JPH05146294A - Cancer-related protein - Google Patents

Abstract

PURPOSE:To obtain a new cancer-related protein, having the ability to bind to a silencer sequence present on the upstream side of a rat hepatic catalase gene and useful for early diagnosis of cancer diseases as an objective antigen in an immunoassay system. CONSTITUTION:A protein composed of a specific amino acid sequence originating from a rat hepatic cancerous cell AH-60C strain or a substance having the same effects as those of the protein in which its amino acid residue is deficient, substituted or inserted. This protein is obtained by preparing a cDNA library for an mRNA purified from, e.g. a cell derived from rat hepatic cancer, using an oligonucleotide corresponding to a silencer sequence on the upstream side of a rat hepatic catalase gene as a probe, then selecting a cDNA having the ability to bind to the probe from the cDNA library, integrating the cDNA into an expression vector, subsequently transforming a host with the resultant expression vector and expressing the cDNA.

Description

Detailed Description of the Invention

[Object of the Invention]

[0002]

TECHNICAL FIELD The present invention relates to a cancer-related protein, a DNA encoding the protein, a DNA expression vector comprising the DNA, a host transformed with the expression vector, and a method for producing the protein. ..

[0003]

2. Description of the Related Art Cancer can be diagnosed by palpation of a mass, general symptoms such as fever, pleural effusion or ascites, fibrinolysis and leukocyte increase, or malignant tumor cell Secreted substances, α-fetoprotein, carcinoembryonic antigen (Carcino Enbryonic Antigen
: CEA), immunoglobulin, gastrin, insulin, serotonin, erythropoietin, etc. are generally used for diagnosis based on abnormal increase in blood.

However, with the recent advances in biotechnology, it is expected that more sensitive and highly specific diagnostic methods will be developed. This is because the use of such means enables more accurate and objective diagnosis.

When cancer growth progresses in a cancer-bearing living body,
It is known that living organisms fall into the state of epidemics (caquexia) and eventually die. In the tumor-bearing rat, the catalase activity of the liver is remarkably reduced, and further, when the cancer tissue is extracted from the tumor-bearing rat, the catalase activity is reduced.
It was reported by Greenstein in the 1940s that the zeal activity was restored (Greenstein, JP, Jenrett.
e, WV, and White, J .; J. Natl. Cancer Inst. 2 , 2
8, (1941)). From this fact, it was considered that some factor is secreted from the cancer tissue and the factor suppresses the catalase activity in the liver.

In 1948, Nakahara et al. Revealed that injection of an extract of cancer tissue into mice suppressed hepatic catalase activity (Nakahara, W. and Fukuoka,
F .; Japan Med. J. 1 , 271, (1948)) failed to purify the factors involved in the suppression.

[0007]

DISCLOSURE OF THE INVENTION The present inventors have investigated these phenomena from the viewpoint of regulating the expression of the catalase gene, and as a result, have found that they bind to a specific silencer sequence existing upstream of the gene. The present invention was completed by finding that a protein having the function has a relationship with cancer and determining the amino acid sequence and nucleotide sequence of the protein and the cDNA encoding the protein.

[Structure of Invention]

[0009]

That is, the present invention is
(1) Amino acid numbers 1 to 463 shown in SEQ ID NO: 2 in the sequence listing
A protein consisting of the amino acid sequence of, or at one or more positions thereof, one or more amino acid residues are deleted, substituted or inserted,
(2) a protein having the amino acid sequence of amino acid numbers 1 to 463 shown in SEQ ID NO: 2 in the sequence listing,
(3) Amino acid numbers 1 to 464 shown in SEQ ID NO: 4 in the sequence listing
A protein consisting of the amino acid sequence of, or at one or more positions thereof, one or more amino acid residues are deleted, substituted or inserted,
(4) A protein comprising the amino acid sequence of amino acid numbers 1 to 464 shown in SEQ ID NO: 4 of the sequence listing, (5) a DNA encoding the protein of (1), (6) (2) DNA coding for the protein described, (7) (3) DNA coding for the protein, (8) (4) DNA coding for the protein, (9) Sequence listing Nucleotide number 1 shown in SEQ ID NO: 1
(5) or (6), which comprises the nucleotide sequence from 1 to 1389, (10) consisting of the nucleotide sequence from nucleotide number 1 to 1392 shown in SEQ ID NO: 3 in the sequence listing (7) or (8)
DNA described in (11) (5), (6), (7), (8), (9) or DN described in (10)
A DNA expression vector, which is composed of A and is contained so that the DNA is expressible and replicable, and a host transformed with the DNA expression vector according to (12) (11), (13) (1), ( 2), (3) or (4) described in the host, by transforming the host with a DNA expression vector comprising a DNA coding for the protein described in (4), such that the DNA is expressible and replicable. The present invention relates to a method for producing the protein according to (1), (2), (3) or (4), wherein the protein according to (1), (2), (3) or (4) is recovered.

The protein of the present invention described in (1), (2), (3) or (4) is a known silencer sequence existing in the upstream of the rat liver catalase gene (Table 1: 50). Presented at the Annual Meeting of the Japanese Cancer Society)).

Of the proteins of the present invention, preferably (2) or
It is the protein described in (4).

[0012]

[Table 1] Table 1 -------------------------------------------------- 5'-GGTTTTGGGGGGGAGGG-3 ' CCCCCCTCCCCCAAA-5 ′ −−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−− DNA encoding the protein of the present invention Can be manufactured by the following steps.

M from animal cells such as rat liver cancer-derived cells
RNA is purified and a cDNA library for it is prepared.

An oligonucleotide corresponding to the above silencer sequence was prepared, and this oligonucleotide was used as a probe.
Regarding the above cDNA library, the SouthWestern method (Miskimins, WK et al .: Pro
c. Natl. Acad. Sci. USA, 82 , 6741, (1985): Singh,
H. et al .: Cell, 52 , 415, (1982))
And a cDNA encoding a protein capable of binding to the probe is selected.

When it is presumed that the cDNA does not encode the whole of the above protein, the cDNA is further used as a probe to screen the above cDNA library by a conventional hybridization method. CDNA of the entire chain length that encodes the entire protein
Select. Thereby, the DNA of the present invention can be obtained.

This finally obtained cDNA is incorporated into a suitable expression vector, and the vector is used to
The protein of the present invention can be obtained by transforming a suitable host in the combination with the vector and expressing the cDNA.

Each of these steps will be described in detail below.

As the cell line used for preparing the cDNA library described in the above, preferably, a cell line derived from rat liver cancer such as AH60c can be used, but other than this, the silencer shown in Table 1 can be used. Any cell line can be used, so long as it is a cell line that actively expresses a protein capable of binding to the sequence. Such cell lines include, for example, rat liver cancer-derived strains AH 66 and Ac2F, human liver cancer-derived strains.
Hep G2 and the like can be mentioned.

A total RNA fraction and further an mRNA fraction were prepared from these cell lines, single-stranded cDNA was synthesized using this mRNA as a template and reverse transcriptase, and then this cD
Double-stranded DNA can be prepared based on NA.

For extraction of total RNA from cancer cells, the guanidine thiocyanate hot phenol method, the guanidine thiocyanate guanidine hydrochloride method and the like can be adopted, but the guanidine thiocyanate phenol -Le Chloroform method (Chomczynski, P. and Sacc
hi, N.: Anal. Biochem. 162 , 156, (1987)) is preferred.

Most of the mRNAs present in the cytoplasm of eukaryotic cells are known to have a poly A sequence at their 3'ends, and this sequence can be used for oligo (dT) cellulosic column. The mRNA fraction can be purified by adsorbing the mRNA and subsequently eluting it. Furthermore, mRNA can be further fractionated by a sucrose density gradient centrifugation method or the like.

