JP2003289876A - Preventive and/or therapeutic agent for cancer - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To obtain an excellent anticancer agent. <P>SOLUTION: This invention provides an oligonucleotide comprising a base sequence equal or substantially equal to a base sequence inducing apoptosis to cancer cells and provides a preventive and/or therapeutic agent for cancer which contains the oligonucleotide. A method for screening a compound or a salt thereof promoting or inhibiting a transcription-regulating activity of a DNA having a base sequence equal or substantially equal to a specific base sequence is provided. The anti-sense oligonucleotide is useful as an excellent anticancer agent. <P>COPYRIGHT: (C)2004,JPO

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to antisense oligonucleotides and the like for prevention and treatment of cancer.

[0002]

2. Description of the Related Art In chemotherapy for cancer, the life-prolonging effect is improved by the development of new anti-cancer agents, and the number of cases toward cure is increasing. However, most of the currently used anti-cancer agents are highly cytotoxic agents that damage DNA or inhibit cell division, and therefore damage normal cells to some extent, especially cells. Strong side effects often appear in the bone marrow where the division is active. From these things, a drug that targets a molecule specifically expressed in cancer cells and can inhibit the growth of cancer cells or induce apoptosis is desired. An antisense oligonucleotide is an RNA having a complementary sequence when introduced into a cell.
And induces RNA degradation by RNaseH to inhibit protein translation, or direct hybridization inhibits protein synthesis. Since it is possible to specifically suppress the target gene function, it has been frequently used as a means for analyzing the function of a gene, and some antisense oligonucleotides are being developed for clinical application. The protein tyrosine kinase gene group forms the largest gene family among oncogenes and is deeply involved in cell proliferation and differentiation and morphogenesis (Harvey Lect. 94, 81-119, 1998-99). Have been picked up as an ideal target gene for the development of anticancer drugs. Among them, receptor tyrosine kinase (RTK)
EGF receptor, HER2 receptor or VE classified into
Drug development targeting the GF receptor has produced anticancer drugs at a level that is actually tested in humans, and among protein tyrosine kinases, RTK is considered to be a subfamily with a high probability of success as a drug. R
Among TK, Eph (erythropoietin-producing human
The hepatocellular carcinoma cell line) receptor gene family is currently composed of 14 members and has the largest scale (Cell 90, 403-404, 1997).
Year). Ephrin identified as a ligand for the Eph receptor
The gene family has also been reported to consist of at least eight members. Eph receptor and
Regarding the physiological function of Ephrin ligand, its involvement in the morphogenesis during the nascent stage or the proper projection of nerve axons has been suggested from the extremely localized expression pattern in the embryonic period and the brain (Annu. Rev. Neurosci. 21). : 309-345, 1998)
However, their role in cancer has been attracting attention in recent years. For example,
Upregulation of Eph receptor expression in various cancers reported (Onco
Gene 19: 5614-5619, 2000). Furthermore, their involvement in angiogenesis has been suggested (Gene
s & Dev. 13: 1055-1066, 1999).

[0003]

There is a great need for a drug that targets a molecule expressed in cancer cells and can inhibit the growth of cancer cells or induce apoptosis.

[0004]

[Means for Solving the Problems] As a result of intensive studies to solve the above-mentioned problems, the present inventors have found that the cancer is suppressed by suppressing the expression of the NEPHA gene expressed in cancer cells. Find that cells undergo apoptosis,
As a result of further studies based on this finding, the present invention has been completed.

That is, the present invention provides (1) SEQ ID NO:
13 or an oligonucleotide containing the same or substantially the same base sequence as the base sequence represented by SEQ ID NO: 14, (2) The above-mentioned (1) containing the base sequence represented by SEQ ID NO: 13 Oligonucleotides, (3)
The oligonucleotide according to (1) above, which contains the nucleotide sequence represented by SEQ ID NO: 14, (4) the oligonucleotide according to (1) above, which is an antisense oligonucleotide, and (5) above (1), which is a DNA. The oligonucleotide according to (6), a medicine comprising the oligonucleotide according to (1) above, (7) the medicine according to (5) above, which is a preventive and / or therapeutic agent for cancer.
(8) A DNA containing a base sequence identical or substantially identical to the base sequence represented by SEQ ID NO: 20, (9)
The DNA according to (8) above, which is a transcription control region of a DNA containing the same or substantially the same base sequence as the base sequence represented by SEQ ID NO: 12, and (10) the transcription control region is a promoter region ( 9) described DNA,
(11) A recombinant vector containing the DNA according to (8) above, (12) a DN containing the same or substantially the same base sequence as the base sequence represented by SEQ ID NO: 12.
D having a reporter gene downstream of the transcriptional regulatory region of A
A recombinant vector according to (11) above, which contains NA,
(13) A transformant containing the recombinant vector according to (11) above, or (14) the same or substantially the same as the nucleotide sequence represented by SEQ ID NO: 12, characterized by using the DNA according to (8) above. For screening a compound or a salt thereof that promotes or inhibits the transcriptional regulatory activity of DNAs containing the same nucleotide sequence, (15) above (1)
3) The screening method according to the above (14) using the transformant according to the above (16), which contains the DNA according to the above (8), and is the same as or substantially the same as the nucleotide sequence represented by SEQ ID NO: 12. Kit for screening a compound or a salt thereof that promotes or inhibits the transcriptional regulatory activity of DNAs containing the same nucleotide sequence, (17) The screening method according to (14) above or (1) above.
6) A compound or a salt thereof, which promotes or inhibits 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, which is obtained by using the screening kit described in (). 18) A medicament comprising the compound according to the above (17) or a salt thereof, (19) a medicament according to the above (18), which is an agent for preventing and / or treating cancer.

An oligonucleotide containing a base sequence substantially the same as the base sequence represented by SEQ ID NO: 13 or SEQ ID NO: 14 (hereinafter, abbreviated as the oligonucleotide of the present invention or the antisense oligonucleotide of the present invention). In some cases) is an Eph receptor protein containing the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 11 (hereinafter referred to as the receptor protein of the present invention or the Eph receptor protein of the present invention). (Also sometimes abbreviated as)), can inhibit the synthesis or function of the RNA, or can interact with the receptor protein-related RNA of the present invention to interact with the receptor. It can regulate and control the expression of somatic protein genes. SEQ ID NO: 13 or SEQ ID NO: 1
The oligonucleotide containing a base sequence substantially the same as the base sequence represented by 4 is a base sequence hybridizing with the base sequence represented by SEQ ID NO: 13 or SEQ ID NO: 14 under high stringent conditions. And an oligonucleotide having substantially the same activity as the oligonucleotide having the nucleotide sequence represented by SEQ ID NO: 13 or SEQ ID NO: 14. Examples of substantially the same activity include the activity of inhibiting the expression of the receptor protein of the present invention. Examples of the expression inhibitory activity include transcriptional inhibition, inhibition of splicing of precursor RNA, inhibition of mRNA cytoplasmic transfer, and inhibition of translation into protein. "Substantially the same quality" means that the activities are qualitatively the same. Therefore,
Activities such as 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 inhibitory activity and the like can be measured according to known methods. The inhibitory activity is the transformant containing the receptor protein of the present invention, the in vivo or in vitro gene expression system of the receptor protein of the present invention, or the in vivo or in vitro translation system of the receptor protein of the present invention. Can be checked using. For example, (A) a method for measuring the amount of mRNA of the receptor protein of the present invention or a partial peptide thereof described below,
(B) It can be measured by the method described in the method for quantifying the receptor protein of the present invention or its partial peptide or its salt.

The DNA which can hybridize with the nucleotide sequence represented by SEQ ID NO: 13 or SEQ ID NO: 14 under high stringent conditions is, for example, SEQ ID NO:
13 or the base sequence represented by SEQ ID NO: 14 and about 60
% Or more, more preferably about 70% or more, more preferably about 80% or more, particularly preferably about 90% or more, most preferably about 95% or more, a DNA containing a base sequence having homology is used. Hybridization can be carried out by a method known per se or a method analogous thereto, for example, molecular cloning (Molecular Clonin
g) 2nd (J. Sambrook et al., Cold Spring Harbor La
b. Press, 1989). Also, when using a commercially available library,
It can be performed according to the method described in the attached instruction manual. More preferably, it can be carried out under high stringent conditions. Highly stringent conditions include, for example, a sodium concentration of about 19 to 40 mM,
Preferably about 19-20 mM and temperature about 50-70.
C., preferably about 60 to 65.degree. Particularly, the case where the sodium concentration is about 19 mM and the temperature is about 65 ° C. is most preferable.

"Oligonucleotides" include polydeoxynucleotides containing 2-deoxy-D-ribose, polydeoxynucleotides containing D-ribose, purines or other N-glycosides of pyrimidine bases. Type of oligonucleotide, or other polymers with non-nucleotide backbones (eg commercially available protein nucleic acids and synthetic sequence-specific nucleic acid polymers) or other polymers containing special linkages (provided that the polymer is in DNA or RNA). Containing a nucleotide having a configuration that allows pairing of bases and attachment of bases, etc.). These can be double-stranded DNAs, single-stranded DNAs, double-stranded RNAs, single-stranded RNAs, and even DNA: RNA hybrids, as well as unmodified oligonucleotides (or unmodified oligonucleotides), as well as known Modified, eg labeled, capped, methylated, one or more natural nucleotides replaced by an analogue, intramolecular, as known in the art. Nucleotide-modified, such as uncharged bonds (eg, methylphosphonates, phosphotriesters,
Those with phosphoramidates, carbamates, etc.,
Those having a charged bond or a sulfur-containing bond (eg, phosphorothioate, phosphorodithioate, etc.), such as a protein (nuclease, nuclease inhibitor, toxin, antibody, signal peptide, poly-
L-lysine, etc.), sugars (eg, monosaccharides, etc.) having side chain groups, intercurrent compounds (eg, acridine, psoralen, etc.), chelate compounds (eg, metals, radioactivity) Metal, a boron, an oxidative metal, etc.), an alkylating agent, or a modified bond (eg, α-anomeric nucleic acid).
Here, the term “nucleoside”, “nucleotide” and “nucleic acid” may include those having not only purine and pyrimidine bases but also other modified heterocyclic bases. Such modifications may include methylated purines and pyrimidines, acylated purines and pyrimidines, or other heterocycles. Modified nucleotides and modified nucleotides may also be modified on the sugar moiety, eg, 1
One or more hydroxyl groups may be substituted with halogen, an aliphatic group or the like, or may be converted into a functional group such as ether or amine. Further, the oligonucleotide of the present invention may be a modified nucleic acid (RNA, DNA). Specific examples of the modified nucleic acid include sulfur derivatives of nucleic acids, thiophosphate derivatives, and those resistant to degradation of polynucleoside amides or oligonucleoside amides. Specific examples of the oligonucleotide of the present invention include DNA containing the base sequence represented by SEQ ID NO: 13, DNA containing the base sequence represented by SEQ ID NO: 14, and the like. The oligonucleotide of the present invention is produced by a known method, for example, a solid phase or liquid phase synthesis method (eg, automated nucleic acid synthesizer, liquid phase technique, etc.). Alternatively, it may be produced according to a known genetic engineering technique (eg, use of reverse transcriptase, PCR synthesis, etc.).

As the amino acid sequence substantially the same as the amino acid sequence represented by SEQ ID NO: 11, for example, about 55% or more of the amino acid sequence represented by SEQ ID NO: 11,
An amino acid sequence having a homology of preferably about 60% or more, more preferably about 70% or more, further preferably about 80% or more, particularly preferably about 90% or more, most preferably about 95% or more, and the like can be mentioned. . The protein containing an amino acid sequence substantially the same as the amino acid sequence represented by SEQ ID NO: 11 of the present invention has, for example, an amino acid sequence substantially the same as 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 preferable. As substantially the same quality of activity, for example, ligand binding activity, signal transduction activity, cell death suppression activity,
Examples include cell adhesion activity and cell motility regulation activity. "Substantially the same quality" means that their activities are qualitatively the same. Therefore, activities such as ligand binding activity, signal transduction activity, cell death inhibitory activity, cell adhesion activity and cell motility regulatory activity are equivalent (eg, about 0.0
1 to 100 times, 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 are different. May be. The ligand binding activity, signal transduction activity, cell death inhibitory activity, cell adhesion activity, cell motility regulatory activity and the like can be measured according to known methods.

In the amino acid sequence of the receptor protein in the present specification, the left end is the N-terminal (amino terminal) and the right end is the C-terminal (carboxyl terminal) according to the convention of peptide notation. Receptor protein of the present invention, C-terminal, carboxyl group (-COOH), a carboxylate (-COO ^-),
Amide (-CONH ₂₎ or an ester (-COOR)
It may be any of Here, R in the ester is, for example, C _1-6 alkyl group such as methyl, ethyl, n-propyl, isopropyl or n-butyl, for example, C _3-8 cycloalkyl group such as cyclopentyl or cyclohexyl, for example, phenyl. , A C _6-12 aryl group such as α-naphthyl, for example, a phenyl-C _1-2 alkyl group such as benzyl and phenethyl, or an α-naphthyl-C _1-2 alkyl group such as α-naphthylmethyl.
_In addition to the _7-14 aralkyl group, a pivaloyloxymethyl group commonly used as an oral ester is used. When the receptor protein of the present invention has a carboxyl group (or carboxylate) in addition to the C-terminal, those in which the carboxyl group is amidated or esterified are also included in the receptor protein of the present invention. As the ester in this case, for example, the above-mentioned C-terminal ester or the like is used. Further, in the receptor protein of the present invention, in the above-mentioned protein, the amino group of the N-terminal methionine residue is a protecting group (for example, C _1-6 acyl such as C _2-6 alkanoyl group such as formyl group and acetyl). Group, etc.), the N-terminal side is cleaved in vivo to form a glutamyl group produced by pyroglutamine oxidation, a substituent on the side chain of an amino acid in the molecule (for example, —OH, —SH, amino). Group, imidazole group, indole group, guanidino group, etc. are suitable protecting groups (for example, formyl group, C such as acetyl, etc.)
_2-6 alkanoyl groups and other C _1-6 acyl groups, etc.), or complex proteins such as so-called glycoproteins to which sugar chains are bound are also included. As a specific example of the receptor protein of the present invention, for example, a receptor protein containing the amino acid sequence represented by SEQ ID NO: 11 is used.

