology and Monoclonal Antibodie
s)) (above, published by Academic Press). As described above, by using the antibody of the present invention, the protein or the like of the present invention can be quantified with high sensitivity. 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, obesity It can be diagnosed as having a disease such as illness, diabetes, or having a high possibility of contracting in the future. On the other hand, when an increase 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, it is a disease such as obesity, or a future disease. It is possible to diagnose that there is a high possibility that Further, the antibody of the present invention can be used for detecting the protein of the present invention present in a subject such as body fluid or 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 during purification, analysis of the behavior of the protein of the present invention in test cells, etc. Can be used for

[4] Pharmaceutical containing the antibody of the present invention The antibody of the present invention having an action of neutralizing the activity of the protein of the present invention or its partial peptide is, for example, an overexpression of the protein of the present invention or its partial peptide. It can be used as a medicine such as a prophylactic / therapeutic drug for a disease (eg, obesity) caused by. The therapeutic / prophylactic agent for the above-mentioned diseases containing the antibody of the present invention, as a liquid formulation as it is, or as a pharmaceutical composition in an appropriate dosage form, human or mammal (eg, rat, rabbit, sheep, pig, cow, cat, Can be orally or parenterally administered to dogs, monkeys, etc.). The dose varies depending on the administration subject, target disease, symptom, administration route, etc., but when it is used for an adult, for example, the antibody of the present invention is usually administered in a dose of about 0.01 to 20 mg / kg body weight. , Preferably 0.1
-10 mg / kg body weight, more preferably 0.1-
It is convenient to administer about 5 mg / kg body weight by intravenous injection about 1 to 5 times a day, preferably about 1 to 3 times a day. In the case of other parenteral administration and oral administration, an amount similar to this can be administered. If the symptoms are particularly severe, the dose may be increased according to the symptoms. The antibody of the present invention can be administered per se or as an appropriate 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 a composition is
It is provided as a dosage form suitable for oral or parenteral administration.
That is, for example, as a composition for oral administration,
Solid or liquid dosage forms, such as tablets (including sugar-coated tablets and film-coated tablets), pills, granules, powders, capsules (including soft capsules), syrups, emulsions, suspensions, etc. To be Such a composition is produced by a method known per se and contains a carrier, a diluent or an excipient which is usually used in the field of formulation. For example, lactose, starch, sucrose, magnesium stearate and the like are used as carriers and excipients for tablets.

As the composition for parenteral administration, for example, injections, suppositories, etc. are used, and the injections are intravenous injection, subcutaneous injection, intradermal injection, intramuscular injection, drip injection, etc. Dosage forms of Such an injection is prepared according to a method known per se, for example, by dissolving, suspending or emulsifying the above-mentioned antibody or a salt thereof in a sterile aqueous or oily liquid usually used for injection. As the aqueous solution for injection, for example, physiological saline, isotonic solution containing glucose and other adjuvants, etc. are used.
For example, alcohol (eg, ethanol), polyalcohol (eg, propylene glycol, polyethylene glycol), nonionic surfactant [eg, polysorbate 80,
HCO-50 (polyoxyethylene (50mol) adduct of
hydrogenated castor oil)] etc.
As the oily liquid, for example, sesame oil, soybean oil and the like are used, and solubilizing agents such as benzyl benzoate and benzyl alcohol may be used in combination. The prepared injection solution is usually filled in a suitable ampoule. The suppository used for rectal administration is prepared by mixing the above antibody or a salt thereof with an ordinary suppository base. The oral or parenteral pharmaceutical compositions described above are conveniently prepared in dosage unit form for administration of the active ingredient.
Examples of the dosage form of such a dosage unit include tablets, pills, capsules, injections (ampoules), suppositories, etc., usually 5 to 500 mg per dosage unit dosage form, especially 5 to 100 mg for injections, 10-2 for other dosage forms
It is preferable to contain 50 mg of the above antibody.
Each composition described above may contain other active ingredients as long as the composition does not cause an unfavorable interaction with the antibody.

[5] Gene Diagnostic Agent The DNA or antisense DNA of the present invention is, for example,
By using as a probe, the protein of the present invention or a portion thereof in a warm-blooded animal (eg, human, rat, mouse, guinea pig, rabbit, bird, sheep, pig, cow, horse, cat, dog, monkey, chimpanzee, etc.) Since it is possible to detect an abnormality (gene abnormality) in the DNA or mRNA encoding the peptide, for example, genetic diagnosis of damage, mutation or decreased expression of the DNA or mRNA, increase or excessive expression of the DNA or mRNA, etc. It is useful as an agent. The above-mentioned gene diagnosis using the DNA of the present invention can be carried out, for example, by known Northern hybridization or PCR-SSCP.
Method (Genomics), Volume 5, 874-87
Page 9 (1989), Proceedings of the National Academy of Sciences of
USA (Proceedings of the Natinal Academy of
Sciences of the United States of America), 8th
Vol. 6, pages 2766-2770 (1989)) and the like. For example, when decreased expression is detected by Northern hybridization or PCR
-If a DNA mutation is detected by the SSCP method, it can be diagnosed as being likely to be a disease such as arteriosclerosis, hyperlipidemia, obesity, and diabetes.

[6] Pharmaceutical Agent Containing Antisense Polynucleotide The present invention is capable of binding complementarily to the polynucleotide (eg, DNA) of the present invention and suppressing the expression of the polynucleotide (eg, DNA). Since the antisense polynucleotide has low toxicity and can suppress the function of the protein of the present invention, its partial peptide or its salt, or the polynucleotide (eg, DNA) of the present invention in vivo, for example, the present invention Can be used as a prophylactic / therapeutic agent for diseases (eg, obesity) caused by overexpression of the protein or its partial peptide. When the antisense polynucleotide is used as the therapeutic / prophylactic agent, the antisense polynucleotide can be formulated in the same manner as in the case of the polynucleotide of the present invention. The preparation thus obtained has low toxicity and should be orally or parenterally administered to humans or mammals (eg, rat, rabbit, sheep, pig, cow, cat, dog, monkey, etc.) You can The antisense polynucleotide can be administered as it is or with a physiologically acceptable carrier such as an auxiliary agent 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 administration route, etc. k
It is about 0.1 to 100 mg per day for g). Further, the antisense polynucleotide can also be used as a diagnostic oligonucleotide probe for investigating the presence of the DNA of the present invention or the expression state thereof in tissues or cells.

The present invention further comprises a double-stranded RNA containing a part of RNA encoding the protein of the present invention or a partial peptide thereof and RNA complementary thereto, a medicament comprising the double-stranded RNA, There is provided a ribozyme containing a part of RNA encoding the protein of the present invention or a partial peptide thereof, and a medicament comprising the ribozyme. These double-stranded RNA (RNAi; RNA interference method),
Ribozymes and the like can suppress the expression of the polynucleotide (eg, DNA) of the present invention in the same manner as the above antisense polynucleotides, and thus the protein of the present invention or its partial peptide or its salt in vivo, or the present invention Since it can suppress the function of the polynucleotide (eg, DNA), it can be used as a prophylactic / therapeutic agent for a disease (eg, obesity) caused by overexpression of the protein of the present invention or its partial peptide. it can. Double-stranded RNA can be prepared by a known method (eg, Nature, Vol. 411,
Page 494, 2001), and can be designed and produced based on the sequence of the polynucleotide of the present invention. Ribozymes can be prepared by known methods (eg, TRENDS in Molecular Medicin
e, Vol. 7, p. 221, 2001), and can be designed and manufactured based on the sequence of the polynucleotide of the present invention.
For example, it can be produced by linking a known ribozyme to a part of RNA encoding the protein of the present invention or its partial peptide. Examples of a part of RNA encoding the protein of the present invention or a partial peptide thereof include a portion (RNA fragment) close to the cleavage site on the RNA of the present invention which can be cleaved by a known ribozyme. When the above-mentioned double-stranded RNA or ribozyme is used as the above-mentioned preventive / therapeutic agent, it can be formulated and administered in the same manner as the antisense polynucleotide.

