JPH09299092A - New protein and its dna - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain a new protein, comprising a specific amino acid sequence and useful as a screening reagent for compounds capable of promoting protein chinases or for prevention, treatment, etc., of diseases concerned in a hematopoietic system. SOLUTION: This new protein is a new p26 protein having the same or substantially the same amino acid sequence as an amino acid sequence represented by formula I, II or III and is produced by a cell participating in a hematopoietic system or a blood corpuscular cell. The new protein is useful as a screening reagent for compounds having protein chinase promoting or inhibiting activities or inhibiting activities against phosphatases, as a preventing and therapeutic agent for diseases concerned in the hematopoietic system such as aplastic anemia, acute myelogeneic leukemia, T cell leukemia, etc. The protein is obtained by screening a cDNA library derived from a human thymus with a part of a cDNA capable of coding a rat p26 protein as a probe, integrating the resultant gene into a vector and expressing the gene in a host cell.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a novel p2 responsible for signal transduction, which is produced by cells involved in hematopoiesis and cells of the blood cell lineage.
6 protein and DNA encoding the same.

[0002]

2. Description of the Related Art In recent years, with the development of molecular biology, the existence of various proteins involved in intracellular signal transduction system for extracellular stimulation and control of cell cycle, which could not be the targets of conventional pharmacology, The intermolecular interactions of these proteins have become clear. Along with this, research on drugs that specifically act on signal transduction systems and cell cycle control proteins is progressing. FK with immunosuppressive action
506 (Takenori Ochiai, Medical Immunology (Medic
al Immunol.) 14, 631-636 (1987)) at the time of discovery, it was completely unknown what mechanism of action exerts immunosuppressive action, but as research progressed, FK506 was present in T lymphocytes. FK506 binding protein (FKBP)
It has been found that it binds to Ca ²⁺ / calmodulin-dependent phosphatase, calcineurin, and suppresses its activity. Therefore, the Ca 2 ⁺ -mediated system of the intracellular signal transduction system involved in the activation of T lymphocytes is blocked, and the production of cytokines such as interleukin 2 necessary for immune reaction is suppressed, resulting in immunosuppression. It became clear that the action was triggered [Fruman,
DA et al., FASEB J. 8, 39
1-400 (1994)]. Furthermore, identification of individual molecules involved in intracellular signal transduction is also progressing rapidly. Particularly, the elucidation of a series of cascades from the receptor tyrosine kinase stimulation to the oncogene Ras has been revealed by the discovery of Grb2 / Ash [Matsuoka, K, et al.・ The National Academy Sciences of the USA (Proceedings of
the Natinal Academy of Sciences of the United Stat
es of America) 89, 9015-9019 (1992)], from the extracellular stimulus to the transcription factor, it can be said that it belongs to the most elucidated class of the signal transduction system.
Moreover, the pathway from the G protein-coupled receptor that is coupled to the αβγ heterotrimeric GTP-binding protein to the MAP kinase has been clarified. However, such an example is only a part of the signal transduction system, and there is no clue how many of the signal transduction systems have not yet been elucidated at present.

Also in hematopoietic stem cells and blood cells, various peptide hormones, cytokines, cell growth factors,
Cell proliferation and differentiation are finely controlled by stimulation from cell adhesion factors. However, in spite of the development of complex information transmission networks caused by these stimuli, at present, the organic integration of the information transmission molecules forming them is hardly done. Hematopoietic stem cells have their surface antigen, CD34
Although it can be concentrated from bone marrow, peripheral blood, etc. as an index, its content is very small. Therefore, recently, in order to clone a gene specifically expressed in hematopoietic stem cells, hematopoietic stem cells were enriched with surface antigens from fetal hepatocytes such as mice, and a region conserved in a signal transduction factor was used as a probe. It is being used to identify new genes. A novel receptor tyrosine kinase FLK2 specifically expressed in hematopoietic stem cells has been cloned by such a method [Matthew, W. et.
al. Cell 65, 1143-1152 (1991)]. On the other hand, protein kinase C is activated by phospholipids and phospholipid metabolites and phosphorylates serine and threonine residues of various nuclear proteins, cytoskeletal proteins, etc. It is known to control proliferation and differentiation.

[0004]

[Problems to be Solved by the Invention] In cells involved in hematopoietic system and cells of hematopoietic system, isolation and purification of a protein responsible for signal transduction makes it possible to develop a new drug for controlling hematopoietic action. Become. It has been desired to find a protein that is responsible for information transmission in cells involved in the hematopoietic system and cells of the hematopoietic system, and develop a method for producing it in large amounts by gene recombination technology.

[0005]

[Means for Solving the Problems] As a result of intensive studies to solve the above problems, the present inventors have found that a rat brain-derived cDNA library, a human thymus-derived cDNA library, and a mouse brain-derived cDNA library. From
We succeeded in cloning cDNAs each having a novel nucleotide sequence, and the protein encoded by them was named p26. As a result of further research to elucidate the in vivo significance of the p26 protein, the p26 protein was 1) most frequently expressed in the fetal liver and peripheral blood in vivo, and the hematopoietic organ of the fetal liver was found. Fetus 1 whose role as a bone is transferred to bone marrow and spleen
Expression decreases sharply after 7 days, 2) in cultured cells, a large amount is expressed in cultured cells of T cell leukemia differentiated from hematopoietic stem cells, 3) protein kinase C
Is a cytoplasmic protein that is phosphorylated by
It was found to be a signal transduction factor that plays an important role in differentiation and proliferation into cells. Therefore,
Development of a drug that regulates this p26 protein has a mechanism of action completely different from that of the past, aplastic anemia,
It is a therapeutic drug for hematopoietic diseases such as acute myelogenous leukemia and T cell leukemia. The present inventors have further studied based on these findings, and have completed the present invention.

That is, the present invention provides (1) SEQ ID NO:
1, a protein or a salt thereof containing the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 2 or SEQ ID NO: 3, (2) SEQ ID NO:
1, the amino acid sequence represented by SEQ ID NO: 2 or SEQ ID NO: 3 has substantially the same amino acid sequence as SEQ ID NO:
The protein according to item (1), which is an amino acid sequence having the 55th to 77th and 137th to 149th amino acid sequences of the amino acid sequence represented by 1. (3) SEQ ID NO: 1, SEQ ID NO: 2 or sequence The amino acid sequence which is substantially the same as the amino acid sequence represented by NO: 3 has the 4th to 20th, 22nd to 27th, 29th to 39th and 41st to 52nd amino acid sequences represented by SEQ ID NO: 1. Th, 54th to 79th, 85th to 91st, 93rd to 9th
The protein according to item (1), which is an amino acid sequence having the 9th, 101st to 114th, 116th to 154th, 157th to 163rd and 166th to 181st amino acid sequences, and (4) the (1) (5) a partial peptide of the protein or a salt thereof, (5) the protein of (1), a DNA containing a DNA having a base sequence encoding the partial peptide of (4), (6) sequence No .: 4, SEQ ID NO: 5 or SEQ ID NO: 6, the DNA according to item (5), which has the nucleotide sequence represented by: (7) The recombinant vector containing the DNA according to item (5),
(8) A transformant carrying the recombinant vector according to item (7), (9) a transformant according to item (8), and
A method for producing a protein or a salt thereof according to item (1), which comprises producing and accumulating the protein or salt thereof according to item (1), and collecting the protein or salt, and (10) item (1). , A drug comprising the partial peptide according to item (4) or a salt thereof, (11) a drug comprising the DNA according to item (5), (12) aplastic anemia, acute The drug according to item (10) or (11), which is a therapeutic / preventive agent or immunomodulator for myeloid leukemia or T cell leukemia,

(13) The protein according to item (1), the antibody against the partial peptide according to item (4) or a salt thereof, (14) the protein according to item (1), (4)
Item 15. A method for screening a compound having phosphatase inhibitory activity or a salt thereof, which comprises using the partial peptide according to the item or a salt thereof, (15) The protein according to item (1), and the item (4). Kit for screening a compound having a phosphatase inhibitory activity or a salt thereof, which comprises a partial peptide of
6) A compound having a phosphatase inhibitory activity or a salt thereof obtained by using the screening method described in (14) or the screening kit described in (15), and (17) phosphorylated (1) ) The protein, the partial peptide according to (4) or a salt thereof, or a pharmaceutical agent containing a compound having an activity of inhibiting dephosphorylation by a dephosphorylation enzyme, and (1)
8) A method for screening a compound having protein kinase inhibitory activity or a salt thereof, which comprises using the protein according to item (1), the partial peptide according to item (4) or a salt thereof.

Furthermore, the present invention is for screening a compound having a protein kinase inhibitory activity containing the protein according to (19) (1), the partial peptide according to (4) or a salt thereof, or a salt thereof. kit,
(20) A compound having a protein kinase inhibitory activity or a salt thereof obtained by using the screening method according to item (14) or the screening kit according to item (15), and (21) the item (1) according to protein kinase.
(22) (1) A pharmaceutical comprising a compound having the activity of inhibiting the phosphorylation of the protein according to item (4), the partial peptide according to item (4) or a salt thereof,
Item 23. A method for screening a compound having a protein kinase promoting activity or a salt thereof, which comprises using the protein according to item (4) or the partial peptide according to item (4), or the protein according to item (23). , A kit for screening a compound having a protein kinase promoting activity or a salt thereof, which comprises the partial peptide according to item (4) or a salt thereof, (24) the screening method according to item (22) or the item (23). A compound having a protein kinase-promoting activity or a salt thereof, which is obtained by using the screening kit as described above, (25) the protein according to (1) above, which comprises a protein kinase,
A drug comprising a compound having the activity of promoting phosphorylation of the partial peptide according to item (4) or a salt thereof, or a salt thereof, (26) a signal transducing substance in cells involved in hematopoiesis or cells of hematopoietic lineage The protein according to (1) above, (27) the protein according to (1) above, which is a protein phosphorylated by an intracellular kinase, (28) the α sub of αβγ heterotrimeric GTP-binding protein The protein according to item (1), which is a protein that binds to a unit (Gα protein) and promotes the hydrolytic activity of Gα protein from GTP to GDP, and (29) is involved in desensitization of G protein-coupled receptors The protein according to item (1), which is a signal transduction control protein, is provided.

The p26 protein of the present invention has the SEQ ID NO:
1, a protein containing the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 2 or SEQ ID NO: 3, and more preferably SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO: Containing an amino acid sequence substantially identical to the amino acid sequence represented by 3,
A protein having substantially the same activity as the protein containing the amino acid sequence represented by SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO: 3. The p26 protein of the present invention is used in humans and mammals (eg, guinea pig, rat, mouse, chicken, rabbit, pig, sheep, cow,
Cells involved in hematopoiesis of monkeys (eg, PA-6,
ST-2, etc.) or hematopoietic cells (eg, ME
L, M1, CTLL-2, HT-2, WEHI-3, H
L-60, JOSK-1, K562, ML-1, MOL
T-3, MOLT-4, MOLT-10, CCRF-C
EM, TALL-1, Jurkat, CCRT-HSB
-2, KE-37, SKW-3, HUT-78, HUT
-102, H9, U937, THP-1, HEL, JK
-1, CMK, KU-812, MEG-01, etc.) or any tissue in which those cells are present, for example, the brain and each part of the brain (eg, olfactory bulb, amygdala, basal sphere, hippocampus, thalamus, hypothalamus). , Cerebral cortex, medulla oblongata, cerebellum), spinal cord,
Pituitary, stomach, pituitary, pancreas, kidney, liver, gonad, thyroid, gallbladder, bone marrow, adrenal gland, skin, muscle, lung, digestive tract, blood vessel, heart, thymus, spleen, submandibular gland, peripheral blood, intestinal tract, It may be a protein derived from the prostate, testis, ovary, placenta, uterus, etc., or may be a synthetic protein.
The amino acid sequence substantially the same as the amino acid sequence represented by SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO: 3 means, for example, the amino acid represented by SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO: 3. Sequence and about 40% or more, preferably about 70% or more, more preferably about 80% or more, further preferably about 90% or more, most preferably about 95%.
The amino acid sequences having the above homology are shown.

Specific examples of the amino acid sequence substantially the same as the amino acid sequence represented by SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO: 3 include, for example, the 55th amino acid sequence represented by SEQ ID NO: 1. ~ 77th and 13th
An amino acid sequence having the 7th to 149th amino acid sequences and the like can be mentioned. More preferred specific examples include, for example, the 54th to the amino acid sequences represented by SEQ ID NO: 1.
The amino acid sequence having the 79th amino acid sequence and the 131st to 149th amino acid sequence is more preferably, for example, the 4th to 2nd amino acid sequences represented by SEQ ID NO: 1.
0th, 22nd-27th, 29th-39th, 41st
~ 52nd, 54th to 79th, 85th to 91st, 93rd to 99th, 101st to 114th, 116th to 1st
54th, 157th to 163rd and 166th to 18th
Examples include an amino acid sequence having the first amino acid sequence. Substantially the same activity includes, for example, an action of being phosphorylated by a phosphorylating enzyme, a signal transduction action in the above-mentioned cells involved in the hematopoietic system or cells of the hematopoietic system, an α sub of αβγ heterotrimeric GTP-binding protein. The action includes binding to a unit (Gα protein) to promote the hydrolytic activity of Gα protein from GTP to GDP, and desensitization of G protein-coupled receptor. Substantially the same indicates that their activities are the same in nature. Therefore, the action of receiving phosphorylation by a phosphorylating enzyme, the signal transduction action in the cells involved in the above-mentioned hematopoietic system or the cells of the hematopoietic system, G of Gα protein
The activity of promoting hydrolytic activity from TP to GDP and the activity of desensitizing G protein-coupled receptors are equivalent (eg,
It is preferably about 0.5 to 2 times), but quantitative factors such as the degree of activity and the molecular weight of the protein may be different.

The p26 protein of the present invention includes SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO: 3.
An amino acid sequence in which 1 or 2 or more (for example, 2 or more and 20 or so, preferably 2 to 9 and more preferably several) amino acids in the amino acid sequence represented by are deleted, SEQ ID NO: 1, sequence Number: 2 or SEQ ID NO: 3
An amino acid sequence in which 1 or 2 or more (for example, 2 or more and 20 or so, preferably 2 to 9 and more preferably several) amino acids are added to the amino acid sequence represented by
1 or 2 or more (for example, 2) in the amino acid sequence represented by SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO: 3
It may be a protein or the like containing an amino acid sequence in which about 20 or more, preferably about 2 to 9, more preferably several) amino acids are substituted with other amino acids. When the amino acid sequence is deleted or substituted as described above, the position of the deletion or substitution is not particularly limited. For example, the amino acid sequence represented by SEQ ID NO: 1 may be the 55-77th position or the Positions other than the 137th to 149th amino acid sequences, preferably SEQ ID NO:
Positions other than the 54th to 79th or 131st to 149th amino acid sequences of the amino acid sequence represented by 1, more preferably the 4th to 20th and the 22nd to 27th positions of the amino acid sequence represented by SEQ ID NO: 1. , 29th to 39th, 41st to 52nd, 54th to 79th, 85th to 9th
1st, 93rd to 99th, 101st to 114th, 116th to 154th, 157th to 163rd or 1st
Positions other than the 66th to 181st amino acid sequences are included. The 4th to 20th, 22nd to 27th, 29th to 3rd amino acid sequences represented by SEQ ID NO: 1
9th, 41st to 52nd, 54th to 79th, 85th
~ 91th, 93rd to 99th, 101st to 114th, 116th to 154th, 157th to 163rd and 166th to 181st amino acid sequences are SEQ ID NOs:
It is a partial amino acid sequence common to the amino acid sequence represented by 1, the amino acid sequence represented by SEQ ID NO: 2 and the amino acid sequence represented by SEQ ID NO: 3.

Further, the p26 protein of the present invention comprises
N-terminal methionine residue protected with a protecting group (eg, C _1-6 acyl group such as formyl group, acetyl group, etc.), N-terminal side of glutamic acid residue is cleaved in vivo to form pyroglutamine Such as those in which the side chain of an amino acid in the molecule is protected by a suitable protecting group (for example, C _1-6 acyl group such as formyl group, acetyl group, etc.), or so-called glycoprotein to which a sugar chain is bound. Complex proteins and the like are also included. In the protein in this specification, the left end is the N-terminus (amino terminus) and the right end is the C-terminus (carboxyl terminus) in accordance with 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 carboxylate (—COO ⁻ ) at the C-terminus, but has an amide at the C-terminus. (-CON
H ₂ ) or an ester (—COOR).
Here, R in the ester is, for example, methyl,
C _1-6 alkyl groups such as ethyl, n-propyl, isopropyl or n-butyl, for example cyclopentyl,
C _3-8 cycloalkyl group such as cyclohexyl, for example, C _6-12 aryl group such as phenyl and α-naphthyl,
For example, phenyl-C _1-2 such as benzyl and phenethyl.
In addition to an alkyl group or a C _7-14 aralkyl group such as an α-naphthyl-C _1-2 alkyl group such as α-naphthylmethyl, a pivaloyloxymethyl ester commonly used as an oral ester is used. When the protein of the present invention has a carboxyl group (or carboxylate) other than the C-terminus, the protein of the present invention includes a carboxyl group amidated or esterified. As the ester in this case, for example, the above-mentioned C-terminal ester or the like is used.

More preferred specific examples of the p26 protein of the present invention include rat brain-derived p26 protein containing the amino acid sequence represented by SEQ ID NO: 1 and human thymus containing the amino acid sequence represented by SEQ ID NO: 2. And a mouse brain-derived p26 protein containing the amino acid sequence represented by SEQ ID NO: 3. As the salt of the p26 protein of the present invention, a physiologically acceptable acid addition salt is particularly preferable. Examples of such salts include salts with inorganic acids (eg, hydrochloric acid, phosphoric acid, hydrobromic acid, sulfuric acid) and organic acids (eg, acetic acid, formic acid, propionic acid, fumaric acid, maleic acid, succinic acid) And tartaric acid, citric acid, malic acid, oxalic acid, benzoic acid, methanesulfonic acid and benzenesulfonic acid). The p26 protein of the present invention or a salt thereof can be produced from the cells or tissues of humans or warm-blooded animals described above by a method known per se for protein purification, or a transformation containing a DNA encoding the p26 protein described below. It can also be produced by culturing the body. Further, it can also be produced according to the peptide synthesis method described later. When producing from human or mammalian tissues or cells, homogenize human or mammalian tissues or cells, then extract with acid or the like, and subject the extract to chromatography such as reverse phase chromatography or ion exchange chromatography. Can be purified and isolated by combining The activity of the p26 protein of the present invention, that is, the action of receiving phosphorylation by a phosphorylating enzyme, the signal transduction action in the cells involved in the hematopoietic system or the cells of the hematopoietic system, the α subunit of the αβγ heterotrimeric GTP-binding protein ( G
G protein from GTP to G
The action of promoting the hydrolytic activity to DP, the desensitization of G protein-coupled receptor, and the like can be measured according to methods known per se. For example, phosphorylation by a phosphorylating enzyme can be measured by measuring [ ³² P] radioactivity described in the method for screening a compound having a protease inhibitory activity or a salt thereof described below.