A partial peptide of the receptor protein of the present invention (hereinafter sometimes abbreviated as a partial peptide of the present invention)
Can also be used. The partial peptide of the present invention may be any one as long as it is a partial peptide of the above-mentioned receptor protein of the present invention.For example, among the receptor protein molecules of the present invention, it is exposed outside the cell membrane. And a portion located inside the cell membrane, which has substantially the same activity. Here, the “substantially the same activity” means, for example, a ligand binding activity, a signal transduction activity, a cell death inhibitory activity, a cell adhesion activity and a cell motility regulatory activity. Ligand binding activity,
The signal transduction activity, cell death inhibitory activity, cell adhesion activity, cell motility control activity and the like can be measured according to known methods. The amino acid number of the partial peptide of the present invention is at least 5 or more, preferably 10 or more, preferably 50 or more, more preferably 100 or more amino acid sequences among the constituent amino acid sequences of the receptor protein of the present invention described above. And the like are preferred.
A substantially identical amino acid sequence means about 50% or more, preferably about 60% or more, more preferably about 70% or more, further preferably about 80% or more, and particularly preferably about 90% or more with these amino acid sequences. Most preferably about 95
An amino acid sequence having homology of not less than% is shown.

The partial peptide of the present invention is (i) one or more (preferably 1) in the above amino acid sequence.
About 10 to about 10, more preferably several (1 to 5) amino acids are deleted, and (ii) 1 or 2 or more (preferably about 1 to 20 and more preferably 1) in the amino acid sequence. About 10 pieces, more preferably several pieces (1 to 5 pieces)
Or (iii) 1 or 2 or more (preferably about 1 to 10) in the above amino acid sequence,
(More preferably several, more preferably about 1 to 5)
The amino acid of may be substituted with another amino acid. As a specific example of the partial peptide of the present invention, SEQ ID NO: 11
Examples include peptides containing the 60th to 73rd or 491st to 504th amino acid sequences of the amino acid sequence represented by.

The partial peptide of the present invention has a C-terminal carboxyl group (-COOH), carboxylate (-C).
OO ^-), amide (-CONH ₂₎ or ester (-C
OOR) (R has the same meaning as described above).
When the partial peptide of the present invention has a carboxyl group (or carboxylate) in addition to the C-terminal, those in which the carboxyl group is amidated or esterified are also included in the partial peptide of the present invention. As the ester in this case, for example, the above-mentioned C-terminal ester or the like is used. Further, in the partial peptide of the present invention, as in the above-described receptor protein of the present invention, the amino group of the N-terminal methionine residue is protected by a protecting group, and the N-terminal side is cleaved in vivo. Pyroglutamine-oxidized glutamyl groups, those in which the substituents on the side chains of amino acids in the molecule are protected by appropriate protecting groups, or complex peptides such as so-called glycopeptides to which sugar chains are bound are also included. . Examples of the salt of the receptor protein of the present invention or its partial peptide include physiologically acceptable salts with acids or bases, and physiologically acceptable acid addition salts are particularly preferable. Examples of such salts include salts with inorganic acids (eg, hydrochloric acid, phosphoric acid, hydrobromic acid, sulfuric acid), or organic acids (eg, acetic acid, formic acid, propionic acid, fumaric acid, maleic acid, succinic acid). Acid, tartaric acid, citric acid, malic acid, oxalic acid, benzoic acid, methanesulfonic acid, benzenesulfonic acid) and salts thereof are used.

The receptor protein of the present invention or a salt thereof can be produced from the above-mentioned human or mammalian cells or tissues by a known method for purifying the receptor protein,
DNA encoding the receptor protein of the present invention described later
It can also be produced by culturing a transformant containing In addition, it can also be produced by a protein synthesis method described later or a method similar thereto. 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 extract is subjected to chromatography such as reverse phase chromatography or ion exchange chromatography. Can be purified and isolated by combining

For the synthesis of the receptor protein of the present invention or its partial peptide or its salt or its amide, a commercially available resin for protein synthesis can be used. Examples of such a resin include chloromethyl resin, hydroxymethyl resin, benzhydrylamine resin, aminomethyl resin, 4-benzyloxybenzyl alcohol resin, 4-methylbenzhydrylamine resin, PAM resin, 4-hydroxymethylmethyl resin. Phenylacetamide methyl resin, polyacrylamide resin, 4- (2 ', 4'
-Dimethoxyphenyl-hydroxymethyl) phenoxy resin, 4- (2 ', 4'-dimethoxyphenyl-Fmoc
Aminoethyl) phenoxy resin and the like can be mentioned. Using such a resin, an amino acid having an α-amino group and a side chain functional group appropriately protected is condensed on the resin according to various known condensation methods according to the amino acid sequence of the desired protein or peptide. At the end of the reaction, the protein or peptide is cleaved from the resin, at the same time various protecting groups are removed, and an intramolecular disulfide bond-forming reaction is carried out in a highly diluted solution to obtain the desired protein or partial peptide or its amide. Regarding the condensation of the protected amino acid described above, various activating reagents that can be used for protein synthesis can be used, but carbodiimides are particularly preferable. As carbodiimides, DCC, N, N'-diisopropylcarbodiimide, N-ethyl-N '-(3-
Dimethylaminoprolyl) carbodiimide and the like are used. For activation by these, a protected amino acid is added directly to the resin together with a racemization-inhibiting additive (eg, HOBt, HOOBt), or a symmetrical acid anhydride or H
The protected amino acid can be added to the resin after activation of the protected amino acid as an OBt ester or a HOOBt ester.

The solvent used for activation of the protected amino acid or condensation with the resin can be appropriately selected from solvents known to be usable for protein condensation reaction. For example,
Acid amides such as N, N-dimethylformamide, N, N-dimethylacetamide and N-methylpyrrolidone, halogenated hydrocarbons such as methylene chloride and chloroform,
Alcohols such as trifluoroethanol, sulfoxides such as 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. Are used. The reaction temperature is appropriately selected from the range known to be applicable to protein bond-forming reactions, and is usually appropriately selected from the range of about -20 ° C to 50 ° C. The activated amino acid derivative is usually used in a 1.5 to 4-fold excess. As a result of the test using the ninhydrin reaction, when the condensation is insufficient, sufficient condensation can be performed by repeating the condensation reaction without removing the protecting group. When sufficient condensation cannot be obtained even after repeating the reaction, unreacted amino acid can be acetylated using acetic anhydride or acetylimidazole.

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

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

Protection of a functional group which should not be involved in the reaction of the raw material, removal of the protective group and removal of the protective group, activation of the functional group involved in the reaction, etc. can be appropriately selected from known groups or known means. . As another method for obtaining an amide form of a protein, for example, first, the carboxy terminal amino acid α-
After protecting the carboxyl group by amidation, after extending the peptide (protein) chain to the amino group side to the desired chain length,
A protein in which only the protecting group for the α-amino group at the N-terminus of the peptide chain was removed and a protein in which only the protecting group for the carboxyl group at the C-terminus was removed were prepared, and both proteins were mixed in a mixed solvent as described above. To condense. The details of the condensation reaction are the same as above. After purifying the protected protein obtained by condensation, all the protecting groups can be removed by the above method to obtain the desired crude protein. This crude protein can be purified by making full use of various known purification means, and the main fraction can be lyophilized to obtain the amide form of the desired protein. To obtain an ester form of a protein, for example, the α-carboxyl group of a carboxy-terminal amino acid is condensed with a desired alcohol to form an amino acid ester, and then an ester form of the desired protein is obtained in the same manner as the amide form of the protein. be able to.

The partial peptide of the protein of the present invention or a salt thereof can be produced according to a known peptide synthesis method or by cleaving the protein of the present invention with an appropriate peptidase. The peptide synthesis method includes:
For example, either solid phase synthesis method or liquid phase synthesis method may be used. That is, the target peptide can be produced by condensing a partial peptide or amino acid that can form the protein of the present invention with the remaining portion and removing the protecting group if the product has a protecting group. Examples of known condensation methods and removal of protective groups include the methods described in (i) to (v) below. (I) M. Bodanszky and MA Ondetti, Peptide Synthesis, Interscience Publisher
s, New York (1966) (ii) Schroeder and Luebke, The Peptid
e), Academic Press, New York (1965) (iii) Nobuo Izumiya et al., Fundamentals and Experiments of Peptide Synthesis, Maruzen
(1975) (iv) Haruaki Yajima and Shunpei Sakakibara, Laboratory of Biochemistry 1, Protein Chemistry IV, 205, (1977) (v) Supervision of Haruaki Yajima, Development of Pharmaceuticals Volume 14 Peptide After the reaction, the partial peptide of the present invention can be purified and isolated by a combination of ordinary purification methods such as solvent extraction, distillation, column chromatography, liquid chromatography, and recrystallization. When the partial peptide obtained by the above method is a free form, it can be converted into an appropriate salt by a known method, and conversely, when it is obtained as a salt, it can be converted into a free form by a known method. You can

Any polynucleotide can be used as the polynucleotide encoding the receptor protein of the present invention, so long as it contains the nucleotide sequence (DNA or RNA, preferably DNA) encoding the receptor protein of the present invention. May be. The polynucleotide is DNA such as DNA encoding the receptor protein of the present invention or RNA such as mRNA, and may be double-stranded or single-stranded. In the case of double-stranded, double-stranded DNA, double-stranded RNA or DNA:
It may be a hybrid of RNA. In the case of a single strand, it may be a sense strand (that is, a coding strand) or an antisense strand (that is, a non-coding strand). The DNA encoding the receptor protein of the present invention includes genomic DN
A, genomic DNA library, cDNA derived from the cells / tissues described above, cDNA library derived from the cells / tissues described above, or synthetic DNA may be used. The vector used for the library may be any of bacteriophage, plasmid, cosmid, phagemid and the like. In addition, total RNA or mRNA from the above cells / tissues
Direct reverse transcribing using prepared NA fraction
ptase Polymerase Chain Reaction (hereinafter RT-PC
DNA can also be amplified by the R method).

Specifically, as the DNA encoding the receptor protein of the present invention, for example, DNA containing the nucleotide sequence represented by SEQ ID NO: 12 or SEQ ID NO: 1
It has a DNA that hybridizes with the DNA containing the nucleotide sequence represented by 2 under high stringent conditions, and has substantially the same activity as the receptor protein of the present invention (eg, ligand binding activity, signal transduction activity). , A cell death-suppressing activity, a cell adhesion activity, a cell motility controlling activity, etc.).

The DNA that hybridizes with the DNA having the nucleotide sequence represented by SEQ ID NO: 12 under high stringent conditions is, for example, about 55% or more, preferably about 50%, the nucleotide sequence represented by SEQ ID NO: 12. 60
% Or more, preferably about 70% or more, preferably about 80%
DN containing a base sequence having homology of at least 90%, more preferably at least 90%, and even more preferably at least 95%.
A or the like is used.

Hybridization can be carried out by a known method or a method analogous thereto, for example, Molecular Cloning 2nd (J. Sambrook et al.
al., Cold Spring Harbor Lab. Press, 1989), and when using a commercially available library, the method described in the attached instruction manual can be used. More preferably, hybridization can be performed under high stringent conditions. The high stringent conditions include, for example, a sodium concentration of about 19 to 40 mM, preferably about 19 to 2
At 0 mM, the temperature is about 50 to 70 ° C., preferably about 60.
The conditions of ˜65 ° C. are shown. Especially, the sodium concentration is about 19
Most preferred is mM and a temperature of about 65 ° C.

DNA encoding the partial peptide of the present invention
Any may be used as long as it contains the nucleotide sequence encoding the partial peptide of the present invention. Further, any of genomic DNA, genomic DNA library, cDNA derived from the cells / tissues described above, cDNA library derived from the cells / tissues described above, and synthetic DNA may be used. The vector used for the library may be any of bacteriophage, plasmid, cosmid, phagemid and the like. In addition, RT-P can be directly used by using an mRNA fraction prepared from the cells and tissues described above.
DNA can also be amplified by the CR method. Specifically, the DNA encoding the partial peptide of the present invention includes, for example, DNA having a partial base sequence of DNA containing the base sequence represented by SEQ ID NO: 12 or DNA represented by SEQ ID NO: 12. A partial base sequence of a DNA containing a DNA that hybridizes under high stringent conditions and encoding a protein having substantially the same activity as the protein peptide of the present invention, for example, a ligand binding activity, a signal transduction action, etc. Having DNA
Are used.

As a means for cloning a DNA which 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), the receptor protein of the present invention is coded. DNA fragment that encodes a part or the whole region of the receptor protein of the present invention, which is amplified by the PCR method using a synthetic DNA primer having a partial base sequence of the base sequence of the DNA Or synthetic D
It can be selected by hybridization with that labeled with NA.

