JP2001161384A - Method for detecting carcinoma - Google Patents

Abstract

PROBLEM TO BE SOLVED: To utilize a protein bearing an important role on the long term survival of the cranial nerves cell and a DNA encoding the protein on the detection of carcinoma. SOLUTION: The detection of carcinoma, e.g. large bowel carcinoma, skin carcinoma or the like, is carried out by using the DNA encoding the bradion protein or its analogue derived from human beings and the antibody immunoreactive with the protein or its analogue.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to a DNA encoding a protein involved in long-term survival of brain nerve cells and a method of using an antibody against the protein in detecting cancer. Specifically, the present invention relates to human-derived Bradion (Brad).
eion) A method of using a DNA encoding a protein or an analog thereof and an antibody immunoreactive with the protein or a derivative thereof in the detection of cancer.

[0002]

2. Description of the Related Art Brain nerve cells (neurons) are the main elements that govern the survival of individuals of higher organisms. They do not divide at all, but only fall off and necrotic every moment. This loss of neurons occurs in normal conditions, but is greatly accelerated in genetic diseases, cerebral ischemia, status epilepticus, and hypotrophic / hypoxic conditions. The cerebral neuropathy (blur etc.) due to aging is due to the shortage of the absolute amount of functional neurons due to the accumulation of these neurons.
Regeneration of neurons is considered to be the most urgent issue in aging.

[0003] After the differentiation induction phase in the developmental process, brain neurons do not divide thereafter, survive while maintaining their functions (or with a gradual decline in function) until the end of the life of the individual, and have a unique mitotic arrest mechanism and It is presumed that a function maintenance mechanism exists, but the mechanism has not been elucidated yet. The central nervous system is a treasure trove of unexplained proteins and signal transmitters (both stimulators and receptors), and there are a large number of substances that are currently awaiting elucidation. In particular, there are a large number of stimulators and their receptors involved in the brain and specific signal transduction, and the full picture has not yet been elucidated.

[0004] Many studies on such important factors controlling survival of brain nerve cells have been conducted on a world level.
The material and molecular elucidation of the substance itself has not yet been elucidated, and the development of analytical techniques had to be started. Recently, a group of Dr. Masashi Yanagisawa of the Howard Hughes Foundation Certified Medical Institute at the University of Texas at the United States has succeeded in developing a technology to randomly probe such neuropeptides and their receptors using cultured cells. As an example, we succeeded in discovering a substance (orexin) that directly binds and stimulates the hypothalamus appetite stimulus center and elucidated the function of its receptor (Cell, 9).
2 , 573-585 (1998)). However, attempts to search for such a system of substances have been rare, and at present the stimulators involved in brain-specific signal transduction and their receptors have not yet been sufficiently elucidated. Under such circumstances, the present inventors have developed an improved expression gene (cDNA) library, developed a systematic screening technique, and succeeded in extracting and selecting brain neuron-specific genes. Was completed.

[0005]

SUMMARY OF THE INVENTION An object of the present invention is to use a DNA encoding a bradion protein involved in long-term survival of brain nerve cells or an antibody against the protein for detecting cancer.

[0006]

The present invention provides the following properties: (1) a membrane protein, (2) Kyte-Doolit.
By the hydropathy analysis by the tle method, it has a structure characteristic of the interleukin receptor including the transmembrane portion (once), and (3) strong expression is observed in the human adult brain. (4) Overexpression in cultured undifferentiated human neurons induces cell death, (5) Overexpression in cultured normal human cells (6) presents in the cytoplasm in the process of cell death, forms intracellular aggregates, accumulates in mitochondria within 24 hours, and (7) human colon cancer cell lines and humans The present invention relates to a human-derived bradion protein or an analog thereof, which is specifically expressed in a skin cancer cell line.

[0007] The protein of the present invention includes SEQ ID NO: 2 in the sequence listing.
Or the amino acid sequence shown in 4, and includes proteins referred to as α-bradion and β-bradion, respectively.
These proteins are considered to be expression products by alternative splicing during transcription. In addition to the above properties, α-bradion exhibits the property of inducing cell death when a gene encoding α-bradion or a DNA encoding an analog thereof is introduced into cultured cancer cells. In such cases, no such cell death is observed.

[0008] In the present specification, the term "analog" refers to properties substantially equivalent to human-derived bradion, especially at least (1), (2), (3), (6) and (7) Or an amino acid sequence having at least one amino acid deleted, substituted or added in the amino acid sequence shown in SEQ ID NO: 2 or 4. It is desirable that the analog has 90% or more, preferably 95% or more, more preferably 97% or more homology with the amino acid sequence shown in SEQ ID NO: 2 or 4. Analogs of the present invention include:
Amino acid variants or variants of the human-derived Bradion protein, as well as Bradion proteins of comparable properties derived from non-human mammals are included. For example, in the mouse brain, only β-type was found among the two types derived from human, but the homology with human-derived β-bradion was 9%.
4%. The analog of the present invention can be obtained by a DNA recombination technique using an artificial modification method such as a site-directed mutagenesis method as long as the biological activity of human bradion is not impaired. It may not be included. Further, it may be chemically modified with a water-soluble polymer such as polyethylene glycol. The present invention also relates to a DNA comprising a base sequence encoding the Bradion protein or an analog thereof as defined above, or a fragment thereof.

Specific examples of such a DNA or a fragment thereof include a DNA consisting of the nucleotide sequence from position 129 to position 1943 of SEQ ID NO: 1 (that is, a DNA encoding human α-bradion) or 15 or more of the nucleotide sequence A DNA fragment having a sequence consisting of preferably 20 or more, more preferably 30 or more consecutive nucleotides, or a DNA consisting of the entire base sequence shown in SEQ ID NO: 1, and 129 to 1562 in SEQ ID NO: 3. (I.e., DNA encoding human β-bradion)
A) or a DNA fragment having a sequence consisting of 15 or more, preferably 20 or more, more preferably 30 or more consecutive nucleotides of the base sequence, or SEQ ID NO: 3
And DNA consisting of the entire base sequence shown in the above. Furthermore, DNAs that can hybridize with these DNAs under stringent conditions are also included in the present invention. Here, the stringent condition refers to a homology of 90% or more, preferably 95% or more with the nucleotide sequence shown in SEQ ID NO: 1 (positions 129 to 1943) or SEQ ID NO: 3 (positions 129 to 1562). Preferably, hybridization occurs only when 97% or more homology exists between the sequences. Normal,
About 5 ° C to about 3 degrees above the melting temperature (Tm) of the complete hybrid
This refers to the case where hybridization occurs at a temperature of 0 ° C., preferably about 10 ° C. to about 25 ° C. lower. For stringent conditions, see Sambrook et al., Mol.
ecological Cloning, Alaborator
y Manual, Second Edition,
Cold Spring Harbor Labora
tory Press (1989), especially section 11.45 “C
indications for Hybridizati
on of Oligonucleotide Pro
and the conditions described herein can be used. The DNA or a fragment thereof of the present invention can be used not only for expression of the Bradion protein but also as a probe for hybridization and as a primer for PCR. Can also be used.

[0010] The present invention further relates to a vector containing a DNA encoding the bradion protein. The vector is
It contains at least a promoter so that the DNA can be expressed, and additionally contains an origin of replication, an enhancer, a ribosome binding site, a transcription terminator (terminator),
Elements such as selectable markers, RNA splice sites, polyadenylation signals, etc., can be included. The present invention further relates to host cells transformed or transfected with such vectors. The host cell may be either prokaryotic or eukaryotic, but is preferably a eukaryotic cell, more preferably a mammalian cell, especially a human cell line.

