HK1112481B - Modified human hepatitis c virus genomic rna having autonomous replicative competence - Google Patents

Description

Modified human hepatitis C virus genomic RNA having autonomous replication ability

Technical Field

The present invention relates to a method for autonomously replicating human Hepatitis C Virus (HCV) of various genotypes in a cultured cell line, and a modified HCV genomic RNA used for the method, and a cell replicating the HCV genomic RNA.

Background

It has been known from recent studies that hepatitis c viruses can be classified into various types according to genotype or serotype. As a currently mainstream HCV genotype classification method, HCV is classified into 6 types in a systematic analysis (phyloanalysis) of the base sequence of HCV strain used by Simmonds and the like: genotype 1a, genotype 1b, genotype 2a, genotype 2b, genotype 3a, and genotype 3b (non-patent document 1), and the respective types thereof are divided into several subtypes. Therefore, the nucleotide sequence of the entire genome of HCV has also been determined for a plurality of genotypes of HCV (patent document 1 and non-patent documents 2 to 4).

HCV causes chronic hepatitis through persistent infection. The main cause of chronic hepatitis, which is now considered worldwide, is persistent infection with HCV. In fact, about 50% of persistently infected patients suffer from chronic hepatitis, and about 20% of them turn into cirrhosis after 10 to 20 years, and partially develop into a fatal disease such as liver cancer.

The current treatment of hepatitis c is primarily through interferon- α, interferon- β, and therapeutic approaches using interferon- α in combination with ribavirin, which is a purine-nucleoside derivative. However, even with this treatment, only 60% of all the patients treated showed a therapeutic effect, and if the treatment was stopped after the effect was produced, half or more of the patients relapsed. It is known that the therapeutic effect of interferon is related to the genotype of HCV, and it is said that the effect on genotype 1b is low and the effect on genotype 2a is high (non-patent document 5). Furthermore, the substrate specificity of HCV for protease differs depending on the genotype, and the inhibitory activity of an inhibitor developed using NS3 protease of genotype 1b against NS3 protease of another genotype is reduced by 50 times or more (non-patent document 6). Therefore, in order to develop an HCV therapeutic agent with high efficiency, it is necessary to develop the agent while confirming the reactivity in each genotype of HCV.

Recently, it has become possible to analyze the replication structure of HCV using cultured cells by preparing an RNA having an autonomous replication ability from an HCV subgenomic RNA replicon (patent documents 2 and 3, and non-patent documents 7 to 9). The HCV subgenomic RNA replicon is obtained by replacing a downstream structural protein of HCV IRES present in the 5' -untranslated region of HCV genomic RNA with a neomycin resistance gene and EMCV-IRES ligated downstream thereof. The autonomous replication of the RNA replicon in Huh7 cells was confirmed by introducing the RNA replicon into a hepatoma cell Huh7 and culturing the RNA replicon in the presence of neomycin. Furthermore, it was confirmed that a part of HCV subgenomic RNA autonomously replicates not only in Huh7 cells but also in human cervical cancer cells HeLa, human liver cancer cells HepG2, and the like (patent document 3).

However, such an intracellular RNA replication system for HCV is not prepared by using only limited genomic RNA of HCV, rather than a system for a limited genotype. Therefore, it is extremely difficult to discuss differences in therapeutic effects of developed HCV therapeutic drugs due to differences in genotypes among HCV having a large number of genotypes. Furthermore, an RNA replicon is an experimental system capable of evaluating only viral RNA replication in the process of propagation and replication of HCV virus, and cannot evaluate the process of formation of HCV virus particles in infected cells, release of HCV virus particles to the outside of cells, and infection of new cells.

Currently, methods for evaluating the process of formation and extracellular release of HCV viral particles and infection of new cells are limited to experimental systems using animals such as chimpanzees (non-patent document 10). However, an experimental system using a living body such as an animal as it is complicated to operate and very difficult to analyze. Therefore, it is necessary to establish an extremely simple experimental system capable of reproducing the process of forming and releasing HCV virus particles to the outside of cells and further infecting new cells, that is, to construct an HCV virus particle replication system using a cultured cell line, and to analyze the process and develop an anti-HCV drug that inhibits the process as a mechanism of action.

Furthermore, if stable HCV viral particles can be provided by culturing cell lines, the virus can also be attenuated, or non-infectious HCV can be prepared using molecular biological methods and used in vaccines. However, since the protein sequence of HCV differs depending on the genotype, the antigenicity of HCV also differs depending on the genotype. In fact, the presence of multiple genotypes has been a major obstacle in HCV vaccine production (non-patent document 11). Therefore, in order to produce HCV vaccines with high efficiency, it is also desired to stably produce HCV viral particles of each genotype by culturing cell lines.

HCV is known as spherical particles of 55 to 65nm and is present in the blood of HCV-infected patients. As methods for purifying HCV present in human serum, affinity chromatography using lectin (non-patent document 12) and chromatography using heparin (non-patent document 13) are known. However, these methods can purify only viruses with a concentration of less than 1mL, about 1M copies/mL, and are not suitable for industrial use at all.

To date, methods for purifying viruses other than HCV have been invented (for example, patent documents 4, 5, and 6). However, as is known from these documents, the nature of the viral particles varies and no reference is provided for the most suitable purification method for human hepatitis C virus. Further, regarding hepatitis a virus which is also hepatitis virus, patent document 7 discloses that hepatitis a virus can be purified by removing DNA by anion exchange chromatography. However, although hepatitis a virus is also hepatitis virus, it is a virus having DNA as a gene, and since hepatitis c virus is known to be a virus having RNA as a gene, there is no point of similarity between them at all, and thus no reference information is provided on the purification method. Since a large amount of high-purity human hepatitis c virus particles are required for industrial application to vaccines and the like in the future, development of a purification method is urgently needed.

Patent document 1: japanese laid-open patent publication No. 2002-171978

Patent document 2: japanese unexamined patent application publication No. 2001-17187

Patent document 3: WO2004/104198A1

Patent document 4: patent No. 3313117

Patent document 5: tekke Table 2002-

Patent document 6: special table 2000-510682

Patent document 7: special Fair 6-48980

Non-patent document 1: simmonds, P.et al, Hepatology, 10(1994) p1321-1324

Non-patent document 2: choo, Q.L et al, Science, 244(1989) p359-362

Non-patent document 3: okamoto, H et al, j.gen.virol., 73(1992) p673-679

Non-patent document 4: mori, S.et al biochem.Biophis.Res.Commun.183(1992) p334-342

Non-patent document 5: yoshioka, K.et al, Hepatology, 16(1992) p293-299

Non-patent document 6: thibeault, d.et al, j.virol., 78(2004) p7352-7359

Non-patent document 7: blight et al, Science, 290(2000) p1972-1974

Non-patent document 8: friebe et al, J.Virol, 75(2001) p12047-12057

Non-patent document 9: kato, T.et al., Gastroenterology, 125(2003) p1808-1817

Non-patent document 10: kolykhalov et al, Science 277(1997) p570-574

Non-patent document 11: farci, P., et al, Semin Liver Dis 20(2000) p103-126

Non-patent document 12: virology, 196(1993) p354-357

Non-patent document 13: journal of General Virology 86(2005) p 677-

Disclosure of Invention

The present invention aims at providing a method for replicating and propagating hepatitis C virus of various genotypes by using a culture cell line.

The present inventors have intensively studied to solve the above problems and found that a modified hepatitis c virus genomic RNA capable of autonomous replication in a cell system is prepared by combining a genomic RNA of HCV JFH1 strain having an autonomous replication ability with a genomic RNA of HCV strain having no autonomous replication ability in vitro (in vitro). Specifically, the present inventors have found that HCV genomic RNA that does not have the ability to autonomously replicate in vitro can be changed to RNA that can autonomously replicate in a cultured cell line by introducing the genome of JFH1 strain from the NS3 protein-encoding sequence to the 3' end.

That is, the present invention relates to a modified hepatitis c virus genomic RNA containing two or more of the 5 'untranslated region, core (core) protein coding sequence, E1 protein coding sequence, E2 protein coding sequence, p7 protein coding sequence, NS2 protein coding sequence of hepatitis c virus genomic RNA, and the base sequences of NS3, NS4A, NS4B, NS5A, NS5B protein coding sequence, and 3' untranslated region of JFH1 strain, and having an autonomous replication ability.

Specifically, one embodiment of the present invention provides a modified hepatitis c virus genomic RNA having an autonomous replication ability, wherein the RNA partial sequences (RNA sequences obtained by substituting U for T in the sequence indicated by positions 3867 and 9678 in the DNA sequence of Genbank accession No. ab047639) encoding the NS3, NS4, NS5A, and NS5B proteins of the JFH1 strain shown in sequence No. 1 are substituted for the genomic sequence NS5B protein coding sequence from the NS3 protein coding sequence to the 3' terminus of the hepatitis c virus genomic RNA.

In another embodiment, a modified hepatitis c virus genomic RNA having an autonomous replication ability is provided by substituting the NS5B protein-encoding sequence of the hepatitis c virus genomic RNA with the NS5B protein-encoding sequence of JFH1 strain shown in sequence No. 2.

As the 2 or more types of hepatitis C virus, preferably using genotype 1b nuclear genotype 1a hepatitis C virus. The virus strain of genotype 1b includes, for example, HCV-con1 strain, HCV-TH strain, HCV-J strain, HCV-JT strain, and HCV-BK strain; furthermore, examples of the virus strain of genotype 1a include the HCV-J6 strain, the HCV-JFH1 strain and the HCV-JCH1 strain.

The modified hepatitis c virus genomic RNA of the present invention may also further comprise at least one selectable marker gene and/or at least one reporter gene, and at least one IRES sequence.

In this case, the modified hepatitis C virus genomic RNA contains, in order from 5 'to 3', the above-mentioned 5 'untranslated region, at least one selectable marker gene and/or at least one reporter gene, at least one IRES sequence, a core protein coding sequence, an E1 protein coding sequence, an E2 protein coding sequence, a p7 protein coding sequence, an NS2 protein coding sequence, an NS3 protein coding sequence, an NS4A protein coding sequence, an NS4B protein coding sequence, an NS5A protein coding sequence, an NS5B protein coding sequence, and a 3' untranslated region.

In the present specification, the modified hepatitis C virus genomic RNA includes, for example

(a) RNA having the base sequence represented by SEQ ID NO. 11, or

(b) An RNA comprising a base sequence in which 1 or more, preferably 100, more preferably 50, and still more preferably 10 bases of the base sequence represented by SEQ ID NO. 11 are deleted, substituted, or added, and a modified hepatitis C virus genomic RNA having an autonomous replication ability and a hepatitis C virus particle production ability.

The present invention also provides a cell into which the modified hepatitis C virus genomic RNA of the present invention has been introduced, which can replicate the hepatitis C virus and produce viral particles. In the present invention, the host cell is preferably a proliferating cell, and particularly preferably a eukaryotic cell, for example, a human liver-derived cell, a human cervix-derived cell, or a human embryonic kidney-derived cell such as Huh7 cell, HepG2 cell, IMY-N9 cell, HeLa cell, or 293 cell.

The present invention also provides a method for producing hepatitis C virus particles characterized by culturing the above-mentioned cells and recovering virus particles from the culture medium, and hepatitis C virus particles produced by the method.

The present invention also provides a method for producing a hepatitis C virus-infected cell characterized by culturing the above-mentioned cell and infecting other cells with viral particles in the culture, and a hepatitis C virus-infected cell produced by the method. The present invention can obtain HCV viral particles having purity applicable to industry and medical treatment by purifying HCV viral particles using column chromatography and/or density gradient centrifugation. The column chromatography used at this time is one or more selected from the group consisting of ion exchange chromatography, gel filtration chromatography, and affinity chromatography. Density gradient centrifugation is density gradient centrifugation using a solvent containing one or more solutes selected from cesium chloride, sucrose, and sugar polymers to purify HCV.

The present invention also provides a method for screening an anti-hepatitis C virus substance using the cell of the present invention or a hepatitis C virus-infected cell. The method is characterized by culturing the cells of the present invention or hepatitis C virus-infected cells in the presence of a test substance, and detecting hepatitis C virus RNA or virus particles in the culture to evaluate the anti-hepatitis C virus effect of the test substance.

Further, the present invention provides a method for producing a hepatitis c vaccine using the hepatitis c virus particle of the present invention or a part thereof as an antigen.

The present invention also provides a method for replicating and/or expressing a foreign gene in a cell, and a viral vector containing a hepatocyte targeted to the modified hepatitis c virus genomic RNA of the present invention, characterized in that RNA encoding the foreign gene is inserted into the modified hepatitis c virus genomic RNA of the present invention, introduced into a target cell, and replicated or expressed.

According to the present invention, infectious HCV virus particles can be produced using a cultured cell line. Furthermore, even in the case of an HCV strain isolated from a patient and having no autonomous replication ability, it can be autonomously replicated in vitro by replacing the region from the NS3 region to the 3' end with the viral genomic RNA of JFH1 or replacing the NS5B region with the NS5B region of JFH 1. Therefore, it is possible to prepare viral particles of HCV strains of various genotypes by culturing cell lines, which is useful for studying HCV infection processes, screening various substances affecting HCV infection, preparing HCV vaccines, and the like.

Drawings

FIG. 1 is a schematic diagram showing the steps for constructing a template DNA for preparing the HCV genomic RNA of the present invention, and shows the structure of plasmid clone pJFH1 prepared by inserting the full-length HCV genome downstream of the T7 promoter. The labels in the figure are as follows: t7: a T7RNA promoter; 5' UTR: a 5' untranslated region; c: a core protein; e1, E2: envelope proteins; NS2, NS3, NS4A, NS4B, NS5A, NS 5B: a non-structural protein; 3' UTR: a 3' untranslated region; AgeI, PmeI, XbaI: restriction sites for restriction enzymes AgeI, PmeI, and XbaI; GDP: the amino acid motif GDP of the active center of the NS5B protein.

FIG. 2 is a photograph showing the Northern blot results, which suggests that rJFH1 replicates in Huh7 cells into which rJFH1, which is HCV genomic RNA, has been introduced.

FIG. 3 shows the results of detection of HCV core protein, NS3 protein, NS5A protein, and E2 protein in the medium.

FIG. 4 shows the results of release of core protein over time into the medium of cells into which HCV genomic RNA had been introduced.

FIG. 5 is a graph showing the amounts of HCV core protein and HCV genomic RNA in each fraction isolated from the culture supernatant of Huh7 cells into which rJFH1 had been introduced by sucrose density gradient centrifugation. Black circles indicate HCV Core (Core) protein, white circles indicate HCV genomic RNA. FIG. 5A shows untreated cells, FIG. 5B shows RNase treatment, FIG. 5C shows NP40 treatment, and FIG. 5D shows rJFH 1-introduced Huh7 cells after NP40+ RNase treatment.

FIG. 6 shows infectivity of viral particles secreted into a culture medium of Huh7 cells into which rJFH1 was introduced. Fig. 6A is a photograph showing the results of immunostaining by an anti-Core antibody (left) and an anti-NS 5A antibody (right). FIG. 6B is a graph showing the number of cells stained positive by an anti-Core antibody. Fig. 6C is a graph showing the change with time in the amount of HCV RNA in the cell (left) and the supernatant (right).

FIG. 7 is a graph showing infectivity of viral particles secreted into a culture medium of Huh7 cells into which rJCH1/NS5B (jfh1) was introduced. FIG. 7A is a graph showing the amplification of HCV RNA in naive Huh7 cells of virus particles secreted into a culture medium of Huh7 cells into which JCH1/NS5B (jfh1) was introduced. FIG. 7B is a graph showing the number of cells stained positive by an anti-Core antibody.

FIG. 8 shows the structure of a TH/JFH1 chimeric replicon

FIG. 9 is the observation result of colony formation by transfection of TH/JFH1 chimeric replicon RNA.

FIG. 10 shows the results of colony formation by infection of culture supernatant of TH/JFH1 chimeric replicon.

FIG. 11 is a graph showing elution in gel filtration chromatography. The vertical axis represents the absorbance at a wavelength of 490 nm. S-300, S-400 and S-500 are Sephacryl (registered trademark) S-300, S-400 and S-500, respectively. In addition, the horizontal axis represents the amount of the solution eluted from the column.

FIG. 12 is a graph showing elution in ion exchange chromatography. The vertical axis represents the amount of core protein of HCV viral particles.

FIG. 13 is a graph showing elution in lectin affinity chromatography. The vertical axis represents the amount of core protein of HCV viral particles.

FIG. 14 is a graph showing elution in heparin, Sulfate cellulofine chromatography. The vertical axis represents the absorbance at a wavelength of 490 nm.

FIG. 15 is a graph showing elution in blue dye affinity chromatography. The vertical axis represents the amount of core protein of HCV viral particles.

FIG. 16 shows a purification scheme combining column chromatography and sucrose density gradient centrifugation. The vertical axis represents the amount of core protein of HCV viral particles. In addition, the amount of the core protein of HCV virus particles and the density of each part of the solution in sucrose density gradient centrifugation are shown in the vertical axis.

The present specification includes the contents described in the specifications of Japanese patent application No. 2004-.

Detailed description of the preferred embodiments

The present invention will be described in detail below.

1. Modified chimeric hepatitis c virus genomic RNA

The genome of Hepatitis C Virus (HCV) is a (+) strand single-stranded RNA consisting of 9600 nucleotides. The genomic RNA is composed of a 5 '-untranslated region (also referred to as 5' -NTR or 5 '-UTR), a translated region composed of a structural region and an unstructured region, and a 3' -untranslated region (also referred to as 3 '-NTR or 3' -UTR). The structural region encodes structural proteins of HCV, and the non-structural region encodes various non-structural proteins of HCV.