As a method for synthesizing a single strand using the obtained mRNA as a template using a reverse transcriptase and then synthesizing a double stranded DNA based on this cDNA, the S1 nuclease method (Efstratiadis, A . et al. (1976) Cell 7, 279-288), Ok
ayama-Berg method (Okayama, H. and Berg, P. (1982) Mol.Cell.Bi
ol. 2 , 161-170), Land method (Land, H. et al . (1981) Nucleic
Acids Res. 9 , 2251-2266), O.Joon Yoo method (Yoo, OJ et a
l. (1983) Proc.Natl.Acad.Sci.USA 79, 1049-1053) , etc. may be employed but, for the purposes of the present invention is suitably Land method.

The resulting double-stranded DNA is added to EcoRI adapter
To connect Ec of Lambda Phase (λφ) Vector
integrated by inserting into oRI cleavage site, cDNA
A library can be created. The lambda fuzz vector used here is preferably .lambda.gt11 or .lambda.ZAP.

Preparation of oligonucleotides corresponding to the silencer sequences of Table 1 described in, for example, the phosphite triester method (Hunkapiller, M. et al . (1984) Na.
ture 310 , 105-111) and the like, and can be produced by chemical synthesis of nucleic acid. This cDNA was labeled with ³² P or the like and used as a probe to prepare a cDNA library described in South Western (South Western).
) Method can be used for screening.

That is, the described recombinant lambda far.
The vector is infected with E. coli and then cultured on agar medium to form plaques. In addition, IPTG (Iso-p
ropyl thio β-D-galactoside) or other promoter-inducing agent to induce the phage vector,
Express a foreign protein in the mouse.

In the present invention, the target cDNA is λgt
Since it is located under the control of 11 lactose operators, it is possible to express the cDNA by inducing the promoter with an inducer such as IPTG.

The foreign protein thus expressed can be immobilized on a nitrocellulose membrane and subjected to the following screening operation.

That is, using the above ³² P-labeled synthetic oligonucleotide, a clone expressing a protein capable of binding to the oligonucleotide probe among clones attached to the above nitrocellulose membrane. Select the option.

From this positive clone, a phage DNA containing the cDNA of interest was isolated, and the phage DNA was isolated.
A desired DNA can be obtained by cutting out the target double-stranded DNA from

In this screening, in order to increase the binding ability and the screening efficiency, a polymer obtained by ligating a plurality of the above-mentioned synthetic probes is labeled with ³² P. It is more preferable to use.

The DNA thus obtained can be sequenced by, for example, the Maxam-Gilbert chemical modification method (Maxam,
AMand Gilbert, W. (1980): “Methods in Enzymology”
65 , 499-559) and the dideoxynucleotide chain termination method using M13 phage (Messing, J. and Vieira, J. (1982) Gene 1
9 , 269-276) etc.

The determination of whether or not the obtained cDNA was a full-length cDNA encoding the entire desired protein was determined by using the cDNA as a labeling probe and using it as an initial material. The total RNA fraction was prepared from animal cells and analyzed by Northern blotting to determine the corresponding mR
This can be done by comparing the molecular weight of NA with that of the cDNA.

When the cDNA is not the full-length cDNA, the secondary screen is carried out by the method described in.
Can be done.

That is, the plaque (λZAP obtained by
) Is transferred to a Hybond filter (manufactured by Amersham). On the other hand, the partial cDN obtained in
A is labeled with ³² P, and this is used as a probe to select positive clones from the above plaques by the technique of plaque hybridization.

Further, according to the method described above, Table 1
It is possible to obtain a full-length double-stranded DNA encoding a protein capable of binding to the silencer sequence of

Further, the DNA of the present invention can be obtained by the method described below.

(A) Prepared by polymerase chain reaction
Screening method using probe Using all or part of the amino acid sequence of the target protein determined above, oligonucleotides of sense strand and antisense strand corresponding to a part of the amino acid are synthesized, and these are combined to form a polymerase chain. Reaction (Sai
ki, RK et al . (1988) Science 239 , 487-491),
A DNA fragment encoding the target protein or its equivalent is amplified. The template DNA used here was synthesized by reverse transcription from mRNA of cells producing the protein.
cDNA or genomic DNA can be used.
The DNA fragment thus prepared is labeled with ³² P or ³⁵ S, and is used as a probe to perform colony hybridization or plaque hybridization to select a target clone.

(B) Selection using an antibody against the target protein
In advance, the cDNA is incorporated into an expression vector, a protein is produced in the transformant, and the desired protein-producing strain is detected using an antibody against the protein and a secondary antibody against the antibody, and the target strain is selected. To do.

(C) Selective hybridization
Using the translation system, the cDNA obtained from the transformant is blotted on a nitrocellulose filter, hybridized with the mRNA from the target protein-producing cells, and the mRNA bound to the cDNA is dissociated and recovered. To do. Recovered mRNA
A protein translation system, for example, injection into Xenopus oocytes or a cell-free system such as rabbit reticulocyte lysate or wheat germ, which is detected using an antibody against the target protein, and the target strain is detected. Select.

DNA obtained by such various methods
As a DNA comprising the nucleotide sequence of nucleotide numbers 1 to 1389 shown in SEQ ID NO: 1 of the sequence listing, or
A DNA consisting of the nucleotide sequence of nucleotide numbers 1 to 1392 shown in SEQ ID NO: 3 can be exemplified, but the present invention is not limited to this, and the protein described in (1), (2), (3) or (4) can be used. All the coded DNA is included in the present invention.

The fragment thus cloned, which contains the gene encoding the protein capable of binding to the silencer sequence of Table 1, is reintegrated into an appropriate vector DNA to give another prokaryotic or eukaryotic organism. Host cells can be transformed. Furthermore, by introducing an appropriate promoter and sequences involved in expression into these vectors, it is possible to express the gene in each host cell.

As a host of prokaryotic cells, for example, Escherichia coli (E
scherichia coli) and Bacillus subtilis. To express the gene of interest in these host cells, a replicon from a species compatible with the host,
That is, a host cell may be transformed with a plasmid vector containing an origin of replication and regulatory sequences. Further, the vector preferably has a sequence capable of imparting phenotypic (phenotypic) selectivity to the transformed cell.

For example, E. coli K12 strain and the like are often used as Escherichia coli, and pBR322 and pUC type plasmids are often used as vectors. However, the present invention is not limited to this, and various known strains and vectors can be used. Available.
As the promoter, in E. coli, tryptophan (trp) promoter, lactose (lac) promoter, tryptophan lactose (tac) promoter,
Lipoprotein (lpp) promoter, the lambda-derived bacteriophage (lambda) P _L promoter, polypeptide chain elongation factor Tu (tufB) promoter and the like, any promoters in Table 1 of the present invention silencer - of binding to sequences It can be used to produce the proteins that it has.

As Bacillus subtilis, for example, strain 207-25 is preferable, and as a vector, pTUB228 (Ohmura, K. et al . (1984)
J. Biochem. 95 , 87-93) and the like are used, but not limited thereto. As a promoter, α of Bacillus subtilis
-The regulatory sequence of the amylase gene is often used, and if necessary, by ligating the DNA sequence encoding the signal peptide sequence of α-amylase, the secretory expression outside the cells becomes possible. Eukaryotic host cells include cells such as vertebrates, insects, yeasts, and examples of vertebrate cells include COS cells (Gluzman, Y. (1
981) Cell 23 , 175-182) and Chinese hamster ovary cell (CHO) dihydrofolate reductase-deficient strain (Urlaub,
G. and Chasin, LA (1980) Proc.Natl.Acad.Sci.USA 77 , 4
216-4220) and the like are often used, but the invention is not limited to these.

