JP2003116584A - Prophylactic, therapeutic, and diagnostic agent for cancer - Google Patents

Abstract

PROBLEM TO BE SOLVED: To develop an anticancer agent and an antimetastatic agent targeting a molecule specifically expressed in a cancer cell. SOLUTION: A medicine targeting type II core-binding factor alpha subunit 1 (CBFAI) specifically expressed in some cancer tissues such as a mammary cancer tissue is suitable as the anticancer agent, the antimetastatic agent, a diagnostic agent, and a testing agent. Thereby, a compound useful as a prophylactic and therapeutic agent, a metastatic inhibitor or a metastatic prophylactic agent for cancer can be obtained. The cancer or its metastasis can be diagnosed or tested by using the DNA.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a preventive / therapeutic agent for cancer, a metastasis inhibitor, a metastasis preventive agent, a diagnostic agent and the like.

[0002]

2. Description of the Related Art CBFA1 (Core-Binding)
Factor Alpha subunit 1) is R
PEBP2α, which is a transcription factor with an unt domain
1, AML3, OSF2, Til-1 and Runx2
Also called. Nucleotide sequence of mouse PEBP2α1 cDNA (Proc. Natl. Acad. Sci. USA, 90, 6859-6)
863, 1993), nucleotide sequence of human AML3 cDNA (Genomics, 23, 425-432, 1994), mouse OS
Nucleotide sequence of F2 cDNA (Cell, 89, 747-754, 1
997), and the nucleotide sequence of mouse Til-1 cDNA (Proc. Natl. Acad. Sci. USA, 94, 8464-8651, 19).
1997) has already been reported. CBFA1 knockout mouse and human studies have revealed that CBFA1 is a transcription factor essential for bone formation. CBFA1 is a transcription factor capable of binding DNA,
CBFB (Core-Binding Factor B)
It is known to promote transcription by binding to eta subunit) and the like. In addition, CBFA1 has many splicing variants (sometimes referred to as isoforms). Enomoto et al. (J. Biol. Chem.,
275, 8695-8702, 2000) is a splicing variant of mouse cDNA starting from exon 2 (PE
BP2α1 / AML3), TypeI CBFA, a splicing variant starting from exon 1 (OSF)
2 / Til1) is called TypeIICBFA1 respectively. Mundlos et al. (Cell, 89, 773-779, 1997.
Et al.) Reported that similar splicing variants also exist in humans, and the corresponding mouse exons 1 and 2 are referred to as exons 0 and 1, respectively. All of these are names that focus on the difference in the translation initiation exons, and they have a common structure up to the following exons including the translation stop codon. In the present specification, CBFA1 starting from exon 1 is referred to as Ty regardless of species such as human and mouse according to the designation of Mundlos et al.
peI CBFA1 and CBFA1 starting from exon 0 (SEQ ID NO: 1) are collectively referred to as Type II CBFA1. On the other hand, CBFA1 has been reported to be expressed in two types of cancer cell lines (Mol. Cell Biol. Res.
Com., Vol. 3, pp. 218-223, 2000), PCR primers for confirming CBFA1 expression used in this paper include Type I CBFA1 and Type II CBF.
A1 cannot be identified and which isoform is expressed cannot be distinguished. Moreover, the question remains whether expression in systematic cell lines is reflected in actual cancer tissues.

[0003]

Conventional anticancer agents (eg, fluorouracil, etc.) act on cells other than cancer cells, and since many of them show toxicity, they are molecules specifically expressed in cancer cells. The development of anti-cancer and anti-metastatic agents targeting the drug has been earnestly desired from the viewpoint of avoidance of toxicity and side effects.

[0004]

[Means for Solving the Problems] As a result of intensive studies to solve the above problems, the present inventors have found that
TypeII CBFA specifically in some cancer tissues
It was found that the up-regulation of 1 is occurring, and Type I
A drug targeting I CBFA1 is considered to be suitable as an anti-cancer agent, an anti-metastatic agent, a diagnostic agent and a test agent,
As a result of further studies, the present invention has been completed.

That is, the present invention provides (1) SEQ ID NO:
(2) A method for screening a compound or its salt that suppresses the expression of a gene containing a DNA, which comprises using a DNA containing the same or substantially the same nucleotide sequence as the nucleotide sequence represented by 1. A cell having the ability to produce a protein or a salt thereof containing the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 2 is cultured in the presence and absence of the test compound, The screening method according to (1) above, which comprises measuring the amount of the protein or the amount of mRNA encoding the protein in the presence and absence of the protein, respectively (3) SEQ ID NO:
The cell having the ability to produce a protein or a salt thereof containing the same or substantially the same amino acid sequence as the amino acid sequence represented by 2, is the same or substantially the same as the base sequence represented by SEQ ID NO: 1. The screening method according to (2) above, which is a transformant transformed with a recombinant vector containing a DNA containing the nucleotide sequence of
(4) The above (1) to which the DNA is chromosomal DNA
(3) the screening method described, (5) SEQ ID NO:
A method for screening a compound or its salt that suppresses the activity of the protein, which comprises using a protein containing the same or substantially the same amino acid sequence as the amino acid sequence represented by 2. (6) The activity is a transcription activity (7) The screening method according to (5) above, which is
(8) A preventive / therapeutic agent for cancer, a cancer metastasis inhibitor or a cancer metastasis preventive agent, which comprises the compound or a salt thereof obtained by the screening method according to any one of (1) to (6) above.
A diagnostic agent for cancer or its metastasis, which comprises a DNA containing the same or substantially the same base sequence as SEQ ID NO: 1, (9) the same as the base sequence shown in SEQ ID NO: 1. Alternatively, a method of diagnosing cancer or metastasis thereof using DNA containing substantially the same nucleotide sequence, (1
0) A drug containing an antisense polynucleotide containing a base sequence complementary to the base sequence represented by SEQ ID NO: 1 or the same or substantially the same base sequence, or (11) prevention of cancer The drug according to (10) above, which is a therapeutic agent, a cancer metastasis suppressor or a cancer metastasis preventive agent,
(12) DNA containing a nucleotide sequence identical or substantially identical to the nucleotide sequence represented by SEQ ID NO: 3, SEQ ID NO: 4 or SEQ ID NO: 5, (13) SEQ ID NO: 3,
The DNA according to (12) above, which comprises the nucleotide sequence represented by SEQ ID NO: 4 or SEQ ID NO: 5, (14) above (1
2) A recombinant vector containing the DNA described in (15)
The transformant transformed with the recombinant vector according to (14) above, (16) the transformant according to (15) above is cultured, and the same or substantially the same amino acid sequence as represented by SEQ ID NO: 2 is obtained. A method for producing the protein or a salt thereof, which comprises producing a protein containing the same amino acid sequence, (17) containing the same or substantially the same base sequence as the base sequence represented by SEQ ID NO: 1. Wherein the DNA to be used is the DNA according to (12) above, (18) a compound obtained by the screening method according to any one of (1) to (6) above for mammals Or a method for preventing or treating cancer, a method for suppressing cancer metastasis or a method for preventing cancer metastasis, which comprises administering an effective amount of a salt thereof, (19) prevention of cancer
There is provided use of a compound or a salt thereof obtained by the screening method according to any one of (1) to (6) above for producing a therapeutic agent, a cancer metastasis inhibitor or a cancer metastasis preventive agent.

[0006]

BEST MODE FOR CARRYING OUT THE INVENTION A protein containing the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 2 (hereinafter also referred to as a protein used in the present invention) has a sequence. It contains an amino acid sequence encoded by a DNA containing the same or substantially the same base sequence as the base sequence represented by the number 1. The protein used in the present invention is a human or other warm-blooded animal (eg, guinea pig, rat, mouse, chicken, rabbit, pig, sheep, cow, monkey, etc.) cell (eg, hepatocyte, splenocyte, nerve cell). , Glial cells, pancreatic β cells, bone marrow cells, mesangial cells, Langerhans cells, epidermal cells, epithelial cells, goblet cells, endothelial cells, smooth muscle cells, fibroblasts, fiber cells, muscle cells, adipocytes, immune cells (eg. , Macrophages, T cells, B cells, natural killer cells, mast cells, neutrophils, basophils, eosinophils, monocytes), megakaryocytes, synovial cells, chondrocytes, osteocytes, osteoblasts, rupture Bone cells, mammary gland cells, hepatocytes or stromal cells, or progenitor cells of these cells, stem cells or cancer cells) or any tissue in which these cells are present, for example, brain, each of the brain Place (for example, the olfactory bulb, amygdala,
Basal sphere, hippocampus, thalamus, hypothalamus, cerebral cortex, medulla oblongata,
Cerebellum), spinal cord, pituitary gland, stomach, pancreas, kidney, liver, gonad, thyroid, gallbladder, bone marrow, adrenal gland, skin, muscle, lung, digestive tract (eg, large intestine, small intestine), blood vessel, heart, thymus, spleen, Submandibular gland, peripheral blood, prostate, testicles, ovaries, placenta, uterus, bones,
It may be a protein derived from a joint, skeletal muscle, or the like, or may be a synthetic protein.

An amino acid sequence substantially identical to the amino acid sequence represented by SEQ ID NO: 2 is about 85% or more, preferably about 90% of the amino acid sequence represented by SEQ ID NO: 2.
% Or more, more preferably about 95% or more, further preferably about 98% or more homologous amino acid sequences and the like. Examples of the protein containing the amino acid sequence substantially the same as the amino acid sequence represented by SEQ ID NO: 2 include, for example, the amino acid sequence substantially the same as the amino acid sequence represented by SEQ ID NO: 2. A protein having substantially the same activity as the protein containing the amino acid sequence represented by No. 2 is preferable. Examples of the “substantially the same activity” include a transcription promoting activity and the like. The term “substantially the same” means that the activity is qualitatively (eg, physiologically or pharmacologically) the same. Therefore, for example, the transcription promoting activity is equivalent (eg, about 0.01 to 100 times, preferably about 0.1 to 1 times).
It is preferably 0 times, more preferably 0.5 to 2 times), but quantitative factors such as the degree of this activity and the molecular weight of the protein may be different.

(1) 1 or 2 or more (preferably 1 to 30) of the amino acid sequence represented by SEQ ID NO: 2
About 2, preferably 1 to 10, more preferably 1 to 5) amino acid sequence, (2)
1 or 2 or more (preferably about 1 to 30, preferably 1 to 10) in the amino acid sequence represented by SEQ ID NO: 2.
(3, more preferably 1 to 5) amino acid sequence, 1 or 2 or more (preferably 1 to 5) amino acid sequence represented by (3) SEQ ID NO: 2
Amino acid sequence in which (1) or two or more (preferably 1 to 5) amino acids in the amino acid sequence represented by SEQ ID NO: 2 are substituted with other amino acids. Or (5) so-called muteins such as proteins containing an amino acid sequence combining these are also included in the proteins used in the present invention.

The protein in the present specification is a peptide
According to the convention of the mark, the left end is the N-terminal (amino terminal), the right end
Is the C terminal (carboxyl terminal). Used in the present invention
In the protein, the C-terminal has a carboxyl group (-CO
OH), carboxylate (-COO^-), Amide (-C
ONH₂) Or ester (-COOR)
May be. Here, as R in the ester, for example,
For example, methyl, ethyl, n-propyl, isopropyl, n
-C such as butyl_1-6An alkyl group, for example cyclopen
C such as chill and cyclohexyl_3-8Cycloalkyl group,
For example, C such as phenyl and α-naphthyl_6-12Aryl
Groups, for example phenyl-C, such as benzyl, phenethyl, etc.
_1-2Alkyl group or α-, such as α-naphthylmethyl
Naphthyl-C_1-2C such as alkyl group_7-14Aralkyl
Group, pivaloyloxymethyl group and the like are used. Starting
The protein used in Ming is carboxyl other than C-terminal
If it has a group (or carboxylate),
Those in which the voxyl group is amidated or esterified
Is also included in the proteins used in the present invention. in this case
Examples of the ester include the C-terminal ester described above.
Are used. Furthermore, the tamper used in the present invention
N-terminal amino acid residues (eg, methionine residue)
Amino group is a protecting group (eg, formyl group, acetyl group)
C such as ru radical _1-6C such as alkanoyl_1-6Acyl group
Protected by, etc., generated by cutting in vivo
The N-terminal glutamine residue was oxidized by pyroglutamine.
A substituent on the side chain of an amino acid in the molecule (for example, -O
H, -SH, amino group, imidazole group, indole
Groups, guanidino groups, etc.) are suitable protecting groups (eg,
C such as mil group and acetyl group_1-6Such as alkanoyl groups
C_1-6Protected with an acyl group), or
Complex tamper such as so-called glycoprotein in which sugar chains are bound
It also includes quality. Of the proteins used in the present invention
As a specific example, for example, the amino acid represented by SEQ ID NO: 2
Examples thereof include proteins containing a no acid sequence.