The present invention also relates to antibodies which are immunoreactive with the Bradion protein as defined above or an analogue thereof.
Preferably, the antibody specifically immunoreacts with the Bradion protein or an analog thereof, and may be either a polyclonal or monoclonal antibody.

The invention further relates to the use of a DNA or a fragment thereof as defined above or an antibody as defined above in the detection of cancer. Specifically, the present invention provides a method for using the following DNA or a fragment thereof, or an antibody in detecting cancer: (1) a human-derived bradion having the amino acid sequence shown in SEQ ID NO: 2 or SEQ ID NO: 4 D encoding protein
NA or a fragment thereof comprising a sequence of 15 or more consecutive nucleotides; (2) having an amino acid sequence in which at least one amino acid is deleted, substituted or added in the amino acid sequence shown in SEQ ID NO: 2 or SEQ ID NO: 4; And the following properties: (a) a membrane protein; (b) Kyte-Doolitt
According to the hydropathy analysis by the le method, a strong expression is observed in the human adult brain, which has a structure characteristic of the interleukin receptor including the transmembrane portion (one time). It is expressed but not expressed in other organs or human fetuses. (D) When overexpressed in undifferentiated human cultured neurons, it induces cell death. (E) When overexpressed in cultured normal human cells. Induces senescence and division arrest, (f) forms intracellular aggregates in the cytoplasm in the process of cell death, accumulates in mitochondria within 24 hours, and (g) human colon cancer cell line or skin DNA having specific expression in a cancer cell line
DNA encoding a human-derived bradion protein analog obtainable by recombinant techniques, or a fragment thereof comprising a sequence of 15 or more contiguous nucleotides,

(3) Positions 129 to 19 shown in SEQ ID NO: 1
A DNA consisting of the nucleotide sequence at position 43 or a DNA hybridizable therewith under stringent conditions, or a fragment thereof comprising a sequence of 15 or more consecutive nucleotides; (4) positions 129 to 1562 shown in SEQ ID NO: 3 A DNA consisting of a base sequence or a DNA hybridizable therewith under stringent conditions, or a fragment thereof consisting of a sequence of 15 or more consecutive nucleotides,

(5) DNA encoding bradion contained in the deposited microorganism FERM BP-6922, or a fragment thereof comprising a sequence of 15 or more consecutive nucleotides, or (6) the above (1) to (5) An antibody against the human-derived bradion protein encoded by the DNA or fragment thereof according to any one of the above, or an analog thereof, or a fragment thereof. Here, the cancer is preferably human colon cancer or human skin cancer. In addition, detection can be performed by hybridization or immunoassay.

[0015]

BEST MODE FOR CARRYING OUT THE INVENTION The cDNA encoding the bradion protein of the present invention can be obtained by the following method. First, brain tissue is homogenized with a phenol or phenol-chloroform solution containing guanidine isothiocyanate, separated into an aqueous layer and an organic layer by high-speed centrifugation, and then total RNA contained in the aqueous layer is precipitated by adding it to isopropanol. Alternatively, it is recovered by sucrose or cesium chloride density gradient centrifugation.
This total RNA is subjected to oligo (dT) cellulose chromatography to purify the mRNA (ie, poly (A) RNA).

Next, cDNA is synthesized from mRNA in the presence of reverse transcriptase, and an appropriate restriction enzyme site is prepared so as to be ligable to a phage or plasmid vector. Ligated into a vector and transformed or transfected into Escherichia coli with the vector thus obtained.
Create an NA library.

Since the cloned cDNA library contains various DNA fragments having different information in addition to the target DNA, it is necessary to select the target DNA. As a selection method, for example, a method such as plaque hybridization or colony hybridization can be used. According to this method, a plaque (in the case of a phage vector) or a colony (in the case of a plasmid vector) formed on agar is transferred to a nitrocellulose membrane or a nylon membrane, treated with an alkaline solution, and then treated with a target DNA. A radioactive (32 P) or fluorescently labeled DNA probe capable of hybridizing with the DNA is added and bound, and sensitized on an X-ray film, thereby detecting and recovering a plaque or clone containing the target DNA. Alternatively, the obtained clones were
-Forced expression of a protein with an inducer such as -thio-β-D-galactoside (referred to as IPTG), transfer of the protein to a nylon membrane or a cellulose membrane, and immunologically select a corresponding clone using a specific antibody against the target protein It is also possible.

For the cDNA of interest recovered from plaques or clones that showed a positive reaction to the probe,
Maxiam-Gilber
t) Method or Sanger-Coulson (Sanger-Co
(ulson) method. For cloning methods and sequencing, see, eg, Sambrook et al.,
Molecular Cloning (supra), Aus
ubel et al., Current Protocols in
Molecular Biology, Green
Publishing Company Assoc.
and John Wiley Interscien
ce, NY, 1992 and the like.

Specifically, as described in Examples below, a human adult brain cDNA library was constructed, and a cDNA encoding the bradion protein of the present invention was obtained from a positive clone thereof. As a result of sequence analysis, two types of α-type and β-type bradion genes presumed to be due to alternative splicing were found. These nucleotide sequences are shown in SEQ ID NO: 1 and SEQ ID NO: 3, respectively, and the coding regions were at positions 129 to 1943 and 129 to 1562, respectively. In addition, α found from the base sequence
Type and β type bradion proteins were found to have the amino acid sequences shown in SEQ ID NO: 2 and SEQ ID NO: 4, respectively. That is, α-type bradion DNA consists of 1815 nucleotides and encodes a protein consisting of 605 amino acids. Also, β-type bradion DNA
Encodes a protein consisting of 1434 nucleotides and consisting of 478 amino acids. However, Met at position 1 in the amino acid sequence shown in SEQ ID NO: 2 or 4 may be deleted. Scrutiny of these sequences by GeneBank revealed that both α-type and β-type were novel genes and proteins.

The α and β bradion proteins are Kyte
-Doolittle method (J. Mol. Biol., 1
57 (1): 105-132, 1982), it was found that it has a structure characteristic of an interleukin receptor including a transmembrane portion (once). That is, it was shown to have a transmembrane receptor structure expected to be involved in the intracellular signal transduction mechanism. Furthermore, there are two types of expression modes, α-type and β-type, one of which is also persistent in various organisms, which is similar to the relationship between the tumor suppressor genes p53 / p73. In addition, intracellular aggregate formation shows extremely high homology to that seen in human neurodegenerative mutations caused by triplet repeat gene expression substances (Igarashi et al., Nature).
Genetics, 111-117, 1998; Ma.
rtindale et al., Nature Genetics,
150-154, 1998), this normal gene expression state is considered to be greatly involved in human-specific arrest of neuronal cell division and maintenance of function in brain nerve cells after development and differentiation.

The human-derived bradion protein or its analog of the present invention can be obtained, for example, by using a gene recombination technique as follows. Based on the base sequence shown in SEQ ID NO: 1 or 3 or the amino acid sequence shown in SEQ ID NO: 2 or 4, any number of consecutive 15 or more, preferably about 20 to about 50 A hybridization probe consisting of the following bases is prepared, and using this, a DNA encoding the bradion protein can be screened from a genomic DNA library or a cDNA library derived from human or non-human mammalian brain tissue. . The library is, for example, λZA
It can be prepared using a commercially available vector such as PII, pBluescript (registered trademark) cloning vector (Stratagene Cloning Systems). Plaques or clones containing the target DNA are selected by plaque hybridization or colony hybridization.