The substitution of the nucleotide sequence of DNA is carried out by using PCR or a known kit such as Mutan ^™ -superExpress Km (Takara Shuzo) or Mutan ^™ -K (Takara Shuzo).
It can be carried out according to a known method such as A-LA PCR method, gapped duplex method, Kunkel method, or a method similar thereto. DN encoding a cloned receptor protein
Depending on the purpose, A can be used as it is, or if desired, after digestion with a restriction enzyme or addition of a linker. The DNA may have ATG as a translation initiation codon at the 5′-terminal side and TAA, TGA or TAG as a translation termination codon at the 3′-terminal side. These translation initiation codon and translation termination codon can be added using an appropriate synthetic DNA adaptor. The DNA encoding the receptor protein of the present invention includes, for example, (a) the DNA encoding the receptor protein of the present invention. It can be incorporated into an expression vector by ligating it downstream of a promoter in an appropriate expression vector.

As a vector for cloning or expression, a plasmid derived from E. coli (eg pC
R4, pCR2.1, pBR322, pBR325, pUC12, pUC13), Bacillus subtilis-derived plasmid (eg, pUB110, pTP5, pC194), yeast-derived plasmid (eg, pSH19, pSH15), bacteriophage such as λ phage, retrovirus In addition to animal viruses such as vaccinia virus and baculovirus, pA1-11, pXT1, pRc / CMV, pRc / RSV, pcDNAI / Neo and the like are used. The promoter may be any promoter as long as it is suitable for the host used for gene expression. For example, when an animal cell is used as a host, SRα promoter, SV40 promoter, LTR promoter, CMV promoter, HS
V-TK promoter etc. are mentioned. Among these, it is preferable to use the CMV promoter, the SRα promoter and the like. When the host is Escherichia, the trp promoter, lac promoter, rec
A promoter, λP _L promoter, lpp promoter, etc., when the host is Bacillus, SPO
1 promoter, SPO2 promoter, penP promoter, etc., when the host is yeast, PHO5 promoter, PGK promoter, GAP promoter,
The ADH promoter and the like are preferable. When the host is an insect cell, polyhedrin promoter, P10 promoter and the like are preferable.

In addition to the above, the expression vector may optionally contain an enhancer, a splicing signal, a poly A addition signal, a selection marker, an SV40 replication origin (hereinafter sometimes abbreviated as SV40 ori) and the like. Can be used. Examples of the selectable marker include, for example, dihydrofolate reductase (hereinafter sometimes abbreviated as dhfr) gene [methotrexate (MTX)
Resistance], ampicillin resistance gene (hereinafter sometimes abbreviated as Amp ^r ), neomycin resistance gene (hereinafter,
Sometimes abbreviated as Neo ^r, include G418 resistance) and the like. In particular, when the dhfr gene is used as a selection marker using CHO (dhfr ⁻ ) cells, the target gene can be selected by a medium containing no thymidine. Also,
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. When the host is Escherichia, PhoA signal sequence, OmpA signal sequence, etc., when the host is Bacillus, α-amylase signal sequence, subtilisin signal sequence, etc., the host is yeast For MFα · signal sequence, SUC2 · signal sequence, etc., when the host is an animal cell, an insulin signal sequence, α-interferon signal sequence, antibody molecule / signal sequence, etc. can be used. A transformant can be produced using the vector containing the DNA encoding the receptor protein of the present invention thus constructed.

Examples of the host used in the present invention include Escherichia, Bacillus, yeast, insect cells, insects and animal cells. Specific examples of the Escherichia bacterium include Escherichia col.
i) K12 DH1 [Proc. Natl. Acad. Sci. USA], Volume 60, 16
0 (1968)], JM103 [Nucleic Acids Research, Volume 9, 309 (1981)], J
A221 [Journal of Molecular Biology, 120, 517 (197)
8)], HB101 [Journal of Molecular Biology, 41, 459 (1969)], C600 [Genetics, 39, 440 (1954)], DH5α [Inoue,
H., Nojima, H. and Okayama, H., Gene, 96, 23-28 (1
990)], DH10B [Procedures of the National Academy of Sciences of the
USA (Proc. Natl. Acad. Sci. USA), Vol. 87,
4645-4649 (1990)] is used. Examples of the genus Bacillus include, for example, Bacillus s.
ubtilis) MI114 [Gene, 24, 255 (1983)], 207-2
1 〔Journal of Biochemistry
f Biochemistry), Vol. 95, 87 (1984)]. Examples of yeast include Saccharomyces cerevisiae ^{(Saccharomyces cerevisiae) AH22, AH22R -} , 0NA
87-11A, DKD-5D, 20B-12, Schizosaccharomyces pombe NCYC1913, NCYC2036,
Pichia pastoris and the like are used.

Examples of insect cells include virus A
In the case of cNPV, larvae of the night-thief moth (Spodop
tera frugiperda cell; Sf cell), Trichoplusia ni
MG1 cells from the midgut of the mouse, H from the eggs of Trichoplusia ni
igh Five ^TM cells, cells from Mamestra brassicae or
Cells derived from Estigmena acrea are used. When the virus is BmNPV, the silkworm-derived cell line (Bombyx mor
i N; BmN cells) and the like are used. Examples of the Sf cells include Sf9 cells (ATCC CRL1711), Sf21 cells (above, Vaughn, JL et al., In Vivo,
13, 213-217 (1977)) are used. Examples of insects include silkworm larvae [Maeda et al.
Nature, Volume 315, 592 (1985)]. Examples of animal cells include monkey cells COS-7, Vero,
Chinese hamster cell CHO (hereinafter abbreviated as CHO cell), dhfr gene-deficient Chinese hamster cell CHO (hereinafter abbreviated as CHO (dhfr ⁻ ) cell), mouse L cell, mouse AtT-20, mouse myeloma cell, rat GH3, human FL cells and the like are used.

For transforming Escherichia, for example, Proc. Natl. Acad. Sci. USA, Volume 69, Proceedings of the National Academy of Sciences of the USA. ， 2110 (197
2) and Gene, 17, vol. 107 (1982). To transform Bacillus, for example, Molecular & General Genetics (Molecular & General Genetics
s), Volume 168, 111 (1979) and the like. For transformation of yeast, for example, Methods in Enzymolology (Methods in Enzymol
ogy), 194, 182-187 (1991), Proc.
Of the National Academy of Sciences of the USA (Proc. Natl. Acad. Sc
i. USA), Vol. 75, 1929 (1978) and the like. For transforming insect cells or insects, for example, Bio / Techno
logy), 6, 47-55 (1988) and the like. To transform animal cells, see, eg, Cell Engineering Supplement 8, New Cell Engineering Experimental Protocol. 26
3-267 (1995) (published by Shujunsha), Virology
gy), 52, 456 (1973). Thus, a transformant transformed with the expression vector containing the DNA encoding the Eph receptor protein is obtained. When the host is culturing a transformant that is a bacterium of the genus Escherichia, a bacterium of the genus Bacillus, a liquid medium is suitable as a medium used for the culture, and among them, a carbon source necessary for the growth of the transformant, A nitrogen source, an inorganic substance, etc. are contained. Examples of the carbon source include glucose, dextrin, soluble starch, sucrose and the like, and examples of the nitrogen source include ammonium salts, nitrates,
Examples of the inorganic or organic substances such as corn steep liquor, peptone, casein, meat extract, soybean meal, and potato extract, and inorganic substances include calcium chloride, sodium dihydrogen phosphate, magnesium chloride and the like. In addition, yeast extract, vitamins, growth promoting factor and the like may be added. The pH of the medium is preferably about 5-8.

As a medium for culturing Escherichia, for example, M9 medium containing glucose and casamino acid [Miller, Journal of Experiments in Molecular Genetics (Journa
l of Experiments in Molecular Genetics), 431-43
3, Cold Spring Harbor Laboratory, New York 1972]
Is preferred. If necessary, a drug such as 3β-indolylacrylic acid can be added to the promoter in order to work it efficiently. When the host is a bacterium belonging to the genus Escherichia, the culturing is usually carried out at about 15 to 43 ° C. for about 3 to 24 hours, and if necessary, aeration and stirring can be added.
When the host is a bacterium of the genus Bacillus, the culture is usually carried out at about 30 to 40 ° C. for about 6 to 24 hours, and aeration and agitation can be added if necessary. When culturing a transformant whose host is yeast,
Examples of the medium include Burkholde (Burkholde).
r) Minimal medium [Bostian, KL, et al., "Proc. Natl. Acad., Proc. of the National Academy of Sciences of the USA.
Sci. USA), 77, 4505 (1980)]] and SD medium containing 0.5% casamino acid [Bitter, GA et al., "Procedures of the National Academy of Sciences of the・ USA (Proc. Natl.
Acad. Sci. USA), 81, 5330 (1984)]]. The pH of the medium is preferably adjusted to about 5-8. Culturing is usually performed at about 20 ° C to 35 ° C for about 24 to 72 hours, and aeration and agitation are added if necessary.

When culturing a transformant whose host is an insect cell or an insect, the medium is Grace's Insect Mediu.
m (Grace, TCC, Nature, 195, 788 (1
962)) to which an additive such as immobilized 10% bovine serum is appropriately added is used. The pH of the medium is preferably adjusted to about 6.2-6.4. Culturing is usually at about 27 ° C for about 3-5
Do it for a day and add aeration and agitation as needed. When a transformant whose host is an animal cell is cultured, examples of the medium include MEM medium containing about 5 to 20% fetal bovine serum [Science, Volume 122, 501 (1952)], DM.
EM medium [Virology, Volume 8, 396 (195
9)], RPMI1640 medium [The Journal of the American Medical Association (The Journal
of the American Medical Association) 199, 519 (1
967)], 199 medium [Procedure of the Society for the Biological Medicine (Pr.
oceeding of the Society for the Biological Medicin
e), Vol. 73, 1 (1950)]. pH is about 6
It is preferably -8. Culturing is usually performed at about 30-40 ° C.
Perform for 15 to 60 hours, add aeration and agitation as needed.
As described above, the receptor protein of the present invention can be produced inside the transformant cell, on the cell membrane or outside the cell.

Separation and purification of the receptor protein of the present invention from the above culture can be carried out, for example, by the following method. To extract the receptor protein of the present invention from cultured bacterial cells or cells, after the culture, the bacterial cells or cells are collected by a known method, suspended in an appropriate buffer, and then subjected to ultrasonic wave, lysozyme and / or freezing. A method of obtaining a crude extract of the receptor protein by disrupting the cells or cells by thawing and then centrifuging or filtering is appropriately used. The buffer solution may contain a protein denaturing agent such as urea or guanidine hydrochloride, or a surfactant such as Triton X-100 ^™ . When the receptor protein is secreted into the culture medium, after the completion of the culture, the 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 the extract can be carried out by appropriately combining known separation / purification methods. Known separation and purification methods for these are methods utilizing solubility such as salting out and solvent precipitation, methods utilizing mainly differences in molecular weight such as dialysis, ultrafiltration, and gel filtration, and ion exchange. Chromatography, etc., charge difference, affinity chromatography, etc., specific affinity, reverse phase high performance liquid chromatography, etc., hydrophobicity, isoelectric focusing, etc. The method of utilizing the difference of the isoelectric points is used.

When the receptor protein thus obtained is obtained as a free form, it can be converted into a salt by a known method or a method analogous thereto, and conversely, when it is obtained as a salt, it is known. Can be converted into a free form or other salt by the method described above or a method similar thereto. The receptor protein produced by the recombinant is treated with an appropriate protein-modifying enzyme before or after purification,
It may be optionally modified or the polypeptide may be partially removed. Examples of the protein-modifying enzyme include trypsin, chymotrypsin, arginyl endopeptidase, protein kinase, glycosidase and the like. 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 and an enzyme immunoassay using a specific antibody.

The antibody against the receptor protein of the present invention or its partial peptide or its salt is either a polyclonal antibody or a monoclonal antibody as long as it is an antibody capable of recognizing the receptor protein of the present invention or its partial peptide or its salt. It may be. 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) is
It can be produced according to a known method for producing an antibody or antiserum using the receptor protein or the like of the present invention as an antigen.

[Preparation of Monoclonal Antibody] (a) Preparation of Monoclonal Antibody-Producing Cell The receptor protein or the like of the present invention is administered to a mammal at a site where antibody production can be performed by itself or with a carrier or diluent. To be done. Complete Freund's adjuvant or incomplete Freund's adjuvant may be administered to enhance the antibody-producing ability upon administration. The administration is usually performed once every 2 to 6 weeks, about 2 to 10 times in total. Examples of mammals used include monkeys, rabbits, dogs, guinea pigs, mice, rats, sheep, and goats, and mice and rats are preferably used. For preparation of monoclonal antibody-producing cells, warm-blooded animals immunized with an antigen, for example, individuals with an antibody titer were selected from mice, and 2 to 5 days after the final immunization, spleens or lymph nodes were collected and By fusing the antibody-producing cells contained therein 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 described below with the antiserum, and then measuring the activity of the labeling agent bound to the antibody. The fusion operation can be carried out according to a known method, for example, the method of Kohler and Milstein [Nature, 256, 495 (1975)]. As the fusion promoter, for example,
Examples thereof include polyethylene glycol (PEG) and Sendai virus, but PEG is preferably used.
Examples of myeloma cells include NS-1, P3U1, S
Examples include P2 / 0, but P3U1 is preferably used. The preferred ratio of the number of antibody-producing cells (spleen cells) to the number of myeloma cells used is about 1: 1 to 20: 1, and PEG (preferably PEG1000 to PEG60) is used.
00) is added at a concentration of about 10 to 80%, and about 20 to
Cell fusion can be efficiently performed by incubating at 40 ° C., preferably about 30 to 37 ° C. for about 1 to 10 minutes.

