JP2002348299A - Irap binding protein - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain an IRAP binding protein or the like. SOLUTION: The IRAP binding protein, a medicine containing the protein, a screening method for a compound inhibiting the bond between the protein and the IRAP, and the compound obtained by the screening method, and the like can be obtained. This protein is useful for a preventive or therapeutic agent of a disease such as hypoglycemia and a reagent for a screening of a compound inhibiting the binding of the protein and an IRAP(insulin responsive aminopeptidase) or a GLUT4(glucose transporter 4), and a compound inhibiting the protein and the IRAP or the GLUT4 is useful for a preventive or therapeutic agent of a disease such as hyperglycemia or diabetes.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The present invention relates to a protein or a salt thereof, which binds to insulin responsive aminopeptidase (IRAP) or glucose transporter 4, and a method using the protein or a salt thereof.
The present invention relates to a method for screening a preventive / therapeutic agent for hyperglycemia or diabetes, a compound obtained by the screening method, use of the compound, and the like.

[0002]

BACKGROUND OF THE INVENTION Blood sugar levels are regulated by the action of insulin and by the uptake of glucose in skeletal muscle and adipose tissue. Diabetes is caused by persistent hyperglycemia due to this reduced effect. In order for glucose to be taken into cells, the intervention of a membrane protein called a glucose transporter (sugar transport carrier) is necessary.
Currently, eight types of glucose transporters, GLUT1-8, are known (Bell et al., J. Bio.
l. Chem., 268, 3352-3356, 1993; Olson & Pessin, An
nu. Rev. Nutr. 16, 235-256, 1996; Ibberson et a
l., J. Biol. Chem., 275, 4607-4612, 2000; Doege et.
al., J. Biol. Chem., 275, 16275-16280). Among them, those strongly involved in the sugar transport activity by insulin are:
GLUT4 mainly expressed in skeletal muscle and adipose tissue (Fukumoto et al., Proc. Natl. Acad. Sci. US
A., 85, 5434-5438, 1988; Birnbaum et al., Cell, 5
7, 305-315, 1989). Normally, GLUT4 is G
Although present in intracellular vesicles called LUT4 endoplasmic reticulum, it is thought that when blood sugar rises, insulin is transferred to the cell membrane (translocation) by the action of insulin to promote glucose uptake (Bell et a).
l., Diabetes Care, 13, 198-208, 1990; Czech et a
l., Trends. Biochem. Sci., 17, 197-201, 1992). In order to elucidate the molecular mechanism of the GLUT4 endoplasmic reticulum transfer, identification of not only GLUT4 itself but also other proteins constituting the GLUT4 endoplasmic reticulum has been performed. At present, as molecules constituting the GLUT4 endoplasmic reticulum, VMAPs (vesicle-assosiated membrane protei
ns; Cain et al., J. Biol. Chem., 267, 11681-1163
4, 1992), SCAMPs (secretory component-assac)
itated membrane proteins; Thiodis et al, J. Biol.
Chem., 268, 11691-11696, 1993; Laurie et al., J. Bi.
ol. Chem, 268, 19110-19117, 1993), phosphatidylin
ositol 4-kinase (Del Vacchio & Pilch, J. Biol. Che
m, 266, 13278-13283, 1991), the low molecular weight GTP-binding protein Rab4 (Cormont et al., J. Biol. Chem.,
268, 1949-19497, 1993) and IRAP (insuli)
n-responsive aminopeptidase; Kandror & Pilch, Pro
c. Natl. Acad. Sci. USA, 91, 8017-8021, 1994, Kand.
ror et al., J. Biol. Chem. 269, 30777-30780, 199
4, Keller et al, J. Biol. Chem., 270, 23612-2361
8, 1995). IRAP is a one-time transmembrane membrane protein also called gp160, and is localized in the GLUT4 endoplasmic reticulum in intracellular organelles. In terms of protein structure, 109 amino acids at the amino terminus (N-terminus) are a cytoplasmic domain, followed by a transmembrane domain of 22 amino acids, and an extracellular domain consisting of 785 amino acids at the carboxy terminus (C-terminus) ( Kandror &
Pilch, Proc. Natl. Acad. Sci. USA, 91, 8017-8021,
1994; Keller et al, J. Biol. Chem., 270, 23612-236.
18, 1995). The extracellular domain is a zinc-dependent protease (aminopeptidase) whose activity has been confirmed (Kandror et al., J. Biol. Chem. 269, 30777-30).
780, 1994). When a peptide corresponding to the N-terminal domain (cytoplasmic domain) of these domains is injected into cells, GLUT4 endoplasmic reticulum is transferred to the cell surface. Is expected to be a protein that binds to IRAP (Walters et al., J. Biol. Chem, 272,
23323-23327, 1997). The cDNA obtained in the present invention is a human very long chain acyl-CoA deh
ydrogenase (VLCAD) sequence (Anders
en et al., Hum. Mol. Benet., 5, 461-472, 1996),
No literature reports that this protein binds to IRAP or reports that it is directly involved in blood glucose regulation.

[0003]

SUMMARY OF THE INVENTION The present invention provides a VLCAD
Provided are a method for screening a compound having a blood glucose lowering effect by utilizing the protein-IRAP protein interaction, and a compound obtained by the screening method.

[0004]

Means for Solving the Problems The present inventors have made intensive studies to solve the above-mentioned problems, and as a result, the yeast two-hybrid
brid method (Fields & Strenglanz, Trends. Genet., 10, 28
6-292, 1994; Brent & Finley, Annu. Rev. Genet., 3
VLCA as an IRAP-binding protein from a human skeletal muscle-derived cDNA library using
D was successfully cloned. The present inventors have further studied and completed the present invention.

That is, the present invention relates to (1) a protein comprising the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 1, which binds to insulin responsive aminopeptidase or glucose transporter 4; Or a salt thereof, (2) a medicament comprising the protein of the above (1) or a salt thereof, and (3) D encoding the protein of the above (1).
A drug containing NA-containing DNA, (4) the drug according to the above (2) or (3), which is a binding agent to insulin-responsive aminopeptidase or glucose transporter 4, (5) hypoglycemia Prevention
The medicament according to the above (2) or (3), which is a therapeutic agent.
(6) DNA encoding the protein of (1) above
(7) a method for preventing hypoglycemia, which comprises administering to a non-human mammal an effective amount of the protein or salt thereof described in (1) above. A therapeutic method, (8) use of the protein or the salt thereof according to the above (1) for producing a prophylactic or therapeutic agent for hypoglycemia, (9) a base sequence of a DNA encoding the protein according to the above (1). A medicine comprising an antisense DNA containing a complementary or substantially complementary nucleotide sequence or a part thereof, (10) a medicine comprising an antibody against the protein or salt thereof according to the above (1), 1
1) The medicament according to the above (9) or (10), which is a prophylactic / therapeutic agent for hyperglycemia or diabetes; and (12) an antibody against the protein or the salt thereof according to the above (1) against a non-human mammal. (13) A method for preventing or treating hyperglycemia or diabetes, which comprises administering an effective amount, to (13) a protein or a salt thereof according to the above (1) for producing an agent for preventing or treating hyperglycemia or diabetes. Use of an antibody, (14) a diagnostic agent comprising an antibody against the protein of (1) or a salt thereof, (15) an amino acid sequence identical or substantially identical to the amino acid sequence represented by SEQ ID NO: 1 A method for screening a compound having a blood glucose increasing effect or a blood glucose lowering effect or a salt thereof, which comprises using a protein, a partial peptide thereof, or a salt thereof containing (16) a blood sugar increasing effect, characterized by using a DNA containing a DNA containing the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 1 or a DNA encoding a partial peptide thereof; Or a method for screening for a compound having a blood glucose lowering effect or a salt thereof, (17) a protein containing an amino acid sequence identical or substantially identical to the amino acid sequence represented by SEQ ID NO: 1, a partial peptide thereof, or a salt thereof. A screening kit for a compound having a blood sugar increasing effect or a blood sugar lowering effect or a salt thereof, comprising (18) a protein containing an amino acid sequence identical or substantially identical to the amino acid sequence represented by SEQ ID NO: 1 Or DNA containing DNA encoding the partial peptide thereof. A compound or screening kit of the salts thereof glucose increasing activity or hypoglycemic action, (19) above (15) or (16), wherein the screening method or the (17)
Or a compound having a blood glucose increasing effect or a salt thereof, which can be obtained by using the screening kit according to (18), (20) a medicament comprising the compound or a salt thereof according to (19), It is obtained by using the medicament according to the above (20), which is a preventive or therapeutic agent for glycemia, (22) the screening method according to the above (15) or (16), or the screening kit according to the above (17) or (18). A compound having a hypoglycemic action or a salt thereof, (23) a medicament comprising the compound of the above (22) or a salt thereof, or (24) a drug for preventing or treating hyperglycemia or diabetes. 23) the medicament according to the above,
(25) A protein represented by SEQ ID NO: 1, characterized by using a protein having the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 1, a partial peptide thereof, or a salt thereof. Of a protein or a salt thereof, which promotes or inhibits the binding of a protein, a partial peptide thereof, or a salt thereof to an insulin-responsive aminopeptidase or glucose transporter 4, which contains the same or substantially the same amino acid sequence as A screening method, (26) using a cell having the ability to produce a protein containing the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 1 or a partial peptide thereof ( 25) the screening method according to the above,
(27) a protein having the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 1, one of which is immobilized on a solid phase, a partial peptide thereof,
Or a screening method according to (25), wherein a test compound is added to a complex of the salt thereof and insulin-responsive aminopeptidase or glucose transporter 4, and a free substance is detected. A) a DNA encoding a partial peptide corresponding to the cytoplasmic domain of insulin responsive aminopeptidase or a partial peptide corresponding to the cytoplasmic domain of glucose transporter 4 fused with a reporter gene binding domain and a reporter gene transcriptionally active domain Transformed with a DNA encoding a protein containing the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 1 or a partial peptide thereof in the presence and absence of the test compound Culturing, and detecting the expression of the reporter gene in each case; (29) the screening method according to (25), wherein the partial peptide corresponding to the cytoplasmic domain of insulin-responsive aminopeptidase is SEQ ID NO: 5. A partial peptide containing the 55th to 82nd amino acid sequence of the amino acid sequence represented by 5, wherein the partial peptide corresponding to the cytoplasmic domain of glucose transporter 4 contains the amino acid sequence represented by SEQ ID NO: 7. The screening method according to the above (28), which is a partial peptide,
(30) an amino acid sequence represented by SEQ ID NO: 1 containing a protein having the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 1, a partial peptide thereof, or a salt thereof; A screening kit for a compound or a salt thereof that inhibits the binding of a protein containing the same or substantially the same amino acid sequence, a partial peptide thereof, or a salt thereof to insulin-responsive aminopeptidase or glucose transporter 4; ) SEQ ID NO: which can be obtained using the screening method according to (25) to (29) or the screening kit according to (30).
A protein containing the same or substantially the same amino acid sequence as the amino acid sequence represented by No. 1, a partial peptide thereof, or a salt thereof and insulin responsive aminopeptidase or glucose transporter 4
(32) a compound comprising the compound or a salt thereof described in (31) above, or (33) a drug for preventing or treating hyperglycemia or diabetes, wherein (32) (34) a method for preventing or treating hyperglycemia or diabetes, which comprises administering to a non-human mammal an effective amount of the compound or a salt thereof according to (31); Use of the compound of the above (31) or a salt thereof for producing a prophylactic / therapeutic agent for hyperglycemia or diabetes, (36) the screening method of the above (25) to (29), or the above (30) SEQ ID NO: 1 obtained using the screening kit of
A protein containing an amino acid sequence identical or substantially identical to the amino acid sequence represented by the following, a partial peptide thereof, or a salt thereof and insulin responsive aminopeptidase or glucose transporter 4
(37) a medicament comprising the compound of (36) or a salt thereof, (38) a drug for preventing or treating hypoglycemia,
(4) a method of preventing or treating hypoglycemia, which comprises administering an effective amount of the compound or a salt thereof according to (3) to a non-human mammal;
0) The above (3) for producing a prophylactic / therapeutic agent for hypoglycemia
6) use of the compound or a salt thereof according to (41), wherein a protein having the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 1 or a salt thereof is used. A method for screening a compound or a salt thereof that promotes or suppresses protein expression, (42) Transcription regulation of DNA encoding a protein containing an amino acid sequence identical or substantially identical to the amino acid sequence represented by SEQ ID NO: 1 (41) The method according to the above (41), wherein the cells transformed with the DNA in which the reporter gene is fused to the region are cultured in the presence and absence of the test compound, and the expression of the reporter gene in each case is detected. Screening method, (4
3) Screening of a compound or a salt thereof, which comprises or contains a protein having the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 1 or a salt thereof, and which promotes or suppresses the expression of the protein. Kit (44) identical or substantially identical to the amino acid sequence represented by SEQ ID NO: 1, which can be obtained by using the screening method described in (41) or (42) or the screening kit described in (43). Or a salt thereof, which suppresses the expression of a protein having the same amino acid sequence as described above, (45) a medicament comprising the compound or a salt thereof described in (44), (46) prevention of hyperglycemia or diabetes, The medicament according to the above (45), which is a therapeutic agent,
(47) identical or substantially identical to the amino acid sequence represented by SEQ ID NO: 1, which can be obtained by using the screening method described in (41) or (42) or the screening kit described in (43). A compound or a salt thereof that promotes the expression of a protein containing an amino acid sequence, (48) a medicament containing the compound of the above (47) or a salt thereof, and (49) a medicament for preventing or treating hypoglycemia. (48) the method of preventing or treating hypoglycemia, which comprises administering an effective amount of the compound or a salt thereof according to (47) to a non-human mammal; The present invention also relates to the use of the compound described in (47) or a salt thereof for producing a prophylactic / therapeutic agent for hypoglycemia.

[0006]

BEST MODE FOR CARRYING OUT THE INVENTION A protein having the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 1 of the present invention (hereinafter sometimes referred to as the protein of the present invention) is a human or a human. Other warm-blooded animals (eg, guinea pigs, rats, mice, chickens, rabbits,
Cells of pigs, sheep, cattle, monkeys, etc. (eg, hepatocytes, spleen cells, 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, fibrocytes, muscle cells, adipocytes, immune cells (eg, macrophages, T cells, B cells, natural killer cells, mast cells, neutrophils, basophils, eosinophils, Monocytes), megakaryocytes, synovial cells, chondrocytes, osteocytes, osteoblasts, osteoclasts, mammary cells, hepatocytes or stromal cells, or precursors, stem cells or cancer cells of these cells, or the like Any tissue where cells exist, such as the brain,
Various parts of the brain (eg, olfactory bulb, amygdala, basal sphere, hippocampus, thalamus, hypothalamus, cerebral cortex, medulla, cerebellum), spinal cord, pituitary, stomach, pancreas, kidney, liver, gonad, thyroid, gall bladder,
Bone marrow, adrenal gland, skin, muscle, lung, digestive tract (eg, large intestine, small intestine), blood vessels, heart, thymus, spleen, submandibular gland, peripheral blood, prostate, testicle, ovary, placenta, uterus, bone, joint, skeleton It may be a protein derived from muscle or the like, or may be a synthetic protein.

The amino acid sequence substantially identical to the amino acid sequence represented by SEQ ID NO: 1 is about 70% or more, preferably about 80% or more, of the amino acid sequence represented by SEQ ID NO: 1.
% Or more, more preferably about 90% or more, still more preferably about 95% or more. Examples of the protein having an amino acid sequence substantially the same as the amino acid sequence represented by SEQ ID NO: 1 of the present invention include, for example, a protein having the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 1 , SEQ ID NO: 1
A protein having substantially the same properties as the protein having the amino acid sequence represented by is preferred. Properties of substantially the same quality include, for example, IRAP or GLU
T4 binding activity and the like. Substantially the same means that those properties are qualitatively the same. Therefore, it is preferable that the binding activity to IRAP or GLUT4 is equivalent, but quantitative factors such as the degree of these properties and the molecular weight of the protein may be different.
As the amino acid sequence substantially identical to the amino acid sequence represented by SEQ ID NO: 1 of the present invention, more specifically, for example, the amino acid sequence represented by SEQ ID NO: 9;
And the amino acid sequence represented by SEQ ID NO: 11.

[0008] The protein of the present invention includes, for example, one or more (preferably, one or more) in the amino acid sequence represented by SEQ ID NO: 1, SEQ ID NO: 9, SEQ ID NO: 10 or SEQ ID NO: 11. An amino acid sequence in which about 1 to 25, preferably about 1 to 10, and more preferably number (1 to 5) amino acids have been deleted, SEQ ID NO: 1, SEQ ID NO: 9, SEQ ID NO: 10 or sequence No. 1 or 2 or more (preferably,
An amino acid sequence to which about 1 to 25, preferably about 1 to 10, and more preferably number (1 to 5) amino acids have been added, SEQ ID NO: 1, SEQ ID NO: 9, SEQ ID NO: 1
0 or 1 or 2 or more amino acids in the amino acid sequence represented by SEQ ID NO: 11 (preferably, about 1 to 25, preferably about 1 to 10, more preferably number (1 to 5))
Amino acid sequence having the amino acid inserted therein, SEQ ID NO:
1, SEQ ID NO: 9, SEQ ID NO: 10 or SEQ ID NO: 1
1 or 2 or more (preferably about 1 to 25, preferably about 1 to 10,
More preferably, a so-called mutein such as a protein containing an amino acid sequence in which several (1 to 5) amino acids are substituted with another amino acid, or an amino acid sequence obtained by combining them is also included.

In the present specification, the left end is the N-terminus (amino terminal) and the right end is the C-terminus (carboxyl terminus) according to the convention of peptide labeling. The protein of the present invention, including the protein containing the amino acid sequence represented by SEQ ID NO: 1, usually has a carboxyl group (—COOH) or a carboxylate (—COO)
^-), but it is, C-terminal, may be an amide (-CONH ₂₎ or ester (-COOR). Here, as R in the ester, for example, methyl, ethyl, n-
C _1-6 alkyl groups such as propyl, isopropyl, n-butyl, etc., for example, C _3-8 cycloalkyl groups such as cyclopentyl, cyclohexyl, eg, phenyl, α
A C _6-12 aryl group such as _-naphthyl , for example, a phenyl-C _1-2 alkyl group such as benzyl, phenethyl or α-naphthyl-C such as α-naphthylmethyl;
A C _7-14 aralkyl group such as a _1-2 alkyl group, a pivaloyloxymethyl group, and the like are used.

When the protein of the present invention has a carboxyl group (or carboxylate) other than the C-terminus, those in which the carboxyl group is amidated or esterified are also included in the protein of the present invention. As the ester in this case, for example, the above-mentioned C-terminal ester and the like are used. Further, the protein of the present invention includes N
When the amino group of the terminal amino acid residue (eg, methionine residue) is a protecting group (for example, C
_1-6 alkanoyl or other C _1-6 acyl group), N-terminal glutamine residue generated by cleavage in vivo, pyroglutamine oxidation, on the side chain of amino acid in the molecule (For example, -OH, -S
H, amino group, imidazole group, indole group, guanidino group, etc.) a suitable protecting group (e.g., formyl group, such as _{C 1-6} alkanoyl group such as acetyl group _{C 1-6}
(Eg, an acyl group) or a complex protein such as a so-called glycoprotein to which a sugar chain is bound. Specific examples of the protein of the present invention include:
For example, a protein derived from human skeletal muscle having the amino acid sequence represented by SEQ ID NO: 1, that is, VLC described in the literature
AD (Andersen et al., Hum. Mol. Benet., 5, 461-47
2, 1996).