As the expression vector for vertebrate cells, one having a promoter located upstream of the gene to be expressed, an RNA splice site, a polyadenylation site, a transcription termination sequence and the like can be used. It may have an origin of replication. An example of the expression vector is pSV2dh having an SV40 early promoter.
fr (Subramani, S. et al . (1981) Mol.Cell.Biol. 1 , 854-86
4) and the like can be exemplified, but not limited thereto.

As the eukaryotic microorganism, yeast is commonly used, and among them, yeast of the genus Saccharomyces, for example, Saccharomyces cerevisiae is preferable. Examples of the expression vector for eukaryotic microorganisms such as yeast include, for example, the promoter of the alcohol dehydrogenase gene (Bennetzen, JLand H
all, BD (1982) J. Biol. Chem. 257 , 3018-3025) and the promoter of the acid phosphatase gene (Miyanohara, A. et a .
l . (1983) Proc.Natl.Acad.Sci.USA 80 , 1-5) and the like can be preferably used.

Taking the case of using COS cells as a host cell, the expression vector has an SV40 origin of replication, is capable of autonomous growth in COS cells, and further comprises a transcription promoter, a transcription termination signal and an RNA splice. It is possible to use a device having a part. The expression vector is DEAE-dextran method (Luthman, H. and Magnu
sson, G. (1983) Nucleic Acids Res. 11 , 1295-1308), calcium phosphate-DNA coprecipitation method (Graham, FL and van der
Ed, AJ (1973) Virology 52 , 456-457) and the electric pulse perforation method (Neumann, E. et al . (1982) EMBO J. 1 , 841-845) can be incorporated into COS cells, Thus, the desired transformed cell can be obtained. When CHO cells are used as host cells, G4
18 A vector capable of expressing the neo gene that functions as a resistance marker, for example pRSVneo (Sambrook, J. et al . (1989):
“Molecular Cloning-A Laboratory Manual” Cold Spr
ingHarbor Laboratory, NY) and pSV2-neo (Southern, PJa
nd Berg, P. (1982) J. Mol. Appl. Genet. 1 , 327-341) etc., and has the ability to bind to the silencer sequence of Table 1 by selecting G418 resistant colonies. Transformed cells that stably produce the protein can be obtained.

The desired transformant obtained above can be cultured according to a conventional method, and the protein capable of binding to the silencer sequence of Table 1 is produced inside or outside the cell by the culture. As the medium used for the culturing, various kinds of medium commonly used can be appropriately selected according to the adopted host cell.For example, in the case of the above COS cells, RPMI-1640 medium or Dulbecco's modified Eagle minimum essential medium (DMEM) etc. The medium may be supplemented with a serum component such as fetal bovine serum (FBS) if necessary.

As described above, the protein capable of binding to the silencer sequence of Table 1 produced intracellularly or extracellularly of the transformant has a physical property of the protein capable of binding to the silencer sequence of Table 1. It can be separated and purified from them by various known separation procedures utilizing chemical properties, immunological properties and the like.

Specific examples of the method include treatment with an ordinary protein precipitant, ultrafiltration, molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, affinity chromatography, high performance liquid. Examples include various liquid chromatography such as chromatography (HPLC), dialysis method, and combinations thereof.

By the above-mentioned method, a protein having a desired ability to bind to the silencer sequence of Table 1 can be easily produced on an industrial scale with high yield and high purity. The properties of the thus obtained protein having the ability to bind to the silencer sequence of Table 1 of the present invention are as described in detail in the Examples below.

[0052]

The substance obtained by the genetic engineering method using the DNA of the present invention is the silencer of Table 1.
In order to have the ability to bind to the sequence, the amino acid sequence of amino acid numbers 1 to 463 shown in SEQ ID NO: 2 in the sequence listing or the amino acid number 1 shown in SEQ ID NO: 4
It does not have to have all of the amino acid sequences of ~ 464, but for example, a partial sequence thereof, as long as it exhibits the binding ability to the silencer sequence of Table 1, proteins having those amino acid sequences are also included in the present invention. Included in. Further, the DNA encoding the protein is also included in the present invention.

In general, eukaryotic genes are polymorphic (polym
orphism) (for example, Nishi, T. et al . (1
985) J. Biochem. 97 , 153-159), this polymorphism may result in the replacement of one or more amino acids, or a nucleotide sequence change but no amino acid changes at all. There is also.

A protein consisting of the amino acid sequence of amino acid numbers 1 to 463 shown in SEQ ID NO: 2 in the sequence listing or SEQ ID NO: 4
In the amino acid sequence of the protein other than the protein consisting of the amino acid sequence of amino acid numbers 1 to 464 shown in, at one or more sites, one or more amino acid residues are missing, Even the inserted or substituted protein may have the ability to bind to the silencer sequence in Table 1. In the present invention, these proteins are
It is called the same effect substance of the protein. All such naturally occurring or artificially synthesized proteins are included in the present invention as long as they have the ability to bind to the silencer sequences of Table 1.

The present invention also includes all DNAs having the same nucleotide sequence that code for these proteins.

Such various DNAs of the present invention can be synthesized, for example, by the phosphite triester method (Hunkapille method) based on the above-mentioned information on the amino acid sequence and nucleotide sequence.
r, M. et al . (1984) Nature 310 , 105-111) and the like, and can also be produced by chemical synthesis of nucleic acid.

The codon for the desired amino acid is known per se, and its selection may be arbitrary. For example, it can be determined according to a conventional method in consideration of the frequency of codon usage of the host used (Grantham, R. et al . (1981). ) Nucleic Acids Res. 9 r43-
r74). Furthermore, partial modification of the codons of these nucleotide sequences is carried out according to a conventional method by using a primer composed of a synthetic oligonucleotide encoding a desired modification.
agenesis) (Mark, DF et al . (1984) Proc.Natl.Acad.Sc
i.USA8 1 , 5662-5666) etc.

The DNA of the present invention has the ability to strongly hybridize to the mRNA corresponding to the DNA expressed in cancer tissues or normal tissues in a tumor-bearing living body, regardless of species. Therefore, by using the DNA as a probe and examining the expression level of the corresponding mRNA in each tissue extracted from the living body, it is effective for early diagnosis of cancer diseases. As such a tissue, for example, a minute amount of tissue obtained from human liver by an operation such as biopsy, or peripheral blood lymphocyte obtained from human blood can be used.

The protein of the present invention is also actively expressed in cancer tissues or normal tissues in a tumor-bearing living body, regardless of species. Therefore, by investigating the dynamics of the protein in vivo, it becomes possible to make an early diagnosis of cancer disease.

The protein of the present invention is also used as an essential protein when establishing an immunoassay system useful for such early diagnosis.

That is, a monochrome specific for the protein
It is used as an antigen when producing a null antibody or a polyclonal antibody. Polyclonal antibodies are prepared using animals such as rabbits and horses, and monoclonal antibodies are prepared using
Using animals such as mice and rats, any of them can be performed by methods well known to those skilled in the art. In this case, an antibody against the protein of the present invention can be obtained by immunizing the above-mentioned various animals with the protein amount of 10 to 1,000 μg in several divided doses.

In such an assay system, the protein of the present invention is used as a target antigen. In the assay system, the protein or the polyclonal or monoclonal antibody obtained above is labeled with a fluorescent reagent, an enzyme, a radioisotope or the like by a method well known to those skilled in the art. Any of a liquid phase method using a competitive reaction and the like, a solid phase method using a sandwich method by a one-step reaction or a two-step reaction, and the like can be adopted.

