WO2000060073A1 - Gene of irg27 polypeptide, antibody against the same and therapeutic utilization thereof - Google Patents

Abstract

Novel diagnostics for cancer, a diagnostic method, etc. with the use of a gene encoding a polypeptide called IRG27 the expression of which is induced by the inactivation of p53 and elevated in various human cancers such as esophageal cancer, stomach cancer, lung cancer, kidney cancer, thyroid cancer, parotid cancer, ureteral cancer, bladder cancer, uterus cancer, liver cancer, mammary cancer, ovarian cancer and tubal cancer, and an antibody against IRG27.

Description

 Specification

Genes and antibodies to IRG27 polypeptide and their use for diagnosis

 The present invention relates to the use of a gene that evolves when P53 force, which is a tumor suppressor, is inactivated, or an antibody against a polypeptide encoded by the gene, for cancer diagnosis. Specifically, the expression of P53 is induced by inactivation of P53, and esophageal esophagus cancer, stomach cancer, lung cancer, kidney cancer, thyroid cancer, parotid gland cancer, ureteral cancer, bladder cancer, uterine cancer, liver cancer, breast cancer A gene encoding a polypeptide called IRG27, which is expressed in various human cancers such as ovarian cancer and fallopian tube cancer, and a novel cancer using the antibody against IRG27. Diagnostic agents and diagnostic methods. Background art

 Heretofore, there has been a diagnostic method in which a protein which is produced during ontogeny but is significantly increased in the production of cancer cells, such as CE II and α-fetoprotein, is used as a diagnostic method. However, due to the large number of false-negative and false-positive powers, and the existence of cancer types that do not produce these antigenic proteins (for example, --fetoprotein, which is a protein restricted to esophageal cancer), It is less than 50% of the total cancer. Therefore, there is a need for a method of diagnosing cancer with a higher probability, and there is a need for the establishment of a new diagnostic method that can detect cancer even at a low stage of evil. Disclosure of the invention

The object of the present invention is to provide a diagnostic agent for cancer and a method for diagnosing cancer, using a gene whose expression is increased upon inactivation by force or an antibody against a polypeptide encoded by the gene. I do. That is, the expression power is induced by inactivation of the present invention 53, and Eating m, stomach cancer, lung cancer, 肾 cancer, ¾ »¾, parotid, ureteral cancer, bladder cancer, uterine cancer, liver cancer, breast cancer, sickle, fallopian tube, etc. I, RG

An object of the present invention is to provide a novel diagnostic agent for cancer, a method for diagnosing cancer, and the like using a gene encoding a polypeptide designated as No. 27 and an antibody against IRG27. The present inventors have considered using P53, which is a tumor suppressor gene product, for the development of a novel cancer diagnostic technique (tumor marker 1).

 P53 gene The gene with the highest mutation level in a variety of human cancers. In 1989, the wild-type p53 gene was cloned and its function was confirmed. As a result, wild-type p53 suppressed transformation (carcinogenesis), and the introduction of wild-type p53 resulted in tumorigenicity. In other words, it is defined as a gene that has been deleted or inactivated in cancer cells, and the normal gene has the function of returning cancer cells to normal cells. It was found to be one of the “genes” (protein nucleic acid enzyme, 35: 54-55, 1990).

 53 genes {^ 1 ^, abnormalities (mostly missense mutations and ^ body deletions) are found in about 50% of cancers of all tissues, including liver and bladder cancers, and abnormalities in the p53 gene Is widely known to be deeply involved in the development and progression of cancer (Murakami I, eta 1., Am. J. Respirr. Crit. Carre. Med., 154, 1117-1123, 1996, Kang MS, eta 1., Int. J. Cancer, 67, 898-902, 1996, Uchida T, eta 1., J. Urol., 153, 1097-1104, 1995). In addition, it has been shown that the p53 gene occurs more frequently during the carcinogenesis process before the cancer eradicates. (Michiko Takaki, et al., Applied Medicine, 10, 964-968, 1992, Miyak i M, eta 1., Cancer Res., 50, 7166-7173, 1990). Furthermore, it has been reported that abnormalities of the p53 gene become highly recognized as the cancer progresses (Ucida T, etal., J. Urol., 153, 1097-1). 104, 1 995).

The present inventors have found high MJ¾ gene abnormalities in such various cancers Focusing on P53, we examined factors that increase in expression due to p53 gene abnormality (p53 inactivation). That is, as a result of selecting genes that are specifically expressed in the fibril trait ^ ffl cells in which P53 was inactivated, we succeeded in obtaining a plurality of genes, and of these genes, IRG 27 (imm ortaliz at i It was found that the gene of the factor named on rel at edgene 27) was evolved along with inactivation of p53 and was elevated ± in each S cell. Furthermore, the present inventors obtained the finding that 17 of the 24 cancer patients (approximately 70%) showed that the IRG27 gene was elevated in the tumor part compared to the non-tumor part, and We found that it can be used for diagnosis of various cancers. Furthermore, the present inventors have prepared an antibody corresponding to the IRG27 polypeptide, and have shown that an antibody against the IRG27 polypeptide is also effective for cancer diagnosis.

As a result of the homology search, the RG27 gene had high homology (98.1%) with the ISG20 gene whose expression was induced by interferon. It has been reported that it co-localizes with n SG 1 ear body along with I SG2C PM L and SP 100, but its function is unknown (Gongo ra C, eta 1., J. Β iο 1. Chem , 272, 19457- 19463, 1997). ISG20 from ^ BD audi ^ ^ IRG27 derived from human peripheral blood because it is a protein with two amino acid residues deleted compared to IRG27 from human peripheral blood leukocytes The nucleotide sequences of the three clones encoding were cloned, and no amino acid sequence deletion was found in any of the clones. On the other hand, since mouse IRG27 had the same number of amino acid residues as human IRG27, IRG27 derived from human peripheral blood leukocytes had the original amino acid sequence, ISG20 obtained from Daudi cells was thought to have an amino acid sequence unique to Daudi cells. Furthermore, as a protein having the same amino acid sequence as IRG27 (base sequence is 8 different salts), HEM45 (Pent ecost BT, J. Steroid Biochem. l. Biol., 64, 25-33, 1998). As described above, it was a protein having an amino ^ S sequence similar to or identical to that of IRG27I known proteins, IS020 and HEM45. However, the relationship between the ISO 20 and HEM 45 was not known at all. That is, it has not been clarified that the expression is induced by the inactivation of P53 and that it is useful for diagnosis of various cancers.

 The present invention has been completed based on the above findings.

 That is, the present invention

 (1) Single- or double-stranded DNA consisting of at least 17 contiguous bases of the positive and reverse strands of the gene having the following (a) and (b), or the DNA of A diagnostic agent for cancer that uses a standard enzyme as an active ingredient.

 (a) Expression is induced by inactivation of tumor suppressor p53

 (b) Increased expression in cancer fibers compared to normal fibers

 (2) Single- or double-stranded DNA consisting of at least 17 contiguous bases of the normal and reverse strands of the gene having the following (a), (b) and (c), or DNA target ^ #: The active ingredient, cancer diagnostics,

 (a) Inducibility is induced by inactivation of tumor suppressor p53

 (b) Increased expression in cancer fibers compared to normal fibers

 (c) contains the nucleotide sequence of any one of SEQ ID NO: 14 to SEQ ID NO: 29

 (3) Single-stranded or double-stranded DN capable of specifically detecting mRNA for a gene consisting of the nucleotide sequence of any one of SEQ ID NOS: 3 to 5 or allele mutant gene thereof

A, or a diagnostic agent for cancer, comprising the target of these DNAs as the active ingredient.

 (4) SEQ ID NO: 3 to SEQ ID NO: 5, consisting of a sequence consisting of at least M 17 bases of the positive and reverse chains of the gene consisting of the base sequence described in the sequence or the allele mutant gene thereof A diagnostic agent for cancer, comprising strand or double-stranded DNA, or the labeling of these DNAs as an active ingredient.

(5) The diagnostic reagent for cancer according to any one of (1) to (4), wherein the probe is a probe for a hybridization reaction or a primer for a PCR reaction, (6) The diagnostic agent for cancer according to (5), wherein the length of the primer for the PCR reaction is difficult to be 17 to 50 bases.

 (7) The cancer according to (5) or (6), wherein the following A, B, or C primer set, or a set of said primers comprising a sequence of at least 17 bases or more of these primers is used as an active ingredient. Diagnostics,

 (A)

 5 'primer sequence TGAGGGCGCAGAGGCAGGCAGCAT (SEQ ID NO: 8)

 3 'primer sequence CCGAGCTGTGTCCAAGCAGGCTGT (SEQ ID NO: 9)

 (B)

 5 'primer sequence AAAGGCAAGCTGGTGGTGGGTCAT (SEQ ID NO: 10)

 3 'primer sequence CTGTCCCAAAAAGCCGAAAGCCTC (SEQ ID NO: 11)

 (C)

 5'-side primer, rooster TTCCGCCCCTGACTTCACTTGATAACAAAC (SEQ ID NO: 12) 3'-side primer, rooster CAGGCCGGATGAACTTGTCGT (SEQ ID NO: 13)

 (8) an antibody, an antibody fragment, or a derivative thereof that specifically recognizes a polypeptide encoded by a gene having the following (a) and (b):

 (a) Induced by inactivation of tumor suppressor p53

 (b) Increased expression in cancer fibers compared to normal fibers

 (9) SEQ ID NO: 14 to SEQ ID NO: 29, an antibody, an antibody fragment, or a derivative thereof that specifically recognizes a polypeptide encoded by a gene containing the nucleotide sequence described in any one of the above sequences;

 (10) An antibody, antibody fragment, or antibody thereof that specifically recognizes a polypeptide having the amino acid sequence of SEQ ID NO: 1 or (iffi, SEQ ID NO: 2, or an allelic variant thereof)

 (11) A diagnostic agent for cancer, comprising the antibody, antibody fragment, or derivative thereof according to any of (8) to (10) as an active ingredient.