Various methods can be used for screening a hybridoma producing a monoclonal antibody. For example, a hybridoma culture supernatant is added to a solid phase (eg, a microplate) on which an antigen such as a receptor protein is directly or adsorbed with a carrier. Then, a monoclonal antibody bound to a solid phase by adding an anti-immunoglobulin antibody labeled with a radioactive substance or an enzyme (if the cell used for cell fusion is a mouse, an anti-mouse immunoglobulin antibody is used) or protein A Method, the hybridoma culture supernatant is added to the solid phase on which the anti-immunoglobulin antibody or protein A is adsorbed, the receptor protein labeled with a radioactive substance or enzyme is added, and the monoclonal antibody bound to the solid phase is added. Examples include a method of detecting. The selection of the monoclonal antibody can be carried out by a known method or a method analogous thereto, but usually it can be carried out in a medium for animal cells supplemented with HAT (hypoxanthine, aminopterin, thymidine). As the selection and breeding medium, any medium can be used as long as the hybridoma can grow. For example, RPMI1640 medium containing 1 to 20%, preferably 10 to 20% fetal bovine serum, 1 to
GIT medium (Wako Pure Chemical Industries, Ltd.) containing 10% fetal bovine serum or serum-free medium for culturing hybridoma (SF
M-101, Nissui Pharmaceutical Co., Ltd., etc. can be used. The culture temperature is usually 20 to 40 ° C, preferably about 37 ° C. Cultivation time is usually 5 days to 3 weeks, preferably 1 week to 2 weeks. Culturing can usually be performed under 5% carbon dioxide gas. The antibody titer of the hybridoma culture supernatant can be measured in the same manner as the above antibody titer measurement in antiserum.

(B) Purification of Monoclonal Antibody The isolation and purification of the monoclonal antibody can be carried out by the same method as the isolation and purification of ordinary polyclonal antibodies [eg, salting out method, alcohol precipitation method, isoelectric focusing method, Electrophoresis method, adsorption / desorption method with ion exchanger (eg, DEAE), ultracentrifugation method, gel filtration method, antigen-binding solid phase or active antibody such as protein A or protein G is used to dissociate the antibody. Specific Purification Method of Obtaining Antibody to Obtain Antibody]

[Preparation of Polyclonal Antibody] The polyclonal antibody of the present invention can be manufactured by publicly known methods or modifications thereof. For example, a complex of an immunizing antigen (an antigen such as the protein of the present invention) and a carrier protein is formed, and a mammal is immunized in the same manner as in the above-mentioned method for producing a monoclonal antibody, and the receptor protein of the present invention is immunized from the immunized animal. It can be produced by collecting the antibody-containing substance against the above and isolating and purifying the antibody. Regarding a complex of an immunizing antigen and a carrier protein used for immunizing a mammal, the kind of the carrier protein and the mixing ratio of the carrier and the hapten are such that the antibody against the hapten immunized by crosslinking the carrier can be efficiently prepared. What kind of thing may be crosslinked at any ratio, but, for example, bovine serum albumin, bovine thyroglobulin, keyhole limpet hemocyanin, etc. in a weight ratio of about 0.1 to 20 with respect to 1 hapten. Preferably, a method of coupling at a ratio of about 1 to 5 is used. Further, for the coupling of the hapten and the carrier, various condensing agents can be used, but glutaraldehyde, carbodiimide,
An active ester reagent containing a maleimide active ester, a thiol group, a dithiopyridyl group or the like is used. The condensation product is administered to a warm-blooded animal at a site where antibodies can be produced, by itself or together with a carrier and a diluent. Complete Freund's adjuvant or incomplete Freund's adjuvant may be administered to enhance the antibody-producing ability upon administration. Administration is usually about once every 2 to 6 weeks, about 3 to 3 in total.
Can be done about 10 times. The polyclonal antibody is
Blood, ascites, etc., of mammals immunized by the above method, preferably blood, can be collected. The polyclonal antibody titer in the antiserum can be measured in the same manner as the above-mentioned antibody titer in the serum. Separation and purification of the polyclonal antibody can be performed according to the same immunoglobulin separation and purification method as the above-described separation and purification of the monoclonal antibody.

(A) Measurement of mRNA amount of receptor protein of the present invention or partial peptide thereof mRN of receptor protein of the present invention or partial peptide thereof
The following method may be mentioned as a specific example of the measurement of the amount A. (I) Normal or disease model non-human mammal (eg, mouse, rat, rabbit, sheep, pig, cow, cat, dog, monkey, etc., more specifically, demented rat, obese mouse, arteriosclerotic rabbit, cancer bearing) Drugs (eg anti-dementia drugs, blood pressure lowering drugs,
Anti-cancer drug, anti-obesity drug, etc.) or physical stress (eg flooding stress, electric shock, light / darkness, low temperature, etc.), and after a certain period of time, blood, or a specific organ (eg, brain, lung, large intestine, etc.) ), Or tissue or cells isolated from an organ. The mRNA of the receptor protein of the present invention or its partial peptide contained in the obtained cells can be isolated from cells such as mRNA by a conventional method.
NA can be extracted and quantified by using a technique such as TaqMan PCR, and can also be analyzed by performing Northern blotting by a known means. (Ii) A transformant expressing the receptor protein of the present invention or its partial peptide is prepared according to the above method, and mRNA of the receptor protein of the present invention or its partial peptide contained in the transformant is similarly treated. It can be quantified and analyzed.

(B) Quantification of Receptor Protein of the Present Invention or Partial Peptide or Salt Thereof Since the antibody of the present invention can specifically recognize the receptor protein of the present invention, etc. It can be used for quantification of the receptor protein and the like of the present invention, particularly for quantification by sandwich immunoassay. For example, (i) the antibody of the present invention is allowed to react competitively with a test solution, a labeled receptor protein or the like, and the ratio of the labeled receptor protein or the like bound to the antibody is measured A method for quantifying the receptor protein or the like of the present invention in a test solution, (ii) reacting the test solution with the antibody of the present invention insolubilized on a carrier and the labeled antibody of the present invention simultaneously or successively After that, the activity of the labeling agent on the insolubilized carrier is measured, and a method for quantifying the receptor protein or the like of the present invention in a test solution can be mentioned. Above (i
In i), it is preferable that one antibody recognizes the N-terminal portion of the receptor protein of the present invention and the other antibody reacts with the C-terminal portion of the receptor protein of the present invention. .

A monoclonal antibody against the receptor protein or the like of the present invention (hereinafter sometimes referred to as the monoclonal antibody of the present invention) can be used to measure the receptor protein or the like of the present invention, or to detect by tissue staining or the like. You can also 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 measuring 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 antigen (eg, receptor protein amount) in the liquid to be measured. Any measuring method may be used as long as it is a measuring method in which 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, competitive method, immunometric method and sandwich method are preferably used, but sensitivity,
In terms of specificity, it is particularly preferable to use the sandwich method described later. As the labeling agent used in the measuring method using the labeling substance, for example, a radioisotope, an enzyme, a fluorescent substance, a luminescent substance, etc. are used. Examples of radioisotopes include, for example, [ ¹²⁵ I], [ ¹³¹ I], [ ³ H], [ ¹⁴
C] or the like is used. As the above-mentioned enzyme, those having a stable and large specific activity are preferable, and for example, β-galactosidase, β-glucosidase, alkaline phosphatase, peroxidase, malate dehydrogenase and the like are used. As the fluorescent substance, for example, fluorescamine, fluorescein isothiocyanate, etc. are used. As the luminescent substance, for example, luminol, luminol derivative, luciferin, lucigenin and the like are used. Furthermore, the biotin-avidin system can be used for binding the antibody or the antigen to the labeling agent.

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

The monoclonal antibody of the present invention can be used in a measuring system other than the sandwich method, for example, a competitive method, an immunometric method or nephrometry. In the competitive method, the antigen in the test liquid and the labeled antigen are reacted competitively with the antibody, and then the unreacted labeled antigen (F) and the labeled antigen (B) bound to the antibody are separated. (B
/ F separation), the labeled amount of either B or F is measured to quantify the amount of antigen in the test liquid. In this reaction method, a soluble antibody is used as an antibody, B / F separation is performed by polyethylene glycol, a liquid phase method using a second antibody or the like against the above antibody, and a solid phase antibody is used as the first antibody, or The first antibody is soluble and the second antibody is a solid-phased antibody. In the immunometric method, the antigen in the test liquid and the solid-phased antigen are allowed to undergo a competitive reaction with a fixed amount of the labeled antibody, and then the solid phase and the liquid phase are separated, or
Alternatively, the antigen in the test liquid is reacted with an excess amount of the labeled antibody, and then the immobilized antigen is added to bind the unreacted labeled antibody to the solid phase, and then the solid phase and the liquid phase are separated. To do. Next, the labeled amount of either phase is measured to quantify the amount of antigen in the test liquid. In nephrometry, the amount of insoluble precipitate generated as a result of the antigen-antibody reaction in the gel or in the solution is measured. Even when the amount of antigen in the test liquid is small and only a small amount of precipitate can be obtained, laser nephrometry utilizing laser scattering is preferably used.

In applying these individual immunological assay methods to the assay method of the present invention, it is not necessary to set special conditions, operations and the like. The assay system for the receptor protein of the present invention or a salt thereof may be constructed by adding ordinary technical consideration to those skilled in the art to ordinary conditions and operating methods in each method. For details of these general technical means, reference can be made to reviews, books, etc. [eg Irie
Hiroshi "Radioimmunoassay" (Kodansha, published in 1974), Hiroshi Irie "Radioimmunoassay" (Kodansha, published in 1979), Eiji Ishikawa et al. "Enzyme Immunoassay"
(Medical Shoin, published in 1978), Eiji Ishikawa et al. “Enzyme Immunoassay” (2nd edition) (Medical Shoin, published in 1982), Eiji Ishikawa et al. “Enzyme Immunoassay” (3rd edition) (Medical Shoin, published in 1987), "Methods in ENZYMOLOGY" Vol. 70 (Immunochemica)
l Techniques (Part A)), ibid Vol. 73 (Immunochemi
cal Techniques (Part B)), ibid Vol. 74 (Immunoche
mical Techniques (Part C)), ibid Vol. 84 (Immunoche
mical Techniques (Part D: Selected Immunoassays)),
Ibid Vol. 92 (Immunochemical Techniques (Part E: M
onoclonal Antibodies and General Immunoassay Method
s)), ibid Vol. 121 (Immunochemical Techniques (Par
t I: Hybridoma TechnoloGy and Monoclonal Antibodie
s)) (above, issued by Academic Press) etc.). 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) Pharmaceutical containing antisense oligonucleotide The oligonucleotide of the present invention is stable in cells,
It has high cell permeability, high affinity for the target sense strand, and low toxicity. The polynucleotide of the present invention is, for example, a disease associated with overexpression of the receptor protein of the present invention [eg, cancer (eg, non-small cell lung cancer, ovarian cancer, prostate cancer, gastric cancer, bladder cancer, breast cancer, cervical cervix Cancer, colon cancer, rectal cancer, etc.) and the like]. The antisense oligonucleotides of the present invention may contain altered or modified sugars, bases, linkages, are provided in a special form such as liposomes, microspheres, or applied by gene therapy. , Can be given in added form. As an additive form, a polycationic substance such as polylysine that acts to neutralize the charge of the phosphate skeleton, a lipid that enhances the interaction with the cell membrane or the uptake of nucleic acid (for example, , Phospholipids, cholesterol, etc.) and hydrophobic ones. Preferred lipids to add include cholesterol and its derivatives (eg cholesteryl chloroformate, cholic acid, etc.). These can be attached to the 3'end or 5'end of the nucleic acid, and can be attached via a base, a sugar, or an intramolecular nucleoside bond. Other groups include
A capping group specifically arranged at the 3'-end or the 5'-end of a nucleic acid, which includes a group for preventing decomposition by nucleases such as exonuclease and RNase. Examples of such a capping group include, but are not limited to, hydroxyl group-protecting groups known in the art including glycols such as polyethylene glycol and tetraethylene glycol. When the antisense oligonucleotide of the present invention is used as the above-mentioned medicine, it can be formulated and administered according to a known method. For example, in the case of using the antisense oligonucleotide, 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 then, according to a conventional method, a human or non-human It can be administered orally or parenterally to a human mammal (eg, rat, rabbit, sheep, pig, cow, cat, dog, monkey, etc.). The antisense oligonucleotide can be administered as it is or in the form of a formulation with a physiologically acceptable carrier such as an auxiliary agent for promoting ingestion, and can be administered by a gene gun or a catheter such as a hydrogel catheter. The dose of the antisense oligonucleotide varies depending on the target disease, the administration subject, the administration route, etc.
When the antisense oligonucleotide of the present invention is locally administered to an organ (eg, liver, lung, heart, kidney, etc.) for the purpose of cancer treatment, the antisense oligonucleotide is generally administered per day in an adult (body weight 60 kg). About 0.1 to 100 mg of oligonucleotide is administered. Furthermore, the antisense oligonucleotide can also be used as a diagnostic oligonucleotide probe for examining the presence or expression of the polynucleotide (eg, DNA) of the present invention in tissues or cells.

