WO2003085106A1

WO2003085106A1 - Preventives and/or remedies for cancer

Info

Publication number: WO2003085106A1
Application number: PCT/JP2003/004272
Authority: WO
Inventors: Yuichi Hikichi; Koji Yoshimura; Isao Kaieda
Original assignee: Takeda Chemical Industries, Ltd.
Priority date: 2002-04-05
Filing date: 2003-04-03
Publication date: 2003-10-16
Also published as: JP2003289876A; AU2003236358A1

Abstract

Oligonucleotides having base sequences which are the same or substantially the same as a base sequence represented by SEQ ID NO:13 or 14 are useful as excellent anticancer agents.

Description

Description Cancer preventive and / or therapeutic agent

The present invention relates to antisense oligonucleotides useful for cancer prevention and treatment, screening for cancer prevention and treatment agents, and the like. Background art

In cancer chemotherapy, the development of new anticancer drugs has improved the life-span effect and is increasing the number of cases toward cure. However, most of the currently used anticancer drugs are highly cytotoxic drugs that damage DNA or inhibit cell division. Strong side effects often appear in the rapidly dividing bone marrow. For these reasons, there is a strong need for drugs that target molecules that are specifically expressed in cancer cells and that can inhibit the growth of cancer cells or induce apoptosis.

Antisense oligonucleotides, when introduced into cells, hybridize with RNA having a complementary sequence and induce RNA degradation by RNaseH to inhibit protein translation, or hybridize to inhibit direct protein synthesis. Also bring. Since it is possible to specifically suppress the function of the target gene, it is frequently used as a means for analyzing the function of genes, and some antisense oligonucleotides are being developed for clinical application.

Protein tyrosine kinase genes form the largest family of oncogenes and are deeply involved in cell proliferation and differentiation and morphogenesis (Harvey Lecto. 94, 81-119, 1998- 1999), it has been picked up as a good target gene for developing botanicals. In particular, drug development targeting the EGF receptor, HER2 receptor or VEGF receptor, which is classified as receptor tyrosine kinase (RTK), has produced anticancer drugs at a level that can be tested in humans. Among the tyrosine kinases, RTK is considered to be one of the subfamilies with a high probability of success as pharmaceuticals. At present, among the RTKs, the Eph (erythropoietin producing human hepatocellular carcinoma cell line) receptor gene family is currently composed of 14 members and has the largest size (Cell 90, 403-404, 1997). ). The Ephr in gene family, identified as ligands for the Eph receptor, has also been reported to consist of at least eight members. Regarding the physiological functions of Eph receptors and Ephr in ligands, extremely localized expression in embryos and the brain suggests their involvement in morphogenesis during development and proper projection of nerve axons ( Annu. Rev. Neurosci. 21, 309-345 (1998)), but its role in cancer has also attracted attention in recent years. For example, increased expression of Eph receptor in various cancers has been reported

(Oncogene 19, 5614-5619, 2000). Furthermore, its involvement in angiogenesis has been suggested (Genes & Dev. 13, 1055-1066, 1999). There is a strong need for drugs that target molecules expressed in cancer cells and that can inhibit the growth of cancer cells or induce apoptosis. Disclosure of the invention

The present inventors have conducted intensive studies to solve the above-mentioned problems, and as a result, found that cancer cells cause apoptosis by suppressing the expression of NEPHA gene expressed in cancer cells. Further studies based on this finding have led to the completion of the present invention.

That is, the present invention

(1) an oligonucleotide having a nucleotide sequence identical or substantially identical to the nucleotide sequence represented by SEQ ID NO: 13 or SEQ ID NO: 14,

(2) an oligonucleotide having a base sequence represented by SEQ ID NO: 13;

(3) an oligonucleotide consisting of the nucleotide sequence represented by SEQ ID NO: 14, (4) the oligonucleotide according to (1) above, which is an antisense oligonucleotide,

(5) the oligonucleotide according to the above (1), which is DNA;

(6) a medicine comprising the oligonucleotide according to the above (1),

(7) The medicament according to the above (6), which is an agent for preventing and / or treating cancer. (8) a DNA containing the same or substantially the same base sequence as the base sequence represented by SEQ ID NO: 20,

(9) The DNA according to the above (8), which is a transcription control region of a DNA containing the same or substantially the same nucleotide sequence as the nucleotide sequence represented by SEQ ID NO: 12.

(10) the DNA according to the above (9), wherein the transcription regulatory region is a promoter region;

(11) a recombinant vector containing the DNA of (8) above,

(12) The method according to the above (11), wherein the DNA comprises a DNA having a reporter gene downstream of a transcriptional regulatory region of the DNA having the same or substantially the same nucleotide sequence as the nucleotide sequence represented by SEQ ID NO: 12. Described recombination vector,

(13) a transformant containing the recombinant vector according to the above (11),

(14) The use of the DNA according to (8) above, which promotes the transcription control activity of DNA containing the same or substantially the same nucleotide sequence as the nucleotide sequence represented by SEQ ID NO: 12. Or a method of screening for a compound or a salt thereof that inhibits

(15) The screening method according to the above (14) using the transformant according to the above (13),

(16) Promotes the activity of regulating the transcription of DNA having the same or substantially the same nucleotide sequence as the nucleotide sequence represented by SEQ ID NO: 12, characterized by containing the DNA of (8) above. Or a screening kit for a compound that inhibits or a salt thereof,

(17) Contains the same or substantially the same nucleotide sequence as the nucleotide sequence represented by SEQ ID NO: 12 obtained by using the screening method according to (14) or the screening kit according to (16). Compounds or salts thereof that promote or inhibit the activity of regulating the transcription of DNA,

(17a) It contains the same or substantially the same nucleotide sequence as the nucleotide sequence represented by SEQ ID NO: 12 obtained by using the screening method described in (14) or the screening kit described in (16). A compound or a salt thereof that inhibits the transcription regulation activity of DNA,

(18) a medicament comprising the compound according to (17) or a salt thereof, (18a) a medicament comprising the compound according to (17a) or a salt thereof, (19) the medicament according to the above (18), which is an agent for preventing and / or treating cancer; (19a) the medicament according to the above (18a), which is an agent for preventing and / or treating cancer; A method for preventing and treating cancer, which comprises administering to an animal an effective amount of the oligonucleotide according to (1).

(21) Use of the oligonucleotide described in (1) above for the manufacture of an agent for preventing or treating cancer. BEST MODE FOR CARRYING OUT THE INVENTION

Oligonucleotide containing a base sequence substantially identical to the base sequence represented by SEQ ID NO: 13 or SEQ ID NO: 14 (hereinafter, abbreviated as oligonucleotide of the present invention or antisense oligonucleotide of the present invention) Is an Eph receptor protein having the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 11 (hereinafter, the receptor protein of the present invention or the Eph receptor protein of the present invention) (May be abbreviated as) and can inhibit the synthesis or function of the RNA, or through the interaction with the receptor protein-related RNA of the present invention. The expression of the receptor protein gene can be regulated and / or controlled.

Oligonucleotides containing a base sequence substantially identical to the base sequence represented by SEQ ID NO: 13 or SEQ ID NO: 14 include nucleotides represented by SEQ ID NO: 13 or SEQ ID NO: 14. Any oligonucleotide having a nucleotide sequence that hybridizes under stringent conditions and having substantially the same activity as the oligonucleotide having the nucleotide sequence represented by SEQ ID NO: 13 or SEQ ID NO: 14 It may be something.

Examples of substantially equivalent activities include, for example, the activity of inhibiting the expression of the receptor protein of the present invention. Examples of the expression inhibiting activity include transcription inhibition, splicing inhibition of precursor RNA, inhibition of mRNA translocation to the cytoplasm, and inhibition of translation into protein.

Substantially the same indicates that the activity is the same in nature. Therefore, activities such as the above-mentioned expression inhibitory activity are equivalent (eg, about 0.01 to 100 times, preferably about (0.5 to 20 times, more preferably about 0.5 to 2 times). The measurement of the above-mentioned inhibitory activity and the like can be performed according to a known method. The inhibitory activity is determined by using a transformant containing the receptor protein of the present invention, an in vivo or in vitro gene expression system of the receptor protein of the present invention, or an in vivo or in vitro translation system of the receptor protein of the present invention. Can be checked using For example, the method described in (A) the method for measuring the mRNA amount of the receptor protein of the present invention or the partial peptide thereof, and the method described in (B) the method for quantifying the receptor protein or the partial peptide or a salt thereof of the present invention described below are used. , Can be measured.

Examples of DNA that can hybridize with the nucleotide sequence represented by SEQ ID NO: 13 or SEQ ID NO: 14 under high stringent conditions include, for example, SEQ ID NO: 13 or SEQ ID NO: 14 A base having about 60% or more, more preferably about 70% or more, more preferably about 80% or more, particularly preferably about 90% or more, and most preferably about 95% or more homology with the base sequence. DNA containing a sequence is used. .

Hybridization can be performed by a method known per se or a method analogous thereto, for example, a method described in Molecular Cloning 2nd (J. Sambrook et al., Cold Spring Harbor Lab. Press, 1989). Etc. When a commercially available library is used, the procedure can be performed according to the method described in the attached instruction manual. More preferably, it can be carried out under high stringency conditions.

High stringency conditions include, for example, a sodium concentration of about 19 to 40 mM, preferably about 19 to 20 mM, and a temperature of about 50 to 70, preferably about 60 to 60. The condition of 5 t is shown. Particularly, the case where the sodium concentration is about 19 mM and the temperature is about 65 is most preferable.

"Oligonucleotides" are polydeoxynucleotides containing 2-dexoxy-D-reports, polydeoxynucleotides containing D-reports, N-glycosides of purine or pyrimidine bases. Other types of oligonucleotides or other polymers with non-nucleotide backbones (eg, commercially available protein nucleic acids and synthetic sequence-specific nucleic acid polymers) or specialty Other polymers containing a suitable bond (these polymers include base pairing as found in DNA and RNA, and contain a nucleotide having a configuration permitting base attachment). . These can be double-stranded DNA, single-stranded DNA, double-stranded RNA, single-stranded RNA, and even DNA: RNA hybrids, and can also be unmodified oligonucleotides (or unmodified oligonucleotides). Nucleotides), as well as those with known modifications, such as labeled, capped, methylated, one or more natural nucleotides, as known in the art. Is substituted with an analog, modified with an intramolecular nucleotide, for example, has an uncharged bond (eg, methylphosphonate, phosphotriester, phosphoramidite, thiolbamate, etc.), a charged bond or sulfur Those having contained bonds (for example, phosphorothioate, phosphorodithioate, etc.), for example, proteins (nucleases, nucleases / inhibitors) , Toxins, antibodies, signal peptides, poly-L-lysine, etc.) or sugars (for example, monosaccharides), etc., or those having an intermolecular compound (for example, acridine, psoralen, etc.) , Compounds containing chelating compounds (eg, metals, radioactive metals, boron, oxidizable metals, etc.), those containing alkylating agents, those with modified bonds (eg, α-anomers) Type nucleic acid). Here, “nucleoside”, “nucleotide” and “nucleic acid” may include not only those containing purine and pyrimidine bases but also those having other modified heterocyclic bases. Such modifications may include methylated purines and pyrimidines, acylated purines and pyrimidines, or other heterocycles. Modified nucleotides and modified nucleotides may also be modified at the sugar moiety, e.g., where one or more hydroxyl groups have been replaced with halogens, aliphatic groups, etc., or functional groups such as ethers, amines, etc. It may be converted to a base. The oligonucleotide of the present invention may be a modified nucleic acid (RNA, DNA). Specific examples of the modified nucleic acid include a sulfur derivative, a thiophosphate derivative, and a nucleic acid that is resistant to degradation of polynucleoside amide or oligonucleoside amide.

As a specific example of the oligonucleotide of the present invention, a base represented by SEQ ID NO: 13 DNA containing a sequence, DNA containing the base sequence represented by SEQ ID NO: 14 and the like are used.

The oligonucleotide of the present invention is produced according to a known method, for example, a solid phase or liquid phase synthesis method (eg, an automated nucleic acid synthesizer, liquid phase technology, etc.). Alternatively, it may be produced according to known gene engineering techniques (eg, use of reverse transcriptase, PCR synthesis, etc.).

Examples of the amino acid sequence substantially the same as the amino acid sequence represented by SEQ ID NO: 11 include, for example, about 55% or more, and preferably about 60% or more of the amino acid sequence represented by SEQ ID NO: 11, More preferably, an amino acid sequence having a homology of about 70% or more, more preferably about 80% or more, particularly preferably about 90% or more, and most preferably about 95% or more. .

Examples of a protein having an amino acid sequence substantially identical to the amino acid sequence represented by SEQ ID NO: 11 include, for example, a protein having an amino acid sequence substantially identical to the amino acid sequence represented by SEQ ID NO: 11 A protein having substantially the same activity as the amino acid sequence represented by SEQ ID NO: 11 is preferred.

