JP2003219880A - New protein and dna thereof - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a new protein having an activity of exchange transport of K<SP>+</SP>ion and Na<SP>+</SP>ion, a DNA encoding the protein, a method for screening a compound promoting or inhibiting the activity of the protein and a compound obtained through the method for screening. <P>SOLUTION: The protein described in the invention is useful for a diagnostic marker for heart disease, kidney disease, central nervous disease, inflammatory disease, autoimmune disease, allergic disease, thymic disease, splenic disease, immune deficiency, genital disease and/or immunological rejection after organ transplantation. The compound prompting or inhibiting the activity of the protein obtained by the method for screening using the protein is allowed to be used as a preventing agent, a therapeutic agent and an inhibitor for the diseases. <P>COPYRIGHT: (C)2003,JPO

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a novel Na, K-
Provided are an ATPase protein, a polynucleotide encoding the protein, a method for screening a compound that promotes or inhibits the activity of the protein, a compound obtained by the screening method, and the like.

[0002]

2. Description of the Related Art Na, K-ATPase is a P-type ATP
It is a type of ase and uses the energy of ATP to K
^It is a membrane-type enzyme that is responsible for the exchange and transport of ⁺ and Na ⁺ ions. Also called sodium pump, it is present in the cytoplasmic membrane of many cells in vivo. This enzyme plays a central role in maintaining homeostasis in cells by controlling the concentration gradient of K ⁺ and Na ⁺ ions inside and outside the cell (Curr. Opin. Nephro. Hypertens. Vol. 6,
434-439, 1997). In addition, Na, K-ATPase
The Na ⁺ concentration gradient caused by the ionization produces energy for transporting other ions and nutrients, and this energy is used for the expression of cell functions such as muscle contraction, nerve signal transmission, and renal Na ⁺ reabsorption. It is used. Furthermore, Na, K-ATPase is a drug target of cardiac glycosides that are often used in heart disease, and Na, K-ATP
The activity of ase is inhibited by ouabain. Na, K
-ATPase is composed of an α subunit and a β subunit. The α subunit has a 10-transmembrane structure, and its N-terminus and C-terminus are located intracellularly. In contrast, the β subunit is a single-transmembrane glycoprotein, has a short intracellular domain and a large extracellular domain. The α subunit is responsible for the enzymatic activity of Na, K-ATPase, which decomposes ATP and transports K ⁺ and Na ⁺ ions. The β subunit is believed to be the regulatory molecule required for the α subunit to function. So far, four types of α molecules and three types of β molecules have been identified. The α1 isoform is ubiquitous in many cells, the α2 isoform is mainly present in heart muscle, skeletal muscle, brain, and adipocytes, and the α3 isoform is mainly present in neural tissues and ovaries (Am. J. . Phy
siol., 275, F633, 1998).

[0003]

Problems to be Solved by the Invention Na, K-ATPas
Multiple isoforms of e have different expression sites.
Difference between these isoforms and Na, K-ATPa
The relationship with the function of se is almost unknown.
Although Na, K-ATPase has many important functions for cells, clarifying the relationship between the specific function and the isoform composition is useful for heart disease, kidney disease, central nervous system disease, inflammatory disease, Autoimmune disease, allergic disease, thymus disease, spleen disease, immunodeficiency, genital disease, cancer,
Or Na, KA in rejection after organ transplantation
This leads to clarifying the role of TPase.

[0004]

Means for Solving the Problems The inventors of the present invention have conducted extensive studies to solve the above problems, and as a result, new Na,
The K-ATPase protein was found. This novel protein is Na, K-ATPase at the amino acid level.
It showed 79% homology with the α2 subunit. As a method of suppressing the protein, for example, inhibition of exchange transport of K ⁺ ion and Na ⁺ ion, reduction of the expression level by transcriptional suppression of the protein gene, and the like are considered. As a method of activating the protein, for example, promotion of exchange transport of K ⁺ ions and Na ⁺ ions, activation of a promoter of the protein gene, and enhancement of expression level by stabilizing mRNA can be considered. 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 a salt thereof containing the same or substantially the same amino acid sequence as the amino acid sequence represented by
(2) The protein or salt thereof according to (1), which comprises the amino acid sequence represented by SEQ ID NO: 1 or SEQ ID NO: 33, (3) SEQ ID NO: 24 or SEQ ID NO: 34.
(1) The protein or salt thereof described in (1) above, which contains an amino acid sequence represented by 4) A polynucleotide containing a polynucleotide encoding the partial peptide, (6) The polynucleotide according to (5), which is DNA, (7) SEQ ID NO: 2, SEQ ID NO: 29, SEQ ID NO: 30, The DNA according to (6) above, which comprises the base sequence represented by SEQ ID NO: 31 or SEQ ID NO: 32,
(8) SEQ ID NO: 25, SEQ ID NO: 35, SEQ ID NO: 3
6 or the DNA according to (6) above, which contains the nucleotide sequence represented by SEQ ID NO: 37, (9) the DN according to (6) above
A recombinant vector containing A, (10) a transformant transformed with the recombinant vector according to (9), (1)
1) The transformant described in (10) above is cultured to produce and accumulate the protein described in (1) above or the partial peptide described in (4) above, and this is collected (1) above. The described protein or the above (4)
(12) A method for producing the partial peptide or the salt thereof, (12) the protein comprising the protein according to (1) or the partial peptide according to (4) or a salt thereof, (13).
A pharmaceutical comprising the DNA according to (6) above, (14)
An antibody against the protein according to (1) above, the partial peptide according to (4) above or a salt thereof, (15) the protein according to (1) above, or the partial peptide according to (4) above or a salt thereof is used. A method for screening a compound or a salt thereof that promotes or inhibits the activity of the protein according to (1) above, the partial peptide according to (4) above, or a salt thereof, (16) the protein according to (1) above or ) The partial peptide or the salt thereof according to the above (1), the protein according to the above (1) or the partial peptide according to the above (4) or a compound for promoting or inhibiting the activity of the salt thereof, or a kit for screening the salt thereof, ( 17) The above (15)
A compound or a salt thereof which promotes or inhibits the activity of the protein according to (1), the partial peptide according to (4) or a salt thereof, which is obtained by using the screening method according to claim 16 or the screening kit according to (16). (18) A drug comprising the compound according to (17) above or a salt thereof, (19) the expression of the protein gene according to (1) above, which comprises using the polynucleotide according to (5) above. (20) A method for screening a compound or a salt thereof, which promotes or inhibits the expression of a compound or a salt thereof, which comprises the polynucleotide according to (5) above and which promotes or inhibits the expression of the protein gene according to (1) above. Screening kit,
(21) A compound or a salt thereof which promotes or inhibits the expression of the protein gene according to (1), which is obtained by using the screening method according to (19) or the screening kit according to (20), (22) A drug comprising the compound according to (21) or a salt thereof, (23) a diagnostic agent comprising the antibody according to (14), (24) an antibody comprising (14). (25) A method for quantifying a protein according to (1) above, which comprises using the antibody according to (25) above.
(26) A method for diagnosing a disease associated with the function of the protein according to (1), characterized by using the quantification method according to (25), and (27) using the antibody according to (14). And (28) a method for screening a compound or a salt thereof that promotes or inhibits the expression of the protein according to (1) above, (28) the expression of the protein according to (1) above, which comprises the antibody according to (14) above (29) A screening kit for a compound or a salt thereof that promotes or inhibits the expression of the protein according to (1), which is obtained using the screening method according to (27) or the screening kit according to (28). Or a salt thereof that promotes or inhibits (30) above (2)
9) A pharmaceutical comprising the compound or the salt thereof according to
(31) Preventive / therapeutic agent for heart disease, kidney disease, central disease, inflammatory disease, autoimmune disease, allergic disease, thymus disease, spleen disease, immunodeficiency, genital disease or cancer, or suppression of rejection after organ transplantation The agent (12), (1
3), (18), (22), (24) or (30) the pharmaceutical agent, (32) a mammal, the above (17),
(21) or (29) or an effective amount of the salt thereof is administered, heart disease, kidney disease, central disease, inflammatory disease, autoimmune disease, allergic disease,
Thymic disease, spleen disease, immunodeficiency, reproductive organ disease or cancer prevention / treatment method, or rejection suppression method after organ transplantation, (33) heart disease, kidney disease, central disease, inflammatory disease, autoimmune disease, allergy The compound according to the above (17), (21) or (29) for producing a preventive / therapeutic agent for diseases, thymus diseases, spleen diseases, immunodeficiency, reproductive organ diseases or cancer, or a rejection inhibitor after organ transplantation. Or the use of the salt is provided.

The present invention further comprises (34) a diagnostic agent comprising the DNA according to (6) above, (35) a nucleotide sequence complementary to the polynucleotide according to (5) above or a part thereof. Antisense polynucleotide, (3
6) A medicine comprising the antisense polynucleotide according to (35), (37) a non-human mammal having the DNA according to claim 6 or a mutant DNA thereof, (38).
The animal according to (37) above, wherein the non-human mammal is a rodent, (39) the animal above (38), wherein the rodent is a mouse or rat, (40) D according to (6) above.
A recombinant vector containing NA or a mutant DNA thereof and capable of being expressed in a mammal, (41) D according to claim 6.
NA-inactivated non-human mammalian embryonic stem cells, (4
2) The DNA is a reporter gene (eg β derived from E. coli)
-Galactosidase gene) -inactivated by introducing the embryonic stem cell according to (41), (43) the embryonic stem cell according to (41) which is resistant to neomycin, (4)
4) The embryonic stem cell according to (41) above, wherein the non-human mammal is a rodent, (45) the embryonic stem cell according to (44) above, wherein the rodent is a mouse, (46) D according to (6) above.
The non-human mammal deficient in DNA expression in which NA is inactivated, (47) the DNA is inactivated by introducing a reporter gene (eg, β-galactosidase gene derived from Escherichia coli), and the reporter gene is Invention DN
(4) which can be expressed under the control of a promoter for A.
6) The non-human mammal described above, (48) The non-human mammal described in (46) above, wherein the non-human mammal is a rodent.
(49) The non-human mammal according to the above (48), wherein the rodent is a mouse. (50) The animal according to (47) above,
Provided is a method for screening a compound or its salt that promotes or inhibits the promoter activity for the DNA of the present invention, which comprises administering a test compound and detecting the expression of a reporter gene.

[0007]

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 (hereinafter also referred to as the protein of the present invention or the protein used in the present invention. ) Is for humans and other warm-blooded animals (eg, guinea pigs, rats,
Mouse, chicken, rabbit, pig, sheep, cow, monkey, etc.) cells (eg, hepatocytes, splenocytes, nerve cells, glial cells, pancreatic β cells, bone marrow cells, mesangial cells,
Langerhans cells, epidermal cells, epithelial cells, goblet cells, endothelial cells, smooth muscle cells, fibroblasts, fiber cells, muscle cells,
Adipocytes, immune cells (eg macrophages, T cells, B
Cells, natural killer cells, mast cells, neutrophils, basophils, eosinophils, monocytes), megakaryocytes, synovial cells, chondrocytes,
Bone cells, osteoblasts, osteoclasts, mammary gland cells, hepatocytes or stromal cells, or precursors of these cells, stem cells or cancer cells) or any tissue in which these cells are present, eg, brain, brain Each site (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, Adrenals, skin, muscles, lungs,
Digestive tract (eg, large intestine, small intestine), blood vessel, heart, thymus, spleen,
Submandibular gland, peripheral blood, prostate, testicles, ovaries, placenta, uterus,
It may be a protein derived from bone, joint, skeletal muscle, or the like, or may be a synthetic protein.

The amino acid sequence which is substantially the same as the amino acid sequence represented by SEQ ID NO: 1 is about 85% or more, preferably about 8% of the amino acid sequence represented by SEQ ID NO: 1.
Examples include amino acid sequences having a homology of 7% or more, more preferably about 90% or more, particularly preferably about 95% or more, and most preferably about 97% or more. SEQ ID NO: 1
As the protein containing the amino acid sequence substantially the same as the amino acid sequence represented by, for example, containing the amino acid sequence substantially the same amino acid sequence represented by the above-mentioned SEQ ID NO: 1, SEQ ID NO: A protein having substantially the same activity as the protein having the amino acid sequence represented by 1 is preferable. Substantially equivalent activity includes:
For example, the exchange transport activity of K ⁺ ions and Na ⁺ ions can be mentioned. The term “substantially the same” means that those properties are qualitatively (eg, physiologically or pharmacologically) the same. Therefore, the exchange transport activity of K ⁺ ion and Na ⁺ ion is equivalent (eg, about 0.01 to 100 times, preferably about 0.1 to 10 times, more preferably 0.5 to 2 times). Although preferable, quantitative factors such as the degree of these activities and the molecular weight of the protein may be different. K ⁺
The activity such as the exchange transport activity of ions and Na ⁺ ions may be measured according to a known method, for example, J. Biol.
Chem. Vol. 275, 1976-1986, 2000, or a method analogous thereto or a method analogous thereto.

The protein used in the present invention is, for example, 1 or 2 or more (eg, about 1 to 150, preferably about 1 to 100, preferably about 1 to 100 in the amino acid sequence represented by SEQ ID NO: 1. Is about 1 to 50,
1 to 2 is added to the amino acid sequence represented by SEQ ID NO: 1, preferably about 1 to 30, preferably about 1 to 10 and more preferably several (1 to 5) amino acids. More than one (for example, about 1 to 150,
Preferably about 1 to 100, preferably about 1 to 50, preferably about 1 to 30, preferably 1 to 10.
1 or 2 or more (for example, about 1 to 150, preferably 1 to 2 in the amino acid sequence represented by SEQ ID NO: 1, which is an amino acid sequence in which about 1, more preferably several (1 to 5) amino acids are added. About 100, preferably 1 to 50
About 1, preferably about 1 to 30, preferably 1 to
Amino acid sequence in which about 10 amino acids, more preferably several (1 to 5) amino acids are inserted, 1 or 2 or more (for example, 1 to 1) in the amino acid sequence represented by SEQ ID NO: 1.
About 50, preferably about 1 to 100, preferably about 1 to 50, preferably about 1 to 30, preferably about 1 to 10, more preferably a number (1 to 5).
The so-called mutein such as a protein containing an amino acid sequence in which each amino acid) is replaced with another amino acid, or an amino acid sequence in which they are combined. When the amino acid sequence is inserted, deleted or substituted as described above, the position of the insertion, deletion or substitution is not particularly limited.

The protein in the present specification is a peptide
According to the convention of notation, the left end is the N-terminal (amino terminal), the right end
Is the C terminal (carboxyl terminal). SEQ ID NO: 1
Including proteins containing the represented amino acid sequences
The protein used in the present invention has a C-terminal cal
Boxyl group (-COOH), carboxylate (-CO
O^-), Amide (-CONH₂) Or ester (-CO
OR). Where in the ester
Examples of R include methyl, ethyl, n-propyl,
C such as isopropyl and n-butyl_1-6Alkyl group, eg
For example, C such as cyclopentyl and cyclohexyl_3-8Shi
Chloroalkyl groups such as phenyl and α-naphthyl
C_6-12Aryl groups such as benzyl, phenethyl
Which Phenyl-C_1-2Alkyl group or α-naphthyl
Α-naphthyl-C such as methyl_1-2C such as alkyl group
_7-14For aralkyl group, pivaloyloxymethyl group, etc.
Can be The protein used in the present invention is other than the C-terminal
Has a carboxyl group (or carboxylate)
The carboxyl group is amidated or esterified
Included in the proteins used in the present invention
Be done. Examples of the ester in this case include C described above.
A terminal ester or the like is used. Furthermore, for use in the present invention
Included proteins have N-terminal amino acid residues (eg,
The amino group of the methionine residue is a protecting group (eg
C such as ruly group and acetyl group _1-6C such as alkanoyl_1-6
Those protected by an acyl group, etc., that are cleaved in vivo
And the generated N-terminal glutamine residue is pyroglutamine.
Oxidized substances, substituents on the side chains of amino acids in the molecule (eg
For example, -OH, -SH, amino group, imidazole group, in
Suitable protecting groups (eg dol group, guanidino group, etc.)
For example, C such as formyl group and acetyl group_1-6Alkanoyl
C such as group_1-6Protected with an acyl group),
Alternatively, compounds such as so-called glycoproteins in which sugar chains are linked
It also includes synthetic proteins. Tan used in the present invention
Specific examples of the protein include, for example, those represented by SEQ ID NO: 1.
Protein containing the amino acid sequence shown in SEQ ID NO: 3
Protein containing the amino acid sequence represented by 3, sequence
A protein containing the amino acid sequence represented by No. 24
Quality, contains the amino acid sequence represented by SEQ ID NO: 34
Examples include proteins.

The partial peptide of the protein used in the present invention is a partial peptide of the protein used in the present invention, and preferably has the same properties as the protein used in the present invention. Any one will do. For example, at least 20 of the constituent amino acid sequences of the protein used in the present invention
Or more, preferably 50 or more, more preferably 70
Peptides containing an amino acid sequence of 1 or more, more preferably 100 or more, and most preferably 200 or more are used. The partial peptide used in the present invention has 1 or 2 or more amino acids in its amino acid sequence (preferably about 1 to 10 and more preferably about 1 to 5).
1 or 2 or more (preferably 1 to 20 or so, more preferably 1 to 10 or so, still more preferably a number (1 to 2)
5) amino acids are added, or 1 or 2 or more (preferably about 1 to 20 amino acids) is added to the amino acid sequence,
More preferably about 1 to 10, more preferably a number (1 to 5) amino acid is inserted, or 1 or 2 or more (preferably 1 to 1) in the amino acid sequence is inserted.
About 0, more preferably several, and further preferably 1 to
About 5 amino acids) may be substituted with other amino acids. The partial peptide used in the present invention, the C-terminus is a carboxyl group (-COOH), a carboxylate (-COO ^-), may be any of an amide (-CONH ₂₎ or an ester (-COOR). Furthermore, the partial peptides used in the present invention include those having a carboxyl group (or carboxylate) in addition to the C-terminal, and N-terminal amino acid residues (eg, the same as the above-mentioned protein used in the present invention). , A methionine residue) whose amino group is protected by a protecting group, a glutamine residue produced by cleavage at the N-terminal side in vivo is pyroglutamine-oxidized, and a substituent on the side chain of an amino acid in the molecule is Those that are protected by a suitable protecting group, or complex peptides such as so-called glycopeptides to which sugar chains are bound are also included. The partial peptide used in the present invention can also be used as an antigen for antibody production. Specific examples of the partial peptide used in the present invention include, for example, SEQ ID NO: 24.
Examples of the amino acid sequence represented by are peptides containing the 412 to 431st, 528 to 543th, and 883 to 902nd amino acid sequences.

The salts of the protein or partial peptide used in the present invention include physiologically acceptable acids (eg,
A salt with an inorganic acid, an organic acid) or a base (eg, an alkali metal salt) is used, and a physiologically acceptable acid addition salt is particularly preferable. Examples of such salts include salts with inorganic acids (for example, hydrochloric acid, phosphoric acid, hydrobromic acid, sulfuric acid),
Alternatively, organic acids (eg, acetic acid, formic acid, propionic acid,
Fumaric acid, maleic acid, succinic acid, tartaric acid, citric acid,
Malic acid, oxalic acid, benzoic acid, methanesulfonic acid, benzenesulfonic acid) and the like salts are used. The protein or its partial peptide or salt thereof used in the present invention can be produced from the cells or tissues of the above-mentioned humans and other warm-blooded animals by a known protein purification method, or a DNA encoding the protein can be obtained. It can also be produced by culturing the transformant containing it. It can also be produced according to the peptide synthesis method described below. When producing from tissues or cells of humans or other mammals, homogenize tissues or cells of humans or other mammals, then extract with acid, etc., and extract the resulting extract by reverse phase chromatography or ion exchange. Purification and isolation can be achieved by combining chromatography such as chromatography.

For the synthesis of the protein or partial peptide used in the present invention or a salt thereof, or an amide thereof, a commercially available resin for protein synthesis can be used. Examples of such a resin include chloromethyl resin, hydroxymethyl resin, benzhydrylamine resin, aminomethyl resin, 4-benzyloxybenzyl alcohol resin, 4-methylbenzhydrylamine resin, PAM resin, 4-hydroxymethylmethyl resin. Phenylacetamide methyl resin, polyacrylamide resin, 4
Examples thereof include-(2 ', 4'-dimethoxyphenyl-hydroxymethyl) phenoxy resin and 4- (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 various known condensation methods according to the sequence of the target protein. At the end of the reaction, the protein or partial peptide is cleaved from the resin, at the same time various protecting groups are removed, and then an intramolecular disulfide bond formation reaction is performed in a highly diluted solution to obtain the target protein or partial peptide or amide of them. To do. 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 carbodiimides, D
CC, N, N'-diisopropylcarbodiimide, N-
Ethyl-N '-(3-dimethylaminoprolyl) carbodiimide and the like are used. For activation by these, a protected amino acid is directly added to the resin together with a racemization-inhibiting additive (eg, HOBt, HOOBt), or the corresponding acid anhydride or HOBt ester or HOO is added.
It can be added to the resin after previously performing activation of the protected amino acid as a Bt 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 dimethylsulfoxide, 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 protective 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 esterified (for example,
Linear, branched or cyclic alkyl esterification such as methyl, ethyl, propyl, butyl, t-butyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, 2-adamantyl and the like, aralkyl esterification (for example, benzyl ester, 4-nitrobenzyl ester, 4-methoxybenzyl ester, 4-chlorobenzyl ester, benzhydryl esterification), phenacyl esterification, benzyloxycarbonylhydrazideation, t-butoxycarbonylhydrazideation, tritylhydrazideation, etc. You can The hydroxyl group of serine can be protected by, for example, 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. Moreover, examples of a group suitable for etherification include a benzyl group, a tetrahydropyranyl group, and a t-butyl group. Examples of the protective group for the phenolic hydroxyl group of tyrosine include Bz
1, Cl ₂ -Bzl, 2-nitrobenzyl, Br-Z,
t-Butyl or the like is used. Examples of the protecting group for imidazole of histidine include Tos and 4-methoxy-
2,3,6-trimethylbenzenesulfonyl, DNP,
Benzyloxymethyl, Bum, Boc, Trt, Fm
oc or the like is 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- Dinitrophenol, cyanomethyl alcohol, paranitrophenol, HONB, N-hydroxysuccinide, N
-Hydroxyphthalimide, ester with HOBt)]
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 Pd
-Catalytic reduction in a hydrogen stream in the presence of a catalyst such as black or Pd-carbon, or acid with anhydrous hydrogen fluoride, methanesulfonic acid, trifluoromethanesulfonic acid, trifluoroacetic acid, or a mixture thereof. Treatment, base treatment with diisopropylethylamine, triethylamine, piperidine, piperazine, etc., and reduction with sodium in liquid ammonia are also used. The elimination reaction by the acid treatment is generally carried out at a temperature of about -20 ° C to 40 ° C. In the acid treatment, for example, anisole, phenol, thioanisole, metacresol,
The addition of cation trapping agents such as para-cresol, dimethyl sulfide, 1,4-butanedithiol, 1,2-ethanedithiol is effective. Further, 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 functional groups which should not participate in the reaction of the starting materials, elimination of the protective groups and activation of functional groups involved in the reaction are appropriately carried out by known means, and the protecting groups used are appropriately selected from known groups. Selected and applied. 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 peptide is condensed in a mixed solvent as described above. The details of the condensation reaction are the same as above. After purifying the protected protein or peptide obtained by condensation, all protecting groups can be removed by the above method to obtain the desired crude protein or peptide. The crude protein or peptide can be purified by utilizing various known purification means, and the main fraction can be lyophilized to obtain the amide of the desired protein or peptide. To obtain an ester form of a protein or peptide, for example, after condensing the α-carboxyl group of the carboxy terminal amino acid with a desired alcohol to form an amino acid ester, the desired protein An ester form of the peptide can be obtained.

The partial peptide used in the present invention or a salt thereof can be produced according to a known peptide synthesis method or by cleaving the protein used in 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 used in the present invention with the residual portion and removing the protective group when the product has a protective group. it can. Examples of known condensation methods and removal of protective groups include the methods described in (a) to (e) below. (A) M. Bodanszky and MA Ondetti, peptides
Synthesis (Peptide Synthesis), Interscience Publi
shers, New York (1966) (b) Schroeder and Luebke, The Pepti
de), Academic Press, New York (1965) (c) Nobuo Izumiya et al., Fundamentals and experiments of peptide synthesis, Maruzen
(1975) (d) Haruaki Yajima and Shunpei Sakakibara, Laboratory for Biochemistry 1,
Protein Chemistry IV, 205, (1977) (e) Supervised by Haruaki Yajima, Development of Pharmaceuticals, Volume 14, Peptide Synthesis, Hirokawa Shoten Also, after the reaction, ordinary purification methods such as solvent extraction / distillation / The partial peptide used in the present invention can be purified and isolated by combining column chromatography, liquid chromatography, recrystallization and the like. When the partial peptide obtained by the above method is a free form, it can be converted into an appropriate salt by a known method or a method analogous thereto, and conversely, when a partial peptide is obtained, a known method or a method similar thereto is used. It can be converted into the educt or other salt by the method.

The polynucleotide encoding the protein used in the present invention may be any polynucleotide containing the nucleotide sequence encoding the protein used in the present invention. Preferably DNA
Is. As DNA, genomic DNA, genomic DNA
Library, cDNA derived from the cells / tissues described above, cDNA library derived from the cells / tissues described above, synthetic D
Any of NA may be used. The vector used for the library may be any of bacteriophage, plasmid, cosmid, phagemid and the like. In addition, reverse Transcriptase Polymera can be directly prepared by using a total RNA or mRNA fraction prepared from the cells and tissues described above.
It can also be amplified by se Chain Reaction (hereinafter abbreviated as RT-PCR method). As the DNA encoding the protein used in the present invention, for example,
A DNA containing the nucleotide sequence represented by SEQ ID NO: 29,
Alternatively, a DNA or sequence containing a nucleotide sequence that hybridizes with the nucleotide sequence represented by SEQ ID NO: 29 under high stringent conditions and that encodes a protein having substantially the same properties as the protein used in the present invention A DNA containing the nucleotide sequence represented by No. 2 or a nucleotide sequence hybridizing with the nucleotide sequence represented by SEQ ID NO: 2 under high stringent conditions, which is substantially the same as the protein used in the present invention. Which encodes a protein having similar homologous properties, DNA containing the nucleotide sequence represented by SEQ ID NO: 25, or SEQ ID NO:
Any DNA encoding a protein containing a nucleotide sequence that hybridizes with the nucleotide sequence represented by 25 under high stringent conditions and that encodes a protein having substantially the same properties as the protein used in the present invention But it's okay.

