JP2003079378A - Novel endothelial zinc finger protein ezi and gene thereof - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a novel zinc finger protein (EZI) and the gene thereof. SOLUTION: Mouse and human-originating novel zinc finger protein EZI (endothelial zinc finger protein), the EZI gene encoding the protein, the recombinant vector including the gene, the transformant including the recombinant vector and a method of producing the EZI protein are disclosed.

Description

Detailed Description of the Invention

[0001]

The present invention relates to the mouse and human origin of a novel zinc finger protein EZI (e ndotheli
al zi nc finger protein), which encodes the protein
The present invention relates to an EZI gene, a recombinant vector containing the gene, a transformant containing the recombinant vector, a method for producing an EZI protein, and the like.

[0002]

Oncostatin M (OSM) is a protein secreted by stimulating a macrophage cell line with PMA (phorbol 12-myristate 13-acetate) and initially does not affect normal cells. Identified as a factor that inhibits the growth of tumor cells such as melanoma cell line (A375) [Malik, N. et al .: Mol.Cell.Biol., 9: 2847-
2853 (1989)]. Subsequent studies have revealed that OSM is a multifunctional cytokine with a variety of activities including growth stimulatory activity in Kaposi's sarcoma, endothelial cells and muscle cells.

In the process of embryonic development in mice, hematopoiesis is initiated in the yolk sac of the embryo about 7.5 days after mating. After initiation of hematopoiesis, a period of primitive hematopoiesis in which specific expression of embryonic globin in nucleated red blood cells was observed, and
Reproductive ridge-Mesorenal region [AGM (aorta-gonad-mesonephros)
Region], adult secondary hematopoiesis begins. In this AGM region, it is known that vascular endothelial cells proliferate to form clusters and blood cells are produced by the action of the OSM. However, there are many unclear points about intracellular events triggered by the action of OSM.

[0004]

DISCLOSURE OF THE INVENTION The present invention provides a novel zinc finger protein mouse EZI (mouse E, which is expressed by stimulation with OSM in a cell line derived from the AGM region).
ZI protein) and its human homologue protein (also called human EZI protein), EZI gene encoding the protein, recombinant vector containing the gene, transformant containing the recombinant vector, and method for producing EZI protein Etc. are intended to be provided.

[0005]

[Means for Solving the Problems] As a result of intensive studies to solve the above-mentioned problems, the present inventors have found that the OSM was determined by a cDNA subtraction method using a cell line LO derived from the AGM region.
We identified and isolated a novel gene mouse EZI induced by the human homologue gene (human
We succeeded in demonstrating that the EZI gene was obtained) and completed the present invention. That is, the present invention is the following (1) to (21).

(1) A protein containing an amino acid sequence identical or substantially identical to the amino acid sequence represented by SEQ ID NO: 2 or SEQ ID NO: 9 or a salt thereof. (2) The protein according to (1) or a salt thereof, wherein the protein has a C2H2-type zinc finger domain and has a binding ability to a STAT protein. (3) The STAT protein is STAT3 protein or STAT5
The protein according to the above (2) or a salt thereof, which is a protein.

(4) A partial peptide of the protein according to any one of (1) to (3) above or a salt thereof. (5) The protein according to any one of (1) to (3) above or (4) above
A polynucleotide comprising a base sequence encoding the peptide of. (6) The polynucleotide of (5) above, wherein the polynucleotide is DNA.

(7) A polynucleotide having a nucleotide sequence represented by SEQ ID NO: 1 or SEQ ID NO: 8 or a nucleotide sequence hybridizing with the nucleotide sequence under stringent conditions. (8) The polynucleotide of (7) above, wherein the polynucleotide is DNA. (9) A recombinant vector containing the polynucleotide according to any one of (5) to (8) above.

(10) A transformant containing the recombinant vector according to (9) above. (11) The transformant of (10) is cultured to produce the protein of any of (1) to (3) or the peptide of (4) above.
Accumulate and collect this (1)
To (3) the protein or salt thereof, or the peptide of (4) or a salt thereof. (12) An antibody against the protein or salt thereof according to any one of (1) to (3), or the partial peptide according to (4) or salt thereof.

(13) The antibody according to (1) above with respect to the antibody according to (12) above
~ Any of the proteins of (3) or a salt thereof or the above
(4) a test solution containing the partial peptide or a salt thereof, and the labeled (1) to (3) any of the proteins or salts thereof or the labeled partial peptide of (4) or Characterized in that the salt reacts competitively
A method for quantifying the protein or salt thereof according to any one of (1) to (3) or the partial peptide or salt thereof according to (4) above. (14) A protein or salt thereof according to any one of (1) to (3) or a partial peptide or salt thereof according to (4) above is used, wherein the protein or salt thereof or the partial peptide or thereof A method for screening a compound that inhibits or promotes the binding between a salt and a STAT protein.

(15) The screening method comprises labeling the protein according to any one of (1) to (3) or a salt thereof or the labeled partial peptide according to (4) or a salt thereof with STA
When the T protein is contacted in the absence of the test compound, and the labeled protein or salt thereof or the labeled partial peptide or salt thereof is contacted with the STAT protein in the presence of the test compound. In case of
The screening method according to (14) above, which further comprises a step of measuring and comparing the amount of binding of the labeled protein or salt thereof or the labeled partial peptide or salt thereof to the STAT protein.

(16) The STAT protein is STAT3 or S
The screening method according to (14) or (15) above, which is TAT5. (17) The protein or salt thereof or a portion thereof, which comprises the protein or salt thereof according to any one of (1) to (3) and / or the partial peptide or salt thereof of (4) as a constituent element. A kit for screening a compound that inhibits or promotes the binding between a peptide or a salt thereof and a STAT protein. (18) The screening kit according to (17) above, which further comprises a STAT protein as a component.

(19) The screening kit according to (18) above, wherein the STAT protein is STAT3 protein or STAT5 protein. (20) Any one of (1) to (3), which is obtained by using the screening method according to any one of (14) to (16) or the screening kit according to any one of (17) to (19). A compound that inhibits or promotes the binding between the protein or a salt thereof, the partial peptide of (4) or a salt thereof, and a STAT protein. (21) A pharmaceutical composition comprising the compound of (20). Hereinafter, the present invention will be described in detail.

[0014]

BEST MODE FOR CARRYING OUT THE INVENTION The protein newly found in the present invention is a zinc finger protein mouse EZI and its human homologue protein which are induced to be expressed when a mouse embryo AGM region-derived cell line LO is stimulated with oncostatin M. It is a human EZI protein. The amino acid sequence of mouse EZI protein is represented by SEQ ID NO: 2, the amino acid sequence of human EZI protein is represented by SEQ ID NO: 9, and a protein containing an amino acid sequence substantially the same as the amino acid sequence is also included in the scope of the present invention. .

1. The protein of the present invention and its partial peptide: Examples of the protein containing an amino acid sequence substantially the same as the amino acid sequence represented by SEQ ID NO: 2 or SEQ ID NO: 9 of the present invention include, for example, humans and warm-blooded animals (eg, mouse). , Rat, guinea pig, chicken, rabbit, pig, sheep, cow, monkey, etc.) cells (eg, hepatocytes, splenocytes, nerve cells, glial cells, pancreatic β cells, bone marrow cells, mesangial cells, Langerhans cells, epidermal cells) , Epithelial cells, endothelial cells, fibroblasts, fibrocytes, muscle cells, adipocytes, immune cells, megakaryocytes, synovial cells, chondrocytes, osteocytes, osteoblasts, osteoclasts, mammary cells, or interstitium Cells, or precursor cells of these cells, or any tissue in which these cells exist (eg, brain, spinal cord, pituitary gland, stomach, pancreas, kidneys, liver, gonads, insteps) Gland, gall bladder,
Bone marrow, adrenal gland, skin, muscle, lung, digestive tract, blood vessel, heart, thymus, spleen, submandibular gland, peripheral blood, prostate, testis, ovary, placenta, uterus, bone, joint, skeletal muscle, etc. Alternatively, it may be a protein derived from its cultured cell line or the like, or may be a synthetic protein.

The protein containing an amino acid sequence substantially identical to the amino acid sequence represented by SEQ ID NO: 2 or 9 is about 50% or more, preferably about 50% or more, of the amino acid sequence represented by SEQ ID NO: 2 or SEQ ID NO: 9. Include proteins having amino acid sequences with about 70% or more, more preferably about 90% or more, and most preferably about 95% or more homology. The protein containing an amino acid sequence substantially the same as the amino acid sequence represented by SEQ ID NO: 2 or 9 of the present invention is, for example, substantially the same as the amino acid sequence represented by SEQ ID NO: 2 or SEQ ID NO: 9. A protein having the same amino acid sequence as the above and having substantially the same physiological activity as the protein containing the amino acid sequence represented by SEQ ID NO: 2 or SEQ ID NO: 9 is preferable.

The physiological activity which is substantially the same as that of the protein containing the amino acid sequence represented by SEQ ID NO: 2 or SEQ ID NO: 9 means the physiological activity originally possessed by the protein in vivo and the biological activity A physiologically equivalent activity, for example, an activity that activates a promoter activated by the STAT3 protein (eg, APRE promoter, β-casein promoter, CIS promoter, OSM promoter, etc.) in cooperation with the STAT3 protein, STAT5 A promoter activated by a protein (for example, APRE promoter, β-casein promoter, CIS promoter,
OSM promoter etc.) and the activity of co-activating with STAT5 protein, binding affinity with STAT3 protein, binding affinity with STAT5 protein and the like. Therefore, STAT
It is preferable that activities such as the strength of binding affinity with 3 protein and / or STAT5 protein are equivalent (for example, about 0.1 to 20 times, preferably about 0.5 to 2 times), but the strength of these activities is Quantitative factors such as the molecular weight of the protein may be different. For example, STAT3 protein and / or S
The binding affinity with the TAT5 protein can be measured according to a method known per se, for example, it can be measured according to the method described in the section of the screening method described later.

The protein of the present invention is, for example, 1 or 2 or more (preferably about 1 to 30 and more preferably 1 to 10) in the amino acid sequence represented by SEQ ID NO: 2 or SEQ ID NO: 9. 1 or 2 or more (preferably 1 to 30 or more, more preferably 1) in the amino acid sequence represented by SEQ ID NO: 2 or SEQ ID NO: 9 in which the number of amino acids is deleted, more preferably several amino acids. ~ About 10
More preferably several amino acids are added or inserted, or 1 or 2 or more (preferably 1 to 2) in the amino acid sequence represented by SEQ ID NO: 2 or SEQ ID NO: 9.
A so-called mutein such as a protein containing an amino acid sequence in which about 30 amino acids, more preferably about 1 to 10 amino acids, and further preferably several amino acids are substituted with other amino acids, or an amino acid sequence combining them is also included. .

The protein in the present specification has the N-terminal (amino terminal) at the left end and the C-terminal (carboxyl terminal) at the right end according to the convention of peptide notation. In the protein of the present invention, including the protein containing the amino acid sequence represented by SEQ ID NO: 2 or SEQ ID NO: 9, the C-terminal is usually a carboxyl group (-COOH) or a carboxylate (-
COO ⁻ ), but C-terminal may be amide (—CONH ₂ ) or ester (—COOR). Here, R in the ester is, for example, methyl, ethyl, n-propyl,
C _1-6 alkyl group such as isopropyl or n-butyl, for example, C such as cyclopentyl or cyclohexyl
_{In addition to 3-8} cycloalkyl groups, for example, C _6-12 aryl groups such as phenyl and α-naphthyl, for example, C _6-12 aryl-C _1-2 alkyl groups such as benzyl, phenethyl and α-naphthylmethyl, Pivaloyloxymethyl ester, which is widely used as an oral ester, is used.

When the protein of the present invention has a carboxyl group (or carboxylate) in addition to the C-terminal,
Those in which the carboxyl group is amidated or esterified are also included in the protein of the present invention. As the ester in this case, for example, the above-mentioned C-terminal ester or the like is used. Furthermore, in the protein of the present invention, the amino group of the N-terminal methionine residue is protected by a protecting group (eg, C _1-6 acyl group such as formyl group, acetyl group, etc.), cleaved in vivo. The resulting N-terminal glutamic acid residue is pyroglutaminated, and is present on the side chain of an amino acid in the molecule, for example, OH, COOH, NH ₂ , S
H and the like protected with a suitable protecting group (eg, formyl group, C _1-6 acyl group such as acetyl group, etc.),
Alternatively, a complex protein such as a so-called glycoprotein having a sugar chain bound thereto is also included.

Specific examples of the protein of the present invention include proteins containing the amino acid sequence represented by SEQ ID NO: 2 or SEQ ID NO: 9. The protein of the present invention has a C2H2-type zinc finger motif, preferably 12
It has a C2H2-type zinc finger motif. The protein of the present invention binds to the STAT protein, and the APRE promoter [Takeda, T. et al .: J. Immunol.
153: 4573-4582 (1994)], β-casein promoter [Waka
o, H. et al .: EMBO J, 13: 2182-2192 (1994)], OSM promoter [Yoshimura A. et al .: EMBO J, 15: 1055-1063 (19)
96)], CIS promoter [Matsumoto, A. et al .: Blood,
89: 3148-3154 (1997)] and so on. Examples of STAT protein include STAT3 protein and STAT5 protein, and the protein of the present invention particularly strongly binds to STAT3 protein.