(12) The diagnostic agent for cancer according to any one of (1) to (7) or (11) is used. A method of diagnosing cancer,

 (13) The method for diagnosing cancer according to the above (12), wherein the cell is used as a diagnostic object.

 (14) The method for diagnosing cancer according to (12), wherein blood or saliva-containing operative night or urine is used as a diagnosis target.

 (15) any one of the above (12) to (14) for diagnosing fixation;

 (16) The method for diagnosing cancer according to any one of (12) to (15) above, for diagnosing renal cancer or urinary cancer including erectal cancer.

 (17) a gene comprising the nucleotide sequence of any one of SEQ ID NOs: 14 to 29 and whose expression is induced by inactivation of tumor suppressor Ρ53, and

(18) a gene which comprises the nucleotide sequence of any one of SEQ ID NOs: 30 to 41 and is regulated by inactivation of the tumor suppressor factor 53. "DNA", which is an active ingredient of a diagnostic agent for cancer in the present invention, is expressed by (a) inactivation of tumor suppressor P53, and (b) cancer compared to normal thread Refers to a DNA having a sequence of at least 17 bases or more of a gene having the following:

 Here, "(a) a gene whose expression is induced by inactivation of the tumor suppressor p53" can be isolated, for example, by a method including the following steps A to E.

 A. A step of obtaining a transformed cell in which the function of normal P53 is inactivated by expressing a mutant or deleted p53 gene in iiij in a human normal cultured cell line.

 B. A. A. trait wheel Step of preparing total RNA.

 C. By performing differential display using mRNΑ derived from a trait or a cell as type II, a gene that is specifically expressed in the inactivation of p53 is detected by PCR, and the i »S identity of the gene is determined. Step of performing.

D. Perform RT-PCR on p53 inactivation-specific ^ I genes in each swelling ^ iffl cell A step of confirming the expression.

 E. Cloning the full-length cDNA (gene) from a human cDNA library.

The mutant or deleted p53 gene used in the above step A is a normal p53 gene (Mol.Cell.Biol., 7, p961 (1987), Japanese Cancer Research Resources Bank, Depo sit No.C0104). And the following mutations or deletions. That is, mutations from arginine at position 175 to histidine, mutations from arginine at position 248 to tryptophan, or mutations from arginine at position 273 to histidine, which are mutations found in high ^ TO in human cancers ( The mutant P53 gene can be obtained by applying Lasky T. et a 1., Environ. Health Perspect., 104, 1324-1331 (1996)) or the like at the gene level. In addition, the portion other than amino acid residues 302 to 393 (Mol. Cell. Biol., 12, p5 581-5592 (1992)), which is a region involved in the polymerization (tetramer formation) of p53, By deleting at the gene level, the above-mentioned deleted p53 gene can be obtained. Here, regarding the acquisition of the mutant p53 gene, the normal type p53 gene (Japanese Cancer Research Resources Bank, Deposit No. C0104) was used as type 錡, and the 5 'side was set so that the mutation would enter the target site. The primer and the 3 '* ¾ {"primer of the normal P53 gene, and the 3'-side primer and the 5'-end primer of the normal p53 gene, which were designed to introduce mutations into the target site, respectively PCR reaction, and then, to ligate the amplified fragments on the 5 'and 3' sides, mix both fragments, and perform PCR using primers on the 5 'and 3' sides of the normal P53 gene. Re-amplification to obtain the desired mutant p53 gene. Further, acquisition of a deletion type p53 gene, for example, a primer for amplifying the 302nd to 393rd positions of ρ53 may be prepared and amplified by a PCR reaction according to a conventional method. In addition, it is preferable to add an appropriate restriction enzyme cleavage site to the primer sequence in order to introduce the mutant and deleted P53 genes into the expression vector. In addition to the mutations introduced by PCR as described above, in vitro mutage ne sis (Met hods in Enzymology, 100, p448 (1983)) by the Kunke 1 method In addition, gene mutation can be introduced.

Variant or deletion type _P 5 3 gene mutant P 5 3 gene and the deletion type (obtained in order to obtain a transformant ¾ί ^ ¾Β vesicles were expressed transliteration p 5 in Step A 3 gene p CAG GS _{(Gene, 108, P 193-200 (} 1991)) was inserted into a mammalian expression base Kuta one such building a謹example onset expression vector, which was introduced into human normal cultured cells, and culture may be When PCAGGS is used as an expression vector, a blasticidin S resistance gene (bsr) may be introduced in advance in order to reduce the size of the transformed cells by adding a drug. The human normal cultured cells to be used may be any human normal cultured cells, but examples include human normal lung double blast cell line, HEL 299 (Dainippon Pharmaceutical). Phosphoric acid co-precipitation method, ribosome DNA molecules can be introduced using standard methods (ribosome method, lipofectin method, lipofectamine method, HV J-ribosome method), electrophoresis method, microinjection method, etc. Sffl vesicles (i, for example, can be selected by adding a drug such as 2 g Ζπι1 of blasticidin S).

By culturing the transformed MB vesicles prepared as described above, a transformed cell overexpressing the mutant or deleted P53 gene can be obtained. Although p53 is known to function by forming a tetramer, normal tetramerization is achieved by expressing mutant or deleted P53 in f! j in the transformed vesicle. Inhibition of body formation and consequent inactivation of normal p53 function. Inactivation of p53 function maintains cell division compared to control transformed cells into which only one vector has been introduced. Preparation of total RNA contained in step B It may be performed according to a conventional method. For example, there is a method of degrading cells by exposing cells in the presence of a surfactant such as SDS, NP-40, Triton-X100, or phenol. In addition, cells are disrupted by a physical method such as a homogenizer and the cells are crushed with guanidine thiosinate, and then the total RNA is denatured with a cesium chloride density gradient. After the acid test, the phenolic thigh (Oki Gua) Total RNA can be prepared by the diphenyl thiocyanate monophenol chromatography method).

In order to select genes for p53-inactivated bffl-vesicle-specific expression using the differential display method included in step C, first, RT kits such as RNAmap kit (GenHunter) were used. — After the synthesis of single-stranded DNA by PCR, the amplified fragment is labeled with [a- ³⁵ S] d ATP, and subjected to 6% denaturing polyacrylamide gel electrophoresis. After electrophoresis, the gel is evaporated to dryness, and P53-inactivated bffl cells (mutated or deleted P53) are compared to normal P53-transformed cells (cells into which only the vector has been introduced). After detecting DNA whose expression has been increased in the transfected cells, that is, DNA corresponding to the target p53 inactivation-specific gene, the DNA fragment is recovered from the gel. Then, after re-amplification using PCR again, the PCR fragments are collected, introduced into a PCR clone vector such as pT7Blue (R) T (Novagen), and the nucleotide sequence is determined according to a conventional method. I do. In this manner, a DNA fragment of a gene that is specifically expressed by inactivating P53 can be obtained.

Step D This is for confirming the expression of the DNA selected in Step C. In step I, an appropriate primer portion was set based on the salt of the clone obtained, and the total RNA of the normal (mutated vector), mutant and deleted p53 gene-transformed cells was determined. Perform a PCR reaction by a conventional method as type 铸. In order to examine gene expression by PCR, it is necessary to set conditions for quantitatively detecting fragments, such as controlling the number of amplifications. At this time, a method of labeling the DNA fragment in the presence of [a- ³² P] dCTP or labeling the DNA fragment with a primer labeled at the end of the primer to detect the DNA fragment amplified to It is simple and convenient. The amplification frequency set in this way is preferably 16 to 22 times $. By performing the above PCR, it can be confirmed that the DNA obtained in the step C is a DNA that is specifically expressed in the P53 inactivation.

After cloning of the full-length cDNA (gene) in step E, the expression in each human MffiM was examined. Using a DNA fragment as a probe, The full-length cDNA can be cloned. Preparation of a cDNA library (Use a commercially available cDNA synthesis kit to convert the mRNA from the origin into type I. A human tissue-derived cDNA library (use a commercially available cDNA library) Is also possible.

 The gene of the present invention together with the 赚 of the above (a), a force which also has the characteristic of “(b) an increase in expression s ′ in cancer fibers compared to normal fibers” m E. Using an appropriate part of the gene obtained by E as a probe, RNA prepared from tumors 5 and non-tumor of the same patient (for example, Hum an Tumor Panel B lots of In Vitrogen, etc.) Use), or RNA prepared from the same fiber of a patient and a healthy person, by performing a lino-san blot analysis using a conventional method.

 The DNA comprising at least a continuous sequence of at least 17 bases of the gene obtained as described above is the `` DNA '' of the present invention, which is an active ingredient of a diagnostic agent for cancer, and the mRNA of the fia fi gene is As long as its presence can be specifically detected, it may be single-stranded or double-stranded, and may be any length of DNA at any position in the gene. Further, the DNA may be labeled such that the single-stranded or double-stranded DNA can be diagnosed. Regarding what kind of DNA can specifically detect the presence of the corresponding mRNA of the pre-S gene, it can be easily performed by actually performing the PCR method and the hybridization method as described below. You can judge it.

 The limitation of "at least 17 bases" is stochastically based on the fact that at least 17 bases are sufficient to identify one gene. If such a DNA is short, it can be synthesized using a DNA synthesizer, if it is long, it can be prepared by PCR, or ii if there is an appropriate restriction enzyme site, it can be prepared using S. it can.

Specific examples of the DNA of the present invention include the genes having the above-mentioned difficulties (a) and (b) and containing the base sequence described in any of the following: Single-stranded or double-stranded DNA consisting of at least 17 nucleotides of reverse strand, or Is the value of these characteristics. Here, the nucleotide sequences described in SEQ ID NOs: 14 to 29 are nucleotide sequences corresponding to a part of the novel gene obtained by the above A to E. A gene containing the nucleotide sequence of any one of SEQ ID NOs: 14 to 29 can be cloned according to the method described in A to E in the above-mentioned H. More simply, SEQ ID NO: 14 to SEQ ID NO: 29 By using an appropriate part of the DNA as a hybridization probe based on the body of the salt, and screening a cDNA library derived from a cell that expresses the DNA well. I can get it. The specific method at that time can be performed by referring to a basic book such as {^ Molecular Cloning 2nd Edt., Cold Spring Harbor Laboratory Press (1989).