For the synthesis of the protein of the present invention, a salt thereof or an amide thereof, a commercially available resin for protein synthesis can be used. Examples of such a resin include chloromethyl resin, hydroxymethyl resin, benzhydrylamine resin, aminomethyl resin, 4-benzyloxybenzyl alcohol resin, 4-methylbenzhydrylamine resin, PAM resin, and 4-hydroxymethylmethyl. Phenylacetamidomethyl resin, polyacrylamide resin, 4- (2 ′, 4′-dimethoxyphenyl-hydroxymethyl) phenoxy resin, 4- (2 ′, 4′-dimethoxyphenyl-Fmocaminoethyl) phenoxy resin, and the like. it can. 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 the target protein according to various known condensation methods. At the end of the reaction, the protein 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 or an amide thereof. For the condensation of the protected amino acids described above, various activating reagents that can be used for protein synthesis can be used, and carbodiimides are particularly preferable. As the carbodiimides, DCC, N, N′-diisopropylcarbodiimide, N-ethyl-N ′-(3-dimethylaminoprolyl) carbodiimide and the like are used. For activation by these, the protected amino acid was added directly to the resin together with a racemization inhibitor additive (eg, HOBt, HOOBt), or the protected amino acid was previously activated as a symmetrical acid anhydride or HOBt ester or HOOBt ester. It can be added to the resin later. The solvent used for activation of the protected amino acid or condensation with the resin can be appropriately selected from solvents known to be usable for protein condensation reaction. For example, N, N-dimethylformamide, N, N-
Acid amides such as dimethylacetamide and N-methylpyrrolidone, halogenated hydrocarbons such as methylene chloride and chloroform, alcohols such as trifluoroethanol, sulfoxides such as dimethyl sulfoxide, tertiary amines such as pyridine, dioxane, Ethers such as 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 performed by repeating the condensation reaction without removing the protecting group. When sufficient condensation cannot be obtained even by repeating the reaction, unreacted amino acids can be acetylated using acetic anhydride or acetylimidazole.

Examples of the protective group for the amino group of the raw material include Z, Boc, tertiary pentyloxycarbonyl, isobornyloxycarbonyl, 4-methoxybenzyloxycarbonyl, Cl-Z, Br-Z, adamantyloxycarbonyl, Trifluoroacetyl, phthaloyl, formyl, 2-nitrophenylsulfenyl, diphenylphosphinothioyl oil, Fmoc and the like are used. The carboxyl group is, for example, alkyl esterified (for example, methyl, ethyl, propyl, butyl, tertiary butyl,
Linear, branched or cyclic alkyl esterification such as cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, 2-adamantyl, etc., aralkyl esterification (for example, benzyl ester, 4-nitrobenzyl ester, 4-methoxybenzyl ester, 4-chlorobenzyl ester, benzhydryl esterification), phenacyl esterification, benzyloxycarbonyl hydrazide formation, tertiary butoxycarbonyl hydrazide formation,
It can be protected by trityl hydrazide formation and the like. The hydroxyl group of serine can be protected, for example, by esterification or etherification. Examples of groups suitable for the esterification include lower alkanoyl groups such as acetyl group, aroyl groups such as benzoyl group, groups derived from carbonic acid such as benzyloxycarbonyl group and ethoxycarbonyl group. Examples of the group suitable for etherification include a benzyl group, a tetrahydropyranyl group, a t-butyl group and the like. Examples of the protective group for the phenolic hydroxyl group of tyrosine include Bzl and Cl ₂
-Bzl, 2-nitrobenzyl, Br-Z, tertiary butyl and the like are used. Examples of the protecting group for imidazole of histidine include Tos, 4-methoxy-2,3,6-trimethylbenzenesulfonyl, DNP, benzyloxymethyl, B
um, Boc, Trt, Fmoc, etc. are used.

The activated carboxyl group of the raw material includes, for example, corresponding acid anhydrides, azides, active esters [alcohols (for example, pentachlorophenol, 2,4,5-trichlorophenol, 2,4- Ester with dinitrophenol, cyanomethyl alcohol, para-nitrophenol, HONB, N-hydroxysuccinimide, N-hydroxyphthalimide, HOBt)] and the like 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 black or Pd-
Catalytic reduction in a stream of hydrogen in the presence of a catalyst such as carbon, acid treatment with anhydrous hydrogen fluoride, methanesulfonic acid, trifluoromethanesulfonic acid, trifluoroacetic acid or a mixture thereof, diisopropylethylamine Base treatment with triethylamine, piperidine, piperazine, etc., reduction with sodium in liquid ammonia, and the like are also used. The elimination reaction by the above-mentioned acid treatment is generally carried out at a temperature of about -20 ° C to 40 ° C. Addition of a cation scavenger such as 1,2-ethanedithiol or 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 1,2-ethanedithiol, 1,1,
In addition to deprotection by acid treatment in the presence of 4-butanedithiol or the like, it is also removed by alkali treatment with dilute sodium hydroxide solution, dilute ammonia or the like.

Protection of a functional group which should not be involved in the reaction of the starting material, removal of the protective group, removal of the protective group, activation of the functional group involved in the reaction and the like can be appropriately selected from known groups or known means. . As another method for obtaining an amide form of a protein, for example, first, after protecting the α-carboxyl group of the carboxy terminal amino acid by amidation, the peptide (protein) chain is extended to a desired chain length on the amino group side, A protein from which only the protecting group for the N-terminal α-amino group of the peptide chain has been removed and a protein from which only the protecting group for the carboxyl group at the C-terminal has been removed, and these proteins are mixed in a mixed solvent as described above. To condense. Details of the condensation reaction are the same as described above. After purifying the protected protein obtained by the condensation, all the protecting groups are removed by the above-mentioned method, and a desired crude protein can be obtained. This crude protein is purified by various known purification means, and the main fraction is freeze-dried to obtain an amide of the desired protein. In order to obtain an ester of a protein, for example, after condensing the α-carboxyl group of the carboxy terminal amino acid with a desired alcohol to form an amino acid ester, an ester of the desired protein is obtained in the same manner as the amide of the protein be able to.

The partial peptide of the p26 protein of the present invention may be any peptide as long as it is a partial peptide of the above-mentioned protein of the present invention.
A peptide having the amino acid sequence of the 55th to 77th amino acids or (and) the 137th to 149th amino acids of the amino acid sequence represented by SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO: 3 is used. More preferably, SEQ ID NO:
1, the amino acid sequence represented by SEQ ID NO: 2 or SEQ ID NO: 3, the 54th to 79th amino acid sequence or (and) the 131st to 149th amino acid sequence (preferably 116th to 154th amino acid sequence). The amino acid sequence of the second amino acid sequence) is used, and more preferably, for example, the 4th to 20th amino acid sequences represented by SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO: 3,
22nd to 27th, 29th to 39th, 41st to 52nd, 54th to 79th, 85th to 91st, 93rd to 9th
9th, 101st to 114th, 116th to 154th, 157th to 163rd or (and) 166th
A peptide having the 181st amino acid sequence is used. The C-terminal of the partial peptide of the present invention is usually a carboxyl group (-COOH) or a carboxylate.
(-COO ^-) a but, as in the protein of the present invention described above, C-terminal, it may be an amide (-CONH ₂₎ or ester (-COOR). As the salt of the partial peptide of p26 protein of the present invention, the salt of p26 protein of the present invention described above and the like can be used.

The partial peptide of the p26 protein of the present invention or a salt thereof can be produced by publicly known methods for peptide synthesis or by cleaving the p26 protein of the present invention with an appropriate peptidase.
The peptide synthesis method may be either a solid phase synthesis method or a liquid phase synthesis method. That is, the target peptide can be produced by condensing a partial peptide or amino acid capable of constituting the protein of the present invention with the remaining portion and, when the product has a protecting group, removing the protecting group. Examples of known condensation methods and elimination of protective groups include the methods described in the following (1) to (3). M. Bodanszky and MA Ondetti, Peptide Synthesis, Interscience Publisher
s, New York (1966) Schroeder and Luebke, The Peptide,
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 for Biochemistry 1, Protein Chemistry IV, 205, (1977) Supervised by Haruaki Yajima, Development of Pharmaceuticals Volume 14 Peptide Synthesis Hirokawa Shoten The protein of the present invention can be purified and isolated by a combination of purification methods such as solvent extraction, distillation, column chromatography, liquid chromatography, recrystallization and the like. When the protein obtained by the above method is a free form, it can be converted into a suitable salt by a known method, and conversely, when it is obtained with a salt,
It can be converted into an educt by a known method.

The DNA encoding the p26 protein of the present invention or its partial peptide may be any DNA as long as it contains the nucleotide sequence encoding the p26 protein of the present invention or its partial peptide. In addition, any of genomic DNA, genomic DNA library, cDNA derived from the above-described cells / tissues, cDNA library derived from the aforementioned cells / tissues, 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, using the mRNA fraction prepared from the cells / tissues described above,
e Transcriptase Polymerase Chain Reaction (hereinafter
It is abbreviated as RT-PCR method. ). Specifically, a DNA encoding a protein having the amino acid sequence represented by SEQ ID NO: 1 of the present invention
Include, for example, a base sequence represented by SEQ ID NO: 4 or a base sequence hybridizing with the base sequence under high stringent conditions and having the same activity as that of the protein having the amino acid sequence represented by SEQ ID NO: 1, for example, , An action of being phosphorylated by a phosphatase, a signal transduction action in the cells involved in the above-mentioned hematopoietic system or cells of a hematopoietic system, and a Gα protein bound to an α subunit (Gα protein) of an αβγ heterotrimeric GTP-binding protein Containing a nucleotide sequence encoding a protein having an action of promoting the hydrolytic activity of GTP from GTP to GDP, a desensitization of G protein-coupled receptor, etc.
Any NA may be used. The hybridizing base sequence includes, for example, a base sequence having a homology of about 70 to 80% or more, preferably about 90% or more, more preferably about 95% or more with the base sequence represented by SEQ ID NO: 4. Used. D encoding a protein having the amino acid sequence represented by SEQ ID NO: 2 of the present invention
Examples of NA include, for example, a base sequence represented by SEQ ID NO: 5 or a base sequence hybridizing with the base sequence under high stringent conditions and having the same activity as that of the protein having the amino acid sequence represented by SEQ ID NO: 2, For example, the action of being phosphorylated by a phosphorylating enzyme, the signal transduction action in the above-mentioned cells involved in the hematopoietic system or the cells of the hematopoietic system, binding to the α subunit (Gα protein) of the αβγ heterotrimeric GTP-binding protein, and Gα
Any DNA may be used as long as it contains a nucleotide sequence encoding a protein having an action of promoting the hydrolytic activity of a protein from GTP to GDP and an activity of desensitizing a G protein-coupled receptor. The hybridizing base sequence is, for example, about 70 to 80% or more, preferably about 90% of the base sequence represented by SEQ ID NO: 5.
Above, more preferably, a nucleotide sequence having a homology of about 95% or more is used.

The DNA encoding the protein having the amino acid sequence represented by SEQ ID NO: 3 of the present invention includes, for example, the nucleotide sequence represented by SEQ ID NO: 6 or a nucleotide sequence hybridizing with the nucleotide sequence under high stringent conditions. Having the same activity as that of the protein having the amino acid sequence represented by SEQ ID NO: 3, for example, an action of being phosphorylated by a phosphorylating enzyme, an information transduction action in the above-mentioned cells involved in hematopoietic system or cells of hematopoietic system. , A protein having an activity of binding to the α subunit (Gα protein) of an αβγ heterotrimeric GTP-binding protein to promote the hydrolytic activity of Gα protein from GTP to GDP, and an activity of desensitizing a G protein-coupled receptor Any DNA may be used as long as it contains a nucleotide sequence encoding Examples of the hybridizing base sequence include a base sequence having a homology of about 70 to 80% or more, preferably about 90% or more, more preferably about 95% or more with the base sequence represented by SEQ ID NO: 6. Used. Hybridization can be performed according to a method known per se or a method analogous thereto, and for example, Molecular Cloning 2nd (ed .; J.
Sambrooket al., Cold Spring Harbor Lab. Press, 198
It can be performed according to the method described in 9). High stringency conditions include, for example, a sodium concentration of about 19 to 40 mM, preferably about 19 to 20 mM.
At a temperature of about 50-70 ° C, preferably about 60-65 ° C
Are shown. 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 rat brain-derived p26 protein containing the amino acid sequence of SEQ ID NO: 1, DNA having the nucleotide sequence represented by SEQ ID NO: 4 or the like is used. The DNA encoding the human thymus-derived p26 protein containing the amino acid sequence of SEQ ID NO: 2 has the nucleotide sequence represented by SEQ ID NO: 5.
Are used. DN encoding mouse brain-derived p26 protein containing the amino acid sequence of SEQ ID NO: 3
As A, DNA having the base sequence represented by SEQ ID NO: 6 or the like is used.

The DNA encoding the partial peptide having the amino acid sequence of the 55th to 77th amino acids or (and) the 137th to 149th amino acids of the amino acid sequence represented by SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO: 3 is: For example, a DNA having the 163rd to 231st nucleotide sequence and / or the 409th to 447th nucleotide sequence in the nucleotide sequence represented by SEQ ID NO: 4, SEQ ID NO: 5 or SEQ ID NO: 6.
Are used. Nos. 54 to 79 of the amino acid sequence represented by SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO: 3.
Examples of the DNA encoding the partial peptide having the thirteenth (and / or) thirteenth to 149th amino acid sequence include, for example, 160th to 237th of the base sequence represented by SEQ ID NO: 4, SEQ ID NO: 5 or SEQ ID NO: 6. A DNA having the thirteenth (and / or) thirty-ninth to 447th nucleotide sequence is used. SEQ ID NO: 1, SEQ ID NO:
2 or the fourth of the amino acid sequence represented by SEQ ID NO: 3
~ 20th, 22nd to 27th, 29th to 39th, 41st to 52nd, 54th to 79th, 85th to 91st, 93rd to 99th, 101st to 114th, 11th
The DNA encoding the partial peptide having the 6th to 154th position, the 157th to 163rd position or (and) the 166th to 181st amino acid sequence is, for example, SEQ ID NO: 4, SEQ ID NO: 5 or SEQ ID NO: 6. 10th to 60th, 64th to 81st, 8th of the represented nucleotide sequence
5th to 117th, 121st to 156th, 160th to 2nd
37th, 253-273, 277-297, 301-342, 346-462, 469-489 or (and) 496-543
DNA having the th base sequence is used.

As a means for cloning the DNA encoding the p26 protein or its partial peptide of the present invention, a synthetic DNA primer having a partial base sequence of the DNA encoding the p26 protein is used, and the DNA library or the like is prepared by PCR. To the desired DN
A amplified or integrated into an appropriate vector D
NA can be selected by hybridization with a DNA fragment having a part or the whole region of p26 protein or labeled with a synthetic DNA. Hybridization can be performed, for example, with Molecular Cl
oning 2nd (ed .; J. Sambrook et al., Cold Spring H
arbor Lab. Press, 1989). When using a commercially available library, hybridization can be performed according to the method described in the attached instruction manual. DNA
The conversion of the nucleotide sequence of is known kit, for example, Mutan ^™
-G (Takara Shuzo Co., Ltd., hereinafter TM means a registered trademark), Mutan ^TM- K (Takara Shuzo Co., Ltd.), etc.
It can be performed according to a method known per se such as the ped duplex method or the Kunkel method or a method similar thereto. The DNA encoding the cloned p26 protein or a partial peptide thereof (hereinafter sometimes abbreviated as p26 protein etc.) may be used as it is, or if desired, after digestion with a restriction enzyme or addition of a linker. Can be used. The DNA has ATG as a translation initiation codon at the 5'terminal side, and TAA, TGA or TA as a translation termination codon at the 3'terminal side.
It may have G. These translation initiation codon and translation termination codon can also be added using a suitable synthetic DNA adapter. The expression vector of the DNA encoding the p26 protein or the like is, for example, (a) p2 of the present invention.
Target DNA from DNA encoding 6 proteins, etc.
It can be produced by cutting out the A fragment and (b) ligating the DNA fragment downstream of the promoter in an appropriate expression vector.

As the vector, a plasmid derived from Escherichia coli (eg, pBR322, pBR325, pUC12, pBR322)
UC13), a plasmid derived from Bacillus subtilis (eg, pUB11)
0, pTP5, pC194), yeast-derived plasmids (eg, pSH19, pSH15), bacteriophages such as λ phage, animal viruses such as retrovirus, vaccinia virus, baculovirus, etc.
A1-11, pXT1, pRc / CMV, pRc / RS
V, pcDNAI / Neo, etc. are used. The promoter used in the present invention may be any promoter as long as it is appropriate for the host used for gene expression. For example, when animal cells are used as hosts, the SRα promoter, SV40 promoter, L
TR promoter, CMV promoter, HSV-TK
Promoters and the like. Of these, CMV
It is preferable to use a promoter, SRα promoter or the like. When the host is Escherichia, trp
Promoter, lac promoter, recA promoter, λP _L promoter, lpp promoter, etc., when the host is Bacillus sp., SPO1 promoter, SPO2 promoter, penP promoter, etc .; , GAP promoter, ADH
Promoters and the like are preferred. When the host is an insect cell, a polyhedrin promoter, a P10 promoter and the like are preferable.

In addition to the above, the expression vector may optionally contain an enhancer, a splicing signal, a poly A addition signal, a selection marker, an SV40 replication origin (hereinafter sometimes abbreviated as SV40 ori) and the like. Can be used. 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 ), neomycin resistance gene (hereinafter sometimes Neo, G418 resistance)
And the like. In particular, when the dhfr gene is used as a selection marker using CHO (dhfr ⁻ ) cells, selection can also be performed using a thymidine-free medium. In addition, if necessary, a signal sequence suitable for the host may be added to p2.
6 protein etc. are added to the N-terminal side. If the host is a genus Escherichia, the phoA signal sequence, omp
When the host is a bacterium of the genus Bacillus, the A. signal sequence, α-amylase signal sequence, subtilisin
When the host is yeast, the signal sequence etc. is MFα.
When the host is an animal cell, such as a signal sequence and SUC2 signal sequence, for example, an insulin signal sequence, an α-interferon signal sequence, an antibody molecule / signal sequence, etc. can be used. DN encoding the p26 protein and the like constructed in this manner
A transformant can be produced by introducing a vector containing A into cells.