The partial peptide of the protein of the present invention may be any of the above-mentioned partial peptides of the protein of the present invention as long as it has the physiological activity of the protein of the present invention. For example, 100 or more, preferably 250 or more, more preferably 350 or more, more preferably 450 or more, most preferably 550 or more amino acid sequence of the constituent amino acid sequence of the protein of the present invention, EZI Biological activity of protein [Promoter activated by STAT3 protein (eg, APRE promoter, β-casein promoter, CIS promoter,
OSM promoter, etc.) that activates in cooperation with STAT3 protein, and promoters activated by STAT5 protein (eg APRE promoter, β-casein promoter, CIS promoter, OSM promoter, etc.)
Peptides having the activity of co-activating with STAT5 protein, the binding affinity with STAT3 protein, the binding affinity with STAT5 protein, etc. are used.

The partial peptide of the present invention has a deletion of one or more (preferably about 1 to 10, more preferably several) amino acids in its amino acid sequence, or its partial amino acid sequence. 1 or 2 or more (preferably about 1 to 20, more preferably about 1 to 10, more preferably several) amino acids are added, or 1 or 2 or more (preferably in the amino acid sequence) Is 1
About 10 amino acids, more preferably about several amino acids, still more preferably about 1 to 5 amino acids) may be substituted with other amino acids. The C-terminal of the partial peptide of the present invention is usually a carboxyl group (-COOH) or a carboxylate (-COOH).
O ^-) a but, as in the protein of the present invention described above,
C-terminal may be an amide (-CONH ₂₎ or ester (-COOR). Further, in the partial peptide of the present invention, similar to the above-mentioned protein of the present invention, the amino group of the N-terminal methionine residue is protected by a protective group, and the N-terminal side is cleaved in vivo. Also included are those in which the glutamyl group is pyroglutaminated, those in which the substituents on the side chains of amino acids in the molecule are protected by appropriate protecting groups, or complex peptides such as so-called glycopeptides to which sugar chains are bound. The partial peptide of the present invention has binding ability to STAT protein (for example, STAT3 protein, STAT5 protein), and particularly has higher binding ability to STAT3 protein.

The salt of the protein of the present invention or its partial peptide is preferably a physiologically acceptable acid addition salt. Examples of such salts include salts with inorganic acids (eg, hydrochloric acid, phosphoric acid, hydrobromic acid, sulfuric acid), or organic acids (eg, acetic acid, formic acid, propionic acid, fumaric acid, maleic acid, succinic acid). , Tartaric acid, citric acid, malic acid, oxalic acid, benzoic acid, methanesulfonic acid, benzenesulfonic acid) and the like. In addition, salts with inorganic bases (eg, alkali metals such as sodium and potassium, alkaline earth metals such as calcium and magnesium, aluminum or ammonium), organic bases (eg, trimethylamine, triethylamine, pyridine, picoline, 2,6 -Lutidine, ethanolamine, diethanolamine, triethanolamine, cyclohexylamine, dicyclohexylamine, N, N'-
Dibenzyl ethylenediamine etc.) and the like are also used.

The protein of the present invention or a salt thereof can be produced from the cells or tissues of the above-mentioned humans or warm-blooded animals (for example, mice) by a method known per se, or a DNA encoding the protein described below. It can also be produced by culturing a transformant containing It can also be produced according to the peptide synthesis method described below. When producing from human or mammalian (eg, mouse) tissues or cells, after homogenizing human or mammalian (eg, mouse) tissues or cells,
Extraction with an acid or the like can be performed, and the extract can be isolated and purified by combining chromatography such as reverse phase chromatography and ion exchange chromatography.

For the synthesis of the protein of the present invention, a partial peptide thereof, a salt thereof or an amide thereof, a commercially available resin for protein synthesis can be used.
Examples of such a resin include chloromethyl resin,
Hydroxymethyl resin, benzhydrylamine resin, aminomethyl resin, 4-benzyloxybenzyl alcohol resin, 4-methylbenzhydrylamine resin, PAM resin, 4
-Hydroxymethylphenylacetamide methyl resin,
Examples thereof include polyacrylamide resin, 4- (2 ′, 4′-dimethoxyphenylhydroxymethyl) phenoxy resin, and 4- (2 ′, 4′-dimethoxyphenyl-Fmoc aminoethyl) phenoxy resin. Using such a resin, α-
An amino acid having an amino group and a side chain functional group appropriately protected is condensed on the resin according to the sequence of the target protein according to various condensation methods known per se.

At the end of the reaction, the protein is cleaved from the resin, at the same time various protecting groups are removed, and an intramolecular disulfide bond-forming reaction is carried out in a highly diluted solution to give the desired protein, its partial peptide or their amides. get. Regarding the condensation of the above-mentioned protected amino acids, various activation reagents that can be used for protein synthesis can be used, but carbodiimides are particularly preferable. As the carbodiimides, DCC, N, N′-diisopropylcarbodiimide, N-ethyl-N ′-(3-dimethylaminopropyl) carbodiimide and the like are used. For activation by these, the protected amino acid is directly added to the resin together with a racemization inhibitor additive (eg, HOBt, HOOBt), or the protected amino acid is activated beforehand as a symmetrical acid anhydride or HOBt ester or HOOBt ester. After that, it can be added to the resin. The solvent used for activation of the protected amino acid 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-
Dimethylacetamide, acid amides such as N-methylpyrrolidone, halogenated hydrocarbons such as methylene chloride and chloroform, alcohols such as trifluoroethanol, sulfoxides such as dimethyl sulfoxide, ethers such as pyridine, dioxane, and tetrahydrofuran, Nitriles such as acetonitrile and propionitrile, esters such as methyl acetate and ethyl acetate, or an appropriate mixture thereof are used. The reaction temperature is appropriately selected from the range known to be applicable to protein bond-forming reactions, and is usually selected in the range of approximately -20 ° C to 50 ° C. Activated amino acid derivatives are usually
Used in 1.5-4 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 after repeating the reaction, unreacted amino acid can be acetylated using acetic anhydride or acetylimidazole.

Examples of the protective group for the amino group of the raw material include Z, Boc, tert-pentyloxycarbonyl, isobornyloxycarbonyl, 4-methoxybenzyloxycarbonyl, Cl-Z, Br-Z, adamantyloxycarbonyl, Trifluoroacetyl, phthaloyl, formyl, 2-
Nitrophenylsulfenyl, diphenylphosphinothioyl oil, Fmoc, etc. are used. Carboxyl groups are, for example, alkyl esterified (eg, methyl, ethyl,
Propyl, butyl, tert-butyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, 2-adamantyl and other linear, branched or cyclic alkyl esterification), aralkyl esterification, (eg benzyl ester, 4-nitro Benzyl ester, 4-methoxybenzyl ester, 4-chlorobenzyl ester, benzhydryl esterification), phenacyl esterification, benzyloxycarbonyl hydrazide formation, tert-butoxycarbonyl hydrazide formation, trityl hydrazide formation and the like. The hydroxyl group of serine is, for example,
It can be protected by esterification or etherification. Examples of groups suitable for this 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.

The group suitable for etherification is, for example, a benzyl group, a tetrahydropyranyl group or a tert-butyl group. Examples of the protective group for the phenolic hydroxyl group of tyrosine include Bzl, Cl ₂ -Bzl, 2-nitrobenzyl, Br-Z, tert-butyl and the like. Examples of the imidazole protecting group of histidine include Tos, 4-methoxy-2,3,6-trimethylbenzenesulfonyl, DNP, benzyloxymethyl, Bum, Boc, Trt, Fmoc and the like. The activated carboxyl group of the raw material includes, for example, corresponding acid anhydrides, azides, active esters [alcohols (for example, pentachlorophenol, 2,
4,5-trichlorophenol, 2,4-dinitrophenol,
Cyanomethyl alcohol, para-nitrophenol, HON
B, N-hydroxysuccinimide, ester with HOBt)] and the like are used. As the activated amino group of the raw material, for example, the corresponding phosphoric acid amide is used.

Examples of the method for removing (eliminating) the protecting group include catalytic reduction in a hydrogen stream in the presence of a catalyst such as Pd black or Pd-carbon, or anhydrous hydrogen fluoride or methanesulfonic acid. Acid treatment with trifluoromethanesulfonic acid, trifluoroacetic acid, or a mixture thereof,
A base treatment with diisopropylethylamine, triethylamine, piperidine, piperazine or the like, or reduction with sodium in liquid ammonia can also be used. The elimination reaction by the acid treatment is generally performed at a temperature of about −20 ° C. to 40 ° C., but in the acid treatment, for example, anisole, phenol, thioanisole, metacresol, paracresol, dimethyl sulfide, 1,4 -The addition of cation scavengers such as butanedithiol and 1,2-ethanedithiol is effective.

Further, the 2,4-dinitrophenyl group used as the imidazole protecting group of histidine is removed by thiophenol treatment, and the formyl group used as the indole protecting group of tryptophan is the above 1,2-ethanedithiol, 1,4 -In addition to deprotection by acid treatment in the presence of butanedithiol, etc., it is also removed by alkali treatment with dilute sodium hydroxide solution, dilute ammonia, etc. Protection of a functional group which should not be involved in the reaction of the raw 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 or its partial peptide, for example, first, the α-carboxyl group of the carboxyl terminal amino acid is amidated and protected, and then the peptide (protein) chain is attached to the amino group side to the desired chain length. After extending to the peptide chain, a protein (partial peptide) from which only the protecting group for the α-amino group at the N-terminus of the peptide chain has been removed
And a protein (partial peptide) from which only the protecting group of the C-terminal carboxyl group has been removed, and both proteins (partial peptide) are condensed in the mixed solvent as described above. The details of the condensation reaction are the same as above.

After purifying the protected protein obtained by condensation, all the protecting groups can be removed by the above method to obtain the desired crude protein (partial peptide).
This crude protein (partial peptide) can be purified by making full use of various known purification means, and the main fraction can be lyophilized to obtain the amide of the desired protein (partial peptide). To obtain an ester form of a protein or a partial peptide thereof, for example, after condensing an α-carboxyl group of a carboxyl terminal amino acid with a desired alcohol to form an amino acid ester, the same procedure as the amide form of a protein (partial peptide) is carried out, An ester form of a desired protein (partial peptide) can be obtained.

The partial peptide of the present invention or a salt thereof can be produced by publicly known methods for peptide synthesis, or by cleaving the protein of the present invention with an appropriate peptidase. The peptide synthesis method may be, for example, either a solid phase synthesis method or a liquid phase synthesis method. That is, the target peptide can be produced by condensing a partial peptide or amino acid that can form the protein of the present invention with the remaining portion and removing the protecting group when the product has a protecting group. Known condensation methods and removal of protecting groups include, for example, M. Bodans
zky and MA Ondetti, Peptide Synthesi
s), Interscience Publishers, New York (1966),
Schroeder and Luebke, The Peptid
e), Academic Press, New York (1965), Nobuo Izumiya et al., Fundamentals and Experiments of Peptide Synthesis, Maruzen Co., Ltd. (1975)
It is possible to adopt the method described in the above. In addition, after the reaction, the protein of the present invention or its partial peptide can be isolated and purified by combining ordinary purification methods such as solvent extraction, distillation, column chromatography, liquid chromatography, and recrystallization. When the protein or its partial peptide obtained by the above method is a free form, it can be converted into an appropriate salt by a known method,
Conversely, when it is obtained as a salt, it can be converted into a free form by a known method.

2. Polynucleotide encoding protein of the present invention or partial peptide thereof As the polynucleotide (preferably DNA) encoding the protein of the present invention, any polynucleotide containing the above-mentioned nucleotide sequence encoding the protein of the present invention can be used. It may be one. Further, it may be any of the above-mentioned cell / tissue-derived cDNA, one isolated from the above-mentioned cell / tissue-derived cDNA library by a method known per se, and synthetic DNA. The vector used for the library may be any of bacteriophage, plasmid, cosmid, phagemid and the like. In addition, using the total RNA fraction or mRNA fraction prepared from the cells / tissues described above, the reverse transcriptase polymerase chain rea
can also be isolated by ction (hereinafter, abbreviated as RT-PCR method).

For example, DNA (SEQ ID NO: 1) encoding the full-length sequence of mouse EZI protein (SEQ ID NO: 2) was prepared by stimulating a mouse embryo AGM region-derived cell line (eg, LO cells) with oncostatin M, and MRNA was prepared from 5'-atgaga using the cDNA synthesized against the obtained mRNA as a template.
gagaccctggaggc-3 '(SEQ ID NO: 3) and 5'-ttagaaaaagat
It can be easily prepared by PCR by using a primer pair having a base sequence of tggaggtg -3 '(SEQ ID NO: 4). Similarly, DNA (SEQ ID NO: 8) encoding the full-length sequence of human EZI protein (SEQ ID NO: 9)
MRNA was prepared from cells of human origin, 5'-atgattcggaaggt was used as a template for the cDNA synthesized for the obtained mRNA.
gaaggt-3 '(SEQ ID NO: 10) and 5'-tcagaagaagagcggggg
It can be easily prepared by PCR by using a primer pair having the base sequence of cg-3 '(SEQ ID NO: 11).