[7] DNA Transfer Animal The present invention provides a DNA encoding an exogenous protein of the present invention (hereinafter abbreviated as exogenous DNA of the present invention) or a mutant DNA thereof (exogenous mutated DNA of the present invention). Sometimes abbreviated). That is, the present invention provides (1) a non-human mammal having the exogenous DNA of the present invention or a mutant DNA thereof, (2) an animal according to (1), wherein the non-human mammal is a rodent, (3)
(2) An 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. . Exogenous DNA of the present invention
Alternatively, a non-human mammal having the mutant DNA (hereinafter,
The abbreviated DNA-transferred animal of the present invention) is, for unfertilized eggs, fertilized eggs, germ cells including sperm and its progenitor cells, preferably the stage of embryogenesis in the development of non-human mammals (more preferably At the single-cell or fertilized egg cell stage and generally before the 8-cell stage), calcium phosphate method, electric pulse method, lipofection method, agglutination method, microinjection method, particle gun method, DE
It can be produced by transferring the target DNA by the AE-dextran method or the like. Also, the D
The foreign DNA of the present invention can be transferred to somatic cells, living organs, tissue cells and the like by the NA transfer method and used for cell culture, tissue culture and the like. The DNA-transferred animal of the present invention can also be produced by fusing cells with a cell fusion method known per se.

Non-human mammals include, for example, bovine,
Pigs, sheep, goats, rabbits, dogs, cats, guinea pigs, hamsters, mice, rats and the like are used. Among them, rodents, especially mice (for example, C57 as a pure line), which have relatively short ontogeny and biological cycle from the viewpoint of creating a pathological animal model system, and are easy to reproduce.
BL6 line, DBA2 line, etc.
C3F ₁ system, BDF ₁ system, B6D2F ₁ system, BAL
B / c strain, ICR strain, etc.) or rat (eg,
(Wistar, SD, etc.) and the like are preferable. "Mammal" in a recombinant vector that can be expressed in a mammal
Examples of the non-human mammal include humans and the like. The exogenous DNA of the present invention does not mean the DNA of the present invention originally possessed by the non-human mammal, but the DNA of the present invention once isolated and extracted from the mammal. The mutant DNA of the present invention is a DNA having a mutation (eg, mutation) in the base sequence of the original DNA of the present invention, specifically, addition of a base, deletion, substitution with another base, etc. The generated DNA or the like is used, and abnormal DNA is also included. The abnormal DNA means a DNA that expresses an abnormal protein of the present invention. For example, D that expresses a protein that suppresses the function of the normal protein of the present invention.
NA or the like is used. The exogenous DNA of the present invention may be derived from mammals of the same or different species as the target animal. 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 that can be expressed in animal cells. For example, when transferring the human DNA of the present invention, various mammals having the DNA of the present invention having high homology with the human DNA (eg, rabbit,
Fertilized eggs of a target mammal, to which the DNA construct (eg, vector) in which the human DNA of the present invention is bound downstream of various promoters capable of expressing DNA derived from dog, cat, guinea pig, hamster, rat, mouse, etc.,
For example, a DNA-transferred mammal highly expressing the DNA of the present invention can be produced by microinjection into fertilized mouse eggs.

The expression vector of the protein of the present invention includes Escherichia coli-derived plasmid, Bacillus subtilis-derived plasmid, yeast-derived plasmid, bacteriophage such as λ phage, retrovirus such as Moloney leukemia virus, vaccinia virus or baculovirus. The animal viruses and the like are used. Of these, a plasmid derived from Escherichia coli, a plasmid derived from Bacillus subtilis, a plasmid derived from yeast, and the like are preferably used. DN above
Examples of the promoter that regulates A expression include:
DNA promoters derived from viruses (eg, simian virus, cytomegalovirus, Moloney leukemia virus, JC virus, breast cancer virus, poliovirus, etc.), various mammals (human, rabbit, dog, cat,
Promoters derived from guinea pig, hamster, rat, mouse, etc., such as albumin, insulin II,
Uroplakin II, elastase, erythropoietin,
Endothelin, muscle creatine kinase, glial fibrillary acidic protein, glutathione S-transferase, platelet-derived growth factor β, keratins K1, K10 and K14, collagen type I and type II, cyclic AMP-dependent protein kinase βI subunit, dystrophin, Tartrate-resistant alkaline phosphatase,
Atrial natriuretic factor, endothelial receptor tyrosine kinase (generally abbreviated as Tie2), sodium potassium adenosine 3 phosphatase (Na, K-ATPas)
e), neurofilament light chain, metallothionein I
And IIA, metalloproteinase 1 tissue inhibitor, MHC class I antigen (H-2L), H-ras, renin, dopamine β-hydroxylase, thyroid peroxidase (TPO), polypeptide chain elongation factor 1α (EF-
1α), β actin, α and β myosin heavy chain, myosin light chain 1 and 2, myelin basic protein, thyroglobulin, Thy-1, immunoglobulin, H chain variable region (V
NP), serum amyloid P component, myoglobin, troponin C, smooth muscle α-actin, preproenkephalin A, vasopressin, and other promoters. Of these, a cytomegalovirus promoter, a human polypeptide chain elongation factor 1α (EF-1α) promoter, a human and chicken β actin promoter, which can be highly expressed systemically, are preferable.

The above vector preferably has a sequence (generally called terminator) that terminates the transcription of the desired messenger RNA in a DNA-transferred mammal. For example, DNAs derived from viruses and various mammals Can be used, and preferably the SV40 terminator of simian virus is used. In addition, a splicing signal of each DNA for the purpose of further highly expressing the foreign DNA of interest,
It is also possible to connect an enhancer region, a part of an intron of eukaryotic DNA, or the like 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 livers, kidneys, thyroid cells, fibroblasts derived from various mammals (eg, human, rabbit, dog, cat, guinea pig, hamster, rat, mouse, etc.).
It can be obtained as a whole or a part of genomic DNA from NA and various commercially available genomic DNA libraries, or as a starting material complementary DNA prepared by a known method from RNA derived from liver, kidney, thyroid cells, and fibroblasts. . In addition, an exogenous abnormal DNA can be prepared by mutating a translation region of a normal protein obtained from the above-mentioned cells or tissues by a point mutagenesis method. The translation region can be expressed in metastatic animals D
The NA construct can be prepared by a usual DNA engineering method in which it is ligated to the downstream of the promoter and, if desired, the upstream of the transcription termination site. The transfer of the foreign DNA of the present invention at the fertilized egg cell stage is ensured to be present in all germinal cells and somatic cells of the target mammal. The presence of the exogenous DNA of the present invention in the embryonic cell of the produced animal after DNA transfer means that all the progeny of the produced animal retain the exogenous DNA of the present invention in all of its embryonic cells and somatic cells. means. The offspring of this type of animal that inherits the exogenous DNA of the present invention has the exogenous DNA of the present invention in all of its germ cells and somatic cells.