Alternatively, based on the nucleotide sequence shown at positions 129 to 1943 of SEQ ID NO: 1 or 129 to 1562 of SEQ ID NO: 3, a continuous DNA sequence of usually 15 to 100 nucleotides complementary thereto is prepared as a primer, Using this, a genomic DNA library or a cDNA library derived from human or other mammalian brain tissue is subjected to polymerase chain reaction (PCR) to obtain the desired D
NA can be specifically amplified. For example, PCR can be performed under the conditions of 20 cycles or more, preferably 30 cycles or more with denaturation at 94 ° C for 1 minute; annealing at 57 ° C for 2 minutes; and extension at 70 ° C for 3 minutes. PCR
For example, techniques described in Protein Nucleic Acid Enzyme “The Forefront of the PCR Method—From Basic Technology to Application—”, Vol. 4, No. 5, April 1996, extra edition, Kyoritsu Shuppan can be used. After cloning or amplifying the DNA of interest, the DNA of interest is recovered and integrated into a suitable available expression vector,
Further, the protein can be transformed into a suitable host cell, cultured and expressed in a suitable medium, and the target protein can be recovered and purified.

The analogs of the present invention have substantially the same properties as human-derived bradion, in particular, at least the properties (1), (2), (3), (6) and (7) among the above properties. And those having an amino acid sequence in which one or more amino acids are deleted, substituted or added in the amino acid sequence shown in SEQ ID NO: 2 or 4. As an analog, 90% or more, preferably 95% or more, more preferably 9% or more of the amino acid sequence shown in SEQ ID NO: 2 or 4
Those having a homology of 7% or more are desirable. This means that the amino acid sequence may have at least one amino acid deletion, substitution or addition modification as long as it has a biological activity substantially equivalent to that of human α or β bradion. Means that. Well-known techniques such as, for example, site-directed mutagenesis / PCR (SN Ho et al., Ge
ne 77 , 51 (1989); translated by Kaoru Saigo and Yumiko Sano, CURRENT PROTOCOLS Compact Edition, Molecular Biology Experiment Protocol I, June 1997,
(Maruzen), it is also possible to introduce a mutation into the amino acid sequence shown in SEQ ID NO: 2 or 4 by genetic engineering. Examples of substitution include substitution between hydrophobic amino acids (Ala, Val, Leu, Ile, etc.), Se
substitution between r and Thr, substitution between Asp and Glu, A
a substitution between sn and Gln, a substitution between Lys and Arg,
Substitution between Phe and Tyr is mentioned. Examples of the addition include addition of Met to the N-terminus of the mature protein observed in the expression product in a bacterial host, a His-tag at the N-terminus for facilitating induction into the mature protein, Met-Lys or Met- And the addition of an Arg sequence. It is also possible to truncate some amino acid residues at the carboxyl or amino terminus of the bradion protein within a range that does not lead to loss of biological activity.

In addition, when a prokaryotic cell such as a bacterium is used as a host cell when expressing a DNA encoding a bradion protein or an analog thereof by a gene recombination technique, a protein having no sugar chain is produced. When expressed in eukaryotic cells such as yeast, insect cells, plant cells, and mammalian cells, products having sugar chains may be obtained. The introduction of a sugar chain can be carried out by arbitrarily adding an N-linked sugar chain site (Asn-Xa
a-Thr / Ser, where Xaa is any amino acid other than Pro). In any case, the resulting analog should have substantially the same biological activity as human bradion.

The human bradion protein or its analog of the present invention can be chemically bonded to a water-soluble polymer such as polyethylene glycol (PEG) to the N-terminal amino group or the ε-amino group of lysine. PEGylation is known to reduce or suppress antigenicity or immunogenicity upon administration to a living body. The present invention further provides the aforementioned DNA
And a host cell transformed or transfected with this vector.

The vector of the present invention may be in the form of a plasmid, phage, virus or the like. For example, bacterial plasmids (such as pBR322, pKC30, pCFM536), phage DNA (such as lambda phage), yeast plasmids (such as pG-1), baculovirus, vaccinia virus, and adenovirus as vectors for mammalian cells; Any other vector can be used so long as it is replicable in the host cell, such as SV40 and its derivatives. The vector contains an origin of replication, a selectable marker, and a promoter, and may contain, if necessary, an enhancer, a transcription termination sequence (terminator), a ribosome binding site, a polyadenylation signal, and the like.

As a replication origin, for example, those derived from ColE1, R-factor, and F-factor with respect to E. coli vector
For yeast vectors, for example, 2 μm DNA, AR
With respect to the vector derived from S1, the vector derived from SV40, adenovirus, or bovine papilloma virus can be used with respect to the vector for mammalian cells. A promoter is a regulatory sequence for directing the synthesis of mRNA encoding the DNA of the present invention, representative examples of which include the adenovirus or SV40 promoter, E. coli lac or trp.
Promoter, phage lambda PL promoter, ADH, PHO5, GPD, PGK, AOX for yeast
1 promoter, a nuclear polyhedrosis virus-derived promoter for silkworm cells.

The selection marker is a gene for imparting a phenotype for selecting a transformed host cell to the host. For example, a vector for Escherichia coli includes a kanamycin resistance gene, an ampicillin resistance gene, and a tetracycline resistance gene. And the like, yeast vectors include Leu2,
Trp1, Ura3 genes and the like, and vectors for mammalian cells include neomycin resistance gene, thymidine kinase gene, dihydrofolate reductase gene and the like.

As the vector, commercially available vectors can be used. Examples of such vectors include pQE70, pQE60, and pQE70 for bacterial cells.
-9 (Qiagen), pBluescript II
KS, ptrc99a, pKK223-3, pDR5
40, pRIT2T (Pharmacia), pET-
11a (Novagen); and pX for eukaryotic ones
T1, pSG5 (Stratagene), pSVK
3, pBPV, pMSG, pSVL SV40 (Pha
rmacia).

The DNA of the present invention can be introduced into a vector by any means. Desirably, the vector contains a polylinker with various restriction sites therein, or contains a single restriction site. A specific restriction site in the vector is cleaved by a specific restriction endonuclease, and the DNA of the present invention is inserted into the cleavage site. The expression vector containing the DNA and the regulatory sequence of the present invention can be used for transforming or transfecting a suitable host cell to cause the host cell to express and produce the human bradion protein of the present invention or an analog thereof.

Examples of host cells include bacterial cells such as Escherichia coli, Streptomyces and Bacillus subtilis; fungal cells such as strains of the genus Aspergillus; yeast cells such as baker's yeast and methanol-assimilating yeast; insect cells such as Drosophila S2, Spodoptera Sf9, etc .; mammalian cells including human cultured cells, such as CHO, COS, BHK,
3T3, C127 and the like. Transformation or transfection can be performed by a known method such as a calcium chloride / rubidium chloride method, a calcium phosphate method, DEAE-dextran-mediated transfection, and electroporation.

The human-derived bradion or an analog thereof of the present invention is obtained by expressing a host cell transformed or transfected as described above under the control of a promoter, and recovering the produced target protein. For expression,
After growing or growing the host cells to an appropriate cell density,
Promoters can be temperature shifted or chemically induced (IPTG
Etc.), and the cells are cultured for a further period of time. When the target protein is secreted extracellularly, it is directly from the medium. When it is present inside the cell, physical means (ultrasonic disruption or mechanical disruption) or chemical means (lysozyme or cell lysing agent) After destroying the cells by
Purify the desired protein. For example, proteins can be obtained from a recombinant cell culture medium or an extract thereof by ammonium sulfate or ethanol precipitation, acid extraction, anion or cation exchange chromatography, hydrophobic interaction chromatography, affinity chromatography, gel filtration chromatography, HPLC, It can be partially or completely purified by a combination of conventional techniques such as electrophoresis and chromatofocusing.