Substantially the same activity includes, for example, ligand binding activity, signal transduction activity, cell death inhibitory activity, cell adhesion activity, and cell motility regulating activity. Substantially the same indicates that their activities are the same in nature. Therefore, activities such as ligand binding activity, signal transduction activity, cell death inhibitory activity, cell adhesion activity and cell motility control activity are equivalent (eg, about 0.01 to 100 times, preferably Is preferably about 0.5 to 20 times, more preferably about 0.5 to 2 times), but the quantitative factors such as the degree of activity and the molecular weight of the protein may be different.

Measurement of ligand binding activity, signal transduction activity, cell death inhibitory activity, cell adhesion activity, cell motility control activity, and the like can be performed according to known methods. In the present specification, the amino acid sequence of the receptor protein is the N-terminus (amino terminus) at the left end and the C-terminus (aminopropyl end) at the right end, according to the convention of peptide notation. Receptor protein of the present invention, C-terminal, the force Rupokishiru group (-C00H), Karupokishire bets (-C00-), amide (- C0NH ₂₎ or ester (- C00R) may be either. Here, as R in the ester, e.g., methyl, Echiru, n- propyl, C _1H5 alkyl group such as isopropyl or n- butyl, cyclopentyl Le, C ₃ _ ₈ cycloalkyl group such as cyclohexyl, for example, phenyl, (3 ₆ _ ₁₂ Ariru groups, such as single-naphthyl shed, for example, benzyl, phenylene route C DOO ₂ alkyl or α- naphthylmethyl etc. α- naphthyl such as phenethyl - C, such as _ ₂ alkyl groups in addition to C ₇ _ ₁₄ Ararukiru group, Viva port Iruo Kishimechiru group commonly used as an oral ester.

When the receptor protein of the present invention has a lipoxyl group (or carboxylate) other than the C-terminus, the receptor protein of the present invention also includes those in which the lipoxyl group is amidated or esterified. . As the ester in this case, for example, the above-mentioned C-terminal ester and the like are used.

Furthermore, the receptor protein of the present invention is the protein mentioned above, Amino group protecting groups methylate Onin residues of N-terminal (e.g., formyl group, C ₂ such Asechiru - such Ashiru group such as ₆ Al Kanoiru group ), The N-terminal side is cleaved in vivo, and the daryumil group formed is pyroglutamine-oxidized, the substituent on the side chain of the amino acid in the molecule (eg, -0H, -SH, amino group, imidazole group, indole group, Guanijino group, etc.) a suitable protecting group (e.g., formyl group, C ₂ such Asechiru - those protected by ₆ etc. Ashiru groups such Arukanoiru group), a sugar chain Complex proteins such as bound so-called glycoproteins are also included.

Specific examples of the receptor protein of the present invention include, for example, a receptor protein containing the amino acid sequence represented by SEQ ID NO: 11 and the like.

Further, a partial peptide of the receptor protein of the present invention (hereinafter, sometimes abbreviated as the partial peptide of the present invention) can also be used. The partial peptide of the present invention may be any peptide as long as it is a partial peptide of the above-described receptor protein of the present invention. For example, of the receptor protein molecules of the present invention, For example, a portion having a substantially identical activity or a portion located inside the cell membrane is used.

Here, “substantially the same activity” refers to, for example, ligand binding activity, signal information transmission activity, cell death inhibitory activity, cell adhesion activity, and cell motility control activity. You. Ligand binding activity, signal transduction activity, cell death suppressing activity, measurements, such as cell adhesion activity and cell motility control activity, the amino acid number of the partial base peptide of the _c the present invention can be carried out according to known methods, Peptides having an amino acid sequence of at least 5 or more, preferably 10 or more, preferably 50 or more, more preferably 100 or more of the amino acid sequences of the above-described receptor protein of the present invention are preferable. . A substantially identical amino acid sequence is at least about 50%, preferably at least about 60%, more preferably at least about 70%, still more preferably at least about 80%, and most preferably at least about 50% of these amino acid sequences. An amino acid sequence having about 90% or more, most preferably about 95% or more homology is shown.

In addition, the partial peptide of the present invention comprises: (i) one or more (preferably about 1 to 10, more preferably several (1 to 5)) amino acids in the above amino acid sequence; (Ii) one or two or more (preferably about 1 to 20, more preferably about 1 to 10, and more preferably several (1 to 5)) (Iii) one or more (preferably about 1 to 10, more preferably several, more preferably about 1 to 5) amino acids in the above amino acid sequence It may be substituted with an amino acid. Specific examples of the partial peptide of the present invention include the 60th to 73rd or 491st to 504th amino acid sequences of the amino acid sequence represented by SEQ ID NO: 11. Examples of such peptides include:

In the partial peptide, the C-terminus carboxyl group of the present invention (- C00H), Karupoki Shireto (- C00-), amide (- C0NH ₂₎ or esters may be a (-C00R) (R is as defined above Is shown). When the partial peptide of the present invention has a carboxyl group (or carboxylate) other than the C-terminus, those in which the carbonyl group is amidated or esterified are also included in the partial peptide of the present invention. As the ester in this case, for example, the above-mentioned C-terminal ester and the like are used.

Further, similar to the above-described receptor protein of the present invention, the partial peptide of the present invention has a N-terminal methionine residue in which the amino group of the methionine residue is protected with a protecting group, and the N-terminal side is cleaved in vivo to produce Dartamyl group is pyroglutamine-oxidized, amino acid side chain in the molecule is protected by a suitable protecting group, or sugar chain Conjugated peptides, such as so-called glycopeptides, to which is bound.

Examples of the salt of the receptor protein or its partial peptide of the present invention include a physiologically acceptable salt with an acid or a base, and a physiologically acceptable acid addition salt is particularly preferable. Such salts include, for example, salts with inorganic acids (eg, hydrochloric acid, phosphoric acid, hydrobromic acid, sulfuric acid) or organic acids (eg, acetic acid, formic acid, propionic acid, fumaric acid, maleic acid, succinic acid) Acids, tartaric acid, citric acid, malic acid, oxalic acid, benzoic acid, methanesulfonic acid, benzenesulfonic acid) and the like are used.

The receptor protein of the present invention or a salt thereof can be produced from the above-described human or mammalian cells or tissues by a known method for purifying a receptor protein, or encodes the receptor protein of the present invention described later. It can also be produced by culturing a transformant containing DNA. Also, the protein can be produced by the protein synthesis method described later or according to this method.

When producing from human or mammalian tissues or cells, the human or mammalian tissues or cells are homogenized and then extracted with an acid or the like, and the resulting extract is subjected to reverse phase chromatography, ion exchange chromatography, etc. Purification and isolation can be carried out by combining the above chromatography methods.

For the synthesis of the receptor protein of the present invention, its partial peptide, its salt or its amide, a commercially available resin for protein synthesis can be usually used. Examples of such resins include chloromethyl resin, hydroxymethyl resin, benzhydrylamine resin, aminomethyl resin, 4-benzyloxybenzyl alcohol resin, 4-methylbenzhydrylamine resin, PAM resin, and 4-hydroxy resin. Methylmethylphenylacetamidomethyl resin, polyacrylamide resin, 4- (2 ', 4'dimethoxyphenylhydroxymethyl) phenoxy resin, 4- (2', 4'dimethyloxyphenyl Fmocaminoethyl) Phenoxy resins and the like can be mentioned. Using such a resin, amino acids having appropriately protected amino groups and side chain functional groups are condensed on the resin according to various known condensation methods in accordance with the amino acid sequence of the target protein or peptide. At the end of the reaction, a protein or peptide is cleaved from the resin, and at the same time, various protecting groups are removed.In addition, an intramolecular disulfide bond formation reaction is performed in a highly diluted solution to obtain a target protein or partial peptide. Or its amide form.

Regarding the condensation of the above protected amino acids, various activating reagents that can be used for protein synthesis can be used, and carbodiimides are particularly preferable. Examples of the carbopimides include DCC, Ν, Ν′-diisopropyl carbopimide, and ェ -ethyl-Ν ′-(3-dimethylaminoprolyl) carbopimide. Activation by these involves the addition of a protected amino acid directly to the resin along with a racemization inhibitor additive (eg, HOBt, HOOBt), or a symmetrical acid anhydride or HOBt ester or H〇〇Bt ester. Can be added to the resin after activation of the protected amino acid in advance.

The solvent used for activating the protected amino acid or for condensing with the resin can be appropriately selected from solvents known to be usable for the protein condensation reaction. For example, acid amides such as N, N-dimethylformamide, N, N-dimethylacetamide, and N-methylpyrrolidone; halogenated hydrocarbons such as methylene chloride and chloroform; alcohols such as trifluoroethanol. And sulfoxides such as dimethylsulfoxide, ethers such as pyridine, dioxane and tetrahydrofuran, nitriles such as acetonitrile and propionitrile, esters such as methyl acetate and ethyl acetate, or an appropriate mixture thereof. The reaction temperature is appropriately selected from the range known to be usable for the protein bond formation reaction, and is usually 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 a sufficient condensation cannot be obtained even by repeating the reaction, the unreacted amino acid can be acetylated using acetic anhydride or acetylimidazole.

Examples of the protecting group for the amino group of the starting material include Z, Boc, succinyl-pentyloxycarbonyl, isoporiloxycarbonyl, 4-methoxybenzyloxycarbonyl, C1-Z, Br-Z, Adamantyloxycarbonyl, trifluoroacetyl, phthaloyl, formyl, 2-ditrophenylsulfenyl, diphenylphosphinothioyl, Fmoc and the like are used. The lipoxyl group may be, for example, an alkyl esterified (eg, methyl, ether, propyl, butyl, tert-butyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, 2-adamantyl, etc.) Or cyclic alkyl esterification), aralkyl esterification (eg, benzyl ester, 4-nitrobenzyl ester, 4-methoxybenzyl ester, 4-cyclobenzyl ester, benzhydryl esterification), phenacyl ester Can be protected by benzyloxycarbonyl hydrazide, short-lived butoxycarbonyl hydrazide, trityl hydrazide, etc.

The hydroxyl group of serine can be protected, for example, by esterification or etherification. As a group suitable for the esterification, for example, a lower alkanol group such as an acetyl group, an aroyl group such as a benzoyl group, a group derived from carbonic acid such as a benzyloxycarbonyl group, an ethoxycarbonyl group and the like are used. In addition, groups suitable for etherification include, for example, a benzyl group, a tetrahydropyranyl group, and a t-peptizole group.

The protecting group of the phenolic hydroxyl group of tyrosine, for _{example, B zl, C 1 2 -} B z 1, 2 twelve Torobenjiru, B r- Z, such as tertiary butyl is used. Examples of the protecting group for histidine imidazole include Tos, 4-methoxy-2,3,6-trimethylbenzenesulfonyl, DNP, benzyloxymethyl, Bum, Boc, Trt, and Fmoc. Can be

Activated carbonyl groups of the raw materials include, for example, corresponding acid anhydrides, azides, active esters [alcohols (eg, pentachlorophenol, 2,4,5-trichloromouth phenol, 2,4-dinitro Phenol, cyanomethyl alcohol, paranitrophenol, H〇NB, N-hydroxysuccinimide, N-hydroxyphthalimide, and esters with HOBt). As the activated amino group of the raw material, for example, a corresponding phosphoric amide is used.

Methods for removing (eliminating) the protecting group include, for example, catalytic reduction in a hydrogen stream in the presence of a catalyst such as Pd-black or Pd-carbon, or hydrogen fluoride anhydride or methanesulfonic acid. , Trifluoromethanesulfonic acid, trifluoroacetic acid or this Acid treatment with a mixed solution thereof, base treatment with diisopropylethylamine, triethylamine, piperidine, piperazine, and the like, and reduction with sodium in liquid ammonia are also used. The elimination reaction by the above acid treatment generally takes about one

The reaction is carried out at a temperature of 20 ° C to 40 ° C. In the acid treatment, for example, anisol, phenol, thioanisole, metacresol, paracresol, dimethylsulfide, 1,4-butanedithiol, 1,2 —Addition of cation scavenger such as ethanedithiol is effective. 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 tributofan is 1,2-ethanedithiol, 1,4 —In addition to deprotection by acid treatment in the presence of butanedithiol, etc., it is also removed by alkali treatment with dilute sodium hydroxide solution, dilute ammonia, etc.

The protection of the functional group which should not be involved in the reaction of the raw materials, the protecting group, the elimination of the protective group, the activation of the functional group involved in the reaction, and the like can be appropriately selected from known groups or known means.

As another method for obtaining an amide form of a protein, for example, first, after amidating and protecting the α-hydroxyl group of the carboxy-terminal amino acid, a peptide (protein) chain is added to the amino group side to a desired chain length. After the elongation, a protein was prepared by removing only the protecting group of the α-amino group at the Ν-terminal of the peptide chain and a protein was obtained by removing only the protecting group of the carboxy group at the C-terminus. In such a mixed solvent. Details of the condensation reaction are the same as described above. After purifying the protected protein obtained by the condensation, all the protecting groups are removed by the above-mentioned method, and a desired crude protein can be obtained. This crude protein is purified by various known purification means, and the main fraction is freeze-dried to obtain an amide of the desired protein.

