JP3484727B2 - Polypeptide derived from hepatitis B virus and gene encoding the same - Google Patents

Description

Detailed Description of the Invention

[0001]

FIELD OF THE INVENTION The present invention relates to a novel hepatitis B virus (hereinafter abbreviated as "HBV") polypeptide and a gene encoding the same, and more particularly to a polypeptide derived from HBV X protein. , The gene that encodes it,
And their subfragments.

[0002]

2. Description of the Related Art Human hepatitis viruses of type A, type B, type C, type D and type E have been discovered so far, and serological diagnostic methods have been established. Has been done. That is, A in the mid 1970s
Hepatitis B virus (HAV) and hepatitis B virus (H
BV, hepatitis D virus (HD
V), hepatitis C virus (HCV) in the late 1980s
And a specific diagnostic technique to detect hepatitis E virus (HEV) infection has been developed, and now post-transfusion hepatitis and sporadic hepatitis due to vertical or horizontal infection from these carriers are drastically reduced worldwide. It is the actual situation.

In Japan, there are almost no patients infected with hepatitis D and E viruses, and hepatitis A, B, and C viruses are the cause of 99.9% of all hepatitis patients. The number of patients has been drastically reduced due to the benefits of detecting and diagnosing HBV and HCV infections. However, about 20% of sporadic hepatitis and about 2% of post-transfusion hepatitis still exist in the human society as hepatitis of unknown cause due to a negative diagnostic method for detecting HAV, HBV and HCV infections, which is a problem. At present, hepatitis of unknown cause is considered to be transmitted by a virus based on its infectious disease state (Internal Medicine, 69 (6), 1157-1159, 199).
2). Currently, as a method for detecting and diagnosing HBV infection,
B surface antigen (HBs-Ag) and HB core antibody (HBc-
It has been determined by the detection of Ab).
B surface antibody (HBs-Ab), HBe antigen (HBe-A
g) and HBe antibody (HBe-Ab) have also been detected, and HBV was detected by comprehensive implementation of these detection systems.
Has been determined to be infected. That is, HBV infection is not determined when all of these 5 types of HBV detection systems are negative. When a hepatitis patient is determined to be negative by these five types of HBV infection detection and diagnosis methods and HAV and HCV infection detection and diagnosis methods, viral hepatitis of unknown cause is diagnosed. For example, if HBV or HCV infection is found, it is possible to receive treatment with interferon or the like, but if the cause is unknown, it is not possible. In addition, if there is a healthy person (carrier) who has a hepatitis virus that cannot be detected by known hepatitis virus infection detection methods, in addition to hepatitis patients, indiscriminately infect other people by some method. There is. For example, the prevention and treatment of infection with HBV vaccines and HBV immunoglobulins, and the spread of hepatitis virus due to the result of serodiagnosis of blood donation, etc. are not currently prevented against the hepatitis virus of unknown cause. is there.

These existing HAV, HBV and HC
A report suggesting the existence of unknown hepatitis F virus or hepatitis G virus as a cause of hepatitis virus that cannot be determined by the detection and diagnosis method of V infection (Clinician, 18 (5), 624-).
625, 1992), but this alone did not reveal all the causes. In addition, the existence of HCV that has been mutated and cannot be detected by existing HCV detection diagnostic methods has been reported (Shimonotono et al., Proceedings of National Academy of Sciences USA (pro).
c. Natl. Acad. Sci. USA), 87 , 9
524-9528, 1990; Takamizawa et al., Journal
Of virology (Journal of Virolo
gy), 65 (3), 1105-1113, 1991).
However, such cases alone cannot explain all unexplained hepatitis. There have been many reports of mutants of HBV, such as William F. Carman et al.
Reported an example in which HBV in which the 145th amino acid of HBs was mutated from Gly to Arg was administered although the vaccine derived from V surface antigen (HBs-Ag) was administered to induce HBs antibody (HBs-Ab) (The Lancet (The Lancet), 336 , 325-3
29, 1990). In addition, Omata et al. Have reported an example in which HBV infection is fulminant due to mutation in the HBV precore region (Nippon Clinic, 51 (2), 29-33, 1993). Furthermore, Ehata et al. Reported the amino acid sequence of a mutation in the core protein of HBV isolated from a chronically HBV-infected patient (Journal of Clinical Investigation).
Investigation), 89 , 332-33.
8, 1992). However, all of these HBV mutant viruses were detected by the known HBV infection detection diagnostic method.
It can be judged as V infection.