The partial peptide of the protein of the present invention (hereinafter sometimes abbreviated as the partial peptide of the present invention) is the partial peptide of the protein of the present invention described above, preferably the protein of the present invention described above. Any material may be used as long as it has the same properties as described above. For example, a peptide having at least 20 or more, preferably 50 or more, more preferably 70 or more, more preferably 100 or more, and most preferably 200 or more amino acid sequences among the constituent amino acid sequences of the protein of the present invention. Is used. Particularly preferably, a peptide having an amino acid sequence consisting of 200 to less than 655 (more preferably, 200 to less than 620) amino acid residues consecutive from the C-terminus of the protein of the present invention is used. In the partial peptide of the present invention, one or more (preferably, about 1 to 10, more preferably, about 1 to 5) amino acids in the amino acid sequence are deleted, or One or two or more (preferably about 1 to 10, more preferably number (1 to 5)) amino acids are added to the amino acid sequence, or one or two or more (preferably, About 1 to 10, more preferably number (1 to 5) amino acids are inserted, or 1 or 2 or more (preferably about 1 to 10 and more preferably number ( 1-5) amino acids may be substituted with another amino acid. More specifically, as the partial peptide of the present invention, 36th (Ala) to 655th (Phe) from the N-terminal of the amino acid sequence represented by SEQ ID NO: 1
And a partial peptide containing the amino acid sequence represented by

The partial peptide of the present invention usually has a carboxyl group (—COOH) or a carboxylate (—COO ⁻ ) at the C-terminus, but has an amide (—CONN) at the C-terminus as in the protein of the present invention.
H ₂ ) or an ester (—COOR).
Further, the partial peptide of the present invention includes, as in the protein protein of the present invention described above, one in which the amino group of the N-terminal amino acid residue (eg, methionine residue) is protected by a protecting group, and Glutamine residue produced by cleavage in vivo, pyroglutamine oxidation, substituent on the side chain of the amino acid in the molecule is protected with an appropriate protecting group, or so-called glycopeptide having a sugar chain attached And the like. The partial peptide of the present invention can be used as an antigen for preparing an antibody, and can also be used for screening for a compound that inhibits the binding of the protein of the present invention to IRAP or GLUT4.

The protein or partial peptide of the present invention
Salts include physiologically acceptable acids (eg, inorganic acids,
Salts with organic acids) or bases (eg, alkali metal salts)
And especially preferred are physiologically acceptable acid addition salts.
No. Such salts include, for example, inorganic acids (for example, salts
Acid, phosphoric acid, hydrobromic acid, sulfuric acid) or organic
Acids (eg, acetic acid, formic acid, propionic acid, fumaric acid,
Oleic, succinic, tartaric, citric, malic, oxalic
Acid, benzoic acid, methanesulfonic acid, benzenesulfone
Acid) and the like. Proteins and parts of the present invention
The peptide or its salt can be used for human or other
Methods for Purifying Known Proteins from Blood Animal Cells or Tissues
Can be produced by
Transformation containing DNA encoding a protein or peptide
It can also be produced by culturing the transformant.
It can also be produced according to the peptide synthesis method described below.
Wear. Books from human or other mammalian tissues or cells
Production of the protein of the invention, its partial peptide or its salt
To produce human or other mammalian tissues or cells
After homogenizing, extraction with acid etc.
The extracted solution is subjected to reverse phase chromatography, ion exchange chromatography.
Combine chromatography such as torography
Thus, it can be purified and isolated.

For the synthesis of the protein, partial peptide or amide thereof or a salt thereof of the present invention, a commercially available resin for protein synthesis can be usually used. Such resins include, for example, chloromethyl resin, hydroxymethyl resin, benzhydrylamine resin, aminomethyl resin, 4-benzyloxybenzyl alcohol resin,
4-methylbenzhydrylamine resin, PAM resin, 4
-Hydroxymethylmethylphenylacetamidomethyl resin, polyacrylamide resin, 4- (2 ', 4'-dimethoxyphenyl-hydroxymethyl) phenoxy resin, 4- (2', 4'-dimethoxyphenyl-Fmoc
Aminoethyl) phenoxy resin and the like. Using such a resin, an amino acid having an α-amino group and a side chain functional group appropriately protected is condensed on the resin in accordance with the sequence of a target protein or peptide according to various known condensation methods. At the end of the reaction, a protein or peptide is cleaved from the resin, and at the same time, various protecting groups are removed. Further, an intramolecular disulfide bond formation reaction is carried out in a highly diluted solution to obtain a target protein, peptide or an amide thereof.

With respect to the condensation of the above protected amino acids,
Various activating reagents that can be used for protein synthesis can be used, and carbodiimides are particularly preferable. As the carbodiimides, DCC, N, N'-diisopropylcarbodiimide, N-ethyl-N '-(3-dimethylaminoprolyl) carbodiimide and the like are used. Activation by these includes racemization inhibiting additives (eg, H
OBt, HOBt) together with the protected amino acid directly to the resin or the corresponding acid anhydride or HOBt
The protected amino acid can be added to the resin after activation of the protected amino acid in advance as an ester or a HOOBt ester.

The solvent used for activating the protected amino acid or condensing with the resin can be appropriately selected from solvents known to be usable for the protein condensation reaction. For example, acid amides such as N, N-dimethylformamide, N, N-dimethylacetamide, N-methylpyrrolidone, halogenated hydrocarbons such as methylene chloride and chloroform, alcohols such as trifluoroethanol,
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; or an appropriate mixture thereof are used. The reaction temperature is appropriately selected from a range known to be usable for a protein bond forming reaction, and is usually appropriately selected from a range of about -20 ° C to 50 ° C. The activated amino acid derivative is usually used in a 1.5 to 4-fold excess. As a result of the test using the ninhydrin reaction, when the condensation is insufficient, sufficient condensation can be carried out by repeating the condensation reaction without removing the protecting group. When a sufficient condensation cannot be obtained even by repeating the reaction, the unreacted amino acid can be acetylated using acetic anhydride or acetylimidazole to prevent the subsequent reaction from being affected.

Examples of the protecting group for the amino group of the starting material include Z, Boc, t-pentyloxycarbonyl, isobornyloxycarbonyl, 4-methoxybenzyloxycarbonyl, Cl-Z, Br-Z, adamantyloxycarbonyl, Trifluoroacetyl, phthaloyl, formyl, 2-nitrophenylsulfenyl, diphenylphosphinothioyl, Fmoc and the like are used. Carboxyl groups can be, for example, alkyl esterified (eg,
Linear, branched or cyclic alkyl esterification such as methyl, ethyl, propyl, butyl, t-butyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, 2-adamantyl), aralkyl esterification (for example, benzyl ester, 4-nitrobenzyl ester, 4-methoxybenzyl ester, 4-chlorobenzyl ester, benzhydryl esterification), phenacyl esterification, benzyloxycarbonyl hydrazide, t-butoxycarbonyl hydrazide, trityl hydrazide, etc. Can be. The hydroxyl group of serine can be protected, for example, by esterification or etherification. As a group suitable for this esterification, for example, a lower (C _1-6 ) group such as an acetyl group
An aroyl group such as an alkanoyl group and a benzoyl group, and a group derived from carbonic acid such as a benzyloxycarbonyl group and an ethoxycarbonyl group are used. Examples of the group suitable for etherification include a benzyl group, a tetrahydropyranyl group, and a t-butyl group. Examples of the protecting group for the phenolic hydroxyl group of tyrosine include Bz
1, Cl ₂ -Bzl, 2-nitrobenzyl, Br-Z,
T-butyl and the like are used. As a protecting group for imidazole of histidine, for example, Tos, 4-methoxy-
2,3,6-trimethylbenzenesulfonyl, DNP,
Benzyloxymethyl, Bum, Boc, Trt, Fm
oc or the like is used.

Examples of the activated carboxyl group of the raw material include, for example, a corresponding acid anhydride, azide, active ester [alcohol (eg, pentachlorophenol, 2,4,5-trichlorophenol, 2,4- Dinitrophenol, cyanomethyl alcohol, paranitrophenol, HONB, N-hydroxysuccinimide, N
-Ester with hydroxyphthalimide, HOBt)]
Are used. As the activated amino group of the raw material, for example, a corresponding phosphoric amide is used. As a method for removing (eliminating) the protecting group, for example, Pd
-Catalytic reduction in a stream of hydrogen in the presence of a catalyst such as black or Pd-carbon, or acid with anhydrous hydrogen fluoride, methanesulfonic acid, trifluoromethanesulfonic acid, trifluoroacetic acid or a mixture thereof. Treatment, base treatment with diisopropylethylamine, triethylamine, piperidine, piperazine, and the like, reduction with sodium in liquid ammonia, and the like are also used. The elimination reaction by the acid treatment is generally performed at a temperature of about -20 ° C to 40 ° C. In the acid treatment, for example, anisole, phenol, thioanisole, metacresol, paracresol, dimethylsulfide, 1,4 -Addition of a cation scavenger such as butanedithiol, 1,2-ethanedithiol and the like is effective. Further, the 2,4-dinitrophenyl group used as an imidazole protecting group of histidine is removed by thiophenol treatment, and the formyl group used as an indole protecting group of tryptophan is the above 1,2-ethanedithiol, 1,4-butane. In addition to deprotection by acid treatment in the presence of dithiol,
It is also removed by an alkali treatment using a dilute sodium hydroxide solution, dilute ammonia or the like.

The protection of the functional group which should not be involved in the reaction of the raw materials, the protecting group, the elimination of the protective group, the activation of the functional group involved in the reaction, and the like can be appropriately selected from known groups or known means. . As another method for obtaining an amide form of the target protein or peptide, for example, first, after protecting the α-carboxyl group of the carboxy terminal amino acid by amidation, a peptide (protein) chain is added to the amino group side of the desired chain. After extending the length, a protein or peptide from which only the protecting group for the N-terminal α-amino group of the peptide chain has been removed, and a protein or peptide from which only the protecting group for the C-terminal carboxyl group has been removed,
The two proteins or peptides are condensed in a mixed solvent as described above. Details of the condensation reaction are the same as described above. After purifying the protected protein or peptide obtained by the condensation, all the protecting groups are removed by the above-mentioned method to obtain a desired crude protein or peptide. This crude protein or peptide is purified by various known purification means, and the main fraction is lyophilized to obtain an amide of the desired protein or peptide. To obtain an ester of the target 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, Can be obtained.

The partial peptide of the present invention or a salt thereof can be produced according to a known peptide synthesis method or by cleaving the protein of the present invention with an appropriate peptidase. As a method for synthesizing a peptide, for example, any of a solid phase synthesis method and a liquid phase synthesis method may be used.
That is, a partial peptide or amino acid capable of constituting the partial peptide of the present invention is condensed with the remaining portion (peptide or amino acid), and when the product has a protecting group, the protecting group is eliminated to produce the desired peptide. can do. Known methods for condensation and elimination of protecting groups include, for example, the methods described in (1) to (4) below. 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 Biochemical Experiments 1, Chemistry of Protein IV, 205, (1977) Supervised by Haruaki Yajima, Development of Continuing Pharmaceuticals, Vol. 14, Peptide Synthesis, Hirokawa Shoten Thereafter, the protein or peptide of the present invention can be purified and isolated by a combination of ordinary purification methods such as solvent extraction, distillation, column chromatography, liquid chromatography, and recrystallization. When the protein or peptide obtained by the above method is a free form,
It can be converted to an appropriate salt by a known method or a method analogous thereto, and conversely, when obtained with a salt,
It can be converted to a free form or another salt by a known method or a method analogous thereto.

The DNA encoding the protein of the present invention may be any DNA containing the above-described nucleotide sequence encoding the protein of the present invention. Genomic DNA, genomic DNA library,
Any of the above-described cell / tissue-derived cDNA, the above-described cell / tissue-derived cDNA library, and synthetic DNA may be used. The vector used for the library may be any of bacteriophage, plasmid, cosmid, phagemid and the like. In addition, from the cells and tissues described above,
Reverse Transcriptase Polymerase Chain React directly using prepared total RNA or mRNA fraction
It can also be amplified by ion (hereinafter abbreviated as RT-PCR method).

The DNA encoding the protein of the present invention is, for example, a DNA containing the nucleotide sequence represented by SEQ ID NO: 2 or a DNA having the nucleotide sequence represented by SEQ ID NO: 2, which is highly stringent. D, which encodes a protein having a nucleotide sequence that hybridizes under conditions and having substantially the same properties as the protein of the present invention.
Any NA may be used. Examples of the DNA that can hybridize with the DNA having the nucleotide sequence represented by SEQ ID NO: 2 under high stringency conditions include, for example, about 70% or more of the nucleotide sequence represented by SEQ ID NO: 2,
Preferably, a DNA containing a base sequence having about 99% or more homology is used. Hybridization is performed by a known method or a method analogous thereto, for example,
Molecular Cloning 2n
d (J. Sambrook et al., Cold Spring Harbor Lab. Pre.
ss, 1989). When a commercially available library is used, it can be performed according to the method described in the attached instruction manual.
More preferably, it can be performed under high stringency conditions. The high stringent conditions are, for example, conditions in which the sodium concentration is about 19 to 40 mM, preferably about 19 to 20 mM, and the temperature is about 50 to 70 ° C, preferably about 60 to 65 ° C. Particularly, the case where the sodium concentration is about 19 mM and the temperature is about 65 ° C. is most preferable. More specifically, as the DNA encoding the protein having the amino acid sequence represented by SEQ ID NO: 1, a DNA having the base sequence represented by SEQ ID NO: 2 or the like is used.

DNA encoding the partial peptide of the present invention
May be any as long as it contains a base sequence encoding the above-described partial peptide of the present invention. In addition, any of genomic DNA, genomic DNA library, cDNA derived from the above-mentioned cells / tissues, cDNA library derived from the aforementioned cells / tissues, and synthetic DNA may be used. DNA encoding the partial peptide of the present invention
For example, a DNA having a part of the base sequence represented by SEQ ID NO: 2 or a base sequence that hybridizes under high stringent conditions to a DNA having the base sequence represented by SEQ ID NO: 2 Alternatively, a DNA having a part of a DNA encoding a protein having substantially the same activity as the protein of the present invention may be used. The DNA capable of hybridizing with the DNA having the nucleotide sequence represented by SEQ ID NO: 2 has the same significance as described above.

As a means for cloning DNA encoding the protein and partial peptide of the present invention (hereinafter, these may be simply abbreviated to the protein of the present invention), synthetic D having a partial base sequence of the protein of the present invention is used.
Amplify by PCR using NA primers,
Alternatively, the DNA can be selected by hybridization with a DNA incorporated into an appropriate vector and labeled with a DNA fragment or a synthetic DNA encoding a part or the entire region of the protein of the present invention. Hybridization methods are described, for example, in Molecular Cloning 2nd (J. Sambrook e).
tal., Cold Spring Harbor Lab. Press, 1989). When a commercially available library is used, it can be performed according to the method described in the attached instruction manual. The DNA base sequence can be replaced using a known kit, for example, Mutan ^™ -G (Takara Shuzo), Mutan ^™ -K (Takara Shuzo), or the like.
It can be performed according to a known method such as the ex method or the Kunkel method, or a method analogous thereto. The cloned DNA encoding the protein of the present invention can be used as it is depending on the purpose, or may be used after digesting with a restriction enzyme or adding a linker, if desired. The DNA
Has ATG at the 5 'end as a translation initiation codon and TA at the 3' end as a translation stop codon.
A, TGA or TAG. These translation initiation codons and translation termination codons are appropriately synthesized DNA
It can also be added using an adapter. The expression vector for the protein of the present invention can be prepared, for example, by (A) cutting out a DNA fragment of interest from a DNA encoding the protein of the present invention, for example, cDNA, and (B) converting the DNA fragment downstream of a promoter in an appropriate expression vector. It can be manufactured by connecting to.

As a vector, a plasmid derived from Escherichia coli (eg, pBR322, pBR325, pUC12,
UC13), a plasmid derived from Bacillus subtilis (eg, pUB11)
0, pTP5, pC194), yeast-derived plasmids (eg, pSH19, pSH15), bacteriophages such as phage λ, 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 appropriate for the host used for gene expression. For example, when animal cells are used as hosts, SRα promoter, SV40 promoter, L
TR promoter, CMV promoter, HSV-TK
Promoters and the like. Of these, CMV
(Cytomegalovirus) promoter, SRα promoter and the like are preferably used. When the host is Escherichia, trp promoter, lac promoter, recA promoter, .lambda.P _L promoter,
When the host is a bacterium of the genus Bacillus, the SPO1 promoter, the S7 promoter,
When the host is yeast, such as a PO2 promoter and a penP promoter, a PHO5 promoter, a PGK promoter, a GAP promoter, an ADH promoter, and the like are preferable. When the host is an insect cell, a polyhedrin promoter, a P10 promoter and the like are preferable.

[0026] In addition to the above, an expression vector containing an enhancer, a splicing signal, a polyA addition signal, a selection marker, an SV40 replication origin (hereinafter sometimes abbreviated as SV40 ori) and the like, if desired, may be used. Can be used. As a selection marker, for example, dihydrofolate reductase (hereinafter, dhfr)
Gene (sometimes abbreviated as “methotrexate (M
TX) resistance], an ampicillin resistance gene (hereinafter referred to as Amp
^r ), a 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 dhfr gene-deficient Chinese hamster cells, the target gene can be selected using a thymidine-free medium. If necessary, a DNA encoding a signal sequence suitable for the host may be
It is added to the 5 'terminal side of the DNA encoding the protein of the present invention. When the host is Escherichia, Ph.
oA / signal sequence, OmpA / signal sequence, etc.
When the host is a bacterium belonging to the genus Bacillus, an α-amylase signal sequence, a subtilisin signal sequence, and the like, and when the host is a yeast, an MFα signal sequence, SUC2
If the host is an animal cell, such as a signal sequence,
Insulin signal sequence, α-interferon
Signal sequences such as a signal sequence and an antibody molecule / signal sequence can be used. A transformant can be produced using the vector containing the DNA encoding the protein of the present invention thus constructed.

As the host, for example, Escherichia, Bacillus, yeast, insect cells, insects, animal cells and the like are used. Specific examples of Escherichia bacteria include
For example, Escherichia coli K1
2. DH1 [Processings of the National Academy of Sciences of the USA (Proc. Natl. Acad. Sci. USA), 60 volumes, 1
60 (1968)], JM103 [Nucleic Acids Research, (Nucleic Acids Research), 9, 3
09 (1981)], JA221 [Journal of MolecularBiol.
ogy)], Volume 120, 517 (1978)], HB101
[Journal of Molecular Biology, 4
1, 459 (1969)], C600 [Genetics, 39, 440 (1954)] and the like. Examples of Bacillus bacteria include, for example, Bacillus
Bacillus subtilis MI114 [Gene, 24, 255 (1983)], 207-21 [Journal of Biochemistry
emistry), Vol. 95, 87 (1984)]. Examples of yeast include Saccharomyces cerevisiae AH22, AH22
R ⁻ , NA87-11A, DKD-5D, 20B-1
2, Y190, Schizo Saccharomyces pombe (Schizo
saccharomyces pombe) NCYC1913, NCYC2
036, Pichia pastoris KM
71 or the like is used.

As insect cells, for example, the virus is A
In the case of cNPV, a cell line derived from night larvae of larvae (Spodop
tera frugiperda cell (Sf cell), Trichoplusia ni
MG1 cells from the midgut, H from eggs of Trichoplusia ni
igh Five ^TM cells, cells from Mamestra brassicae or
Estigmena acrea-derived cells are used. When the virus is BmNPV, a silkworm-derived cell line (Bombyx mor
iN cells; BmN cells) and the like. Examples of the Sf cells include Sf9 cells (ATCC CRL1711), Sf9
21 cells (Vaughn, JL et al., In Vivo
ivo), 13, 213-217, (1977)) and the like. As insects, for example, silkworm larvae are used [Maeda et al., Nature, 315, 592 (19).
85)]. As animal cells, for example, monkey cells COS
-7, Vero, Chinese hamster cell CHO
(Hereinafter abbreviated as CHO cells), dhfr gene-deficient Chinese hamster cells CHO (hereinafter, CHO (dhf
r ⁻ ) cells), mouse L cells, mouse AtT-2
0, mouse myeloma cells, rat GH3, human FL cells, and the like. For transformation of Escherichia sp., For example, Procagings of the National Academy of Sciences of the USA (Proc. Natl. Acad. Sci. USA), Vol. 69,
2110 (1972) and Gene, Volume 17,
107 (1982).

To transform Bacillus sp., For example, Molecular & General Genetics, Vol.
111 (1979). To transform yeast, for example, Methods in Enzymolog
y), 194, 182-187 (1991), Processings of the National Academy of Sciences of the USA (Proc. Na)
Acad. Sci. USA), 75, 1929 (1978).
And the like. Insect cells or insects can be transformed, for example, according to the method described in Bio / Technology, 6, 47-55 (1988). To transform animal cells, for example, see Cell Engineering Separate Volume 8, New Cell Engineering Experiment Protocol. 263-267 (1995)
(Published by Shujunsha), Virology, 52 volumes,
456 (1973). Thus, a transformant transformed with the expression vector containing the DNA encoding the protein of the present invention can be obtained. When culturing a transformant whose host is a genus Escherichia or Bacillus, a liquid medium is suitable as a medium to be used for the culture. Nitrogen sources, inorganic substances, etc. are contained. As a carbon source, for example, glucose, dextrin, soluble starch, sucrose, etc.As a nitrogen source, for example, ammonium salts, nitrates, corn steep liquor, peptone, casein,
Meat extract, soybean meal, inorganic or organic substances such as potato extract, as the inorganic substance, for example, calcium chloride,
Examples include sodium dihydrogen phosphate and magnesium chloride. In addition, yeast, vitamins, growth promoting factors and the like may be added. The pH of the medium is preferably about 5 to 8.