The non-human mammal to which the exogenous normal DNA of the present invention has been transferred is confirmed to stably retain the exogenous DNA by mating, and then subcultured in a normal breeding environment as the DNA-carrying animal. You can The 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 cells and somatic cells of the target mammal. Excessive presence of the exogenous DNA of the present invention in the germinal cells of the producing animal after DNA transfer means that all the progeny of the producing animal have the exogenous DNA of the present invention in all of their germinal cells and somatic cells. Means The offspring of this type of animal that inherited the exogenous DNA of the present invention has the exogenous DNA of the present invention in all of its germ cells and somatic cells.
Have an excess. By obtaining a homozygous animal having the introduced DNA in both homologous chromosomes and mating the male and female animals, all offspring can be passaged so that the DNA has an excess. The non-human mammal having the normal DNA of the present invention is highly expressed with the normal DNA of the present invention, and by promoting the function of the endogenous normal DNA, finally, the hyperactivity of the protein of the present invention is suppressed. It may develop, and can be used as a model animal for its pathological condition. For example, using the normal DNA-transferred animal of the present invention, it is possible to elucidate the pathological mechanism of hyperactivity of the protein of the present invention and diseases related to the protein of the present invention, and to investigate treatment methods for these diseases. It is possible. In addition, since the mammal to which the exogenous normal 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 screening tests for therapeutic agents for diseases related to the protein of the present invention. is there.

On the other hand, the non-human mammal having the exogenous abnormal DNA of the present invention should be subcultured in a normal breeding environment as the DNA-bearing animal after confirming that the exogenous DNA is stably retained by mating. Can be done. Furthermore, the foreign DNA of interest can be incorporated into the above-mentioned plasmid and used as a protozoa. The DNA construct with the promoter can be prepared by a usual DNA engineering technique. Abnormality D of the present invention at the fertilized egg cell stage
NA transfer is ensured to be present in all germinal and somatic cells of the subject mammal. The presence of the abnormal DNA of the present invention in the germinal cells of the producing animal after DNA transfer means that all the progeny of the producing animal have the abnormal DNA of the present invention in all of its germinal cells and somatic cells. The offspring of this type of animal that inherits the exogenous DNA of the present invention has the abnormal DNA of the present invention in all of its germ cells and somatic cells. By obtaining a homozygous animal having the introduced DNA in both homologous chromosomes and mating the male and female animals, all the progeny can be passaged so that they have the DNA. The non-human mammal having the abnormal DNA of the present invention is highly expressed with the abnormal DNA of the present invention, and finally inhibits the function of the endogenous normal DNA, thereby finally deactivating the protein of the present invention. It may become refractory and can be used as a model animal for its pathological condition. For example, by using the abnormal DNA-transferred animal of the present invention, it is possible to elucidate the pathological mechanism of the functionally inactive refractory disease of the protein of the present invention and to examine the method for treating this disease. Further, as a specific applicability, the animal with high expression of abnormal DNA of the present invention is characterized by inhibition of normal protein function (dominant negat) by the abnormal protein of the present invention in functionally inactive refractory disease of the protein of the present invention.
ive action) becomes a model to elucidate. Further, since the mammal to 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 functionally inactive refractory disease of the protein of the present invention. Is.

Further, other uses of the above-mentioned two kinds of DNA-transferred animals of the present invention include, for example, use as a cell source for tissue culture, DNA or R in tissues of the DNA-transferred animals of the present invention.
Analysis of the relationship with a protein specifically expressed or activated by the protein of the present invention by directly analyzing NA or analyzing a protein tissue expressed by DNA, The cells 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, screening for agents that enhance the function of cells by using the cells described above, and It is conceivable to isolate and purify the mutant protein and produce its antibody. Furthermore, the DNA-transferred animal of the present invention can be used to investigate clinical symptoms of diseases related to the protein of the present invention, including functionally inactive refractory disease of the protein of the present invention, and the protein of the present invention. More detailed pathological findings in each organ of a disease model associated with thyroid gland can be obtained, which can contribute to the development of new treatment methods and further to the study and treatment of secondary diseases caused by the disease. Further, each organ was taken out from the DNA-transferred animal of the present invention, cut into small pieces, and then released by a proteolytic enzyme such as trypsin.
It is possible to obtain NA-transferred cells, culture them, or systematize the cultured cells. Furthermore, it is possible to investigate the specification of the protein-producing cell of the present invention, its relation to differentiation or proliferation, or the signal transduction mechanism in them, and to investigate their abnormalities. It becomes an effective research material.
Furthermore, in order to develop a therapeutic agent for a disease related to the protein of the present invention, including a functionally inactive refractory disease of the protein of the present invention, using the DNA-transferred animal of the present invention, the above-mentioned test method and quantification It is possible to provide an effective and rapid screening method for the drug for treating the disease using the method. Further, it is possible to study and develop a DNA therapeutic method for diseases associated with the protein of the present invention, using the DNA-transferred animal of the present invention or the foreign DNA expression vector of the present invention.

[8] Knockout Animal The present invention provides a non-human mammal embryonic stem cell in which the DNA of the present invention is inactivated and a DNA expression-deficient non-human mammal of the present invention. That is, the present invention provides (1) a non-human mammal embryonic stem cell in which the DNA of the present invention is inactivated,
(2) The embryonic stem cell according to item (1), wherein the DNA is inactivated by introducing a reporter gene (eg, β-galactosidase gene derived from Escherichia coli), (3) neomycin-resistant item (1) The embryonic stem cell according to item (4), the embryonic stem cell according to item (1), wherein the non-human mammal is a rodent, (5) the embryonic stem cell according to item (4), wherein the rodent is a mouse, ( 6) The DNA expression-deficient non-human mammal in which the DNA of the present invention is inactivated, (7) The DNA is inactivated by introducing a reporter gene (eg, β-galactosidase gene derived from Escherichia coli), The non-human mammal according to item (6), wherein the reporter gene can be expressed under the control of a promoter for the DNA of the present invention,
(8) The non-human mammal according to item (6), wherein the non-human mammal is a rodent, (9) the non-human mammal according to item (8), wherein the rodent is a mouse, and (10) ) D of the present invention, which comprises administering a test compound to the animal according to item (7) and detecting the expression of a reporter gene.
Provided is a screening method for a compound or its salt that promotes or inhibits the promoter activity against NA.

The non-human mammal embryonic stem cell in which the DNA of the present invention is inactivated means whether the expression ability of the DNA is suppressed by artificially mutating the DNA of the present invention possessed by the non-human mammal. Alternatively, the DNA does not substantially have the ability to express the protein of the present invention by substantially losing the activity of the protein of the present invention encoded by the DNA (hereinafter, referred to as the knockout DNA of the present invention. Occasionally) non-human mammalian embryonic stem cells (hereinafter ES
Abbreviated as cell). As the non-human mammal, the same ones as described above are used. Examples of the method for artificially mutating the DNA of the present invention include, for example, deletion of a part or all of the DNA sequence by a genetic engineering method, other DNA
By inserting or replacing. Due to these mutations, the knockout DNA of the present invention may be prepared, for example, by shifting the reading frame of the codon or disrupting the function of the promoter or exon. Specific examples of the non-human mammalian embryonic stem cell in which the DNA of the present invention is inactivated (hereinafter abbreviated as the DNA inactivated ES cell of the present invention or the knockout ES cell of the present invention) include, for example, The DNA of the present invention contained in a non-human mammal is isolated, and its exon portion has a neomycin resistance gene, a drug resistance gene represented by a hygromycin resistance gene, lacZ (β-galactosidase gene), cat (chloramphenicol acetyl). A DNA sequence (eg, polyA) that disrupts the function of an exon by inserting a reporter gene typified by a transferase gene) or terminates gene transcription at an intron portion between exons.
Insert additional signals, etc., and complete messenger R
By disabling NA synthesis, a DNA chain having a DNA sequence constructed so as to destroy the gene as a result (hereinafter abbreviated as targeting vector) is introduced into the chromosome of the animal by, for example, homologous recombination method. , D on the Southern hybridization analysis or targeting vector of the obtained ES cells using the DNA sequence of the present invention or a DNA sequence in the vicinity thereof as a probe
The knockout E of the present invention was analyzed by the PCR method using the NA sequence and the DNA sequence of the neighboring region other than the DNA of the present invention used for the preparation of the targeting vector as a primer.
It can be obtained by selecting S cells.

