JPH05192160A - C type hepatitis virus genome rna, cdna and polypeptide - Google Patents

Abstract

PURPOSE:To enable the screening of a diagnostic reagent and an antiviral medicine by adding a gene containing a prescribed basic sequence or its fragment. CONSTITUTION:A gene having a basic sequence of the formula obtained by the purification of RNA produced by extracting the serum of a C type hepatitis patient or the fragment of the gene is added to obtain (A) a C type hepatitis virus (HCV) genome RNA. The component A is reacted with a reverse transcriptase, etc., to produce (B) a cDNA complementary or homologous to the component A. The component B is introduced into a plasmid to obtain (C) a recombined DNA containing an expressed plasmid (e.g. pSR 2541). The component C is introduced into a host cell (e.g. COS-1 cell), and the produced transformant strain is cultured to produce (D) a HCV protein. The blood of a C type hepatitis patient is subjected to an immunoassay using the component D as an antigen to detect a HCV antigen in the blood.

Description

Detailed Description of the Invention

[0001]

FIELD OF THE INVENTION The present invention relates to hepatitis C virus genes and polypeptides. More specifically, the present invention is a set comprising a gene encoding a hepatitis C trypsin-like serine protease and its substrate, which can be used for diagnosing hepatitis caused by hepatitis C virus and screening for hepatitis C virus-specific antiviral drugs. It relates to a modified DNA and the same viral polypeptide.

[0002]

2. Description of the Related Art Hepatitis A virus belonging to picornavirus and hepatitis B virus belonging to hepadnavirus have already been identified as the causative viruses of viral hepatitis, and immunoserologic diagnostic methods have been established for each of them and put to practical use. Have been offered to.

However, it is gradually becoming clear that most hepatitis associated with blood transfusion is caused by a pathogen different from hepatitis A and B viruses and other viruses that grow in hepatocytes, and non-A non-B hepatitis. Was named.

At present, in Japan or the United States, 90% or more of post-transfusion hepatitis and about 5 cases of sporadic hepatitis without transfusion.
This hepatitis accounts for 0%.

In recent years, the non-A non-B hepatitis virus was successfully cloned by Chiron Corporation of the United States and named Hepatitis C virus (HCV). After that, the HCV gene was cloned in Europe, America and Asia.
It has been revealed that the nucleotide sequence of the V gene differs considerably depending on the region (for example, in the Japanese type and the American type,
10-40% difference).

At present, an antiviral antibody detection system using a part of the HCV virus antigen is used for diagnosing hepatitis (hepatitis C) caused by HCV.
It is desired to develop an effective and more general diagnostic method capable of accurately detecting V clones. Further, at present, no method has been found for measuring physiological activity such as enzyme activity of HCV, but if this method is established, HCV replication,
Since it becomes a marker showing the degree of proliferation, it is considered to be extremely useful in clinical diagnosis. Moreover, since a substance that selectively inhibits such an enzyme activity can be an HCV-specific antiviral drug, the enzyme activity assay method is also useful as a screen for an antiviral drug.

[0007]

In view of the current state of viral hepatitis, the problem to be solved by the present invention is to provide an effective diagnostic method for hepatitis caused by hepatitis C virus and a screening method for therapeutic agents. Is.

[0008]

[Means for Solving the Problems] In order to solve the above problems, the present inventors have isolated a non-A non-B hepatitis virus gene and have succeeded in producing an antigen protein. The viral gene of the present invention includes a serine protease and its substrate part among the constituent parts of the HCV gene. At present, HCV is classified as a flavivirus or a flavivirus based on the homology of its amino acid sequence and the like.
The flavivirus viral genome is a single-stranded RNA with positive polarity. The open reading frame of the viral gene is one over the entire length of the genome. The structural protein genes are arranged on the 5 ′ side, and the nonstructural protein genes are arranged on the 3 ′ side. First, a huge protein corresponding to the full length of the reading frame is read, and this is divided by the intracellular enzyme and the enzyme activity inherent in the viral protein itself, resulting in nucleocapsid proteins, coat proteins, and nonstructural proteins called NS1-NS5. To be processed. Of these nonstructural proteins, NS3
Has been shown to have serine protease activity in yellow fever virus and dengue virus closely related to HCV,
In the amino acid sequence of NS3 in HCV, three pairs of catalytic residues consisting of serine, aspartic acid, and histidine necessary for the activity of serine protease are conserved, and the amino acid sequence serving as a substrate for serine protease is C of NS2.
It is well conserved from the end to the N-terminal part of NS3. More recently, in a yellow fever virus mutant strain with a substitution mutation in the catalytic residue portion of this serine protease, virus particles were not formed, indicating that this protease activity is essential for viral replication. Was done.

[0009] On the other hand, as described above, HCV has been reported to have diversity, but in general, different strains of the same virus have different immunoserologic properties, and the same diagnostic method shows that Differences in detection sensitivity may occur between strains, and the problem of false positives may occur.

At present, an immunoserologic diagnostic method is being developed for hepatitis C as well, but it has a number of problems such as specificity and false positives, which are thought to be due to the diversity of HCV. Under such circumstances, the inventors of the present invention have an essential serine protease which is presumed to have extremely high homology in its nucleotide sequence and amino acid sequence among various HCV strains because it is essential for viral replication. The HCV gene was cloned for the purpose of using the substrate region for detection of HCV antibody and endogenous protease activity, and the gene fragment was expressed by gene recombination technology to obtain the obtained expression. Product reacts specifically with serum of hepatitis C patient and HCV by protease
The processing of the polypeptide was confirmed, and the present invention was completed. That is, the present invention provides RNA and recombinant DNA containing the gene encoding the hepatitis C trypsin-like serine protease and its substrate, and the viral polypeptide.