The polynucleotide encoding the protein of the present invention includes, for example, SEQ ID NO: 1 or SEQ ID NO: 8
Which has a base sequence which hybridizes with a DNA containing the base sequence represented by or the base sequence represented by SEQ ID NO: 1 or SEQ ID NO: 8 under stringent conditions.
Alternatively, a physiological activity substantially the same as the protein having the amino acid sequence represented by SEQ ID NO: 9, for example, a promoter activated by the STAT3 protein (eg, APRE
Promoter, β-casein promoter, CIS promoter, OSM promoter, etc.) that activates in cooperation with STAT3 protein, promoters activated by STAT5 protein (eg APRE promoter, β-casei
n promoter, CIS promoter, OSM promoter, etc.)
Examples thereof include DNA encoding a protein having binding affinity with T3 protein, binding affinity with STAT5 protein, and the like. As the DNA that can hybridize with the base sequence represented by SEQ ID NO: 8 under stringent conditions,
For example, the nucleotide sequence represented by SEQ ID NO: 8 and about 70% or more,
Preferably about 80% or more, more preferably about 90% or more,
Most preferably, DNA containing a nucleotide sequence having a homology of about 95% or more is used.

Hybridization can be carried out by a method known per se or a method analogous thereto, for example, molecular
Cloning (Molecular Cloning) 2nd [J. Sambrook et
al., Cold Spring Harbor Lab. Press, 1989] and the like. When a commercially available library is used, it can be performed according to the method described in the attached instruction manual. More preferably, it can be carried out under stringent conditions. Stringent conditions include, for example, sodium concentration of about 19-40 m
M, preferably about 19 to 20 mM, and a temperature of about 50 to 70 ° C, preferably about 60 to 65 ° C. Most preferably, the sodium concentration is about 19 mM and the temperature is about 65 ° C. More specifically, as the DNA encoding the protein containing the amino acid sequence of SEQ ID NO: 2, DNA having the base sequence represented by SEQ ID NO: 1 is used, and the protein containing the amino acid sequence of SEQ ID NO: 9 is used. As the DNA that encodes, a DNA having the base sequence represented by SEQ ID NO: 8 or the like is used.

The DNA encoding the partial peptide of the present invention may be any DNA as long as it contains the nucleotide sequence encoding the partial peptide of the present invention described above. In addition, genomic DNA, genomic DNA library, cDNA derived from the above-mentioned cells / tissues, cDNA derived from the above-mentioned cells / tissues
It may be either a cDNA isolated from a cDNA library by a method known per se or a synthetic DNA. As the DNA encoding the partial peptide of the present invention, for example, a DNA containing a part of the nucleotide sequence represented by SEQ ID NO: 1 or SEQ ID NO: 8,
Alternatively, a protein having a base sequence that hybridizes with the base sequence represented by SEQ ID NO: 1 or SEQ ID NO: 8 under stringent conditions and substantially having the amino acid sequence represented by SEQ ID NO: 2 or SEQ ID NO: 9 DNA having a partial base sequence of DNA encoding proteins having the same activity
Are used. The same hybridization method and stringent conditions as described above are used.

As a means for cloning a DNA encoding the protein of the present invention or a partial peptide thereof (hereinafter also simply referred to as the protein of the present invention), a synthetic method having a nucleotide sequence encoding a partial sequence of the protein of the present invention can be used. Amplify by PCR using DNA primer,
Alternatively, a genomic DNA, a genomic DNA library, a cDNA derived from the cells / tissues described above, or a part of the protein of the present invention isolated from a cDNA library derived from the cells / tissues described above by a method known per se is used. Code a region
It can be isolated by hybridization with a DNA fragment or that labeled with a synthetic DNA. Hybridization can be performed, for example, by molecular cloning (Molecular Cloning) 2nd [J. Sambrook e
t.al., Cold Spring Harbor Lab. Press, 1989] and the like. When a commercially available library is used, it can be performed according to the method described in the attached instruction manual. Conversion of the nucleotide sequence of DNA can be carried out using a known kit, for example, Mutant ^TM -G (Takara Shuzo Co., Ltd.), Mutant ^TM -K (Takara Shuzo Co., Ltd.), etc.
It can be carried out according to a method known per se such as the Gupped duplex method or the Kunkel method or a method similar thereto.

The cloned DNA encoding the protein of the present invention can be used as it is, or if desired, after digestion with a restriction enzyme or addition of a linker, depending on the purpose. The DNA may have ATG as a translation initiation codon at the 5'-terminal side and TAA, TGA or TAG as a translation termination codon at the 3'-terminal side. These translation initiation codon and translation termination codon can be added using an appropriate synthetic DNA adaptor.

3. Recombinant vector of the present invention and transformant containing the same The expression vector of the protein of the present invention can be obtained by, for example, cutting out a target DNA fragment from the DNA encoding the protein of the present invention, and converting the DNA fragment into a suitable expression vector. It can be produced by ligating to the downstream of the promoter. Vectors include E. coli-derived plasmids (eg, pBR322, pBR325, pUC18, pUC19), Bacillus subtilis-derived plasmids (eg, pUB110, pTP5, pC194), yeast-derived plasmids (eg, YEp13, YEp24, YCp50), λ. Bacteriophage such as phage, retrovirus, vaccinia virus, animal virus such as baculovirus, pA1-11, pXT1, pRc / CMV, pRc / RSV, pcDNAI / N
eo is used. The promoter used in the present invention may be any promoter as long as it is suitable for the host used for gene expression.

For example, when an animal cell is used as a host, SV40-derived promoter, retrovirus promoter, metallothionein promoter, heat shock promoter, cytomegalovirus promoter,
Examples include SRα promoter. Of these, the cytomegalovirus promoter, SRα promoter and the like are preferably used. If the host is Escherichia, trp promoter, lac promoter, recA promoter, λP _L promoter, lpp promoter, etc., if the host is Bacillus, SP01 promoter, SP02 promoter, penP promoter, etc. In the case of yeast, pH05 promoter, PGK promoter, GAP promoter, ADH promoter and the like are preferable. When the host is an insect cell, polyhedrin promoter, P10 promoter and the like are preferable.

In addition to the above, the expression vector may optionally include an enhancer, a splicing signal, a poly A addition signal, a selection marker, an SV40 origin of replication (hereinafter referred to as SV40or).
abbreviated as i) and the like can be used. Examples of selection markers include dihydrofolate reductase (hereinafter abbreviated as dhfr) gene [methotrexate (MTX) resistance], ampicillin resistance gene (hereinafter abbreviated as Amp ^r ), neomycin resistance gene (hereinafter Neo) abbreviated as ^r and sometimes abbreviated as G418 resistance). In particular, CHO (dhfr ^-) when dhfr gene is used as the selection marker using cells,
The transformant containing the target gene can also be selected by using a thymidine-free medium.

If necessary, a signal sequence suitable for the host can be added to the N-terminal side of the protein of the present invention. If the host is Escherichia, alkaline phosphatase signal sequence, OmpA signal sequence, etc., if the host is Bacillus, α-amylase signal sequence, subtilisin signal sequence, etc., the host is yeast. In some cases, a mating factor α signal sequence, invertase signal sequence, etc., and when the host is an animal cell, for example, an insulin signal sequence, α-interferon signal sequence, antibody molecule signal sequence, etc. can be used, respectively. A transformant can be produced using the vector containing the DNA encoding the protein of the present invention thus constructed.

As the host, for example, Escherichia, Bacillus, yeast, insect cells, insects, animal cells and the like are used. Specific examples of Escherichia bacteria include:
Escherichia coli (Escherichia coli) JM103 [Nuclei
c Acids Research, 9: 309 (1981), JA221 [J. Mol. Bio
l., 120: 517 (1978)], HB101 [J. Mol. Biol., 41: 459 (19
69)], C600 [Genetics, 39: 440 (1954)] and the like are used. Examples of the bacterium of the genus Bacillus include Bacillus subtilis MI114 [Gene, 24: 255 (1983)],
207-221 [J. Biochem., 95:87 (1984)] and the like are used. Examples of yeast include Saccharomyces cerevisiae ^{(Saccharomyces cerevisiae) AH22, AH22R -} , NA87
-11A, DKD-5D, 20B-12, Schizosaccharomyces pombe NCYC1913, NCYC2036,
Such as Pichia pastoris (Pichia pastoris) is used.

As the insect cell, for example, the virus is Ac
In the case of NPV, the cell line derived from the larvae of the night-throwing moth ( Spodoptera
frugiperda cell; Sf cell), MG1 cell from Trichoplusia ni midgut, High Five ^TM from Trichoplusia ni egg
Cells, Mamestra brassicae-derived cells or Estigmena
Cells derived from acrea are used. When the virus is BmNPV, silkworm-derived cell lines ( Bombyx mori N cells; BmN cells) and the like are used. Examples of the Sf cell include Sf
9 cells, Sf21 cells, etc. are used. Examples of insects include silkworm larvae [Nature, 315: 592].
(1985)]. Examples of animal cells include monkey cells COS-7,
Vero, Chinese hamster cell CHO, DHFR gene deficient Chinese hamster cell CHO (dhfr ^- CHO cell),
Mouse L cells, mouse AtT-20, mouse myeloma cells,
Rat GH3, human FL cells, etc. are used.

Transformation of Escherichia can be carried out, for example, by
Method using calcium ion [Proc. Natl. Acad. Sc
i. USA, 69: 2110 (1972)], electroporation method and the like. Transformation of Bacillus
It can be performed by the protoplast method or the like. Transformation of yeast is performed by the electroporation method [Methods. Enzymo
L., 194: 182-187 (1991)], Spheroplast method [Proc. N
atl. Acad. Sci. USA, 75: 1929 (1978)], lithium acetate method
[J. Bacteriol., 153: 163 (1983)] and the like. Transformation of insect cells can be performed by, for example, the calcium phosphate method, lipofection method, electroporation method, or the like. Transformation of animal cells can be performed by an electroporation method, a calcium phosphate method, a lipofection method, or the like. Thus, a transformant transformed with the expression vector containing the DNA encoding the protein of the present invention can be obtained.

4. Protein of the present invention, a method for producing a partial peptide thereof or a salt thereof, when the host is a transformant which is a bacterium of the genus Escherichia or a bacterium of the genus Bacillus, a liquid medium is suitable as the medium used for the culture, and A carbon source, a nitrogen source, an inorganic substance and other components necessary for the growth of the transformant are contained therein.
Examples of carbon sources include glucose, dextrin,
Examples of nitrogen sources such as soluble starch and sucrose include ammonium salts, nitrates, corn steep liquor,
Examples of inorganic or organic nitrogen sources such as peptone, casein, meat extract, soybean meal, and potato extract, and inorganic substances include calcium chloride, sodium dihydrogen phosphate, magnesium chloride and the like. Also, if necessary,
Yeast extract, vitamins, growth promoting factors and the like may be added. The pH of the medium is preferably about 5-8.

As a medium for culturing Escherichia, for example, M9 medium containing glucose and casamino acid [J
ournal of Experiments in Molecular Genetics, 431-4
33, Cold Spring Harbor Laboratory, New York 1972] is preferable. If necessary, a drug such as 3β-indolylacrylic acid can be added to the promoter so that the promoter works efficiently. When the host is a genus Escherichia, the culture is usually performed at about 15 to 43 ° C for about 3 to 24 hours,
If necessary, aeration and stirring can be added. When the host is a bacterium of the genus Bacillus, the culture is usually performed at about 30 to 40 ° C and about 6 to
It is performed for 24 hours, and if necessary, aeration and stirring can be added. When culturing a transformant in which the host is yeast, the medium is, for example, SD medium containing 0.5% casamino acid.
[Bitter, GA et al .: Proc. Natl. Acad. Sci. USA, 8
1: 5330 (1984)]. The pH of the medium is preferably adjusted to about 5-8. Culturing is usually about 20 ~ 35 ℃ about 24 ~
Perform for 72 hours, add aeration and agitation as needed.

When culturing a transformant whose host is an insect cell or an insect, the medium is Grace's Insect Medi
Immobilized to um [Grace, TCC: Nature, 195: 788 (1962)]
For example, those to which additives such as 10% bovine serum are appropriately added are used. The pH of the medium is preferably adjusted to about 6.2-6.4.
Culturing is usually carried out at about 27 ° C. for about 3 to 5 days, and aeration and agitation are added if necessary. When a transformant whose host is an animal cell is cultured, examples of the medium include MEM medium containing about 5 to 20% fetal bovine serum [Science, 122: 501 (1952)], DMEM.
Medium [Virology, 8: 396 (1959)], RPMI 1640 Medium [Journal
of the American Medical Association, 199: 519 (196
7)], 199 medium [Proceeding of the Society for the Biol
ogical Medicine, 73: 1 (1950)] and the like are used. The pH is preferably about 6-8. Culturing is usually about 30-40 ℃
For about 15 to 60 hours, and add aeration and agitation as needed. As described above, the protein of the present invention can be produced in the culture medium or the transformant.

