JP2003000266A - New protein and its dna - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain a new protein having a Ca<+2> channel activity, a DNA encoding the protein, to provide a method for screening a compound capable of promoting or inhibiting the activity of the protein and to obtain the compound, etc., obtained by the method for screening. SOLUTION: The protein containing an amino acid sequence which is the same or substantially the same as an amino acid sequence represented by a specific sequence is useful as diagnostic marker, to the like, for central nervous system diseases or cerebrovascular diseases. The compound obtained by the method for screening using the protein and capable of promoting or inhibiting the activity of the protein can be used as a prophylatic or a therapeutic agent for diseases, e.g. myopathies, central nervous system diseases or cerebrovascular diseases.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a novel voltage difference-dependent Ca ²⁺ channel γ subunit protein, a DNA encoding the protein, a screening method for a compound that promotes or inhibits the activity of the protein, and the screening method. The resulting compounds and the like are provided.

[0002]

2. Description of the Related Art Potential-difference-dependent Ca ²⁺ channels are roughly classified into transient / low-threshold active type and persistent / high-threshold active type based on the difference in electrophysiological opening time and activation potential threshold. It
The former belongs to T-type channel, and the latter belongs to L-type, N-type, P-type.
Type, Q type, and R type channels. These voltage-gated Ca ²⁺ channels basically consist of α1, α ₂ δ, β
It is composed of three subunits, but recently it has become clear that there is an additional γ subunit.
The γ subunit is considered to be a 4-transmembrane type membrane protein, and it has been reported so far that γ1 to γ8 subtypes exist. Among them, γ1 is considered to associate with α1S to form an L-type Ca ²⁺ channel in skeletal muscle. In addition, γ2 and α1A, γ3 and α1B, γ
There are also reports suggesting associations between 4 and α1E and γ5 and α1G. These γ subunits are voltage-dependent C
In the a ²⁺ channel, a role of regulating the amount of current flowing and the rate of activation is assumed. It is also known that in Stargazer mice that exhibit epileptiform abnormalities, mutation of the γ2 subunit causes this abnormality.

[0003]

A large number of molecular species exist for Ca ²⁺ channels, and their expression sites are also diverse. In addition, one cell often expresses multiple Ca ²⁺ channels. The characteristics of Ca ²⁺ current due to the opening of individual Ca ²⁺ channels have become quite clear, but the nature of the signal when multiple Ca ²⁺ channels work and the interaction between each Ca ²⁺ channel. There is little knowledge about the regulatory action of sucrose. Ca ²⁺ channels have many important functions for cells, but it is necessary to clarify the relationship between the specific function and each molecular species such as central nervous system disease, cerebrovascular disease, muscle disease, heart disease, etc. Leading to the elucidation of the role of Ca ²⁺ channels in

[0004]

[Means for Solving the Problems] As a result of intensive studies to solve the above problems, the present inventors have found that a novel voltage difference-dependent Ca ²⁺ channel γ subunit protein (human voltage difference-dependent Ca ²⁺ Channel gamma subunit homolog)
Found. The human voltage difference-dependent Ca ²⁺ channel γ subunit homologue shows a partial 70% homology with the human voltage difference-dependent Ca ²⁺ channel γ subunit at the amino acid level, and can function as a Ca ²⁺ channel. Is. Examples of the method for suppressing the voltage-gated Ca ²⁺ channel γ subunit homologue include, for example, inhibition of Ca ²⁺ ion permeation, and suppression of the transcription of the voltage-gated Ca ²⁺ channel γ subunit homologue to the expression level Is considered to be lowered. As a method of activating a potential difference dependent Ca ²⁺ channel γ subunit homologs, for example, to facilitate penetration of Ca ²⁺ ions, the potential difference dependent Ca ²⁺
It is considered that the expression level is enhanced by activating the promoter of the channel γ subunit homolog or stabilizing the mRNA. 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 according to (1) above, which comprises the amino acid sequence represented by SEQ ID NO: 1, or a salt thereof, (3) the protein described above (1), which comprises the amino acid sequence represented by SEQ ID NO: 15. Or a salt thereof, (4) a partial peptide of the protein according to (1) or a salt thereof,
(5) A polynucleotide containing a polynucleotide encoding the protein according to (1) above or the partial peptide according to (4) above, (6) a DNA above (5)
The polynucleotide described above, (7) the DNA described in (5) above, which comprises the base sequence represented by SEQ ID NO: 2, (8)
The DNA according to (5) above, which contains the base sequence represented by SEQ ID NO: 16, (9) the DNA according to (5), which contains the base sequence represented by SEQ ID NO: 14, (10)
A recombinant vector containing the DNA according to (6) above,
(11) A transformant transformed with the recombinant vector according to (10) above, (12) a transformant according to (11) above is cultured, and the protein according to (1) above or (4) above is described. (3) The method for producing the protein according to (1) above, the partial peptide according to (4) above, or a salt thereof, which comprises producing and accumulating the partial peptide according to (1), and (13) above. A medicine comprising a protein or the partial peptide according to (4) above or a salt thereof, (14) a medicine comprising the DNA according to (6) above, (15) the protein according to (1) above or ( 4) An antibody against the partial peptide or a salt thereof, (16) the protein according to (1) above, or the partial peptide or a salt thereof according to (4) above is used, (1) The method for screening a compound or a salt thereof which promotes or inhibits the activity of the protein according to (4) or the partial peptide according to (4) or a salt thereof, (17) the protein according to (1) above or (4) above A kit for screening a compound or a salt thereof, which comprises a partial peptide or a salt thereof, which promotes or inhibits the activity of the protein according to (1) above, or the partial peptide according to (4) above or a salt thereof,
(18) Promoting the activity of the protein according to (1) above, the partial peptide according to (4) above, or a salt thereof obtained by using the screening method according to (16) above or the screening kit according to (17) above. Or a compound or salt thereof which inhibits, (19) above (1
The above-mentioned (1) obtained by using the screening method described in 6) or the screening kit described in (17) above.
(20) A medicament comprising a compound which promotes or inhibits the activity of the protein according to (4) or the partial peptide according to (4) or a salt thereof, or (20) a prophylactic / therapeutic agent for central nervous system disease or cerebrovascular disease There is provided the pharmaceutical according to (13) or (14), and the pharmaceutical according to (19), which is a prophylactic / therapeutic agent for (21) central nervous system disease or cerebrovascular disease.

[0006]

BEST MODE FOR CARRYING OUT THE INVENTION SEQ ID NO: 1 used in the present invention
A protein containing the same or substantially the same amino acid sequence as the amino acid sequence represented by (hereinafter sometimes referred to as the protein of the present invention or the protein used in the present invention) is a human or other warm-blooded animal ( For example, guinea pig, rat, mouse, chicken, rabbit, pig,
Sheep cells, bovine cells, monkey cells, etc.) (eg, hepatocytes, splenocytes, nerve cells, glial cells, pancreatic β cells, bone marrow cells,
Mesangial cells, Langerhans cells, epidermal cells, epithelial cells, 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, synovium Cells, chondrocytes, osteocytes, osteoblasts, osteoclasts, mammary gland cells, hepatocytes or stromal cells, or precursors of these cells, stem cells or cancer cells) or any tissue in which those cells are present, eg , Brain, various parts of the brain (eg, olfactory bulb, amygdala, basal sphere, hippocampus, thalamus, hypothalamus, cerebral cortex, medulla oblongata, cerebellum), spinal cord, pituitary gland, stomach, pancreas, kidney, liver, gonad, thyroid gland , Gallbladder, bone marrow, adrenal gland, skin, muscle, lung, digestive tract (eg, large intestine, small intestine), blood vessel, heart, thymus, spleen, submandibular gland, peripheral blood, prostate, testis, ovary, placenta, uterus, bone, Even proteins derived from joints, skeletal muscle, etc. Ku, may be a synthetic protein.