A preferred embodiment of the RNA of the present invention, the following sequence as the gene (SEQ ID NO: 1) 5'GACCAAGGUC AUCACCUGGG GGGCAGACAC CGCGGCGUGU GGGGACAUUA UCUCAGGUCU ACCCGUCUCC GCCCGAAGGG GGAAGGAGAU ACUUUUGGGA CCGGCCGAUA GUUUUGAAGG GCAGGGGUGG CGACUCCUUG CGCCCAUCAC GGCCUACUCC CAACAGACGC GGGGCCUACU UGGUUGCAUU AUCACUAGUC UCACGGGCCG GGACAAGAAC CAGGUUGAGG GGGAGGUUCA AGUGGUCUCC ACCGCAACAC AAUCUUUCCU GGCGACCUGU GU AACGGCG CGUGCUGGAC UGUCUUUCAC GGUGCCGGCU CAAAGACCUU AGCCGGCCCA AAAGGCCCAA UCACCCAAAU GUACACCAAU GUAGACCAGG ACCUCGUCGG CUGGCCAGCG CCCCCCGGGG CGCGUUCCUU AACACCGUGC ACCUGCGGCA GCUCGGACCU UUACCUGGUC ACGAGACAUG CUGAUGUCAU CCCGGUGCGC CGGCGGGGCG ACACUAGGGG GAGCUUGCUC UCCCCUAGAC CCAUCUCCUA CUUGAAGGGC UCUUCGGGUG GUCCAUUGCU CUGCCCCUCG GGGCACGUUG UGGGCAUCUU CCGGG CUGCC GUGUGCACCC GGGGGGUCGC GAAGGCGGUG GACUUCAUAC CCGUUGAAUC UAUGGAAACC ACUAUGCGGU CUCCGGUAUU CACAGACAAC UCAACCCCCC CGGCCGUACC GCAGACCUUC CAAGUGGCCC AUCUACACGC CCCCACUGGC AGCGGUAAAA GCACUAAGGU GCCGGCUGCA UAUGCAGCCC AAGGGUACAA GGUGCUCGUC CUGAACCCGU CCGUUGCUGC CACCCUGGGU UUUGGGGCGU or those having a fragment thereof, and the following sequence as the gene (SEQ ID NO: 2) 5'CUGCUGAUAG CCCAGGCCGA GGCCGCCUUA GAGA ACUUGG UGGUCCUCAA UGCGGCGUCC GUGGCCGGAG CGCAUGGCAU UCUCCCCUUC UUUAUGUUCU UCUGUGCCGC CUGGUACAUU AAGGGCAGGC UGGUCCCUGG AGCGGCAUAC GCUUUCUACG GCGUAUGGCC GCUGCUCCUG CUCUUGCUAG CAUUACCACC ACGAGCUUAC GCCAUGGACC GGGAGAUGGC UGCAUCUUGC GGAGGGGGGG UUUUUGUAGG UCUAGCACUC CUGACCUUGU CACCAUACUA UAAAGUGUUC CUCGCUAGGC UCAUAUGGUG GUUACAAUAU UUUAUCACCA AAGCCGAGGC GCAUUU CAA GUGUGGGUCC CCCCCCUCAA CGUUCGAGGC GGCCGCGAUG CCAUCAUCCU CCUCAUGUGC GCGGUCCACC CAGAGCUAAU CUUUGACAUC ACCAAACUUC UGCUCUCCAU ACUCGGUCCG CUCAUGGUGC UCCAAGCUAG UUUAAUCCGA GUGCCGUACU UCGUGCGCGC UCAAGGGCUC AUUCGCGCAU GCAUGUUGGU GCGGAAAGCU GCCGGGGGCC AUUAUGUCCA AAUGGCCUUC GUGAAGCUAG CUGCGCUGAC AGGCACGUAC GUUUAUGACC ACCUCACUCC ACUGCAGGAU UGGGCCCAUG UGGGCCUAC AGACCUUGCG GUGGCAGUAG AGCCCGUUGU CUUUUCUGCC AUGGAGACCA AGGUCAUCAC CUGGGGGGCA GACACCGCGG CGUGUGGGGA CAUUAUCUCA GGUCUACCCG UCUCCGCCCG AAGGGGGAAG GAGAUACUUU UGGGACCGGC CGAUAGUUUU GAAGGGCAGG GGUGGCGACU CCUUGCGCCC AUCACGGCCU ACUCCCAACA GACGCGGGGC CUACUUGGUU GCAUUAUCAC UAGUCUCACG GGCCGGGACA AGAACCAGGU UGAGGGGGAG GUUCAAGUGG UCUCCACCGC AACACAAUCU UUCCUGGCGA C UGUGUCAA CGGCGCGUGC UGGACUGUCU UUCACGGUGC CGGCUCAAAG ACCUUAGCCG GCCCAAAAGG CCCAAUCACC CAAAUGUACA CCAAUGUAGA CCAGGACCUC GUCGGCUGGC CAGCGCCCCC CGGGGCGCGU UCCUUAACAC CGUGCACCUG CGGCAGCUCG GACCUUUACC UGGUCACGAG ACAUGCUGAU GUCAUCCCGG UGCGCCGGCG GGGCGACACU AGGGGGAGCU UGCUCUCCCC UAGACCCAUC UCCUACUUGA AGGGCUCUUC GGGUGGUCCA UUGCUCUGCC CCUCGGGGCA CGUUGUGGGC AUCU CCGGG CUGCCGUGUG CACCCGGGGG GUCGCGAAGG CGGUGGACUU CAUACCCGUU GAAUCUAUGG AAACCACUAU GCGGUCUCCG GUAUUCACAG ACAACUCAAC CCCCCCGGCC GUACCGCAGA CCUUCCAAGU GGCCCAUCUA CACGCCCCCA CUGGCAGCGG UAAAAGCACU AAGGUGCCGG CUGCAUAUGC AGCCCAAGGG UACAAGGUGC UCGUCCUGAA CCCGUCCGUU GCUGCCACCC UGGGUUUUGG GGCGU'3 or has a fragment thereof. Further, the DN of the present invention
A preferred embodiment of A is a gene having a DNA complementary or homologous to the RNA of SEQ ID NO: 1 or 2 or a fragment thereof as a gene. Further, preferred embodiments of the DNA of the present invention has the following sequence (SEQ ID NO: 3) Thr Lys Val Ile Thr Trp Gly Ala Asp Thr Ala Ala Cys Gly Asp Ile Ile Ser Gly Leu Pro Val Ser Ala Arg Arg Gly Lys Glu Ile Leu Leu Gly Pro Ala Asp Ser Phe Glu Gly Gln Gly Trp Arg Leu Leu Ala Pro Ile Thr Ala Tyr Ser Gln Gln Thr Arg Gly Leu Leu Gly Cys Ile Ile Thr Ser Leu Thr Gly Arg Asp Lys Asn Gln Val Glu Gly Glu Val Gln Val Val Ser Thr Ala Thr Gln Ser Phe Leu Al a Thr Cys Val Asn Gly Ala Cys Trp Thr Val Phe His Gly Ala Gly Ser Lys Thr Leu Ala Gly Pro Lys Gly Pro Ile Thr Gln Met Tyr Thr Asn Val Asp Gln Asp Leu Val Gly Trp Pro Ala Pro Pro Gly Ala Arg Ser Leu Thr Pro Cys Thr Cys Gly Ser Ser Asp Leu Tyr Leu Val Thr Arg His Ala Asp Val Ile Pro Val Arg Arg Arg Gly Asp Thr Arg Gly Ser Leu Leu Ser Pro Arg Pro Ile Ser Tyr Leu Lys Gly Ser Ser Gly Gly Pro Leu Leu Cy s Pro Ser