In order to obtain a protein ester, for example, after condensing the carboxyl group of the carboxy terminal amino acid with a desired alcohol to form an amino acid ester, the ester of the desired protein is converted in the same manner as the amide of the protein. Obtainable.

The partial peptide of the receptor protein of the present invention or a salt thereof can be obtained by cleaving the receptor protein of the present invention with an appropriate peptidase according to a known peptide synthesis method. And can be manufactured by As a peptide synthesis method, for example, any of a solid phase synthesis method and a liquid phase synthesis method may be used. That is, the objective peptide is produced by condensing a partial peptide or amino acid capable of constituting the receptor protein of the present invention with the remaining portion, and if the product has a protecting group, removing the protecting group to produce the desired peptide. Can be. Known condensation methods and elimination of protecting groups include, for example, the following (i) to

(V).

(i). Bodanszky and M.A. Ondetti, Peptide Synthesis;, Interscience Publishers, New York (1966)

(ii) Schroeder and Luebke, The Peptide, Academic Press, New York (1965)

(iii) Nobuo Izumiya et al., Fundamentals and experiments of peptide synthesis, Maruzen Co., Ltd. (1975)

(iv) Haruaki Yajima and Shunpei Sakakibara, Laboratory of Biochemistry 1, Protein Chemistry IV, 205, (1977)

(V) Supervised by Haruaki Yajima, Continuing Drug Development

After the reaction, the partial peptide of the present invention can be purified and isolated by a combination of ordinary purification methods, for example, solvent extraction, distillation, column chromatography, 'liquid chromatography', and recrystallization. When the partial peptide obtained by the above method is a free form, it can be converted to an appropriate salt by a known method, and conversely, when it is obtained as a salt, it is converted to a free form by a known method. be able to.

The polynucleotide encoding the receptor protein of the present invention is not particularly limited as long as it contains the above-described nucleotide sequence (DNA or RNA, preferably DNA) encoding the receptor protein of the present invention. Is also good. The polynucleotide is RNA such as DNA or mRNA encoding the receptor protein of the present invention, and may be double-stranded or single-stranded. In the case of double-stranded, it may be double-stranded DNA, double-stranded RNA or DNA: RNA hybrid. In the case of a single strand, it may be a sense strand (ie, a coding strand) or an antisense strand (ie, a non-coding strand).

Examples of the DNA encoding the receptor protein of the present invention include genomic DNA, genomic DNA library, cDNA derived from the above-described cells and tissues, and cells derived from the above-described cells. It may be either a conventional cDNA library or synthetic DNA. The vector used for the library may be any of bacteriophage, plasmid, cosmid, phagemid and the like. Alternatively, DNA may be directly amplified by reverse transcriptase polymerase chain reaction (hereinafter abbreviated as RT-PCR) using a preparation of all RNA or mRNA fractions from the above-mentioned cells' tissues. it can. Specifically, as the DNA encoding the receptor protein of the present invention, for example, a DNA containing the nucleotide sequence represented by SEQ ID NO: 12 or the nucleotide represented by SEQ ID NO: 12 Has DNA that hybridizes under high stringent conditions to DNA containing the sequence, and has substantially the same activity as the receptor protein of the present invention (eg, ligand binding activity, signal transduction activity, cell death) Any DNA may be used as long as it encodes a receptor protein having inhibitory activity, cell adhesion activity, and cell motility regulating activity.

Examples of the DNA which hybridizes with the DNA having the nucleotide sequence represented by SEQ ID NO: 12 under high stringent conditions include, for example, about 55% or more of the nucleotide sequence represented by SEQ ID NO: 12; Preferably containing a base sequence having a homology of about 60% or more, preferably about 70% or more, preferably about 80% or more, more preferably about 90% or more, and still more preferably about 95% or more. DNA or the like is used.

Hybridization can be performed by a known method or a method analogous thereto, for example, according to the method described in Molecular Cloning 2nd (J. Sambrook et al., Cold Spring Harbor Lab. Press, 1989). When a commercially available library is used, it can be carried out according to the method described in the attached instruction manual. More preferably, hybridization can be performed under high stringency conditions.

The high stringency conditions include, for example, a sodium concentration of about 19 to 40 mM, preferably about 19 to 20 mM, and a temperature of about 50 to 70 ° C, preferably about 60 to 70 ° C. The condition of 65 T: is shown. In particular, the case where the sodium concentration is about 19 mM and the temperature is about 65 ° C is most preferable.

The DNA encoding the partial peptide of the present invention may be any DNA containing the above-described nucleotide sequence encoding the partial peptide of the present invention. Also, any of genomic DNA, genomic DNA library, cDNA derived from the above-described cells and tissues, cDNA library derived from the above-described cells and tissues, and synthetic DNA may be used. The vector used for the library may be any of bacteriophage, plasmid, cosmid, phagemid and the like. DNA can also be directly amplified by the RT-PCR method using an mRNA fraction prepared from the cells and tissues described above.

Specifically, the DNA encoding the partial peptide of the present invention includes, for example, DNA having a partial nucleotide sequence of DNA containing the nucleotide sequence represented by SEQ ID NO: 12, or DNA represented by SEQ ID NO: 12. A protein containing DNA that hybridizes under high stringent conditions with the DNA to be obtained and having substantially the same activity as the receptor protein of the present invention, for example, ligand binding activity, signal transduction activity, etc. For example, a DNA having a partial nucleotide sequence of the DNA to be used is used.

As a means for cloning a DNA that completely encodes the receptor protein of the present invention or a partial peptide thereof (hereinafter, sometimes abbreviated as the receptor protein of the present invention), a DNA encoding the receptor protein of the present invention may be used. Amplification by the PCR method using a synthetic DNA primer having a partial nucleotide sequence of the nucleotide sequence, or DNA incorporating the DNA into a suitable vector is performed for partial or whole region of the receptor protein of the present invention. Selection can be performed by hybridization with a DNA fragment to be coded or labeled with a synthetic DNA.

The DNA base sequence can be replaced by ODA-LA PCR using PCR or a known kit, for example, Mutan ™ -super Express Km (Takara Shuzo Co., Ltd.), Mutan ™ -K (Takara Shuzo Co., Ltd.), etc. The method can be performed according to known methods such as the gapped duplex method and the Kunkel method, or a method analogous thereto.

The DNA encoding the cloned receptor protein can be used as it is depending on the purpose, or can be used by digesting with a restriction enzyme or adding a linker if desired. The DNA may have ATG as a translation initiation codon at the 5 'end, and TAA, TGA or TAG as a translation stop codon at the 3' end. These translation initiation codon and translation termination codon can also be added using a suitable synthetic DNA adapter. The DNA encoding the receptor protein of the present invention includes, for example, (a) a DNA fragment encoding the receptor protein of the present invention; Can be incorporated into an expression vector by ligating the DNA downstream of the promoter in an appropriate expression vector.

Examples of vectors for cloning or expression include plasmids derived from E. coli (eg, pCR4, pCR2.K pBR322, pBR325, pUC12, pUC13), and plasmids derived from Bacillus subtilis (eg, pUB110, pTP5, pC194) ), Yeast-derived plasmids (eg, pSH19,

PSH15), bacteriophage such as λ phage, animal viruses such as retrovirus, vaccinia virus, baculovirus, etc., as well as pAl-11, XTK pRc / CMV, pRc / RSV, pcDNAI / Neo, etc. .

The promoter may be any promoter as long as it is appropriate for the host used for gene expression. For example, when an animal cell is used as a host, SRo; a promoter, an SV40 promoter, an LTR promoter, a CMV promoter, an HSV-TK promoter, and the like can be mentioned. Of these, it is preferable to use CMV promoter, SRa promoter and the like.

When the host is Escherichia, trp promoter, 1 ac promoter, recA promoter, λ プロモーター ^ promoter, 1 ρ promoter, etc., and when the host is Bacillus, S S When the host is yeast, such as 〇1 promoter, SΡΟ2 promoter, peηΡ promoter, etc., ΡΗ05 promoter, PGK promoter, GAP promoter, ADH promoter and the like are preferable. If the host is an insect cell, the polyhedrin promoter and the P10 promoter

—I prefer evening.

In addition to the above, expression vectors include, if desired, an enhancer, a splicing signal, a polyA addition signal, a selection marker, and an SV40 replication origin (hereinafter, referred to as the

Examples of the _c- selectable marker that can be used include a gene for dihydrofolate reductase (hereinafter sometimes abbreviated as dhfr) gene [methotrexate (MTX) resistance ], An ampicillin resistance gene (hereinafter sometimes abbreviated as Amp ¹ ), a neomycin resistance gene (hereinafter sometimes abbreviated as Ne of, G418 resistance), and the like. When the dhfr gene is used as a selection marker using (dhfr) cells, the target gene can be selected even with a thym-free medium.

If necessary, a signal sequence suitable for the host can be added to the N-terminal side of the receptor protein of the present invention. If the host is a genus Escherichia, the Ph A signal sequence, the 〇mp A signal sequence, etc., if the host is a Bacillus genus, the α-amylase signal sequence, subtilisin signal sequence, etc. If the host is yeast, MF, signal sequence, SUC2, signal sequence, etc.If the host is an animal cell, insulin signal sequence, Hi-interferon, signal sequence, antibody molecule, signal sequence, etc. Are available respectively.

Using the vector containing the DNA encoding the receptor protein of the present invention thus constructed, a transformant can be produced.

Examples of the host used in the present invention include Escherichia bacteria, Bacillus bacteria, yeast, insect cells, insects, animal cells, and the like.

Specific examples of the bacterium belonging to the genus Escherichia include Escherichia coli K12-DH1 [Proc. Natl. Acad. Sci. USA, 60, 160 (1968)], JM103.

CNucleic Acids Research, 9, 309 (1981)], JA221 [Journal of Molecular Biology, 120, 517 (1978)], HB101 [Journal of Molecular Biology, 41, 459 (1969)], C600 [Genetics, 39 Vol., 440 (1954)], DH5a [Gene, 96, 23-28 (1990)], DHIOB [Proc. Natl. Acad. Sci. USA, 87, 645-4649 (1990)], etc. .

Bacillus bacteria include, for example, Bacillus subtilis (Bacillus

subtilis) MI114 [Gene, 24, 255 (1983)], 207-21 [Journal of

Biochemistry, 95, 87 (1984)).

Examples of yeast include, for example, Saccharomyces cerevisiae AH22, AH22R-, ONA87-11A, DKD-5D, 20B-12, Schizosaccharomyces pombe NCYC1913, NCYC2036 Pichia pastoris (Pichia pastoris) ) Is used.

Insect cells include, for example, when the virus is AcNPV, a cell line derived from a larva of Spodoptera (Spodoptera frugiperda cell; Si cell), or the midgut of Trichoplusia ni Original MG1 cells, High Five ™ cells derived from Trichoplus ia ni eggs, Mames tra

Cells derived from brass icae or cells derived from Estigmena acrea are used. When the virus is BmNPV, a cell line derived from silkworm (Bombyx mori N; BmN cell) is used. As the Sf cells, for example, Sf9 cells (ATCC CRL1711), Sf21 cells (hereinafter, In Vivo, 13, 213-217 (1977)) and the like are used.

As insects, for example, silkworm larvae are used [Nature, vol. 315, 592 (1985)].

Examples of animal cells include monkey cells COS-7, Vero, Chinese Hams Yuichi cell CH0 (hereinafter abbreviated as CH0 cells), dhfr gene-deficient Chinese Hams Yuichi cells CH0 (hereinafter abbreviated as CHO (dhfr) cells). ), Mouse L cells, mouse AtT-20, mouse myeloma cells, rat GH3, human FL cells, and the like.

Transformation of a bacterium belonging to the genus Escherichia can be performed, for example, according to the method described in Proc. Natl. Acad. Sci. USA, 69, 2110 (1972) or Gene, 17, 107 (1982). it can.

Transformation of Bacillus can be performed, for example, according to the method described in Molecular & General Genetics, Vol. 168, 111 (1979).

Transformation of yeast can be performed, for example, according to the method described in Methods in Enzymology, Vol. 194, 182-187 (1991), Proc. Natl. Acad. Sci. USA, Vol. 75, 1929 (1978). Can be.

Insect cells or insects can be transformed, for example, according to the method described in Bio / Technology, 6, 47-55 (1988).

To transform animal cells, for example, see Cell Engineering Separate Volume 8 New Cell Engineering Experimental Protocol. 263-267 (1995) (published by Shujunsha), Virology, Vol. 52, 456 (1973). It can be performed according to the method.

Thus, a transformant transformed with the expression vector containing the DNA encoding the Eph receptor protein is obtained.

When culturing a transformant whose host is a bacterium belonging to the genus Escherichia or Bacillus, a liquid medium is suitable as a medium used for the cultivation, and a carbon source necessary for the growth of the transformant is contained therein. , Nitrogen sources, inorganic substances and others. As a carbon source, For example, glucose, dextrin, soluble starch, sucrose, etc.Nitrogen sources include, for example, inorganic or nitric acid salts such as ammonium salts, nitrates, corn steep liquor, peptone, power zein, meat extract, soybean meal, potato extract, etc. Examples of the organic substance and the inorganic substance include calcium chloride, sodium dihydrogen phosphate, magnesium chloride, and the like. In addition, yeast extract, vitamins, growth promoting factors and the like may be added. The pH of the medium is preferably about 5-8.