The amino acid sequence substantially identical to the amino acid sequence represented by SEQ ID NO: 1 is about 75% or more, preferably about 80%, with the amino acid sequence represented by SEQ ID NO: 1.
% Or more, more preferably about 85% or more, particularly preferably about 90% or more, and most preferably about 95% or more homologous amino acid sequences. A protein containing an amino acid sequence substantially the same as the amino acid sequence represented by SEQ ID NO: 1 includes, for example, an amino acid sequence substantially the same as the amino acid sequence represented by SEQ ID NO: 1 above. , A protein having substantially the same activity as the protein containing the amino acid sequence represented by SEQ ID NO: 1 is preferable. Examples of substantially the same activity include Ca ²⁺ ion permeation activity. Substantially homogenous means that their properties are qualitatively (eg,
It indicates homogeneity (physiologically or pharmacologically). Therefore, Ca ²⁺ ion permeation activity is equivalent (eg,
About 0.01 to 100 times, preferably about 0.1 to 10 times,
It is more preferably 0.5 to 2 times), but quantitative factors such as the degree of activity and the molecular weight of the protein may be different. Activity such as Ca ²⁺ ion permeation activity can be determined by known methods, for example, J. Biol. Chem. 27.
5: 12237-12242, 2000, or a method similar thereto.

The protein used in the present invention is, for example, 1 or 2 or more (preferably about 1 to 30) in the amino acid sequence represented by SEQ ID NO: 1.
Preferably about 1 to 10, more preferably a number (1 to
5) The amino acid sequence in which the amino acid is deleted, or 1 or 2 or more (preferably about 1 to 30, preferably about 1 to 10 and more preferably about 1 to 2 in the amino acid sequence represented by SEQ ID NO: 1. 1 or 2 or more (preferably 1 to 30 or so, preferably 1 to 10 or so) in the amino acid sequence represented by SEQ ID NO: 1, in which an amino acid number (1 to 5) is added. More preferably, an amino acid sequence in which several (1 to 5) amino acids are inserted,
1 or 2 in the amino acid sequence represented by SEQ ID NO: 1
1 or more (preferably about 1 to 30 and preferably 1 to 30)
A so-called mutein such as a protein containing an amino acid sequence in which about 10 amino acids, more preferably several (1 to 5) amino acids are substituted with other amino acids, or an amino acid sequence combining these is also included. 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, SEQ ID NO: 1 or
Human containing the amino acid sequence represented by SEQ ID NO: 15
Examples include proteins derived from the brain.

The partial peptide of the protein used in the present invention is a partial peptide of the above-mentioned protein used in the present invention, and preferably has the same properties as the above-mentioned 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.

[0011] In the partial peptide used in the present invention C-terminal carboxyl group (-COOH), a carboxylate (-COO ^-), may be any of an amide (-CONH ₂₎ or an ester (-COOR) . Further, the partial peptide used in the present invention has a carboxyl group (or carboxylate) in addition to the C-terminal, like the above-mentioned protein used in the present invention,
Amino acid residue of N-terminal amino acid residue (eg, methionine residue) protected with a protecting group, glutamine residue cleaved in vivo at the N-terminal side is pyroglutamine-oxidized, amino acid in molecule Those in which the substituent on the side chain of is protected by a suitable protecting group, or a complex peptide such as a so-called glycopeptide to which a sugar chain is bound are included.
The partial peptide used in the present invention can also be used as an antigen for antibody production. For the purpose of preparing the antibody of the present invention, for example, a peptide containing the 31st to 104th and 153rd to 172nd amino acid sequences in the amino acid sequence represented by SEQ ID NO: 1 can be mentioned.

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 its salt 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 method for purifying the protein, or contains a DNA encoding the protein. It can also be produced by culturing the transformant. It can also be produced according to the peptide synthesis method described below. When producing from tissues or cells of humans or other mammals, the tissues or cells of humans or other mammals are homogenized and then extracted with an acid or the like, and the extract is subjected to reverse phase chromatography or ion exchange chromatography. Purification and isolation can be achieved by combining chromatography such as.

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
-(2 ', 4'-dimethoxyphenyl-hydroxymethyl)
Phenoxy resin, 4- (2 ', 4'-dimethoxyphenyl-
Fmoc aminoethyl) phenoxy resin and the like can be mentioned. 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. The carbodiimides include DCC,
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 protected amino acid is activated in advance as a corresponding acid anhydride or HOBt ester or HOOBt ester. After that, it can be added to the resin.

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

Examples of the 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, Fmo
c 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)] and the like 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 protective group include catalytic reduction in a hydrogen stream in the presence of a catalyst such as Pd-black or Pd-carbon, or anhydrous hydrogen fluoride, methanesulfonic acid, or trifluoride. Acid treatment with methanesulfonic acid, trifluoroacetic acid or a mixture thereof, base treatment with diisopropylethylamine, triethylamine, piperidine, piperazine, etc., reduction with sodium in liquid ammonia, and the like are also used. The elimination reaction by the 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, paracresol, dimethyl sulfide, 1,4 -Addition of a cation scavenger such as butanedithiol, 1,2-ethanedithiol is effective. Also, the 2,4-dinitrophenyl group used as the imidazole protecting group of histidine is removed by thiophenol treatment, and the formyl group used as the indole protecting group of tryptophan is the above-mentioned 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 the following (1) to (3). M. Bodanszky and MA Ondetti, Peptide Synthesis, Interscience Publisher
s, New York (1966) Schroeder and Luebke, The Peptid
e), Academic Press, New York (1965) Nobuo Izumiya et al., Fundamentals and experiments of peptide synthesis, Maruzen Co., Ltd.
(1975) Haruaki Yajima and Shunpei Sakakibara, Laboratory of Biochemistry 1, Protein Chemistry IV, 205, (1977) Supervised by Haruaki Yajima, Development of Pharmaceuticals, Volume 14, Peptide Synthesis, Hirokawa Shoten After that, the partial peptide used in the present invention can be purified and isolated by combining ordinary purification methods such as solvent extraction, distillation, column chromatography, liquid chromatography, and recrystallization. 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 the polynucleotide, RNA such as DNA or mRNA encoding the protein used in the present invention
And may be double-stranded or single-stranded. When it is double-stranded, it may be double-stranded DNA, double-stranded RNA or a hybrid of DNA: RNA. In the case of a single strand, it may be a sense strand (that is, a coding strand) or an antisense strand (that is, a non-coding strand). As the DNA, genomic DNA, genomic DNA library, cDNA derived from the cells / tissues described above, cDNA library derived from the cells / tissues described above, synthetic DNA
Any of The vector used for the library may be any of bacteriophage, plasmid, cosmid, phagemid and the like. In addition, reverse Transcriptase Polymerase can be directly used by preparing total RNA or mRNA fraction from the above-mentioned cells / tissues.
Chain Reaction (hereinafter abbreviated as RT-PCR method)
Can also be amplified by. The DNA encoding the protein used in the present invention is, for example, a DNA containing the base sequence represented by SEQ ID NO: 2, or a hybrid with the base sequence represented by SEQ ID NO: 2 under high stringent conditions. DNA containing a soybean base sequence and encoding a protein having substantially the same properties as the protein used in the present invention, SEQ ID NO: 16
DNA containing the base sequence represented by or the base sequence represented by SEQ ID NO: 16 and containing a base sequence which hybridizes under high stringent conditions, and is substantially homologous to the protein used in the present invention. Any DNA may be used as long as it encodes a protein having the above property. DN containing the nucleotide sequence represented by SEQ ID NO: 2
Examples of A include DNA containing the nucleotide sequence represented by SEQ ID NO: 14.