As the host, for example, Escherichia, Bacillus, yeast, insects, animal cells and the like are used. Specific examples of the genus Escherichia include, for example, Escherichia coli K12.DH1
[Proceedings of the National Academy of Sciences o
f the United States of America), 60, 160 (1968)], JM103 [Nucleic Acids Research, (Nucleic Acids Research), 9, 3]
09 (1981)], JA221 [Journal of Molecular Biology (Journal o
f Molecular Biology)], 120
Vol. 517 (1978)], HB101 [Journal of Molecular Biology, 41, 45.
9 (1969)], C600 [Genetics, 39, 440 (1954).
Year)] is used. Examples of the genus Bacillus include Bacillus subtilis MI1.
14 [Gene, 24, 255 (1983)], 2
07-21 [Journal of Biochemistry, Vol. 95, p. 87 (19
1984)] and the like are used. As yeast, for example,
Saccharomyces cerevi
^{siae) AH22, AH22R -, NA87-11A} , DK
D-5D, 20B-12, etc. and Schizosaccharomyces pombe NCYC19
13, NCYC2036, etc. are used. As the insect cell, for example, when the virus is AcNPV, a cell line derived from the larva of Spodoptera frugiperda cel.
l; Sf cells), MG1 cells derived from the midgut of Trichoplusia ni, High Five ^TM cells derived from eggs of Trichoplusia ni, Mame
Cells derived from strabrassicae or cells derived from Estigmena acrea are used. When the virus is BmNPV, a silkworm-derived cell line (Bombyx mori N; BmN cell) or the like is used. Examples of the Sf cells include Sf9
Cells (ATCC CRL1711), Sf21 cells [above, Vaughn,
JL et al., In Vitro, Volume 13, 213
-217 (1977)] and the like are used. As the insects, for example, silkworm larvae are used [Maeda et al., Nature, 315, p. 592 (19).
1985)].

Animal cells include, for example, monkey cell CO
S-7, Vero cell, Chinese hamster cell C
HO (hereinafter abbreviated as CHO cell), dhfr gene-deficient Chinese hamster cell CHO (hereinafter CHO (d
hfr ^-) cells and abbreviated), L cells, myeloma cells, human FL cells, 293 cells, C127 cells, mouse cells,
BALB3T3 cells, Sp-2 / O cells and the like are used. Among these, CHO cells, CHO (dhf
r ^-) cells, such as 293 cells are preferred. In order to transform Escherichia, for example,
Proceedings of the N Academy of Sciences of the USA
atinal Academy of Sciences of the United States of
America), 69, 2110 (1972) and Gene, 17: 107 (1982). In order to transform Bacillus sp., For example, Molecular & General Genetics (Molecular & General G)
enetics), 168, page 111 (1979) and the like. For transforming yeast, for example, Methods in Enzymology, 194, 182-18.
It can be performed according to the method described on page 7 (1991). To transform insect cells or insects,
For example, Bio / Technology, 6
Vol. 47-55 (1988) and the like. For transforming animal cells, for example, Cell Engineering Supplement 8, New Cell Engineering Experimental Protocol, pp. 263-267 (1995) (published by Shujunsha)
It can be performed according to the method described in.

The expression vector can be introduced into cells by, for example, the lipofection method [Felgner, PL et a.
l. Proceedings of the NatinalAcademy of Sciences of the National Academy of Sciences of the USA
the United States of America), 84, 7413
(1987)], calcium phosphate method [Graham, F.
L. and van der Eb, AJ Virology,
52, 456-467 (1973)], electroporation [Nuemann, E. et al. Embo Journal (EMBO
J.), 1, 841-845 (1982)]. Thus, a transformant transformed with the expression vector containing the DNA encoding the protein or the like of the present invention is obtained. In addition, as a method for stably expressing the protein of the present invention using animal cells,
There is a method of selecting cells in which the expression vector introduced into the animal cell has been integrated into the chromosome by clonal selection. Specifically, a transformant is selected using the above-mentioned selection marker as an index. Furthermore, a stable animal cell line having a high expression ability of the protein of the present invention or the like can be obtained by repeatedly performing clonal selection on the animal cells obtained using the selection marker. When the dhfr gene is used as a selection marker,
By culturing the MTX concentration gradually and selecting a resistant strain, the DNA encoding the protein or the like of the present invention can be amplified in the cell together with the dhfr gene to obtain a more highly expressed animal cell line. it can. The above transformant is cultured under conditions in which a DNA encoding the protein or the like of the present invention can be expressed to produce the protein or the like of the present invention.
By accumulating the protein, the protein of the present invention can be produced.

When culturing a transformant whose host is a bacterium of the genus Escherichia or a bacterium of the genus Bacillus, a liquid medium is suitable as a medium used for the culture, and among them, a liquid medium is necessary for the growth of the transformant. A carbon source, a nitrogen source, an inorganic substance, etc. are contained. As the carbon source, for example, glucose, dextrin, soluble starch, sucrose, etc., as the nitrogen source, for example, ammonium salts, nitrates, corn steep liquor, peptone, casein, meat extract, soybean meal, potato extract, etc. As the inorganic or organic substance and the inorganic substance, for example, calcium chloride, sodium dihydrogen phosphate, magnesium chloride and the like are used. Also,
Yeast extract, vitamins, growth promoting factor and the like may be added. The pH of the medium is preferably about 5-8. As a medium for culturing Escherichia bacteria, for example, M9 medium containing glucose and casamino acid [Miller, Journal of Experiments in Molar Genetics (Journal of Experiments in Mo
lecular Genetics), pp. 431-433, Cold Spring
Harbor Laboratory, New York (1972)] is preferable. If necessary, an agent such as 3β-indolyl acrylic 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 Bacillus, the culture is usually about 30-4.
It is carried out at 0 ° C. for about 6 to 24 hours, and aeration and stirring can be added if necessary.

When culturing a transformant whose host is yeast, the medium is, for example, Burkholder.
lder) Minimal medium [Bostian, KL et al., “Proceedings of the National Academy of Sciences of the USA”
the Natinal Academy of Sciences of the United Sta
tes of America, 77, 4505 (1980)
Year)] or SD medium containing 0.5% casamino acid [Bitte
r, GA et al., "Procedures of the National Academy of Sciences of the
USA (Proceedings of the Natinal Academy o
f Sciences of the United States of America), 81
Vol., Page 5330 (1984)]. Preferably, the pH of the medium is adjusted to about 5-8. Culturing is usually carried out at about 20 ° C to 35 ° C for about 24 to 72 hours, and aeration and agitation are added if necessary. When culturing a transformant whose host is an insect cell, the medium is Grace's Insect Mediu
m [Grace, TCC, Nature, Volume 195,
Pp. 788 (1962)] to which appropriate additives such as immobilized 10% bovine serum have been added. The pH of the medium is preferably adjusted to about 6.2 to 6.4. Culturing is usually performed at about 27 ° C. for about 3 to 5 days, and aeration and agitation are added if necessary. When culturing a transformant whose host is an animal cell, the medium is, for example, MEM medium containing about 5 to 20% fetal bovine serum [Science,
122, 501 (1952)], DMEM medium [Virology, 8, 396 (195).
9)], RPMI 1640 medium [Journal of the American Medical Association (Th
e Jounal of the American Medical Association) 19
9, p. 519 (1967)], 199 medium [Proceeding of the Society Medicine]
for the Biological Medicine, 73, 1 (19
50 years)] is used. Preferably, the pH is between about 6-8. Culturing is usually performed at about 30 to 40 ° C for about 15 to 7
Perform for 2 hours, add aeration and agitation as needed. In particular, when CHO (dhfr ⁻ ) cells and the dhfr gene are used as selection markers, it is preferable to use DMEM medium containing dialyzed fetal bovine serum containing almost no thymidine.

Separation and purification of p26 protein and the like from the above culture can be performed, for example, by the following method. When extracting p26 protein or the like from cultured bacterial cells or cells, the bacterial cells or cells are collected by a known method after culturing, suspended in an appropriate buffer, and then ultrasonically, lysozyme and / or freeze-thaw is used. After destroying the cells or cells, a method of obtaining a crude extract such as p26 protein by centrifugation or filtration can be appropriately used. The buffer solution may contain a protein denaturing agent such as urea or guanidine hydrochloride, or a surfactant such as Triton X-100 ^™ . When the p26 protein or the like is secreted into the culture medium, after the completion of the culture, the cells or cells are separated from the supernatant by a method known per se, and the supernatant is collected. The culture supernatant thus obtained or the p26 protein or the like contained in the extract can be purified by an appropriate combination of separation / purification methods known per se. These known separation and purification methods include methods utilizing solubility such as salting out and solvent precipitation, dialysis, ultrafiltration, gel filtration, and SDS-polyacrylamide gel electrophoresis, and the like. Method using difference in charge, method using charge difference such as ion exchange chromatography, method using specific novelty such as affinity chromatography, use of difference in hydrophobicity such as reversed-phase high-performance liquid chromatography And a method utilizing the difference in isoelectric points such as isoelectric focusing.

When the thus obtained p26 protein or the like is obtained as a free form, it can be converted into a salt by a method known per se or a method analogous thereto, and conversely, when it is obtained as a salt, it is known per se. The free form or other salt can be converted by a method or a method analogous thereto. In addition, the p26 protein or the like produced by the recombinant can be optionally modified or the polypeptide can be partially removed by acting an appropriate protein-modifying enzyme before or after the purification. Examples of the protein-modifying enzyme include trypsin, chymotrypsin, arginyl endopeptidase, protein kinase, glycosidase and the like. The presence of the p26 protein and the like thus produced can be measured by an enzyme immunoassay using a specific antibody.

Antibodies against the p26 protein of the present invention, a partial peptide thereof or a salt thereof include the p26 protein,
Any of a polyclonal antibody and a monoclonal antibody may be used as long as the antibody can recognize the partial peptide or a salt thereof. An antibody against the p26 protein, a partial peptide thereof or a salt thereof (hereinafter sometimes simply abbreviated as p26 protein) of the present invention is produced by using the p26 protein as an antigen according to a method for producing an antibody or antiserum known per se. be able to. [Preparation of Monoclonal Antibody] (a) Preparation of Monoclonal Antibody-Producing Cell The p26 protein is administered to a warm-blooded animal at a site where antibody production is possible by itself or together with a carrier and a diluent. Complete Freund's adjuvant or incomplete Freund's adjuvant may be administered in order to enhance the antibody-producing ability upon administration. The administration is usually performed once every 2 to 6 weeks, about 2 to 10 times in total. 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, warm-blooded animals immunized with the antigen, for example, individuals with antibody titers are selected from mice, and spleens or lymph nodes are collected 2 to 5 days after the final immunization. A monoclonal antibody-producing hybridoma can be prepared by fusing the contained antibody-producing cells with myeloma cells. The antibody titer in the antiserum is measured, for example, by reacting the labeled p26 protein described below with the antiserum and then measuring the activity of the labeling agent bound to the antibody. The fusion operation can be carried out according to a known method, for example, the method of Koehler and Milstein [Nature, 256, 495 (1975)]. As the fusion promoter, for example, polyethylene glycol (PEG) or Sendai virus is used, but PEG is preferably used. Examples of myeloma cells include NS-1, P3U1, SP2 / 0, A
P-1 and the like are mentioned, but P3U1 is preferably used. The preferred ratio of the number of antibody-producing cells (spleen cells) to the number of myeloma cells used is about 1: 1 to 20: 1, and PEG (preferably PEG1000 to PEG600) is used.
0) is added at a concentration of about 10 to 80%, and 20 to 40
Cell fusion can be efficiently carried out by incubating at 1 ° C., preferably 30 to 37 ° C. for 1 to 10 minutes.

Various methods can be used for screening anti-p26 protein antibody-producing hybridomas. For example, the hybridoma culture supernatant is added to a solid phase (eg, a microplate) on which the p26 protein antigen is directly or adsorbed with a carrier. Then, an anti-immunoglobulin antibody labeled with a radioactive substance or an enzyme (an anti-mouse immunoglobulin antibody is used when the cells used for cell fusion are mice) or protein A is added, and the anti-p26 protein bound to the solid phase is added. Antibody detection method, hybridoma culture supernatant is added to a solid phase on which anti-immunoglobulin antibody or protein A is adsorbed, p26 protein labeled with a radioactive substance or enzyme is added, and anti-p2 bound to the solid phase is added.
For example, a method for detecting a 6-protein monoclonal antibody is used. The anti-p26 protein monoclonal antibody can be selected by a method known per se or a method analogous thereto. It is usually carried out in a medium for animal cells supplemented with HAT (hypoxanthine, aminopterin, thymidine). As a selection and breeding medium, any medium can be used as long as a hybridoma can grow. For example, RPMI 1640 medium containing 1-20%, preferably 10-20% fetal calf serum, 1-10%
Medium containing fetal calf serum (Wako Pure Chemical Industries, Ltd.)
Alternatively, a serum-free medium for hybridoma culture (SFM-1
01, Nissui Pharmaceutical Co., Ltd., etc. can be used. The culturing temperature is usually 20 to 40 ° C, preferably about 37 ° C. Cultivation time is usually 5 days to 3 weeks, preferably 1 week to 2 weeks. 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 above-mentioned measurement of anti-p26 protein antibody titer in antiserum.

(B) Purification of monoclonal antibody Separation and purification of the anti-p26 protein monoclonal antibody
Immunoglobulin separation and purification method similar to normal polyclonal antibody separation and purification [eg, salting out method, alcohol precipitation method,
Isoelectric focusing method, electrophoresis method, ion exchanger (eg, DEA)
E) Adsorption / desorption method, ultracentrifugation method, gel filtration method, specific purification method in which only antibody is collected by an antigen-bonded solid phase or an active adsorbent such as protein A or protein G, and the bond is dissociated to obtain the antibody] Can be done according to. [Preparation of Polyclonal Antibody] The polyclonal antibody against the p26 protein of the present invention can be manufactured by publicly known methods or modifications thereof. For example, a complex of an immunizing antigen (p26 protein antigen) and a carrier protein is formed, a warm-blooded animal is immunized in the same manner as in the above-described method for producing a monoclonal antibody, and an antibody-containing substance against the p26 protein is collected from the immunized animal. Then, it can be produced by separating and purifying the antibody. Regarding a complex of an immunogen and a carrier protein used for immunizing a warm-blooded animal, the kind of the carrier protein and the mixing ratio of the carrier and the hapten are:
As long as the antibody can efficiently crosslink to the carrier-immunized hapten, any kind may be crosslinked in any proportion, for example, bovine serum albumin, bovine thyroglobulin, or hemocyanin in a weight ratio. A method of coupling to about 1 to about 0.1 to 20, preferably about 1 to 5, 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 about once every 2 to 6 weeks, about 3 to 1 in total.
It is performed about 0 times. The polyclonal antibody is collected from the blood, ascites, etc., preferably from the blood of a warm-blooded animal immunized by the above method. The antibody titer of the anti-p26 protein in the antiserum can be measured in the same manner as the antibody titer of the hybridoma culture supernatant. Antibody separation and purification can be performed according to the same immunoglobulin separation and purification method as the above-described monoclonal antibody separation and purification.

The p26 protein of the present invention is an αβγ heterotrimeric GTP-binding protein α that is coupled to a G protein-coupled receptor (7-transmembrane receptor).
By binding to the subunit (Gα protein) and promoting the hydrolytic activity of Gα protein from GTP to GDP, the signal transduction triggered by the binding of the ligand to the G protein-coupled receptor is negatively regulated. , A signal transduction control protein involved in so-called desensitization. In addition, the phosphorylation of the p26 protein is involved in the binding between the p26 protein and the Gα protein, and by phosphorylating the p26 protein,
The bond between the p26 protein and the Gα protein is dissociated,
Desensitization is released by suppressing the hydrolytic activity of GTP bound to Gα protein to GDP. At present, two proteins known to be involved in the desensitization of G protein-coupled receptors are G protein-coupled receptor kinases and arrestin, and these antagonists are G protein-coupled receptor receptors. It is considered to be a therapeutic drug for diseases associated with desensitization. From these facts, the binding inhibitor of the p26 protein of the present invention to the Gα protein, the p2 of the present invention
The 6-protein phosphorylation promoter and the p26 protein dephosphorylation inhibitor of the present invention are β-adrenergic receptor blockers administered in the case of diseases associated with desensitization of G protein-coupled receptors, for example, heart failure. It is considered to be an agent for improving the effects such as a decrease in the effect of the long-term administration of morphine and a decrease in the sensitivity due to the long-term administration of morphine. Therefore, the p26 protein of the present invention is an inhibitor of the binding of the p26 protein of the present invention to the Gα protein, p2 of the present invention.
It is useful as a reagent for screening the phosphorylation promoter of 6 protein and the dephosphorylation inhibitor of p26 protein of the present invention. Further, the compound that inhibits the phosphorylation of p26 protein of the present invention by protein kinase becomes a drug such as an anticancer drug. Therefore, the p26 protein of the present invention is also useful as a reagent for screening protein kinase inhibitors.

Furthermore, the p26 protein of the present invention is
1) In vivo, it is most frequently expressed in fetal liver and peripheral blood, and the role of fetal liver as a hematopoietic organ is that expression rapidly decreases after 17 days of transfer to the bone marrow and spleen. 2) In cultured cells, many are expressed in cultured cells of T-cell leukemia differentiated from hematopoietic stem cells, and 3) because they are cytoplasmic proteins phosphorylated by protein kinase C. It can be seen that it is a signal transmitter that plays an important role in the differentiation of stem cells into T cells. Therefore, the p26 protein of the present invention is useful as a therapeutic / preventive agent for various diseases related to the hematopoietic system. The p26 protein of the present invention is also useful as a reagent for screening drug candidate compounds for various diseases related to the hematopoietic system. The use of the p26 protein of the present invention or a salt thereof, the DNA encoding the p26 protein of the present invention, and the antibody against the p26 protein of the present invention or a salt thereof will be described in more detail below.