 Further, as a preferred level of the DNA of the present invention, a single strand or a single strand capable of specifically detecting the corresponding mRNA of the IRG27 gene consisting of SEQ ID NO: 3 to SEQ ID NO: 5, Examples include double-stranded DNA, or の ^^ 'of these DNAs. More specifically (a single-stranded or double-stranded DNA consisting of at least the 17-base sequence of the IRG27 gene, or a target I ^ thereof. Here, SEQ ID NOs: 3 and 4 The nucleotide sequence of the human IRG27 gene, which has the same nucleotide sequence except that both nucleotide sequences in the untranslated region are partially different from those of the human IRG27 gene. These are the nucleotide sequences of the mouse IRG27 gene These genes can be obtained by the methods described in the above-mentioned steps A to E. Also, an appropriate probe is prepared based on the sequence information of SEQ ID NOS: 3 to 5, for example, from leukocytes. Cloning can also be achieved by screening the cDNA library of IRG27. In addition, the IRG27 gene can be cloned from the known HEM45 gene (accession No. U88964; Pent ecost BT, J. Steroid). Bio chem. Mo l. Bio l., 64, 25-33, 1998) and the same sequence (SEQ ID NO: 3 at positions 522 and 711). I can create the gene for IRG27.

In addition, there is a possibility that allelic variants or genetic polymorphisms may exist in the IRG27 gene described in SEQ ID NO: 3 to SEQ ID NO: 5, and inheritance of the allelic variants or genetic polymorphisms is also possible. DNA consisting of at least a 17-strand assassin and its indicator can also be used as an active ingredient in cancer diagnostics.

 As described above, the DNA of the present invention is used as a probe for the reaction of PCR or a primer for the reaction of PCR for diagnosing cancer. Here, when the DNA of the present invention is used as a PCR primer, a preferred length is from 17 bases to 50 salts Sgj¾. A preferred length of ^ used as a probe for hybridization is a length S of 50 to 500 bases.

 Specific examples of PCR primers include a set of PCR primers derived from the IRG27 gene described in Α, Β or C below, or a set of primers consisting of a sequence of at least 17 bases or more of these primers. No.

 (A)

 5 'primer sequence TGAGGGCGCAGAGGCAGGCAGCAT (SEQ ID NO: 8)

 3 'primer sequence CCGAGCTGTGTCCAAGCAGGCTGT (SEQ ID NO: 9)

 (B)

 5 'primer sequence AAAGGCAAGCTGGTGGTGGGTCAT (SEQ ID NO: 10)

 3 'primer sequence CTGTCCCAAAAAGCCGAAAGCCTC (SEQ ID NO: 11)

 (C)

 5 'primer sequence TTCCGCCCCTGACTTCACTTGATAACAAAC (SEQ ID NO: 12) 3' primer sequence CAGGCCGGATGAACTTGTCGT (SEQ ID NO: 13)

 By dissolving the DNA of the present invention in an appropriate buffer that does not hinder the reaction, it can be used as an active ingredient of a diagnostic agent for cancer. For example, it is Rikikawa no to use in the form of a kit that also contains transcriptase, Taq polymerase, dNTP, reaction stop solution, etc.

 Methods for diagnosing cancer using the diagnostic agent include the following methods.

1) Using the single-stranded DNA of the present invention (paired one-strand and reverse-strand) as a PCR primer, total RNA obtained from M ^ ffl thread cells, blood, urine, etc. collected from the subject to be diagnosed (Poly (A) RNA type 錡) 2) Label the single-stranded or double-stranded DNA of the present invention, and use this as a probe to diagnose total RNA or poly (A) RNA obtained from ¾JM cells, blood, urine, etc. by Northern blot. How to do.

 The diagnostic method of the above 1) and 2) can be performed based on a basic book such as (^ Molecular Cloning 2nd Edt., Cold Spring Harbor Laboratory Press (1989)).

 Specific examples are shown below.

 As a specific method of diagnosis by PCR in 1), there is, for example, the method of i 挙 げ T. First, the primer DNA of the present invention is synthesized by a conventional method, and this is used as a diagnostic agent. Next, total RNA or poly (A) RNA is prepared from ¾JM cells, blood, urine, etc. by the method described above, and this is used as type 錡. Is prepared. The preparation of the single-stranded cDNA can be easily performed by using, for example, {^ Superscript pre-Sir 1 cation system (Life Technologies, Gaithersburg, MD). After that, the primer is added and the PCR reaction is carried out in the usual manner. The PCR reaction conditions include, for example, 30 cycles of 95 ° C for 1 minute, 60 ° C for 1 minute, and 72 ° C for 2 minutes, followed by heating at 72 ° C for 10 minutes. Is electrophoresed on a suitable agarose gel to detect the presence or absence of the expression of mRNA of IRG27.

 In addition, a measurement method based on the following principle, which is frequently used in clinical diagnosis, is based on the PCR method.

 First, RNA is extracted and purified from blood or the like in the same manner as described above, and amplification (PCR reaction) of the target sample using the biotinylated primer is performed in the same manner as described above. Thereafter, the re-amplification product is unified with an alkaline solution and hybridized with the solid phase on which the complementary DNA probe is immobilized. After that, the solid phase is washed and reacted with enzyme-labeled avidin. After washing the solid phase, the enzyme substrate is added to perform a color reaction, and the absorbance is measured to detect the presence or absence of IRG27 mRNA.

In addition, the ability to make a diagnosis is also possible by using the in situ PCR method (Fernandez et al., Mol. Carcinog, 20, 317-326, 1997). As a specific method of the diagnosis by Northern plot of 2), for example, the following method can be used. First, the single-stranded or double-stranded DNA of the present invention is labeled; Prepare and use this as a diagnostic reagent Double-stranded DNA, for example, ³² P-labeled PCR reaction product prepared by the method described in 1) above, using the nick translation method or the random prime labeling method. Next, total RNA or poly (A) RNA is prepared from the subject's fibrous cells, blood, urine, etc. by the same method as described above, and is subjected to formaldehyde gel electrophoresis and DNA digestion by a conventional method. By performing blotting on the membrane, hybridization of the membrane with the probe can detect the presence or absence of expression of IRG27 mRNA. When using double-stranded DNA as a probe, for example, 50% (v / v) formamide, 1M NaCl, 10% (w / v) dextran, 1% (wZv) SDS, 100 μg / m1 salmon After hybridizing at 42 ° C for 16-24 hours under sperm DNA conditions, wash twice in 2XSSC at room temperature for 10 minutes, and then wash twice in 60 ° C, 2XSSC, 1% SDS for 20 minutes. Such conditions are mentioned.

 Furthermore, diagnosis using a DNA chip is also possible. That is, first, the DNA of the present invention is synthesized on a DNA chip. Next, total RNA or (poly (A) RNA is prepared from fiber cells, blood, urine, etc. for fiber, and this is labeled with fluorescence or the like. It is possible to diagnose cancer by detecting the fluorescence of DNA, etc. Here, the DNA chip to which the DNA or polynucleotide of the present invention is bound can be obtained by ordering from Affymetrix in the United States. It is possible, and individual specific means of the above-mentioned diagnostic method can be performed with reference to Nature Genetics, 21, 1-60 (1999) and the like.

 In addition, the present invention is applied not only to detection and diagnosis using the primers and the probe, but also to tissue tissue hybridazion of tissue.

Hereafter, the solid DNA is derived from a gene induced by inactivation of p53.DNA derived from a gene regulated by inactivation of P53 is also a similar cancer. It can be used as a diagnostic agent for Here, the genes whose expression is controlled by the inactivation of p53 are exactly the same except that the gene whose expression is specifically controlled by the inactivation of p53 is detected in the differential display in the step C. By going through steps A to E, it is possible to “close”. The partial sequence of the novel gene thus found is shown in SEQ ID NO: 30 to SEQ ID NO: 1.

 In addition, SEQ ID NO: 14 to SEQ ID NO: 29 disclosed herein are a group of DNAs whose expression is induced by inactivation of p53, and SEQ ID NO: 30 to SEQ ID NO: 4. 1 is a group of DNAs whose expression is regulated by inactivation of p53. These are all novel DNAs, and a gene containing the nucleotide sequence is also included in the bacterium of the present invention.

 In the present invention, the antibody specifically recognizes the antibody and the polypeptide encoded by the above-described gene of the present invention (hereinafter, sometimes referred to as the polypeptide of the present invention). That is, (a) expression is induced by inactivation of the tumor suppressor p53, and (b) the expression is increased in cancer S compared to normal. An antibody that specifically recognizes a polypeptide. Specifically, an antibody capable of specifically recognizing a polypeptide encoded by a gene containing the nucleotide sequence described in any one of SEQ ID NO: 14 to NO: 29 is more preferable. SEQ ID NO: 1 Antibodies specifically recognizing human IRG27 comprising the amino acid sequence described, mouse IRG27 comprising the amino acid sequence represented by SEQ ID NO: 2, or an allelic variant thereof.

 These antibodies can be easily prepared by the method described in, for example, Antibodies; A Laboratory Manual, Lane, HD et al., Cold Spring Harbor Laboratory Press (1989), New Cell Engineering ^^ Protocol, Shujunsha (1993), etc. Produced. That is, the antibody of the present invention can be produced by appropriately immunizing an animal by a conventional method using the polypeptide of the present invention or a part thereof as an immunogen.