DNA capable of hybridizing with the nucleotide sequence represented by SEQ ID NO: 2 under high stringent conditions
Is, for example, about 75% or more, preferably about 80% or more, more preferably about 85% or more, particularly preferably about 90% or more, most preferably about 95% with the base sequence represented by SEQ ID NO: 2. A DNA containing a base sequence having the above homology is used. As the DNA capable of hybridizing with the base sequence represented by SEQ ID NO: 16 under high stringent conditions, for example, SEQ ID NO: 16
And the nucleotide sequence represented by about 75% or more, preferably about 80%
% Or more, more preferably about 85% or more, particularly preferably about 90% or more, most preferably about 95% or more homologous DNA containing a base sequence is used. Hybridization can be carried out by a known method or a method analogous thereto, for example, molecular cloning (Mole
cular Cloning) 2nd (J. Sambrook et al., Cold Sprin
g Harbor Lab. Press, 1989). When a commercially available library is used, it can be performed according to the method described in the attached instruction manual. More preferably, it can be carried out under high stringent conditions. High stringent conditions mean, for example, that the sodium concentration is about 19-40.
mM, preferably about 19-20 mM, at a temperature of about 50-
The conditions are 70 ° C., preferably about 60 to 65 ° C. Particularly, the case where the sodium concentration is about 19 mM and the temperature is about 65 ° C. is most preferable. More specifically, as the DNA encoding the protein containing the amino acid sequence represented by SEQ ID NO: 1, DNA containing the nucleotide sequence represented by SEQ ID NO: 2 is represented by SEQ ID NO: 15. As a DNA encoding a protein containing the amino acid sequence of SEQ ID NO: 16 containing the nucleotide sequence represented by SEQ ID NO: 16
A or the like is used.

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, a DNA containing a part of the DNA containing the base sequence represented by SEQ ID NO: 2, or a base sequence represented by SEQ ID NO: 2 A DNA containing a base sequence that hybridizes under stringent conditions and containing a part of a DNA encoding a protein having substantially the same activity as the protein of the present invention is used. Sequence number:
DNA capable of hybridizing with the nucleotide sequence represented by 2
Indicates 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 a PCR method using a primer, or selection by hybridization with a DNA incorporated into an appropriate vector and labeled with a DNA fragment encoding 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. The substitution of the nucleotide sequence of DNA is carried out by PCR or a known kit, for example, Mutan ^™ -superExpress Km (Takara Shuzo), Mutan ^™ -K (Takara Shuzo), etc., using the ODA-LA PCR method, Gapped duplex method, Kunkel method, etc. Can be performed according to known methods or methods similar thereto. The cloned DNA encoding the protein of the present invention can be used as it is, or if desired, digested with a restriction enzyme or added with a linker depending on the purpose. 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) A DNA fragment containing a DNA encoding the protein of the present invention, for example, is produced by cutting out a desired DNA fragment from cDNA and ligating the DNA fragment downstream of a promoter in a suitable expression vector. You can

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, 1 [Procedures of the National Academy of Sciences of the USA]
60 (1968)], JM103 [Nukuirek Acids.
Research, (Nucleic Acids Research), Volume 9, 309 (1
981)], JA221 [Journal of Molecular Biology], 12
0, 517 (1978)], HB101 [Journal of
Molecular Biology, 41, 459 (1969)], C
600 [Genetics, Volume 39, 440 (1
954)] 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, larvae of the night-thief moth (Spodop
tera frugiperda cell; Sf cell), Trichoplusia ni
MG1 cells from the midgut of the mouse, H from the eggs of Trichoplusia ni
igh Five ^TM cells, cells from Mamestra brassicae or
Cells derived from Estigmena acrea are used. When the virus is BmNPV, the silkworm-derived cell line (Bombyx mor
i N cells; BmN cells) and the like are used. Examples of the Sf cells include Sf9 cells (ATCC CRL1711), Sf
21 cells (above Vaughn, JL et al., In Vivo
ivo), 13, 213-217, (1977)) is used. As the insect, for example, silkworm larvae are used [Maeda et al., Nature, 315, 592 (1985)]. Examples of animal cells include monkey cells COS-7 and Ver.
o, Chinese hamster cell CHO (hereinafter, CHO
Cells), dhfr gene-deficient Chinese hamster cells CHO (hereinafter abbreviated as CHO (dhfr ⁻ ) cells), mouse L cells, mouse AtT-20, mouse myeloma cells, rat GH3, human FL cells and the like. To transform Escherichia, for example,
Proceedings of the National Academy of Sciences of the USA (Pr)
oc. Natl. Acad. Sci. USA), Vol. 69, 2110 (1972), Gene, Vol. 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), 75
Vol., 1929 (1978) and the like. For transforming insect cells or insects, for example, Bio / Technology, 6, 47-
55 (1988)) and the like. To transform animal cells, see, eg, Cell Engineering Supplement 8, New Cell Engineering Experimental Protocol. 263-267 (1995)
(Published by Shujunsha), Virology, Volume 52, 45
6 (1973). 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 and corn steep.
Examples of inorganic or organic substances such as liquor, peptone, casein, meat extract, soybean meal, and potato extract, and inorganic substances 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 other 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 Kohler and Milstein [Nature, 256, 495 (197).
5)]. 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),
Or, as the antisense nucleotide containing a substantially complementary base sequence, it contains a base sequence complementary to or substantially complementary to the base sequence of the DNA of the present invention,
Any antisense nucleotide may be used as long as it has an action of suppressing the expression of the DNA, but antisense DNA is preferable. The nucleotide sequence substantially complementary to the DNA of the present invention means, for example, the DN of the present invention.
About 70% or more, preferably about 80% or more, more preferably about 90% of the total or partial base sequence of the base sequence complementary to A (that is, the complementary strand of the DNA of the present invention).
As mentioned above, the most preferable example is a nucleotide sequence having a homology of about 95% 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 nucleotide containing a base sequence complementary to or substantially complementary to the base sequence of DNA containing the base sequence represented by SEQ ID NO: 2, or a part thereof, preferably, for example, , D containing the nucleotide sequence represented by SEQ ID NO: 2
Examples include an antisense nucleotide containing a base sequence complementary to the base sequence of NA, or a part thereof. The antisense nucleotide is usually composed of about 10 to 40 bases, preferably about 15 to 30 bases. In order to prevent degradation by a hydrolase such as nuclease, the phosphate residue (phosphate) of each nucleotide constituting the antisense DNA is changed to, for example, a chemically modified phosphate residue such as phosphorothioate, methylphosphonate, or phosphorodithionate. It may be substituted. These antisense nucleotides can be produced using a known DNA synthesizer or the like.

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 referred to as the DNA of the present invention and 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 an antisense nucleotide of the DNA of the present invention (hereinafter referred to as the anti-nucleotide of the present invention). (Sometimes abbreviated as “sense nucleotide”).

Since the drug containing the compound or its salt that inhibits the activity of the protein of the present invention can suppress the Ca ²⁺ channel activity, it can suppress the generation of the membrane potential based on the Ca ²⁺ current. For example, central nervous system diseases,
It can be used as a therapeutic / preventive agent for cerebrovascular disease, muscle disease, heart disease and the like. On the other hand, a drug containing a compound or a salt thereof that promotes the activity of the protein of the present invention can promote, for example, Ca ²⁺ channel activity and promote the generation of membrane potential based on Ca ²⁺ current. It is associated with improved function and can be used as a therapeutic / preventive agent for central nervous system diseases, cerebrovascular diseases, muscle diseases, heart diseases and the like.