As a result of examining the function of the bradion protein by introducing the DNA of the present invention into various human cell lines and cancer cell lines, the following was found in addition to the above findings. (1) The DNA of the present invention is used for culturing human cultured neural cells NT2neu, which is undifferentiated human cultured neural cells
ron (Stratagene) to Superfec
When the gene was introduced using t-reagent (manufactured by Qiagen) and overexpressed, cell death was induced within 18 to 24 hours. (2) Overexpression in cultured human normal cells in a similar manner induced senescence and mitotic arrest. (3) Gene transfer into cultured cancer cells induced cell death only in α-type, but no cell death was observed in β-type. (4) During the process leading to cell death, the bradion protein was present in the cytoplasm, formed intracellular aggregates, and accumulated in mitochondria within 24 hours. Furthermore, in studies using cultured human cancer cells, bradion was specifically detected in colorectal cancer cell lines and melanoma (skin cancer) cell lines, and along with canceration in tissues other than the cerebral nervous system. It was found that the function was specifically expressed.

Based on the above findings, it is considered that the bradion protein of the present invention enables non-divisional survival of central cerebral nerve cells through transmission of nerve stimulation. This is thought to be important in order to arrest cell lifespan in gene overexpression and to maintain the α and β type expression ratio (10: 1 in normal brain nerve cells). It was also suggested that a change in the expression ratio (for example, 1: 1) induces canceration. Therefore, Bradion protein is presumed to act as a cell life control / cancer control factor that determines long-term non-dividing survival after development / differentiation of brain nerve cells, and monitoring of neuronal loss with aging, It is useful for elucidating neuronal necrosis that occurs in cerebral ischemia and status epilepticus, for understanding the survival mechanism of nerve cells in the central nervous system, and for understanding the pathology of the brain.It is also useful for treating genetic diseases and creating new drugs. is there. Further, application to gene diagnosis and gene therapy for cancer is also possible.

Therefore, the protein of the present invention or a fragment thereof, an antibody thereto, or the DNA or a fragment thereof of the present invention can be used for cancer (for example, human colon cancer or human skin cancer).
Can be used for diagnosis or treatment for controlling cell lifespan and canceration. For such a purpose, a polyclonal or monoclonal antibody that monospecifically recognizes the protein is useful. Antibodies, particularly monoclonal antibodies, can be used in diagnostics, vaccines, drug delivery systems, and the like. For production of antibodies, see, for example, Nao Matsuhashi, Biochemistry Experimental Method 15, Introduction to Immunological Experiments (1982
Tatsuo Iwasaki et al., Monoclonal antibody-hybridoma and ELISA (1987), Kodansha Scientific, and the like.

The polyclonal antibody is prepared by using the protein of the present invention or a fragment thereof as an antigen, mixing the antigen solution with Freund's complete adjuvant to form an emulsion, and then preparing rabbit, mouse, goat, cow, horse, etc. The antigen can be obtained as an antiserum by subcutaneously injecting into a mammal, and about 2 weeks later, similarly injecting the antigen solution emulsified in Freund's incomplete adjuvant into the animal, boosting if necessary, and collecting blood. Further, IgG was obtained from the antiserum by salting out with ammonium sulfate, ion exchange chromatography using DEAE cellulose, etc., and the protein of the present invention or a fragment thereof was purified from CNBr.
The IgG fraction is applied to affinity chromatography bound to activated Sephadex or Sepharose, and the target antibody bound by immunoreaction can be purified monospecifically.

The monoclonal antibody was prepared by mixing the antigen-adjuvant emulsion prepared in the same manner as described above with a mouse (BALB / c
Etc.) into the intraperitoneal cavity to immunize mice, remove the spleen,
After obtaining the spleen cells, these cells were transformed into myeloma cells (X6
3, NS-1 etc.) and polyethylene glycol (PEG4
000), antibody-producing hybridomas are selected in a HAT medium, and the desired monoclonal antibody can be obtained from the cloning method or from ascites after intraperitoneal injection in mice. See also Teng et al., Proc. Nat
l. Acad. Sci. USA (1983), 8
0 : 7308-7312, Kozbor et al., Immun.
logic Today (1983), 4 (3): 72
It is also possible to produce humanized monoclonal antibodies using known techniques as described in -79. As an immunological method when the above antibody is used for diagnosis of cancer or the like,
Conventional immunoassays such as enzyme immunoassay, radioimmunoassay, and fluorescent antibody method can be used.

Further, when the DNA of the present invention or a fragment thereof is used as a probe or a primer, SEQ ID NO: 1
(Positions 129 to 1943) or SEQ ID NO: 3 (129 to 15
A probe or primer composed of any 15 or more, preferably 20 or more, more preferably 30 or more nucleotides in the nucleotide sequence shown at position 62) is prepared using an automatic DNA synthesizer, and isotopes, After labeling with a fluorescent substance or the like, it can be used as a diagnostic probe or PCR primer. Conditions for performing hybridization are described in Sambrook et al., Mole.
Cultural Cloning (supra); M. Aus
ubel et al., Short Protocols In
Molecular Biology, Third E
conditions, John Wiley & Sons.

[0039]

The present invention will be described in detail with reference to the following examples, but the present invention is not limited to these examples. Example 1 Cloning and Sequencing of cDNA Encoding Human Bradion First, a plasmid vector pCMV SPORT that can be connected to a CMV promoter for expression in eukaryotic cells
1 (Life Technologies (USA)) was used to construct a human adult brain cDNA library. At this time, the adult brain was a specimen from a 36-year-old Caucasian white woman in the United States, and Life Techno
logis Trizol reagent (trade name)
RNA (poly (A) RNA) extraction, message maker reagent (Life Technologies)
MRNA (poly (A) RNA) was purified using a Superscript plasmid system, and double-stranded cDNA synthesis / library construction was started.

The above-prepared mRNA (poly (A)
RNA) to the NotI primer adapter at the 3 'end, and then used as a reverse transcriptase SuperScr.
Using iptII and T4 DNA polymerase, double-stranded cDNA was synthesized and prepared according to a standard method. Then, cDN
A SalI adapter was added to the 5 ′ end of the A chain, and the 3 ′ end was treated with a restriction enzyme NotI to obtain a cDNA fragment having a restriction site Sal1-NotI at both ends. This cDNA was separated by size by gel filtration chromatography, and cDN having a size of 1 kb or more was obtained.
A was selected and sorted. The obtained cDNA fragment group was inserted into a plasmid vector pCMV SPORT1 similarly digested with Sal1-NotI, and a circular plasmid was prepared by a conventional method. This was introduced into Escherichia coli DH12S cells (Life Technologies) by electroporation, proliferated, and a library was constructed.

This group of Escherichia coli was grown on LB agar medium supplemented with antibiotic ampicillin to form colonies. A Biodyne A nylon membrane (Pall, USA) treated with 10 mM IPTG was closely adhered to the colonies and allowed to stand at 37 ° C. for 2 hours, and then the antibody CE5 [Nature, which recognizes the nylon membrane specifically for brain nerve cells] , 296 ,
34-38, (1982)].
ueTM Immunoscreening Kit
(STRATAGENE, USA) to select positive clones.