Gly His Val Val Gly Ile Phe Arg Ala Ala Val Cys Thr Arg Gly Val Ala Lys Ala Val Asp Phe Ile Pro Val Glu Ser Met Glu Thr Thr Met Arg Ser Pro Val Phe Thr Asp Asn Ser Thr Pro Pro Ala Val Pro C type having Gln Thr Phe Gln Val Ala His Leu His Ala Pro Thr Gly Ser Gly Lys Ser Thr Lys Val Pro Ala Ala Tyr Ala Ala Gln Gly Tyr Lys Val Leu Val Leu Asn Pro Ser Val Ala Ala Thr Leu Gly Phe Gly Ala Hepatitis virus polype Cide, the following sequence (SEQ ID NO: 4) Gly Arg Leu Val Pro Gly Ala Ala Tyr Ala Phe Tyr Gly Val Try Pro Leu Leu Leu Leu Leu Leu Ala Leu Pro Pro Arg Ala Tyr Ala Met Asp Arg Glu Met Ala Ala Ser Cys Gly Gly Val Phe Val Gly Leu Ala Leu Leu Thr Leu Ser Pro Tyr T r Lys Val Phe Leu Ala Arg Leu Ile Trp Trp Leu Gln Tyr Phe Ile Thr Lys Ala Glu Ala His Leu Gln Val Trp Val Pro Pro Leu Asn Val Arg Gly Gly Arg Asp Ala Ile Ile Leu Leu Met Cys Ala Val His Pro Glu Leu Ile Phe Asp Ile Thr Lys Leu Leu Leu Ser Ile Leu Gly Pro Leu Met Val Leu Gln Ala Ser Leu Ile Arg Val Pro Tyr Phe Val Arg Ala Gln Gly Leu Ile Arg Ala Cys Met Leu Val Arg Lys Ala Ala Gly Gly His Tyr Val G n Met Ala Phe Val Lys Leu Ala Ala Leu Thr Gly Thr Tyr Val Tyr Asp His Leu Thr Pro Leu Gln Asp Trp Ala His Val Gly Leu Arg Asp Leu Ala Val Ala Val Glu Pro Val Val Phe Ser Ala Met Glu Thr Lys Val Ile Thr Trp Gly Ala Asp Thr Ala Ala Cys Gly Asp Ile Ile Ser Gly Leu Pro Val Ser Ala Arg Arg Gly Lys Glu Ile Leu Leu Gly Pro Ala Asp Ser Phe Glu Gly Gln Gly Trp Arg Leu Leu Ala Pro Ile Thr Ala Tyr Ser Gln G n Thr Arg Gly Leu Leu Gly Cys Ile Ile Thr Ser Leu Thr Gly Arg Asp Lys Asn Gln Val Glu Gly Glu Val Gln Val Val Ser Thr Ala Thr Gln Ser Phe Leu Ala Thr Cys Val Asn Gly Ala Cys Trp Thr Val Phe His Gly Ala Gly Ser Lys Thr Leu Ala Gly Pro Lys Gly Pro Ile Thr Gln Met Tyr Thr Asn Val Asp Gln Asp Leu Val Gly Trp Pro Ala Pro Pro Gly Ala Arg Ser Leu Thr Pro Cys Thr Cys Gly Ser Ser Asp Leu Tyr Leu Val Thr A g His Ala Asp Val Ile Pro Val Arg Arg Arg Gly Asp Thr Arg Gly Ser Leu Leu Ser Pro Arg Pro Ile Ser Tyr Leu Lys Gly Ser Ser Gly Gly Pro Leu Leu Cys Pro Ser Gly His Val Val Gly Ile Phe Arg Ala Ala Val Cys Thr Arg Gly Val Ala Lys Ala Val Asp Phe Ile Pro Val Glu Ser Met Glu Thr Thr Met Arg Ser Pro Val Phe Thr Asp Asn Ser Thr Pro Pro Ala Val Pro Gln Thr Phe Gln Val Ala His Leu His Ala Pro Thr Gly Ser G y Lys Ser Thr Lys Val Pro Ala Ala Tyr Ala Ala Gln Gly Tyr Lys Val Leu Val Leu Asn Pro Ser Val Ala Ala Thr Leu Gly Phe Gly Ala Leu Leu Ile Ala Gln Ala Glu Ala Ala Ile Glu Asn Leu Val Val Leu Asn Ala Ala Ser Val Ala Gly Ala His Gly Ile Leu Pro Phe Phe Met Phe Phe Cys Ala Ala Trp Tyr Ile Lys Gly Arg Leu Val Pro Gly Ala Ala Tyr Ala Phe Tyr Gly Val Try Pro Leu Leu Leu Leu Leu Leu Ala Leu Pro Pro Arg A a Tyr Ala Met Asp Arg Glu Met Ala Ala Ser Cys Gly Gly Val Phe Val Gly Leu Ala Leu Leu Thr Leu Ser Pro Tyr Tyr Lys Val Phe Leu Ala Arg Leu Ile Trp Trp Leu Gln Tyr Phe Ile Thr Lys Ala Glu Ala His Leu Gln Val Trp Val Pro Pro Leu Asn Val Arg Gly Gly Arg Asp Ala Ile Ile Leu Leu Met Cys Ala Val His Pro Glu Leu Ile Phe Asp Ile Thr Lys Leu Leu Leu Ser Ile Leu Gly Pro Leu Met Val Leu Gln Ala Ser Leu Ile A g Val Pro Tyr Phe Val Arg Ala Gln Gly Leu Ile Arg Ala Cys Met Leu Val Arg Lys Ala Ala Gly Gly His Tyr Val Gln Met Ala Phe Val Lys Leu Ala Ala Leu Thr Gly Thr Tyr Val Tyr Asp His Leu Thr Pro Leu Gln Asp Trp Ala His Val Gly Leu Arg Asp Leu Ala Val Ala Val Glu Pro Val Val Phe Ser Ala Met Glu C-type hepatitis virus polypeptide or following sequence (SEQ ID NO: 5) Asn Gly Ala Cys Trp Thr Val Phe His Gly Ala Gly Ser Lys Thr Leu Ala Gly ro Lys Gly Pro Ile Thr Gln Met Tyr Thr Asn Val Asp Gln Asp Leu Val Gly Trp Pro Ala Pro Pro Gly Ala Arg Ser Leu Thr Pro Cys Thr Cys Gly Ser Ser Asp Leu Tyr Leu Val Thr Arg His Ala Asp Val Ile Pro Val Arg Arg Arg Gly Asp Thr Arg Gly Ser Leu Leu Ser Pro Pro Arg Pro Ile Ser Tyr Le Le Lys Gly Ser Ser Al Gy Ser Ly Ger Pro Ser Le Ger Pro Gy Pro Ser Le Gur Pro Gy Pro Ser Le Gur Gur DN coding Nucleotide sequence or recombinant DN, characterized in that it comprises a fragment thereof
It is A.