As a medium for culturing the genus Escherichia, for example, an M9 medium containing glucose and casamino acid [Miller, Journal of Experiments in Molecular Genetics (Journa)
l of Experiments in Molecular Genetics), 431-
433, Cold Spring Harbor Laboratory, New York 1
972] is preferred. If necessary, an agent such as 3β-indolylacrylic acid can be added in order to make the promoter work efficiently. When the host is a bacterium belonging to the genus Escherichia, the cultivation is usually carried out at about 15 to 43 ° C. for about 3 to 24 hours, and if necessary, aeration and stirring can be added. When the host is a bacterium belonging to the genus Bacillus, the cultivation is usually performed at about 30 to 40 ° C. for about 6 to 24 hours, and if necessary, aeration and stirring can be added. When culturing a transformant in which the host is yeast, as a medium, for example, Burkholder minimum medium [Bostian, KL et al.
Prossings of the National Academy of Sciences of the USA (Pr
Natl. Acad. Sci. USA), 77, 4505 (19
80)] and SD medium containing 0.5% casamino acid [Bitt
er, GA, et al., Processings of the National Academy of Sciences of the USA (Proc. Natl. Acad. Sci. USA), Volume 81,
5330 (1984)]. Preferably, the pH of the medium is adjusted to about 5-8. Culture is usually about 20 ° C ~
Perform at 35 ° C. for about 24-72 hours, adding aeration and agitation as needed. When culturing a transformant in which the host is an insect cell or an insect, the medium used is Grace's Insect Med.
ium (Grace, TCC, Nature, 195, 788
(1962)) to which an additive such as immobilized 10% bovine serum is appropriately added. The pH of the medium is about 6.2
It is preferable to adjust to 6.4. Culture is usually about 27 ° C
For about 3 to 5 days, and aeration and stirring are added as necessary. When culturing a transformant whose animal host is an animal cell, for example, as a medium, a MEM medium containing about 5 to 20% fetal bovine serum [Science, 122, 50
1 (1952)], DMEM medium [Virolo
gy), 8, 396 (1959)], RPMI 1640
Medium [The Journal of the American Medical Association
dical Association) Vol. 199, 519 (1967)],
199 medium [Proceeding of the Society for the Biological Medicine (Proceedi
ng of the Society for the Biological Medicine), 7
3 (1950)]. pH is about 6
~ 8 is preferred. Culture is usually about 30 ° C to 40 ° C
For about 15 to 60 hours, and aeration and stirring are added as necessary. As described above, the protein of the present invention can be produced in the cell, in the cell membrane, or outside the cell of the transformant.

The protein of the present invention can be separated and purified from the above culture by, for example, the following method. When extracting the protein of the present invention from cultured cells or cells, after culturing, the cells or cells are collected by a known method, suspended in an appropriate buffer, and subjected to sonication, lysozyme and / or freeze-thawing. After the cells or cells are destroyed by the method, a method of obtaining a crude extract of the protein by centrifugation or filtration is used as appropriate. The buffer may contain a protein denaturant such as urea or guanidine hydrochloride, or a surfactant such as Triton X-100 ^™ . When the protein of the present invention is secreted into the culture solution, after completion of the culture, the supernatant is separated from the cells or cells by a known method, and the supernatant is collected. Purification of the protein of the present invention contained in the thus obtained culture supernatant or extract can be performed by appropriately combining known separation and purification methods. As these known separation and purification methods, methods utilizing solubility such as salting out and solvent precipitation, dialysis, ultrafiltration, gel filtration,
And methods using mainly differences in molecular weight such as SDS-polyacrylamide gel electrophoresis, methods using differences in charge such as ion exchange chromatography, methods using specific affinity such as affinity chromatography, reverse phase A method using a difference in hydrophobicity such as high performance liquid chromatography, a method using a difference in isoelectric point such as isoelectric focusing, and the like are used.

When the protein of the present invention thus obtained is obtained in a free form, it can be converted into a salt by a known method or a method analogous thereto. It can be converted to a free form or another salt by a known method or a method analogous thereto. In addition, the protein produced by the recombinant can be arbitrarily modified or the polypeptide can be partially removed by applying an appropriate protein modifying enzyme before or after purification. As a protein modifying enzyme,
For example, trypsin, chymotrypsin, arginyl endopeptidase, protein kinase, glycosidase and the like are used. The presence of the protein of the present invention thus produced can be measured by an enzyme immunoassay using a specific antibody, Western blotting, or the like.

The antibody against the protein, the partial peptide or a salt thereof of the present invention may be any of a polyclonal antibody and a monoclonal antibody as long as it can recognize the protein of the present invention or a salt thereof. Antibodies against the protein, the partial peptide of the present invention, or a salt thereof (hereinafter, these may be simply abbreviated as the protein of the present invention) can be obtained by using the protein of the present invention as an antigen according to a known antibody or antiserum production method. Can be manufactured.

[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 the antibody can be produced by administration to itself or together with a carrier and a diluent. You. Complete Freund's adjuvant or incomplete Freund's adjuvant may be administered in order to enhance the antibody-producing ability upon administration. Administration is usually performed once every 2 to 6 weeks, for a total of about 2 to 10 times. Examples of the warm-blooded animal used include monkeys, rabbits, dogs, guinea pigs, mice, rats, sheep, goats, and chickens, and mice and rats are preferably used. When preparing monoclonal antibody-producing cells, a warm-blooded animal immunized with an antigen, for example, an individual having an antibody titer is selected from a mouse, and the spleen or lymph node is collected 2 to 5 days after the final immunization and contained in the spleen or lymph node. By fusing the antibody-producing cells thus obtained with myeloma cells of the same or different species, a monoclonal antibody-producing hybridoma can be prepared. The antibody titer in the antiserum can be measured, for example, by reacting a labeled protein described below with the antiserum, and then measuring the activity of a labeling agent bound to the antibody. The fusion operation is performed by known methods, for example, the method of Kohler and Milstein [Nature, 256, 495].
(1975)]. Examples of the fusion promoter include polyethylene glycol (PEG) and Sendai virus, and preferably PE.
G is used.

Examples of myeloma cells include NS-1,
Myeloma cells of warm-blooded animals such as P3U1, SP2 / 0 and AP-1 can be mentioned, but P3U1 is preferably used. The preferred ratio between the number of antibody-producing cells (spleen cells) and the number of myeloma cells used is about 1: 1 to 20: 1,
PEG (preferably PEG1000 to PEG6000)
Is added at a concentration of about 10 to 80%,
Preferably, cell fusion can be carried out efficiently by incubating at 30 to 37 ° C for 1 to 10 minutes. Various methods can be used to screen for monoclonal antibody-producing hybridomas. For example, a hybridoma culture supernatant is added to a solid phase (eg, a microplate) on which a protein antigen is adsorbed directly or together with a carrier, and then radioactive substances or An anti-immunoglobulin antibody labeled with an enzyme or the like (if the cell used for cell fusion is a mouse, an anti-mouse immunoglobulin antibody is used) or protein A
A method for detecting a monoclonal antibody bound to a solid phase, adding a hybridoma culture supernatant to a solid phase to which an anti-immunoglobulin antibody or protein A is adsorbed, adding a protein labeled with a radioactive substance or an enzyme, and A method for detecting a monoclonal antibody bound to a phase is exemplified. The selection of the monoclonal antibody can be performed according to a known method or a method analogous thereto. Normal HAT
(Hypoxanthine, aminopterin, thymidine) in an animal cell culture medium. As a selection and breeding medium, any medium can be used as long as a hybridoma can grow. For example, 1-2
RP containing 0%, preferably 10-20% fetal calf serum
MI 1640 medium, G containing 1-10% fetal calf serum
An IT medium (Wako Pure Chemical Industries, Ltd.) or a serum-free medium for hybridoma culture (SFM-101, Nissui Pharmaceutical Co., Ltd.) can be used. The culturing temperature is usually 20 to 40 ° C, preferably about 37 ° C. The culturing time is usually 5 days to 3 weeks, preferably 1 week to 2 weeks. The culture can be usually performed under 5% carbon dioxide. The antibody titer of the hybridoma culture supernatant can be measured in the same manner as the measurement of the antibody titer in the antiserum described above.

(B) Purification of Monoclonal Antibody Separation and purification of monoclonal antibodies can be performed by known methods, for example, immunoglobulin separation and purification methods (eg, salting out method, alcohol precipitation method, isoelectric point precipitation method, electrophoresis method). , Ion-exchanger (eg, DEAE) adsorption / desorption method, ultracentrifugation method, gel filtration method, antigen-binding solid phase or active antibody such as protein A or protein G to collect only antibody and dissociate the antibody Specific purification method for obtaining

[Preparation of Polyclonal Antibody] The polyclonal antibody of the present invention can be produced according to known methods or modifications thereof. For example, an immunizing 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 a substance and separating and purifying the antibody. Regarding a complex of an immunizing antigen and a carrier protein used to immunize a warm-blooded animal, the type of carrier protein and the mixing ratio between the carrier and the hapten are such that the antibody is efficiently cross-linked to the hapten immunized by cross-linking with the carrier. If possible, any material may be cross-linked at any ratio. For example, bovine serum albumin, bovine thyroglobulin, hemocyanin, etc. may be used in a weight ratio of about 0.1 to 20, preferably about 1 to 20, relative to hapten 1. A method of coupling at a rate of ５5 is used. Various coupling agents can be used for coupling the hapten and the carrier. For example, glutaraldehyde, carbodiimide, a maleimide active ester, an active ester reagent containing a thiol group or a dithioviridyl group, or the like is used. The condensation product is administered to a warm-blooded animal itself or together with a carrier or diluent at a site where antibody production is possible.
Complete Freund's adjuvant or incomplete Freund's adjuvant may be administered in order to enhance the antibody-producing ability upon administration. The administration is usually performed once every about 2 to 6 weeks, about 3 to 10 times in total. The polyclonal antibody can be collected from the blood, ascites, etc., preferably from the blood of a warm-blooded animal immunized by the above method. The measurement of the polyclonal antibody titer in the antiserum can be performed in the same manner as the measurement of the antibody titer in the antiserum described above. The 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.

DNAs encoding the proteins and partial peptides of the present invention (hereinafter, these DNAs are referred to as the DNAs of the present invention)
May be abbreviated as a), or as an antisense DNA having a base sequence complementary or substantially complementary to:
Any antisense DNA may be used as long as it has a base sequence complementary or substantially complementary to the DNA of the present invention and has an action capable of suppressing the expression of the DNA. The nucleotide sequence substantially complementary to the DNA of the present invention is, for example, about 70% of the total nucleotide sequence or a partial nucleotide sequence of the nucleotide sequence complementary to the DNA of the present invention (that is, the complementary strand of the DNA of the present invention). % Or more, preferably about 80% or more, more preferably about 90% or more, and most preferably about 9% or more.
A base sequence having a homology of 5% or more is exemplified.
In particular, of the entire base sequence of the complementary strand of the DNA of the present invention, the base sequence of the portion encoding the N-terminal portion of the protein of the present invention (for example, the base sequence near the start codon, etc.) is at least about 70% or more of the complementary strand, An antisense DNA having a homology of preferably about 80% or more, more preferably about 90% or more, and most preferably about 95% or more is suitable. These antisense DNAs can be produced using a known DNA synthesizer or the like.

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

(1) Agent for Prevention / Treatment of Various Diseases Related to the Protein of the Present Invention The protein of the present invention binds to GLUT4 endoplasmic reticulum (GLUT4, IRAP, VAMP) by binding to IRAP.
(the endoplasmic reticulum in which proteins such as s, SCAMPs, and Rab4 are localized) remain in the cells, and increase the blood glucose level by suppressing the incorporation of blood sugar into muscle cells and fat cells. Therefore, the protein of the present invention or the DNA of the present invention can be used as a medicament such as an agent for preventing or treating various diseases such as hypoglycemia. For example, when there is a patient in which the homeostasis of the living body or the biological defense mechanism is not sufficiently or normally exerted because the protein or the like of the present invention is reduced or deleted in the living body, (a) the present invention By administering DNA to the patient and expressing the protein of the present invention in vivo, (b)
After inserting the DNA of the present invention into cells and expressing the protein of the present invention, transplanting the cells into the patient, or (c) administering the protein of the present invention to the patient, Can sufficiently or normally exert the role of the protein of the present invention. When the DNA of the present invention is used as the above-mentioned prophylactic / therapeutic agent, the DNA is inserted alone or into an appropriate vector such as a retrovirus vector, an adenovirus vector, an adenovirus associated virus vector, and the like. It can be administered to humans or warm-blooded animals. The DNA of the present invention can be administered as it is or in the form of a formulation together with a physiologically acceptable carrier such as an adjuvant for promoting uptake, and administered with a gene gun or a catheter such as a hydrogel catheter. When the protein of the present invention is used as the above-mentioned prophylactic / therapeutic agent, it is at least 90%, preferably 95% or more, more preferably 98% or more, and still more preferably 99% or more.
% Or more is preferably used.

The protein of the present invention can be administered, for example, orally as tablets, capsules, elixirs, microcapsules and the like, optionally coated with sugar, or in the form of an aerosolized inhalant, or in the form of water or water. It can be used parenterally in the form of injections, such as sterile solutions with other pharmaceutically acceptable liquids, or suspensions. For example,
Manufactured by mixing the protein of the present invention with physiologically acceptable carriers, flavoring agents, excipients, vehicles, preservatives, stabilizers, binders, etc. in generally accepted unit dosage forms required for the practice of formulations. can do. The amount of the active ingredient in these preparations is such that a proper dosage in the specified range can be obtained. Examples of additives that can be incorporated into tablets, capsules, and the like include binders such as gelatin, corn starch, tragacanth, and acacia, excipients such as crystalline cellulose, corn starch, gelatin, and alginic acid. Swelling agents, lubricants such as magnesium stearate,
Sweetening agents such as sucrose, lactose or saccharin, flavoring agents such as peppermint, reddish oil or cherry and the like are used. When the preparation unit form is a capsule, a liquid carrier such as fats and oils can be further contained in the above-mentioned type of material. Sterile compositions for injection can be formulated according to conventional pharmaceutical manufacturing methods such as dissolving or suspending the active substance in vehicles such as water for injection, and naturally occurring vegetable oils such as sesame oil, coconut oil and the like. . Examples of aqueous liquids for injection include physiological saline, isotonic solutions containing glucose and other adjuvants (eg, D-sorbitol, D-mannitol, sodium chloride, etc.)
And suitable solubilizers such as alcohols (eg, ethanol), polyalcohols (eg, propylene glycol, polyethylene glycol, etc.), nonionic surfactants (eg, Polysorbate 80 ^™ , HCO-50). Etc.). Examples of the oily liquid include sesame oil and soybean oil, and may be used in combination with a solubilizing agent such as benzyl benzoate or benzyl alcohol. Also, a buffer (for example,
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), antioxidants and the like. The prepared injection is usually filled in a suitable ampoule. The vector into which the DNA of the present invention has been inserted is also formulated in the same manner as described above, and is usually used parenterally.

The preparations thus obtained are safe and have low toxicity, and are, for example, human or other warm-blooded animals (for example, rats, mice, guinea pigs, rabbits, birds, sheep, pigs, cows, horses, Cats, dogs, monkeys, chimpanzees, etc.). The dose of the protein of the present invention varies depending on the target disease, the subject of administration, the administration route, and the like. For example, when the protein of the present invention is orally administered for the purpose of treating hypoglycemia, it is generally adult (60 kg). )), About 0.1 mg to 100 mg, preferably about 1.0 to 50 mg, more preferably about 1.0 to 20 mg of the protein per day.
Administer. When administered parenterally, the single dose of the protein varies depending on the administration subject, target disease, and the like. For example, the protein of the present invention is injected into an adult (body weight) for the purpose of treating hypoglycemia. 60 kg), the protein may be administered in an amount of about 0.01-30 mg per day.
It is convenient to administer the drug by injecting into the affected part, preferably about 0.1 to 20 mg, more preferably about 0.1 to 10 mg. For other animals, 60
An amount converted per kg can be administered.

(2) Screening for Binding Inhibitors The protein of the present invention binds to the cytoplasmic domain of IRAP, retains the GLUT4 endoplasmic reticulum in cells, and suppresses the uptake of blood sugar into muscle cells and fat cells. . Therefore, a compound that inhibits the binding of the protein of the present invention to IRAP, preferably a compound that inhibits the binding of the protein of the present invention to the cytoplasmic domain of IRAP, is obtained by incorporating glucose in blood into muscle cells and fat cells. And can lower blood glucose levels, for example, hyperglycemia, diabetes (eg, type 1 diabetes, type 2 diabetes, gestational diabetes, etc.), impaired glucose tolerance [IGT (Impaired Glucose Tolerance)],
Diabetic complications [eg, neuropathy, nephropathy, retinopathy, cataract, macrovascular disease, osteopenia, diabetic hyperosmotic coma, infections (eg, respiratory infections, urinary tract infections, gastrointestinal Infection,
Skin and soft tissue infections, lower limb infections, etc.), diabetic gangrene,
And the like, and pharmaceuticals such as an agent for suppressing the transition from impaired glucose tolerance to diabetes mellitus. Regarding the criterion of diabetes, a new criterion was reported by the Japanese Diabetes Association in 1999. According to this report,
Diabetes refers to a fasting blood glucose level (glucose concentration in venous plasma) of 126 mg / dl or more, a 75-hour oral glucose tolerance test (75 g OGTT) 2-hour value (glucose concentration in venous plasma) of 200 mg / dl or more, Glucose concentration in plasma) 200 mg /
dl or more. In addition, it does not correspond to the above-mentioned diabetes, and “Fasting blood glucose level (glucose concentration in venous plasma) is less than 110 mg / dl or 75 mg / dl.
g Oral glucose tolerance test (75 g OGTT) 2-hour value (glucose concentration in venous plasma) is 140 mg / d
A state that is not “a state showing less than 1” (normal type) is called a “boundary type”. Regarding the criteria for judging diabetes, 19
A new criterion was reported by ADA (American Diabetes Association) in 1997 and by WHO in 1998. According to these reports, diabetes is defined as a fasting blood glucose level (glucose concentration in venous plasma) of 126 mg / dl or more and a 2-hour 75 g oral glucose tolerance test (glucose concentration in venous plasma) of 200 mg / dl. dl or more. According to the above report, impaired glucose tolerance refers to a fasting blood glucose level (glucose concentration in venous plasma) of less than 126 mg / dl and a 2-hour 75 g oral glucose tolerance test (glucose concentration in venous plasma). Is 140 mg / dl or more and less than 200 mg / dl. Furthermore, according to the report of ADA, the fasting blood glucose level (glucose concentration in venous plasma) was 11%.
IFG for the condition of 0 mg / dl or more and less than 126 mg / dl
(Impaired Fasting Glucose). On the other hand, according to the report of WHO, the IFG (Impaired Fasting Glucose)
Among them, a state in which the 75-hour oral glucose tolerance test 2-hour value (glucose concentration in venous plasma) is less than 140 mg / dl is called IFG (Impaired Fasting Glycemia). Compounds that inhibit the binding between the protein of the present invention and IRAP include diabetes, borderline type, impaired glucose tolerance, and IFG (Impaired Fasting) determined by the above-described new criteria.
Glucose) and IFG (Impaired Fasting Glycemi)
It is also used as a prophylactic / therapeutic agent in a). Furthermore, compounds that inhibit the binding of the protein of the present invention to IRAP include boundary type, impaired glucose tolerance, and IFG (Impaired Fasting G).
lucose) or IFG (Impaired Fasting Glycemia)
It can also prevent progression to diabetes. The compound that inhibits the binding between the protein of the present invention and IRAP includes, for example, a compound represented by the formula:

Embedded image Or

Embedded image And the like. Further, the protein of the present invention also binds to the cytoplasmic domain of GLUT4 (amino acids 468 to 510 of GLUT4; SEQ ID NO: 7), and retains the GLUT4 endoplasmic reticulum in the cell, thereby improving blood sugar muscle cells and Suppresses uptake into fat cells. Therefore, a compound that inhibits the binding of the protein of the present invention to GLUT4, preferably a compound that inhibits the binding of the protein of the present invention to the cytoplasmic domain of GLUT4, is a compound that inhibits the binding of the protein of the present invention to IRAP. Similarly to the above, it is useful as a medicament such as a prophylactic / therapeutic agent for diseases such as hyperglycemia and diabetes.