On the other hand, the HBV X protein consists of 154 amino acid residues, and it has been confirmed that an antibody against this X protein is detected in the sera of patients initially infected with HBV. I don't really understand. In recent years, the relationship between the gene encoding the X protein and liver cancer has been suggested, and it has been attracting attention, but the detection system and diagnostic system for it have not yet been established.

As described above, no effective antiviral agent or vaccine has been developed for the hepatitis virus that is not detected by the existing hepatitis virus infection detection and diagnosis method because the target is unknown. Similarly, effective diagnostic agents against these viruses have not been developed. Therefore, hepatitis virus-infected patients who are not detected by the existing methods for detecting and detecting hepatitis virus infection may not be effectively selected as the treatment method. Further, a person who may be in contact with a hepatitis virus carrier that is not detected by the existing hepatitis virus infection detection / diagnosis method may not be able to execute an effective method such as a vaccine from the viewpoint of infection prevention. Furthermore, blood or the like contaminated with hepatitis virus, which is not detected by the existing diagnostic method for detecting hepatitis virus infection, cannot be disposed of by an effective method and may cause the transmission of virus infection to other people.

[0007]

[Means for Solving the Problems] The present inventors have been discovered so far for the purpose of developing a method for diagnosing, treating and preventing hepatitis virus-infected patients and carriers that are not detected by existing methods for detecting and detecting hepatitis virus infection. A novel diagnosis that acquires a viral gene of a different nature from the virus and has a character different from the diagnostic system developed so far, especially in the range that could not be detected until now, or a higher rate of detecting hepatitis virus infection For the development of the method, for the development of the vaccine having the immunity acquisition ability superior to the HBV vaccines developed up to now, and for the development of the evaluation system for the development of the better antiviral agent than ever, As a result of repeated studies, a novel HBV-derived gene was found and it was obtained.

That is, the gist of the present invention resides in a polypeptide derived from hepatitis B virus X protein represented by the amino acid sequence shown in SEQ ID NO: 1 of the sequence listing, a gene encoding the same, and partial fragments thereof. The present invention will be described in detail below. The novel HBV-X protein-derived polypeptide of the present invention and the gene encoded thereby are those shown in SEQ ID NO: 1 in the Sequence Listing, or those containing all or a part thereof, and the polypeptide is an existing hepatitis. Detects viral infection and immunochemically reacts specifically with sera from patients infected with hepatitis virus that are not detected by diagnostic methods. Naturally, in the present invention, modification such as removal, insertion, modification or addition of a part of amino acids or nucleic acids within a range that does not impair the reactivity with the serum of a hepatitis virus-infected patient that is not detected by the existing diagnostic method for detecting hepatitis virus infection. Those which have been carried out are also included in the novel polypeptide of the present invention. Further, a gene encoding this modified polypeptide is also included in the scope of the present invention.

[0009] In addition, the polypeptide shown in SEQ ID NO: 1 of the sequence listing, or a polypeptide containing all or a part thereof is immunized with a specificity different from that of sera of a patient infected with hepatitis virus, which is not detected by the existing diagnostic method for detecting hepatitis virus infection. Chemically reactive substances are not detected by such existing hepatitis virus infection detection diagnostic methods, but some amino acids are removed, inserted, without impairing the reactivity with serum of hepatitis virus infected patients,
The novel polypeptide of the present invention also includes those modified or added. Further, a gene encoding this modified polypeptide is also included in the scope of the present invention.