Next, the method for determining the nucleotide sequence of the HCV gene, the method for preparing the recombinant DNA, the method for detecting the HCV antibody, and the method for detecting the HCV protease activity will be described to explain the present invention in more detail. After extracting RNA using plasma of a hepatitis C patient as a material for cloning a gene as the object of the present invention, Kato et al. [Proc. Natl. Acad. Sci. USA 8
7 , 9524-9528 (1990)] reported HC
Based on the nucleotide sequence of the V gene, a primer was prepared and R
NA was reverse transcribed and the cDNA of the region containing the HCV protease and its substrate portion was selectively amplified by the PCR (Polymerase Chain Reaction) method.

The number of DNA fragments amplified by the PCR method is 2
Fractionated by% agarose gel electrophoresis and purified. This was inserted into the SmaI site of plasmid pUC19,
Obtained plasmids pUC3241 and pUC2533
Escherichia coli JM109 strain was transformed with Escherichia coli and the nucleotide sequence was determined by the dideoxy chain termination method. In order to express the thus obtained HCV gene in animal cells, for example, the expression plasmid pcDLSRα
When 296 is used, recombinant DNA can be prepared as follows.

Harada et al. [J. Virol. 65 ,
3015-3021 (1991)], which has been reported to express the HCV core protein.
After blunting the HgiAI site downstream of the translation initiation codon at the N-terminus of the HCV gene of 316, the NotI linker was ligated to change to the NotI site.
And Asp718 to prepare a 3.4 kb fragment containing SRα promoter, translation initiation codon and poly A addition signal. On the other hand, pUC32 containing the HCV gene of the present invention
41 was digested with BamHI to blunt the ends and NotI
After ligating with the linker, it was digested with NotI and Asp718, and 0.9 kb NotI-A containing HCV gene was digested.
Prepare the sp718 fragment. 3.4k thus obtained
The expression plasmid pSR3241 is obtained by ligating b with the 0.9 kb fragment. Furthermore, this pSR3
After digesting 241 with NotI, the ends were blunted, XbaI linker was ligated, and digested with SacII and XbaI,
Prepare a 4.2 kb DNA fragment. On the other hand, H of the present invention
SacI was added to the plasmid pUC2533 containing the CV gene.
0.8k containing HCV gene digested with I and XbaI
The DNA fragment of b was prepared and the 4.2 kb fragment and 0.8 kb
Expression plasmid pSR25 by ligating with the fragment
41 is obtained. The expression vector is not limited to this example, and for example, SV40, cytomegalovirus promoter, metallothionein promoter, poly A addition signal and the like may be used.

Next, a host cell (for example, COS-1 cell)
The HCV protein of the present invention can be obtained by transfecting the expression plasmid prepared in 1. and recovering the cells 2 days later. Using the thus obtained HCV protein as an antigen, an HCV antibody in the blood of a hepatitis C patient can be detected by an immunoassay such as Western blotting, and an HCV protein fragment cleaved by an HCV protease can be detected. You can

[0016]

EXAMPLES The present invention will be described in more detail with reference to examples, but the present invention is not limited to these examples. In addition, DNA and RN in the examples
A, various enzymes, E. coli, culture cells, etc. are handled by Molecular Cloning a Lab
oratory Manual, Second Ed
edition, T.I. Maniatis et al. Ed. (198
9 years) Cold Spring Harbor La
The gallery was used as a reference. Example 1 Cloning of HCV cDNA C-100, which is an HVs antigen-negative and HCV-infected marker.
Antibody (Ortho; Ortho-anti C-10
0 antibody ELISA assay systems
150) by positive hepatitis C patients (according to tem)
500 μl of guanidinium isothiocyanate solution (6 M guanidinium isothiocyanate, 5 mM)
Sodium citrate PH 7.0, 0.1 M β-mercaptoethanol, 0.5% sodium lauroyl sarcosinate) was added to denature the protein, and then 50 μl.
2M sodium acetate (pH 4.0) was added, and the mixture was extracted with phenol / chloroform, and an equal volume of 100% isopropanol was added.
It was precipitated by the After washing the pellet with 70% ethanol, 200 μl of sodium dodecyl sulfate (SDS) solution (0.2% SDS, 10 mM Tris-HCl p
H8, 1 mM EDTA). Furthermore, 20
0 μl urea solution (7M urea, 1% SDS, 0.35
M NaCl, 10 mM Tris-HCl pH7.
4, 10 mM EDTA) was added and extracted with phenol / chloroform, and then 1/10 amount of 2 M sodium acetate was added to precipitate with ethanol. Next, add 10 μl of the pellet to 1
What was dissolved in mM dithiothreitol (DTT) was added to R
CDNA was prepared as an NA solution. First, the base sequence (5'-CCATGTGCCTTGGACATA-
3 ') having the antisense primer-413JA, a reverse transcription reaction was performed according to the following composition to obtain (-) chain cDNA.
A was synthesized.