(2) Reporter Gene Assay 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. , A method for screening a compound that promotes or inhibits the transcriptional regulatory activity, which comprises measuring and comparing reporter activities of the test compound in the presence and absence of a test compound, and the method. Also provided is a screening kit. Examples of the transcriptional regulatory region of the gene of the receptor protein of the present invention include DNA containing the same or substantially the same base sequence as the base sequence represented by SEQ ID NO: 20. D containing a base sequence substantially identical to the base sequence represented by SEQ ID NO: 20
As the NA, any DNA can be used as long as it has a nucleotide sequence that hybridizes under high stringent conditions and has a transcriptional regulatory activity (eg, promoter activity) of substantially the same quality as SEQ ID NO: 20. Good. Sequence number:
The DNA that can hybridize with the base sequence represented by 20 under high stringent conditions is, for example, about 50% or more, preferably about 60% or more, more preferably the base sequence represented by SEQ ID NO: 20. About 70% or more,
More preferably about 80% or more, and particularly preferably about 90%.
As described above, most preferably, a DNA containing a nucleotide sequence having a homology of about 95% or more is used. Hybridization can be carried out by a method known per se or a method analogous thereto, for example, molecular cloning (Molecula
r Cloning) 2nd (J. Sambrook et al., Cold Spring Ha
rbor Lab. Press, 1989). When a commercially available library is used, it can be performed according to the method described in the attached instruction manual. More preferably, it can be carried out under high stringent conditions. High stringent conditions include, for example, sodium concentration of about 19 to 4
0 mM, preferably about 19-20 mM, at a temperature of about 50
The conditions are: ~ 70 ° C, preferably about 60-65 ° C. Particularly, the case where the sodium concentration is about 19 mM and the temperature is about 65 ° C. is most preferable. The transcriptional regulatory region (eg, DNA containing a base sequence substantially the same as the base sequence represented by SEQ ID NO: 20) is substantially the same as the base sequence represented by SEQ ID NO: 20. 5'to the base sequence
Sequences to which an upstream genomic DNA sequence is added are also included (preferably promoter region). The number of bases in the base sequence 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 is also included. Examples of the reporter gene include lacZ (β-galactosidase gene), chloramphenicol acetyltransferase (CAT), luciferase, growth factor,
β-glucuronidase, alkaline phosphatase, Gree
n fluorescent protein (GFP), β-lactamase and the like are used. Reporter gene product (eg mRNA,
Protein) is measured by a known method to control (particularly promote) the transcriptional regulatory activity (preferably promoter activity) of the receptor protein of the present invention with a test compound that increases the amount of the reporter gene product. It can be selected as a compound having an action, that is, a compound having an activity of promoting the expression of the receptor protein of the present invention. Conversely, a test compound that reduces the amount of the reporter gene product has a function of controlling (particularly inhibiting) the transcriptional regulatory activity (preferably promoter activity) of the receptor protein of the present invention, that is, the receptor protein of the present invention. It can be selected as a compound having an activity of inhibiting expression. The test compound is, for example, a compound selected from peptides, proteins, non-peptide compounds, synthetic compounds, fermentation products, cell extracts, plant extracts, animal tissue extracts, plasma and the like. As the salt of the compound, the same salts as the salts of the receptor protein of the present invention can be used. Cultivation of the transformant can be carried out in the same manner as the transformant containing the receptor protein of the present invention. The vector construction of the reporter gene and the assay method can follow known techniques (eg, Molecular Biotechnology 13, 29-43, 199).
9).

The compound having the 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 pharmaceutical. The compound having the activity of inhibiting the expression of the peptide of the present invention or a salt thereof is a safe and low-toxic pharmaceutical agent for suppressing the physiological activity of the receptor protein of the present invention,
For example, it is useful as a preventive and / or therapeutic agent for cancer (for example, 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 salts of the receptor protein of the present invention described above are used. When the above compound is used as a medicine, it can be formulated according to a conventional method. For example, the compound is orally as a sugar-coated tablet, capsule, elixir, microcapsule, or the like, or aseptic solution with water or other pharmaceutically acceptable liquid, Alternatively, it can be used parenterally in the form of injection such as suspension. For example, admixing the compound with a known physiologically acceptable carrier, flavoring agent, excipient, vehicle, preservative, stabilizer, binder and the like in a unit dose form generally required for the implementation of the formulation. Can be manufactured by. The amount of active ingredient in these preparations is such that a suitable dose within the indicated range can be obtained.

The preventive and / or therapeutic agent is
For example, buffer (eg, phosphate buffer, sodium acetate buffer), soothing agent (eg, benzalkonium chloride, procaine hydrochloride, etc.), stabilizer (eg, human serum albumin, polyethylene glycol, etc.), preservation You may mix | blend with agents (for example, benzyl alcohol, phenol, etc.), antioxidants, etc. The prepared injection solution is usually filled in a suitable ampoule. Since the preparation thus obtained is safe and has low toxicity, it should be administered to, for example, humans or mammals (eg, rat, mouse, rabbit, sheep, pig, cow, cat, dog, monkey). You can The dose of the compound or a salt thereof varies depending on the administration subject, target organ, symptom, administration method, and the like, but in the case of oral administration, generally, for example, cancer patients (60
in kg), about 0.1 to 100 m per day
g, preferably about 1.0 to 50 mg, more preferably about 1.0
~ 20 mg. Part 1 when administered parenterally
Although the dose may vary depending on the administration subject, target organ, condition, administration method, etc., for example, in the form of injection, for example, in patients with cancer (as 60 kg), about 0.01 to 30 mg per day, It is convenient to administer about 0.1 to 20 mg, preferably about 0.1 to 10 mg by intravenous injection. 60 for other animals
An amount converted per kg can be administered.

In the present specification, when an abbreviation is used for a base or amino acid, the abbreviation is IUPAC-IUB Commissi
Based on abbreviations based on on Biochemical Nomenclature or abbreviations commonly used in the field. An example is shown below. When amino acids may have optical isomers, the L form is shown unless otherwise specified. Gly: Glycine Ala: Alanine Val: Valine Leu: Leucine Ile: Isoleucine Ser: Serine Thr: Threonine Cys: Cysteine Met: Methionine Glu: Glutamic acid Asp: Aspartic acid Lys: Lysine Arg: Arginine His: Histidine The: Phenyl Tyr: Tyr: Tyr. : Tryptophan Pro: Proline Asn: Asparagine Gln: Glutamine pGlu: Pyroglutamic acid Me: Methyl group Et: Ethyl group Bu: Butyl group Ph: Phenyl group TC: Thiazolidine-4 (R) -carboxamide group

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

The sequence numbers in the sequence listing in this specification show the following sequences. [SEQ ID NO: 1] This shows a part of the nucleotide sequence (5 'side) of the cDNA obtained in Reference Example 1 below. [SEQ ID NO: 2] This shows the base sequence of primer 1 used in the PCR reaction of Reference Example 1 below. [SEQ ID NO: 3] This shows the base sequence of primer 2 used in the PCR reaction of Reference Example 1 below. [SEQ ID NO: 4] This shows a part (central portion) of the base sequence of the cDNA obtained in Reference Example 2 below. [SEQ ID NO: 5] This shows the base sequence of primer 3 used in the PCR reaction of Reference Example 2 below. [SEQ ID NO: 6] This shows the base sequence of primer 4 used in the PCR reaction of Reference Example 2 below. [SEQ ID NO: 7] This shows a part of the base sequence (3 ′ side) of the cDNA obtained in Reference Example 3 below. [SEQ ID NO: 8] This shows the base sequence of primer 5 used in the PCR reaction of Reference Example 3 below. [SEQ ID NO: 9] This shows the base sequence of primer 6 used in the PCR reaction of Reference Example 3 below. [SEQ ID NO: 10] This shows the base sequence of primer 7 used in the PCR reaction of Reference Example 3 below. [SEQ ID NO: 11] This shows the amino acid sequence of the human brain-derived novel receptor protein NEPHA obtained in Reference Example 5. [SEQ ID NO: 12] Novel receptor protein NEP derived from human brain
The nucleotide sequence of cDNA encoding HA is shown. [SEQ ID NO: 13] This shows the base sequence of the antisense oligonucleotide of the present invention. [SEQ ID NO: 14] This shows the base sequence of the antisense oligonucleotide of the present invention. [SEQ ID NO: 15] This shows the base sequence of the oligonucleotide used in EXAMPLE 1 below. [SEQ ID NO: 16] This shows the base sequence of the oligonucleotide used in EXAMPLE 1 below. [SEQ ID NO: 17] This shows the base sequence of the primer used in the PCR reaction of Example 1 below. [SEQ ID NO: 18] This shows the base sequence of the primer used in the PCR reaction of Example 1 below. [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] This shows the base sequence of transcription regulatory region of NEPHA.

Escherichia coli obtained in Reference Example 5 (Esch.
erichia coli) TOP10 / pTB2230 is 200
From May 24, 1st, 1-1, Higashi, Tsukuba, Ibaraki, Japan
1 Chuo No. 6 (Postal Code 305-8566), National Institute of Advanced Industrial Science and Technology, Japan Patent Organism Depositary, deposit number FERM BP-7606, April 2, 2001
From the 4th, under the consignment number IFO 16623, 2-17-85, Jusohonmachi, Yodogawa-ku, Osaka-shi, Osaka (postal code 532-8)
686) has been deposited with the Institute for Fermentation (IFO).

[0057]

BEST MODE FOR CARRYING OUT THE INVENTION Hereinafter, the present invention will be described in more detail with reference to Reference Examples and Examples, but the present invention is not limited thereto.

EXAMPLES Reference Example 1 Determination of 5'partial base sequence of cDNA encoding human brain-derived novel receptor protein Human brain cDNA (CL included in MTC Panel I)
A PCR reaction was carried out using 2 primers, Primer 1 (SEQ ID NO: 2) and Primer 2 (SEQ ID NO: 3), using ONTECH as a template. The composition of the reaction solution in the reaction was such that 0.5 μl of the above-mentioned cDNA was used as a template and Pfu Turbo DNA Polymer was used.
se (STRATAGENE) 1U, primer 1
(SEQ ID NO: 2) and primer 2 (SEQ ID NO: 3) each 1 μM, dNTPs 200 μM, and 2XGC B
10 μl of user I (Takara Shuzo) was added to make a volume of 20 μl. PCR reaction at 96 ° C, 1 minute later, 96 ° C,
The cycle of 1 minute, 60 ° C., 1 minute, 72 ° C., 1 minute was repeated 40 times. Next, the P
After purifying the CR reaction product, it was purified using Zero Blun.
t TOPO PCR Cloning Kit (Invit
plasmid vector pCR according to the prescription of Rogen.
-BluntII-TOPO (Invitrogen
Company). E. coli TOP10
And selected on LB agar containing kanamycin. As a result of analyzing the sequence of each clone, a nucleotide sequence (SEQ ID NO: 1) of a cDNA encoding a part of the novel receptor protein was obtained.

Reference Example 2 Determination of Central Partial Base Sequence of cDNA Encoding Novel Receptor Protein Derived from Human Brain Human brain cDNA (CLCTECH MTC Pan)
elI) as a template, two primers, primer 3
PCR reaction was performed using (SEQ ID NO: 5) and primer 4 (SEQ ID NO: 6). The composition of the reaction solution in the reaction was such that 1 μl of the above cDNA was used as a template and Pfu was used.
Turbo DNA Polymerase (STRA
2.5U, primer 3 (SEQ ID NO:
5) and primer 4 (SEQ ID NO: 6) at 1 μM each, d
200 μM of NTPs, and 2XGC Buffer I
(Takara Shuzo) was added in an amount of 25 μl to give a liquid volume of 50 μl. P
CR reaction is 96 ° C, 1 minute, 96 ° C, 1 minute, 60
The cycle of 1 minute at 72 ° C and 2 minutes at 72 ° C was repeated 40 times. Next, after purifying the PCR reaction product by agarose gel electrophoresis, it was used for Zero Blunt TOP
O PCR Cloning Kit (Invitroge
n company) according to the prescription of plasmid vector pCR-Blue
It was subcloned into ntII-TOPO (Invitrogen). This was introduced into E. coli TOP10,
Selection was done on LB agar containing kanamycin. As a result of analyzing the sequences of the individual clones, 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 Derived from Human Brain Human brain cDNA (CLCTECH MTC Pan)
elI) as a template, two primers, primer 5
PCR reaction was performed using (SEQ ID NO: 8) and primer 6 (SEQ ID NO: 9). The composition of the reaction solution in the reaction was such that 1 μl of the above cDNA was used as a template and Pfu was used.
Turbo DNA Polymerase (STRA
2.5U of TAGENE, primer 5 (SEQ ID NO:
8) and primer 6 (SEQ ID NO: 9) at 1 μM each, d
200 μM of NTPs, and 2XGC Buffer I
(Takara Shuzo) was added in an amount of 25 μl to give a liquid volume of 50 μl. P
CR reaction is 96 ° C, 1 minute, 96 ° C, 1 minute, 60
The cycle of 1 minute at 72 ° C and 2 minutes at 72 ° C was repeated 40 times. Next, using 5 μl of the PCR reaction product as a template, Pfu Turbo DNA Polymers was used.
e (STRATAGENE) 2.5U, primer 5
(SEQ ID NO: 8) and primer 7 (SEQ ID NO: 10)
1 μM each, dNTPs 200 μM, and 2XGC
Buffer I (Takara Shuzo) was added in an amount of 25 μl to give a liquid volume of 50 μl. PCR reaction was performed at 96 ° C. for 1 minute and then 96
℃, 1 minute, 60 ℃, 1 minute, 72 ℃, 2 minutes cycle 4
Repeated 5 times. Next, after purifying the PCR reaction product by agarose gel electrophoresis, this was purified with Zero Bl.
unt TOPO PCR Cloning Kit (In
plasmid vector pCR-BluntII-TOPO (Invitrog) according to the prescription of Vitrogen.
en). E. coli TOP
Introduced into 10 and selected in LB agar containing kanamycin. As a result of analyzing the sequences of the individual clones, a nucleotide sequence (SEQ ID NO: 7) of a cDNA encoding a part of the novel receptor protein was obtained.