[1] Remedies for Preventing and Treating Various Diseases Involved by the Protein of the Present Invention The protein of the present invention is one of the subunits of the Ca ²⁺ channel complex and contributes to Ca ²⁺ ion permeation. , Plays a central role in the generation of cell membrane potential. Further, the membrane potential generation by the protein of the present invention is
It plays an important role in the expression of cell functions such as neurotransmission, muscle contraction, and secretion of physiologically active substances. The protein of the present invention has a high homology to the Ca ²⁺ channel γ subunit protein CACNG7 at the N-terminal side when compared in amino acid sequence. 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, muscle disease, central nervous system disease, heart disease Various diseases such as Therefore, the protein of the present invention and the DNA of the present invention are
For example, central nervous system diseases (eg, headache, pain, cerebral lobe dysfunction, movement disorders, multiple sclerosis, schizophrenia, dementia, etc.), cerebrovascular diseases (eg, ischemic syndrome, hemorrhagic syndrome, etc.) ), Muscle diseases, heart diseases and the like, and can be used as a medicine for treating or preventing diseases. Preferred is a therapeutic / preventive agent for diseases such as central nervous system diseases and cerebrovascular diseases. For example, when there is a patient whose Ca ²⁺ ion permeation is not sufficiently or normally exerted due to the decrease or deficiency of the protein of the present invention in vivo, (a) the DNA of the present invention By expressing the protein of the present invention in vivo,
(B) by inserting the DNA of the present invention into a cell and expressing the protein of the present invention, and then transplanting the cell into a patient, or (c) administering the protein of the present invention to the patient, etc. The role of the protein of the present invention in the patient can be fully or normally exerted. When the DNA of the present invention is used as the above-mentioned therapeutic / prophylactic 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 therapeutic / prophylactic agent, at least 90
%, Preferably 95% or more, more preferably 98% or more, still more 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. It can be manufactured by The amount of active ingredient in these preparations is such that a suitable dose within the indicated range can be obtained. Additives that can be mixed with tablets, capsules, etc. include, for example, binders such as gelatin, corn starch, tragacanth, acacia, excipients such as crystalline cellulose, corn starch, gelatin, alginic acid, etc. Puffing agents, lubricants such as magnesium stearate, sweeteners such as sucrose, lactose or saccharin, flavoring agents such as peppermint, red oil or cherry and the like are used. When the unit dosage form is a capsule, a liquid carrier such as fat may be included in addition to materials of the above type. Sterile compositions for injection can be formulated according to conventional pharmaceutical practices such as dissolving or suspending the active substance in a vehicle such as water for injection, naturally occurring vegetable oils such as sesame oil, coconut oil and the like. Examples of the aqueous solution for injection include physiological saline, isotonic solution containing glucose and other adjuvants (for example, D-sorbitol, D-mannitol, sodium chloride, etc.), and a suitable solubilizing agent. For example, it may be used in combination with alcohol (eg, ethanol), polyalcohol (eg, propylene glycol, polyethylene glycol, etc.), nonionic surfactant (eg, Polysorbate 80 ^™ , HCO-50, etc.). Examples of the oily liquid include sesame oil, soybean oil and the like, which may be used in combination with solubilizing agents such as benzyl benzoate and benzyl alcohol. In addition, buffers (eg, phosphate buffer, sodium acetate buffer, etc.), soothing agents (eg, benzalkonium chloride, procaine hydrochloride, etc.), stabilizers (eg, human serum albumin, polyethylene glycol, etc.), Preservatives (eg,
(Benzyl alcohol, phenol, etc.), antioxidant, etc. The prepared injection solution is usually filled in a suitable ampoule. The vector into which the DNA of the present invention has been inserted is also formulated as in the above, and is usually used parenterally.

The preparation thus obtained is safe and has low toxicity. Dog, monkey, chimpanzee, etc.). The dose of the protein or the like of the present invention varies depending on the target disease, the administration subject, the administration route, etc., but when the protein or the like of the present invention is orally administered for the purpose of treating muscle disease, it is generally adult (60 kg In the above), about 0.1 mg to 100 mg, preferably about 1.0
50 mg, more preferably about 1.0-20 mg is administered. When administered parenterally, the single dose of the protein or the like varies 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 (with a body weight of 60 kg) in the form of an injection for the purpose of treating muscle diseases, the protein or the like is administered in an amount of about 0.01 to 30 mg per day.
Conveniently, about 0.1 to 20 mg, more preferably about 0.1 to 10 mg, is administered by injecting into the affected area. 60 for other animals
An amount converted per 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. That is, the present invention is
(1) A compound or a salt thereof that promotes or inhibits the activity of the protein of the present invention (for example, Ca ²⁺ ion permeation activity), which is characterized by using the protein of the present invention (hereinafter, a promoter and an inhibitor, respectively) May be abbreviated)), more specifically,
For example, (2) (i) Ca ²⁺ ion permeation activity of cells capable of producing the protein of the present invention and (ii) Ca ² of a mixture of cells and a test compound capable of producing the protein of the present invention. Provided is a method for screening a promoter or an inhibitor, which comprises comparing the permeability of ⁺ ions. Specifically, in the above screening method, for example, in the cases of (i) and (ii), C
a ²⁺ ion current patch clamp method (eg FEBS Lette
rs, 470, 189-197, 2000 etc.)
FLIPR for comparison as an index of a ²⁺ ion permeation activity
(Molecular Device) and FLIPR MembraneP
An otential Assay kit (manufactured by Molecular Devices) is used to compare the changes in the membrane potential caused by the permeation of Ca ²⁺ ions as an index.

Examples of test compounds include peptides,
Examples include proteins, non-peptidic compounds, synthetic compounds, fermentation products, cell extracts, plant extracts, animal tissue extracts, etc. 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 may be used as long as it does not inhibit the Ca ²⁺ ion permeation activity of the protein of the present invention, such as a phosphate buffer or a borate buffer (preferably having a pH of about 6 to 8). 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 Ca ²⁺ ion permeation activity of the protein of the present invention can be determined by a known method, for example, J. Biol. Chem. 275: 12
It can be measured according to the method described in 237-12242, 2000 or a method similar thereto. For example, above (ii)
The Ca ²⁺ ion permeation activity in the case of
In comparison with the above case, a test compound that promotes about 20% or more, preferably 30% or more, more preferably about 50% or more can be selected as a compound or a salt thereof that promotes the activity of the protein of the present invention. Further, for example, the Ca ²⁺ ion permeation activity in the case of (ii) above is about 20% or more, preferably 30% or more, as compared with the case of (i) above.
A test compound that inhibits (or suppresses) by% or more, more preferably about 50% or more can be selected as a compound that inhibits the activity of the protein of the present invention or a salt thereof.
In addition, a gene such as secretory alkaline phosphatase or luciferase is inserted downstream of the promoter of the protein voltage-gated Ca ²⁺ channel γ subunit homolog gene of the present invention, and the gene is expressed in the various cells described above, and the test compound is added to the cells. The expression or expression of the protein of the present invention (potential difference-dependent Ca ²⁺ channel γ subunit homolog) is promoted or suppressed by searching for a compound or a salt thereof that activates or inhibits the enzyme activity when contacted (ie, the present invention). (Promoting or inhibiting the activity of the protein of 1.) or a salt thereof can be screened.

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 having the ability to produce 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, a peptide, protein, non-peptide compound, synthetic compound, fermentation product, cell extract, A compound or a salt thereof selected from a plant extract, an animal tissue extract, plasma, etc., which promotes or inhibits the activity (eg, Ca ²⁺ ion permeation activity) of the protein of the present invention or a salt thereof. is there. 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 its salt that promotes the activity of the protein of the present invention is, for example, a central nervous system disease (eg,
Treatment for headache, pain, cerebral lobe dysfunction, movement disorder, multiple sclerosis, schizophrenia, dementia, etc., cerebrovascular disease (eg, ischemic syndrome, hemorrhagic syndrome, etc.), muscle disease, heart disease -Useful as a medicine such as preventive agent. Also,
The compound or its salt that inhibits the activity of the protein of the present invention is, for example, a central nervous system disease (eg, headache, pain, cerebral lobe dysfunction, movement disorder, multiple sclerosis, schizophrenia, dementia, etc.) , Cerebrovascular diseases (eg, ischemic syndrome, hemorrhagic syndrome, etc.), muscle diseases, heart diseases, and other therapeutic / preventive agents.