Composition (A) RNA solution 2 μl 5 × RT buffer- ¹⁾ 2 μl 413JA (1 μl / μg) 1 μl H ₂ O 2.5 μl reverse transcriptase 0.5 μl 1): 250 mM Tris-HCl pH 8.8,5
0 mM MgCl ₂ , 700 mM KCl, 50 mM
DTT After incubating this reaction solution at 42 ° C. for 2 hours, 1 μl of 1 mg
/ Ml RNaseA was added, and the solution having the following composition (B) was further added to this solution to perform polymerase-chain reaction (PCR) at 94 ° C for 1 minute, 55 ° C for 1 minute, and 77 ° C.
For 30 minutes under the condition of 2 minutes.

Composition (B) 2.5 mM dNTPs 3 μl 10 × PCR buffer- ²⁾ 5 μl sense primer-312JS ³⁾ (1 μg / μl) 1 μl H ₂ O 30 μl Taq polymerase 0.5 μl mineral oil 35 μl 2) 100 mM Tris -HCl pH 8.3, 50
0 mM KCl, 15 mM MgCl ₂ , 0.1% gelatin 3) 5′-GTCCAAATGGCCTTCATGAA
-3 'Next, take 1/50 amount of the reaction solution of composition (B) to obtain antisense primer-413JA and the base sequence (5'-GT
Sense primer-323JS having CCAAATGGCCTTTATGAA-3 ') was added, and PCR was further performed for 30 cycles.

2.0% of the product obtained by the above reaction
When separated by agarose gel electrophoresis and stained with ethidium bromide, a DNA band was observed at a position of 0.9 kb. After fractionating this band and purifying the DNA, the plasmid pUC19 digested with SmaI was cloned to obtain plasmid pUC3241, which was subjected to dideoxy chain termination to determine the nucleotide sequence of the inserted DNA region (complementary or homologous to SEQ ID NO: 1). DNA) was determined.

Furthermore, 5'of the protease and substrate regions
In order to clone the HCV gene in the terminal region, a primer having the following nucleotide sequence was prepared, and cDNA cloning was performed in the same manner as above. Primer- J248S: 5'-GCGTGGGAGCACAGGGCT-3 'J253S: 5'-GTGCCTGCTTTGTGGATGAGTG-3'331JA: 5'-CCTGAGATAATGTCCCCACA-3' 336JA: 5'-JTCATCCTCACA-3 '336JA: 5'-JAGCTCTCACA. Chain cd
NA was synthesized, J248S was further added, and PCR was performed.
A second PCR was performed using the 1/50 amount and J253S and 331JA. The obtained product is 2.0% agar
A 0.8 kb band was detected after separation by sgel electrophoresis and staining. This was fractionated, purified, and cloned into SmaI-digested plasmid pCU19 to obtain plasmid pUC2533. The nucleotide sequence of the inserted HCV gene was determined by dideoxy chain termination (DNA complementary or homologous to the sequence from the 1st to the 762nd nucleotide in SEQ ID NO: 2).

When the amino acid sequence was deduced from the obtained nucleotide sequence, the region from amino acids 337 to 447 was similar to the amino acid sequence of the trypsin-like serine protease region present in flavivirus and pestivirus,
The amino acid sequence conserved in the active region of serine protease was completely conserved. The site presumed to be cleaved by serine protease is amino acid 2
It was conserved between the 68th and 269th positions (amino acid number according to SEQ ID NO: 4). Creating SRα promoter Example 2 Plasmid PSR3241 - data -, the HigAI site downstream of the HCV gene translation initiation codon of plasmid pSR316 with poly A addition signal of SV40 was cut, T _4-DNA
The ends were blunted by the action of polymerase. This fragment and T ₄ - polynucleotide Kina - NotI were phosphorylated at the 5 'end with zero linker - the T _4-DNA Riga - coupled with zero, after digestion with NotI and Asp718, 1% agarose - subjected to Sugeru electrophoresis A 3.4 kb DNA fragment was isolated. In addition, the plasmid pUC3241 was cloned into Bam
After digesting with HI, blunt the ends as above, then Not
I linkers were ligated and digested with NotI and Asp718. This is subjected to 1% agarose gel electrophoresis,
A 0.9 kb DNA fragment was isolated. Two isolated D
Mixed NA fragment, T _4-DNA Riga - was coupled with zero. E. coli JM109 strain was transformed to 50 μg / ml
The cells were spread and cultured on solid LB medium containing ampicillin.
A plasmid was obtained from the resulting clone, digested with BamHI, and subjected to gel electrophoresis to obtain DNs of about 1 kb and about 3.3 kb.
The A fragment was confirmed, and this plasmid was named pSR3241. Example 3 Construction of plasmid pSR2541 Plasmid pSR3241 was digested with NotI to give T ₄ −
DNA polymerase - a blunted by zero, the fragment T ₄ - polynucleotide Kina - XbaI linker 5 'ends were phosphorylated at zero - the T _4-DNA Riga - coupled with zero, after digestion with SacII XbaI Digested with and subjected to gel electrophoresis to isolate a 4.2 kb fragment.