The structural proteins (Core, E1, and E2) and nonstructural proteins (NS2, NS3, NS4A, NS4B, NS5A, and NS5B) of HCV are produced by translation from the translated region into a single continuous polyprotein and then released by restriction digestion with a protease. Among these structural and non-structural proteins (i.e., viral proteins of HCV), Core is the Core protein, and E1 and E2 are envelope proteins. The nonstructural protein is a protein involved in the replication of the virus itself, and it is known that NS2 has a metalloproteinase activity, and NS3 has a serine protease (one third of the N-terminal side) activity and a helicase (two thirds of the C-terminal side) activity. In addition, it has been reported that NS4 is a cofactor against the protease activity of NS3, and NS5B has the activity of RNA-dependent RNA polymerase.

Currently, as genotypes of HCV, at least types 1 to 6 are known, and Simmonds and the like (see Simmonds, p.et al, Hepatology, (1994)10, p.1321 to 1324) classify HCV into various genotypes according to their sequences in the international classification (HCV1a, HCV1b, HCV2a, HCV2b and the like). In the present invention, HCV genomic RNA that is not autonomously replicable is not limited to these known viral types, but includes all HCV genomic RNA that is not autonomously replicable, i.e., has no ability to release infectious particles outside the cell. In the present invention, the term "RNA" has an autonomous replication ability "or" autonomously replicates "means that HCV genomic RNA has an ability to self-proliferate, that is, an ability to release infectious particles outside cells, after the HCV genomic RNA is introduced into the cells.

In the present specification, RNA including HCV genomic RNA having the autonomous replication ability of the cultured cell line as described above is referred to as "replicon RNA" or "RNA replicon". In the present specification, the replicon RNA of the present invention containing the full length of the replicon RNA is referred to as "full-length HCV replicon RNA". The full length HCV replicon RNA of the present invention has the ability to produce viral particles. In addition, the modified hepatitis c virus genomic RNA of the present invention is a full-length HCV replicon RNA.

The modified hepatitis C virus genomic RNA of the present invention comprises base sequences of the 5 'untranslated region, the core protein coding sequence, the E1 protein coding sequence, the E2 protein coding sequence, the p7 protein coding sequence, the NS2 protein coding sequence of 2 or more types of hepatitis C virus genomic RNA, and the NS3, NS4A, NS4B, NS5A, and NS5B protein coding sequences of JFH1 strain, and the 3' untranslated region, and has an autonomous replication ability. Specifically, one embodiment of the present invention relates to a modified hepatitis C virus genomic RNA having an autonomously replicating ability, which is obtained by substituting a genomic sequence NS5B protein coding sequence from the NS3 protein coding sequence to the 3' -end of the hepatitis C virus genomic RNA with an RNA partial sequence (an RNA sequence obtained by substituting U for T in the sequence represented by 3867-9678 in the DNA sequence of GenBank Accession No. AB047639) encoding the JFH1 strain NS3, NS4, NS5A, and NS5B proteins, which is represented by SEQ ID NO. 1.

In another embodiment, a modified hepatitis C virus genomic RNA having an autonomous replication ability is provided by substituting the NS5B protein coding sequence of the hepatitis C virus genomic RNA with the NS5B protein coding sequence of the JFH1 strain shown in SEQ ID NO. 2.

More preferably, the hepatitis C virus genome RNA obtained by using the hepatitis C viruses of genotype 1b and genotype 2a is a modified hepatitis C virus genome RNA which comprises a nucleotide sequence including a 5 'untranslated region, a core protein coding sequence, an E1 protein coding sequence, an E2 protein coding sequence, a p7 protein coding sequence, an NS2 protein coding sequence, NS3, NS4A, NS4B, NS5A, and NS5B protein coding sequences of JFH1 strain, and a 3' untranslated region, and which has an autonomous replication ability.

The modified hepatitis C virus genomic RNA may further contain at least one selectable marker gene and/or at least one reporter gene, and at least one IRES sequence.

In the present invention, by combining an HCV strain having an autonomous replication ability in a cultured cell line and an HCV strain having no autonomous replication ability in a cultured cell line as 2 or more types of hepatitis c viruses, an HCV strain having no autonomous replication ability can be converted into an HCV strain capable of autonomous replication. Alternatively, an HCV strain having a low autonomous replication ability can be converted into an HCV strain having a high autonomous replication ability.

As HCV strains, specifically, HCV-1 strain, HCV-H strain, HCV-J1 strain and the like among type 1a are known; HCV-con1 strain, HCV-TH strain, HCV-J strain, HCV-JT strain, HCV-BK strain, etc., among type 1 b; HCV-J6 strain, HFH-1 strain, JCH1 strain among type 2a, and the like; strain HC-J8 in type 2 b; e-b1 strain in type 3a, and the like. The structure of these viruses is essentially composed of the 5 '-UTR, Core, E1, E2, p7, NS2, NS3, NS4A, NS4a, NS5a, NS5b, 3' -UTR (as described above). The nucleotide sequences of the respective regions of the above-mentioned HCV strains have been clarified, and for example, the regions of Core, E1, E2, p7 and NS2 have been clarified in the full-length sequence of the TH strain. In addition, the regions of Core, E1, E2, p7 and NS2 have been identified in HCV-JT strain. As an embodiment of the replicon RNA related to the present invention, for example, chimeric replication of the JFH1 strain of HCV type 2a and strains other than the JFH-1 strain, such as HCV-1 strain, HCV-H strain, HCV-J1 strain, HCV-con1 strain, HCV-TH strain (Wa kita et al, J.biol.chem., (1994), 269, p.14205-14210, and Mordapour et al, biochem.Biophys.Res.Commun., (1998)246, p.920-924), HCV-J strain, HCV-JT strain, HCV-BK strain, HCV-J6 strain, JCH1, HC-J8, E-b1 strain, on RNA is used.

Further, as a preferable embodiment of the modified HCV genomic RNA of the present invention, for example, HCV genomic RNA is obtained by substituting the region from the NS3 region to the 3' -side in the HCV genomic RNA of the hepatitis c virus JFH1 strain with viral genomic RNA of JFH1, or by substituting or inserting the NS5B protein-encoding sequence with the NS5B protein-encoding sequence in another HCV genomic RNA. For example, the region from the NS3 region to the 3' -side of JCH1(ref), which is known to be replication-incompetent in vitro, is replaced with viral genomic RNA of JFH1, whereby the HCV genomic RNA can be converted to HCV genomic RNA that can autonomously replicate the HCV genomic RNA.

Furthermore, the RNA sequences encoding the NS4, NS5A, and NS5B proteins from NS3 of the Con-1 clone (ref) (EMBL Accession No. AJ238799) of HCV genotype 1b as HCV genomic RNA were substituted with the RNA sequences encoding the NS4, NS5A, and NS5B proteins from NS3 of the JFH1 strain, or only the sequence encoding the NS5B protein of the Con-1 clone (ref) of HCV genotype 1b was substituted with the sequence encoding the NS5B protein of the JFH1 strain, whereby the HCV genomic RNA could be converted into HCV genomic RNA capable of autonomously replicating HCV genomic RNA.

The full-length replicon using the gene cloned by Con-1 did not form HCV virus particles although it had an autonomous replication ability (see Pietschmann et al, Journal of Virology, (2002)76, p.4008-4021). However, as shown in the examples of the present invention, particles can be formed by replacing the RNA sequences encoding the NS4, NS5A, and NS5B proteins from NS3 with the RNA sequences encoding the NS4, NS5A, and NS5B proteins from NS3 of the JFH1 strain. That is, according to the method of the present invention, hepatitis C virus genomic RNA that cannot form particles despite having an autonomous replication ability can be converted into modified hepatitis C virus genomic RNA that can form particles.

Furthermore, even in HCV in which a replicon having an autonomous replication ability cannot be produced, such as the TH strain and the JCH strain, particles can be formed by preparing a chimeric gene thereof with the JFH-1 strain as shown in the examples of the present invention. Therefore, HCV genomic RNA that cannot autonomously replicate can be converted into a modified hepatitis C virus genomic RNA that can form particles.

Furthermore, since introduction of a mutation into NS5B in the RNA sequence of the JFH strain stopped the growth of HCV genomic RNA and the production of HCV particles, NS5B was found to be important for imparting autonomous replication ability and particle production ability.

Currently, Simmonds et al (see Simmonds, P.et al, Hepatology, (1994)10, p.1321-1324) classify HCV into various genotypes according to the international classification (HCV1a, HCV1b, HCV2a, HCV2b, etc.) based on their sequences. In the present invention, the HCV genomic RNA having no autonomous replication ability is not limited to these known viral types, but includes all HCV genomic RNAs having no autonomous replication ability.

The sequence encoding the NS5B protein referred to herein is a sequence encoding the NS5B protein derived from the JFH1 strain (SEQ ID NO: 3), and has the base sequence shown in SEQ ID NO. 2. However, any nucleotide sequence that encodes an amino acid that functions as the NS5B protein (for example, NS5B protein containing conservative substitutions) is included in the NS5B protein-encoding sequence of the present invention, as long as it can hybridize to the nucleotide sequence represented by seq id No. 2 under stringent conditions.

The stringent conditions are, for example, conditions in which the sodium concentration is 300 to 2000mM, the temperature is 40 to 75 ℃, preferably 600 to 900mM, and the temperature is 65 ℃. Those skilled in the art can refer to Molecular Cloning (Sambrook, J.et. al., Molecular Cloning: a Laboratory Manual 2)^nded., Cold Spring harbor laboratory Press, 10 Skyline Drive Plainiew, NY (1989)), and the like readily obtain homologs of NS5B such as those described above.

The HCV genomic RNA of the present invention has an RNA sequence encoding NS4, NS5A, and NS5B proteins from NS3, or has a sequence encoding NS5B protein, among JFH1HCV genomic RNAs.

In one embodiment, the HCV genomic RNA of the present invention is an RNA comprising a base sequence of a 5 'untranslated region, a core protein coding sequence, an E1 protein coding sequence, an E2 protein coding sequence, an NS2 protein coding sequence, an NS3 protein coding sequence, an NS4A protein coding sequence, an NS4B protein coding sequence, an NS5A protein coding sequence, an NS5B protein coding sequence, and a 3' untranslated region on a genomic RNA of a hepatitis c virus strain, and the RNA sequence encoding NS4, NS5A, and NS5B proteins starting from NS3 is an RNA sequence portion encoding NS4, NS5A, and NS5B proteins starting from NS3 derived from the exogenously introduced JFH1HCV genomic RNA. Preferably, the sequence encoding the NS5B protein is an RNA sequence encoding the NS5B protein derived from an exogenously introduced JFH1HCV genomic RNA.

In the present specification, "5 'untranslated region (5' NTR or 5 'UTR)", "Core protein coding sequence (Core region or C region)", "E1 protein coding sequence (E1 region)", "E2 protein coding sequence (E2 region)", "NS 2 protein coding sequence (NS2 region)", "NS 3 protein coding sequence (NS3 region)", "NS 4A protein coding sequence (NS4A region)", "NS 4B protein coding sequence (NS4B region)", "NS 5A protein coding sequence (NS5A region)", "NS 5B protein coding sequence (NS5B region)", "and 3' untranslated region (3 'NTR or 3' UTR)", and other specific regions or sites have been known in various genotypes. As for the above-mentioned regions or sites of unknown HCV strains, they can be easily determined by aligning the known HVC full-length genomic RNA sequence with the full-length RNA sequence of the HCV strain.

In the present invention, the "selectable marker" refers to a gene that confers cell selectivity such that only cells expressing the gene can be selected. As a general example of a selection marker gene, an antibiotic resistance gene is exemplified. In the present invention, examples of suitable selectable marker genes include a neomycin resistance gene, a thymidine kinase gene, a kanamycin resistance gene, a pyrithione resistance gene, an adenylyltransferase gene, a hygromycin resistance gene, a puromycin resistance gene, and the like, and the neomycin resistance gene and the thymidine kinase gene are preferable, and the neomycin resistance gene is more preferable. However, the selectable marker gene of the present invention is not limited thereto.

In the present invention, the "reporter gene" refers to a marker gene encoding a gene product that is an index for the expression of the gene. Typical examples of the reporter gene include structural genes of enzymes that catalyze luminescence reactions or color reactions. In the present invention, examples of suitable reporter genes include chloramphenicol acetyltransferase derived from transposon Tn9, β -glucuronidase or β -galactosidase gene derived from Escherichia coli, luciferase gene, green fluorescent protein gene, jellyfish luminescent protein gene derived from jellyfish, and secretory placental alkaline phosphatase (SEAP) gene. However, the reporter gene of the present invention is not limited thereto.

The above-mentioned selectable marker gene and reporter gene may be contained in the replicon RNA alone or in both. The selectable marker gene or the reporter gene may be contained in one kind or two or more kinds of the modified hepatitis C virus genomic RNA.

The HCV genomic RNA of the present invention may further contain RNA encoding a foreign gene that is desired to be expressed in the cell into which the full-length HCV genomic RNA is introduced. The RNA encoding the foreign gene may be ligated downstream of the 5 'untranslated region, or may be inserted upstream of the 3' untranslated region. In addition, the sequence may be between any two of the core protein coding sequence, the E1 protein coding sequence, the E2 protein coding sequence, the NS2 protein coding sequence, the NS3 protein coding sequence, the NS4A protein coding sequence, the NS4B protein coding sequence, the NS5A protein coding sequence, and the NS5B protein coding sequence.

When HCV genomic RNA containing RNA encoding a foreign gene is translated in the introduced cell, the gene product encoded by the foreign gene can be expressed. Therefore, HCV genomic RNA containing RNA encoding a foreign gene can also be suitably used for the purpose of producing a gene product of the foreign gene in a cell.

In the HCV genomic RNA of the present invention, the sequence encoding the viral protein and the linkage of a foreign gene and the like are ensured to be translated in a correct reading frame from the HCV genomic RNA. Preferably, the proteins encoded by HCV genomic RNA are linked to each other via a protease cleavage site or the like, so that each protein can be cleaved and released by the protease after the proteins are translated and expressed as one continuous polypeptide.

When the HCV genomic RNA containing the RNA sequence portion of the JFH1 strain encoding NS4, NS5A, and NS5B proteins from NS3, which is prepared in this manner, is introduced into an appropriate host cell, a recombinant cell capable of autonomous replication, preferably continuous autonomous replication of the HCV genomic RNA (i.e., having the ability of autonomous replication of the HCV genomic RNA) can be obtained. Hereinafter, in the present specification, a recombinant cell having the ability to replicate HCV genomic RNA, which contains an RNA sequence portion of the JFH1 strain encoding NS4, NS5A, and NS5B proteins from NS3, is referred to as an "HCV genomic RNA replicating cell".

The host cell used for the "HCV genomic RNA-replicating cell" is not particularly limited as long as it is a cell capable of subculture, but is preferably a eukaryotic cell, more preferably a human cell, and yet more preferably a human liver-derived cell, a human cervix-derived cell, or a human embryonic kidney (fetaldney) -derived cell. Furthermore, the cell is preferably a proliferative cell including a cancer cell or a stem cell, and particularly preferably Huh7 cell, HepG2 cell, IMY-N9 cell, HeLa cell, 293 cell or the like. These cells may be commercially available, may be obtained from a cell depository, or may be obtained from any cell line (e.g., cancer cells or stem cells).

Introduction of HCV genomic RNA into a host cell can be carried out using any known technique. Examples of the introduction method include electroporation, particle gun method, lipofection method, calcium phosphate method, microinjection method, and DEAE-agarose method. The method by electroporation is particularly preferred.

The HCV genomic RNA may be introduced alone or in combination with other nucleic acids. When the amount of HCV genomic RNA to be introduced is changed while the amount of RNA to be introduced is desired to be constant, the desired amount of HCV genomic RNA to be introduced may be mixed with total cellular RNA extracted from the introduced cells to obtain a constant total amount of RNA, and the mixture may be introduced into the cells. The amount of HCV genomic RN to be introduced into the cell may be determined according to the introduction method used, and is preferably 1 picogram to 100 micrograms, more preferably 10 picograms to 10 micrograms.

The replication of HCV genomic RNA in "HCV genomic RNA-replicating cells" can be confirmed by any known RNA detection method, and for example, a method of Northern hybridization of total RNA extracted from cells using a DNA fragment specific to introduced HCV genomic RNA as a probe, or an RT-PCR method using a primer specific to introduced HCV genomic RNA can be used.

Furthermore, if an HCV protein is detected from the proteins extracted from the "HCV genomic RNA replicating cell", it can be determined that the cell is replicating HCV genomic RNA. The detection of HCV genomic RNA can be performed according to any known protein detection method, and for example, can be performed by reacting an antibody against HCV protein expressed from introduced HCV genomic RNA with protein extracted from cells. More specifically, for example, the following method can be used: a protein sample extracted from cells is blotted on nitrocellulose, and an anti-HCV protein antibody (for example, an anti-NS 3-specific antibody, or antiserum collected from a hepatitis c patient) is reacted therewith, thereby detecting the anti-HCV protein antibody.

The method for confirming the autonomous replication of the HVC genomic RNA is not limited, and can be confirmed, for example, by the following method: the target RNA was transfected into Huh7 cells, the Huh7 cells were cultured, and Northern blot was performed on RNA extracted from the cells in the obtained culture using a probe capable of specifically detecting the introduced RNA. Specific examples of procedures for confirming the autonomous replication ability are presented in the description of confirmation of HCV protein expression, detection of HCV genomic RNA, and the like described in examples of the present specification.

Preparation of HCV viral particles

The HCV genomic RNA-replicating cells prepared as described above can produce HCV viral particles in vitro. That is, HCV viral particles can be easily obtained by culturing the HCV genomic RNA-replicating cells of the present invention in an appropriate medium and collecting the produced viral particles from the culture (preferably, culture solution).

The ability of HCV genomic RNA to produce viral particles can be confirmed by any known virus detection method. For example, when a culture solution of a cell thought to produce viral particles is separated by a sucrose density gradient, and the density of each portion, the concentration of HCV core protein, and the amount of HCV genomic RNA are measured, as a result, the peak of HCV core protein and the peak of HCV genomic RNA match each other, and the density of the portion where the peak is detected is low (for example, 1.15mg to 1.22mg) as compared with the density of the same component when the culture supernatant is separated after being treated with 0.25% NP40 (: polyoxyethylene (9) octylphenylene ether).