Here, as the immunogen, (A) the polypeptide of the present invention, (B) a fusion protein of the polypeptide with GST, etc., (C) a conjugate of an oligopeptide comprising a part of the polypeptide with KLH, etc. I can do it. The polypeptide of the present invention (A) is inserted into an appropriate expression vector, and the gene of the present invention is inserted into Escherichia coli or (Ono-cultured cell line). The polypeptide can be obtained by preparing and purifying the polypeptide in large quantities, and the fusion protein of the above (B), for example, the gene of the invention of the present invention can be added to a GST fusion protein expression vector such as pGEX-6P-1 (Pharmacia). This is introduced into Escherichia coli to obtain a transformant, followed by disruption of the cells by a conventional method, extraction of the fusion protein, and purification by daltathione Sepharose 4B (Pharmacia) or the like. In addition, the conjugate of the above (C) can be obtained by mixing with oligoprotein after synthesis of the oligopeptide: << Specific examples of the conjugate in RG27 IRG27-A consisting of the portion from position 104 to position 127 of the amino acid sequence described in SEQ ID NO: 1, I RG27-B consisting of the portion from position 131 to position 153, from the portion from position 159 to position 181 Each of the following I RG27-C is conjugated to KLH.

 The animal species to be immunized may be a rabbit, a mouse, a rat, a chicken, a rabbit, a donkey, a hidge, a rabbit, or a polyclonal antibody if the antibody specifically recognizes the polypeptide. Alternatively, any of monoclonal antibodies may be used. In order to obtain a purified antibody from the elimination, it is sufficient to carry out rear affinity purification by a conventional method. Specific examples of the Nada body include an antibody corresponding to I RG 27-C comprising the portion of the amino acid sequence of human I RG 27 shown in SEQ ID NO: 1 from position 159 to position 181.

Various antibody fragments can also be prepared based on the antibodies obtained as described above. The body fragments, such Yotsute pepsin digestion ^^ of辱Ru F (ab ') antibody ₂ fragments, F (ab') ₂ Fab 'fragments which can be ^^ by reducing the disulfide bonds of the fragment, and The use of papain and a reducing agent to transform antibodies into 2K Fab and iiFab fragments, which are humiliated, is also included in the scope of the present invention.

Further, various types of antibodies can be prepared based on these antibodies or antibody fragments. Here are the invitations {{for example, chimeric antibodies, humanized antibodies, l-body For example, it can be prepared according to the method described in JP-A-6500 / 47, Nature, 522 (1986). Also included in the category are those in which the antecedent or the dead fragment is labeled with an enzyme or the like. Specific enzyme labeling methods include, for example, the daltaraldehyde method, the perioso method, the maleimide method, and the pyridyl disulfide. Enzymes used for labeling include, for example, small intestine-alkaline phosphatase, horseradish peroxidase, etc. These labeled antibodies, for example (enzyme-linked immunosorbent assay, Medical Shoin ( Those skilled in the art will be able to easily prepare them according to basic textbooks such as 1978), etc. Further, those obtained by biotinylation of the above-mentioned am isomer or body fragment are also included in the category of the derivative.

 It can be used as a diagnostic agent for the above-described antibodies, antibody fragments, or cancer-inducing cancers of the present invention. In the case of IRG27, a typical protein of the present invention, the results of RNA detection by Northern blotting and the results of protein detection by Western blotting were in good agreement. Therefore, similarly to the above-mentioned DNA, the antibody, the antibody fragment, and the derivative thereof of the present invention can be used as a diagnostic agent for cancer. Specifically, the antibody or the like of the present invention can be used as an active ingredient of a cancer diagnostic agent by allowing the antibody or the like of the present invention to be present in an appropriate medium such as serum albumin-containing phosphorus solution (PH7.0) at night. . At that time, depending on the measurement method, it can be used in the form of a kit containing, for example, a brush-labeled secondary antibody, a color former, a color former, a stop solution, a standard product, and the like.

 Examples of a method for performing an immunological diagnosis using the diagnostic agent of the present invention include, for example, solid-state fiber. In addition to a method for detecting the presence of the polypeptide of the present invention in cells, a bodily fluid containing blood or saliva or In other words, a method for detecting the presence of the polypeptide of the present invention can be mentioned, that is, cytotoxic T cells (CTLs) are induced in the body against human cancer cells, and the CTL cells are damaged, resulting in cancer. Since the polypeptide of the present invention, which is specifically expressed in cells, is released into the blood or urine, the polypeptide can be detected in blood, saliva or urine.

画免疫測定法 (R I A) 、 麟免疫測定法 (E L I SA) などが 挙げられる。 特に、 血液や唾液を含む術夜や尿に含まれる該ポリペプチドを検出す る齢に 通常 EL I SAまたは RI A法力撮も簡便である。 これらの診断 いずれも酵素免疫測定法、 医学書院 （1978) 等の基本書に基づき容易に すること 力できる。 E L I S A法の場合、 1次抗体でプレートをコートし、 試料中に存在する I RG27を結合後 2嫌体と反応させ、 I RG 27に結合した抗体を検出すること 力簡便である。 また、 抗体カ¾¾識するェピトープに対するポリペプチドまたはオリゴ ペプチドを用いた競合阻害による測定も可能である。 Specific detection methods include fluorescent # ¾, Western 'plot, immunoprecipitation,

Immunoassay (RIA), Lin immunoassay (ELI SA), etc. No. In particular, normal ELISA or RIA imaging at the operative night including blood or saliva or at the age at which the polypeptide contained in urine is detected is also convenient. All of these diagnoses can be easily performed based on basic books such as enzyme immunoassays and the Medical School (1978). In the case of the ELISA method, it is simple and convenient to coat the plate with a primary antibody, bind the IRG27 present in the sample, and react with the two isotopes to detect the antibody bound to the IRG27. In addition, it is also possible to perform measurement by competitive inhibition using a polypeptide or an oligopeptide for an epitope detected by an antibody.

 Hereinafter, a specific example of the ELISA method using IRG27 will be described.

 First, as a primary antibody, anti-I RG27A IgG (an antibody described in the I RG27-A portion) or anti-I RG27B IgG (an antibody against the I RG27-B portion) is adsorbed on the plate, and then a cancer patient Of urine (collected supernatant after collection). At that time, the same awakening is performed for the positive and sexual controls. Subsequently, as a secondary antibody, biotin kMl RG27C IgG (an antibody that binds to the IRG27-C moiety) is reacted with peroxidase-labeled streptavidin, followed by a color reaction. The same detection can be performed by using the anti-IRG27C IgG directly labeled with enzyme such as peroxidase or alkaline phosphatase.

 It should be noted that it is possible to diagnose various solids by antibody diagnosis using fiber cells, and various antibodies including solid ^ S and leukemia by antibody diagnosis using body fluids such as blood and saliva or urine. It is possible to diagnose cancer. The diagnostic method of the present invention is particularly effectively used for the diagnosis of renal cancer, ureteral cancer, bladder cancer and urine. BRIEF DESCRIPTION OF THE FIGURES

Figure HEL 299 cells (VECTOR in the figure) transfected with the vector alone, and HEL 299 cells (175H, 175H in the figure) transfected with the three mutant p53 and deletion p53 plasmids prepared in Example 1. 248W, 273H and 302-393) are electrophoretic photographs showing the results of differential display. Arrow band Corresponds to fragment 27.

 FIG. 2 is an electrophoresis photograph showing the results of RT-PCR detecting the expression of the IRG27 gene in a vector-transfected cell and three types of mutant p53- and deletion-type p53 plasmid-transfected cells. The lower part of the figure shows the results of a similar experiment using G3PDH as a control. IRG27 was subjected to electrophoresis of 22 cycles of the PCR product and G3PDH was subjected to 20 cycles of the PCR product.

 FIG. 3 is an electrophoretic photograph showing the result of Northern blotting of the expression of the IRG27 gene in normal human fibers. The RNA plots for a, b, and c were obtained using a: Human, b: Human II, and c: Human Immune System of MuOnti eT issue Nort B rn B lot of C ontech, respectively. Used. The arrow indicates the position of the signal of IRG27.

 FIG. 4 is an electrophoresis photograph showing the results of Northern blot analysis of the expression of the IRG27 gene in each human cell line. Human Cancell e Cell Line of Mu 1 tip 1 e Tis ssue No r the rn B l o t of C 1 o n t e ch was used. In the figure, HL60 is a pro-IS leukemia cell line, He La is a cervical carcinoma cell line, K562 is a chronic medullary leukemia cell line, and MOLT 4 acute lymphoblastic leukemia cell line. Raj i (a one-kit lymphoma cell line, SW480; a cell line, an A 549 glutamic carcinoma cell line, and G 361 a malignant melanoma cell line. The arrow indicates the position of the signal of IRG27.

 FIG. 5 is an electrophoretic photograph showing the results of Northern blot analysis of the expression of the IRG27 gene in the tumor part (T in the figure) and non-tumor part (N in the figure) of the cancer patient. Threads are shown on each lane.

 FIG. 6 is an electrophoretic photograph showing the results of Northern blot analysis of the expression of the IRG27 gene in the tumor part (T in the figure) and the non-tumor part (N in the figure) of four cases of lung cancer patients.

FIG. 7 is an electrophoretic photograph showing the result of Western blot analysis using an anti-I RG27 antibody. Electrolyzed sample DLD-1 cell lysate of stable transformed cells in which an IRG27 expression vector was introduced into cells (IRG27 lane in the figure) This is a cell lysate of a stable transformed ^ ffl cell transfected with only a vector (vector lane in the figure). As anti-IRG27 antibodies, three kinds of anti-IRG27 antibodies (A-2, B-2, C-2) before affinity purification and after affinity purification were used.

 Fig. 8 is an electrophoresis photograph showing the results of Northern blotting and estamplotting of the expression of mRNA and protein of IRG27 in various human cell lines. Coomassie protein used for Western plotting The results of staining with Priluent Blue are also shown at the bottom of the figure, where HEL299 and * WI38 are normal lung 2-fold # »blast cells and HepG2 is a liver cancer cell line. 293 is a fetal kidney-derived plasma carcinoma cell line, He La is a cervical carcinoma cell line, Saos-2 is an osteosarcoma Mffl vesicle line, and U937 (! ^ Lymphoma). Cell line, RD myofioma cell line, T24 bladder cancer cell line, ScaBER is bladder cancer cell line, DLD-1 is a general cell line, SW480 Is a colorectal cancer cell line, Raj i (a kitlin follicular cell line, HE p-2 iin a head cancer cell line K U- 2 is a renal cancer cell lines.