In the screening method of the present invention, the protein of the present invention is used.
Peptide or GL corresponding to the cytoplasmic domain of AP
A peptide corresponding to the cytoplasmic domain of UT4 or GLUT4 may be used. Further, the screening method of the present invention relates to a method for producing a cell having the ability to produce the protein of the present invention (preferably, a transformant transformed with the DNA encoding the protein of the present invention (eg, a cell such as a yeast or an animal cell) ) May be used. The transformant contains DNA encoding the protein of the present invention and IR
A transformant transformed with DNA encoding a peptide corresponding to the cytoplasmic domain of AP or IRAP,
Alternatively, the transformant may be a transformant transformed with a DNA encoding the protein of the present invention and a DNA encoding GLUT4 or a peptide corresponding to the cytoplasmic domain of GLUT4. (2-1) Screening by In Vitro Binding Test The protein of the present invention is immobilized on a solid phase (eg, an EIA plate) using an antibody against the protein of the present invention,
Alternatively, the protein of the present invention is a Tag protein (eg,
After fusion with His-Tag, GST (glutathione-S-transferase), and the like, they are immobilized on a solid phase.
When the partial peptide of the present invention is used as the protein of the present invention, a partial peptide having binding activity to IRAP or GLUT4 (such as amino acids 36 to 655 of SEQ ID NO: 1) is preferably used. When immobilizing a protein on a solid phase, nickel is used for His-Tag, and glutathione is used for GST. In addition, IRAP or a partial peptide corresponding to the cytoplasmic domain of IRAP (amino acid sequence represented by SEQ ID NO: 5 or a partial sequence thereof, preferably amino acids 55 to 82 of SEQ ID NO: 5) or GLUT4 or GL
A partial peptide corresponding to the cytoplasmic domain of UT4 (SEQ ID NO: 7) labeled with biotin or the like is added and bound. A test compound is added to this complex, and the IRAP or IRAP partial peptide or GLUT4 or GLUT4 partial peptide released as a result of inhibition of the binding of the protein of the present invention to IRAP or GLUT4 is converted to a commercially available product for detecting a labeled substance such as biotin. Kit or known anti-IRAP antibody or commercially available anti-GLUT4
Detection and quantification are performed using antibodies. IRAP or IR
AP partial peptide or GLUT4 or GLUT4
The compound that releases the partial peptide is selected as a compound that inhibits the binding between the protein of the present invention and IRAP or GLUT4 (hereinafter sometimes abbreviated as a binding inhibitor). In addition, IRAP or a partial peptide corresponding to the IRAP cytoplasmic domain (amino acid sequence represented by SEQ ID NO: 5 or a partial sequence thereof, preferably amino acids 55 to 82 of SEQ ID NO: 5) or GLUT4 or a cytoplasmic domain of GLUT4 Is immobilized on a solid phase, and the protein of the present invention is bound to the immobilized partial peptide (SEQ ID NO: 7). IRAP or IRA
When the P partial peptide or GLUT4 or GLUT4 partial peptide is immobilized on a solid phase, for example, biotin-labeled IRAP or IRAP partial peptide or GLUT4 or GLUT4 partial peptide and avidin-labeled solid phase (eg, plate) Is preferably used. A test compound is added to this complex, and the released protein of the present invention is detected and quantified with an antibody against the protein of the present invention or an antibody against the Tag protein.
In this method, the protein of the present invention may use the protein of the present invention fused with the Tag protein,
In that case, the released protein of the present invention may be detected and quantified with an antibody against the protein of the present invention.
It may be detected and quantified using an antibody against the ag protein.
Compounds that release the protein of the invention are selected as binding inhibitors. The inhibitory activity of the selected compound can be confirmed by a known method such as an immunoprecipitation method using an anti-IRAP antibody, an anti-GLUT4 antibody, an antibody against the protein of the present invention, or an antibody against Tag. In the immunoprecipitation method, an antibody against the protein of the present invention or an antibody against the protein of the present invention, or an antibody against Tag protein, which is released by inhibiting the binding of the protein of the present invention to IRAP or GLUT4 by the selected compound , IRA
It is detected by an antibody against P or GLUT4.

(2-2) Screening by Two-Hybrid method (2-2-1) Yeast Screening by two-hybrid method Yeast (eg, Saccharomyces cerevisiae, more preferably
(S. cerevisiae strain Y190) in which a reporter gene-binding domain is fused with a partial peptide corresponding to the IRAP cytoplasmic domain or a DNA encoding a partial peptide corresponding to the GLUT4 cytoplasmic domain, and a reporter gene transcriptionally active domain is fused. When the DNA encoding the protein of the present invention is expressed, two-hybrid
By the action of brid, phenotypes of the reporter gene β-galactosidase gene and the histidine synthesis system gene HIS3 are expressed. This yeast strain is grown in the presence of the test compound.
After culturing for a certain period of time, a compound that reduces the β-galactosidase activity of the yeast strain or converts the yeast strain to histidine auxotrophy is selected. The yeast strain can be cultured in the same manner as in the culture of the above-described transformant whose host is yeast. β
-The activity of galactosidase was X-Gal (5-bromo-4-ch).
loro-3-indolyl-β-D-galactopyranoside), ONPG
(O-nitrophenyl β-D-galactopyranoside) or C
PRG (chlorophenyl red-β-D-galactopyranoside)
Can be measured according to a known method as a substrate. Expression of the HIS3 phenotype can be measured by culturing the yeast in a minimal medium lacking histidine. Among the selected compounds, cytotoxic compounds and compounds that inhibit the activity of the reporter gene product itself due to interaction with the reporter gene product or the like can be excluded as false positive compounds.

(2-2-2) Animal cells Screening by two-hybrid method Animal cells (eg, Chinese hamster ovary (CH)
O) cells), for example, a chloramphenicol acetyltransferase (CAT) gene or a firefly luciferase gene is introduced as a reporter gene. The transcription regulatory region of the reporter gene is, for example, a promoter in which a transcriptional activation sequence (UAS) of GAL1 is bound downstream of a promoter that functions in animal cells (eg, a minimal promoter (TATA box) derived from adenovirus E1b). GAL4-G of yeast two-hybrid system
The transcriptional regulatory system of AL1 is introduced into animal cells so that reporter gene expression is induced in animal cells. The above I fused to the GAL4-DNA binding domain
Expression of a DNA encoding the protein of the present invention fused to a DNA encoding a partial peptide corresponding to the RAP cytoplasmic domain or a partial peptide corresponding to the GLUT4 cytoplasmic domain, for example, a DNA encoding a VP16 protein derived from herpes simplex virus Then, an animal cell line in which the reporter gene is expressed by the action of two-hybrid is obtained. This cell line is cultured for a certain period of time in the presence of the test compound, the activity of the reporter gene product is measured, and a compound that reduces the activity is selected. The animal cell strain can be cultured in the same manner as the culture of the above-described transformant whose host is an animal cell. The activity of reporter gene products such as CAT and luciferase can be determined according to a known method.
It can be measured using a commercially available kit. Among the selected compounds, cytotoxic compounds and compounds that inhibit the activity of the reporter gene product itself due to interaction with the reporter gene product or the like can be excluded as false positive compounds.

(3) Screening for a compound that promotes or suppresses the expression of the protein of the present invention The transcriptional regulatory region of the DNA of the present invention is cloned, and a reporter gene (eg, β-galactosidase, firefly luciferase, chloramphenicol, Acetyltransferase (CAT) or the like, and is introduced into animal cells (eg, CHO cells). This cell line is cultured for a certain period of time in the presence of the test compound, and a compound that increases or decreases the production of the reporter gene product is selected. The animal cell strain can be cultured in the same manner as the culture of the above-described transformant whose host is an animal cell. The increase or decrease in the production amount of the reporter gene product can be determined, for example, by measuring the activity of the reporter gene product in the culture solution. Among the selected compounds, compounds that increase or decrease the activity of the reporter gene product itself due to interaction with the cytotoxic compound and the reporter gene product can be excluded as false positive compounds.

The test compound includes, for example, a peptide,
Proteins, non-peptidic compounds, synthetic compounds, fermentation products, cell extracts, plant extracts, animal tissue extracts and the like, and these compounds may be novel compounds or known compounds Is also good. Examples of the compound that suppresses the expression of the protein of the present invention include a compound that suppresses the expression of the protein of the present invention obtained by the above screening, the antisense DNA described above, and the DN of the present invention described below.
Compounds that inhibit the promoter activity of A. Examples of the compound that promotes the expression of the protein of the present invention include a compound that promotes the expression of the protein of the present invention obtained by the above screening, and a DNA of the present invention that will be described later.
And the like, which promote the promoter activity of the promoter.

The screening kit of the present invention contains the protein of the present invention and further comprises IRAP or IRAP.
Peptide or GL corresponding to the cytoplasmic domain of AP
It may contain a peptide corresponding to the cytoplasmic domain of UT4 or GLUT4. In addition, the screening kit of the present invention includes a cell capable of producing the protein of the present invention (preferably, a transformant transformed with a DNA encoding the protein of the present invention (eg, a yeast, animal cell, or the like). Cells)). The transformant contains DNA encoding the protein of the present invention and IR
A transformant transformed with DNA encoding a peptide corresponding to the cytoplasmic domain of AP or IRAP,
Alternatively, the transformant may be a transformant transformed with a DNA encoding the protein of the present invention and a DNA encoding GLUT4 or a peptide corresponding to the cytoplasmic domain of GLUT4.

Compounds or salts thereof obtained by using the screening method or screening kit of the present invention may be any of the test compounds described above, for example, peptides, proteins, non-peptide compounds, synthetic compounds, fermentation products, cell extracts, Compounds selected from plant extracts, animal tissue extracts, plasma, and the like, such as compounds that inhibit the binding of the protein of the present invention to IRAP or GLUT4, and compounds that promote or suppress the expression of the protein of the present invention. can give. As the salt of the compound, those similar to the aforementioned salts of the protein of the present invention are used. The compound that inhibits the binding of the protein of the present invention to IRAP or GLUT4 or the compound that suppresses the expression of the protein of the present invention is useful as a medicament such as a prophylactic / therapeutic agent for diseases such as hyperglycemia and diabetes. . The compound that promotes the expression of the protein of the present invention is useful, for example, as a medicament such as a prophylactic or therapeutic agent for diseases such as hypoglycemia.

When a compound or a salt thereof obtained by using the screening method or screening kit of the present invention is used as the above-mentioned prophylactic / therapeutic agent, it can be formulated into a pharmaceutical preparation according to a conventional method. For example, in the same manner as the above-mentioned drug containing the protein of the present invention, it is formulated into tablets, capsules, elixirs, microcapsules, sterile solutions, suspensions and the like, and administered orally or parenterally. be able to. The preparations obtained in this way are safe and of low toxicity, such as, for example, humans or other warm-blooded animals (eg, mice, rats, rabbits, sheep, pigs, cows, horses, birds, cats, dogs, monkeys, Chimpanzees). The dose of the compound or a salt thereof varies depending on its action, target disease, administration subject, administration route and the like. For example, a compound that inhibits the binding of the protein of the present invention to IRAP or GLUT4 for the purpose of treating diabetes Or, when orally administering a compound that suppresses the expression of the protein of the present invention, generally in adults (with a body weight of 60 kg),
About 0.1 to 100 mg, preferably about 1.0 to 50 mg, more preferably about 1.0 to 20 mg of the compound per day
mg. When administered parenterally, the single dose of the compound varies depending on the administration subject, target disease, and the like. For example, a compound that inhibits the binding of the protein of the present invention to IRAP or GLUT4 for the purpose of treating diabetes Alternatively, when a compound that suppresses the expression of the protein of the present invention is usually administered to an adult (as 60 kg) in the form of an injection, the compound is administered in an amount of about 0.01 to 30 mg per day,
It is convenient to administer preferably about 0.1 to 20 mg, more preferably about 0.1 to 10 mg by intravenous injection. In the case of other animals, the amount can be administered in terms of 60 kg. For example, when a compound that promotes expression of the protein of the present invention is orally administered for the purpose of treating hypoglycemia, generally, in an adult (assuming a body weight of 60 kg), the compound is used in an amount of about 0.1 to 10 per day.
0 mg, preferably about 1.0 to 50 mg, more preferably about 1.0 to 20 mg. For example, when a compound that promotes the expression of the protein of the present invention is usually administered to an adult (as 60 kg) in the form of an injection for the treatment of hypoglycemia, the compound is administered in an amount of about 0.01 to 30 mg per day,
It is convenient to administer preferably about 0.1 to 20 mg, more preferably about 0.1 to 10 mg by intravenous injection. In the case of other animals, the amount can be administered in terms of 60 kg.

(4) Quantification of the Protein of the Present Invention An antibody against the protein of the present invention (hereinafter sometimes abbreviated as the antibody of the present invention) can specifically recognize the protein of the present invention. It can be used for quantification of the protein of the present invention in a test solution, particularly for quantification by a sandwich immunoassay. That is, the present invention
(I) reacting the antibody of the present invention with a test solution and a labeled protein of the present invention competitively, and measuring the ratio of the labeled protein of the present invention bound to the antibody. And (ii) simultaneously or sequentially reacting the test solution with the antibody of the present invention insolubilized on a carrier and another antibody of the present invention labeled on a carrier. The present invention provides a method for quantifying the protein of the present invention in a test solution, which comprises measuring the activity of a labeling agent on an insolubilized carrier after the reaction. In the quantitative method (ii), it is preferable that one antibody is an antibody that recognizes the N-terminal of the protein of the present invention and the other antibody is an antibody that reacts with the C-terminal of the protein of the present invention.

In addition to the quantification of the protein of the present invention using a monoclonal antibody against the protein of the present invention (hereinafter sometimes referred to as the monoclonal antibody of the present invention), detection by tissue staining or the like is also possible. . For these purposes, the antibody molecule itself may be used, or F (ab ′) ₂ , Fab ′, or Fab fraction of the antibody molecule may be used. The method for quantifying the protein of the present invention using the antibody of the present invention is not particularly limited, and may be an antibody, an antigen, or an antibody-antigen complex corresponding to the amount of antigen (eg, the amount of protein) in the test solution. Any measurement method may be used as long as the amount is detected by chemical or physical means, and the amount is calculated from a standard curve prepared using a standard solution containing a known amount of antigen. For example, nephelometry, competition method, immunometric method and sandwich method are preferably used, but in terms of sensitivity and specificity, it is particularly preferable to use the sandwich method described later. As a labeling agent used in a measurement method using a labeling substance, for example, a radioisotope, an enzyme, a fluorescent substance, a luminescent substance and the like are used. Examples of the radioisotope include [ ¹²⁵ I], [ ¹³¹ I],
[ ³ H] and [ ¹⁴ C] are used. The enzyme is preferably a stable enzyme having a large specific activity.
β-galactosidase, β-glucosidase, alkaline phosphatase, peroxidase, malate dehydrogenase and the like are used. As the fluorescent substance, for example, fluorescamine, fluorescein isothiocyanate and the like are used. As the luminescent substance, for example, luminol, a luminol derivative, luciferin, lucigenin and the like are used. Further, a biotin-avidin system can be used for binding the antibody or antigen to the labeling agent.

For insolubilization of the antigen or antibody, physical adsorption may be used, or a method using a chemical bond usually used for insolubilizing and immobilizing proteins or enzymes may be used. Examples of the carrier include insoluble polysaccharides such as agarose, dextran, and cellulose; synthetic resins such as polystyrene, polyacrylamide, and silicon; and glass. In the sandwich method, the test solution is reacted with the insolubilized monoclonal antibody of the present invention (primary reaction), and further reacted with another labeled monoclonal antibody of the present invention (secondary reaction). By measuring the activity of the labeling agent, the amount of the protein of the present invention in the test solution can be determined. The primary reaction and the secondary reaction may be performed in the reverse order, may be performed simultaneously, or may be performed at staggered times. The labeling agent and the method of insolubilization can be in accordance with those described above. In addition, in the immunoassay by the sandwich method,
The antibody used for the solid phase antibody or the labeling antibody is not necessarily one kind, and a mixture of two or more kinds of antibodies may be used for the purpose of improving measurement sensitivity and the like. In the method for measuring the protein of the present invention by the sandwich method of the present invention, as the monoclonal antibody of the present invention used in the primary reaction and the secondary reaction, an antibody having a different site to which the protein of the present invention binds is preferably used. That is, when the antibody used in the primary reaction and the secondary reaction recognizes the C-terminal of the protein of the present invention, the antibody used in the primary reaction is preferably For example, 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 measurement system other than the sandwich method, for example, a competition method, an immunometric method, a nephelometry and the like. In the competitive method, an antigen in a test solution and a labeled antigen are allowed to react competitively with an antibody, and then the unreacted labeled antigen is reacted.
(F) is separated from the labeled antigen (B) bound to the antibody (B / F separation), the amount of labeling of either B or F is measured, and the amount of antigen in the test solution is quantified. In this reaction method, a soluble antibody is used as the antibody, B / F separation is performed using polyethylene glycol, a liquid phase method using a second antibody against the antibody, or a solid phase antibody is used as the first antibody, or A solid-phase method using a soluble antibody as the first antibody and using a solid-phased antibody as the second antibody is used. In the immunometric method, an antigen in a test solution and a solid-phased antigen are subjected to a competitive reaction with a fixed amount of a labeled antibody, and then the solid phase and the liquid phase are separated. Is reacted with an excess amount of the labeled antibody, and then the immobilized antigen is added to bind the unreacted labeled antibody to the solid phase, and then the solid phase and the liquid phase are separated.
Next, the amount of label in any phase is measured to determine the amount of antigen in the test solution. In nephelometry, the amount of insoluble precipitates generated as a result of an antigen-antibody reaction in a gel or in a solution is measured. Even when the amount of antigen in the test solution is small and only a small amount of sediment is obtained, laser nephelometry utilizing laser scattering is preferably used.

In applying these individual immunoassays to the quantification method of the present invention, no special conditions, operations, and the like need to be set. The protein measurement system of the present invention may be constructed by adding ordinary technical considerations of those skilled in the art to ordinary conditions and operation methods in each method. For details of these general technical means, reference can be made to reviews, books, and the like. For example, Hiroe Irie, “Radio Immunoassay” (Kodansha, published in 1974), Hiroshi Irie, “Sequence Radioimmunoassay” (Kodansha, published in 1979), Eiji Ishikawa et al., “Enzyme Immunoassay” (Medical Shoin, Showa Ed. Ishikawa et al., “Enzyme Immunoassay” (Second Edition) (Issue Shoin, 1982) Published in 1962, "Methods in ENZYMOLOGY" Vol. 70 (Immunoc
chemical Techniques (Part A)), ibid. Vol. 73 (Immu
nochemical Techniques (Part B)), Ibid. Vol. 74 (I
mmunochemical Techniques (Part C)), ibid. Vol. 84
(Immunochemical Techniques (Part D: Selected Immun
oassays)), Ibid. Vol. 92 (Immunochemical Techniqu)
es (Part E: Monoclonal Antibodies and General Immun
oassay Methods)), Ibid. Vol. 121 (Immunochemical
Techniques (Part I: Hybridoma Technology and Monocl
onal Antibodies)) (above, published by Academic Press). As described above,
By using the antibody of the present invention, the protein of the present invention can be quantified with high sensitivity. Furthermore, by quantifying the concentration of the protein of the present invention using the antibody of the present invention, (1) when an increase in the concentration of the protein of the present invention is detected, for example, in diseases such as hyperglycemia or diabetes (2) If a decrease in the concentration of the protein of the present invention is detected, for example, a disease such as hypoglycemia or a future illness It can be diagnosed that the probability is high. Further, the antibody of the present invention can be used for detecting the protein of the present invention present in a subject such as a body fluid or a tissue. Further, for preparation of an antibody column used for purifying the protein of the present invention, detection of the protein of the present invention in each fraction at the time of purification, analysis of the behavior of the protein of the present invention in test cells, etc. Can be used.

(5) Gene diagnostic agent The DNA of the present invention can be used, for example, as a probe to produce a human or other warm-blooded animal (eg, rat, mouse, guinea pig, rabbit, bird, sheep, pig, cow, Since abnormalities (genetic abnormalities) of DNA or mRNA encoding the protein of the present invention in horses, cats, dogs, monkeys, chimpanzees, etc.) can be detected, for example, damage, mutation or reduced expression of the DNA or mRNA Further, it is useful as a diagnostic agent for a gene such as an increase or excessive expression of the DNA or mRNA. The above-described genetic diagnosis using the DNA of the present invention can be performed, for example, by known Northern hybridization or PCR.
-SSCP method (Genomics, Vol. 5, 8
74-879 (1989), Proceedings of the National Academy of Sciences of USA
Academy of Sciences of the USA), Vol. 86, 27
66-2770 (1989)). For example, when overexpression is detected by Northern hybridization or PCR-SSCP
When DNA mutation is detected by the method, for example, it is possible to diagnose that there is a high possibility of a disease such as hyperglycemia or diabetes or hypoglycemia.