The HCV viral particles released in the culture medium can also be detected using an antibody against the core protein, E1 protein, or E2 protein, for example. Furthermore, it is also possible to immediately detect the presence of HCV virus particles by amplifying HCV genomic RNA contained in HCV virus particles in a culture medium by RT-PCR using specific primers.

3. Infection of other cells by the HCV particle of the present invention

The HCV viral particles produced by the method of the present invention have an ability to infect cells (preferably, HCV-sensitive cells). The present invention provides a method for producing a hepatitis C virus-infected cell, which comprises culturing an HCV genomic RNA-replicating cell and infecting another cell (preferably an HCV-sensitive cell) with a viral particle in the obtained culture (preferably a culture solution). In the present specification, the term "HCV-sensitive cell" refers to a cell that is infectious to HCV, and is preferably a hepatocyte or a lymphocyte, but is not limited thereto. Specifically, as the hepatocytes, for example, primary hepatocytes, Huh7 cells, HepG2 cells, IMY-N9 cells, HeLa cells, 293 cells, and the like; examples of the lymphocytes include Molt4 cells, HPB-Ma cells, and Daudi cells, but are not limited thereto.

If a cell (e.g., an HCV-sensitive cell) is infected with an HCV viral particle produced in an HCV genomic RNA-replicating cell of the present invention, the HCV genomic RNA is replicated in the infected cell to form a viral particle. When the HCV viral particles produced in the HCV genomic RNA-replicating cells of the present invention infect cells, HCV genomic RNA is replicated in the cells, and viral particles can be further produced.

The HCV viral particles produced in the HCV genomic RNA-replicating cells of the present invention can cause HCV-derived hepatitis by infecting an HCV virus-infectable animal such as chimpanzee.

Purification of HCV viral particles

In purifying HCV viral particles, the solution containing the virus may be selected from any one or more of blood derived from HCV-infected patients, HCV-infected cultured cells, and a culture solution and cell homogenate of cells that produce HCV viral particles by gene recombination techniques.

The solution containing HCV virus is centrifuged and/or a filter is used to remove cells and cell debris. The solution from which the residue has been removed can be concentrated by about 10 to 100 times using an ultrafiltration membrane having a molecular weight cutoff of 100000 to 500000.

The HCV solution from which the residue has been removed can be purified by a chromatography and/or density gradient centrifugation described below, in combination in any order, or by single use. Representative chromatography and density gradient centrifugation methods are described below, but the present invention is not limited thereto.

Gel filtration chromatography is capable of purifying HCV virus particles by using a chromatography carrier containing a crosslinked polymer of acryl dextran and N, N' -methylenebisacrylamide as a gel matrix, preferably by using a chromatography composed of Sephacryl (registered trademark) S-300, S-400, and S-500.

The ion exchange chromatography is preferably performed using Q Sepharose (registered trademark) or the like as an anion exchange resin, and preferably using SP Sepharose (registered trademark) or the like as a cation exchange resin, and is capable of purifying HCV virus particles.

The affinity chromatography preferably uses a resin as a carrier to which a matrix selected from heparin, Sulfated cellulose, lectin, and various pigments is bound as a ligand, and can purify HCV virus particles. It is further preferable that the HCV viral particles can be purified using a vector to which HITrap Heparin HP (registered trademark), HITrap Blue HP (registered trademark), HITrap Benzamidine FF (registered trademark), Sulfated cellulose, and LCA, ConA, RCA-120, and WGA are bound. Most preferably, the purification of HCV viral particles using Sulfated cellulofine as a vector is carried out such that the ratio of the total protein mass in the solution to the RNA copy number of HCV reaches 30-fold or more before and after the purification.

Purification by density gradient centrifugation can be preferably performed using a sugar polymer such as cesium chloride, sucrose, Nycodenz (registered trademark), and Ficoll (registered trademark) and Percoll (registered trademark) as a solute for forming a density gradient. Preferably, sucrose is used. As the solvent to be used, water, phosphate buffer, Tris buffer, acetate buffer, or glycine buffer can be preferably used.

The temperature for purification is preferably 0 to 40 ℃, more preferably 0 to 25 ℃, and most preferably 0 to 10 ℃.

The centrifugal force for purification by density gradient centrifugation is preferably 1X 10⁴～1×10⁹g, more preferably 5X 10⁴～1×10⁷g, most preferably 5X 10⁴～5×10⁵g，

The combination of the purification methods may be a combination of density gradient centrifugation and chromatography in any order, preferably a combination of density gradient centrifugation after purification by various column chromatography, more preferably a combination of purification by density gradient centrifugation of a HCV virus particle-containing fraction obtained by anion exchange column followed by affinity chromatography, and most preferably a combination of further purification by applying a Sulfated cellulofine-containing fraction obtained by column chromatography using QSepharose (registered trademark). Furthermore, between the steps of column chromatography and density gradient centrifugation, it is possible to displace the solute of the solution containing HCV viral particles and/or to perform concentration of HCV viral particles by using dialysis or ultrafiltration.

5. Other embodiments of the invention

In the HCV genomic RNA replicating cell of the present invention, the HCV genomic RNA is efficiently replicated. Therefore, the HCV genomic RNA can be efficiently produced using the HCV genomic RNA replicating cell of the present invention.

In the present invention, HCV genomic RNA can be prepared by culturing HCV genomic RNA-replicating cells, extracting RNA from the culture (cultured cells and/or culture medium), separating the RNA by electrophoresis, and separating and purifying the separated HCV genomic RNA. The RNA thus prepared contains the HCV genomic sequence. By providing a method for producing RNA containing HCV genomic sequences, HCV genomes can be analyzed in more detail.

Furthermore, the HCV genomic RNA-replicating cells of the present invention can be suitably used for producing HCV proteins. The HCV protein can be produced by any known method, for example, by introducing HCV genomic RNA into cells to produce recombinant cells, culturing the recombinant cells, and recovering the protein from the obtained culture (cultured cells and/or culture solution) by a conventional method.

HCV viral particles can have hepatic cell targeting. Therefore, the HCV genomic RNA of the present invention can be used to prepare a hepatocyte-targeted viral vector. The viral vector is suitable for gene therapy. In the present invention, by recombining RNA encoding a foreign gene into HCV genomic RNA and introducing the RNA into a cell, the foreign gene can be introduced into the cell and replicated and expressed in the cell.

Furthermore, by preparing RNA in which the coding sequence for the E1 protein and/or the coding sequence for the E2 protein in HCV genomic RNA is changed to the coat protein of a virus derived from another species, and introducing the RNA into cells to prepare viral particles, it is possible to infect cells of various species with the RNA. In this case, a foreign gene can be incorporated into HCV genomic RNA, and the foreign gene can be used as a viral vector for expressing a cell-targeting gene in various cells depending on the targeting property of the recombinant viral coat protein.

The present invention also relates to a method for producing a viral vector containing a foreign gene, which comprises inserting RNA encoding the foreign gene into HCV genomic RNA, introducing the RNA into a cell, and culturing the cell to produce viral particles.

The present invention also provides a method for producing a hepatitis c vaccine or a vaccine for a virus against a recombinant viral coat protein, which comprises the HCV viral particle of the present invention or a part thereof as an antigen, or a particle prepared by recombining a viral coat protein for the purpose of changing cell targeting, or a part thereof as an antigen. Furthermore, an HCV infection-neutralizing antibody can be prepared by using the HCV viral particle or a part thereof according to the present invention as an antigen, or a particle prepared by recombining viral coat proteins for the purpose of changing cell targeting, or a part thereof as an antigen.

The HCV genomic RNA-replicating cell of the present invention or an HCV-infected cell infected with a viral particle produced by the cell can be used as an experimental system for HCV replication, reconstitution of a viral particle, and screening for a substance (anti-hepatitis c virus substance) that promotes or inhibits viral particle release. Specifically, for example, by culturing the cells in the presence of a test substance, detecting HCV genomic RNA or viral particles in the obtained culture, and determining whether or not the test substance promotes or inhibits replication of replicon RNA or HCV genomic RNA or formation or release of viral particles, a substance that promotes or inhibits growth of hepatitis c virus can be screened. In this case, the HCV genomic RNA in the culture can be detected by detecting the amount, ratio or presence/absence of HCV genomic RNA in the RNA extracted from the above-mentioned cells. Detection of viral particles in a culture (mainly, a culture solution) can be performed by detecting the amount, ratio, or presence of HCV protein contained in the culture solution.

The HCV viral particles and HCV-sensitive cells produced in the HCV genomic RNA-replicating cells of the present invention can also be used as an experimental system for screening a substance that promotes or inhibits binding of HCV to cells. Specifically, for example, HCV viral particles produced in the HCV genomic RNA-replicating cells of the present invention are cultured together with HCV-sensitive cells in the presence of a test substance, and HCV genomic RNA or viral particles in the obtained culture are detected, and it is determined whether or not the test substance promotes or inhibits replication of HCV genomic RNA or formation of viral particles, thereby enabling screening of a substance that promotes or inhibits growth of hepatitis c virus.

The HCV genomic RNA or viral particle can be detected by the above-described method or the examples described below. The above-described test system can also be used for the preparation or evaluation of a prophylactic, therapeutic, or diagnostic agent for hepatitis C virus infection.

Specifically, as a utilization example of the experimental system of the present invention, for example, the following example is given:

(1) search for substances that inhibit HCV proliferation and infection

Examples of the substance that inhibits the proliferation and infection of HCV include organic compounds that directly or indirectly affect the proliferation and infection of HCV, and antisense oligonucleotides that directly or indirectly affect the proliferation of HCV or the translation of HCV proteins by hybridizing with a target sequence of HCV genome or its complementary strand.

(2) Evaluation of various substances having antiviral action in cell culture

Examples of the various substances include substances obtained by rational drug design or high-throughput screening (e.g., isolated and purified enzymes).

(3) Identification of novel targets for treatment of HCV-infected patients

For example, the HCV genomic RNA-replicating cells of the present invention can be used to identify host cellular proteins that play an important role in HCV viral replication.

(4) Evaluation of ability of HCV virus to acquire resistance to drugs and the like and identification of mutations involved in the resistance

(5) Development and production of diagnostic or therapeutic drugs for hepatitis C virus infection, and production of viral protein as a potential antigen for evaluation

(6) Development, production, and evaluation of viral proteins used as potential antigens and production of attenuated HCV for vaccine against hepatitis C Virus infection

Examples

The present invention will be described more specifically with reference to the following examples and the accompanying drawings. However, the technical scope of the present invention is not limited to these examples.

EXAMPLE 1 preparation of HCV genomic RNA

1. Construction of expression vectors

From the JFH1 clone (Kato, T., et al, J.Med.Vitrol.64(2001) p334-339) containing the full-length genomic cDNA of the JFH1 strain (genotype 2a) isolated from fulminant hepatitis patients, DNA corresponding to the total genomic region of the virus strain was obtained and inserted downstream of the T7RNA promoter sequence which had been inserted into the pUC19 plasmid. Specifically, the RT-PCR fragment obtained by amplifying the viral RNA of JFH1 strain was cloned into pGEM-T EASY vector (Promega) to obtain pGEM1-258, pGEM44-486, pGEM317-849, pGEM617-1323, pGEM1141-2367, pGEM2285-3509, pGEM3471-4665, pGEM4547-5970, pGEM5883-7003, pGEM6950-8035, pGEM7984-8892, pGEM8680-9283, pGEM9231-9634 and pGEM9594-9678 plasmids (pGto, T., GASTROENTOLOGY, 125 (2003)) p.1808-1817. The viral genomic cdnas contained in the plasmids were ligated by PCR and restriction endonuclease to clone the full-length genomic cDNA, and the T7RNA promoter sequence was inserted upstream to obtain the JFH clone (pJFH1) (fig. 1). The full-length cDNA sequence of pJFH1 was registered in the International DNA database (DDBJ/EMBL/GenBank) under the accession number AB 047639.

Next, a mutant plasmid clone pJFH1/GND was prepared by mutating the NS5B region (nucleotide sequence: SEQ ID NO: 2; amino acid sequence: SEQ ID NO: 3) in pJFH1 to GND, which is the amino acid motif GDD corresponding to the active center of RNA polymerase, and introducing the mutation into GND. Since the active center of the gene encoding NS5B protein was mutated, the mutant plasmid clone pJFH1/GND was unable to express the active NS5B protein necessary for HCV RNA replication.

Next, pJFH 1/. DELTA.E 1-E2 lacking the E1 region through the E2 region of JFH1 was prepared. In addition, full-length HCV cDNA of J6CF strain (GenBank Accession No. af177036) and JCH1(Kato, T., et al, j.med.vitrol.64(2001) p334-339), which are different from JFH1 strain, were inserted downstream of the T7RNA promoter sequence that had been inserted in the pUC19 plasmid, to prepare pJ6CF and p JCH 1. Furthermore, p JCH1/NS5B (JFH1) was prepared by substituting NS5B of JFH1 for the region of p JCH1 encoding NS 5B.

Preparation of HCV genomic RNA

In order to prepare template DNAs for RNA synthesis, pJFH1, pJFH1/GND, pJFH 1/. DELTA.E 1-E2, pJ6CF, p JCH1, and p JCH1/NS5B (jfh1) constructed as described above were each cleaved with restriction enzyme XbaI. Then, 10 to 20. mu.g of these XbaI-digested fragments were incubated with 20U of Mung Bean Nuclear (total reaction solution amount: 50. mu.l) at 30 ℃ for 30 minutes. The Mung Bean nucleic is an enzyme that catalyzes a reaction that selectively decomposes a single-stranded portion of a double-stranded DNA. In general, when RNA synthesis is performed using the XbaI fragment as a template, a 4-base CUGA that is a part of the XbaI recognition sequence adds an extra portion to the 3' -end of the replicon RNA to be synthesized. Thus, in this example, a 4-base CUGA was removed from the XbaI fragment by treating the XbaI fragment with Mung Bean Nuclear. Then, the solution after Mung Bean Nuclear treatment containing the XbaI fragment was deproteinized by a conventional method, and the 4-base CUGA-removed XbaI fragment was purified and used as a template DNA.

Next, RNA was synthesized in vitro using the template DNA. Using MEGAscript available from Ambion, 20. mu.l of a reaction solution containing 0.5 to 1.0. mu.g of template DNA was reacted at 37 ℃ for 3 to 16 hours to synthesize RNA.

After RNA synthesis, DNase (2U) was added to the reaction solution, and after reaction at 37 ℃ for 15 minutes, RNA was extracted with acidic phenol to remove the template DNA. Thus, HCV RNAs synthesized from the above-mentioned template DNAs derived from pJFH1 and pJFH1/GND were designated as rJFH1, rJFH1/GND, rJFH1/Δ E1-E2, rJ6CF, rJCH1, and rJCH1/NS5B (jfh1), respectively.

These HCV RNAs were RNA prepared using as a template the DNA of GenBank Accession No. AB047639, rJFH1/GND was RNA prepared using as a template the DNA obtained by substituting the 8618-th position G of GenBank Accession No. AB047639 with A, rJFH1/Δ E1-E2 was RNA prepared using as a template the DNA obtained by deleting the 989-containing 2401 sequence of GenBank Accession No. AB047639, pJ6CF was RNA prepared using as a template the DNA of GenBank Accession No. 177036, pJCH1 was RNA prepared using as a template the DNA of GenBank Accession No. AB047640, rJCH1/NS 355 (jfh1) was RNA prepared using as a nucleotide sequence of GenBank Accession No. 1-3866 and the DNA of GenBank Accession No. AB047639, and Av047667 restriction enzyme sites were ligated DNA of Av 389639.

EXAMPLE 2 intracellular HCV genomic RNA-replicating cells and production of HCV viral particles

1. Intracellular HCV genome replication and HCV viral particle production

The above-described synthetic full-length HCV genomic RNAs (rJFH1, rJFH1/GND) were prepared so that the total amount of RNA was 10. mu.g. This mixed RNA was then introduced into Huh7 cells by electroporation. The electroporated Huh7 cells were seeded in a petri dish and cultured for 12 hours, 24 hours, 48 hours, and 72 hours, and then the cells were collected, RNA was extracted from the cells, and analysis was performed by the Northern blot method. Northern blot analysis was performed as described in Molecular Cloning, a Laboratory Manual, 2nd edition, J.Sambrook, E.F.Fritsch, T.Maniatis, Cold spring harbor Laboratory Press (1989). RNA extracted from cells was subjected to denaturing agarose gel electrophoresis, and after the electrophoresis was completed, the RNA was transferred to a positively charged nylon membrane. The RNA transferred to the membrane was hybridized with a 32P-labeled DNA or RNA probe prepared from pJFH1, and the membrane was washed and exposed to light, whereby an RNA band specific to the HCV genome was detected.

As shown in FIG. 2, when JFH1/GND was transfected, an introduced RNA band with a weak signal was observed 4 hours after transfection, the signal decreased with time, and the signal of the band was hardly observed 24 hours after transfection.

On the other hand, in the case of rJFH1 transfection, the intensity of the signal of the RNA band introduced was temporarily reduced almost similarly to the case of JFH1/GND introduction 4 to 12 hours after the transfection, but a clear signal of the RNA band could be confirmed even after 24 hours. This signal is specific for HCV. That is, it is considered that a part of the introduced rJFH1 RNA was replicated and proliferated. No replication could be observed in rJFH1/GND in which the active motif of RNA replicase NS5B was mutated, and it was confirmed that the activity of NS5B is important for replication of the full-length RNA of HCV. Furthermore, the inventors carried out the same experiment with strain JCH1(Kato, T., et al, J.Med.Vitrol.69(2001) p334-339) isolated from chronic hepatitis, and could not confirm replication of HCV RNA at all in this strain.