(1) Screening of compound having phosphatase inhibitory activity or salt thereof As described above, the p26 protein of the present invention or salt thereof is phosphorylated by protein kinase (eg, protein kinase C). Therefore, by using the phosphorylated protein of the present invention and the dephosphorylating enzyme, a compound or a salt thereof that inhibits the activity of the dephosphorylating enzyme can be screened.
Thus, the p26 protein of the present invention is useful as a reagent for screening a compound having a phosphatase inhibitory activity or a salt thereof. That is, the present invention includes (i) a compound having a protein kinase inhibitory activity characterized by using a phosphorylated p26 protein of the present invention, a partial peptide thereof or a salt thereof (hereinafter referred to as dephosphorylation enzyme). (Sometimes abbreviated as inhibitor)), more specifically,
For example, (ii) (a) a case where a phosphorylated p26 protein of the present invention, a partial peptide thereof or a salt thereof is contacted with a dephosphorylating enzyme, and (b) a phosphorylated p26 protein of the present invention, A method for screening a phosphatase inhibitor, which comprises performing a comparison between the partial peptide or a salt thereof and a case where the phosphatase and a test compound are contacted. Specifically, in the above screening method, for example, in the cases of (a) and (b), the degree of dephosphorylation of the phosphorylated p26 protein, its partial peptide, or a salt thereof by a dephosphorylating enzyme. Is measured and compared.

A specific description of the screening method of the present invention is given below. The dephosphorylating enzyme may be any one as long as it can dephosphorylate the phosphorylated p26 protein of the present invention, its partial peptide or a salt thereof. For example, protein phosphatase 1 (P
P-1), protein phosphatase 2A (PP2-
A) or the like is used. As a test compound, for example,
Examples include peptides, proteins, non-peptidic compounds, synthetic compounds, fermentation products, cell extracts, plant extracts, animal tissue extracts, and the like.These compounds may be novel compounds or known compounds. It may be. In order to carry out the above-mentioned screening method, a phosphorylated p26 protein of the present invention, a partial peptide thereof or a salt thereof (hereinafter sometimes abbreviated as a phosphorylated p26 protein of the present invention) is used for screening. A standard product such as the phosphorylated p26 protein of the present invention is prepared by suspending it in a suitable buffer. The buffer may be a buffer that does not inhibit the reaction between the phosphorylated p26 protein of the present invention and the dephosphorylating enzyme, such as Tris-hydrochloric acid buffer having a pH of about 4 to 10 (desirably, a pH of about 6 to 8). Any one will do. The degree of dephosphorylation of the phosphorylated p26 protein or the like of the present invention by a dephosphorylating enzyme can be measured by [ ³² P] -labeled p
26 protein and the protein phosphatase of interest are suspended in the above buffer, and after a certain period of time, the decrease in radioactivity of [ ³² P] -labeled p26 protein can be measured. For example, above (b)
The degree of dephosphorylation of the phosphorylated p26 protein or the like of the present invention in the case of
Test compounds that inhibit about 20% or more, preferably 50% or more, can be selected as phosphatase inhibitors.

The screening kit of the present invention contains the p26 protein of the present invention, its partial peptide or its salt. Examples of the screening kit of the present invention include the following. [Screening Reagent] Measurement buffer pH 7.5 Tris-HCl buffer (20 mM Tri
s-HCl (pH 7.5), 0.1 mM CaCl ₂ , 5 m
Labeled with M MgCl ₂ , 0.31 mg / ml L-α-phosphatidyl-L-serine, 0.06 mg / ml 1,2-diolein, 0.03% Triton-X) p26 protein standard [ ³² P]. P26 protein of the present invention, its partial peptide or salts thereof, dephosphorylating enzyme preparation, protein phosphatase 1 10 mg / ml detection [ ³² P] radioactivity measurement [measurement method] [ ³² P] -labeled book Protein phosphatase 1 (10 mg / ml) was added to the assay buffer containing the p26 protein of the invention and the assay buffer containing the p32 protein of the present invention labeled with [ ³² P] and the test compound, respectively. Add and react at 30 ° C. for 2 hours. The reaction solution is subjected to SDS polyacrylamide gel electrophoresis (non-reducing), the gel is dried, and then [ ³² P] radioactivity is measured to measure the degree of phosphorylation.

The compound or its salt obtained by using the screening method or screening kit of the present invention is a test compound described above, for example, a peptide, protein, non-peptidic compound, synthetic compound, fermentation product, cell extract, The compound is a compound selected from a plant extract, an animal tissue extract, and the like, and is a compound that inhibits the activity of dephosphorylating enzyme and inhibits the dephosphorylation of the phosphorylated p26 protein of the present invention. As the salt of the compound, a physiologically acceptable acid addition salt is particularly preferable. As such salts, those similar to the salts of p26 protein described above are used. The compound having a phosphatase inhibitory activity or a salt thereof (a phosphatase inhibitor) obtained by using the screening method or the screening kit of the present invention is involved in desensitization of a G protein-coupled receptor, for example. Useful as a safe and low toxic therapeutic / preventive drug for diseases (eg, decreased effect of long-term administration of β-adrenergic receptor blocker administered in case of heart failure, decreased sensitivity due to long-term administration of morphine, etc.) Is. Further, it is also useful as a drug such as an immunomodulator (eg, an immunosuppressant), a prophylactic / therapeutic agent for terminal leukemia. When the phosphatase inhibitor obtained by using the screening method or the screening kit of the present invention is used as the above-mentioned medicine, it can be carried out in a conventional manner. For example, orally as tablets, capsules, elixirs, microcapsules, etc., optionally sugar-coated, or aseptic solution with water or other pharmaceutically acceptable liquid, or suspension It can be used parenterally in the form of injections such as. For example,
By admixing the compound or a salt thereof with a physiologically acceptable carrier, flavoring agent, excipient, vehicle, preservative, stabilizer, binder and the like in a unit dose form generally required for pharmaceutical practice. It can be manufactured.
The amount of the active ingredient in these preparations is such that an appropriate dose in the specified range can be obtained.

Examples of additives that can be mixed with tablets, capsules and the like include binders such as gelatin, corn starch, tragacanth and gum arabic, excipients such as crystalline cellulose, corn starch, gelatin and alginic acid. Puffing agents such as and the like, lubricants such as magnesium stearate, sweeteners such as sucrose, lactose or saccharin, flavoring agents such as peppermint, red oil or cherry, and the like are used. When the preparation unit form is a capsule, a liquid carrier such as oil and fat can be further contained in the above-mentioned type of material. Sterile compositions for injection can be formulated according to conventional pharmaceutical practices such as dissolving or suspending the active substance in a vehicle such as water for injection, naturally occurring vegetable oils such as sesame oil, coconut oil and the like. As the aqueous solution for injection, for example, physiological saline, isotonic solution containing glucose and other adjuvants (for example, D-sorbitol, D-mannitol, sodium chloride, etc.) and the like are used. For example, alcohol (eg ethanol), polyalcohol (eg propylene glycol, polyethylene glycol), nonionic surfactant (eg polysorbate 80 (TM), HCO-50) and the like may be used in combination. As the oily liquid, for example, sesame oil, soybean oil and the like are used, and as a solubilizing agent, for example, benzyl benzoate, benzyl alcohol and the like may be used in combination.
In addition, buffers (eg, phosphate buffer, sodium acetate buffer), soothing agents (eg, benzalkonium chloride, procaine hydrochloride, etc.), stabilizers (eg, human serum albumin, polyethylene glycol, etc.), preservation You may mix | blend with agents (for example, benzyl alcohol, phenol, etc.), antioxidants, etc. The adjusted injection solution is
Usually, it is filled into a suitable ampoule.

Since the preparation thus obtained is safe and has low toxicity, it can be used, for example, in humans or warm-blooded animals (eg, rat, mouse, guinea pig, bird, rabbit, sheep, pig, cow, horse, cat, Dogs, monkeys, etc.). The dose of the protein, its partial peptide or DNA will vary depending on the symptoms, but in the case of oral administration, it is generally about 0.1 mg to 100 m per day for an adult (as 60 kg).
g, preferably about 1.0 to 50 mg, more preferably about 1.0 to 20 mg. When administered parenterally,
The single dose varies depending on the administration subject, target tissue, symptom, administration method, etc., but, for example, in the form of an injection, usually in an adult (weight 60 kg), about 0.01 to 30 mg per day, Preferably about 0.1 to 20 m
Conveniently, about g, more preferably about 0.1 to 10 mg, will be administered by intravenous injection. In the case of other animals, the dose converted per 60 kg can be administered.

(2) Screening of compound having protein kinase inhibitory activity or salt thereof The p26 protein of the present invention or salt thereof is phosphorylated by protein kinase (eg, casein kinase II, protein kinase C, etc.). Therefore, by using the protein of the present invention and various protein kinases, compounds or salts thereof that inhibit the phosphorylation activity of protein kinases can be screened. Thus, the p26 protein of the present invention is useful as a reagent for screening a compound having protein kinase inhibitory activity or a salt thereof. That is, the present invention provides (i) a compound having a protein kinase inhibitory activity characterized by using the p26 protein of the present invention, a partial peptide thereof or a salt thereof (hereinafter abbreviated as a protein kinase inhibitor) More specifically, for example, (ii) (a) contacting a protein kinase with the p26 protein of the present invention, a partial peptide thereof or a salt thereof, and (b) Provided is a method for screening a protein kinase inhibitor, which comprises performing a comparison between the case where the p26 protein of the present invention, a partial peptide thereof or a salt thereof is contacted with a protein kinase and a test compound. Specifically, in the above screening method, for example, in the cases of (a) and (b),
It is characterized in that the degree of phosphorylation of the p26 protein, its partial peptide or salts thereof by protein kinase is measured and compared.

A detailed description of the screening method of the present invention is given below. The protein kinase may be any protein as long as it can phosphorylate the p26 protein of the present invention, its partial peptide or salts thereof, and examples thereof include casein kinase II, protein kinase C, and cAMP-dependent protein kinase. Is used. As the test compound, for example, a peptide, a protein, a non-peptide compound, a synthetic compound, a fermentation product, a cell extract, a plant extract, an animal tissue extract, etc. are used, and these compounds may be novel compounds. It may be a known compound. To carry out the above-mentioned screening method, the p26 protein of the present invention, a partial peptide thereof or a salt thereof (hereinafter sometimes abbreviated as p26 protein of the present invention, etc.) is suspended in a buffer suitable for screening. To prepare a preparation such as p26 protein of the present invention. The buffer has a pH of about 4
Any buffer that does not inhibit the reaction between the p26 protein of the present invention and the protein kinase, such as a Tris-hydrochloric acid buffer having a pH of about 10 (preferably about 6 to 8), may be used. P of the present invention by protein kinase
The degree of phosphorylation of 26 proteins etc. can be measured by [γ- ³²
It can be carried out by suspending P] ATP and the target protein kinase in the above buffer and measuring the radioactivity of [ ³² P] incorporated into the p26 protein after a certain period of time. Then, a protein kinase inhibitor is selected depending on the degree of phosphorylation of p26 protein when a test compound is added to this system and when it is not added. For example, a test compound that inhibits the degree of phosphorylation of the p26 protein or the like of the present invention in the case of (ii) by about 20% or more, preferably 50% or more as compared with the case of the above (i) is protein kinase inhibition. It can be selected as an agent.

The screening kit of the present invention contains the p26 protein of the present invention, its partial peptide or its salt. Examples of the screening kit of the present invention include the following. [Screening Reagent] Measurement buffer pH 7.5 Tris-HCl buffer (20 mM Tri
s-HCl (pH 7.5), 0.1 mM CaCl ₂ , 5 m
M MgCl ₂ , 0.31 mg / ml L-α-phosphatidyl-L-serine, 0.06 mg / ml 1,2-diolein, 0.03% Triton-X) p26 protein preparation p26 protein preparation of the invention, Partial peptide of the present invention or salt thereof Protein kinase standard casein kinase II 10 mg / ml substrate 100 nM ATP, 6.66 pmol [γ- ³² P] AT
Measurement of Radioactivity for P Detection [ ³² P] [Measurement Method] 100 nM was added to a measurement buffer containing the p26 protein of the present invention and a measurement buffer containing the p26 protein of the present invention and a test compound, respectively.
ATP, 6.66 pmol [γ- ³² P] ATP and casein kinase II (10 mg / ml) were added, and the temperature was 30 ° C.
And react for 2 hours. This reaction solution is subjected to SDS polyacrylamide gel electrophoresis (non-reducing), and the gel is dried.
The radioactivity of [ ³² P] is measured to measure the degree of phosphorylation.

The compound or its salt obtained by using the screening method or the screening kit of the present invention is a test compound described above, for example, a peptide, protein, non-peptidic compound, synthetic compound, fermentation product, cell extract, The compound is a compound selected from a plant extract, an animal tissue extract, and the like, and is a compound that inhibits the activity of protein kinase and inhibits the phosphorylation of p26 protein of the present invention. As the salt of the compound, a physiologically acceptable acid addition salt is particularly preferable. For such salt,
The same salt as the above-mentioned salt of p26 protein is used. The compound having a protein kinase inhibitory activity or a salt thereof (protein kinase inhibitor) obtained by using the screening method or the screening kit of the present invention is useful as a drug such as a safe and low toxic anti-cancer agent. When the protein kinase inhibitor obtained by using the screening method or the screening kit of the present invention is used as the above-mentioned medicine, it can be carried out in the same manner as the above-mentioned phosphatase inhibitor.

(3) Screening for compound having protein kinase promoting activity or salt thereof The p26 protein of the present invention or salt thereof is phosphorylated by protein kinase (eg, casein kinase II, protein kinase C, etc.). Therefore, by using the protein of the present invention and various protein kinases, a compound or its salt that promotes the phosphorylation activity of protein kinase can be screened. Thus, the p26 protein of the present invention is useful as a reagent for screening a compound having a protein kinase promoting activity or a salt thereof. That is, the present invention provides (i) a compound having a protein kinase promoting activity characterized by using the p26 protein of the present invention, a partial peptide thereof or a salt thereof (hereinafter, abbreviated as a protein kinase promoting agent). More specifically, for example, (ii) (a) contacting a protein kinase with the p26 protein of the present invention, a partial peptide thereof or a salt thereof, and (b) There is provided a method for screening a protein kinase promoter, which comprises performing a comparison between the case where the p26 protein of the present invention, a partial peptide thereof or a salt thereof is contacted with a protein kinase and a test compound. Specifically, in the above screening method, for example, in the cases of (a) and (b),
It is characterized in that the degree of phosphorylation of the p26 protein, its partial peptide or salts thereof by protein kinase is measured and compared.

The screening method for the protein kinase promoter of the present invention can be carried out according to the screening method for the protein kinase inhibitor described above.
The protein kinase may be any protein as long as it can phosphorylate the p26 protein of the present invention, its partial peptide or salts thereof, and examples thereof include casein kinase II, protein kinase C, and cAMP-dependent protein kinase. Is used. As the test compound, for example, a peptide, a protein, a non-peptide compound, a synthetic compound, a fermentation product, a cell extract, a plant extract, an animal tissue extract, etc. are used, and these compounds may be novel compounds. It may be a known compound. To carry out the above-mentioned screening method, the p26 protein of the present invention, a partial peptide thereof or a salt thereof (hereinafter sometimes abbreviated as p26 protein of the present invention, etc.) is suspended in a buffer suitable for screening. To prepare a preparation such as p26 protein of the present invention. The buffer should have a pH of about 4-10 (desirably,
Any buffer that does not inhibit the reaction between the p26 protein of the present invention and the protein kinase, such as Tris-hydrochloric acid buffer having a pH of about 6 to 8) may be used. To measure the degree of phosphorylation of p26 protein or the like of the present invention by protein kinase, [γ- ³² P] ATP and the target protein kinase were suspended in the above buffer and incorporated into p26 protein after a certain time [ ³² It can be carried out by measuring the radioactivity of P].
Then, a protein kinase promoter is selected according to the degree of phosphorylation of p26 protein when a test compound is added to this system and when it is not added. For example, a test compound that promotes the degree of phosphorylation of the p26 protein or the like of the present invention in the case of (b) above by about 20% or more, preferably 50% or more compared to the case of (a) above is promoted by a protein kinase. It can be selected as an agent.

The screening kit of the present invention contains the p26 protein of the present invention, its partial peptide or its salt. Examples of the screening kit of the present invention include the following. [Screening Reagent] Measurement buffer pH 7.5 Tris-HCl buffer (20 mM Tri
s-HCl (pH 7.5), 0.1 mM CaCl ₂ , 5 m
M MgCl ₂ , 0.31 mg / ml L-α-phosphatidyl-L-serine, 0.06 mg / ml 1,2-diolein, 0.03% Triton-X) p26 protein preparation p26 protein preparation of the invention, Partial peptide of the present invention or salt thereof Protein kinase standard casein kinase II 10 mg / ml substrate 100 nM ATP, 6.66 pmol [γ- ³² P] AT
Measurement of Radioactivity for P Detection [ ³² P] [Measurement Method] 100 nM was added to a measurement buffer containing the p26 protein of the present invention and a measurement buffer containing the p26 protein of the present invention and a test compound, respectively.
ATP, 6.66 pmol [γ- ³² P] ATP and casein kinase II (10 mg / ml) were added, and the temperature was 30 ° C.
And react for 2 hours. This reaction solution is subjected to SDS polyacrylamide gel electrophoresis (non-reducing), and the gel is dried.
The radioactivity of [ ³² P] is measured to measure the degree of phosphorylation.

The compound or its salt obtained by using the screening method or the screening kit of the present invention is a test compound described above, for example, a peptide, protein, non-peptidic compound, synthetic compound, fermentation product, cell extract, The compound is a compound selected from a plant extract, an animal tissue extract, and the like, and is a compound that promotes the activity of protein kinase and promotes phosphorylation of the p26 protein and the like of the present invention. As the salt of the compound, a physiologically acceptable acid addition salt is particularly preferable. For such salt,
The same salt as the above-mentioned salt of p26 protein is used. A compound having a protein kinase promoting activity or a salt thereof (protein kinase promoting agent) obtained by using the screening method or the screening kit of the present invention is, for example, a disease involved in desensitization of a G protein-coupled receptor (eg, Β-administered in case of heart failure
It is useful as a drug such as a safe and low toxic therapeutic / prophylactic agent against a decrease in the effect of long-term administration of an adrenergic receptor blocker, a decrease in sensitivity due to long-term administration of morphine, and the like. When the protein kinase promoter obtained by using the screening method or the screening kit of the present invention is used as the above-mentioned medicine, it can be carried out in the same manner as the above-mentioned phosphatase inhibitor.