(6) Pharmaceuticals Containing Antisense DNA Antisense DNA that can complementarily bind to the DNA of the present invention and suppress the expression of the DNA suppresses the production of the protein of the present invention in vivo. Therefore, it can be used, for example, as a prophylactic / therapeutic agent for hyperglycemia, diabetes, etc., in the same manner as the above-mentioned compound that suppresses the expression of the protein of the present invention. When the antisense DNA is used as the above-mentioned prophylactic / therapeutic agent, it can be carried out in the same manner as the above-mentioned various prophylactic / therapeutic agents containing the DNA of the present invention. For example, when the antisense DNA is used, the method can be carried out according to a conventional method after inserting the antisense DNA alone or into an appropriate vector such as a retrovirus vector, an adenovirus vector, and an adenovirus associated virus vector. The antisense DNA can be administered as it is or in the form of a formulation together with a physiologically acceptable carrier such as an adjuvant for promoting uptake, and then administered using a gene gun or a catheter such as a hydrogel catheter. Alternatively, it can be aerosolized and administered into the trachea as an inhalant. Further, the antisense D
NA can also be used as a diagnostic oligonucleotide probe for examining the presence or expression status of the DNA of the present invention in tissues or cells.

(7) Pharmaceutical Containing Antibody of the Present Invention The antibody of the present invention, which has an activity of neutralizing the activity of the protein of the present invention, is used as a medicament (prophylactic / therapeutic agent) for diseases such as hyperglycemia and diabetes. Can be used. The prophylactic / therapeutic agent for the above-mentioned diseases containing the antibody of the present invention can be used as it is as a liquid preparation or as a pharmaceutical composition in an appropriate dosage form as a human or other warm-blooded animal (eg, rat, rabbit, sheep, pig, bovine). , Cats, dogs, monkeys, etc.) can be administered orally or parenterally. The dose is
Depending on the administration subject, target disease, symptoms, administration route and the like, for example, when used for prevention and treatment of diabetes in adults, the antibody of the present invention is usually used in a dose of 0.01 to 20 mg as a single dose. / Kg body weight, preferably 0.1 to
About 10 mg / kg body weight, more preferably 0.1 to 5
It is convenient to administer about mg / kg body weight by intravenous injection about 1 to 5 times a day, preferably about 1 to 3 times a day. In the case of other parenteral administration and oral administration, an equivalent amount can be administered. If the symptoms are particularly severe, the dose may be increased according to the symptoms. The antibodies of the present invention can be administered as such or as a suitable pharmaceutical composition. The pharmaceutical composition used for the above administration contains the antibody of the present invention and a pharmacologically acceptable carrier, diluent or excipient. Such compositions are provided in dosage forms suitable for oral or parenteral administration. That is, for example, compositions for oral administration include solid or liquid dosage forms, specifically tablets (including sugar-coated tablets and film-coated tablets), pills, granules, powders, capsules (soft capsules and the like). ), Syrups, emulsions, suspensions and the like. Such a composition is produced by a known method and contains a carrier, diluent or excipient usually used in the field of pharmaceuticals. For example, lactose, starch, sucrose, magnesium stearate and the like are used as carriers and excipients for tablets.

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

(8) DNA-Transferred Animal The present invention relates to a DNA encoding an exogenous protein of the present invention.
A non-human mammal having NA (hereinafter abbreviated as the exogenous DNA of the present invention) or a mutant DNA thereof (sometimes abbreviated as the exogenous mutant DNA of the present invention) is provided. That is, the present invention provides (1) a non-human mammal having the exogenous DNA of the present invention or a mutant DNA thereof, (2) the animal according to (1), wherein the non-human mammal is a rodent, (3) (2) The animal according to (2), wherein the rodent is a mouse or a rat; and (4) a recombinant vector containing the exogenous DNA of the present invention or a mutant DNA thereof and capable of being expressed in a mammal. . Non-human mammals having the exogenous DNA of the present invention or the mutant DNA thereof (hereinafter abbreviated as the DNA-transferred animal of the present invention) can be used for non-fertilized eggs, fertilized eggs, germ cells including spermatozoa and their progenitor cells, and the like. Preferably, the calcium phosphate method, the electric pulse method, the lipofection method, the microinjection method, at the stage of embryonic development in non-human mammal development (more preferably, at the stage of single cells or fertilized egg cells and generally before the 8-cell stage) The target D by the particle gun method, the DEAE-dextran method, the retrovirus method, etc.
It can be created by transferring NA. Further, by the DNA transfer method, the exogenous DNA of the present invention can be transferred to somatic cells, organs of living organisms, tissue cells, and the like, and used for cell culture, tissue culture, and the like. The DNA-transferred animal of the present invention can also be produced by fusing the above-mentioned germ cells with a known cell fusion method.

Non-human mammals include, for example, bovine,
Pigs, sheep, goats, rabbits, dogs, cats, guinea pigs, hamsters, mice, rats and the like are used. Above all, rodents, which are relatively short in ontogeny and biological cycle in terms of preparation of disease animal model systems, and are easy to breed, especially mice (for example, C57 as a pure strain)
As a hybrid line such as BL / 6 line and DBA2 line, B6 line
C3F ₁ system, BDF ₁ system, B6D2F ₁ system, BA
LB / c strain, ICR strain, etc.) or rat (eg, Wistar, SD, etc.) are preferred. "Mammals" in a recombinant vector that can be expressed in mammals include humans and the like in addition to the above-mentioned non-human mammals. The exogenous DNA of the present invention refers to the DNA of the present invention once isolated and extracted from a mammal, not the DNA of the present invention originally possessed by a non-human mammal.
As the mutant DNA of the present invention, those having a mutation (for example, mutation) in the base sequence of the original DNA of the present invention,
Specifically, DNA in which addition or deletion of a base, substitution with another base, or the like has occurred is used, and abnormal DNA is also included. The abnormal DNA means a DNA that expresses an abnormal protein of the present invention, and for example, a DNA that expresses a protein that suppresses the function of the normal protein of the present invention is used. The exogenous DNA of the present invention comprises:
It may be derived from a mammal of the same or different species as the animal of interest. In transferring the DNA of the present invention to a target animal, it is generally advantageous to use the DNA as a DNA construct linked downstream of a promoter capable of being expressed in animal cells. For example, when transferring the human DNA of the present invention, DNAs derived from various mammals (eg, rabbits, dogs, cats, guinea pigs, hamsters, rats, mice, etc.) having the DNA of the present invention having high homology thereto are expressed. Highly expressing the DNA of the present invention by microinjecting a DNA construct (eg, a vector, etc.) in which the human DNA of the present invention is bound downstream of various promoters that can be made into a fertilized egg of a target mammal, for example, a mouse fertilized egg. DNA-transferring mammals can be created.

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

The above-described vector preferably has a sequence that terminates transcription of a messenger RNA of interest in a DNA-transferred mammal (generally called a terminator). Can be used, and preferably, a simian virus SV40 terminator or the like is used. In addition, a splicing signal of each DNA for the purpose of further expressing the desired foreign DNA,
It is also possible to link an enhancer region, a part of an intron of eukaryotic DNA, etc. 5 ′ upstream of the promoter region, between the promoter region and the translation region, or 3 ′ downstream of the translation region. The normal translation region of the protein of the present invention may be human or various mammals (eg, rabbit,
Dogs, cats, guinea pigs, hamsters, rats, mice, etc.) derived from liver, kidney, thyroid cells, fibroblast-derived DNA and various commercially available genomic DNA libraries as all or part of genomic DNA, or liver,
Complementary DNA prepared by known methods from RNA derived from kidney, thyroid cells, and fibroblasts can be obtained as a raw material. In addition, a translation region obtained by mutating a translation region of a normal protein obtained from the above-described cells or tissues by a point mutagenesis method can be used for the exogenous abnormal DNA. The translation region can be prepared as a DNA construct that can be expressed in a transgenic animal by a conventional genetic engineering technique in which it is ligated downstream of the above promoter and, if desired, upstream of the transcription termination site. Transfer of the exogenous DNA of the present invention at the fertilized egg cell stage
It is ensured that it is present in all germ cells and somatic cells of the target mammal. The presence of the exogenous DNA of the present invention in the germ cells of the produced animal after DNA transfer
It means that all progeny of the produced animal will carry the exogenous DNA of the present invention in all of their germinal and somatic cells. The progeny of such animals that have inherited the foreign DNA of the present invention have the foreign DNA of the present invention in all of their germinal and somatic cells.

The non-human mammal to which the exogenous normal DNA of the present invention has been transferred is confirmed to stably maintain the exogenous DNA by mating, and is subcultured as an animal having the DNA in a normal breeding environment. I can do it. Transfer of the exogenous DNA of the present invention at the fertilized egg cell stage is ensured to be present in excess in all germ cells and somatic cells of the target mammal. Excessive presence of the exogenous DNA of the present invention in germ cells of a produced animal after DNA transfer means that all offspring of the produced animal have an excessive amount of the exogenous DNA of the present invention in all of their germ cells and somatic cells. Means The offspring of such an animal inheriting the exogenous DNA of the present invention will have the exogenous DNA of the present invention in all of its germinal and somatic cells.
In excess. By obtaining a homozygous animal having the introduced DNA on both homologous chromosomes and mating the male and female animals, all offspring can be bred to have the DNA in excess. The non-human mammal having the normal DNA of the present invention, in which the normal DNA of the present invention is highly expressed, promotes the function of the endogenous normal DNA, and eventually causes hyperactivity of the protein of the present invention. Occasionally, it can be used as a disease model animal. For example, using the normal DNA-transferred animal of the present invention, it is possible to elucidate the pathological mechanism of hyperactivity of the protein of the present invention and diseases associated with the protein of the present invention, and to examine a method for treating these diseases. It is possible. In addition, since the mammal into which the exogenous normal DNA of the present invention has been transferred has an increased symptom of the free protein of the present invention, it is also used for a screening test for a prophylactic / therapeutic agent for a disease associated with the protein of the present invention. It is possible.

On the other hand, a non-human mammal having the exogenous abnormal DNA of the present invention should be subcultured in a normal breeding environment as an animal having the DNA after confirming that the exogenous DNA is stably maintained by mating. Can be done. Furthermore, the desired foreign DNA can be incorporated into the above-described plasmid and used as a source material. The DNA construct with the promoter can be prepared by a usual genetic engineering technique. Abnormal D of the present invention at the fertilized egg cell stage
NA transfer is ensured to be present in all germinal and somatic cells of the subject mammal. The presence of the abnormal DNA of the present invention in the germinal cells of the produced animal after DNA transfer means that all the offspring of the produced animal have the abnormal DNA of the present invention in all of its germinal and somatic cells. The offspring of such animals that have inherited the exogenous DNA of the present invention have the abnormal DNA of the present invention in all of their germinal and somatic cells. A homozygous animal having the introduced DNA on both homologous chromosomes is obtained, and by crossing these male and female animals, it is possible to breed so that all offspring have the DNA. The non-human mammal having the abnormal DNA of the present invention, in which the abnormal DNA of the present invention is highly expressed, inhibits the function of the endogenous normal DNA, and finally, a functionally inactive form of the protein of the present invention. It may become refractory and can be used as a disease model animal. For example, using the abnormal DNA-transferred animal of the present invention, it is possible to elucidate the pathological mechanism of the function-inactive refractory of the protein of the present invention and to examine a method for treating this disease. In addition, as a specific possibility, an animal in which the abnormal DNA of the present invention is highly expressed is characterized in that the abnormal protein of the present invention inhibits the function of a normal protein (dominant negat) in the function-inactive refractory disease of the protein of the present invention.
ive effect). In addition, since the mammal into which the foreign abnormal DNA of the present invention has been transferred has an increased symptom of the released protein of the present invention, it can be used for a therapeutic drug screening test for inactive refractory disease of the protein of the present invention. It is.

Other possible uses of the above two kinds of DNA-transferred animals of the present invention include, for example, use as a cell source for tissue culture, and DNA or R in the tissues of the DNA-transferred animal of the present invention.
Analysis of the relationship with proteins specifically expressed or activated by the protein of the present invention by directly analyzing NA or analyzing protein tissues expressed by DNA; Cultured by standard tissue culture techniques, using these to study the function of cells from tissues that are generally difficult to culture, screening for agents that enhance cell function by using the cells described above, and Isolation and purification of the mutant protein and production of its antibody can be considered. Furthermore, using the DNA-transferred animal of the present invention, it is possible to examine clinical symptoms of a disease associated with the protein of the present invention, including a functionally inactive refractory disease of the protein of the present invention. More detailed pathological findings in each organ of a disease model related to the disease can be obtained, which can contribute to the development of a new treatment method, and further to the research and treatment of a secondary disease caused by the disease. In addition, each organ was taken out from the DNA-transferred animal of the present invention, cut into small pieces, and D was released by a protease such as trypsin.
It is possible to obtain NA-transferred cells, culture them, or systematize the cultured cells. Furthermore, it is possible to investigate the specificity of the protein-producing cell of the present invention, its relationship with apoptosis, differentiation or proliferation, or its signal transduction mechanism, and to investigate its abnormalities. It is an effective research material for Furthermore, using the DNA-transferred animal of the present invention, the protein of the present invention includes a functionally inactive refractory disease,
In order to develop therapeutic agents for diseases related to the protein of the present invention, using the above-described test methods and quantitative methods,
It is possible to provide an effective and rapid screening method for the therapeutic agent for the disease. Further, using the transgenic animal of the present invention or the exogenous DNA expression vector of the present invention, a method for treating a DNA associated with the protein of the present invention was investigated.
It is possible to develop.

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

A non-human mammalian embryonic stem cell in which the DNA of the present invention has been inactivated is defined as a DNA obtained by artificially mutating the DNA of the present invention possessed by the non-human mammal to suppress the DNA expression ability. Alternatively, the DNA substantially does not have the ability to express the protein of the present invention by substantially losing the activity of the protein of the present invention encoded by the DNA (hereinafter referred to as the knockout DNA of the present invention). Embryonic stem cells of non-human mammals (hereinafter sometimes referred to as ES)
(Abbreviated as cell). As the non-human mammal, the same one as described above is used. As a method for artificially mutating the DNA of the present invention, for example, deletion of a part or all of the DNA sequence by genetic engineering techniques,
Can be inserted or substituted. With these mutations, for example, the knockout DNA of the present invention may be prepared by shifting the codon reading frame or disrupting the function of the promoter or exon. Specific examples of the non-human mammalian embryonic stem cells in which the DNA of the present invention has been inactivated (hereinafter abbreviated as the DNA-inactivated ES cells of the present invention or the knockout ES cells of the present invention) include, for example, The DNA of the present invention possessed by a non-human mammal is isolated and its exon portion is a drug resistance gene typified by a neomycin resistance gene, a hygromycin resistance gene, or lacZ (β-galactosidase gene), cat (chloramphenicol acetyl). A DNA sequence that disrupts the function of exons by inserting a reporter gene or the like typified by a transferase gene) or terminates transcription of a gene in an intron between exons (for example, pol
a DNA sequence that is constructed to disrupt the gene by inserting a complete messenger RNA by inserting
A chain (hereinafter abbreviated as targeting vector)
Is introduced into the chromosome of the animal by, for example, homologous recombination, and the obtained ES cells are subjected to Southern hybridization analysis using a DNA sequence on or near the DNA of the present invention as a probe or to a DNA sequence on a targeting vector. It can be obtained by analyzing by a PCR method using a DNA sequence of a neighboring region other than the DNA of the present invention used for the preparation of the vector as a primer, and selecting a knockout ES cell of the present invention.

Further, the DN of the present invention may be
As the original ES cells for inactivating A, for example, those already established as described above may be used, or newly established ES cells according to the known method of Evans and Kaufman may be used. For example, in the case of mouse ES cells, currently, 129 line ES cells are generally used. However, since the immunological background is not clear, a pure line instead of this is used as an immunological genetic background. For example, C57BL / 6 mice or C57BL
/ 6 egg collection number of lack of usable like even better DBA / 2 and BDF ₁ mice improved by crossing (C57BL / 6 and _{F 1} and DBA / 2) which were established by using a. B
DF ₁ mice are often egg number and egg can be changed to that of a tough, since with C57BL / 6 mice in the background, when the ES cells obtained therefrom, which were generated pathological model mice , C57BL / 6 mice can be advantageously used in that their genetic background can be replaced with C57BL / 6 mice by backcrossing. Also,
When ES cells are established, blastocysts 3.5 days after fertilization are generally used. In addition to this, a large number of initial cells can be efficiently prepared by collecting 8-cell embryos and culturing them up to blastocysts. Embryos can be obtained. Either male or female ES cells may be used, but generally male ES cells are more convenient for producing a germline chimera. It is also desirable to discriminate between males and females as soon as possible in order to reduce cumbersome culture labor. As a method for determining the sex of ES cells, for example, a method of amplifying and detecting a gene in the sex-determining region on the Y chromosome by PCR can be mentioned. Using this method, conventionally, for example G-banding method, requires about 10 ⁶ cells for karyotype analysis, since suffices ES cell number of about 1 colony (about 50), culture The primary selection of ES cells in the early stage can be performed by discriminating between male and female, and by enabling selection of male cells at an early stage, labor in the initial stage of culture can be greatly reduced.

The secondary selection can be performed, for example, by confirming the number of chromosomes by the G-banding method. The number of chromosomes in the obtained ES cells is desirably 100% of the normal number. However, when it is difficult due to physical operations or the like at the time of establishment, after knocking out the gene of the ES cells, the cells can be knocked out of normal cells (for example, chromosome in mice). Number is 2n
= Cells). The embryonic stem cell line obtained in this way usually has very good growth properties, but must be carefully subcultured because it tends to lose its ontogenetic ability. For example, on a suitable feeder cell such as STO fibroblast in the presence of LIF (1-10000 U / ml) in a carbon dioxide incubator (preferably 5% carbon dioxide, 95% air or 5% oxygen, 5% The cells are cultured by a method such as culturing at about 37 ° C. in carbon dioxide gas and 90% air. At the time of subculture, for example, trypsin / EDTA solution (usually 0.001-0.5% trypsin / 0.1-5 mM EDTA, Preferably about 0.1
% Trypsin / 1 mM EDTA), and the cells are seeded on freshly prepared feeder cells. Such passage is usually performed every 1 to 3 days. At this time, it is desirable to observe the cells and, if morphologically abnormal cells are found, discard the cultured cells. ES cells are differentiated into various types of cells, such as parietal, visceral, and cardiac muscles, by monolayer culture up to high density or suspension culture until cell clumps are formed under appropriate conditions. (M.
J. Evans and MH Kaufman, Nature
292, 154, 1981; GR Martin, Processings of the National Academy of Sciences of USA (Proc. Natl. Acad. Sc)
i. USA) 78, 7634, 1981; TCDoetschman
Et al., Journal of Embryology and Experimental Morphology, Vol. 87, p. 27, 19
85 years], the DNA-deficient cell of the present invention obtained by differentiating the ES cell of the present invention is useful in in vitro cell biology of the protein of the present invention. The non-human mammal deficient in DNA expression of the present invention may comprise an mR of the animal.
By measuring the amount of NA using a known method and indirectly comparing the expression level, it is possible to distinguish from normal animals. As the non-human mammal, those similar to the aforementioned can be used.

The non-human mammal deficient in DNA expression of the present invention may be prepared, for example, by introducing the targeting vector prepared as described above into mouse embryonic stem cells or mouse egg cells,
By knocking out the DNA of the present invention, the DNA sequence in which NA has been inactivated is replaced with the DNA of the present invention on the chromosome of mouse embryonic stem cells or mouse egg cells by gene homologous recombination. it can. A cell in which the DNA of the present invention has been knocked out is a DNA sequence of the present invention derived from the mouse used in the targeting vector, the DNA sequence of the DNA of the present invention or a DNA sequence of the targeting vector, or a DNA sequence on or near the DNA of the present invention. It can be determined by analysis by PCR using a DNA sequence of a nearby region other than DNA as a primer. When a non-human mammalian embryonic stem cell is used, a cell line in which the DNA of the present invention has been inactivated by gene homologous recombination is cloned, and the cell is cultured at an appropriate time, for example, at the 8-cell stage of a non-human mammalian stem cell. The chimeric embryo is injected into an animal embryo or a blastocyst, and the produced chimeric embryo is transplanted into the uterus of the pseudopregnant non-human mammal. The produced animal is a chimeric animal comprising both cells having the normal DNA locus of the present invention and cells having the artificially mutated DNA locus of the present invention. When a part of the germ cells of the chimeric animal has a mutated DNA locus of the present invention, all tissues are artificially mutated from a population obtained by crossing such a chimeric individual with a normal individual. D of the present invention added
It can be obtained by selecting individuals composed of cells having NA loci, for example, by judging coat color or the like. The individual thus obtained is usually an individual heterozygously deficient in expression of the protein of the present invention, mated with an individual deficient in heteroexpression of the protein of the present invention, and homozygously expressing the protein of the present invention from their offspring. Defective individuals can be obtained. When using an egg cell, for example, a transgenic non-human mammal having a targeting vector introduced into a chromosome can be obtained by injecting a DNA solution into the egg cell nucleus by a microinjection method,
Compared to these transgenic non-human mammals,
It can be obtained by selecting a DNA locus of the present invention having a mutation by homologous recombination.