 FIG. 9 is a graph showing detection of IRG27 by the ELISA method using an anti-IRG27 antibody. The abscissa indicates the values of the GST-fused IRG27 protein as an antigen and the control GST protein, and the ordinate indicates the absorbance. The measured values when the detection antibody of 1 mg / m1 was used only for the GST protein are shown. BEST MODE FOR CARRYING OUT THE INVENTION

 Hereinafter, the present invention will be described in further detail with reference to examples. The present invention is not limited to these examples of Δδδ.

麵 Example 1

Construction of mutant and deletion type Ρ53 expression plasmid vectors

In the amino acid sequence of P53 (Mol. Cell. Biol., 7, p961 (1987)), clone 175 was converted from arginine to histidine, and position 248 was changed from arginine to tributane. Mutated clone, No. 273: Arginine to Histige The clones which were mutated to the same were prepared. These mutations are all mutations frequently found in human cancers (Nuc. Acid. Res., 25, pl51-157 (1997)).

 Specifically, pro Sp 53 containing a 1.9 Kb normal p53 cDNA gene (provided by Dr. Kiyomitsu Nemoto, Shizuoka Prefectural University; generally, D-marked osit No. from Japanese Cancer Research Resources Bank) (Available as CO 104) or pBS-p53 in which the gene contained in the EcoRlZBamHI cut fragment in the plasmid was introduced into pBuescriptII-SK (+) (Stratagene). As a type, the 5 'side which was set so that the mutation was inserted at the 1st, 75th, 248th, or 273rd position in the amino acid sequence of p53 (175Hismut .: ATG ACG GAG GTT GTG AGG CAC TGC CCC CAT CAT G AG CGC TGC TCA GA, 248Trpmut .: TGC ATG GGC GGC ATG AAC TGG AGG CCT ATC CTC ACC ATC ATC ATC ACA CT, 273Hismut .: GGA CGG AAC AGC TTT GAG GTA CAT GTT TGT GCC TGT CCT GGG AG) 'Side-facing primer (175HisA: TCT GAG CAG CGC TCA TGA T GG GGG CAG TGC CTC ACA ACC TCC GTC AT, 248TrpA: AGT GTG ATG ATG GTG AGG ATA GGC CTC CAG TTC ATG CCG CCC ATG CA, 273HisA: CTC CCA GGA CAG GCA C ACA TG T ACC TCA AAG CTG TTC CGT CC) and 3 'end of open reading frame of normal p53 gene (p53tenn: GTT AAC TCG AGT CAG Using TCT GAG TCA GGC CCT TCT G TC TTG M) and 5'¾ (p53Xho: GTT AAC TCG AGC CAC CAT GGA GGA GCC GCA GTC AGA TCC TAG CGT CGA GC) primers, perform the PCR reaction as follows. I went.

To obtain the p53 gene fragment with mutation at the amino acid at position 175, use the p53Xhoo primer and 175HisA primer, the 175Hismut primer and p53term primer in the usual manner. PCR was performed to amplify the DNA fragments in the 5 'H region and the 3' region of p53. Next, DNA fragments of the desired length were purified by agarose electrophoresis. PCR was performed by a conventional method using the obtained two types of DNA fragments, the p53Xho primer and the p53term primer. The amplified DNA fragment was subjected to 1% agarose Geso HI electrophoresis, and a DNA fragment of a desired length was recovered, cut with XhoI, and introduced into pB1uescript II SK (+). 248th and 27th The p53 gene fragment having a mutation at the amino acid at position 3 was also introduced into pB1uescript II SK (+) using the corresponding primer in the same manner as described above. The normal P53 cDNA was also amplified using primers (p53te rm and p53Xho, respectively) with XhoI cleavage sequences added to the 3 'and 5' sides of the open reading frame, and And introduced it into pB 1 ue script II SK (+).

 As a result of analyzing the nucleotide sequence of each of the obtained clones, the DNA of the clone mutated at position 175 had a deletion of about 200 bp at the 5 'side, and the mutation potential at positions 248 and 273 was i. The DNA of the clone containing the DNA had a deletion of 29 bp each on the 5 'side. Therefore, the 5′-side region of the normal P53 gene was ligated using the NcoI cleavage site contained in the p53 gene to obtain each mutant P53 gene of interest.

 In addition to the mutant P53 gene as described above, a deleted p53 gene consisting only of the p53 domain (nuclear translocation, tetramer formation, non-specific single-stranded DNA binding domain) is also produced. did. That is, a primer (pMINI: GTT AAC TCG AGC CAC CAT GGG GAG CAC TAA GCG AGC ACT GCC CM C CA) in the region containing ATG is provided before the amino acid sequence of p53 at position 302. PCR was performed using the 3 ′ primer (p53terra) of the open reading frame of the normal p53 gene. The fragment obtained in the same manner as described above was incorporated into pCAGGS (Gene, 108, pl93-200 (1991)) to obtain 13 target clones. As a result of determining the base sequence of one of them, it was confirmed that it was the desired target.

The pB1uescript II recombinant vector containing the mutant P53 DNA and the pCAGGS thread male vector containing the deleted P53 DNA obtained as described above are cut at X1101 to cut the insert DNA. separated. Insert these insert DNAs into Pvu II—EcoR I DNA fragment containing the blasticidin S resistance gene (bsr) expression unit of p SV2 — bsr (Health Pharmaceutical Co., Ltd.) into S a 1 I in a pCAGGS vector Each was introduced into the XhoI site of the inserted pCAGGS-bsr. In the clones obtained, 175 amino acid residues were mutated from arginine to histidine. PCAGGS— bsr— 175H, clone 248 amino acid residues arginine mutated to tryptophan pCAGGS— bsr—248W, 273 amino acid residue cloned arginine to histidine P CAGGS— bsr—273H The deletion p53 clone consisting of 302 to 393 amino acid residues was named pCAGG S-bsr-p53-302-393. Subsequently, Escherichia coli (JM109, Toyobo) was transformed with these plasmids. Example 2

Preparation of transformed ^ ffl vesicle and RNA preparation

Three mutant p53 plasmids (pCAGGS-bsr-175H, pCAGGS-bsr-248W, pCAGGS-bsr-273H) and deletion p53 plasmid (pCAGGS-bsr-p53-302-393), respectively 2.2 × 10 ⁶ human normal lung 2ί ^ «| blast cells using an electroporator S SH-10 (manufactured by Shima ^^ Instruments) suspended in 0.4 ml of PBS (-), 200 g each (HEL 299 cells, firewood made by Dai Nippon) by electroporation. The application conditions were 300 V, 0.75 kV / cm, a pulse width of 1000 μs, the number of pulses was 6 times, and a culture solution containing 2 μg / m 1 blasticidin S 24 hours later Was selected. After 5 days from the start of selection, surviving transformed cells were collected. As a control, similar cells into which vector-1 (pCAG GS-b sr) was introduced were also prepared.

 In cells into which the p53 gene (pCAGGS-bsr-175H) with a mutation at the 175 amino acid residue and the deleted p53 gene (pCAGGS-bsrp53-302-393) have been introduced, the vector (pCAGGS — Compared to control cells transfected with bsr), the cells were able to maintain cell division more strongly. On the other hand, cells transfected with the P53 gene (pCAGGS-bsr-248W, pCAGGS-bsr-273H) with mutations at 248 and 273 amino acid residues are more likely to maintain cells than control cells. there were.

In addition, 53 cells have been introduced into these mutant and deleted P53 genes. Western blot analysis using an antibody confirmed that the protein strength {i}, which is considered to have been produced by the introduced gene, was obtained.

 From the above results, normal p53 function was inactivated in HEL 299 cells by i¾ expression of the P53 gene into which the mutation or deletion had been introduced. It was thought that Rin was aroused.

Thereafter, the recovered transformed cells 0. 5% NP- 40, 1 OmM T ris- HC 1 (pH8. 6), 14 OmM NaC l, Karoe a _{1. 5mM MgG l 2, 1000UZ ml} RNa sei nh ibitor the re supernatant by the suspension after centrifugation was collected, equal amounts of _{0. 2M T ris -HC 1 (P} H7. 5), 2% SDS, 25mM EDT a, 0. 3M NaC l, 200 β g / m 1 Proteinase K was added, and the mixture was heated at 37 ° C. for 30 minutes. After adding and extracting an equal amount of phenol: chloroform: isoamyl alcohol (25: 24: 1), total RNA was recovered with ethanol-5 and used in the following procedure. Imperial example 3

Selection of genes specific to P53-inactivated cells by differential display method Total RNA derived from the transformed cells obtained in Example 2 according to the protocol of RNAmapkits A to D from GenHunter. The RT reaction was carried out using the 铸 type. Next, PCR was performed using a total of 80 primer sets. PCR reaction Using GeneAmp PCR system 9600 manufactured by Perkin E 1mer, 94 ° Cx 20 sec -48 ° Cx 1 min-72 ° Cx 30 sec 10 cycles, 94 ° Cx 20 sec- 35 cycles of 40 ° C 1 min-72 ° C x 30 sec were performed, heated at 72 ° C for 5 min, and then cooled to 4 ° C.