Further, as shown in SEQ ID NO: 1 in the sequence listing,
Alternatively, a polypeptide containing all or part thereof is
It acts specifically on the sequences of enhancers and promoters of various genes. Of course, in the present invention, a part of amino acids are removed and inserted within a range that does not impair the action with the sequences of enhancers and promoters of such various genes,
The novel polypeptide of the present invention also includes those modified or added. Further, a gene encoding this modified polypeptide is also included in the scope of the present invention.

[0012] Similarly, the one shown in SEQ ID NO: 1 of the sequence listing, or a polypeptide containing all or a part thereof, which acts on the sequences of enhancers and promoters of various genes with different specificities, is also applicable. Modifications such as removal, insertion, modification or addition of some amino acids are included in the novel polypeptide of the present invention as long as they do not impair the action of various genes on the enhancer and promoter sequences. Further, a gene encoding this modified polypeptide is also included in the scope of the present invention. (1) A method for obtaining a novel HBV cDNA clone derived from the serum of a hepatitis virus-infected patient that is not detected by the existing diagnostic method for detecting hepatitis virus infection shown in SEQ ID NO: 1 of the sequence listing, and a method for determining the nucleotide sequence of the clone. The gene encoding the novel polypeptide represented by the amino acid sequence shown in the sequence table or its DNA fragment can be obtained, for example, by the following method.

That is, as shown in Example 1 described later, nucleic acids are extracted from the serum of a patient infected with hepatitis virus, which is not detected by the existing method for detecting and detecting hepatitis virus infection. The serum is usually HAV antibody, HBs-Ag, HBs-A in a radioimmunoassay test kit manufactured by Dynabot.
b, HBc-Ab, HBe-Ag, HBe-Ab, and PHA test kit HCV antibody manufactured by Dynabot Co., all of which have negative HCV antibody measurements are used. CDNA from such nucleic acid
As a means for obtaining the enzyme, a polymerase chain reaction method (PCR method) developed by Saiki et al. (Nature, 324 , 126 (1986))
Improve and use. Specifically, it is carried out under the conditions as described in the second embodiment. The first obtained in this way
By using the complementary strand DNA (1st cDNA) as a template and performing the PCR method as in Example 2, the desired DNA fragment can be obtained. At this time, the PCR conditions may vary depending on the type and combination of primers, the length of amplification, etc., so the conditions may be appropriately changed depending on the situation.

The DNA fragments thus obtained independently determine the nucleotide sequences of three or more clones for both strands. Fluorescence Sequencer Genesis 2000 (Genesis 2000) manufactured by DuPont
The system can be easily determined by following the protocol for the system from DuPont. When it is difficult to determine the base sequence or when the DNA fragment to be determined has about 180 bases or more, subcloning may be performed according to a conventional method.

The base sequence of the DNA fragment of the present invention thus determined is, for example, as shown in SEQ ID NO: 1 in the sequence listing. The amino acid sequence shown in SEQ ID NO: 1 in the sequence listing clearly differs from the known HBV-X region in Leu at position 73 and Val-Trp- at positions 131 to 134.
It is Arg-Leu (SEQ ID NO: 10 in the sequence listing). Further, although the known X region encodes 154 amino acids, the X gene obtained by such a method encodes 134 amino acids. This is X gene 21
The 7th gene is C in the X gene of the present invention, and the eight gene sequences TGTTACTA from the 395th to the 402nd of the known X gene were deleted, so that the 3'side codon was changed. Due to that. That is, this protein is completely different in antigenicity and physiological function from the conventional HBV X protein.

The X protein can be produced in a large amount using a gene recombination technique. That is, the gene (cDNA) encoding the X protein or a fragment thereof is 5 '
The ends are modified and inserted downstream of the promoter of the expression vector, and then introduced into host cells such as Escherichia coli, yeast and animal cells, and the host cells are cultured to produce recombinant X protein inside and outside the host cells.