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 therapeutic / prophylactic 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 warm-blooded animals (for example, mouse, rat, rabbit, sheep, pig, cow, horse, bird, cat, dog, monkey, chimpanzee, etc.) ) Orally or parenterally. The dose of the compound or its salt varies depending on its action, target disease, administration subject, administration route, etc., but for example, for the purpose of treating muscle disease, the compound or its salt that promotes the activity of the protein of the present invention In the case of oral administration, generally, in an adult person (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 mg per day.
More preferably, about 1.0 to 20 mg is administered. 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 muscle 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. About 0.
It is convenient to administer about 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 An antibody against the protein of the present invention (hereinafter sometimes abbreviated as antibody of the present invention) specifically recognizes the protein of the present invention Therefore, it can be used for quantification of the protein of the present invention in a test solution, particularly for quantification by sandwich immunoassay. That is, the present invention is
(I) An antibody of the present invention is allowed to competitively react with a test solution and a labeled protein of the present invention, and the ratio of the labeled protein of the present invention bound to the antibody is measured. And a method for quantifying the protein of the present invention in a test solution, and (ii) the test solution and the antibody of the present invention insolubilized on a carrier and another labeled antibody of the present invention are simultaneously or consecutively There is provided a method for quantifying the protein of the present invention in a test solution, which comprises measuring the activity of a labeling agent on an insolubilized carrier after the reaction. In the quantification method 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 (ab ′) ₂ of the antibody molecule may be used.
, 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 not particularly limited, and may include an antibody, an antigen or an antibody-antigen complex corresponding to the amount of antigen (for example, the amount of protein) in the liquid to be measured. Any measuring method may be used as long as it is a method of detecting the amount by chemical or physical means and calculating it from a standard curve prepared using a standard solution containing a known amount of antigen. For example, nephrometry, competitive method,
Although the immunometric method and the sandwich method are preferably used, the sandwich method described below is particularly preferably used in terms of sensitivity and specificity. 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 radioisotopes include [ ¹²⁵ I], [ ¹³¹ I],
[ ³ H], [ ¹⁴ C] or the like is 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. Luminol, a luminol derivative, luciferin, lucigenin, etc. are used as a luminescent substance, for example. Furthermore, the biotin-avidin system can be used for binding the antibody or the antigen to the labeling agent.

For the insolubilization of 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 is reacted.
(F) and the labeled antigen (B) bound to the antibody are separated (B
/ F separation), the labeled amount of either B or F is measured to quantify the amount of antigen in the test liquid. In this reaction method, a soluble antibody is used as an antibody, and B / F separation is performed using polyethylene glycol,
A liquid phase method using a second antibody or the like against the antibody, and using a solid phase antibody 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, Hiroshi Irie "Radioimmunoassay" (Kodansha, published in 1974), Hiroshi Irie "Radioimmunoassay" (Kodansha, published in 1979), Eiji Ishikawa et al. "Enzyme Immunoassay" (Medical Shoin, Showa Showa) 1993), Eiji Ishikawa et al., "Enzyme Immunoassay" (second edition) (Medical Shoin, published in 1982), Eiji Ishikawa, et al. "Enzyme Immunoassay" (3rd edition) (Medicine Shoin, Showa) 1987), "Methods in ENZYMOLOGY" Vol. 70 (Immunochem
ical Techniques (Part A)), ibid Vol. 73 (Immunochem
ical Techniques (Part B)), ibid Vol. 74 (Immunochem
ical Techniques (Part C)), ibid Vol. 84 (Immunochem
ical Techniques (Part D: Selected Immunoassays)),
Ibid Vol. 92 (Immunochemical Techniques (Part E: Mono
clonal Antibodies and General Immunoassay Method
s)), ibid Vol. 121 (Immunochemical Techniques (Part
I: Hybridoma Technology and Monoclonal Antibodie
s)) (above, published by Academic Press). As described above, the protein of the present invention can be quantified with high sensitivity by using the antibody of the present invention. Furthermore, by quantifying the concentration of the protein of the present invention using the antibody of the present invention, (1)
When a decrease in the concentration of the protein of the present invention is detected, for example, central nervous system diseases (eg, headache, pain, cerebral lobe dysfunction, movement disorders, multiple sclerosis, schizophrenia, dementia, etc.), It can be diagnosed that there is a high possibility of cerebrovascular disease (eg, ischemic syndrome, hemorrhagic syndrome, etc.), muscle disease, heart disease and the like. (2) On the contrary, for example, when an increase in the concentration of the protein of the present invention is detected, a central nervous system disease (eg, headache, pain, cerebral lobe dysfunction, movement disorder, multiple sclerosis, schizophrenia) Disease, dementia, etc.), cerebrovascular disease (eg, ischemic syndrome, hemorrhagic syndrome, etc.), muscle disease,
It can be diagnosed as having a high possibility of heart disease. 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, central nervous system diseases (eg, headache, pain, cerebral lobe dysfunction, movement disorder, multiple sclerosis, schizophrenia, dementia, etc.), cerebrovascular disease It can be diagnosed as having a high possibility of diseases (eg, ischemic syndrome, hemorrhagic syndrome, etc.), muscle diseases, heart diseases and the like. On the contrary, when decreased expression is detected or a DNA mutation is detected by the PCR-SSCP method, for example, a central nervous system disease (eg, headache, pain,
It may be cerebral lobe dysfunction, movement disorder, multiple sclerosis, schizophrenia, dementia, etc., cerebrovascular disease (eg, ischemic syndrome, hemorrhagic syndrome, etc.), muscle disease, heart disease, etc. It can be diagnosed as high.

[5] Medicament Containing Antisense Nucleotide The antisense nucleotide of the present invention, which can bind to the DNA of the present invention complementarily and suppress the expression of the DNA, has low toxicity. Since the function of the protein of the present invention or the DNA of the present invention (eg, Ca ²⁺ ion permeation activity) can be suppressed, for example, central nervous system diseases (eg, headache, pain, cerebral lobe dysfunction, exercise) It can be used as a therapeutic / preventive agent for disorders, multiple sclerosis, schizophrenia, dementia, etc., cerebrovascular disease (eg, ischemic syndrome, hemorrhagic syndrome, etc.), muscle disease, heart disease and the like. When the above antisense nucleotide is used as the above-mentioned therapeutic / prophylactic 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. Animals (eg, rat,
Rabbit, sheep, pig, cow, cat, dog, monkey, etc.)
Can be administered orally or parenterally. 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. The dose of the antisense nucleotide varies depending on the target disease, the administration subject, the administration route, etc., but, for example, when the antisense nucleotide of the present invention is locally administered to the liver for the purpose of treating muscle disease, it is generally Adult (weight 60k
In g), about 0.1 to 100 mg of the antisense nucleotide is administered per day. Further, the antisense nucleotide can be used as D of the present invention in tissues or cells.
It can also be used as a diagnostic oligonucleotide probe for examining the presence of NA and its expression status.

Furthermore, 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 There is also provided a ribozyme containing a part of RNA encoding ribozyme, a drug containing the ribozyme, an expression vector containing a gene (DNA) encoding the 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 it is possible to suppress the function of the protein used in the present invention or the DNA used in the present invention in vivo, for example, central nervous system diseases (eg, headache, pain, cerebral lobe dysfunction, movement disorder, multiple occurrence) It can be used as a prophylactic / therapeutic agent for sclerosis, schizophrenia, dementia, etc., cerebrovascular disease (eg, ischemic syndrome, hemorrhagic syndrome, etc.), muscle disease, heart disease. Double-stranded RNA is prepared according to a known method (eg, Nature, 411, 494, 2001),
It can be designed and manufactured based on the sequence of the polynucleotide of the present invention. Ribozymes can be prepared by known methods (eg, TREN
DS in Molecular Medicine, Volume 7, 221 pages, 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 sequence near a consensus sequence NUX (wherein N represents all bases, X represents a base other than G) that can be cleaved by a known ribozyme, etc. Can be mentioned. When the above-mentioned double-stranded RNA or ribozyme is used as the above-mentioned preventive / therapeutic agent, it can be formulated and administered in the same manner as the antisense polynucleotide. 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.