In such an immunoassay, the degree of expression of the protein in each tissue or body fluid extracted from the living body is examined, which is effective for early diagnosis of cancer diseases.
As such a tissue, for example, a trace amount of tissue obtained from human liver by an operation such as biopsy or peripheral blood lymphocyte obtained from human blood, and as such a body fluid, serum or the like can be used.

Since the protein of the present invention has the ability to bind to the silencer sequence in Table 1, it also has the ability to bind to the synthetic oligonucleotide corresponding to the sequence. Therefore, a compound that inhibits the binding between the protein and the synthetic oligonucleotide is effective as a drug that restores the decrease in catalase activity associated with cancer disease. That is, the protein of the present invention, when combined with the synthetic oligonucleotide, provides a search system for a remedy for a decrease in catalase activity associated with cancer disease.

[0065]

The present invention will be described in more detail with reference to the following examples, but the present invention is not limited thereto.

Example 1. (1) Extraction of mRNA fraction from rat cell line After culturing the rat cell line AH 60c strain in 10 cm dish, the medium was removed with an aspirator and the following Trypsin-ED
1 ml of TA solution was added to remove cells, and the cells were transferred to a centrifuge tube.

The cells were semi-adherent cells and were completely detached from the dish by this operation.

[0068] Trypsin-EDTA solution Trypsin 0.5 g EDTA 0.2 g KCl 0.4 g NaCl 8.0 g NaHCO 3 0.35 g KH 2 PO 4 0.06 g Na 2 HPO 4 · 7H 2 O 0.09 g Glucose 1.0 g Phenol Red 0.01 g distilled water 1 L To the cell suspension in the centrifuge tube, 0.1 volume of 2 M sodium acetate (pH 4.7) was added, and then the total volume and an equivalent amount of phenol were added and mixed. Further, chloroform: isoamyl alcohol (49: 1) in an amount 0.2 times the total volume here was added, and after mixing, the mixture was allowed to stand at 0 ° C for 15 minutes. After standing, the precipitate obtained by centrifuging at 10,000 xg for 20 minutes at 4 ℃ was dissolved in solution A (4 M guanidine thiocyanate, 25 mM sodium citrate, 0.5% sarcosyl, 0.1% 2-mercaptoethanol). It was extracted with phenol / chloroform and 2.5 times its amount of ethanol was added to the extract to form a precipitate, which was used as a total RNA fraction.

This total RNA fraction was adsorbed on an oligo (dT) cellulosic column according to a conventional method, and the adsorbed fraction was eluted to obtain a poly A RNA fraction.

(2) Preparation of a cDNA library and λgt11 and λZAP were selected as primary screening vectors for clones.
A cDNA library was prepared according to the Land method using the poly A RNA obtained from the strain.

Screening of the target clone from the recombinant phage clones obtained here was carried out by the Southwestern method (Miskimins, WK et al .: Proc. Natl. Acad.
Sci.USA, 82 , 6741, (1985)) with some modifications
Et al. (Singh H. et al: Cell, 52 , 415, (1988))
According to.

Specifically, recombinant λgt11 was transformed into Escherichia coli BB4.
The mixture was mixed with 37 ° C. for 15 minutes to infect it, and then soft agar was added thereto and mixed, and this was spread on an agar plate. Incubate this plate at 42 ° C for 4 hours, and when the phagy plaque begins to grow, put a nitrocellulose filter impregnated with IPTG on it, and further 37
Induction was carried out at 4 ° C for 4 to 14 hours.

After the induction was completed, the nitrocellulose filter was peeled from the plate and the blocking solution (10 mM Tris-HCl (pH 7.5), 50 mM NaCl, 10 mM MgCl 2 was added).
₂ , non-specific adsorption was prevented by immersing it in a Denhart solution containing 0.1 mM EDTA and 5 mM 2-mercaptoethanol. The protein on this filter was then subjected to a specific binding reaction with the probe.

The probe was ³² P-labeled with a plurality of 15-nucleotide (see Table 1) synthetic oligonucleotides containing a silencer region in the 5'untranslated region of the rat catalase gene. Used. Here, the reason that a plurality of oligonucleotides are linked is used in order to increase the binding force between the oligonucleotide and the target protein and increase the screening efficiency. A positive clone SW was obtained by this screening.

C inserted in this positive clone SW
As a result of analysis of the nucleotide sequence of DNA by the dideoxy method, the cDNA had 717 base pairs and the desired cDNA
It was revealed that it did not include the entire chain length of NA.

(3) Secondary Screening In order to obtain a clone containing the desired full-length cDNA, secondary screening was carried out as follows using the technique of plaque hybridization. ..

Specifically, the above recombinant λZAP was contacted with Escherichia coli BB4 for 10 minutes at room temperature to infect it, soft agar was added thereto, and this was spread on an agar plate. Incubate the plate at 37 ° C overnight to form a fuge plaque,
A Hybond C filter (manufactured by Amersham Co.) was placed on it, and the plaque was adsorbed on it.

Subsequently, the filter was removed from the plate, treated with an alkali to denature the DNA, and further neutralized, and then the DNA was fixed on the filter by UV cross-linking and subjected to hybridization. here,
Included in the clone SW obtained by primary screening
cDNA (717 base pairs) labeled with ³² P
It was used as a bush

Thus, clone SW5 (the inserted cDNA has 2813 base pairs), SW6 (the inserted cDNA has 2041 base pairs) containing the target full-length cDNA,
And SW35 (the inserted cDNA was 2710 base pairs). The nucleotide sequences of these cDNAs were analyzed using the dideoxy method. As a result, the insertion of SW35 cD
NA is nucleotide number 1 to SEQ ID NO: 1 of the sequence listing.
It was found that the inserted cDNA of SW5 and SW6 contains the nucleotide sequence consisting of 1389, and the nucleotide sequence consisting of nucleotide numbers 1 to 1392 of SEQ ID NO: 3 in the sequence listing.

Example 2. The cDNA of the present invention can be expressed by the method described below. The expression for clone SW35 will be described below, but the method described here can also be applied to clone SW5 or SW6.

Since the cDNA of the present invention is integrated into phage λZAP, E. coli XL-1 / Blue was infected with this phage plaque and Escherichia coli phage f1 at 37 ° C.
After culturing for 4 to 6 hours, heat at 70 ℃ for 20 minutes and centrifuge. The supernatant is further infected with Escherichia coli XL-1 / Blue and plated on LB agar medium containing a final concentration of 50 μg / ml ampicillin to obtain transformed E. coli colonies.
Among them, Bluescript containing the cDNA of the present invention
The derived plasmid pSW35 is present.

Then, the obtained plasmid pSW35 was partially cleaved with restriction enzymes Hind III and Pst I to give a 1986 base pair fragment containing most of the 3'side of the cDNA of the present invention and the subsequent 3'untranslated region. Cut out the polynucleotide. To fill in the 5 ', oligonucleotides having the sequences shown in Table 2 below are synthesized.

[0083]

[Table 2] Table 2 −−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−− 5′-C ATG GAG ACC GAA CAG CCA GAA GAA ACC TCC 3'- CTC TGG CTT GTC GGT CTT CTT TGG AAG CCT AAC ACC GAA ACC AAT GGT GAA TTT GGT AAA GGA TTG TGG CTT TGG TTA CCA CTT AAA CCA TTT CGC CCT GCA -3 'GCG GG -5 '−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−− In the DNA region of this sequence, one is the restriction enzyme NcoI , The other is the PstI site, and the nucleotide sequence starting from the second nucleotide of the above sequence (ATG GAG
ACC ...) Is the sequence from the translation initiation site when the cDNA of the present invention is expressed.

Then, the expression vector pKK233-2 (black
(Manufactured by Ntech Co., Ltd.) with the restriction enzymes NcoI and HindIII, and ligated with the above-mentioned 1986 base pair fragment and synthetic DNA fragment by T ₄ ligase to obtain the expression vector pSW35-1. These procedures are shown in FIG.