As the expression vector, one containing a promoter at a position where the gene (cDNA) encoding the X protein or a fragment thereof can be transcribed is used. For example, when a microorganism such as Escherichia coli or Bacillus subtilis is used as a host, the expression vector is composed of a promoter, a ribosome binding (SD) sequence, a gene encoding the X protein (cDNA) or a fragment thereof, a transcription termination sequence, and a gene controlling the promoter. It is preferable that

Transformation of host cells can be carried out by a conventional method, and such transformants can be cultured by molecular cloning (Cold Spring Harbor).
Laboratory, 1982). The produced recombinant X protein is isolated and purified from the host cell by a known method.

[0018]

The X protein, which is the HBV X gene product, is
Not only is it useful as an antigen for detecting infection with HBV, but it also functions as an enhancer of HBV itself and enhancers and promoter sequences of other cellular genes through interaction with cellular factors in normal hepatocytes. Finding that it acts on trans and activates transcription,
Participation in liver cancer is drawing attention. The polypeptide of the present invention or a polypeptide containing all or a part thereof,
It is considered to be a novel region specific to the X protein of HBV, and if a hepatitis virus detection system using these polypeptides is developed, it is highly possible that negative ones will be detected as positive by the existing hepatitis virus infection detection method. , Useful as a polypeptide for diagnosis. Similarly, if a transcriptional activity detection system using these polypeptides and genes is developed, it will be useful for the development of effective antiviral agents.

[0019]

The present invention will be described in more detail with reference to the following examples, but the present invention is not limited to the following examples as long as the gist thereof is not exceeded. In addition, in the experimental operations of Examples 1 to 4, basically, in order to pay attention to sample contamination, for example, tips and pipettes used in all operations such as preparation of samples for each reaction solution and reagents were Replace after each use.

Further, the following basic gene manipulations, which were performed without mentioning any particular method, were carried out according to the experimental manuals shown in parentheses after each manipulation and the protocols attached by the reagent vendors. Electrophoresis of nucleic acids (Molecular Cloning, 1
982 Cold Spring Harbor (Cold
(Spring Harbor)), extraction of DNA fragments from acrylamide gels and agarose gels (Molecular Cloning).
g), 1982, Cold Spring Harbor (C
old Spring Harbor)).

Example 1 Extraction of Nucleic Acid from Serum of Hepatitis Virus-Infected Patient Not Detected by Existing Diagnosis Method for Hepatitis Virus Infection 6 Hepatitis Virus-Infected Patients Not Detected by Existing Diagnosis Method for Detection of Hepatitis Virus Infection (A1, A2, A3, respectively) ,
Sera derived from A4, A5, and A6) (these sera are HAV antibodies, HBs-Ag, HBs-Ab in a radioimmunoassay test kit manufactured by Dynabot),
HBc-Ab, HBe-Ag, HBe-Ab, and PHA test kit manufactured by Dynabot Co., Ltd. all showed negative results in HCV antibody measurement) in 1 ml each of Tris buffer (50
mM Tris-HCl (pH 8.0), 1 mM ED
TA, 100 mM NaCl) 1 ml, and after mixing 2
It was centrifuged at 10,000 g and 20 ° C. for 5 hours. A proteinase K solution (1% sodium dodecyl sulfate, 10
mM EDTA, 10 mM Tris-HCl (pH
7.5), Protenase K (Pharmacia)
2 mg / ml, yeast tRNA mixture
6.6 μg) 3 ml was added and dissolved, then 180 ° C for 70 minutes
Incubate at room temperature, add equal amount of phenol / chloroform, mix vigorously, and collect aqueous phase containing nucleic acid by centrifugation (hereinafter abbreviated as "phenol / chloroform treatment") 4 times or more. went.

Further, the chloroform treatment was performed twice or more. The aqueous phase thus obtained has a tenth amount of 3M.
Sodium acetate or an equal amount of 4M ammonium acetate and 2.5 times the volume of ethanol of the aqueous phase are added and mixed, and the mixture is kept at -20 ° C.
Overnight or at -80 ° C for 15 minutes or longer, then switch
41Ti rotor (Beckman) 35,000 rp
After centrifugation at m for 4 hours, the nucleic acid was recovered as a precipitate.