The DN of the present invention can also be obtained by the homologous recombination method or the like.
As the original ES cells for inactivating A, for example, those already established as described above may be used.
It may be newly established according to the method of Evans and Kaufma. For example, in the case of mouse ES cells, currently, 129 ES cells are generally used. However, since the immunological background is not clear, an alternative pure line immunological genetic background is used. For the purpose of obtaining ES cells whose expression is clear, for example, C57BL / 6 mouse and C57BL /
Well as the number of oocytes retrieved the lack of 6 DBA / 2 and BDF ₁ mice improved by crossing (C57BL / 6 and F ₁ and DBA / 2) which were established by using the usable good. B
The DF ₁ mouse has a large number of eggs and has the advantage that the eggs are strong, and since it has a C57BL / 6 mouse as a background, the ES cells obtained using this can be used when a pathological model mouse is produced. , C57BL / 6 mice can be advantageously used in that their genetic background can be changed to that of C57BL / 6 mice. Also,
When ES cells are established, blastocysts on the 3.5th day after fertilization are generally used. In addition to this, by culturing 8-cell stage embryos and culturing them up to the blastocysts, many initial stages can be efficiently performed. The embryo can be obtained. Although either male or female ES cells may be used, male ES cells are usually more convenient for producing a germ line chimera. In addition, it is desirable to distinguish males and females as soon as possible in order to reduce troublesome culturing. As a method for determining the sex of ES cells, for example, a method of amplifying and detecting a gene in the sex determining region on the Y chromosome by the PCR method can be mentioned. Using this method, conventionally, for example G-banding method, requires about 10 ⁶ cells for karyotype analysis, since suffices ES cell number of about 1 colony (about 50), culture It is possible to perform the primary selection of ES cells in the early stage by discriminating males and females, and the early selection of male cells can significantly reduce the labor required in the initial stage of culture.

The secondary selection can be carried out, for example, by confirming the number of chromosomes by the G-banding method. The number of chromosomes of ES cells obtained is preferably 100% of the normal number, but if it is difficult due to physical manipulations during establishment, after knocking out the gene of ES cells, normal cells (for example, in mice, The number is 2n
= 40 cells). The embryonic stem cell line thus obtained usually has a very good proliferative property, but since it is easy to lose the ability to develop ontogeny, it is necessary to carefully subculture. For example, on a suitable feeder cell such as STO fibroblast in the presence of LIF (1-10000 U / ml) in a carbon dioxide incubator (preferably 5% carbon dioxide, 95% air or 5% carbon dioxide).
It is cultivated by a method such as culturing at about 37 ° C. in% oxygen, 5% carbon dioxide gas, 90% air), and at the time of subculture, for example, trypsin / EDTA solution (usually 0.001-0.5% trypsin / 0.1%). 1-5 mM EDTA, preferably about 0.1% trypsin / 1 mM EDTA) to give single cells,
For example, a method of seeding on newly prepared feeder cells can be used. Such passage is usually carried out every 1 to 3 days. At this time, it is desirable to observe the cells and to discard the cultured cells when morphologically abnormal cells are found. ES cells are differentiated into various types of cells such as parietal muscle, visceral muscle, and myocardium by performing monolayer culture until reaching a high density or suspension culture until forming a cell clump under appropriate conditions. It is possible to
Evans and MH Kaufman, Nature No. 292
Volume, 154, 1981; GR Martin Proceedings of National Academy of Sciences USA (Proc. Natl. Acad. Sci. USA) No. 7
8: 7634, 1981; TC Doetschman et al., Journal of Embiology and Experimental Morphology, 87, 27, 1985], obtained by differentiating the ES cell of the present invention. The DNA expression-deficient cells of the present invention are useful in in vitro cell biological studies of the proteins of the present invention. DNA of the present invention
An expression-deficient non-human mammal 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, the same ones as described above are used.

The non-human mammal deficient in expression of the DNA of the present invention can be obtained by, for example, introducing the targeting vector prepared as described above into mouse embryonic stem cells or mouse egg cells, and introducing the targeting vector D of the present invention.
It is possible to knock out the DNA of the present invention by performing homologous recombination in which the DNA sequence in which NA is inactivated replaces the DNA of the present invention on the chromosome of mouse embryonic stem cells or mouse egg cells by gene homologous recombination. it can. The cells in which the DNA of the present invention has been knocked out are the DNA sequences on the Southern hybridization analysis or targeting vector using the DNA sequence on or near the DNA of the present invention as a probe, and the mouse-derived cells of the present invention used for the targeting vector. It can be determined by an analysis by the PCR method using a DNA sequence in a neighboring region other than DNA as a primer. When a non-human mammal embryonic stem cell is used, a cell line in which the DNA of the present invention is inactivated by gene homologous recombination is cloned, and the cell is cloned at an appropriate time, for example, at the 8-cell stage. It is injected into an animal embryo or blastocyst, and the produced chimeric embryo is transplanted into the uterus of the pseudopregnant non-human mammal. The produced animal is a chimeric animal composed of both cells having the normal DNA locus of the present invention and cells having the artificially mutated DNA locus of the present invention. When a part of the germ cells of the chimeric animal has the mutated DNA locus of the present invention, all tissues are artificially mutated from the population obtained by mating such a chimeric individual with a normal individual. D of the present invention added
It can be obtained by selecting individuals composed of cells having the NA locus, for example, by determining the coat color. The individual thus obtained is usually an individual deficient in hetero expression of the protein of the present invention, and heterologous individuals deficient in hetero expression of the protein of the present invention are crossed with each other to homo-express the protein of the present invention from their offspring. Defective individuals can be obtained. When using egg cells, for example, a transgenic non-human mammal having a targeting vector introduced into its chromosome by injecting a DNA solution into the egg cell nucleus by a microinjection method can be obtained,
Compared to these transgenic non-human mammals,
It can be obtained by selecting those having a mutation in the DNA locus of the present invention by homologous recombination.

As described above, for an individual whose DNA of the present invention has been knocked out, the animal individual obtained by mating should be confirmed to have the DNA knocked out, and then the animal should be passaged in a normal breeding environment. You can further,
The acquisition and maintenance of the germ line may be carried out according to the usual method. That is, by mating male and female animals carrying the inactivated DNA, a homozygous animal having the inactivated DNA on both homologous chromosomes can be obtained. The homozygous animals obtained were normal individuals 1,
It can be efficiently obtained by breeding in a state in which a plurality of homozygotes are present. By mating male and female heterozygous animals, homozygous and heterozygous animals having the inactivated DNA are bred. The non-human mammal embryonic stem cell in which the DNA of the present invention is inactivated is very useful in producing the non-human mammal deficient in expression of the DNA of the present invention. Further, the DN of the present invention
Since a non-human mammal deficient in A expression lacks various biological activities that can be induced by the protein of the present invention, it can be a model of diseases caused by inactivation of the biological activity of the protein of the present invention. It is useful for investigating the cause of diseases and studying treatment methods.