From the obtained positive clone, plasmid DN
A was collected and used as a 32P-labeled probe for a human membrane-specific mRNA transcribed nylon membrane (MTN blot,
(Clontech) and tested for brain specificity. The nucleotide sequence of the cDNA was determined by sequence analysis, compared with other sequences having homology from the gene bank, and only those found to be completely novel sequences were registered in the gene bank as being suitable for the purpose. The base sequence of the α-bradion cDNA thus determined is shown in SEQ ID NO: 1 (Genebank accession number: AB002110) in the sequence listing. The coding region was at positions 129-1943. The amino acid sequence of α-bradion determined based on this nucleotide sequence is shown in SEQ ID NO: 2. DNA containing α-Bradion cDNA
FERMP-1 was established on July 14, 1998 by the National Institute of Advanced Industrial Science and Technology (1-1-3 Higashi, Tsukuba, Ibaraki, Japan).
Deposited as 6897.

Based on the sequence of the α-bradion cDNA, a 5 ′ terminal primer (described below) was synthesized, and a systematic search for related genes was performed. For that, Gene Tra
paper Positive Selections
The above-mentioned gene library was screened using synthetic oligonucleotides and magnet beads using ystem (manufactured by Life Technologies). The sequence of the oligonucleotide used was 5′-
ctgagcaagttcgtgaagagattttc-
3 '(SEQ ID NO: 5) and 5'-cagtccctctga
caaccagcagta-3 ′ (SEQ ID NO: 6). As a result, a gene named β-bradion was detected, and its nucleotide sequence was shown in SEQ ID NO: 3 (Genebank accession number: AB008753) in the sequence listing. The coding region was at positions 129-1562. The amino acid sequence of β-bradion determined based on this nucleotide sequence is shown in SEQ ID NO: 4.

Example 2 Characterization of α and β Bradion (1) Hydropathy Analysis The amino acid sequence of the α and β bradion proteins determined in Example 1 was determined using the Kyte-Doolittle method (Kyte, J. and Doolittle). , RF.
J. , J. Mol. Biol. (1982), 15
7 (1): 105-132). This analysis method is one of the methods for estimating the higher-order structure from the amino acid sequence of a protein. The distribution of hydrophobic and hydrophilic parts in the protein can be known, and based on this information, the presence or absence of a three-dimensional structure and the membrane can be determined. It is possible to deduce the presence of the transmembrane domain. FIG. 1a and FIG. 1b show the analysis results, and at the same time, human-derived interleukin (IL)
Analysis results for the 2 receptor, IL3 receptor, IL4 receptor and growth hormone receptor are also shown for comparison. From the figure, it can be seen that the protein having the growth hormone / cytokine receptor sequence is located in the transmembrane region (3 from the left).
The second frame (shown in red) is the assembly of hydrophobic groups (appears as a plus in the calculation formula), the extracellular part in front of it (the second frame from the left (shown in blue)), and the rear cytoplasm It can be seen that it is roughly divided into the inner part (fourth frame from the left (shown in green)). These structures are common to each receptor, and it has been found that the bradion of the present invention has a similar structure. However, Bradion does not have a signal peptide (first frame from the left (shown in yellow)) which is a hydrophobic group portion. As a result, it was found that the bradion of the present invention is a membrane protein and has a structure characteristic of an interleukin receptor including its transmembrane portion (once) in its amino acid sequence.

(2) Localization of Bradion Protein mRNA transcribed from human organ-specific mRNA (MTNb)
(Lot, Clontech) showed strong expression in the human adult brain, and only slight expression (less than 10% of brain) in the heart. It was not expressed in other organs, the human fetus. In the adult brain, the above α and β types were expressed, and it was considered that the difference was caused by alternative splicing. Homologous gene sequences are also present in the mouse brain, but only one, of the two human types, was the beta type (94% homology) only.

(3) Experiments for Introducing α and β Bradion Genes into Cultured Cells α and β Bradion DNAs were transferred to cultured human neurons NT2 neuron (STRATAGENE), respectively.
That is, the undifferentiated human cultured neurons,
The gene was transfected using ct reagent (QIAGEN, USA) and overexpressed. The results are shown in FIGS. 2a, b and c.

Panel a in FIG. 2 includes α (upper row) and β
(Lower) Regarding the labeled image 24 hours after gene transfer of the Bradion gene, the positions of the α (upper) and β (lower) Bradions are shown on the left, the mitochondria are shown on the left, and the left and middle double on the right. The image was shown. All images were viewed with a confocal laser microscope. as a result,
α-Bradion showed the presence consistent with mitochondria (red + green and yellow color), but it was found that β-Bradion did not.

Further, after transfection of the α-bradion gene, 1
Although cell death is induced within 8 to 24 hours, it has been shown that α-bradion forms intracellular aggregates and accumulates in mitochondria in the course of death. Panel b shows cell images at each time indicated after gene transfer, and the left shows human cultured cells NT2neuron (STRATAGENE).
The right is human cancer cells HeLa. As a result, formation of cytoplasmic aggregates and cell death were observed in both cell lines. To confirm this fact, Panel b
As a result of electron microscopic observation using the cells, the presence of apoptotic bodies specific to programmed cell death (apoptosis) was confirmed, as shown in panel c.

(4) Correlation between α and β Bradion genes and cancer The α and β Bradion genes are expressed in normal tissues, although they are expressed only in the brain and very little (about 10% of the brain) in the heart. However, expression in cultured cancer cells was observed. The results are shown in FIGS. 3a, 3b and 3c. Fig. 3a shows the results of Northern blotting for α and β bradion gene expression assays in different cultured human cancer cells. Lane 8 (skin cancer cell line G361) and lane 6
A specific expression reaction (signal) was observed only in (colorectal cancer cell line SW480).

Therefore, when expression tests were searched using human patient specimens (pathological tissue specimens), as shown in FIG. 3b, 10 colon cancers (T1 to T10; described as Ad) and 3 Cancer (T11 to T13; Muc, MM
Specific expression). FIG. 3c
Shows the gene expression confirmation as a tissue staining image. The above results indicate that α and β bradions and the genes encoding them can be used as tumor markers for cancer such as colon cancer and skin cancer.

[0051]

It is considered that the bradion of the present invention and the DNA encoding the same enable non-mitotic survival in central cerebral nerve cells through transmission of neural stimulation. This instructs the arrest of cell life in gene overexpression, and it is considered that the expression ratio of α and β types (10: 1 in normal brain nerve cells) is maintained in vivo. It is also suggested that this change in expression ratio (eg, 1: 1) induces canceration. Therefore, Bradion and the DNA encoding it are presumed to function as cell life control / carcinogenesis controlling factors that determine long-term survival of the brain nerve cells in a non-dividing state after development / differentiation. It is useful for monitoring the loss of nerve cells, elucidating neuronal necrosis that occurs in cerebral ischemia and status epilepticus, understanding the mechanism of neuronal survival in the central nervous system, understanding the pathology of the brain, treating genetic diseases, and treating new diseases. It is also useful for drug discovery. Further, application to gene diagnosis and gene therapy for cancer is also possible.