The protein of the present invention can be separated and purified from the above-mentioned culture, for example, by the following method. When extracting the protein of the present invention from cultured bacterial cells or cells, after culturing, the bacterial cells or cells are collected by a known method, suspended in a suitable buffer solution, ultrasonicated,
A method of disrupting the cells or cells by lysozyme and / or freeze-thawing and then obtaining a crude protein extract by centrifugation or filtration is appropriately used. The buffer solution may contain a protein denaturing agent such as urea or guanidine hydrochloride, or a surfactant such as Triton X-100. If the protein is secreted into the culture medium,
After 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 protein contained in the culture supernatant or the extract thus obtained can be purified by an appropriate combination of separation / purification methods known per se. These known separation and purification methods include solubility-based methods such as salting-out and solvent precipitation methods, dialysis methods, ultrafiltration methods, gel filtration methods, and SDS-polyacrylamide gel electrophoresis methods. The difference in charge, the difference in charge such as ion exchange chromatography, the method using specific affinity such as affinity chromatography, and the difference in hydrophobicity such as reversed-phase high performance liquid chromatography A method utilizing a difference in isoelectric points such as a method and an isoelectric focusing method is used. When the protein thus obtained is obtained as a free form, it can be converted into a salt by a method known per se or a method analogous thereto, and conversely, when it is obtained as a salt, a method known per se or It can be converted into a free form or another salt by a method similar to that.

The protein produced by the recombinant is
Before or after purification, an appropriate protein-modifying enzyme may be allowed to act to arbitrarily modify or partially remove the polypeptide. As a protein-modifying enzyme,
For example, trypsin, chymotrypsin, arginyl endopeptidase, protein kinase, glycosidase and the like are used. The activity of the thus-produced protein of the present invention or a salt thereof is controlled by a promoter activated by the STAT3 protein (eg, APRE promoter, β
-Activities that activate casein promoter, CIS promoter, OSM promoter, etc.), promoters that are activated by STAT5 protein in cooperation with STAT5 protein (eg APRE promoter, β-casein promoter, CIS promoter, OSM promoter, etc.) It can be measured by a measurement experiment of activating activity and an enzyme immunoassay using a specific antibody.

5. Antibodies to the protein of the present invention, its partial peptides or their salts, the antibodies to the proteins of the present invention, their partial peptides or their salts are antibodies that can recognize the proteins of this invention, their partial peptides or their salts, It may be a polyclonal antibody or a monoclonal antibody. An antibody against the protein of the present invention, its partial peptide or a salt thereof can be produced by using the protein of the present invention, its partial peptide or a salt thereof as an antigen, according to a method for producing an antibody or antiserum known per se.

(1) Preparation of monoclonal antibody (i) Preparation of monoclonal antibody-producing cell
Administered with diluent. Complete Freund's adjuvant or incomplete Freund's adjuvant may be administered to enhance the antibody-producing ability upon administration. Administration is usually 2
It is performed once every 6 weeks, for a total of 2 to 10 times. Examples of warm-blooded animals used include monkeys, rabbits, dogs, guinea pigs, mice, rats, sheep, goats, chickens, and the like, and mice and rats are preferably used. When preparing monoclonal antibody-producing cells,
A warm-blooded animal immunized with an antigen, for example, an individual with an antibody titer is selected from mice, and spleens or lymph nodes are collected 2 to 5 days after the final immunization, and the antibody-producing cells contained therein are myeloma cells. A monoclonal antibody-producing hybridoma can be prepared by fusion with

The antibody titer in the antiserum can be measured, for example, by reacting the labeled protein described below with the antiserum and then measuring the activity of the labeling agent bound to the antibody. The fusion operation can be performed according to a known method, for example, the method of Kohler and Milstein [Nature, 256: 495 (1975)]. Examples of the fusion promoter include polyethylene glycol (PEG) and Sendai virus, but PEG is preferably used. Examples of myeloma cells include NS-1, P3U1, SP2 / 0, AP-1 and the like, and P3U1 is preferably used. The preferred ratio between the number of antibody-producing cells (spleen cells) and the number of myeloma cells used is
1: 1 to 20: 1, PEG (preferably PEG1000 to PEG
6000) is added at a concentration of about 10 to 80%, and the cells can be efficiently fused by incubating at 20 to 40 ° C., preferably 30 to 37 ° C. for 1 to 10 minutes. Although various methods can be used for screening an anti-protein antibody-producing hybridoma, for example, a hybridoma culture supernatant is added to a solid phase (for example, a microplate) on which a protein antigen is directly or adsorbed with a carrier, and then a radioactive substance is added. Method for detecting anti-protein monoclonal antibody bound to a solid phase by adding anti-immunoglobulin antibody labeled with an enzyme or enzyme (anti-mouse immunoglobulin antibody is used when cells used for cell fusion are mice) or protein A A method of detecting the anti-protein monoclonal antibody bound to the solid phase by adding the hybridoma culture supernatant to the solid phase on which the anti-immunoglobulin antibody or protein A is adsorbed, adding the protein labeled with a radioactive substance or enzyme, etc. Is mentioned. The anti-protein monoclonal antibody can be selected according to a method known per se or a method analogous thereto.

It can be usually carried out in a medium for animal cells supplemented with HAT (hypoxanthine, aminopterin, thymidine). As the selection and breeding medium, any medium can be used as long as the hybridoma can grow. For example, RPMI 1640 medium containing 1-20%, preferably 10-20% fetal bovine serum, G containing 1-10% fetal bovine serum.
IT medium (Wako Pure Chemical Industries, Ltd.) or serum-free medium for hybridoma culture (SFM-101, Nissui Pharmaceutical Co., Ltd.) can be used. The culture temperature is usually 20 to 40 ° C, preferably about 37 ° C. Cultivation time is usually 5 days to 3 weeks, preferably 1 week to 2 weeks. Culture is usually 5
% It can be performed under carbon dioxide gas. The antibody titer of the hybridoma culture supernatant can be measured in the same manner as the above-mentioned measurement of anti-protein antibody titer in antiserum.

(Ii) Purification of Monoclonal Antibody Separation and purification of the anti-protein monoclonal antibody can be carried out by a method known per se, for example, a method for separating and purifying immunoglobulin (eg salting out method, alcohol precipitation method, isoelectric focusing method, electrolysis method). Specific to obtain antibody by electrophoresis, adsorption / desorption method using ion exchanger, ultracentrifugation method, gel filtration method, antigen-binding solid phase or active adsorbent such as protein A or protein G, and dissociating the bond to obtain antibody Purification method).

(2) Preparation of Polyclonal Antibody The polyclonal antibody of the present invention can be manufactured by publicly known methods or modifications thereof. For example, a complex of an immune antigen (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 protein of the present invention is collected from the immunized animal. Then, it can be produced by separating and purifying the antibody. Regarding the complex of the immunizing antigen and the carrier protein used to immunize a warm-blooded animal, the type of carrier protein and the mixing ratio of the carrier and the hapten are such that the antibody is effective against the hapten immunized by crosslinking the carrier. If possible, any kind may be cross-linked at any ratio, but, for example, bovine serum albumin or hemocyanin at a weight ratio of about 0.1 to 20, preferably about 1 to 5 with respect to 1 hapten. A couple method is used.

For the coupling of the hapten and the carrier, various condensing agents can be used, and glutaraldehyde, carbodiimide, maleimide active ester, active ester reagent containing thiol group and dithiopyridyl group, etc. are used. . The condensation product is administered to warm-blooded animals at a site where antibodies can be produced, by itself or together with a carrier or diluent. Complete Freund's adjuvant or incomplete Freund's adjuvant may be administered to enhance the antibody-producing ability upon administration. The administration is usually performed once every 2 to 6 weeks, about 3 to 10 times in total. The polyclonal antibody can be collected from the blood, ascites, etc., of the warm-blooded animal immunized by the above method, preferably from the blood. The polyclonal antibody titer in the antiserum can be measured in the same manner as the above-mentioned antibody titer in the antiserum. Separation and purification of the polyclonal antibody can be performed according to the same immunoglobulin separation and purification method as the above-described separation and purification of the monoclonal antibody.

(3) Application of the antibody of the present invention Since the antibody of the present invention can specifically recognize the protein of the present invention and the like, it is possible to quantify the protein of the present invention in a test solution, particularly sandwich immunization. It can be used for quantification by a measuring method. That is, the present invention is, for example, an antibody that reacts with the protein or the like of the present invention, competitively reacts with a test solution and a labeled protein of the present invention, and the labeled antibody bound to the antibody Provided is a method for quantifying the protein or the like of the present invention in a test solution, which comprises measuring the ratio of the protein or the like of the invention. The protein or the like of the present invention can be measured using a monoclonal antibody that recognizes the protein or the like of the present invention, or can be detected by tissue staining or the like.

For these purposes, the antibody molecule itself may be used, or F (ab ') ² , Fab' or the antibody molecule of the antibody molecule may be used.
Fab fractions may be used. The method for measuring the protein or the like of the present invention using the antibody of the present invention is not particularly limited, and may be an antibody, an antigen or an antibody-antigen corresponding to the amount of the antigen in the liquid to be measured (for example, the amount of the protein of the present invention). Any measuring method may be used as long as it is a method of detecting the amount of the complex by chemical or physical means and calculating it from a standard curve prepared using a standard solution containing a known amount of antigen. . For example, the competitive method, the immunometric method and the sandwich method are preferably used, but the sandwich method is particularly preferable in terms of sensitivity and specificity.

Labels used in measurement methods using labeled substances
Examples of intelligence agents include radioisotopes, enzymes, fluorescent substances, and luminescence.
Examples include substances. As a radioisotope, for example,
If [ ¹²⁵I], [¹³¹I], [³H], [¹⁴C] etc.
As the above-mentioned enzyme, those with stable and large specific activity are preferable.
For example, peroxidase, alkaline phosphater
Ze, β-galactosidase, β-glucosidase, apple
Acid dehydrogenase, etc. is used as a fluorescent substance.
Isothiocyanate, etc., is a luminescent substance
Phosphorus, luminol, luminol derivatives, etc. are listed respectively.
You can Furthermore, the binding between the antibody or antigen and the labeling agent
Alternatively, the biotin-avidin system can be used. Antigen
Physical adsorption may be used to insolubilize antibodies.
And insolubilize or immobilize proteins or enzymes
The method using the chemical bond used for the conversion may be used.
As the carrier, agarose, dextran, cellulose
Insoluble polysaccharides such as polystyrene, polyacrylamy
, Synthetic resin such as silicon, glass, etc.
Be done.

In the sandwich method, an insolubilized antibody of the present invention (primary antibody) is reacted with a test solution, and a labeled antibody (secondary antibody) against the primary antibody is further reacted. The amount of the protein of the present invention in the test solution can be quantified by measuring the activity. In the competitive method, after the antigen in the test liquid and the labeled antigen are competitively reacted with the antibody, the labeled antigen in the final reaction (F) and the labeled antigen (B) bound to the antibody are added. Are separated (B / F separation), the labeled amount of either B or F is measured, and the amount of antigen in the test liquid is quantified. In the immunometric method, the antigen in the test liquid and the solid-phased antigen are competitively reacted with a fixed amount of the labeled antibody and then the solid phase and the liquid phase are separated, or the antigen in the test liquid is separated. After reacting with an excess amount of the labeled antibody and then adding the immobilized antigen to bind the unreacted labeled antibody to the solid phase, the solid phase and the liquid phase are separated. Next, the labeled amount of either phase is measured to quantify the amount of antigen in the test liquid.

In applying these individual immunological assay methods to the protein assay method of the present invention, it is not necessary to set special conditions, operations and the like. The assay system for the protein or the like of the present invention may be constructed by adding ordinary technical consideration to those skilled in the art to the ordinary condition operating method in each method. For details on these general technical means, see Methods in Enzymology, vol.7, published by Academic Press.
0, Immunochemical Techniques (Part A), Methods in E
nzymology, vol.73, Immunochemical Techniques (Part
B), Methods in Enzymology, vol.74, Immunochemical
Techniques (Part C), Methods in Enzymology, vol.84, I
See mmunochemical Techniques (Part D).
As described above, the protein or the like of the present invention can be quantified with high sensitivity by using the antibody against the protein or the like of the present invention. Furthermore, by quantifying the concentration of the protein or the like of the present invention using an antibody against the protein or the like of the present invention, for example, a disease associated with the protein or the like of the present invention can be diagnosed. Further, the antibody of the present invention can be used to detect the protein of the present invention present in a subject such as a cell or tissue.
In addition, preparation of an antibody column used for purifying the protein of the present invention, detection of the protein of the present invention in each fraction during purification, analysis of behavior of the protein of the present invention in test cells, etc. Can be used for

6. Preventive / therapeutic agent for EZI protein-related diseases The DNA encoding the EZI protein of the present invention can be used as a preventive / therapeutic agent for EZI protein-related diseases. Further, since the protein of the present invention exhibits binding activity to intracellular signal transduction molecules such as STAT protein, particularly STAT3 protein and STAT5 protein, it can be used as a prophylactic / therapeutic agent for diseases mediated by STAT3 protein or STAT5 protein. You can For example, in a leukemia patient whose onset of leukemia is caused by a mutation or deficiency of the EZI protein of the present invention, the DNA encoding the protein of the present invention is administered to the patient and expressed, or hematopoietic stem cells of the present invention are expressed. The effect of the protein of the present invention in the patient can be sufficiently exerted by, for example, transplanting the cell into the patient after inserting and expressing the DNA encoding the protein. Therefore, the DNA encoding the protein of the present invention can be used as a safe and low-toxicity prophylactic / therapeutic agent for EZI protein-related diseases.