Similarly, the plasmid pUC2533 was cloned into Sa
After digestion with cII and XbaI, 1% agarose gel electrophoresis was performed to isolate a 0.8 kb DNA fragment. The two isolated DNA fragments were mixed and the T ₄ -DNA ligation was performed.
It was combined with ze. Transformation of E. coli JM109 strain,
The cells were spread and cultured on solid LB medium containing 50 μg / ml of ampicillin. A plasmid was obtained from the generated clone, digested with BamHI, and subjected to gel electrophoresis to give about 1.7 kb.
And a DNA fragment of about 3.3 kb was confirmed, and this plasmid was named pSR2541. Example 4 Expression of HCV cDNA in Animal Cells The plasmid pSR3241 obtained in Example 2 was prepared by a conventional method, and 20 μg of the plasmid pSR3241 was subcultured on the day before transfection, 1 × 10 ⁶ COS-1 cells (100
mm Phish, DMEM medium) to Cell Phect
Transfection was performed using the Transfection kit (Pharmacia). The cells were collected on the second day after transfection, and then PBS (Phosph
After washing with a gate buffered saline, 10 volumes of sample buffer- (2% SDS, 10
0 mM DDT, 60 mM Tris-HCl pH
6.8, 0.01% bromphenol blue) was added to solubilize the cells. The solubles 10 [mu] l 100 ° C., was treated for 5 minutes, subjected to SDS- polyacrylamide gel electrophoresis, nitrocellulose - was transferred to the scan film (Western Burotte _b ring). The nitrocellulose membrane was washed with water, blocked with 5% skim milk, and then reacted with hepatitis C patient plasma at 4 ° C. overnight as a primary antibody. After washing with Tris buffer containing 0.1% Tween 20, the biotinylated anti-human IgG was reacted at room temperature for 30 minutes and then washed again, and avidin and peroxidase complex were reacted at room temperature for 30 minutes. 4-color
Chloro-1-naphthol, H ₂ O ₂ method was used.

Four patients with chronic hepatitis C (KK, SI, T
U, HI) was subjected to Western blot analysis by the method described above, and a signal was detected at a position of about 32 kDa in all 4 cases. This molecular weight is consistent with the molecular weight of the polypeptide expressed by the transfected HCV cDNA and cleaved by post-translational HCV protease. In addition, this signal was not detected when a lysate of cells not transfected with HCV cDNA was used.

[0024]

INDUSTRIAL APPLICABILITY RNA and cDNA obtained by the present invention
A and protein are used as diagnostic reagents for hepatitis C and H
It is expected to be used as a screening method for CV-specific antiviral drugs.

[0025]

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁵ Identification code Internal reference number FI Technical display location C12N 15/86 C12P 21/02 C 8214-4B C12Q 1/68 Z 8114-4B 1/70 8114- 4B G01N 33/53 D 8310-2J N 8310-2J 33/569 L 9015-2J 33/576 Z 9015-2J // (C12P 21/02 C12R 1:91)

Claims (13)