Example 2 Synthesis of cDNA [1] Preparation of sample After drying each nucleic acid derived from 6 patients obtained in Example 1, 30 μl of water was added and dissolved in each. The cDNA synthesis shown below was performed using this nucleic acid solution. [2] Synthesis of cDNA Using Synthetic Primer According to the method of Saiki et al. [Nature, 324, 126 (1986)] using 10 μl of each nucleic acid aqueous solution derived from 6 patients prepared in [1] DNA having a specific sequence by the so-called PCR method
Was amplified.

That is, each of these DNA samples was 10 μm.
l, 10x PCR buffer (100 mM Tris-HCl
(PH 8.3), 500 mM KCl, 15 mM Mg
Cl2, 1% gelatin) 10 μl, 2.5 mM 4d
NTP 8 μl, synthetic primer p198 (5′-TT
TTGCTCGCAGCCGGTCTG-3 ′: 2 μl (15 pmols / μl) of SEQ ID NO: 2 in Sequence Listing, and synthetic primer p200 (5′-TACGGGTCA)
ATGTCCATGCC-3 ′: SEQ ID NO: 3 in the sequence listing)
(15 pmols / μl) Add water to 2 μl for a total of 1
First, the solution was kept at 95 ° C. for 5 minutes so that it became 00 μl, and then rapidly cooled to 0 ° C. After 1 minute, 0.5 μl of Taq DNA polymerase (7 units / μl, AmpliTaq ™ (Takara Shuzo)) was added and mixed, and then overlaid with mineral oil. This sample was subjected to DNA Thermal Cycler manufactured by Perkin Elmer Cetus at 95 ° C. 1
25 minutes at 40 ° C to 55 ° C for 1 minute, 72 ° C for 1 minute to 5 minutes
Processed twice.

Finally, after incubating at 72 ° C. for 7 minutes, this reaction aqueous solution was treated with phenol / chloroform, and then ethanol precipitation was carried out by a conventional method to obtain an amplified DNA. Next, this amplified DNA fragment was added to p204 (5'-GAGCG
AAAACTTATCGGAACC-3 ′: SEQ ID NO: 4 in Sequence Listing) and p205 (5′-CCAAAGCCACC)
CAAGGCACA-3 ′: Second PC according to the above method using the synthetic primer of SEQ ID NO: 5) in the sequence listing.
Various amplified D derived from each of 6 patients by performing R
I got NA.

Example 3 Cloning of Amplified DNA Fragment and Determination of Nucleotide Sequence 6 hepatitis virus-infected patients obtained by the method of [2] of Example 2 which were not detected by the existing diagnostic method for detecting hepatitis virus infection. Each DNA fragment derived from serum was extracted by electrophoresis using a 2% agarose gel (Molecular Cloning).
g), 1982, Cold Spring Harbor (C
old Spring Harbor)). Genclean II kit (B
IO (Bio) 101 company) was used and the protocol attached to this kit was followed.

The extracted DNA fragment is prepared by a conventional method,
DuPont fluorescent sequencer GENESIS200
The 0 system was used to sequence. P202 (5'-TTCTGCCGTT as a sequence primer
CCGGCCGACCAC-3 ′: SEQ ID NO: 6 in the sequence listing), p201 (5′-GTGGGTCGCGCCGGAA)
CGGCAGAA-3 ′: SEQ ID NO: 7) in the sequence listing, p1
99 (5'-AGGCATACTTCAAAGACTG
TGT-3 ′: SEQ ID NO: 8) and P203 in Sequence Listing
(5'-ACACAGTCTTTTGAAGTATGCC
T-3 ′: Using SEQ ID NO: 9) in the sequence listing, the nucleotide sequences of the + strand and − strand of the HBV-X region portion of the DNA fragment were determined. Six hepatitis virus-infected patients (A1, A2, A3, A
4, A5 and A6) D of each + strand from serum
The NA fragments all matched and had a base sequence as shown in SEQ ID NO: 1 in the sequence listing.