The partial peptide of the protein used in the present invention (hereinafter sometimes referred to as the partial peptide used in the present invention) is the partial peptide of the above-mentioned protein used in the present invention, preferably, Any protein may be used as long as it has the same activity as the protein used in the present invention. In addition, the partial peptide used in the present invention may be used in a meaning including its amide form and ester form. For example,
A peptide containing at least 20 or more, preferably 50 or more, more preferably 70 or more, more preferably 100 or more, and most preferably 150 or more amino acid sequences among the constituent amino acid sequences of the protein used in the present invention. Are used. The partial peptide used in the present invention is 1 or 2 in the amino acid sequence.
1 or more (preferably 1 to 20, more preferably 1 to 10 and more preferably 1 to 5) amino acids are deleted, or 1 or 2 or more (preferably 1) amino acid sequence is deleted. ~ About 20, more preferably 1 ~
About 10 amino acids, more preferably 1 to 5 amino acids are added, or 1 or 2 or more (preferably 1 to 20 and more preferably 1 to 1) amino acid sequence is added.
About 0, more preferably 1 to 5 amino acids are inserted, or 1 or 2 or more (preferably about 1 to 20 and more preferably 1 to 1) amino acid sequence is inserted.
About 0, more preferably 1 to 5) amino acids may be substituted with other amino acids.

[0011] In the partial peptide used in the present invention C-terminal carboxyl group (-COOH), a carboxylate (-COO ^-), amide (-CONH ₂₎ or an ester (-COOR) (R is as above defined Is shown). Further, in the case where the partial peptide used in 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 partial peptide used in the present invention. Be done. As the ester in this case, for example, the above-mentioned C-terminal ester or the like is used. Furthermore, in the partial peptide used in the present invention, the amino group of the N-terminal amino acid residue (eg, methionine residue) is protected by a protecting group, as in the above-mentioned protein used in the present invention, Glutamine residue produced by cleavage at the N-terminal side in vivo is pyroglutamine-oxidized, a substituent on the side chain of an amino acid in the molecule is protected by an appropriate protecting group, or a so-called sugar chain-bonded Complex peptides such as glycopeptides are also included. The partial peptide used in the present invention can also be used as an antigen for antibody production.

The salts of the protein or partial peptide used in the present invention include physiologically acceptable acids (eg,
Inorganic acids, organic acids, etc.), bases (eg, alkali metals, etc.)
And the like are used, and physiologically acceptable acid addition salts are particularly preferable. 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). Acid, tartaric acid, citric acid, malic acid, oxalic acid, benzoic acid, methanesulfonic acid, benzenesulfonic acid) and salts thereof are used. The protein or its partial peptide or salt thereof used in the present invention is produced by a known protein purification method from the cells or tissues of the above-mentioned humans or other warm-blooded animals, or a transformant containing a DNA encoding the protein. It can be produced by culturing.
It can also be produced according to the peptide synthesis method described below. When producing from tissues or cells of humans or other warm-blooded animals, after homogenizing tissues or cells of humans or other warm-blooded animals, extraction with acid or the like, the resulting extract is subjected to reverse phase chromatography, Purification and isolation can be achieved by combining chromatography such as ion exchange chromatography.

For the synthesis of the protein or partial peptide used in the present invention, its salt, or its amide, 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-hydroxymethylmethylphenylacetamidomethyl resin, polyacrylamide resin, 4-
Examples thereof include (2 ', 4'-dimethoxyphenyl-hydroxymethyl) phenoxy resin and 4- (2', 4'-dimethoxyphenyl-Fmocaminoethyl) phenoxy resin. Using such a resin, an amino acid having an α-amino group and a side chain functional group appropriately protected is condensed on the resin according to various known condensation methods according to the sequence of the target protein. At the end of the reaction, the protein 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 obtain the target protein or partial peptide or an amide thereof. 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. Carbodiimides include DCC, N, N'-
Diisopropylcarbodiimide, N-ethyl-N'-
(3-Dimethylaminoprolyl) carbodiimide or the like is used. For activation by these, the protected amino acid was directly added to the resin together with a racemization-inhibiting additive (eg, HOBt, HOOBt), or the protected amino acid was previously activated as a symmetrical acid anhydride or HOBt ester or HOOBt ester. It can later be added to the resin.

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

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

The activated carboxyl group of the raw material includes, for example, corresponding acid anhydrides, azides, active esters [alcohols (for example, pentachlorophenol, 2,4,5-trichlorophenol, 2,4- Dinitrophenol, cyanomethyl alcohol, paranitrophenol, HONB, N-hydroxysuccinide, N
-Hydroxyphthalimide, ester with HOBt)]
Are used. As the activated amino group of the raw material, for example, the corresponding phosphoric acid amide is used. Examples of the method for removing (eliminating) the protecting group include Pd
-Catalytic reduction in a hydrogen stream in the presence of a catalyst such as black or Pd-carbon, acid treatment with anhydrous hydrogen fluoride, methanesulfonic acid, trifluoromethanesulfonic acid, trifluoroacetic acid or a mixture thereof, diisopropyl A base treatment with ethylamine, triethylamine, piperidine, piperazine or the like, reduction with sodium in liquid ammonia and the like are used. The elimination reaction by the acid treatment is generally carried out at a temperature of about -20 ° C to 40 ° C. In the acid treatment, for example, anisole, phenol, thioanisole, metacresol, paracresol, dimethyl sulfide, 1,4 -Butanedithiol,
It is effective to add a cation trapping agent such as 1,2-ethanedithiol. Further, the 2,4-dinitrophenyl group used as the imidazole protecting group of histidine is removed by thiophenol treatment, and the formyl group used as the indole protecting group of tryptophan is the above 1,2-ethanedithiol, 1,4-butane group. In addition to deprotection by acid treatment in the presence of dithiol or the like, it is also removed by alkali treatment with dilute sodium hydroxide solution, dilute ammonia or the like.

The protection of functional groups that should not participate in the reaction of the starting materials, elimination of the protecting groups, activation of the functional groups that participate in the reaction, etc. can be carried out by known means, and of functional groups that should not participate in the reaction of the starting materials. The protective group that can be used for protection can be appropriately selected and applied from known groups. As another method for obtaining an amide form of a protein or partial peptide, for example, first, the α-carboxyl group of the carboxy-terminal amino acid is amidated and protected, and then the peptide (protein) chain is added to the amino group side up to the desired chain length. After extension, the α at the N-terminus of the peptide chain
A protein or partial peptide from which only the amino-protecting group has been removed and a protein or partial peptide from which only the C-terminal carboxyl-protecting group has been removed, and these proteins or partial peptides in a mixed solvent as described above To condense. The details of the condensation reaction are the same as above. After purifying the protected protein or peptide obtained by condensation, all the protecting groups can be removed by the above method to obtain the desired crude protein or partial peptide. The crude protein or partial peptide can be purified using various known purification means, and the main fraction can be lyophilized to obtain the amide of the desired protein or partial peptide. To obtain an ester form of a protein or partial peptide, for example, the α-carboxyl group of the carboxy-terminal amino acid is condensed with a desired alcohol to form an amino acid ester, which is then treated in the same manner as the amide form of the protein or partial peptide. An ester form of a protein or partial peptide can be obtained.

The protein used in the present invention and its partial peptide or a salt thereof can be produced by a known peptide synthesis method or by cleaving the protein used in the present invention with an appropriate peptidase. The peptide synthesis method may be, for example, either a solid phase synthesis method or a liquid phase synthesis method. That is,
The target peptide can be produced by condensing the partial peptide or amino acid that can form the partial peptide used in the present invention with the remaining portion and removing the protecting group when the product has the protecting group. Examples of known condensation methods and removal of protective groups include the methods described in the following (1) to (3). M. Bodanszky and MA Ondetti, Peptide Synthesis, Interscience Publisher
s, New York (1966) Schroeder and Luebke, The Peptid
e), Academic Press, New York (1965) Nobuo Izumiya et al., Fundamentals and experiments of peptide synthesis, Maruzen Co., Ltd.
(1975) Haruaki Yajima and Shunpei Sakakibara, Laboratory of Biochemistry 1, Protein Chemistry IV, 205, (1977) Supervised by Haruaki Yajima, Development of Pharmaceuticals, Volume 14, Peptide Synthesis, Hirokawa Shoten After that, the partial peptide used in the present invention can be purified and isolated by combining ordinary purification methods such as solvent extraction, distillation, column chromatography, liquid chromatography, and recrystallization. When the protein or partial peptide obtained by the above method is a free form, it can be converted into a suitable salt by a known method or a method analogous thereto, and conversely, when it is obtained by a salt, a known method or It can be converted into an educt or other salt by a method analogous thereto.

A DNA containing the same or substantially the same nucleotide sequence as the nucleotide sequence represented by SEQ ID NO: 1 (hereinafter, also referred to as DNA used in the present invention).
May be any of genomic DNA, genomic DNA library, cDNA derived from the cells or tissues described above, cDNA library derived from the cells or tissues described above, or synthetic DNA. The vector used for the library may be any of bacteriophage, plasmid, cosmid, phagemid and the like. In addition, reverse Transcriptase Polymerase Chain can be directly used by preparing total RNA or mRNA fraction from the cells or tissues described above.
It can also be amplified by a reaction (hereinafter, abbreviated as RT-PCR method). The DNA containing the base sequence represented by SEQ ID NO: 1 is a DNA containing the base sequence substantially the same as the base sequence represented by SEQ ID NO: 1.
Any DNA can be used as long as it has a DNA that hybridizes under high stringency conditions and that encodes a protein having substantially the same properties as the protein used in the present invention.

The DNA which can hybridize with the DNA containing the nucleotide sequence represented by SEQ ID NO: 1 under high stringent conditions includes, for example, about 85% or more of the nucleotide sequence represented by SEQ ID NO: 1, Preferably about 90% or more, more preferably about 95%, more preferably about 9%.
A DNA or the like containing a nucleotide sequence having a homology of 8% or more is used. Hybridization can be carried out by a known method or a method analogous thereto, for example, molecular
Cloning (Molecular Cloning) 2nd (J. Sambro
ok et al., Cold Spring Harbor Lab. Press, 1989). When a commercially available library is used, it can be performed according to the method described in the attached instruction manual. More preferably,
It can be performed under high stringent conditions. The high stringent condition means, for example, a sodium concentration of about 19 to 40 mM, preferably about 19 to 20.
mM, temperature about 50-70 ° C, preferably about 60-6
The conditions of 5 ° C. are shown. Especially, the sodium concentration is about 19 mM
Most preferably, the temperature is about 65 ° C.

The DNA encoding the partial peptide used in the present invention may be any DNA as long as it contains the base sequence encoding the partial peptide used in the present invention. Also, genomic DNA,
Genomic DNA library, cDNA derived from the cells or tissues described above, cDNA derived from the cells or tissues described above
Either a library or synthetic DNA may be used. As the DNA encoding the partial peptide used in the present invention,
For example, D containing the base sequence represented by SEQ ID NO: 1
It has a DNA containing a partial base sequence of NA, or a DNA containing the base sequence represented by SEQ ID NO: 1, which hybridizes under high stringent conditions, and is substantially equivalent to the protein used in the present invention. For example, a DNA containing a partial base sequence of a DNA encoding a protein having the same activity is used. The same hybridization method and high stringent conditions as described above are used.

The protein used in the present invention or the partial peptide used in the present invention (hereinafter, in the explanation of cloning and expression of DNA encoding these,
(These may be simply abbreviated as the protein used in the present invention.) As a means for cloning DNA that completely encodes, a synthetic DNA primer containing a part of the nucleotide sequence encoding the protein used in the present invention is used. DNA amplified by a PCR method or incorporated into an appropriate vector, and selected by hybridization with a DNA fragment encoding a part or the whole region of the protein used in the present invention or labeled with a synthetic DNA. be able to. The method of hybridization is described in, for example, Molecular Cloning 2nd (J. Sambrook et al.
al., Cold Spring Harbor Lab. Press, 1989). When a commercially available library is used, it can be performed according to the method described in the attached instruction manual. Replacement of the nucleotide sequence of DNA is performed by PCR or a known kit, for example, Mutan ^™ -superExp
ress Km (Takara Shuzo), Mutan ^TM -K (Takara Shuzo)
ODA-LA PCR method, Gapped duplex method, Kunke
It can be carried out according to a known method such as the method l or a method similar thereto. The cloned DNA encoding the protein 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 has ATG as a translation initiation codon at the 5'-terminal side, and TAA, TGA or TA as a translation stop codon at the 3'-terminal side.
It may have G. These translation initiation codon and translation termination codon can be added using an appropriate synthetic DNA adaptor. The expression vector of the protein used in the present invention is, for example, (a) a DNA fragment of interest is cut out from the DNA encoding the protein used in the present invention, and (b) the DNA fragment of a promoter in an appropriate expression vector is used. It can be manufactured by connecting to the downstream.