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

The 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] This shows the amino acid sequence of human TCH053 protein. [SEQ ID NO: 2] DNA encoding TCH053 protein containing the amino acid sequence represented by SEQ ID NO: 1
The base sequence of is shown. [SEQ ID NO: 3] Primer A7 used in Example 2
The base sequence of is shown. [SEQ ID NO: 4] Primer B5 used in Example 2
The base sequence of is shown. [SEQ ID NO: 5] This shows the base sequence of TaqMan probe T1 used in Example 2. [SEQ ID NO: 6] Primer A3 used in Example 1
The base sequence of is shown. [SEQ ID NO: 7] Primer B4 used in Example 1
The base sequence of is shown. [SEQ ID NO: 8] Primer SP used in Example 1
6 shows the base sequence of 6. [SEQ ID NO: 9] Primer T7 used in Example 1
The base sequence of is shown. [SEQ ID NO: 10] Primer A used in Example 1
2 shows the base sequence of 2. [SEQ ID NO: 11] Primer B 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 R used in Example 1
1 shows the base sequence of 1. [SEQ ID NO: 14] This shows the base sequence of cDNA containing TCH053 full-length gene obtained in Example 1. [SEQ ID NO: 15] This shows the amino acid sequence of TCH053 protein of 333 amino acids obtained in Example 1. [SEQ ID NO: 16] D encoding TCH053 protein containing the amino acid sequence represented by SEQ ID NO: 15
The base sequence of NA is shown.

The transformant Escherichia coli obtained in Example 1 described below was used.
DH10B / pCR-BluntII-TCH053
Since July 16, 2001, the deposit number FERM BP-7665 has been assigned to the Japan Patent Organism Depositary Center, National Institute of Advanced Industrial Science and Technology, Central Research Institute No. 1 (1), 1-1, Higashi, Tsukuba, Ibaraki Prefecture (postal code 305-8566). 2001
2-17-, Jusohonmachi, Yodogawa-ku, Osaka-shi, Osaka from July 3, 2014
It has been deposited with the Fermentation Research Institute (IFO) of 85 (postal code 532-8686) under the deposit number IFO 16674.

[0059]

EXAMPLES The present invention will be described in more detail with reference to the following examples.
However, these do not limit the scope of the invention.
Absent. In addition, the gene manipulation method using E. coli is
Described in Molecular Cloning
I followed the method. Example 1 Cloning of human TCH053 gene cDNA Two kinds of primer DNA, primer A3 (SEQ ID NO:
6) and primer B4 (SEQ ID NO: 7),
P for human brain cDNA library (Takara Shuzo)
Perform CR and plasmid clone pCR-BluntI
Two clones # 1 and # 2 of I-TCH053 were obtained.
This is used as a primer DNA [primer SP6 (sequence number
No .: 8), primer T7 (SEQ ID NO: 9), primer
-A2 (SEQ ID NO: 10), primer B1 (SEQ ID NO:
No .: 11), primer F2 (SEQ ID NO: 12), plastic
Immer R1 (SEQ ID NO: 13)] and BigDye
Terminator Cycle Sequenci
For ng Kit (manufactured by Applied Biosystems)
Reaction is carried out to determine the base sequence of the inserted cDNA fragment.
Rows of DNA sequencer ABI PRISM 370
0 DNA Analyzer (Applied Biosystems)
Manufactured). As a result, 2 clones obtained
Contains the same DNA fragment and has 1400 nucleotide sequences
(SEQ ID NO: 14). In this cDNA fragment
Encodes a 275 amino acid sequence (SEQ ID NO: 1)
(SEQ ID NO: 2) and contains this amino acid sequence
Is designated as human TCH053 protein.
It was In addition, the start of SEQ ID NO: 2 was added to this cDNA fragment.
The start codon in the same reading frame on the 5'side of the codon
Is present and is translated 33
It also encodes the three amino acid sequence (SEQ ID NO: 15).
(SEQ ID NO: 16). Plus containing the above cDNA fragment
The transformant containing the amide was transformed into Escherichia coli (Es
checkeria coli) DH10B / pCR-
It was named BluntII-TCH053. Blast
  P [Nucleic Acid Research (Nucle
ic Acids Res.), 25, 3389.
(1997)] for homology
When searched, the base sequence represented by SEQ ID NO: 14
CDNA containing is a voltage-dependent Ca²⁺Channel γ
A novel gene belonging to a subunit, which is a 4-transmembrane type
It was assumed to have the structure of Human TCH053 is
Voltage-dependent Ca reported in G.²⁺Channel gamma sub
The unit CACNG7 [Genomics (Genom
ics), Vol. 71, p. 339 (2001)], and the end of N
End (from the start codon of CACNG7 to the fourth transmembrane region)
Has a high homology, but is not found in CACNG7.
It had a non-C-terminal cytoplasmic region. CACN
Figure 1 shows a comparison of the amino acid sequences of G7 and human TCH053.
Show. In the figure, CACNG7 is the potential difference reported in humans.
Survival Ca ²⁺Shows the channel γ subunit, TCH05
3 shows the human-derived protein of the present invention. Also transmembrane
Areas are indicated by TM1 to TM4. In the amino acid sequence
The homology was 69.5%.

Example 2 Analysis of tissue distribution of human TCH053 gene product Two primers D designed from the sequence of TCH053
NA, primer A7 (SEQ ID NO: 3) and primer B5 (SEQ ID NO: 4), and TaqMan probe T1
(SEQ ID NO: 5) and each human tissue (heart,
(Brain, placenta, lung, liver, skeletal muscle, kidney, pancreas, spleen, thymus, prostate, testis, ovary, small intestine, large intestine, peripheral blood leukocyte)
The expression level of TCH053 in the cDNA (Human MTC panel I and Human MTC panel II: Clontech) of
It was measured by n PCR. Reaction is TaqMan Gold RT-PCR
AB using the kit (manufactured by Applied Biosystems)
I PRISM 7700 sequence detection system (manufactured by Applied Biosystems) at 50 ° C. for 2 minutes, 95 ° C. for 10 minutes, then 95 ° C. for 15 seconds, 60 ° C.
1 minute is 1 reaction cycle and 40 cycles are repeated.
Detection was performed at the same time. The results are shown in Figure 2. Expression level is cd
It was represented by the copy number per 1 μl of NA solution. TCH05
Three gene products (mRNA) were slightly expressed in placenta, lung, liver, pancreas, thymus, prostate, testis, ovary, large intestine, and peripheral blood leukocytes, and slightly expressed in small intestine, but most strongly expressed in brain. Indicated.

[0061]

EFFECTS OF 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 is a central nervous system disease (eg, headache, pain, cerebral lobe dysfunction, movement disorder). , Multiple sclerosis, schizophrenia, dementia, etc., useful as a diagnostic marker for cerebrovascular disease (eg, ischemic syndrome, hemorrhagic syndrome, etc.), muscle disease, heart disease, etc., and screening using the protein Compounds that promote or inhibit the activity of the protein obtained by the method include, for example, central nervous system diseases (eg, headache, pain, cerebral lobe dysfunction, movement disorder, multiple sclerosis, schizophrenia, dementia, etc.) ), Cerebrovascular disease (eg, ischemic syndrome, hemorrhagic syndrome, etc.), muscle disease, heart disease, and other diseases.