Detection of HCV proteins

Proteins were extracted from cells transfected with rJFH1 or rJFH1/GND RNA with time in a conventional manner and analyzed by SDS-PAGE and Western blot. In the analysis, Huh7 cells were transfected with expression plasmid DNA containing NS3, NS5A, Core, or E2 genes, and the resulting cell extract was used as a positive control (NS3 protein). Furthermore, a protein extracted from untransfected Huh7 cells was used as a negative control. A protein sample extracted from each cell clone was blotted on a PVDF membrane (Immobilon-P, manufactured by Millipore Co., Ltd.), and NS3, NS5A, Core and E2 proteins encoded by JFH1 RNA were detected using an anti-NS 3-specific antibody (Dr. Mordapour gifer; Wolk B, et al, J.virology.2000; 74: 2293-. In addition, actin proteins were detected using anti-actin antibodies as internal controls.

As shown in fig. 3, it was confirmed that NS3, NS5A, Core, and E2 proteins were detected in cells transfected with rJFH1 24 hours after transfection, and the expression level was increased with time. On the other hand, in the cells transfected with rJFH1/GND or in the untransfected Huh7 cells, NS3, NS5A, Core and E2 proteins were not detected, indicating that the transfected rJFH1 expresses its proteins by autonomous replication.

From the above results 1 and 2, it was confirmed that in the cells established by transfection of rJFH1, rJFH1 was replicated.

Detection of HCV core protein in transfected cell culture broth

The HCV core proteins in the culture solution were measured after culturing Huh7 cell inoculum dishes obtained by introducing cells of rJFH1, rJFH1/GND, rJFH 1/. DELTA.E 1-E2, rJ6CF, and rJCH1 by electroporation for 2 hours, 12 hours, 24 hours, 48 hours, and 72 hours. The assay was performed using the Ortho HCV antigen IRMA assay (Aoyagi et al, j. clin. microbiol., 37(1999) p.1802-1808).

As shown in FIG. 4, the core protein was detected in the culture medium from 148 hours to 72 hours after the transfection of rJFH. On the other hand, no HCV core protein was detected in the culture medium of cells transfected with rJFH1/GND, rJ6CF, and rJCH1, and a small amount of HCV core protein was detected in the culture medium of cells transfected with rJFH1/Δ E1-E2. rJFH1/GND, rJ6CF, and rJCH1 were unable to replicate autonomously in Huh7 cells, and rJFH1 and rJFH1/Δ E1-E2 were able to replicate autonomously in Huh7 cells. Thus, it was revealed that autonomous replication of the introduced HCV RNA is essential for release of the core protein, and that E1 and E2 are essential for stable release of the core protein to the outside of the cell in a large amount.

Detection of HCV in transfected cell culture broth

To analyze whether the core protein released into the culture broth in the above examples was secreted as a viral particle, the culture broth after transfection with rJFH16 days was separated with a sucrose density gradient. 2ml of a 60% (w/w) sucrose solution (dissolved in 50mM Tris pH7.5/0.1M NaCl/1mM EDTA), 1ml of a 50% sucrose solution, 1ml of a 40% sucrose solution, 1ml of a 30% sucrose solution, 1ml of a 20% sucrose solution, and 1ml of a 10% sucrose solution were layered in a centrifuge tube, and then 4ml of a sample culture supernatant was layered. After centrifugation at 400000RPM for 16 hours at 4 ℃ using a Beckmann rotor SW41Ti, 0.5ml of each fraction was recovered from the bottom of the tube. The density of each fraction, the density of HCV core protein, and the number of HCV RNA copies were determined. Detection of replicon RNA by quantitative RT-PCR was carried out by detecting RNA in the 5' untranslated region of HCV RNA according to the method of Takeuchi et al (Takeuchi, T., et al, Gastroenterology, 116: 636-642 (1999)). Specifically, replicon RNA contained in RNA extracted from cells was PCR-amplified using the following synthetic primers and EZ rTth RNA kit (Applied Biosystems), and detected by ABI Prism 7700sequence detector system (Applied Biosystems).

R6-130-S17: 5'-CGGGAGAGCCATAGTGG-3' (Serial number 6)

R6-290-R19: 5'-AGTACCACAAGGCCTTTCG-3' (Serial number 7)

TagMan Probe，R6-148-S21FT：

5'-CTGCGGAACCGGTGAGTACAC-3' (Serial number 8)

As shown in FIG. 5, in the fraction of 1.17mg/ml, the core protein coincided with the peak of HCV RNA. This fraction has a density of about 1.17mg/ml, which is less than the specific gravity of previously reported conjugates of core proteins and nucleic acids. A core protein present in a fraction of 1.17mg/ml is considered to be nuclease-resistant if it forms an HCV viral particle structure together with HCV RNA. Thus, the culture broth after the day of rJFH16 transfection was treated with 10. mu.g/ml RNase A for 20 minutes, and then separated by sucrose density gradient.

As a result, as shown in FIG. 5B, HCV RNA was degraded, and peaks of the core protein and HCV RNA were detected in a fraction of 1.17mg/ml as in the case of RNase A. That is, it was confirmed that the core protein present in a fraction of 1.17mg/ml and HCV RNA form an HCV viral particle structure.

Furthermore, the culture broth was treated with 0.25% NP40 and then similarly isolated, so that the peak specific gravity of the core protein and HCV RNA became 1.28mg/ml (FIG. 5C). Furthermore, if the RNase A treatment was performed simultaneously with the treatment with NP40 (0.25%), the peak of HCV RNA disappeared (FIG. 5D). That is, it is considered that the outer membrane having a low lipid specific gravity is peeled off from the surface of the virus by NP40, and becomes Core particles composed of only nucleic acid and Core protein, which do not retain a virus-like structure, and the specific gravity becomes high.

As described above, it was confirmed that when rJFH1 was transfected into Huh7 cells, viral RNA replicated, and viral particles were formed and secreted into the culture medium.

5. Infection test of Virus particles in culture solution

Whether virus particles secreted into the culture medium by transfecting rJFH1 into Huh7 cells were infectious was examined. After transfection of Huh7 cells with rJFH1 or rJFH 1/. DELTA.E 1-E2 for 3 days, culture supernatants were recovered. The collected culture supernatant was centrifuged, and the centrifuged supernatant was collected and filtered through a 0.45 μm filter. In the presence of this culture medium, Huh7 cells that were not transfected with RNA were cultured, and after 48 hours, the cells were immunofluorescent-stained with an anti-Core antibody or an anti-NS 5A antibody. As shown in fig. 6A, the expression of the core protein and NS5A protein was observed in cells cultured in the presence of a culture solution obtained by transfecting rJFH1 into Huh7 cells. On the other hand, in cells cultured in the presence of a culture solution obtained by transfecting rJFH 1/. DELTA.E 1-E2 into Huh7 cells, no expression of the core protein and NS5A protein was observed in the cells (data not shown).

Next, rJFH1 was transfected into Huh7 cells for 3 days, and the culture supernatant was collected and subjected to ultrafiltration using an ultrafiltration membrane (cut off 1X 10)⁵Da) was concentrated 30-fold. Untransfected RNA Huh7 cells, 48 cells, were cultured on 15mm coverslips with 100. mu.l of concentrated HCV virus particle-containing mediumAfter hours, immunostaining with the anti-Core antibody was performed, and the anti-Core antibody staining positive, i.e., infected cells were counted, as shown in fig. 6B, 394.0 ± 26.5 infected cells (0.51% of the total number of cells) were confirmed. Therefore, it was confirmed that the infection was caused by HCV virus particles secreted into the culture medium by transfecting rJFH1 into Huh7 cells. That is, the culture solution for infection was subjected to UV treatment, or using a culture solution prepared without the process of RNA transfection, untransfected Huh7 cells were cultured on a 15mm cover glass, immunostaining was performed with an anti-Core antibody after 48 hours, and infected cells were counted. As a result, the UV treatment abruptly decreased the infected cells, and when a culture solution prepared without the RNA transfection process was used, it was confirmed that there were no infected cells.

Furthermore, it was investigated whether or not the infected HCV viral particles were amplified in RNA in the cells, and new HCV viral particles were released into the culture medium. The culture medium after 48 hours after transfection of rJFH1 into Huh7 cells was concentrated, and the Huh7 cells without RNA transfection were cultured in 100. mu.l of the concentrated culture medium containing HCV virus particles, and the cells and the culture medium were collected daily, and RNA was collected, and HCV RNA was quantified by the above-described method. As a result, as shown in FIG. 6C, a constant amount of HCV RNA was amplified in the cells, and the amount of HCV RNA increased day by day in the supernatant. On the other hand, in the same study using a culture solution obtained by transfecting rJFH 1/. DELTA.E 1-E2 into Huh7 cells, HCVRNA could not be detected both in the cells and in the culture solution.

From these results, it was confirmed that HCV viral particles secreted into the culture medium by transfecting Huh7 cells with rJFH1 have an infection ability, and also have an ability to amplify HCV RNA in infected cells to produce new HCV viral particles.

6. Preparation of HCV virions Using rJCH1/NS5B (jfh1)

Whether or not HCV viral particles could be secreted into the culture medium by transfecting rJCH1/NS5B (jfh1) into Huh7 cells and whether or not the secreted HCV viral particles were infectious was examined. The culture medium 6 days after rJCH1/NS5B (jfh1) was transfected into Huh7 cells was concentrated by the method described in (5). after the culture medium was concentrated, Huh7 cells not transfected with RNA were cultured in the presence of the culture medium, and the amount of HCV RNA was quantified over time, and the amount of HCV RNA in the cells increased over time from 12 hours after the start of the culture (FIG. 7A). Furthermore, Huh7 cells not transfected with RNA were cultured on a 15mm cover glass, cultured for 48 hours in the presence of the concentrated culture solution, immunostained with an anti-Core antibody, and the number of infected cells stained positively with the anti-Core antibody was counted, and as shown in fig. 7B, the infected cells were confirmed. These results show that HCV virus particles secreted into the culture medium by transfecting rJCH1/NS5B (jfh1) into Huh7 cells acquired the infection ability and also had the ability to amplify HCV RNA in infected cells to produce new HCV virus particles.

Therefore, in a strain which does not have an autonomous replication ability in vitro, such as an HCV strain isolated from a patient, it is possible to autonomously replicate in a cultured cell line and produce HCV virus particles by replacing the NS5B region of the strain with NS5B of rJFH 1.

[ example 3]

1. Preparation of HCV viral particles Using Con1/C-NS2/JFH-1

Whether or not HCV viral particles could be secreted into a culture medium by transfecting Huh7 cells with chimeric HCV RNA containing the Con-1 strain of HCV genotype 1b and the NS5B portion of JFH-1, and whether or not the secreted HCV viral particles were infectious was examined.

The following constructs were prepared: the Core, E1, E2, p7 and NS2 regions of the Con-1 strain of HCV genotype 1b were ligated downstream of the 5 'UTR of the JFH-1 strain, the NS3 to NS5b regions of the JFH-1 strain were ligated downstream thereof, and the 3' UTR of the JFH-1 strain was ligated downstream thereof. Using this construct, r Con1/C-NS2/JFH-1 chimeric HCV RNA was prepared as described in 2 of example 1, and the RNA was transfected into Huh7 as described in 1 of example 2.HCV RNA was transfected into Huh7 cells, the core protein in the supernatant was measured over time, and the core protein was detected in the supernatant from about 48 hours later, confirming that HCV viral particles were produced in the cell supernatant. Then, the supernatant was concentrated 20-fold with an ultrafiltration membrane, and the concentrate was added to Huh7 cells. After 48 hours of culture, cells were stained with rabbit anti-NS 3 antibody.

As a result, in mock and rJFH-1/Δ EE1-E2, anti-NS 3 antibody-positive cells were not observed, while in rJFH-1 and rCon1/C-NS2/JFH-1, anti-NS 3 antibody-positive cells were detected. From the above results, it was confirmed that rCon1/C-NS2/JFH-1 can produce infectious HCV virus particles in the same manner as rJFH-1.

Example 4 preparation of full-Length chimeric HCV replicon RNA derived from full-Length chimeric HCV genomic RNA

(1) Construction of expression vectors

Plasmid DNA was prepared by inserting DNA (JFH-1 clone: SEQ ID NO: 9) containing the full-length genomic cDNA of the JFH-1 strain of hepatitis C virus (genotype 2a) isolated from patients with fulminant hepatitis into the downstream of the T7RNA promoter sequence inserted into the pUC19 plasmid.

Specifically, the RT-PCR fragment obtained by amplifying the viral RNA of the JFH-1 strain was cloned into pGEM-T EASY vector (Promega) to obtain plasmid DNAs of pGEM1-258, pGEM44-486, pGEM317-849, pGEM617-1323, pGEM1141-2367, pGEM2285-3509, pGEM3471-4665, pGEM4547-5970, pGEM5883-7003, pGEM6950-8035, pGEM7984-8892, pGEM 928680-9286883, pGEM9231-9634 and pGEM9594-9678 (Kato, T., al, Gastroenterology, (2003)125 p.1808-1817). The cDNA derived from the viral genomic RNA contained in each plasmid was ligated by PCR and restriction enzyme, and the full-length viral genomic cDNA was cloned. The T7RNA promoter sequence was inserted upstream of the full-length viral genome. Hereinafter, the plasmid DNA thus constructed is referred to as pJFH 1. The preparation of the JFH-1 clone is described in JP 2002-171978A and Kato et al (Kato, T., et al, J.Med.Vitrol. (2001)64 (3): p.334-339). The nucleotide sequence of the full-length cDNA cloned in pJFH1 was registered in the International DNA database (DDBJ/EMBL/GenBank) under the accession number AB 047639.

Then, EMCV-IRES (internal ribosome entry site of encephalomyocarditis virus) and a neomycin resistance gene (neo; also referred to as neomycin phosphotransferase gene) were inserted between the 5' untranslated region and the core region of plasmid DNA pJFH1, and plasmid DNApFGEP-JFH 1 was constructed. The construction procedure is in accordance with Ikeda (Ikeda et al, J.Vitrol. (2002)76 (6): p.2997-3006).

(2) Construction of chimeric expression vectors

The JFH strain is HCV derived from HCV type 2a, and chimeric HCV vectors were prepared using TH strain derived from HCV type 1b (Wakita et al, J.biol.chem., (1994)269, p.14205-14210, and Mordapour et al, (biochem.Biophys.Res.Commun., (1998)246, p.920-924). Chimeric HCV and pFGEP-TH/JFH 1 were prepared by substituting the cores, E1, E2 and p7 of pFGEP-JFH 1 prepared above for the cores, E1, E2 and p7 derived from TH strain.

In the present specification, the full length of the JFH1 strain (derived from the JFH-1 clone) and the partial RNA sequence of the TH strain used in the chimera (the partial genomic RNA sequence (1-3748) containing a part of the region from the 5' untranslated region to the NS3 region of the HCV TH strain) are represented by seq id nos 9 and 10 of the sequence listing, respectively. In the full-length genomic RNA sequence (SEQ ID NO: 9) of the JFH-1 strain, the "5' untranslated region" corresponds to 1 to 340, the "core protein coding sequence" corresponds to 341 to 913, the "E1 protein coding sequence" corresponds to 914 to 1489, the "E2 protein coding sequence" corresponds to 1490 to 2590, the "NS 2 protein coding sequence" corresponds to 2780 to 3430, the "NS 3 protein coding sequence" corresponds to 3431 to 5323, the "NS 4A protein coding sequence" corresponds to 5324 to 5486, the "NS 4B protein coding sequence" corresponds to 5487 to 6268, the "NS 5A protein coding sequence" corresponds to 6269 to 7663, and the "NS 5B protein coding sequence" corresponds to 7664 to 9442.

(3) Preparation of full-Length chimeric HCV replicon RNA

In order to prepare a template DNA for synthesizing the full-length chimeric HCV replicon RNA, the expression vector pFGEP-TH/JFH 1 constructed as described above was cleaved with the restriction enzyme XbaI. Then, 10 to 20. mu.g of the XbaI fragment was used to prepare 50. mu.l of a reaction solution, and 20U of Mung Bean Nuclear was used to incubate at 30 ℃ for 30 minutes, thereby further processing. A MungBean Nuclear is an enzyme that catalyzes a selective decomposition reaction of a single-stranded portion of a double-stranded DNA. In general, when RNA synthesis is performed using the XbaI fragment as a template, a 4-base CUGA that is a part of the XbaI recognition sequence adds an extra portion to the 3' -end of the replicon RNA to be synthesized. Thus, in this example, a 4-base CUGA was removed from the XbaI fragment by treating the XbaI fragment with Mung Bean Nuclear. Then, the solution after Mung Bean Nuclear treatment containing the XbaI fragment was deproteinized by a conventional method, and the 4-base CUGA-removed XbaI fragment was purified and used as a template DNA.

Next, RNA was synthesized in vitro from the template DNA using T7RNA polymerase. MEGAscript from Ambion was used for RNA synthesis. Mu.l of a reaction solution containing 0.5 to 1.0. mu.g of template DNA was reacted according to the manufacturer's instructions.

After RNA synthesis, DNAse (2U) was added to the reaction solution, and after reaction at 37 ℃ for 15 minutes, RNA was extracted with acidic phenol to remove the template DNA. Thus, HCV RNA synthesized from the above-mentioned template DNA derived from pFGRPH-TH/JFH 1 was designated as rFGREP-TH/JFH 1. The nucleotide sequence of the chimeric HCV genomic RNA of rFGREP-TH/JFH1 is represented by SEQ ID NO. 11. rFGREP-TH/JFH1 is an example of the full-length chimeric HCV replicon RNA of the present invention.