The DNA hybridizable with the nucleotide sequence represented by SEQ ID NO: 29 or SEQ ID NO: 2 under high stringent conditions is, for example, SEQ ID NO: 2
9 or the nucleotide sequence represented by SEQ ID NO: 2 is about 80% or more, preferably about 85% or more, more preferably about 90%.
Above all, particularly preferably, a DNA containing a nucleotide sequence having a homology of about 95% or more is used. As a specific example, D containing the nucleotide sequence represented by SEQ ID NO: 29
NA, DN containing the nucleotide sequence represented by SEQ ID NO: 2
A, DN containing the nucleotide sequence represented by SEQ ID NO: 30
A, DN containing the nucleotide sequence represented by SEQ ID NO: 31
A, DN containing the nucleotide sequence represented by SEQ ID NO: 32
A etc. are mentioned. The DNA that can hybridize with the base sequence represented by SEQ ID NO: 25 under high stringent conditions is, for example, about 80% or more, preferably about 85% or more, with the base sequence represented by SEQ ID NO: 25.
More preferably about 90% or more, and particularly preferably about 95%.
A DNA containing a base sequence having the above homology is used. Specific examples include DNA containing the nucleotide sequence represented by SEQ ID NO: 25, DNA containing the nucleotide sequence represented by SEQ ID NO: 35, DNA containing the nucleotide sequence represented by SEQ ID NO: 36, Examples thereof include DNA containing the nucleotide sequence represented by SEQ ID NO: 37. Hybridization can be carried out by a known method or a method analogous thereto, for example, molecular cloning (Molecula
r Cloning) 2nd (J. Sambrook et al., Cold Spring Ha
rbor Lab. Press, 1989). When a commercially available library is used, it can be performed according to the method described in the attached instruction manual. More preferably, it can be carried out under high stringent conditions. Highly stringent conditions include, for example, a sodium concentration of about 19 to 40 m.
M, preferably about 19-20 mM, at a temperature of about 50-7.
The conditions are 0 ° C., preferably about 60 to 65 ° C. In particular,
Most preferably, the sodium concentration is about 19 mM and the temperature is about 65 ° C. More specifically, DN encoding a protein containing the amino acid sequence represented by SEQ ID NO: 1.
A is a protein containing the amino acid sequence represented by SEQ ID NO: 33, such as a DNA containing the base sequence represented by SEQ ID NO: 29, SEQ ID NO: 2, SEQ ID NO: 30 or SEQ ID NO: 31. The DNA encoding the DNA is DN containing the nucleotide sequence represented by SEQ ID NO: 32.
The DNA encoding the protein containing the amino acid sequence represented by SEQ ID NO: 24, such as A, includes DNA containing the nucleotide sequence represented by SEQ ID NO: 25, SEQ ID NO: 36 or SEQ ID NO: 37. Is SEQ ID NO: 34
Examples of the DNA encoding the protein containing the amino acid sequence represented by: include the DNA containing the nucleotide sequence represented by SEQ ID NO: 35.

The DNA encoding the partial peptide used in the present invention may be any DNA as long as it contains the nucleotide sequence encoding the partial peptide used in the present invention. In addition, genomic DNA, genomic DNA library, c derived from the cells / tissues described above
It may be DNA, a cDNA library derived from the cells / tissues described above, or synthetic DNA. As the DNA encoding the partial peptide used in the present invention, for example,
(1) SEQ ID NO: 29, SEQ ID NO: 2 or SEQ ID NO:
DNA containing a part of the DNA containing the nucleotide sequence represented by 25, or (2) under high stringent conditions with the nucleotide sequence represented by SEQ ID NO: 29, SEQ ID NO: 2 or SEQ ID NO: 25 A DNA containing a hybridizing base sequence and containing a part of a DNA encoding a protein having substantially the same activity as the protein of the present invention is used. The DNA hybridizable with the base sequence represented by SEQ ID NO: 29, SEQ ID NO: 2 or SEQ ID NO: 25 has the same meaning as described above. The same hybridization method and high stringent conditions as described above are used.

D which completely encodes the protein or partial peptide used in the present invention (hereinafter, in the description of cloning and expression of the DNA encoding them, these are sometimes simply referred to as the protein of the present invention)
As a means for cloning NA, a synthetic DNA containing a part of the nucleotide sequence encoding the protein of the present invention is used.
Amplification by PCR using a primer, or selection by hybridization with a DNA incorporated into an appropriate vector and labeled with a DNA fragment coding for a part or the whole region of the protein of the present invention or a synthetic DNA. To be Hybridization methods include, for example, molecular cloning (Molecu
lar Cloning) 2nd (J. Sambrook et al., Cold Spring
Harbor Lab. Press, 1989). When a commercially available library is used, it can be performed according to the method described in the attached instruction manual. Replacement of the nucleotide sequence of DNA is carried out by PCR or a known kit such as Mutan ^™ -superExpress Km (Takara Shuzo) or Mutan ^™ -K (Takara Shuzo).
Method, Gapped duplex method, Kunkel method or the like or a method similar thereto can be used. Cloned DNA encoding the protein of the invention
Can be used as it is, or if desired, after digestion with a restriction enzyme or addition of a linker. The DNA may have ATG as a translation initiation codon at the 5′-terminal side and TAA, TGA or TAG as a translation termination codon at the 3′-terminal side. 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 is, for example,
(A) by excising a target DNA fragment from a DNA (eg, cDNA) containing the DNA encoding the protein of the present invention, and (b) ligating the DNA fragment downstream of a promoter in an appropriate expression vector. It can be manufactured.

As a vector, a plasmid derived from E. 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, the trp promoter, the lac promoter, the recA promoter, the λP _L 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 belonging to the genus Escherichia, the PhoA signal sequence, the OmpA signal sequence, etc., and when the host is a bacterium belonging to the genus Bacillus, the α-amylase signal sequence, the subtilisin signal sequence, etc.
When the host is yeast, MFα signal sequence, SUC
2 If the host is an animal cell, such as a signal sequence,
Insulin signal sequences, α-interferon signal sequences, antibody molecule signal sequences, etc. can be used. 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], Volume 60, 160. [Procedures of the National Academy of Sciences of the USA]
(1968)], JM103 [Nucleic Acids Research], Volume 9, 309 (198).
1)], JA221 [Journal of Molecular
Biology (Journal of Molecular Biology), 120
Vol. 517 (1978)], HB101 [Journal of Molecular Biology, Vol. 41, 459 (1969)], C6.
00 [Genetics, 39, 440 (195
4)] is used. Examples of the bacterium of the genus Bacillus include Bacillus subtilis MI.
114 [Gene, 24, 255 (1983)], 207-21 [Journal of Bioch
emistry), Vol. 95, 87 (1984)]. Examples of yeast include Saccharomyces cerevisiae (Sa
^{ccharomyces cerevisiae) AH22, AH22R -,} N
A87-11A, DKD-5D, 20B-12, Schizosaccharomyces pombe
e) NCYC1913, NCYC2036, Pichia pastoris KM71, etc. are used.

Examples of insect cells include virus A
In the case of cNPV, the cell line derived from the larva of Spodoptera frugiperda (Spod
optera frugiperda cell; Sf cell), Trichoplusia n
MG1 cells from the midgut of i, from the eggs of Trichoplusia ni
High 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
mori N cells; BmN cells) and the like are used. The Sf
Examples of the cells include Sf9 cells (ATCC CRL1711),
Sf21 cells (above Vaughn, JL et al., In Vivo, 13, 213-217, (1977)) and the like are used.
Examples of insects include silkworm larvae [Maeda et al., Nature, 315, 592 (198).
Five)〕. 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 transformation of Escherichia, for example, Proc. Natl. Acad. Sci. USA, 69, 21.
10 (1972) and Gene (17), 107 (1982) and the like.

To transform Bacillus, for example, Molecular & General Genetics, 168, 111
(1979) and the like. For transforming yeast, for example, Methods in Enzymology, 194, 182.
−187 (1991), Procedures of the National Academy of Sciences of the
USA (Proc. Natl. Acad. Sci. USA), Volume 75,
It can be performed according to the method described in 1929 (1978). To transform insect cells or insects, for example,
Bio / Technology, 6, 47-55
(1988) and the like.
To transform animal cells, for example, see Cell Engineering Supplement 8
New cell engineering protocol. 263-267 (1995) (published by Shujunsha), Virology, Volume 52, 456 (1
It can be performed according to the method described in 973). In this way, a transformant transformed with the expression vector containing the DNA encoding the protein can be obtained. 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), 431-43
3, Cold Spring Harbor Laboratory, New York 1972]
Is preferred. 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 performed at about 15 to 43 ° C. for about 3 to 24.
If necessary, aeration and agitation can be added over time. When the host is Bacillus, the culture is usually about 30-
It is carried out at 40 ° C. for about 6 to 24 hours, and if necessary, aeration and stirring can be added. When culturing a transformant whose host is yeast, examples of the medium include Burkholder minimum medium [Bostian, KL, et al., Procedures of the National Academy of Sciences of Science]. The USA (Proc. Na
Acad. Sci. USA), 77, 4505 (1980)] and 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. USA), 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 10% immobilized on Grace's Insect Medium (Grace, TCC, Nature, 195,788 (1962)).
For example, those to which additives such as bovine serum are appropriately added are used. The pH of the medium is preferably adjusted to about 6.2 to 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, about 5
MEM medium containing 20% fetal bovine serum [Science (Sc
ience), Volume 122, 501 (1952)], DMEM medium [Virology, Volume 8, 396 (1959)], RPMI
1640 medium [The Journal of the American Medical Association (The Journal of the Ame
rican Medical Association) 199, 519 (1967)], 1
99 Medium [Procedure of the Society
For the Biological Medicine (Proceeding
of the Society for the Biological Medicine), 73
Vol. 1, 1 (1950)], etc. are used. The pH is preferably about 6-8. Culturing is usually performed at about 30 to 40 ° C for about 15
Perform for ~ 60 hours, add 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-mentioned culture, for example, by the following method. When extracting the protein of the present invention from cultured bacterial cells or cells, after culturing, the bacterial cells or cells are collected by a known method, suspended in a suitable buffer solution, ultrasonicated,
A method of disrupting the cells or cells by lysozyme and / or freeze-thawing and then 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 known method, and the supernatant is collected. The protein contained in the culture supernatant or the extract thus obtained can be purified by appropriately combining known separation / purification methods. Known separation and purification methods for these are methods utilizing solubility such as salting out and solvent precipitation, dialysis,
Ultrafiltration, gel filtration, SDS-polyacrylamide gel electrophoresis and other methods that mainly utilize the difference in molecular weight, ion exchange chromatography and other methods that utilize the difference in charge, affinity chromatography and other specific methods A method of using affinity, a method of utilizing difference in hydrophobicity such as reversed-phase high performance liquid chromatography, a method of utilizing difference in isoelectric point such as isoelectric focusing are used.

When the protein thus obtained is obtained as a free form, it can be converted into a salt by a known method or a method analogous thereto, and conversely, when it is obtained as a salt, a known method. Alternatively, it can be converted into a free form or another salt by a method similar thereto. 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 of the protein of the present invention thus produced can be measured by an enzyme immunoassay using a specific antibody, Western blotting, or the like.

The antibody against the protein or partial peptide or salt thereof used in the present invention may be either a polyclonal antibody or a monoclonal antibody as long as it is an antibody capable of recognizing the protein or partial peptide or salt thereof used in the present invention. Good. Antibodies against the protein or partial peptide or a salt thereof used in the present invention (hereinafter, these may be simply referred to as the protein of the present invention in the description of the antibody) are known by using the protein of the present invention as an antigen. It can be produced according to the method for producing an antibody or antiserum. [Preparation of Monoclonal Antibody] (a) Preparation of Monoclonal Antibody-Producing Cell The protein of the present invention is administered to a warm-blooded animal 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 Koehler and Milstein [Nature, 256, 495 (19
75)]. 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. Selection of the monoclonal antibody can be performed according to a known method or a method similar thereto. Normal HAT
(Hypoxanthine, aminopterin, thymidine) can be added to the medium for animal cells. As the medium for selection and breeding, any medium may be used as long as the hybridoma can grow. For example, 1 to 20
%, Preferably 10-20% fetal calf serum
I 1640 medium, GI containing 1-10% fetal bovine serum
T medium (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 4
It is 0 ° C, preferably about 37 ° C. Cultivation time is usually 5
Day to 3 weeks, preferably 1 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 known method, 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 and dissociate the binding Specific Purification Method to Obtain]].

[Preparation of Polyclonal Antibody] The polyclonal antibody of the present invention can be manufactured by publicly known methods or modifications thereof. For example, an immune antigen (protein antigen) 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 product and separating and purifying the antibody.
Regarding the complex of the immunizing antigen and the carrier protein used for immunizing warm-blooded animals, the type of carrier protein and the mixing ratio of the carrier and the 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 at any ratio. For example, bovine serum albumin, bovine thyroglobulin, hemocyanin, etc. may be cross-linked in a weight ratio of hapten 1.
On the other hand, a method of coupling at a ratio of about 0.1 to 20, preferably about 1 to 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 about once every 2 to 6 weeks, about 3 to 1 in total.
It is performed about 0 times. 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.

Complementary to the nucleotide sequence of the DNA encoding the protein or partial peptide used in the present invention (hereinafter, these DNAs may be abbreviated as the DNA of the present invention in the description of antisense nucleotides),
Alternatively, the antisense nucleotide containing a substantially complementary nucleotide sequence or a part thereof is the DN of the present invention.
Any antisense nucleotide as long as it contains a base sequence complementary to or substantially complementary to the base sequence of A or a part thereof and has an action of suppressing the expression of the DNA However, antisense DNA is preferable. The term “substantially complementary nucleotide sequence to the DNA of the present invention” refers to, for example, a total nucleotide sequence or a partial nucleotide sequence of a nucleotide sequence complementary to the DNA of the present invention (that is, a complementary strand of the DNA of the present invention) and about 70 nucleotides. % Or more, preferably about 80% or more, more preferably about 90% or more, most preferably about 9% or more.
Examples include base sequences having a homology of 5% or more.
In particular, of the total base sequence of the complementary strand of the DNA of the present invention, about 70% or more with the complementary strand of the base sequence of the portion encoding the N-terminal portion of the protein of the present invention (for example, the base sequence near the start codon). Antisense nucleotides having a homology of preferably about 80% or more, more preferably about 90% or more, and most preferably about 95% or more are suitable. Specifically, an antisense containing a base sequence complementary to or substantially complementary to the base sequence of DNA containing the base sequence represented by SEQ ID NO: 29 or SEQ ID NO: 2, or a part thereof. Nucleotides and the like.
About 10 to 40 antisense nucleotides are usually used,
It is preferably composed of about 15 to 30 bases. In order to prevent degradation by a hydrolase such as nuclease, the phosphate residue (phosphate) of each nucleotide constituting the antisense DNA is changed to, for example, a chemically modified phosphate residue such as phosphorothioate, methylphosphonate, and phosphorodithionate. It may be substituted. These antisense nucleotides can be produced using a known DNA synthesizer or the like. According to the invention,
Antisense polynucleotide (nucleic acid) capable of inhibiting the replication or expression of the protein gene of the present invention
Can be designed and synthesized based on the nucleotide sequence information of the DNA encoding the cloned or determined protein. Such a polynucleotide (nucleic acid) can hybridize with RNA of the protein gene of the present invention, can inhibit the synthesis or function of the RNA, or can interact with the protein-related RNA of the present invention. Regulate the expression of the protein gene of the present invention
Can be controlled. Protein-related RNA of the present invention
The polynucleotide complementary to the selected sequence of the above and a polynucleotide capable of specifically hybridizing with the protein-related RNA of the present invention regulate and control the expression of the protein gene of the present invention in vivo and in vitro. It is also useful for treating or diagnosing diseases and the like. The term "corresponding" means having homology or being complementary to a specific sequence of nucleotides, base sequences or nucleic acids including genes. Nucleotides, nucleotide sequences or nucleic acids and peptides (proteins)
“Corresponding” between and usually refers to the amino acid of a peptide (protein) in a command derived from the sequence of nucleotides (nucleic acid) or its complement. 5′-end hairpin loop of protein gene, 5′-end 6-base pair repeat, 5′-end untranslated region, polypeptide translation initiation codon, protein coding region, ORF translation stop codon, 3′-end untranslated region, 3 ′ The end palindromic region and the 3 ′ end hairpin loop can be selected as the preferred target region, but any region within the protein gene can be selected as the target. It can be said that 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 are “antisense”.
Antisense polynucleotides are 2-deoxy-D
-Polynucleotides containing ribose, polynucleotides containing D-ribose, other types of polynucleotides that are N-glycosides of purine or pyrimidine bases, or other polymers having a non-nucleotide backbone (eg, Commercially available protein nucleic acids and synthetic sequence-specific nucleic acid polymers) or other polymers containing special linkages, provided that the polymer is DN
A or nucleotide containing a nucleotide having a configuration that allows base pairing or base attachment as found in RNA). They include double-stranded DNA, single-stranded DNA, double-stranded RNA, single-stranded RNA, and DN.
A: RNA hybrid, and may further be an unmodified polynucleotide (or an unmodified oligonucleotide), and also with a known modification, eg, with a label known in the art, capped , Methylated, substituted with one or more natural nucleotides by analogs, modified with an intramolecular nucleotide, such as an uncharged bond (eg, methylphosphonate, phosphotriester, phosphoramidate) , Carbamates, etc., charged bonds or sulfur-containing bonds (eg phosphorothioates, phosphorodithioates, etc.), eg proteins (nucleases, nuclease inhibitors, toxins,
Antibodies, signal peptides, poly-L-lysine, etc.), sugars (eg, monosaccharides, etc.) having side chain groups, intercalating compounds (eg, acridine, psoralen, etc.), chelates Those containing compounds (eg, metals, radioactive metals, boron, oxidizing metals, etc.), those containing alkylating agents, those having modified bonds (eg, α-anomer type nucleic acids, etc.) May be 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. The modified nucleotide and the modified nucleotide may also have a modified sugar moiety, for example, one or more hydroxyl groups are substituted with halogen, an aliphatic group, or the like,
Alternatively, it may be converted into a functional group such as ether or 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, has a greater affinity for the target sense strand, and if it is toxic, Make sense nucleic acid less toxic. Many such 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, polycationic substances such as polylysine that act to neutralize the charge of the phosphate group, lipids that enhance the interaction with cell membranes and increase the uptake of nucleic acids ( Examples thereof include hydrophobic ones such as phospholipids 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. Examples of the other group include a capping group specifically arranged at the 3'-end or the 5'-end of a nucleic acid, which is 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 known methods.

Hereinafter, the protein or partial peptide of the present invention or a salt thereof (hereinafter sometimes abbreviated as the protein of the present invention), DNA encoding the protein or partial peptide of the present invention (hereinafter, DNA of the present invention)
May be abbreviated), an antibody against the protein or partial peptide of the present invention or a salt thereof (hereinafter sometimes abbreviated as the antibody of the present invention), and D of the present invention.
The use of the antisense nucleotide of NA (hereinafter sometimes abbreviated as the antisense nucleotide of the present invention) will be described. A drug containing a compound that inhibits the activity of the protein of the present invention or a salt thereof is Na, K-ATPas.
Since e activity can be suppressed and K ⁺ ion and Na ⁺ ion exchange transport can be suppressed, for example, heart disease (eg, heart failure, cardiac hypertrophy), renal disease (eg, renal failure, interventricular kidney) Diseases, etc., central nervous system diseases (eg, Alzheimer's disease, Parkinson's syndrome, schizophrenia, etc.), inflammatory diseases (eg,
Chronic obstructive pulmonary disease, etc., autoimmune disease (eg, myasthenia gravis, glomerulonephritis, multiple sclerosis, Sjogren's syndrome, insulin resistant diabetes, rheumatoid arthritis, systemic lupus erythematosus, etc.), allergic disease (eg , Bronchial asthma, hay fever, allergic rhinitis, anaphylactic shock, atopic dermatitis, etc.), thymic disease, spleen disease (eg, hypersplenism, etc.), immunodeficiency (eg, leukocyte abnormality, splenic or thymic abnormality) Immunodeficiency associated with),
Reproductive organ diseases (eg, testicular hypersensitivity, ovarian insufficiency, etc.) and cancers (eg, pancreatic cancer, lung cancer, kidney cancer, liver cancer, non-small cell lung cancer, ovarian cancer, prostate cancer, gastric cancer, bladder cancer, breast cancer, cervix) It can be used as a therapeutic / preventive agent for cancer, colon cancer, rectal cancer, etc., or a rejection inhibitor after organ transplantation. On the other hand, a drug containing a compound or a salt thereof that promotes the activity of the protein of the present invention is, for example, Na, K
-It is possible to promote ATPase activity and promote exchange transport of K ⁺ and Na ⁺ ions, and thus, for example, heart disease (eg, heart failure, cardiac hypertrophy), kidney disease (eg, renal failure, interventricularity) Kidney disease, etc., central nervous system disease (eg, Alzheimer's disease, Parkinson's syndrome, schizophrenia, etc.), inflammatory disease (eg, chronic obstructive pulmonary disease, etc.), autoimmune disease (eg, myasthenia gravis, glomerulus) Nephritis, multiple sclerosis, Sjogren's syndrome, insulin resistant diabetes, rheumatoid arthritis, systemic lupus erythematosus, etc., allergic diseases (eg, bronchial asthma, hay fever, allergic rhinitis, anaphylactic shock, atopic dermatitis, etc.), Thymic disease, spleen disease (eg, hypersplenism, etc.), immunodeficiency (eg, white blood cell abnormalities, immunodeficiency associated with splenic or thymic abnormalities), genital organs Diseases (eg, testicular hypersensitivity, ovarian dysfunction, etc.) and cancers (eg, pancreatic cancer, lung cancer, kidney cancer,
As a therapeutic / preventive agent for liver cancer, non-small cell lung cancer, ovarian cancer, prostate cancer, gastric cancer, bladder cancer, breast cancer, cervical cancer, colon cancer, rectal cancer, etc., or a rejection inhibitor after organ transplantation Can be used.

[1] Preventive / therapeutic agent for various diseases related to the protein of the present invention The protein of the present invention has Na, K-ATPase activity and contributes to exchange transport of K ⁺ ion and Na ⁺ ion. , Plays a central role in maintaining homeostasis of cells. In addition, the Na ⁺ concentration gradient by the protein of the present invention produces energy for transport of other ions and transport of nutrients, and cell functions such as muscle contraction, nerve signal transmission, and Na ⁺ reabsorption in the kidney. Plays an important role in the expression of. 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 decreased, for example, heart disease (eg, heart failure, cardiac hypertrophy, etc.) ), Renal disease (eg, renal failure, interventricular renal disease, etc.), central nervous system disease (eg, Alzheimer's disease, Parkinson's syndrome, schizophrenia, etc.), inflammatory disease (eg, chronic obstructive pulmonary disease, etc.), Autoimmune disease (eg, myasthenia gravis, glomerulonephritis, multiple sclerosis, Sjogren's syndrome,
Insulin-resistant diabetes, rheumatoid arthritis, systemic lupus erythematosus, etc., allergic disease (eg, bronchial asthma, hay fever, allergic rhinitis, anaphylactic shock, atopic dermatitis, etc.), thymic disease, spleen disease (eg, splenic function) Hypersensitivity), immunodeficiency (eg, white blood cell abnormalities, immunodeficiency associated with splenic or thymic abnormalities),
Reproductive organ diseases (eg, testicular hypersensitivity, ovarian insufficiency, etc.) and cancers (eg, pancreatic cancer, lung cancer, kidney cancer, liver cancer, non-small cell lung cancer, ovarian cancer, prostate cancer, gastric cancer, bladder cancer, breast cancer, cervix) Cancer, colon cancer, rectal cancer, etc.) or rejection after organ transplantation. Therefore,
The protein of the present invention and the DNA of the present invention are, for example,
Heart disease (eg, heart failure, cardiac hypertrophy, etc.), kidney disease (eg, renal failure, interventricular kidney disease, etc.), central nervous system disease (eg, Alzheimer's disease, Parkinson's syndrome, schizophrenia, etc.),
Inflammatory diseases (eg, chronic obstructive pulmonary disease, etc.), autoimmune diseases (eg, myasthenia gravis, glomerulonephritis, multiple sclerosis, Sjogren's syndrome, insulin resistant diabetes, rheumatoid arthritis, systemic lupus erythematosus, etc. ), Allergic disease (eg, bronchial asthma, hay fever, allergic rhinitis, anaphylactic shock, atopic dermatitis, etc.), thymus disease, spleen disease (eg, hypersplenism, etc.), immunodeficiency (eg, leukocyte abnormality, Spleen insufficiency or immunodeficiency associated with thymic abnormalities), genital diseases (eg, testicular hypersensitivity, ovarian insufficiency, etc.) and cancers (eg, pancreatic cancer, lung cancer, kidney cancer,
Prevention and treatment of diseases such as liver cancer, non-small cell lung cancer, ovarian cancer, prostate cancer, gastric cancer, bladder cancer, breast cancer, cervical cancer, colon cancer, rectal cancer, etc., or suppression of rejection after organ transplantation It can be used as a medicine such as an agent. For example, in the case where there is a patient in whom exchange transport of K ⁺ ions and Na ⁺ ions is not sufficiently or normally exerted due to the decrease or deficiency of the protein of the present invention in vivo,
(B) by administering the DNA of the present invention to the patient and expressing the protein of the present invention in vivo, (b) after inserting the DNA of the present invention into cells and expressing the protein of the present invention, The role of the protein of the present invention in the patient can be sufficiently or normally exerted by transplanting the cells into the patient or (c) administering the protein of the present invention to the patient.
When the DNA of the present invention is used as the above-mentioned preventive / therapeutic agent, the DNA of the present invention is used alone or after being inserted into an appropriate vector such as a retrovirus vector, an adenovirus vector, an adenovirus associated virus vector, etc. Can be administered to humans or other warm-blooded animals according to. 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 of the present invention is used as the above-mentioned preventive / therapeutic agent, it is at least 90%, preferably 95%.
% Or more, more preferably 98% or more, and further preferably 99% or more is preferably used.

The protein and the like of the present invention include, for example, tablets, capsules, elixirs, and 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 implementation. 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 etc. of the present invention varies depending on the target disease, administration subject, administration route, etc. For example, when the protein etc. of the present invention is orally administered for the purpose of treating heart failure disease, it is generally an adult (60 kg In the above), about 0.1 mg to 100 mg, 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., for example, when the protein or the like of the present invention is administered to an adult (as a body weight of 60 kg) in the form of an injection for the purpose of treating heart failure, the protein or the like per day About 0.01 to 30 m
It is convenient to administer about g, preferably about 0.1 to 20 mg, more preferably about 0.1 to 10 mg by injection into the affected area. 6 for other animals
An amount converted per 0 kg can be administered.

[2] Screening of drug candidate compounds against diseases The protein of the present invention is useful as a reagent for screening a compound or its salt that promotes or inhibits the activity of the protein of the present invention. The present invention includes (1) a compound or a salt thereof that promotes or inhibits the activity of the protein of the present invention (for example, exchange transport of K ⁺ ion and Na ⁺ ion) characterized by using the protein of the present invention (hereinafter , And may be abbreviated as an accelerator and an inhibitor, respectively). More specifically, for example, (2) (i) K ⁺ ion and Na ⁺ ion exchange transport activity of cells having the ability to produce the protein of the present invention, and (ii) the ability to produce the protein of the present invention, Provided is a screening method for promoters or inhibitors, which comprises comparing the exchange transport activity of K ⁺ ions and Na ⁺ ions of a mixture of cells with test compounds. Specifically, in the above screening method, for example,
In the cases (i) and (ii), the exchange transport activity of K ⁺ and Na ⁺ ions was measured by the patch clamp method, and K ⁺
It is characterized by making a comparison as an index of the exchange transport activity of ions and Na ⁺ ions.

Examples of test compounds include peptides,
Examples include proteins, non-peptidic compounds, synthetic compounds, fermentation products, cell extracts, plant extracts, animal tissue extracts, and the like, and these compounds may be novel compounds or known compounds. Good. To carry out the above-mentioned screening method, cells capable of producing the protein of the present invention are suspended in a buffer suitable for screening and prepared. The pH of the buffer is about 4-10.
Any buffer (preferably about pH 6 to 8) such as a phosphate buffer or a borate buffer may be used as long as it does not inhibit the exchange transport activity of K ⁺ ion and Na ⁺ ion of the protein of the present invention. As the cell having the ability to produce the protein of the present invention, for example, a host (transformant) transformed with the vector containing the DNA encoding the protein of the present invention is used. Animal cells such as CHO cells are preferably used as the host. For the screening, for example, a transformant in which the protein of the present invention is expressed on the cell membrane by culturing by the method described above is preferably used.