[0052]

[Sequence List] SEQUENCE LISTING <110> Agency of Industrial Science and Technology <120> Cancer detecting methods <130><140><141><160> 6 <170> Windows 95 <210> 1 <211> 2274 <212> DNA <213> Homo sapiens <220><221> mat peptide <222> (129) ··· (1943) <400> 1 gaaaggagca agccaggaag ccagacaaca acagcatcaa aacaaggctg tttctgtgtg 60 tgaggaactt tgcctgggag ataaaattag acctagagct ttctgacagg gagtctgaag 120 cgtgggacat ggaccgttca ctgggatggc aagggaattc tgtccctgag gacaggactg 180 aacctgggat caaccgtttc ctggaggaca ccacggatga tggagaactg agcaagttcg 240 tgaaggattt ctcaggaaat gcgagctgcc acccaccaga ggctaagacc tgggcatcca 300 ggccccaagt cccggagcca aggccccagg ccccggacct ctatgatgat gacctggagt 360 tcagaccccc ctcgcggccc cagtcctctg acaaccagca gtacttctgt gccccagccc 420 ctctcagccc atctgccagg ccccgcagcc catgggggga gcttgatccc tatgattcct 480 ctgaggtaga gcctccagcc ctgcctttgc ctttcagtgg gctgctgcag gaagaccggg 540 ggcagggagc aggaatgtgt gtgtgtgtgt gtgtgtgtgt gtgtgtgtgt gtgtttgtgt 600 gtgtgtgtat ctgggaccca tttcagtcct gtgtcagccc tagctccaaa atatctgccc 660 ccaagggcac tggaaatttg cagtttcagc aagggcagga ggcccagctg gtggcctcag 720 atgggaactc acagaagtct ggcactgctt ttttaaggct ggggcaaagg cctgaaaggg 780 agagaagatt ggcgctgggt gccggggccc ctttggctcc tcac cgtgat gcattctgcc 840 ttcctgtcta ctacgatgac aaggagtatg tgggctttgc aaccctcccc aaccaagtcc 900 accgaaagtc cgtgaagaaa ggctttgact ttaccctcat ggtggcagga gagtctggcc 960 tgggcaaatc cacacttgtc aatagcctct tcctcactga tctgtaccgg gaccggaaac 1020 ttcttggtgc tgaagaaagg atcatgcaaa ctgtggagat cactaagcat gcagtggaca 1080 tagaaaaaaa aggtgtgagg ctgcggctca ccattgtgga cacaccaagt tttggggatg 1140 cagtcaacaa cacagagtgt atgtctgact ggaagcctgt ggcagaatac attgatcagc 1200 agtttgagca gtatttccga gacgagagtg gcctgaaccg aaagaacatc caagacaaca 1260 gggtgcactg ctgcctgtac ttcatctcac ccttcggcca tgggctccgg ccattggatg 1320 ttgaattcat gaaggccctg catcagcggg tcaacatcgt gcctatcctg gctaaggcag 1380 acacactgac acctcccgaa gtggaccaca agaaacgcaa aatccgggag gagattgagc 1440 attttggaat caagatctat caattcccag actgtgactc tgatgaggat gaggacttca 1500 aattgcagga ccaagcccta aaggaaagca tcccatttgc agtaattggc agcaacactg 1560 tagtagaggc cagagggcgg cgagttcggg gtcgactcta cccctggggc atcgtggaag 1620 tggaaaaccc agggcactgc gactttgtga agctgaggac aatgctggta cg tacccaca 1680 tgcaggacct gaaggatgtg acacgggaga cacattatga gaactaccgg gcacagtgca 1740 tccagagcat gacccgcctg gtggtgaatg aacggaatcg caagtatgac cagaagccag 1800 gacaaagctg gcagggggag atcccaagcc tagccttggg tgagaccaag ccctactttt 1860 gttcttctat aggccctggg ctcaatctaa gcgggtgctg gggtcctcct cgccttatca 1920 acccttttct ccctttagca aactgactcg ggaaagtggt accgacttcc ccatccctgc 1980 tgtcccacca gggacagatc cagaaactga gaagcttatc ccagagaaag attaggagct 2040 gcggcggata cacgagatac tacaccaaat accaaaacag ataaaggaga actatttact 2100 ggctttcagc cctggatatt taaatctcct cctcttcttc ctgtccatgc cggcccctcc 2160 cagcaccagc tctgctcagg ccccttcagc tactgccact tcgccttaca tccctgctga 2220 ctgcccagag actcagagga aataaagttt aataaatctg taggtggctt ctgg 2274 <210> 2 <211> 605 <212> PRT <213> Homo sapiens <400> 2 Met Asp Arg Ser Leu Gly Trp Gln Gly Asn Ser Val Pro Glu Asp Arg 1 5 10 15 Thr Glu Pro Gly Ile Asn Arg Phe Leu Glu Asp Thr Thr Asp Asp Gly 20 25 30 Glu Leu Ser Lys Phe Val Lys Asp Phe Ser Gly Asn Ala Ser Cys His 35 40 45 Pro Pro Glu Ala Lys Thr Trp Ala Ser Arg Pro Gln Val Pro Glu Pro 50 55 60 Arg Pro Gln Ala Pro Asp Leu Tyr Asp Asp Asp Leu Glu Phe Arg Pro 65 70 75 80 Pro Ser Arg Pro Gln Ser Ser Asp Asn Gln Gln Tyr Phe Cys Ala Pro 85 90 95 Ala Pro Leu Ser Pro Ser Ala Arg Pro Arg Ser Pro Trp Gly Glu Leu 100 105 110 Asp Pro Tyr Asp Ser Ser Glu Val Glu Pro Pro Ala Leu Pro Leu Pro 115 120 125 Phe Ser Gly Leu Leu Gln Glu Asp Arg Gly Gln Gly Ala Gly Met Cys 130 135 140 Val Cys Val Cys Val Cys Val Cys Val Cys Val Phe Val Cys Val Cys 145 150 155 160 Ile Trp Asp Pro Phe Gln Ser Cys Val Ser Pro Ser Ser Lys Ile Ser 165 170 175 Ala Pro Lys Gly Thr Gly Asn Leu Gln Phe Gln Gln Gly Gln Glu Ala 180 185 190 Gln Leu Val Ala Ser Asp Gly Asn Ser Gln Lys Ser Gly Thr Ala Phe 195 200 205 Leu Arg Leu Gly Gln Arg Pro Glu Arg Glu Arg Arg Leu Ala Leu Gly 210 215 220 Ala Gly Ala Pro Leu Ala Pro His Arg Asp Ala Phe Cys Leu Pro Val 225 230 235 240 Tyr Tyr Asp Asp Lys Glu Tlu Val Gly Phe Ala Thr Leu Pro Asn Gln 245 250 255 Val His Arg Lys S er Val Lys Lys Gly Phe Asp Phe Thr Leu Met Val 260 265 270 Ala Gly Glu Ser Gly Leu Gly Lys Ser Thr Leu Val Asn Ser Leu Phe 275 280 285 Leu Thr Asp Leu Tyr Arg Asp Arg Lys Leu Leu Gly Ala Glu Glu Arg 290 295 300 Ile Met Gln Thr Val Glu Ile Thr Lys His Ala Val Asp Ile Glu Lys 305 310 315 320 Lys Gly Val Arg Leu Arg Leu Thr Ile Val Asp Thr Pro Ser Phe Gly 325 330 335 Asp Ala Val Asn Asn Thr Glu Cys Met Ser Asp Trp Lys Pro Val Ala 340 345 350 Glu Tyr Ile Asp Gln Gln Phe Glu Gln Tyr Phe Arg Asp Glu Ser Gly 355 360 365 Leu Asn Arg Lys Asn Ile Gln Asp Asn Arg Val His Cys Cys Leu Tyr 370 375 380 380 Ile Ser Pro Phe Gly His Gly Leu Arg Pro Leu Asp Val Glu Phe 385 390 395 400 400 Met Lys Ala Leu His Gln Arg Val Asn Ile Val Pro Ile Leu Ala Lys 405 410 415 Ala Asp Thr Leu Thr Pro Pro Glu Val Asp His Lys Lys Arg Lys Ile 420 425 430 Arg Glu Glu Ile Glu His Phe Gly Ile Lys Ile Tyr Gln Phe Pro Asp 435 440 445 Cys Asp Ser Asp Glu Asp Glu Asp Phe Lys Leu Gln Asp Gln Ala Leu 450 455 460 Lys Glu Ser Ile Pro P he Ala Val Ile Gly Ser Asn Thr Val Val Glu 465 470 475 480 Ala Arg Gly Arg Arg Val Arg Gly Arg Leu Tyr Pro Trp Gly Ile Val 485 490 495 Glu Val Glu Asn Pro Gly His Cys Asp Phe Val Lys