EXAMPLE 5 preparation of replicating cells of full-Length chimeric HCV replicon RNA and establishment of cell clone

(1) Introduction of full-Length chimeric HCV genomic RNA into cells

The full-length chimeric HCV replicon RNA (rFGREP-TH/JFH1) synthesized as described above was mixed with total cellular RNA extracted from Huh7 cells at various doses to a total amount of 10. mu.g. Then, the RNA was introduced into Huh7 cells by electroporation. After 16 to 24 hours of incubation, G418 was added at various concentrations. The culture medium was changed 2 times per week and the culture was continued. After 21 days of culture, the surviving cells were stained with crystal violet. The stained colonies were measured and the number of colonies obtained per unit weight of transfected RNA was calculated. In addition, a portion of the surviving cell colonies in the culture dish were cloned and cultured. After extracting RNA, genomic DNA, and protein from each of the cloned cells, detection of full-length chimeric HCV replicon RNA, presence or absence of neomycin-resistant gene recombination into genomic DNA, and expression of HCV protein were examined. The details of these results are shown below.

(2) Colony Forming ability

As a result of the transfection, the formation of cell colonies was observed even at a G418 concentration of 1.0 mg/ml. This is considered to be because the autonomous replication of rFGREP-TH/JFH1 replicon RNA, the sustained expression of the neomycin resistance gene, and the sustained maintenance of G418 resistance resulted in the ability to form colonies in Huh7 cells transfected with rFGREP-TH/JFH1 replicon RNA, and the cells were able to proliferate.

Example 6 infection of chimeric HCV Virus in culture supernatant

Infection experiment of chimeric HCV viral particles in culture supernatant

A culture supernatant of a full-length chimeric HCV replicon RNA-replicating cell clone established by transfecting rFGREP-TH/JFH1 into Huh7 cells was recovered, and this culture supernatant was further added to uninfected Huh7 cells to infect Huh7 cells with viral particles in the culture supernatant. On the next day of infection, 0.3mg/ml of G418 was added to the culture medium of Huh7 cells, followed by culture for another 21 days. After the completion of the culture, the cells were fixed and stained with crystal violet, and then colony formation was observed in infected cells using a culture supernatant of a full-length chimeric HCV replicon RNA-replicating cell clone obtained by transfection with rFGREP-TH/JFH 1. This result indicates that infectious HCV was produced in the full-length chimeric HCV replicon RNA-replicating cell clone obtained by transfection of rFGREP-TH/JFH1, and that the HCV maintained infectivity for new cells.

EXAMPLE 7 purification of HCV viral particles

(1) Gel filtration

The distribution of HCV viral particles in each fraction by gel filtration chromatography is shown in fig. 11. The gel carriers used were Sephacryl (registered trademark) S300, S400 and S500. The solution containing HCV viral particles used for column chromatography is purified by column chromatography using the above gel carriers, respectively. The buffer used for purification was 10mM Tris hydrochloride, 1mM ethylenediaminetetraacetic acid and 100mM sodium chloride (pH 8.0). As a result, when Sephacryl (registered trademark) S300 is used, since HCV virus particles are obtained in the pass fraction called the Void fraction, it is possible to separate proteins having a small molecular weight and change the salt concentration of the solution by using Sephacryl (registered trademark) S300. At this time, the ratio of HCV core protein/total protein mass became 3.78, and the ratio of HCV particles in the total protein was increased compared to HCV viral particles before purification. On the other hand, when Sephacryl (registered trademark) S400 and S500 are used, HCV exists in a fraction eluted depending on the molecular weight, and therefore, it can be separated from proteins having other molecular weights.

(2) Ion exchange chromatography

The distribution of HCV viral particles in each fraction by ion exchange chromatography is shown in fig. 12. The carriers used were SP Sepharose HP (registered trademark) and Q Sepharose HP (registered trademark).

When a column using SP Sepharose HP (registered trade mark) was used, the column was equilibrated with 50mM citric acid buffer solution (pH 6.2). A solution containing HCV virus particles, which is concentrated using an ultrafiltration membrane having a molecular weight cut-off of 100000 to 500000 and diluted with 50mM citric acid buffer solution (pH6.2), is applied to a column, and then the 50mM citric acid buffer solution (pH6.2) is allowed to flow through the column at about 10-fold column capacity. Then, 50mM citric acid buffer (pH6.2) to which 0.1M NaCl, 0.3M NaCl, or 1M NaCl was added was sequentially passed through the column at about 3 times the column capacity. Then, 50mM citric acid buffer (pH6.2) to which 1M NaCl was added was passed through the column at about 5 times the column capacity (1M NaCl W fraction). As a result, HCV virus particles were eluted in a 50mM citrate buffer (pH6.2) containing 0.1M NaCl.

When Q Sepharose HP (registered trade mark) was used, the column was equilibrated with 50mM Tris-HCl buffer (pH 8.0). A solution containing HCV virus particles, which is concentrated using an ultrafiltration membrane having a molecular weight cut-off of 100000 to 500000 and diluted with 50mM Tris-HCl buffer (pH8.0), is applied to a column, and then 50mM Tris-HCl buffer (pH8.0) is allowed to flow through the column at about 10-fold column capacity. Then, 50mM Tris-HCl buffer (pH8.0) to which 0.1M NaCl, 0.3M NaCl, or 1M NaCl was added was sequentially passed through the column at about 3 times the column capacity. Then, 50mM Tris-HCl buffer (pH8.0) to which 1M NaCl was added was passed through the column at about 5 times the column capacity (1M NaCl W fraction). As a result, HCV virus particles were eluted in a 50mM Tris-HCl buffer (pH8.0) to which 0.3M NaCl was added. At this time, the ratio of HCV core protein/total protein mass became 2.32, and the ratio of HCV particles in the total protein was increased compared to HCV viral particles before purification.

(3) Affinity chromatography

The distribution of HCV viral particles in each fraction by affinity chromatography is shown in fig. 13. Affinity chromatography uses a vector to which RCA-120, ConA, LCA, and WGA are ligated, respectively.

In the case of ConA, LCA, and WGA affinity chromatography, the column was equilibrated with phosphate buffered saline. A solution containing HCV virus particles, which is concentrated using an ultrafiltration membrane having a molecular weight cut-off of 100000 to 500000 and diluted with a phosphate-buffered saline, is applied to a column, and then the phosphate-buffered saline is passed through the column at about 10 times the column capacity. Then, phosphate buffered saline to which 0.35M lactose was added was passed through the column at about 3 times the column capacity. Then, phosphate buffered saline to which 0.5M lactose was added was passed through the column at about 5 times the column capacity. As a result, HCV did not bind specifically to the vector when affinity chromatography was performed using LCA and ConA. When WGA affinity chromatography is used, HCV viral particles are eluted in a phosphate buffered saline fraction to which 0.35M lactose is added.

In RCA-120 affinity chromatography, the column was equilibrated with phosphate buffered saline. A solution containing HCV virus particles, which is concentrated using an ultrafiltration membrane having a molecular weight cut-off of 100000 to 500000 and diluted with a phosphate-buffered saline, is applied to a column, and then the phosphate-buffered saline is passed through the column at about 10 times the column capacity. Then, phosphate buffered saline to which 0.38M lactose was added was passed through the column at about 3 times the column capacity. Then, phosphate buffered saline to which 0.38M lactose was added was passed through the column at about 5 times the column capacity. In RCA-120 affinity chromatography, HCV virus particles were eluted in a phosphate buffered saline fraction supplemented with 0.38M lactose.

The distribution of HCV virus particles in each fraction by heparin, sulfonated cellulose chromatography is shown in fig. 14.

In each chromatography, the column was equilibrated with 20mM phosphate buffer (pH 7.0). A solution containing HCV virus particles, which is concentrated using an ultrafiltration membrane having a molecular weight cut-off of 100000 to 500000 and diluted with 20mM phosphate buffer (pH7.0), is applied to a column, and then 20mM phosphate buffer (pH7.0) is passed through the column at about 10-fold column capacity. Then, 20mM phosphate buffer (pH7.0) to which 0.5M NaCl, 0.3M NaCl, or 1M NaCl was added was sequentially passed through the column at about 3 times the column capacity. Then, 20mM phosphate buffer (pH7.0) to which 1M NaCl was added was passed through the column at about 5 times the column capacity. As a result, when heparin affinity chromatography was used, HCV virus particles were eluted in a 20mM phosphate buffer (pH7.0) containing 0.3M NaCl. At this time, the ratio of HCV core protein/total protein mass was 0.36, and the ratio of HCV particles in the total protein was reduced compared to HCV viral particles before purification. In the case of using sulfocelllulofine chromatography, HCV virus particles were eluted in a 20mM phosphate buffer solution (pH7.0) to which 0.1M NaCl was added.

The distribution of HCV virus particles in each fraction by blue dye affinity chromatography (blue dye affinity chromatography) is shown in fig. 15.

In Blue dye affinity chromatography, Cibacron Blue F3G-A was bound to a support of agarose particles for column. The column was equilibrated with 20mM phosphate buffer (pH 7.0). A solution containing HCV virus particles, which is concentrated using an ultrafiltration membrane having a molecular weight cut-off of 100000 to 500000 and diluted with 20mM phosphate buffer solution (pH7.0), is applied to a column, and then phosphate-buffered physiological saline is passed through the column at about 10 times the column capacity. Then, 20mM phosphate buffer (pH7.0) to which 1M NaCl or 2M NaCl was added was sequentially passed through the column at about 3 times the column capacity. Then, 20mM phosphate buffer (pH7.0) to which 2M NaCl was added was passed through the column at about 5 times the column capacity. As a result, HCV virus particles were eluted in the column non-binding portion. At this time, the ratio of HCV core protein/total protein mass became 3.33, and the ratio of HCV particles in the total protein decreased more than that of HCV viral particles before purification.

(4) Sucrose density gradient centrifugation

With reference to the above examples, purification of HCV viral particles was performed by combining column chromatography and sucrose density gradient centrifugation.

First, HCV virus particles were purified using Q Sepharose HP (registered trademark). The column was equilibrated with 50mM Tris-HCl buffer (pH 8.0). A solution containing HCV virus particles, which is concentrated using an ultrafiltration membrane having a molecular weight cut-off of 100000 to 500000 and diluted with 50mM Tris-HCl buffer (pH8.0), is applied to a column, and then 50mM Tris-HCl buffer (pH8.0) is allowed to flow through the column at about 10-fold column capacity. Then, 50mM Tris-HCl buffer (pH8.0) to which 0.1M NaCl, 0.3M NaCl, or 1M NaCl was added was sequentially passed through the column at about 3 times the column capacity. Then, 50mM Tris-HCl buffer (pH8.0) to which 1M NaCl was added was passed through the column at about 5 times the column capacity (1M NaCl W fraction). As shown in FIG. 16A, HCV virus particles eluted in 50mM Tris-HCl buffer (pH8.0) to which 0.3M NaCl was added, 50mM Tris-HCl buffer (pH8.0) to which 1M NaCl was added, and 1M NaClW. The fractions containing HCV virus particles were collected, and at this time, the ratio of HCV core protein/total protein mass became 2.29, and the ratio of HCV particles to the total protein was increased compared to HCV virus particles before purification.

HCV was then purified using Sulfated cellulofine chromatography. In each chromatography, the column was equilibrated with 20mM phosphate buffer (pH 7.0). An HCV particle-containing fraction purified by QSepharose HP (registered trademark) is concentrated by an ultrafiltration membrane having a cut-off molecular weight of 100000-500000, diluted with 20mM phosphate buffer solution (pH7.0), and the diluted HCV particle-containing solution is applied to a column, and then the phosphate buffer solution (pH7.0) is allowed to flow through the column at about 10-fold column capacity. Then, 20mM phosphate buffer (pH7.0) to which 0.25M NaCl or 1M NaCl was added was sequentially passed through the column at about 3 times the column capacity. Then, 20mM phosphate buffer (pH7.0) to which 1M NaCl was added was passed through the column at about 5 times the column capacity. As shown in FIG. 16B, HCV virus particles were mainly eluted in a portion of 20mM phosphate buffer solution (pH7.0) to which 1M NaCl was added. In a 20mM phosphate buffer (pH7.0) containing 1M NaCl, the ratio of HCV core protein/total protein mass was 31.4, and the ratio of HCV particles to the total protein was increased as compared with that of HCV virus particles before purification.

Further, sucrose density gradient centrifugation was performed. A portion of 20mM phosphate buffer (pH7.0) to which 1M NaCl was added after chromatography using Sulfated cellulofine was concentrated using an ultrafiltration membrane having a molecular weight cut-off of 100000 to 500000, and diluted with TEN buffer (10mM Tris hydrochloric acid buffer (pH8.0), 0.1M sodium chloride, 1mM ethylenediaminetetraacetic acid (pH 8.0)). The solution layer containing HCV virus particles was added to the layered 60%, 50%, 40%, 30%, 20%, 10% sucrose solution and centrifuged at 390 kXg at 4 ℃ for 18 hours. Since HCV is concentrated in a portion having a specific gravity of about 1.2, the portion is collected. In the collected fraction, the ratio of HCV core protein/total protein mass became 1.69, and the ratio of HCV particles in the total protein was increased compared to HCV viral particles before purification.

The fraction containing HCV viral particles purified by sucrose density gradient centrifugation was purified to a ratio of HCV core protein/total protein mass of about 120 times as compared to that before the start of purification. In the final fraction, 10 is contained⁹copies/mL of HCV.

All publications, patents and patent applications cited in this specification are herein incorporated by reference as if fully set forth.

Industrial applicability

According to the present invention, viral particles of HCV strains of various genotypes can be produced in a cultured cell line. That is, even in an HCV strain isolated from a patient, which does not have an ability to autonomously replicate in vitro, by replacing the RNA sequence portion encoding the NS4, NS5A, and NS5B proteins from NS3 with the RNA sequence portion encoding the NS4, NS5A, and NS5B proteins from NS3 from JFH1, it is possible to autonomously replicate in a cultured cell system and produce HCV virus particles. The HCV viral particles purified in accordance with the present invention can be directly used as a vaccine for medical use. The HCV genomic RNA or viral particle provided by the present invention can also be used as a viral vector for foreign genes. In addition, the method of the present invention can also be used as a system for studying the HCV infection process and screening various substances affecting the HCV infection process.

Sequence Listing content

Sequence number 1: RNA partial sequences (cDNA sequences) of JFH1 strain encoding NS3, NS4, NS5A, and NS5B proteins

Sequence number 2: sequences (cDNAs) encoding NS3 to NS5 proteins of JFH1

Sequence number 3: NS5B protein of JFH1

Sequence number 4: synthetic peptide designed based on JFH 1E 2 region

Sequence number 5: synthetic peptide designed based on JFH 1E 2 region

Sequence number 6: primer (R6-130-S17)

Sequence number 7: primer (R6-290-S19)

Sequence number 8: TaqMan probe (R6-148-S21FT)

Sequence number 9: full-Length genomic RNA of HCV JFH1 Strain (JFH-1 clone)

Sequence number 10: genomic RNA of HCV TH Strain comprising a portion from the 5' untranslated region to the NS3 region

Sequence number 11: a chimeric HCV genomic RNA comprising the genomic RNA of the HCV JFH1 strain (JFH-1 clone) and the genomic RNA of the HCVTH strain.

Sequence listing

<110>Tokyo Metropolitan Organization for Medical Research

Toray Industries Inc.

<120> modified human hepatitis C Virus genomic RNA having autonomous replication ability