The amino acid sequence of SEQ ID NO: 1 in the Sequence Listing clearly differs from the known HBV-X region in Leu at position 73 and Val-Tr from position 131 to position 134.
It was p-Arg-Leu (SEQ ID NO: 10 in the sequence listing). Further, although the known X region coded 154 amino acids, the obtained X gene coded 134 amino acids. This is because the 217th gene of the known X gene is C in the X gene of the present invention, and the TGTTACTA of 8 gene sequences from the 395th to the 402nd of the known X gene is deleted. Was due to. That is, it was found that the nucleotide sequence obtained here is specific to the hepatitis virus which is not detected by the existing hepatitis virus infection detection and diagnosis method.

[0029]

[Sequence list]

SEQ ID NO: 1 Sequence length: 405 Sequence type: Nucleic acid Number of chains: double-stranded Topology: linear Sequence type: cDNA to genomic RNA Origin: Hepatitis B virus Direct origin Patient sera negative for known hepatitis virus detection diagnostic methods Array   ATG GCT GCT AGG GTG TGC TGC CAA CTG GAT CCT GCG CGG GAC GTC CTT 48   Met Ala Ala Arg Val Cys Cys Gln Leu Asp Pro Ala Arg Asp Val Leu     1 5 10 15   TGT CTA CGT CCC GTC GGC GCT GAA TCC CGC GGA CGA CCC GTC TCG GGG 96   Cys Leu Arg Pro Val Gly Ala Glu Ser Arg Gly Arg Pro Val Ser Gly                20 25 30   CCG TTT GGG GCT CTA CCG TCC CCT TCT TCT TCT GCC GTT CCG GCC GAC 144   Pro Phe Gly Ala Leu Pro Ser Pro Ser Ser Ser Ala Val Pro Ala Asp            35 40 45   CAC GGG GCG CAC CTC TCT TTA CGC GGT CTC CCC GTC TGT GCC TTC TCA 192   His Gly Ala His Leu Ser Leu Arg Gly Leu Pro Val Cys Ala Phe Ser        50 55 60   TCT GCC GGA CCG TGT GCA CTT CGC CTC ACC TCT GCA CGT CGC ATG GAG 240   Ser Ala Gly Pro Cys Ala Leu Arg Leu Thr Ser Ala Arg Arg Met Glu    65 70 75 80   ACC ACC GTG AAC GCC CAC CAG GTC TTG CCC AAG GTC TTA CAT AAG AGG 288   Thr Thr Val Asn Ala His Gln Val Leu Pro Lys Val Leu His Lys Arg                    85 90 95   ACT CTT GGA CTC TCA GCC ATG TCA ACG ACC GAC CTT GAG GCA TAC TTC 336   Thr Leu Gly Leu Ser Ala Met Ser Thr Thr Asp Leu Glu Ala Tyr Phe               100 105 110   AAA GAC TGT TTG TTT AAA GAC TGG GAG GAG TTG GGG GAG GAG ATT AGG 384   Lys Asp Cys Leu Phe Lys Asp Trp Glu Glu Leu Gly Glu Glu Ile Arg           115 120 125   TTA AAG GTC TGG AGG CTG TAG 405   Leu Lys Val Trp Arg Leu stop       130 135

SEQ ID NO: 2 Sequence length: 20 Sequence type: Nucleic acid Topology: linear Sequence type: Other nucleic acids Synthetic DNA Array   TTTTGCTCGC AGCCGGTCTG 20

SEQ ID NO: 3 Sequence length: 20 Sequence type: Nucleic acid Topology: linear Sequence type: Other nucleic acids Synthetic DNA Array   TACGGGTCAA TGTCCATGCC 20

SEQ ID NO: 4 Sequence length: 20 Sequence type: Nucleic acid Topology: linear Sequence type: Other nucleic acids Synthetic DNA Array   GAGCGAAACT TATCGGAACC 20

SEQ ID NO: 5 Sequence length: 20 Sequence type: Nucleic acid Topology: linear Sequence type: Other nucleic acids Synthetic DNA Array   CCAAAGCCAC CCAAGGCACA 20