The pSW35-1 constructed here was introduced into Escherichia coli XL-1 / Blue according to a conventional method, and the final concentration was 50 μg.
Culture in LB medium containing / ml ampicillin to obtain the target recombinant E. coli.

By culturing this recombinant according to a conventional method, the target protein coded by clone pSW35-1 is produced as insoluble granules in the cells. The resulting cells were disrupted by ultrasonic waves and then at 10,000 xg.
After 20 minutes of centrifugation, the increase body is obtained.

50% of the obtained increase body was used.
It can be dissolved in a solution of mM Tris-HCl (pH 7.5), 10 mM EDTA, 6 M guanidine hydrochloride and reformed according to a conventional method to produce a protein.

Subsequently, the target protein can be purified from this solution by a method used for purification of a usual protein such as gel filtration, ion exchange chromatography, and chromatofocusing. At that time, immunoassays such as SDS-PAGE (SDS polyacrylamide gel electrophoresis) and immunoblotting are effective as a method for identifying the protein.

[Effects of the Invention]

[0090]

[Effect] The cDNA of the present invention has the property of hybridizing with mRNA strongly expressed in cancer across species,
By using all or part of this cDNA as a probe, the expression of the corresponding mRNA in the liver tissue collected by biopsy can be measured by Northern blotting or in situ hybridization. And, since the corresponding mRNA is extremely strongly expressed in cancer, such measurement has an effective effect on the diagnosis of cancer.

The results of the Northern blotting analysis are shown below in Test Examples.

Test Example 1. (1) Cell line, tissue Degree of expression of mRNA corresponding to the cDNA of the present invention,
It was examined in rat hepatoma cell line, rat breast cancer cell line and human hepatoma cell line, and in rat normal tissue transplanted with breast cancer cells. The normal tissue used here was extracted from the rat. Rat Breast Cancer Strain Walker 256 Carcinosarcoma was obtained from Wister rat. Rat breast cancer-derived cell line AH66, Ac2F, human liver cancer-derived H
epG2 is a known cell line.

(2) Separation of total RNA and When using tissues, 10 times its amount of solution A (described in Example 1) was added, homogenized, and transferred to a centrifuge tube. Also,
When using cultured cells, the cells were detached from the dish by the method described in Example 1 and transferred to a centrifuge tube. In addition, total RNA was extracted from each tissue and cell line by the method described in Example 1.

(3) Northern hybridization Rat normal liver tissue, liver cancer cell lines such as AH66, Ac2F and HepG2, whole RN collected from rat liver transplanted with breast cancer cells
Fraction A was applied to 1% agarose / 18% (V / V) formaldehyde gel in an amount of 10 μg of RNA, respectively,
Electrophoresis was performed.

After thorough migration, Hybond N + was placed on the gel.
Place a filter (Amersham) on the gel RN
RNA was fixed to the filter by transferring A to the filter and then cross-linking the filter. Subsequently, the RNA fraction on this filter was hybridized with ³² P-labeled clone SW35 cDNA, and autoradiography was performed. The result is shown in FIG.

In FIG. 2, lane 1: rat normal liver lane 2: AH 66 strain lane 3: Ac2F strain lane 4: HepG2 strain lane 5: Walker strain, respectively. The result of Northern blotting of the RNA fraction extracted from the liver of the above is shown.

The rat liver used in lane 5 was histologically normal.

In this result, the cancer cell lines AH66, Ac2F
Two dark bands with lengths of 3.0 Kb and 2.2 Kb were observed in 3 liver strains of HepG2 and HepG2 and in normal liver of tumor-bearing rats, but almost no corresponding bands were found in normal liver of rats. ..

From this, the cDNA obtained in the present invention
It has been revealed that the mRNA corresponding to is strongly expressed in cancer regardless of cancer type regardless of species.

That is, it was revealed that the DNA of the present invention and the protein encoded by the DNA have relevance to not only liver cancer but also other cancer diseases.

[0101]