The PCR solution subjected to the thermal MS was electrophoresed on a 6% sequence denaturing gel (Long Ranger 6% premix gel; FMC). After drying by JE, X-ray film (Bio Max MR; Kodak) was Exposure for ~ 3 days. Figure 1 shows the results of autoradiography. In addition to DNA fragments that appear specifically in P53-inactivated cells, we also searched for DNA fragments whose expression decreased with p53 inactivation, and matched the target band in the autoradiogram. The dried gel portion was cut out. Table 1 shows the strength of expression of each selected DNA fragment in each medullary cell. Each excised gel portion was immersed in 100 1 water for 10 minutes, heated at 100 ° C for 15 minutes, centrifuged, and 50 g of daricogen was added to the supernatant, and the amplification product was recovered with ethanol. Using the recovered DNA solution, re-amplify by PCR, purify the DNA fragment in a SUPREC-02P Gongai ¾ centrifuge tube (Takara), and use the plasmid vector pT7B1ue (R) (Nov age n ).

得られた 64種の断片を含むクローン群の塩基配列の決定を行い、 GenB an kZEMBL遺伝子データべ一ス （DNAS I S CD-ROM 033 ;日立ソ フトウェアエンジニアリング社） によってホモロジ一検索を行った結果、 新規あるい は公知の種々の遺伝子が同定された。 結果をまとめて表 2に示す。 表中の 「Nove 1」 力新規な遺伝子に相当する。 これら新規な遺伝子のうち P 53不活化細胞特異的 に出現する DN Aの塩基配列を配列番号： 14〜29に、 また p 53不活化に伴って 懇カ '減少する DNAの塩基配列を配列番号： 30〜配列番号： 41に記載した。 なお、 p 53の不活化によって発現力減少する遺伝子群の中に、 p 53によって遺 伝子の発現力％1導される p 21 WAF— 1力 ¾1別されていたことより （フラグメント 61に相当） 、 本発明で用いたディファレンシャルディスプレイの 件 P 5 3の不活化によって発現が上昇あるいは減少する遺伝子群を解斤するのに適したもの であると判断された。

The nucleotide sequence of the clone group containing the obtained 64 types of fragments was determined, and a homology search was performed using a GenBank ZEMBL gene database (DNAS IS CD-ROM 033; Hitachi Software Engineering Co., Ltd.). Various new or known genes have been identified. Table 2 summarizes the results. “Nove 1” in the table corresponds to a novel gene. Among these novel genes, the nucleotide sequence of DNA that appears specifically in P53-inactivated cells is shown in SEQ ID NOS: 14 to 29, and the nucleotide sequence of DNA that decreases with p53 inactivation is shown in SEQ ID NO: : 30 to SEQ ID NO: 41. In addition, among the gene groups whose expression level is reduced by the inactivation of p53, p21 WAF-1 is derived from p53 by the expression level of the gene by% 1. However, the differential display used in the present invention was determined to be suitable for unraveling genes whose expression increases or decreases due to inactivation of P53.

Among these DNA fragments, p53-inactivated cells were most prominent; S ± elevation was observed (DNA of fragment 27).

Fragment Homologous by Fragment size Homologous by

 Fragment No. SEQ ID No. Fragment No. SEQ ID No. to

 size (bo) comouter search (bp) computer search

 1 14 197 Novel 2 34 208 Novel

 5 15 335 Novel 8 21 139 Novel

 6 16 254 Novel 9 107 myc related zinc finger mRNA

 10 30 111 Novel 12 35 206, 97 Novel, DAD

11 326 T / A stretch ⁸ 14 22 111 Novel

 16 97 Ribosomal protein 12 19 36 208, 173 Novel, c-myc

 31 Λβ Novel rt j f wovei, lysu oxiaase

 ert Na ATPase β-subunit

 17 Novel I 9

 Novel iiiiiociionana gene, r 1

27-1 121 Novel ^b 31 23 175, 320 Novel, Phorbolin 1

 18 F27

27-2 121 Novel ^b 35 24 313 Novel

 29 121 Novel- 36 187 KIAA

30 ^c 338 p57kip2-XPC fuslon ^d 37 155 a-tublin

 > 290 T / A stretch * 38 25 89 Novel

 32 258 Phorbolin I 41 96, 171 Rab 13, Cystathionine ^ -synthase

33 217 p57kip2 43 96, 153, 133 Rab 13, tFN-inducibte 56kDa, MHC 1

34 32 148 Novel 44 38 103 Novel

 42 172 Rab13 46 39 307 Novel

 49 33 273 Novel 47 40 202 Novel

 53 326 Signal recognition particle 9 50 41 264 Novel

 54 19 346 Novel 51 26 132 Novel

 57 20 153 Novel 60 27 88 Novel

 61 123 p21waf-1 62 59 IGF-BP5

 3C-1 28 209 Novel

 3C-2 29 232 Novel

a) A / T base sequence (art if act) b) Same fi gene 27 difragment 27)

c) 338bp = 6c I ones.> 290bp = 3c I ones d) art fact

Difficult case 4

 Examination of the expression of the gene corresponding to 27 in transformed cells

 The following experiment was performed to confirm that the gene corresponding to 27 was significantly increased in p53-inactivated cells.

Using a commercially available primer for G3PDH gene detection (manufactured by CIontech) as a control for examining the appropriate amplification conditions for the fragment 27-compatible gene by PCR, all the transformed cells were analyzed. Using the RNA as type II, RT-PCR was performed. The 5 'end of the primer was labeled with ³² P, and the number of amplification cycles was changed to 16, 18, 20, and 22 times. As a result of electrophoresis of the PCR solution and measurement of the signal intensity of the amplified DNA fragment, it was shown that the DNA fragment of G3PDH was microamplified in any of the transformed cells in 16 to 22 cycles. It was confirmed that the total RNA samples used for RT-PCR were almost equivalent.

 The DNA sequence corresponding to fragment 27 was found from the EST database (accession on No. AA 150500 and accession on No. AA 1567 04), and the primers that can specifically detect the gene corresponding to fragment 27 were used. The primary sequence was selected and the same RT-PCR was performed. When the number of cycles was increased from 18 to 22, the logarithmic amplification was shown. The primer used (see below).

(1) Primer sequence on the 5 side: GCATGGCTGGGAGCGTGATGTGGT (SEQ ID NO: 6) (2) Primer sequence on the 3 ′ side: CTCTTGTGCAGGAGGCGCTCACTCAG (SEQ ID NO: 7) Total RNA obtained from the P53-inactivated transformed cells Figure 2 shows the results of RT-PCR after 22 cycles of amplification of type III. In all the p53-inactivated cells, the expression of the fragment 27-corresponding gene was significantly increased. The gene corresponding to fragment 27 was named immortalization-related gene I RG27. Difficult case 5 I RG27 gene expression! Examination of ¾

 To determine the source of the library for cloning the full-length cDNA of IRG27, Northern blot analysis was performed to determine the IRG2

The expression of 7 was examined. In order to obtain DNA to be used as a probe, a target DNA fragment was amplified using a primer that specifically detects IRG27, and then cloned into pT7B1ue (R) (Novagen¾). The base sequence of the primer (see below).

 (1) Primer sequence on the 5 'side: TGAGGGCGCAGAGGCAGGCAGCAT (SEQ ID NO: 8)

(2) Primer sequence on the 3 'side: CCGAGCTGTGTCCAAGCAGGCTGT (SEQ ID NO: 9) Amplified and cloned gene fragment Cut out from the recombinant plasmid vector using a restriction enzyme and purified by Prep A gene (BioRad) , using be a BEST DNA labeling kit (Tak ar a company) labeled with [a- ³² P] dCT P, as a probe.

 Northern blotting was carried out by a conventional method using a filter from which RNA derived from each cell and cells was blotted and purchased from C1onetech. Figure 3 shows the results of examining the expression in various human normal threads. Strong expression and expression of IRG27 were observed in lymphoid fibers such as peripheral blood leukocytes, spleen, and thymus. Fibers other than the lymphoid fiber were relatively strong in the small intestine, but this may be due to the small intestine's ability to possess its own immune fibers (lymphocytes). In normal humans except for the above-mentioned threadwork, IRG27 was not expressed, or even if it was expressed, the expression was extremely low. The size of the mRNA was 0.9 Kb, and transcripts of 1.8 Kb, 3 Kb, and 4.5 Kb were detected, though weakly.

Fig. 4 shows the results of examining the expression in various human cancer cell lines. Expression was observed in most of the cancer cell lines examined. In particular, high frequency expression was observed in a lymphoma-derived cell line (Raj i), and strong expression was also observed in a large cell line (SW480) and a cervical lining cell line (HeLa). Furthermore, the expression was high in the fetal liver thread, but extremely low in the adult liver (Fig. 3). This means that the IRG27 gene This suggests that, like Rikiichi CE A, he is closely related to Ganji-Dani. Difficult case 6

 I RG27 full-length c DNA Cloning

From the above, the gene corresponding to IRG27 was found to have extremely high expression in peripheral blood leukocytes. Therefore, screening of IRG27 cDNA was performed using a human leukocyte cDNA library (Gibco BRL). 2. RESULTS subjected to colony High Priestess die See Chillon using the probes prepared in Example 5 to 7x l 0 ⁶ colonies, 8 clones by three subscription-learning is selected.

 All the nucleotide sequences were determined for three clones, I RG27-3-1, I RG27-4-1, and I RG27-9-11, each having an insertion sequence of 1 Kb. As a result, the open reading frame portions corresponding to the 181 amino acid residues of the three clones were identical. However, the sequence 5 ′ upstream of the start codon of IRG27-4-1 was different from that of IRG27-3-1 and IRG27-9-1. The nucleotide sequences contained in the clones I RG27-3-1 and IRG27-4-1 are shown in SEQ ID NOs: 3 and 4. The amino acid sequence deduced from these nucleotide sequences is shown in SEQ ID NO: 1. It should be noted that PCR using body DNA as a type III is expected to be caused by the above-mentioned DNA sequence difference a 1 t rennativessplicnicg.

As a result of a homology search using the GenBankZEMBL gene database (DNAS IS CD-ROM 035; Hitachi Software Engineering Co., Ltd.), a deletion ability of 2 amino acid residues was found in the amino acid sequence of IRG27 of the present invention. ISG20 gene encoding a polypeptide (accession No. X89773; Gonorra C, et al., J. Biol. Chem., 272, 19457— 19463, 1997), and the I RG of the present invention HEM45 gene (accession on No. U88964; Pentecost BT, J. Steroid id Biochi) em. Mo 1. Biol., 64, 25-33, 1998). It was revealed that the nucleotide sequence of the 5'-end page region of HEM45 was of type IRG27-3-1.