When the DNA of the present invention is used as the above therapeutic agent, the DNA is used alone or after being inserted into an appropriate vector such as a retrovirus vector, an adenovirus vector, an adenovirus associated virus vector, etc. can do. For example,
Orally as sugar-coated tablets, capsules, elixirs, microcapsules and the like, or aseptic solutions with water or other pharmaceutically acceptable liquids, suspensions, etc. It can be used parenterally in the form of injection. For example, admixing the DNA of the present invention together with physiologically acceptable carriers, flavoring agents, excipients, vehicles, preservatives, stabilizers, binders and the like in a unit dose form required for generally accepted formulation practice. Can be manufactured by. The amount of active ingredient in these preparations is such that a suitable amount within the indicated 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, alginic acid and the like. Such as a leavening agent, a lubricant such as magnesium stearate, a sweetener such as sucrose, lactose or saccharin, and a flavoring agent such as peppermint, red oil or cherry. When the unit dosage form is a capsule, a liquid carrier such as fat may be included in addition to materials of the above type. Sterile compositions for injection can be formulated according to conventional pharmaceutical practices such as dissolving or suspending the active substance in a vehicle such as water for injection, naturally occurring vegetable oils such as sesame oil, coconut oil and the like. Examples of the aqueous solution for injection include physiological saline, isotonic solution containing glucose and other adjuvants (for example, D-sorbitol, D-mannitol, sodium chloride, etc.), and a suitable solubilizing agent, for example, You may use together with alcohol (for example, ethanol), polyalcohol (for example, propylene glycol, polyethylene glycol), etc.

Examples of the oily liquid include sesame oil and soybean oil, which may be used in combination with solubilizing agents such as benzyl benzoate and benzyl alcohol. In addition, buffers (eg, phosphate buffer, sodium acetate buffer), 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 filled in a suitable ampoule. Since the preparation thus obtained is safe and has low toxicity, it can be administered to, for example, warm-blooded mammals (eg, rat, rabbit, sheep, pig, cow, cat, dog, monkey, human). it can. Although the dose of the DNA varies depending on the symptoms and the like, in the case of oral administration, it is generally about 0.1 to 100 mg, preferably about 1.0 to 50 mg, and more preferably, about 0.1 to 100 mg per day in an adult (weight 60 kg). It is about 1.0 to 20 mg. When administered parenterally, the single dose varies depending on the administration subject, target organs, symptoms, administration method, etc., but in the case of an injection, for example, for an adult (body weight 60 kg) per day, About 0.01-30m
g, preferably about 0.1 to 20 mg, more preferably 0.1
It is convenient to administer about 10 mg intravenously.

7. Screening method and screening kit for compounds that inhibit or promote the binding between EZI protein and STAT protein (1) Screening method By comparing the binding between EZI protein and STAT protein in the absence and presence of a test compound By using a competitive binding assay system, EZI protein and S
A method for screening a compound (eg, peptide, protein, non-peptidic compound, synthetic compound, fermentation product, etc.) or a salt thereof that inhibits or promotes binding to a TAT protein is provided.

Specific examples of the present screening method include the following. That is, the labeled EZI protein or a salt thereof is brought into contact with the STAT protein, and the labeled EZI protein or a salt thereof and a test compound are brought into contact with the STAT protein. STAT expressed by intracellularly expressing a labeled EZI protein or a salt thereof by culturing a transformant containing a DNA encoding the STAT protein.
In the case of contacting with a protein, and in the case of contacting a labeled EZI protein or a salt thereof and a test compound with a STAT protein expressed intracellularly by culturing a transformant containing a DNA encoding the STAT protein, A labeled EZI protein or a salt thereof is measured for binding amount to STAT protein, a screening method characterized by comparing, and a host cell introduced with a plasmid for expressing EZI protein and a plasmid for expressing STAT protein, EZ in the host cell when the host cell is contacted with a test compound
It is a screening method characterized by comparing the expression of genes whose expression is regulated by the interaction of I protein and STAT protein.

In the above-mentioned screening method, the labeled EZI proteins include [ ³ H], [ ¹²⁵ I], [ ¹⁴
C], [ ¹³⁵ S], etc. labeled EZI protein and EZ
I protein analog compounds and the like are used. Also, S
As the TAT protein, those extracted from cells of warm-blooded animal can be used, but those expressed in large amounts by using gene recombination technology are suitable. In the screening method of the present invention, EZI protein and / or STAT
When a cell containing a protein is used, the cell may be fixed with glutaraldehyde, formalin or the like. Immobilization methods are known in the art.

For example, the screening method described above
Specifically, it can be performed as follows. First,
A STAT protein preparation is prepared by suspending cells containing STAT protein in an appropriate buffer. Any buffer may be used as long as it does not inhibit the binding between EZI protein and STAT protein, such as a phosphate buffer having a pH of 4 to 10 (preferably a pH of 6 to 8) and a Tris-hydrochloric acid buffer. Further, a surfactant such as CHAPS, digitonin or deoxycholate may be added to the buffer for the purpose of reducing non-specific binding. Furthermore, protease inhibitors such as PMSF, leupeptin, pepstatin, etc. can be added for the purpose of suppressing the degradation of STAT protein and EZI protein by protease.

A fixed amount (5,000 to 500,000 cpm) of the labeled EZI protein was added to 0.01 to ¹⁰ ml of the STAT protein solution, and 10 ^-4 to 10 ^-10 M of the test compound was coexisted at the same time. A large excess of unlabeled EZI for non-specific binding
Prepare a reaction tube containing protein. Reaction is 0
~ 50 ° C, preferably 4-37 ° C for 20 minutes to 24 hours, preferably 30 minutes to 3 hours. After the reaction, the mixture is filtered through a glass fiber filter paper or the like, washed with an appropriate amount of the same buffer, and the radioactivity remaining on the glass fiber filter paper is measured with a liquid scintillation counter or a γ-counter. When the count (B0-NSB) obtained by subtracting the nonspecific binding amount (NSB) from the count (B0) when there is no antagonist is 100%, the specific binding amount (B-NSB) is, for example, 50% or less. Can be selected as a candidate compound with competitive inhibition ability, while a test compound with a specific binding amount (B-NSB) of, for example, 150% or more is selected as a candidate compound with binding promoting ability. be able to.

(2) Screening Kit The screening method described in (1) above can be used as a kit. The components of the kit include a protein containing the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 9 or a salt thereof, and / or
Alternatively, a partial peptide of a protein containing the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 9 or a salt thereof can be mentioned. Other kits include STAT proteins (eg STAT3 protein, STAT5
Protein) is preferably added. In addition, a buffer solution or the like can be added if necessary.

8. Screening method of the present invention Medical and pharmaceutical application of the obtained compound Examples of the compound obtained by using the screening method or the screening kit described in 7 above include peptides, proteins, non-peptidic compounds, synthetic compounds and fermentation products. These compounds may be novel compounds or known compounds. Compound is STAT
Compounds having gene expression promoting activity or gene expression suppressing activity of genes whose expression is activated by 3 proteins (for example, APRE gene, β-casein gene, CIS gene, OSM gene) are included. Examples of the protein that binds to the protein of the present invention include STAT3 protein and STAT5 protein. Aberrant activity of STAT3 and STAT5 proteins is known to be involved in a wide range of malignancies including hematological, breast, prostate and cervical cancers [Turkson, J. et al .: Oncogene, 19 : 6613-6626 (200
0)]. Therefore, the EZI protein having the binding activity to the STAT3 protein and the STAT5 protein and working in cooperation with these proteins is useful as a target for the prevention and treatment of the malignant tumor, and the EZI protein found in 7 above. A compound that regulates the binding between STAT protein and STAT protein is useful as a prophylactic / therapeutic agent for malignant tumor (eg, hematological cancer, breast cancer, prostate cancer, cervical cancer, etc.).

When the compound or its salt obtained by using the screening method or the screening kit of the present invention is used as a pharmaceutical composition, it can be carried out according to a usual means. For example, tablets, capsules, elixirs, microcapsules, etc., which are optionally sugar-coated, orally, or aseptic solution with water or other pharmaceutically acceptable liquid, or suspension. It can be used parenterally in the form of injections such as solutions. For example, the compound or salt thereof is admixed with a physiologically acceptable carrier, flavoring agent, excipient, vehicle, preservative, stabilizer, binder and the like in a unit dosage form generally required for pharmaceutical practice. It can be manufactured by The amount of active ingredient in these preparations is such that a suitable amount within the indicated range can be obtained.

Additives that can be mixed with tablets, capsules and the like include, for example, gelatin, corn starch, tragacanth, binders such as gum arabic, excipients such as crystalline cellulose, corn starch, gelatin, alginic acid and the like. Such as a leavening agent, a lubricant such as magnesium stearate, a sweetener such as sucrose, lactose or saccharin, and a flavoring agent such as peppermint, red oil or cherry. When the dosage unit form is a capsule, a liquid carrier such as fat may be added to the above-mentioned materials. 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 8)
0 ^TM , HCO-50) and the like. As the oily liquid, for example, sesame oil, soybean oil and the like are used, and solubilizing agents such as benzyl benzoate and benzyl alcohol may be used in combination. 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 filled in a suitable ampoule.

Since the preparation thus obtained is safe and has low toxicity, it is administered to, for example, mammals (eg, rat, rabbit, sheep, pig, cow, cat, dog, monkey, human). be able to. The dose of the compound or its salt varies depending on the symptoms, etc., but in the case of oral administration, it is generally about 0.1 to 100 mg, preferably about 1.0 to 50 mg per day for an adult (as 60 kg). , More preferably about 1.0-20 mg. When administered parenterally, the single dose varies depending on the administration subject, target organ, symptom administration method, etc., but in the form of an injection, it is usually a day for adults (60 kg). It is convenient to administer about 0.01 to 30 mg, preferably about 0.1 to 20 mg, more preferably about 0.1 to 10 mg by intravenous injection. In the case of animals other than humans, it is possible to administer the same amount when converted per 60 kg.

[0081]

The present invention will be described in more detail below with reference to examples, but the present invention is not limited thereto. [Example 1] Cloning of cDNA encoding mouse EZI protein by subtraction method cDN encoding mouse EZI protein as follows
A (also called mouse EZI gene) was cloned.
First, a cDNA library was prepared from OSM-reactive cell line LO derived from the AGM region before and after OSM stimulation.
Using these two cDNA libraries, subtraction was performed in the direction of subtracting the library before OSM stimulation from the library for one hour after OSM stimulation. Specifically, first the library before OSM stimulation of the one to be drawn is labeled with biotin, mixed with the library for one hour after OSM stimulation of the one to be drawn, and denatured the double-stranded DNA into single strands by high temperature treatment. Then, they were allowed to associate under the condition of 68 ° C. Next, streptavidin was added to this mixed solution, and DNA derived from the library before OSM stimulation and DNA derived from the library after OSM stimulation associated with the library before OSM stimulation were removed by butanol extraction. The remaining DNA derived from the library for one hour after OSM stimulation was used as a subtracted library. Clones were arbitrarily picked up from this subtracted library and sequenced with a fluorescent automatic DNA sequencer to obtain the mouse EZI gene. The mouse EZI gene is a DN that encodes a protein having an amino acid sequence represented by SEQ ID NO: 2 and composed of 594 amino acids.
It was A (SEQ ID NO: 1).

[Example 2] Structural analysis of mouse EZI protein The structure of mouse EZI protein encoded by the mouse EZI gene cloned in Example 1 was analyzed using known motif analysis software. As a result, mouse EZ
The I protein was found to be a zinc finger protein containing 12 zinc finger motifs in the sequence. In general, zinc finger proteins are based on the amino acid sequences that make up the zinc finger region,
It is classified into four types: C2-H2, CH3, C3H and C4.
Mouse EZI protein contains a sequence containing two cysteine residues at the N-terminal side of its zinc finger region (CXXC sequence) and a sequence containing two histidine residues at the C-terminal side (HXXXH sequence), It was found to be a C2-H2 type zinc finger protein. To date, EKLF and LKLF have been identified as belonging to the C2-H2 type zinc finger protein.
And other Kruppel-like transcription factors are known and many of them have been reported to be involved in the developmental process.
[Nuez, B. et al .: Nature, 375: 316-318 (1995)], these target genes have not yet been identified. In addition, the mouse EZI protein has a nuclear localization signal (in FIG. 1,
It was also found to include the part indicated by the box).

[Example 3] Induction of expression and tissue distribution of mouse EZI gene The mouse EZI gene was cloned into oncostatin M (oncostatin M ( OSM) was identified as a gene that is induced when stimulated. Based on this finding, the induction of mouse EZI mRNA in LO cells was investigated in detail. As a result of Northern hybridization, it was revealed that the expression level of mouse EZI gene was rapidly increased 0.5 hours after OSM stimulation, and the expression level returned to the basal level 1 hour after OSM stimulation. Next, the results of examining the tissue distribution of mouse EZI mRNA by RT-PCR are shown in FIG. For RT-PCR, the following primer pair: 5'-gcactcctgctcagggca-3 '(SEQ ID NO: 5), 5'-
cttgtcgcactctgagca-3 '(SEQ ID NO: 6) was used. As shown in Figure 2, mouse EZI mRNA was detected in all tissues tested. These results suggest that the mouse EZI protein may be responsible for maintaining common intracellular functions in normal adult tissues, although it may have unique functions in certain cases.