As a vector, a plasmid derived from E. coli (eg pBR322, pBR325, pUC12, pBR322
UC13), a Bacillus subtilis-derived plasmid (eg, pUB11)
0, pTP5, pC194), yeast-derived plasmids (eg, pSH19, pSH15), bacteriophages such as λ phage, animal viruses such as retrovirus, vaccinia virus, baculovirus, etc.
A1-11, pXT1, pRc / CMV, pRc / RS
V, pcDNAI / Neo, etc. are used. The promoter used in the present invention may be any promoter as long as it is suitable for the host used for gene expression. For example, when an animal cell is used as a host, SRα promoter, SV40 promoter, L promoter
TR promoter, CMV promoter, HSV-TK
Examples include promoters. Of these, CMV
It is preferable to use (cytomegalovirus) promoter, SRα promoter and the like. When the host is a bacterium belonging to the genus Escherichia, the trp promoter, the lac promoter, the recA promoter, the λP _L promoter,
lpp promoter, T7 promoter, etc., when the host is a Bacillus genus, SPO1 promoter, S
When the host is yeast such as PO2 promoter and penP promoter, PHO5 promoter, PGK promoter, GAP promoter, ADH promoter and the like are preferable. When the host is an insect cell, polyhedrin promoter, P10 promoter and the like are preferable.

In addition to the above, the expression vector may optionally contain an enhancer, a splicing signal, a poly A addition signal, a selection marker, an SV40 replication origin (hereinafter sometimes abbreviated as SV40 ori) and the like. Can be used. Examples of selection markers include dihydrofolate reductase (hereinafter, dhfr).
The gene [methotrexate (M
TX) resistance], ampicillin resistance gene (hereinafter referred to as Amp
^r ), neomycin resistance gene (hereinafter sometimes abbreviated as Neo ^r , G418 resistance) and the like. In particular, when the dhfr gene is used as a selection marker using Chinese hamster cells lacking the dhfr gene, the target gene can also be selected by a thymidine-free medium. In addition, if necessary, a signal sequence suitable for the host is added to the N-terminal side of the protein used in the present invention. When the host is Escherichia, PhoA / signal sequence, OmpA
When the host is a bacterium of the genus Bacillus, the signal sequence and the like are α-amylase signal sequence and subtilisin signal sequence, and when the host is yeast, MFα.
When the host is an animal cell such as a signal sequence or SUC2 signal sequence, an insulin signal sequence,
α-interferon / signal sequence, antibody molecule / signal sequence, etc. can be used. D encoding the protein used in the present invention constructed in this way
A transformant can be produced using the vector containing NA.

As the host, for example, Escherichia, Bacillus, yeast, insect cells, insects, animal cells and the like are used. Specific examples of Escherichia bacteria include:
For example, Escherichia coli K1
2. DH1 [Proc. Natl. Acad. Sci. USA], Volume 60, 1
60 (1968)], JM103 [Nucleic Acids Research], Volume 9, 3.
09 (1981)], JA221 [Journal of Molecular Biol
ogy), 120, 517 (1978)], HB101
[Journal of Molecular Biology, 4
1 volume, 459 (1969)], C600 [Genetics, 39 volume, 440 (1954)] and the like. Examples of Bacillus include Bacillus
Bacillus subtilis MI114 [Gene, 24, 255 (1983)], 207-21 [Journal of Bioch
emistry), Vol. 95, 87 (1984)]. Examples of the yeast include Saccharomyces cerevisiae AH22 and AH22.
^{R -, NA87-11A, DKD-5D} , 20B-1
2. Schizosaccharomy
ces pombe) NCYC1913, NCYC2036, Pichia pastoris KM71 and the like are used.

Examples of insect cells include virus A
In the case of cNPV, the cell line derived from the larva of Spodoptera frugiperda (Spod
optera frugiperda cell; Sf cell), Trichoplusia n
MG1 cells from the midgut of i, from the eggs of Trichoplusia ni
High Five ^TM cells, cells from Mamestra brassicae or
Cells derived from Estigmena acrea are used. When the virus is BmNPV, the silkworm-derived cell line (Bombyx
mori N cells; BmN cells) and the like are used. The S
Examples of f cells include Sf9 cells (ATCCCRL171
1), Sf21 cells (above Vaughn, JL et al., In Vivo, 13, 213-217, (1977)) and the like are used. Examples of insects include silkworm larvae [Maeda et al., Nature, 315, 59.
2 (1985)]. Examples of animal cells include monkey cells COS-7, Vero, Chinese hamster cells C
HO (hereinafter, abbreviated as CHO cell), dhfr gene-deficient Chinese hamster cell CHO (hereinafter, CHO (d
hfr ^-) cell), mouse L cells, mouse AtT
-20, mouse myeloma cells, rat GH3, human F
L cells, H9c2 cells and the like are used. For transforming Escherichia, for example,
Of the National Academy of Sciences of the USA (Proc. Natl. Acad. Sc
i. USA), 69, 2110 (1972), Gene (Ge
ne), Vol. 17, 107 (1982) and the like.

For transforming Bacillus, for example, Molecular & General Genetics, 168,
111 (1979) and the like. To transform yeast, for example,
Methods in Enzymology,
194, 182-187 (1991), Procedures of the National Academy of Sciences of the USA (Proc. Natl. Ac
Ad. Sci. USA), Vol. 75, 1929 (1978) and the like. Transformation of insect cells or insects can be performed according to the method described in, for example, Bio / Technology, 6, 47-55 (1988)). To transform animal cells, see, eg, Cell Engineering Supplement 8, New Cell Engineering Experimental Protocol. 263-267 (1995) (published by Shujunsha), Virology, Volume 52, 456 (1)
It can be performed according to the method described in 973). In this way, a transformant transformed with the expression vector containing the DNA encoding the protein can be obtained. When the host is culturing a transformant which is a bacterium of the genus Escherichia, a bacterium of the genus Bacillus, a liquid medium is suitable as a medium used for the culture, in which a carbon source necessary for the growth of the transformant, A nitrogen source, an inorganic substance, etc. are contained. Examples of the carbon source include glucose, dextrin, soluble starch and sucrose, and examples of the nitrogen source include:
For example, ammonium salts, nitrates, corn steep liquor, peptone, casein, meat extract, soybean meal, potato extract and other inorganic or organic substances, as inorganic substances, for example, calcium chloride, sodium dihydrogen phosphate, magnesium chloride. And so on. In addition, yeast extract, vitamins, growth promoting factor and the like may be added. The pH of the medium is preferably about 5-8.

As a medium for culturing Escherichia, for example, M9 medium containing glucose and casamino acid [Miller, Journal of Experiments in Molecular Genetics (Journa
l of Experiments in Molecular Genetics), 431-
433, Cold Spring Harbor Laboratory, New York1
972] is preferable. If necessary, a drug such as 3β-indolylacrylic acid can be added to the promoter so as to work efficiently. When the host is a bacterium belonging to the genus Escherichia, the culture is usually carried out at about 15 to 43 ° C. for about 3 to 24 hours, and if necessary, aeration and stirring can be added. When the host is a bacterium of the genus Bacillus, the culture is usually carried out at about 30 to 40 ° C. for about 6 to 24 hours, and aeration and stirring can be added if necessary. When culturing a transformant whose host is yeast, examples of the medium include Burkholder's minimum medium [Bostian, KL
Et al., Proc. Natl. Acad. Sci. USA, 77, 4505.
(1980)] and SD medium containing 0.5% casamino acid [Bitter, GA et al., Procedures of the National Academy of Sciences of
The USA (Proc. Natl. Acad. Sci. USA), 8
1, 5330 (1984)]. P of medium
H is preferably adjusted to about 5-8. Culture is usually about 2
It is carried out at 0 ° C to 35 ° C for about 24 to 72 hours, and aeration and stirring are added if necessary. When culturing a transformant whose host is an insect cell or an insect, the medium is Grace's Ins.
ect Medium (Grace, TCC, Nature), 19
5, 788 (1962)) to which an additive such as immobilized 10% bovine serum is appropriately added. The pH of the medium is preferably adjusted to about 6.2 to 6.4. Culturing is usually performed at about 27 ° C. for about 3 to 5 days, and aeration and agitation are added if necessary. When culturing a transformant whose host is an animal cell, the medium is, for example, MEM medium containing about 5 to 20% fetal bovine serum [Science, 122.
Vol. 501 (1952)], DMEM medium [Virology, Vol. 8, 396 (1959)], RPMI
1640 medium [The Journal of the American Medical Association (The Journal of the Ame
rican Medical Association) Volume 199, 519 (196
7)], 199 medium [Proceeding of the Society for the Biological Medi
cine), 73, 1 (1950)] and the like. p
H is preferably about 6-8. Culture is usually about 30 ℃
Perform at about -40 ° C for about 15-60 hours, adding aeration and agitation as needed. As described above, the protein used in the present invention can be produced inside or outside the cytoplasm of the transformant.

The protein used in the present invention can be separated and purified from the above culture by the following method, for example. When extracting the protein used in the present invention from cultured bacterial cells or cells, after the culture, the bacterial cells or cells are collected by a known method, suspended in an appropriate buffer, and then subjected to ultrasonic wave, lysozyme and / or freezing. A method of obtaining a crude protein extract by centrifugation or filtration after disrupting cells or cells by thawing or the like is appropriately used. The buffer solution may contain a protein denaturing agent such as urea or guanidine hydrochloride, or a surfactant such as Triton X-100 ^™ . When the protein is secreted into the culture medium, after the culture is completed, the cells or cells are separated from the supernatant by a known method, and the supernatant is collected. The protein contained in the culture supernatant or the extract thus obtained can be purified by appropriately combining known separation / purification methods. Known separation and purification methods for these are mainly methods using solubility such as salting out and solvent precipitation, dialysis, ultrafiltration, gel filtration, and SDS-polyacrylamide gel electrophoresis. 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 known method or a method analogous thereto, and conversely, when it is obtained as a salt, a known method. Alternatively, it can be converted into a free form or another salt by a method similar thereto. In addition, the protein produced by the recombinant can be optionally modified or the polypeptide can be partially removed by acting an appropriate protein-modifying enzyme before or after the purification. Examples of the protein-modifying enzyme include trypsin, chymotrypsin, arginyl endopeptidase, protein kinase, glycosidase and the like. The presence of the thus-produced protein used in the present invention can be measured by an enzyme immunoassay using a specific antibody, Western blotting or the like.

The DNA used in the present invention is SEQ ID NO:
It may be any DNA containing a DNA containing the same or substantially the same base sequence as the base sequence represented by 1, and preferably contains a DNA containing the base sequence represented by SEQ ID NO: 1. There is DNA. The DNA used in the present invention includes cDNA, chromosomal DNA, synthetic D
It may be any of NA, and may include a promoter region and an enhancer region. Specific examples of the DNA include DNA containing (1) exons 0 to 7 of Type II CBFA1 (SEQ ID NO: 3; full-length Type).
eII CBFA1 DNA),
(2) DNA containing exons 0-5 and 7 (SEQ ID NO: 4; exon 6-deleted Type II CBFA1
(Sometimes referred to as DNA), (3) exon 0
DNA containing 3, 5 and 7 (SEQ ID NO: 5; DN of exon 4 and 6 deficient Type II CBFA1
(Sometimes referred to as A)) and the like.