 As described above, it was determined that the protein had the same amino acid sequence as that of IRG27 HEM45 and that was different from ISG20 in two amino acid residues. HEM45 is known to be upregulated by estrogen, and ISG20 is known to be upregulated by type I and II interferon. However, it is not known that the expression is increased by inactivating ρ53. Difficult case 7

Cloning of mouse type I R_27:

 Partial sequence of mouse type I RG27 (ml RG27) gene Deduced from EST database, base deletion in the region corresponding to open reading frame of hI RG27, translated into amino acid sequence as is Then, the protein encoded by the mIRG27 gene becomes a polypeptide having a C-terminal deletion compared to the hIRG27 protein. Therefore, in order to clarify the amino acid sequence of the protein encoded by the mlRG27 gene, a primer was set based on the nucleotide sequence of the EST database, and a mouse spleen cDNA library (C1tech) The DNA fragment was amplified by PCR using the 铸 as a 铸, and introduced into pGEM_Teasy Vector (Promega). As a result, eight types of clones were obtained, of which the total nucleotide sequences of two types were determined. In addition, the nucleotide sequence was determined by the direct method using the amplified PCR fragment, and the correct nucleotide sequence of the mlRG27c DNA gene was determined.

Comparison of the determined nucleotide sequence with the sequence obtained from the EST database revealed that there were substitutions, deletions and insertions at four positions. The homology of the open reading frame region of the mIRG27 gene and the hIRG27 gene is 83.2%, indicating that the mIRG27 and hIRG27 proteins were deduced from the nucleotide sequence of the gene. The amino acid sequence had a homology of 82.3%. M I RG2 as well as h I RG27 7 was also 181 amino acid. The determined nucleotide sequence of mI RG27 is shown in SEQ ID NO: 5, and the deduced amino acid sequence is shown in SEQ ID NO: 2. Example 8

 Examination of expression of I RG27 in cancer

In order to examine the expression of IRG27 in the tumor fiber, Northern plot defoliation was performed using RNA prepared from tumor and non-tumor fibers of the same patient. Using N col-Sac I fragment of hI RG27 cDNA (SEQ ID NO: 3) as a probe, Hum an Tumor Panel B lots ( Invitrogen) was used for rehybridization by a conventional method. The results are shown in FIGS. I RG27 It was revealed that the gene expression in the tumor was elevated in a high degree. As a result of comparing the increase in the IRG27 gene in the tumor region with the carcinoma, 70% of the carcinomas had increased the expression level of IRG27. Specifically, esophageal cancer, stomach cancer, body, kidney cancer, instep W¾, parotid, ureteral cancer, bladder cancer, uterine cancer, liver cancer,? IRG27 expression was elevated in human, ovarian, and fallopian tube cancers. In addition, in 3 out of 4 patients (Fig. 6), and in 2 out of 2 renal cancer patients (Fig. 5), the rise of IRG27 was observed. Tables 3 and 4 summarize the rise of S ± in the tumor area. (In the table, the more Byeons, the greater the expression in the tumor area than in the non-tumor area. This indicates that the expression is lower in the iiffl's tumor than in the non-tumor's). Table 3 corresponds to the results in Figure 5, and Table 4 corresponds to the results in Figure 6. Table 3

Table 4

以上のように、 p 5 3の不活化にともない発現カ^:昇する遺伝子としてクロ一ニン グされた I R G 2 7遺伝子の発現上昇 ί 7 0 %¾8の腫瘍部で認められたが、 この 値は、 癌患者において Ρ 5 3カ変異している割合 （約 5 0 %、 Cel l , 88， _P323-331 ( 1997) ) より高 、値であった。 また、 図 5に示された 2 0症例の中で非腫瘍部ではほ とんど発現がなく腫瘍部で発現力 s '認められた症例に限定すると、 2 0症 』の内、 腎癌 ( 2症例） 、 肝癌、 甲状腺癌、 耳下腺癌、 尿管癌、 膀胱、 子宮癌、 卵巣癌でその傾向 (顕著であった。 頻度は 4 5 %であった。

As described above, the expression of the IRG27 gene, which was cloned as a gene that increases with the inactivation of p53, was observed in tumors of ί70% ¾8. the proportion that [rho 5 3 months mutations in cancer patients than the height (about 5 0%, Cel l, 88 , P 323-331 (1997)), was a value. In addition, among the 20 cases shown in Fig. 5, if the expression was not observed in the non-tumor area and was expressed in the tumor area s', the renal cancer (20 cases) 2 cases), liver cancer, thyroid cancer, parotid gland cancer, ureteral cancer, bladder, uterine cancer, ovarian cancer (the tendency was remarkable. The frequency was 45%.

As a result, the cases examined showed that the cancer could be detected at a rate higher than the diagnosis rate of the p53 mutation in the cancer. When P53 itself is used as a diagnostic agent for cancer, it cannot be diagnosed until a patient whose P53 expression is no longer observed due to mutation of p53. By using IRG27 of the present invention, it is possible to diagnose such patients, and therefore, use IRG27 rather than using P53 as a cancer diagnostic. It was thought that doing it would be more useful. Wei example 9

Production of antibodies to hIRG27 polypeptide

 In order to prepare an antibody for detecting the I RG27 protein, three regions were selected from the amino acid sequence of hi RG27 shown in SEQ ID NO: 1, and an oligopeptide was synthesized. Specifically, I RG27-A consisting of the portion at positions 104 to 127 of the amino acid sequence described in SEQ ID NO: 1, I RG27-B consisting of the portion at positions 131 to 153, and position 159 To 181st position of IRG27-C were synthesized. Then, the oligopeptide was conjugated to KLH, and this was used as an antigen to immunize rabbits in the usual manner. Egrets were prepared.

RG27 protein was recognized.

Next, 2-fluoro-1-methylpyridinium toluene-14-sulfonic acid-activated Cellulofine (Seikagaku Corporation) was used as the carrier for the peptide-immobilized column, and the oligos previously used as antigens were used. Peptides were coupled to the carrier. Next, of the previously prepared iitlill purified I RG27 A-1 and 2, 110278-1 and 2 and I RG27C-1 and 2, purified I RG27 A-2, I RG27B-2 and I RG27C-2 Was added to the affinity column and mixed by inversion. Then, the mixture was left overnight at 4 ° C, washed with TBS, and eluted with 0.1 M glycine-HC1 (pH 2.5). Measurement of the I _g G amount of an antibody of § full Initi After one purification, anti-I RG27A- 2 I gG, anti I RG27B- 2 I g G, anti-I RG 27 C- 2 I gG ^ each approximately 0.2 mg / m 1, lmgXm 1, 0.2 mgZml. FIG. 7 shows the results of Western plot clarification using δ-purified and purified antibodies. SDS—Polyacrylamide electrophoresed protein p cDNA3.1 (+) (I η V itroge η) with the IRG27 gene inserted into the DLD-1 cell (Dainihon m ± ) Is a cell lysate of a stable transformed cell obtained by transfection, and a cell lysate of a stable transformed cell into which only the vector is introduced. As evident from Figure 7

り、 ゥ エスタンブロットに使用する抗体として最適であると考えられた。

, Especially anti-I RG27C—2 I

、 It was considered to be optimal as an antibody for use in easter blot.

Detection of I RG27 using antibodies

 Correlation between gene sex and protein production using RNA and proteins obtained from normal cells HEL299 and WI38, fetal kidney-derived transformed cells 293 and various cancer cells It was examined whether or not was observed.

 1) Northern blotting

Preparation of total RNA from various cells GI BCO-BRL was performed using a Trizol kit. 20 g of total RNA is formalin-containing 1% agarose per ligation! / «Electrophoresis and transferred to NEN nylon membrane (Gene Screen P 1 us), then 1 MNaCl, 10% dextran Prehybridization was performed using 1% SDS, 50% formamide, and 100 ^ 6 g / ml salmon sperm DNA. Using the Nco IZS ac I fragment of IRG27 cDNA (SEQ ID NO: 3) labeled with ³² P using BcaBEST Labeling Kit from Takara as a probe, linogen hybridization was performed by a conventional method. I went to one session. Washing was performed twice at room temperature for 10 minutes with 2 XSSC, twice at 60 ° C for 20 minutes with 2 XSSC and 1% SDS, and then at room temperature for 10 minutes with 0.1 x SSC and 0.1% SDS once. Detection was performed using Kodak's BI OMAX MS film.

2) Western blotting

Preparation of proteins from cells was performed by lysing the cells with 31.25 niM Tris-HCl (pH 6.8), 7.5% glycerol, and 2% SDS.The protein iU¾ was obtained using Pierce's BCA protein measurement kit. Were determined. Add 5% (V / V) 2-mercaptoethanol and 0.01% (W / V) BPB to the protein solution of No. 10, and add oat barley at 100 ° C for 5 minutes, 15¾SDS-polyacrylamide gel 1 / S It was subjected to electrophoresis. After being electrically transferred to a Millipore PVDF membrane (Immobilon P), TBS-T (2 OmM TrisHCl (pH 7.5) containing 1% normal goat serum (Vector), 15 OmM NaC 1, 0.05% (v / v) T Ween 20) solution and blocked with 200-fold diluted affinity-purified anti-I RG27 antibody (anti-I RG27C-2 IgG). Then, a 5000-fold diluted secondary antibody (Kappel's HRP standard heron IgG goat antibody) was reacted, and IRG27 was detected with Amersham's ECL Plus.