[8a] Method for screening compound having therapeutic / prophylactic effect against diseases caused by DNA deficiency or damage of the present invention The non-human mammal deficient in DNA expression of the present invention is D
It can be used for screening a compound having a therapeutic / preventive effect on diseases caused by NA deficiency or damage (eg, arteriosclerosis, hyperlipidemia, obesity, diabetes, etc.). That is, the present invention results from a defect or damage of the DNA of the present invention, which comprises administering a test compound to the non-human mammal deficient in DNA expression of the present invention and observing and measuring the change in the animal. Provided is a method for screening a compound having a therapeutic / preventive effect against a disease or a salt thereof. 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. Examples of test compounds include peptides, proteins, non-peptidic compounds, synthetic compounds,
Fermentation products, cell extracts, plant extracts, animal tissue extracts, plasma and the like are mentioned, and these compounds may be novel compounds or known compounds. Specifically, the non-human mammal deficient in DNA expression of the present invention is treated with a test compound and compared with an untreated control animal, and tested using changes in each organ, tissue, disease symptoms, etc. of the animal as an index. The therapeutic / prophylactic effect of a compound can be tested. As a method of treating a test animal with a test compound, for example,
Oral administration, intravenous injection, etc. are used to
It can be appropriately selected depending on the properties of the test compound. Further, 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 non-human mammal deficient in DNA expression of the present invention is subjected to glucose loading treatment, and a test compound is administered before or after glucose loading treatment, The blood glucose level and body weight change of the animal are measured over time. In the screening method, when a test compound is administered to a test animal and 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, It can be selected as a compound having a therapeutic / preventive effect on sclerosis.

The compound obtained by using the screening method of the present invention is a compound selected from the above-mentioned test compounds, and is a disease caused by a defect or damage of the protein of the present invention (eg, arteriosclerosis, etc.) Since it has a therapeutic / prophylactic effect against, it can be used as a medicine such as a safe and low-toxic therapeutic / prophylactic agent for the disease. Furthermore, compounds derived from the compounds obtained by the above screening can be used as well. The compound obtained by the screening method may form a salt, and the salt of the compound may be a physiologically acceptable acid (eg, inorganic acid, organic acid) or base (eg, alkali metal) and the like. Are used, and physiologically acceptable acid addition salts are particularly preferable. Examples of such salts include 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). Acid, tartaric acid, citric acid, malic acid, oxalic acid, benzoic acid, methanesulfonic acid, benzenesulfonic acid) and salts thereof are used. The drug containing the compound or its salt obtained by the screening method can be manufactured in the same manner as the drug containing the protein of the present invention. Since the preparation thus obtained is safe and has low toxicity, it can be used in mammals (for example, humans, rats, mice, guinea pigs, rabbits, sheep, pigs, cows, horses, cats, dogs, monkeys, etc.). Can be administered. Although the dose of the compound or its salt varies depending on the target disease, administration subject, administration route, etc., for example, when the compound is orally administered for the purpose of treating arteriosclerosis, an adult (body weight 60
(as kg), the compound is about 0.
1-100 mg, preferably about 1.0-50 mg, more preferably about 1.0-20 mg is administered. When administered parenterally, the single dose of the compound varies depending on the administration subject, target disease, etc., but for example, for the purpose of treating arteriosclerosis, the compound is usually in the form of an injection (as 60 kg). ) To about 0.0% of the compound per day.
It is convenient to administer about 1 to 30 mg, preferably about 0.1 to 20 mg, and more preferably about 0.1 to 10 mg by intravenous injection. For other animals,
The amount converted per 60 kg can be administered.

[8b] Method for Screening Compound Promoting Activity of Promoter for DNA of the Present Invention The present invention provides a non-human mammal deficient in expression of the DNA of the present invention,
Provided is a method for screening a compound or its salt that promotes or inhibits the activity of a promoter for the DNA of the present invention, which comprises administering a test compound and detecting the expression of a reporter gene. In the above screening method, the non-human mammal deficient in DNA expression of the present invention, among the non-human mammal deficient in DNA expression of the present invention, the DNA of the present invention is inactivated by introducing a reporter gene, A reporter gene that can be expressed under the control of a promoter for the DNA of the present invention is used. Examples of the test compound include the same ones as described above. As the reporter gene, the same ones as described above are used, and the β-galactosidase gene (lacZ), the soluble alkaline phosphatase gene, the luciferase gene and the like are preferable. DN of the present invention in which the DNA of the present invention is replaced with a reporter gene
In a non-human mammal deficient in A expression, the reporter gene exists under the control of the promoter for the DNA of the present invention, and thus the activity of the promoter can be detected by tracing the expression of the substance encoded by the reporter gene.

For example, when a part of the DNA region encoding the protein of the present invention is replaced with the β-galactosidase gene (lacZ) derived from Escherichia coli, the tissue of the present invention is originally expressed in the tissue of the present invention. Β-galactosidase is expressed instead of protein. Thus, for example, 5-bromo-4-chloro-3-indolyl-β-
By staining with a reagent that serves as a substrate for β-galactosidase such as galactopyranoside (X-gal), the expression state of the protein of the present invention in an animal body can be easily observed. Specifically, the protein-deficient mouse of the present invention or a tissue section thereof is fixed with glutaraldehyde or the like, and a phosphate buffered saline (PB
After washing with S), it is reacted with a staining solution containing X-gal at room temperature or around 37 ° C. for about 30 minutes to 1 hour, and then the tissue specimen is washed with 1 mM EDTA / PBS solution to give β-galactosidase. The reaction may be stopped and the color may be observed. In addition, mRNA encoding lacZ may be detected according to a conventional method.

The compound or its salt obtained by the above screening method is a compound selected from the above-mentioned test compounds and is a compound which promotes or inhibits the promoter activity for the DNA of the present invention. The compound obtained by the screening method may form a salt,
The salt of the compound includes a physiologically acceptable acid (eg,
Inorganic acids) and bases (eg organic acids) are used as salts,
Particularly, physiologically acceptable acid addition salts are preferable. Examples of such salts include 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). Acid, tartaric acid, citric acid, malic acid, oxalic acid, benzoic acid, methanesulfonic acid, benzenesulfonic acid) and salts thereof are used. The compound or its salt that promotes the promoter activity for the DNA of the present invention can promote the expression of the protein of the present invention and the function of the protein, and thus, for example, arteriosclerosis, hyperlipidemia,
It is useful as a medicine such as a safe and low toxic therapeutic / prophylactic agent against diseases such as obesity and diabetes. On the other hand, the compound or its salt that inhibits the promoter activity for the DNA of the present invention can inhibit the expression of the protein of the present invention and the function of the protein.
It is useful as a medicine such as a safe and low toxic therapeutic / prophylactic agent for diseases such as obesity.