[Claims] 1. A following base sequence 5'GACCAAGGUC AUCACCUGGG GGGCAGACAC CGCGGCGUGU GGGGACAUUA UCUCAGGUCU ACCCGUCUCC GCCCGAAGGG GGAAGGAGAU ACUUUUGGGA CCGGCCGAUA GUUUUGAAGG GCAGGGGUGG CGACUCCUUG CGCCCAUCAC GGCCUACUCC CAACAGACGC GGGGCCUACU UGGUUGCAUU AUCACUAGUC UCACGGGCCG GGACAAGAAC CAGGUUGAGG GGGAGGUUCA AGUGGUCUCC ACCGCAACAC AAUCUUUCCU GGCGACCUGU GUCAACGGCG CGUGCUGGAC UGUCUUUCA GGUGCCGGCU CAAAGACCUU AGCCGGCCCA AAAGGCCCAA UCACCCAAAU GUACACCAAU GUAGACCAGG ACCUCGUCGG CUGGCCAGCG CCCCCCGGGG CGCGUUCCUU AACACCGUGC ACCUGCGGCA GCUCGGACCU UUACCUGGUC ACGAGACAUG CUGAUGUCAU CCCGGUGCGC CGGCGGGGCG ACACUAGGGG GAGCUUGCUC UCCCCUAGAC CCAUCUCCUA CUUGAAGGGC UCUUCGGGUG GUCCAUUGCU CUGCCCCUCG GGGCACGUUG UGGGCAUCUU CCGGGCUGCC GUGUGCACCC GGGGGGUCGC GA AGGCGGUG GACUUCAUAC CCGUUGAAUC UAUGGAAACC ACUAUGCGGU CUCCGGUAUU CACAGACAAC UCAACCCCCC CGGCCGUACC GCAGACCUUC CAAGUGGCCC AUCUACACGC CCCCACUGGC AGCGGUAAAA GCACUAAGGU GCCGGCUGCA UAUGCAGCCC AAGGGUACAA GGUGCUCGUC CUGAACCCGU CCGUUGCUGC CACCCUGGGU gene having UUUGGGGCGU or C, which comprises a fragment thereof
Hepatitis virus genomic RNA. 2. A following base sequence 5'CUGCUGAUAG CCCAGGCCGA GGCCGCCUUA GAGAACUUGG UGGUCCUCAA UGCGGCGUCC GUGGCCGGAG CGCAUGGCAU UCUCCCCUUC UUUAUGUUCU UCUGUGCCGC CUGGUACAUU AAGGGCAGGC UGGUCCCUGG AGCGGCAUAC GCUUUCUACG GCGUAUGGCC GCUGCUCCUG CUCUUGCUAG CAUUACCACC ACGAGCUUAC GCCAUGGACC GGGAGAUGGC UGCAUCUUGC GGAGGGGGGG UUUUUGUAGG UCUAGCACUC CUGACCUUGU CACCAUACUA UAAAGUGUUC CUCGCUAGG UCAUAUGGUG GUUACAAUAU UUUAUCACCA AAGCCGAGGC GCAUUUGCAA GUGUGGGUCC CCCCCCUCAA CGUUCGAGGC GGCCGCGAUG CCAUCAUCCU CCUCAUGUGC GCGGUCCACC CAGAGCUAAU CUUUGACAUC ACCAAACUUC UGCUCUCCAU ACUCGGUCCG CUCAUGGUGC UCCAAGCUAG UUUAAUCCGA GUGCCGUACU UCGUGCGCGC UCAAGGGCUC AUUCGCGCAU GCAUGUUGGU GCGGAAAGCU GCCGGGGGCC AUUAUGUCCA AAUGGCCUUC GUGAAGCUAG CUGCGCUGAC AGGCACGUAC GU UUAUGACC ACCUCACUCC ACUGCAGGAU UGGGCCCAUG UGGGCCUACG AGACCUUGCG GUGGCAGUAG AGCCCGUUGU CUUUUCUGCC AUGGAGACCA AGGUCAUCAC CUGGGGGGCA GACACCGCGG CGUGUGGGGA CAUUAUCUCA GGUCUACCCG UCUCCGCCCG AAGGGGGAAG GAGAUACUUU UGGGACCGGC CGAUAGUUUU GAAGGGCAGG GGUGGCGACU CCUUGCGCCC AUCACGGCCU ACUCCCAACA GACGCGGGGC CUACUUGGUU GCAUUAUCAC UAGUCUCACG GGCCGGGACA AGAACCAGGU UGAG GGGAG GUUCAAGUGG UCUCCACCGC AACACAAUCU UUCCUGGCGA CCUGUGUCAA CGGCGCGUGC UGGACUGUCU UUCACGGUGC CGGCUCAAAG ACCUUAGCCG GCCCAAAAGG CCCAAUCACC CAAAUGUACA CCAAUGUAGA CCAGGACCUC GUCGGCUGGC CAGCGCCCCC CGGGGCGCGU UCCUUAACAC CGUGCACCUG CGGCAGCUCG GACCUUUACC UGGUCACGAG ACAUGCUGAU GUCAUCCCGG UGCGCCGGCG GGGCGACACU AGGGGGAGCU UGCUCUCCCC UAGACCCAUC UCCUACUUGA AGGGCUC Gene or a fragment thereof having UC GGGUGGUCCA UUGCUCUGCC CCUCGGGGCA CGUUGUGGGC AUCUUCCGGG CUGCCGUGUG CACCCGGGGG GUCGCGAAGG CGGUGGACUU CAUACCCGUU GAAUCUAUGG AAACCACUAU GCGGUCUCCG GUAUUCACAG ACAACUCAAC CCCCCCGGCC GUACCGCAGA CCUUCCAAGU GGCCCAUCUA CACGCCCCCA CUGGCAGCGG UAAAAGCACU AAGGUGCCGG CUGCAUAUGC AGCCCAAGGG UACAAGGUGC UCGUCCUGAA CCCGUCCGUU GCUGCCACCC UGGGUUUUGG GGCGU'3 C, which comprises
Hepatitis virus genomic RNA. 3. A cDNA complementary to the hepatitis C virus genomic RNA according to claim 1 or 2. 4. A cDNA homologous to the hepatitis C virus genomic RNA according to claim 1 or 2. 5. A recombinant DNA comprising the cDNA according to claim 3 or claim 4 or a fragment thereof. 6. The recombinant DNA according to claim 5, wherein the cDNA according to claim 3 or 4 or a fragment thereof is incorporated into a vector DNA. 7. A host transformed with the recombinant DNA according to claim 6. 8. The following sequence Thr Lys Val Ile Thr Trp Gly Ala Asp Thr Ala Ala Cys Gly Asp Ile Ile Ser Gly Leu Pro Val Ser Ala Arg Arg Gly Lys Glu Ile Leu Leu Gly Pro Ala Asp Ser Phe Glu Gly Gln Gly Trp Arg Leu Leu Ala Pro Ile Thr Ala Tyr Ser Gln Gln Thr Arg Gly Leu Leu Gly Cys Ile Ile Thr Ser Leu Thr Gly Arg Asp Lys Asn Gln Val Glu Gly Glu Val Gln Val Val Ser Thr Ala Thr Gln Ser Phe Leu Ala Thr Cys Val Asn Gly Al Cys Trp Thr Val Phe His Gly Ala Gly Ser Lys Thr Leu Ala Gly Pro Lys Gly Pro Ile Thr Gln Met Tyr Thr Asn Val Asp Gln Asp Leu Val Gly Trp Pro Ala Pro Pro Gly Ala Arg Ser Leu Thr Pro Cys Thr Cys Gly Ser Ser Asp Leu Tyr Leu Val Thr Arg His Ala Asp Val Ile Pro Val Arg Arg Arg Gly Asp Thr Arg Gly Ser Leu Leu Ser Pro Arg Pro Ile Ser Tyr Leu Lys Gly Ser Ser Gly Gly Pro Leu Leu Cys Pro Ser Gly His Val Va Gly Ile Phe Arg Ala Ala Val Cys Thr Arg Gly Val Ala Lys Ala Val Asp Phe Ile Pro Val Glu Ser Met Glu Thr Thr Met Arg Ser