<130>PH-2565-PCT

<140>

<141>

<150>JP 2004-290801

<151>2004-10-01

<150>JP 2004-243975

<151>2004-08-24

<150>JP 2005-069527

<151>2005-03-11

<150>JP 2005-069725

<151>2005-03-11

<160>11

<170>PatentIn Ver.3.1

<210>1

<211>6013

<212>DNA

<213> hepatitis C Virus

<220>

<223>Inventor：Wakita，Takaji

Inventor：Kato，Takanobu

Inventor：Date，Tomoko

Inventor：Bartenchlager，Ralf

Inventor：Tanabe，Junichi

Inventor：Sone，Saburou

<220>

<223> sequences (cDNA sequences) encoding NS3 to NS5 proteins of JFH1

<400>1

gctcccatca ctgcttatgc ccagcaaaca cgaggcctcc tgggcgccat agtggtgagt 60

atgacggggc gtgacaggac agaacaggcc ggggaagtcc aaatcctgtc cacagtctct 120

cagtccttcc tcggaacaac catctcgggg gttttgtgga ctgtttacca cggagctggc 180

aacaagactc tagccggctt acggggtccg gtcacgcaga tgtactcgag tgctgagggg 240

gacttggtag gctggcccag cccccctggg accaagtctt tggagccgtg caagtgtgga 300

gccgtcgacc tatatctggt cacgcggaac gctgatgtca tcccggctcg gagacgcggg 360

gacaagcggg gagcattgct ctccccgaga cccatttcga ccttgaaggg gtcctcgggg 420

gggccggtgc tctgccctag gggccacgtc gttgggctct tccgagcagc tgtgtgctct 480

cggggcgtgg ccaaatccat cgatttcatc cccgttgaga cactcgacgt tgttacaagg 540

tctcccactt tcagtgacaa cagcacgcca ccggctgtgc cccagaccta tcaggtcggg 600

tacttgcatg ctccaactgg cagtggaaag agcaccaagg tccctgtcgc gtatgccgcc 660

caggggtaca aagtactagt gcttaacccc tcggtagctg ccaccctggg gtttggggcg 720

tacctatcca aggcacatgg catcaatccc aacattagga ctggagtcag gaccgtgatg 780

accggggagg ccatcacgta ctccacatat ggcaaatttc tcgccgatgg gggctgcgct 840

agcggcgcct atgacatcat catatgcgat gaatgccacg ctgtggatgc tacctccatt 900

ctcggcatcg gaacggtcct tgatcaagca gagacagccg gggtcagact aactgtgctg 960

gctacggcca caccccccgg gtcagtgaca accccccatc ccgatataga agaggtaggc 1020

ctcgggcggg agggtgagat ccccttctat gggagggcga ttcccctatc ctgcatcaag 1080

ggagggagac acctgatttt ctgccactca aagaaaaagt gtgacgagct cgcggcggcc 1140

cttcggggca tgggcttgaa tgccgtggca tactatagag ggttggacgt ctccataata 1200

ccagctcagg gagatgtggt ggtcgtcgcc accgacgccc tcatgacggg gtacactgga 1260

gactttgact ccgtgatcga ctgcaatgta gcggtcaccc aagctgtcga cttcagcctg 1320

gaccccacct tcactataac cacacagact gtcccacaag acgctgtctc acgcagtcag 1380

cgccgcgggc gcacaggtag aggaagacag ggcacttata ggtatgtttc cactggtgaa 1440

cgagcctcag gaatgtttga cagtgtagtg ctttgtgagt gctacgacgc aggggctgcg 1500

tggtacgatc tcacaccagc ggagaccacc gtcaggctta gagcgtattt caacacgccc 1560

ggcctacccg tgtgtcaaga ccatcttgaa ttttgggagg cagttttcac cggcctcaca 1620

cacatagacg cccacttcct ctcccaaaca aagcaagcgg gggagaactt cgcgtaccta 1680

gtagcctacc aagctacggt gtgcgccaga gccaaggccc ctcccccgtc ctgggacgcc 1740

atgtggaagt gcctggcccg actcaagcct acgcttgcgg gccccacacc tctcctgtac 1800

cgtttgggcc ctattaccaa tgaggtcacc ctcacacacc ctgggacgaa gtacatcgcc 1860

acatgcatgc aagctgacct tgaggtcatg accagcacgt gggtcctagc tggaggagtc 1920

ctggcagccg tcgccgcata ttgcctggcg actggatgcg tttccatcat cggccgcttg 1980

cacgtcaacc agcgagtcgt cgttgcgccg gataaggagg tcctgtatga ggcttttgat 2040

gagatggagg aatgcgcctc tagggcggct ctcatcgaag aggggcagcg gatagccgag 2100

atgttgaagt ccaagatcca aggcttgctg cagcaggcct ctaagcaggc ccaggacata 2160

caacccgcta tgcaggcttc atggcccaaa gtggaacaat tttgggccag acacatgtgg 2220

aacttcatta gcggcatcca atacctcgca ggattgtcaa cactgccagg gaaccccgcg 2280

gtggcttcca tgatggcatt cagtgccgcc ctcaccagtc cgttgtcgac cagtaccacc 2340

atccttctca acatcatggg aggctggtta gcgtcccaga tcgcaccacc cgcgggggcc 2400

accggctttg tcgtcagtgg cctggtgggg gctgccgtgg gcagcatagg cctgggtaag 2460

gtgctggtgg acatcctggc aggatatggt gcgggcattt cgggggccct cgtcgcattc 2520

aagatcatgt ctggcgagaa gccctctatg gaagatgtca tcaatctact gcctgggatc 2580

ctgtctccgg gagccctggt ggtgggggtc atctgcgcgg ccattctgcg ccgccacgtg 2640

ggaccggggg agggcgcggt ccaatggatg aacaggctta ttgcctttgc ttccagagga 2700

aaccacgtcg cccctactca ctacgtgacg gagtcggatg cgtcgcagcg tgtgacccaa 2760

ctacttggct ctcttactat aaccagccta ctcagaagac tccacaattg gataactgag 2820

gactgcccca tcccatgctc cggatcctgg ctccgcgacg tgtgggactg ggtttgcacc 2880

atcttgacag acttcaaaaa ttggctgacc tctaaattgt tccccaagct gcccggcctc 2940

cccttcatct cttgtcaaaa ggggtacaag ggtgtgtggg ccggcactgg catcatgacc 3000

acgcgctgcc cttgcggcgc caacatctct ggcaatgtcc gcctgggctc tatgaggatc 3060

acagggccta aaacctgcat gaacacctgg caggggacct ttcctatcaa ttgctacacg 3120

gagggccagt gcgcgccgaa accccccacg aactacaaga ccgccatctg gagggtggcg 3180

gcctcggagt acgcggaggt gacgcagcat gggtcgtact cctatgtaac aggactgacc 3240

actgacaatc tgaaaattcc ttgccaacta ccttctccag agtttttctc ctgggtggac 3300

ggtgtgcaga tccataggtt tgcacccaca ccaaagccgt ttttccggga tgaggtctcg 3360

ttctgcgttg ggcttaattc ctatgctgtc gggtcccagc ttccctgtga acctgagccc 3420

gacgcagacg tattgaggtc catgctaaca gatccgcccc acatcacggc ggagactgcg 3480

gcgcggcgct tggcacgggg atcacctcca tctgaggcga gctcctcagt gagccagcta 3540

tcagcaccgt cgctgcgggc cacctgcacc acccacagca acacctatga cgtggacatg 3600

gtcgatgcca acctgctcat ggagggcggt gtggctcaga cagagcctga gtccagggtg 3660

cccgttctgg actttctcga gccaatggcc gaggaagaga gcgaccttga gccctcaata 3720

ccatcggagt gcatgctccc caggagcggg tttccacggg ccttaccggc ttgggcacgg 3780

cctgactaca acccgccgct cgtggaatcg tggaggaggc cagattacca accgcccacc 3840

gttgctggtt gtgctctccc cccccccaag aaggccccga cgcctccccc aaggagacgc 3900

cggacagtgg gtctgagcga gagcaccata tcagaagccc tccagcaact ggccatcaag 3960

acctttggcc agcccccctc gagcggtgat gcaggctcgt ccacgggggc gggcgccgcc 4020

gaatccggcg gtccgacgtc ccctggtgag ccggccccct cagagacagg ttccgcctcc 4080

tctatgcccc ccctcgaggg ggagcctgga gatccggacc tggagtctga tcaggtagag 4140

cttcaacctc ccccccaggg ggggggggta gctcccggtt cgggctcggg gtcttggtct 4200

acttgctccg aggaggacga taccaccgtg tgctgctcca tgtcatactc ctggaccggg 4260

gctctaataa ctccctgtag ccccgaagag gaaaagttgc caatcaaccc tttgagtaac 4320

tcgctgttgc gataccataa caaggtgtac tgtacaacat caaagagcgc ctcacagagg 4380

gctaaaaagg taacttttga caggacgcaa gtgctcgacg cccattatga ctcagtctta 4440

aaggacatca agctagcggc ttccaaggtc agcgcaaggc tcctcacctt ggaggaggcg 4500

tgccagttga ctccacccca ttctgcaaga tccaagtatg gattcggggc caaggaggtc 4560

cgcagcttgt ccgggagggc cgttaaccac atcaagtccg tgtggaagga cctcctggaa 4620

gacccacaaa caccaattcc cacaaccatc atggccaaaa atgaggtgtt ctgcgtggac 4680

cccgccaagg ggggtaagaa accagctcgc ctcatcgttt accctgacct cggcgtccgg 4740

gtctgcgaga aaatggccct ctatgacatt acacaaaagc ttcctcaggc ggtaatggga 4800

gcttcctatg gcttccagta ctcccctgcc caacgggtgg agtatctctt gaaagcatgg 4860

gcggaaaaga aggaccccat gggtttttcg tatgataccc gatgcttcga ctcaaccgtc 4920

actgagagag acatcaggac cgaggagtcc atataccagg cctgctccct gcccgaggag 4980

gcccgcactg ccatacactc gctgactgag agactttacg taggagggcc catgttcaac 5040

agcaagggtc aaacctgcgg ttacagacgt tgccgcgcca gcggggtgct aaccactagc 5100

atgggtaaca ccatcacatg ctatgtgaaa gccctagcgg cctgcaaggc tgcggggata 5160

gttgcgccca caatgctggt atgcggcgat gacctagtag tcatctcaga aagccagggg 5220

actgaggagg acgagcggaa cctgagagcc ttcacggagg ccatgaccag gtactctgcc 5280

cctcctggtg atccccccag accggaatat gacctggagc taataacatc ctgttcctca 5340

aatgtgtctg tggcgttggg cccgcggggc cgccgcagat actacctgac cagagaccca 5400

accactccac tcgcccgggc tgcctgggaa acagttagac actcccctat caattcatgg 5460

ctgggaaaca tcatccagta tgctccaacc atatgggttc gcatggtcct aatgacacac 5520

ttcttctcca ttctcatggt ccaagacacc ctggaccaga acctcaactt tgagatgtat 5580

ggatcagtat actccgtgaa tcctttggac cttccagcca taattgagag gttacacggg 5640

cttgacgcct tttctatgca cacatactct caccacgaac tgacgcgggt ggcttcagcc 5700

ctcagaaaac ttggggcgcc acccctcagg gtgtggaaga gtcgggctcg cgcagtcagg 5760

gcgtccctca tctcccgtgg agggaaagcg gccgtttgcg gccgatatct cttcaattgg 5820

gcggtgaaga ccaagctcaa actcactcca ttgccggagg cgcgcctact ggacttatcc 5880

agttggttca ccgtcggcgc cggcgggggc gacatttttc acagcgtgtc gcgcgcccga 5940

ccccgctcat tactcttcgg cctactccta cttttcgtag gggtaggcct cttcctactc 6000

cccgctcggt aga 6013

<210>2

<211>1773

<212>DNA

<213> hepatitis C Virus

<220>

<221>CDS

<222>(1)..(1773)