The drug containing the compound or its salt obtained by the screening method can be produced in the same manner as the above-mentioned drug containing the protein of the present invention or its salt. Since the preparation thus obtained is safe and has low toxicity, it is, for example, a mammal (for example, human,
Rat, mouse, guinea pig, rabbit, sheep, pig,
Cow, horse, cat, dog, monkey, etc.). The dose of the compound or a salt thereof varies depending on the target disease, administration subject, administration route, etc.,
For example, when a compound that promotes the promoter activity for the DNA of the present invention is orally administered for the purpose of treating arteriosclerosis, generally in adults (as a body weight of 60 kg),
The daily amount of the compound is about 0.1-100 mg, preferably about 1.0-50 mg, more preferably about 1.0-20.
administer mg. When administered parenterally, the single dose of the compound varies depending on the administration subject, target disease and the like, but for example, a compound that promotes the promoter activity for the DNA of the present invention is injected for the purpose of treating arteriosclerosis. When administered in the form of a drug to an adult (as 60 kg), the amount of the compound is about 0.01 to 30 mg per day, preferably about 0.1 to 20 mg, more preferably about 0.1 to 1 mg.
It is convenient to administer about 0 mg intravenously. In the case of other animals, the dose converted per 60 kg can be administered. As described above, the non-human mammal deficient in DNA expression of the present invention is extremely useful for screening a compound or its salt that promotes or inhibits the activity of the promoter for the DNA of the present invention, and It can greatly contribute to the investigation of the causes of various diseases caused or the development of preventive / therapeutic drugs. In addition, by using DNA containing the promoter region of the protein of the present invention, genes encoding various proteins are ligated downstream of the DNA, and this is injected into an egg cell of an animal to obtain a so-called transgenic animal (transgenic animal). Once created, it is possible to specifically synthesize the protein and study its action in the living body. Furthermore, if a suitable reporter gene is bound to the above promoter portion and a cell line that expresses this is established, a low molecular weight compound having an action of specifically promoting or suppressing the production ability of the protein of the present invention itself in the body. Can be used as a search system for. It is also possible to find new cis elements and transcription factors that bind to them by analyzing the promoter portion.

In the present specification and drawings, when an abbreviation is used for a base or amino acid, IUPAC-IUB
It is based on the abbreviations by the Commission on Biochemical Nomenclature or the abbreviations commonly used in this field, and examples are given below. When amino acids may have optical isomers, the L form is shown unless otherwise specified. DNA: deoxyribonucleic acid cDNA: complementary deoxyribonucleic acid A: adenine T: thymine G: guanine C: cytosine RNA: ribonucleic acid mRNA: messenger ribonucleic acid dATP: deoxyadenosine triphosphate dTTP: deoxythymidine triphosphate dGTP: deoxyguanosine tri Phosphate dCTP: Deoxycytidine triphosphate ATP: Adenosine triphosphate EDTA: Ethylenediaminetetraacetic acid SDS: Sodium dodecyl sulfate

Gly: Glycine Ala: Alanine Val: Valine Leu: Leucine Ile: Isoleucine Ser: Serine Thr: Threonine Cys: Cysteine Met: Methionine Glu: Glutamic acid Asp: Aspartic acid Lys: Lysine Arg: Arginine His: Histidine Phe: Phenylalanine Tyr: Tyrosine Trp: Tryptophan Pro: Proline Asn: Asparagine Gln: Glutamine pGlu: pyroglutamic acid

Further, substituents, protecting groups and reagents commonly used in the present specification are represented by the following symbols. Me: Methyl group Et: Ethyl group Bu: Butyl group Ph: Phenyl group TC: Thiazolidine-4 (R) -carboxamide group Tos: p-toluenesulfonyl CHO: Formyl Bzl: Benzyl Cl ₂ Bzl: 2,6-dichlorobenzyl Bom : Benzyloxymethyl Z: benzyloxycarbonyl Cl-Z: 2-chlorobenzyloxycarbonyl Br-Z: 2-bromobenzyloxycarbonyl Boc: t-butoxycarbonyl DNP: dinitrophenyl Trt: trityl Bum: t-butoxymethyl Fmoc: N-9-fluorenylmethoxycarbonyl HOBt: 1-hydroxybenztriazole HOOBt: 3,4-dihydro-3-hydroxy-4-oxo-1,2,3-benzotriazine HONB: 1-hydroxy-5-nor Borne-2,3-dicarboximide DCC: N, N'-dicyclohexylcarbodiimide

The sequence numbers in the sequence listing in the present specification show the following sequences. [SEQ ID NO: 1] This shows the amino acid sequence of the human adipocyte-derived protein (LIP-5) of the present invention. [SEQ ID NO: 2] Human adipocyte-derived protein (LI of the present invention having the amino acid sequence represented by SEQ ID NO: 1
The nucleotide sequence of DNA encoding P-5) is shown. [SEQ ID NO: 3] This shows the base sequence of the synthetic primer used for cloning the DNA encoding the human-derived protein of the present invention in Example 1. [SEQ ID NO: 4] This shows the base sequence of the synthetic primer used for cloning the DNA encoding the human-derived protein of the present invention in Example 1. [SEQ ID NO: 5] This shows the base sequence of the synthetic primer used in the analysis of the expression distribution in human tissues of Example 2. [SEQ ID NO: 6] This shows the base sequence of the synthetic primer used in the analysis of the expression distribution in human tissues of Example 2.

The plasmid pT obtained in Example 1 described later
Escherichia col holding B2177
i DH5α / pTB2177 is November 13, 2000
From 1 to 1 Higashi 1-1-chome, Tsukuba-shi, Ibaraki, Japan Central Institute of Industrial Science and Technology, Institute of Advanced Industrial Science and Technology Patent Biological Depositary Center (Former Institute of Biotechnology, National Institute of Advanced Industrial Science and Technology (NIBH)) ) Deposit number FE
RM BP-7364 since October 31, 2000 2-17-85, Jusohonmachi, Yodogawa-ku, Osaka, Japan
No. IFO1 to the Fermentation Research Institute (IFO)
Deposited as 6495.

[0088]

The present invention will be described in more detail below with reference to examples, but the present invention is not limited thereto. In addition, the gene manipulation method using Escherichia coli followed the method described in Molecular Cloning.

Example 1 Cloning of cDNA encoding human adipocyte-derived lipolytic protein and determination of nucleotide sequence Using human adipocyte Marathon-Ready cDNA (CLONTECH) as a template, two primers, primer 1 (SEQ ID NO: :
PCR reaction was performed using 3) and primer 2 (SEQ ID NO: 4). The composition of the reaction solution in the reaction is 2 μl of the above cDNA.
l as a template, Pfu Turbo DNA polymerase (STR
ATAGENE) 0.2 μl volume, Primer 1 (SEQ ID NO: 3) and Primer 2 (SEQ ID NO: 4) 0.5 μM each, dNTPs 200 μM each, and 2 μl of the buffer attached to the enzyme,
The liquid volume was 20 μl. PCR reaction at 94 ° C for 2 minutes, then 94 ° C
-The cycle of 1 minute, 65 ° C / 1 minute, 72 ° C / 2 minutes was repeated 35 times, and finally the extension reaction was carried out at 72 ° C / 5 minutes. The PCR reaction product was extruded with adenine at the 3 ′ end using ExTaq (Takara Shuzo), and then the plasmid vector pCR2.1 (Invitrogen) according to the prescription of DNA Ligation Kit Ver.2 (Takara Shuzo).
Subcloned into This was introduced into Escherichia coli DH5α, and a clone having a cDNA (insert) was selected in LB agar medium containing ampicillin. As a result of analyzing the nucleotide sequences of the individual clones, clones containing the DNA having the nucleotide sequence represented by SEQ ID NO: 2 and containing the cDNA (insert) encoding the novel neutral lipolytic enzyme protein were obtained. This clone (transformant) was transformed into Escherichia coli (Escherichi
a coli) DH5α / pTB2177. Also, SEQ ID NO:
The novel lipolytic enzyme protein containing the amino acid sequence (SEQ ID NO: 1) derived from the nucleotide sequence represented by 2 was named LIP-5.