In addition, mouse EZ during mouse embryo development
ImRNA expression was examined by RT-PCR. The results are shown in Fig. 3. In FIG. 3, E7.5, E11.5, E14.5, and E18.5 are embryonic day 7.5 embryos, embryonic day 11.5 embryos, and embryonic day 14.5, respectively.
It is a result when using RNA extracted from the day 1 embryo and embryo 18.5 day embryo. As is clear from FIG. 3, mouse EZIm
The expression level of RNA increased with the progress of development.

Then, each tissue of the mouse embryo on day 11.5 (AG
The expression of mouse EZI mRNA in M region, liver, brain and leg) was examined by RT-PCR. The results are shown in Fig. 4. As is clear from FIG. 4, particularly high expression was observed in the AGM region. Then, AGM was cultured and the expression of mouse EZI mRNA was examined over time (1 day, 3 days, 5 days, and 7 days after the start of culture). The results are shown in Fig. 5. As shown in FIG.
Although the expression level of mouse EZI mRNA was slight 1 day after the start of the culture, it increased with the elapse of the culture time and reached its maximum 7 days after the start of the culture.

Example 4 Intracellular Localization of Mouse EZI Protein The EZI protein is a member of the zinc finger protein and is considered to function as a transcription factor. To confirm this, first, the intracellular localization of the mouse EZI protein was examined. That is, Myc peptide tag (t
COS7 cells were transfected with an expression vector containing DNA encoding the mouse EZI protein with ag) added.
Then, the mouse EZI protein tagged with Myc peptide was
Visualization was performed using a Myc tag antibody and FITC-labeled anti-mouse antibody, and mouse EZI protein was detected using a confocal microscope. Nuclei were counterstained with propidium iodide. The staining pattern of mouse EZI completely matched the staining pattern of propidium iodide, demonstrating that the mouse EZI protein was localized in the nucleus in the transient expression system using COS7.

[Example 5] Transcription promoting activity of EZI protein Transcriptional regulation is a major function of zinc finger proteins. Therefore, it was investigated whether the EZI protein has such a function. That is, first mouse EZ
Mouse EZI for investigating the transcription promoting activity of I protein
The protein expression vector was transfected into COS cells together with a vector containing DNA in which a luciferase gene (used as a reporter gene) was linked downstream of various different promoters (SRα, FOS, APRE, β-cas, OSM, CIS promoter). . Two days after the transfection, the cells were harvested and the obtained cells were lysed with a lysis buffer. The luciferase activity of the lysate was measured. The measurement results are shown in FIG. As a control (mock), the luciferase activity of COS cells transfected with the vector pME18S was measured.

As is clear from FIG. 6, APRE, β-cas,
Only when the luciferase gene was ligated downstream of the OSM and CIS promoters, higher luciferase activity was observed compared to the control. This indicates that the APRE, β-cas, OSM and CIS promoters were activated by the EZI protein expressed in COS cells. A
PRE, β-cas, OSM and CIS promoters are all STA
It is known to be a promoter activated by T3 protein and STAT5 protein. Therefore, EZ
It was speculated that the I protein cooperates with the STAT protein to activate transcription.

A gel shift assay was performed to reveal the DNA binding sequence of the mouse EZI protein. The β-cas promoter sequence, whose transcription is activated by the mouse EZI protein, was used in this assay. However, no sequence was found to bind specifically to the mouse EZI protein. This suggests that EZI protein alone does not bind to DNA and may function in the form of a transcription complex.

In order to investigate the possibility that the EZI protein forms a transcription complex with other proteins, the above-mentioned experiments pointed out a synergistic action with the EZI protein.
Immunoprecipitation experiments with proteins were performed. That is, first
An expression vector incorporating the EZI gene and the STAT3 gene was introduced into COS cells by the lipofection method, and these proteins were forcibly expressed in the cells. On the second day after the introduction, COS was performed using a cell lysis buffer for protein extraction.
Protein was extracted from the cells. An anti-Myc antibody against the Myc label added to EZI was added to this extract, followed by the addition of protein A sepharose that specifically binds to the Fc region of the antibody to selectively remove the Myc-labeled EZI protein. Allowed to settle. The precipitate was electrophoresed on an acrylamide gel under denaturing conditions and transferred onto a protein-adsorbing membrane. When this membrane was reacted in an anti-STAT3 antibody solution and then detected, a STAT3 band was detected. This result indicates that the EZI protein complex precipitated with the anti-Myc antibody contains STAT3. From the above experimental results, it was revealed that the EZI protein has a binding ability with the STAT3 protein.

Example 6 Antibody against Mouse EZI Protein An antibody against mouse EZI protein was prepared as follows. That is, first, the mouse EZI protein C
Peptide consisting of 12 amino acids in the 12th zinc finger motif on the terminal side N-Lys Ser Phe Ser Arg Lys
Thr His Leu Val Arg His Gln Arg Ile-C (SEQ ID NO: 7) was chemically synthesized. Then, a rabbit was immunized with the obtained synthetic peptide. Whole serum was collected 9 weeks after the first immunization. The finally obtained whole serum was affinity purified using an NHS activated Hi-trap column (AP-Biotech) to obtain an antibody against the mouse EZI protein.

[Example 7] Search for human EZI gene Next, the human homolog gene of mouse EZI gene was replaced with Ensem.
We searched from the bl database (http://www.ensembl.org/). As a result, a gene (SEQ ID NO: 8) encoding a protein (SEQ ID NO: 9) having 86% homology with the mouse EZI protein was found. FIG. 7 shows an alignment of the amino acid sequences of mouse EZI protein (SEQ ID NO: 2) and human EZI protein (SEQ ID NO: 9). Furthermore, the structure of the amino acid sequence of the obtained human EZI protein was analyzed using motif analysis software.
As a result, as shown in FIG. 8, the human EZI protein, like the mouse EZI protein, contained 12 zinc finger motifs in the sequence, and at the fourth zinc finger motif counted from the N-terminal side, It was found to be a C2-H2 type zinc finger protein containing a translocation signal (portion indicated by a box in Fig. 8).

[0093]

INDUSTRIAL APPLICABILITY According to the present invention, novel zinc finger protein EZI derived from mouse and human, EZI gene encoding the protein, recombinant vector containing the gene, transformant containing the recombinant vector, and EZI protein And a method for manufacturing the same are provided. INDUSTRIAL APPLICABILITY The present invention is useful for screening candidate drugs for therapeutic agents for diseases associated with EZI protein.

[0094]