<220>

<223> sequences (cDNA sequences) encoding NS3 to NS5 proteins of JFH1

<400>2

tcc atg tca tac tcc tgg acc ggg gct cta ata act ccc tgt agc ccc 48

Ser Met Ser Tyr Ser Trp Thr Gly Ala Leu Ile Thr Pro Cys Ser Pro

1 5 10 15

gaa gag gaa aag ttg cca atc aac cct ttg agt aac tcg ctg ttg cga 96

Glu Glu Glu Lys Leu Pro Ile Asn Pro Leu Ser Asn Ser Leu Leu Arg

20 25 30

tac cat aac aag gtg tac tgt aca aca tca aag agc gcc tca cag agg 144

Tyr His Asn Lys Val Tyr Cys Thr Thr Ser Lys Ser Ala Ser Gln Arg

35 40 45

gct aaa aag gta act ttt gac agg acg caa gtg ctc gac gcc cat tat 192

Ala Lys Lys Val Thr Phe Asp Arg Thr Gln Val Leu Asp Ala His Tyr

50 55 60

gac tca gtc tta aag gac atc aag cta gcg gct tcc aag gtc agc gca 240

Asp Ser Val Leu Lys Asp Ile Lys Leu Ala Ala Ser Lys Val Ser Ala

65 70 75 80

agg ctc ctc acc ttg gag gag gcg tgc cag ttg act cca ccc cat tct 288

Arg Leu Leu Thr Leu Glu Glu Ala Cys Gln Leu Thr Pro Pro His Ser

85 90 95

gca aga tcc aag tat gga ttc ggg gcc aag gag gtc cgc agc ttg tcc 336

Ala Arg Ser Lys Tyr Gly Phe Gly Ala Lys Glu Val Arg Ser Leu Ser

100 105 110

ggg agg gcc gtt aac cac atc aag tcc gtg tgg aag gac ctc ctg gaa 384

Gly Arg Ala Val Asn His Ile Lys Ser Val Trp Lys Asp Leu Leu Glu

115 120 125

gac cca caa aca cca att ccc aca acc atc atg gcc aaa aat gag gtg 432

Asp Pro Gln Thr Pro Ile Pro Thr Thr Ile Met Ala Lys Asn Glu Val

130 135 140

ttc tgc gtg gac ccc gcc aag ggg ggt aag aaa cca gct cgc ctc atc 480

Phe Cys Val Asp Pro Ala Lys Gly Gly Lys Lys Pro Ala Arg Leu Ile

145 150 155 160

gtt tac cct gac ctc ggc gtc cgg gtc tgc gag aaa atg gcc ctc tat 528

Val Tyr Pro Asp Leu Gly Val Arg Val Cys Glu Lys Met Ala Leu Tyr

165 170 175

gac att aca caa aag ctt cct cag gcg gta atg gga gct tcc tat ggc 576

Asp Ile Thr Gln Lys Leu Pro Gln Ala Val Met Gly Ala Ser Tyr Gly

180 185 190

ttc cag tac tcc cct gcc caa cgg gtg gag tat ctc ttg aaa gca tgg 624

Phe Gln Tyr Ser Pro Ala Gln Arg Val Glu Tyr Leu Leu Lys Ala Trp

195 200 205

gcg gaa aag aag gac ccc atg ggt ttt tcg tat gat acc cga tgc ttc 672

Ala Glu Lys Lys Asp Pro Met Gly Phe Ser Tyr Asp Thr Arg Cys Phe

210 215 220

gac tca acc gtc act gag aga gac atc agg acc gag gag tcc ata tac 720

Asp Ser Thr Val Thr Glu Arg Asp Ile Arg Thr Glu Glu Ser Ile Tyr

225 230 235 240

cag gcc tgc tcc ctg ccc gag gag gcc cgc act gcc ata cac tcg ctg 768

Gln Ala Cys Ser Leu Pro Glu Glu Ala Arg Thr Ala Ile His Ser Leu

245 250 255

act gag aga ctt tac gta gga ggg ccc atg ttc aac agc aag ggt caa 816

Thr Glu Arg Leu Tyr Val Gly Gly Pro Met Phe Asn Ser Lys Gly Gln

260 265 270

acc tgc ggt tac aga cgt tgc cgc gcc agc ggg gtg cta acc act agc 864

Thr Cys Gly Tyr Arg Arg Cys Arg Ala Ser Gly Val Leu Thr Thr Ser

275 280 285

atg ggt aac acc atc aca tgc tat gtg aaa gcc cta gcg gcc tgc aag 912

Met Gly Asn Thr Ile Thr Cys Tyr Val Lys Ala Leu Ala Ala Cys Lys

290 295 300

gct gcg ggg ata gtt gcg ccc aca atg ctg gta tgc ggc gat gac cta 960

Ala Ala Gly Ile Val Ala Pro Thr Met Leu Val Cys Gly Asp Asp Leu

305 310 315 320

gta gtc atc tca gaa agc cag ggg act gag gag gac gag cgg aac ctg 1008

Val Val Ile Ser Glu Ser Gln Gly Thr Glu Glu Asp Glu Arg Asn Leu

325 330 335

aga gcc ttc acg gag gcc atg acc agg tac tct gcc cct cct ggt gat 1056

Arg Ala Phe Thr Glu Ala Met Thr Arg Tyr Ser Ala Pro Pro Gly Asp

340 345 350

ccc ccc aga ccg gaa tat gac ctg gag cta ata aca tcc tgt tcc tca 1104

Pro Pro Arg Pro Glu Tyr Asp Leu Glu Leu Ile Thr Ser Cys Ser Ser

355 360 365

aat gtg tct gtg gcg ttg ggc ccg cgg ggc cgc cgc aga tac tac ctg 1152

Asn Val Ser Val Ala Leu Gly Pro Arg Gly Arg Arg Arg Tyr Tyr Leu

370 375 380

acc aga gac cca acc act cca ctc gcc cgg gct gcc tgg gaa aca gtt 1200

Thr Arg Asp Pro Thr Thr Pro Leu Ala Arg Ala Ala Trp Glu Thr Val

385 390 395 400

aga cac tcc cct atc aat tca tgg ctg gga aac atc atc cag tat gct 1248

Arg His Ser Pro Ile Asn Ser Trp Leu Gly Asn Ile Ile Gln Tyr Ala

405 410 415

cca acc ata tgg gtt cgc atg gtc cta atg aca cac ttc ttc tcc att 1296

Pro Thr Ile Trp Val Arg Met Val Leu Met Thr His Phe Phe Ser Ile

420 425 430

ctc atg gtc caa gac acc ctg gac cag aac ctc aac ttt gag atg tat 1344

Leu Met Val Gln Asp Thr Leu Asp Gln Asn Leu Asn Phe Glu Met Tyr

435 440 445

gga tca gta tac tcc gtg aat cct ttg gac ctt cca gcc ata att gag 1392

Gly Ser Val Tyr Ser Val Asn Pro Leu Asp Leu Pro Ala Ile Ile Glu

450 455 460

agg tta cac ggg ctt gac gcc ttt tct atg cac aca tac tct cac cac 1440

Arg Leu His Gly Leu Asp Ala Phe Ser Met His Thr Tyr Ser His His

465 470 475 480

gaa ctg acg cgg gtg gct tca gcc ctc aga aaa ctt ggg gcg cca ccc 1488

Glu Leu Thr Arg Val Ala Ser Ala Leu Arg Lys Leu Gly Ala Pro Pro

485 490 495

ctc agg gtg tgg aag agt cgg gct cgc gca gtc agg gcg tcc ctc atc 1536

Leu Arg Val Trp Lys Ser Arg Ala Arg Ala Val Arg Ala Ser Leu Ile

500 505 510

tcc cgt gga ggg aaa gcg gcc gtt tgc ggc cga tat ctc ttc aat tgg 1584

Ser Arg Gly Gly Lys Ala Ala Val Cys Gly Arg Tyr Leu Phe Asn Trp

515 520 525

gcg gtg aag acc aag ctc aaa ctc act cca ttg ccg gag gcg cgc cta 1632

Ala Val Lys Thr Lys Leu Lys Leu Thr Pro Leu Pro Glu Ala Arg Leu

530 535 540

ctg gac tta tcc agt tgg ttc acc gtc ggc gcc ggc ggg ggc gac att 1680

Leu Asp Leu Ser Ser Trp Phe Thr Val Gly Ala Gly Gly Gly Asp Ile

545 550 555 560

ttt cac agc gtg tcg cgc gcc cga ccc cgc tca tta ctc ttc ggc cta 1728

Phe His Ser Val Ser Arg Ala Arg Pro Arg Ser Leu Leu Phe Gly Leu

565 570 575

ctc cta ctt ttc gta ggg gta ggc ctc ttc cta ctc ccc gct cgg 1773

Leu Leu Leu Phe Val Gly Val Gly Leu Phe Leu Leu Pro Ala Arg

580 585 590

<210>3

<211>591

<212>PRT

<213> hepatitis C Virus

<220>

<223> NS5B protein of JFH1

<400>3

Ser Met Ser Tyr Ser Trp Thr Gly Ala Leu Ile Thr Pro Cys Ser Pro

1 5 10 15

Glu Glu Glu Lys Leu Pro Ile Asn Pro Leu Ser Asn Ser Leu Leu Arg

20 25 30

Tyr His Asn Lys Val Tyr Cys Thr Thr Ser Lys Ser Ala Ser Gln Arg

35 40 45

Ala Lys Lys Val Thr Phe Asp Arg Thr Gln Val Leu Asp Ala His Tyr

50 55 60

Asp Ser Val Leu Lys Asp Ile Lys Leu Ala Ala Ser Lys Val Ser Ala

65 70 75 80

Arg Leu Leu Thr Leu Glu Glu Ala Cys Gln Leu Thr Pro Pro His Ser

85 90 95

Ala Arg Ser Lys Tyr Gly Phe Gly Ala Lys Glu Val Arg Ser Leu Ser

100 105 110

Gly Arg Ala Val Asn His Ile Lys Ser Val Trp Lys Asp Leu Leu Glu

115 120 125

Asp Pro Gln Thr Pro Ile Pro Thr Thr Ile Met Ala Lys Asn Glu Val

130 135 140

Phe Cys Val Asp Pro Ala Lys Gly Gly Lys Lys Pro Ala Arg Leu Ile

145 150 155 160

Val Tyr Pro Asp Leu Gly Val Arg Val Cys Glu Lys Met Ala Leu Tyr

165 170 175

Asp Ile Thr Gln Lys Leu Pro Gln Ala Val Met Gly Ala Ser Tyr Gly

180 185 190

Phe Gln Tyr Ser Pro Ala Gln Arg Val Glu Tyr Leu Leu Lys Ala Trp

195 200 205

Ala Glu Lys Lys Asp Pro Met Gly Phe Ser Tyr Asp Thr Arg Cys Phe

210 215 220

Asp Ser Thr Val Thr Glu Arg Asp Ile Arg Thr Glu Glu Ser Ile Tyr

225 230 235 240

Gln Ala Cys Ser Leu Pro Glu Glu Ala Arg Thr Ala Ile His Ser Leu

245 250 255

Thr Glu Arg Leu Tyr Val Gly Gly Pro Met Phe Asn Ser Lys Gly Gln

260 265 270

Thr Cys Gly Tyr Arg Arg Cys Arg Ala Ser Gly Val Leu Thr Thr Ser

275 280 285

Met Gly Asn Thr Ile Thr Cys Tyr Val Lys Ala Leu Ala Ala Cys Lys

290 295 300

Ala Ala Gly Ile Val Ala Pro Thr Met Leu Val Cys Gly Asp Asp Leu

305 310 315 320

Val Val Ile Ser Glu Ser Gln Gly Thr Glu Glu Asp Glu Arg Asn Leu

325 330 335

Arg Ala Phe Thr Glu Ala Met Thr Arg Tyr Ser Ala Pro Pro Gly Asp

340 345 350

Pro Pro Arg Pro Glu Tyr Asp Leu Glu Leu Ile Thr Ser Cys Ser Ser

355 360 365

Asn Val Ser Val Ala Leu Gly Pro Arg Gly Arg Arg Arg Tyr Tyr Leu

370 375 380

Thr Arg Asp Pro Thr Thr Pro Leu Ala Arg Ala Ala Trp Glu Thr Val

385 390 395 400

Arg His Ser Pro Ile Asn Ser Trp Leu Gly Asn Ile Ile Gln Tyr Ala

405 410 415

Pro Thr Ile Trp Val Arg Met Val Leu Met Thr His Phe Phe Ser Ile

420 425 430

Leu Met Val Gln Asp Thr Leu Asp Gln Asn Leu Asn Phe Glu Met Tyr

435 440 445

Gly Ser Val Tyr Ser Val Asn Pro Leu Asp Leu Pro Ala Ile Ile Glu

450 455 460

Arg Leu His Gly Leu Asp Ala Phe Ser Met His Thr Tyr Ser His His

465 470 475 480

Glu Leu Thr Arg Val Ala Ser Ala Leu Arg Lys Leu Gly Ala Pro Pro

485 490 495

Leu Arg Val Trp Lys Ser Arg Ala Arg Ala Val Arg Ala Ser Leu Ile

500 505 510

Ser Arg Gly Gly Lys Ala Ala Val Cys Gly Arg Tyr Leu Phe Asn Trp

515 520 525

Ala Val Lys Thr Lys Leu Lys Leu Thr Pro Leu Pro Glu Ala Arg Leu

530 535 540

Leu Asp Leu Ser Ser Trp Phe Thr Val Gly Ala Gly Gly Gly Asp Ile

545 550 555 560

Phe His Ser Val Ser Arg Ala Arg Pro Arg Ser Leu Leu Phe Gly Leu

565 570 575

Leu Leu Leu Phe Val Gly Val Gly Leu Phe Leu Leu Pro Ala Arg

580 585 590

<210>4

<211>14

<212>PRT

<213> Artificial sequence

<220>

<223> description of artificial sequences: synthetic peptide designed based on JFH 1E 2 fragment

<400>4

Gly Thr Thr Thr Val Gly Gly Ala Val Ala Arg Ser Thr Asn

1 5 10

<210>5

<211>14

<212>PRT

<213> Artificial sequence

<220>

<223> description of artificial sequences: synthetic peptide designed based on JFH 1E 2 fragment

<400>5

Cys Asp Leu Glu Asp Arg Asp Arg Ser Gln Leu Ser Pro Leu

1 5 10

<210>6

<211>17

<212>DNA

<213> Artificial sequence

<220>

<223> description of artificial sequences: primer (R6-130-S17)

<400>6

cgggagagcc atagtgg 17

<210>7

<211>19

<212>DNA

<213> Artificial sequence

<220>

<223> description of artificial sequences: primer (R6-290-R19)

<400>7

agtaccacaa ggcctttcg 19

<210>8

<211>21

<212>DNA

<213> Artificial sequence

<220>

<223> description of artificial sequences: TaqMan probe (R6-148-S21FT)

<400>8

ctgcggaacc ggtgagtaca c 21

<210>9

<211>9707

<212>RNA

<213> Intelligent (Homo sapiens)

<220>

<223> full-Length hepatitis C Virus genomic RNA derived from JFH1 Strain (JFH-1 clone)