The exchange transport activity of K ⁺ ion and Na ⁺ ion of the protein of the present invention can be determined by known methods, for example, J. Biol.
Chem. Vol. 275, 1976-1986, 2000, or a method analogous thereto or a method analogous thereto.
For example, the exchange transport activity of K ⁺ ions and Na ⁺ ions in the case of the above (ii) is about 20% or more, preferably 30% or more, more preferably about 50% or more as compared with the case of the above (i). A test compound that promotes can be selected as a compound that promotes the activity of the protein of the present invention or a salt thereof. Also, for example, K in the case of (ii) above
The exchange transport activity of ⁺ ion and Na ⁺ ion is about 20% or more, preferably 30% or more, as compared with the case (i).
More preferably, a test compound that inhibits (or suppresses) about 50% or more can be selected as a compound that inhibits the activity of the protein of the present invention or a salt thereof. In addition, a gene for secretory alkaline phosphatase, luciferase or the like is inserted downstream of the promoter of the protein gene of the present invention, expressed in the various cells described above, and the enzyme activity is activated when the test compound is contacted with the cells or Promote or suppress the expression of the protein of the present invention by searching for a compound or a salt thereof that inhibits (ie,
A compound or its salt that promotes or inhibits the activity of the protein of the present invention can be screened.

The polynucleotide encoding the protein of the present invention is useful as a reagent for screening a compound or its salt that promotes or inhibits the expression of the protein gene of the present invention. The present invention uses (3) a polynucleotide encoding the protein of the present invention, which promotes or inhibits the expression of the protein gene of the present invention or a salt thereof (hereinafter, abbreviated as promoter and inhibitor, respectively). In some cases, more specifically, for example, (4) (iii)
Promotion characterized by performing a comparison between the case of culturing cells having the ability to produce the protein of the present invention and (iv) the case of culturing a mixture of cells having the ability to produce the protein of the present invention and a test compound A method for screening an agent or an inhibitor is provided. In the above screening method, for example, in the cases of (iii) and (iv), the expression level of the protein gene of the present invention (specifically, the amount of the protein of the present invention or the mR encoding the protein)
NA amount) is measured and compared. As a test compound,
For example, peptides, proteins, non-peptidic compounds,
Examples thereof include synthetic compounds, fermentation products, cell extracts, plant extracts, animal tissue extracts, etc. These compounds may be novel compounds or known compounds.
To carry out the above-mentioned screening method, cells capable of producing the protein of the present invention are suspended in a buffer suitable for screening and prepared. The buffer may be a buffer that does not inhibit the exchange transport of K ⁺ ions and Na ⁺ ions of the protein of the present invention, such as a phosphate buffer or a borate buffer having a pH of about 4 to 10 (desirably, a pH of about 6 to 8). It doesn't matter. Examples of cells having the ability to produce the protein of the present invention include:
A host (transformant) transformed with the vector containing the above-mentioned DNA encoding the protein of the present invention is used. Animal cells such as CHO cells are preferably used as the host. The screening includes
For example, a transformant in which the protein of the present invention is expressed on the cell membrane by culturing by the method described above is preferably used. The amount of the protein of the present invention can be measured by a known method, for example, using an antibody that recognizes the protein of the present invention to measure the protein present in a cell extract or the like,
It can be measured by a method such as Western analysis or ELISA method or a method similar thereto. The expression level of the protein gene of the present invention can be determined by a known method such as Northern blotting or Reverse transcription-polymerase.
chain reaction (RT-PCR), real-time PC
R analysis system (ABI, TaqMan polymerase chai
nreaction) or the like or a method similar thereto. For example, a test for accelerating the expression level of the protein gene of the present invention in the case of (iv) above by about 20% or more, preferably 30% or more, more preferably about 50% or more as compared with the case of (iii) above. The compound can be selected as a compound that promotes the expression of the protein gene of the present invention or a salt thereof. For example, the expression level of the protein gene of the present invention in the case of (iv) above is about 20% compared to the case of (iii) above.
Or more, preferably 30% or more, more preferably about 50%
The test compound that inhibits the above can be selected as a compound that inhibits the expression of the protein gene of the present invention or a salt thereof. Furthermore, the antibody of the present invention is useful as a reagent for screening a compound or its salt that promotes or inhibits the expression of the protein of the present invention. The present invention provides (5) a compound or a salt thereof that promotes or inhibits the expression of the protein of the present invention, which is characterized by using the antibody of the present invention (hereinafter, may be abbreviated as “promoter” or “inhibitor”, respectively). A screening method is provided, and more specifically, for example, (6) (v) when a cell capable of producing the protein of the present invention is cultured, and (vi) a cell capable of producing the protein of the present invention And a case of culturing a mixture of the test compound and the test compound are provided.
In the above screening method, for example, in the cases of (v) and (vi), the expression level of the protein of the present invention (specifically, the protein amount of the present invention) is measured using the antibody (of the protein of the present invention The expression is detected, the expression level of the protein of the present invention is quantified, etc.) and compared. 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, and these compounds may be novel compounds. It may be a known compound. To carry out the above-mentioned screening method, cells capable of producing the protein of the present invention are suspended in a buffer suitable for screening and prepared. The buffer has a pH of about 4 to 10 (desirably, a pH of about 6 to
Any buffer may be used as long as it does not inhibit the exchange transport of K ⁺ ions and Na ⁺ ions of the protein of the present invention, such as the phosphate buffer and borate buffer of 8). Cells having the ability to produce the protein of the present invention include:
For example, DN encoding the above-mentioned protein of the present invention
A host (transformant) transformed with the vector containing A is used. Animal cells such as CHO cells are preferably used as the host. In the screening, for example, by culturing by the method described above,
A transformant in which the protein of the present invention is expressed on the cell membrane is preferably used. The amount of the protein of the present invention can be measured by a known method, for example, by using an antibody that recognizes the protein of the present invention, the protein present in a cell extract or the like is subjected to a method such as Western analysis or ELISA, or a method similar thereto. It can be measured according to the method. For example, the expression level of the protein of the present invention in the case of (vi) above is about 20% or more compared to the case of (v) above,
A test compound that promotes preferably 30% or more, more preferably about 50% or more can be selected as a compound or its salt that promotes the expression of the protein of the present invention. For example, the expression level of the protein of the present invention in the case of (vi) above is about 20% or more compared to the case of (v) above,
A test compound that inhibits preferably 30% or more, more preferably about 50% or more can be selected as a compound that inhibits the expression of the protein of the present invention or a salt thereof.

The screening kit of the present invention contains the protein or partial peptide used in the present invention or a salt thereof, or a cell capable of producing the protein or partial peptide used in the present invention. 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, peptide, protein, non-peptide compound, synthetic compound, fermentation product, cell extract, plant extract. , A compound or a salt thereof selected from animal tissue extract, plasma, etc., which promotes or inhibits the activity of the protein of the present invention (eg, K ⁺ ion-Na ⁺ ion exchange transport activity) or a salt thereof. Is. As the salt of the compound, those similar to the salts of the protein of the present invention described above are used. The compound or a salt thereof that promotes the activity of the protein of the present invention is, for example, heart disease (eg, heart failure, cardiac hypertrophy, etc.), kidney disease (eg, renal failure, interventricular kidney disease, etc.), central nervous disease (eg, , Alzheimer's disease, Parkinson's syndrome, schizophrenia, etc., inflammatory diseases (eg, chronic obstructive pulmonary disease, etc.), autoimmune diseases (eg, myasthenia gravis, glomerulonephritis, multiple sclerosis, Sjogren's syndrome, Insulin-resistant diabetes, rheumatoid arthritis, systemic lupus erythematosus, etc., allergic disease (eg, bronchial asthma, hay fever, allergic rhinitis, anaphylactic shock, atopic dermatitis, etc.), thymic disease, spleen disease (eg, splenic function) Hypersensitivity), immunodeficiency (eg, white blood cell abnormalities, immunodeficiency associated with splenic or thymic abnormalities), genital disease (eg, testicular hypersensitivity, ovarian insufficiency) Etc.) and cancer (e.g., pancreatic cancer, lung cancer, kidney cancer,
Prophylactic / therapeutic agent for liver cancer, non-small cell lung cancer, ovarian cancer, prostate cancer, gastric cancer, bladder cancer, breast cancer, cervical cancer, colon cancer, rectal cancer, etc., or a rejection inhibitor after organ transplantation, etc. Is useful as a medicine. In addition, compounds or salts thereof that inhibit the activity of the protein of the present invention include, for example, heart disease (eg, heart failure, cardiac hypertrophy, etc.), kidney disease (eg, renal failure, interventricular kidney disease, etc.), central nervous disease (Eg, Alzheimer's disease, Parkinson's syndrome, schizophrenia, etc.), inflammatory disease (eg, chronic obstructive pulmonary disease, etc.), autoimmune disease (eg, myasthenia gravis, glomerulonephritis, multiple sclerosis, Sjogren's disease) Syndrome, insulin resistant diabetes, rheumatoid arthritis, systemic lupus erythematosus, etc., allergic disease (eg, bronchial asthma, hay fever, allergic rhinitis, anaphylactic shock, atopic dermatitis, etc.), thymic disease, spleen disease (eg, Hypersplenism, etc.), immunodeficiency (eg, white blood cell abnormalities, immunodeficiency associated with splenic or thymic abnormalities), genital disease (eg, testicular hypersensitivity, ovary) Ability insufficiency, etc.) and cancer (e.g., pancreatic cancer, lung cancer, kidney cancer,
Prophylactic / therapeutic agent for liver cancer, non-small cell lung cancer, ovarian cancer, prostate cancer, gastric cancer, bladder cancer, breast cancer, cervical cancer, colon cancer, rectal cancer, etc., or a rejection inhibitor after organ transplantation, etc. Is useful as a medicine. The compound or a salt thereof that promotes the expression of the protein gene of the present invention is, for example, a heart disease (eg, heart failure, cardiac hypertrophy, etc.), a kidney disease (eg, renal failure, an interventricular kidney disease, etc.), a central nervous disease ( Eg, Alzheimer's disease, Parkinson's syndrome, schizophrenia, inflammatory diseases (eg, chronic obstructive pulmonary disease, etc.), autoimmune diseases (eg, myasthenia gravis, glomerulonephritis, multiple sclerosis, Sjogren's syndrome) , Insulin resistant diabetes, rheumatoid arthritis, systemic lupus erythematosus, etc., allergic diseases (eg, bronchial asthma, hay fever, allergic rhinitis, anaphylactic shock, atopic dermatitis, etc.), thymic diseases, spleen diseases (eg, splenic) Hyperfunction, etc., immunodeficiency (eg, white blood cell abnormalities, immunodeficiency associated with splenic or thymic abnormalities), genital disease (eg, testicular hypersensitivity, ovary) Ability insufficiency, etc.) and cancer (e.g., pancreatic cancer, lung cancer, kidney cancer,
Prophylactic / therapeutic agent for liver cancer, non-small cell lung cancer, ovarian cancer, prostate cancer, gastric cancer, bladder cancer, breast cancer, cervical cancer, colon cancer, rectal cancer, etc., or a rejection inhibitor after organ transplantation, etc. Is useful as a medicine. Further, the compound or its salt that inhibits the expression of the protein gene of the present invention can be used, for example, in heart disease (eg, heart failure, cardiac hypertrophy, etc.), kidney disease (eg, renal failure, interventricular kidney disease, etc.), central nervous system. Diseases (eg, Alzheimer's disease, Parkinson's syndrome, schizophrenia, etc.), inflammatory diseases (eg, chronic obstructive pulmonary disease, etc.), autoimmune diseases (eg, myasthenia gravis, glomerulonephritis,
Multiple sclerosis, Sjogren's syndrome, insulin-resistant diabetes, rheumatoid arthritis, systemic lupus erythematosus, etc.), allergic diseases (eg, bronchial asthma, hay fever, allergic rhinitis, anaphylactic shock, atopic dermatitis, etc.), thymic disease , Spleen disease (eg, hypersplenism, etc.), immunodeficiency (eg, white blood cell abnormalities, immunodeficiency associated with splenic or thymic abnormalities), genital disease (eg, testicular hypersensitivity, ovarian dysfunction, etc.) and cancer ( For example, a prophylactic / therapeutic agent for pancreatic cancer, lung cancer, kidney cancer, liver cancer, non-small cell lung cancer, ovarian cancer, prostate cancer, gastric cancer, bladder cancer, breast cancer, cervical cancer, colon cancer, rectal cancer, etc., It is also useful as a drug such as a rejection inhibitor after organ transplantation. The compound or a salt thereof that promotes the expression of the protein of the present invention is, for example, a heart disease (eg, heart failure, cardiac hypertrophy, etc.), kidney disease (eg, renal failure, interventricular kidney disease, etc.), central nervous disease (eg, , Alzheimer's disease, Parkinson's syndrome, schizophrenia, etc., inflammatory diseases (eg, chronic obstructive pulmonary disease, etc.), autoimmune diseases (eg, myasthenia gravis, glomerulonephritis,
Multiple sclerosis, Sjogren's syndrome, insulin-resistant diabetes, rheumatoid arthritis, systemic lupus erythematosus, etc.), allergic diseases (eg, bronchial asthma, hay fever, allergic rhinitis, anaphylactic shock, atopic dermatitis, etc.), thymic disease , Spleen disease (eg, hypersplenism, etc.), immunodeficiency (eg, white blood cell abnormalities, immunodeficiency associated with splenic or thymic abnormalities), genital disease (eg, testicular hypersensitivity, ovarian dysfunction, etc.) and cancer ( For example, a prophylactic / therapeutic agent for pancreatic cancer, lung cancer, kidney cancer, liver cancer, non-small cell lung cancer, ovarian cancer, prostate cancer, gastric cancer, bladder cancer, breast cancer, cervical cancer, colon cancer, rectal cancer, etc., It is also useful as a drug such as a rejection inhibitor after organ transplantation. In addition, the compound or its salt that inhibits the expression of the protein of the present invention is, for example, a heart disease (eg, heart failure, cardiac hypertrophy, etc.), kidney disease (eg, renal failure, interventricular kidney disease, etc.), central nervous disease (Eg, Alzheimer's disease, Parkinson's syndrome, schizophrenia, etc.), inflammatory disease (eg, chronic obstructive pulmonary disease, etc.), autoimmune disease (eg, myasthenia gravis, glomerulonephritis, multiple sclerosis, Sjogren's disease) Syndrome, insulin resistant diabetes, rheumatoid arthritis, systemic lupus erythematosus, etc., allergic disease (eg, bronchial asthma, hay fever, allergic rhinitis, anaphylactic shock, atopic dermatitis, etc.), thymic disease, spleen disease (eg, Hypersplenism, etc.), immunodeficiency (eg, white blood cell abnormalities, immunodeficiency associated with splenic or thymic abnormalities), genital disease (eg, testicular hypersensitivity, ovary) Dysfunction) and cancer (eg, pancreatic cancer, lung cancer, kidney cancer, liver cancer, non-small cell lung cancer, ovarian cancer, prostate cancer, gastric cancer, bladder cancer, breast cancer, cervical cancer, colon cancer, rectal cancer, etc.) It is useful as a preventive / therapeutic agent for the above, or a drug such as a rejection inhibitor after organ transplantation.

When the compound or its salt obtained by using the screening method or the screening kit of the present invention is used as the above-mentioned prophylactic / therapeutic agent, it can be formulated according to a conventional method. For example, tablets, capsules, elixirs, microcapsules, sterile solutions, suspensions and the like can be used. Since the preparation thus obtained is safe and has low toxicity, for example, humans or other warm-blooded animals (for example, mouse, rat, rabbit, sheep, pig, cow, horse, bird, cat, dog,
It can be administered orally or parenterally to monkeys, chimpanzees, etc.). The dose of the compound or its salt varies depending on its action, target disease, administration subject, administration route, etc. When administered, generally in adults (as a body weight of 60 kg), the compound or its salt is about 0.1 to 100 mg, preferably about 1.0 to 50 m per day.
g, more preferably about 1.0-20 mg. When administered parenterally, the single dose of the compound or its salt varies depending on the administration subject, target disease, etc., but for example, a compound that promotes the activity of the protein of the present invention for the purpose of treating heart failure disease or When the salt is usually administered to an adult (body weight 60 kg) in the form of an injection, the compound or salt thereof is about 0.01 to 30 mg, preferably about 0.1 to 20 mg, and more preferably one day. It is convenient to administer about 0.1 to 10 mg by intravenous injection. Also in the case of other animals, the dose converted per 60 kg of body weight can be administered.

[3] Quantification of protein of the present invention, partial peptide or salt thereof Since the antibody of the present invention can specifically recognize the protein of the present invention, It can be used for quantification, especially by sandwich immunoassay. That is, the present invention provides (i) a ratio of the labeled protein of the present invention bound to the antibody by competitively reacting the antibody of the present invention with a test solution and the labeled protein of the present invention. A method for quantifying the protein of the present invention in a test liquid, which comprises measuring (ii) the test liquid and the antibody of the present invention insolubilized on a carrier and another antibody of the present invention labeled The present invention provides a method for quantifying the protein of the present invention in a test solution, which comprises reacting at the same time or successively and measuring the activity of the labeling agent on the insolubilized carrier. In the quantification method of (ii) above, 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.

Further, the protein of the present invention can be quantified by using a monoclonal antibody against the protein of the present invention (hereinafter sometimes referred to as the monoclonal antibody of the present invention), and the protein of the present invention can be detected by tissue staining or the like. You can also do For these purposes, the antibody molecule itself may be used, or the F (a
b ′) ₂ , Fab ′, or Fab fraction may be used.
The method for quantifying the protein of the present invention using the antibody of the present invention is
It is not particularly limited, and the amount of the antibody, the antigen or the antibody-antigen complex corresponding to the amount of the antigen (for example, the amount of protein) in the liquid to be measured is detected by chemical or physical means, and this is known. Any measuring method may be used as long as it is a measuring method calculated from a standard curve prepared using a standard solution containing an amount of antigen. For example, nephrometry, competitive method, immunometric method and sandwich method are preferably used, but from the viewpoint of sensitivity and specificity, the sandwich method described later is particularly preferably used. 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] and the like are used. The enzyme is preferably stable and has a large specific activity,
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. As the luminescent substance, for example,
Luminol, luminol derivative, luciferin, lucigenin and the like are used. Furthermore, the biotin-avidin system can be used for binding the antibody or the antigen to the labeling agent.

For the insolubilization of the antigen or antibody, physical adsorption may be used, or a method using a chemical bond 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 antibodies of the present invention used in the primary reaction and the secondary reaction are preferably antibodies having different binding sites for the protein of the present invention. That is, 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 and the secondary reaction is preferably an antibody used in the primary reaction. 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 (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.

In 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 protein measurement system of the present invention may be constructed by adding ordinary technical consideration to those skilled in the art to the ordinary 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, Hiro Irie's "Radioimmunoassay" (Kodansha, published in 1974), Hiroshi Irie's "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) Published in 1987), "Methods in ENZYMOLOGY" Vol. 70 (Immunoch
emical Techniques (Part A)), ibid Vol. 73 (Immunoch
emical Techniques (Part B)), ibid Vol. 74 (Immunoch
emical Techniques (Part C)), ibid Vol. 84 (Immunoch
emical Techniques (Part D: Selected Immunoassays)),
Ibid Vol. 92 (Immunochemical Techniques (Part E: Mon
oclonal Antibodiesand 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, the protein of the present invention can be quantified with high sensitivity by using the antibody of the present invention. Furthermore, when a decrease in the concentration of the protein of the present invention is detected by quantifying the concentration of the protein of the present invention using the antibody of the present invention, for example,
Heart disease (eg, heart failure, cardiac hypertrophy, etc.), kidney disease (eg, renal failure, interventricular kidney disease, etc.), central nervous system disease (eg, Alzheimer's disease, Parkinson's syndrome, schizophrenia, etc.),
Inflammatory diseases (eg, chronic obstructive pulmonary disease, etc.), autoimmune diseases (eg, myasthenia gravis, glomerulonephritis, multiple sclerosis, Sjogren's syndrome, insulin resistant diabetes, rheumatoid arthritis, systemic lupus erythematosus, etc. ), Allergic disease (eg, bronchial asthma, hay fever, allergic rhinitis, anaphylactic shock, atopic dermatitis, etc.), thymus disease, spleen disease (eg, hypersplenism, etc.), immunodeficiency (eg, leukocyte abnormality, Spleen insufficiency or immunodeficiency associated with thymic abnormalities), genital diseases (eg, testicular hypersensitivity, ovarian insufficiency, etc.) and cancers (eg, pancreatic cancer, lung cancer, kidney cancer,
Diagnosis of high likelihood of liver cancer, non-small cell lung cancer, ovarian cancer, prostate cancer, gastric cancer, bladder cancer, breast cancer, cervical cancer, colon cancer, rectal cancer, etc.) or rejection after organ transplantation be able to. When an increase in the concentration of the protein of the present invention is detected, for example, heart disease (eg, heart failure, cardiac hypertrophy, etc.), kidney disease (eg, renal failure, interventricular kidney disease, etc.),
Central nervous system diseases (eg, Alzheimer's disease, Parkinson's syndrome, schizophrenia, etc.), inflammatory diseases (eg, chronic obstructive pulmonary disease, etc.), autoimmune diseases (eg, myasthenia gravis, glomerulonephritis, multiple sclerosis) Disease, Sjogren's syndrome, insulin resistant diabetes, rheumatoid arthritis, systemic lupus erythematosus, etc., allergic disease (eg, bronchial asthma, hay fever, allergic rhinitis, anaphylactic shock, atopic dermatitis, etc.), thymic disease, spleen disease (Eg, hypersplenism, etc.), immunodeficiency (eg, white blood cell abnormalities, immunodeficiency associated with splenic or thymic abnormalities), genital disease (eg, testicular hypersensitivity, ovarian dysfunction, etc.) and cancer (eg pancreas) Cancer, lung cancer, kidney cancer, liver cancer, non-small cell lung cancer, ovarian cancer, prostate cancer, gastric cancer, bladder cancer, breast cancer, cervical cancer, colon cancer, rectal cancer, etc.) Or it may be diagnosed as likely, such as rejection after organ transplantation. 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, for preparation of an antibody column used for purifying the protein of the present invention, detection of the protein of the present invention in each fraction during purification, analysis of the behavior of the protein of the present invention in a test cell, etc. Can be used.

[4] Gene Diagnostic Agent The DNA of the present invention can be used as a probe, for example, to prepare humans or other warm-blooded animals (eg, rat, mouse, guinea pig, rabbit, bird, sheep, pig, cow, It is possible to detect an abnormality (gene abnormality) of DNA or mRNA encoding the protein of the present invention or a partial peptide thereof in a horse, cat, dog, monkey, chimpanzee, etc.). It is useful as a gene diagnostic agent for mutation or decreased expression, increased DNA or mRNA, or excessive expression. 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, Vol. 5, pp. 874-879 (1989), Procedures of the National Academy of Sciences of USA (Proceedings
of the National Academy of Sciences of the United S
tates of America, Vol. 86, pp. 2766-2770 (1989)
Year)) etc. For example, when increased expression is detected by Northern hybridization, for example, heart disease (eg, heart failure, cardiac hypertrophy, etc.),
Kidney disease (eg, renal failure, interventricular kidney disease, etc.), central nervous system disease (eg, Alzheimer's disease, Parkinson's syndrome, schizophrenia, etc.), inflammatory disease (eg, chronic obstructive pulmonary disease, etc.), autoimmunity Disease (eg, myasthenia gravis, glomerulonephritis,
Multiple sclerosis, Sjogren's syndrome, insulin resistant diabetes, rheumatoid arthritis, systemic lupus erythematosus, etc.), allergic diseases (eg, bronchial asthma, hay fever, allergic rhinitis, anaphylactic shock, atopic dermatitis, etc.), thymic disease , Spleen disease (eg, hypersplenism, etc.), immunodeficiency (eg, white blood cell abnormalities, immunodeficiency associated with splenic or thymic abnormalities), genital disease (eg, testicular hypersensitivity, ovarian dysfunction) and cancer (eg For example, pancreatic cancer, lung cancer, kidney cancer, liver cancer, non-small cell lung cancer, ovarian cancer, prostate cancer, gastric cancer, bladder cancer, breast cancer, cervical cancer, colon cancer, rectal cancer, etc.), or rejection after organ transplantation It can be diagnosed that there is a high possibility of reaction. In addition, when decreased expression is detected or when a DNA mutation is detected by the PCR-SSCP method, for example, heart disease (eg, heart failure,
Cardiac hypertrophy, kidney disease (eg, renal failure, interventricular kidney disease), central nervous system disease (eg, Alzheimer's disease, Parkinson's syndrome, schizophrenia), inflammatory disease (eg, chronic obstructive pulmonary disease) Etc.), autoimmune diseases (eg, myasthenia gravis, glomerulonephritis, multiple sclerosis, Sjogren's syndrome,
Insulin-resistant diabetes, rheumatoid arthritis, systemic lupus erythematosus, etc., allergic disease (eg, bronchial asthma, hay fever, allergic rhinitis, anaphylactic shock, atopic dermatitis, etc.), thymic disease, spleen disease (eg, splenic function) Hypersensitivity), immunodeficiency (eg, white blood cell abnormalities, immunodeficiency associated with splenic or thymic abnormalities),
Reproductive organ diseases (eg, testicular hypersensitivity, ovarian insufficiency, etc.) and cancers (eg, pancreatic cancer, lung cancer, kidney cancer, liver cancer, non-small cell lung cancer, ovarian cancer, prostate cancer, gastric cancer, bladder cancer, breast cancer, cervix) Cancer, colon cancer, rectal cancer, etc.), or rejection after organ transplantation.

[5] Medicament Containing Antisense Nucleotide The antisense nucleotide of the present invention capable of binding to the DNA of the present invention in a complementary manner and suppressing the expression of the DNA has low toxicity, and can be used in vivo. function of the DNA of the protein or the present invention of the invention it is possible to suppress (including examples ,, K ⁺ ions and Na ⁺ exchange transport activity of ions), for example, heart disease (eg, heart failure, cardiac hypertrophy, etc.), kidney Diseases (eg, renal failure, interventricular kidney disease, etc.), central nervous system diseases (eg, Alzheimer's disease, Parkinson's syndrome, schizophrenia, etc.), inflammatory diseases (eg, chronic obstructive pulmonary disease, etc.), autoimmune diseases (Eg myasthenia gravis, glomerulonephritis,
Multiple sclerosis, Sjogren's syndrome, insulin resistant diabetes, rheumatoid arthritis, systemic lupus erythematosus, etc.), allergic diseases (eg, bronchial asthma, hay fever, allergic rhinitis, anaphylactic shock, atopic dermatitis, etc.), thymic disease , Spleen disease (eg, hypersplenism, etc.), immunodeficiency (eg, white blood cell abnormalities, immunodeficiency associated with splenic or thymic abnormalities), genital disease (eg, testicular hypersensitivity, ovarian dysfunction) and cancer (eg For example, a prophylactic / therapeutic agent for pancreatic cancer, lung cancer, kidney cancer, liver cancer, non-small cell lung cancer, ovarian cancer, prostate cancer, gastric cancer, bladder cancer, breast cancer, cervical cancer, colon cancer, rectal cancer, etc., Alternatively, it can be used as a rejection inhibitor after organ transplantation. When the above antisense nucleotide is used as the above-mentioned prophylactic / therapeutic agent, it can be formulated and administered according to a known method. For example, when the antisense nucleotide is used, the antisense nucleotide alone or after being inserted into an appropriate vector such as a retrovirus vector, an adenovirus vector, an adenovirus associated virus vector, etc., is subjected to human or other mammals according to a conventional method. It can be administered orally or parenterally to animals (eg, rat, rabbit, sheep, pig, cow, cat, dog, monkey, etc.). The antisense nucleotide can be administered as it is or in the form of a formulation with a physiologically acceptable carrier such as an auxiliary agent for promoting ingestion, and can be administered by a gene gun or a catheter such as a hydrogel catheter. Although the dose of the antisense nucleotide varies depending on the target disease, administration subject, administration route, etc., for example, when the antisense nucleotide of the present invention is locally administered to the heart for the purpose of treating heart failure, it is generally adult. (Body weight 60 kg), the antisense nucleotide is added in an amount of about 0.1 to 100 m per day.
g. Further, the antisense nucleotide is
It can also be used as a diagnostic oligonucleotide probe for investigating the presence of the DNA of the present invention or its expression in tissues or cells.