Example 2 Investigation 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. White blood cell, 10. Liver, 11. Lung, 12. Mammary gland, 13. Ovary, 14. Pancreas, 15. Prostate, 1
6. Skeletal muscle, 17. Small intestine, 18. Spleen, 19. Testis, 20. Thymus, 21.
Marathon-Ready cDNA derived from human tissue of uterus, 22. adrenal gland)
(CLONTECH) as a template, two primers, primer 1 (SEQ ID NO: 5) and primer 2 (SEQ ID NO :)
6) was used to carry out PCR reaction. The composition of the reaction solution in the reaction was such that 0.5 μl of the above cDNA was used as a template, 0.1 μl of ExTaq (Takara Shuzo), 0.5 μM each of Primer 1 (SEQ ID NO: 5) and Primer 2 (SEQ ID NO: 6), and dNTPs. 200μ each
2 μl of the buffer attached to M and the enzyme was added to make a volume of 20 μl. PCR reaction is 94 ° C for 1 minute, then 94 ° C for 30 seconds,
A cycle of 60 ° C for 30 seconds and 72 ° C for 2 minutes was repeated 35 times, and finally an extension reaction at 72 ° C for 5 minutes was performed. 5 μl of the obtained reaction solution was analyzed by 1.5% agarose gel electrophoresis. The length of the PCR product was 324 bp. As a result, LI
P-5 is adipose tissue, aorta, large intestine, kidney, mammary gland, ovary,
Expression was observed in the prostate, small intestine, spleen, testis, uterus and adrenal gland. Figure 1 shows the results of LIP-5 expression distribution.

[0091]

INDUSTRIAL APPLICABILITY The protein of the present invention and the DNA encoding the same can be used as a therapeutic / preventive agent for diseases such as arteriosclerosis, hyperlipidemia, obesity and diabetes. Moreover, cells having the ability to express the protein of the present invention or the gene of the protein of the present invention are useful as reagents for screening compounds or salts thereof that promote or inhibit the esterase activity of the protein of the present invention. Furthermore, since the 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.

[0092]

[Sequence list] SEQUENCE LISTING <110> Takeda Chemical Industries, Ltd. <120> Novel Protein and its DNA <130> B01413 <150> JP 2000-335899 <151> 2000-11-02 <150> JP 2000-357997 <151> 2000-11-24 <160> 6 <210> 1 <211> 340 <212> PRT <213> Human <400> 1 Met Pro Lys Phe Ile Arg Phe Leu His Asp Ser Val Arg Ile Lys Lys                  5 10 15 Asp Pro Glu Leu Val Val Thr Asp Leu Arg Phe Gly Thr Ile Pro Val              20 25 30 Arg Leu Phe Gln Pro Lys Ala Ala Ser Ser Arg Pro Arg Arg Gly Ile          35 40 45 Ile Phe Tyr His Gly Gly Ala Thr Val Phe Gly Ser Leu Asp Cys Tyr      50 55 60 His Gly Leu Cys Asn Tyr Leu Ala Arg Glu Thr Glu Ser Val Leu Leu  65 70 75 80 Met Ile Gly Tyr Arg Lys Leu Pro Asp His His Ser Pro Ala Leu Phe                  85 90 95 Gln Asp Cys Met Asn Ala Ser Ile His Phe Leu Lys Ala Leu Glu Thr             100 105 110 Tyr Gly Val Asp Pro Ser Arg Val Val Val Cys Gly Glu Ser Val Gly         115 120 125 Gly Ala Ala Val Ala Ala Ile Thr Gln Ala Leu Val Gly Arg Ser Asp     130 135 140 Leu Pro Arg Ile Arg Ala Gln Val Leu Ile Tyr Pro Val Val Gln Ala 145 150 155 160 Phe Cys Leu Gln Leu Pro Ser Phe Gln Gln Asn Gln Asn Val Pro Leu                 165 170 175 Leu Ser Arg Lys Phe Met Val Thr Ser Leu Cys Asn Tyr Leu Ala Ile             180 185 190 Asp Leu Ser Trp Arg Asp Ala Ile Leu Asn Gly Thr Cys Val Pro Pro         195 200 205 Asp Val Trp Arg Lys Tyr Glu Lys Trp Leu Ser Pro Asp Asn Ile Pro     210 215 220 Lys Lys Phe Lys Asn Arg Gly Tyr Gln Pro Trp Ser Pro Gly Pro Phe 225 230 235 240 Asn Glu Ala Ala Tyr Leu Glu Ala Lys His Met Leu Asp Val Glu Asn                 245 250 255 Ser Pro Leu Ile Ala Asp Asp Glu Val Ile Ala Gln Leu Pro Glu Ala             260 265 270 Phe Leu Val Ser Cys Glu Asn Asp Ile Leu Arg Asp Asp Ser Leu Leu         275 280 285 Tyr Lys Lys Arg Leu Glu Asp Gln Gly Val Arg Val Thr Trp Tyr His     290 295 300 Leu Tyr Asp Gly Phe His Gly Ser Ile Ile Phe Phe Asp Lys Lys Ala 305 310 315 320 Leu Ser Phe Pro Cys Ser Leu Lys Ile Val Asn Ala Val Val Ser Tyr                 325 330 335 Ile Lys Gly Ile             340 <210> 2 <211> 1020 <212> DNA <213> Human <400> 2 atgcccaaat ttattcgttt tttacatgat agcgtgagaa ttaaaaagga ccctgaactt 60 gtggtgaccg acctgcgttt tgggacgata cccgtgaggc tgttccagcc gaaggcagca 120 tcctccagac cccggcgagg catcatcttc taccatggag gggccacagt atttgggagc 180 ctggattgtt accatggcct gtgcaattat ctggcccggg agactgaatc tgtacttctg 240 atgattgggt accgcaagct tcctgaccac cattcccctg cccttttcca agactgcatg 300 aatgcctcca ttcacttcct gaaggccctg gaaacctatg gggtggaccc ctccagggtt 360 gtggtctgtg gagaaagcgt cggaggtgca gcggtggccg ccatcaccca ggccttggtg 420 ggcagatcag atcttccccg gatccgggct caggttctga tttatccagt tgtccaggca 480 ttctgtttgc agttgccatc ctttcagcag aaccaaaatg tcccattact ttcccggaag 540 ttcatggtga cttctctgtg taactatctg gccattgacc tctcctggcg tgacgccatc 600 ttgaacggca cttgtgtacc cccagacgtc tggaggaagt acgagaagtg gctcagccct 660 gacaacatcc ccaagaaatt taagaacaga ggctaccaac cctggtctcc cggccctttt 720 aatgaagctg cctatctaga agccaaacat atgctggatg tagaaaattc acccctgata 780 gcagatgatg aggtcatcgc tcagcttcct gaggccttcc tggtgagctg tgagaatgac 840 atactccgtg atgacagctt gctctataag aagcgcttgg aggaccaggg ggtccgcgtg 900 acatggtacc acctgtatga tggttttcac ggatccatta tcttttttga taagaaggct 960 ctctctttcc catgttccct gaagattgtg aatgctgtag tcagttatat aaagggcata 1020 <210> 3 <211> 24 <212> DNA <213> Artificial Sequence <223> Designed oligonucleotide primer to amplify DNA encoding LIP-5 <400> 3 ccaagctagg ctaccctttg gcaa 24 <210> 4 <211> 27 <212> DNA <213> Artificial Sequence <223> Designed oligonucleotide primer to amplify DNA encoding LIP-5 <400> 4 aagcacccgg tttctggtag agtccat 27 <210> 5 <211> 24 <212> DNA <213> Artificial Sequence <223> Designed oligonucleotide primer to amplify DNA encoding LIP-5 <400> 5 taccgcaagc ttcctgacca ccat 24 <210> 6 <211> 24 <212> DNA <213> Artificial Sequence <223> Designed oligonucleotide primer to amplify DNA encoding LIP-5 <400> 6 ggccagatag ttacacagag aagt 24

[Brief description of drawings]

FIG. 1 shows the results of LIP-5 expression distribution in human 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 fetal brain, lane 7 is heart, lane 8 is kidney, lane 9 is white blood cell, la
ne 10 is liver, lane 11 is lung, lane 12 is mammary gland, lane 13 is ovary, lane 14 is pancreas, lane 15 is prostate, lane 16 is skeletal muscle, lane 17 is small intestine, lane 18 is spleen, lane 19 is testis, l
ane 20 is thymus, lane 21 is uterus, lane 22 is adrenal gland, M is 100
Indicates a bp marker.