The antibody against the protein or partial peptide or salt thereof used in the present invention may be either a polyclonal antibody or a monoclonal antibody as long as it is an antibody capable of recognizing the protein or partial peptide or salt thereof used in the present invention. Good. Antibodies against the proteins or partial peptides or salts thereof used in the present invention (hereinafter, these may be simply referred to as proteins used in the present invention in the description of antibodies) include antibodies using the proteins used in the present invention as antigens. Used,
It can be produced according to a known antibody or antiserum production method. [Preparation of Monoclonal Antibody] (a) Preparation of Monoclonal Antibody-Producing Cell The protein used in the present invention is administered to a warm-blooded animal at a site where antibody production can be performed by itself or together with a carrier or diluent. Complete Freund's adjuvant or incomplete Freund's adjuvant may be administered to enhance the antibody-producing ability upon administration. The administration is usually performed once every 2 to 6 weeks, about 2 to 10 times in total. Examples of warm-blooded animals used include monkeys, rabbits, dogs, guinea pigs, mice, rats, sheep, goats, and chickens, and mice and rats are preferably used. When preparing monoclonal antibody-producing cells, warm-blooded animals immunized with the antigen, for example, individuals with antibody titers are selected from mice, and spleens or lymph nodes are collected 2 to 5 days after the final immunization. A monoclonal antibody-producing hybridoma can be prepared by fusing the resulting antibody-producing cells with myeloma cells of the same or different species. The antibody titer in the antiserum can be measured, for example, by reacting the labeled protein described below with the antiserum and then measuring the activity of the labeling agent bound to the antibody. The fusion operation is a known method, for example, the method of Koehler and Milstein [Nature, 2
56, 495 (1975)]. Examples of the fusion accelerator include polyethylene glycol (PE
G) and Sendai virus can be mentioned, but PEG is preferably used.

Examples of myeloma cells include NS-1,
Examples include myeloma cells of warm-blooded animals such as P3U1, SP2 / 0 and AP-1, but P3U1 is preferably used. The preferred ratio of the number of antibody-producing cells (spleen cells) to the number of myeloma cells used is about 1: 1 to 20: 1,
PEG (preferably PEG1000-PEG6000)
Is added at a concentration of about 10 to 80%, and 20 to 40 ° C.,
Cell fusion can be efficiently performed by preferably incubating at 30 to 37 ° C. for 1 to 10 minutes. Although various methods can be used for screening a monoclonal antibody-producing hybridoma, for example, a hybridoma culture supernatant is added to a solid phase (eg, a microplate) on which a protein antigen is directly or adsorbed with a carrier, and then a radioactive substance or Anti-immunoglobulin antibody labeled with an enzyme (when the cells used for cell fusion are mice, anti-mouse immunoglobulin antibody is used) or protein A
And detecting the monoclonal antibody bound to the solid phase, adding the hybridoma culture supernatant to the solid phase adsorbing the anti-immunoglobulin antibody or protein A, adding the protein labeled with a radioactive substance or enzyme, Examples include a method of detecting a monoclonal antibody bound to a phase. Selection of the monoclonal antibody can be performed according to a known method or a method similar thereto. Normal HAT
(Hypoxanthine, aminopterin, thymidine) can be added to the medium for animal cells. As the medium for selection and breeding, any medium may be used as long as the hybridoma can grow. For example, 1 to 20
%, Preferably 10-20% fetal calf serum
I 1640 medium, GI containing 1-10% fetal bovine serum
T medium (Wako Pure Chemical Industries, Ltd.) or serum-free medium for hybridoma culture (SFM-101, Nissui Pharmaceutical Co., Ltd.)
Etc. can be used. The culture temperature is usually 20 to 4
It is 0 ° C, preferably about 37 ° C. Cultivation time is usually 5
Day to 3 weeks, preferably 1 to 2 weeks. Culturing can usually be performed under 5% carbon dioxide gas. The antibody titer of the hybridoma culture supernatant can be measured in the same manner as the above antibody titer measurement in antiserum.

(B) Purification of Monoclonal Antibody Monoclonal antibody can be separated and purified by a known method, for example, a method for separating and purifying immunoglobulin [eg, salting out method, alcohol precipitation method, isoelectric focusing method, electrophoresis method, Adsorption / desorption method using ion exchanger (eg, DEAE), ultracentrifugation method, gel filtration method, antigen binding solid phase or active adsorbent such as protein A or protein G to collect only antibody and dissociate the binding Specific Purification Method to Obtain]].

[Preparation of Polyclonal Antibody] The polyclonal antibody used in the present invention can be manufactured by publicly known methods or modifications thereof. For example, a complex of an immune antigen (an antigen such as a protein used in the present invention) and a carrier protein is formed, and a warm-blooded animal is immunized in the same manner as in the above-described method for producing a monoclonal antibody,
It can be produced by collecting an antibody-containing substance against the protein used in the present invention from the immunized animal and separating and purifying the antibody. Regarding the complex of the immunizing antigen and the carrier protein used for immunizing warm-blooded animals, the type of carrier protein and the mixing ratio of the carrier and the hapten are such that the antibody is effective against the hapten immunized by crosslinking with the carrier. If possible, what kind may be crosslinked in any proportion, for example,
Bovine serum albumin, bovine thyroglobulin, hemocyanin, etc. in a weight ratio of about 0.1 to 20 to 1 hapten,
Preferably, a method of coupling at a ratio of about 1 to 5 is used. Also, for the coupling of the hapten and carrier,
Although various condensing agents can be used, glutaraldehyde, carbodiimide, maleimide active ester, active ester reagent containing a thiol group, dithiopyridyl group, etc. are used. The condensation product is administered to a warm-blooded animal at a site where antibodies can be produced, by itself or together with a carrier and a diluent. Complete Freund's adjuvant or incomplete Freund's adjuvant may be administered to enhance the antibody-producing ability upon administration. The administration is usually performed once every about 2 to 6 weeks, about 3 to 10 times in total. The polyclonal antibody can be collected from the blood, ascites, etc., 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.

DNA complementary to or substantially the same as the DNA encoding the protein or partial peptide used in the present invention (hereinafter, these DNAs are abbreviated as the DNA used in the present invention in the description of antisense polynucleotides). The antisense polynucleotide containing a nucleotide sequence complementary to has a nucleotide sequence complementary to or substantially complementary to the DNA used in the present invention, and has an action of suppressing the expression of the DNA. If you have
Although any antisense polynucleotide may be used, antisense DNA is preferred. The nucleotide sequence substantially complementary to the DNA used in the present invention means, for example, the entire nucleotide sequence of the nucleotide sequence complementary to the DNA used in the present invention (that is, the complementary strand of the DNA used in the present invention) or Partial base sequence and about 97% or more, preferably about 98
% And more preferably about 99% or more homologous base sequences. In particular, of the total nucleotide sequence of the complementary strand of the DNA used in the present invention, about the same as the complementary strand of the nucleotide sequence of the portion encoding the N-terminal portion of the protein used in the present invention (for example, the nucleotide sequence near the start codon). Antisense polynucleotides having a homology of 97% or more, preferably about 98% or more, more preferably about 99% or more are suitable. The antisense polynucleotide is usually composed of about 10 to 40 bases, preferably about 15 to 30 bases. In order to prevent degradation by a hydrolase such as nuclease, the phosphate residue (phosphate) of each nucleotide constituting the antisense polynucleotide is, for example, a chemically modified phosphate residue such as phosphorothioate, methylphosphonate, or phosphorodithionate. May be substituted with. These antisense polynucleotides can be produced using a known DNA synthesizer or the like.

According to the present invention, an antisense polynucleotide (nucleic acid) capable of inhibiting the replication or expression of the protein gene of the present invention has been cloned, or the DNA encoding a protein whose amino acid sequence has been determined. It can be designed and synthesized based on the nucleotide sequence information. Such an antisense polynucleotide (nucleic acid) can hybridize with RNA of the protein gene of the present invention, inhibit the synthesis or function of the RNA, or interact with the protein-related RNA of the present invention. The expression of the protein gene of the present invention can be regulated / controlled via Protein-related RN of the present invention
The polynucleotide complementary to the selected sequence of A and the polynucleotide capable of specifically hybridizing with the protein-related RNA of the present invention regulate the expression of the protein gene of the present invention in vivo and in vitro. It is useful for controlling, and is useful for treating or diagnosing diseases and the like. The term "corresponding" means having homology or being complementary to a specific sequence of nucleotides, base sequences or nucleic acids including genes. “Corresponding” between a nucleotide, a nucleotide sequence or a nucleic acid and a peptide (protein) means a nucleotide (nucleic acid)
Usually refers to the amino acid of a peptide (protein) translated from the sequence or its complement. 5'end hairpin loop of protein gene, 5'end 6-base pair repeat, 5'end untranslated region, polypeptide translation initiation codon, protein coding region, ORF translation stop codon,
The 3'-end untranslated region, the 3'-end palindromic region, and the 3'-end hairpin loop can be selected as preferred regions of interest, but any region within the protein gene can be selected as a subject. It can be said that the relationship between the target nucleic acid and the polynucleotide complementary to at least a part of the target region, and the relationship between the polynucleotide capable of hybridizing with the target region are “antisense”. Antisense polynucleotides include polynucleotides containing 2-deoxy-D-ribose, polynucleotides containing D-ribose, other types of polynucleotides that are N-glycosides of purines or pyrimidine bases, or Other polymers with non-nucleotide backbones (eg commercially available proteins, nucleic acids and synthetic sequence-specific nucleic acid polymers) or other polymers containing special linkages, provided that such polymers are found in DNA or RNA. Containing a nucleotide having a configuration that allows pairing of bases and attachment of bases) and the like. They are double-stranded DNA, single-stranded DN
A, double-stranded RNA, single-stranded RNA, and further DNA: RN
A hybrid, which can be an unmodified polynucleotide (or an unmodified oligonucleotide), and also an addition of a known modification, for example, a label known in the art, a capped one, Methylated, substituted with one or more natural nucleotides by analogs, modified with an intramolecular nucleotide, such as an uncharged bond (eg, methylphosphonate,
With phosphotriesters, phosphoramidates, carbamates, etc., with charged bonds or with sulfur-containing bonds (eg phosphorothioates, phosphorodithioates, etc.), eg proteins (nucleases, nuclease inhibitors, toxins, Those having side chain groups such as antibodies, signal peptides, poly-L-lysine) and sugars (eg, monosaccharides), intercalating compounds (eg, acridine,
Those having psoralen, etc., chelate compounds (eg,
Metal, radioactive metal, boron, oxidizing metal, etc.), alkylating agent, modified bond (eg, α-anomer type nucleic acid, etc.) Good. As used herein, the term "nucleoside", "nucleotide" and "nucleic acid" may include those having not only purine and pyrimidine bases but also other modified heterocyclic bases. Such modifications include methylated purines and pyrimidines,
It may include acylated purines and pyrimidines, or other heterocycles. The modified nucleotide and modified nucleotide may also have a modified sugar moiety, for example, one or more hydroxyl groups are substituted with halogen, an aliphatic group, or a functional group such as ether or amine. It may have been converted.

The antisense polynucleotide (nucleic acid) of the present invention is RNA, DNA, or a modified nucleic acid (RNA, DNA). Specific examples of the modified nucleic acid include, but are not limited to, sulfur derivatives and thiophosphate derivatives of nucleic acids, and those resistant to degradation of polynucleoside amides and oligonucleoside amides. The antisense nucleic acid of the present invention can be preferably designed according to the following policy. That is, it makes the antisense nucleic acid more stable in the cell, increases the cell permeability of the antisense nucleic acid, increases the affinity for the target sense strand, and Make sense nucleic acid less toxic. Thus many modifications are known in the art, for example J. Kawakami et al., Pharm Tech Japan, Vo.
l. 8, pp. 247, 1992; Vol. 8, pp. 395, 1992; ST Cr
ooke et al. ed., Antisense Research and Applicatio
ns, CRC Press, 1993 etc. The antisense nucleic acid of the present invention has an altered or modified sugar,
It may contain a base or a bond, and may be provided in a special form such as liposome, microsphere, applied by gene therapy, or provided in an added form. Thus, as an additive form, polycationic substances such as polylysine that act to neutralize the charge of the phosphate group, lipids that enhance the interaction with cell membranes and increase the uptake of nucleic acids ( Examples thereof include hydrophobic ones such as phospholipids and cholesterol). Preferred lipids to add include cholesterol and its derivatives (eg cholesteryl chloroformate, cholic acid, etc.). These can be attached to the 3'-end or 5'-end of the nucleic acid, and can be attached via a base, a sugar, or an intramolecular nucleoside bond. Other groups include
A capping group specifically arranged at the 3'-end or the 5'-end of a nucleic acid, which includes a group for preventing decomposition by nucleases such as exonuclease and RNase. Examples of such a capping group include, but are not limited to, hydroxyl group-protecting groups known in the art including glycols such as polyethylene glycol and tetraethylene glycol. The inhibitory activity of the antisense nucleic acid can be examined using the transformant of the present invention, the in vivo or in vitro gene expression system of the present invention, or the in vivo or in vitro translation system of the protein of the present invention. The nucleic acid can be applied to cells by various known methods.