In the individual in which the DNA of the present invention has been knocked out as described above, it is necessary to confirm that the DNA has been knocked out in the animal individual obtained by mating, and to carry out subculture in a normal breeding environment. Can be. further,
The germline can be obtained and maintained in accordance with a conventional method. That is, by crossing male and female animals having the inactivated DNA, homozygous animals having the inactivated DNA on both homologous chromosomes can be obtained. The obtained homozygous animal was compared to the mother animal by one normal individual,
It can be obtained efficiently by breeding in a state where a plurality of homozygotes are formed. By mating male and female heterozygous animals, homozygous and heterozygous animals having the inactivated DNA are bred and passaged. The non-human mammalian embryonic stem cells in which the DNA of the present invention has been inactivated are extremely useful for producing a non-human mammal deficient in expressing the DNA of the present invention. In addition, the DN of the present invention
Since non-human mammals deficient in A expression lack various biological activities that can be induced by the protein of the present invention, they can serve as models for diseases caused by inactivation of the biological activity of the protein of the present invention. It is useful for investigating the cause of disease and examining treatment methods.

(10) A method for screening for a compound having a preventive / therapeutic effect against diseases caused by DNA deficiency or damage according to the present invention.
It can be used for screening for a compound having a preventive / therapeutic effect on diseases caused by NA deficiency or damage (eg, hypoglycemia, etc.). That is, the present invention is characterized by administering a test compound to a non-human mammal deficient in expressing the DNA of the present invention, and observing and measuring changes in the animal. Provided is a method for screening a compound or a salt thereof having a preventive / therapeutic effect on a disease. Examples of the non-human mammal deficient in expressing DNA of the present invention used in the screening method include the same ones as described above. Test compounds include, for example, peptides, proteins, non-peptidic compounds, synthetic compounds, fermentation products, cell extracts, plant extracts, animal tissue extracts, and plasma, and these compounds are novel compounds. Or a known compound. Specifically, a non-human mammal deficient in expression of the DNA of the present invention is treated with a test compound, compared with an untreated control animal, and tested using changes in each organ, tissue, disease symptoms, etc. of the animal as an index. The prophylactic and therapeutic effects of the compounds can be tested. As a method of treating a test animal with a test compound, for example, oral administration, intravenous injection and the like are used, and it can be appropriately selected according to the symptoms of the test animal, properties of the test compound, and the like. The dose of the test compound can be appropriately selected according to the administration method, the properties of the test compound, and the like.

The compound obtained by using the screening method of the present invention is a compound selected from the test compounds described above, and is used for a disease (eg, hypoglycemia, etc.) caused by decreased expression of the protein of the present invention. Prevention
Since it has a therapeutic effect, it can be used as a drug such as a safe and low-toxic preventive / therapeutic agent for the disease.
Furthermore, compounds derived from the compounds obtained by the above screening can be used in the same manner. The compound obtained by the screening method may form a salt, and the salt of the compound may include a physiologically acceptable acid (eg, an inorganic acid, an organic acid) and a base (eg, an alkali metal). Are used, and especially preferred are physiologically acceptable acid addition salts. Examples of such salts include salts with inorganic acids (eg, 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,
For example, salts with malic acid, oxalic acid, benzoic acid, methanesulfonic acid, and benzenesulfonic acid) are used. A drug containing the compound or a salt thereof obtained by the screening method can be produced in the same manner as the above-mentioned drug containing the protein of the present invention. The preparations obtained in this way are safe and have low toxicity, for example, human or warm-blooded animals (eg, rat, mouse, guinea pig, rabbit, sheep, pig, cow, horse, cat, dog, monkey, etc.) Can be administered. The dose of the compound or a salt thereof varies depending on the target disease, the subject to be administered, the administration route, and the like.
g), the compound is added at about 0.1 per day.
-100 mg, preferably about 1.0-50 mg, more preferably about 1.0-20 mg. When administered parenterally, the single dose of the compound varies depending on the administration subject, target disease, and the like. For example, for the purpose of treating hypoglycemia, the compound is usually administered in the form of an injection to an adult (60 kg). )), The compound is administered in an amount of about 0.01 per day.
About 30 mg, preferably about 0.1 to 20 mg,
More preferably, about 0.1 to 10 mg is administered by intravenous injection. For other animals, 60
An amount converted per kg can be administered.

(11) Method for screening for a compound that promotes or inhibits the activity of the promoter for the DNA of the present invention The present invention relates to a method for screening a non-human mammal deficient in expression of the DNA of the present invention.
The present invention provides a method for screening a compound or a salt thereof which promotes or inhibits the activity of a promoter for DNA of the present invention, which comprises administering a test compound and detecting the expression of a reporter gene. In the above-mentioned screening method, the non-human mammal deficient in expression of DNA of the present invention may be a non-human mammal deficient in expressing DNA of the present invention in which the DNA of the present invention is inactivated by introducing a reporter gene, A reporter gene that can be expressed under the control of a promoter for the DNA of the present invention is used. Examples of the test compound include the same compounds as described above. As the reporter gene, the same one as described above is used, and a β-galactosidase gene (lacZ), a soluble alkaline phosphatase gene, a luciferase gene and the like are preferable. The DN of the present invention in which the DNA of the present invention is replaced with a reporter gene
In a non-human mammal deficient in A expression, since the reporter gene exists under the control of the promoter for the DNA of the present invention, the activity of the promoter can be detected by tracing the expression of the substance encoded by the reporter gene.

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

The compound or a salt thereof obtained by using the above-mentioned screening method is a compound selected from the above-mentioned test compounds, and is a compound that promotes or inhibits the promoter activity for the DNA of the present invention. The compound obtained by the screening method may form a salt,
Salts of the compound include physiologically acceptable acids (eg,
Salts with inorganic acids) and bases (eg, organic acids),
Particularly preferred are physiologically acceptable acid addition salts. Such salts include, for example, salts with inorganic acids (eg, hydrochloric acid, phosphoric acid, hydrobromic acid, sulfuric acid) or organic acids (eg, acetic acid, formic acid, propionic acid, fumaric acid, maleic acid, succinic acid) Acids, tartaric acid, citric acid, malic acid, oxalic acid, benzoic acid, methanesulfonic acid, benzenesulfonic acid) and the like are used. The compound or a salt thereof that promotes the promoter activity of the DNA of the present invention can promote the expression of the protein of the present invention and promote the activity of the protein. It is useful as a medicament such as a toxic prophylactic or therapeutic agent. On the other hand, compounds or salts thereof that inhibit the promoter activity of the DNA of the present invention can inhibit the expression of the protein of the present invention and inhibit the activity of the protein. It is useful as a medicament such as a safe and low toxic prophylactic / therapeutic agent. Furthermore, compounds derived from the compounds obtained by the above screening can be used in the same manner.

A drug containing the compound or a salt thereof obtained by the screening method can be produced in the same manner as the above-mentioned drug containing the protein of the present invention. The preparations obtained in this way are safe and have low toxicity, for example, human or warm-blooded animals (eg, rat, mouse, guinea pig, rabbit, sheep, pig, cow, horse, cat, dog, monkey, etc.) Can be administered. The dose of the compound or a salt thereof varies depending on the target disease, the administration subject, the administration route, and the like.For example, when a compound that inhibits the promoter activity of the DNA of the present invention for the purpose of treating diabetes is orally administered, In an adult (assuming a body weight of 60 kg), the compound is used in an amount of about 0.1 to 100 mg, preferably about 1.0 to 50 mg, more preferably about 1.0 to 20 mg per day.
Administer. When administered parenterally, the single dose of the compound varies depending on the administration subject, target disease, and the like.
For example, when a compound that inhibits the promoter activity of the DNA of the present invention for the purpose of treating diabetes is usually administered to an adult (as 60 kg) in the form of an injection, about 0.01 to 30 mg of the compound per day is preferable. Is about 0.1
It may be convenient to administer about 20 mg, more preferably about 0.1-10 mg, by intravenous injection. In the case of other animals, the amount can be administered in terms of 60 kg. On the other hand, for example, when a compound that promotes the promoter activity for DNA of the present invention is orally administered for the purpose of treating hypoglycemia, generally, in an adult (with a body weight of 60 kg), the compound is used in an amount of about 0.1 per day. -10
0 mg, preferably about 1.0 to 50 mg, more preferably about 1.0 to 20 mg. When administered parenterally, the single dose of the compound varies depending on the administration subject, target disease, and the like. For example, a compound that promotes the promoter activity of the DNA of the present invention for the purpose of treating hypoglycemia is injected. When administered to a normal adult (as 60 kg) in the form of a preparation, the compound is administered in an amount of about 0.01 to 30 mg, preferably about 0.1 to 20 mg, more preferably about 0.1 to 10 mg per day. It is conveniently administered by intravenous injection. In the case of other animals, the amount can be administered in terms of 60 kg. As described above, the non-human mammal deficient in expressing the DNA of the present invention
It is extremely useful in screening for a compound or a salt thereof that promotes or inhibits the activity of a promoter against, and greatly contributes to the investigation of the causes of various diseases caused by DNA expression deficiency of the present invention or the development of preventive / therapeutic agents. it can. Further, using a DNA containing the promoter region of the protein of the present invention, genes encoding various proteins are ligated downstream thereof and injected into egg cells of an animal to produce a so-called transgenic animal (gene-transferred animal). Once created, it will also be possible to specifically synthesize the protein and study its effects in living organisms.
Furthermore, if a suitable reporter gene is linked to the above-mentioned promoter portion and a cell line in which this is expressed is established, a low-molecular compound having an action of specifically promoting or suppressing the ability of the protein itself of the present invention to produce in the body. Can be used as a search system. By analyzing the promoter portion, it is also possible to find a new cis element and a transcription factor binding thereto.

In the present specification and drawings, when bases, amino acids and the like are represented by abbreviations, IUPAC-IUB
It is based on the abbreviation by the Commission on Biochemical Nomenclature or the abbreviation commonly used in the relevant field. When an amino acid can have an optical isomer, the L-form is indicated unless otherwise specified. DNA: deoxyribonucleic acid cDNA: complementary deoxyribonucleic acid A: adenine T: thymine G: guanine C: cytosine RNA: ribonucleic acid mRNA: messenger ribonucleic acid dATP: deoxyadenosine triphosphate dTTP: deoxythymidine triphosphate dGTP: deoxyguanosine triphosphate Phosphate dCTP: Deoxycytidine triphosphate ATP: Adenosine triphosphate EDTA: Ethylenediaminetetraacetic acid SDS: Sodium dodecyl sulfate Gly: Glycine Ala: Alanine Val: Valine Leu: Leucine Ile: Isoleucine Ser: Serine Thr: thyronine : Methionine Glu: Glutamic acid Asp: Aspartic acid Lys: Lysine Arg: Arginine His: Histidine Phe Phenylalanine Tyr: tyrosine Trp: tryptophan Pro: proline Asn: asparagine Gln: glutamine pGlu: pyroglutamic acid

The substituents, protecting groups and reagents frequently 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 indicate the following sequences. [SEQ ID NO: 1] This shows the amino acid sequence of human MD25 (VLCAD). [SEQ ID NO: 2] This shows the base sequence of human MD25 (VLCAD) gene (cDNA). [SEQ ID NO: 3] This shows the base sequence of the primer used in EXAMPLE 1. [SEQ ID NO: 4] This shows the base sequence of the primer used in EXAMPLE 1. [SEQ ID NO: 5] This shows the amino acid sequence of N-terminal 109 amino acid residues of IRAP. [SEQ ID NO: 6] This shows the base sequence of DNA encoding the amino acid sequence of N-terminal 109 amino acid residues of IRAP. [SEQ ID NO: 7] This shows the amino acid sequence of the 468th to 510th amino acid residues of GLUT4. [SEQ ID NO: 8] This shows the base sequence of DNA encoding the amino acid sequence of the 468th to 510th amino acid residues of GLUT4. [SEQ ID NO: 9] This shows the amino acid sequence of rat VLCAD. [SEQ ID NO: 10] This shows the amino acid sequence of mouse VLCAD. [SEQ ID NO: 11] This shows the amino acid sequence of bovine VLCAD. Plasmid pTB21 containing the cDNA base sequence of MD25 (SEQ ID NO: 2) obtained in Example 1 described below.
Escherichia coli carrying Escherichia coli 24
ichia coli) DH5α / pTB2124 has been available since September 4, 2000, 1-1 1-1 Higashi, Tsukuba City, Ibaraki Pref., National Institute of Advanced Industrial Science and Technology (AIST) Deposit No. FERM BP-729 with the National Institute of Advanced Industrial Science and Technology (NIBH)
It has been deposited as a deposit No. IFO 16469 from August 24, 2000 to 2-17-85 Jusanhoncho, Yodogawa-ku, Osaka-shi, Osaka, Japan, under the deposit number IFO 16469.

[0083]

EXAMPLES The present invention will be described in more detail with reference to the following Examples, but it should not be construed that the present invention is limited thereto. In addition, the genetic manipulation method using Escherichia coli followed the method described in Molecular cloning.

Example 1 IRA by yeast two-hybrid method
Cloning of cDNA encoding P-binding protein cDNA encoding a protein that binds to insulin-responsive aminopeptidase (IRAP) is a yeast two-hybrid
Cloned by the method. The yeast two-hybrid method was basically performed using a MATCHMAKER two-hybrid system manufactured by Clontech. A polypeptide from amino acid residue 55 to position 82 of IRAP (Keller et al, J. Biol. Chem.,
270, 23612-23618, 1995; a DNA fragment encoding SEQ ID NO: 5 amino acids 55-82) was chemically synthesized,
This is called GAL4-DNA under the control of the ADH1 promoter.
The fusion domain was inserted into a plasmid pGBT9 (Clontech) expressing the binding domain (GAL4-BD) in a form fused thereto,
This was designated as a bait vector, pBait-2. As a cDNA library to be screened, a human skeletal muscle-derived cDNA library manufactured by Clontech was used. This library contains GAL4 under the control of the ADH1 promoter.
The library cDNA is constructed to be expressed in yeast in a form fused with the transcription activation domain (GAL4-AD). The host yeast is Saccharomyces cerevisi
ae Y190 was used. This yeast strain contains GAL1 TATA box and UAS (upstream ac) as reporter genes.
β-galactosidase gene (LacZ) and histidine synthesis gene (H
IS3) on its chromosome. S. cerevisia
e Introduced pBait-2 (TRP1 marker) and a human skeletal muscle-derived cDNA library plasmid (LEU2 marker) into Y190, have both plasmids, and
A transformant yeast expressing HIS3, one of the hybrid reporter genes, was transformed into a 60 mM 3
-Selection was made on SD medium without aminotriazole and without tryptophan, leucine and histidine. The selected transformant colonies were transferred to a nylon membrane by a replica method, and the yeast cell wall was crushed by freezing and thawing with liquid nitrogen, followed by X-Gal (5-bromo-4-chloro-β-D-galactosid.
Staining was performed in e), and a strain exhibiting β-galactosidase activity was defined as a primary candidate strain. Screened 10 ⁷ or more libraries cDNA in the above manner, it was obtained candidate genes 12 clones. A cell extract was prepared from these yeasts using Zymolyase (manufactured by Seikagaku Corporation),
Escherichia coli requiring leucine using the DNA fraction
Escherichia coli HB101 was transformed. The transformed Escherichia coli was spread on a leucine-free M9 medium, Escherichia coli strains having a library plasmid (LEU2 marker) were selected, and the plasmid was extracted therefrom. Using the extracted library plasmid and pBait-2 which is an IRAP bit vector, S. cerevis was again used.
iae Y190 was transformed, and the resulting transformant was examined for histidine requirement and β-galactosidase activity,
Five clones showing reproducibility were obtained. Among these, β-
A clone having strong galactosidase activity (MD25 strain) was selected. The nucleotide sequence of MD25 is human very-long chain
acyl-CoA dehydrogenase (Genbank D43682; hereafter VLC
(Referred to as AD). The matched part was downstream from the 36th amino acid in the 655 amino acids of VLCAD (SEQ ID NO: 1). VLCA
The cDNA sequence containing the N-terminus of D is obtained from the polymerase chain reaction using human skeletal muscle cDNA library as a template.
(PCR). The primer sequences used for this PCR are shown below. (1) 5'-AGAGATTCGGAGATGCAGGCGGCT-3 '(SEQ ID NO: 3) (2) 5'-AGGGTAATGCCCACGCCAAGGTCA-3' (SEQ ID NO: 4) Restriction of this PCR fragment and a partial fragment of VLCAD cloned by yeast two-hybrid method It was ligated with the enzyme SphI to obtain a full-length MD25 cDNA (pTB
2124). MD25 cDNA base sequence (SEQ ID NO:
2) and the amino acid sequence (SEQ ID NO: 1)
Shown in

Example 2 Confirmation of Binding Activity by Measurement of β-Galactosidase Activity To quantitatively confirm the binding of MD25 to IRAP, C
Β-galactosidase activity was measured using PRG (chlorophenolred-β-D-galactopyranoside) as a substrate.
The yeast holding both the Bait and prey vectors was subjected to liquid culture, the cells were collected, and the cell wall was disrupted by freezing and thawing with liquid nitrogen. The suspension of the crushed cells
PRG was added and the absorbance at 578 nm of the samples was measured as β-galactosidase activity. The unit of β-galactosidase activity is that one yeast cell per minute converts 1 μmol of CPRG into chlorophenol red and D-galactose.
The enzymatic activity hydrolyzed to oside was defined as one unit. Bai
The t sequence includes IRAP (55-82; amino acids 55 to 82 of SEQ ID NO: 5), and the prey sequence includes MAP.
Among the D25 cDNA sequences, a sequence directly isolated by the yeast two-hybrid method (a sequence corresponding to the 3 ′ side from base number 118 in FIGS. 1 to 3) was used. Further, as a negative control, a vector pGBT9 expressing a non-fused sequence of GAL4-BD with bait was used. S. cerevis using plasmids with these sequences
iae Y190 was transformed, and the β-galactosidase activity of the reconstructed yeast transformant was measured. MD25 cD
The transformant having NA has IRAP (55-82) as b
When expressed as ait, β-galactosidase activity of about 11 units was exhibited. On the other hand, GAL4-BD alone showed almost no binding activity to a protein having no bait sequence. In addition, MD25 cDNA which is a prey sequence
The value of β-galactosidase activity in the experiment using the strain having no was below the detection limit (FIG. 4).

Example 3 Histidine Non-auxotrophic Test The parent strain S. cerevisiae Y190 was originally a histidine auxotroph, but when bait and prey were bound, HIS3, one of the reporter genes, was expressed.
The yeast strain becomes non-histidine-requiring. I as bait
The strain into which RAP was introduced and MD25 was introduced as a prey grew on an SD medium (minimum medium) supplemented with 40 mM 3-aminotriazole and without tryptophan, leucine, or histidine. This confirmed that IRAP and MD25 bind.

Example 4 Distribution of Expression of MD25 mRNA in Human Tissue The distribution of expression of MD25 mRNA in human tissue was detected by Northern blotting. That is, poly (A) ⁺ RNA (2 μg each) of each human tissue was MD25 cD
Northern blotting was performed using NA (base numbers 479-2049 of SEQ ID NO: 3) as a probe. As a nylon membrane to which mRNA of each human tissue was transferred, an MTN blot (human 12 lane) manufactured by Clontech was used. An MD25 cDNA probe labeled with ³² P was hybridized and washed under stringent conditions, and detected with an image analyzer BAS2000II (manufactured by Fuji Film Co., Ltd.). As shown in FIG. 5, MD25 mRNA was about 2.4.
Strong expression was observed in the heart, skeletal muscle, kidney and liver with a length of kb.