[0062]

[Sequence list]                                SEQUENCE LISTING <110> Takeda Chemical Industries, Ltd. <120> Novel Protein and its DNA <130> P2001-022A <140> <141> <150> JP 2001-29443 <151> 2001-2-6 <160> 16 <170> PatentIn Ver. 2.1 <210> 1 <211> 275 <212> PRT <213> Human <400> 1 Met Ser His Cys Ser Ser Arg Ala Leu Thr Leu Leu Ser Ser Val Phe   1 5 10 15 Gly Ala Cys Gly Leu Leu Leu Val Gly Ile Ala Val Ser Thr Asp Tyr              20 25 30 Trp Leu Tyr Met Glu Glu Gly Thr Val Leu Pro Gln Asn Gln Thr Thr          35 40 45 Glu Val Lys Met Ala Leu His Ala Gly Leu Trp Arg Val Cys Phe Phe      50 55 60 Ala Gly Arg Glu Lys Gly Arg Cys Val Ala Ser Glu Tyr Phe Leu Glu  65 70 75 80 Pro Glu Ile Asn Leu Val Thr Glu Asn Thr Glu Asn Ile Leu Lys Thr                  85 90 95 Val Arg Thr Ala Thr Pro Phe Pro Met Val Ser Leu Phe Leu Val Phe             100 105 110 Thr Ala Phe Val Ile Ser Asn Ile Gly His Ile Arg Pro Gln Arg Thr         115 120 125 Ile Leu Ala Phe Val Ser Gly Ile Phe Phe Ile Leu Ser Gly Leu Ser     130 135 140 Leu Val Val Gly Leu Val Leu Tyr Ile Ser Ser Ile Asn Asp Glu Val 145 150 155 160 Met Asn Arg Pro Ser Ser Ser Glu Gln Tyr Phe His Tyr Arg Tyr Gly                 165 170 175 Trp Ser Phe Ala Phe Ala Ala Ser Ser Phe Leu Leu Lys Glu Gly Ala             180 185 190 Gly Val Met Ser Val Tyr Leu Phe Thr Lys Arg Tyr Ala Glu Glu Glu         195 200 205 Met Tyr Arg Pro His Pro Ala Phe Tyr Arg Pro Arg Leu Ser Asp Cys     210 215 220 Ser Asp Tyr Ser Gly Gln Phe Leu Gln Pro Glu Ala Trp Arg Arg Gly 225 230 235 240 Arg Ser Pro Ser Asp Ile Ser Ser Asp Val Ser Ile Gln Met Thr Gln                 245 250 255 Asn Tyr Pro Pro Ala Ile Lys Tyr Pro Asp His Leu His Ile Ser Thr             260 265 270 Ser Pro Cys         275 <210> 2 <211> 825 <212> DNA <213> Human <400> 2 atgagtcact gcagcagccg cgccctgacc ctgctgagca gcgtgtttgg tgcgtgtggc 60 ctgctcctgg taggcatcgc ggtcagcact gactactggc tgtacatgga agaaggcaca 120 gtgctaccgc agaaccagac caccgaggtc aagatggccc tgcacgccgg cctctggcga 180 gtctgcttct ttgcaggtcg ggagaaaggt cgctgtgtgg cctcagaata ttttcttgaa 240 ccggagatca atttggtgac ggaaaacacg gagaatattc tgaagacagt gcgcacggcc 300 acccccttcc ccatggtcag cctcttcctc gtgttcacgg ccttcgtcat cagcaacatc 360 ggccacatcc gcccgcagag gaccattctg gcttttgtct ctggcatctt cttcatacta 420 tcgggcctct ccttggtggt gggcttggtt ctttacatct ccagcatcaa cgacgaggtc 480 atgaacaggc ccagcagctc tgagcagtat tttcattatc gctacgggtg gtcttttgcc 540 ttcgccgctt cctccttcct actcaaagag ggggccggcg tgatgtccgt gtacctgttc 600 accaagcgct acgcggagga ggagatgtac cgtccacacc cggccttcta ccgcccgcgt 660 ctcagcgact gctccgacta ctcgggccag ttcctgcagc ccgaggcgtg gcgccgcggc 720 cggagcccct ccgacatctc cagcgacgtg tccatccaaa tgacgcagaa ctaccctccc 780 gccatcaagt acccggacca cctgcacatc tccacctcgc cctgc 825 <210> 3 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Primer A7 used in Example 2 <400> 3 gcgtgatgtc cgtgtacctg 20 <210> 4 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Primer B5 used in Example 2 <400> 4 cgggtgtgga cggtacatct 20 <210> 5 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> TaqMan Probe T1 used in Example 2 <400> 5 tcaccaagcg ctacgcggag g 21 <210> 6 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Primer A3 used in Example 1 <400> 6 tgggcctggg ctgaaagttg tgat 24 <210> 7 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Primer B4 used in Example 1 <400> 7 aaggggagga gtcatttggg agtg 24 <210> 8 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> Primer SP6 used in Example 1 <400> 8 atttaggtga cactatag 18 <210> 9 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> Primer T7 used in Example 1 <400> 9 aatacgactc actataggg 19 <210> 10 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Primer A2 used in Example 1 <400> 10 catggaagaa ggcacagtgc 20 <210> 11 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Primer B1 used in Example 1 <400> 11 gttgctgatg acgaaggccg tgaa 24 <210> 12 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Primer F2 used in Example 1 <400> 12 ttcgccgctt cctccttcct a 21 <210> 13 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Primer R1 used in Example 1 <400> 13 gggagacccc ctaagacgag 20 <210> 14 <211> 1400 <212> DNA <213> Human <400> 14 tgggcctggg ctgaaagttg tgattagaaa tctaattctg gggttgggga tctggatctg 60 gagctagacc acaggcagca ggtttgggga gcccagccct gaggctgagg ctcaacagtt 120 ggtgtctctg gtcagactct agggttgggg tcatgggtca ggccacggag gtcagatctg 180 agatttcgat ttgggagtca gtgtctctgg ctagggccca gcatcccggg ttgctgcatg 240 gggtcaagca ctctggtcgg tcccatggag ctctgcactg tagcttccct tccacaggcc 300 ccgcagggcg ccccctgcct ctgaggatga gtcactgcag cagccgcgcc ctgaccctgc 360 tgagcagcgt gtttggtgcg tgtggcctgc tcctggtagg catcgcggtc agcactgact 420 actggctgta catggaagaa ggcacagtgc taccgcagaa ccagaccacc gaggtcaaga 480 tggccctgca cgccggcctc tggcgagtct gcttctttgc aggtcgggag aaaggtcgct 540 gtgtggcctc agaatatttt cttgaaccgg agatcaattt ggtgacggaa aacacggaga 600 atattctgaa gacagtgcgc acggccaccc ccttccccat ggtcagcctc ttcctcgtgt 660 tcacggcctt cgtcatcagc aacatcggcc acatccgccc gcagaggacc attctggctt 720 ttgtctctgg catcttcttc atactatcgg gcctctcctt ggtggtgggc ttggttcttt 780 acatctccag catcaacgac gaggtcatga acaggcccag cagctctgag cagtattttc 840 attatcgcta cgggtggtct tttgccttcg ccgcttcctc cttcctactc aaagaggggg 900 ccggcgtgat gtccgtgtac ctgttcacca agcgctacgc ggaggaggag atgtaccgtc 960 cacacccggc cttctaccgc ccgcgtctca gcgactgctc cgactactcg ggccagttcc 1020 tgcagcccga ggcgtggcgc cgcggccgga gcccctccga catctccagc gacgtgtcca 1080 tccaaatgac gcagaactac cctcccgcca tcaagtaccc ggaccacctg cacatctcca 1140 cctcgccctg ctgaggcccg cccctcggag ctccccctgc ctcctcctcc tcctcgtctt 1200 agggggtctc cctgcaatgc agcgccccct tccgtcctcg ggactcctcg ctcccacccg 1260 gaggaggctg cgccagcttt aggccccgcc ctcctcccaa tggctccgcc cacagactcc 1320 cttatttcaa tggccgcgcc ctcttttccc gacctctcct tttcattggt ccctctcact 1380 cccaaatgac tcctcccctt 1400 <210> 15 <211> 333 <212> PRT <213> Human <400> 15 Met Gly Gln Ala Thr Glu Val Arg Ser Glu Ile Ser Ile Trp Glu Ser                  5 10 15 Val Ser Leu Ala Arg Ala Gln His Pro Gly Leu Leu His Gly Val Lys              20 25 30 His Ser Gly Arg Ser His Gly Ala Leu His Cys Ser Phe Pro Ser Thr          35 40 45 Gly Pro Ala Gly Arg Pro Leu Pro Leu Arg Met Ser His Cys Ser Ser      50 55 60 Arg Ala Leu Thr Leu Leu Ser Ser Val Phe Gly Ala Cys Gly Leu Leu  65 70 75 80 Leu Val Gly Ile Ala Val Ser Thr Asp Tyr Trp Leu Tyr Met Glu Glu                  85 90 95 Gly Thr Val Leu Pro Gln Asn Gln Thr Thr Glu Val Lys Met Ala Leu             100 105 110 His Ala Gly Leu Trp Arg Val Cys Phe Phe Ala Gly Arg Glu Lys Gly         115 120 125 Arg Cys Val Ala Ser Glu Tyr Phe Leu Glu Pro Glu Ile Asn Leu Val     130 135 140 Thr Glu Asn Thr Glu Asn Ile Leu Lys Thr Val Arg Thr Ala Thr Pro 145 150 155 160 Phe Pro Met Val Ser Leu Phe Leu Val Phe Thr Ala Phe Val Ile Ser                 165 170 175 Asn Ile Gly His Ile Arg Pro Gln Arg Thr Ile Leu Ala Phe Val Ser             180 185 190 Gly Ile Phe Phe Ile Leu Ser Gly Leu Ser Leu Val Val Gly Leu Val         195 200 205 Leu Tyr Ile Ser Ser Ile Asn Asp Glu Val Met Asn Arg Pro Ser Ser     210 215 220 Ser Glu Gln Tyr Phe His Tyr Arg Tyr Gly Trp Ser Phe Ala Phe Ala 225 230 235 240 Ala Ser Ser Phe Leu Leu Lys Glu Gly Ala Gly Val Met Ser Val Tyr                 245 250 255 Leu Phe Thr Lys Arg Tyr Ala Glu Glu Glu Met Tyr Arg Pro His Pro             260 265 270 Ala Phe Tyr Arg Pro Arg Leu Ser Asp Cys Ser Asp Tyr Ser Gly Gln         275 280 285 Phe Leu Gln Pro Glu Ala Trp Arg Arg Gly Arg Ser Pro Ser Asp Ile     290 295 300 Ser Ser Asp Val Ser Ile Gln Met Thr Gln Asn Tyr Pro Pro Ala Ile 305 310 315 320 Lys Tyr Pro Asp His Leu His Ile Ser Thr Ser Pro Cys                 325 330 <210> 16 <211> 999 <212> DNA <213> Human <400> 16 atgggtcagg ccacggaggt cagatctgag atttcgattt gggagtcagt gtctctggct 60 agggcccagc atcccgggtt gctgcatggg gtcaagcact ctggtcggtc ccatggagct 120 ctgcactgta gcttcccttc cacaggcccc gcagggcgcc ccctgcctct gaggatgagt 180 cactgcagca gccgcgccct gaccctgctg agcagcgtgt ttggtgcgtg tggcctgctc 240 ctggtaggca tcgcggtcag cactgactac tggctgtaca tggaagaagg cacagtgcta 300 ccgcagaacc agaccaccga ggtcaagatg gccctgcacg ccggcctctg gcgagtctgc 360 ttctttgcag gtcgggagaa aggtcgctgt gtggcctcag aatattttct tgaaccggag 420 atcaatttgg tgacggaaaa cacggagaat attctgaaga cagtgcgcac ggccaccccc 480 ttccccatgg tcagcctctt cctcgtgttc acggccttcg tcatcagcaa catcggccac 540 atccgcccgc agaggaccat tctggctttt gtctctggca tcttcttcat actatcgggc 600 ctctccttgg tggtgggctt ggttctttac atctccagca tcaacgacga ggtcatgaac 660 aggcccagca gctctgagca gtattttcat tatcgctacg ggtggtcttt tgccttcgcc 720 gcttcctcct tcctactcaa agagggggcc ggcgtgatgt ccgtgtacct gttcaccaag 780 cgctacgcgg aggaggagat gtaccgtcca cacccggcct tctaccgccc gcgtctcagc 840 gactgctccg actactcggg ccagttcctg cagcccgagg cgtggcgccg cggccggagc 900 ccctccgaca tctccagcga cgtgtccatc caaatgacgc agaactaccc tcccgccatc 960 aagtacccgg accacctgca catctccacc tcgccctgc 999