Hereinafter, the protein or partial peptide used in the present invention or a salt thereof (hereinafter sometimes abbreviated as the protein used in the present invention), D encoding the protein or partial peptide used in the present invention
NA (hereinafter sometimes abbreviated as DNA used in the present invention), an antibody against the protein or partial peptide used in the present invention or a salt thereof (hereinafter sometimes abbreviated as antibody used in the present invention), and The use of the DNA antisense polynucleotide used in the present invention (hereinafter sometimes abbreviated as the antisense polynucleotide used in the present invention) will be described.

[1] Screening of Drug Candidate Compounds against Diseases The DNA used in the present invention causes specific upregulation of the protein encoded by the gene containing the DNA in some cancer tissues such as breast cancer tissues. Therefore, a compound or a salt thereof that suppresses its expression, and a compound or a salt thereof that suppresses the activity of the protein used in the present invention are cancer (eg, colon cancer, breast cancer, lung cancer, prostate cancer, esophageal cancer, Gastric cancer, liver cancer, biliary tract cancer, spleen cancer, kidney cancer, bladder cancer,
It is useful as a prophylactic / therapeutic agent for uterine cancer, testicular cancer, thyroid cancer, pancreatic cancer, brain tumor, blood tumor, etc., metastasis inhibitor, and metastasis preventive agent. Therefore, the DNA and protein used in the present invention are useful for screening the compound or a salt thereof. That is, the present invention relates to a compound or its salt that suppresses the expression of the gene using the DNA used in the present invention, and a screening method for the compound or its salt that suppresses the activity of the protein using the protein used in the present invention ( Hereinafter, it may be abbreviated as the screening method of the present invention). Specific examples of the screening method of the present invention include:
(I) when culturing cells capable of producing the protein used in the present invention, and (ii) when culturing cells capable of producing the protein used in the present invention in the presence of a test compound. Make a comparison. In the above method, in the cases (i) and (ii) (presence or absence of a test compound), the expression level of the DNA used in the present invention (specifically, the amount of the protein used in the present invention or the mRNA encoding the protein) Amount) or the activity of the protein used in the present invention (eg, transcription activity) is measured and compared. The amount of the protein can be measured by a known method, for example, by using an antibody that recognizes SEQ ID NO: 2 to analyze the protein present in a cell extract or the like according to a method such as Western analysis or ELISA or a method similar thereto. Can be measured. The amount of mRNA is measured by a known method, for example, Northern hybridization using a nucleic acid containing SEQ ID NO: 1 or a part thereof as a probe, or a nucleic acid containing SEQ ID NO: 1 or a part thereof as a primer. It can be measured according to the PCR method or a method similar thereto. Here, as a diffusion containing a part of SEQ ID NO: 1, SEQ ID NO:
Nucleic acid containing the base sequence represented by 12.
The base sequence represented by SEQ ID NO: 12 is the 68th to 124th base sequence of the base sequence represented by SEQ ID NO: 1.

For example, the expression of the gene in the case of (ii) (addition of test compound) is about 20% or more, preferably 30% or more, as compared with the case of (i) (no addition of test compound). More preferably, a test compound that inhibits by about 50% or more can be selected as a compound that suppresses the expression of the gene used in the present invention. The transcription activity is measured by a known method, for example, a DNA containing a nucleotide sequence of a DNA recognized by the protein used in the present invention.
Can be ligated upstream of a reporter gene (eg, lacZ, alkaline phosphatase, luciferase, etc.) and its reporter activity can be used as an indicator, or the amount of the gene or its product that induces expression of the protein in cells can be measured. It can be measured according to a method such as the above or a method similar thereto. For example, the transcription activity in the case of (ii) above (addition of test compound) is about 20% or more, preferably 30% or more, more preferably about 50% compared to the case of (i) above (no addition of test compound). A test compound that inhibits by% or more can be selected as a compound that inhibits the activity of the protein used in the present invention. Further, examples of the screening method of the present invention include a method of obtaining a compound that binds to the protein used in the present invention and inhibits binding of a protein that promotes transcriptional activity in a coordinated manner. Specifically, (i) a) a protein used in the present invention and b) a protein that binds to the protein used in the present invention and promotes transcriptional activity in a coordinated manner, and (ii) a) test “A protein used in the present invention” in the case of contacting a compound, b) a protein used in the present invention, and c) a protein that binds to the protein used in the present invention and cooperatively promotes transcription activity
And "the protein that binds to the protein used in the present invention and promotes the transcriptional activity in a coordinated manner" are compared. Furthermore, as the screening method of the present invention,
A method for obtaining a compound that binds to the protein used in the present invention and suppresses its transcription activity is also included. The binding activity is measured by a known method, for example, a yeast or mammal two-hybrid method, an immunoprecipitation method, a Biacore method, a sandwich ELISA method, or the like. For example, in the above screening method, a test compound which inhibits the binding activity in the absence of the test compound by about 20% or more, preferably about 30% or more, more preferably about 50% or more in the presence of the test compound is used in the present invention. It can be selected as a compound that inhibits the binding of a protein that binds to the protein used and promotes transcription activity in a coordinated manner.

The test compound used in the screening method of the present invention includes, for example, peptides, proteins, biological non-peptide compounds (sugars, lipids, etc.), synthetic compounds, microbial cultures, cell extracts, plant extracts, Examples thereof include animal tissue extracts, and these compounds may be novel compounds or known compounds. In the screening method of the present invention, cells capable of producing the protein used in the present invention are cultured in a medium suitable for screening. Any medium may be used as long as it does not affect the gene expression of the protein used in the present invention. Examples of cells having the ability to produce the protein used in the present invention include cancer cells (eg, breast cancer cells), fetus-derived cells, and the like. Further, for the purpose of large-scale expression of the protein used in the present invention, a cell obtained by transforming the above-mentioned recombinant vector containing the DNA of the present invention into a host (eg, animal cell such as CHO cell) is preferable. Used.

The screening kit of the present invention contains the protein or gene used in the present invention or cells having the ability to produce the protein used in the present invention. "Salt" of compound or salt thereof obtained by using the screening method or screening kit of the present invention
The same salt as the above-mentioned protein salt used in the present invention can be used.

The compound or its salt obtained by using the screening method or the screening kit of the present invention is, for example, cancer (eg, colon cancer, breast cancer, lung cancer, prostate cancer,
Esophageal cancer, gastric cancer, liver cancer, biliary tract cancer, spleen cancer, kidney cancer, bladder cancer, uterine cancer, testicular cancer, thyroid cancer, pancreatic cancer, brain tumor, blood tumor, etc.) It is useful as an agent. When used as the above-mentioned preventive / therapeutic agent, metastasis inhibitor, or metastasis preventive agent, it can be formulated according to a conventional method. For example, tablets, capsules, elixirs, microcapsules, sterile solutions, suspensions and the like can be used. When the above compound or its salt is used as the above-mentioned preventive / therapeutic agent, it can be prepared into a pharmaceutical preparation according to a conventional method. For example, tablets, capsules,
It may be an elixir, a microcapsule, a sterile solution, a suspension or the like. For example, orally as a tablet, capsule, elixir, microcapsule, etc., optionally sugar-coated, or aseptic solution with water or other pharmaceutically acceptable liquid, or suspension It can be used parenterally in the form of injections such as. For example, the above compound or a salt thereof is mixed with a physiologically acceptable carrier, flavoring agent, excipient, vehicle, preservative, stabilizer, binder and the like in a unit dosage form generally required for the implementation of the formulation. It can be manufactured by The amount of active ingredient in these preparations is such that a suitable dose within the indicated range can be obtained. Additives that can be mixed with tablets, capsules, etc. include, for example, binders such as gelatin, corn starch, tragacanth, acacia, excipients such as crystalline cellulose, corn starch, gelatin, alginic acid, etc. Puffing agents, lubricants such as magnesium stearate, sweeteners such as sucrose, lactose or saccharin, flavoring agents such as peppermint, red oil or cherry and the like are used. When the unit dosage form is a capsule, a liquid carrier such as fat may be included in addition to materials of the above type. Sterile compositions for injection consist of the active substance in a vehicle such as water for injection, sesame oil,
It can be formulated according to conventional formulation practices such as dissolving or suspending naturally occurring vegetable oils such as coconut oil and the like. Aqueous solutions for injection include, for example, saline, isotonic solutions containing glucose and other adjuvants (eg,
D-sorbitol, D-mannitol, sodium chloride, etc., and the like, and a suitable solubilizing agent such as alcohol (eg, ethanol), polyalcohol (eg, propylene glycol, polyethylene glycol, etc.), nonionic interface. You may use together with an activator (For example, polysorbate ^80TM , HCO-50, etc.). Examples of the oily liquid include sesame oil, soybean oil and the like, which may be used in combination with solubilizing agents such as benzyl benzoate and benzyl alcohol. In addition, buffers (eg, phosphate buffer, sodium acetate buffer, etc.), soothing agents (eg, benzalkonium chloride, procaine hydrochloride, etc.), stabilizers (eg, human serum albumin, polyethylene glycol, etc.), You may mix | blend with preservatives (for example, benzyl alcohol, phenol, etc.), antioxidants, etc. The prepared injection solution is usually filled in a suitable ampoule. Since the preparation thus obtained is safe and has low toxicity, for example, humans or other warm-blooded animals (for example, mouse, rat, rabbit, sheep, pig,
Bovine, equine, avian, feline, canine, simian, chimpanzee, etc.) or parenterally. The dose of the compound or its salt varies depending on its action, target disease, administration subject, administration route, etc., but when the compound or its salt is orally administered for the purpose of treating breast cancer, it is generally an adult. (Weight 60 kg
In the above), about 0.1 to 100 mg, preferably about 1.0 to 50, of the above-mentioned compound or salt thereof per day.
mg, more preferably about 1.0-20 mg. In the case of parenteral administration, the single dose of the compound or its salt varies depending on the administration subject, target disease and the like. For example, when the compound or a salt thereof is administered to an adult (as 60 kg) in the form of an injection for the purpose of treating breast cancer, the amount of the compound or a salt is about 0.01 to 30 m per day.
About g, preferably about 0.1 to 20 mg, more preferably about 0.1 to 10 mg is preferably administered by intravenous injection. In the case of other animals, the dose converted per 60 kg can be administered.

[2] Quantification of protein used in the present invention, partial peptide or salt thereof The antibody used in the present invention can specifically recognize the protein used in the present invention. Can be used for quantification of the protein used in the present invention, in particular, quantification by sandwich immunoassay.
That is, the present invention provides (i) the labeled invention of the present invention bound to the antibody by competitively reacting the antibody used in the present invention with the test solution and the labeled protein of the present invention. Method for quantifying the protein used in the present invention in a test solution, which comprises measuring the ratio of the protein used in (4), and (ii) the antibody and the label used in the present invention insolubilized on the test solution and the carrier. The protein used in the present invention in a test solution, which is characterized by measuring the activity of a labeling agent on an insolubilized carrier after simultaneous or continuous reaction with a different antibody used in the present invention. To provide a quantitative method of. In the quantification method of (ii) above,
It is desirable that one antibody recognizes the N-terminal portion of the protein used in the present invention and the other antibody reacts with the C-terminal portion of the protein used in the present invention.

Further, using a monoclonal antibody against the protein used in the present invention (hereinafter sometimes referred to as the monoclonal antibody used in the present invention), the protein used in the present invention is quantified and detected by tissue staining or the like. You can also For these purposes, the antibody molecule itself may be used, or the F (a
b ′) ₂ , Fab ′, or Fab fraction may be used. The method for quantifying the protein used in the present invention using the antibody used in the present invention is not particularly limited, and the antibody, the antigen or the antibody-corresponding to the amount of the antigen (for example, the amount of protein) in the test liquid can be used. Any measurement method can be used as long as it is a method of detecting the amount of the antigen complex by chemical or physical means and calculating it from a standard curve prepared using a standard solution containing a known amount of antigen. Good. For example, nephrometry, competitive method, immunometric method and sandwich method are preferably used. From the viewpoint of sensitivity and specificity, it is particularly preferable to use the sandwich method described later. As the labeling agent used in the measuring method using the labeling substance, for example, a radioisotope, an enzyme, a fluorescent substance, a luminescent substance, etc. are used. As the radioisotope, for example, [ ¹²⁵ I], [ ¹³¹ I], [ ³ H], [ ¹⁴ C] and the like are used. The enzyme is preferably stable and has a large specific activity, for example, β-galactosidase, β-
Glucosidase, alkaline phosphatase, peroxidase, malate dehydrogenase and the like are used. As the fluorescent substance, for example, fluorescamine, fluorescein isothiocyanate, etc. are used. Luminol, a luminol derivative, luciferin, lucigenin, etc. are used as a luminescent substance, for example. Furthermore, the biotin-avidin system can be used for binding the antibody or the antigen to the labeling agent.