[Sequence Listing] SEQ ID NO: 1 Sequence length: 1389 Sequence type: Nucleic acid Number of strands: Double-stranded Topology :: Linear Sequence type: cDNA to mRNA Hypothetical sequence: No Antisense: No Origin Cell type: Rat hepatoma cell line name: AH 60c Sequence characteristics 1-1389 E CDS sequence ATG GAG ACC GAA CAG CCA GAA GAA ACC TTC CCT AAC ACC GAA ACC AAT 48 Met Glu Thr Glu Gln Pro Glu Glu Thr Phe Pro Asn Thr Glu Thr Asn 1 5 10 15 GGT GAA TTT GGT AAA CGC CCT GCA GAA GAT ATG GAA GAG GAA CAA GCC 96 Gly Glu Phe Gly Lys Arg Pro Ala Glu Asp Met Glu Glu Glu Gln Ala 20 25 30 TTT AAA AGA TCT AGA AAT ACT GAT GAG ATG GTT GAA TTG CGC ATT TTG 144 Phe Lys Arg Ser Arg Asn Thr Asp Glu Met Val Glu Leu Arg Ile Leu 35 40 45 CTT CAG AGC AAG AAT GCT GGG GCA GTG ATT GGA AAA GGA GGC AAG AAT 192 Leu Gln Ser Lys Asn Ala Gly Ala Val Ile Gly Lys Gly Gly Lys Asn 50 55 60 ATT AAG GCT CTC CGT ACA GAC TAC AAT GCC AGT GTT TCA GTC CCA GAC 240 Ile Lys Ala Leu Arg Thr Asp Tyr Asn Ala Ser Val Ser Val Pro Asp 65 70 75 80 AGC AGT GGC CCC GAG CGC ATA CTG AGT ATC AGT GCT GAT ATT GAG ACG 288 Ser Ser Gly Pro Glu Arg Ile Leu Ser Ile Ser Ala Asp Ile Glu Thr 85 90 95 ATT GGA GAA ATT CTG AAG AAA ATC ATC CCT ACT TTG GAA GAG GGC CTG 336 Ile Gly Glu Ile Leu Lys Lys Ile Ile Pro Thr Leu Glu Glu Gly Leu 100 105 110 CAG TTG CCA TCA CCC ACT GCA ACC AGC CAG CTC CCG CTC GAA TCT GAT 384 Gln Leu Pro Ser Pro Thr Ala Thr Ser Gln Leu Pro Leu Glu Ser Asp 115 120 125 GCT GTG GAA TGC TTA AAT TAC CAG CAT TAT AAA GGG AGT GAC TTC GAC 432 Ala Val Glu Cys Leu Asn Tyr Gln His Tyr Lys Gly Ser Asp Phe Asp 130 135 140 TGC GAG CTG AGA CTG TTG ATT CAT CAA AGT CTG GCC GGA GGA ATT ATT 480 Cys Glu Leu Arg Leu Leu Ile His Gln Ser Leu Ala Gly Gly Ile Ile 145 150 155 160 GGT GTT AAA GGT GCT AAA ATC AAA GAA CTT CGA GAA AAC ACT CAG ACA 528 Gly Val Lys Gly Ala Lys Ile Lys Glu Leu Arg Glu Asn Thr Gln Thr 165 170 175 ACA ATC AAG CTT TTC CAG GAA TGC TGC CCT CAT TCT ACT GAC AGA GTT 576 Thr Ile Lys Leu Phe Gln Glu Cys Cy s Pro His Ser Thr Asp Arg Val 180 185 190 GTT CTT ATT GGA GGA AAG CCC GAT AGG GTT GTA GAA TGC ATC AAG ATC 624 Val Leu Ile Gly Gly Lys Pro Asp Arg Val Val Glu Cys Ile Lys Ile 195 200 205 ATC CTT GAC CTT ATA TCT GAG TCT CCC ATC AAA GGA CGT GCA CAA CCT 672 Ile Leu Asp Leu Ile Ser Glu Ser Pro Ile Lys Gly Arg Ala Gln Pro 210 215 220 TAT GAT CCC AAC TTC TAT GAT GAA ACC TAT GAT TAT GGT GGT TTT ACA 720 Tyr Asp Pro Asn Phe Tyr Asp Glu Thr Tyr Asp Tyr Gly Gly Phe Thr 225 230 235 240 ATG ATG TTT GAT GAC CGC CGA GGA CGA CCT GTG GGA TTC CCC ATG CGG 768 Met Met Phe Asp Asp Arg Arg Gly Arg Pro Val Gly Phe Pro Met Arg 245 250 255 GGA AGA GGT GGT TTT GAC AGA ATG CCT CCT GGT CGG GGT GGG CGT CCC 816 Gly Arg Gly Gly Phe Asp Arg Met Pro Pro Gly Arg Gly Gly Arg Pro 260 265 270 ATG CCT CCT TCT AGA AGA GAT TAT GAT GAT ATG AGC CCT CGT CGA GGA 864 Met Pro Pro Ser Arg Arg Asp Tyr Asp Asp Met Ser Pro Arg Arg Gly 275 280 285 285 CCA CCA CCA CCA CCA CCT GGT CGA GGT GGA CGG GGT GGC AGC AGA GCT 912 Pro Pro Pro Pro Pro Pr o Gly Arg Gly Gly Arg Gly Gly Ser Arg Ala 290 295 300 CGG AAT CTT CCT CTT CCT CCA CCA CCA CCA CCC AGA GGG GGA GAT CTC 960 Arg Asn Leu Pro Leu Pro Pro Pro Pro Pro Pro Arg Gly Gly Asp Leu 305 310 315 320 ATG GCT TAC GAC AGA AGA GGA CGG CCT GGA GAC CGC TAT GAT GGC ATG 1008 Met Ala Tyr Asp Arg Arg Gly Arg Pro Gly Asp Arg Tyr Asp Gly Met 325 330 335 GTT GGG TTC AGT GCT GAT GAA ACT TGG GAC TCT GCA ATT GAC ACA TGG 1056 Val Gly Phe Ser Ala Asp Glu Thr Trp Asp Ser Ala Ile Asp Thr Trp 340 345 350 AGC CCA TCA GAA TGG CAA ATG GCT TAT GAA CCA CAG GGT GGT TCT GGA 1104 Ser Pro Ser Glu Trp Gln Met Ala Tyr Glu Pro Gln Gly Gly Ser Gly 355 360 365 TAT GAT TAT TCT TAT GCA GGG GGC CGT GGC TCG TAT GGT GAT CTT GGC 1152 Tyr Asp Tyr Ser Tyr Ala Gly Gly Arg Gly Ser Tyr Gly Asp Leu Gly 370 375 380 GGA CCT ATT ATC ACT ACA CAA GTA ACT ATT CCC AAA GAT TTG GCT GGA 1200 Gly Pro Ile Ile Thr Thr Gln Val Thr Ile Pro Lys Asp Leu Ala Gly 385 390 395 400 TCT ATT ATT GGC AAA GGT GGT CAG CGG ATT AAA CAA ATT CGT CAT GAG 1248 Ser Ile Ile Gly Lys Gly Gly Gln Arg Ile Lys Gln Ile Arg His Glu 405 410 415 TCT GGA GCA TCT ATC AAA ATT GAT GAA CCT TTA GAA GGA TCT GAA GAT 1296 Ser Gly Ala Ser Ile Lys Ile Asp Glu Pro Leu Glu Gly Ser Glu Asp 420 425 430 CGG ATC ATT ACC ATT ACG GGA ACA CAG GAC CAG ATA CAG AAC GCA CAG 1344 Arg Ile Ile Thr Ile Thr Gly Thr Gln Asp Gln Ile Gln Asn Ala Gln 435 440 445 TAT TTG CTG CAG AAC AGT GTG AAG CAG TAT TCT GGA AAG TTT TTC 1389 Tyr Leu Leu Gln Asn Ser Val Lys Gln Tyr Ser Gly Lys Phe Phe 450 455 460 SEQ ID NO: 2 Sequence Length: 463 Sequence Type: Amino Acid Number of Chains: Single Chain Topology −: Type of linear sequence: Protein hypothetical sequence: Yes Origin of cell type: Rat hepatoma cell line: AH 60c sequence Met Glu Thr Glu Gln Pro Glu Glu Thr Phe Pro Asn Thr Glu Thr Asn 1 5 10 15 Gly Glu Phe Gly Lys Arg Pro Ala Glu Asp Met Glu Glu Glu Gln Ala 20 25 30 Phe Lys Arg Ser Arg Asn Thr Asp Glu Met Val Glu Leu Arg Ile Leu 35 40 45 Leu G ln Ser Lys Asn Ala Gly Ala Val Ile Gly Lys Gly Gly Lys Asn 50 55 60 Ile Lys Ala Leu Arg Thr Asp Tyr Asn Ala Ser Val Ser Val Pro Asp 65 70 75 80 Ser Ser Gly Pro Glu Arg Ile Leu Ser Ile Ser Ala Asp Ile Glu Thr 85 90 95 Ile Gly Glu Ile Leu Lys Lys Ile Ile Pro Thr Leu Glu Glu Gly Leu 100 105 110 Gln Leu Pro Ser Pro Thr Ala Thr Ser Gln Leu Pro Leu Glu Ser Asp 115 120 125 Ala Val Glu Cys Leu Asn Tyr Gln His Tyr Lys Gly Ser Asp Phe Asp 130 135 140 Cys Glu Leu Arg Leu Leu Ile His Gln Ser Leu Ala Gly Gly Ile Ile 145 150 155 160 Gly Val Lys Gly Ala Lys Ile Lys Glu Leu Arg Glu Asn Thr Gln Thr 165 170 175 Thr Ile Lys Leu Phe Gln Glu Cys Cys Pro His Ser Thr Asp Arg Val 180 185 190 Val Leu Ile Gly Gly Lys Pro Asp Arg Val Val Glu Cys Ile Lys Ile 195 200 205 Ile Leu Asp Leu Ile Ser Glu Ser Pro Ile Lys Gly Arg Ala Gln Pro 210 215 220 Tyr Asp Pro Asn Phe Tyr Asp Glu Thr Tyr Asp Tyr Gly Gly Phe Thr 225 230 235 240 Met Met Phe Asp Asp Arg Arg Gly Arg Pro Val Gly Phe Pro Met Arg 245 250 255 Gly Arg Gly Gl y Phe Asp Arg Met Pro Pro Gly Arg Gly Gly Arg Pro 260 265 270 Met Pro Pro Ser Arg Arg Asp Tyr Asp Asp Met Ser Pro Arg Arg Gly 275 280 285 Pro Pro Pro Pro Pro Pro Gly Arg Gly Gly Arg Gly Gly Ser Arg Ala 290 295 300 Arg Asn Leu Pro Leu Pro Pro Pro Pro Pro Pro Arg Gly Gly Asp Leu 305 310 315 320 Met Ala Tyr Asp Arg Arg Gly Arg Pro Gly Asp Arg Tyr Asp Gly Met 325 330 335 Val Gly Phe Ser Ala Asp Glu Thr Trp Asp Ser Ala Ile Asp Thr Trp 340 345 350 Ser Pro Ser Glu Trp Gln Met Ala Tyr Glu Pro Gln Gly Gly Ser Gly 355 360 365 Tyr Asp Tyr Ser Tyr Ala Gly Gly Arg Gly Ser Tyr Gly Asp Leu Gly 370 375 380 Gly Pro Ile Ile Thr Thr Gln Val Thr Ile Pro Lys Asp Leu Ala Gly 385 390 395 400 Ser Ile Ile Gly Lys Gly Gly Gln Arg Ile Lys Gln Ile Arg His Glu 405 410 415 Ser Gly Ala Ser Ile Lys Ile Asp Glu Pro Leu Glu Gly Ser Glu Asp 420 425 430 Arg Ile Ile Thr Ile Thr Gly Thr Gln Asp Gln Ile Gln Asn Ala Gln 435 440 445 Tyr Leu Leu Gln Asn Ser Val Lys Gln Tyr Ser Gly Lys Phe Phe 