SEQ ID NO: 6 Sequence length: 22 Sequence type: Nucleic acid Topology: linear Sequence type: Other nucleic acids Synthetic DNA Array   TTCTGCCGTT CCGGCCGACC AC 22

SEQ ID NO: 7 Sequence length: 22 Sequence type: Nucleic acid Topology: linear Sequence type: Other nucleic acids Synthetic DNA Array   GTGGTCGGCC GGAACGGCAG AA 22

SEQ ID NO: 8 Sequence length: 22 Sequence type: Nucleic acid Topology: linear Sequence type: Other nucleic acids Synthetic DNA Array   AGGCATACTT CAAAGACTGT GT 22

SEQ ID NO: 9 Sequence length: 22 Sequence type: Nucleic acid Topology: linear Sequence type: Other nucleic acids Synthetic DNA Array   ACACAGTCTT TGAAGTATGC CT 22

SEQ ID NO: 10 Sequence length: 4 Sequence type: Amino acid Topology: linear Sequence type: Peptide Array Val Trp Arg Leu

─────────────────────────────────────────────────── ─── Continuation of the front page (56) References JP-A-4-126085 (JP, A) International Publication 91/019800 (WO, A1) Jpn. J. Exp. Med., 1987, Vol. 57, No. 4, p. 231-236 J. Gen. Virol. , 1988, Vol. 69, p. 2575-2583 (58) Fields investigated (Int.Cl. ⁷ , DB name) C07K 14/02 C12N 15/09 ZNA SwissProt / PIR / GeneSeq GenBank / EMBL / DDBJ / GeneSeq BIOSIS / WPI (DIALOG)

Claims (3)

(57) [Claims] 1. A polypeptide derived from hepatitis B virus X protein represented by the following amino acid sequences. Met Ala Ala Arg Val Cys Cys Gln Leu Asp Pro Ala Arg Asp Val Leu 1 5 10 15 Cys Leu Arg Pro Val Gly Ala Glu Ser Arg Gly Arg Pro Val Ser Gly 20 25 30 Pro Phe Gly Ala Leu Pro Ser Pro Ser Ser Ser Ser Ala Val Pro Ala Asp 35 40 45 His Gly Ala His Leu Ser Leu Arg Gly Leu Pro Val Cys Ala Phe Ser 50 55 60 Ser Ala Gly Pro Cys Ala Leu Arg Leu Thr Ser Ala Arg Arg Met Glu 65 70 75 80 Thr Thr Val Asn Ala His Gln Val Leu Pro Lys Val Leu His Lys Arg 85 90 95 Thr Leu Gly Leu Ser Ala Met Ser Thr Thr Asp Leu Glu Ala Tyr Phe 100 105 110 Lys Asp Cys Leu Phe Lys Asp Trp Glu Glu Leu Gly Glu Glu Ile Arg 115 120 125 Leu Lys Val Trp Arg Leu 130 2. A gene encoding the polypeptide according to claim 1. 3. A gene encoding a polypeptide derived from hepatitis B virus X protein represented by the following base sequence. ATGGCTGCTA GGGTGTGCTG CCAACTGGAT CCTGCGCGGG ACGTCCTTTG TCTACGTCCC 60 GTCGGCGCTG AATCCCGCGG ACGACCCGTC TCGGGGCCGT TTGGGGCTCT ACCGTCCCCT 120 TCTTCTTCTG CCGTTCCGGC CGACCACGGG GCGCACCTCT CTTTACGCGG TCTCCCCGTC 180 TGTGCCTTCT CATCTGCCGG ACCGTGTGCA CTTCGCCTCA CCTCTGCACG TCGCATGGAG 240 ACCACCGTGA ACGCCCACCA GGTCTTGCCC AAGGTCTTAC ATAAGAGGAC TCTTGGACTC 300 TCAGCCATGT CAACGACCGA CCTTGAGGCA TACTTCAAAG ACTGTTTGTT TAAAGACTGG 360 GAGGAGTTGG GGGAGGAGAT TAGGTTAAAG GTCTGGAGGC TGTAG 405