Pro Val Phe Thr Asp Asn Ser Thr Pro Pro Ala Val Pro Gln Thr Phe Gln Val Ala His Leu His Ala Pro Thr Gly Ser Gly Lys Ser Thr Lys Val Pro Pro Ala Ala Tyr Ala Ala Gln Gly Tyr Al Ly Gal Ly Gal Ly Pro Val Le Le Syr Lys Val Leu Val Pro Le La SV Ly. 9. The following sequence Leu Leu Ile Ala Gln Ala Glu Ala Ala Ile Glu Asn Leu Val Val Leu Asn Ala Ala Ser Val Ala Gly Ala HisGly Ile Leu Pro Phe Phe Met Phe Phe Cys Ala Ala Trp Tyr Ile Lys Gly Arg Leu Val Pro Gly Ala Ala Tyr Ala Phe Tyr Gly Val Try Pro Leu Leu Leu Leu Leu Leu Ala Leu Pro Pro Arg Ala Tyr Ala Met Asp Arg Glu Met Ala Ala Ser Cys Gly Gly Val Phe Val Gly Leu Ala Leu Leu Thr Leu Ser Pro Tyr Tyr Lys Val Phe Leu Ala Arg Leu Ile Trp Trp Leu Gln Tyr Phe Ile Thr Lys Ala Glu Ala His Leu Gln Val Trp Val Pro Pro Leu Asn Val Arg Gly Gly Arg Asp Ala Ile Ile Leu Leu Met Cys Ala Val His Pro Glu Leu Ile Phe Asp Ile Thr Lys Leu Leu Leu Ser Ile Leu Gly Pro Leu Met Val Leu Gln Ala Ser Leu Ile Arg Val Pro Tyr Phe Val Arg Ala Gln Gly Leu Ile Arg Ala Cys Met Leu Val Arg Lys Ala Ala Gly Gly His Tyr Val Gln MetAla Phe Val Lys Leu Ala Ala Leu Thr Gly Thr Tyr Val Tyr Asp His Leu Thr Pro Leu Gln Asp Trp Ala His Val Gly Leu Arg Asp Leu Ala Val Ala Val Glu Pro Val Val Phe Ser Ala Met Glu Thr Lys Val Ile Thr Trp Gly Ala Asp Thr Ala Ala Cys Gly Asp Ile Ile Ser Gly Leu Pro Val Ser Ala Arg Arg Gly Lys Glu Ile Leu Leu Gly Pro Ala Asp Ser Phe Glu Gly Gln Gly Trp Arg Leu Leu Ala Pro Ile Thr Ala Tyr Ser Gln Gln Thr Arg Gly Leu Leu Gly Cys Ile Ile Thr Ser Leu Thr Gly Arg Asp Lys Asn Gln Val Glu Gly Glu Val Gln Val Val Ser Thr Ala Thr Gln Ser Phe Leu Ala Thr Cys Val Asn Gly Ala Cys Trp Thr Val Phe His Gly Ala Gly Ser Lys Thr Leu Ala Gly Pro Lys Gly Pro Ile Thr Gln Met Tyr Thr Asn Val Asp Gln Asp Leu Val Gly Trp Pro Ala Pro Pro Gly Ala Arg Ser Leu Thr Pro Cys Thr Cys Gly Ser Ser Asp Leu Tyr Leu Val Thr Arg His Ala Asp Val Ile Pro Val Arg Arg Arg Gly Asp Thr Arg Gly Ser Leu Leu Ser Pro Arg Pro Ile Ser Tyr Leu Lys Gly Ser Ser Gly Gly Pro Leu Leu Cys Pro Ser Gly His Val Val Gly Ile Phe Arg Ala Ala Val Cys Thr Arg Gly Val Ala Lys Ala Val Asp Phe Ile Pro Val Glu Ser Met Glu Thr Thr Met Arg Ser Pro Val Phe Thr Asp Asn Ser Thr Pro Pro Ala Val Pro Gln Thr Phe Gln Val Ala His Leu His Ala Pro Thr Gly Ser Gly Lys Ser Thr Lys Val Pro Ala Ala Tyr Ala Ala Gln Gly Tyr Lys Val Leu Val Leu Asn Pro Ser Val Ala Ala Thr Leu Gly Phe Gly Ala Leu Leu Ile Ala Gln Ala Glu Ala Ala Ile Glu Asn Leu Val Val Leu Asn Ala Ala Ser Val Ala Gly Ala His Gly Ile Leu Pro Phe Phe Met Phe Phe Cys Ala Ala Trp Tyr Ile Lys Gly Arg Leu Val Pro Gly Ala Ala Tyr Ala Phe Tyr Gly Val Try Pro Leu Leu Leu Leu Leu Leu Ala Leu Pro Pro Arg Ala Tyr Ala Met Asp Arg Glu Met Ala Ala Ser Cys Gly Gly Val Phe Val Gly Leu Ala Leu Leu Thr Leu Ser Pro Tyr Tyr Lys Val Phe Leu Ala Arg Leu Ile Trp Trp Leu Gln Tyr Phe Ile Thr Lys Ala Glu Ala His Leu Gln Val Trp Val Pro Pro Leu Asn Val Arg Gly Gly Arg Asp Ala Ile Ile Leu Leu Met Cys Ala Val His Pro Glu Leu Ile Phe Asp Ile Thr Lys Leu Leu Leu Ser Ile Leu Gly Pro Leu Met Val Leu Gln Ala Ser Leu Ile Arg Va Pro Tyr Phe Val Arg Ala Gln Gly Leu Ile Arg Ala Cys Met Leu Val Arg Lys Ala Ala Gly Gly His Tyr Val Gln Met Ala Phe Val Lys Leu Ala Ala Leu Thr Gly Thr Tyr Val Tyr Asp His Leu Thr Pro Leu Gln Asp Trp A hepatitis C virus polypeptide having Ala His Val Gly Leu Arg Asp Leu Ala Val Ala Val Glu Pro Val Val Phe Ser Ala Met Glu. 10. C virus protease - following sequence having a peptidase activity Asn Gly Ala Cys Trp Thr Val Phe His Gly Ala Gly Ser Lys Thr Leu Ala Gly Pro Lys Gly Pro Ile Thr Gln Met Tyr Thr Asn Val Asp Gln Asp Leu Val Gly Trp Pro Ala Pro Pro Gly Ala Arg Ser Leu Thr Pro Cys Thr Cys Gly Ser Ser Asp Leu Tyr Leu Val Thr Arg His Ala Asp Val Ile Pro Val Arg Arg Arg Gly Asp Thr Arg Gly Ser Leu Leu Ser Pro Arg Pro Ile Ser Tyr Leu Lys Gly Ser Ser ly Gly Pro Leu Leu Cys Pro Ser Gly His Val Val Gly Ile Phe Arg Ala Ala Val C type hepatitis virus polypeptide of Cys. 11. A recombinant DN comprising a DNA base sequence encoding the hepatitis C virus polypeptide according to claim 5, claim 6 or claim 7, or a fragment thereof.
A. 12. A reagent for diagnosing hepatitis C, which uses a nucleic acid having the nucleotide sequence according to claim 1, claim 2 or claim 3. 13. A reagent for diagnosing hepatitis C, which comprises the polypeptide according to claim 8, 9, or 10.