Further, the present invention provides a double-stranded RNA containing a part of RNA encoding the protein of the present invention and RNA complementary thereto, a medicament comprising said double-stranded RNA, protein of the present invention Also provided are a ribozyme containing a part of RNA encoding the above, a drug containing the above ribozyme, an expression vector containing a gene (DNA) encoding the above ribozyme, and the like. Like the above antisense polynucleotide, the double-stranded R
NA, ribozyme and the like can also destroy RNA transcribed from the DNA of the present invention or suppress its function,
Since the function of the protein used in the present invention or the DNA used in the present invention can be suppressed in vivo, for example, heart disease (eg, heart failure, cardiac hypertrophy, etc.), kidney disease (eg, renal failure, interventricular chamber) Renal disease, etc., central nervous system disease (eg, Alzheimer's disease, Parkinson's syndrome, schizophrenia, etc.), inflammatory disease (eg, chronic obstructive pulmonary disease, etc.), autoimmune disease (eg, myasthenia gravis, thread) Glomerulonephritis,
Multiple sclerosis, Sjogren's syndrome, insulin resistant diabetes, rheumatoid arthritis, systemic lupus erythematosus, etc.), allergic diseases (eg, bronchial asthma, hay fever, allergic rhinitis, anaphylactic shock, atopic dermatitis, etc.), thymic disease , Spleen disease (eg, hypersplenism, etc.), immunodeficiency (eg, white blood cell abnormalities, immunodeficiency associated with splenic or thymic abnormalities), genital disease (eg, testicular hypersensitivity, ovarian dysfunction) and cancer (eg For example, a prophylactic / therapeutic agent for pancreatic cancer, lung cancer, kidney cancer, liver cancer, non-small cell lung cancer, ovarian cancer, prostate cancer, gastric cancer, bladder cancer, breast cancer, cervical cancer, colon cancer, rectal cancer, etc., Alternatively, it can be used as a rejection inhibitor after organ transplantation. Double-stranded RNA can be prepared by a known method (eg, Nature, 411, 494, 20).
2001), and can be designed and manufactured based on the sequence of the polynucleotide of the present invention. Ribozymes can be prepared by known methods (eg, TRENDS in Molecular Medicine, Volume 7, 221
Page, 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 substituting a part of the known ribozyme sequence with a part of RNA encoding the protein of the present invention. As a part of RNA encoding the protein of the present invention, a consensus sequence NUX that can be cleaved by a known ribozyme (wherein N is all bases,
X represents a base other than G) and the like. The above-mentioned prevention of double-stranded RNA or ribozyme
When used as a therapeutic agent, it can be formulated and administered in the same manner as the antisense polynucleotide. The expression vector is used in the same manner as a known gene therapy method and the like, and used as the preventive / therapeutic agent.

[6] Production of Animal Having DNA of the Present Invention The present invention is D which encodes the protein of the present invention which is exogenous.
Provided is a non-human mammal having NA (hereinafter abbreviated as exogenous DNA of the present invention) or a mutant DNA thereof (may be abbreviated as exogenous mutant DNA of the present invention). 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) the animal according to (1) above, wherein the non-human mammal is a rodent, (3) A recombinant vector and the like containing the animal according to the above (2), wherein the rodent is a mouse or rat, and (4) containing the exogenous DNA of the present invention or a mutant DNA thereof and capable of being expressed in a mammal. A non-human mammal having the exogenous DNA of the present invention or a mutant DNA thereof (hereinafter, abbreviated as the DNA-introduced animal of the present invention) is used for embryo cells such as unfertilized eggs, fertilized eggs, sperms and their progenitor cells. Preferably, at the stage of embryogenesis in development of non-human mammals (more preferably at the stage of single cells or fertilized egg cells and generally before the 8-cell stage), the calcium phosphate method, electric pulse method, lipofection method, agglutination method, It can be produced by introducing the target DNA by the microinjection method, particle gun method, DEAE-dextran method, or the like. In addition, somatic cells,
It is also possible to introduce the foreign DNA of the present invention into an organ, tissue cell or the like of a living body, and use it for cell culture, tissue culture, etc., and further, by using these cells with the above-mentioned embryo cells by a known cell fusion method. By fusing the DN of the present invention
It is also possible to create an A-introduced animal. Examples of non-human mammals include cow, pig, sheep, goat, rabbit,
Dogs, cats, guinea pigs, hamsters, mice, rats, etc. are used. Among them, rodents that have relatively short ontogenesis and biological cycle from the viewpoint of creating a pathological animal model system and are easy to reproduce, especially mice (for example, C57BL / 6 strain and DBA2 strain as pure strains). B6C3F ₁ system, BDF ₁ system, B
6D2F ₁ system, BALB / c system, ICR system, etc.)
Alternatively, a rat (eg, Wistar, SD, etc.) and the like are preferable. Examples of the “mammal” in the recombinant vector that can be expressed in mammals include humans in addition to the above-mentioned non-human mammals.

The exogenous DNA of the present invention refers to the DNA of the present invention once isolated and extracted from mammals, not the DNA of the present invention originally possessed by non-human mammals.
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, DNA in which a base is added, deleted, or replaced with another base is used, and abnormal DNA is also included. The abnormal DNA means a DNA that expresses the abnormal protein of the present invention, and for example, a DNA that expresses a protein that suppresses the function of the normal protein of the present invention is used. The exogenous DNA of the present invention is
It may be derived from mammals of the same or different species from the target animal. When the DNA of the present invention is introduced into a target animal, it is generally advantageous to use the DNA as a DNA construct in which the DNA is linked downstream of a promoter that can be expressed in animal cells. For example, when the human DNA of the present invention is introduced, various mammals having the DNA of the present invention having high homology therewith (for example, 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-introduced mammal that highly expresses 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 K
14, collagen type I and II, cyclic AM
P-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.
It is preferable that the vector has a sequence (generally called a terminator) that terminates transcription of a target mRNA in a DNA-introduced mammal. For example, sequences of DNAs derived from viruses and various mammals are used. And preferably S of simian virus
V40 terminator or the like is used.

In addition, for the purpose of further highly expressing the desired foreign DNA, the splicing signal of each DNA, the enhancer region, a part of the intron of eukaryotic DNA and the like are provided 5'upstream of the promoter region, between the promoter region and the translation region. Alternatively, it may be linked to the 3'downstream of the translation region depending on the purpose. The normal translation region of the protein of the present invention includes liver-derived, kidney-derived, thyroid-derived, fibroblast-derived DNA derived from humans or various mammals (eg, rabbit, dog, cat, guinea pig, hamster, rat, mouse, etc.) and commercially available Can be obtained as a whole or a part of the genomic DNA from various genomic DNA libraries or a complementary DNA prepared from a liver, kidney, thyroid cell, or fibroblast-derived RNA by a known method as a raw material. In addition, an exogenous abnormal DNA can be produced by mutating a translation region of a normal polypeptide obtained from the above cells or tissues by a point mutagenesis method. The translation region can be prepared as a DNA construct that can be expressed in an introduced animal by a conventional DNA engineering method in which it is linked to the downstream of the above promoter and, if desired, the upstream of the transcription termination site. The introduction 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 germinal cells of the produced animal after the introduction of DNA means that all the progeny of the produced animal retain the exogenous DNA of the present invention in all of its germinal 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. Exogenous normal DNA of the present invention
The non-human mammal introduced with the
After confirming that the DNA is stably maintained, the DNA-carrying animal can be subcultured in a normal breeding environment. The introduction of the exogenous DNA of the present invention at the fertilized egg cell stage is ensured so as to be present in excess in all the germinal cells and somatic cells of the target mammal. The excessive presence of the exogenous DNA of the present invention in the germinal cells of the producing animal after the introduction of the DNA 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 have the exogenous DNA of the present invention in excess in all of their 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 offspring can be passaged so that the DNA has an excess.

In the non-human mammal having the normal DNA of the present invention, the normal DNA of the present invention is highly expressed, and the function of the protein of the present invention is finally obtained by promoting the function of the endogenous normal DNA. It may develop hyperactivity and can be used as a model animal for its pathological condition.
For example, using the normal DNA-introduced animal of the present invention, it is possible to elucidate the pathological mechanism of hyperfunction 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 into which the exogenous normal DNA of the present invention has been introduced has an increased symptom of the released protein of the present invention, it can be used for screening tests for therapeutic agents for diseases related to the protein of the present invention. . On the other hand, the non-human mammal having the exogenous abnormal DNA of the present invention can be subcultured as a DNA-carrying animal in a normal breeding environment after confirming that the exogenous DNA is stably retained by mating.
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. The introduction of the abnormal 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. D
The presence of the abnormal DNA of the present invention in the germinal cells of the producing animal after NA 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 offspring can obtain the DNA.
Can be passaged for breeding.

In the non-human mammal having the abnormal DNA of the present invention, the abnormal DNA of the present invention is highly expressed, and the function of the protein of the present invention is finally obtained by inhibiting the function of the endogenous normal DNA. It may become inactive refractory and can be used as a model animal for its pathological condition. For example, using the abnormal DNA-introduced 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 treatment method for this disease. In addition, as a specific applicability, the animal with high expression of the abnormal DNA of the present invention shows the functional inhibition (dominant negative action) of the normal protein by the abnormal protein of the present invention in the functionally inactive refractory disease of the protein of the present invention. It becomes a model to elucidate. In addition, the foreign abnormal DNA of the present invention
Since the mammals into which the protein of the present invention has an increased symptom of the released protein of the present invention, it can be used in a therapeutic drug screening test for the protein of the present invention or its functionally inactive refractory disease. Further, other applications of the above-mentioned two kinds of DNA-introduced animals of the present invention include, for example, use as a cell source for tissue culture, DNA or R in tissues of the DNA-introduced animals of the present invention.
Analysis of the association with a protein that is specifically expressed or activated by the protein of the present invention by directly analyzing NA or analyzing a polypeptide tissue expressed by DNA; Culturing by standard tissue culture techniques and using them to study the function of cells from tissues that are generally difficult to culture, screening for agents that enhance cell function by using the cells described above, and It is conceivable to isolate and purify the mutant protein and produce its antibody. Furthermore, by using the DNA-introduced animal of the present invention, it is possible to investigate clinical symptoms of diseases related to the protein of the present invention, including functionally inactive refractory diseases of the protein of the present invention, and 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. In addition, each organ was taken out from the DNA-introduced animal of the present invention, and after being minced, D released by a protease such as trypsin was released.
It is possible to obtain NA-introduced cells, culture the cells, 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 apoptosis, differentiation or proliferation, or the signal transduction mechanism in them, and to investigate their abnormalities. It becomes an effective research material for Furthermore, using the DNA-introduced animal of the present invention, a functionally inactive refractory disease of the protein of the present invention is included.
In order to develop a therapeutic agent for a disease associated with the protein of the present invention, using the above-mentioned test method and quantification method,
It is possible to provide an effective and rapid screening method for the therapeutic drug for the disease. Further, using the DNA-introduced animal of the present invention or the foreign DNA expression vector of the present invention, a method for treating DNA for diseases associated with the protein of the present invention is examined,
It is possible to develop.

[7] Knockout Animal The present invention provides a non-human mammal embryonic stem cell in which the DNA of the present invention is inactivated and a 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 (1) above, which is inactivated by introducing a reporter gene (eg, β-galactosidase gene derived from Escherichia coli), and (3) above, wherein the DNA is neomycin resistant. Embryo stem cells of (4), the non-human mammal is a rodent, the embryo stem cell of (1) above, (5) the rodent is a mouse of (4) above, (6) the present invention Non-human mammal deficient in expression of DNA, wherein (7) the DNA is inactivated by introducing a reporter gene (eg, β-galactosidase gene derived from Escherichia coli), and the reporter gene is The non-human mammal according to (6) above, which can be expressed under the control of a promoter for the DNA of the present invention,
(8) The non-human mammal according to (6) above, wherein the non-human mammal is a rodent, (9) the non-human mammal according to (8) above, wherein the rodent is a mouse, and (10) above. The DN of the present invention, which comprises administering a test compound to the animal described in (7) and detecting the expression of a reporter gene.
Provided is a method for screening a compound or its salt that promotes or inhibits the promoter activity for A. The non-human mammal embryonic stem cell in which the DNA of the present invention is inactivated means that the expression ability of the DNA is suppressed by artificially mutating the DNA of the present invention which the non-human mammal has. Due to the substantial loss of the activity of the protein of the present invention encoded by the DNA, the DNA has substantially no ability to express the protein of the present invention (hereinafter, this may be referred to as the knockout DNA of the present invention. )
Non-human mammalian embryonic stem cells (hereinafter abbreviated as ES cells). As the non-human mammal, the same ones as described above are used.

As a method for artificially mutating the DNA of the present invention, for example, the DNA can be prepared by a genetic engineering method.
It can be carried out by deleting a part or all of the sequence or inserting or substituting another DNA. 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). Transferase gene) or a reporter gene typified by destroying the function of the exon, or insert a DNA sequence that terminates the transcription of the gene in the intron portion between the exons (for example, polyA addition signal), By making it impossible to synthesize complete mRNA, A DNA chain having a DNA sequence constructed so as to disrupt the gene (hereinafter, abbreviated as a targeting vector) is introduced into the chromosome of the animal by, for example, homologous recombination method, and the obtained ES cell is used in the present invention. Hybridization analysis using a DNA sequence on or near the target DNA as a probe, or by a PCR method using a DNA sequence on the targeting vector and a DNA sequence in the near region other than the DNA of the present invention used for preparing the targeting vector as primers. Then, it can be obtained by selecting the knockout ES cells of the present invention.

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, those already established as described above may be used, or those newly established according to the known method of Evans and Kaufman. 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
/ 6 egg collection number of lack of usable like even better DBA / 2 and BDF ₁ mice improved by crossing (C57BL / 6 and F ₁ and DBA / 2) which were established by using a. 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 performed, 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% oxygen, 5% It is cultured by a method such as culturing at about 37 ° C. in carbon dioxide gas, 90% air), and at the time of passage, for example, trypsin / EDTA solution (usually 0.001-0.5% trypsin / 0.1-5 mM EDTA, Preferably about 0.1
% Trypsin / 1 mM EDTA) treatment to obtain single cells and seeding on newly prepared feeder cells. 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 make (M.
J. Evans and MH Kaufman, Nature No.
Volume 292, 154, 1981; GR Martin Proceedings of National Academy of Sciences USA (Proc. Natl. Acad. Sci. USA)
78, 7634, 1981; TC Doetschman et al., Journal of Embiology and Experimental Morphology, 87, 27, 1985], obtained by differentiating ES cells of the present invention. The DNA expression-deficient cells of the present invention thus obtained are useful for in vitro cell biological studies of the protein of the present invention. DN of the present invention
A non-human mammal deficient in A expression 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.

In the non-human mammal deficient in DNA expression of the present invention, for example, the targeting vector prepared as described above is introduced into mouse embryonic stem cells or mouse egg cells, and the targeting vector D of the present invention is introduced.
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 bred to homozygous expression of the protein of the present invention from their offspring. Defective individuals can be obtained. When using an egg cell, 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. In this way, an individual whose DNA of the present invention has been knocked out can be passaged in a normal breeding environment after confirming that the DNA of the animal individual obtained by mating is also knocked out. Furthermore, the acquisition and maintenance of the germ line may be carried out according to the conventional method. That is, by crossing male and female animals carrying the inactivated DNA, the inactivated D
A homozygous animal having NA on both homologous chromosomes can be obtained. The obtained homozygous animal can be efficiently obtained by raising the mother animal in such a state that there are 1 normal individual and 1 homozygous animal.
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 non-human mammal deficient in DNA expression of the present invention,
Since it lacks various biological activities that can be induced by the protein of the present invention, it can serve as a model for diseases caused by inactivation of the biological activity of the protein of the present invention. It is useful for examination.

[7a] Method for screening compound having therapeutic / prophylactic effect against diseases caused by DNA deficiency or damage of the present invention The DNA expression-deficient non-human mammal 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. 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 a 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 or a salt thereof having a therapeutic / prophylactic effect against diseases. 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, etc., and these compounds are novel compounds. Or a known compound may be used. Specifically, the non-human mammal deficient in DNA expression of the present invention is
The therapeutic / prophylactic effect of the test compound can be tested by using the change in each organ, tissue, disease symptom, etc. of the animal treated with the test compound as compared with a control animal which is not treated.
As a method of treating a test animal with a test compound, for example, oral administration, intravenous injection, etc. are used, and it can be appropriately selected depending on the symptoms of the test animal, the properties of the test compound, and the like. 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 preventive / therapeutic effect on heart failure, the DN of the present invention is used.
A test compound is administered to a non-human mammal deficient in A expression, and the cardiac function, electrocardiogram, heart weight, etc. of the animal are measured over time.
The compound obtained by using the screening method is a compound selected from the above-mentioned test compounds and has a prophylactic / therapeutic effect on a disease caused by a deficiency or damage of the protein of the present invention. It can be used as a medicine such as a safe and low toxic preventive / therapeutic agent. 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. As the salt of the compound, a physiologically acceptable acid (eg, inorganic acid, organic acid, etc.) or base (eg, salt) is used. A salt with an alkali metal or the like) is used, and a physiologically acceptable acid addition salt is particularly preferable. Examples of such salts include salts with inorganic acids (for example, hydrochloric acid, phosphoric acid, hydrobromic acid, sulfuric acid, etc.), or organic acids (for example, acetic acid, formic acid, propionic acid, fumaric acid, maleic acid, Succinic acid, tartaric acid, citric acid, malic acid, oxalic acid, benzoic acid, methanesulfonic acid, benzenesulfonic acid, etc.) and the like salts 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. The formulation thus obtained is
Since it is safe and has low toxicity, it can be administered to, for example, humans or other mammals (eg, rat, mouse, guinea pig, rabbit, sheep, pig, cow, horse, cat, dog, monkey, etc.) . The dose of the compound or its salt varies depending on the target disease, administration subject, administration route and the like. , About 0.1 to 10 times a day of the compound
0 mg, preferably about 1.0-50 mg, more preferably about 1.0-20 mg. When administered parenterally, the single dose of the compound varies depending on the administration subject, target disease and the like, but for example, the compound is usually administered in the form of an injection to an adult (as 60 kg) heart failure patient. In that case, it is convenient to administer about 0.01 to 30 mg, preferably about 0.1 to 20 mg, more preferably about 0.1 to 10 mg of the compound by intravenous injection per day. In the case of other animals, the dose converted per 60 kg can be administered.

[7b] Method of Screening for Compound that Promotes or Inhibits Promoter Activity 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.

In the non-human mammal deficient in expression of the DNA of the present invention in which the DNA of the present invention is substituted with the reporter gene, the reporter gene exists under the control of the promoter for the DNA of the present invention. The activity of the promoter can be detected by tracing the expression. For example, a part of the DNA region encoding the protein of the present invention is a β derived from Escherichia coli.
-When it is replaced with the galactosidase gene (lacZ), β-galactosidase is originally expressed in the tissue in which the protein of the present invention is expressed instead of the protein of the present invention. Thus, for example, 5-bromo-4-chloro-3
-Indolyl-β-galactopyranoside (X-gal)
The expression state of the protein of the present invention in an animal body can be easily observed by staining with a reagent that serves as a substrate for β-galactosidase. Specifically, the protein-deficient mouse of the present invention or a tissue section thereof is fixed with glutaraldehyde or the like, washed with phosphate buffered saline (PBS), and then stained with X-gal at room temperature or around 37 ° C. After reacting for about 30 minutes to 1 hour, the tissue sample may be washed with a 1 mM EDTA / PBS solution to stop the β-galactosidase reaction and observe the coloration. In addition, mRNA encoding lacZ may be detected according to a conventional method.
The compound or its salt obtained by using the above screening method is a compound selected from the above-mentioned test compounds, and is a compound that promotes or inhibits the promoter activity for the DNA of the present invention.

The compound obtained by the screening method may form a salt. As the salt of the compound, a physiologically acceptable acid (eg, inorganic acid) or base (eg, organic acid) is used. ) And the like are used, and physiologically acceptable acid addition salts are particularly preferable. For such salt,
For example, inorganic acids (for example, hydrochloric acid, phosphoric acid, hydrobromic acid,
Sulfates) or organic acids (eg acetic acid, formic acid, propionic acid, fumaric acid, maleic acid, succinic acid,
Tartaric acid, citric acid, malic acid, oxalic acid, benzoic acid, methanesulfonic acid, benzenesulfonic acid, etc.) and the like are used. The compound or a salt thereof 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, heart disease (eg, heart failure, cardiac hypertrophy, etc.). ), Renal disease (eg, renal failure, interventricular renal disease, etc.), central nervous system disease (eg, Alzheimer's disease, Parkinson's syndrome, schizophrenia, etc.), inflammatory disease (eg, chronic obstructive pulmonary disease, etc.), Autoimmune diseases (eg, myasthenia gravis, glomerulonephritis, multiple sclerosis, Sjogren's syndrome, insulin resistant diabetes, rheumatoid arthritis, systemic lupus erythematosus, etc.), allergic diseases (eg, bronchial asthma, hay fever, allergies) Rhinitis, anaphylactic shock, atopic dermatitis, etc., thymus disease, spleen disease (eg, hypersplenism, etc.), immunodeficiency (eg, white) Ball abnormalities, immunodeficiency associated with splenic or thymic abnormalities, genital diseases (eg, testicular hypersensitivity, ovarian insufficiency) and cancers (eg, pancreatic cancer, lung cancer, kidney cancer, liver cancer, non-small cell lung cancer, It is useful as a preventive / therapeutic agent for ovarian cancer, prostate cancer, gastric cancer, bladder cancer, breast cancer, cervical cancer, colon cancer, rectal cancer, etc., or a drug for suppressing rejection after organ transplantation.

Further, the compound or its salt which 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, and therefore, for example, heart disease (eg heart failure). , Cardiac hypertrophy, etc.), kidney disease (eg, renal failure, interventricular kidney disease, etc.),
Central nervous system diseases (eg, Alzheimer's disease, Parkinson's syndrome, schizophrenia, etc.), inflammatory diseases (eg, chronic obstructive pulmonary disease, etc.), autoimmune diseases (eg, myasthenia gravis, glomerulonephritis, multiple sclerosis) Disease, Sjogren's syndrome, insulin resistant diabetes, rheumatoid arthritis, systemic lupus erythematosus, etc., allergic disease (eg, bronchial asthma, hay fever, allergic rhinitis, anaphylactic shock, atopic dermatitis, etc.), thymic disease, spleen disease (Eg, hypersplenism, etc.), immunodeficiency (eg, white blood cell abnormalities, immunodeficiency associated with splenic or thymic abnormalities), genital disease (eg, testicular hypersensitivity, ovarian dysfunction, etc.) and cancer (eg pancreas) Cancer, lung cancer, kidney cancer, liver cancer, non-small cell lung cancer, ovarian cancer, prostate cancer, gastric cancer, bladder cancer, breast cancer, cervical cancer, colon cancer, rectal cancer, etc.) Which prophylactic or therapeutic agent, or useful as pharmaceuticals such as rejection inhibitors after organ transplantation. Furthermore, compounds derived from the compounds obtained by the above screening can be used as well. 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, for example, humans or other mammals (for example, rat, mouse, guinea pig, rabbit, sheep, pig, cow, horse, cat, dog, monkey, etc.) ). The dose of the compound or a salt thereof may vary depending on the target disease, the administration subject, the administration route, etc.
When the compound which promotes the promoter activity for the DNA of the present invention is orally administered, it is generally adult (body weight 60 kg
In a patient with heart failure (1), the compound is about 0.1 to 100 mg, preferably about 1.0 to 50 mg per day.
g, more preferably about 1.0-20 mg. When administered parenterally, the single dose of the compound varies depending on the administration subject, target disease, etc., but for example, a compound that promotes the promoter activity for the DNA of the present invention in the form of an injection is usually used in adults ( When administered to a patient with heart failure (as 60 kg), about 0.01 to about
It is convenient to administer about 30 mg, preferably about 0.1 to 20 mg, more preferably about 0.1 to 10 mg by intravenous injection. 60k for other animals
An amount converted per g can be administered.

On the other hand, for example, when a compound that inhibits the promoter activity for the DNA of the present invention is orally administered,
Generally, in an adult patient (with a body weight of 60 kg) with heart failure, the compound is administered in an amount of about 0.1 to 100 m per day.
g, preferably about 1.0-50 mg, more preferably about 1.0-20 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 inhibits the promoter activity against the DNA of the present invention is usually administered in the form of an injection ( When administered to patients with heart failure (as 60 kg),
The daily amount of the compound is about 0.01 to 30 mg, preferably about 0.1 to 20 mg, more preferably about 0.1.
It is convenient to administer about 1 to 10 mg by intravenous injection. In the case of other animals, the dose converted per 60 kg can be administered. Thus, the DN of the present invention
The non-human mammal deficient in A expression 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 the cause of various diseases caused by the deficiency of DNA expression of the present invention is investigated. Or it can make a great contribution to the development of preventive and 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 thereof, and this is injected into an egg cell of an animal to obtain a so-called transgenic animal (transgenic animal).
, Then specifically synthesize the polypeptide,
It is also possible to study the action in the living body. Further, by binding an appropriate reporter gene to the above promoter portion and establishing a cell line capable of expressing this, 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.

In the present specification and drawings, when an abbreviation is used for a base, amino acid, etc., IUPAC-IUB Commission
Based on the abbreviations based on on Biochemical Nomenclature or abbreviations commonly used in this field, 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: Cysteine Met: Cysteine Met : Methionine Glu: Glutamic acid Asp: Aspartic acid Lys: Lysine Arg: Arginine His: Histidine Phe: Phenylalanine T r: 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-hydro Shi-5-norbornene-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] Human TCH115 obtained in Example 1
The amino acid sequence of the protein is shown. [SEQ ID NO: 2] D encoding human TCH115 protein containing the amino acid sequence represented by SEQ ID NO: 1
The base sequence of NA is shown. [SEQ ID NO: 3] Primer A5 used in Example 1
The base sequence of is shown. [SEQ ID NO: 4] Primer B3 used in Example 1
The base sequence of is shown. [SEQ ID NO: 5] Primer U1 used in Example 1
9 shows the nucleotide sequence of 9. [SEQ ID NO: 6] Primer T7 used in Example 1
The base sequence of is shown. [SEQ ID NO: 7] Primer A1 used in Example 1
The base sequence of is shown. [SEQ ID NO: 8] Primer A2 used in Example 1
The base sequence of is shown. [SEQ ID NO: 9] Primer B1 used in Example 1
The base sequence of is shown. [SEQ ID NO: 10] Primer B used in Example 1
2 shows the base sequence of 2. [SEQ ID NO: 11] Primer F used in Example 1
1 shows the base sequence of 1. [SEQ ID NO: 12] Primer F used in Example 1
2 shows the base sequence of 2. [SEQ ID NO: 13] Primer F used in Example 1
3 shows the nucleotide sequence of 3. [SEQ ID NO: 14] Primer F used in Example 1
4 shows the base sequence of 4. [SEQ ID NO: 15] Primer F used in Example 1
5 shows the base sequence of 5. [SEQ ID NO: 16] Primer F used in Example 1
6 shows the base sequence of 6. [SEQ ID NO: 17] Primer R used in Example 1
1 shows the base sequence of 1. [SEQ ID NO: 18] Primer R used in Example 1
2 shows the base sequence of 2. [SEQ ID NO: 19] Primer R used in Example 1
4 shows the base sequence of 4. [SEQ ID NO: 20] Primer R used in Example 1
5 shows the base sequence of 5. [SEQ ID NO: 21] Primer R used in Example 1
6 shows the base sequence of 6. [SEQ ID NO: 22] Primer R used in Example 1
7 shows the base sequence of 7. [SEQ ID NO: 23] This shows the base sequence of cDNA containing the TCH115 full-length gene obtained in Example 1. [SEQ ID NO: 24] This shows the amino acid sequence of human TCH115 protein containing 1029 amino acids obtained in Example 1. [SEQ ID NO: 25] D encoding the TCH115 protein containing the amino acid sequence represented by SEQ ID NO: 24
The base sequence of NA is shown. [SEQ ID NO: 26] Primer A used in Example 2
6 shows the base sequence of 6. [SEQ ID NO: 27] Primer B used in Example 2
6 shows the base sequence of 6. [SEQ ID NO: 28] TaqMan used in Example 2
The base sequence of probe T1 is shown. [SEQ ID NO: 29] This shows the base sequence of DNA encoding human TCH115 protein containing the amino acid sequence represented by SEQ ID NO: 1. [SEQ ID NO: 30] This shows the base sequence of DNA encoding human TCH115 protein containing the amino acid sequence represented by SEQ ID NO: 1. [SEQ ID NO: 31] This shows the base sequence of DNA encoding human TCH115 protein containing the amino acid sequence represented by SEQ ID NO: 1. [SEQ ID NO: 32] This shows the base sequence of DNA encoding human TCH115 protein containing the amino acid sequence represented by SEQ ID NO: 33. [SEQ ID NO: 33] Human TCH115 containing the amino acid sequence encoded by the nucleotide sequence represented by SEQ ID NO: 32.
The amino acid sequence of the protein is shown. [SEQ ID NO: 34] Human T containing 1029 amino acids
1 shows the amino acid sequence of CH115 protein. [SEQ ID NO: 35] D encoding the TCH115 protein containing the amino acid sequence represented by SEQ ID NO: 34
The base sequence of NA is shown. [SEQ ID NO: 36] D encoding the TCH115 protein containing the amino acid sequence represented by SEQ ID NO: 24
The base sequence of NA is shown. [SEQ ID NO: 37] D encoding the TCH115 protein containing the amino acid sequence represented by SEQ ID NO: 24
The base sequence of NA is shown.