For the insolubilization of the antigen or antibody, physical adsorption may be used, or a method using a chemical bond usually used for insolubilizing or immobilizing protein or enzyme. As the carrier, agarose, dextran,
Examples thereof include insoluble polysaccharides such as cellulose, polystyrene, polyacrylamide, synthetic resins such as silicon, and glass. In the sandwich method, an insolubilized monoclonal antibody used in the present invention is reacted with a test solution (first reaction), and then another labeled monoclonal antibody used in the present invention is reacted (second reaction). By measuring the activity of the labeling agent on the insolubilized carrier, the amount of the protein used in the present invention in the test solution can be quantified. The first reaction and the second reaction may be performed in the reverse order, may be performed at the same time, or may be performed at different times. The labeling agent and the method of insolubilization can be the same as those described above. In the sandwich method, the antibody used for the solid phase antibody or the labeling antibody does not necessarily have to be one type, and a mixture of two or more types of antibodies may be used for the purpose of improving the measurement sensitivity. In the method for measuring the protein used in the present invention by the sandwich method, the monoclonal antibodies used in the present invention used in the primary reaction and the secondary reaction are preferably antibodies having different binding sites for the protein used in the present invention. Used. That is, as the antibody used in the primary reaction and the secondary reaction, for example, when the antibody used in the secondary reaction recognizes the C-terminal portion of the protein used in the present invention, the antibody used in the primary reaction is An antibody that recognizes other than the C-terminal portion, for example, the N-terminal portion is preferably used.

In the competitive method, the antigen in the test liquid and the labeled antigen are allowed to react competitively with the antibody, and then the unreacted labeled antigen (F) and the labeled antigen (B) bound to the antibody. And 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. This way,
A soluble antibody is used as the antibody, B / F separation is performed by a liquid phase method using polyethylene glycol, a second antibody against the antibody, etc., and a solid phase antibody is used as the first antibody, or the first antibody is soluble. A solid phase method using an antibody and a solid phase antibody as the second antibody is used. 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. Further, in nephrometry, the amount of insoluble precipitate produced as a result of the antigen-antibody reaction in the gel or in the solution is measured. The amount of antigen in the test solution is small,
When only a small amount of sediment can be obtained, laser nephrometry utilizing laser scattering is preferably used.

When applying these immunological measurement methods to the quantification method of the present invention, it is not necessary to set special conditions, operations and the like. The assay system for the protein used in the present invention may be constructed by adding ordinary technical consideration of those skilled in the art to the usual conditions and operating methods in each method. For details of these general technical means, reference can be made to reviews, books, and the like. For example, Hiro Irie's "Radioimmunoassay" (Kodansha, published in 1974), Hiroshi Irie's "Radioimmunoassay" (Kodansha, Showa 5)
4 years), Eiji Ishikawa et al. “Enzyme Immunoassay” (medical booklet, published in 1978), Eiji Ishikawa et al. “Enzyme Immunoassay” (2nd edition) (medical booklet, published in 1982), Eiji Ishikawa et al., "Enzyme Immunoassay" (3rd edition) (Medical Shoin, published in 1987), "Methods in ENZYMOLOGY" Vol. 70 (Imm
unochemical Techniques (Part A)), `` Methods in ENZY
MOLOGY "Vol. 73 (Immunochemical Techniques (Part
B)), `` Methods in ENZYMOLOGY '' Vol. 74 (Immunochemi
cal Techniques (Part C)), "Methods in ENZYMOLOGY"
Vol.84 (Immunochemical Techniques (Part D: Selected
Immunoassays)), "Methods in ENZYMOLOGY" Vol. 92 (I
mmunochemical Techniques (Part E: Monoclonal Antib
odies and General Immunoassay Methods)), `` Methods
in ENZYMOLOGY ”Vol. 121 (Immunochemical Technique
s (Part I: Hybridoma Technology and Monoclonal Antib
odies)) (issued by Academic Press Co., Ltd.) etc. can be referred to. As described above, the protein used in the present invention can be quantified with high sensitivity by using the antibody used in the present invention. Moreover,
By quantifying the concentration of the protein used in the present invention using the antibody used in the present invention, the protein used in the present invention is detected, or when an increase in the concentration is observed, for example, cancer (eg, Colorectal cancer, breast cancer, lung cancer, prostate cancer, esophageal cancer, gastric cancer, liver cancer, biliary tract cancer, spleen cancer, kidney cancer, bladder cancer, uterine cancer, testicular cancer, thyroid cancer, pancreatic cancer, brain cancer, blood tumor, etc.) , Or are likely to be affected by these diseases in the future. Also,
The antibody used in the present invention can be used to detect the protein used in the present invention present in a subject such as a body fluid or a tissue. Further, preparation of an antibody column used for purifying the protein used in the present invention, detection of the protein used in the present invention in each fraction during purification, behavior of the protein used in the present invention in a test cell Can be used for analysis, etc.

[3] Genetic Diagnosis The DNA used in the present invention can be used, for example, as a probe to analyze humans and other warm-blooded animals (eg, rat, mouse, guinea pig, rabbit, bird, sheep, pig, cow). , Horses, cats, dogs, monkeys, chimpanzees, etc.) encoding the protein or partial peptide thereof used in the present invention.
Since it is possible to detect the abnormality (gene abnormality), it is useful for gene diagnosis such as damage, mutation or decreased expression of the DNA or mRNA, or increase or excessive expression of the DNA or mRNA. Specifically, cancer (eg, colon cancer, breast cancer, lung cancer, prostate cancer, esophageal cancer,
It is useful for diagnosis (examination) of gastric cancer, liver cancer, biliary tract cancer, spleen cancer, kidney cancer, bladder cancer, uterine cancer, testicular cancer, thyroid cancer, pancreatic cancer, brain tumor, blood tumor, etc. and its metastasis. The above-mentioned gene diagnosis using the DNA used in the present invention can be carried out, for example, by known Northern hybridization, PCR.
-SSCP method (Genomics, Volume 5, 8
Pages 74-879 (1989), Proceedings of the Nationa (Proceedings of the Nationa)
l Academy of Sciences of the United States of Amer
ica), 86, 2766-2770 (1989).
Year)) etc. For example, when overexpression of a gene (for example, a gene containing at least exon 0) is detected by Northern hybridization, or when a DNA mutation is detected by the PCR-SSCP method, for example, cancer (eg, colon) is detected. Cancer, breast cancer, lung cancer, prostate cancer, esophageal cancer, gastric cancer, liver cancer, biliary tract cancer,
It can be diagnosed as being likely to be a disease such as spleen cancer, renal cancer, bladder cancer, uterine cancer, testicular cancer, thyroid cancer, pancreatic cancer, brain tumor, blood tumor).

[4] Drug containing antisense polynucleotide Complementarily binds to the DNA used in the present invention,
The antisense polynucleotide used in the present invention capable of suppressing the expression of A has low toxicity and can suppress the function of the protein used in the present invention or the DNA used in the present invention in vivo. , Cancer (eg, colon cancer, breast cancer, lung cancer, prostate cancer, esophageal cancer,
Gastric cancer, liver cancer, biliary tract cancer, spleen cancer, kidney cancer, bladder cancer, uterine cancer, testicular cancer, thyroid cancer, pancreatic cancer, brain tumor, blood tumor, etc.) Can be used. When the above antisense polynucleotide is used as the above-mentioned prophylactic / therapeutic agent, metastasis inhibitor, or metastasis preventive agent, it can be formulated and administered according to a known method. For example, when the antisense polynucleotide is used, the antisense polynucleotide is inserted alone or in an appropriate vector such as a retrovirus vector, an adenovirus vector, or an adenovirus associated virus vector, and then the human or other Can be orally or parenterally administered to a warm-blooded animal (eg, mouse, rat, rabbit, sheep, pig, cow, horse, bird, cat, dog, monkey, chimpanzee, etc.). The antisense polynucleotide can be administered as it is or in the form of a formulation with a physiologically acceptable carrier such as an auxiliary agent for promoting ingestion, and can be administered by a gene gun or a catheter such as a hydrogel catheter. The dose of the antisense polynucleotide varies depending on the target disease, the administration subject, the administration route, etc., but, for example,
When the antisense polynucleotide used in the present invention is orally administered for the purpose of treating breast cancer, an adult (body weight 60
(kg), about 0.1 to 100 mg of the antisense polynucleotide is administered per day. Furthermore, the antisense polynucleotide can also be used as a diagnostic oligonucleotide probe for investigating the presence of the DNA used in the present invention and its expression in tissues and cells. Similarly to the above antisense polynucleotide, double-stranded RNA containing a part of RNA encoding the protein of the present invention, ribozyme containing a part of RNA encoding the protein of the present invention, and the like are also genes of the present invention. Can be suppressed and the function of the protein used in the present invention or the DNA used in the present invention can be suppressed in vivo, and thus, for example, cancer (eg, colon cancer, breast cancer, lung cancer, prostate cancer) can be suppressed. , Esophageal cancer,
Gastric cancer, liver cancer, biliary tract cancer, spleen cancer, kidney cancer, bladder cancer, uterine cancer, testicular cancer, thyroid cancer, pancreatic cancer, brain tumor, blood tumor, etc.) preventive / therapeutic agent, metastasis inhibitor, metastasis prevention It can be used as an agent. Double-stranded RNA can be designed and produced based on the sequence of the polynucleotide of the present invention according to a known method (eg, Nature, 411, 494, 2001). Ribozymes can be prepared by known methods (eg,
TRENDS in Molecular Medicine, Volume 7, 221 pages, 2001)
According to the above, it can be designed and produced based on the sequence of the polynucleotide of the present invention. For example, it can be produced by linking a known ribozyme to a part of RNA encoding the protein of the present invention. As a part of RNA encoding the protein of the present invention, a portion (RNA fragment) close to the cleavage site on the RNA of the present invention which can be cleaved by a known ribozyme can be mentioned. When the above-mentioned double-stranded RNA or ribozyme is used as the above-mentioned preventive / therapeutic agent, it can be formulated and administered in the same manner as the antisense polynucleotide.

[5] Pharmaceutical containing the antibody used in the present invention The antibody used in the present invention having an activity of neutralizing the activity of the protein used in the present invention is, for example, cancer (eg, colon cancer, breast cancer, Lung cancer, prostate cancer, esophageal cancer, gastric cancer, liver cancer,
Bile duct cancer, spleen cancer, renal cancer, bladder cancer, uterine cancer, testicular cancer, thyroid cancer, pancreatic cancer, brain tumor, blood tumor, etc.) it can. The above-mentioned prophylactic / therapeutic agent, metastasis inhibitor, metastasis preventive agent, etc. containing the antibody used in the present invention has low toxicity, and as a liquid formulation as it is or as a pharmaceutical composition in a suitable dosage form, human or other warm blood It can be administered orally or parenterally to animals (eg, mouse, rat, rabbit, sheep, pig, cow, horse, bird, cat, dog, monkey, chimpanzee, etc.). Although the dose varies depending on the administration subject, target disease, symptom, administration route, etc., for example, when used for treatment / prevention of adult breast cancer, the antibody used in the present invention is used as a single dose, Usually about 0.01 to 20 mg / kg body weight, preferably about 0.1 to 10 mg / kg body weight, more preferably about 0.1 to 5 mg / kg body weight, about 1 to 5 times a day, preferably 1 day a day. It is convenient to administer the drug by intravenous injection about 3 times. In the case of other parenteral administration and oral administration, an amount similar to this can be administered. If the symptoms are particularly severe, the dose may be increased according to the symptoms. The antibody used in the present invention can be administered per se or as an appropriate pharmaceutical composition. The pharmaceutical composition used for the above administration contains the above antibody or a salt thereof and a pharmacologically acceptable carrier, diluent or excipient. Such a composition is provided as a dosage form suitable for oral or parenteral administration. Thus, for example, a composition for oral administration may be a solid or liquid dosage form,
Specific examples include tablets (including sugar-coated tablets and film-coated tablets), pills, granules, powders, capsules (including soft capsules), syrups, emulsions, suspensions and the like. Such a composition is produced by a known method and contains a carrier, diluent or excipient usually used in the field of formulation. For example, lactose, starch, sucrose, magnesium stearate and the like are used as carriers and excipients for tablets.