Reference Example 4 Full-length cDNA encoding human brain-derived novel receptor protein
Based on the base sequences (SEQ ID NO: 1 and SEQ ID NO: 4 and SEQ ID NO: 7) of the partial cDNAs encoding the novel receptor proteins obtained in Reference Example 1, Reference Example 2 and Reference Example 3 The nucleotide sequence of the full-length cDNA (SEQ ID NO: 12) was determined.
SEQ ID NO: 12 is the sequence from 71st to 789th of SEQ ID NO: 1 and 284th to 113th of SEQ ID NO: 4
Sequences up to the fourth and SEQ ID NO: 7 from 473 to 1
It corresponds to the 926th sequence. The amino acid sequence of the protein predicted from the nucleotide sequence of full-length cDNA (SEQ ID NO: 12) is shown in SEQ ID NO: 11. A protein containing the amino acid sequence represented by SEQ ID NO: 11 was prepared as NEPHA (no
vel EphA). NEPHA and human Ep
The hrin receptor EphA7 is 5 at the amino acid level.
It has a homology of 0.7%. The gene encoding the full length is obtained by cleaving the cDNA fragments obtained in Reference Example 1, Reference Example 2 and Reference Example 3 with an appropriate restriction enzyme and ligating with T4 ligase. Alternatively, c that encodes the full length based on the nucleotide sequences clarified in Reference Examples 1 and 3
A primer that amplifies DNA is designed and obtained by the PCR method.

Reference Example 5 Full-length cDNA encoding human brain-derived novel receptor protein
Preparation of a plasmid having the nucleotide sequence of SEQ ID NO: 4 The plasmids containing the nucleotide sequences (SEQ ID NO: 4 and SEQ ID NO: 7) of the cDNA obtained in Reference Examples 2 and 3 were cleaved with a restriction enzyme. The composition of the reaction solution is SEQ ID NO:
1 μg of a plasmid containing the nucleotide sequence of the cDNA represented by 4 or a plasmid containing the nucleotide sequence of the cDNA represented by SEQ ID NO: 7, XhoI (Takara Shuzo) 5U, and 1
Add 5 μl of 0xH Buffer (Takara Shuzo) and add 50 μl
Of the liquid. The restriction enzyme reaction was performed at 37 ° C. for 1 hour, and 5 U of BstEII (Takara Shuzo) was added to the reaction product, and the reaction was performed at 60 ° C. for 1 hour. Next, SEQ ID NO: 4
After purifying the reaction product corresponding to 941 bp from the plasmid containing the cDNA nucleotide sequence represented by and the plasmid corresponding to 5273 bp from the plasmid containing the cDNA nucleotide sequence represented by SEQ ID NO: 7, by agarose electrophoresis, DNA Ligation Kit Ver. They were combined according to the prescription of 2 (Takara Shuzo). E. coli TOP1
0 introduced and selected in LB agar containing kanamycin. As a result of analyzing the sequences of the individual clones, the base sequence of the cDNA represented by SEQ ID NO: 13 was obtained. Further, the plasmid containing the cDNA base sequence (SEQ ID NO: 1) obtained in Reference Example 1 and the plasmid containing the cDNA base sequence represented by SEQ ID NO: 13 were cleaved with a restriction enzyme. The composition of the reaction solution was 1 μg of a plasmid containing the cDNA nucleotide sequence represented by SEQ ID NO: 1 or a plasmid containing the cDNA nucleotide sequence represented by SEQ ID NO: 13, PvuI (Takara Shuzo) 10U, and 10xK.
Buffer (Takara Shuzo) was added to make a liquid volume of 50 μl.
The restriction enzyme reaction was performed at 37 ° C. for 1 hour. Next, from the plasmid containing the nucleotide sequence of cDNA represented by SEQ ID NO: 1, 743 bp and the cDNA represented by SEQ ID NO: 13
The reaction product corresponding to 5977 bp was purified from the plasmid containing the nucleotide sequence of A by agarose gel electrophoresis, and then the DNA ligation kit Ve
r. They were combined according to the prescription of 2 (Takara Shuzo). This was introduced into E. coli TOP10 and selected in LB agar medium containing kanamycin. As a result of analyzing the sequences of individual clones, full-length cDNA encoding a novel receptor protein derived from human brain
A plasmid having the nucleotide sequence of NA (SEQ ID NO: 12) was obtained. The plasmid having the SEQ ID NO: 12 obtained here was designated as pTB2230, and the obtained transformant was designated as Escherichia coli TOP10 / pTB223.
It was named 0.

Example 1 In order to analyze the influence of the NEPHA gene on apoptosis, an antisense oligonucleotide introduction experiment was conducted. First, antisense to the base sequence represented by SEQ ID NO: 12 (SEQ ID NO: 13 and SEQ ID NO: 1)
After designing 4), a phosphorothioate-modified oligonucleotide was synthesized, purified by HPLC and used in an introduction experiment. As the control oligonucleotide, the reverse sequence of the nucleotide sequence represented by SEQ ID NO: 13 (SEQ ID NO: 15) and the reverse sequence of the nucleotide sequence represented by SEQ ID NO: 14 (SEQ ID NO: 16) are similarly phosphorothioated,
It was used after being purified by HPLC. Breast cancer cell line ZR-75-1 (Cancer Res, 38 (10), 3352-3364 as test cells
(Page, 1978, purchased from ATCC), 2 × 10 ⁴ cells were seeded in a 24-well plate (Falcon) on the day before the introduction of the oligonucleotide. OligofectAMINE (manufactured by Invitrogen) was used for oligonucleotide introduction, and the protocol was followed. 20 hours after introduction RNeasy mini
RNA was extracted according to the protocol of the kit (Qiagen), and c was obtained using the TaqMan RT kit (Perkin-Elmer).
DNA was prepared. At the same time, the same reaction was carried out without adding reverse transcriptase to serve as a non-reverse transcription control. Two
Species primers (SEQ ID NO: 17 and SEQ ID NO: 1)
8), and NEP using the probe (SEQ ID NO: 19)
TaqMan β-ac for correction of HA gene expression level
β using tin Control Reagents (Perkin-Elmer)
Let's look at the expression level of actin.
PCR was performed according to the manual (manufactured by er). NEPH
The expression amount of the A gene was subtracted from the data obtained using cDNA from the data obtained using a non-reverse transcription control, and then corrected by the β-actin expression amount for comparison. On the other hand, by introducing antisense oligonucleotide, NEP
In order to see the effect on the apoptosis when the expression of the HA gene was suppressed, the cells were compared with the control oligonucleotide-introduced sample using Cell death detection ELISA (Roche) 3 days after the introduction of the antisense oligonucleotide. As a result, when antisense to the NEPHA gene (SEQ ID NO: 13) was used, the expression level of the NEPHA gene was 53% at 20 hours after the introduction of the antisense oligonucleotide, compared with the control oligonucleotide (SEQ ID NO: 15). And when antisense (SEQ ID NO: 14) was used,
The control oligonucleotide (SEQ ID NO: 1
Compared to 6), the expression level of NEPHA gene was reduced to 57%. On the third day after the introduction of the antisense oligonucleotide, apoptosis was 224% when the antisense oligonucleotide (SEQ ID NO: 13) was introduced, and the control oligonucleotide ( When SEQ ID NO: 16) was set to 100%, it increased to 185% when the antisense oligonucleotide (SEQ ID NO: 14) was introduced. From these, it was confirmed that cancer cells undergo apoptosis by suppressing the expression of NEPHA gene expressed in cancer cells.
It was revealed that the A gene is involved in cancer cell proliferation and apoptosis.

Example 2 Analysis of 5'upstream region of NEPHA Using the nucleotide sequence represented by SEQ ID NO: 1, BLAST search was performed on the human genomic sequence of NCBI. The nucleotide sequence of No. 176 is the genomic sequence of human chromosome 1 (Accession number is NT # 004568,
VERSION is NT # 004568.8 (GI: 18548377), DEFINITION is
Homo sapiens chromosome 1 working draft sequence s
egment). Therefore, from this genome sequence, NE
Obtain the genomic sequence (SEQ ID NO: 20) 5'upstream of the start codon of PHA (about 2 Kb), and use the transcription factor prediction software TR
The transcription factor binding region was predicted in ANFAC Matrix version 1.3. As a result, as shown in Tables 1 to 11, binding positions of various transcription factors are predicted, and this region is NEPHA.
It was suggested that this is a transcriptional regulatory region.

[Table 1]

[Table 2]

[Table 3]

[Table 4]

[Table 5]

[Table 6]

[Table 7]

[Table 8]

[Table 9]

[Table 10]

[Table 11]

[0064]

INDUSTRIAL APPLICABILITY The oligonucleotide of the present invention has low toxicity and is useful as an excellent prophylactic and / or therapeutic agent for cancer. Further, a compound obtained by screening using 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 receptor protein gene of the present invention, preferably the present invention The compound having the activity of promoting the expression of the receptor protein or the salt thereof, which has low toxicity, is also useful as an excellent prophylactic and / or therapeutic agent for cancer.

[0065]