The transformant Escherichia coli obtained in Example 1 described later.
TOP10 / pT7Blue-TCH115 is 200
From August 13, 1st, 1-1, Higashi 1-chome, Tsukuba City, Ibaraki Prefecture
Chuo No. 6 (Postal code 305-8566), National Institute of Advanced Industrial Science and Technology, Patent Biological Depository Center, deposit number FERM BP-7705, from August 2, 2001, 2nd, 13 Sanchon-cho, Yodogawa-ku, Osaka-shi, Osaka It has been deposited at the Fermentation Research Institute (IFO) of No. 17-85 (zip code 532-8686) under the deposit number IFO 16687.

[0078]

The present invention will be described in more detail with reference to the following examples, which are not intended to limit the scope of the present invention. The gene manipulation method using Escherichia coli followed the method described in Molecular Cloning. Example 1 Acquisition of human TCH115 gene cDNA Two kinds of primer DNA, primer A5 (SEQ ID NO:
3) and primer B3 (SEQ ID NO: 4),
PC for human testis cDNA library (Clontech) by Pyrobest DNA polymerase (Takara Shuzo)
Perform R and amplify the amplified fragment with ExTaq DNA polymerase (Takara Shuzo)
After the addition of dA by the method described above, cloning was performed using pT7Blue T-vector (Novagen). One to three clones were obtained from each of the four PCR reactions, and a total of eight clones (from the first PCR reaction, clones # 3, # 11, and # 24 were cloned into 2 clones).
Clone # 28 from the 3rd PCR reaction
Clones # 63, # 70, and # A7 were obtained from the R reaction, and clone # B8) was obtained from the fourth PCR reaction. This was used as a primer DNA [primer U19 (SEQ ID NO:
5), primer T7 (SEQ ID NO: 6), primer A
1 (SEQ ID NO: 7), primer A2 (SEQ ID NO:
8), primer B1 (SEQ ID NO: 9), primer B
2 (SEQ ID NO: 10), primer F1 (SEQ ID NO: 1)
1), primer F2 (SEQ ID NO: 12), primer F3 (SEQ ID NO: 13), primer F4 (SEQ ID NO :)
14), primer F5 (SEQ ID NO: 15), primer F6 (SEQ ID NO: 16), primer R1 (SEQ ID NO: 17), primer R2 (SEQ ID NO: 18),
Primer R4 (SEQ ID NO: 19), primer R5
(SEQ ID NO: 20), primer R6 (SEQ ID NO: 2)
1), primer R7 (SEQ ID NO: 22)] and BigD
The reaction was performed using the ye Terminator Cycle Sequencing Kit (Applied Biosystems), and the inserted c
The base sequence of the DNA fragment is the DNA sequencer ABIP.
Determined using RISM 3100 DNA Analyzer (Applied Biosystems). As a result, the cd
The NA fragment had a nucleotide sequence of 3257 (SEQ ID NO: 23). A transformant having a plasmid containing the cDNA fragment was prepared as Escherichia coli (Escherichia coli).
hia coli) TOP10 / pT7Blue-TC
It was named H115. The cDNA fragment encodes a 1000 amino acid sequence (SEQ ID NO: 1) (SEQ ID NO: 29), and the protein containing the amino acid sequence was named human TCH115 protein. The cDNA fragment also has a start codon in the same reading frame on the 5 ′ side of the start codon of SEQ ID NO: 29, and the 1029 amino acid sequence (SEQ ID NO: 24) translated from the start codon is also present. It was encoded (SEQ ID NO: 25). Of the 8 clones obtained above, 4 clones (clone #
11, # 24, # 70, # B8) had a nucleotide sequence corresponding to SEQ ID NO: 23, but other clones had 6 positions (95th nucleotide of the nucleotide sequence represented by SEQ ID NO: 23, Two
Base substitutions were observed at positions 74, 1064, 1106, 1143 and 2155). G95A found in 2 or more clones out of 8 clones (substitution of 95th G to A in the nucleotide sequence represented by SEQ ID NO: 23. The same applies hereinafter), C1064T and G11.
The 06A substitution did not change the amino acid. However, A27 found only in clone # 28
4G (Asp → Gly), G1143C (Glu → Gl)
n) and G2155C (Gly → Ala) each had the amino acid substitution shown in parentheses. These base substitutions may be derived from genetic polymorphisms (SNPs), especially G95A, C1064T and G1106.
It is highly possible that the three sites of A are derived from gene polymorphisms (SNPs). OR in the nucleotide sequences of 2 clones (clone # 3, # 63) of the above 8 clones
One clone of F in SEQ ID NO: 30 (clone # A7)
The ORF in the nucleotide sequence of SEQ ID NO: 31 is the ORF in the nucleotide sequence of 1 clone (clone # 28).
Are shown in SEQ ID NO: 32, respectively. The amino acid sequence encoded by the base sequences represented by SEQ ID NO: 30 and SEQ ID NO: 31 is the same as the amino acid sequence represented by SEQ ID NO: 1. The amino acid sequence encoded by the base sequence represented by SEQ ID NO: 32 is shown in SEQ ID NO: 33. The amino acid sequence of 1029 amino acids derived from the same gene as SEQ ID NO: 32 is shown in SEQ ID NO: 34. The nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 34 is shown in SEQ ID NO: 35.
A nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 24, wherein the nucleotide sequence derived from the same gene as SEQ ID NO: 30 is SEQ ID NO: 36, and the nucleotide sequence derived from the same gene as SEQ ID NO: 31 is SEQ ID NO: 37. Shown in. A summary of these sequences is shown in [Table 1].

[0079]

[Table 1]

In the base sequence represented by SEQ ID NO: 30, the 993rd G of the base sequence represented by SEQ ID NO: 29 is A.
Has been replaced by. In the base sequence represented by SEQ ID NO: 31, the 951st C is replaced by T and the 993th G is replaced by A in the base sequence represented by SEQ ID NO: 29. The base sequence represented by SEQ ID NO: 32 is SEQ ID NO:
The 161st A of the nucleotide sequence represented by 29 is G and 95
The first C is replaced with T, the 993th G is replaced with A, the 1030th G is replaced with C, and the 2042nd G is replaced with C. The nucleotide sequence represented by SEQ ID NO: 29 in which the 951th C is replaced with T is SEQ ID NO: 2
Shown in. In the base sequence represented by SEQ ID NO: 36, the 69th G in the base sequence represented by SEQ ID NO: 25 is A and 1
The 080th G is replaced with A, respectively. In the nucleotide sequence represented by SEQ ID NO: 37, the 69th G in the nucleotide sequence represented by SEQ ID NO: 25 is A and the 1038th C is C.
Is replaced with T, and the 1080th G is replaced with A. The base sequence represented by SEQ ID NO: 32 is SEQ ID NO:
The 161st A of the nucleotide sequence represented by 29 is G and 95
The first C is replaced with T, the 993rd G is replaced with A, the 1030th G is replaced with C, and the 2042nd G is replaced with C. The nucleotide sequence represented by SEQ ID NO: 35 has the 69th G in the nucleotide sequence represented by SEQ ID NO: 25 as A,
248th A to G, 1038th C to T, 10
80th G is A, 1117th G is C, 212
The 9th G is replaced with a C, respectively. Sequence number:
In the amino acid sequence represented by 33, the 54th Asp of SEQ ID NO: 1 is Gly, and the 344th Glu is Gln.
In addition, the 681st Gly is replaced with Ala. In the amino acid sequence represented by SEQ ID NO: 34, Asp at position 83 in SEQ ID NO: 24 is replaced with Gly, Glu at 373 position is replaced with Gln, and Gly at 710 position is replaced with Ala. Blast [Nucleic Acids Res.] Vol. 25, 3389
Page, 1997], a homology search was performed on a known database, and it was found that the cDNA having the nucleotide sequence represented by SEQ ID NO: 23 is a novel gene belonging to Na, K-ATPase. (FIGS. 1 and 2). In the figure, the transmembrane regions are indicated by TM1 to 10. Na, K which is Na, K-ATPase reported in humans
-ATPase α2 subunit [J. Biol. Chem.
, 264, 17532, 1989] at the base level.
%, 79% homology at the amino acid level, human TC
The H115 protein was speculated to have a 10-transmembrane structure.

Example 2 Analysis of Tissue Distribution of Human TCH115 Gene Product Two kinds of primer DNA, primer A6 (SEQ ID NO:
26) and primer B6 (SEQ ID NO: 27), and T
Using the aqMan probe T1 (SEQ ID NO: 28),
CDNA (Human MTC p) of each human tissue (heart, brain, placenta, lung, liver, skeletal muscle, kidney, pancreas, spleen, thymus, prostate, testis, ovary, small intestine, large intestine, peripheral blood leukocyte)
annel I and Human MTC panel I
(I: Clontech), the expression level of human TCH115 was measured by TaqMan PCR. The reaction is TaqMan
Using the Gold RT-PCR kit (Applied Biosystems), the ABI PRISM 7900 sequence detection system (Applied Biosystems) was first placed at 50 ° C. for 2 minutes, then at 95 ° C. for 10 minutes, and then at 95 ° C. for 15 seconds, 60 seconds.
40 cycles were repeated with 1 minute at 1 ° C. as one reaction cycle, and detection was performed simultaneously. The results are shown in Fig. 3. Human TC
The H115 gene product (mRNA) was slightly expressed in brain, liver, pancreas, spleen, thymus, ovary, small intestine, large intestine, and also in prostate and peripheral blood leukocytes, and showed the strongest expression in testis.

[0082]

INDUSTRIAL APPLICABILITY The protein, polynucleotide, antibody and the like of the present invention are, for example, heart disease (eg, heart failure, cardiac hypertrophy, etc.), kidney disease (eg, renal failure, interventricular kidney disease, etc.), central nervous system disease. (Eg, Alzheimer's disease, Parkinson's syndrome, schizophrenia, etc.), inflammatory disease (eg, chronic obstructive pulmonary disease, etc.), autoimmune disease (eg, myasthenia gravis, glomerulonephritis, multiple sclerosis, Sjogren's disease) syndrome,
Insulin-resistant diabetes, rheumatoid arthritis, systemic lupus erythematosus, etc., allergic disease (eg, bronchial asthma, hay fever, allergic rhinitis, anaphylactic shock, atopic dermatitis, etc.), thymic disease, spleen disease (eg, splenic function) Hypersensitivity), immunodeficiency (eg, white blood cell abnormalities, immunodeficiency associated with splenic or thymic abnormalities),
Reproductive organ diseases (eg, testicular hypersensitivity, ovarian insufficiency, etc.) and cancers (eg, pancreatic cancer, lung cancer, kidney cancer, liver cancer, non-small cell lung cancer, ovarian cancer, prostate cancer, gastric cancer, bladder cancer, breast cancer, cervix) Cancer, colon cancer, rectal cancer, etc.) or a diagnostic marker such as rejection after organ transplantation. Compounds that promote or inhibit the activity of the protein obtained by screening using the protein polynucleotide or antibody, compounds that promote or inhibit the expression of the protein gene, compounds that promote or inhibit the expression of the protein, and the like, for example, Heart disease (eg, heart failure, cardiac hypertrophy, etc.), kidney disease (eg, renal failure, interventricular kidney disease, etc.), central nervous system disease (eg, Alzheimer's disease, Parkinson's syndrome, schizophrenia, etc.), inflammatory disease (eg Eg, chronic obstructive pulmonary disease), autoimmune disease (eg, myasthenia gravis, glomerulonephritis, multiple sclerosis, Sjogren's syndrome,
Insulin-resistant diabetes, rheumatoid arthritis, systemic lupus erythematosus, etc., allergic disease (eg, bronchial asthma, hay fever, allergic rhinitis, anaphylactic shock, atopic dermatitis, etc.), thymic disease, spleen disease (eg, splenic function) Hypersensitivity), immunodeficiency (eg, white blood cell abnormalities, immunodeficiency associated with splenic or thymic abnormalities),
Reproductive organ diseases (eg, testicular hypersensitivity, ovarian insufficiency, etc.) and cancers (eg, pancreatic cancer, lung cancer, kidney cancer, liver cancer, non-small cell lung cancer, ovarian cancer, prostate cancer, gastric cancer, bladder cancer, breast cancer, cervix) It can be used as a prophylactic / therapeutic agent for cancer, colon cancer, rectal cancer, etc., or a rejection inhibitor after organ transplantation.

[0083]