As the composition for parenteral administration, for example, injections, suppositories and the like are used. The injections include intravenous injections, subcutaneous injections, intradermal injections, intramuscular injections, drip injections and the like. The injection is prepared according to a known method, for example, by dissolving, suspending or emulsifying the above-mentioned antibody or a salt thereof in a sterile aqueous or oily liquid usually used for injection. As the aqueous solution for injection, for example, an isotonic solution containing physiological saline, glucose, and other adjuvants is used, and a suitable solubilizing agent, for example, alcohol (eg, ethanol), polyalcohol (eg, Propylene glycol, polyethylene glycol), nonionic surfactants [eg polysorbate 80,
HCO-50 (polyoxyethylene (50 mol) adduct of h
ydrogenated castor oil)] etc. As the oily liquid, for example, sesame oil, soybean oil and the like are used. You may use together benzyl benzoate, benzyl alcohol, etc. as a solubilizing agent. The prepared injection solution is usually filled in a suitable ampoule. The suppository used for rectal administration is prepared by mixing the above antibody or a salt thereof with an ordinary suppository base.

The above-mentioned oral or parenteral pharmaceutical composition is conveniently prepared in a unit dosage form suitable for the dose of the active ingredient. Examples of the dosage form of such a dosage unit include tablets, pills, capsules, injections (ampoules), suppositories, etc., and usually 5 to 500 mg per dosage unit dosage form, especially 5 to 10 injections.
It is preferable that the above antibody is contained in 0 mg, and in other dosage forms, 10 to 250 mg. Each composition described above may contain other active ingredients as long as the composition does not cause an unfavorable interaction with the antibody.

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

The substituents, protecting groups and reagents commonly used in the present specification are represented by the following symbols. Me: methyl Et: ethyl Bu: butyl Ph: phenyl TC: thiazolidine -4 (R) - carboxamide Tos: p-toluenesulfonyl CHO: formyl Bzl: benzyl Cl ₂ -Bzl: 2,6-dichlorobenzyl Bom: benzyloxymethyl Z: benzyloxycarbonyl Cl-Z: 2-chlorobenzyloxycarbonyl Br-Z: 2-bromobenzyloxycarbonyl Boc: t-butoxycarbonyl DNP: dinitrophenyl Trt: trityl Bum: t-butoxymethyl Fmoc: N-9-. Fluorenylmethoxycarbonyl HOBt: 1-hydroxybenztriazole HOOBt: 3,4-dihydro-3-hydroxy-4-oxo-1,2,3-benzotriazine HONB: 1-hydroxy 5-norbornene-2,3-dicarboxylic Kishiimido DCC: N, N'- dicyclohexylcarbodiimide

The sequence numbers in the sequence listing in the present specification show the following sequences. [SEQ ID NO: 1] This shows the base sequence of exon 0 of Type II CBFA1. [SEQ ID NO: 2] This shows the amino acid sequence of the protein encoded by the base sequence represented by SEQ ID NO: 12. [SEQ ID NO: 3] Full length Type II obtained in Example 2
1 shows the nucleotide sequence of CBFA1 DNA. [SEQ ID NO: 4] This shows the base sequence of exon 6-deficient Type II CBFA1 DNA obtained in Example 2. [SEQ ID NO: 5] This shows the base sequence of exon 4 and 6 deficient Type II CBFA1 DNA obtained in Example 2. [SEQ ID NO: 6] This shows the base sequence of the primer used in EXAMPLE 1. [SEQ ID NO: 7] This shows the base sequence of the primer used in EXAMPLE 1. [SEQ ID NO: 8] This shows the base sequence of the primer used in EXAMPLE 1. [SEQ ID NO: 9] This shows the base sequence of the primer used in EXAMPLE 1. [SEQ ID NO: 10] This shows the base sequence of the primer used in EXAMPLE 2. [SEQ ID NO: 11] This shows the base sequence of the primer used in EXAMPLE 2. [SEQ ID NO: 12] This shows the 68th to 124th base sequence of the base sequence represented by SEQ ID NO: 1.

Transformant Es obtained in Example 2 described later
cherichia coli TOP10 / pTB22
27 is 1 East, Tsukuba City, Ibaraki Prefecture since May 17, 2001
Chome 1 Address 1 Central No. 6 (Postal Code 305-8566)
20 of the National Institute of Advanced Industrial Science and Technology, the Patent Biological Depository Center, with the deposit number FERM BP-7597
From April 24, 2001, 2-Juzonhoncho, Yodogawa-ku, Osaka-shi, Osaka
It has been deposited with the Fermentation Research Institute (IFO) of 17-85 (zip code 532-8686) under the deposit number IFO 16621. The transformant Escherichia coli TOP10 / pT obtained in Example 2 described later
B2226 has been available from May 17, 2001, 1-chome, 1-chome, 1-chome, Tsukuba, Ibaraki prefecture, Chuo No. 6 (zip code 305-85)
66) at the National Institute of Advanced Industrial Science and Technology, Patent Biological Depository Center, under the deposit number FERM BP-7596, from April 24, 2001, 2-17-85, Jusohonmachi, Yodogawa-ku, Osaka-shi, Osaka (postal code 532-). 8686) Fermentation Research Institute (IFO) with deposit number IFO 166
Deposited as 20. Transformant Escherichia coli TOP1 obtained in Example 2 described later
0 / pTB2225 has been available from May 17, 2001, 1-chome, 1-chome, 1-chome, Tsukuba, Ibaraki prefecture, 6th central (postal code 30
5-8566), National Institute of Advanced Industrial Science and Technology, Patent Organism Depositary, deposit number FERM BP-759
As of 5, from April 24, 2001, 2-17-85, Jusohonmachi, Yodogawa-ku, Osaka-shi, Osaka (postal code 532-868)
6) Deposit No. IFO to Fermentation Research Institute (IFO)
Deposited as 16619.

[0059]

The present invention will be described in more detail below with reference to examples, but the present invention is not limited thereto. The gene manipulation method using Escherichia coli is described in Molecular Cloning, 2nd
(J. Sambrook et al., Cold Spring Harbor Lab. Pres
s, 1989). Example 1 1) Expression of CBFA1 in major cancer tissues Matched cDNA pair which can compare gene expression in lesioned part and normal part in the same cancer patient
Primer 1 (SEQ ID NO: 6) and primer 2 (SEQ ID NO :) using (CLONTECH) as a template
PCR was performed according to the manual using 7). Primer 1 and primer 2 were designed based on exon 4 and exon 7, respectively. The results are shown in Fig. 1.
From this, 5 out of 14 cases (2, 3 and 4 of breast cancer patients, 3 of lung cancer patients, and 3 of colon cancer patients) only in the cancer tissue, 1 case (5 of colon cancer patients) It was found that the expression was elevated in cancer tissues. 2) Expression of Type II CBFA1 in breast cancer tissue In the above 1), CB was frequently found in 3 out of 4 breast cancer patients.
Since it was found that FA1 was expressed, Bre
ast Tumor cDNA panel (BIOCH
AIN), PCR is performed in the same manner, and C in breast cancer
The expression of BFA1 was examined. The results are shown in Figure 2. From this, it was found that CBFA1 was expressed in 5 out of 8 breast cancer patients (2nd, 3rd, 5th, 6th and 8th).
Next, as a Type I CBFA1-specific sequence, primer 2 (SEQ ID NO: 7) and primer 3 (SEQ ID NO: 8) were designed based on exon 1 and exon 7, respectively. Primer 2 (SEQ ID NO: 7) and primer 4 (SEQ ID NO: 9) were designed based on exon 0 and exon 7, respectively, as Type II CBFA1-specific sequences. With respect to the above 5 breast cancer patients, PCR was similarly performed using Primer 2 and Primer 3 to examine the expression of Type I CBFA1. Further, PCR was similarly performed using Primer 2 and Primer 4 to examine the expression of Type II CBFA1. The results are shown in Fig. 3. TypeII CB
Expression of FA1 was observed in all 5 cases. Thus, CBFA1 expressed in breast cancer patients is Type II C
BFA1 was found to be major. The cDNA used here was an amount corrected based on the β-actin expression level.

Example 2 First, cDNA was prepared using total RNA derived from breast cancer tissues (3 cases) purchased from BIO-CLINICAL PARTNERS. For the cDNA preparation, a random 6-mer was used and ThermoScript RT-
It was performed according to the protocol attached to the PCR system (GIBCO). Using the obtained cDNA as a template, two types of primers (SEQ ID NO: 10 and SEQ ID NO: 11), P
fuTURBO DNA polymerase (STRATA
PCR is performed using GENE) and pCRblunt (INVI
E. coli TOP10 (INVITR
After the introduction into OGEN), the base sequence was determined. As a result, from 2 out of 3 breast cancer patients, exon 4 and 6 deficient Ty
peII CBFA1 (SEQ ID NO: 5) was exon 6-deleted Type II CBFA1 (SEQ ID NO: 5) from one example.
4) was obtained, and all were found to be novel isoforms as Type II CBFA1. Each transformant was transformed into Escherichia col.
i TOP10 / pTB2227, Escherich
It was named ia coli TOP10 / pTB2226. On the other hand, human normal chondrocyte-derived RNA (BIOCHAIN)
Was used for full length cloning in the same manner to obtain a clone containing all exons 0 to 7. The nucleotide sequence of the DNA contained in the clone is shown in SEQ ID NO: 3. The transformant was designated as Escherichia coli TOP.
It was named 10 / pTB2225. It was also found that the lengths of exon-1 (upstream of exon 0) of SEQ ID NO: 3, SEQ ID NO: 4 and SEQ ID NO: 5 are all different.

[0061]

The compound obtained by the screening method of the present invention, the antibody of the present invention, the antisense polynucleotide of the present invention and the like are, for example, cancer (eg, colon cancer, breast cancer, lung cancer, prostate cancer, esophageal cancer, gastric cancer, Compounds useful as prophylactic / therapeutic agents, metastasis suppressors or metastasis preventive agents for liver cancer, biliary tract cancer, spleen cancer, renal cancer, bladder cancer, uterine cancer, testicular cancer, thyroid cancer, pancreatic cancer, brain cancer, blood tumor, etc.) Obtainable. In addition, the DNA used in the present invention, the antibody of the present invention,
By using the antisense polynucleotide or the like of the present invention, cancer (eg, colon cancer, breast cancer, lung cancer, prostate cancer, esophageal cancer, gastric cancer, liver cancer, biliary tract cancer, spleen cancer, renal cancer, bladder cancer,
Uterine cancer, testicular cancer, thyroid cancer, pancreatic cancer, brain tumor, hematological tumor, etc.) and metastases thereof can be diagnosed or examined.

[0062]