[Sequence list] SEQUENCE LISTING <110> Takeda Chemical Industries, Ltd. <120> Preventing and treating agent for cancer <130> B02103 <160> 20 <210> 1 <211> 789 <212> DNA <213> Human <400> 1 ggcattgctc agcggtgcta ggctggcgcg gcttgagccg ccgccggact gacagctcgg 60 tctgcggacc atggagacct gcgccggtcc acacccgctg cgcctcttcc tctgccggat 120 gcagctctgt ctcgcgctgc ttttgggacc ctggcggcct gggaccgccg aggaagttat 180 cctcctggat tccaaagcct cccaggccga gctgggctgg actgcactgc caagtaatgg 240 gtgggaggag atcagcggcg tggatgaaca cgaccgtccc atccgcacgt accaagtgtg 300 caatgtgctg gagcccaacc aggacaactg gctgcagact ggctggataa gccgtggccg 360 cgggcagcgc atcttcgtgg aactgcagtt cacactccgt gactgcagca gcatccctgg 420 cgccgcgggt acctgcaagg agaccttcaa cgtctactac ctggaaactg aggccgacct 480 gggccgtggg cgtccccgcc taggcggcag ccggccccgc aaaatcgaca cgatcgcggc 540 ggacgagagc ttcacgcagg gcgacctggg tgagcgcaag atgaagctga acacagaggt 600 gcgcgagatc ggaccgctca gccggcgggg tttccacctg gcctttcagg acgtgggcgc 660 atgcgtggcg cttgtctcgg tgcgcgtcta ctacaagcag tgccgcgcca ccgtgcgggg 720 cctggccacg ttcccagcca ccgcagccga gagcgccttc tccacactgg tggaagtggc 780 cggaacgtg 789 <210> 2 <211> 24 <212> DNA <213> Artificial Sequence <223> Primer <400> 2 ggcattgctc agcggtgcta ggct 24 <210> 3 <211> 23 <212> DNA <213> Artificial Sequence <223> Primer <400> 3 cacgttccgg ccacttccac cag 23 <210> 4 <211> 1134 <212> DNA <213> Human <400> 4 gcagccggcc ccgcaaaatc gacacgatcg cggcggacga gagcttcacg cagggcgacc 60 tgggtgagcg caagatgaag ctgaacacag aggtgcgcga gatcggaccg ctcagccggc 120 ggggtttcca cctggccttt caggacgtgg gcgcatgcgt ggcgcttgtc tcggtgcgcg 180 tctactacaa gcagtgccgc gccaccgtgc ggggcctggc cacgttccca gccaccgcag 240 ccgagagcgc cttctccaca ctggtggaag tggccggaac gtgcgtggcg cactcggaag 300 gggagcctgg cagcccccca cgcatgcact gcggcgccga cggcgagtgg ctggtgcctg 360 tgggccgctg cagctgcagc gcgggattcc aggagcgtgg tgacttctgc gaagcctgtc 420 ccccagggtt ttacaaggtg tccccgcggc ggcccctctg ctcaccgtgc ccagagcaca 480 gccgggccct ggaaaacgcc tccaccttct gcgtgtgcca ggacagctat gcgcgctcac 540 ccaccgaccc gccctcggct tcctgcaccc ggccgccgtc ggcgccgcgg gacctgcagt 600 acagcctgag ccgctcgccg ctggtgctgc gactgcgctg gctgccgccg gccgactcgg 660 gaggccgctc ggacgtcacc tactcgctgc tgtgcctgcg ctgcggccgc gagggcccgg 720 cgggcgcctg cgagccgtgc gggccgcgcg tggccttcct accgcgccag gcagggctgc 780 gggagcgagc cgccacgctg ctgcacctgc ggcccggcgc gcgctacacc gtgcgcgtgg 840 ccgcgctcaa cggcgtctcg ggcccggcgg ccgccgcggg aaccacctac gcgcaggtca 900 ccgtctccac cgggcccggg gcgccctggg aggaggatga gatccgcagg gaccgagtgg 960 aaccccagag cgtgtccctg tcgtggcggg agcccatccc tgccggagcc cctggggcca 1020 atgacacgga gtacgagatc cgatactacg agaagggtca gagtgagcag acttactcca 1080 tggtgaagac aggggcgccc acagtcaccg tcaccaacct gaagccggct accc 1134 <210> 5 <211> 20 <212> DNA <213> Artificial Sequence <223> Primer <400> 5 gcagccggcc ccgcaaaatc 20 <210> 6 <211> 23 <212> DNA <213> Artificial Sequence <223> Primer <400> 6 gggtagccgg cttcaggttg gtg 23 <210> 7 <211> 2033 <212> DNA <213> Human <400> 7 ggccgctcgg acgtcaccta ctcgctgctg tgcctgcgct gcggccgcga gggcccggcg 60 ggcgcctgcg agccgtgcgg gccgcgcgtg gccttcctac cgcgccaggc agggctgcgg 120 gagcgagccg ccacgctgct gcacctgcgg cccggcgcgc gctacaccgt gcgcgtggcc 180 gcgctcaacg gcgtctcggg cccggcggcc gccgcgggaa ccacctacgc gcaggtcacc 240 gtctccaccg ggcccggggc gccctgggag gaggatgaga tccgcaggga ccgagtggaa 300 ccccagagcg tgtccctgtc gtggcgggag cccatccctg ccggagcccc tggggccaat 360 gacacggagt acgagatccg atactacgag aagggtcaga gtgagcagac ttactccatg 420 gtgaagacag gggcgcccac agtcaccgtc accaacctga agccggctac ccgctacgtc 480 tttcagatcc gggccgcttc cccggggcca tcctgggagg cccagagttt taaccccagc 540 attgaagtac agaccctggg ggaggctgcc tcagggtcca gggaccagag ccccgccatt 600 gtcgtcaccg tagtgaccat ctcggccctc ctcgtcctgg gctccgtgat gagtgtgctg 660 gccatttgga ggaggccctg cagctatggc aaaggaggag gggatgccca tgatgaagag 720 gagctgtatt tccacttcaa agtcccaaca cgtcgcacat tcctggaccc ccagagctgt 780 ggggacctgc tgcaggctgt gcatctgttc gccaaggaac tggatgcgaa aagcgtcacg 840 ctggagagga gccttggagg agggcggttt ggggagctgt gctgtggctg cttgcagctc 900 cccggtcgcc aggagctgct cgtagccgtg catatgctga gggacagcgc ctccgactca 960 cagaggctcg gcttcctggc cgaggccctc acgctgggcc agtttgacca tagccacatc 1020 gtgcggctgg agggcgttgt tacccgagga agcaccttga tgattgtcac cgagtacatg 1080 agccatgggg ccctggacgg cttcctcagg cggcacgagg ggcagctggt ggctgggcaa 1140 ctgatggggt tgctgcctgg gctggcatca gccatgaagt atctgtcaga gatgggctac 1200 gttcaccggg gcctggcagc tcgccatgtg ctggtcagca gcgaccttgt ctgcaagatc 1260 tctggcttcg ggcggggccc ccgggaccga tcagaggctg tctacaccac tatgagtggc 1320 cggagcccag cgctatgggc cgctcccgag acacttcagt ttggccactt cagctctgcc 1380 agtgacgtgt ggagcttcgg catcatcatg tgggaggtga tggcctttgg ggagcggcct 1440 tactgggaca tgtctggcca agacgtgatc aaggctgtgg aggatggctt ccggctgcca 1500 ccccccagga actgtcctaa ccttctgcac cgactaatgc tcgactgctg gcagaaggac 1560 ccaggtgagc ggcccaggtt ctcccagatc cacagcatcc tgagcaagat ggtgcaggac 1620 ccagagcccc ccaagtgtgc cctgactacc tgtcccaggc ctcccacccc actagcggac 1680 cgtgccttct ccaccttccc ctcctttggc tctgtgggcg cgtggctgga ggccctggac 1740 ctgtgccgct acaaggacag cttcgcggct gctggctatg ggagcctgga ggccgtggcc 1800 gagatgactg cccaggacct ggtgagccta ggcatctctt tggctgaaca tcgagaggcc 1860 ctcctcagcg ggatcagcgc cctgcaggca cgagtgctcc agctgcaggg ccagggggtg 1920 caggtgtgag tggaccccat tcttccaagg caggactccg gtgggggtcc agtcccccag 1980 ccctgcccaa ggaccgtggc aagctgcgct ccagcagtgt gggagggagc gct 2033 <210> 8 <211> 23 <212> DNA <213> Artificial Sequence <223> Primer <400> 8 ggccgctcgg acgtcaccta ctc 23 <210> 9 <211> 30 <212> DNA <213> Artificial Sequence <223> Primer <400> 9 tcgtgaaaat gggaggggca ggcagtgaaa 30 <210> 10 <211> 20 <212> DNA <213> Artificial Sequence <223> Primer <400> 10 agcgctccct cccacactgc 20 <210> 11 <211> 1008 <212> PRT <213> Human <400> 11 Met Glu Thr Cys Ala Gly Pro His Pro Leu Arg Leu Phe Leu Cys Arg                  5 10 15 Met Gln Leu Cys Leu Ala Leu Leu Leu Gly Pro Trp Arg Pro Gly Thr              20 25 30 Ala Glu Glu Val Ile Leu Leu Asp Ser Lys Ala Ser Gln Ala Glu Leu          35 40 45 Gly Trp Thr Ala Leu Pro Ser Asn Gly Trp Glu Glu Ile Ser Gly Val      50 55 60 Asp Glu His Asp Arg Pro Ile Arg Thr Tyr Gln Val Cys Asn Val Leu  65 70 75 80 Glu Pro Asn Gln Asp Asn Trp Leu Gln Thr Gly Trp Ile Ser Arg Gly                  85 90 95 Arg Gly Gln Arg Ile Phe Val Glu Leu Gln Phe Thr Leu Arg Asp Cys             100 105 110 Ser Ser Ile Pro Gly Ala Ala Gly Thr Cys Lys Glu Thr Phe Asn Val         115 120 125 Tyr Tyr Leu Glu Thr Glu Ala Asp Leu Gly Arg Gly Arg Pro Arg Leu     130 135 140 Gly Gly Ser Arg Pro Arg Lys Ile Asp Thr Ile Ala Ala Asp Glu Ser 145 150 155 160 Phe Thr Gln Gly Asp Leu Gly Glu Arg Lys Met Lys Leu Asn Thr Glu                 165 170 175 Val Arg Glu Ile Gly Pro Leu Ser Arg Arg Gly Phe His Leu Ala Phe             180 185 190 Gln Asp Val Gly Ala Cys Val Ala Leu Val Ser Val Arg Val Tyr Tyr         195 200 205 Lys Gln Cys Arg Ala Thr Val Arg Gly Leu Ala Thr Phe Pro Ala Thr     210 215 220 Ala Ala Glu Ser Ala Phe Ser Thr Leu Val Glu Val Ala Gly Thr Cys 225 230 235 240 Val Ala His Ser Glu Gly Glu Pro Gly Ser Pro Pro Arg Met His Cys                 245 250 255 Gly Ala Asp Gly Glu Trp Leu Val Pro Val Gly Arg Cys Ser Cys Ser             260 265 270 Ala Gly Phe Gln Glu Arg Gly Asp Phe Cys Glu Ala Cys Pro Pro Gly         275 280 285 Phe Tyr Lys Val Ser Pro Arg Arg Pro Leu Cys Ser Pro Cys Pro Glu     290 295 300 His Ser Arg Ala Leu Glu Asn Ala Ser Thr Phe Cys Val Cys Gln Asp 305 310 315 320 Ser Tyr Ala Arg Ser Pro Thr Asp Pro Pro Ser Ala Ser Cys Thr Arg                 325 330 335 Pro Pro Ser Ala Pro Arg Asp Leu Gln Tyr Ser Leu Ser Arg Ser Pro             340 345 350 Leu Val Leu Arg Leu Arg Trp Leu Pro Pro Ala Asp Ser Gly Gly Arg         355 360 365 Ser Asp Val Thr Tyr Ser Leu Leu Cys Leu Arg Cys Gly Arg Glu Gly     370 375 380 Pro Ala Gly Ala Cys Glu Pro Cys Gly Pro Arg Val Ala Phe Leu Pro 385 390 395 400 Arg Gln Ala Gly Leu Arg Glu Arg Ala Ala Thr Leu Leu His Leu Arg                 405 410 415 Pro Gly Ala Arg Tyr Thr Val Arg Val Ala Ala Leu Asn Gly Val Ser             420 425 430 Gly Pro Ala Ala Ala Ala Gly Thr Thr Tyr Ala Gln Val Thr Val Ser         435 440 445 Thr Gly Pro Gly Ala Pro Trp Glu Glu Asp Glu Ile Arg Arg Asp Arg     450 455 460 Val Glu Pro Gln Ser Val Ser Leu Ser Trp Arg Glu Pro Ile Pro Ala 465 470 475 480 Gly Ala Pro Gly Ala Asn Asp Thr Glu Tyr Glu Ile Arg Tyr Tyr Glu                 485 490 495 Lys Gly Gln Ser Glu Gln Thr Tyr Ser Met Val Lys Thr Gly Ala Pro             500 505 510 Thr Val Thr Val Thr Asn Leu Lys Pro Ala Thr Arg Tyr Val Phe Gln         515 520 525 Ile Arg Ala Ala Ser Pro Gly Pro Ser Trp Glu Ala Gln Ser Phe Asn     530 535 540 Pro Ser Ile Glu Val Gln Thr Leu Gly Glu Ala Ala Ser Gly Ser Arg 545 550 555 560 Asp Gln Ser Pro Ala Ile Val Val Thr Val Val Thr Ile Ser Ala Leu                 565 570 575 Leu Val Leu Gly Ser Val Met Ser Val Leu Ala Ile Trp Arg Arg Pro             580 585 590 Cys Ser Tyr Gly Lys Gly Gly Gly Asp Ala His Asp Glu Glu Glu Leu         595 600 605 Tyr Phe His Phe Lys Val Pro Thr Arg Arg Thr Phe Leu Asp Pro Gln     610 615 620 Ser Cys Gly Asp Leu Leu Gln Ala Val His Leu Phe Ala Lys Glu Leu 625 630 635 640 Asp Ala Lys Ser Val Thr Leu Glu Arg Ser Leu Gly Gly Gly Arg Phe                 645 650 655 Gly Glu Leu Cys Cys Gly Cys Leu Gln Leu Pro Gly Arg Gln Glu Leu             660 665 670 Leu Val Ala Val His Met Leu Arg Asp Ser Ala Ser Asp Ser Gln Arg         675 680 685 Leu Gly Phe Leu Ala Glu Ala Leu Thr Leu Gly Gln Phe Asp His Ser     690 695 700 His Ile Val Arg Leu Glu Gly Val Val Thr Arg Gly Ser Thr Leu Met 705 710 715 720 Ile Val Thr Glu Tyr Met Ser His Gly Ala Leu Asp Gly Phe Leu Arg                 725 730 735 Arg His Glu Gly Gln Leu Val Ala Gly Gln Leu Met Gly Leu Leu Pro             740 745 750 Gly Leu Ala Ser Ala Met Lys Tyr Leu Ser Glu Met Gly Tyr Val His         755 760 765 Arg Gly Leu Ala Ala Arg His Val Leu Val Ser Ser Asp Leu Val Cys     770 775 780 Lys Ile Ser Gly Phe Gly Arg Gly Pro Arg Asp Arg Ser Glu Ala Val 785 790 795 800 Tyr Thr Thr Met Ser Gly Arg Ser Pro Ala Leu Trp Ala Ala Pro Glu                 805 810 815 Thr Leu Gln Phe Gly His Phe Ser Ser Ala Ser Asp Val Trp Ser Phe             820 825 830 Gly Ile Ile Met Trp Glu Val Met Ala Phe Gly Glu Arg Pro Tyr Trp         835 840 845 Asp Met Ser Gly Gln Asp Val Ile Lys Ala Val Glu Asp Gly Phe Arg     850 855 860 Leu Pro Pro Pro Arg Asn Cys Pro Asn Leu Leu His Arg Leu Met Leu 865 870 875 880 Asp Cys Trp Gln Lys Asp Pro Gly Glu Arg Pro Arg Phe Ser Gln Ile                 885 890 895 His Ser Ile Leu Ser Lys Met Val Gln Asp Pro Glu Pro Pro Lys Cys             900 905 910 Ala Leu Thr Thr Cys Pro Arg Pro Pro Thr Pro Leu Ala Asp Arg Ala         915 920 925 Phe Ser Thr Phe Pro Ser Phe Gly Ser Val Gly Ala Trp Leu Glu Ala     930 935 940 Leu Asp Leu Cys Arg Tyr Lys Asp Ser Phe Ala Ala Ala Gly Tyr Gly 945 950 955 960 Ser Leu Glu Ala Val Ala Glu Met Thr Ala Gln Asp Leu Val Ser Leu                 965 970 975 Gly Ile Ser Leu Ala Glu His Arg Glu Ala Leu Leu Ser Gly Ile Ser             980 985 990 Ala Leu Gln Ala Arg Val Leu Gln Leu Gln Gly Gln Gly Val Gln Val         995 1000 1005 <210> 12 <211> 3024 <212> DNA <213> Human <400> 12 atggagacct gcgccggtcc acacccgctg cgcctcttcc tctgccggat gcagctctgt 60 ctcgcgctgc ttttgggacc ctggcggcct gggaccgccg aggaagttat cctcctggat 120 tccaaagcct cccaggccga gctgggctgg actgcactgc caagtaatgg gtgggaggag 180 atcagcggcg tggatgaaca cgaccgtccc atccgcacgt accaagtgtg caatgtgctg 240 gagcccaacc aggacaactg gctgcagact ggctggataa gccgtggccg cgggcagcgc 300 atcttcgtgg aactgcagtt cacactccgt gactgcagca gcatccctgg cgccgcgggt 360 acctgcaagg agaccttcaa cgtctactac ctggaaactg aggccgacct gggccgtggg 420 cgtccccgcc taggcggcag ccggccccgc aaaatcgaca cgatcgcggc ggacgagagc 480 ttcacgcagg gcgacctggg tgagcgcaag atgaagctga acacagaggt gcgcgagatc 540 ggaccgctca gccggcgggg tttccacctg gcctttcagg acgtgggcgc atgcgtggcg 600 cttgtctcgg tgcgcgtcta ctacaagcag tgccgcgcca ccgtgcgggg cctggccacg 660 ttcccagcca ccgcagccga gagcgccttc tccacactgg tggaagtggc cggaacgtgc 720 gtggcgcact cggaagggga gcctggcagc cccccacgca tgcactgcgg cgccgacggc 780 gagtggctgg tgcctgtggg ccgctgcagc tgcagcgcgg gattccagga gcgtggtgac 840 ttctgcgaag cctgtccccc agggttttac aaggtgtccc cgcggcggcc cctctgctca 900 ccgtgcccag agcacagccg ggccctggaa aacgcctcca ccttctgcgt gtgccaggac 960 agctatgcgc gctcacccac cgacccgccc tcggcttcct gcacccggcc gccgtcggcg 1020 ccgcgggacc tgcagtacag cctgagccgc tcgccgctgg tgctgcgact gcgctggctg 1080 ccgccggccg actcgggagg ccgctcggac gtcacctact cgctgctgtg cctgcgctgc 1140 ggccgcgagg gcccggcggg cgcctgcgag ccgtgcgggc cgcgcgtggc cttcctaccg 1200 cgccaggcag ggctgcggga gcgagccgcc acgctgctgc acctgcggcc cggcgcgcgc 1260 tacaccgtgc gcgtggccgc gctcaacggc gtctcgggcc cggcggccgc cgcgggaacc 1320 acctacgcgc aggtcaccgt ctccaccggg cccggggcgc cctgggagga ggatgagatc 1380 cgcagggacc gagtggaacc ccagagcgtg tccctgtcgt ggcgggagcc catccctgcc 1440 ggagcccctg gggccaatga cacggagtac gagatccgat actacgagaa gggtcagagt 1500 gagcagactt actccatggt gaagacaggg gcgcccacag tcaccgtcac caacctgaag 1560 ccggctaccc gctacgtctt tcagatccgg gccgcttccc cggggccatc ctgggaggcc 1620 cagagtttta accccagcat tgaagtacag accctggggg aggctgcctc agggtccagg 1680 gaccagagcc ccgccattgt cgtcaccgta gtgaccatct cggccctcct cgtcctgggc 1740 tccgtgatga gtgtgctggc catttggagg aggccctgca gctatggcaa aggaggaggg 1800 gatgcccatg atgaagagga gctgtatttc cacttcaaag tcccaacacg tcgcacattc 1860 ctggaccccc agagctgtgg ggacctgctg caggctgtgc atctgttcgc caaggaactg 1920 gatgcgaaaa gcgtcacgct ggagaggagc cttggaggag ggcggtttgg ggagctgtgc 1980 tgtggctgct tgcagctccc cggtcgccag gagctgctcg tagccgtgca tatgctgagg 2040 gacagcgcct ccgactcaca gaggctcggc ttcctggccg aggccctcac gctgggccag 2100 tttgaccata gccacatcgt gcggctggag ggcgttgtta cccgaggaag caccttgatg 2160 attgtcaccg agtacatgag ccatggggcc ctggacggct tcctcaggcg gcacgagggg 2220 cagctggtgg ctgggcaact gatggggttg ctgcctgggc tggcatcagc catgaagtat 2280 ctgtcagaga tgggctacgt tcaccggggc ctggcagctc gccatgtgct ggtcagcagc 2340 gaccttgtct gcaagatctc tggcttcggg cggggccccc gggaccgatc agaggctgtc 2400 tacaccacta tgagtggccg gagcccagcg ctatgggccg ctcccgagac acttcagttt 2460 ggccacttca gctctgccag tgacgtgtgg agcttcggca tcatcatgtg ggaggtgatg 2520 gcctttgggg agcggcctta ctgggacatg tctggccaag acgtgatcaa ggctgtggag 2580 gatggcttcc ggctgccacc ccccaggaac tgtcctaacc ttctgcaccg actaatgctc 2640 gactgctggc agaaggaccc aggtgagcgg cccaggttct cccagatcca cagcatcctg 2700 agcaagatgg tgcaggaccc agagcccccc aagtgtgccc tgactacctg tcccaggcct 2760 cccaccccac tagcggaccg tgccttctcc accttcccct cctttggctc tgtgggcgcg 2820 tggctggagg ccctggacct gtgccgctac aaggacagct tcgcggctgc tggctatggg 2880 agcctggagg ccgtggccga gatgactgcc caggacctgg tgagcctagg catctctttg 2940 gctgaacatc gagaggccct cctcagcggg atcagcgccc tgcaggcacg agtgctccag 3000 ctgcagggcc agggggtgca ggtg 3024 <210> 13 <211> 20 <212> DNA <213> Artificial Sequence <223> <400> 13 cacctcccac atgatgatgc 20 <210> 14 <211> 20 <212> DNA <213> Artificial Sequence <223> <400> 14 catcctccac agccttgatc 20 <210> 15 <211> 20 <212> DNA <213> Artificial Sequence <223> <400> 15 cgtagtagta caccctccac 20 <210> 16 <211> 20 <212> DNA <213> Artificial Sequence <223> <400> 16 ctagttccga cacctcctac 20 <210> 17 <211> 16 <212> DNA <213> Artificial Sequence <223> Primer <400> 17 gccgtggccg agatga 16 <210> 18 <211> 21 <212> DNA <213> Artificial Sequence <223> Primer <400> 18 cctctcgatg ttcagccaaa g 21 <210> 19 <211> 24 <212> DNA <213> Artificial Sequence <223> Probe <400> 19 tgcccaggac ctggtgagcc tagg 24 <210> 20 <211> 2050 <212> DNA <213> Human <400> 20 ggttcagagg aagtctgtat tttgttttag caccgagggt cagaacctct gggctgaagt 60 ccctggagct tcagtaagaa gccatgattg gtgccttaga gcctgctaag gggatggaac 120 ccggttttaa agaggagtga ggggtggtgg agtgaggact cattttgaaa tgaaaggagt 180 cctgggtctc taagcagctt ctaaaaatac catctcccag aggtggaggc agggggaggg 240 gagaaatcga gcgtgaggag aatgaagatg gaaaggaggt ggcagtgtga gaacccttcc 300 tcaggtaggt ctggttttag aagtcccgcc tgcatccaag gactggaggg atgccacacc 360 tccttctgct tcctcagggg gcttgtctcc tgtggtgtcc aagccacagc ctctcctgat 420 gacctttctt tctgcaggta aagaagacca attcctccca cctactcctc ctattgctac 480 tgtccagcaa gtatgtctga gtgggcacgt gtgcatttac tcgcacatat acacaagcac 540 acacgcagaa atggcacatg aaagcgggtt gtaaatggcc agtgatctac gatgccagga 600 atcctcctta cactcggcgt tcaaagaggt atggactctg gcagagacag cgtggttcac 660 cagggcatcc gcagcacaga gcatggcgcc cagtgagggc tgggtagagg ggtgagggat 720 acatatatgc aaaagcacag acactcagac cgcaggggcc aagtagtcac aggactcaca 780 ctcactctcg aggtttctca ggctatattc gcttccaaat agagaggggg tgggtccccc 840 aagaaaagag ctcggaaaag ccacgaggtg ggtggagctt tctcccctca agttcagccc 900 agatggtggc gctcagtagc cgcctccctc gtcctggagg gccccaaccc ttcccaccac 960 caaagtgtta aaccctcagc ttgcacataa atgaccttca ctagacagag gtcttgttcc 1020 acgttttctt gcggcttctt cagcgccggc tgaactctgc caggattgct ccgcctctct 1080 cgcagatgtc tccgattcct ggcctctgtg ctgttcacca gtcccctggg gtgggcggag 1140 tggggtgatc aaagagggga acccccgact ctgagcagac tgagggcatg ggtggttgtg 1200 aggtgtgctg cttgtatctt gacagcagga gcaagaacct gaggaaccga tcgttcatgc 1260 aaaaccgcca ccgcccccat cccagcgtcc acatggtttc ccagcctcgg aagcgcgttg 1320 gtagggcagg accccgtccc tcactttgct ctcctttccc gagcagctcc cgggatcgtt 1380 cggcccctcc ttcctgccgc tgtgtgtgtg tgtgtgtgtg tgtgtgtgtg tgtgtgtgtg 1440 tgtgtgatga gggcgggaag agaaaggggg ggatgagcta ggagacaccg ggagcctcag 1500 acacaggaac agacctaggg cagggagaca ggagagctcc ccagagcttg gggtcaggac 1560 ataaagcttc aaaaccccgc aagtcctggg aacaagggag ctttcagact agctcagagg 1620 ctgcaagtgt tgctgcctcc ccgagggtcg cccctcctta agccaagccc accttgcctg 1680 cctacttctc aggaatgagg agctaaggtt tagcgctctg ggccaccgat gccagctcat 1740 tcccctctcg cagaagccct gccagctccc acagctgccc caccccccac ggtttggaga 1800 tctgcgggac cccttccttt ccccaccagt tgggtgttcc gcccctggcc tatgaaactc 1860 caccttccca ccctctgctg gcggggattg gttggcatcc cgcggcggtg cctagtgatt 1920 ggttcccatg gatgatggtg agcggtgaga ctccgccccc cgctggctct ggggtctggg 1980 ggcattgctc agcggtgcta ggctggcgcg gcttgagccg ccgccggact gacagctcgg 2040 tctgcggacc 2050