[Sequence list]                                SEQUENCE LISTING <110> Takeda Chemical Industries, Ltd. <120> Novel Protein and its DNA <130> P01-0293 <150> JP2001-022512 <151> 2001-01-30 <150> JP2001-358228 <151> 2001-11-22 <160> 37 <210> 1 <211> 1000 <212> PRT <213> Human <400> 1 Met Val Lys Arg Glu Lys Gln Lys Arg Asn Met Glu Glu Leu Lys Lys   1 5 10 15 Glu Val Val Met Asp Asp His Lys Leu Thr Leu Glu Glu Leu Ser Thr              20 25 30 Lys Tyr Ser Val Asp Leu Thr Lys Gly His Ser His Gln Arg Ala Lys          35 40 45 Glu Ile Leu Thr Arg Asp Gly Pro Asn Thr Val Thr Pro Pro Pro Thr      50 55 60 Thr Pro Glu Trp Val Lys Phe Cys Lys Gln Leu Phe Gly Gly Phe Ser  65 70 75 80 Leu Leu Leu Trp Thr Gly Ala Ile Leu Cys Phe Val Ala Tyr Ser Ile                  85 90 95 Gln Ile Tyr Phe Asn Glu Glu Pro Thr Lys Asp Asn Leu Tyr Leu Ser             100 105 110 Ile Val Leu Ser Val Val Val Ile Val Thr Gly Cys Phe Ser Tyr Tyr         115 120 125 Gln Glu Ala Lys Ser Ser Lys Ile Met Glu Ser Phe Lys Asn Met Val     130 135 140 Pro Gln Gln Ala Leu Val Ile Arg Gly Gly Glu Lys Met Gln Ile Asn 145 150 155 160 Val Gln Glu Val Val Leu Gly Asp Leu Val Glu Ile Lys Gly Gly Asp                 165 170 175 Arg Val Pro Ala Asp Leu Arg Leu Ile Ser Ala Gln Gly Cys Lys Val             180 185 190 Asp Asn Ser Ser Leu Thr Gly Glu Ser Glu Pro Gln Ser Arg Ser Pro         195 200 205 Asp Phe Thr His Glu Asn Pro Leu Glu Thr Arg Asn Ile Cys Phe Phe     210 215 220 Ser Thr Asn Cys Val Glu Gly Thr Ala Arg Gly Ile Val Ile Ala Thr 225 230 235 240 Gly Asp Ser Thr Val Met Gly Arg Ile Ala Ser Leu Thr Ser Gly Leu                 245 250 255 Ala Val Gly Gln Thr Pro Ile Ala Ala Glu Ile Glu His Phe Ile His             260 265 270 Leu Ile Thr Val Val Ala Val Phe Leu Gly Val Thr Phe Phe Ala Leu         275 280 285 Ser Leu Leu Leu Gly Tyr Gly Trp Leu Glu Ala Ile Ile Phe Leu Ile     290 295 300 Gly Ile Ile Val Ala Asn Val Pro Glu Gly Leu Leu Ala Thr Val Thr 305 310 315 320 Val Cys Leu Thr Leu Thr Ala Lys Arg Met Ala Arg Lys Asn Cys Leu                 325 330 335 Val Lys Asn Leu Glu Ala Val Glu Thr Leu Gly Ser Thr Ser Thr Ile             340 345 350 Cys Ser Asp Lys Thr Gly Thr Leu Thr Gln Asn Arg Met Thr Val Ala         355 360 365 His Met Trp Phe Asp Met Thr Val Tyr Glu Ala Asp Thr Thr Glu Glu     370 375 380 Gln Thr Gly Lys Thr Phe Thr Lys Ser Ser Asp Thr Trp Phe Met Leu 385 390 395 400 Ala Arg Ile Ala Gly Leu Cys Asn Arg Ala Asp Phe Lys Ala Asn Gln                 405 410 415 Glu Ile Leu Pro Ile Ala Lys Arg Ala Thr Thr Gly Asp Ala Ser Glu             420 425 430 Ser Ala Leu Leu Lys Phe Ile Glu Gln Ser Tyr Ser Ser Val Ala Glu         435 440 445 Met Arg Glu Lys Asn Pro Lys Val Ala Glu Ile Pro Phe Asn Ser Thr     450 455 460 Asn Lys Tyr Gln Met Ser Ile His Leu Arg Glu Asp Ser Ser Gln Thr 465 470 475 480 His Val Leu Met Met Lys Gly Ala Pro Glu Arg Ile Leu Glu Phe Cys                 485 490 495 Ser Thr Phe Leu Leu Asn Gly Gln Glu Tyr Ser Met Asn Asp Glu Met             500 505 510 Lys Glu Ala Phe Gln Asn Ala Tyr Leu Glu Leu Gly Gly Leu Gly Glu         515 520 525 Arg Val Leu Gly Phe Cys Phe Leu Asn Leu Pro Ser Ser Phe Ser Lys     530 535 540 Gly Phe Pro Phe Asn Thr Asp Glu Ile Asn Phe Pro Met Asp Asn Leu 545 550 555 560 Cys Phe Val Gly Leu Ile Ser Met Ile Asp Pro Pro Arg Ala Ala Val                 565 570 575 Pro Asp Ala Val Ser Lys Cys Arg Ser Ala Gly Ile Lys Val Ile Met             580 585 590 Val Thr Gly Asp His Pro Ile Thr Ala Lys Ala Ile Ala Lys Gly Val         595 600 605 Gly Ile Ile Ser Glu Gly Thr Glu Thr Ala Glu Glu Val Ala Ala Arg     610 615 620 Leu Lys Ile Pro Ile Ser Lys Val Asp Ala Ser Ala Ala Lys Ala Ile 625 630 635 640 Val Val His Gly Ala Glu Leu Lys Asp Ile Gln Ser Lys Gln Leu Asp                 645 650 655 Gln Ile Leu Gln Asn His Pro Glu Ile Val Phe Ala Arg Thr Ser Pro             660 665 670 Gln Gln Lys Leu Ile Ile Val Glu Gly Cys Gln Arg Leu Gly Ala Val         675 680 685 Val Ala Val Thr Gly Asp Gly Val Asn Asp Ser Pro Ala Leu Lys Lys     690 695 700 Ala Asp Ile Gly Ile Ala Met Gly Ile Ser Gly Ser Asp Val Ser Lys 705 710 715 720 Gln Ala Ala Asp Met Ile Leu Leu Asp Asp Asn Phe Ala Ser Ile Val                 725 730 735 Thr Gly Val Glu Glu Gly Arg Leu Ile Phe Asp Asn Leu Lys Lys Ser             740 745 750 Ile Met Tyr Thr Leu Thr Ser Asn Ile Pro Glu Ile Thr Pro Phe Leu         755 760 765 Met Phe Ile Ile Leu Gly Ile Pro Leu Pro Leu Gly Thr Ile Thr Ile     770 775 780 Leu Cys Ile Asp Leu Gly Thr Asp Met Val Pro Ala Ile Ser Leu Ala 785 790 795 800 Tyr Glu Ser Ala Glu Ser Asp Ile Met Lys Arg Leu Pro Arg Asn Pro                 805 810 815 Lys Thr Asp Asn Leu Val Asn His Arg Leu Ile Gly Met Ala Tyr Gly             820 825 830 Gln Ile Gly Met Ile Gln Ala Leu Ala Gly Phe Phe Thr Tyr Phe Val         835 840 845 Ile Leu Ala Glu Asn Gly Phe Arg Pro Val Asp Leu Leu Gly Ile Arg     850 855 860 Leu His Trp Glu Asp Lys Tyr Leu Asn Asp Leu Glu Asp Ser Tyr Gly 865 870 875 880 Gln Gln Trp Thr Tyr Glu Gln Arg Lys Val Val Glu Phe Thr Cys Gln                 885 890 895 Thr Ala Phe Phe Val Thr Ile Val Val Val Gln Trp Ala Asp Leu Ile             900 905 910 Ile Ser Lys Thr Arg Arg Asn Ser Leu Phe Gln Gln Gly Met Arg Asn         915 920 925 Lys Val Leu Ile Phe Gly Ile Leu Glu Glu Thr Leu Leu Ala Ala Phe     930 935 940 Leu Ser Tyr Thr Pro Gly Met Asp Val Ala Leu Arg Met Tyr Pro Leu 945 950 955 960 Lys Ile Thr Trp Trp Leu Cys Ala Ile Pro Tyr Ser Ile Leu Ile Phe                 965 970 975 Val Tyr Asp Glu Ile Arg Lys Leu Leu Ile Arg Gln His Pro Asp Gly             980 985 990 Trp Val Glu Arg Glu Thr Tyr Tyr                            1000 <210> 2 <211> 3000 <212> DNA <213> Human <400> 2 atggtgaaga gggaaaaaca gaagcgcaat atggaggaac tgaagaagga agtggtcatg 60 gatgatcaca aattaacctt ggaagagctg agcaccaagt actccgtgga cctgacaaag 120 ggccatagcc accaaagggc aaaggaaatc ctgactcgag atggacccaa tactgttacc 180 ccacccccca ccactccaga atgggtcaaa ttctgtaagc aactgttcgg aggcttctcc 240 ctcctactat ggactggggc cattctctgc tttgtggcct acagcatcca gatatatttc 300 aatgaggagc ctaccaaaga caacctctac ctgagcatcg tactgtccgt cgtggtcatc 360 gtcactggct gcttctccta ttatcaggag gccaagagct ccaagatcat ggagtctttt 420 aagaacatgg tgcctcagca agctctggta attcgaggag gagagaagat gcaaattaat 480 gtacaagagg tggtgttggg agacctggtg gaaatcaagg gtggagaccg agtccctgct 540 gacctccggc ttatctctgc acaaggatgt aaggtggaca actcatcctt gactggggag 600 tcagaacccc agagccgctc ccctgacttc acccatgaga accctctgga gacccgaaac 660 atctgcttct tttccaccaa ctgtgtggaa ggaaccgccc ggggtattgt gattgctacg 720 ggagactcca cagtgatggg cagaattgcc tccctgacgt caggcctggc ggttggccag 780 acacctatcg ctgctgagat cgaacacttc atccatctga tcactgtggt ggccgtcttc 840 cttggtgtca ctttttttgc gctctcactt ctcttgggct atggttggct ggaggctatc 900 atttttctca ttggcatcat tgtggccaat gtgcctgagg ggctgttggc tacagtcact 960 gtgtgcctga ccctcacagc caagcgcatg gcgcggaaga actgcctggt gaagaacctg 1020 gaggcggtgg agacgctggg ctccacgtcc accatctgct cagacaagac gggcaccctc 1080 acccagaacc gcatgaccgt cgcccacatg tggtttgata tgaccgtgta tgaggccgac 1140 accactgaag aacagactgg aaaaacattt accaagagct ctgatacctg gtttatgctg 1200 gcccgaatcg ctggcctctg caaccgggct gactttaagg ctaatcagga gatcctgccc 1260 attgctaaga gggccacaac aggtgatgct tccgagtcag ccctcctcaa gttcatcgag 1320 cagtcttaca gctctgtggc ggagatgaga gagaaaaacc ccaaggtggc agagattccc 1380 tttaattcta ccaacaagta ccagatgtcc atccaccttc gggaggacag ctcccagacc 1440 cacgtactga tgatgaaggg tgctccggag aggatcttgg agttttgttc tacctttctt 1500 ctgaatgggc aggagtactc aatgaacgat gaaatgaagg aagccttcca aaatgcctat 1560 ttagaactgg gaggtctggg ggaacgtgtg ctaggcttct gcttcttgaa tctgcctagc 1620 agcttctcca agggattccc atttaataca gatgaaataa atttccccat ggacaacctt 1680 tgttttgtgg gcctcatatc catgattgac cctccccgag ctgcagtgcc tgatgctgtg 1740 agcaagtgtc gcagtgcagg aattaaggtg atcatggtaa caggagatca tcccattaca 1800 gctaaggcca ttgccaaggg tgtgggcatc atctcagaag gcactgagac ggcagaggaa 1860 gtcgctgccc ggcttaagat ccctatcagc aaggtcgatg ccagtgctgc caaagccatt 1920 gtggtgcatg gtgcagaact gaaggacata cagtccaagc agcttgatca gatcctccag 1980 aaccaccctg agatcgtgtt tgctcggacc tcccctcagc agaagctcat cattgtcgag 2040 ggatgtcaga ggctgggagc cgttgtggcc gtgacaggtg acggggtgaa cgactcccct 2100 gcgctgaaga aggctgacat tggcattgcc atgggcatct ctggctctga cgtctctaag 2160 caggcagccg acatgatcct gctggatgac aactttgcct ccatcgtcac gggggtggag 2220 gagggccgcc tgatctttga caacctgaag aaatccatca tgtacaccct gaccagcaac 2280 atccccgaga tcacgccctt cctgatgttc atcatcctcg gtatacccct gcctctggga 2340 accataacca tcctctgcat tgatctcggc actgacatgg tccctgccat ctccttggct 2400 tatgagtcag ctgaaagcga catcatgaag aggcttccaa ggaacccaaa gacggataat 2460 ctggtgaacc accgtctcat tggcatggcc tatggacaga ttgggatgat ccaggctctg 2520 gctggattct ttacctactt tgtaatcctg gctgagaatg gttttaggcc tgttgatctg 2580 ctgggcatcc gcctccactg ggaagataaa tacttgaatg acctggagga cagctacgga 2640 cagcagtgga cctatgagca acgaaaagtt gtggagttca catgccaaac ggcctttttt 2700 gtcaccatcg tggttgtgca gtgggcggat ctcatcatct ccaagactcg ccgcaactca 2760 cttttccagc agggcatgag aaacaaagtc ttaatatttg ggatcctgga ggagacactc 2820 ttggctgcat ttctgtccta cactccaggc atggacgtgg ccctgcgaat gtacccactc 2880 aagataacct ggtggctctg tgccattccc tacagtattc tcatcttcgt ctatgatgaa 2940 atcagaaaac tcctcatccg tcagcacccg gatggctggg tggaaaggga gacgtactac 3000 <210> 3 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 3 tcgggcagct ctttctgggg atagc 25 <210> 4 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 4 aagccagccc aggattgccc atctt 25 <210> 5 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 5 gttttcccag tcacgacgt 19 <210> 6 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 6 aatacgactc actataggg 19 <210> 7 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 7 gggcaggagt actcaatgaa cgat 24 <210> 8 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 8 gctaatcagg agatcctgcc 20 <210> 9 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 9 agcactggca tcgaccttgc tgat 24 <210> 10 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 10 tgggaatccc ttggagaagc 20 <210> 11 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 11 agcctaccaa agacaacctc 20 <210> 12 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 12 ctgcttcttt tccaccaact g 21 <210> 13 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 13 tccacgtcca ccatctgctc a 21 <210> 14 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 14 gcagaagctc atcattgtcg 20 <210> 15 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 15 aaggaaccca aagacggata a 21 <210> 16 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 16 cctgcgaatg tacccactca a 21 <210> 17 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 17 cgacggacag tacgatgctc a 21 <210> 18 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 18 tctcagcagc gataggtgtc t 21 <210> 19 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 19 ggagtcgttc accccgtcac 20 <210> 20 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 20 gattatccgt ctttgggttc c 21 <210> 21 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 21 gagccaccag gttatcttga 20 <210> 22 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 22 ctcggaagca tcacctgttg t 21 <210> 23 <211> 3257 <212> DNA <213> Human <400> 23 tcgggcagct ctttctgggg atagctatgg ggctttgggg gaagaaaggg acagtggctc 60 cccatgacca gagtccaaga cgaagaccta aaaaggggct tatcaagaaa aaaatggtga 120 agagggaaaa acagaagcgc aatatggagg aactgaagaa ggaagtggtc atggatgatc 180 acaaattaac cttggaagag ctgagcacca agtactccgt ggacctgaca aagggccata 240 gccaccaaag ggcaaaggaa atcctgactc gagatggacc caatactgtt accccacccc 300 ccaccactcc agaatgggtc aaattctgta agcaactgtt cggaggcttc tccctcctac 360 tatggactgg ggccattctc tgctttgtgg cctacagcat ccagatatat ttcaatgagg 420 agcctaccaa agacaacctc tacctgagca tcgtactgtc cgtcgtggtc atcgtcactg 480 gctgcttctc ctattatcag gaggccaaga gctccaagat catggagtct tttaagaaca 540 tggtgcctca gcaagctctg gtaattcgag gaggagagaa gatgcaaatt aatgtacaag 600 aggtggtgtt gggagacctg gtggaaatca agggtggaga ccgagtccct gctgacctcc 660 ggcttatctc tgcacaagga tgtaaggtgg acaactcatc cttgactggg gagtcagaac 720 cccagagccg ctcccctgac ttcacccatg agaaccctct ggagacccga aacatctgct 780 tcttttccac caactgtgtg gaaggaaccg cccggggtat tgtgattgct acgggagact 840 ccacagtgat gggcagaatt gcctccctga cgtcaggcct ggcggttggc cagacaccta 900 tcgctgctga gatcgaacac ttcatccatc tgatcactgt ggtggccgtc ttccttggtg 960 tcactttttt tgcgctctca cttctcttgg gctatggttg gctggaggct atcatttttc 1020 tcattggcat cattgtggcc aatgtgcctg aggggctgtt ggccacagtc actgtgtgcc 1080 tgaccctcac agccaagcgc atggcgcgga agaactgcct ggtgaagaac ctggaggcgg 1140 tggagacgct gggctccacg tccaccatct gctcagacaa gacgggcacc ctcacccaga 1200 accgcatgac cgtcgcccac atgtggtttg atatgaccgt gtatgaggcc gacaccactg 1260 aagaacagac tggaaaaaca tttaccaaga gctctgatac ctggtttatg ctggcccgaa 1320 tcgctggcct ctgcaaccgg gctgacttta aggctaatca ggagatcctg cccattgcta 1380 agagggccac aacaggtgat gcttccgagt cagccctcct caagttcatc gagcagtctt 1440 acagctctgt ggcggagatg agagagaaaa accccaaggt ggcagagatt ccctttaatt 1500 ctaccaacaa gtaccagatg tccatccacc ttcgggagga cagctcccag acccacgtac 1560 tgatgatgaa gggtgctccg gagaggatct tggagttttg ttctaccttt cttctgaatg 1620 ggcaggagta ctcaatgaac gatgaaatga aggaagcctt ccaaaatgcc tatttagaac 1680 tgggaggtct gggggaacgt gtgctaggct tctgcttctt gaatctgcct agcagcttct 1740 ccaagggatt cccatttaat acagatgaaa taaatttccc catggacaac ctttgttttg 1800 tgggcctcat atccatgatt gaccctcccc gagctgcagt gcctgatgct gtgagcaagt 1860 gtcgcagtgc aggaattaag gtgatcatgg taacaggaga tcatcccatt acagctaagg 1920 ccattgccaa gggtgtgggc atcatctcag aaggcactga gacggcagag gaagtcgctg 1980 cccggcttaa gatccctatc agcaaggtcg atgccagtgc tgccaaagcc attgtggtgc 2040 atggtgcaga actgaaggac atacagtcca agcagcttga tcagatcctc cagaaccacc 2100 ctgagatcgt gtttgctcgg acctcccctc agcagaagct catcattgtc gagggatgtc 2160 agaggctggg agccgttgtg gccgtgacag gtgacggggt gaacgactcc cctgcgctga 2220 agaaggctga cattggcatt gccatgggca tctctggctc tgacgtctct aagcaggcag 2280 ccgacatgat cctgctggat gacaactttg cctccatcgt cacgggggtg gaggagggcc 2340 gcctgatctt tgacaacctg aagaaatcca tcatgtacac cctgaccagc aacatccccg 2400 agatcacgcc cttcctgatg ttcatcatcc tcggtatacc cctgcctctg ggaaccataa 2460 ccatcctctg cattgatctc ggcactgaca tggtccctgc catctccttg gcttatgagt 2520 cagctgaaag cgacatcatg aagaggcttc caaggaaccc aaagacggat aatctggtga 2580 accaccgtct cattggcatg gcctatggac agattgggat gatccaggct ctggctggat 2640 tctttaccta ctttgtaatc ctggctgaga atggttttag gcctgttgat ctgctgggca 2700 tccgcctcca ctgggaagat aaatacttga atgacctgga ggacagctac ggacagcagt 2760 ggacctatga gcaacgaaaa gttgtggagt tcacatgcca aacggccttt tttgtcacca 2820 tcgtggttgt gcagtgggcg gatctcatca tctccaagac tcgccgcaac tcacttttcc 2880 agcagggcat gagaaacaaa gtcttaatat ttgggatcct ggaggagaca ctcttggctg 2940 catttctgtc ctacactcca ggcatggacg tggccctgcg aatgtaccca ctcaagataa 3000 cctggtggct ctgtgccatt ccctacagta ttctcatctt cgtctatgat gaaatcagaa 3060 aactcctcat ccgtcagcac ccggatggct gggtggaaag ggagacgtac tactaaactc 3120 agcagatgaa gagcttcatg tgacacaggg gtgttgtgag agctgggatg gggccagaga 3180 ttataagttt gacacaacat ctgagacact aggatgaatt atcttggatg agaaagatgg 3240 gcaatcctgg gctggct 3257 <210> 24 <211> 1029 <212> PRT <213> Human <400> 24 Met Gly Leu Trp Gly Lys Lys Gly Thr Val Ala Pro His Asp Gln Ser                  5 10 15 Pro Arg Arg Arg Pro Lys Lys Gly Leu Ile Lys Lys Lys Met Val Lys              20 25 30 Arg Glu Lys Gln Lys Arg Asn Met Glu Glu Leu Lys Lys Glu Val Val          35 40 45 Met Asp Asp His Lys Leu Thr Leu Glu Glu Leu Ser Thr Lys Tyr Ser      50 55 60 Val Asp Leu Thr Lys Gly His Ser His Gln Arg Ala Lys Glu Ile Leu  65 70 75 80 Thr Arg Asp Gly Pro Asn Thr Val Thr Pro Pro Pro Thr Thr Pro Glu                  85 90 95 Trp Val Lys Phe Cys Lys Gln Leu Phe Gly Gly Phe Ser Leu Leu Leu             100 105 110 Trp Thr Gly Ala Ile Leu Cys Phe Val Ala Tyr Ser Ile Gln Ile Tyr         115 120 125 Phe Asn Glu Glu Pro Thr Lys Asp Asn Leu Tyr Leu Ser Ile Val Leu     130 135 140 Ser Val Val Val Ile Val Thr Gly Cys Phe Ser Tyr Tyr Gln Glu Ala 145 150 155 160 Lys Ser Ser Lys Ile Met Glu Ser Phe Lys Asn Met Val Pro Gln Gln                 165 170 175 Ala Leu Val Ile Arg Gly Gly Glu Lys Met Gln Ile Asn Val Gln Glu             180 185 190 Val Val Leu Gly Asp Leu Val Glu Ile Lys Gly Gly Asp Arg Val Pro         195 200 205 Ala Asp Leu Arg Leu Ile Ser Ala Gln Gly Cys Lys Val Asp Asn Ser     210 215 220 Ser Leu Thr Gly Glu Ser Glu Pro Gln Ser Arg Ser Pro Asp Phe Thr 225 230 235 240 His Glu Asn Pro Leu Glu Thr Arg Asn Ile Cys Phe Phe Ser Thr Asn                 245 250 255 Cys Val Glu Gly Thr Ala Arg Gly Ile Val Ile Ala Thr Gly Asp Ser             260 265 270 Thr Val Met Gly Arg Ile Ala Ser Leu Thr Ser Gly Leu Ala Val Gly         275 280 285 Gln Thr Pro Ile Ala Ala Glu Ile Glu His Phe Ile His Leu Ile Thr     290 295 300 Val Val Ala Val Phe Leu Gly Val Thr Phe Phe Ala Leu Ser Leu Leu 305 310 315 320 Leu Gly Tyr Gly Trp Leu Glu Ala Ile Ile Phe Leu Ile Gly Ile Ile                 325 330 335 Val Ala Asn Val Pro Glu Gly Leu Leu Ala Thr Val Thr Val Cys Leu             340 345 350 Thr Leu Thr Ala Lys Arg Met Ala Arg Lys Asn Cys Leu Val Lys Asn         355 360 365 Leu Glu Ala Val Glu Thr Leu Gly Ser Thr Ser Thr Ile Cys Ser Asp     370 375 380 Lys Thr Gly Thr Leu Thr Gln Asn Arg Met Thr Val Ala His Met Trp 385 390 395 400 Phe Asp Met Thr Val Tyr Glu Ala Asp Thr Thr Glu Glu Gln Thr Gly                 405 410 415 Lys Thr Phe Thr Lys Ser Ser Asp Thr Trp Phe Met Leu Ala Arg Ile             420 425 430 Ala Gly Leu Cys Asn Arg Ala Asp Phe Lys Ala Asn Gln Glu Ile Leu         435 440 445 Pro Ile Ala Lys Arg Ala Thr Thr Gly Asp Ala Ser Glu Ser Ala Leu     450 455 460 Leu Lys Phe Ile Glu Gln Ser Tyr Ser Ser Val Ala Glu Met Arg Glu 465 470 475 480 Lys Asn Pro Lys Val Ala Glu Ile Pro Phe Asn Ser Thr Asn Lys Tyr                 485 490 495 Gln Met Ser Ile His Leu Arg Glu Asp Ser Ser Gln Thr His Val Leu             500 505 510 Met Met Lys Gly Ala Pro Glu Arg Ile Leu Glu Phe Cys Ser Thr Phe         515 520 525 Leu Leu Asn Gly Gln Glu Tyr Ser Met Asn Asp Glu Met Lys Glu Ala     530 535 540 Phe Gln Asn Ala Tyr Leu Glu Leu Gly Gly Leu Gly Glu Arg Val Leu 545 550 555 560 Gly Phe Cys Phe Leu Asn Leu Pro Ser Ser Phe Ser Lys Gly Phe Pro                 565 570 575 Phe Asn Thr Asp Glu Ile Asn Phe Pro Met Asp Asn Leu Cys Phe Val             580 585 590 Gly Leu Ile Ser Met Ile Asp Pro Pro Arg Ala Ala Val Pro Asp Ala         595 600 605 Val Ser Lys Cys Arg Ser Ala Gly Ile Lys Val Ile Met Val Thr Gly     610 615 620 Asp His Pro Ile Thr Ala Lys Ala Ile Ala Lys Gly Val Gly Ile Ile 625 630 635 640 Ser Glu Gly Thr Glu Thr Ala Glu Glu Val Ala Ala Arg Leu Lys Ile                 645 650 655 Pro Ile Ser Lys Val Asp Ala Ser Ala Ala Lys Ala Ile Val Val His             660 665 670 Gly Ala Glu Leu Lys Asp Ile Gln Ser Lys Gln Leu Asp Gln Ile Leu         675 680 685 Gln Asn His Pro Glu Ile Val Phe Ala Arg Thr Ser Pro Gln Gln Lys     690 695 700 Leu Ile Ile Val Glu Gly Cys Gln Arg Leu Gly Ala Val Val Ala Val 705 710 715 720 Thr Gly Asp Gly Val Asn Asp Ser Pro Ala Leu Lys Lys Ala Asp Ile                 725 730 735 Gly Ile Ala Met Gly Ile Ser Gly Ser Asp Val Ser Lys Gln Ala Ala             740 745 750 Asp Met Ile Leu Leu Asp Asp Asn Phe Ala Ser Ile Val Thr Gly Val         755 760 765 Glu Glu Gly Arg Leu Ile Phe Asp Asn Leu Lys Lys Ser Ile Met Tyr     770 775 780 Thr Leu Thr Ser Asn Ile Pro Glu Ile Thr Pro Phe Leu Met Phe Ile 785 790 795 800 Ile Leu Gly Ile Pro Leu Pro Leu Gly Thr Ile Thr Ile Leu Cys Ile                 805 810 815 Asp Leu Gly Thr Asp Met Val Pro Ala Ile Ser Leu Ala Tyr Glu Ser             820 825 830 Ala Glu Ser Asp Ile Met Lys Arg Leu Pro Arg Asn Pro Lys Thr Asp         835 840 845 Asn Leu Val Asn His Arg Leu Ile Gly Met Ala Tyr Gly Gln Ile Gly     850 855 860 Met Ile Gln Ala Leu Ala Gly Phe Phe Thr Tyr Phe Val Ile Leu Ala 865 870 875 880 Glu Asn Gly Phe Arg Pro Val Asp Leu Leu Gly Ile Arg Leu His Trp                 885 890 895 Glu Asp Lys Tyr Leu Asn Asp Leu Glu Asp Ser Tyr Gly Gln Gln Trp             900 905 910 Thr Tyr Glu Gln Arg Lys Val Val Glu Phe Thr Cys Gln Thr Ala Phe         915 920 925 Phe Val Thr Ile Val Val Val Gln Trp Ala Asp Leu Ile Ile Ser Lys     930 935 940 Thr Arg Arg Asn Ser Leu Phe Gln Gln Gly Met Arg Asn Lys Val Leu 945 950 955 960 Ile Phe Gly Ile Leu Glu Glu Thr Leu Leu Ala Ala Phe Leu Ser Tyr                 965 970 975 Thr Pro Gly Met Asp Val Ala Leu Arg Met Tyr Pro Leu Lys Ile Thr             980 985 990 Trp Trp Leu Cys Ala Ile Pro Tyr Ser Ile Leu Ile Phe Val Tyr Asp         995 1000 1005 Glu Ile Arg Lys Leu Leu Ile Arg Gln His Pro Asp Gly Trp Val Glu    1010 1015 1020 Arg Glu Thr Tyr Tyr 1025 <210> 25 <211> 3087 <212> DNA <213> Human <400> 25 atggggcttt gggggaagaa agggacagtg gctccccatg accagagtcc aagacgaaga 60 cctaaaaagg ggcttatcaa gaaaaaaatg gtgaagaggg aaaaacagaa gcgcaatatg 120 gaggaactga agaaggaagt ggtcatggat gatcacaaat taaccttgga agagctgagc 180 accaagtact ccgtggacct gacaaagggc catagccacc aaagggcaaa ggaaatcctg 240 actcgagatg gacccaatac tgttacccca ccccccacca ctccagaatg ggtcaaattc 300 tgtaagcaac tgttcggagg cttctccctc ctactatgga ctggggccat tctctgcttt 360 gtggcctaca gcatccagat atatttcaat gaggagccta ccaaagacaa cctctacctg 420 agcatcgtac tgtccgtcgt ggtcatcgtc actggctgct tctcctatta tcaggaggcc 480 aagagctcca agatcatgga gtcttttaag aacatggtgc ctcagcaagc tctggtaatt 540 cgaggaggag agaagatgca aattaatgta caagaggtgg tgttgggaga cctggtggaa 600 atcaagggtg gagaccgagt ccctgctgac ctccggctta tctctgcaca aggatgtaag 660 gtggacaact catccttgac tggggagtca gaaccccaga gccgctcccc tgacttcacc 720 catgagaacc ctctggagac ccgaaacatc tgcttctttt ccaccaactg tgtggaagga 780 accgcccggg gtattgtgat tgctacggga gactccacag tgatgggcag aattgcctcc 840 ctgacgtcag gcctggcggt tggccagaca cctatcgctg ctgagatcga acacttcatc 900 catctgatca ctgtggtggc cgtcttcctt ggtgtcactt tttttgcgct ctcacttctc 960 ttgggctatg gttggctgga ggctatcatt tttctcattg gcatcattgt ggccaatgtg 1020 cctgaggggc tgttggccac agtcactgtg tgcctgaccc tcacagccaa gcgcatggcg 1080 cggaagaact gcctggtgaa gaacctggag gcggtggaga cgctgggctc cacgtccacc 1140 atctgctcag acaagacggg caccctcacc cagaaccgca tgaccgtcgc ccacatgtgg 1200 tttgatatga ccgtgtatga ggccgacacc actgaagaac agactggaaa aacatttacc 1260 aagagctctg atacctggtt tatgctggcc cgaatcgctg gcctctgcaa ccgggctgac 1320 tttaaggcta atcaggagat cctgcccatt gctaagaggg ccacaacagg tgatgcttcc 1380 gagtcagccc tcctcaagtt catcgagcag tcttacagct ctgtggcgga gatgagagag 1440 aaaaacccca aggtggcaga gattcccttt aattctacca acaagtacca gatgtccatc 1500 caccttcggg aggacagctc ccagacccac gtactgatga tgaagggtgc tccggagagg 1560 atcttggagt tttgttctac ctttcttctg aatgggcagg agtactcaat gaacgatgaa 1620 atgaaggaag ccttccaaaa tgcctattta gaactgggag gtctggggga acgtgtgcta 1680 ggcttctgct tcttgaatct gcctagcagc ttctccaagg gattcccatt taatacagat 1740 gaaataaatt tccccatgga caacctttgt tttgtgggcc tcatatccat gattgaccct 1800 ccccgagctg cagtgcctga tgctgtgagc aagtgtcgca gtgcaggaat taaggtgatc 1860 atggtaacag gagatcatcc cattacagct aaggccattg ccaagggtgt gggcatcatc 1920 tcagaaggca ctgagacggc agaggaagtc gctgcccggc ttaagatccc tatcagcaag 1980 gtcgatgcca gtgctgccaa agccattgtg gtgcatggtg cagaactgaa ggacatacag 2040 tccaagcagc ttgatcagat cctccagaac caccctgaga tcgtgtttgc tcggacctcc 2100 cctcagcaga agctcatcat tgtcgaggga tgtcagaggc tgggagccgt tgtggccgtg 2160 acaggtgacg gggtgaacga ctcccctgcg ctgaagaagg ctgacattgg cattgccatg 2220 ggcatctctg gctctgacgt ctctaagcag gcagccgaca tgatcctgct ggatgacaac 2280 tttgcctcca tcgtcacggg ggtggaggag ggccgcctga tctttgacaa cctgaagaaa 2340 tccatcatgt acaccctgac cagcaacatc cccgagatca cgcccttcct gatgttcatc 2400 atcctcggta tacccctgcc tctgggaacc ataaccatcc tctgcattga tctcggcact 2460 gacatggtcc ctgccatctc cttggcttat gagtcagctg aaagcgacat catgaagagg 2520 cttccaagga acccaaagac ggataatctg gtgaaccacc gtctcattgg catggcctat 2580 ggacagattg ggatgatcca ggctctggct ggattcttta cctactttgt aatcctggct 2640 gagaatggtt ttaggcctgt tgatctgctg ggcatccgcc tccactggga agataaatac 2700 ttgaatgacc tggaggacag ctacggacag cagtggacct atgagcaacg aaaagttgtg 2760 gagttcacat gccaaacggc cttttttgtc accatcgtgg ttgtgcagtg ggcggatctc 2820 atcatctcca agactcgccg caactcactt ttccagcagg gcatgagaaa caaagtctta 2880 atatttggga tcctggagga gacactcttg gctgcatttc tgtcctacac tccaggcatg 2940 gacgtggccc tgcgaatgta cccactcaag ataacctggt ggctctgtgc cattccctac 3000 agtattctca tcttcgtcta tgatgaaatc agaaaactcc tcatccgtca gcacccggat 3060 ggctgggtgg aaagggagac gtactac 3087 <210> 26 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 26 aaggcactga gacggcaga 19 <210> 27 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 27 tggcatcgac cttgctgat 19 <210> 28 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Prpbe <400> 28 tcgctgcccg gcttaagatc cc 22 <210> 29 <211> 3000 <212> DNA <213> Human <400> 29 atggtgaaga gggaaaaaca gaagcgcaat atggaggaac tgaagaagga agtggtcatg 60 gatgatcaca aattaacctt ggaagagctg agcaccaagt actccgtgga cctgacaaag 120 ggccatagcc accaaagggc aaaggaaatc ctgactcgag atggacccaa tactgttacc 180 ccacccccca ccactccaga atgggtcaaa ttctgtaagc aactgttcgg aggcttctcc 240 ctcctactat ggactggggc cattctctgc tttgtggcct acagcatcca gatatatttc 300 aatgaggagc ctaccaaaga caacctctac ctgagcatcg tactgtccgt cgtggtcatc 360 gtcactggct gcttctccta ttatcaggag gccaagagct ccaagatcat ggagtctttt 420 aagaacatgg tgcctcagca agctctggta attcgaggag gagagaagat gcaaattaat 480 gtacaagagg tggtgttggg agacctggtg gaaatcaagg gtggagaccg agtccctgct 540 gacctccggc ttatctctgc acaaggatgt aaggtggaca actcatcctt gactggggag 600 tcagaacccc agagccgctc ccctgacttc acccatgaga accctctgga gacccgaaac 660 atctgcttct tttccaccaa ctgtgtggaa ggaaccgccc ggggtattgt gattgctacg 720 ggagactcca cagtgatggg cagaattgcc tccctgacgt caggcctggc ggttggccag 780 acacctatcg ctgctgagat cgaacacttc atccatctga tcactgtggt ggccgtcttc 840 cttggtgtca ctttttttgc gctctcactt ctcttgggct atggttggct ggaggctatc 900 atttttctca ttggcatcat tgtggccaat gtgcctgagg ggctgttggc cacagtcact 960 gtgtgcctga ccctcacagc caagcgcatg gcgcggaaga actgcctggt gaagaacctg 1020 gaggcggtgg agacgctggg ctccacgtcc accatctgct cagacaagac gggcaccctc 1080 acccagaacc gcatgaccgt cgcccacatg tggtttgata tgaccgtgta tgaggccgac 1140 accactgaag aacagactgg aaaaacattt accaagagct ctgatacctg gtttatgctg 1200 gcccgaatcg ctggcctctg caaccgggct gactttaagg ctaatcagga gatcctgccc 1260 attgctaaga gggccacaac aggtgatgct tccgagtcag ccctcctcaa gttcatcgag 1320 cagtcttaca gctctgtggc ggagatgaga gagaaaaacc ccaaggtggc agagattccc 1380 tttaattcta ccaacaagta ccagatgtcc atccaccttc gggaggacag ctcccagacc 1440 cacgtactga tgatgaaggg tgctccggag aggatcttgg agttttgttc tacctttctt 1500 ctgaatgggc aggagtactc aatgaacgat gaaatgaagg aagccttcca aaatgcctat 1560 ttagaactgg gaggtctggg ggaacgtgtg ctaggcttct gcttcttgaa tctgcctagc 1620 agcttctcca agggattccc atttaataca gatgaaataa atttccccat ggacaacctt 1680 tgttttgtgg gcctcatatc catgattgac cctccccgag ctgcagtgcc tgatgctgtg 1740 agcaagtgtc gcagtgcagg aattaaggtg atcatggtaa caggagatca tcccattaca 1800 gctaaggcca ttgccaaggg tgtgggcatc atctcagaag gcactgagac ggcagaggaa 1860 gtcgctgccc ggcttaagat ccctatcagc aaggtcgatg ccagtgctgc caaagccatt 1920 gtggtgcatg gtgcagaact gaaggacata cagtccaagc agcttgatca gatcctccag 1980 aaccaccctg agatcgtgtt tgctcggacc tcccctcagc agaagctcat cattgtcgag 2040 ggatgtcaga ggctgggagc cgttgtggcc gtgacaggtg acggggtgaa cgactcccct 2100 gcgctgaaga aggctgacat tggcattgcc atgggcatct ctggctctga cgtctctaag 2160 caggcagccg acatgatcct gctggatgac aactttgcct ccatcgtcac gggggtggag 2220 gagggccgcc tgatctttga caacctgaag aaatccatca tgtacaccct gaccagcaac 2280 atccccgaga tcacgccctt cctgatgttc atcatcctcg gtatacccct gcctctggga 2340 accataacca tcctctgcat tgatctcggc actgacatgg tccctgccat ctccttggct 2400 tatgagtcag ctgaaagcga catcatgaag aggcttccaa ggaacccaaa gacggataat 2460 ctggtgaacc accgtctcat tggcatggcc tatggacaga ttgggatgat ccaggctctg 2520 gctggattct ttacctactt tgtaatcctg gctgagaatg gttttaggcc tgttgatctg 2580 ctgggcatcc gcctccactg ggaagataaa tacttgaatg acctggagga cagctacgga 2640 cagcagtgga cctatgagca acgaaaagtt gtggagttca catgccaaac ggcctttttt 2700 gtcaccatcg tggttgtgca gtgggcggat ctcatcatct ccaagactcg ccgcaactca 2760 cttttccagc agggcatgag aaacaaagtc ttaatatttg ggatcctgga ggagacactc 2820 ttggctgcat ttctgtccta cactccaggc atggacgtgg ccctgcgaat gtacccactc 2880 aagataacct ggtggctctg tgccattccc tacagtattc tcatcttcgt ctatgatgaa 2940 atcagaaaac tcctcatccg tcagcacccg gatggctggg tggaaaggga gacgtactac 3000 <210> 30 <211> 3000 <212> DNA <213> Human <400> 30 atggtgaaga gggaaaaaca gaagcgcaat atggaggaac tgaagaagga agtggtcatg 60 gatgatcaca aattaacctt ggaagagctg agcaccaagt actccgtgga cctgacaaag 120 ggccatagcc accaaagggc aaaggaaatc ctgactcgag atggacccaa tactgttacc 180 ccacccccca ccactccaga atgggtcaaa ttctgtaagc aactgttcgg aggcttctcc 240 ctcctactat ggactggggc cattctctgc tttgtggcct acagcatcca gatatatttc 300 aatgaggagc ctaccaaaga caacctctac ctgagcatcg tactgtccgt cgtggtcatc 360 gtcactggct gcttctccta ttatcaggag gccaagagct ccaagatcat ggagtctttt 420 aagaacatgg tgcctcagca agctctggta attcgaggag gagagaagat gcaaattaat 480 gtacaagagg tggtgttggg agacctggtg gaaatcaagg gtggagaccg agtccctgct 540 gacctccggc ttatctctgc acaaggatgt aaggtggaca actcatcctt gactggggag 600 tcagaacccc agagccgctc ccctgacttc acccatgaga accctctgga gacccgaaac 660 atctgcttct tttccaccaa ctgtgtggaa ggaaccgccc ggggtattgt gattgctacg 720 ggagactcca cagtgatggg cagaattgcc tccctgacgt caggcctggc ggttggccag 780 acacctatcg ctgctgagat cgaacacttc atccatctga tcactgtggt ggccgtcttc 840 cttggtgtca ctttttttgc gctctcactt ctcttgggct atggttggct ggaggctatc 900 atttttctca ttggcatcat tgtggccaat gtgcctgagg ggctgttggc cacagtcact 960 gtgtgcctga ccctcacagc caagcgcatg gcacggaaga actgcctggt gaagaacctg 1020 gaggcggtgg agacgctggg ctccacgtcc accatctgct cagacaagac gggcaccctc 1080 acccagaacc gcatgaccgt cgcccacatg tggtttgata tgaccgtgta tgaggccgac 1140 accactgaag aacagactgg aaaaacattt accaagagct ctgatacctg gtttatgctg 1200 gcccgaatcg ctggcctctg caaccgggct gactttaagg ctaatcagga gatcctgccc 1260 attgctaaga gggccacaac aggtgatgct tccgagtcag ccctcctcaa gttcatcgag 1320 cagtcttaca gctctgtggc ggagatgaga gagaaaaacc ccaaggtggc agagattccc 1380 tttaattcta ccaacaagta ccagatgtcc atccaccttc gggaggacag ctcccagacc 1440 cacgtactga tgatgaaggg tgctccggag aggatcttgg agttttgttc tacctttctt 1500 ctgaatgggc aggagtactc aatgaacgat gaaatgaagg aagccttcca aaatgcctat 1560 ttagaactgg gaggtctggg ggaacgtgtg ctaggcttct gcttcttgaa tctgcctagc 1620 agcttctcca agggattccc atttaataca gatgaaataa atttccccat ggacaacctt 1680 tgttttgtgg gcctcatatc catgattgac cctccccgag ctgcagtgcc tgatgctgtg 1740 agcaagtgtc gcagtgcagg aattaaggtg atcatggtaa caggagatca tcccattaca 1800 gctaaggcca ttgccaaggg tgtgggcatc atctcagaag gcactgagac ggcagaggaa 1860 gtcgctgccc ggcttaagat ccctatcagc aaggtcgatg ccagtgctgc caaagccatt 1920 gtggtgcatg gtgcagaact gaaggacata cagtccaagc