[0088]

The protein of the present invention is strongly expressed in heart, skeletal muscle, kidney and liver. The protein of the present invention increases the blood glucose level by binding to IRAP, and thus is useful as a preventive / therapeutic agent for hypoglycemia. Further, the protein of the present invention is different from the protein of the present invention by IR
It can be used in a screening method for inhibiting the binding to AP or GLUT4. The protein of the present invention and I
Compounds that inhibit the binding to RAP or GLUT4 are useful as agents for preventing or treating diseases such as hyperglycemia and diabetes.

[0089]

[SEQUENCE LISTING]

 <110> Takeda Chemical Industries, Ltd. <120> IRAP Binding Protein <130> P2001-144 <150> JP 2000-254263 <151> 2000-08-21 <150> JP 2000-276633 <151> 2000-09- 07 <160> 11 <210> 1 <211> 655 <212> PRT <213> Human <400> 1 Met Gln Ala Ala Arg Met Ala Ala Ser Leu Gly Arg Gln Leu Leu Arg 1 5 10 15 Leu Gly Gly Gly Ser Ser Arg Leu Thr Ala Leu Leu Gly Gln Pro Arg 20 25 30 Pro Gly Pro Ala Arg Arg Pro Tyr Ala Gly Gly Ala Ala Gln Leu Ala 35 40 45 Leu Asp Lys Ser Asp Ser His Pro Ser Asp Ala Leu Thr Arg Lys Lys 50 55 60 Pro Ala Lys Ala Glu Ser Lys Ser Phe Ala Val Gly Met Phe Lys Gly 65 70 75 80 Gln Leu Thr Thr Asp Gln Val Phe Pro Tyr Pro Ser Val Leu Asn Glu 85 90 95 Glu Gln Thr Gln Phe Leu Lys Glu Leu Val Glu Pro Val Ser Arg Phe 100 105 110 Phe Glu Glu Val Asn Asp Pro Ala Lys Asn Asp Ala Leu Glu Met Val 115 120 125 Glu Glu Thr Thr Trp Gln Gly Leu Lys Glu Leu Gly Ala Phe Gly Leu 130 135 140 Gln Val Pro Ser Glu Leu Gly Gly Val Gly Leu Cys Asn Thr Gln Tyr 145 150 155 160 Ala Arg Leu Val Glu Ile Val Gly Met His Asp Leu Gly Val Gly Ile 165 170 175 Thr Leu Gly Ala His Gln Ser Ile Gly Phe Lys Gly Ile Leu Leu Phe 180 185 190 Gly Thr Lys Ala Gln Lys Glu Lys Tyr Leu Pro Lys Leu Ala Ser Gly 195 200 205 Glu Thr Val Ala Ala Phe Cys Leu Thr Glu Pro Ser Ser Gly Ser Asp 210 215 220 Ala Ala Ser Ile Arg Thr Ser Ala Val Pro Ser Pro Cys Gly Lys Tyr 225 230 235 240 Tyr Thr Leu Asn Gly Ser Lys Leu Trp Ile Ser Asn Gly Gly Leu Ala 245 250 255 Asp Ile Phe Thr Val Phe Ala Lys Thr Pro Val Thr Asp Pro Ala Thr 260 265 270 Gly Ala Val Lys Glu Lys Ile Thr Ala Phe Val Val Glu Arg Gly Phe 275 280 285 Gly Gly Ile Thr His Gly Pro Pro Glu Lys Lys Met Gly Ile Lys Ala 290 295 300 Ser Asn Thr Ala Glu Val Phe Phe Asp Gly Val Arg Val Pro Ser Glu 305 310 315 320 Asn Val Leu Gly Glu Val Gly Ser Gly Phe Lys Val Ala Met His Ile 325 330 335 Leu Asn Asn Gly Arg Phe Gly Met Ala Ala Ala Leu Ala Gly Thr Met 340 345 350 Arg Gly Ile Ile Ala Lys Ala Val Asp His Ala Thr Asn Arg Thr Gln 355 360 365 Phe Gly Glu Lys Ile His Asn Phe Gly Leu Ile Gln Glu Lys Leu Ala 370 375 380 Arg Met Val Met Leu Gln Tyr Val Thr Glu Ser Met Ala Tyr Met Val 385 390 395 400 Ser Ala Asn Met Asp Gln Gly Ala Thr Asp Phe Gln Ile Glu Ala Ala 405 410 415 Ile Ser Lys Ile Phe Gly Ser Glu Ala Ala Trp Lys Val Thr Asp Glu 420 425 430 Cys Ile Gln Ile Met Gly Gly Met Gly Phe Met Lys Glu Pro Gly Val 435 440 445 Glu Arg Val Leu Arg Asp Leu Arg Ile Phe Arg Ile Phe Glu Gly Thr 450 455 460 Asn Asp Ile Leu Arg Leu Phe Val Ala Leu Gln Gly Cys Met Asp Lys 465 470 475 480 Gly Lys Glu Leu Ser Gly Leu Gly Ser Ala Leu Lys Asn Pro Phe Gly 485 490 495 Asn Ala Gly Leu Leu Leu Gly Glu Ala Gly Lys Gln Leu Arg Arg Arg 500 505 510 Ala Gly Leu Gly Ser Gly Leu Ser Leu Ser Gly Leu Val His Pro Glu 515 520 525 Leu Ser Arg Ser Gly Glu Leu Ala Val Arg Ala Leu Glu Gln Phe Ala 530 535 540 Thr Val Val Glu Ala Lys Leu Ile Lys His Lys Lys Gly Ile Val Asn 545 550 555 555 560 Glu Gln Phe Leu Leu Gln Arg Leu Ala Asp Gly Ala Ile Asp Leu Tyr 565 570 575 Ala Met Val Val Val Leu Ser Arg Ala Ser Arg Ser Leu Ser Glu Gly 580 585 590 His Pro Thr Ala Gln His Glu Lys Met Leu Cys Asp Thr Trp Cys Ile 595 600 605 Glu Ala Ala Ala Arg Ile Arg Glu Gly Met Ala Ala Leu Gln Ser Asp 610 615 615 620 Pro Trp Gln Gln Glu Leu Tyr Arg Asn Phe Lys Ser Ile Ser Lys Ala 625 630 635 640 Leu Val Glu Arg Gly Gly Val Val Thr Ser Asn Pro Leu Gly Phe 645 650 655 <210> 2 <211> 1965 <212> DNA <213> Human <400> 2 ATGCAGGCGG CTCGGATGGC CGCGAGCTTG GGGCGGCAGC TGCTGAGGCT CGGGGGCGGA 60 AGCTCGCGGC TCACGGCGCT CCTGGGGCAG CCCCGGCCCG GCCCTGCCCG GCGGCCCTAT 120 GCCGGGGGTG CCGCTCAGCT GGCTCTGGAC AAGTCAGATT CCCACCCCTC TGACGCTCTG 180 ACCAGGAAAA AACCGGCCAA GGCGGAATCT AAGTCCTTTG CTGTGGGAAT GTTCAAAGGC 240 CAGCTCACCA CAGATCAGGT GTTCCCATAC CCGTCCGTGC TCAACGAAGA GCAGACACAG 300 TTTCTTAAAG AGCTGGTGGA GCCTGTGTCC CGTTTCTTCG AGGAAGTGAA CGATCCCGCC 360 AAGAATGACG CTCTGGAGAT GGTGGAGGAG ACCACTTGGC AGGGCCTCAA GGAGCTGGGG 420 GCCTTTGGTC TGCAAGTGCC CAGTGAGCTG GGTGGTGTGG GCCTTTGCAA CACCCAGTAC 480 GCCCGTTTGG TGGAGATCGT GGGCATGCAT GACCTTGGCG TGGGCAT TAC CCTGGGGGCC 540 CATCAGAGCA TCGGTTTCAA AGGCATCCTG CTCTTTGGCA CAAAGGCCCA GAAAGAAAAA 600 TACCTCCCCA AGCTGGCATC TGGGGAGACT GTGGCCGCTT TCTGTCTAAC CGAGCCCTCA 660 AGCGGGTCAG ATGCAGCCTC CATCCGAACC TCTGCTGTGC CCAGCCCCTG TGGAAAATAC 720 TATACCCTCA ATGGAAGCAA GCTTTGGATC AGTAATGGGG GCCTAGCAGA CATCTTCACG 780 GTCTTTGCCA AGACACCAGT TACAGATCCA GCCACAGGAG CCGTGAAGGA GAAGATCACA 840 GCTTTTGTGG TGGAGAGGGG CTTCGGGGGC ATTACCCATG GGCCCCCTGA GAAGAAGATG 900 GGCATCAAGG CTTCAAACAC AGCAGAGGTG TTCTTTGATG GAGTACGGGT GCCATCGGAG 960 AACGTGCTGG GTGAGGTTGG GAGTGGCTTC AAGGTTGCCA TGCACATCCT CAACAATGGA 1020 AGGTTTGGCA TGGCTGCGGC CCTGGCAGGT ACCATGAGAG GCATCATTGC TAAGGCGGTA 1080 GATCATGCCA CTAATCGTAC CCAGTTTGGG GAGAAAATTC ACAACTTTGG GCTGATCCAG 1140 GAGAAGCTGG CACGGATGGT TATGCTGCAG TATGTAACTG AGTCCATGGC TTACATGGTG 1200 AGTGCTAACA TGGACCAGGG AGCCACGGAC TTCCAGATAG AGGCCGCCAT CAGCAAAATC 1260 TTTGGCTCGG AGGCAGCCTG GAAGGTGACA GATGAATGCA TCCAAATCAT GGGGGGTATG 1320 GGCTTCATGA AGGAACCTGG AGTAGAGCGT GTGCTCCGAG ATCTTCGCAT CTTCCGGATC 1380 TTTGAGGGGA CAAATGACAT TCTTCGGCTG TTTGTGGCTC TGCAGGGCTG TATGGACAAA 1440 GGAAAGGAGC TCTCTGGGCT TGGCAGTGCT CTAAAGAATC CCTTTGGGAA TGCTGGCCTC 1500 CTGCTAGGAG AGGCAGGCAA ACAGCTGAGG CGGCGGGCAG GGCTGGGCAG CGGCCTGAGT 1560 CTCAGCGGAC TTGTCCACCC GGAGTTGAGT CGGAGTGGCG AGCTGGCAGT ACGGGCTCTG 1620 GAGCAGTTTG CCACTGTGGT GGAGGCCAAG CTGATAAAAC ACAAGAAGGG GATTGTCAAT 1680 GAACAGTTTC TGCTGCAGCG GCTGGCAGAC GGGGCCATCG ACCTCTATGC CATGGTGGTG 1740 GTTCTCTCGA GGGCCTCAAG ATCCCTGAGT GAGGGCCACC CCACGGCCCA GCATGAGAAA 1800 ATGCTCTGTG ACACCTGGTG TATCGAGGCT GCAGCTCGGA TCCGAGAGGG CATGGCCGCC 1860 CTGCAGTCTG ACCCCTGGCA GCAAGAGCTC TACCGCAACT TCAAAAGCAT CTCCAAGGCC 1920 TTGGTGGAGC GGGGTGGTGT GGTCACCAGC AACCCACTTG GCTTC 1965 <AG> AG> AG> AG> AG> AGC GGCT 24 <210> 4 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 4 AGGGTAATGC CCACGCCAAG GTCA 24 <210> 5 <211> 109 <212> PRT <213> Human <400> 5 Met Glu Thr Phe Thr Asn Asp Arg Leu Gln Leu Pro Arg Asn Met Ile 1 5 10 15 Glu Asn Ser Met Phe Glu Glu Glu Pro Asp Val Val Asp Leu Ala Lys 20 25 30 Glu Pro Cys Leu His Pro Leu Glu Pro Asp Glu Val Glu Tyr Glu Pro 35 40 45 Arg Gly Ser Arg Leu Leu Val Arg Gly Leu Gly Glu His Glu Met Asp 50 55 60 Glu Asp Glu Glu Asp Tyr Glu Ser Ser Ala Lys Leu Leu Gly Met Ser 65 70 75 80 Phe Met Asn Arg Ser Ser Gly Leu Arg Asn Ser Ala Thr Gly Tyr Arg 85 90 95 Gln Ser Pro Asp Gly Thr Cys Ser Val Pro Ser Ala Arg 100 105 <210> 6 <211> 327 <212> DNA <213> Human <400> 6 ATGGAGACCT TTACCAATGA TCGACTTCAG CTTCCAAGGA ATATGATTGA AAACAGCATG 60 TTTGAAGAAG AACCAGATGT GGTAGATTTA GCCAAAGAAC CTTGTTTACA TCCTCTGGAA 120 CCTGATGAAG TTGAATATGA GCCCCGAGGT TCGAGGCTTC TGGTGAGAGG TCTTGGTGAG 180 CATGAGATGG ATGAGGATGA AGAGGATTAT GAGTCATCTG CCAAGCTGCT GGGCATGTCC 240 TTCATGAACA GAAGCTCAGG CCTTCGGAAC AGTGCAACAG GCTACAGGCA GAGTCCAGAT 300 GGGACTTGTT CAGTACCCTC TGCCAGG 327 <210> 7 <211> 43 <212> PRT <213> Human <400 > 7 Lys Val Pro Glu Thr Arg Gly Arg Thr Phe Asp Gln Ile Ser Ala Ala 1 5 10 15 Phe Arg Arg Thr Pro Ser Leu Leu Glu Gln Glu Val Lys Pro Ser Thr 20 25 30 Glu Leu Glu Tyr Leu Gly Pro Asp Glu Asn Asp 35 40 <210> 8 <211> 129 <212> DNA <213> Human <400> 8 AAAGTGCCTG AAACCAGAGG CCGGACGTTT GACCAGATCT CAGCTGCCTT CCGACGGACA 60 CCTTCCCTTT TAGAGCAGGA GGTGAAACCC AGTACAGAAC TTGAATACTT AGGGCCAGAT 120 GAGAATGAC 129 <210> 9 <211> 653 <212> PRT Ser Ag Met Thr Pro Ser Val Gly Arg Gln Leu Leu Arg 1 5 10 15 Leu Gly Ala Arg Ser Ser Arg Ser Ala Ala Leu Gln Gly Gln Pro Arg 20 25 30 Pro Thr Ser Ala Gln Arg Leu Tyr Ala Ser Glu Ala Thr Gln Ala Val 35 40 45 Leu Glu Lys Pro Glu Thr Leu Ser Ser Asp Ala Ser Thr Arg Glu Lys 50 55 60 Pro Ala Arg Ala Glu Ser Lys Ser Phe Ala Val Gly Met Phe Lys Gly 65 70 75 80 Gln Leu Thr Thr Asp Gln Val Phe Pro Tyr Pro Ser Val Leu Asn Glu 85 90 95 Gly Gln Thr Gln Phe Leu Lys Glu Leu Val Gly Pro Val Ala Arg Phe 100 105 110 Phe Glu Glu Val Asn Asp Pro Ala Lys Asn Asp Ser Leu Glu Lys Val 115 120 125 Glu Glu Asp T hr Leu Gln Gly Leu Lys Glu Leu Gly Ala Phe Gly Leu 130 135 140 Gln Val Pro Ser Glu Leu Gly Gly Leu Gly Leu Ser Asn Thr Gln Tyr 145 150 155 160 Ala Arg Leu Ala Glu Ile Val Gly Met His Asp Leu Gly Val Ser Val 165 170 175 Thr Leu Gly Ala His Gln Ser Ile Gly Phe Lys Gly Ile Leu Leu Tyr 180 185 190 Gly Thr Lys Ala Gln Lys Glu Lys Tyr Leu Pro Arg Val Ala Ser Gly 195 200 205 Gln Ala Leu Ala Ala Phe Cys Leu Thr Glu Pro Ser Ser Gly Ser Asp 210 215 220 Val Ala Ser Ile Arg Ser Ser Ala Val Pro Ser Pro Cys Gly Lys Tyr 225 230 235 240 Tyr Thr Leu Asn Gly Ser Lys Ile Trp Ile Ser Asn Gly Gly Leu Ala 245 250 255 Asp Ile Phe Thr Val Phe Ala Lys Thr Pro Ile Lys Asp Ala Ala Thr 260 265 270 Gly Ala Val Lys Glu Lys Ile Thr Ala Phe Val Val Glu Arg Ser Phe 275 280 285 Gly Gly Val Thr His Gly Leu Pro Glu Lys Lys Met Gly Ile Lys Ala 290 295 300 Ser Asn Thr Ser Glu Val Tyr Phe Asp Gly Val Lys Val Pro Ala Glu 305 310 315 320 Asn Val Leu Gly Glu Val Gly Asp Gly Phe Lys Val Ala Val Asn Ile 325 330 335 Leu Asn Asn G ly Arg Phe Gly Met Ala Ala Thr Leu Ala Gly Thr Met 340 345 350 Lys Ala Ile Ile Ala Lys Ala Val Asp His Ala Thr Asn Arg Thr Gln 355 360 365 Phe Gly Asp Lys Ile His Asn Phe Gly Val Ile Gln Glu Lys Leu Ala 370 375 380 Arg Met Ala Ile Leu Gln Tyr Val Thr Glu Ser Met Ala Tyr Met Leu 385 390 395 400 Ser Ala Asn Met Asp Gln Gly Phe Lys Asp Phe Gln Ile Glu Ala Ala 405 410 415 Ile Ser Lys Ile Phe Gly Ser Glu Ala Ala Trp Lys Val Thr Asp Glu 420 425 430 Cys Ile Gln Ile Met Gly Gly Met Gly Phe Met Lys Glu Pro Gly Val 435 440 445 Glu Arg Val Leu Arg Asp Ile Arg Ile Phe Arg Ile Phe Glu Gly Thr 450 455 460 Asn Asp Ile Leu Arg Leu Phe Val Ala Leu Gln Gly Cys Met Asp Lys 465 470 475 480 Gly Lys Glu Leu Thr Gly Leu Gly Asn Ala Leu Lys Asn Pro Leu Gly 485 490 490 495 Asn Val Gly Leu Leu Ile Gly Glu Ala Ser Lys Gln Leu Arg Arg Arg 500 505 510 Thr Gly Ile Gly Ser Gly Leu Ser Leu Ser Gly Ile Val His Pro Glu 515 520 525 Leu Ser Arg Ser Gly Glu Leu Ala Val Gln Ala Leu Glu Gln Phe Ala 530 535 540 Thr Val Val Glu A la Lys Leu Met Lys His Lys Lys Gly Ile Val Asn 545 550 555 560 Glu Gln Phe Leu Leu Gln Arg Leu Ala Asp Gly Ala Ile Asp Leu Tyr 565 570 575 Ala Met Val Val Val Leu Ser Arg Ala Ser Arg Ser Leu Ser Glu Gly 580 585 590 Tyr Pro Thr Ala Gln His Glu Lys Met Leu Cys Asp Ser Trp Cys Ile 595 600 605 Glu Ala Ala Thr Arg Ile Arg Glu Asn Met Ala Ser Leu Gln Ser Asn 610 615 620 620 Pro Gln Gln Gln Glu Leu Phe Arg Asn Phe Arg Ser Ile Ser Lys Ala 625 630 635 640 Met Val Glu Asn Gly Gly Leu Val Thr Ser Asn Pro Leu 645 650 653 <210> 10 <211> 655 <212> PRT <213> Mouse <400> 10 Met Gln Ser Ala Arg Met Thr Pro Ser Val Gly Arg Gln Leu Leu Arg 1 5 10 15 Leu Gly Ala Arg Ser Ser Arg Ser Thr Thr Val Leu Gln Gly Gln Pro 20 25 30 Arg Pro Ile Ser Ala Gln Arg Leu Tyr Ala Arg Glu Ala Thr Gln Ala 35 40 45 Val Leu Asp Lys Pro Glu Thr Leu Ser Ser Asp Ala Ser Thr Arg Glu 50 55 60 Lys Pro Ala Arg Ala Glu Ser Lys Ser Phe Ala Val Gly Met Phe Lys 65 70 75 80 Gly Gln Leu Thr Ile Asp Gln Val Phe Pro Tyr Pro Ser Val Leu Ser 85 90 95 Glu Glu Gln Ala Gln Phe Leu Lys Glu Leu Val Gly Pro Val Ala Arg 100 105 110 Phe Phe Glu Glu Val Asn Asp Pro Ala Lys Asn Asp Ala Leu Glu Lys 115 120 125 Val Glu Asp Asp Thr Leu Gln Gly Leu Lys Glu Leu Gly Ala Phe Gly 130 135 140 Leu Gln Val Pro Ser Glu Leu Gly Gly Leu Gly Leu Ser Asn Thr Gln 145 150 155 160 Tyr Ala Arg Leu Ala Glu Ile Val Gly Met His Asp Leu Gly Val Ser 165 170 175 Val Thr Leu Gly Ala His Gln Ser Ile Gly Phe Lys Gly Ile Leu Leu 180 185 190 Tyr Gly Thr Lys Ala Gln Arg Glu Lys Tyr Leu Pro Arg Val Ala Ser 195 200 205 Gly Gln Ala Leu Ala Ala Phe Cys Leu Thr Glu Pro Ser Ser Gly Ser 210 215 220 Asp Val Ala Ser Ile Arg Ser Ser Ala Ile Pro Ser Pro Cys Gly Lys 225 230 235 240 Tyr Tyr Thr Leu Asn Gly Ser Lys Ile Trp Ile Ser Asn Gly Gly Leu 245 250 255 Ala Asp Ile Phe Thr Val Phe Ala Lys Thr Pro Ile Lys Asp Ala Ala 260 265 270 270 Thr Gly Ala Val Lys Glu Lys Ile Thr Ala Phe Val Val Glu Arg Ser 275 280 285 Phe Gly Gly Val Thr His Gly Leu Pro Glu Lys Lys Met Gly Ile Lys 290 295 300 A la Ser Asn Thr Ser Glu Val Tyr Phe Asp Gly Val Lys Val Pro Ser 305 310 315 320 Glu Asn Val Leu Gly Glu Val Gly Asp Gly Phe Lys Val Ala Val Asn 325 330 335 Ile Leu Asn Asn Gly Arg Phe Gly Met Ala Ala Thr Leu Ala Gly Thr 340 345 350 Met Lys Ser Leu Ile Ala Lys Ala Val Asp His Ala Thr Asn Arg Thr 355 360 365 Gln Phe Gly Asp Lys Ile His Asn Phe Gly Val Ile Gln Glu Lys Leu 370 375 380 Ala Arg Met Ala Ile Leu Gln Tyr Val Thr Glu Ser Met Ala Tyr Met 385 390 395 400 400 Leu Ser Ala Asn Met Asp Gln Gly Phe Lys Asp Phe Gln Ile Glu Ala 405 410 415 Ala Ile Ser Lys Ile Phe Cys Ser Glu Ala Ala Trp Lys Val Ala Asp 420 425 430 Glu Cys Ile Gln Ile Met Gly Gly Met Gly Phe Met Lys Glu Pro Gly 435 440 445 Val Glu Arg Val Leu Arg Asp Ile Arg Ile Phe Arg Ile Phe Glu Gly 450 455 460 Ala Asn Asp Ile Leu Arg Leu Phe Val Ala Leu Gln Gly Cys Met Asp 465 470 475 480 480 Lys Gly Lys Glu Leu Thr Gly Leu Gly Asn Ala Leu Lys Asn Pro Phe 485 490 495 Gly Asn Val Gly Leu Leu Met Gly Glu Ala Gly Lys Gln Leu Arg Arg 500 505 510 510 A rg Thr Gly Ile Gly Ser Gly Leu Ser Leu Ser Gly Ile Val His Pro 515 520 525 Glu Leu Ser Arg Ser Gly Glu Leu Ala Val Gln Ala Leu Asp Gln Phe 530 535 540 Ala Thr Val Val Glu Ala Lys Leu Val Lys His Lys Lys Gly Ile Val 545 550 555 560 Asn Glu Gln Phe Leu Leu Gln Arg Leu Ala Asp Gly Ala Ile Asp Leu 565 570 575 Tyr Ala Met Val Val Val Leu Ser Arg Ala Ser Arg Ser Leu Ser Glu 580 585 585 590 Gly Tyr Pro Thr Ala Gln His Glu Lys Met Leu Cys Asp Ser Trp Cys 595 600 605 Ile Glu Ala Ala Thr Arg Ile Arg Glu Asn Met Ala Ser Leu Gln Ser 610 615 620 Ser Pro Gln His Gln Glu Leu Phe Arg Asn Phe Arg Ser Ile Ser Lys 625 630 635 640 Ala Met Val Glu Asn Gly Gly Leu Val Thr Gly Asn Pro Leu Gly 645 650 655 <210> 11 <211> 655 <212> PRT <213> Bovine <400> 11 Met Gln Ala Ala Arg Met Thr Ala Ser Leu Gly Arg Thr Leu Leu Arg 1 5 10 15 Leu Arg Gly Val Ser Ser Trp Pro Gly Glu Leu Leu Gly Gln Pro Arg 20 25 30 Pro Gly Pro Ala Pro Arg Pro Tyr Ala Ser Gly Val Ala Gln Ala Ala 35 40 45 Val Asp Gln Ser Asp Ser Gln Pro Ser Glu Al a Ser Thr Arg Glu Lys 50 55 60 Arg Ala Asn Ser Val Ser Lys Ser Phe Ala Val Gly Thr Phe Lys Gly 65 70 75 80 Gln Leu Thr Thr Asp Gln Val Phe Pro Tyr Pro Ser Val Leu Asn Glu 85 90 95 Asp Gln Thr Gln Phe Leu Lys Glu Leu Val Gly Pro Val Thr Arg Phe 100 105 110 Phe Glu Glu Val Asn Asp Ala Ala Lys Asn Asp Met Leu Glu Arg Val 115 120 125 Glu Glu Thr Glu Thr Met Gln Gly Leu Lys Glu Leu Gly Ala Phe Gly Leu 130 135 140 Gln Val Pro Asn Glu Leu Gly Gly Val Gly Leu Cys Asn Thr Gln Tyr 145 150 155 160 Ala Arg Leu Val Glu Ile Val Gly Met Tyr Asp Leu Gly Val Gly Ile 165 170 175 Val Leu Gly Ala His Gln Ser Ile Gly Phe Lys Gly Ile Leu Leu Phe 180 185 190 Gly Thr Lys Ala Gln Lys Glu Lys Tyr Leu Pro Lys Leu Ala Ser Gly 195 200 205 Glu Thr Ile Ala Ala Phe Cys Leu Thr Glu Pro Ser Ser Gly Ser Asp 210 215 220 Ala Ala Ser Ile Arg Ser Ser Ala Val Pro Ser Pro Cys Gly Lys Tyr 225 230 235 240 Tyr Thr Leu Asn Gly Ser Lys Ile Trp Ile Ser Asn Gly Gly Leu Ala 245 250 255 Asp Ile Phe Thr Val Phe Ala Lys Thr Pro Val Thr Asp Thr Ala Thr 260 265 270 Gly Ala Val Lys Glu Lys Ile Thr Ala Phe Val Val Glu Arg Ser Phe 275 280 285 Gly Gly Val Thr His Gly Pro Pro Glu Lys Lys Met Gly Ile Lys Ala 290 295 300 Ser Asn Thr Ala Glu Val Tyr Phe Asp Gly Val Arg Val Pro Ala Glu 305 310 315 320 Asn Val Leu Gly Glu Val Gly Gly Gly Phe Lys Val Ala Met His Ile 325 330 335 Leu Asn Asn Gly Arg Phe Gly Met Ala Ala Ala Leu Ala Gly Thr Met 340 345 350 Lys Gly Ile Ile Ala Lys Ala Val Asp His Ala Ala Asn Arg Thr Gln 355 360 365 Phe Gly Glu Lys Ile His Asn Phe Gly Leu Ile Gln Glu Lys Leu Ala 370 375 380 Arg Met Ala Met Leu Gln Tyr Val Thr Glu Ser Met Ala Tyr Met Val 385 390 395 400 400 Ser Ala Asn Met Asp Gln Gly Ser Thr Asp Phe Gln Ile Glu Ala Ala 405 410 415 Ile Ser Lys Ile Phe Gly Ser Glu Ala Ala Trp Lys Val Thr Asp Glu 420 425 430 Cys Ile Gln Ile Met Gly Gly Met Gly Phe Met Lys Glu Pro Gly Val 435 440 445 Glu Arg Val Leu Arg Asp Leu Arg Ile Phe Arg Ile Phe Glu Gly Thr 450 455 460 Asn Asp Ile Leu Arg Leu Phe Val Ala Leu Gln Gly Cys MetAsp Lys 465 470 475 480 480 Gly Lys Glu Leu Ser Gly Leu Gly Asn Ala Leu Lys Asn Pro Phe Gly 485 490 495 Asn Ala Gly Leu Leu Leu Gly Glu Ala Gly Lys Gln Leu Arg Arg Arg 500 505 510 Ala Gly Leu Gly Ser Gly Leu Ser Leu Ser Gly Ile Val His Gln Glu 515 520 525 Leu Ser Arg Ser Gly Glu Leu Ala Val Gln Ala Leu Glu Gln Phe Ala 530 535 540 Thr Val Val Glu Ala Lys Leu Ile Lys His Lys Lys Asp Ile Ile Asn 545 550 555 560 Glu Gln Phe Leu Leu Gln Arg Leu Ala Asp Ser Ala Ile Asp Leu Tyr 565 570 575 Ala Met Val Val Val Leu Ser Arg Ala Ser Arg Ser Leu Ser Glu Gly 580 585 590 His Pro Thr Ala Gln His Glu Lys Met Leu Cys Asp Ser Trp Cys Ile 595 600 605 Glu Ala Ala Ala Arg Ile Arg Glu Asn Met Thr Ala Leu Gln Ser Asp 610 615 620 Pro Gln Gln Gln Glu Leu Phe Arg Asn Phe Lys Ser Ile Ser Lys Ala 625 630 635 635 640 Leu Val Glu Arg Gly Gly Val Val Thr Ser Asn Pro Leu Gly Phe 645 650 655