3) Result

 As shown in FIG. 8, the results of detection of RNA by Northern plotting and the results of protein detection by stamp lotting were in good agreement. In addition, although protein expression was not detected in DLD-1 cells where mRNA expression was not observed, it is probable that as shown in the results of CBB staining, the amount of sampno applied to electrophoresis was small. As described above, the anti-IRG27 antibody thus produced was shown to be capable of quantitatively detecting IRG27. Example 1 1

Ij £ of the ELISA method using anti-I RG27 antibody

 In order to detect the IRG27 protein present in body fluids including blood and saliva and urine, a sandwich ELISA using an anti-IRG27 antibody? did.

The IRG protein used in the present invention was prepared as a fusion protein with GST as follows. That is, first, the cDNA of human I RG 27 (SEQ ID NO: 3) cloned in Example 6 was inserted into pGEX-6 合 う -1 (Pharmacia) so that the reading frame matched it. A GST fusion I RG27 protein expression plasmid was prepared, and then the host E. coli JM109 (Toyobo) was transformed with the expression plasmid and cultured at 37 ° C. 1 mM IPTG was added and the cells were cultured for 1 hour * ¾ and the cells were recirculated 4. The cells were buffered with PBS containing ImM PMSF, 1 β g / m 1 pepstatin A, and lgZml leptin. — Suspended in E (PBS, 5mM EDTA), sonicated, and added Triton X-100 to a final concentration of 1% as a control. Glutathione Sepharose 4B (Pharmacia) was added to the supernatant, and GST-fused IRG 27 proteins were bound. The daltathione Sepharose was washed three times with PBS-E, and the GST-fused IRG27 protein was eluted with 50 mM TrisHCl (pH 8.0) buffer containing lQmM original glutathione. The purified GST fusion I RG27 protein was quantified using the Protein Assay Kit (BI ORAD).

 Using the GST-fused IRG27 and anti-IRG27 antibody prepared as described above, the ELISA method was performed as follows.

First, the anti-I RG27 B-2 IgG prepared in Example 9 was adsorbed onto a 96-well plate, Immunoplate II (Nunc), and then the GST fusion I prepared at various concentrations prepared above. RG27 was reacted. Subsequently, the biotin bMl RG 27 C-2IgG prepared using the biotin labeling kit of Boehringer Mannheim was reacted, and peroxidase-labeled streptavidin (B10 SOURCE I NTERNAT ION AL) After the reaction, a color reaction was carried out using TMB Microcell II Perox i das e Sub strat e System (KPL). The results are shown in FIG. The anti-IRG27 antibody of the present invention did not react with GST, but reacted with GST-fused IRG27 in a manner dependent on the antigen iJS and the detection antibody. From these results, the ability to perform ELISA using the anti-IRG27 antibody of the present invention became clear.

Detection of IRG27 protein in blood and urine of cancer-bearing mice by ELISA

A human lymphoma-derived cancer cell such as Raji cell is administered to mouse J »such as C57B6, and the cancer cell is proliferated by. As a result, it is induced in the cytotoxic T cell (CTL) mouse against human cancer cells in the same manner as in the induction of CTL force on cancer cells in human cancer patients, and damages the CTL-powered cancer cells. However, it is possible that IRG27 derived from human cancer cells is released into fil and urine. Therefore, the blood and urine can be woven at any time after inoculation of the cancer cells and subjected to the ELISA method described in Example 11 above, whereby the protein of IRG27 can be detected. mi 3

 Detection of IRG27 protein by ELISA using urine of cancer patients

 By subjecting the urine of a cancer patient and a healthy subject to ELISA in the same manner as in {5} Example 12, it is possible to determine that the urine of a cancer patient has significant IRG27 protein control.

 Specifically, for example, (^ the urine of a patient with bladder cancer ゃ renal cancer patient was collected, centrifuged, the supernatant was collected, and the amount of patient urine to be detected was gradually increased, as described in Example 11 Perform EL ISA Significantly detect IRG27 protein in S cancer patients compared to healthy individuals, so that it is possible to select patients with cancer from among the examinees.

 The 122nd and 136th bases of the base sequence described in SEQ ID NO: 4 {The base could not be determined.

 ^ S at the 131st position in the base sequence described in SEQ ID NO: 19 could not be determined.

 The 74th and 87th nucleotides in the nucleotide sequence of SEQ ID NO: 20 could not be determined.

 The 24th, 34th, 45th, 65th, 66th, 70th, 73rd, 74th, 84th, 84th, and 86th positions of the nucleotide sequence described in SEQ ID NO: 21 The 89th, 109th, 114th and 123rd bases The base could not be determined.

 The 17th, 62nd, 99th, 11th, 125th, 129th and 170th bases of the base sequence described in SEQ ID NO: 23 could not be determined.

 The 17th, 62nd, 99th, 11th, 125th, 129th, and 170th bases of the base sequence described in SEQ ID NO: 24 bases that could not be determined.

The eighth, 41st and 87th amino acids in the nucleotide sequence of SEQ ID NO: 26 The base could not be determined.

 The 63rd, 75th, 108th, 158th, 179th and 189th bases of the base sequence described in SEQ ID NO: 28 can be determined. No bases 15th, 28th, and 41st bases in the base sequence of SEQ ID NO: 30 I Bases that could not be determined.

 1st, 1st, 1st, 2nd, 68th, 70th, 89th, 146th, 169th, 1st, 1st The 77th, 179th, 186th, 191st and 196th bases are bases that could not be determined SEQ ID NO: 36 10 2nd, 1st 1st, 1st 3rd, 1st 150th, 1st 58th, 1st 167th and 170th bases bases that could not be determined .

 Rooster No .: bases described in 39. Bases No. 108, No. 157, No. 181, and No. 209 in base of rooster. These bases could not be determined. .

 SEQ ID NO: 41 2nd 21st ^ S of the nucleotide sequence described in SEQ ID NO: 41 is a nucleotide that could not be determined. Industrial applicability

 Antibodies against the IRG27 gene of the present invention or a part thereof, or the IRG27 protein are useful for diagnosis of a wide variety of cancers.

Claims

The scope of the claims 1. Effective single- or double-stranded DNA consisting of at least 17 nucleotides of the normal and reverse strands of the gene having the following (a) and (b), or the target of these DNAs. As a component, a diagnostic agent for cancer.  (a) Expression is induced by inactivation of tumor suppressor p53  (b) Increased expression of S in cancer fiber compared to normal thread! ^ 2. Single- or double-stranded DNA consisting of at least the 17i # S sequence of the positive and reverse chains of the gene having the following (a), (b) and (c), or A diagnostic agent for cancer, comprising the separation of DNA as an active ingredient.  (a) Expression is induced by inactivation of tumor suppressor p53  (b) Cancer thread compared to normal fiber! Increased expression of S in ^  (c) SEQ ID NO: 14 to 29 3. Single- or double-stranded DNA capable of specifically detecting mRNA for a gene consisting of the nucleotide sequence of any one of SEQ ID NO: 3 to SEQ ID NO: 5 or an allele mutant gene thereof, or A diagnostic agent for cancer, comprising a labeled DNA as an active ingredient. 4. SEQ ID NO: 3 to SEQ ID NO: 5 V Single-stranded sequence consisting of a sequence consisting of at least 17 consecutive bases of the gene consisting of the base sequence described in the deviation force or the positive and reverse chains of the allele mutant gene thereof Or double-stranded DNA or the target of these DNAs as an active ingredient, 5. The diagnostic agent for cancer according to claim 1, wherein the diagnostic agent is a probe for an amplification reaction or a primer for a PCR reaction. 6. The cancer diagnostic agent according to claim ⁵ , wherein the length of the primer for the PCR reaction is 17 to 50 bases. 7. The diagnostic agent for cancer according to claim 5 or 6, wherein the following A, B or C primer set, or the primer set consisting of a sequence of at least 17 bases or more of these primers is used as an active ingredient. .  (A)  5 'primer sequence TGAGGGCGCAGAGGCAGGCAGCAT (SEQ ID NO: 8)  3'-side primer sequence CCGAGCTGTGTCCAAGCAGGCTGT (Rooster column number: 9)  (B)  5 'primer sequence AAAGGCAAGCTGGTGGTGGGTCAT (SEQ ID NO: 10)  3 'primer Primer's line CTGTCCCAAAAAGCCGAAAGCCTC (SEQ ID NO: 1 1)  (C)  5, side primer sequence TTCCGCCCCTGACTTCACTTGATAACAAAC (SEQ ID NO: 12) 3 'primer sequence CAGGCCGGATGAACTTGTCGT (SEQ ID NO: 13) 8. An antibody, antibody fragment, or derivative thereof that specifically recognizes a polypeptide encoded by a gene having the following (a) and (b):  (a) Expression is induced by inactivation of tumor suppressor p53  (b) Increased expression in cancer thread compared to normal, ^ 9. An antibody, an antibody fragment, or an antibody thereof specifically recognizing a polypeptide encoded by a gene containing the nucleotide sequence of any one of SEQ ID NOs: 14 to 29. 10. An antibody, antibody fragment, or derivative thereof that specifically recognizes a polypeptide comprising the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2 or an allelic variant thereof body. 11. A diagnostic agent for cancer, comprising the antibody, the antibody fragment or the derivative thereof according to any one of claims 8 to 10V as an active ingredient. 12. A method for diagnosing cancer, comprising using the diagnostic agent for cancer according to any one of claims 1 to 7 or claim 11. 13. The method for diagnosing cancer according to claim 12, wherein a thread or a cell is used as a diagnosis target. 14. The method for diagnosing cancer according to claim 12, wherein blood or saliva or urine is used as a diagnosis target. 15. The method for diagnosing cancer according to any one of claims 12 to 14 for diagnosing inflection. 16. The method for diagnosing cancer according to any one of claims 12 to 15, for diagnosing urinary system cancers including renal cancer and ephedra cancer. 17. A gene containing the nucleotide sequence of any one of SEQ ID NOs: 14 to 29 and whose expression is induced by inactivation of the tumor suppressor 因子 53. 18. A gene comprising the nucleotide sequence of any one of SEQ ID NO: 30 to SEQ ID NO: 1, and whose expression is regulated by inactivation of the tumor suppressor factor 53.