As a medium for culturing the genus Escherichia, for example, an M9 medium containing glucose and casamino acids (Journal of Experiments in Molecular Genetics, 431-433, Cold Spring Harbor Laboratory, New York 1972) is preferable. If necessary, an agent such as 3-indolylacrylic acid can be added to make the promoter work efficiently.

When the host is a bacterium belonging to the genus Escherichia, the cultivation is usually performed at about 15 to 43 ° C for about 3 to 24 hours, and if necessary, aeration and stirring may be added.

When the host is a bacterium belonging to the genus Bacillus, the cultivation is usually performed for about 30 to 4 (TC for about 6 to 24 hours, and if necessary, aeration and stirring may be applied.

When culturing a transformant in which the host is yeast, the culture medium may be, for example, a Burkholder minimum medium [Proc. Natl. Acad. Sci. USA, 77, 4505 (1980)] or 0.5 Natl. Acad. Sci. USA, 81, 5330 (1984)]]. The pH of the medium is preferably adjusted to about 5-8. Cultivation is usually carried out at about 20 ° C to 35 for about 24 to 72 hours, with aeration and agitation as necessary.

When culturing an insect cell or a transformant in which the host is an insect, a medium such as Grace's Insect Medium (Nature, 195, 788 (1962)) is supplemented as appropriate with an additive such as immobilized 10% serum. Are used. Preferably, the pH of the medium is adjusted to about 6.2-4. Culture is usually performed at about 27 ° C. for about 3 to 5 days, and if necessary, aeration and agitation are added.

When culturing a transformant whose animal host is an animal cell, for example, a MEM medium containing about 5 to 20% fetal bovine serum [Science, 122, 501 (1952)], a DMEM medium [Virology , 8 vol., 396 (1959)], RPMI1640 medium [The Journal of the Amer i can Med i cal Assoc i ation, Volume 199, 519 (1967)], 199 medium [Proceeding of the Socie ty for or the Biologi cal Med ici ne, Volume 73, 1 (1950) ] Etc. are used. Preferably, the pH is about 6-8. Culture is usually performed at about 30 ° C to 4 (TC for about 15 to 60 hours, and aeration and agitation are added as necessary.

As described above, the receptor protein of the present invention can be produced in the cells, in the cell membrane, or outside the cells of the transformant.

Separation and purification of the receptor protein of the present invention from the above culture can be performed, for example, by the following method.

In order to extract the receptor protein of the present invention from cultured cells or cells, after culturing, the cells or cells are collected by a known method, suspended in an appropriate buffer, and subjected to ultrasonication, lysozyme and / or lysozyme. Alternatively, a method of destroying bacterial cells or cells by freeze-thawing or the like, and then obtaining a crude extract of the receptor protein by centrifugation or filtration is used as appropriate. The buffer may contain a protein denaturant such as urea or guanidine hydrochloride, or a surfactant such as Triton X-100 ™. When the receptor protein is secreted into the culture solution, after the culture is completed, a culture supernatant containing the receptor protein is collected by a known method.

Purification of the receptor protein contained in the thus obtained culture supernatant or extract can be carried out by appropriately combining known separation and purification methods. These known separation and purification methods include methods that utilize solubility such as salting out and solvent precipitation, and methods that mainly use the difference in molecular weight, such as dialysis, ultrafiltration, and gel filtration. A method that utilizes a difference in charge such as ion exchange chromatography; a method that utilizes a specific affinity such as affinity chromatography; a method that utilizes a difference in hydrophobicity such as reverse-phase high-performance liquid chromatography; A method utilizing the difference between isoelectric points such as point electrophoresis is used.

When the receptor protein thus obtained is obtained as a free form, it can be converted to a salt by a known method or a method analogous thereto, and conversely, when the receptor protein is obtained as a salt, the known method is used. Alternatively, it can be converted to a free form or another salt by a method analogous thereto.

The receptor protein produced by the recombinant can be arbitrarily modified or partially modified by the action of an appropriate protein-modifying enzyme before or after purification. Can also be removed. As the protein modifying enzyme, for example, trypsin, chymotrypsin, arginyl endopeptidase, protein kinase, glycosidase and the like are used.

The activity of the receptor protein of the present invention or a salt thereof thus produced can be measured by a binding experiment with a labeled ligand, an enzymimnoassay using a specific antibody, or the like.

The antibody against the receptor protein or its partial peptide or its salt of the present invention may be any of a polyclonal antibody and a monoclonal antibody as long as it is an antibody that can recognize the receptor protein or its partial peptide or its salt of the present invention. Is also good.

An antibody against the receptor protein of the present invention or a partial peptide thereof or a salt thereof (hereinafter, sometimes abbreviated as the receptor protein of the present invention) may be a known antibody using the receptor protein or the like of the present invention as an antigen. Alternatively, it can be produced according to a method for producing an antiserum.

[Preparation of monoclonal antibody]

(a) Preparation of monoclonal antibody-producing cells

The receptor protein or the like of the present invention is administered to a mammal at a site capable of producing an antibody by administration itself or together with a carrier or a diluent. Complete Freund's adjuvant / incomplete Freund's adjuvant may be administered in order to enhance antibody production upon administration. The administration is usually performed once every 2 to 6 weeks, for a total of about 2 to 10 times. Examples of mammals to be used include monkeys, rabbits, dogs, guinea pigs, mice, rats, sheep, and goats, and mice and rats are preferably used.

When producing monoclonal antibody-producing cells, a warm-blooded animal immunized with the antigen, for example, a mouse with an antibody titer is selected from a mouse, and the spleen or lymph node is collected 2 to 5 days after the final immunization. By fusing the antibody-producing cells contained in the above with myeloma cells, a monoclonal antibody-producing hybridoma can be prepared. The antibody titer in the antiserum can be measured, for example, by reacting the labeled receptor protein or the like described below with antiserum, and then measuring the activity of the labeling agent bound to the antibody. . The fusion operation is performed in a known manner, for example, in the case of Keller and Milstein. Act (Nature, 256, 495, 1975). Examples of the fusion promoter include polyethylene glycol (PEG) and Sendai virus, and PEG is preferably used.

Examples of myeloma cells include NS-1, P3UK SP2 / 0 and the like, and P3U1 is preferably used. The preferred ratio between the number of somatic cells (spleen cells) and the number of myeloma cells used is about 1: 1 to 20: 1, and PEG (preferably PEG1000 to

PEG6000) is added at a concentration of about 10 to 80%, and the cell fusion can be carried out efficiently by incubating at about 20 to 40 ° (: preferably at about 30 to 37 ° C for about 1 to 10 minutes).

Various methods can be used to screen for monoclonal antibodies producing hybridomas. For example, hybridomas are cultured on a solid phase (eg, microplate) onto which an antigen such as a receptor protein is adsorbed directly or together with a carrier. Then, an anti-immunoglobulin antibody (anti-mouse immunoglobulin antibody is used if the cell used for cell fusion is a mouse) or protein A, which is labeled with a radioactive substance or an enzyme, is added. A method for detecting monoclonal antibodies bound to a solid phase, adding a hybridoma culture supernatant to a solid phase to which an anti-immunoglobulin antibody or protein A is adsorbed, and adding a receptor protein or the like labeled with a radioactive substance, an enzyme, etc. And a method for detecting a monoclonal antibody bound to a solid phase.

The selection of the monoclonal antibody can be carried out according to a known method or a method analogous thereto. Usually, it can be carried out in a medium for animal cells to which HAT (hypoxanthine, aminopterin, thymidine) is added. As a selection and breeding medium, any medium can be used as long as it can grow a hybridoma. For example, RPMI 1640 medium containing 1-20%, preferably 10-20% fetal bovine serum, GIT medium containing 1-10% fetal bovine serum (Wako Pure Chemical Industries, Ltd.) or hybridoma culture medium Serum medium (SFM-101, Nissui Pharmaceutical Co., Ltd.) or the like can be used. The cultivation temperature is usually 20 to 40 ° (:, preferably about 37. The cultivation time is usually 5 days to 3 weeks, preferably 1 week to 2 weeks. The cultivation is usually 5% CO 2 gas. The antibody titer of the culture supernatant of the hybridoma can be measured in the same manner as the measurement of the antibody titer in the antiserum described above. (b) Purification of monoclonal antibody

Monoclonal antibodies can be separated and purified in the same manner as normal polyclonal antibodies. [Examples: salting out, alcohol precipitation, isoelectric focusing, electrophoresis, ion exchangers (ex. , DEAE) adsorption / desorption method, ultracentrifugation method, gel filtration method, antigen-binding solid phase or specific purification by collecting only the antibody using an active adsorbent such as protein A or protein G and dissociating the bond to obtain the antibody Act].

(Preparation of polyclonal antibody)

The polyclonal antibody of the present invention can be produced according to a known method or a method analogous thereto. For example, a complex of an immunizing antigen (an antigen such as the receptor protein of the present invention) and a carrier-protein is formed, and a mammal is immunized in the same manner as in the above-described method for producing a monoclonal antibody. The antibody can be produced by collecting an antibody-containing substance against the receptor protein or the like and separating and purifying the antibody.

Regarding the complex of an immunizing antigen and a carrier protein used for immunizing mammals, the type of carrier protein and the mixing ratio of carrier to hapten are determined by the ratio of hapten immunized by cross-linking to carrier protein. Any antibody may be cross-linked at any ratio as long as it can be efficiently produced.For example, serum serum alpmin, shishiro glopurin, keyhole, limpet, hemocyanin, etc. are weight ratios. A method of coupling the hapten 1 at a ratio of about 0.1 to 20 and preferably about 1 to 5 is used.

Various condensing agents can be used for force-coupling between the hapten and the carrier-protein, but daltaraldehyde, carbodiimide, maleimide active ester, an active ester reagent containing a thiol group or a dithioviridyl group, etc. are used. . The condensation product is administered to a warm-blooded animal itself or together with a carrier or diluent at a site where antibody production is possible. Complete Freund's adjuvant / incomplete Freund's adjuvant may be administered in order to enhance the antibody-producing ability upon administration. The administration can usually be performed once every about 2 to 6 weeks, for a total of about 3 to 10 times.

The polyclonal antibody can be collected from blood, ascites, etc., preferably from blood, of the mammal immunized by the above method. The measurement of the polyclonal antibody titer in the antiserum can be performed in the same manner as the measurement of the antibody titer in the serum described above. Separation and purification of the polyclonal antibody can be carried out according to the same immunoglobulin separation and purification method as the above-mentioned separation and purification of the monoclonal antibody.

(A) Measurement of mRNA Amount of Receptor Protein or Partial Peptide of the Receptor Protein of the Present Invention Specific examples of measurement of mRNA amount of the receptor protein of the present invention or a partial peptide thereof include the following methods.

(0 normal or disease model non-human mammals (e.g., mouse, rat, rabbit, sheep, pigeon, pig, cat, dog, monkey, etc., more specifically, dementia rat, obese mouse, atherosclerotic rabbit, For cancer-bearing mice) or human cells

(E.g., anti-dementia drugs, anti-hypertensive drugs, anti-cancer drugs, anti-obesity drugs, etc.) or physical stress (e.g., flooding stress, electric shock, light / dark, low temperature, etc.) Alternatively, a specific organ (eg, brain, lung, large intestine, etc.) or a tissue or cell isolated from the organ is obtained.

The mRNA of the receptor protein of the present invention or its partial peptide contained in the obtained cells is quantified by, for example, extracting mRNA from cells or the like by a usual method, and using, for example, a technique such as TaqMan PCR. It can also be analyzed by performing Northern blotting by known means.

(ii) A transformant expressing the receptor protein of the present invention or a partial peptide thereof is prepared according to the above method, and the mRNA of the receptor protein of the present invention or the partial peptide thereof contained in the transformant is prepared. It can be quantified and analyzed in the same way.

(B) Quantification of the receptor protein of the present invention or a partial peptide thereof or a salt thereof The antibody of the present invention can specifically recognize the receptor protein of the present invention and the like. Can be used for quantification of the receptor protein and the like, particularly for quantification by sandwich immunoassay. For example,

(i) reacting the antibody of the present invention with a test wave and a labeled receptor protein or the like in a competitive manner, and measuring the ratio of the labeled receptor protein or the like bound to the antibody; Method for quantifying the receptor protein of the present invention in a test solution,

(ii) the antibody of the present invention insolubilized on the test solution and the carrier and the antibody of the present invention A method for measuring the activity of a labeling agent on an insolubilized carrier after simultaneous or continuous reaction with an antibody, and a method for quantifying the receptor protein of the present invention in a test solution, and the like.

In the above (ii), one antibody is an antibody that recognizes the N-terminal of the receptor protein or the like of the present invention, and the other antibody is an antibody that reacts with the C-terminal of the receptor protein or the like of the present invention. Is preferred.