(4) Therapeutic / Preventive Agent for Diseases Related to Hematopoietic System The p26 protein of the present invention is a signal transmitter which plays an important role in hematopoietic stem cells or in the differentiation of hematopoietic stem cells into T cells. Therefore, p of the present invention
When 26 protein or DNA encoding it is abnormal or defective, or its expression level is decreased or increased, various diseases related to hematopoietic system (eg, aplastic anemia, acute bone marrow) It is believed that sexual leukemia, T cell leukemia, etc.) develop. Therefore, the p26 protein of the present invention and the DNA encoding the p26 protein can be used for treating diseases related to the hematopoietic system, such as aplastic anemia, acute myelogenous leukemia, and T cell leukemia.
It can be used as a medicine such as a prophylactic agent. For example, patients in whom p26 protein is reduced or deficient in vivo, so that information transmission in cells involved in hematopoiesis, cells derived from hematopoietic stem cells, cells involved in blood system, etc. is not sufficiently or normally exerted. (B) by administering the DNA encoding the p26 protein of the present invention to the patient and expressing the p26 protein in vivo, or (b) encoding the p26 protein of the present invention in hematopoietic stem cells and the like. P26 in the patient by, for example, transplanting the cells into the patient after inserting the DNA for expressing the p26 protein into the patient.
The role of protein can be fully or normally exerted. Furthermore, the p26 protein of the present invention and the DNA encoding the same can be used as a medicine such as an immunomodulator (eg, an immunosuppressant).

D encoding the p26 protein of the invention
When NA is used as the above-mentioned therapeutic / prophylactic agent, the D
NA may be used alone or after it is inserted into an appropriate vector such as a retrovirus vector, an adenovirus vector, an adenovirus associated virus vector or the like, and then it can be carried out by a conventional method. For example, orally as a sugar-coated tablet, capsule, elixir, microcapsule, etc., or aseptic solution with water or other pharmaceutically acceptable liquid, if necessary.
Alternatively, it can be used parenterally in the form of injection such as suspension. For example, the DNA of the present invention is admixed with physiologically acceptable carriers, flavoring agents, excipients, vehicles, preservatives, stabilizers, binders and the like in a unit dose form generally required for the implementation of the formulation. Can be manufactured by. The amount of the active ingredient in these preparations is such that an appropriate dose in the specified range can be obtained. tablet,
Examples of additives that can be mixed with capsules include gelatin, corn starch, tragacanth,
Binders such as acacia, excipients such as crystalline cellulose, swelling agents such as corn starch, gelatin, alginic acid, lubricants such as magnesium stearate, sweeteners such as sucrose, lactose or saccharin. ,
A flavoring agent such as peppermint, red oil or cherry is used. When the preparation unit form is a capsule, a liquid carrier such as oil and fat can be further contained in the above-mentioned type of material. Sterile compositions for injection can be formulated according to conventional pharmaceutical practices such as dissolving or suspending the active substance in a vehicle such as water for injection, naturally occurring vegetable oils such as sesame oil, coconut oil and the like. Examples of the aqueous solution for injection include physiological saline, isotonic solution containing glucose and other adjuvants (eg, D-sorbitol, D-mannitol, sodium chloride).
And the like, and a suitable solubilizing agent such as alcohol (eg, ethanol), polyalcohol (eg,
Propylene glycol, polyethylene glycol), non-ionic surfactants (eg polysorbate 80 (T
You may use together with M), HCO-50), etc. As the oily liquid, for example, sesame oil, soybean oil and the like are used, and as a solubilizing agent, for example, benzyl benzoate, benzyl alcohol and the like may be used in combination. In addition, buffers (eg, phosphate buffer, sodium acetate buffer), soothing agents (eg, benzalkonium chloride, procaine hydrochloride, etc.), stabilizers (eg, human serum albumin, polyethylene glycol, etc.), preservation You may mix | blend with agents (for example, benzyl alcohol, phenol, etc.), antioxidants, etc. The prepared injection solution is usually filled into a suitable ampoule.

The preparation thus obtained is safe and has low toxicity, and therefore, for example, humans or warm-blooded animals (eg, rat, mouse, guinea pig, rabbit, bird, sheep, pig, cow, horse, cat, Dogs, monkeys, etc.). Although the dose of the DNA varies depending on the symptoms and the like, when administered orally, it is generally about 0.1 m per day for an adult (as 60 kg).
g to 100 mg, preferably about 1.0 to 50 mg, more preferably about 1.0 to 20 mg. When administered parenterally, the single dose varies depending on the administration subject, target tissue, symptom, administration method, etc., but, for example, in the form of an injection, usually in adults (60 kg) per day, About 0.01 to 30 mg, preferably about 0.1
It is convenient to administer about -20 mg, more preferably about 0.1-10 mg by intravenous injection. In the case of other animals, the dose converted per 60 kg can be administered.

(5) Quantification of p26 protein, its partial peptide or salts thereof The antibody against the p26 protein, its partial peptide or salts thereof of the present invention is a p26 protein, its partial peptide or salts thereof (hereinafter, simply p26 (Sometimes abbreviated as “protein”), it can be used for quantification of p26 protein in a test solution, particularly for quantification by sandwich immunoassay. That is, the present invention comprises (i) measuring the ratio of the labeled p26 protein bound to the antibody by competitively reacting the antibody against the p26 protein of the present invention with the test solution and the labeled p26 protein. A characteristic method for quantifying p26 protein in a test solution, and (ii) reacting the test solution with the antibody insolubilized on the carrier and the labeled antibody simultaneously or continuously, and then on the insolubilized carrier A method for quantifying p26 protein in a test solution, which comprises measuring the activity of a labeling agent. In the quantification method of (ii) above, one antibody is N of p26 protein.
It is desirable that the other antibody recognizes the end and the other antibody reacts with the C-end of the p26 protein.

The monoclonal antibody to the p26 protein of the present invention (hereinafter sometimes referred to as anti-p26 protein antibody) can be used to quantify the p26 protein, and it can also be detected by tissue staining or the like. For these purposes, the antibody molecule itself may be used, or F (ab ′) ₂ , Fab ′, or Fab fraction of the antibody molecule may be used. P using the antibody of the present invention
The method for quantifying 26 protein is not particularly limited, and the amount of antibody, antigen or antibody-antigen complex corresponding to the amount of antigen (eg, amount of p26 protein) in the liquid to be measured can be determined chemically or physically. Any measuring method may be used as long as it is detected by a means and is calculated from a standard curve prepared using a standard solution containing a known amount of antigen. For example, nephelometry, a competitive method, an immunometric method, and a sandwich method are suitably used, but in terms of sensitivity and specificity, it is particularly preferable to use a sandwich method described later. As a labeling agent used in a measurement method using a labeling substance, a radioisotope, an enzyme, a fluorescent substance, a luminescent substance and the like are used. As the radioisotope, for example, [ ¹²⁵ I], [ ¹³¹ I], [ ³ H], [ ¹⁴ C] and the like are preferable, and as the above enzyme, those having stable and large specific activity are preferable, for example, β-galactosidase , Β-glucosidase, alkaline phosphatase, peroxidase, malate dehydrogenase, etc., as the fluorescent substance, for example, fluorescamine, fluorescein isothiocyanate, etc., as the luminescent substance, for example, luminol, luminol derivative, luciferin. , Lucigenin, etc. are used respectively. Further, a biotin-avidin system can be used for binding the antibody or antigen to the labeling agent.

For the insolubilization of the antigen or antibody, physical adsorption may be used, or a method using a chemical bond which is usually used for insolubilizing or immobilizing proteins or enzymes. Examples of the carrier include insoluble polysaccharides such as agarose, dextran and cellulose, synthetic resins such as polystyrene, polyacrylamide and silicon, and glass. In the sandwich method, the insolubilized anti-p26 protein antibody is reacted with a test solution (first reaction), and further labeled anti-p26 protein antibody is reacted (second reaction), and then the activity of the labeling agent on the insolubilized carrier is increased. By measuring, the amount of p26 protein in the test liquid can be quantified. 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 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 is used for the purpose of improving measurement sensitivity and the like. You may. In the p26 protein assay method by the sandwich method of the present invention, the anti-p26 protein antibodies used in the primary reaction and the secondary reaction are preferably antibodies having different p26 protein binding sites. That is, when the antibody used in the secondary reaction recognizes the C-terminal portion of p26 protein, for example, the antibody used in the primary reaction and the secondary reaction is preferably the C-terminal. Other than the region, for example, an antibody that recognizes the N-terminal is used.

The p26 protein antibody of the present invention can be used in a measuring system other than the sandwich method, for example, a competitive method, an immunometric method or nephrometry. In the competition method, an antigen in a test solution and a labeled antigen are allowed to competitively react with an antibody, and then the unreacted labeled antigen is separated from (F) and the labeled antigen (B) bound to the antibody. (B / F separation), the amount of any of B and 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, a liquid phase method using polyethylene glycol, a second antibody against the antibody is used for B / F separation, and a solid phase antibody is used as the first antibody, or The first antibody is soluble and the second antibody is a solid-phased antibody. In the immunometric method, 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 labeled amount of either phase is measured to quantify the amount of antigen in the test liquid. 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 immunological measurement methods to the quantification method of the present invention, it is not necessary to set special conditions, operations and the like. In addition to the usual conditions and operating methods in each method, with ordinary technical consideration of those skilled in the art, p2
A measurement system for 6 proteins may be constructed. 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, "Radio Immunoassay" (Kodansha, published in 1979), Eiji Ishikawa et al., "Enzyme Immunoassay" (Medical Shoin, Showa Ed. Ishikawa et al., “Enzyme Immunoassay” (2nd edition) (Issue Shoin, 1982), Eiji Ishikawa et al. “Enzyme Immunoassay” (Third Edition), 3rd edition (Medical Shoin, Showa) Published in 1962, "Methods in ENZYMOLOGY" Vol. 70 (Immunochem
ical Techniques (Part A)), ibid.Vol. 73 (Immunochem
ical Techniques (Part B)), ibid Vol. 74 (Immunochem
ical Techniques (Part C)), ibid.Vol. 84 (Immunochem
ical Techniques (Part D: Selected Immunoassays)),
Ibid.Vol. 92 (Immunochemical Techniques (Part E: Mono
clonal Antibodies and General Immunoassay Method
s)), ibid.Vol. 121 (Immunochemical Techniques (Part
I: Hybridoma Technology and Monoclonal Antibodie
s)) (above, published by Academic Press). As described above, the p26 protein of the present invention can be quantified with high sensitivity by using the p26 protein antibody of the present invention. Furthermore, the antibody of the present invention can be used to detect the p26 protein of the present invention present in a subject such as body fluid or tissue. In addition, preparation of an antibody column used for purifying the p26 protein of the present invention, detection of the p26 protein of the present invention in each fraction during purification, analysis of the behavior of the p26 protein of the present invention in test cells, etc. Can be used for.

(6) Screening of Drug Candidate Compounds for Diseases Related to Hematopoiesis Since the p26 protein of the present invention has a role as an information transmitter in cells involved in hematopoiesis, cells in blood system, etc. The compound or its salt that promotes or inhibits the function of p26 protein of the present invention is used as a therapeutic / preventive agent for diseases associated with the hematopoietic system (eg, aplastic anemia, acute myelogenous leukemia, T cell leukemia, etc.). It can be used as a medicine. Furthermore, it can also be used as a medicine such as an immunomodulator (for example, an immunosuppressant). Therefore, the p26 protein of the present invention is useful as a reagent for screening drug candidate compounds such as therapeutic / preventive agents for diseases related to hematopoietic system or immunomodulators.

In the present specification and drawings, when bases, amino acids and the like are represented by abbreviations, IUPAC-IUB
Abbreviations based on Commision on Biochemical Nomenclature or common abbreviations in the field,
An example is given below. 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 Phosphoric acid dCTP: Deoxycytidine triphosphate ATP: Adenosine triphosphate EDTA: Ethylenediaminetetraacetic acid SDS: Sodium dodecyl sulfate

Gly: Glycine Ala: Alanine Val: Valine Leu: Leucine Ile: Isoleucine Ser: Serine Thr: Threonine Cys: Cysteine Met: Methionine Glu: Glutamic acid Asp: Aspartic acid Phythine arginine: Lys: Arginine: Lys: Pyr: Arginine: Lys: Pyr: arginine: Lys :: : Tyrosine Trp: Tryptophan Pro: Proline Asn: Asparagine Gln: Glutamine pGlu: Pyroglutamic acid Me: Methyl group Et: Ethyl group Bu: Butyl group Ph: Phenyl group TC: Thiazolidine-4 (R) -carboxamide group

The substituents, protecting groups and reagents frequently used in the present specification are represented by the following symbols. Tos: p-toluenesulfonyl CHO: formyl Bzl: benzyl Cl ₂ Bzl: 2,6-dichlorobenzyl Bom: benzyloxymethyl Z: benzyloxycarbonyl Cl-Z: 2-chlorobenzyloxycarbonyl Br-Z: 2-bromobenzyl Oxycarbonyl Boc: t-Butyloxycarbonyl DNP: Dinitrophenol Trt: Trityl Fmoc: N-9-fluorenylmethoxycarbonyl HOBt: 1-hydroxybenztriazole HOOBt: 3,4-dihydro-3-hydroxy-4-oxo- 1,2,3-benzotriazine HONB: 1-hydroxy-5-norbornene-2,3-dicarboximide DCC: N, N'-dicyclohexylcarbodiimide

The sequence numbers in the sequence listing in this specification show the following sequences. [SEQ ID NO: 1] This shows the amino acid sequence of rat-derived p26 protein. [SEQ ID NO: 2] This shows the amino acid sequence of human-derived p26 protein. [SEQ ID NO: 3] This shows the amino acid sequence of mouse-derived p26 protein. [SEQ ID NO: 4] This shows the base sequence of DNA encoding rat-derived p26 protein. [SEQ ID NO: 5] This shows the base sequence of DNA encoding human-derived p26 protein. [SEQ ID NO: 6] This shows the base sequence of DNA encoding mouse-derived p26 protein. [SEQ ID NO: 7] This shows the base sequence of the synthetic primer used for cloning the DNA encoding rat p26 protein. [SEQ ID NO: 8] This shows the base sequence of the synthetic primer used for cloning the DNA encoding rat p26 protein. [SEQ ID NO: 9] This shows the base sequence of synthetic primer used for cloning DNA encoding rat p26 protein. [SEQ ID NO: 10] This shows the base sequence of synthetic primer used for cloning DNA encoding rat p26 protein.

[SEQ ID NO: 11] Synthetic An used for cloning DNA encoding rat p26 protein
The nucleotide sequence of the chored primer is shown. [SEQ ID NO: 12] β-galactosidase-rat p2
6 shows a synthetic primer designed to contain only the translation region excluding methionine at the initiation codon using the plasmid pTS822 as a template in order to construct an expression plasmid for the 6 fusion protein. [SEQ ID NO: 13] β-galactosidase-rat p2
6 shows a synthetic primer designed to contain only the translation region excluding methionine at the initiation codon using the plasmid pTS822 as a template in order to construct an expression plasmid for the 6 fusion protein. [SEQ ID NO: 14] To construct the expression plasmid of rat p26 protein, plasmid pTS822 was used.
2 shows a synthetic primer designed to include only the N-terminal portion of the translation region using as a template. [SEQ ID NO: 15] To construct the expression plasmid of rat p26 protein, plasmid pTS822 was constructed.
2 shows a synthetic primer designed to include only the N-terminal portion of the translation region using as a template. [SEQ ID NO: 16] This shows the synthetic DNA inserted into pTS823 obtained in Example 1 to prepare pTS825. [SEQ ID NO: 17] This shows the synthetic DNA inserted into pTS823 obtained in Example 1 to prepare pTS825.

The transformant Escherichia coli XL1-Blue M obtained in Example 1 described later.
RF '/ pTS834 has been deposited with the deposit number FERM BP-5448 at the Institute of Biotechnology, National Institute of Advanced Industrial Science and Technology (NIBH) since March 6, 1996. The transformant Escherichia coli XL1-Blue MRF '/ p obtained in Example 2 described later
TS855 has been deposited with the deposit number F at the Institute of Biotechnology, National Institute of Advanced Industrial Science and Technology (NIBH) since March 6, 1996.
Deposited as ERM BP-5449. The transformant Escherichia coli (Es) obtained in Example 7 described later
cherichia coli) DH10B / pTS842 is available in 1996
Depositary No. FERM BP-545 to the Institute of Biotechnology, National Institute of Advanced Industrial Science and Technology (NIBH) from March 6, 2014
Deposited as 0.

[0067]

EXAMPLES Hereinafter, the present invention will be described more specifically with reference to Reference Examples and Examples, but the present invention is not limited thereto. Gene manipulation using Escherichia coli is performed by molecular cloning.
Followed the method described in.

[0068]

Example 1 cD encoding rat p26 protein
Cloning of NA After infecting the Escherichia coli Y1090r ⁻ strain with λ phage of a rat brain-derived cDNA library (Clontech), about 3 × 10 ⁴ plaques were plated on a soft agar plate and incubated at 42 ° C. overnight. Plaques were formed. After transferring the plaque onto a nitrocellulose filter (Schleicher & Schuell), a denaturing solution [0.5N sodium hydroxide, 1.5M sodium chloride],
Neutralization solution [0.5M Tris-hydrochloric acid (pH.7.0), 1.5M sodium chloride], 3 × SSC [20 × SSC = 3M sodium chloride, 0.
3M sodium citrate], air-dried at 80 ℃
After baking for 3 hours, the phage DNA was immobilized on a nitrocellulose filter. On the other hand, as a probe, two synthetic oligonucleotides (SEQ ID NO: 7, SEQ ID NO: 8) were used for screening a cDNA library. To label the probe, first, use two oligonucleotides having these partially complementary sequences at 70 ° C.
After denaturing for 10 minutes at 70 ° C., the temperature was gradually lowered from 70 ° C. to anneal two oligonucleotides. Next, [α- ³² P] dCTP (DuPont / NEN 6000C
i / mmol) and Klenow fragment (Takara Shuzo) were used for labeling by the primer extension method for synthesizing the complementary strand of the single-stranded portion to finally obtain an 81-mer oligonucleotide probe. Hybridization was carried out using a hybridization buffer containing a labeled probe [2 × SSPE [20 × SSPE = 3.6M sodium chloride, 0.2M
Sodium phosphate (pH 7.7), 20 mM EDTA], 10 x Denhard
t's solution, 150 μg / ml heat-denatured salmon sperm DNA, 667 μg / m
l yeast RNA] at 50 ° C. The filter is finally 2 x SSC, 0.1% S
After washing in DS solution at 48 ° C., an autoradiogram was taken to search for plaques that hybridize with the probe.