[Sequence list]                                SEQUENCE LISTING           <110> PharmaDesign, Inc.    <120> A Novel Zinc Finger Protein EZI.    <130> PDP-0014    <160> 11    <170> PatentIn Ver. 2.1    <210> 1 <211> 1785 <212> DNA <213> Mus musculus    <220> <221> CDS <222> (1) .. (1782)    <400> 1 atg aga gag acc ctg gag gcc ctg aac tcc ttg gga ttc tca gtg gga 48 Met Arg Glu Thr Leu Glu Ala Leu Asn Ser Leu Gly Phe Ser Val Gly   1 5 10 15 cag cca gag atg gct ccc cag agt gag ccc agg gat ggg ttc agt aat 96 Gln Pro Glu Met Ala Pro Gln Ser Glu Pro Arg Asp Gly Phe Ser Asn              20 25 30 gcc cag gag aag atg tca tcc aga gga gag agc aca ctg cac tcc tgc 144 Ala Gln Glu Lys Met Ser Ser Arg Gly Glu Ser Thr Leu His Ser Cys          35 40 45 tca ggg cat gag act cct ggc cag aaa gaa ggc atc cac aca gaa caa 192 Ser Gly His Glu Thr Pro Gly Gln Lys Glu Gly Ile His Thr Glu Gln      50 55 60 gct gaa gct cct tgt atg ggc agc cag gcc agt acc cca cag aag gcc 240 Ala Glu Ala Pro Cys Met Gly Ser Gln Ala Ser Thr Pro Gln Lys Ala  65 70 75 80 gaa cct gca ggc tct gtc cca ggg gag gaa tgg atg att cgg aag gtg 288 Glu Pro Ala Gly Ser Val Pro Gly Glu Glu Trp Met Ile Arg Lys Val                  85 90 95 aag gta gag gat gag gat cag gag gca gag gag gag gtg gaa tgg ccc 336 Lys Val Glu Asp Glu Asp Gln Glu Ala Glu Glu Glu Val Glu Trp Pro             100 105 110 cag cat ctc tcc ttc ctt ccc agt cct ttt ccc act cct gac ttg ggt 384 Gln His Leu Ser Phe Leu Pro Ser Pro Phe Pro Thr Pro Asp Leu Gly         115 120 125 cag ttg gct gtt acg tac aaa ctg gag cca ggg act ccg gga gca cta 432 Gln Leu Ala Val Thr Tyr Lys Leu Glu Pro Gly Thr Pro Gly Ala Leu     130 135 140 ggt gga atc gcg ctg tcc ggg tgg gcc ccg atc cct gag aag cct tat 480 Gly Gly Ile Ala Leu Ser Gly Trp Ala Pro Ile Pro Glu Lys Pro Tyr 145 150 155 160 ggc tgc gag gaa tgc gag cgg cgt ttc cgg gat cag cta act ctg cgg 528 Gly Cys Glu Glu Cys Glu Arg Arg Phe Arg Asp Gln Leu Thr Leu Arg                 165 170 175 ctc cac cag agg ttg cac cgg ggt gag ggt cct tgt gcc tgc ccg gac 576 Leu His Gln Arg Leu His Arg Gly Glu Gly Pro Cys Ala Cys Pro Asp             180 185 190 tgt ggc cgc agc ttc acg cag cgt gct cat atg ctc ctg cac caa cgc 624 Cys Gly Arg Ser Phe Thr Gln Arg Ala His Met Leu Leu His Gln Arg         195 200 205 agc cac cgc gga gag cgt ccc ttc ccg tgc tca gag tgc gac aag cgc 672 Ser His Arg Gly Glu Arg Pro Phe Pro Cys Ser Glu Cys Asp Lys Arg     210 215 220 ttc agc aag aag gcc cac ctg acc cgc cac ctg cgc acg cac act ggc 720 Phe Ser Lys Lys Ala His Leu Thr Arg His Leu Arg Thr His Thr Gly 225 230 235 240 gag cgt ccc tac ccg tgc gct gag tgc ggc aaa cgc ttc agc cag aag 768 Glu Arg Pro Tyr Pro Cys Ala Glu Cys Gly Lys Arg Phe Ser Gln Lys                 245 250 255 att cac cta ggc tcg cat cag aag acc cac acc gga gag cgg ccc ttc 816 Ile His Leu Gly Ser His Gln Lys Thr His Thr Gly Glu Arg Pro Phe             260 265 270 ccc tgc acc gaa tgc gag aaa cgt ttc cgc aag aag acg cat ctg atc 864 Pro Cys Thr Glu Cys Glu Lys Arg Phe Arg Lys Lys Thr His Leu Ile         275 280 285 cgc cac cag cgt atc cac acc ggc gag agg ccc tac cag tgc acc cag 912 Arg His Gln Arg Ile His Thr Gly Glu Arg Pro Tyr Gln Cys Thr Gln     290 295 300 tgc acg cgc agc ttc aca cac aag caa cac ctg gtg cgg cac cag agg 960 Cys Thr Arg Ser Phe Thr His Lys Gln His Leu Val Arg His Gln Arg 305 310 315 320 gtg cac gat gct gct agc cgc acc cgg tcc tct cca gac att cct gtt 1008 Val His Asp Ala Ala Ser Arg Thr Arg Ser Ser Pro Asp Ile Pro Val                 325 330 335 gct ccc cat tcc ccc acc gcg tct ctt acc ccg tcc cct cct ggg ccc 1056 Ala Pro His Ser Pro Thr Ala Ser Leu Thr Pro Ser Pro Pro Gly Pro             340 345 350 aag cct ttc gct tgt tcc cac tgc ggg cag agc ttc ggc tgg aaa aag 1104 Lys Pro Phe Ala Cys Ser His Cys Gly Gln Ser Phe Gly Trp Lys Lys         355 360 365 aac ctc gct acg cac cag agt ctg cat ctc acc gag ggt cgt cct ttt 1152 Asn Leu Ala Thr His Gln Ser Leu His Leu Thr Glu Gly Arg Pro Phe     370 375 380 ggg tgc gat gaa tgt gca ctc ggc acc aac gtg gac ccc gcc gcc gag 1200 Gly Cys Asp Glu Cys Ala Leu Gly Thr Asn Val Asp Pro Ala Ala Glu 385 390 395 400 ccc tcg gcc tgc act ccc cat gcg cct gac tgt gga ccg ggt tcg ggg 1248 Pro Ser Ala Cys Thr Pro His Ala Pro Asp Cys Gly Pro Gly Ser Gly                 405 410 415 ccc gcg gca ccc cag cgc acc acc tcc agc gag cgc tcc ttc ttc tgc 1296 Pro Ala Ala Pro Gln Arg Thr Thr Ser Ser Glu Arg Ser Phe Phe Cys             420 425 430 ccg gac tgc ggg cga ggc ttt gcc cac ggg cag cac ctg gcc cgc cac 1344 Pro Asp Cys Gly Arg Gly Phe Ala His Gly Gln His Leu Ala Arg His         435 440 445 cgg cgg gtg cac acg ggc gaa agg cca ttt gcc tgt gct cag tgt ggc 1392 Arg Arg Val His Thr Gly Glu Arg Pro Phe Ala Cys Ala Gln Cys Gly     450 455 460 cgc cgc ttc ggc tca cga ccc aat cta gtc gcc cac tcc cgg gcc cat 1440 Arg Arg Phe Gly Ser Arg Pro Asn Leu Val Ala His Ser Arg Ala His 465 470 475 480 agc ggc gcc aga cct ttt gcc tgt gcg caa tgt ggc cgc cgc ttc agt 1488 Ser Gly Ala Arg Pro Phe Ala Cys Ala Gln Cys Gly Arg Arg Phe Ser                 485 490 495 cgc aaa tct cac ctg ggc cgc cac cag gca gtg cac act ggt agt cgc 1536 Arg Lys Ser His Leu Gly Arg His Gln Ala Val His Thr Gly Ser Arg             500 505 510 ccc cac gcc tgc gcc gtc tgc gcc cgc tgc ttc agc tct aaa acc aac 1584 Pro His Ala Cys Ala Val Cys Ala Arg Cys Phe Ser Ser Lys Thr Asn         515 520 525 ctg gtc cgc cat cag gca atc cat aca ggt tcc cgc ccc ttt tcc tgc 1632 Leu Val Arg His Gln Ala Ile His Thr Gly Ser Arg Pro Phe Ser Cys     530 535 540 cct cag tgc gcc aag agc ttc agc cgc aag acc cat ctg gtg cgg cac 1680 Pro Gln Cys Ala Lys Ser Phe Ser Arg Lys Thr His Leu Val Arg His 545 550 555 560 caa cgc atc cat ggg gac gcg gcc ctc cca gcc cca gcc tcg aac ctc 1728 Gln Arg Ile His Gly Asp Ala Ala Leu Pro Ala Pro Ala Ser Asn Leu                 565 570 575 tct gct cca gcc tgg tcc aat ccc tcc gag gtg gta cca cct cca atc 1776 Ser Ala Pro Ala Trp Ser Asn Pro Ser Glu Val Val Pro Pro Pro Ile             580 585 590 ttt ttc taa 1785 Phe Phe       <210> 2 <211> 594 <212> PRT <213> Mus musculus    <400> 2 Met Arg Glu Thr Leu Glu Ala Leu Asn Ser Leu Gly Phe Ser Val Gly   1 5 10 15 Gln Pro Glu Met Ala Pro Gln Ser Glu Pro Arg Asp Gly Phe Ser Asn              20 25 30 Ala Gln Glu Lys Met Ser Ser Arg Gly Glu Ser Thr Leu His Ser Cys          35 40 45 Ser Gly His Glu Thr Pro Gly Gln Lys Glu Gly Ile His Thr Glu Gln      50 55 60 Ala Glu Ala Pro Cys Met Gly Ser Gln Ala Ser Thr Pro Gln Lys Ala  65 70 75 80 Glu Pro Ala Gly Ser Val Pro Gly Glu Glu Trp Met Ile Arg Lys Val                  85 90 95 Lys Val Glu Asp Glu Asp Gln Glu Ala Glu Glu Glu Val Glu Trp Pro             100 105 110 Gln His Leu Ser Phe Leu Pro Ser Pro Phe Pro Thr Pro Asp Leu Gly         115 120 125 Gln Leu Ala Val Thr Tyr Lys Leu Glu Pro Gly Thr Pro Gly Ala Leu     130 135 140 Gly Gly Ile Ala Leu Ser Gly Trp Ala Pro Ile Pro Glu Lys Pro Tyr 145 150 155 160 Gly Cys Glu Glu Cys Glu Arg Arg Phe Arg Asp Gln Leu Thr Leu Arg                 165 170 175 Leu His Gln Arg Leu His Arg Gly Glu Gly Pro Cys Ala Cys Pro Asp             180 185 190 Cys Gly Arg Ser Phe Thr Gln Arg Ala His Met Leu Leu His Gln Arg         195 200 205 Ser His Arg Gly Glu Arg Pro Phe Pro Cys Ser Glu Cys Asp Lys Arg     210 215 220 Phe Ser Lys Lys Ala His Leu Thr Arg His Leu Arg Thr His Thr Gly 225 230 235 240 Glu Arg Pro Tyr Pro Cys Ala Glu Cys Gly Lys Arg Phe Ser Gln Lys                 245 250 255 Ile His Leu Gly Ser His Gln Lys Thr His Thr Gly Glu Arg Pro Phe             260 265 270 Pro Cys Thr Glu Cys Glu Lys Arg Phe Arg Lys Lys Thr His Leu Ile         275 280 285 Arg His Gln Arg Ile His Thr Gly Glu Arg Pro Tyr Gln Cys Thr Gln     290 295 300 Cys Thr Arg Ser Phe Thr His Lys Gln His Leu Val Arg His Gln Arg 305 310 315 320 Val His Asp Ala Ala Ser Arg Thr Arg Ser Ser Pro Asp Ile Pro Val                 325 330 335 Ala Pro His Ser Pro Thr Ala Ser Leu Thr Pro Ser Pro Pro Gly Pro             340 345 350 Lys Pro Phe Ala Cys Ser His Cys Gly Gln Ser Phe Gly Trp Lys Lys         355 360 365 Asn Leu Ala Thr His Gln Ser Leu His Leu Thr Glu Gly Arg Pro Phe     370 375 380 Gly Cys Asp Glu Cys Ala Leu Gly Thr Asn Val Asp Pro Ala Ala Glu 385 390 395 400 Pro Ser Ala Cys Thr Pro His Ala Pro Asp Cys Gly Pro Gly Ser Gly                 405 410 415 Pro Ala Ala Pro Gln Arg Thr Thr Ser Ser Glu Arg Ser Phe Phe Cys             420 425 430 Pro Asp Cys Gly Arg Gly Phe Ala His Gly Gln His Leu Ala Arg His         435 440 445 Arg Arg Val His Thr Gly Glu Arg Pro Phe Ala Cys Ala Gln Cys Gly     450 455 460 Arg Arg Phe Gly Ser Arg Pro Asn Leu Val Ala His Ser Arg Ala His 465 470 475 480 Ser Gly Ala Arg Pro Phe Ala Cys Ala Gln Cys Gly Arg Arg Phe Ser                 485 490 495 Arg Lys Ser His Leu Gly Arg His Gln Ala Val His Thr Gly Ser Arg             500 505 510 Pro His Ala Cys Ala Val Cys Ala Arg Cys Phe Ser Ser Lys Thr Asn         515 520 525 Leu Val Arg His Gln Ala Ile His Thr Gly Ser Arg Pro Phe Ser Cys     530 535 540 Pro Gln Cys Ala Lys Ser Phe Ser Arg Lys Thr His Leu Val Arg His 545 550 555 560 Gln Arg Ile His Gly Asp Ala Ala Leu Pro Ala Pro Ala Ser Asn Leu                 565 570 575 Ser Ala Pro Ala Trp Ser Asn Pro Ser Glu Val Val Pro Pro Pro Ile             580 585 590 Phe Phe                                                                      <210> 3 <211> 20 <212> DNA <213> Artificial Sequence    <220> <223> Description of Artificial Sequence: synthetic DNA    <400> 3 atgagagaga ccctggaggc 20       <210> 4 <211> 20 <212> DNA <213> Artificial Sequence    <220> <223> Description of Artificial Sequence: synthetic DNA    <400> 4 ttagaaaaag attggaggtg 20       <210> 5 <211> 18 <212> DNA <213> Artificial Sequence    <220> <223> Description of Artificial Sequence: synthetic DNA    <400> 5 gcactcctgc tcagggca 18       <210> 6 <211> 18 <212> DNA <213> Artificial Sequence    <220> <223> Description of Artificial Sequence: synthetic DNA    <400> 6 cttgtcgcac tctgagca 18       <210> 7 <211> 15 <212> PRT <213> Artificial Sequence    <220> <223> Description of Artificial Sequence: synthetic peptide    <400> 7 Lys Ser Phe Ser Arg Lys Thr His Leu Val Arg His Gln Arg Ile   1 5 10 15       <210> 8 <211> 1515 <212> DNA <213> Homo sapiens    <220> <221> CDS <222> (1) .. (1515)    <400> 8 atg att cgg aag gtg aag gtg gag gac gaa gat cag gag gca gaa gag 48 Met Ile Arg Lys Val Lys Val Glu Asp Glu Asp Gln Glu Ala Glu Glu   1 5 10 15 gag gtc gaa tgg ccc cag cat cta tcg tta ctt ccc agc ccc ttt ccc 96 Glu Val Glu Trp Pro Gln His Leu Ser Leu Leu Pro Ser Pro Phe Pro              20 25 30 gcg cct gac ctg ggg cat ctg gct gcc gcg tac aaa ctg gag cca ggg 144 Ala Pro Asp Leu Gly His Leu Ala Ala Ala Tyr Lys Leu Glu Pro Gly          35 40 45 gcc ccg ggg gca ctg agt ggg ctc gcg ctg tct ggg tgg ggt ccg atg 192 Ala Pro Gly Ala Leu Ser Gly Leu Ala Leu Ser Gly Trp Gly Pro Met      50 55 60 ccg gag aag ccc tac ggc tgc ggg gag tgt gag cgg cgc ttc cgg gac 240 Pro Glu Lys Pro Tyr Gly Cys Gly Glu Cys Glu Arg Arg Phe Arg Asp  65 70 75 80 cag ctg acg ttg cga ctg cac cag cgg ctg cac cgg ggc gag ggc ccc 288 Gln Leu Thr Leu Arg Leu His Gln Arg Leu His Arg Gly Glu Gly Pro                  85 90 95 tgc gcc tgc ccg gac tgc ggc cgc agc ttc acg cag cgc gcc cac atg 336 Cys Ala Cys Pro Asp Cys Gly Arg Ser Phe Thr Gln Arg Ala His Met             100 105 110 cta ctg cat cag cgc agc cac cgc ggc gag cgg cct ttc ccg tgc tcc 384 Leu Leu His Gln Arg Ser His Arg Gly Glu Arg Pro Phe Pro Cys Ser         115 120 125 gag tgc gac aag cgc ttc agc aag aag gcc cat ctg acc cgc cac ctg 432 Glu Cys Asp Lys Arg Phe Ser Lys Lys Ala His Leu Thr Arg His Leu     130 135 140 cgc acg cac acg ggc gag cgg ccc tac ccg tgc gcg gag tgc ggc aag 480 Arg Thr His Thr Gly Glu Arg Pro Tyr Pro Cys Ala Glu Cys Gly Lys 145 150 155 160 cgc ttc agc cag aag atc cac ctg ggc tcg cac caa aag acc cac acc 528 Arg Phe Ser Gln Lys Ile His Leu Gly Ser His Gln Lys Thr His Thr                 165 170 175 ggc gag cgg ccc ttc ccc tgc acg gaa tgc gag aag cgc ttt cgc aag 576 Gly Glu Arg Pro Phe Pro Cys Thr Glu Cys Glu Lys Arg Phe Arg Lys             180 185 190 aag acg cac ttg att cgg cac cag cgc atc cat acg ggc gag agg ccc 624 Lys Thr His Leu Ile Arg His Gln Arg Ile His Thr Gly Glu Arg Pro         195 200 205 tac cag tgc gca cag tgc gca cgc agc ttc acg cac aag cag cac ttg 672 Tyr Gln Cys Ala Gln Cys Ala Arg Ser Phe Thr His Lys Gln His Leu     210 215 220 gtg cgg cac caa agg gtg cac cag acg gcc ggc ccg gcc agg ccc tct 720 Val Arg His Gln Arg Val His Gln Thr Ala Gly Pro Ala Arg Pro Ser 225 230 235 240 ccc gac tcg tcc gct tct cct cat tcc act gcc ccg tcc ccg acc cca 768 Pro Asp Ser Ser Ala Ser Pro His Ser Thr Ala Pro Ser Pro Thr Pro                 245 250 255 tcc ttt ccc ggg cca aag cct ttc gcg tgc tcc gac tgc ggc ttg agc 816 Ser Phe Pro Gly Pro Lys Pro Phe Ala Cys Ser Asp Cys Gly Leu Ser             260 265 270 ttc ggc tgg aaa aag aac ctc gcc acg cac cag tgt ctg cac cgc agc 864 Phe Gly Trp Lys Lys Asn Leu Ala Thr His Gln Cys Leu His Arg Ser         275 280 285 gag ggt cgc ccc ttt ggg tgc gat gag tgc gca ctg ggc gcc acc gtg 912 Glu Gly Arg Pro Phe Gly Cys Asp Glu Cys Ala Leu Gly Ala Thr Val     290 295 300 gat gcc ccc gcc gcc aag ccc ctg gcc agc gcg cct ggc gga ccg ggc 960 Asp Ala Pro Ala Ala Lys Pro Leu Ala Ser Ala Pro Gly Gly Pro Gly 305 310 315 320 tgc ggc cca gga tcc gat ccc gtg gtg ccc cag cgc gcc ccc tcg ggc 1008 Cys Gly Pro Gly Ser Asp Pro Val Val Pro Gln Arg Ala Pro Ser Gly                 325 330 335 gag cgg tcc ttc ttc tgc ccg gac tgc ggg cgc ggc ttc tcc cat ggg 1056 Glu Arg Ser Phe Phe Cys Pro Asp Cys Gly Arg Gly Phe Ser His Gly             340 345 350 cag cac ctg gcg cgg cac ccg cgc gtg cac acg ggc gaa cgg ccc ttc 1104 Gln His Leu Ala Arg His Pro Arg Val His Thr Gly Glu Arg Pro Phe         355 360 365 gcc tgc acg cag tgt gac cgc cgc ttc ggc tcg cgg cct aat ctg gtc 1152 Ala Cys Thr Gln Cys Asp Arg Arg Phe Gly Ser Arg Pro Asn Leu Val     370 375 380 gcc cac tcc agg gcc cac agc ggc gcc agg cct ttc gcc tgc gct cag 1200 Ala His Ser Arg Ala His Ser Gly Ala Arg Pro Phe Ala Cys Ala Gln 385 390 395 400 tgc ggc cgc cgc ttc agc cgc aag tcg cac ctg ggc cgc cac cag gcg 1248 Cys Gly Arg Arg Phe Ser Arg Lys Ser His Leu Gly Arg His Gln Ala                 405 410 415 gtg cac act ggc agc cgc ccc cac gcc tgc gcc gtc tgc gcc cgc agc 1296 Val His Thr Gly Ser Arg Pro His Ala Cys Ala Val Cys Ala Arg Ser             420 425 430 ttc agc tcc aaa acc aac cta gtc cgc cac cag gcg atc cac aca ggc 1344 Phe Ser Ser Lys Thr Asn Leu Val Arg His Gln Ala Ile His Thr Gly         435 440 445 tcc cgc ccc ttc tcc tgc ccg cag tgc gga aag agc ttc agc cgc aag 1392 Ser Arg Pro Phe Ser Cys Pro Gln Cys Gly Lys Ser Phe Ser Arg Lys     450 455 460 acc cac ctg gtg cgg cac cag ctc att cac ggc gaa gcc gcc cac gcg 1440 Thr His Leu Val Arg His Gln Leu Ile His Gly Glu Ala Ala His Ala 465 470 475 480 gcc ccg gac gcc gcc ctt gcg gcc cca gcc tgg tcc gct ccc ccc gag 1488 Ala Pro Asp Ala Ala Leu Ala Ala Pro Ala Trp Ser Ala Pro Pro Glu                 485 490 495 gtg gcg ccg ccc ccg ctc ttc ttc tga 1515 Val Ala Pro Pro Pro Leu Phe Phe             500 505       <210> 9 <211> 504 <212> PRT <213> Homo sapiens    <400> 9 Met Ile Arg Lys Val Lys Val Glu Asp Glu Asp Gln Glu Ala Glu Glu   1 5 10 15 Glu Val Glu Trp Pro Gln His Leu Ser Leu Leu Pro Ser Pro Phe Pro              20 25 30 Ala Pro Asp Leu Gly His Leu Ala Ala Ala Tyr Lys Leu Glu Pro Gly          35 40 45 Ala Pro Gly Ala Leu Ser Gly Leu Ala Leu Ser Gly Trp Gly Pro Met      50 55 60 Pro Glu Lys Pro Tyr Gly Cys Gly Glu Cys Glu Arg Arg Phe Arg Asp  65 70 75 80 Gln Leu Thr Leu Arg Leu His Gln Arg Leu His Arg Gly Glu Gly Pro                  85 90 95 Cys Ala Cys Pro Asp Cys Gly Arg Ser Phe Thr Gln Arg Ala His Met             100 105 110 Leu Leu His Gln Arg Ser His Arg Gly Glu Arg Pro Phe Pro Cys Ser         115 120 125 Glu Cys Asp Lys Arg Phe Ser Lys Lys Ala His Leu Thr Arg His Leu     130 135 140 Arg Thr His Thr Gly Glu Arg Pro Tyr Pro Cys Ala Glu Cys Gly Lys 145 150 155 160 Arg Phe Ser Gln Lys Ile His Leu Gly Ser His Gln Lys Thr His Thr                 165 170 175 Gly Glu Arg Pro Phe Pro Cys Thr Glu Cys Glu Lys Arg Phe Arg Lys             180 185 190 Lys Thr His Leu Ile Arg His Gln Arg Ile His Thr Gly Glu Arg Pro         195 200 205 Tyr Gln Cys Ala Gln Cys Ala Arg Ser Phe Thr His Lys Gln His Leu     210 215 220 Val Arg His Gln Arg Val His Gln Thr Ala Gly Pro Ala Arg Pro Ser 225 230 235 240 Pro Asp Ser Ser Ala Ser Pro His Ser Thr Ala Pro Ser Pro Thr Pro                 245 250 255 Ser Phe Pro Gly Pro Lys Pro Phe Ala Cys Ser Asp Cys Gly Leu Ser             260 265 270 Phe Gly Trp Lys Lys Asn Leu Ala Thr His Gln Cys Leu His Arg Ser         275 280 285 Glu Gly Arg Pro Phe Gly Cys Asp Glu Cys Ala Leu Gly Ala Thr Val     290 295 300 Asp Ala Pro Ala Ala Lys Pro Leu Ala Ser Ala Pro Gly Gly Pro Gly 305 310 315 320 Cys Gly Pro Gly Ser Asp Pro Val Val Pro Gln Arg Ala Pro Ser Gly                 325 330 335 Glu Arg Ser Phe Phe Cys Pro Asp Cys Gly Arg Gly Phe Ser His Gly             340 345 350 Gln His Leu Ala Arg His Pro Arg Val His Thr Gly Glu Arg Pro Phe         355 360 365 Ala Cys Thr Gln Cys Asp Arg Arg Phe Gly Ser Arg Pro Asn Leu Val     370 375 380 Ala His Ser Arg Ala His Ser Gly Ala Arg Pro Phe Ala Cys Ala Gln 385 390 395 400 Cys Gly Arg Arg Phe Ser Arg Lys Ser His Leu Gly Arg His Gln Ala                 405 410 415 Val His Thr Gly Ser Arg Pro His Ala Cys Ala Val Cys Ala Arg Ser             420 425 430 Phe Ser Ser Lys Thr Asn Leu Val Arg His Gln Ala Ile His Thr Gly         435 440 445 Ser Arg Pro Phe Ser Cys Pro Gln Cys Gly Lys Ser Phe Ser Arg Lys     450 455 460 Thr His Leu Val Arg His Gln Leu Ile His Gly Glu Ala Ala His Ala 465 470 475 480 Ala Pro Asp Ala Ala Leu Ala Ala Pro Ala Trp Ser Ala Pro Pro Glu                 485 490 495 Val Ala Pro Pro Pro Leu Phe Phe             500       <210> 10 <211> 20 <212> DNA <213> Artificial Sequence    <220> <223> Description of Artificial Sequence: synthetic DNA    <400> 10 atgattcgga aggtgaaggt 20       <210> 11 <211> 20 <212> DNA <213> Artificial Sequence    <220> <223> Description of Artificial Sequence: synthetic DNA    <400> 11 tcagaagaag agcgggggcg 20