The receptor protein and the like of the present invention can be measured using a monoclonal antibody against the receptor protein and the like of the present invention (hereinafter sometimes referred to as the monoclonal antibody of the present invention), and detection by tissue staining and the like can be performed. You can do it too. For these purposes, the antibody molecule itself may be used, or F (ab ') ₂ , Fab', or Fab fraction of the antibody molecule may be used. The measurement method using an antibody against the receptor protein or the like of the present invention is not particularly limited, and may be an antibody, an antigen, or an antibody-antigen complex corresponding to the amount of an antigen (for example, the amount of a receptor protein) in a liquid to be measured. Any measurement method may be used as long as the amount of the body is detected by chemical or physical means, and the amount is calculated from a standard curve prepared using a standard solution containing a known amount of antigen. . For example, nephrometry, a competitive method, an immunometric method, and a sandwich method are suitably used, but in terms of sensitivity and specificity, it is particularly preferable to use a sandwich method described later.

As a labeling agent used in a measurement method using a labeling substance, for example, a radioisotope, an enzyme, a fluorescent substance, a luminescent substance and the like are used. Radioisotopes, if example embodiment, ^[125 1], ¹³¹ 1], ¾], "c], etc. As the enzyme, large preferably stable and specific activity, e.g., beta -. Galactosidase one , Β-glucosidase, alkaline phosphatase, peroxidase, malate dehydrogenase, etc. As the fluorescent substance, for example, fluorescamine, fluorescein isothiocyanate, etc., is used. For example, luminol, luminol derivatives, luciferin, lucigenin, etc. In addition, a biotin-avidin system may be used for binding an antibody or antigen to a labeling agent.

For insolubilization of the antigen or antibody, physical adsorption may be used. A method using a chemical bond used for insolubilizing or immobilizing white matter or an enzyme may be used. As the carrier, for example, insoluble polysaccharides such as agarose, dextran, and cellulose; synthetic resins such as polystyrene, polyacrylamide, and silicon; and glass are used.

In the sandwich method, the test solution is reacted with the insolubilized monoclonal antibody of the present invention (primary reaction), and further reacted with the labeled monoclonal antibody of the present invention (secondary reaction). By measuring the activity of the receptor protein, the amount of the receptor protein of the present invention in the test solution can be determined. The primary reaction and the secondary reaction may be performed in the reverse order, may be performed simultaneously, or may be performed at staggered times. The labeling agent and the method of insolubilization can be in accordance with those described above.

In the immunoassay by the sandwich method, the antibody used for the solid phase antibody or the labeling antibody is not necessarily one kind, and a mixture of two or more kinds of antibodies is used for the purpose of improving measurement sensitivity and the like. May be used.

In the method for measuring a receptor protein or the like by the sandwich method of the present invention, the monoclonal antibody of the present invention used in the primary reaction and the secondary reaction is preferably an antibody having a different binding site to the receptor protein or the like. Can be That is, the antibody used in the primary reaction and the secondary reaction is, for example, when the antibody used in the secondary reaction recognizes the C-terminal of the receptor protein, the antibody used in the primary reaction is preferably the C-terminal. For example, an antibody that recognizes other than the N-terminal part is used.

The monoclonal body of the present invention can be used in a measurement system other than the sandwich method, for example, a competition method, an immunometric method, or a nephrometry. In the competition method, the antigen in the test solution and the labeled antigen are allowed to react competitively with the antibody, and then the unreacted labeled antigen is separated from (F) and the labeled antigen (B) bound to the antibody. Then (BZF separation), the amount of labeling of either B or F is measured, and the amount of Bobara in the test solution is quantified. In this reaction method, a soluble antibody is used as the antibody, B / F separation is performed using polyethylene glycol, a liquid phase method using a second antibody against the above antibody, or a solid phase antibody is used as the first antibody. Alternatively, a solid phase method using a soluble first antibody and a solid phase antibody as the second antibody is used.

In the trick method, the antigen in the test solution and the immobilized antigen After a competitive reaction with the body, the solid phase and the liquid phase are separated, or the antigen in the test solution is allowed to react with an excessive amount of the labeled antibody, and then the immobilized antigen is added and unreacted After binding the labeled antibody to the solid phase, the solid phase and the liquid phase are separated. Next, the amount of label in any phase is measured to determine the amount of antigen in the test solution.

In nephelometry, the amount of insoluble sediment generated as a result of an antigen-antibody reaction in a gel or in a solution is measured. Even when the amount of antigen in the test solution is small and only a small amount of precipitate is obtained, laser nephrometry utilizing laser scattering is preferably used.

In applying these individual immunological measurement methods to the measurement method of the present invention, no special conditions, operations, and the like need to be set. What is necessary is just to construct the measuring system of the receptor protein of the present invention or its salt by adding ordinary technical considerations of those skilled in the art to ordinary conditions and operation methods in each method. For details of these general technical means, it is possible to refer to reviews and compendiums. [For example, Hiroshi Irie “Radio Nonoassy” (Kodansha, published in 1974), Hiroshi Irie “ "Radio Imnoatsay" (Kodansha, published in 1979), "Enzyme immunoassay" edited by Eiji Ishikawa et al. (Medical Publishing, published in 1978), "Enzyme immunoassay" edited by Eiji Ishikawa et al. (Edition) (Medical Shoin, published in 1982), edited by Eiji Ishikawa et al., "Enzyme Immunoassay" (3rd edition) (published in 1987), "Methods in ENZYMOLOGY" Vol. 70

(Immunochemical Techniques (Part A)), ibid.Vol. 73 (Immunochemical Techniques (Part B)), ibid.Vol. 74 (Immunochemical Techniques (Part C)), ibid.Vol. 84 (Immunochemical Techniques (Part D: Selected Immunoassays)) ), Ibid., Vol. 92 (Immunochemi cal Techniques (Part E: Monoclonal Ant ibodies and General Immunoassay Methods)), ibid., Vol. 121 (Immunochemical).

Techniques (Part I: Hybridoma TechnoGy and Monoclonal Ant ibodies)) (see Academic Press).

As described above, by using the antibody of the present invention, the receptor protein of the present invention or a salt thereof can be quantified with high sensitivity.

(1) a drug containing an antisense oligonucleotide The oligonucleotide of the present invention is stable in cells, has high cell permeability, has high affinity for the target sense strand, and has low toxicity.

The polynucleotide of the present invention may be, for example, a disease associated with overexpression of the receptor protein of the present invention, such as cancer (eg, non-small cell lung cancer, ovarian cancer, prostate cancer, gastric cancer, bladder cancer, breast cancer, cervix) Cancer, colon cancer, rectal cancer, etc.), and can be used as a medicament such as a prophylactic or Z or therapeutic agent.

The antisense oligonucleotides of the present invention may contain altered or modified sugars, bases, or bonds, may be provided in special forms such as ribosomes, microspheres, or may be applied by gene therapy. , Can be provided in an added form. Additional forms include polycations such as polylysine, which acts to neutralize the charge on the phosphate backbone, and lipids that enhance interaction with cell membranes or increase nucleic acid uptake (eg, Hydrophobic substances such as phospholipids and cholesterol). Preferred lipids for addition 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, sugar, or intramolecular nucleoside bond. Other groups include capping groups specifically arranged at the 3 'end or 5' end of nucleic acids for preventing degradation by nucleases such as exonuclease and RNase. Examples of such capping groups include, but are not limited to, hydroxyl-protecting groups known in the art, including glycols such as polyethylene glycol and tetraethylene glycol.

When the antisense oligonucleotide of the present invention is used as the above medicine, it can be formulated and administered according to a known method.

For example, when the antisense oligonucleotide is used, the antisense oligonucleotide is inserted alone or into an appropriate vector such as a retrovirus vector, an adenovirus vector, an adenovirus associated virus vector, and the like. Oral or non-human administration to human or non-human mammals (eg, rats, gray egrets, sheep, higgs, bush, red sea lions, cats, dogs, monkeys, etc.) It can be administered orally. The antisense oligonucleotide can be administered as it is or in the form of a formulation together with a physiologically acceptable carrier such as an adjuvant for promoting uptake, and then administered using a gene gun or a catheter such as a hydrogel catheter.

The dose of the antisense oligonucleotide varies depending on the target disease, the subject of administration, the route of administration, and the like. For example, the antisense oligonucleotide of the present invention may be administered to an organ (eg, liver, lung, When topically administered to the heart, kidney, etc., generally, for an adult (body weight 60 kg), about 0.1 to 100 mg of the antisense oligonucleotide is administered per day.

Furthermore, the antisense oligonucleotide can also be used as a diagnostic oligonucleotide probe for examining the presence of the polynucleotide (eg, DNA) of the present invention in tissues or cells and the state of expression thereof.

(2) Repo One Night One Gene Atsushi

Furthermore, the present invention provides a transformant transformed with a recombinant DNA in which a reporter gene is ligated downstream (under expression control) of the transcriptional regulatory region of the gene of the receptor protein of the present invention in the presence of a test compound. And a method for screening for a compound that promotes or inhibits the transcriptional regulatory activity, comprising measuring and comparing each repo overnight activity when cultured in the absence and presence of the same, and a script for this method. A kit for single use is also provided.

Examples of the transcription regulatory region of the gene of the receptor protein of the present invention include a DNA having the same or substantially the same nucleotide sequence as the nucleotide sequence represented by SEQ ID NO: 20.

The DNA containing a base sequence substantially identical to the base sequence represented by SEQ ID NO: 20 has a base sequence that hybridizes under high stringency end conditions, and has substantially the same sequence as SEQ ID NO: 20. Any DNA may be used as long as it has the same transcriptional regulatory activity (eg, promoter-one activity).

Examples of DNA that can hybridize with the nucleotide sequence represented by SEQ ID NO: 20 under high stringent conditions include, for example, the nucleotide sequence represented by SEQ ID NO: 20 A base having a homology of about 50% or more, preferably about 60% or more, more preferably about 70% or more, more preferably about 80% or more, particularly preferably about 90% or more, and most preferably about 95% or more. A DNA containing the sequence is used.

Hybridization is performed according to a method known per se or a method analogous thereto, for example, the method described in Molecular Cloning 2nd (J. Sambrook et al., Cold Spring Harbor Lab. Press, 1989). be able to. When a commercially available library is used, it can be performed according to the method described in the attached instruction manual. More preferably, the reaction can be performed under high stringency conditions.

The high stringent end conditions include, for example, a sodium concentration of about 19 to 40 mM, preferably about 19 to 2 OmM, and a temperature of about 50 to 70: preferably about 60 to 65. In particular, it is most preferable that the sodium concentration is about 19 mM and the temperature is about 65 ° C.

In addition, the transcription regulatory region (eg, DNA containing a nucleotide sequence substantially identical to the nucleotide sequence represented by SEQ ID NO: 20) is substantially the same as the nucleotide sequence represented by SEQ ID NO: 20 A sequence obtained by further adding a genomic DNA sequence at the 5 ′ upstream to the same base sequence (preferably a promoter region) is also included. The number of bases to be added is, for example, about 10 Kb or less, preferably about 5 Kb or less, more preferably about 2 Kb or less, and most preferably about 1 Kb or less. Furthermore, a sequence in which an intron sequence of the gene of the receptor protein of the present invention is further added to the nucleotide sequence substantially the same as the nucleotide sequence represented by SEQ ID NO: 20, etc. Reporter genes include, for example, lacZ (yogalactosidase gene), chloramphenicol acetyltransferase (CAT), luciferase, growth factor,] 3-glucuronidase, alphospholiphosphatase, Green fluorescent protein (GFP), / 3-lacquerase and the like are used.

By measuring the amount of the repo overnight gene product (eg, mRNA, protein) using a known method, a test compound that increases the amount of the repo overnight gene product can be used as a test compound for the receptor protein of the present invention. A compound having an action of controlling (particularly promoting) transcriptional regulatory activity (preferably promoter activity), ie, promoting expression of the receptor protein of the present invention. Can be selected as a compound having a certain activity. Conversely, a test compound that reduces the amount of a reporter gene product is a compound that has the activity of controlling (particularly inhibiting) the transcriptional regulatory activity (preferably, overnight promoter) of the receptor protein of the present invention, that is, the compound of the present invention. The compound can be selected as a compound having an activity of inhibiting the expression of the receptor protein. The test compound is, for example, a compound selected from peptides, proteins, non-peptidic compounds, synthetic compounds, fermentation products, cell extracts, plant extracts, animal tissue extracts, plasma, and the like. As the salt of the compound, those similar to the aforementioned salts of the receptor protein of the present invention are used.

The transformant can be cultured in the same manner as the above-described transformant containing the receptor protein of the present invention. The vector construction of the repo overnight gene and the Atsey method can be performed according to known techniques (eg, Molecular Biotechnology 13, 29-43, 1999).

The compound having an activity of promoting the expression of the receptor protein of the present invention or a salt thereof is useful as a safe and low-toxic drug.

A compound having an activity of inhibiting the expression of the peptide of the present invention or a salt thereof is a safe and low-toxic drug for suppressing the physiological activity of the receptor protein of the present invention, for example, cancer (eg, non-small cell lung cancer) Ovarian cancer, prostate cancer, gastric cancer, bladder cancer, breast cancer, cervical cancer, colon cancer, rectal cancer, etc.).