After extracting the phage DNA from the phage clone obtained by this method, the cDNA fragment was cut out by digesting with the restriction enzyme EcoRI to obtain the pUC118 plasmid.
Insertion into the EcoRI site gave pTS819. When the nucleotide sequence of the inserted cDNA fragment was determined by a usual method using [α- ³² P] dCTP, it was found that this cDNA fragment consisted of 545 bp containing poly (A) ⁺ chain. However, since this clone was considered to be a clone in which the 5'region was crossed, the cDNA of BglII-Eco47III of 374 bp of the cDNA newly obtained here was used as a probe, and the cDNA derived from rat brain was again obtained. The library was screened. The above-mentioned cDNA fragment was labeled with [α- ³² P] dCTP and a random primer labeling kit (Amersham) to label the probe. Hybridization is carried out by using a hybridization buffer containing a labeled probe [5 x SSPE, 5 x Denhard
t's solution, 0.1% SDS, 100 μg / ml heat-denatured salmon sperm DN
A] in 65 ° C.
The filter was finally washed in 0.2 × SSC, 0.1% SDS solution at 65 ° C., and an autoradiogram was taken to search for plaques that hybridize with the probe. As a result, 56 positive clones were obtained. After extracting phage DNA from the phage clones for 4 of these clones, pUC119
Inserted into the HincII site of the plasmid, [α- ³² P] dCT
The nucleotide sequence was determined by the usual method using P (pTS8
20-pTS823). The cDNA fragments obtained here were ligated together to determine 842 bp containing a poly (A) ⁺ chain.

Next, in order to further obtain a nucleotide sequence on the 5'end side, 5'RACE (Rapid Amplification of cDNA E
nd). After fractionating total RNA from rat brain by the guanidine thiocyanate method, this total RNA was fractionated.
Was applied to an oligo (dT) span column (Pharmacia) to prepare poly (A) ⁺ RNA. 1 μg of this poly (A) ⁺ RNA
Was synthesized using a primer 1 (SEQ ID NO: 9) complementary to rat p26 protein cDNA, and then synthesized with terminal deoxyribonucleotidyl transferase (TdT) (Gibco) Poly (A) was added to the 5'end of the cDNA. Next, the anchor primer (SEQ ID NO: 11) having poly (T) following the restriction enzyme site and the primer 1 (SEQ ID NO:
PCR was performed using the primer 2 (SEQ ID NO: 10) located slightly 5 ′ upstream of 9). 5'R mentioned above
The 842 bp cDNA fragment obtained by ACE was ligated together to form a poly (A) ⁺ chain-containing cDNA with a total length of 875 bp.
The NA fragment was inserted into the HincII-SmaI site of pUC119 to obtain pTS834. Cd contained in this plasmid
When the base sequence of the NA fragment was determined, it was revealed to have the base sequence represented by SEQ ID NO: 4 [Fig. 1]. This cDNA contains 1 represented by SEQ ID NO: 1.
A rat p26 protein consisting of 81 amino acids was encoded, and it was found that this rat p26 protein has little homology with amino acids of known proteins [Fig. 1]. The obtained pTS834 was transformed into Escherichia coli (Esch
erichia coli) XL1-Blue MRF 'and transformed: Escherichia coli XL1-Blue M
RF '/ pTS834 was obtained.

[0071]

Example 2 cDNA encoding human p26 protein
Cloning of A Human thymus-derived cDNA library (Clontech)
The E. coli Y1090r ⁻ strain was infected with the λ phage of Example 1 and then spread on 120 soft agar plates and incubated overnight at 42 ° C. to form about 1.9 × 10 ⁶ plaques. After transferring the plaques onto a nitrocellulose filter, a denaturing solution [0.5N sodium hydroxide, 1.5M sodium chloride], a neutralizing solution [0.5M Tris-hydrochloric acid (pH 7.0), 1.5
M sodium chloride], sequentially treated with 3 × SSC, air-dried and baked at 80 ° C. for 3 hours to immobilize phage DNA on a nitrocellulose filter. On the other hand, a cDNA encoding rat p26 protein as a probe
The NotI-Aor51HI 732 bp fragment of (pTS834)
It was used for screening a DNA library. The probe was labeled with the above-mentioned cDNA fragment by [α- ³² P] dCTP.
And a random primer labeling kit (Amersham). Hybridization is carried out by using a hybridization buffer solution (0.
5M sodium phosphate (pH7.4), 7% SDS, 1% BSA, 1mM
Hybridization was performed in EDTA] at 60 ° C. The filter was finally washed in 2 × SSC, 0.1% SDS solution at 65 ° C., and an autoradiogram was taken to search for plaques that hybridize with the probe.
As a result, it was found that 38 clones were positive.

After extracting the phage DNA from the phage clone of No. 6 which is considered to have the longest insert among these, the cDNA fragment was excised by digesting with restriction enzymes AluI and EcoRI, and the pGEM9Zf (-) plasmid ( PM by inserting into SacI-EcoRI site of Promega)
S855 was obtained. The nucleotide sequence of the inserted cDNA fragment was determined by an ordinary method using [α- ³² P] dCTP. As a result, it was found that the cDNA fragment containing poly (A) ⁺ chain was 889 It was found to have the nucleotide sequence of [Fig. 2]. The cDNA fragment has the SEQ ID NO:
Human p26 consisting of 181 amino acids represented by 2
The protein is encoded [Fig. 2], rat p26
The homology with the protein (SEQ ID NO: 1) was found to be very well conserved at 93% at the amino acid level [Fig. 4]. Further, from the amino acid sequence, it was found that the rat, human and mouse p26 proteins have multiple sites where casein kinase II and protein kinase C may be phosphorylated (FIG. 4). The obtained pTS855 was transformed into Escherichia coli XL
1-Blue MRF 'was introduced, and transformant: E. coli (Esc
herichia coli) XL1-BlueMRF ′ / pTS855 was obtained. On the other hand, c which encodes mouse p26 protein
DNA cloning was performed in the same manner. The cDNA
Has a base sequence represented by SEQ ID NO: 6,
The cDNA was found to encode the 181 amino acids represented by SEQ ID NO: 3 [Fig. 3].

[0073]

[Example 3] Northern hybridization of rat brain regions 8 weeks old (♀) Sprague Dawley rat (Japan Charles Rive
r) each part of the brain, ie olfactory bulb, a
mygdala (amygdala), basal gangria (basal ganglia), hi
ppocampus (hippocampus), thalamus (thalamus), hypothalamus
(Hypothalamus), cerebral cortex, medulla
(Medulla oblongata), cerebellum (cerebellum), spinal cord (spinal cord), pituitary (pituitary gland) was fractionated by the guanidine thiocyanate method, and the total RNA was applied to an oligo (dT) span column (Pharmacia) to obtain poly.
(A) ⁺ RNA was prepared. 1.5% of this poly (A) + RNA 5 μg
After subjecting it to formalin-denaturing agarose gel electrophoresis, it was blotted on a nylon membrane filter (Nihon Pall Ltd., Biodyne A) by capillary blotting for 16 hours. After fixing the RNA blotted with this nylon membrane filter by UV treatment, hybridization buffer [50% formamide, 5xSSPE, 5x Denhardt's solution, 0.1% SDS, 100μ
g / ml heat-denatured salmon sperm DNA] at 42 ° C. On the other hand, Ec of cDNA (pTS834) encoding rat p26 as a probe
oRI-NcoI 512 bp cDNA fragment was [α- ³² P] dCT
Labeling was performed using P and a random primer labeling kit (Amersham). Hybridization is carried out using a hybridization buffer solution [50
% Formamide, 5 x SSPE, 5 x Denhardt's solution, 0.1% SD
S, 100 μg / ml heat-denatured salmon sperm DNA] at 42 ° C for 1
Hybridization was performed for 6 hours. The filter was finally washed in 0.1 × SSC, 0.1% SDS solution at 50 ° C., and then an autoradiogram was taken to detect a band hybridizing with the probe. As a result, in the brain, the size of rat p26 mRNA was about 1.1 Kb,
The expression level was highest in the hippocampus and cerebral cortex, followed by olfactory bulb, amygdala, and basal ganglia. Expression was weak in the thalamus, hypothalamus, and spinal cord [Fig. 5].

[0074]

[Example 4] Northern hybridization of human tissues 2 μg of poly (A) ⁺ RNA (Clontech) of human tissues
Was subjected to electrophoresis on a 1.5% formalin-denatured agarose gel, and then blotted on a nylon membrane filter (Biodyne B, Japan Pall Co.) for 16 hours by capillary blotting. R by blotting this nylon membrane filter by UV treatment
After fixing NA, hybridization buffer [50% formamide, 5 x SSPE, 5 x Denhardt's solution, 0.1%
SDS, 100 μg / ml heat-denatured salmon sperm DNA] in 42 ℃
Pre-hybridization was performed. On the other hand, cDNA encoding human p26 protein as a probe
The Bpu1 102I 688 bp cDNA fragment of (pTS855) was labeled with [α- ³² P] dCTP and a random primer labeling kit (Amersham). Hybridization was carried out using a hybridization buffer containing a labeled probe [50% formamide, 5 × SSPE, 5 × De
nhardt's solution, 0.1% SDS, 100 μg / ml heat-denatured salmon sperm D
NA] for 16 hours at 42 ° C. The filter is finally 0.1 × SSC, 0.1% SDS
After washing in the liquid at 50 ° C., an autoradiogram was taken to detect a band hybridizing with the probe. As a result, the mRNA coding for the human p26 protein of about 1.0 Kb was expressed in most tissues, and was most frequently expressed in peripheral blood and fetal liver [Fig. 6].

[0075]

Example 5 Southern Hybridization of Human Genomic DNA 8 μg of human genomic DNA was digested with various restriction enzymes (EcoRI, Hind).
(III, BamHI, PstI, BglII), and then subjected to agarose gel electrophoresis, and then subjected to capillary blotting for 16 hours on a nylon membrane filter (Nihon Pall Co., Biodyne B). After immobilizing the DNA blotted on this nylon membrane filter by UV treatment, hybridization buffer [0.5M sodium phosphate (pH7.4), 7% S
DS, 1% BSA, 1 mM EDTA] at 65 ° C. On the other hand, as a probe, human p
CDNA encoding 26 proteins (pTS855)
BanII-Bpu1102I 167 bp cDNA fragment of [α- ³²
Labeling was performed using P] dCTP and a random primer labeling kit (Amersham). Hybridization is carried out using a hybridization buffer containing a labeled probe [0.5M sodium phosphate (pH7.4), 7% SDS,
Hybridization was performed in 1% BSA, 1 mM EDTA] at 65 ° C. for 16 hours. The filter is finally 0.1 x S
After washing in SC, 0.1% SDS solution at 50 ° C., an autoradiogram was taken to detect a band hybridizing with the probe. As a result, EcoRI, HindIII, BamHI, Pst
Since one band was observed in each of the lanes cut with I and BglII, it was revealed that the human p26 gene was present in one copy per haploid cell [FIG. 7].

[0076]

Example 6 Determination of Chromosome Number of Human p26 Gene DNA obtained from hybrid cells containing various human chromosomes was treated with BamHI.
The membrane filter that has been fixed with the hybridization buffer solution [0.5M
Sodium phosphate (pH7.4), 7% SDS, 1% BSA, 1mM EDT
In A], prehybridization was performed at 65 ° C. On the other hand, BanII-Bpu1102I 1 of cDNA (pTS855) encoding human p26 protein as a probe
The 67 bp cDNA fragment was labeled with [α- ³² P] dCTP and a random primer labeling kit (Amersham). Hybridization buffer containing labeled probe [0.5M sodium phosphate (pH7.4), 7% SD
S, 1% BSA, 1 mM EDTA] at 65 ° C. for 16 hours. The filter is finally 0.
After washing in 1 × SSC, 0.1% SDS solution at 50 ° C., an autoradiogram was taken to detect a band hybridizing with the probe. As a result, the male human genome DN
A, including female human genomic DNA, human chromosome 10 s
It was revealed that the human p26 gene is present on chromosome 10 from the fact that one hybridizing band was observed only in the lane of DNA taken from the omatic-hybrid cell, and therefore, it was revealed that the human p26 gene exists on chromosome 10.

[0077]

Example 7 Construction of expression plasmid for rat p26 protein in E. coli In order to express rat p26 protein in E. coli in large amounts, rat p26 protein was first expressed in the form of a fusion protein with β-galactosidase, and then expressed in rat. p2
It was considered to cut out only the 6-protein portion. PCR was carried out using the plasmid pTS822 containing the rat p26 cDNA fragment obtained in Example 1 as a template and two primers designed to contain only the translation region excluding methionine at the initiation codon. Among the primers used at this time, the forward primer was Sal I site followed by
4 of Ile-Glu-Gly-Arg which is the recognition site of blood coagulation factor X
It has a nucleotide sequence corresponding to an amino acid and is 2 in rat p26.
50th having a nucleotide sequence corresponding to the 10th amino acid: arginine from the 10th amino acid: phenylalanine
mer (SEQ ID NO: 12), the reverse primer is the codon AGA → CGT of the 177th arginine in the nucleotide sequence from the 173rd arginine to the termination codon TAA,
It is a 46mer in which the codon of the 181th threonine was changed so that ACA → ACC, and another stop codon TGA was introduced next to the stop codon TAA to have a HindIII site at the end (SEQ ID NO: : 13). After double-digesting the DNA fragment obtained by PCR with Sal I and Hind III, p
It was introduced into the SalI-HindIII site of UC119 (pTS83
2), the base sequence of the DNA fragment obtained by PCR was confirmed using [α- ³² P] dCTP by a usual method. Next, this fragment was constructed into a β-galactosidase fusion protein expression vector pUR290 having a lac promoter.
Β-galactosidase-introduced at Sal I-Hind III site
The expression plasmid pTS833 of rat p26 protein fusion protein was obtained [FIG. 9].

Next, an expression vector was constructed for direct expression of rat p26 protein in E. coli. PCR was performed using the plasmid pTS822 containing the novel cDNA fragment obtained in Example 1 as a template and two primers designed to include only the N-terminal portion of the translation region. Among the primers used in this case, the forward primer was the XbaI and NdeI sites (corresponding to methionine at the start codon), and the base sequence corresponding to the 10th amino acid: serine was changed to the optimal codon of E. coli. 43me
r (SEQ ID NO: 14), the reverse primer is 7
It is a 27mer having a nucleotide sequence corresponding to the third alanine to the 82nd aspartic acid (SEQ ID NO: 1).
5). The DNA fragment obtained here was double-digested with XbaI and NheI, and then XbaI and Nh of pTS832 constructed when the fusion protein with β-galactosidase was expressed earlier.
After inserting into eI (pTS841), the nucleotide sequence of the DNA portion obtained by PCR was confirmed by the usual method using [α- ³² P] dCTP. Next, pTS841 is Nd
The fragment double-digested with eI and HindIII was introduced into the NdeI-HindIII site of the expression vector pRSETB having the T7 promoter, and the rat p26 protein expression plasmid pTS8
42 (FIG. 10) was obtained.

[0079]

Example 8 Construction of Expression Vector of Rat p26 Protein in Animal Cells An expression vector for transiently expressing rat p26 protein in animal cells was constructed. First, pTS823 No containing the rat p26 cDNA fragment obtained in Example 1
The upstream region from the tI site was double-digested with restriction enzymes XbaI and NotI.
Synthetic DNA with restriction enzyme sites for dIII and SfiI
(SEQ ID NOs: 16 and 17) were introduced and pTS825
I got This pTS825 is a restriction enzyme HindIII, Aor51HI
Double digested with the resulting 780 bp fragment of pcDNA
This was introduced into HindIII and EcoRI sites to obtain a transient expression vector pTS829 in animal cells.

[0080]

Example 9 Recombinant rat p from E. coli expressing the β-galactosidase-rat p26 fusion protein
Purification of 26 protein and production of anti-rat p26 protein antibody using the protein 50 Escherichia coli JM109 strain transformed with the β-galactosidase-rat p26 fusion protein expression plasmid pTS833 obtained in Example 7 was used.
It was cultured at 37 ° C. in an LB medium containing μg / ml Biccillin (Meiji Seika), and when OD ₆₀₀ ≈0.6, isopropyl-β-D-thiogalactopyranoside (IPT
G) The expression was induced by adding 1 mM (final concentration), and after continuing the culture for 6 hours, the bacterial cells were collected. The cells were disrupted with lysozyme and ultrasonic waves and then subjected to ultracentrifugation to obtain a soluble fraction. This soluble fraction was applied to an anti-β-galactosidase monoclonal antibody column (Promega) to purify the β-galactosidase-rat p26 fusion protein. Next, purified β-galactosidase-rat p2
The 6 fusion protein was treated with activated blood coagulation factor X (New England Biolabs) to excise rat p26 protein. The excised rat p26 protein was sequentially subjected to cation exchange column (S-sepharose FF;
Pharmacia), hydrophobic column (Phenyl-superose;
(Pharmacia), a hydroxyapatite column (Mitsui Toatsu Kagaku), and a cation exchange column (MONO S; Pharmacia) were used for SDS-PAGE / Coomassie brilliant blue staining to obtain a nearly single purification (FIG. 11).

The recombinant rat p26 protein thus obtained was immunized with New Zealand White Rabbit (Kitayama Labes) to prepare anti-rat p26 protein serum. For the first immunization, 50 μg of recombinant rat p26 protein was mixed with Freund's complete adjuvant (Difco), and then 50 μg from the second to the fifth.
Recombinant rat p26 protein was mixed with Freund's incomplete adjuvant (Difco), and subcutaneous injection was performed 5 times in total. Using the obtained antiserum, β-galactosidase-rat p26 fusion protein, cleaved from the fusion protein with active blood coagulation factor X (New England Biolabs), and directly purified in rat p26 protein and E. coli When Western blotting was performed on the rat p26 protein expressed in the expression system, it was found that the obtained antiserum specifically recognizes these proteins [FIG. 12]. In Western blotting, a sample suspended in a sample buffer is first subjected to SDS-PAGE, and a semi-dried lot system (Biometra)
PVDF membrane (Trans-Blot Transfer Memb
rane, Bio-Rad). 5% of this membrane
After blocking with BSA and treatment with antiserum diluted 200 times, color was developed by colloidal gold staining (Amersham). β-galactosidase-rat p26
The difference in the molecular weight between the rat p26 protein purified from the fusion protein and the rat p26 protein expressed in the direct expression system in E. coli is due to the initiation codon methionine of the rat p26 protein expressed in the direct expression system in E. coli. It is considered to be due to addition.