Leu Arg Thr Met 500 505 510 Leu Val Arg Thr His Met Gln Asp Leu Lys Asp Val Thr Arg Glu Thr 515 520 525 His Tyr Glu Asn Tyr Arg Ala Gln Cys Ile Gln Ser Met Thr Arg Leu 530 535 540 Val Val Asn Glu Arg Asn Arg Lys Tyr Asp Gln Lys Pro Gly Gln Ser 545 550 555 560 Trp Gln Gly Glu Ile Pro Ser Leu Ala Leu Gly Glu Thr Lys Pro Tyr 565 570 575 Phe Cys Ser Ser Ile Gly Pro Gly Leu Asn Leu Ser Gly Cys Trp Gly 580 585 590 590 Pro Pro Arg Leu Ile Asn Pro Phe Leu Pro Leu Ala Asn 595 600 605 <210> 3 <211> 1735 <212> DNA <213> Homo sapiens <220><221> mat peptide <222> (129) ··· (1562) <400> 3 gaaaggagca agccaggaag ccagacaaca acagcatcaa aacaaggctg tttctgtgtg 60 tgaggaactt tgcctgggag ataaaattag acctagagct ttctgacagg gagtctgaag 120 cgtgggacat ggaccgttca ctgggatggc aagggaattc tgtccctgag gacaggactg 180 aagctgggat caagcgtttc ctggaggaca ccacggatga tggagaactg agcaagttcg 240 tgaaggattt ctcaggaaat gcgagctgcc acccaccaga ggctaagacc tgggcatcca 300 ggccccaagt cccggagcca aggccccagg ccccggacct ctatgatgat gacctggagt 360 tcagaccccc ctcgcggccc cagtcctctg acaaccagca gtacttctgt gccccagccc 420 ctctcagccc atctgccagg ccccgcagcc catggggcaa gcttgatccc tatgattcct 480 ctgaggatga caaggagtat gtgggctttg caaccctccc caaccaagtc caccgaaagt 540 ccgtgaagaa aggctttgac tttaccctca tggtggcagg agagtctggc ctgggcaaat 600 ccacacttgt caatagcctc ttcctcactg atctgtaccg ggaccggaaa cttcttggtg 660 ctgaagagag gatcatgcaa actgtggaga tcactaagca tgcagtggac atagaagaga 720 agggtgtgag gctgcggctc accattgtgg acacaccagg ttttggggat gcagtcaaca 780 acacagagtg ctggaagcct gtggcagaat acattgatca gcag tttgag cagtatttcc 840 gagacgagag tggcctgaac cgaaagaaca tccaagacaa cagggtgcac tgctgcctgt 900 acttcatctc acccttcggc catgggctcc ggccattgga tgttgaattc atgaaggccc 960 tgcatcagcg ggtcaacatc gtgcctatcc tggctaaggc agacacactg acacctcccg 1020 aagtggacca caagaaacgc aaaatccggg aggagattga gcattttgga atcaagatct 1080 atcaattccc agactgtgac tctgatgagg atgaggactt caaattgcag gaccaagccc 1140 taaaggaaag catcccattt gcagtaattg gcagcaacac tgtagtagag gccagagggc 1200 ggcgagttcg gggtcgactc tacccctggg gcatcgtgga agtggaaaac ccagggcact 1260 gcgactttgt gaagctgagg acaatgctgg tacgtaccca catgcaggac ctgaaggatg 1320 tgacacggga gacacattat gagaactacc gggcacagtg catccagagc atgacccgcc 1380 tggtggtgaa ggaacggaat cgcaacaaac tgactcggga aagtggtacc gacttcccca 1440 tccctgctgt cccaccaggg acagatccag aaactgagaa gcttatccga gagaaagatg 1500 aggagctgcg gcggatgcag gagatgctac acaaaataca aaaacagatg aaggagaact 1560 attaactggc tttcagccct ggatatttaa atctcctcct cttcttcctg tccatgccgg 1620 cccctcccag caccagctct gctcaggccc cttcagctac tgccacttcg cc taacatcc 1680 ctgctgactg cccagagact cagaggaaat aaagtttaat aaatctgtag gtggc 1735 <210> 4 <211> 478 <212> PRT <213> Homo sapiens <400> 4 Met Asp Arg Ser Leu Gly Trp Gln Gly Asn Ser Val Pro Glu Asp Arg 5 Thr Glu Ala Gly Ile Lys Arg Phe Leu Glu Asp Thr Thr Asp Asp Gly 20 25 30 Glu Leu Ser Lys Phe Val Lys Asp Phe Ser Gly Asn Ala Ser Cys His 35 40 45 Pro Pro Glu Ala Lys Thr Trp Ala Ser Arg Pro Gln Val Pro Glu Pro 50 55 60 Arg Pro Gln Ala Pro Asp Leu Tyr Asp Asp Asp Leu Glu Phe Arg Pro 65 70 75 80 Pro Ser Arg Pro Gln Ser Ser Asp Asn Gln Gln Tyr Phe Cys Ala Pro 85 90 95 Ala Pro Leu Ser Pro Ser Ala Arg Pro Arg Ser Pro Trp Gly Lys Leu 100 105 110 Asp Pro Tyr Asp Ser Ser Glu Asp Asp Lys Glu Tyr Val Gly Phe Ala 115 120 125 Thr Leu Pro Asn Gln Val His Arg Lys Ser Val Lys Lys Gly Phe Asp 130 135 140 Phe Thr Leu Met Val Ala Gly Glu Ser Gly Leu Gly Lys Ser Thr Leu 145 150 155 160 Val Asn Ser Leu Phe Leu Thr Asp Leu Tyr Arg Asp Arg Lys Leu Leu 165 170 175 Gly Ala Glu Glu Arg Ile Met Gln Thr Val Glu Ile Thr Lys His Ala 180 185 190 Val Asp Ile Glu Glu Lys Gly Val Arg Leu Arg Leu Thr Ile Val Asp 195 200 205 Thr Pro Gly Phe Gly Asp Ala Val Asn Asn Thr Glu Cys Val Lys Pro 210 215 220 Val Ala Glu Tyr Ile Asp Gln Gln Phe Glu Gln Tyr Phe Arg Asp Glu 225 230 235 240 Ser Gly Leu Asn Arg Lys Asn Ile Gln Asp Asn Arg Val His Cys Cys 245 250 255 Leu Tyr Phe Ile Ser Pro Phe Gly His Gly Leu Arg Pro Leu Asp Val 260 265 270 Glu Phe Met Lys Ala Leu His Gln Arg Val Asn Ile Val Pro Ile Leu 275 280 285 Ala Lys Ala Asp Thr Leu Thr Pro Pro Glu Val Asp His Lys Lys Arg 290 295 300 Lys Ile Arg Glu Glu Ile Glu His Phe Gly Ile Lys Ile Tyr Gln Phe 305 310 315 320 Pro Asp Cys Asp Ser Asp Glu Asp Glu Asp Phe Lys Leu Gln Asp Gln 325 330 335 Ala Leu Lys Glu Ser Ile Pro Phe Ala Val Ile Gly Ser Asn Thr Val 340 345 350 Val Glu Ala Arg Gly Arg Arg Val Arg Gly Arg Leu Tyr Pro Trp Gly 355 360 365 Ile Val Glu Val Glu Asn Pro Gly His Cys Asp Phe Val Lys Leu Arg 370 375 380 Thr Met Leu Val Arg Thr His Met Gln Asp Leu Lys Asp Val Thr Arg 385 390 395 400 400 Glu Thr His Tyr Glu Asn Tyr Arg Ala Gln Cys Ile Gln Ser Met Thr 405 410 415 Arg Leu Val Val Lys Glu Arg Asn Arg Asn Lys Leu Thr Arg Glu Ser 420 425 430 Gly Thr Asp Phe Pro Ile Pro Ala Val Pro Pro Gly Thr Asp Pro Glu 435 440 445 Thr Glu Lys Leu Ile Arg Glu Lys Asp Glu Glu Leu Arg Arg Met Asp 450 455 460 Glu Met Leu His Lys Ile Gln Lys Gln Met Lys Glu Asn Tyr 465 470 475 <210> 5 <211> 24 <212> DNA <213> Artificial Sequence <220><223> Description of the artificial sequence: a 5'-end primer synthesize d based on the nucleotide sequence of cDNA for alpha-Bradeion <400> 5 ctgagcaagt tcgtgaagga tttc 24 <210> 6 <211> 23 <212> DNA <213> Artificial Sequence <220><223> Description of the artificial sequence: a 5'-end primer synthesize d based on the nucleotide. sequence of cDNA for alpha-Bradeion. <400> 6 cagtcctctg acaaccagca gta 23