As the salt, those similar to the aforementioned salts of the receptor protein of the present invention are used. When the above compound is used as a medicine, it can be formulated according to conventional means.

For example, the compound can be used as a sugar-coated tablet, capsule, elixir, microcapsule or the like as needed, orally, or aseptic solution with water or another pharmaceutically acceptable liquid. It can be used parenterally or in the form of injections such as suspensions. For example, the compound is mixed with known physiologically acceptable carriers, flavoring agents, excipients, vehicles, preservatives, stabilizers, binders, and the like in a unit dosage form generally required for the practice of pharmaceutical preparations. It can be manufactured by The amount of the active ingredient in these preparations is such that a suitable dosage in the specified range can be obtained. Examples of the prophylactic and therapeutic agents include, but are not limited to, a buffer (eg, a phosphate buffer, a sodium acetate buffer), a soothing agent (eg, benzalkonidum chloride, proforce hydrochloride, etc.), a stabilizer (eg, , Human serum albumin, polyethylene glycol, etc.), preservatives (eg, benzyl alcohol, phenol, etc.), antioxidants and the like. The prepared injection solution is usually filled into a suitable ampoule.

The preparations obtained in this way are safe and low toxic, so they can be used, for example, in humans and mammals (eg, rats, mice, egrets, higgs, bushes, cats, cats, dogs, dogs, etc.). Can be administered.

The dose of the compound or a salt thereof varies depending on the administration subject, target organ, symptom, administration method, and the like. However, in the case of oral administration, for example, in a cancer patient (with a body weight of 60 kg), About 0.1 to 100 nig per day, preferably about 1.0 to 50 mg, more preferably about 1.0 to 20 nig. In the case of parenteral administration, the single dose varies depending on the subject of administration, target organ, symptoms, administration method, etc. For example, in the case of injection, it is usually used, for example, in cancer patients (with a body weight of 60 kg). It is convenient to administer about 0.01 to 30 mg, preferably about 0.1 to 20 mg, more preferably about 0.1 to 10 mg per day by intravenous injection. In the case of other animals, the dose can be administered in terms of weight per 60 kg. In the present specification, when a base, an amino acid, or the like is indicated by an abbreviation, the indication is based on an abbreviation by IUPAC-IUB Communication on Biochemical Nomenclature or a conventional abbreviation in the field. An example is shown below. When an amino acid can have optical isomers, the L-form is indicated unless otherwise specified.

Gly: Glycine

Ala: Alanin

Val:) Lynn

Leu: Leucine

l ie: Isolloy

Ser: Serine Thr: Threonine

Cys: Cystine

Met: Methionin

Glu: Glutamic acid

Asp: Aspartic acid

Lys: Lysine

Arg: Arginine

Hi s: Histidine

Phe: feniralanin

Tyr: Tyrosine

Trp: Tryptophan

Pro: Proline

Asn: Asparagine

Gin: Dal Yu Min

pGlu: pyroglutamic acid

Me: methyl group

Et: ethyl group

Bu: butyl group

Ph: phenyl group

TC: Thiazolidine-400-carboxamide

The substituents, protecting groups and reagents frequently used in the present specification are represented by the following symbols.

Tos: p-toluenesulfonyl

CH0: Formyl

Bz l

Cl ₂ Bz l 2, 6-

Bom benzyloxymethyl

Z benzyloxycarponyl

Cl-Z: 22--Ventyloxycarponyl Br-Z 2-bromobenzyloxycarbonyl

Boc t-butoxycarbonyl

DNP dinitrophenol

Tr t Trityl

Bum t-butoxymethyl

Fmoc N-9_fluorenylmethoxycarbonyl

HOBt

HOOBt 3,4-Dihydro-3-hydroxy-4-oxo-1,2,3-benzotriazine HONB 1-Hydroxy-5-norporene-2,3-dicarpoxyimide

DCC Ν, Ν'-dicyclohexylcarposimide SEQ ID NOs in the Sequence Listing of the present specification indicate the following sequences.

[SEQ ID NO: 1]

The following shows a part of the nucleotide sequence (5 'side) of cDNΑ obtained in Reference Example 1 below.

[SEQ ID NO: 2]

Indicates the base sequence of primer 11 used in the PCR reaction in Reference Example 1 below c [SEQ ID NO: 3]

Indicates the nucleotide sequence of primer 2 used in the PCR reaction in Reference Example 1 below c [SEQ ID NO: 4]

A part (center) of the nucleotide sequence of cDNA obtained in Reference Example 2 below is shown.

[SEQ ID NO: 5]

Indicates the base sequence of primer 3 used in the PCR reaction in Reference Example 2 below c [SEQ ID NO: 6]

Indicates the base sequence of primer 4 used in the PCR reaction in Reference Example 2 below c [SEQ ID NO: 7]

FIG. 9 shows a part of the nucleotide sequence (3 ′ side) of cDNA obtained in Reference Example 3 below.

[SEQ ID NO: 8]

The base sequence of primer 5 used in the PCR reaction in Reference Example 3 below is shown. [SEQ ID NO: 9] This shows the base sequence of primer 16 used in the PCR reaction in Reference Example 3 below [SEQ ID NO: 10]

_C represents the base sequence of primer 7 used in the PCR reaction in Reference Example 3 below [SEQ ID NO: 11]

7 shows the amino acid sequence of the human brain-derived novel receptor protein NEPHA obtained in Reference Example 5.

[SEQ ID NO: 12]

The nucleotide sequence of cDNA which encodes a novel receptor protein NEPHA derived from human brain is shown.

[SEQ ID NO: 13]

1 shows the base sequence of the antisense oligonucleotide of the present invention.

[SEQ ID NO: 14]

[SEQ ID NO: 15]

1 shows the base sequence of the oligonucleotide used in Example 1 below.

[SEQ ID NO: 16]

1 shows the base sequence of the oligonucleotide used in Example 1 below.

[SEQ ID NO: 17]

1 shows the nucleotide sequence of a primer used in the PCR reaction in Example 1 below. [SEQ ID NO: 18]

The base sequence of the primer used in the PCR reaction in Example 1 below is shown. [SEQ ID NO: 19] This shows the base sequence of the probe used in the PCR reaction in Example 1 below.

[SEQ ID NO: 20]

2 shows the nucleotide sequence of the transcription control region of NEPHA. Escherichia coli TOP10 / pTB2230 obtained in Reference Example 5 described below has been established on May 24, 2001 by the establishment of 1-1 Chukoku No. 6 Tsukuba Totoro, Ibaraki, Japan (zip code 305-8566). Deposited number at Patent Organism Depositary, National Institute of Advanced Industrial Science and Technology From April 24, 2001, FERM BP-7606 was given to the Fermentation Research Institute (IF0) of 2-17-85, Jusanhoncho, Yodogawa-ku, Osaka-shi (Osaka Prefecture) under the accession number IF0-16623. Has been deposited. Hereinafter, the present invention will be more specifically described with reference examples and examples, but the present invention is not limited thereto.

Reference example 1

Encoding a novel receptor protein from human brain. Determination of the 5 'partial nucleotide sequence of cDNA.

The human brain cDNA (CL0NTECH) contained in MTC Panel I was type III, and PCR was carried out using two primers, primer 1 (SEQ ID NO: 2) and primer 2 (SEQ ID NO: 3). . The composition of the reaction solution used in the reaction was the above cDNA 0.51 as type III, 1 U of Pfu Turbo DNA Polymerase (STRATAGENE), primer 1 (SEQ ID NO: 2) and primer 1 (SEQ ID NO: 3) 1 M each, 200 l of dNTPs, and 101 of 2XGC Buf ferl (Takara Shuzo) were added to give a liquid volume of 201. The PCR reaction was repeated 96 times at 96X3 for 1 minute, followed by a cycle of 96 ° C, 1 minute, 60 ° C, 1 minute, 72 ° C, and 1 minute 40 times. Next, the PCR reaction product was purified by agarose gel electrophoresis, and then purified using the plasmid vector pCR-Bluntil-T0P0 (Invitrogen) according to the prescription of Zero Blunt T0P0 PCR Cloning Kit (Invitrogen). ). This was introduced into E. coli TOP10 and selected on LB agar medium containing kanamycin. As a result of analyzing the sequence of each clone, a nucleotide sequence (SEQ ID NO: 1) of cDNA encoding a part of the novel receptor protein was obtained. Reference example 2

Determination of the central partial nucleotide sequence of cDNA encoding a novel receptor protein from human brain.

Using human brain cDNA (CL0NTECH II MTC Panel I) as type II, PCR was carried out using two primers, primer 3 (SEQ ID NO: 5) and primer 4 (SEQ ID NO: 6). The composition of the reaction solution used in the reaction was the above-mentioned cDNA 11 as type III, and 2.5 U of Pfu Turbo DNA Polymerase (STRATAGENE), primer 3 (SEQ ID NO: 5) and primer 4 (SEQ ID NO: 6). 1 M each, 200 M dNTPs, and Add 25 1 of 2XGC Bufferl (Takara Shuzo) to make a volume of 50 ^ 1. After 96 minutes, the PCR reaction was repeated 40 times at 96 ° C, 1 minute, 60 ° C, 1 minute, 72 minutes and 2 minutes. Next, after purifying the PCR reaction product by agarose gel electrophoresis, the PCR product is transferred to a plasmid vector pCR-Bl until-T0P0 (Invitrogen) according to the instructions of Zero Blunt T0P0 PCR Cloning Kit (Invitrogen). Sub-cloned. This was introduced into E. coli T0P10 and selected on LB agar medium containing kanamycin. As a result of analyzing the sequence of each clone, a nucleotide sequence (SEQ ID NO: 4) of a cDNA encoding a part of the novel receptor protein was obtained. Reference example 3

Determination of 3 'partial nucleotide sequence of cDNA encoding novel receptor protein from human brain

Using human brain cDNA (CL0NTECH MTC Panel1) as type I, PCR was performed using two primers, Primer 5 (SEQ ID NO: 8) and Primer 1 (SEQ ID NO: 9). The composition of the reaction solution used in the reaction was the above cDNA II as type III, and 2.5 U of Pfu Turbo DNA Polymerase (STRATAGENE), Primer 5 (SEQ ID NO: 8) and Primer 6 (SEQ ID NO: 9) 1 M, dNTPs were added to 200 XM, and 2XGC Buffer1 (Takara Shuzo) to make a liquid volume of 501. After 1 minute at 96 ° C, the PCR reaction was repeated 40 times at 96, 1 minute, and 60 times for 1 minute, 72 ° C, and 2 minutes. Next, 5 l of the PCR reaction product was used as type 、, and Pfu Turbo DNA Polymerase was used.

(STRATAGENE) Add 2.5 U, Primer-5 (SEQ ID NO: 8) and Primer-7 (SEQ ID NO: 10) at 1 M each, dNTPs at 200 M, and 25 l of 2XGC Bufferl (Takara Shuzo), and add 50 1 Liquid volume. The PCR reaction was repeated 45 times at 96 ° C., 1 minute, 60 ° C., 1 minute, 72 ° C., 2 minutes after 96 minutes. Next, the PCR reaction product was purified by agarose gel electrophoresis, and then purified using the plasmid vector pCR-BluntII-T0P0 according to the procedure of Zero Blunt T0P0 PCR Cloning Kit (Invitrogen).