[Sequence list] SEQUENCE LISTING <110> Takeda Chemical Industries, Ltd. <120> Prophylactic, remedy and diagnostic agent for cancer <130> P02-0085 <150> JP2001-217186 <151> 2001-07-17 <160> 12 <210> 1 <211> 126 <212> DNA <213> Human <400> 1 tttaaggctg caagcagtat ttacaacaga gggtacaagt tctatctgaa aaaaaaaagg 60 agggactatg gcatcaaaca gcctcttcag cacagtgaca ccatgtcagc aaaacttctt 120 ttggga 126 <210> 2 <211> 19 <212> PRT <213> Human <400> 2 Met Ala Ser Asn Ser Leu Phe Ser Thr Val Thr Pro Cys Gln Gln Asn                  5 10 15 Phe Phe Trp <210> 3 <211> 1694 <212> DNA <213> Human <400> 3 actaccagcc accgagacca acagatttaa ggctgcaagc agtatttaca acagagggta 60 caagttctat ctgaaaaaaa aaaggaggga ctatggcatc aaacagcctc ttcagcacag 120 tgacaccatg tcagcaaaac ttcttttggg atccgagcac cagccggcgc ttcagccccc 180 cctccagcag cctgcagccc ggcaaaatga gcgacgtgag cccggtggtg gctgcgcaac 240 agcagcagca acagcagcag cagcaacagc agcagcagca gcagcaacag cagcagcagc 300 agcaggaggc ggcggcggcg gctgcggcgg cagcggcggc tgcggcggcg gcagctgcag 360 tgccccggtt gcggccgccc cacgacaacc gcaccatggt ggagatcatc gccgaccacc 420 cggccgaact cgtccgcacc gacagcccca acttcctgtg ctcggtgctg ccctcgcact 480 ggcgctgcaa caagaccctg cccgtggcct tcaaggtggt agccctcgga gaggtaccag 540 atgggactgt ggttactgtc atggcgggta acgatgaaaa ttattctgct gagctccgga 600 atgcctctgc tgttatgaaa aaccaagtag caaggttcaa cgatctgaga tttgtgggcc 660 ggagtggacg aggcaagagt ttcaccttga ccataaccgt cttcacaaat cctccccaag 720 tagctaccta tcacagagca attaaagtta cagtagatgg acctcgggaa cccagaaggc 780 acagacagaa gcttgatgac tctaaaccta gtttgttctc tgaccgcctc agtgatttag 840 ggcgcattcc tcatcccagt atgagagtag gtgtcccgcc tcagaaccca cggccctccc 900 tgaactctgc accaagtcct tttaatccac aaggacagag tcagattaca gaccccagac 960 aggcacagtc ttccccgccg tggtcctatg accagtctta cccctcctac ctgagccaga 1020 tgacgtcccc gtccatccac tctaccaccc cgctgtcttc cacacggggc actgggcttc 1080 ctgccatcac cgatgtgcct aggcgcattt cagatgatga cactgccacc tctgacttct 1140 gcctctggcc ttccactctc agtaagaaga gccaggcagg tgcttcagaa ctgggccctt 1200 tttcagaccc caggcagttc ccaagcattt catccctcac tgagagccgc ttctccaacc 1260 cacgaatgca ctatccagcc acctttactt acaccccgcc agtcacctca ggcatgtccc 1320 tcggtatgtc cgccaccact cactaccaca cctacctgcc accaccctac cccggctctt 1380 cccaaagcca gagtggaccc ttccagacca gcagcactcc atatctctac tatggcactt 1440 cgtcaggatc ctatcagttt cccatggtgc cggggggaga ccggtctcct tccagaatgc 1500 ttccgccatg caccaccacc tcgaatggca gcacgctatt aaatccaaat ttgcctaacc 1560 agaatgatgg tgttgacgct gatggaagcc acagcagttc cccaactgtt ttgaattcta 1620 gtggcagaat ggatgaatct gtttggcgac catattgaaa ttcctcagca gtggcccagt 1680 ggtatctggg ggcc 1694 <210> 4 <211> 1631 <212> DNA <213> Human <400> 4 actaccagcc accgagacca acagagtcat ttaaggctgc aagcagtatt tacaacagag 60 ggtacaagtt ctatctgaaa aaaaaaggag ggactatggc atcaaacagc ctcttcagca 120 cagtgacacc atgtcagcaa aacttctttt gggatccgag caccagccgg cgcttcagcc 180 ccccctccag cagcctgcag cccggcaaaa tgagcgacgt gagcccggtg gtggctgcgc 240 aacagcagca gcaacagcag cagcagcaac agcagcagca gcagcagcaa cagcagcagc 300 agcagcagga ggcggcggcg gcggctgcgg cggcggcggc ggctgcggcg gcggcagctg 360 cagtgccccg gttgcggccg ccccacgaca accgcaccat ggtggagatc atcgccgacc 420 acccggccga actcgtccgc accgacagca ccaacttcct gtgctcggtg ctgccctcgc 480 actggcgctg caacaagacc ctgcccgtgg ccttcaaggt ggtagccctc ggagaggtac 540 cagatgggac tgtggttact gtcatggcgg gtaacgatga aaattattct gctgagctcc 600 ggaatgcctc tgctgttatg aaaaaccaag tagcaaggtt caacgatctg agatttgtgg 660 gccggagtgg acgaggcaag agtttcacct tgaccataac cgtcttcaca aatcctcccc 720 aagtagctac ctatcacaga gcaattaaag ttacagtaga tggacctcgg gaacccagaa 780 ggcacagaca gaagcttgat gactctaaac ctagtttgtt ctctgaccgc ctcagtgatt 840 tagggcgcat tcctcatccc agtatgagag taggtgtccc gcctcagaac ccacggccct 900 ccctgaactc tgcaccaagt ccttttaatc cacaaggaca gagtcagatt acagacccca 960 ggcaggcaca gtcttccccg ccgtggtcct atgaccagtc ttacccctcc tacctgagcc 1020 agatgacgtc cccgtccatc cactctacca ccccgctgtc ttccacacgg ggcactgggc 1080 ttcctgccat caccgatgtg cctaggcgca tttcaggtgc ttcagaactg ggcccttttt 1140 cagaccccag gcagttccca agcatttcat ccctcactga gagccgcttc tccaacccac 1200 gaatgcacta tccagccacc tttacttaca ccccgccagt cacctcaggc atgtccctcg 1260 gtatgtccgc caccactcac taccacacct acctgccacc accctacccc ggctcttccc 1320 aaagccagag tggacccttc cagaccagca gcactccata tctctactat ggcacttcgt 1380 caggatccta tcagtttccc atggtgccgg ggggagaccg gtctccttcc agaatgcttc 1440 cgccatgcac caccacctcg aatggcagca cgctattaaa tccaaatttg cctaaccaga 1500 atgatggtgt tgacgctgat ggaagccaca gcagttcccc aactgttttg aattctagtg 1560 gcagaatgga tgaatctgtt tggcgaccat attgaaattc ctcagcagtg gcccagtggt 1620 atctgggggc c 1631 <210> 5 <211> 1492 <212> DNA <213> Human <400> 5 actaccagcc accgagacca acagagtcag tgagtgctct ctaaccacag tctatgcagt 60 aatatttaag gctgcaagca gtatttacaa cagagggtac aagttctatc tgaaaaaaaa 120 aggagggact atggcatcaa acagcctctt cagcacagtg acaccatgtc agcaaaactt 180 cttttgggat ccgagcacca gccggcgctt cagccccccc tccagcagcc tgcagcccgg 240 caaaatgagc gacgtgagcc cggtggtggc tgcgcaacag cagcagcaac agcagcagca 300 gcaacagcag cagcagcagc agcaacagca gcagcagcag caggaggcgg cggcggcggc 360 tgcggcggcg gcggcggctg cggcggcggc agctgcagtg ccccggttgc ggccgcccca 420 cgacaaccgc accatggtgg agatcatcgc cgaccacccg gccgaactcg tccgcaccga 480 cagccccaac ttcctgtgct cggtgctgcc ctcgcactgg cgctgcaaca agaccctgcc 540 cgtggccttc aaggtggtag ccctcggaga ggtaccagat gggactgtgg ttactgtcat 600 ggcgggtaac gatgaaaatt attctgctga gctccggaat gcctctgctg ttatgaaaaa 660 ccaagtagca aggttcaacg atctgagatt tgtgggccgg agtggacgag gcaagagttt 720 caccttgacc ataaccgtct tcacaaatcc tccccaagta gctacctatc acagagcaat 780 taaagttaca gtagatggac ctcgggaacc cagaaacccc aggcaggcac agtcttcccc 840 gccgtggtcc tatgaccagt cttacccctc ctacctgagc cagatgacgt ccccgtccat 900 ccactctacc accccgctgt cttccacacg gggcactggg cttcctgcca tcaccgatgt 960 gcctaggcgc atttcaggtg cttcagaact gggccctttt tcagacccca ggcagttccc 1020 aagcatttca tccctcactg agagccgctt ctccaaccca cgaatgcact atccagccac 1080 ctttacttac accccgccag tcacctcagg catgtccctc ggtatgtccg ccaccactca 1140 ctaccacacc tacctgccac caccctaccc cggctcttcc caaagccaga gtggaccctt 1200 ccagaccagc agcactccat atctctacta tggcacttcg tcaggatcct atcagtttcc 1260 catggtgccg gggggagacc ggtctccttc cagaatgctt ccgccatgca ccaccacctc 1320 gaatggcagc acgctattaa atccaaattt gcctaaccag aatgatggtg ttgacgctga 1380 tggaagccac agcagttccc caactgtttt gaattctagt ggcagaatgg atgaatctgt 1440 ttggcgacca tattgaaatt cctcagcagt ggcccagtgg tatctggggg cc 1492 <210> 6 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Primer used in Example 1 <400> 6 gaacccacgg ccctccctga ac 22 <210> 7 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> Primer used in Example 1 <400> 7 ccgggcacca tgggaaactg atagga 26 <210> 8 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Primer used in Example 1 <400> 8 tcccccggcc acttcgctaa cttg 24 <210> 9 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Primer used in Example 1 <400> 9 ggagggacta tggcatcaaa cagc 24 <210> 10 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Primer used in Example 1 <400> 10 actaccagcc accgagacca acag 24 <210> 11 <211> 17 <212> DNA <213> Artificial Sequence <220> <223> Primer used in Example 1 <400> 11 ggcccccaga taccact 17 <210> 12 <211> 57 <212> DNA <213> Human <400> 12 atggcatcaa acagcctctt cagcacagtg acaccatgtc agcaaaactt cttttgg 57

[Brief description of drawings]

FIG. 1 shows the results of gene expression in the lesioned part and the normal part in the same cancer patient performed in Example 1.

FIG. 2 CBFA1 in breast cancer performed in Example 1
The result of expression of is shown.

FIG. 3 Type in Breast Cancer Patients Performed in Example 1
The expression result of eI CBFA1 is shown.

Front page continuation (51) Int.Cl. ⁷ Identification code FI theme code (reference) A61P 35/04 C12N 1/15 4C084 C12N 1/15 1/19 4C086 1/19 1/21 1/21 C12P 21/02 C 5/10 C12Q 1/68 A C12P 21/02 G01N 33/15 Z C12Q 1/68 33/50 Z G01N 33/15 33/574 Z 33/50 C12N 15/00 ZNAA 33/574 5/00 AF term (reference) 2G045 AA40 DA78 4B024 AA01 AA11 CA04 DA06 EA04 HA01 4B063 QA01 QA05 QA18 QQ53 QR48 QR51 QR75 QR77 QS33 QX02 QX07 4B064 AG01 CA02 CA10 CC24 DA01 DA13 4B065 AA26X AA93Y AB01 BA02 CA24 CA44 CA46 4C084 AA13 AA17 BA35 BA44 CA11 CA53 CA56 CA59 NA14 ZB262 4C086 AA01 AA03 EA16 MA01 MA04 NA13 NA14 ZB26

Claims (19)

[Claims] 1. A compound which suppresses the expression of a gene containing the DNA, which comprises using a DNA containing the same or substantially the same nucleotide sequence as the nucleotide sequence represented by SEQ ID NO: 1, or a compound thereof. Salt screening method. 2. A cell having the ability to produce a protein containing the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 2 or a salt thereof,
2. The screening according to claim 1, wherein the amount of the protein or the amount of mRNA encoding the protein in the presence or absence of the test compound is measured in the presence or absence of the test compound, respectively. Method. 3. A cell having the ability to produce a protein containing the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 2 or a salt thereof,
The screening method according to claim 2, which is a transformant transformed with a recombinant vector containing a DNA containing the same or substantially the same nucleotide sequence as the nucleotide sequence represented by SEQ ID NO: 1. 4. The method according to claim 1, wherein the DNA is chromosomal DNA.
3. The screening method described in 3. 5. A method for screening a compound or its salt that suppresses the activity of a protein, which comprises using a protein containing the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 2. .. 6. The screening method according to claim 5, wherein the activity is transcription activity. 7. A preventive / therapeutic agent for cancer, a cancer metastasis inhibitor, or a cancer metastasis preventive agent, which comprises the compound obtained by the screening method according to claim 1 or a salt thereof. 8. A diagnostic agent for cancer or metastasis thereof, which comprises a DNA containing a base sequence identical or substantially identical to the base sequence represented by SEQ ID NO: 1. 9. A method for diagnosing cancer or metastasis thereof, which uses a DNA containing the same or substantially the same base sequence as the base sequence represented by SEQ ID NO: 1. 10. A medicament containing an antisense polynucleotide containing a base sequence complementary to the same or substantially the same base sequence as the base sequence represented by SEQ ID NO: 1, or a part thereof. 11. The medicine according to claim 10, which is a preventive / therapeutic agent for cancer, a cancer metastasis inhibitor or a cancer metastasis preventive agent. 12. A DNA containing the same or substantially the same base sequence as the base sequence represented by SEQ ID NO: 3, SEQ ID NO: 4 or SEQ ID NO: 5. 13. The DNA according to claim 12, which comprises the nucleotide sequence represented by SEQ ID NO: 3, SEQ ID NO: 4 or SEQ ID NO: 5. 14. A recombinant vector containing the DNA according to claim 12. 15. A transformant transformed with the recombinant vector according to claim 14. 16. The transformant according to claim 15 is cultured to produce a protein containing the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 2. A method for producing a protein or a salt thereof. 17. A DNA containing the same or substantially the same base sequence as the base sequence represented by SEQ ID NO: 1.
Is the DNA according to claim 12, and the screening method according to claim 1. 18. A method for preventing / treating cancer, cancer metastasis, which comprises administering an effective amount of the compound or its salt obtained by the screening method according to claim 1 to a mammal. Suppression method or cancer metastasis prevention method. 19. Use of a compound or a salt thereof obtained by the screening method according to any one of claims 1 to 6 for producing a preventive / therapeutic agent for cancer, a cancer metastasis inhibitor or a cancer metastasis preventive agent.