[Brief description of drawings]

FIG. 1 shows a comparison of the amino acid sequences of CACNG7 and human TCH053.

FIG. 2 shows the expression level of human TCH053 gene in each human tissue.

─────────────────────────────────────────────────── ─── Continuation of front page (51) Int.Cl. ⁷ Identification code FI theme code (reference) A61P 21/00 A61P 25/04 4C085 25/00 25/06 4H045 25/04 25/18 25/06 25 / 28 25/18 C07K 14/47 25/28 16/18 C07K 14/47 C12N 1/15 16/18 1/19 C12N 1/15 1/21 1/19 C12P 21/02 C 1/21 G01N 33/15 Z 5/10 33/50 Z C12P 21/02 A61K 39/395 D G01N 33/15 N 33/50 C12N 15/00 ZNAA // A61K 39/395 5/00 A A61K 37/02 F term (reference) 2G045 AA40 BB20 CA25 CA26 CB03 CB21 DA12 DA13 DA14 DA36 FB02 FB03 FB04 FB07 FB12 4B024 AA01 AA11 BA80 CA04 DA02 DA05 DA06 DA11 DA12 EA02 EA04 GA11 HA11 4B064. AA02 AA03 AA07 AA 13 AA17 BA01 BA02 BA22 NA14 ZA022 ZA082 ZA152 ZA182 ZA362 ZA942 4C085 AA13 AA14 DD62 4H045 AA10 AA11 AA20 AA30 BA10 CA40 DA75 EA21 EA50 FA72 FA74

Claims (21)

[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 a salt thereof. 3. The protein according to claim 1, which comprises the amino acid sequence represented by SEQ ID NO: 15, 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 5, which contains the base sequence represented by SEQ ID NO: 2. 8. The DNA according to claim 5, which contains the base sequence represented by SEQ ID NO: 16. 9. The DNA according to claim 5, which contains the nucleotide sequence represented by SEQ ID NO: 14. 10. A recombinant vector containing the DNA according to claim 6. 11. A transformant transformed with the recombinant vector according to claim 10. 12. The method according to claim 1, wherein the transformant according to claim 11 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. 13. A pharmaceutical comprising the protein according to claim 1, the partial peptide according to claim 4, or a salt thereof. 14. A medicine comprising the DNA according to claim 6. 15. An antibody against the protein according to claim 1, the partial peptide according to claim 4, or a salt thereof. 16. The activity of the protein according to claim 1, the partial peptide according to claim 4, or the salt thereof, which comprises 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. 17. The activity of the protein according to claim 1, the partial peptide according to claim 4, or the partial peptide or 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. 18. The activity of the protein according to claim 1, the partial peptide according to claim 4, or the partial peptide obtained by using the screening method according to claim 16 or the screening kit according to claim 17, is promoted or inhibited. Or a salt thereof. 19. The protein according to claim 1 or the protein according to claim 1 obtained by using the screening method according to claim 16 or the screening kit according to claim 17.
A pharmaceutical comprising a compound or a salt thereof that promotes or inhibits the activity of the partial peptide or the salt thereof. 20. The medicine according to claim 13 or 14, which is a prophylactic / therapeutic agent for central nervous system diseases or cerebrovascular diseases. 21. The medicine according to claim 19, which is a prophylactic / therapeutic agent for central nervous system diseases or cerebrovascular diseases.