(Invitrogen). This was introduced into E. coli T0P10 and selected on LB agar medium containing kanamycin. As a result of analyzing the sequence of each clone, the nucleotide sequence (SEQ ID NO: 7) of cDNA encoding a part of the novel receptor protein was obtained. Reference example 4

Determination of the nucleotide sequence of full-length cMA encoding a novel receptor protein from human brain

Based on the nucleotide sequence of the partial cDNA encoding the novel receptor protein obtained in Reference Example 1, Reference Example 2 and Reference Example 3 (SEQ ID NO: 1, SEQ ID NO: 4 and SEQ ID NO: 7), The sequence (SEQ ID NO: 12) was determined. SEQ ID NO: 12 corresponds to the 71st to 789th sequence of SEQ ID NO: 1, the 284th to 1134th sequence of SEQ ID NO: 4 and the 473th to 1926th sequence of SEQ ID NO: 7 . The amino acid sequence of the protein predicted from the nucleotide sequence of the full-length cDNA (SEQ ID NO: 12) is shown in SEQ ID NO: 11. The protein containing the amino acid sequence represented by SEQ ID NO: 11 was named NEPHA (novel EphA). NEPHA and the human Ephr in receptor EphA7 have 50.7% homology at the amino acid level. The gene encoding the full length is obtained by cleaving the cDNA fragments obtained in Reference Examples 1, 2 and 3 with an appropriate restriction enzyme and ligating them with T4 ligase or the like. Alternatively, a primer is designed to amplify the cDNA encoding the full length based on the nucleotide sequences identified in Reference Examples 1 and 3, and the primers are obtained by PCR. Reference example 5

Construction of a plasmid having a full-length cDNA sequence encoding a novel receptor protein derived from human brain

A plasmid containing the nucleotide sequences of the cDNAs obtained in Reference Examples 2 and 3 (SEQ ID NO: 4 and SEQ ID NO: 7) was digested with restriction enzymes. The composition of the reaction mixture was lg, Xol (Takara Shuzo) 5U, and 含む Buf, containing a plasmid containing the nucleotide sequence of the cDNA represented by SEQ ID NO: 4 or a plasmid containing the nucleotide sequence of the cDNA represented by SEQ ID NO: 7. fer (Takara Shuzo) 5 x I was added to make a liquid volume of 501. The restriction enzyme reaction was performed at 37 ° C. for 1 hour, and 5 U of BstEI I (Takara Shuzo) was added to the reaction product, followed by a reaction at 60 ° C. for 1 hour. Next, the reaction product corresponding to 941 bp from the plasmid containing the nucleotide sequence of cMA represented by SEQ ID NO: 4 and 5273 bp from the plasmid containing the nucleotide sequence of cDNA represented by SEQ ID NO: 7 was subjected to agarose electrophoresis. After purification, they were bound according to the prescription of DNA Ligation Kit Ver. 2 (Takara Shuzo). This is E. coli And selected in LB agar medium containing kanamycin. As a result of analyzing the sequence of each clone, the nucleotide sequence of cDNA represented by SEQ ID NO: 13 was obtained. Further, a plasmid containing the nucleotide sequence of the cDNA obtained in Reference Example 1 (SEQ ID NO: 1) and a plasmid containing the nucleotide sequence of the cDNA represented by SEQ ID NO: 13 were digested with restriction enzymes. The composition of the reaction solution is lfig, Pvul (Takara Shuzo) 10 U, and plasmid containing the nucleotide sequence of the cDNA represented by SEQ ID NO: 1 or plasmid containing the nucleotide sequence of the cDNA represented by SEQ ID NO: 13. (Takara Shuzo) to make a liquid volume of 50〃1. The restriction enzyme reaction was performed at 37 ° (1 hour. Next, the plasmid containing the nucleotide sequence of the cDNA represented by SEQ ID NO: 1 contains 743 bp and the nucleotide sequence of the cDNA represented by SEQ ID NO: 13 The reaction product corresponding to 5977 bp was purified from the plasmid by agarose gel electrophoresis, and these were ligated according to the prescription of DNA Ligation Kit Ver.2 (Takara Shuzo). Selection was performed in LB agar medium containing kanamycin As a result of analyzing the sequence of each clone, a plasmid having the nucleotide sequence of the full-length cDNA encoding the novel human brain-derived receptor protein (SEQ ID NO: 12) was obtained. The resulting plasmid having SEQ ID NO: 12 was named PTB2230, and the resulting transformant was named Escherichia coli TOP10 / pTB2230.

In order to analyze the effect of the NEPHA gene on apo1 cis, an antisense oligonucleotide introduction experiment was performed.

First, after designing an antisense (SEQ ID NO: 13 and SEQ ID NO: 14) for the base sequence represented by SEQ ID NO: 12, a phosphorothioated oligonucleotide was synthesized, purified by HPLC, and used for an introduction experiment. As control oligonucleotides, the reverse sequence of the base sequence represented by SEQ ID NO: 13 (SEQ ID NO: 15) and the reverse sequence of the base sequence represented by SEQ ID NO: 14 (SEQ ID NO: 16) were similarly used. And purified by HPLC for use.

As test cells, breast cancer cell line ZR-75-1 (Cancer Res, 38 (10), 3352-3364, purchased from ATCC, 1978) was used. Falcon). For oligonucleotide introduction, OligofectAMINE (Invitrogen) was used and the protocol was followed. Twenty hours after the introduction, RNA was extracted according to the protocol of the RNeasy mini kit (Qiagen), and cDNA was prepared using the TaqManRT kit (Perkin-Elmer). At the same time, the same reaction was carried out without adding reverse transcriptase to obtain a non-reverse transcription control.

Using two primers (SEQ ID NO: 17 and SEQ ID NO: 18) and a probe (SEQ ID NO: 19), the expression level of the NEPHA gene was corrected, and TaqMan / 3-actin Control Reagents (Perkin - using Elmer Co., Ltd.) and watching i3 Akuchin expression level, expression level of _c NEPHA gene PCR was performed according to the manual of ABI 7900 (Perkin Elmer Inc.) from the data obtained using the cDNA After subtracting the data obtained using the non-reverse transcription control, the results were corrected for i3 actin expression and compared.

On the other hand, on the third day after the introduction of the antisense oligonucleotide, a Cell death detection ELISA (Roche) was used to examine the effect on the apoptosis when the expression of the NEPHA gene was suppressed by introducing the antisense oligonucleotide. The control oligonucleotide was used to compare with the sample introduced with the oligonucleotide.

As a result, when the antisense to the NEPHA gene (SEQ ID NO: 13) was used, the expression of the NEPHA gene was 20 hours after the introduction of the antisense oligonucleotide compared to the control oligonucleotide (SEQ ID NO: 15). The amount of expression of the NEPHA gene was reduced to 57% when the antisense (SEQ ID NO: 14) was used, compared to the control oligonucleotide (SEQ ID NO: 16). Was.

On the third day after the introduction of the antisense oligonucleotide, apoptosis was calculated to be 224% when the control oligonucleotide (SEQ ID NO: 13) was introduced and the control oligonucleotide (SEQ ID NO: 13) was assumed to be 100%. Assuming that the oligonucleotide (SEQ ID NO: 16) was 100%, it was increased to 185% when the antisense oligonucleotide (SEQ ID NO: 14) was introduced.

These results confirm that suppressing the expression of the NEPHA gene expressed in cancer cells causes cancer cells to undergo apoptosis, and that the NEPHA gene is involved in cancer cell proliferation and apoptosis. It became clear that we were doing. Example 2

NEPHA 5, upstream analysis

When a BLAST search was performed on the NCBI human genomic sequence using the nucleotide sequence represented by SEQ ID NO: 1, the nucleotide sequence from No. 1 to 176 of SEQ ID NO: 1 was the genomic sequence of human chromosome 1 ( ACCESSION number is NT # 004568, VERSION is ΝΤ # 004568 · 8

(GI: 18548377), DEFINITION was consistent with Homo sapiens chromosome 1 working draft sequence segment). Therefore, the genomic sequence (SEQ ID NO: 20) 5 'upstream (about 2 Kb) of the NEPHA start codon was obtained from this genomic sequence, and the transcription factor binding region was predicted using the transcription factor prediction software Matrix version 1.3 of TRANFAC. did. As a result, as shown in Table 1, the binding positions of various transcription factors were predicted, suggesting that this region is a transcriptional regulatory region of NEPHA.

[Table 1] Storage core matrix recognition sequence Transcription factor

Match match

4 (+) 0.993 0.97 tcagAGGAAgtct c-Ets-l (p54)

5 (+) 1 0.924 cagAGGAAgtctgt c-Ets-2

6 ω 1 0.95 agAGGAAgtctgtat c-Ets-1

7 (+) 0.837 0.785 gaGGAAGtct Ncx

14 (-) 0.885 0.913 tctgtATTTTgtgttt FAC1

14 (-) 1 0.937 tctGTATTttgttt MRF-2

16 (-) 0.926 0.865 tgtatTTTGTttta FAC1

18 (-) 0.966 0.961 tatTTTGTttta SRY

18 (-) 0.842 0.812 tATTTTgttttagca Evi-1

19 (-) 0.978 0.868 attttGTTTTagca FAC1

24 (-) 0.859 0.89 gttttaGCACCga C / EBP

44 (+) 0.777 0.725 aacctctgGGCTGaagtc Pax-8

67 (-) 0.94 0.936 agcttcaGTAAGaa C / EBPalpha

72 (+) 0.993 0.903 caGTAAGaag Ncx 74 (-) 0.893 0.766 gtaagaagccATGATtggt Pax- 2

83 (+) 0.997 0.991 catGATTGgt GATA-1

84 (-) 1 0.961 atgATTGGtgc NF-Y

93 (+) 0.908 0.752 gccttagaGCCTGctaag Pax-8

104 (+) 0.836 0.803 tgCTAAGggg Ncx

110 (+) 1 0.99 gggGATGGaa GATA-1

127 (+) 0.905 0.761 ttaaagagGAGTGagggg Pax-8

129 (+) 0.897 0.902 aaagaggagTGAGG GKLF

130 (+) 1 0.977 aagaGGAGTgaggg p300

131 (+) 0.981 0.887 agaggagtgAGGGG GKLF

133 (+) 0.796 0.736 aggaGTGAGgggtggtgga Pax- 2

133 ω 0.881 0.852 aggagTGAGGgg t gg t ggag t Pax-4

134 (+) 0.87 0.736 ggagtgagGGGTGgtgga Pax-8

135 (+) 1 0.893 gagtgAGGGGtgg MZF1

141 (-) 1 0.983 gGGGTGgtggag Pax-4

142 (+) 0.905 0.729 gggtggtgGAGTGaggac Pax-8

1 4 (-) 0.903 0.893 gTGGTGgagtga Pax-4

145 (+) 1 0.952 tggtGGAGTgagga p300

148 (+) 0.796 0.732 tggaGTGAGgactcatttt Pax-2

155 (+) 0.918 0.918 aggaCTCATtttgaaat YY1

160 (+) 0.996 0.975 tcattttGAAATga C / EBPbeta

160 (-) 0.929 0.936 tcattttGAAATga C / EBPalpha

172 (+) 1 0.95 gaaaGGAGTcctgg p300

175 (+) 0.825 0.73 aggaGTCCTgggtctctaa Pax-2

194 (+) 0.807 gcagcttctaaAAATAccatct MEF-2

194 (+) 0.898 gcagcttctaaAAATAccatct MEF-2

197 (+) 0.902 gcttcTAAAAataccatc aMEF-2

205 (+) 0.949 aaatACCATctcccaga YY1

208 (-) 0.992 taCCATCtcc GATA-1

220 (-) 0.903 0.897 g AGGTGgaggca Pax-4

226 (+) 1 0.869 gaggcAGGGGgag MZF1 227 (-) 0.992 0.888 aggcaGGGGGag AP-2

231 (+) 0.927 0.924 agggGGAGGggag Spl

231 (+) 0.956 0.911 agggGGAGGggaga GC box

232 (+) 1 0.931 gggggAGGGGaga MZF1

232 (+) 0.935 0.939 gggggAGGGGaga AZR

242 (+) 0.758 0.795 agaaaTCGAGcgtgaggagaa Pax- 4

242 (+) 0.715 0.782 agaaATCGAgc gt gaggag Pax-2

243 (-) 0.84 0.798 gaaatcgagcGTGAGgaga Pax- 2

243 (+) 1 0.862 gaaatcgaGCGTGaggag Pax- 8

243 (-) 0.75 0.713 gaaatCGAGCgt gaggag Pax- 8

244 (-) 1 0.864 aaa t cgagCGTGAggagaatg Pax- 3

252 (+) 0.868 0.73 cgtgaggaGAATGaagat Pax-8

254 (+) 0.952 0.872 t gaggagaATGAAg t ggaaagg OCT- 1

259 (+) 0.673 0.707 agaatgaaGATGGaaagg Pax-8

260 (+) 0.948 0.86 gaa t gaagATGGAaaggagg t gg OCT-1

260 (-) 1 0.974 gaatgaagATGGAaagg YY1

264 (+) 1 0.985 gaaGATG Gaa GATA-1

264 (+) 0.888 0.876 gaagatGGAAAggag OCT-1

265 (+) 0.852 0.867 aaGATGGaaagga C / EBP

268 (+) 0.979 0.964 atggAAAGGaggtgg Barbie Box

276 (-) 0.903 0.897 gAGGTGgcagtg Pax-4

277 (+) 0.846 0.719 aggtggcaGTGTGagaac Pax-8

278 (+) 0.944 0.937 ggtgGCAGTgtgag p300

284 (+) 0.94 0.909 agTGTGAgaaccc C / EBP

296 (+) 0.81 0.738 cttcCTCAGgtaggtctgg Pax-2

299 (-) 1 0.965 cctCAGGTaggtc AREB6

303 (+) 1 0.949 aggtaggTCTGGtttt Handl / E47

305 (+) 0.822 0.777 gtagGTCTGgttttagaag Pax-2

319 (+) 0.833 0.744 agaaGTCCCgcctgcatcc Pax-2

319 (-) 0.984 0.931 agaagtcCCGCCtgc Spzl

320 (+) 0.908 0.726 gaagtcccGCCTGcatcc Pax-8 bbo 〇

532 (-) 1 0.926 cacaagcACACAcgca Poly A 534 (-) 0.986 0.942 caaGCACAcacgcagaaa! GR 537 (-) 1 0.857 gcacaCACGCagaaatgg Pax-8 543 (-) 0.998 0.937 acgcagaaATGGCacat YY1 548 (-) 0.857 0.782 gGAtagcagGAcAgcAgAgAgcAgAgC +) 0.86 0.709 acatgaaaGCGGGttgta Pax- 8 566 (+) 0.872 0.88 ggGTTGTaaatgg C / EBP 567 (-) 0.998 0.963 ggttgtaaATGGCcagt YY1 569 (+) 0.969 0.948 ttGTAAAtgg Ncx 572 (-) 0.902 0.733 taaatggccaTG7 0.75 tctacgatGCCAGgaatc Pax-8 604 (-) 0.993 0.9 ctcCTTACac Ncx 630 (+) 1 0.935 tatggacTCTGGcaga Handl / E47 642 (+) 1 0.709 cagagacaGCGTGgttca Pax-8 663 (-) 0.993 0.985 ggGCATCcgc GATA-1671g (GATA-1671g) at ggc gc GR 673 (+) 0.963 0.752 age ac agaGCATGgc gcc Pax-8 675 (-) 0.998 0.908 cacagagcATGGCgccc YY1 690 (+) 0.777 0.708 ccagtgagGGCTGggtag Pax-8 702 (+) 0.87 0.728 gggtagagGGGTGaggga Pax-8 0.916 ggtagAGGGGtga MZF1 706 (+) 0.765 0.718 agaggggtGAGGGataca Pax-8 709 (-) 1 0.971 gGGGTGagggat Pax-4 710 (+) 0.844 0.847 gggtgA GGGAtac MZF1 711 (+) 0.847 0.822 ggtgagggATACAtatatgcaaa OCT-1 719 (+) 1 0.936 atacatatATGCAaaagcacaga OCT-1 723 (-) 0.972 0.941 atatatGCAAAag C / EBP 723 (+) 1 0.892 atatatGCAAAagca OCT-1719c TACTA 0.99 Tg / EBP 732 (-) 0.986 0.938 aaaGCACAgacac tcagac GR 741 (one) 0.753 0.706 acactCAGACcgcagggg Pax-8 CO to t

o o

oo

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oo

—4 oo oo

• ■ 0 no p Szl

GC b

p Sl circle

i OAV;