[Brief description of the drawings]

FIG. 1. Human VLCAD (MD25) gene (cDN
The base sequence and the predicted amino acid sequence of A) are shown. (Continued to Fig. 2)

FIG. 2: Human VLCAD (MD25) gene (cDN
The base sequence and the predicted amino acid sequence of A) are shown. (Continued from FIG. 1 and continued to FIG. 3)

FIG. 3. Human VLCAD (MD25) gene (cDN
The base sequence and the predicted amino acid sequence of A) are shown.

FIG. 4. IRA by quantification of β-galactosidase activity
3 shows the interaction between P and VLCAD. In the figure,-of the bit
Indicates a GAL4-DNABD sequence alone, and IRAP indicates a sequence obtained by fusing IRAP (55-82) thereto. In addition,-of the prey represents a control without the prey vector, and MD25 represents 118 of the VLCAD in GAL4-AD.
Shows the sequence fused after the 3rd base. IRAP / MD
At 25, significant β-galactosidase activity is observed. The resulting values are expressed in β-galactosidase activity units (mean ±
Standard deviation).

FIG. 5 shows the tissue distribution of MD25 mRNA. In the figure, b
rain is brain, heart is heart, skeletal muscle is skeletal muscle, co
lon means colon, thymus means thymus, spleen means spleen, kidney means kidney, liver means liver, small intestine means small intestine, placenta means placenta, lung means lung and leulocyute means leukocyte. Note that R
The NA size (kb) is shown on the left end.

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI Theme coat ゛ (Reference) A61K 39/395 A61K 48/00 4C086 45/00 A61P 3/10 4H045 48/00 5/48 A61P 3/10 5/50 5/48 43/00 111 5/50 C12Q 1/02 43/00 111 1/68 Z C12Q 1/02 G01N 33/15 Z 1/68 33/50 Z G01N 33/15 A61K 37/02 33 / 50 37/64 // C12N 15/09 C12N 15/00 A F term (reference) 2G045 AA40 DA12 DA13 DA36 FB02 FB03 4B024 AA01 AA11 BA80 CA04 CA07 CA11 DA02 DA05 DA11 EA02 EA03 EA04 FA02 GA11 HA01 HA11 4B063 QA01 QA08 QAQ Q18 QQ13 QQ30 QR15 QR33 QR57 QR60 QR62 QR74 QR80 QS05 QS36 QX02 4C084 AA02 AA06 AA07 AA13 AA17 BA01 BA02 BA08 BA22 BA23 CA53 CA56 DC32 NA14 ZC022 ZC202 ZC352 4C085 AA13 AA14 BB11 BB22 A03A01 A01A01 6 MA01 MA04 NA14 ZC02 ZC19 ZC20 ZC35 4H045 AA10 AA11 AA20 AA30 BA10 CA40 DA00 EA20 EA50 FA72 FA74

Claims (51)

[Claims] 1. A protein containing an amino acid sequence identical or substantially identical to the amino acid sequence represented by SEQ ID NO: 1, which binds to insulin responsive aminopeptidase or glucose transporter 4, or a salt thereof. 2. A pharmaceutical comprising the protein according to claim 1 or a salt thereof. 3. A medicament comprising a DNA containing the DNA encoding the protein of claim 1. 4. The medicament according to claim 2 or 3, which is a binding agent to insulin responsive aminopeptidase or glucose transporter 4. 5. The medicament according to claim 2, which is an agent for preventing or treating hypoglycemia. 6. A diagnostic agent comprising a DNA containing the DNA encoding the protein according to claim 1. 7. A method for preventing or treating hypoglycemia, which comprises administering an effective amount of the protein or a salt thereof according to claim 1 to a non-human mammal. 8. Use of the protein according to claim 1 or a salt thereof for producing an agent for preventing or treating hypoglycemia. 9. A medicament comprising an antisense DNA containing a nucleotide sequence complementary or substantially complementary to the nucleotide sequence of the DNA encoding the protein according to claim 1, or a part thereof. 10. A pharmaceutical comprising an antibody against the protein according to claim 1 or a salt thereof. 11. The medicament according to claim 9, which is an agent for preventing or treating hyperglycemia or diabetes. 12. A method for preventing or preventing hyperglycemia or diabetes, comprising administering an effective amount of an antibody against the protein or the salt thereof according to claim 1 to a non-human mammal.
Method of treatment. 13. Use of an antibody against the protein or its salt according to claim 1, for producing a prophylactic / therapeutic agent for hyperglycemia or diabetes. 14. A diagnostic agent comprising an antibody against the protein according to claim 1 or a salt thereof. 15. An effect of increasing blood sugar or blood sugar, characterized by using a protein having the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 1, a partial peptide thereof, or a salt thereof. A method for screening a compound having a reducing effect or a salt thereof. 16. A DNA encoding a protein containing the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 1, or a partial peptide thereof
A method for screening a compound having a blood glucose increasing effect or a blood glucose lowering effect or a salt thereof, which comprises using a DNA containing 17. A hyperglycemic or hypoglycemic effect comprising a protein having the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 1, a partial peptide thereof, or a salt thereof. Or a salt thereof for screening. 18. A DNA encoding a protein containing the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 1, or a partial peptide thereof
A screening kit for a compound having a blood glucose increasing effect or a blood glucose lowering effect or a salt thereof, comprising a DNA containing 19. A compound having a blood sugar increasing effect or a salt thereof, which is obtainable by using the screening method according to claim 15 or 16 or the screening kit according to claim 17 or 18. 20. A pharmaceutical comprising the compound according to claim 19 or a salt thereof. 21. The medicament according to claim 20, which is a prophylactic / therapeutic agent for hypoglycemia. 22. A compound having a hypoglycemic effect or a salt thereof, which is obtainable by using the screening method according to claim 15 or 16 or the screening kit according to claim 17 or 18. 23. A pharmaceutical comprising the compound according to claim 22 or a salt thereof. 24. The medicament according to claim 23, which is an agent for preventing or treating hyperglycemia or diabetes. 25. A protein comprising the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 1, a partial peptide thereof, or a salt thereof, wherein A protein containing the same or substantially the same amino acid sequence as the represented amino acid sequence, a partial peptide thereof, or a salt thereof and a compound that promotes or inhibits the binding of insulin responsive aminopeptidase or glucose transporter 4, or a compound thereof; Salt screening method. 26. The method according to claim 25, wherein a cell having the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 1 or a cell capable of producing a partial peptide thereof is used. The screening method as described above. 27. Labeled with a protein having the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 1, a partial peptide thereof, or a salt thereof, one of which is immobilized on a solid phase. 26. The screening method according to claim 25, wherein a test compound is added to a complex with insulin-responsive aminopeptidase or glucose transporter 4, and a free substance is detected. 28. Transcription of a DNA encoding a partial peptide corresponding to the cytoplasmic domain of insulin responsive aminopeptidase or a partial peptide corresponding to the cytoplasmic domain of glucose transporter 4 fused with a reporter gene binding domain and reporter gene transcription Cells transformed with a DNA encoding a protein containing the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 1 or a partial peptide thereof fused with an active domain can be used in the presence and absence of a test compound. 26. The screening method according to claim 25, wherein the method is performed by culturing in the presence and detecting the expression of a reporter gene in each case. 29. A partial peptide corresponding to the cytoplasmic domain of insulin responsive aminopeptidase is a fragment of the amino acid sequence represented by SEQ ID NO: 5
29. The screening method according to claim 28, wherein in the partial peptide containing the second amino acid sequence, the partial peptide corresponding to the cytoplasmic domain of glucose transporter 4 is a partial peptide containing the amino acid sequence represented by SEQ ID NO: 7. . 30. An amino acid represented by SEQ ID NO: 1 containing a protein having the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 1, a partial peptide thereof, or a salt thereof A protein containing an amino acid sequence identical or substantially identical to the sequence,
A kit for screening a compound or a salt thereof that inhibits the binding of the partial peptide or a salt thereof to insulin-responsive aminopeptidase or glucose transporter 4. 31. An amino acid identical or substantially identical to the amino acid sequence represented by SEQ ID NO: 1, which can be obtained by using the screening method according to claims 25 to 29 or the screening kit according to claim 30. A compound or a salt thereof that inhibits binding between a protein containing a sequence, a partial peptide thereof, or a salt thereof and insulin-responsive aminopeptidase or glucose transporter 4. A pharmaceutical comprising the compound according to claim 31 or a salt thereof. 33. The medicament according to claim 32, which is an agent for preventing or treating hyperglycemia or diabetes. 34. The method according to claim 31, wherein the non-human mammal is a non-human mammal.
A method for preventing or treating hyperglycemia or diabetes, which comprises administering an effective amount of the compound or a salt thereof. 35. Use of the compound according to claim 31 or a salt thereof for producing a prophylactic / therapeutic agent for hyperglycemia or diabetes. 36. An amino acid sequence identical or substantially identical to the amino acid sequence represented by SEQ ID NO: 1 obtained by using the screening method according to claim 25 or 29 or the screening kit according to claim 30. Or a salt thereof, which promotes the binding between a protein, a partial peptide thereof, or a salt thereof and insulin-responsive aminopeptidase or glucose transporter 4. 37. A medicament comprising the compound according to claim 36 or a salt thereof. 38. The medicament according to claim 37, which is an agent for preventing or treating hypoglycemia. 39. The method according to claim 36, wherein the non-human mammal is a non-human mammal.
A method for preventing or treating hypoglycemia, which comprises administering an effective amount of the compound or a salt thereof described above. 40. Use of the compound according to claim 36 or a salt thereof for producing a prophylactic / therapeutic agent for hypoglycemia. 41. A compound that promotes or suppresses the expression of a protein, which comprises using a protein having the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 1 or a salt thereof. Or a method of screening for a salt thereof. 42. A cell transformed with a DNA obtained by fusing a reporter gene to a transcription regulatory region of a DNA encoding a protein having the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 1. 42. The screening method according to claim 41, wherein the is cultured in the presence and absence of a test compound, and the expression of the reporter gene is detected in each case. 43. A compound comprising a protein having the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 1 or a salt thereof, which promotes or suppresses the expression of the protein, or a compound thereof Salt screening kit. 44. An amino acid sequence identical or substantially identical to the amino acid sequence represented by SEQ ID NO: 1, which can be obtained by using the screening method according to claim 41 or 42 or the screening kit according to claim 43. Or a salt thereof that suppresses the expression of a protein containing A pharmaceutical comprising the compound according to claim 44 or a salt thereof. 46. The medicament according to claim 45, which is a prophylactic / therapeutic agent for hyperglycemia or diabetes. 47. An amino acid sequence identical or substantially identical to the amino acid sequence represented by SEQ ID NO: 1, which can be obtained by using the screening method according to claim 41 or 42 or the screening kit according to claim 43. Or a salt thereof that promotes the expression of a protein containing 48. A medicament comprising the compound according to claim 47 or a salt thereof. 49. The medicament according to claim 48, which is a preventive / therapeutic agent for hypoglycemia. 50. The method according to claim 47, wherein the non-human mammal is a non-human mammal.
A method for preventing or treating hypoglycemia, which comprises administering an effective amount of the compound or a salt thereof described above. 51. Use of the compound or the salt thereof according to claim 47 for producing a prophylactic / therapeutic agent for hypoglycemia.