[0095]

[Sequence list free text] SEQ ID NO: 3: Synthetic DNA SEQ ID NO: 4: Synthetic DNA SEQ ID NO: 5: Synthetic DNA SEQ ID NO: 6: Synthetic DNA SEQ ID NO: 7: synthetic peptide SEQ ID NO: 10: Synthetic DNA SEQ ID NO: 11: synthetic DNA

[Brief description of drawings]

FIG. 1 shows the amino acid sequence of mouse EZI protein.

FIG. 2 shows the tissue distribution of mouse EZI mRNA.

FIG. 3 is a diagram showing changes over time in the expression level of EZI mRNA during embryogenesis.

FIG. 4 is a view showing EZI mRNA expression levels in each tissue of 11.5 day embryos.

FIG. 5 is a diagram showing changes over time in EZI mRNA expression levels in AGM cultures.

FIG. 6 is a diagram showing the activation ability of mouse EZI protein for each promoter.

FIG. 7: Amino acid sequence of mouse EZI protein and human EZI
It is the figure which showed the alignment of the amino acid sequence of protein.

FIG. 8 shows the amino acid sequence of human EZI protein.

─────────────────────────────────────────────────── ─── Continued Front Page (51) Int.Cl. ⁷ Identification Code FI Theme Coat (Reference) C12N 1/15 C12N 1/19 4H045 1/19 1/21 1/21 C12P 21/02 C 5/10 G01N 33/15 Z C12P 21/02 33/50 Z G01N 33/15 33/53 D 33/50 33/58 Z 33/53 C12N 15/00 ZNAA 33/58 5/00 AF Term (reference) 2G045 AA40 BA13 BB03 BB16 BB20 BB29 CB01 CB21 DA12 DA13 DA14 DA36 FB02 FB03 FB04 FB05 FB06 FB07 4B024 AA01 AA11 BA80 CA04 DA02 DA05 DA06 DA11 DA12 EA02 EA04 GA11 HA11 4B064 AG01 CA02 CA05 CA06 CA10 CA19 CC24 DA01 DA13 4B065 AA01X AA57X AA72X AA90X AA91Y AA93Y AB01 BA02 CA24 CA44 CA46 4C084 AA17 NA14 ZB272 4H045 AA10 AA11 AA20 AA30 BA10 CA40 DA75 EA20 EA50 FA72 FA74

Claims (21)

[Claims] 1. A protein comprising an amino acid sequence identical or substantially identical to the amino acid sequence represented by SEQ ID NO: 2 or SEQ ID NO: 9 or a salt thereof. 2. The protein or a salt thereof according to claim 1, wherein the protein has a C2H2-type zinc finger domain and has a binding ability to a STAT protein. 3. The protein or a salt thereof according to claim 2, wherein the STAT protein is STAT3 protein or STAT5 protein. 4. A partial peptide of the protein according to claim 1, or a salt thereof. 5. A polynucleotide comprising a nucleotide sequence encoding the protein according to any one of claims 1 to 3 or the peptide according to claim 4. 6. The polynucleotide according to claim 5, wherein the polynucleotide is DNA. 7. A polynucleotide having the nucleotide sequence represented by SEQ ID NO: 1 or SEQ ID NO: 8 or a nucleotide sequence hybridizing with them under stringent conditions. 8. The polynucleotide according to claim 7, wherein the polynucleotide is DNA. 9. A recombinant vector containing the polynucleotide according to claim 5. 10. A transformant containing the recombinant vector according to claim 9. 11. A method comprising culturing the transformant according to claim 10, producing and accumulating the protein according to any one of claims 1 to 3 or the peptide according to claim 4, and collecting the protein. The method for producing the protein or salt thereof according to any one of claims 1 to 3 or the peptide or salt thereof according to claim 4. 12. An antibody against the protein according to any one of claims 1 to 3 or a salt thereof, or the partial peptide according to claim 4 or a salt thereof. 13. A test liquid containing the protein according to any one of claims 1 to 3 or a salt thereof or the partial peptide according to claim 4 or a salt thereof against the antibody according to claim 12, and a label. 4. The protein according to any one of claims 1 to 3 or a salt thereof, or the labeled partial peptide according to claim 4 or a salt thereof, which is reacted competitively. A method for quantifying the protein or salt thereof according to any one of claims or the partial peptide or salt thereof according to claim 4. 14. A protein or a salt thereof according to any one of claims 1 to 3 or a partial peptide or a salt thereof according to claim 4 is used, wherein the protein or a salt thereof or a partial peptide thereof or A method for screening a compound that inhibits or promotes the binding between its salt and a STAT protein. 15. The test method, wherein the screening method comprises labeling the protein or salt thereof according to any one of claims 1 to 3 or the labeled partial peptide or salt thereof according to claim 4 and a STAT protein. The labeled protein when contacted in the absence of the compound, and when the labeled protein or salt thereof or the labeled partial peptide or salt thereof and STAT protein are contacted in the presence of a test compound 15. The screening method according to claim 14, further comprising the step of measuring and comparing the amount of binding of the salt, the labeled partial peptide or the salt thereof to the STAT protein, respectively. 16. The STAT protein is STAT3 or STA
The screening method according to claim 14 or 15, wherein the screening method is T5. 17. A protein or a salt thereof, or a salt thereof, which comprises the protein or a salt thereof according to any one of claims 1 to 3 and / or the partial peptide or a salt thereof according to claim 4 as a constituent element. A kit for screening a compound that inhibits or promotes the binding between a partial peptide or a salt thereof and a STAT protein. 18. The screening kit according to claim 17, further comprising a STAT protein as a component. 19. The screening kit according to claim 18, wherein the STAT protein is STAT3 protein or STAT5 protein. 20. The method according to any one of claims 1 to 3, which is obtained by using the screening method according to any one of claims 14 to 16 or the screening kit according to any one of claims 17 to 19. Protein or a salt thereof or the partial peptide or a salt thereof according to claim 4, and STAT
A compound that inhibits or promotes binding to a protein. 21. A pharmaceutical composition comprising a compound according to claim 20.