[0082]

Example 10 Purification of recombinant rat p26 protein expressed in Escherichia coli in a direct expression system and production of anti-rat p26 protein antibody using the protein The rat p26 protein expression plasmid pTS842 obtained in Example 7 was used. Escherichia coli transformed with
a coli) BL21 (DE3) / pLysS strain with 1.0M sorbitol (Wako Pure Chemical Industries), 2.5 mM betaine (Wako Pure Chemical Industries), 50 μg / ml Vicillin (Meiji Seika), 25 μg /
ml was cultured in LB medium containing chloramphenicol (Wako Pure Chemical Industries) at 20 ° C, and when OD ₆₀₀ ≈ 0.6, IP
Expression was induced by the addition of 0.4 mM TG (final concentration). After inducing expression, continue culturing for 4 hours, then collect the cells and
The cells were suspended in a buffer containing mM MES (pH 6.0), 10 mM EDTA, 1 mM APM SF. The suspension was freeze-thawed, ultrasonically disrupted to disrupt the cells, and then ultracentrifuged to prepare a soluble fraction. Soluble fractions obtained here are sequentially cation exchange column (SP-sepharose FF; Pharmacia), hydrophobic column (Phenyl-Sepharose HP; Pharmacia), fluoroapatite column (Pentax), cation exchange column. (MONO S; Pharmacia) was used to purify the recombinant rat p26 protein by SDS-PAGE / Coomassie Brilliant Blue staining to almost a single unit [FIG. 1].
3]. N of purified recombinant rat p26 protein
The terminal amino acid sequence and the amino acid sequence of the fragment peptide obtained by cleaving the recombinant rat p26 protein with various endopeptidases (eg, cyanogen bromide, etc.) were combined by mass spectrometry and Edman degradation sequence analysis. When determined, it was exactly the same as the amino acid sequence predicted from DNA.

The recombinant rat p26 protein thus obtained was immunized with New Zealand White Rabbit (Kitayama Labes) to prepare anti-rat p26 protein serum. Immunization should be performed by subcutaneously injecting 200 μg of recombinant rat p26 protein with Freund's complete adjuvant for the first time and 100 μg of recombinant rat p26 protein with Freund's incomplete adjuvant for the second to fifth doses. Was carried out 5 times in total. This antiserum was used as an antigen column (recombinant rat p26
It was purified using protein-bound CNBr-Sepharose CL-4B (Pharmacia) to purify an antigen-specific anti-rat p26 protein antibody. Western blotting was performed on COS-7 cells transfected with pTS829 and pcDNA obtained in Example 5 using the purified antibody and serum collected from a rabbit before immunization. As a result, only about 26 kDa was obtained by using purified anti-rat p26 protein antibody against CTS-7 cells transfected with pTS829.
A specific band was obtained at this position, indicating that this antibody specifically recognizes rat p26 protein [Fig. 14]. Therefore, using this antibody, 8 weeks old Sprague
Western blotting was performed to examine the distribution of p26 protein in the tissues of Dawley rats (Japan Charles River), and expression of p26 protein was confirmed mainly in the hippocampus, thymus, and spleen [Fig. 15].
In Western blotting, a sample suspended in a sample buffer was first subjected to SDS-PAGE, and transferred to a nitrocellulose membrane (Schleicher & Schuell) by a semi-driving system (Biometra). This membrane was blocked with 5% BSA, treated with 200 times diluted antiserum, and then detected by ECL Western blotting system (Amersham).

[0084]

Example 11 Search for Human Cultured Cells Expressing p26 Protein Western human blotting was carried out using the anti-p26 antibody obtained in Example 10 to search for human culture cells necessary for examining the function of p26 protein. I went and looked up. Example 10
As a result of Western blotting of various hematopoietic cells and glial cells in the same manner as described above, expression of HL-60 and THP-1 in the hematopoietic cells was low, and human T-cell leukemia cells (MOLT -4, Jurkat, CCRF-HSB-2, H
A remarkable expression of p26 protein was observed in 9) [Fig. 16]. Next, using this antibody, human T-cell leukemia cells
An attempt was made to immunoprecipitate human p26 protein from Jurkat. Human T-cell leukemia cells Jurkat cultured in RPMI 1640 medium + 10% FBS medium (Hyklon) to confluence (about 2.4 × 10 ⁶ cells / ml) were subcultured in the same medium to 70% confluence the day before. . The next day, after washing the cells with RPMI1640 medium without methionine, RPMI1640 medium without methionine + 10% dialyzed FBS
After changing the medium to (Hyklon), it was spread on a 6-well plate and [ ³⁵ S] -methionine (Amersham, 10 mCi / m).
l) was added to a final concentration of 100 μCi / ml, and CO ₂ in
Cultured in a cubator for 3 hours. After washing the metabolically labeled cells with PBS, TSE buffer [50 mM
Tris-hydrochloric acid (pH 8.0), 0.1% Softes-12 (Lion), 1 mM EDTA, 150 mM sodium chloride].
The supernatant obtained by centrifugation of this was added to the anti-p2 obtained in Example 10.
6 Protein A-Sepharose CL-4B conjugated with purified antibody,
Or Protein A- Sepharo to which antibody before immunization was bound
se CL-4B was added and reacted at 4 ° C. for 2 hours. Then, Protein A-Sepharose CL-4B to which each antibody was bound was washed with TSE buffer, suspended in a sample buffer, and subjected to SDS-PAGE. After the electrophoresis, the gel was dried and an autoradiogram was taken using BAS2000 (Fuji Film). As a result, a band was specifically obtained at about 26 kDa only in the lane of Protein A-Sepharose CL-4B conjugated with the anti-rat p26 purified antibody, and it was revealed that this antibody can specifically immunoprecipitate human p26 protein. [Fig. 17].

[0085]

Example 12 Subcellular localization of p26 protein Human T-cell leukemia cells Jurkat 4 × 10 ⁷ cells were treated with 0.25M.
Low-concentration buffer containing sucrose (Wako Pure Chemical) [10 mM MES
(pH 6.0), 1 mM EDTA] and homogenized, followed by stepwise centrifugation, and each fraction obtained by fractionating intracellular organelles into 5 steps was subjected to Western blotting in the same manner as in Example 10. It was confirmed that the p26 protein was localized in the cytoplasm [Fig. 18].

[0086]

[Example 13] In vitro phosphorylation of p26 protein p26 having an amino acid site presumed to be phosphorylated
To investigate whether the protein is actually phosphorylated in the cell, human T-cell leukemia cell Jurkat cell [
^It was metabolically labeled with ³² P] -orthophosphate and immunoprecipitated using an anti-rat p26 protein antibody. RP
Human T-cell leukemia cells Jurkat cultured in MI 1640 medium + 10% FBS medium (Hyklon) to confluence (approximately 2.4 x 10 ⁶ cells / ml) were continued in the same medium to the previous day so that they were 7.0% confluent. Substitute On the next day, the cells were washed with RPMI1640 medium containing no phosphate, and the medium was replaced with RPMI1640 medium containing no phosphate + 10% dialyzed FBS (Hyklon), and then spread on a 6-well plate, and then [ ³² P] -Orthophosphoric acid (DuPont / NEN, 150mCi / ml) final concentration 1mCi / m
The cells were added so that the amount became 1 and the cells were cultured in a CO ₂ incubator for 4 hours. Phorbol 12-Myristate 13-Acetate (TPA)
When adding (Wako Pure Chemicals) to stimulate cells, [ ³² P]-
After adding orthophosphoric acid and culturing for 2 hours, final concentration 10n
TPA was added so that the concentration would be g / ml, and the culture was continued for another 2 hours. After washing the metabolically labeled cells with PBS, TSE buffer [50 mM Tris-hydrochloric acid (pH 8.0), 0.
1% Softes-12 (Lion), 1 mM EDTA, 150 mM sodium chloride]. The supernatant obtained by centrifuging this was bound with the anti-rat p26 purified antibody obtained in Example 10 pr
oteinA-Sepharose CL-4B was added and reacted at 4 ° C. for 2 hours. And Prot conjugated with anti-rat p26 purified antibody
After washing ein A-Sepharose CL-4B with TSE buffer,
The cells were suspended in sample buffer and subjected to SDS-PAGE. After the electrophoresis, the gel was dried and an autoradiogram was taken using BAS2000 (Fuji Film). As a result, it was revealed that in human T-cell leukemia cells Jurkat, the p26 protein was phosphorylated even in the unstimulated state. Also, T
Since phosphorylation proceeds further by PA stimulation, p
26 proteins were found to be phosphorylated by protein kinase C [Fig. 19].

[0087]

[Example 14] Age-related changes in rat p26 protein expression in the liver In order to observe age-related changes in rat p26 protein expression in the liver, Northern blotting and Western blotting were carried out to encode p26 protein m.
We saw changes in both RNA and p26 protein. First, the total R from the 17.5 day fetus, newborn baby, two weeks old, eight weeks old liver of Sprague Dawley rat (Charles River Japan, Inc.) was measured by the guanidine thiocyanate method.
After fractionating NA, this total RNA was applied to an oligo (dT) span column (Pharmacia) to prepare poly (A) ⁺ RNA. ² μg of this poly (A) ⁺ RNA was subjected to 1.5% formalin-denaturing agarose gel electrophoresis, and then blotted on a nylon membrane filter (Biodyne B, Japan Pall Co.) for 16 hours by capillary blotting. This nylon membrane filter was fixed by blotting RNA by UV treatment, and then hybridization buffer [50% formamide, 5 x SSP
Prehybridization was performed at 42 ° C. in E, 5 × Denhardt's solution, 0.1% SDS, 100 μg / ml heat-denatured salmon sperm DNA]. As a probe, SacII-Aor51HI 77 of cDNA (pTS834) encoding rat p26
The 5 bp cDNA fragment was labeled with [α- ³² P] dCTP and a random primer-labeling kit (Amersham). Hybridization is carried out using a hybridization buffer containing a labeled probe [50% formamide, 5xSSPE, 5x Denhardt's solution, 0.1% SDS, 100 µg / ml.
Hybridization in heat-denatured salmon sperm DNA] at 42 ° C. for 16 hours. The filter finally
After washing in 0.1 × SSC, 0.1% SDS solution at 60 ° C., an autoradiogram was taken to detect a band hybridizing with the probe. As a result, the mRNA coding for p26 protein was most abundantly expressed in the 17.5 day fetus, and the expression level gradually decreased with development, and the expression was slightly observed in the second and eighth weeks of life. It was only [Fig. 20]. Next, the liver of the same rat was removed with time, stained with Coomassie Brilliant Blue and Example 10
Western blotting was carried out using an anti-rat p26 protein antibody in the same manner as described in 1. above, and changes in protein amount were observed. As a result, the amount of p26 protein decreased with age, and almost no expression was observed in 8-week-old liver [Fig. 21]. This is the mR encoding p26 protein by Northern blot analysis.
Correlation with the expression pattern of NA showed that a large amount of p26 protein was expressed in the fetal period when the liver functions as a hematopoietic organ.

[0088]

Example 15 In vitro phosphorylation of rat p26 protein by casein kinase II p26 having an amino acid site presumed to be phosphorylated
To confirm whether the protein is phosphorylated by casein kinase II, phosphorylation by casein kinase II was examined in vitro. The recombinant rat p26 protein obtained in Example 10 contained 238 pmol of 100 n
MATP, 6.66 pmol [γ- ³² P] ATP (Dupon
t / NEN, 3000Ci / mmol, 10mCi / ml), recombinant casein kinase II 1000units (New England Biolabs (UK))
20 mM Tris (pH 7.5), 50 mM
In KCl and 10 mM MgCl ₂ solution,
The reaction was carried out at 30 ° C. for 2.5, 10, 30, 60 and 120 minutes. The reaction was stopped by suspending it in sample buffer and subjecting the reaction to SDS-PAGE.
After the electrophoresis, it was stained with Coomassie Brilliant Blue to confirm that approximately the same amount of p26 protein was loaded in each lane, and then the gel was dried, and autoradiogram was taken to confirm phosphorylation of p26 protein. as a result,
As shown in [FIG. 22], the band of the autoradiogram became darker as the reaction time elapsed, indicating that rat p26 protein serves as a substrate for casein kinase II in vitro.

[0089]

INDUSTRIAL APPLICABILITY The p26 protein of the present invention is useful as a reagent for screening a compound or its salt having an activity of promoting or inhibiting protein kinase and an activity of inhibiting dephosphorylation enzyme. Further, the p26 protein of the present invention and the DNA encoding the same
Is useful as a prophylactic / therapeutic agent for diseases associated with the hematopoietic system such as aplastic anemia, acute myelogenous leukemia, and T cell leukemia. Furthermore, since the antibody against the p26 protein of the present invention can specifically recognize the p26 protein, it can be used for quantification of the p26 protein in a test solution.

[0090]

[Sequence list]

[SEQ ID NO: 1] Sequence length: 181 Sequence type: Amino acid Topology: Linear Sequence type: Peptide Sequence Met Phe Thr Arg Ala Val Ser Arg Leu Ser Arg Lys Arg Pro Pro Ser 1 5 10 15 Asp Ile His Asp Gly Asp Gly Ser Ser Ser Ser Gly His Gln Ser Leu 20 25 30 Lys Ser Thr Ala Lys Trp Ala Ser Ser Leu Glu Asn Leu Leu Glu Asp 35 40 45 Pro Glu Gly Val Lys Arg Phe Arg Glu Phe Leu Lys Lys Glu Phe Ser 50 55 60 Glu Glu Asn Val Leu Phe Trp Leu Ala Cys Glu Asp Phe Lys Lys Thr 65 70 75 80 Glu Asp Lys Lys Gln Met Gln Glu Lys Ala Lys Lys Ile Tyr Met Thr 85 90 95 Phe Leu Ser Asn Lys Ala Ser Ser Gln Val Asn Val Glu Gly Gln Ser 100 105 110 Arg Leu Thr Glu Lys Ile Leu Glu Glu Pro His Pro Leu Met Phe Gln 115 120 125 Lys Leu Gln Asp Gln Ile Phe Asn Leu Met Lys Tyr Asp Ser Tyr Ser 130 135 140 Arg Phe Leu Lys Ser Asp Leu Phe Leu Lys His Arg Arg Thr Glu Glu 145 150 155 160 Glu Glu Glu Asp Pro Pro Asp Ala Gln Thr Ala Ala Lys Arg Ala Ser 165 170 175 Arg Ile Tyr Asn Thr 180

[0091]

[SEQ ID NO: 2] Sequence length: 181 Sequence type: Amino acid Topology: Linear Sequence type: Peptide sequence Met Phe Asn Arg Ala Val Ser Arg Leu Ser Arg Lys Arg Pro Pro Ser 1 5 10 15 Asp Ile His Asp Ser Asp Gly Ser Ser Ser Ser Ser His Gln Ser Leu 20 25 30 Lys Ser Thr Ala Lys Trp Ala Ala Ser Leu Glu Asn Leu Leu Glu Asp 35 40 45 Pro Glu Gly Val Lys Arg Phe Arg Glu Phe Leu Lys Lys Glu Phe Ser 50 55 60 Glu Glu Asn Val Leu Phe Trp Leu Ala Cys Glu Asp Phe Lys Lys Met 65 70 75 80 Gln Asp Lys Thr Gln Met Gln Glu Lys Ala Lys Glu Ile Tyr Met Thr 85 90 95 Phe Leu Ser Ser Lys Ala Ser Ser Gln Val Asn Val Glu Gly Gln Ser 100 105 110 Arg Leu Asn Glu Lys Ile Leu Glu Glu Pro His Pro Leu Met Phe Gln 115 120 125 Lys Leu Gln Asp Gln Ile Phe Asn Leu Met Lys Tyr Asp Ser Tyr Ser 130 135 140 Arg Phe Leu Lys Ser Asp Leu Phe Leu Lys His Lys Arg Thr Glu Glu 145 150 155 160 Glu Glu Glu Asp Leu Pro Asp Ala Gln Thr Ala Ala Lys Arg Ala Ser 165 170 175 Arg Ile Tyr Asn Thr 180

[0092]

[SEQ ID NO: 3] Sequence length: 181 Sequence type: Amino acid Topology: Linear Sequence type: Peptide sequence Met Phe Thr Arg Ala Val Ser Arg Leu Ser Arg Lys Arg Pro Pro Ser 1 5 10 15 Asp Ile His Asp Gly Asp Gly Ser Ser Ser Ser Gly His Gln Ser Leu 20 25 30 Lys Ser Thr Ala Lys Trp Ala Ser Ser Leu Glu Asn Leu Leu Glu Asp 35 40 45 Pro Glu Gly Val Gln Arg Phe Arg Glu Phe Leu Lys Lys Glu Phe Ser 50 55 60 Glu Glu Asn Val Leu Phe Trp Leu Ala Cys Glu Asp Phe Lys Lys Thr 65 70 75 80 Glu Asp Arg Lys Gln Met Gln Glu Lys Ala Lys Lys Ile Tyr Met Thr 85 90 95 Phe Leu Ser Asn Lys Ala Ser Ser Gln Val Asn Val Glu Gly Gln Ser 100 105 110 Arg Leu Thr Glu Lys Ile Leu Glu Glu Pro His Pro Leu Met Phe Gln 115 120 125 Lys Leu Gln Asp Gln Ile Phe Asn Leu Met Lys Tyr Asp Ser Tyr Ser 130 135 140 Arg Phe Leu Lys Ser Asp Leu Phe Leu Lys Pro Arg Arg Thr Glu Glu 145 150 155 160 Glu Glu Glu Glu Pro Pro Asp Ala Gln Thr Ala Ala Lys Arg Ala Ser 165 170 175 Arg Ile Tyr Asn Thr 180

[0093]

[SEQ ID NO: 4] Sequence length: 543 Sequence type: Nucleic acid Number of strands: Double stranded Topology: Linear Sequence type: cDNA Characterization method: S sequence ATGTTCACCC GCGCCGTGAG CCGACTGAGC AGGAAGCGGC CGCCGTCTGA TATCCACGAC 60 GGCGATGGGA GCTCAAGCAG CGGCCACCAG AGCCTCAAGA GCACAGCCAA GTGGGCGTCC 120 TCCCTGGAGA ATCTTCTGGA AGATCCAGAG GGCGTGAAGA GATTCAGGGA ATTTCTGAAA 180 AAGGAATTCA GTGAGGAAAA CGTCTTGTTT TGGCTAGCGT GTGAAGATTT CAAGAAAACG 240 GAGGACAAGA AGCAGATGCA GGAAAAGGCC AAGAAGATCT ACATGACCTT CCTGTCCAAT 300 AAGGCCTCTT CACAAGTCAA TGTGGAGGGG CAGTCTCGGC TCACTGAAAA GATTCTGGAA 360 GAACCACACC CTCTGATGTT CCAAAAGCTC CAGGACCAGA TCTTCAATCT CATGAAGTAT 420 GACAGCTACA GCCGCTTCTT GAAGTCTGAC TTGTTTCTGA AGCACCGGCG GACTGAGGAA 480 GAGGAAGAGG ATCCTCCGGA CGCTCAAACT GCAGCTAAGC GAGCTTCCAG AATCTACAAC 540 ACA 543

[0094]