─────────────────────────────────────────────────── ─── Continuation of front page (51) Int.Cl. ⁷ Identification code FI theme code (reference) A61K 39/395 A61K 48/00 4C084 45/00 A61P 3/04 4C085 48/00 3/06 4C086 A61P 3 / 04 3/10 4H045 3/06 9/10 101 3/10 C07K 16/40 9/10 101 C12N 1/15 C07K 16/40 1/19 C12N 1/15 1/21 1/19 9/20 1/21 C12Q 1/44 5/10 1/68 A 9/20 G01N 33/15 Z C12Q 1/44 33/50 Z 1/68 33/573 A G01N 33/15 C12N 15/00 ZNAA 33/50 5/00 A 33/573 B A61K 37/02 (72) Inventor Kao Taniyama 1-7-9 Kasuga, Tsukuba, Ibaraki Prefecture Takeda Kasuga Heights No. 501 F term (reference) 2G045 AA28 AA29 AA40 BB20 CB01 CB17 DA12 DA13 DA14 DA20 DA36 FB01 FB03 FB07 4B024 AA01 BA11 BA44 CA04 CA11 DA01 DA02 DA05 DA11 EA01 EA02 EA03 EA04 FA02 FA10 GA11 HA01 HA11 4B050 CC01 CC03 DD07 LL01 4B063 QA01 QA08 QA18 QQ05 QQ08 QQ13 QQ32 QQ95 QR08 QR12 QR33 QR42 AB42 AQAQAQAQAQAQAQAQAQAQAQAQAQAQAQA CA25 CA31 CA44 CA46 4C084 AA02 AA06 AA07 AA13 AA17 BA01 BA08 BA22 BA23 MA01 NA14 ZA452 ZA702 ZC332 ZC352 4C085 AA13 AA14 CC32 DD34 DD35 DD36 DD38 DD41 DD43 DD62 EA01 A45 A35 A45 A45 A45 A45 A45 A45 A45 A45 A45 A45 NA45 CA40 DA76 EA20 EA50 FA72 FA74

Claims (44)

[Claims] 1. A protein containing the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 1 or a salt thereof. 2. The protein according to claim 1, which comprises the amino acid sequence represented by SEQ ID NO: 1, or a salt thereof. 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. The protein according to claim 1 or claim 2.
A polynucleotide containing a polynucleotide encoding the described partial peptide. 6. The polynucleotide according to claim 5, which is DNA. 7. The DNA according to claim 6, which contains the base 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 transformant according to claim 9 is cultured, the protein according to claim 1 or the partial peptide according to claim 2 is produced and accumulated, and this is collected. A method for producing the protein according to claim 3 or the partial peptide according to claim 3 or a salt thereof. 11. A pharmaceutical comprising the protein according to claim 1, the partial peptide according to claim 3, or a salt thereof. 12. A medicine comprising the polynucleotide according to claim 5. 13. The medicine according to claim 11 or 12, which is a therapeutic / prophylactic agent for 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 pharmaceutical comprising the antibody according to claim 14. 16. A diagnostic agent comprising the antibody according to claim 14. 17. An antisense DN comprising a base sequence complementary to a DNA encoding the protein according to claim 1 or the partial peptide according to claim 3 or a part thereof.
A. 18. A medicine comprising the antisense DNA according to claim 17. 19. A diagnostic agent comprising the antisense DNA according to claim 17. 20. The esterase activity of the protein according to claim 1, the partial peptide according to claim 2, or the partial peptide according to claim 2, or a salt thereof, wherein the protein according to claim 1 or the partial peptide according to claim 3 or a salt thereof is used. A method for screening a compound or its salt having an activity of promoting or inhibiting. 21. The esterase activity of the protein according to claim 1 or the partial peptide according to claim 3 or the partial peptide or salt thereof, which comprises the protein according to claim 1, the partial peptide according to claim 3, or a salt thereof, or A kit for screening a compound or its salt having an inhibitory activity. 22. The protein according to claim 1 or claim 3 obtained using the screening method according to claim 20 or the screening kit according to claim 21.
A compound or salt thereof having an activity of promoting or inhibiting the esterase activity of the partial peptide or salt thereof described. [23] An activity of promoting the esterase activity of the protein according to [1] or the partial peptide according to [3] or a salt thereof obtained by using the screening method according to [20] or the screening kit according to [21]; A pharmaceutical comprising a compound having: or a salt thereof. 24. The medicine according to claim 23, which is a therapeutic / prophylactic agent for arteriosclerosis, hyperlipidemia, obesity or diabetes. [25] An activity of inhibiting the esterase activity of the protein according to [1] or the partial peptide according to [3] or the salt thereof obtained by using the screening method according to [20] or the screening kit according to [21]; A pharmaceutical comprising a compound having: or a salt thereof. 26. A therapeutic / prophylactic agent for obesity.
The described medicine. 27. The protein according to claim 1 or the partial peptide according to claim 3, or those obtainable by using the screening method according to claim 20 or the screening kit according to claim 21 for a mammal. A method for preventing / treating arteriosclerosis, hyperlipidemia, obesity or diabetes, which comprises administering an effective amount of a compound having an activity of promoting esterase activity of the salt or a salt thereof. 28. The protein according to claim 1 or the partial peptide according to claim 3, or those obtainable by using the screening method according to claim 20 or the screening kit according to claim 21 for mammals. A method for preventing or treating obesity, which comprises administering an effective amount of a compound having an activity of inhibiting the esterase activity of the salt or 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 the compound having the activity of promoting the esterase activity of the protein according to claim 1, the partial peptide according to claim 3, or a salt thereof, or a salt thereof. 30. The screening method according to claim 20 or 21 for producing a prophylactic / therapeutic agent for obesity.
Use of the compound having the activity of inhibiting the esterase activity of the protein according to claim 1, the partial peptide according to claim 3, or a salt thereof, which can be obtained by using the screening kit according to claim 1, 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 according to claim 31, wherein the non-human mammal is a rodent. 33. The animal according to 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 the DNA encoding the protein according to claim 1 or the partial peptide according to claim 3 is inactivated. 36. The embryonic stem cell according to claim 35, wherein the DNA is inactivated by introducing a reporter gene. 37. The embryonic stem cell according to claim 35, which is resistant to neomycin. 38. The embryonic stem cell according to 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 expression of the DNA, wherein the DNA encoding the protein according to claim 1 or the partial peptide according to claim 3 is inactivated. 41. The DNA is inactivated by introducing a reporter gene, wherein the reporter gene is the DN.
5. Expression possible under the control of a promoter for A.
The non-human mammal according to 0. 42. The non-human mammal according to claim 40, wherein the non-human mammal is a rodent. 43. The non-human mammal according to claim 42, wherein the rodent is a mouse. 44. A method for screening a compound or its salt which promotes or inhibits the promoter activity to said DNA, which comprises administering a test compound to the animal of claim 41 and detecting the expression of a reporter gene.