agcttgatca gatcctccag 1980 aaccaccctg agatcgtgtt tgctcggacc tcccctcagc agaagctcat cattgtcgag 2040 ggatgtcaga ggctgggagc cgttgtggcc gtgacaggtg acggggtgaa cgactcccct 2100 gcgctgaaga aggctgacat tggcattgcc atgggcatct ctggctctga cgtctctaag 2160 caggcagccg acatgatcct gctggatgac aactttgcct ccatcgtcac gggggtggag 2220 gagggccgcc tgatctttga caacctgaag aaatccatca tgtacaccct gaccagcaac 2280 atccccgaga tcacgccctt cctgatgttc atcatcctcg gtatacccct gcctctggga 2340 accataacca tcctctgcat tgatctcggc actgacatgg tccctgccat ctccttggct 2400 tatgagtcag ctgaaagcga catcatgaag aggcttccaa ggaacccaaa gacggataat 2460 ctggtgaacc accgtctcat tggcatggcc tatggacaga ttgggatgat ccaggctctg 2520 gctggattct ttacctactt tgtaatcctg gctgagaatg gttttaggcc tgttgatctg 2580 ctgggcatcc gcctccactg ggaagataaa tacttgaatg acctggagga cagctacgga 2640 cagcagtgga cctatgagca acgaaaagtt gtggagttca catgccaaac ggcctttttt 2700 gtcaccatcg tggttgtgca gtgggcggat ctcatcatct ccaagactcg ccgcaactca 2760 cttttccagc agggcatgag aaacaaagtc ttaatatttg ggatcctgga ggagacactc 2820 ttggctgcat ttctgtccta cactccaggc atggacgtgg ccctgcgaat gtacccactc 2880 aagataacct ggtggctctg tgccattccc tacagtattc tcatcttcgt ctatgatgaa 2940 atcagaaaac tcctcatccg tcagcacccg gatggctggg tggaaaggga gacgtactac 3000 <210> 31 <211> 3000 <212> DNA <213> Human <400> 31 atggtgaaga gggaaaaaca gaagcgcaat atggaggaac tgaagaagga agtggtcatg 60 gatgatcaca aattaacctt ggaagagctg agcaccaagt actccgtgga cctgacaaag 120 ggccatagcc accaaagggc aaaggaaatc ctgactcgag atggacccaa tactgttacc 180 ccacccccca ccactccaga atgggtcaaa ttctgtaagc aactgttcgg aggcttctcc 240 ctcctactat ggactggggc cattctctgc tttgtggcct acagcatcca gatatatttc 300 aatgaggagc ctaccaaaga caacctctac ctgagcatcg tactgtccgt cgtggtcatc 360 gtcactggct gcttctccta ttatcaggag gccaagagct ccaagatcat ggagtctttt 420 aagaacatgg tgcctcagca agctctggta attcgaggag gagagaagat gcaaattaat 480 gtacaagagg tggtgttggg agacctggtg gaaatcaagg gtggagaccg agtccctgct 540 gacctccggc ttatctctgc acaaggatgt aaggtggaca actcatcctt gactggggag 600 tcagaacccc agagccgctc ccctgacttc acccatgaga accctctgga gacccgaaac 660 atctgcttct tttccaccaa ctgtgtggaa ggaaccgccc ggggtattgt gattgctacg 720 ggagactcca cagtgatggg cagaattgcc tccctgacgt caggcctggc ggttggccag 780 acacctatcg ctgctgagat cgaacacttc atccatctga tcactgtggt ggccgtcttc 840 cttggtgtca ctttttttgc gctctcactt ctcttgggct atggttggct ggaggctatc 900 atttttctca ttggcatcat tgtggccaat gtgcctgagg ggctgttggc tacagtcact 960 gtgtgcctga ccctcacagc caagcgcatg gcacggaaga actgcctggt gaagaacctg 1020 gaggcggtgg agacgctggg ctccacgtcc accatctgct cagacaagac gggcaccctc 1080 acccagaacc gcatgaccgt cgcccacatg tggtttgata tgaccgtgta tgaggccgac 1140 accactgaag aacagactgg aaaaacattt accaagagct ctgatacctg gtttatgctg 1200 gcccgaatcg ctggcctctg caaccgggct gactttaagg ctaatcagga gatcctgccc 1260 attgctaaga gggccacaac aggtgatgct tccgagtcag ccctcctcaa gttcatcgag 1320 cagtcttaca gctctgtggc ggagatgaga gagaaaaacc ccaaggtggc agagattccc 1380 tttaattcta ccaacaagta ccagatgtcc atccaccttc gggaggacag ctcccagacc 1440 cacgtactga tgatgaaggg tgctccggag aggatcttgg agttttgttc tacctttctt 1500 ctgaatgggc aggagtactc aatgaacgat gaaatgaagg aagccttcca aaatgcctat 1560 ttagaactgg gaggtctggg ggaacgtgtg ctaggcttct gcttcttgaa tctgcctagc 1620 agcttctcca agggattccc atttaataca gatgaaataa atttccccat ggacaacctt 1680 tgttttgtgg gcctcatatc catgattgac cctccccgag ctgcagtgcc tgatgctgtg 1740 agcaagtgtc gcagtgcagg aattaaggtg atcatggtaa caggagatca tcccattaca 1800 gctaaggcca ttgccaaggg tgtgggcatc atctcagaag gcactgagac ggcagaggaa 1860 gtcgctgccc ggcttaagat ccctatcagc aaggtcgatg ccagtgctgc caaagccatt 1920 gtggtgcatg gtgcagaact gaaggacata cagtccaagc agcttgatca gatcctccag 1980 aaccaccctg agatcgtgtt tgctcggacc tcccctcagc agaagctcat cattgtcgag 2040 ggatgtcaga ggctgggagc cgttgtggcc gtgacaggtg acggggtgaa cgactcccct 2100 gcgctgaaga aggctgacat tggcattgcc atgggcatct ctggctctga cgtctctaag 2160 caggcagccg acatgatcct gctggatgac aactttgcct ccatcgtcac gggggtggag 2220 gagggccgcc tgatctttga caacctgaag aaatccatca tgtacaccct gaccagcaac 2280 atccccgaga tcacgccctt cctgatgttc atcatcctcg gtatacccct gcctctggga 2340 accataacca tcctctgcat tgatctcggc actgacatgg tccctgccat ctccttggct 2400 tatgagtcag ctgaaagcga catcatgaag aggcttccaa ggaacccaaa gacggataat 2460 ctggtgaacc accgtctcat tggcatggcc tatggacaga ttgggatgat ccaggctctg 2520 gctggattct ttacctactt tgtaatcctg gctgagaatg gttttaggcc tgttgatctg 2580 ctgggcatcc gcctccactg ggaagataaa tacttgaatg acctggagga cagctacgga 2640 cagcagtgga cctatgagca acgaaaagtt gtggagttca catgccaaac ggcctttttt 2700 gtcaccatcg tggttgtgca gtgggcggat ctcatcatct ccaagactcg ccgcaactca 2760 cttttccagc agggcatgag aaacaaagtc ttaatatttg ggatcctgga ggagacactc 2820 ttggctgcat ttctgtccta cactccaggc atggacgtgg ccctgcgaat gtacccactc 2880 aagataacct ggtggctctg tgccattccc tacagtattc tcatcttcgt ctatgatgaa 2940 atcagaaaac tcctcatccg tcagcacccg gatggctggg tggaaaggga gacgtactac 3000 <210> 32 <211> 3000 <212> DNA <213> Human <400> 32 atggtgaaga gggaaaaaca gaagcgcaat atggaggaac tgaagaagga agtggtcatg 60 gatgatcaca aattaacctt ggaagagctg agcaccaagt actccgtgga cctgacaaag 120 ggccatagcc accaaagggc aaaggaaatc ctgactcgag gtggacccaa tactgttacc 180 ccacccccca ccactccaga atgggtcaaa ttctgtaagc aactgttcgg aggcttctcc 240 ctcctactat ggactggggc cattctctgc tttgtggcct acagcatcca gatatatttc 300 aatgaggagc ctaccaaaga caacctctac ctgagcatcg tactgtccgt cgtggtcatc 360 gtcactggct gcttctccta ttatcaggag gccaagagct ccaagatcat ggagtctttt 420 aagaacatgg tgcctcagca agctctggta attcgaggag gagagaagat gcaaattaat 480 gtacaagagg tggtgttggg agacctggtg gaaatcaagg gtggagaccg agtccctgct 540 gacctccggc ttatctctgc acaaggatgt aaggtggaca actcatcctt gactggggag 600 tcagaacccc agagccgctc ccctgacttc acccatgaga accctctgga gacccgaaac 660 atctgcttct tttccaccaa ctgtgtggaa ggaaccgccc ggggtattgt gattgctacg 720 ggagactcca cagtgatggg cagaattgcc tccctgacgt caggcctggc ggttggccag 780 acacctatcg ctgctgagat cgaacacttc atccatctga tcactgtggt ggccgtcttc 840 cttggtgtca ctttttttgc gctctcactt ctcttgggct atggttggct ggaggctatc 900 atttttctca ttggcatcat tgtggccaat gtgcctgagg ggctgttggc tacagtcact 960 gtgtgcctga ccctcacagc caagcgcatg gcacggaaga actgcctggt gaagaacctg 1020 gaggcggtgc agacgctggg ctccacgtcc accatctgct cagacaagac gggcaccctc 1080 acccagaacc gcatgaccgt cgcccacatg tggtttgata tgaccgtgta tgaggccgac 1140 accactgaag aacagactgg aaaaacattt accaagagct ctgatacctg gtttatgctg 1200 gcccgaatcg ctggcctctg caaccgggct gactttaagg ctaatcagga gatcctgccc 1260 attgctaaga gggccacaac aggtgatgct tccgagtcag ccctcctcaa gttcatcgag 1320 cagtcttaca gctctgtggc ggagatgaga gagaaaaacc ccaaggtggc agagattccc 1380 tttaattcta ccaacaagta ccagatgtcc atccaccttc gggaggacag ctcccagacc 1440 cacgtactga tgatgaaggg tgctccggag aggatcttgg agttttgttc tacctttctt 1500 ctgaatgggc aggagtactc aatgaacgat gaaatgaagg aagccttcca aaatgcctat 1560 ttagaactgg gaggtctggg ggaacgtgtg ctaggcttct gcttcttgaa tctgcctagc 1620 agcttctcca agggattccc atttaataca gatgaaataa atttccccat ggacaacctt 1680 tgttttgtgg gcctcatatc catgattgac cctccccgag ctgcagtgcc tgatgctgtg 1740 agcaagtgtc gcagtgcagg aattaaggtg atcatggtaa caggagatca tcccattaca 1800 gctaaggcca ttgccaaggg tgtgggcatc atctcagaag gcactgagac ggcagaggaa 1860 gtcgctgccc ggcttaagat ccctatcagc aaggtcgatg ccagtgctgc caaagccatt 1920 gtggtgcatg gtgcagaact gaaggacata cagtccaagc agcttgatca gatcctccag 1980 aaccaccctg agatcgtgtt tgctcggacc tcccctcagc agaagctcat cattgtcgag 2040 gcatgtcaga ggctgggagc cgttgtggcc gtgacaggtg acggggtgaa cgactcccct 2100 gcgctgaaga aggctgacat tggcattgcc atgggcatct ctggctctga cgtctctaag 2160 caggcagccg acatgatcct gctggatgac aactttgcct ccatcgtcac gggggtggag 2220 gagggccgcc tgatctttga caacctgaag aaatccatca tgtacaccct gaccagcaac 2280 atccccgaga tcacgccctt cctgatgttc atcatcctcg gtatacccct gcctctggga 2340 accataacca tcctctgcat tgatctcggc actgacatgg tccctgccat ctccttggct 2400 tatgagtcag ctgaaagcga catcatgaag aggcttccaa ggaacccaaa gacggataat 2460 ctggtgaacc accgtctcat tggcatggcc tatggacaga ttgggatgat ccaggctctg 2520 gctggattct ttacctactt tgtaatcctg gctgagaatg gttttaggcc tgttgatctg 2580 ctgggcatcc gcctccactg ggaagataaa tacttgaatg acctggagga cagctacgga 2640 cagcagtgga cctatgagca acgaaaagtt gtggagttca catgccaaac ggcctttttt 2700 gtcaccatcg tggttgtgca gtgggcggat ctcatcatct ccaagactcg ccgcaactca 2760 cttttccagc agggcatgag aaacaaagtc ttaatatttg ggatcctgga ggagacactc 2820 ttggctgcat ttctgtccta cactccaggc atggacgtgg ccctgcgaat gtacccactc 2880 aagataacct ggtggctctg tgccattccc tacagtattc tcatcttcgt ctatgatgaa 2940 atcagaaaac tcctcatccg tcagcacccg gatggctggg tggaaaggga gacgtactac 3000 <210> 33 <211> 1000 <212> PRT <213> Human <400> 33 Met Val Lys Arg Glu Lys Gln Lys Arg Asn Met Glu Glu Leu Lys Lys   1 5 10 15 Glu Val Val Met Asp Asp His Lys Leu Thr Leu Glu Glu Leu Ser Thr              20 25 30 Lys Tyr Ser Val Asp Leu Thr Lys Gly His Ser His Gln Arg Ala Lys          35 40 45 Glu Ile Leu Thr Arg Gly Gly Pro Asn Thr Val Thr Pro Pro Pro Thr      50 55 60 Thr Pro Glu Trp Val Lys Phe Cys Lys Gln Leu Phe Gly Gly Phe Ser  65 70 75 80 Leu Leu Leu Trp Thr Gly Ala Ile Leu Cys Phe Val Ala Tyr Ser Ile                  85 90 95 Gln Ile Tyr Phe Asn Glu Glu Pro Thr Lys Asp Asn Leu Tyr Leu Ser             100 105 110 Ile Val Leu Ser Val Val Val Ile Val Thr Gly Cys Phe Ser Tyr Tyr         115 120 125 Gln Glu Ala Lys Ser Ser Lys Ile Met Glu Ser Phe Lys Asn Met Val     130 135 140 Pro Gln Gln Ala Leu Val Ile Arg Gly Gly Glu Lys Met Gln Ile Asn 145 150 155 160 Val Gln Glu Val Val Leu Gly Asp Leu Val Glu Ile Lys Gly Gly Asp                 165 170 175 Arg Val Pro Ala Asp Leu Arg Leu Ile Ser Ala Gln Gly Cys Lys Val             180 185 190 Asp Asn Ser Ser Leu Thr Gly Glu Ser Glu Pro Gln Ser Arg Ser Pro         195 200 205 Asp Phe Thr His Glu Asn Pro Leu Glu Thr Arg Asn Ile Cys Phe Phe     210 215 220 Ser Thr Asn Cys Val Glu Gly Thr Ala Arg Gly Ile Val Ile Ala Thr 225 230 235 240 Gly Asp Ser Thr Val Met Gly Arg Ile Ala Ser Leu Thr Ser Gly Leu                 245 250 255 Ala Val Gly Gln Thr Pro Ile Ala Ala Glu Ile Glu His Phe Ile His             260 265 270 Leu Ile Thr Val Val Ala Val Phe Leu Gly Val Thr Phe Phe Ala Leu         275 280 285 Ser Leu Leu Leu Gly Tyr Gly Trp Leu Glu Ala Ile Ile Phe Leu Ile     290 295 300 Gly Ile Ile Val Ala Asn Val Pro Glu Gly Leu Leu Ala Thr Val Thr 305 310 315 320 Val Cys Leu Thr Leu Thr Ala Lys Arg Met Ala Arg Lys Asn Cys Leu                 325 330 335 Val Lys Asn Leu Glu Ala Val Gln Thr Leu Gly Ser Thr Ser Thr Ile             340 345 350 Cys Ser Asp Lys Thr Gly Thr Leu Thr Gln Asn Arg Met Thr Val Ala         355 360 365 His Met Trp Phe Asp Met Thr Val Tyr Glu Ala Asp Thr Thr Glu Glu     370 375 380 Gln Thr Gly Lys Thr Phe Thr Lys Ser Ser Asp Thr Trp Phe Met Leu 385 390 395 400 Ala Arg Ile Ala Gly Leu Cys Asn Arg Ala Asp Phe Lys Ala Asn Gln                 405 410 415 Glu Ile Leu Pro Ile Ala Lys Arg Ala Thr Thr Gly Asp Ala Ser Glu             420 425 430 Ser Ala Leu Leu Lys Phe Ile Glu Gln Ser Tyr Ser Ser Val Ala Glu         435 440 445 Met Arg Glu Lys Asn Pro Lys Val Ala Glu Ile Pro Phe Asn Ser Thr     450 455 460 Asn Lys Tyr Gln Met Ser Ile His Leu Arg Glu Asp Ser Ser Gln Thr 465 470 475 480 His Val Leu Met Met Lys Gly Ala Pro Glu Arg Ile Leu Glu Phe Cys                 485 490 495 Ser Thr Phe Leu Leu Asn Gly Gln Glu Tyr Ser Met Asn Asp Glu Met             500 505 510 Lys Glu Ala Phe Gln Asn Ala Tyr Leu Glu Leu Gly Gly Leu Gly Glu         515 520 525 Arg Val Leu Gly Phe Cys Phe Leu Asn Leu Pro Ser Ser Phe Ser Lys     530 535 540 Gly Phe Pro Phe Asn Thr Asp Glu Ile Asn Phe Pro Met Asp Asn Leu 545 550 555 560 Cys Phe Val Gly Leu Ile Ser Met Ile Asp Pro Pro Arg Ala Ala Val                 565 570 575 Pro Asp Ala Val Ser Lys Cys Arg Ser Ala Gly Ile Lys Val Ile Met             580 585 590 Val Thr Gly Asp His Pro Ile Thr Ala Lys Ala Ile Ala Lys Gly Val         595 600 605 Gly Ile Ile Ser Glu Gly Thr Glu Thr Ala Glu Glu Val Ala Ala Arg     610 615 620 Leu Lys Ile Pro Ile Ser Lys Val Asp Ala Ser Ala Ala Lys Ala Ile 625 630 635 640 Val Val His Gly Ala Glu Leu Lys Asp Ile Gln Ser Lys Gln Leu Asp                 645 650 655 Gln Ile Leu Gln Asn His Pro Glu Ile Val Phe Ala Arg Thr Ser Pro             660 665 670 Gln Gln Lys Leu Ile Ile Val Glu Ala Cys Gln Arg Leu Gly Ala Val         675 680 685 Val Ala Val Thr Gly Asp Gly Val Asn Asp Ser Pro Ala Leu Lys Lys     690 695 700 Ala Asp Ile Gly Ile Ala Met Gly Ile Ser Gly Ser Asp Val Ser Lys 705 710 715 720 Gln Ala Ala Asp Met Ile Leu Leu Asp Asp Asn Phe Ala Ser Ile Val                 725 730 735 Thr Gly Val Glu Glu Gly Arg Leu Ile Phe Asp Asn Leu Lys Lys Ser             740 745 750 Ile Met Tyr Thr Leu Thr Ser Asn Ile Pro Glu Ile Thr Pro Phe Leu         755 760 765 Met Phe Ile Ile Leu Gly Ile Pro Leu Pro Leu Gly Thr Ile Thr Ile     770 775 780 Leu Cys Ile Asp Leu Gly Thr Asp Met Val Pro Ala Ile Ser Leu Ala 785 790 795 800 Tyr Glu Ser Ala Glu Ser Asp Ile Met Lys Arg Leu Pro Arg Asn Pro                 805 810 815 Lys Thr Asp Asn Leu Val Asn His Arg Leu Ile Gly Met Ala Tyr Gly             820 825 830 Gln Ile Gly Met Ile Gln Ala Leu Ala Gly Phe Phe Thr Tyr Phe Val         835 840 845 Ile Leu Ala Glu Asn Gly Phe Arg Pro Val Asp Leu Leu Gly Ile Arg     850 855 860 Leu His Trp Glu Asp Lys Tyr Leu Asn Asp Leu Glu Asp Ser Tyr Gly 865 870 875 880 Gln Gln Trp Thr Tyr Glu Gln Arg Lys Val Val Glu Phe Thr Cys Gln                 885 890 895 Thr Ala Phe Phe Val Thr Ile Val Val Val Gln Trp Ala Asp Leu Ile             900 905 910 Ile Ser Lys Thr Arg Arg Asn Ser Leu Phe Gln Gln Gly Met Arg Asn         915 920 925 Lys Val Leu Ile Phe Gly Ile Leu Glu Glu Thr Leu Leu Ala Ala Phe     930 935 940 Leu Ser Tyr Thr Pro Gly Met Asp Val Ala Leu Arg Met Tyr Pro Leu 945 950 955 960 Lys Ile Thr Trp Trp Leu Cys Ala Ile Pro Tyr Ser Ile Leu Ile Phe                 965 970 975 Val Tyr Asp Glu Ile Arg Lys Leu Leu Ile Arg Gln His Pro Asp Gly             980 985 990 Trp Val Glu Arg Glu Thr Tyr Tyr         995 1000 <210> 34 <211> 1029 <212> PRT <213> Human <400> 34 Met Gly Leu Trp Gly Lys Lys Gly Thr Val Ala Pro His Asp Gln Ser                  5 10 15 Pro Arg Arg Arg Pro Lys Lys Gly Leu Ile Lys Lys Lys Met Val Lys              20 25 30 Arg Glu Lys Gln Lys Arg Asn Met Glu Glu Leu Lys Lys Glu Val Val          35 40 45 Met Asp Asp His Lys Leu Thr Leu Glu Glu Leu Ser Thr Lys Tyr Ser      50 55 60 Val Asp Leu Thr Lys Gly His Ser His Gln Arg Ala Lys Glu Ile Leu  65 70 75 80 Thr Arg Gly Gly Pro Asn Thr Val Thr Pro Pro Pro Thr Thr Pro Glu                  85 90 95 Trp Val Lys Phe Cys Lys Gln Leu Phe Gly Gly Phe Ser Leu Leu Leu             100 105 110 Trp Thr Gly Ala Ile Leu Cys Phe Val Ala Tyr Ser Ile Gln Ile Tyr         115 120 125 Phe Asn Glu Glu Pro Thr Lys Asp Asn Leu Tyr Leu Ser Ile Val Leu     130 135 140 Ser Val Val Val Ile Val Thr Gly Cys Phe Ser Tyr Tyr Gln Glu Ala 145 150 155 160 Lys Ser Ser Lys Ile Met Glu Ser Phe Lys Asn Met Val Pro Gln Gln                 165 170 175 Ala Leu Val Ile Arg Gly Gly Glu Lys Met Gln Ile Asn Val Gln Glu             180 185 190 Val Val Leu Gly Asp Leu Val Glu Ile Lys Gly Gly Asp Arg Val Pro         195 200 205 Ala Asp Leu Arg Leu Ile Ser Ala Gln Gly Cys Lys Val Asp Asn Ser     210 215 220 Ser Leu Thr Gly Glu Ser Glu Pro Gln Ser Arg Ser Pro Asp Phe Thr 225 230 235 240 His Glu Asn Pro Leu Glu Thr Arg Asn Ile Cys Phe Phe Ser Thr Asn                 245 250 255 Cys Val Glu Gly Thr Ala Arg Gly Ile Val Ile Ala Thr Gly Asp Ser             260 265 270 Thr Val Met Gly Arg Ile Ala Ser Leu Thr Ser Gly Leu Ala Val Gly         275 280 285 Gln Thr Pro Ile Ala Ala Glu Ile Glu His Phe Ile His Leu Ile Thr     290 295 300 Val Val Ala Val Phe Leu Gly Val Thr Phe Phe Ala Leu Ser Leu Leu 305 310 315 320 Leu Gly Tyr Gly Trp Leu Glu Ala Ile Ile Phe Leu Ile Gly Ile Ile                 325 330 335 Val Ala Asn Val Pro Glu Gly Leu Leu Ala Thr Val Thr Val Cys Leu             340 345 350 Thr Leu Thr Ala Lys Arg Met Ala Arg Lys Asn Cys Leu Val Lys Asn         355 360 365 Leu Glu Ala Val Gln Thr Leu Gly Ser Thr Ser Thr Ile Cys Ser Asp     370 375 380 Lys Thr Gly Thr Leu Thr Gln Asn Arg Met Thr Val Ala His Met Trp 385 390 395 400 Phe Asp Met Thr Val Tyr Glu Ala Asp Thr Thr Glu Glu Gln Thr Gly                 405 410 415 Lys Thr Phe Thr Lys Ser Ser Asp Thr Trp Phe Met Leu Ala Arg Ile             420 425 430 Ala Gly Leu Cys Asn Arg Ala Asp Phe Lys Ala Asn Gln Glu Ile Leu         435 440 445 Pro Ile Ala Lys Arg Ala Thr Thr Gly Asp Ala Ser Glu Ser Ala Leu     450 455 460 Leu Lys Phe Ile Glu Gln Ser Tyr Ser Ser Val Ala Glu Met Arg Glu 465 470 475 480 Lys Asn Pro Lys Val Ala Glu Ile Pro Phe Asn Ser Thr Asn Lys Tyr                 485 490 495 Gln Met Ser Ile His Leu Arg Glu Asp Ser Ser Gln Thr His Val Leu             500 505 510 Met Met Lys Gly Ala Pro Glu Arg Ile Leu Glu Phe Cys Ser Thr Phe         515 520 525 Leu Leu Asn Gly Gln Glu Tyr Ser Met Asn Asp Glu Met Lys Glu Ala     530 535 540 Phe Gln Asn Ala Tyr Leu Glu Leu Gly Gly Leu Gly Glu Arg Val Leu 545 550 555 560 Gly Phe Cys Phe Leu Asn Leu Pro Ser Ser Phe Ser Lys Gly Phe Pro                 565 570 575 Phe Asn Thr Asp Glu Ile Asn Phe Pro Met Asp Asn Leu Cys Phe Val             580 585 590 Gly Leu Ile Ser Met Ile Asp Pro Pro Arg Ala Ala Val Pro Asp Ala         595 600 605 Val Ser Lys Cys Arg Ser Ala Gly Ile Lys Val Ile Met Val Thr Gly     610 615 620 Asp His Pro Ile Thr Ala Lys Ala Ile Ala Lys Gly Val Gly Ile Ile 625 630 635 640 Ser Glu Gly Thr Glu Thr Ala Glu Glu Val Ala Ala Arg Leu Lys Ile                 645 650 655 Pro Ile Ser Lys Val Asp Ala Ser Ala Ala Lys Ala Ile Val Val His             660 665 670 Gly Ala Glu Leu Lys Asp Ile Gln Ser Lys Gln Leu Asp Gln Ile Leu         675 680 685 Gln Asn His Pro Glu Ile Val Phe Ala Arg Thr Ser Pro Gln Gln Lys     690 695 700 Leu Ile Ile Val Glu Ala Cys Gln Arg Leu Gly Ala Val Val Ala Val 705 710 715 720 Thr Gly Asp Gly Val Asn Asp Ser Pro Ala Leu Lys Lys Ala Asp Ile                 725 730 735 Gly Ile Ala Met Gly Ile Ser Gly Ser Asp Val Ser Lys Gln Ala Ala             740 745 750 Asp Met Ile Leu Leu Asp Asp Asn Phe Ala Ser Ile Val Thr Gly Val         755 760 765 Glu Glu Gly Arg Leu Ile Phe Asp Asn Leu Lys Lys Ser Ile Met Tyr     770 775 780 Thr Leu Thr Ser Asn Ile Pro Glu Ile Thr Pro Phe Leu Met Phe Ile 785 790 795 800 Ile Leu Gly Ile Pro Leu Pro Leu Gly Thr Ile Thr Ile Leu Cys Ile                 805 810 815 Asp Leu Gly Thr Asp Met Val Pro Ala Ile Ser Leu Ala Tyr Glu Ser             820 825 830 Ala Glu Ser Asp Ile Met Lys Arg Leu Pro Arg Asn Pro Lys Thr Asp         835 840 845 Asn Leu Val Asn His Arg Leu Ile Gly Met Ala Tyr Gly Gln Ile Gly     850 855 860 Met Ile Gln Ala Leu Ala Gly Phe Phe Thr Tyr Phe Val Ile Leu Ala 865 870 875 880 Glu Asn Gly Phe Arg Pro Val Asp Leu Leu Gly Ile Arg Leu His Trp                 885 890 895 Glu Asp Lys Tyr Leu Asn Asp Leu Glu Asp Ser Tyr Gly Gln Gln Trp             900 905 910 Thr Tyr Glu Gln Arg Lys Val Val Glu Phe Thr Cys Gln Thr Ala Phe         915 920 925 Phe Val Thr Ile Val Val Val Gln Trp Ala Asp Leu Ile Ile Ser Lys     930 935 940 Thr Arg Arg Asn Ser Leu Phe Gln Gln Gly Met Arg Asn Lys Val Leu 945 950 955 960 Ile Phe Gly Ile Leu Glu Glu Thr Leu Leu Ala Ala Phe Leu Ser Tyr                 965 970 975 Thr Pro Gly Met Asp Val Ala Leu Arg Met Tyr Pro Leu Lys Ile Thr             980 985 990 Trp Trp Leu Cys Ala Ile Pro Tyr Ser Ile Leu Ile Phe Val Tyr Asp         995 1000 1005 Glu Ile Arg Lys Leu Leu Ile Arg Gln His Pro Asp Gly Trp Val Glu    1010 1015 1020 Arg Glu Thr Tyr Tyr 1025 <210> 35 <211> 3087 <212> DNA <213> Human <400> 35 atggggcttt gggggaagaa agggacagtg gctccccatg accagagtcc aagacgaaga 60 cctaaaaaag ggcttatcaa gaaaaaaatg gtgaagaggg aaaaacagaa gcgcaatatg 120 gaggaactga agaaggaagt ggtcatggat gatcacaaat taaccttgga agagctgagc 180 accaagtact ccgtggacct gacaaagggc catagccacc aaagggcaaa ggaaatcctg 240 actcgaggtg gacccaatac tgttacccca ccccccacca ctccagaatg ggtcaaattc 300 tgtaagcaac tgttcggagg cttctccctc ctactatgga ctggggccat tctctgcttt 360 gtggcctaca gcatccagat atatttcaat gaggagccta ccaaagacaa cctctacctg 420 agcatcgtac tgtccgtcgt ggtcatcgtc actggctgct tctcctatta tcaggaggcc 480 aagagctcca agatcatgga gtcttttaag aacatggtgc ctcagcaagc tctggtaatt 540 cgaggaggag agaagatgca aattaatgta caagaggtgg tgttgggaga cctggtggaa 600 atcaagggtg gagaccgagt ccctgctgac ctccggctta tctctgcaca aggatgtaag 660 gtggacaact catccttgac tggggagtca gaaccccaga gccgctcccc tgacttcacc 720 catgagaacc ctctggagac ccgaaacatc tgcttctttt ccaccaactg tgtggaagga 780 accgcccggg gtattgtgat tgctacggga gactccacag tgatgggcag aattgcctcc 840 ctgacgtcag gcctggcggt tggccagaca cctatcgctg ctgagatcga acacttcatc 900 catctgatca ctgtggtggc cgtcttcctt ggtgtcactt tttttgcgct ctcacttctc 960 ttgggctatg gttggctgga ggctatcatt tttctcattg gcatcattgt ggccaatgtg 1020 cctgaggggc tgttggctac agtcactgtg tgcctgaccc tcacagccaa gcgcatggca 1080 cggaagaact gcctggtgaa gaacctggag gcggtgcaga cgctgggctc cacgtccacc 1140 atctgctcag acaagacggg caccctcacc cagaaccgca tgaccgtcgc ccacatgtgg 1200 tttgatatga ccgtgtatga ggccgacacc actgaagaac agactggaaa aacatttacc 1260 aagagctctg atacctggtt tatgctggcc cgaatcgctg gcctctgcaa ccgggctgac 1320 tttaaggcta atcaggagat cctgcccatt gctaagaggg ccacaacagg tgatgcttcc 1380 gagtcagccc tcctcaagtt catcgagcag tcttacagct ctgtggcgga gatgagagag 1440 aaaaacccca aggtggcaga gattcccttt aattctacca acaagtacca gatgtccatc 1500 caccttcggg aggacagctc ccagacccac gtactgatga tgaagggtgc tccggagagg 1560 atcttggagt tttgttctac ctttcttctg aatgggcagg agtactcaat gaacgatgaa 1620 atgaaggaag ccttccaaaa tgcctattta gaactgggag gtctggggga acgtgtgcta 1680 ggcttctgct tcttgaatct gcctagcagc ttctccaagg gattcccatt taatacagat 1740 gaaataaatt tccccatgga caacctttgt tttgtgggcc tcatatccat gattgaccct 1800 ccccgagctg cagtgcctga tgctgtgagc aagtgtcgca gtgcaggaat taaggtgatc 1860 atggtaacag gagatcatcc cattacagct aaggccattg ccaagggtgt gggcatcatc 1920 tcagaaggca ctgagacggc agaggaagtc gctgcccggc ttaagatccc tatcagcaag 1980 gtcgatgcca gtgctgccaa agccattgtg gtgcatggtg cagaactgaa ggacatacag 2040 tccaagcagc ttgatcagat cctccagaac caccctgaga tcgtgtttgc tcggacctcc 2100 cctcagcaga agctcatcat tgtcgaggca tgtcagaggc tgggagccgt tgtggccgtg 2160 acaggtgacg gggtgaacga ctcccctgcg ctgaagaagg ctgacattgg cattgccatg 2220 ggcatctctg gctctgacgt ctctaagcag gcagccgaca tgatcctgct ggatgacaac 2280 tttgcctcca tcgtcacggg ggtggaggag ggccgcctga tctttgacaa cctgaagaaa 2340 tccatcatgt acaccctgac cagcaacatc cccgagatca cgcccttcct gatgttcatc 2400 atcctcggta tacccctgcc tctgggaacc ataaccatcc tctgcattga tctcggcact 2460 gacatggtcc ctgccatctc cttggcttat gagtcagctg aaagcgacat catgaagagg 2520 cttccaagga acccaaagac ggataatctg gtgaaccacc gtctcattgg catggcctat 2580 ggacagattg ggatgatcca ggctctggct ggattcttta cctactttgt aatcctggct 2640 gagaatggtt ttaggcctgt tgatctgctg ggcatccgcc tccactggga agataaatac 2700 ttgaatgacc tggaggacag ctacggacag cagtggacct atgagcaacg aaaagttgtg 2760 gagttcacat gccaaacggc cttttttgtc accatcgtgg ttgtgcagtg ggcggatctc 2820 atcatctcca agactcgccg caactcactt ttccagcagg gcatgagaaa caaagtctta 2880 atatttggga tcctggagga gacactcttg gctgcatttc tgtcctacac tccaggcatg 2940 gacgtggccc tgcgaatgta cccactcaag ataacctggt ggctctgtgc cattccctac 3000 agtattctca tcttcgtcta tgatgaaatc agaaaactcc tcatccgtca gcacccggat 3060 ggctgggtgg aaagggagac gtactac 3087 <210> 36 <211> 3087 <212> DNA <213> Human <400> 36 atggggcttt gggggaagaa agggacagtg gctccccatg accagagtcc aagacgaaga 60 cctaaaaaag ggcttatcaa gaaaaaaatg gtgaagaggg aaaaacagaa gcgcaatatg 120 gaggaactga agaaggaagt ggtcatggat gatcacaaat taaccttgga agagctgagc 180 accaagtact ccgtggacct gacaaagggc catagccacc aaagggcaaa ggaaatcctg 240 actcgagatg gacccaatac tgttacccca ccccccacca ctccagaatg ggtcaaattc 300 tgtaagcaac tgttcggagg cttctccctc ctactatgga ctggggccat tctctgcttt 360 gtggcctaca gcatccagat atatttcaat gaggagccta ccaaagacaa cctctacctg 420 agcatcgtac tgtccgtcgt ggtcatcgtc actggctgct tctcctatta tcaggaggcc 480 aagagctcca agatcatgga gtcttttaag aacatggtgc ctcagcaagc tctggtaatt 540 cgaggaggag agaagatgca aattaatgta caagaggtgg tgttgggaga cctggtggaa 600 atcaagggtg gagaccgagt ccctgctgac ctccggctta tctctgcaca aggatgtaag 660 gtggacaact catccttgac tggggagtca gaaccccaga gccgctcccc tgacttcacc 720 catgagaacc ctctggagac ccgaaacatc tgcttctttt ccaccaactg tgtggaagga 780 accgcccggg gtattgtgat tgctacggga gactccacag tgatgggcag aattgcctcc 840 ctgacgtcag gcctggcggt tggccagaca cctatcgctg ctgagatcga acacttcatc 900 catctgatca ctgtggtggc cgtcttcctt ggtgtcactt tttttgcgct ctcacttctc 960 ttgggctatg gttggctgga ggctatcatt tttctcattg gcatcattgt ggccaatgtg 1020 cctgaggggc tgttggccac agtcactgtg tgcctgaccc tcacagccaa gcgcatggca 1080 cggaagaact gcctggtgaa gaacctggag gcggtggaga cgctgggctc cacgtccacc 1140 atctgctcag acaagacggg caccctcacc cagaaccgca tgaccgtcgc ccacatgtgg 1200 tttgatatga ccgtgtatga ggccgacacc actgaagaac agactggaaa aacatttacc 1260 aagagctctg atacctggtt tatgctggcc cgaatcgctg gcctctgcaa ccgggctgac 1320 tttaaggcta atcaggagat cctgcccatt gctaagaggg ccacaacagg tgatgcttcc 1380 gagtcagccc tcctcaagtt catcgagcag tcttacagct ctgtggcgga gatgagagag 1440 aaaaacccca aggtggcaga gattcccttt aattctacca acaagtacca gatgtccatc 1500 caccttcggg aggacagctc ccagacccac gtactgatga tgaagggtgc tccggagagg 1560 atcttggagt tttgttctac ctttcttctg aatgggcagg agtactcaat gaacgatgaa 1620 atgaaggaag ccttccaaaa tgcctattta gaactgggag gtctggggga acgtgtgcta 1680 ggcttctgct tcttgaatct gcctagcagc ttctccaagg gattcccatt taatacagat 1740 gaaataaatt tccccatgga caacctttgt tttgtgggcc tcatatccat gattgaccct 1800 ccccgagctg cagtgcctga tgctgtgagc aagtgtcgca gtgcaggaat taaggtgatc 1860 atggtaacag gagatcatcc cattacagct aaggccattg ccaagggtgt gggcatcatc 1920 tcagaaggca ctgagacggc agaggaagtc gctgcccggc ttaagatccc tatcagcaag 1980 gtcgatgcca gtgctgccaa agccattgtg gtgcatggtg cagaactgaa ggacatacag 2040 tccaagcagc ttgatcagat cctccagaac caccctgaga tcgtgtttgc tcggacctcc 2100 cctcagcaga agctcatcat tgtcgaggga tgtcagaggc tgggagccgt tgtggccgtg 2160 acaggtgacg gggtgaacga ctcccctgcg ctgaagaagg ctgacattgg cattgccatg 2220 ggcatctctg gctctgacgt ctctaagcag gcagccgaca tgatcctgct ggatgacaac 2280 tttgcctcca tcgtcacggg ggtggaggag ggccgcctga tctttgacaa cctgaagaaa 2340 tccatcatgt acaccctgac cagcaacatc cccgagatca cgcccttcct gatgttcatc 2400 atcctcggta tacccctgcc tctgggaacc ataaccatcc tctgcattga tctcggcact 2460 gacatggtcc ctgccatctc cttggcttat gagtcagctg aaagcgacat catgaagagg 2520 cttccaagga acccaaagac ggataatctg gtgaaccacc gtctcattgg catggcctat 2580 ggacagattg ggatgatcca ggctctggct ggattcttta cctactttgt aatcctggct 2640 gagaatggtt ttaggcctgt tgatctgctg ggcatccgcc tccactggga agataaatac 2700 ttgaatgacc tggaggacag ctacggacag cagtggacct atgagcaacg aaaagttgtg 2760 gagttcacat gccaaacggc cttttttgtc accatcgtgg ttgtgcagtg ggcggatctc 2820 atcatctcca agactcgccg caactcactt ttccagcagg gcatgagaaa caaagtctta 2880 atatttggga tcctggagga gacactcttg gctgcatttc tgtcctacac tccaggcatg 2940 gacgtggccc tgcgaatgta cccactcaag ataacctggt ggctctgtgc cattccctac 3000 agtattctca tcttcgtcta tgatgaaatc agaaaactcc tcatccgtca gcacccggat 3060 ggctgggtgg aaagggagac gtactac 3087 <210> 37 <211> 3087 <212> DNA <213> Human <400> 37 atggggcttt gggggaagaa agggacagtg gctccccatg accagagtcc aagacgaaga 60 cctaaaaaag ggcttatcaa gaaaaaaatg gtgaagaggg aaaaacagaa gcgcaatatg 120 gaggaactga agaaggaagt ggtcatggat gatcacaaat taaccttgga agagctgagc 180 accaagtact ccgtggacct gacaaagggc catagccacc aaagggcaaa ggaaatcctg 240 actcgagatg gacccaatac tgttacccca ccccccacca ctccagaatg ggtcaaattc 300 tgtaagcaac tgttcggagg cttctccctc ctactatgga ctggggccat tctctgcttt 360 gtggcctaca gcatccagat atatttcaat gaggagccta ccaaagacaa cctctacctg 420 agcatcgtac tgtccgtcgt ggtcatcgtc actggctgct tctcctatta tcaggaggcc 480 aagagctcca agatcatgga gtcttttaag aacatggtgc ctcagcaagc tctggtaatt 540 cgaggaggag agaagatgca aattaatgta caagaggtgg tgttgggaga cctggtggaa 600 atcaagggtg gagaccgagt ccctgctgac ctccggctta tctctgcaca aggatgtaag 660 gtggacaact catccttgac tggggagtca gaaccccaga gccgctcccc tgacttcacc 720 catgagaacc ctctggagac ccgaaacatc tgcttctttt ccaccaactg tgtggaagga 780 accgcccggg gtattgtgat tgctacggga gactccacag tgatgggcag aattgcctcc 840 ctgacgtcag gcctggcggt tggccagaca cctatcgctg ctgagatcga acacttcatc 900 catctgatca ctgtggtggc cgtcttcctt ggtgtcactt tttttgcgct ctcacttctc 960 ttgggctatg gttggctgga ggctatcatt tttctcattg gcatcattgt ggccaatgtg 1020 cctgaggggc tgttggctac agtcactgtg tgcctgaccc tcacagccaa gcgcatggca 1080 cggaagaact gcctggtgaa gaacctggag gcggtggaga cgctgggctc cacgtccacc 1140 atctgctcag acaagacggg caccctcacc cagaaccgca tgaccgtcgc ccacatgtgg 1200 tttgatatga ccgtgtatga ggccgacacc actgaagaac agactggaaa aacatttacc 1260 aagagctctg atacctggtt tatgctggcc cgaatcgctg gcctctgcaa ccgggctgac 1320 tttaaggcta atcaggagat cctgcccatt gctaagaggg ccacaacagg tgatgcttcc 1380 gagtcagccc tcctcaagtt catcgagcag tcttacagct ctgtggcgga gatgagagag 1440 aaaaacccca aggtggcaga gattcccttt aattctacca acaagtacca gatgtccatc 1500 caccttcggg aggacagctc ccagacccac gtactgatga tgaagggtgc tccggagagg 1560 atcttggagt tttgttctac ctttcttctg aatgggcagg agtactcaat gaacgatgaa 1620 atgaaggaag ccttccaaaa tgcctattta gaactgggag gtctggggga acgtgtgcta 1680 ggcttctgct tcttgaatct gcctagcagc ttctccaagg gattcccatt taatacagat 1740 gaaataaatt tccccatgga caacctttgt tttgtgggcc tcatatccat gattgaccct 1800 ccccgagctg cagtgcctga tgctgtgagc aagtgtcgca gtgcaggaat taaggtgatc 1860 atggtaacag gagatcatcc cattacagct aaggccattg ccaagggtgt gggcatcatc 1920 tcagaaggca ctgagacggc agaggaagtc gctgcccggc ttaagatccc tatcagcaag 1980 gtcgatgcca gtgctgccaa agccattgtg gtgcatggtg cagaactgaa ggacatacag 2040 tccaagcagc ttgatcagat cctccagaac caccctgaga tcgtgtttgc tcggacctcc 2100 cctcagcaga agctcatcat tgtcgaggga tgtcagaggc tgggagccgt tgtggccgtg 2160 acaggtgacg gggtgaacga ctcccctgcg ctgaagaagg ctgacattgg cattgccatg 2220 ggcatctctg gctctgacgt ctctaagcag gcagccgaca tgatcctgct ggatgacaac 2280 tttgcctcca tcgtcacggg ggtggaggag ggccgcctga tctttgacaa cctgaagaaa 2340 tccatcatgt acaccctgac cagcaacatc cccgagatca cgcccttcct gatgttcatc 2400 atcctcggta tacccctgcc tctgggaacc ataaccatcc tctgcattga tctcggcact 2460 gacatggtcc ctgccatctc cttggcttat gagtcagctg aaagcgacat catgaagagg 2520 cttccaagga acccaaagac ggataatctg gtgaaccacc gtctcattgg catggcctat 2580 ggacagattg ggatgatcca ggctctggct ggattcttta cctactttgt aatcctggct 2640 gagaatggtt ttaggcctgt tgatctgctg ggcatccgcc tccactggga agataaatac 2700 ttgaatgacc tggaggacag ctacggacag cagtggacct atgagcaacg aaaagttgtg 2760 gagttcacat gccaaacggc cttttttgtc accatcgtgg ttgtgcagtg ggcggatctc 2820 atcatctcca agactcgccg caactcactt ttccagcagg gcatgagaaa caaagtctta 2880 atatttggga tcctggagga gacactcttg gctgcatttc tgtcctacac tccaggcatg 2940 gacgtggccc tgcgaatgta cccactcaag ataacctggt ggctctgtgc cattccctac 3000 agtattctca tcttcgtcta tgatgaaatc agaaaactcc tcatccgtca gcacccggat 3060 ggctgggtgg aaagggagac gtactac 3087