[SEQ ID NO: 5] Sequence length: 543 Sequence type: Nucleic acid Number of strands: Double strand Topology: Linear Sequence type: cDNA Characterization method: S sequence ATGTTCAACC GCGCCGTGAG CCGGCTGAGC AGGAAGCGGC CGCCGTCAGA CATCCACGAC 60 AGCGATGGCA GTTCCAGCAG CAGCCACCAG AGCCTCAAGA GCACAGCCAA ATGGGCGGCA 120 TCCCTGGAGA ATCTGCTGGA AGACCCAGAA GGCGTGAAAA GATTTAGGGA ATTTTTAAAA 180 AAGGAATTCA GTGAAGAAAA TGTTTTGTTT TGGCTAGCAT GTGAAGATTT TAAGAAAATG 240 CAAGATAAGA CGCAGATGCA GGAAAAGGCA AAGGAGATCT ACATGACCTT TCTGTCCAGC 300 AAGGCCTCAT CACAGGTCAA CGTGGAGGGG CAGTCTCGGC TCAACGAGAA GATCCTGGAA 360 GAACCGCACC CTCTGATGTT CCAGAAACTC CAGGACCAGA TCTTTAATCT CATGAAGTAC 420 GACAGCTACA GCCGCTTCTT AAAGTCTGAC TTGTTTTTAA AACACAAGCG AACCGAGGAA 480 GAGGAAGAAG ATTTGCCTGA TGCTCAAACT GCAGCTAAAA GAGCTTCCAG AATTTATAAC 540 ACA 543

[0095]

[SEQ ID NO: 6] Sequence length: 543 Sequence type: Nucleic acid Number of strands: Double strand Topology: Linear Sequence type: cDNA Characterization method: S sequence ATGTTCACCC GCGCCGTGAG CCGACTGAGC AGGAAGCGGC CGCCGTCTGA TATCCATGAC 60 GGAGATGGGA GCTCAAGCAG CGGCCACCAG AGCCTTAAGA GCACAGCCAA GTGGGCATCC 120 TCCCTGGAGA ATCTTCTGGA AGACCCAGAA GGGGTGCAGA GATTCAGGGA GTTTCTGAAG 180 AAGGAATTCA GCGAAGAGAA TGTCTTGTTT TGGCTAGCGT GTGAAGATTT CAAGAAAACG 240 GAGGACAGGA AGCAGATGCA GGAAAAGGCC AAGGAGATCT ACATGACCTT CCTGTCCAAT 300 AAGGCCTCTT CACAAGTCAA CGTGGAGGGG CAGTCTCGGC TCACTGAAAA GATTCTGGAA 360 GAGCCACACC CTCTGATGTT CCAAAAGCTC CAGGACCAGA TCTTCAATCT CATGAAGTAT 420 GACAGCTACA GCCGCTTCTT GAAGTCTGAC TTGTTTCTGA AACCCAAGCG AACTGAGGAA 480 GAGGAAGAAG AGCCCCCGGA TGCTCAGACC GCAGCTAAGC GAGCCTCCAG AATTTACAAC 540 ACA 543

[0096]

[SEQ ID NO: 7] Sequence length: 51 Sequence type: Nucleic acid Number of strands: Single strand Topology: Linear Sequence type: Synthetic DNA Sequence CACTCGGACG GCATCTTCAC TGACAGCTAC AGCCGCTACC GGAAGCAAAT G 51

[0097]

[SEQ ID NO: 8] Sequence length: 51 Sequence type: Nucleic acid Number of strands: Single strand Topology: Linear Sequence type: Synthetic DNA sequence TAGGACAGCC GCCAAGTATT TCTTAACAGC CATTTGCTTC CGGTAGCGGC T 51

[0098]

[SEQ ID NO: 9] Sequence length: 24 Sequence type: Nucleic acid Number of strands: Single strand Topology: Linear Sequence type: Synthetic DNA Sequence AGATTCTCCA GGGAGGACGC CCAC 24

[0099]

[SEQ ID NO: 10] Sequence length: 24 Sequence type: Nucleic acid Number of strands: Single strand Topology: Linear Sequence type: Synthetic DNA Sequence CTGTGCTCTT GAGGCTCTGG TGGC 24

[0100]

[SEQ ID NO: 11] Sequence length: 37 Sequence type: Nucleic acid Number of strands: Single strand Topology: Linear Sequence type: Synthetic DNA Sequence CTGACTCGAG GTCGACAAGC TTTTTTTTTT TTTTTTT 37

[0101]

[SEQ ID NO: 12] Sequence length: 50 Sequence type: Nucleic acid Number of strands: Single strand Topology: Linear Sequence type: Synthetic DNA sequence ATCCGTCGAC CATCGAAGGT CGTTTCACCC GCGCTGTGAG CCGACTGAGC 50

[0102]

[SEQ ID NO: 13] Sequence length: 46 Sequence type: Nucleic acid Number of strands: Single strand Topology: Linear Sequence type: Synthetic DNA Sequence GCGAGCTTCC CGTATCTACA ACACCTAATG ATC
ATAAGCT TGGGAC 46

[0103]

[SEQ ID NO: 14] Sequence length: 43 Sequence type: Nucleic acid Number of strands: Single strand Topology: Linear Sequence type: Synthetic DNA Sequence CTGGTCTAGA CATATGTTA CCCCGCTGCTGT GAG
CCGTCTG AGC 43

[0104]

[SEQ ID NO: 15] Sequence length: 30 Sequence type: Nucleic acid Number of strands: Single strand Topology: Linear Sequence type: Synthetic DNA Sequence GCGTGTGAAG ATTTCAAGAA AACGGAGGAC 30

[0105]

[SEQ ID NO: 16] Sequence length: 63 Sequence type: Nucleic acid Number of strands: Single strand Topology: Linear Sequence type: Synthetic DNA Sequence CTAGAAAGCT TACGGCCAAG TCGGCCATGT TCA
CCCGCGC CGTGAGCCGA CTGAGCAGGA 60 AGC
63

[0106]

[SEQ ID NO: 17] Sequence length: 63 Sequence type: Nucleic acid Number of strands: Single strand Topology: Linear Sequence type: Synthetic DNA Sequence GGCCGCTTCC TGCTCAGTCG GCTCACGGGCG CGG
GTGAACA TGGCCGAACTT GGCCGTAAGC 60 TTT
63

[0107]

[Brief description of drawings]

FIG. 1 shows the nucleotide sequence of DNA encoding rat p26 protein and the amino acid sequence of rat p26 protein encoded thereby.

FIG. 2 shows the nucleotide sequence of DNA encoding human p26 protein and the amino acid sequence of human p26 protein encoded thereby.

FIG. 3 shows the nucleotide sequence of DNA encoding mouse p26 protein and the amino acid sequence of human p26 protein encoded thereby.

FIG. 4 shows a comparison table of amino acid sequences of human p26 protein (HUMAN p26), rat p26 protein (RAT p26) and mouse p26 protein (MOUSE p26).
The shaded areas indicate homologous amino acids.

FIG. 5: mRNA encoding rat p26 protein
The result of having examined the expression level in each part of the rat brain by Northern hybridization is shown. Olfactory bulb is olfactory bulb, Amygdala is amygdala, Basal gangria is basal ganglia, Hippocampus is hippocampus, Thalamus is thalamus, Hypot
halamus is the hypothalamus, Cerebral cortex is the cerebral cortex,
Medulla is the medulla oblongata, Cerebellum is the cerebellum, Spinal cord is the spinal cord, and Pituitary is the pituitary gland. Left number (k
b) shows the size of the RNA molecular weight marker. G3PD
H is glyceraldehyde 3-, which was taken as an internal marker.
1 shows a pattern of hybridization of phosphate dehydrogenase.

[Fig. 6] Fig. 6 shows the results of examining the expression level of mRNA encoding human p26 protein in each human tissue by Northern hybridization. Heart is the heart, Brain is the brain, Placenta is the placenta, Lung is the lungs, Liver is the liver, S
keltal muscle is skeletal muscle, Kindney is kidney, Pancreas
Is the pancreas, Spleen is the spleen, Thymus is the thymus, Prostate
Is prostate, Testis is testis, Ovary is ovary, Small Int
estine is the small intestine, Colon is the large intestine, Peripheral Blood is the peripheral blood, Fetal Heart is the fetal heart, Fetal Brain is the fetal brain, Fetal Lung is the fetal lung, Fetal Liver is the fetal liver, and Fetal Kindney is the fetal kidney. Indicates. The number (kb) on the left side indicates the size of the RNA molecular weight marker.

FIG. 7 shows the results of Southern hybridization using human genomic DNA and a cDNA fragment encoding human p26 protein. Each lane shows the type of restriction enzyme that cleaved human genomic DNA. The number on the left (kbp) indicates the size of the DNA molecular weight marker.

FIG. 8 shows the results of determination of the chromosome number in which the human p26 gene is present by performing Southern hybridization on DNA obtained from hybrid cells having various human chromosomes using a cDNA fragment encoding the human p26 protein. Show. Malehuman genomic DNA means female human genomic DNA, Male human genomic DNA means male human genome D
NA, Mouse genomic DNA, Ham genomic DNA
ster genomic DNA is hamster genomic DNA
some1 is chromosome 1, chromosome2 is chromosome 2,
chromosome3 is chromosome 3, chromosome4 is chromosome 4, chromosome5 is chromosome 5, and chromosome6 is 6
Chromosome 7 and chromosome 7 chromosome 7
8 is chromosome 8, chromosome 9 is chromosome 9, chro
mosome10 is chromosome 10, chromosome11 is chromosome 11, chromosome12 is chromosome 12, chromoso
me13 is chromosome 13, chromosome14 is chromosome 14, chromosome15 is chromosome 15, chromosome1
6 is chromosome 16, chromosome 17 is chromosome 17, chromosome 18 is chromosome 18, chromosome 19
Is chromosome 19, chromosome 20 is chromosome 20,
chromosome21 is chromosome 21, chromosome22 is 2
Chromosome X, chromosome X X chromosome,
Y represents the Y chromosome. The number (kb) on the left side indicates the size of the molecular weight marker.

FIG. 9 shows a construction diagram of the expression plasmid pTS833 of β-galactosidase-rat p26 fusion protein.
lacZ is a β-galactosidase gene,
26 is rat p26 protein DNA, lac promoter
Indicates the lac promoter, and Amp ^r indicates the ampicillin resistance gene.

FIG. 10: Expression plasmid p of rat p26 protein
A construction drawing of TS842 is shown. Rat p26 is rat p
26 protein DNA, T7 promoter indicates T7 promoter, and Amp ^r indicates ampicillin resistance gene.

FIG. 11 shows the results of SDS-PAGE / Coomassie Brilliant Blue staining at each purification step of the rat p26 protein obtained in Example 9. The horizontal axis shows the protein fraction at each purification step, and the vertical axis shows the size of the molecular weight marker of the protein in KDa. The arrow indicates the band of rat p26 protein.

FIG. 12: Various rat p2 expressed in Escherichia coli using the anti-rat p26 protein serum obtained in Example 9
The results of Western blotting for 6 proteins are shown. CBBR stain shows the result of Coomassie brilliant blue staining, and Immunoblot shows the result of western blotting. The number on the left (KDa) indicates the size of the molecular weight marker of the protein.

FIG. 13 shows the results of SDS-PAGE / Coomassie Brilliant Blue staining at each purification step of the rat p26 protein obtained in Example 10. The horizontal axis shows the protein fraction at each purification step, and the vertical axis shows the size of the molecular weight marker of the protein. The arrow indicates the band of rat p26 protein.

FIG. 14 shows that the anti-rat p26 protein antibody obtained in Example 10 was subjected to CO in Western blotting.
It is shown that the rat p26 protein expressed in S-7 cells is specifically recognized. COS-7 / pcDNAI is p
COS-7 cells transfected with cDNAI
COS-7 / pTS829 indicates CTS-7 cells transfected with pTS829. Pre-immune indicates a sample before immunoprecipitation, and immune indicates a sample after immunoprecipitation. The number on the left (KDa) indicates the molecular weight marker of the protein.

FIG. 15 shows the results of examining the distribution of p26 protein in each rat tissue using the anti-rat p26 protein antibody obtained in Example 10. brain, hippo
campus is hippocampus, thymus is thymus, spleen is spleen, li
ver indicates the liver and testis indicates the testis. Left number (KD
a) shows a molecular weight marker of the protein.

FIG. 16 shows the results of examining the distribution of p26 protein in various human cells using the anti-rat p26 protein antibody obtained in Example 10. The horizontal axis shows the cell type, and the vertical axis shows the molecular weight of the protein. MOLT-4, Jurka
t, CCRF-HSB-2, H9, HL-60, THP-1, MEG-01, U251, KNS
42 each represents a human cell line.

FIG. 17 shows the results of immunoprecipitation of p26 protein from [ ³⁵ S] methionine-labeled human T-cell leukemia cells Jurkat using the anti-rat p26 protein antibody obtained in Example 10. Pre-immune indicates a sample before immunoprecipitation, and immune indicates a sample after immunoprecipitation. Left number (K
Da) indicates the size of the molecular weight marker of the protein.

FIG. 18 shows the results of examining the intracellular localization of p26 protein using the anti-rat p26 protein antibody obtained in Example 10. Lane 1 is the whole protein of human T cell leukemia cells (whole cell), lane 2 is the homogenate fraction of human T cell leukemia cells.
In lane 3, the homogenate fraction of human T-cell leukemia cells was centrifuged at 900 g to give a sedimentation fraction (900 g pellet (nucl
i)), lane 4 is a sedimentation fraction (5000 g pellet (m) obtained by centrifuging the homogenate fraction of human T cell leukemia cells at 5000 g.
Itochondria)), lane 5 is a sedimentation fraction (8000) obtained by centrifuging a homogenate fraction of human T cell leukemia cells at 8000 g.
g pellet (lysosome)), lane 6 is a sedimentation fraction obtained by centrifugation of a homogenate fraction of human T cell leukemia cells at 10000 g (10000 g pellet (Microsome)), and lane 7 is a homogenate fraction of human T cell leukemia cells at 10000 g. The centrifuged supernatant fraction (10000 g SUP (cytosol)) is shown.

FIG. 19 shows that human T-cell leukemia cells Jurkat were transferred to TPA (Phor
p2 when stimulated with bol 12-Myristate 13-Acetate)
The result of having investigated the phosphorylation of 6 protein is shown. Control
Indicates that TPA treatment is not performed, and TPA-treatment indicates TP
The case where A processing is performed is shown. The number on the left (KDa) indicates the size of the molecular weight marker of the protein.

FIG. 20. mRN encoding rat p26 protein
The result of having examined the age-related change of the expression level in A liver by Northern blotting is shown. 17.5 day embryo is 17.5
Newborn is newborn, 2-week is 2 weeks old, and 8-week is 8 weeks old. The number (Kb) on the left side indicates the size of the RNA molecular weight marker. G3PDH shows the pattern of glyceraldehyde 3-phosphate dehydrogenase hybridization taken as an internal marker.

FIG. 21 shows the results of western blotting examination of age-related changes in the expression level of rat p26 protein in the liver. 17.5day embryo is a new born fetus
Indicates a newborn, 2-week indicates 2 weeks old, and 8-week indicates 8 weeks old. Coomassie Stain shows the results of Coomassie Brilliant Blue staining, and Western Blotting shows the results of Western blotting. The number on the left (KDa) indicates the size of the molecular weight marker of the protein.

FIG. 22: p2 by casein kinase II in vitro
The result of having investigated the phosphorylation of 6 protein is shown. A is [ ³²
P] shows the result of autoradiogram of p26 protein phosphorylated, the upper number shows the reaction time, and the number on the left side of A (KDa) shows the size of the molecular weight marker of the protein. B shows the result of staining the same gel as A with Coomassie Brilliant Blue.

─────────────────────────────────────────────────── ─── Continuation of front page (51) Int.Cl. ⁶ Identification code Internal reference number FI Technical indication C07H 21/04 C12P 21/02 C C07K 14/52 ZNA A61K 37/02 ABB 16/24 ACC C12N 1 / 21 ADV C12P 21/02 AED // (C12N 1/21 C12R 1:19) (C12P 21/02 C12R 1:19)

Claims (18)

[Claims] 1. A protein containing an amino acid sequence identical or substantially identical to the amino acid sequence represented by SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO: 3 or a salt thereof. 2. An amino acid sequence which is substantially the same as the amino acid sequence represented by SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO: 3 has the amino acid sequence represented by SEQ ID NO: 1 at positions 55 to 77 and The protein according to claim 1, which is an amino acid sequence having an amino acid sequence of the 137th to 149th amino acids. 3. An amino acid sequence which is substantially the same as the amino acid sequence represented by SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO: 3, is the 4th to 20th amino acid sequence represented by SEQ ID NO: 1, 22nd-27th, 29th-39th, 41st-52nd, 54th-79th, 85th-9th
1st, 93rd to 99th, 101st to 114th, 116th to 154th, 157th to 163rd and 1st
The protein according to claim 1, which has an amino acid sequence having the 66th to 181st amino acid sequences. 4. A partial peptide of the protein according to claim 1, or a salt thereof. 5. The protein according to claim 1 or claim 4.
DN having a nucleotide sequence encoding the described partial peptide
DNA containing A. 6. The DN according to claim 5, which has the nucleotide sequence represented by SEQ ID NO: 4, SEQ ID NO: 5 or SEQ ID NO: 6.
A. 7. A recombinant vector containing the DNA according to claim 5. 8. A transformant carrying the recombinant vector according to claim 7. 9. The protein or salt thereof according to claim 1, which is obtained by culturing the transformant according to claim 8 to produce and accumulate the protein or salt thereof according to claim 1, and collecting the protein or salt. Manufacturing method. [10] A pharmaceutical comprising the protein according to [1], the partial peptide according to [4], or a salt thereof. [11] A pharmaceutical comprising the DNA according to [5]. 12. The medicine according to claim 10 or 11, which is a therapeutic / preventive agent or immunomodulator for aplastic anemia, acute myelogenous leukemia or T cell leukemia. 13. An antibody against the protein according to claim 1, the partial peptide according to claim 4, or a salt thereof. 14. A method for screening a compound having a phosphatase inhibitory activity or a salt thereof, which comprises using the protein according to claim 1, the partial peptide according to claim 4 or a salt thereof. 15. A kit for screening a compound having a phosphatase inhibitory activity or a salt thereof, containing the protein according to claim 1, the partial peptide according to claim 4, or a salt thereof. 16. A compound having a phosphatase inhibitory activity or a salt thereof obtained by using the screening method according to claim 14 or the screening kit according to claim 15. 17. A compound or a salt thereof, which has an activity of inhibiting the dephosphorylation of the protein of claim 1, which is phosphorylated, the partial peptide of claim 4, or a salt thereof by a dephosphorylation enzyme. Medicine. 18. A method for screening a compound having protein kinase inhibitory activity or a salt thereof, which comprises using the protein according to claim 1, the partial peptide according to claim 4 or a salt thereof.