─────────────────────────────────────────────────── ─── Continued Front Page (51) Int.Cl. ⁷ Identification Code FI Theme Coat (Reference) A61P 43/00 105 C12N 1/02 4C086 C12N 1/02 1/15 1/15 1/19 1/19 1 / 21 1/21 C12Q 1/68 A 5/10 G01N 33/15 Z C12Q 1/68 33/50 Z G01N 33/15 C12N 15/00 ZNAA 33/50 5/00 A (72) Inventor Kai Eda Isao Ibaraki 3-12-1, Matsushiro, Tsukuba-shi, Japan Takeda Matsushiro Residence No. 412 F-term (reference) 2G045 AA40 BA13 BB03 BB20 CB01 CB21 DA12 DA13 FB02 FB04 4B024 AA01 AA11 CA02 FA02 HA14 HA17 4Q06BA4QA4 QQ43 BA02B0Q4BA0QB43BQ0 QR4 QR02 QR32 QR32 QR32 QR32 QR32 QR32 QR32 QR32 AA17 MA01 NA14 ZB212 ZB262 4C086 AA01 AA02 AA03 AA04 EA16 MA01 MA04 NA14 ZB21 ZB26

Claims (19)

[Claims] 1. An oligonucleotide containing a base sequence which is the same or substantially the same as the base sequence represented by SEQ ID NO: 13 or SEQ ID NO: 14. 2. The oligonucleotide according to claim 1, which contains the base sequence represented by SEQ ID NO: 13. 3. The oligonucleotide according to claim 1, which contains the 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 DNA. 6. A medicine comprising the oligonucleotide according to claim 1. 7. The medicine according to claim 5, which is a preventive and / or therapeutic agent for 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 claim 8, which is a transcriptional regulatory region of a DNA containing the same or substantially the same base sequence as the base sequence represented by SEQ ID NO: 12. 10. The DNA according to claim 9, wherein the transcriptional regulatory region is a promoter region. 11. A recombinant vector containing the DNA according to claim 8. 12. A DNA containing the same or substantially the same base sequence as the base sequence represented by SEQ ID NO: 12.
Having a reporter gene downstream of the transcriptional regulatory region of Escherichia coli
The recombinant vector according to claim 11, which contains A. 13. A transformant containing the recombinant vector according to claim 11. 14. Use of the DNA according to claim 8 to promote or inhibit the transcriptional regulatory activity of a DNA containing the same or substantially the same base sequence as the base sequence represented by SEQ ID NO: 12. For screening a compound or a salt thereof. 15. The screening method according to claim 14, which uses the transformant according to claim 13. 16. A promoter comprising the DNA according to claim 8, which promotes the transcriptional regulatory activity of a DNA containing the same or substantially the same base sequence as the base sequence represented by SEQ ID NO: 12. A kit for screening a compound that inhibits or a salt thereof. 17. A DNA containing a 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 its salt that promotes or inhibits the transcription regulatory activity. 18. A medicine comprising the compound according to claim 17 or a salt thereof. 19. The medicine according to claim 18, which is a preventive and / or therapeutic agent for cancer.