[0053]

[Sequence List Free Text] SEQ ID NO: 5—Description of synthetic sequence: 5′-end primer synthesized based on base sequence of β-Bradion cDNA SEQ ID NO: 6—Description of synthetic sequence: α-Bradion cDNA base 5'-end primer synthesized based on sequence

[Brief description of the drawings]

FIG. 1A shows the distribution of the hydrophobic and hydrophilic parts of α-bradion in comparison with the IL2, IL3 and IL4 receptors and the growth hormone receptor.
-It is a photograph which shows the result of the hydropathy analysis by the Doolittle method.

FIG. 1B shows the distribution of the hydrophobic and hydrophilic parts of α-bradion in comparison with the IL2, IL3 and IL4 receptors and the growth hormone receptor.
-Photographs showing the results of hydropathy analysis by the Doolittle method (continuation of Fig. 1a).

FIG. 2 shows that α and β bradion genes were cultured in NT2 neuron (undifferentiated human neurons) and He
Labeled image by confocal laser microscope when introduced into La cells and overexpressed (NT2new in the case of panel a)
on; NT2neuron and HeL for panel b
a) and the cells of panel b (18 hrs and 24 hr
It is a photograph which shows the electron microscope image (panel c) about s).
In panel a, EGFP (Enhanced Green)
Fluorescent Protein; Clon
tech.), Mitochondria shows the mitochondrial location of the bradion gene, and Overlay shows the superimposed image of the left label image and the middle label image.

FIG. 3a is a photograph showing equivalent expression of α and β bradion genes in a human cancer cell line. The result of Northern blot analysis using a radiolabeled Bradion gene is shown. Lane 1 shows multiple myelocytic leukemia (po
lymyolyticuclemia), HL6
0; lane 2 is HeLaS3; lane 3 is chronic myologous leukemia (chronic myologen).
ous leukemia), K-562; lane 4
Is lymphoblastic leukemia (lymphoblastic)
laukemia), MOLT-4; lane 5 shows Burkitt's lymphoma.
ma), Raji; lane 6 shows colorectal cancer (colore)
tal adenocarcinoma), SW48
0 SW48021, 22; Lane 7 shows lung cancer (run
gcarcinoma), A549; lane 8 is myeloma, G361. As a positive control, the results of Northern blot analysis using the beta actin gene are shown below.

FIG. 3b shows cancer-specific expression of the bradion gene in colorectal cancer patient samples. RT-PCR method, i
Colorectal cancer (T
1 to T10), skin cancer (T11 to T13), and measurement results of Bradion gene expression in normal cells (N1 to N2). In the figure, * indicates that both the α and β bradion genes were detected and no intragenic mutation was observed, ND indicates that the RNA was undetectable due to degradation of RNA, and Ad (well) indicates that Ad (mod) indicates adenocarcinoma (moderately differentiated), Muc indicates mucin-secreting adenocarcinoma, and MM indicates malignant melanoma.
The standard gene sequence of codon 12 of the K-ras gene is GG
T is described as having T within the gene.
This mutation occurs in one of a pair of chromosomes.

FIG. 3c is a photograph showing the result of in situ hybridization of a human cancer tissue specimen. FIG. 3b
Tissue staining for T13 and T8 in
(sense: positive, Sense: negative control).

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI Theme coat ゛ (Reference) // C07K 14/715 C07K 16/28 16/28 C12P 21/08 C12P 21/08 C12N 15/00 ZNAA

Claims (3)

[Claims] 1. Use of the following DNA or its fragment or antibody in detecting cancer: (1) D encoding human-derived bradion protein having the amino acid sequence shown in SEQ ID NO: 2 or SEQ ID NO: 4
NA or a fragment thereof comprising a sequence of 15 or more consecutive nucleotides; (2) having an amino acid sequence in which at least one amino acid is deleted, substituted or added in the amino acid sequence shown in SEQ ID NO: 2 or SEQ ID NO: 4; And the following properties: (a) it is a membrane protein; (b) it has a structure characteristic of an interleukin receptor including a transmembrane part (once) by hydropathy analysis by the Kyte-Doolittle method; (c) Strong expression is observed in the human adult brain, less than 10% of the brain expression in the heart, but not in other organs or human fetuses. (D) When overexpressed in undifferentiated cultured human neurons ,
Induces cell death, (e) induces senescence and division arrest when overexpressed in cultured normal human cells, (f) forms intracellular aggregates in the cytoplasm in the process of cell death, resulting in 24 hours Accumulate in mitochondria within
And (g) a DNA encoding a human-derived bradion protein analog, which is specifically expressed in a human colon cancer cell line or a skin cancer cell line, and which can be obtained by a DNA recombination technique, or 15 or more A fragment thereof comprising a sequence of consecutive nucleotides; (3) a DNA comprising the nucleotide sequence at positions 129 to 1943 shown in SEQ ID NO: 1 or a DNA hybridizable therewith under stringent conditions, or 15 or more consecutive nucleotides (4) a DNA consisting of the nucleotide sequence at positions 129 to 1562 shown in SEQ ID NO: 3 or a DNA hybridizable therewith under stringent conditions, or a sequence of 15 or more consecutive nucleotides (5) codifying the bradion contained in the deposited microorganism FERM BP-6922 Or a fragment thereof consisting of a sequence of 15 or more consecutive nucleotides, or (6) a human-derived braid encoded by the DNA or a fragment thereof according to any of the above (1) to (5). Antibodies to Dion protein, its analogs, or fragments thereof. 2. The method according to claim 1, wherein the cancer is human colon cancer or human skin cancer. 3. The method according to claim 1, wherein the detection is performed by hybridization or immunoassay.
The method described in.