CO

O O O

O

1155 U. (aggggaaccc, then [c ig Γ d-Λ L

1 C Π QQ7

llo Π9-) 0.897 C t C I g¾b then rAC L then / DDI

11

η ₇ 1

11.808 o 0.989 u.yio atguLrlLr l I Ig rrtg rraag π) 丄) L 丄

,

1189 1 u.yb i b llrgt tglg Γ dX

1194 0.796 Π 70

U. 丄 ggt I ilrAijglglgCl C IΓαλ

0.881 π 00 c

1194 ggL Igl bA IgtgCtgCllg P r vX— / 1

ヽ r>

1202 0.908 Γ ΤΤΡ †

U. ί DO r dX v o ヽ

1209 (-) 0.979 U.oU9 tgcttgtatcii then age a a v—

1217 0.823 U.ί 4 tc t tgaca r and iiragcaa i d o

ヽ

1245 0.864 U.oy aACCGAtcgt CDP CR1

/ ヽ

1247, -0.858 u.oy ccgaTCGTTc CDP CR1

/ 、ヽ

1249 1 gatcgttcATGCAaaaccgccac OCT-1

1250 0.857 n n atcgTTCATgcaaaaccgc Pax-2

(ヽ

1251-) 0.72 Λ u.7Ωyo0 tcgi icatgcAAAACcgcc rax / fl π n 7

I25l, -0.963 tcgttCATGCaaaaccgc Pax-8

ヽ

1252 i) 0.984 I) .94o cgttcatGCAAAac C / EBP

1253 (+) 1 oyo gttcatGCAAAaccg OCT-1 o ヽ n no o

1258 0.984 u.y o tgcaaaaCCGCCacc Spzl

ヽ

1263 (+) 0.92 U.91b aaccgcCACCGc Pax-4,

1266, -1 u.y l cgccaCCGCCccca GC box

(ヽ

l oT 1 Λ QQ7 gccaCCGCCccca Spl i ft i * ヽ

1276 1 cccaTCCCAgcg Ik-2 o n r f ヽ Q

1285) 1 n O gcgtccacATGGTttcc YY1

1 o *, Π QQ / 1

1286 K-) 0.938 U. yo4 cgtccACATGgttt USF ヽ

1286 (+) 0.938 u.yo cgtcCACATggttt USF ヽ Q Q 0 n QQ 0

u.yo tgGTTTCccagcctcgg 'RFXl on r ヽ QQ 1

1295 (-) 1 ggttTCCCAgcc Ik-2

1303 (+) 1 0.941 agcctCGGAAgcgc Elk-1

1311 (-) 1 0.857 aaGCGCGttggta ZF5

1318 (+) 0.823 0.724 ttggtaggGCAGGacccc Pax-8 1333 (-) 0.765 0.707 cccgtCCCTCactttgct Pax-8

1333 (-) 0.945 0.901 CCCGTccctcactt GKLF

1353 (-) 0.978 0.964 ccttTCCCGagc Ik-2 '

1371 (+) 0.995 0.992 cggGATCGtt GATA-1

1381 (-) 1 0.87 cggCCCCTccttc MZF1

1383 (-) 1 0.929 gcccctccTTCCTgc c-Ets-1

1384 (-) 0.981 0.923 CCCCTccttcctgc GKLF

1388 (-) 0.823 0.738 tccttCCTGCcgctgtgt Pax-8

1434 (+) 0.846 0.705 gtgtgtgtGTGTGatgag Pax-8

1441 (+) 0.765 0.707 tgtgtgatGAGGGcggga Pax-8

1441 (+) 0.94 0.942 tgTGTGAtgaggg C / EBP

1445 (+) 0.86 0.716 tgatgaggGCGGGaagag Pax-8

1448 (+) 1 0.928 tgagGGCGGgaag Spl

1449 (+) 0.984 0.937 gagGGCGGgaagaga Spzl

1454 (+) 0.968 0.891 cgggaagagAAAGG GKLF

1455 (+) 1 0.907 gggaagagaAAGGG GKLF

1456 (+) 0.981 0.92 ggaagagaaAGGGG GKLF

1457 (+) 0.946 0.925 gaagagaaaGGGGG GKLF

1458 (+) 0.946 0.915 aagagaaagGGGGG GKLF

1459 (+) 0.946 0.939 agagaaaggGGGGG GKLF

1462 (+) 0.85 0.831 gaaagGGGGGgat MZF1

1463 (+) 0.998 0.925 aaaGGGGGggatgag Spzl

1464 (+) 0.998 0.943 aagGGGGGgatgagc Spzl

1501 (+) 0.993 0.969 acacAGGAAcaga c-Ets-l (p54)

1535 (-) 0.924 0.883 agctccCCAGAgc C / EBP

1538 (+) 0.908 0.762 tccccagaGCTTGgggtc Pax-8

1549 (+) 0.97 0.775 tgggGTCAGgacataaagc Pax- 2

1554 (-) 0.989 0.972 tcaGGACAtaaagcttcaa GR

1558 (-) 0.672 0.734 gacataaagcTTCAAaacc Pax-2

1571 (-) 0.86 0.702 aaaacCCCGCaagtcctg Pax-8

1577 (+) 0.843 0.803 ccGCAAGtcc Ncx 1584 (+) 1 0.98 tccTGGGAacaa Ik-- 2 1594 (-) 0.783 0.789 aaGGGAGct t tea ZF5 1621 (+) 0.843 0.818 ctGCAAGtgt Ncx 1648 (-) 1 0.915 tcgCCCCTcctta MZF1 1654 (-) 0.867 0.795 ctcCTTAAgc Ncx16 caagccCACCTt Pax-4 1668 (+) 0.908 0.743 cccaccttGCCTGcctac Pax-8 1670 (-) 0.843 0.793 cacCTTGCct Ncx 1684 (+) 0.81 0.858 acttCTCAGgaatgaggag Pax-2 1685 (-) 0.831 0.748 cttctcaggaATGAGgaggx Pax -2 1685 (-) -8 1690 (+) 0.844 0.782 caGGAATgag Ncx 1709 (+) 0.944 0.92 ttTAGCGctctg E2F 1733 (-) 0.868 0.737 cagctCATTCccctctcg Pax-8 1736 (-) 0.844 0.791 ctcATTCCcc Ncx 1739 (-) 1 0.947 attCCCCT1cc96c cccctCTCGCagaagccc Pax-8 1775 (-) 0.954 0.957 ctgccCCACCcccc GC box 1775 (+) 1 0.989 ctgcccCACCCc Pax-4 1776 (-) 0.87 0.755 tgcccCACCCcccacggt Pax-8 1776 (-) 0.915 0.932 tgccCCACCcccccccccccccccccccccc1cc (+) 0.984 0.952 ccaccccccaCGGTTt E2 1780 (+) 0.986 0.961 ccaccccccaCGGTTt E2 1791 (+) 1 0.966 ggTTTGGagatct C / EBP 1796 (+) 0.86 0.78 ggagatctGCGGGacccc Pax-8 1810 (-) 0.981 0.907 CCCCTtcctttccc GKLF 1811 (-) 1 0.927 CCCTTcctttcccc GKLF 1818 (+) 0.903 0.905 tttcccCACCAg Pax-18g -Myb 1831 (-) 1 0.901 tGGGTGttccgc Pax-4 cv. . .

P

bD one

o O 〇

CO

1949 (-) 1 0.951 gactCCGCCcccc Spl

1953 (-) 0,915 0.944 ccgcccccCGCTGgctct AP-4

1957 (+) 1 0.93 ccCCCGCtggct AP-2

1968 (+) 1 0.937 tctggggTCTGGgggc Handl / E47

1970 (+) 0.822 0.735 tgggGTCTGggggcattgc Pax- 2

1973 (-) 0.992 0.885 ggtctGGGGGca AP-2

1982 (+) 1 0.919 gcATTGCtcagcgg C / EBP

1982 (+) 0.897 0.894 gcATTGCtcagcg C / EBP

2003 (+) 0.908 0.714 ctggcgcgGCTTGagccg Pax- 8

2027 (-) J 0.974 gacTGACAgct TGIF

Industrial applicability

The oligonucleotide of the present invention has low toxicity and is useful as an excellent agent for preventing and / or treating cancer. Further, it can be obtained by screening using a transformant transformed with a recombinant DNA in which a repo overnight gene is ligated downstream (under expression control) of the transcription regulatory region of the receptor protein gene of the present invention. A compound, preferably a compound having an activity of inhibiting the expression of the receptor protein of the present invention or a salt thereof is also low toxicity and is useful as an excellent agent for preventing and / or treating cancer.

Claims

The scope of the claims

1. An oligonucleotide containing the same or substantially the same nucleotide sequence as the nucleotide sequence represented by SEQ ID NO: 13 or SEQ ID NO: 14.

2. An oligonucleotide having a base sequence represented by SEQ ID NO: 13.

3. An oligonucleotide having a base sequence represented by SEQ ID NO: 14.

4. The oligonucleotide according to claim 1, which is an antisense oligonucleotide.

5. The oligonucleotide according to claim 1, which is a DNA.

6. A pharmaceutical comprising the oligonucleotide according to claim 1.

7. The medicament according to claim 6, which is an agent for preventing and / or treating cancer.

8. DNA containing a nucleotide sequence identical or substantially identical to the nucleotide sequence represented by SEQ ID NO: 20.

9. The DNA according to claim 8, which is a DNA transcriptional regulatory region containing the same or substantially the same nucleotide sequence as the nucleotide sequence represented by SEQ ID NO: 12.

10. The DNA according to claim 9, wherein the transcription control region is a promoter region.

11. A recombinant vector containing the DNA according to claim 8.

12. The method according to claim 11, which comprises a DNA having a repo all-in-one gene downstream of a transcription regulatory region of a DNA containing the same or substantially the same base sequence as the base sequence represented by SEQ ID NO: 12. Recombination vector.

13. A transformant containing the recombinant vector according to claim 11.

14. Use of the DNA of claim 8 to promote or inhibit the transcriptional regulation activity of DNA containing the same or substantially the same nucleotide sequence as the nucleotide sequence represented by SEQ ID NO: 12 A method for screening a compound or a salt thereof.

15. The screening method according to claim 14, wherein the transformant according to claim 13 is used.

16. Promoting or inhibiting the transcriptional regulatory activity of a DNA containing the same or substantially the same nucleotide sequence as the nucleotide sequence represented by SEQ ID NO: 12, characterized by containing the DNA according to claim 8. For screening a compound or a salt thereof.

17. Nucleotide sequence identical or substantially identical to the nucleotide sequence represented by SEQ ID NO: 12 obtained by using the screening method according to claim 14 or the screening kit according to claim 16 A compound or a salt thereof which promotes or inhibits the transcriptional regulatory activity of DNA, comprising:

18. A medicament comprising the compound according to claim 17 or a salt thereof.

19. The medicament according to claim 18, which is a prophylactic and / or therapeutic agent for cancer.

20. A method for preventing and treating cancer, comprising administering an effective amount of the oligonucleotide according to claim 1 to a mammal.

21. Use of the oligonucleotide according to claim 1 for producing an agent for preventing or treating cancer.