450 455 460 SEQ ID NO: 3 Column length: 1392 Sequence type: Nucleic acid Number of strands: Double-stranded Topology-: Linear Sequence type: cDNA to mRNA Hypothetical sequence: No Antisense: No Origin Cell type: Rat hepatoma cell Strain name: AH 60c Sequence characteristics 1-1392 E CDS sequence ATG GAG ACC GAA CAG CCA GAA GAA ACC TTC CCT AAC ACC GAA ACC AAT 48 Met Glu Thr Glu Gln Pro Glu Glu Thr Phe Pro Asn Thr Glu Thr Asn 1 5 10 15 GGT GAA TTT GGT AAA CGC CCT GCA GAA GAT ATG GAA GAG GAA CAA GCC 96 Gly Glu Phe Gly Lys Arg Pro Ala Glu Asp Met Glu Glu Glu Gln Ala 20 25 30 TTT AAA AGA TCT AGA AAT ACT GAT GAG ATG GTT GAA TTG CGC ATT TTG 144 Phe Lys Arg Ser Arg Asn Thr Asp Glu Met Val Glu Leu Arg Ile Leu 35 40 45 CTT CAG AGC AAG AAT GCT GGG GCA GTG ATT GGA AAA GGA GGC AAG AAT 192 Leu Gln Ser Lys Asn Ala Gly Ala Val Ile Gly Lys Gly Gly Lys Asn 50 55 60 ATT AAG GCT CTC CGT ACA GAC TAC AAT GCC AGT GTT TCA GTC CCA GAC 240 Ile Lys Ala Leu Arg Thr Asp Tyr Asn Ala Ser Val Ser Val Pro Asp 65 70 75 80 AGC AGT GGC CCC GAG CGC ATA CTG AGT ATC AGT GCT GAT ATT GAG ACG 288 Ser Ser Gly Pro Glu Arg Ile Leu Ser Ile Ser Ala Asp Ile Glu Thr 85 90 95 ATT GGA GAA ATT CTG AAG AAA ATC ATC CCT ACT TTG GAA GAG GGC CTG 336 Ile Gly Glu Ile Leu Lys Lys Ile Ile Pro Thr Leu Glu Glu Gly Leu 100 105 110 CAG TTG CCA TCA CCC ACT GCA ACC AGC CAG CTC CCG CTC GAA TCT GAT 384 Gln Leu Pro Ser Pro Thr Ala Thr Ser Gln Leu Pro Leu Glu Ser Asp 115 120 125 GCT GTG GAA TGC TTA AAT TAC CAG CAT TAT AAA GGG AGT GAC TTC GAC 432 Ala Val Glu Cys Leu Asn Tyr Gln His Tyr Lys Gly Ser Asp Phe Asp 130 135 140 TGC GAG CTG AGA CTG TTG ATT CAT CAA AGT CTG GCC GGA GGA ATT ATT 480 Cys Glu Leu Arg Leu Leu Ile His Gln Ser Leu Ala Gly Gly Ile Ile 145 150 155 160 GGT GTT AAA GGT GCT AAA ATC AAA GAA CTT CGA GAA AAC ACT CAG ACA 528 Gly Val Lys Gly Ala Lys Ile Lys Glu Leu Arg Glu Asn Thr Gln Thr 165 170 175 ACA ATC AAG CTT TTC CAG GAA TGC TGC CCT CAT TCT ACT GAC AGA GTT 576 Thr Ile Lys Leu Phe Gln Glu Cys Cys Pro His Ser Thr Asp Arg Val 180 18 5 190 GTT CTT ATT GGA GGA AAG CCC GAT AGG GTT GTA GAA TGC ATC AAG ATC 624 Val Leu Ile Gly Gly Lys Pro Asp Arg Val Val Glu Cys Ile Lys Ile 195 200 205 ATC CTT GAC CTT ATA TCT GAG TCT CCC ATC AAA GGA CGT GCA CAA CCT 672 Ile Leu Asp Leu Ile Ser Glu Ser Pro Ile Lys Gly Arg Ala Gln Pro 210 215 220 TAT GAT CCC AAC TTC TAT GAT GAA ACC TAT GAT TAT GGT GGT TTT ACA 720 Tyr Asp Pro Asn Phe Tyr Asp Glu Thr Tyr Asp Tyr Gly Gly Phe Thr 225 230 235 240 ATG ATG TTT GAT GAC CGC CGA GGA CGA CCT GTG GGA TTC CCC ATG CGG 768 Met Met Phe Asp Asp Arg Arg Gly Arg Pro Val Gly Phe Pro Met Arg 245 250 255 GGA AGA GGT GGT TTT GAC AGA ATG CCT CCT GGT CGG GGT GGG CGT CCC 816 Gly Arg Gly Gly Phe Asp Arg Met Pro Pro Gly Arg Gly Gly Arg Pro 260 265 270 ATG CCT CCT TCT AGA AGA GAT TAT GAT GAT ATG AGC CCT CGT CGA GGA 864 Met Pro Pro Ser Arg Arg Asp Tyr Asp Asp Met Ser Pro Arg Arg Gly 275 280 285 CCA CCA CCA CCA CCA CCT GGT CGA GGT GGA CGG GGT GGC AGC AGA GCT 912 Pro Pro Pro Pro Pro Gly Arg Gly Gly Arg Gly Gly Ser Ar g Ala 290 295 300 CGG AAT CTT CCT CTT CCT CCA CCA CCA CCA CCC AGA GGG GGA GAT CTC 960 Arg Asn Leu Pro Leu Pro Pro Pro Pro Pro Pro Arg Gly Gly Asp Leu 305 310 315 320 ATG GCT TAC GAC AGA AGA GGA CGG CCT GGA GAC CGC TAT GAT GGC ATG 1008 Met Ala Tyr Asp Arg Arg Gly Arg Pro Gly Asp Arg Tyr Asp Gly Met 325 330 335 GTT GGG TTC AGT GCT GAT GAA ACT TGG GAC TCT GCA ATT GAC ACA TGG 1056 Val Gly Phe Ser Ala Asp Glu Thr Trp Asp Ser Ala Ile Asp Thr Trp 340 345 350 AGC CCA TCA GAA TGG CAA ATG GCT TAT GAA CCA CAG GGT GGT TCT GGA 1104 Ser Pro Ser Glu Trp Gln Met Ala Tyr Glu Pro Gln Gly Gly Ser Gly 355 360 365 TAT GAT TAT TCT TAT GCA GGG GGC CGT GGC TCG TAT GGT GAT CTT GGC 1152 Tyr Asp Tyr Ser Tyr Ala Gly Gly Arg Gly Ser Tyr Gly Asp Leu Gly 370 375 380 GGA CCT ATT ATC ACT ACA CAA GTA ACT ATT CCC AAA GAT TTG GCT GGA 1200 Gly Pro Ile Ile Thr Thr Gln Val Thr Ile Pro Lys Asp Leu Ala Gly 385 390 395 400 TCT ATT ATT GGC AAA GGT GGT CAG CGG ATT AAA CAA ATT CGT CAT GAG 1248 Ser Ile Ile Gly Lys Gly Gly Gln Arg Ile Lys Gln Ile Arg His Glu 405 410 415 TCT GGA GCA TCT ATC AAA ATT GAT GAA CCT TTA GAA GGA TCT GAA GAT 1296 Ser Gly Ala Ser Ile Lys Ile Asp Glu Pro Leu Glu Gly Ser Glu Asp 420 425 430 CGG ATC ATT ACC ATT ACG GGA ACA CAG GAC CAG ATA CAG AAC GCA CAG 1344 Arg Ile Ile Thr Ile Thr Gly Thr Gln Asp Gln Ile Gln Asn Ala Gln 435 440 445 TAT TTG CTG CAG AAC AGT GTG AAG CAG TAT GCA GAT GTT GAA GGA TTC 1392 Tyr Leu Leu Gln Asn Ser Val Lys Gln Tyr Ala Asp Val Glu Gly Phe 450 455 460 SEQ ID NO: 4 Sequence length: 464 Sequence type: Amino acid Number of chains: Single chain Topology −: Linear Sequence type: Protein Hypothetical Sequence: Yes Origin Cell type: Rat Hepatoma Cell line: AH 60c Sequence Met Glu Thr Glu Gln Pro Glu Glu Thr Phe Pro Asn Thr Glu Thr Asn 1 5 10 15 Gly Glu Phe Gly Lys Arg Pro Ala Glu Asp Met Glu Glu Glu Gln Ala 20 25 30 Phe Lys Arg Ser Arg Asn Thr Asp Glu Met Val Glu Leu Arg Ile Leu 35 40 45 Leu Gln Ser Lys Asn Ala Gly Ala Val Ile Gly Lys Gly Gly Lys Asn 50 55 60 Ile Lys Ala Leu Arg Thr Asp Tyr Asn Ala Ser Val Ser Val Pro Asp 65 70 75 80 Ser Ser Gly Pro Glu Arg Ile Leu Ser Ile Ser Ala Asp Ile Glu Thr 85 90 95 Ile Gly Glu Ile Leu Lys Lys Ile Ile Pro Thr Leu Glu Glu Gly Leu 100 105 110 Gln Leu Pro Ser Pro Thr Ala Thr Ser Gln Leu Pro Leu Glu Ser Asp 115 120 125 Ala Val Glu Cys Leu Asn Tyr Gln His Tyr Lys Gly Ser Asp Phe Asp 130 135 140 Cys Glu Leu Arg Leu Leu Ile His Gln Ser Leu Ala Gly Gly Ile Ile 145 150 155 160 Gly Val Lys Gly Ala Lys Ile Lys Glu Leu Arg Glu Asn Thr Gln Thr 165 170 175 Thr Ile Lys Leu Phe Gln Glu Cys Cys Pro His Ser Thr Asp Arg Val 180 185 190 Val Leu Ile Gly Gly Lys Pro Asp Arg Val Val Glu Cys Ile Lys Ile 195 200 205 Ile Leu Asp Leu Ile Ser Glu Ser Pro Ile Lys Gly Arg Ala Gln Pro 210 215 220 Tyr Asp Pro Asn Phe Tyr Asp Glu Thr Tyr Asp Tyr Gly Gly Phe Thr 225 230 235 240 Met Met Phe Asp Asp Arg Arg Gly Arg Pro Val Gly Phe Pro Met Arg 245 250 255 Gly Arg Gly Gly Phe Asp Arg Met Pro Pro Gly Arg Gly Gly Arg Pro 260 265 270 Met Pro Pro Ser Arg Arg Asp Tyr Asp Asp Met Ser Pro Arg Arg Gly 275 280 285 Pro Pro Pro Pro Pro Pro Gly Arg Gly Gly Arg Gly Gly Ser Arg Ala 290 295 300 Arg Asn Leu Pro Leu Pro Pro Pro Pro Pro Pro Arg Gly Gly Asp Leu 305 310 315 320 Met Ala Tyr Asp Arg Arg Gly Arg Pro Gly Asp Arg Tyr Asp Gly Met 325 330 335 Val Gly Phe Ser Ala Asp Glu Thr Trp Asp Ser Ala Ile Asp Thr Trp 340 345 350 Ser Pro Ser Glu Trp Gln Met Ala Tyr Glu Pro Gln Gly Gly Ser Gly 355 360 365 Tyr Asp Tyr Ser Tyr Ala Gly Gly Arg Gly Ser Tyr Gly Asp Leu Gly 370 375 380 Gly Pro Ile Ile Thr Thr Gln Val Thr Ile Pro Lys Asp Leu Ala Gly 385 390 395 400 Ser Ile Ile Gly Lys Gly Gly Gln Arg Ile Lys Gln Ile Arg His Glu 405 410 415 Ser Gly Ala Ser Ile Lys Ile Asp Glu Pro Leu Glu Gly Ser Glu Asp 420 425 430 Arg Ile Ile Thr Ile Thr Gly Thr Gln Asp Gln Ile Gln Asn Ala Gln 435 440 445 Tyr Leu Leu Gln Asn Ser Val Lys Gln Tyr Ala Asp Val Glu Gly Phe 450 455 460