[Brief description of drawings]

FIG. 1 is a diagram showing a comparison of amino acid sequences of Na, K-ATPase α2 subunit and human TCH115. In the figure, ATP1A2 is Na, K-ATPase.
The amino acid sequence of the α2 subunit, TCH115,
This shows the amino acid sequence represented by SEQ ID NO: 24. The arrow indicates the start codon in SEQ ID NO: 1. TM1
10 indicates a transmembrane region. (Continued to Figure 2)

FIG. 2 is a diagram showing a comparison of amino acid sequences of Na, K-ATPase α2 subunit and human TCH115. In the figure, ATP1A2 is Na, K-ATPase.
The amino acid sequence of the α2 subunit, TCH115,
This shows the amino acid sequence represented by SEQ ID NO: 24. The arrow indicates the start codon in SEQ ID NO: 1. TM1
10 indicates a transmembrane region. (Continued from Figure 1)

FIG. 3 shows the expression level of human TCH115 gene product in each tissue. Human tissue cDNA (Hum
an MTC panel I, II: Clontech)
The expression level of human TCH115 in TaqMan P
It was measured by CR. The expression level was represented by the copy number per 1 μl of the cDNA solution.

─────────────────────────────────────────────────── ─── Continuation of front page (51) Int.Cl. ⁷ Identification code FI theme code (reference) A61P 9/00 A61P 13/12 4C084 9/04 15/00 4C085 13/12 25/00 4C086 15/00 29 / 00 4H045 25/00 35/00 29/00 37/02 35/00 37/06 37/02 37/08 37/06 C07K 16/40 37/08 C12N 1/15 C07K 16/40 1/19 C12N 1 / 15 1/21 1/19 9/14 101 1/21 C12Q 1/02 5/10 G01N 33/15 Z 9/14 101 33/50 Z C12Q 1/02 33/53 D G01N 33/15 M 33 / 50 33/566 33/53 C12N 15/00 ZNAA 5/00 A 33/566 A61K 37/02 F term (reference) 2G045 AA34 AA40 BA11 BB50 DA12 DA13 DA14 DA36 FB02 FB03 4B024 AA01 AA11 BA11 CA04 CA12 HA11 HA17 4B050 CC03 DD11 LL01 LL03 4B063 QA08 QA18 QQ08 QQ30 QR10 QR77 4B065 AA93Y AB01 CA31 CA44 CA46 4C084 AA02 AA07 A A17 BA01 BA08 BA22 BA23 CA17 CA18 CA25 CA59 DC50 NA10 NA14 ZA012 ZA022 ZA362 ZA812 ZB072 ZB092 ZB112 ZB132 ZB262 4C085 AA13 BB11 CC01 CC31 4C086 AA01 AA02 A11 AA02 AA02 AA02 AA02 AA02 AA02 AA02 AA02 AA02 AA02 AA02 AA02 AA02 AA02 AA02 AA02

Claims (33)

[Claims] 1. A protein containing an amino acid sequence which is the same or substantially the same 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 SEQ ID NO: 33, or a salt thereof. 3. The protein according to claim 1, which comprises the amino acid sequence represented by SEQ ID NO: 24 or SEQ ID NO: 34, or a salt thereof. 4. A partial peptide of the protein according to claim 1 or a salt thereof. 5. A polynucleotide containing a polynucleotide encoding the protein according to claim 1 or the partial peptide according to claim 4. 6. The polynucleotide according to claim 5, which is DNA. 7. The DNA according to claim 6, which comprises the nucleotide sequence represented by SEQ ID NO: 2, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31 or SEQ ID NO: 32. 8. The DNA according to claim 6, which contains the nucleotide sequence represented by SEQ ID NO: 25, SEQ ID NO: 35, SEQ ID NO: 36 or SEQ ID NO: 37. 9. A recombinant vector containing the DNA according to claim 6. 10. A transformant transformed with the recombinant vector according to claim 9. 11. The method according to claim 1, wherein the transformant according to claim 10 is cultured, the protein according to claim 1 or the partial peptide according to claim 4 is produced and accumulated, and this is collected. Protein or claim 4
A method for producing the described partial peptide or a salt thereof. 12. A medicine comprising the protein according to claim 1, the partial peptide according to claim 4, or a salt thereof. 13. A medicine comprising the DNA according to claim 6. 14. An antibody against the protein according to claim 1, the partial peptide according to claim 4, or a salt thereof. 15. The activity of the protein according to claim 1, the partial peptide according to claim 4, or the salt thereof is characterized by using the protein according to claim 1, the partial peptide according to claim 4, or a salt thereof. A method for screening a compound or its salt that promotes or inhibits. 16. The activity of the protein according to claim 1, or the partial peptide according to claim 4, or the salt thereof, which comprises the protein according to claim 1, the partial peptide according to claim 4, or a salt thereof, or A kit for screening a compound that inhibits or a salt thereof. 17. The activity of the protein according to claim 1 or the partial peptide according to claim 4 or a salt thereof obtained by using the screening method according to claim 15 or the screening kit according to claim 16 is promoted or inhibited. Or a salt thereof. 18. A medicine comprising the compound according to claim 17 or a salt thereof. 19. A method for screening a compound or a salt thereof that promotes or inhibits the expression of the protein gene according to claim 1, which comprises using the polynucleotide according to claim 5. 20. A kit for screening a compound or a salt thereof which promotes or inhibits the expression of the protein gene according to claim 1, which comprises the polynucleotide according to claim 5. 21. A compound or a salt thereof which promotes or inhibits the expression of the protein gene according to claim 1, which is obtained by using the screening method according to claim 19 or the screening kit according to claim 20. 22. A medicine comprising the compound according to claim 21 or a salt thereof. 23. A diagnostic agent comprising the antibody according to claim 14. 24. A medicine comprising the antibody according to claim 14. 25. The method for quantifying a protein according to claim 1, wherein the antibody according to claim 14 is used. 26. The method for diagnosing a disease associated with the function of the protein according to claim 1, wherein the quantification method according to claim 25 is used. 27. A method for screening a compound or its salt that promotes or inhibits the expression of the protein according to claim 1, characterized by using the antibody according to claim 14. 28. A screening kit for a compound or its salt that promotes or inhibits the expression of the protein according to claim 1, which comprises the antibody according to claim 14. 29. A compound or a salt thereof, which is obtained by using the screening method according to claim 27 or the screening kit according to claim 28, and which promotes or inhibits the expression of the protein according to claim 1. 30. A medicine comprising the compound according to claim 29 or a salt thereof. 31. A prophylactic / therapeutic agent for heart disease, kidney disease, central disease, inflammatory disease, autoimmune disease, allergic disease, thymus disease, spleen disease, immunodeficiency, genital disease or cancer,
Or a rejection inhibitor after organ transplantation,
The medicine according to claim 13, claim 18, claim 22, claim 24 or claim 30. 32. A heart disease, kidney disease, central disease, inflammatory property, which comprises administering an effective amount of the compound according to claim 17, 21 or 29 or a salt thereof to a mammal. Disease, autoimmune disease, allergic disease,
A method for preventing or treating thymic disease, spleen disease, immunodeficiency, genital disease or cancer, or a method for suppressing rejection after organ transplantation. 33. A prophylactic / therapeutic agent for heart disease, kidney disease, central disease, inflammatory disease, autoimmune disease, allergic disease, thymus disease, spleen disease, immunodeficiency, genital disease or cancer,
Alternatively, use of the compound according to claim 17, 21 or 29 or a salt thereof for producing a rejection inhibitor after organ transplantation.