[Brief description of drawings]

FIG. 1 is a construction diagram of expression vector-pSW35-1.

[Explanation of sign]

In the figure, E is EcoRI cleavage site, H is HindIII cleavage site, P
Indicates a PstI cleavage site.

FIG. 2 is a Northern hybridization analysis diagram of RNA fractions of various cancer cells and tissues.

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁵ Identification code Internal reference number FI Technical display location C12P 21/02 8214-4B // (C12N 1/21 C12R 1:19) (C12P 21/02 C12R 1:19)

Claims (13)

[Claims] 1. A protein consisting of the amino acid sequence of amino acid numbers 1 to 463 shown in SEQ ID NO: 2 of the sequence listing, or one or more amino acid residues at one or more positions thereof. , A deleted, substituted or inserted equivalent of the protein. 2. A protein comprising the amino acid sequence of amino acid numbers 1 to 463 shown in SEQ ID NO: 2 in the sequence listing. 3. A protein consisting of the amino acid sequence of amino acid numbers 1 to 464 shown in SEQ ID NO: 4 of the sequence listing, or one or more amino acid residues at one or more positions thereof. , A deleted, substituted or inserted equivalent of the protein. 4. A protein comprising the amino acid sequence of amino acid numbers 1 to 464 shown in SEQ ID NO: 4 in the sequence listing. 5. A DN encoding the protein of claim 1.
A. 6. A DN encoding the protein of claim 2.
A. 7. A DN encoding the protein of claim 3.
A. 8. A DN encoding the protein of claim 4.
A. 9. The DNA according to claim 5 or 6, which comprises the nucleotide sequence of nucleotide numbers 1 to 1389 shown in SEQ ID NO: 1 of the sequence listing. 10. The nucleotide sequence consisting of nucleotide numbers 1 to 1392 shown in SEQ ID NO: 2 in the sequence listing.
Or the DNA according to item 8. 11. A DNA expression vector comprising the DNA according to claim 5, 6, 7, 8, 9 or 10, and being included such that the DNA is expressible and replicable. 12. A host transformed with the DNA expression vector according to claim 11. 13. A host is transformed with a DNA expression vector comprising a DNA encoding the protein according to claim 1, 2, 3 or 4 and containing the DNA such that the DNA is expressible and replicable, The method for producing the protein according to claim 1, 2, 3 or 4, wherein the protein according to claim 1, 2, 3 or 4 is recovered from the host.