<400>9

gaauucuaau acgacucacu auagaccugc cccuaauagg ggcgacacuc cgccaugaau 60

cacuccccug ugaggaacua cugucuucac gcagaaagcg ccuagccaug gcguuaguau 120

gagugucgua cagccuccag gccccccccu cccgggagag ccauaguggu cugcggaacc 180

ggugaguaca ccggaauugc cgggaagacu ggguccuuuc uuggauaaac ccacucuaug 240

cccggccauu ugggcgugcc cccgcaagac ugcuagccga guagcguugg guugcgaaag 300

gccuuguggu acugccugau agggcgcuug cgagugcccc gggaggucuc guagaccgug 360

caccaugagc acaaauccua aaccucaaag aaaaaccaaa agaaacacca accgucgccc 420

agaagacguu aaguucccgg gcggcggcca gaucguuggc ggaguauacu uguugccgcg 480

caggggcccc agguugggug ugcgcacgac aaggaaaacu ucggagcggu cccagccacg 540

ugggagacgc cagcccaucc ccaaagaucg gcgcuccacu ggcaaggccu ggggaaaacc 600

aggucgcccc uggccccuau augggaauga gggacucggc ugggcaggau ggcuccuguc 660

cccccgaggc ucucgccccu ccuggggccc cacugacccc cggcauaggu cgcgcaacgu 720

ggguaaaguc aucgacaccc uaacgugugg cuuugccgac cucauggggu acauccccgu 780

cguaggcgcc ccgcuuagug gcgccgccag agcugucgcg cacggcguga gaguccugga 840

ggacgggguu aauuaugcaa cagggaaccu acccgguuuc cccuuuucua ucuucuugcu 900

ggcccuguug uccugcauca ccguuccggu cucugcugcc caggugaaga auaccaguag 960

cagcuacaug gugaccaaug acugcuccaa ugacagcauc acuuggcagc ucgaggcugc 1020

gguucuccac guccccgggu gcgucccgug cgagagagug gggaauacgu cacgguguug 1080

ggugccaguc ucgccaaaca uggcugugcg gcagcccggu gcccucacgc agggucugcg 1140

gacgcacauc gauaugguug ugauguccgc caccuucugc ucugcucucu acguggggga 1200

ccucuguggc ggggugaugc ucgcggccca gguguucauc gucucgccgc aguaccacug 1260

guuugugcaa gaaugcaauu gcuccaucua cccuggcacc aucacuggac accgcauggc 1320

augggacaug augaugaacu ggucgcccac ggccaccaug auccuggcgu acgugaugcg 1380

cguccccgag gucaucauag acaucguuag cggggcucac uggggcguca uguucggcuu 1440

ggccuacuuc ucuaugcagg gagcgugggc gaaggucauu gucauccuuc ugcuggccgc 1500

ugggguggac gcgggcacca ccaccguugg aggcgcuguu gcacguucca ccaacgugau 1560

ugccggcgug uucagccaug gcccucagca gaacauucag cucauuaaca ccaacggcag 1620

uuggcacauc aaccguacug ccuugaauug caaugacucc uugaacaccg gcuuucucgc 1680

ggccuuguuc uacaccaacc gcuuuaacuc gucagggugu ccagggcgcc uguccgccug 1740

ccgcaacauc gaggcuuucc ggauagggug gggcacccua caguacgagg auaaugucac 1800

caauccagag gauaugaggc cguacugcug gcacuacccc ccaaagccgu guggcguagu 1860

ccccgcgagg ucugugugug gcccagugua cuguuucacc cccagcccgg uaguaguggg 1920

cacgaccgac agacguggag ugcccaccua cacaugggga gagaaugaga cagaugucuu 1980

ccuacugaac agcacccgac cgccgcaggg cucaugguuc ggcugcacgu ggaugaacuc 2040

cacugguuuc accaagacuu guggcgcgcc accuugccgc accagagcug acuucaacgc 2100

cagcacggac uuguugugcc cuacggauug uuuuaggaag cauccugaug ccacuuauau 2160

uaaguguggu ucugggcccu ggcucacacc aaagugccug guccacuacc cuuacagacu 2220

cuggcauuac cccugcacag ucaauuuuac caucuucaag auaagaaugu auguaggggg 2280

gguugagcac aggcucacgg ccgcaugcaa cuucacucgu ggggaucgcu gcgacuugga 2340

ggacagggac aggagucagc ugucuccucu guugcacucu accacggaau gggccauccu 2400

gcccugcacc uacucagacu uacccgcuuu gucaacuggu cuucuccacc uucaccagaa 2460

caucguggac guacaauaca uguauggccu cucaccugcu aucacaaaau acgucguucg 2520

augggagugg gugguacucu uauuccugcu cuuagcggac gccagagucu gcgccugcuu 2580

guggaugcuc aucuuguugg gccaggccga agcagcauug gagaaguugg ucgucuugca 2640

cgcugcgagu gcggcuaacu gccauggccu ccuauauuuu gccaucuucu ucguggcagc 2700

uuggcacauc aggggucggg ugguccccuu gaccaccuau ugccucacug gccuauggcc 2760

cuucugccua cugcucaugg cacugccccg gcaggcuuau gccuaugacg caccugugca 2820

cggacagaua ggcguggguu uguugauauu gaucacccuc uucacacuca ccccggggua 2880

uaagacccuc cucggccagu gucuguggug guugugcuau cuccugaccc ugggggaagc 2940

caugauucag gaguggguac cacccaugca ggugcgcggc ggccgcgaug gcaucgcgug 3000

ggccgucacu auauucugcc cggguguggu guuugacauu accaaauggc uuuuggcguu 3060

gcuugggccu gcuuaccucu uaagggccgc uuugacacau gugccguacu ucgucagagc 3120

ucacgcucug auaaggguau gcgcuuuggu gaagcagcuc gcggggggua gguauguuca 3180

gguggcgcua uuggcccuug gcagguggac uggcaccuac aucuaugacc accucacacc 3240

uaugucggac ugggccgcua gcggccugcg cgacuuagcg gucgccgugg aacccaucau 3300

cuucaguccg auggagaaga aggucaucgu cuggggagcg gagacggcug caugugggga 3360

cauucuacau ggacuucccg uguccgcccg acucggccag gagauccucc ucggcccagc 3420

ugauggcuac accuccaagg gguggaagcu ccuugcuccc aucacugcuu augcccagca 3480

aacacgaggc cuccugggcg ccauaguggu gaguaugacg gggcgugaca ggacagaaca 3540

ggccggggaa guccaaaucc uguccacagu cucucagucc uuccucggaa caaccaucuc 3600

ggggguuuug uggacuguuu accacggagc uggcaacaag acucuagccg gcuuacgggg 3660

uccggucacg cagauguacu cgagugcuga gggggacuug guaggcuggc ccagcccccc 3720

ugggaccaag ucuuuggagc cgugcaagug uggagccguc gaccuauauc uggucacgcg 3780

gaacgcugau gucaucccgg cucggagacg cggggacaag cggggagcau ugcucucccc 3840

gagacccauu ucgaccuuga agggguccuc gggggggccg gugcucugcc cuaggggcca 3900

cgucguuggg cucuuccgag cagcugugug cucucggggc guggccaaau ccaucgauuu 3960

cauccccguu gagacacucg acguuguuac aaggucuccc acuuucagug acaacagcac 4020

gccaccggcu gugccccaga ccuaucaggu cggguacuug caugcuccaa cuggcagugg 4080

aaagagcacc aaggucccug ucgcguaugc cgcccagggg uacaaaguac uagugcuuaa 4140

ccccucggua gcugccaccc ugggguuugg ggcguaccua uccaaggcac auggcaucaa 4200

ucccaacauu aggacuggag ucaggaccgu gaugaccggg gaggccauca cguacuccac 4260

auauggcaaa uuucucgccg augggggcug cgcuagcggc gccuaugaca ucaucauaug 4320

cgaugaaugc cacgcugugg augcuaccuc cauucucggc aucggaacgg uccuugauca 4380

agcagagaca gccgggguca gacuaacugu gcuggcuacg gccacacccc ccgggucagu 4440

gacaaccccc caucccgaua uagaagaggu aggccucggg cgggagggug agauccccuu 4500

cuaugggagg gcgauucccc uauccugcau caagggaggg agacaccuga uuuucugcca 4560

cucaaagaaa aagugugacg agcucgcggc ggcccuucgg ggcaugggcu ugaaugccgu 4620

ggcauacuau agaggguugg acgucuccau aauaccagcu cagggagaug ugguggucgu 4680

cgccaccgac gcccucauga cgggguacac uggagacuuu gacuccguga ucgacugcaa 4740

uguagcgguc acccaagcug ucgacuucag ccuggacccc accuucacua uaaccacaca 4800

gacuguccca caagacgcug ucucacgcag ucagcgccgc gggcgcacag guagaggaag 4860

acagggcacu uauagguaug uuuccacugg ugaacgagcc ucaggaaugu uugacagugu 4920

agugcuuugu gagugcuacg acgcaggggc ugcgugguac gaucucacac cagcggagac 4980

caccgucagg cuuagagcgu auuucaacac gcccggccua cccguguguc aagaccaucu 5040

ugaauuuugg gaggcaguuu ucaccggccu cacacacaua gacgcccacu uccucuccca 5100

aacaaagcaa gcgggggaga acuucgcgua ccuaguagcc uaccaagcua cggugugcgc 5160

cagagccaag gccccucccc cguccuggga cgccaugugg aagugccugg cccgacucaa 5220

gccuacgcuu gcgggcccca caccucuccu guaccguuug ggcccuauua ccaaugaggu 5280

cacccucaca cacccuggga cgaaguacau cgccacaugc augcaagcug accuugaggu 5340

caugaccagc acgugggucc uagcuggagg aguccuggca gccgucgccg cauauugccu 5400

ggcgacugga ugcguuucca ucaucggccg cuugcacguc aaccagcgag ucgucguugc 5460

gccggauaag gagguccugu augaggcuuu ugaugagaug gaggaaugcg ccucuagggc 5520

ggcucucauc gaagaggggc agcggauagc cgagauguug aaguccaaga uccaaggcuu 5580

gcugcagcag gccucuaagc aggcccagga cauacaaccc gcuaugcagg cuucauggcc 5640

caaaguggaa caauuuuggg ccagacacau guggaacuuc auuagcggca uccaauaccu 5700

cgcaggauug ucaacacugc cagggaaccc cgcgguggcu uccaugaugg cauucagugc 5760

cgcccucacc aguccguugu cgaccaguac caccauccuu cucaacauca ugggaggcug 5820

guuagcgucc cagaucgcac cacccgcggg ggccaccggc uuugucguca guggccuggu 5880

gggggcugcc gugggcagca uaggccuggg uaaggugcug guggacaucc uggcaggaua 5940

uggugcgggc auuucggggg cccucgucgc auucaagauc augucuggcg agaagcccuc 6000

uauggaagau gucaucaauc uacugccugg gauccugucu ccgggagccc uggugguggg 6060

ggucaucugc gcggccauuc ugcgccgcca cgugggaccg ggggagggcg cgguccaaug 6120

gaugaacagg cuuauugccu uugcuuccag aggaaaccac gucgccccua cucacuacgu 6180

gacggagucg gaugcgucgc agcgugugac ccaacuacuu ggcucucuua cuauaaccag 6240

ccuacucaga agacuccaca auuggauaac ugaggacugc cccaucccau gcuccggauc 6300

cuggcuccgc gacguguggg acuggguuug caccaucuug acagacuuca aaaauuggcu 6360

gaccucuaaa uuguucccca agcugcccgg ccuccccuuc aucucuuguc aaaaggggua 6420

caagggugug ugggccggca cuggcaucau gaccacgcgc ugcccuugcg gcgccaacau 6480

cucuggcaau guccgccugg gcucuaugag gaucacaggg ccuaaaaccu gcaugaacac 6540

cuggcagggg accuuuccua ucaauugcua cacggagggc cagugcgcgc cgaaaccccc 6600

cacgaacuac aagaccgcca ucuggagggu ggcggccucg gaguacgcgg aggugacgca 6660

gcaugggucg uacuccuaug uaacaggacu gaccacugac aaucugaaaa uuccuugcca 6720

acuaccuucu ccagaguuuu ucuccugggu ggacggugug cagauccaua gguuugcacc 6780

cacaccaaag ccguuuuucc gggaugaggu cucguucugc guugggcuua auuccuaugc 6840

ugucgggucc cagcuucccu gugaaccuga gcccgacgca gacguauuga gguccaugcu 6900

aacagauccg ccccacauca cggcggagac ugcggcgcgg cgcuuggcac ggggaucacc 6960

uccaucugag gcgagcuccu cagugagcca gcuaucagca ccgucgcugc gggccaccug 7020

caccacccac agcaacaccu augacgugga cauggucgau gccaaccugc ucauggaggg 7080

cgguguggcu cagacagagc cugaguccag ggugcccguu cuggacuuuc ucgagccaau 7140

ggccgaggaa gagagcgacc uugagcccuc aauaccaucg gagugcaugc uccccaggag 7200

cggguuucca cgggccuuac cggcuugggc acggccugac uacaacccgc cgcucgugga 7260

aucguggagg aggccagauu accaaccgcc caccguugcu gguugugcuc uccccccccc 7320

caagaaggcc ccgacgccuc ccccaaggag acgccggaca gugggucuga gcgagagcac 7380

cauaucagaa gcccuccagc aacuggccau caagaccuuu ggccagcccc ccucgagcgg 7440

ugaugcaggc ucguccacgg gggcgggcgc cgccgaaucc ggcgguccga cguccccugg 7500

ugagccggcc cccucagaga cagguuccgc cuccucuaug cccccccucg agggggagcc 7560

uggagauccg gaccuggagu cugaucaggu agagcuucaa ccuccccccc aggggggggg 7620

gguagcuccc gguucgggcu cggggucuug gucuacuugc uccgaggagg acgauaccac 7680

cgugugcugc uccaugucau acuccuggac cggggcucua auaacucccu guagccccga 7740

agaggaaaag uugccaauca acccuuugag uaacucgcug uugcgauacc auaacaaggu 7800

guacuguaca acaucaaaga gcgccucaca gagggcuaaa aagguaacuu uugacaggac 7860

gcaagugcuc gacgcccauu augacucagu cuuaaaggac aucaagcuag cggcuuccaa 7920

ggucagcgca aggcuccuca ccuuggagga ggcgugccag uugacuccac cccauucugc 7980

aagauccaag uauggauucg gggccaagga gguccgcagc uuguccggga gggccguuaa 8040

ccacaucaag uccgugugga aggaccuccu ggaagaccca caaacaccaa uucccacaac 8100

caucauggcc aaaaaugagg uguucugcgu ggaccccgcc aaggggggua agaaaccagc 8160

ucgccucauc guuuacccug accucggcgu ccgggucugc gagaaaaugg cccucuauga 8220

cauuacacaa aagcuuccuc aggcgguaau gggagcuucc uauggcuucc aguacucccc 8280

ugcccaacgg guggaguauc ucuugaaagc augggcggaa aagaaggacc ccauggguuu 8340

uucguaugau acccgaugcu ucgacucaac cgucacugag agagacauca ggaccgagga 8400

guccauauac caggccugcu cccugcccga ggaggcccgc acugccauac acucgcugac 8460

ugagagacuu uacguaggag ggcccauguu caacagcaag ggucaaaccu gcgguuacag 8520

acguugccgc gccagcgggg ugcuaaccac uagcaugggu aacaccauca caugcuaugu 8580

gaaagcccua gcggccugca aggcugcggg gauaguugcg cccacaaugc ugguaugcgg 8640

cgaugaccua guagucaucu cagaaagcca ggggacugag gaggacgagc ggaaccugag 8700

agccuucacg gaggccauga ccagguacuc ugccccuccu ggugaucccc ccagaccgga 8760

auaugaccug gagcuaauaa cauccuguuc cucaaaugug ucuguggcgu ugggcccgcg 8820

gggccgccgc agauacuacc ugaccagaga cccaaccacu ccacucgccc gggcugccug 8880

ggaaacaguu agacacuccc cuaucaauuc auggcuggga aacaucaucc aguaugcucc 8940

aaccauaugg guucgcaugg uccuaaugac acacuucuuc uccauucuca ugguccaaga 9000

cacccuggac cagaaccuca acuuugagau guauggauca guauacuccg ugaauccuuu 9060

ggaccuucca gccauaauug agagguuaca cgggcuugac gccuuuucua ugcacacaua 9120

cucucaccac gaacugacgc ggguggcuuc agcccucaga aaacuugggg cgccaccccu 9180

cagggugugg aagagucggg cucgcgcagu cagggcgucc cucaucuccc guggagggaa 9240

agcggccguu ugcggccgau aucucuucaa uugggcggug aagaccaagc ucaaacucac 9300

uccauugccg gaggcgcgcc uacuggacuu auccaguugg uucaccgucg gcgccggcgg 9360

gggcgacauu uuucacagcg ugucgcgcgc ccgaccccgc ucauuacucu ucggccuacu 9420

ccuacuuuuc guagggguag gccucuuccu acuccccgcu cgguagagcg gcacacacua 9480

gguacacucc auagcuaacu guuccuuuuu uuuuuuuuuu uuuuuuuuuu uuuuuuuuuu 9540

uuuuuuuucu uuuuuuuuuu uuucccucuu ucuucccuuc ucaucuuauu cuacuuucuu 9600

ucuugguggc uccaucuuag cccuagucac ggcuagcugu gaaagguccg ugagccgcau 9660

gacugcagag agugccguaa cuggucucuc ugcagaucau gucuaga 9707

<210>10

<211>3748

<212>DNA

<213> hepatitis C Virus

<220>

<223> RNA sequence comprising 5' UTR to NS3 region of TH1 strain

<400>10

gccagccccc gattgggggc gacactccac catagatcac tcccctgtga ggaactactg 60

tcttcacgca gaaagcgtct agccatggcg ttagtatgag tgtcgtgcag cctccaggac 120

cccccctccc gggagagcca tagtggtctg cggaaccggt gagtacaccg gaattgccag 180

gacgaccggg tcctttcttg gatcaacccg ctcaatgcct ggagatttgg gcgtgccccc 240

gcgagactgc tagccgagta gtgttgggtc gcgaaaggcc ttgtggtact gcctgatagg 300

gtgcttgcga gtgccccggg aggtctcgta gaccgtgcac catgagcacg aatcctaaac 360

ctcaaagaaa aaccaaacgt aacaccaacc gccgcccaca ggacgtcaag ttcccgggcg 420

gtggccagat cgttggtgga gtttacctgt tgccgcgcag gggccccagg ttgggtgtgc 480

gcgcgactag gaagacttcc gagcggtcgc aacctcgtgg aaggcgacaa cctatcccca 540

aggatcgccg acccgagggc agggcctggg ctcagcccgg gtacccttgg cccctctatg 600

gcaacgaggg catggggtgg gcaggatggc tcctgtcacc ccgtggctcc cggcctagtt 660

ggggccccaa tgacccccgg cgcaggtcgc gtaatttggg taaagtcatc gataccctta 720

catgcggctt cgccgacctc atggggtaca ttccgctcgt cggcgctccc ttggggggcg 780

ctgccagggc cttggcgcat ggcgtccggg ttctggagga cggcgtgaac tatgcaacag 840

ggaatctgcc cggttgctct ttctctatct tcctcttggc tctgctgtcc tgtctaacca 900

tcccagcttc cgcttatgaa gtgcgcaacg tgtccggggt gtaccatgtc acgaacgact 960

gctccaactc gagcattgtg tacgagacag gggacatgat tatgcacacc cctgggtgcg 1020

tgccctgtgt tcgggagaac aactcctccc gctgctgggc agcgctcact cccacgctcg 1080

cggccaggaa cgccagcgtc cccaccacga caatacggcg ccacgtcgat ttgctcgttg 1140

gggcggctgc tttctgctcc gctatgtacg tgggggatct ctgcggatct gttttcctcg 1200

tctcccagtt gttcaccttc tcgcctcgcc ggcatgagac agtgcaggac tgcaattgtt 1260

caatctatcc cggccacgta tcaggtcacc gcatggcttg ggatatgatg atgaactggt 1320

cacctacaac agccctactg gtatcgcagt tactccggat cccacaagcc gtcgtggaca 1380

tggtggcggg ggcccactgg ggagtcctgg cgggccttgc ctactattcc atggcgggga 1440

actgggctaa ggttttgatt gtgctgctac tctttgccgg cgttgatggg gcgacctacg 1500

tgacgggggg gtcggaagcc agaggggcct ctggcttagc aaacctcttt tcatttgggg 1560

cgtctcagaa gatccagctc ataaatacca acggcagttg gcacatcaat agaactgccc 1620

tgaactgcaa tgactccctc cacactgggt ttcttgccgc gctattctac acacacaaat 1680

tcaacgcgtc cggatgtcca gagcgcatgg ccagctgccg ccccattgaa gagttcgctc 1740

aggggtatgg tcccatcact tatgctgagc cctccccctc ggaccagagg ccctattgct 1800

ggcactacgc gcctcgaccg tgtggtatca tacccgcgtc gcaggtgtgt ggtccagtgt 1860

actgcttcac cccaagccct gttgtggtgg ggacgaccga tcgctccggt gcccccacgt 1920

ataattgggg ggcgaatgag acggacgtgc tgtatctcaa caacacgcgg ccgccgcaag 1980

gcaactggtt cggctgcaca tggatgaatg gcaccgggtt caccaagacg tgcgggggcc 2040

ccccgtgcaa catcgggggg ggcggcaaca acaacacctt gacctgcccc acggactgtt 2100

tccggaaaca ccccgaggcc acctacacca aatgtggttc gggaccttgg ttgacaccta 2160

ggtgcatggt cgactaccca tacaggctct ggcactaccc ctgcaccgtt aactttacca 2220

tctttaaggt taggatgtac gtgggaggtg tggagcacag gctcaacgcc gcatgcaatt 2280

ggacccgagg agagcgttgt aacttagagg acagggatag atcagagctt agcccgctgc 2340

tgctgtcaac aacagagtgg caggtgctac cttgttcctt caccacccta ccggctctgt 2400

ccactggttt gatccatctc caccagaaca tcgtggacgt gcaatacctg tacggtatag 2460

ggtcggcggt tgtctcctat gcaatcaaat gggaatatgt cttgttgctc ttcctcctcc 2520

tggcagacgc gcgcgtctgc gcctgcttgt ggatgatgct gctgatagct caagctgagg 2580

ccgccttaga gaacctggtg gtcctcaatg cggcgtccct ggctggagcg catggccttc 2640

tctctttcct tgtgttcttc tgtgccgctt ggtacatcaa gggcaggttg atccccgggg 2700

cggcgtatgc tttttacggc gtatggccgc tgctcctact cctgctggcg ttaccaccac 2760

gagcatacgc catggaccgg gagatggctg catcgtgcgg aggcgcggtt tttgtaggtc 2820

tggcattcct gaccttgtca ccacactata aggcattcct cgccaagctc atatggtggt 2880

tacaatattt tatcaccaga gccgaggccc atttgcaagt gtggatcccc cccctcaacg 2940

tccggggggg ccgcgatgcc atcatcctcc tcacatgcgc gatccatcca gaccttatct 3000

ttgacatcac caaactcttg ctcgccatgc tcggtccact catggtgctc caggctggca 3060

taactagagt gccgtacttc gtgcgcgctc aagggctcat tcgtgcatgc atgttggtgc 3120

ggaaagtcgc tgggggtcat tatgtccaaa tggccctcat gaagctggcc tcgctgacag 3180

gtacgtacgt ttacgaccat cttactccac tgcgggactg ggcccacggg ggcctacgag 3240

accttgcggt ggcagttgag cccgtcatct tctctgacat ggagaccaaa atcatcactt 3300

ggggagcaga caccgcggcg tgtggggaca tcatctcggg tctgcccgtc tccgcccgaa 3360

gggggaggga gatatttctg ggaccggccg acaagatcag agagcagggg tggcgactcc 3420

ttgcccccat cacggcctat tcccaacaga cgcgaggcct actcggctgc atcatcacta 3480

gcctcacagg ccgggacaag aaccaggtcg agggggaggt tcaagtggtc tctaccgcaa 3540

cgcaatcttt cctggcgacc tgcgtcaacg gcgtgtgttg gactgtctac catggtgccg 3600

gctcgaaaac tctagccggc ccgaagggac caatcaccca aatgtacacc aatgtagacc 3660

aggacctcgt cggctggcag gcgccccccg gggcgcgctc cttaacacca tgcacctgcg 3720

gcagctcgga cctttacttg gtcacgag 3748

<210>11

<211>11102

<212>RNA

<213> Intelligent people

<220>

<223> RNA derived from chimeric hepatitis C Virus gene of HCV JFH1 strain and HCV TH strain (JFH-1 clone)

<400>11

accugccccu aauaggggcg acacuccgcc augaaucacu ccccugugag gaacuacugu 60

cuucacgcag aaagcgccua gccauggcgu uaguaugagu gucguacagc cuccaggccc 120

cccccucccg ggagagccau aguggucugc ggaaccggug aguacaccgg aauugccggg 180

aagacugggu ccuuucuugg auaaacccac ucuaugcccg gccauuuggg cgugcccccg 240

caagacugcu agccgaguag cguuggguug cgaaaggccu ugugguacug ccugauaggg 300

cgcuugcgag ugccccggga ggucucguag accgugcacc augagcacaa auccuaaacc 360

ucaaagaaaa accaaaagaa acaccaaccg ucgcccaaug auugaacaag auggauugca 420

cgcagguucu ccggccgcuu ggguggagag gcuauucggc uaugacuggg cacaacagac 480

aaucggcugc ucugaugccg ccguguuccg gcugucagcg caggggcgcc cgguucuuuu 540

ugucaagacc gaccuguccg gugcccugaa ugaacugcag gacgaggcag cgcggcuauc 600

guggcuggcc acgacgggcg uuccuugcgc agcugugcuc gacguuguca cugaagcggg 660

aagggacugg cugcuauugg gcgaagugcc ggggcaggau cuccugucau cucaccuugc 720

uccugccgag aaaguaucca ucauggcuga ugcaaugcgg cggcugcaua cgcuugaucc 780

ggcuaccugc ccauucgacc accaagcgaa acaucgcauc gagcgagcac guacucggau 840

ggaagccggu cuugucgauc aggaugaucu ggacgaagag caucaggggc ucgcgccagc 900

cgaacuguuc gccaggcuca aggcgcgcau gcccgacggc gaggaucucg ucgugaccca 960

uggcgaugcc ugcuugccga auaucauggu ggaaaauggc cgcuuuucug gauucaucga 1020

cuguggccgg cugggugugg cggaccgcua ucaggacaua gcguuggcua cccgugauau 1080

ugcugaagag cuuggcggcg aaugggcuga ccgcuuccuc gugcuuuacg guaucgccgc 1140

ucccgauucg cagcgcaucg ccuucuaucg ccuucuugac gaguucuucu gaguuuaaac 1200

ccucucccuc cccccccccu aacguuacug gccgaagccg cuuggaauaa ggccggugug 1260

cguuugucua uauguuauuu uccaccauau ugccgucuuu uggcaaugug agggcccgga 1320

aaccuggccc ugucuucuug acgagcauuc cuaggggucu uuccccucuc gccaaaggaa 1380

ugcaaggucu guugaauguc gugaaggaag caguuccucu ggaagcuucu ugaagacaaa 1440

caacgucugu agcgacccuu ugcaggcagc ggaacccccc accuggcgac aggugccucu 1500

gcggccaaaa gccacgugua uaagauacac cugcaaaggc ggcacaaccc cagugccacg 1560

uugugaguug gauaguugug gaaagaguca aauggcucuc cucaagcgua uucaacaagg 1620

ggcugaagga ugcccagaag guaccccauu guaugggauc ugaucugggg ccucggugca 1680

caugcuuuac auguguuuag ucgagguuaa aaaaacgucu aggccccccg aaccacgggg 1740

acgugguuuu ccuuugaaaa acacgaugau accaugagca cgaauccuaa accucaaaga 1800

aaaaccaaac guaacaccaa ccgccgccca caggacguca aguucccggg cgguggccag 1860

aucguuggug gaguuuaccu guugccgcgc aggggcccca gguugggugu gcgcgcgacu 1920

aggaagacuu ccgagcgguc gcaaccucgu ggaaggcgac aaccuauccc caaggaucgc 1980

cgacccgagg gcagggccug ggcucagccc ggguacccuu ggccccucua uggcaacgag 2040

ggcauggggu gggcaggaug gcuccuguca ccccguggcu cccggccuag uuggggcccc 2100

aaugaccccc ggcgcagguc gcguaauuug gguaaaguca ucgauacccu uacaugcggc 2160

uucgccgacc ucauggggua cauuccgcuc gucggcgcuc ccuugggggg cgcugccagg 2220

gccuuggcgc auggcguccg gguucuggag gacggcguga acuaugcaac agggaaucug 2280

cccgguugcu cuuucucuau cuuccucuug gcucugcugu ccugucuaac caucccagcu 2340

uccgcuuaug aagugcgcaa cguguccggg guguaccaug ucacgaacga cugcuccaac 2400

ucgagcauug uguacgagac aggggacaug auuaugcaca ccccugggug cgugcccugu 2460

guucgggaga acaacuccuc ccgcugcugg gcagcgcuca cucccacgcu cgcggccagg 2520

aacgccagcg uccccaccac gacaauacgg cgccacgucg auuugcucgu uggggcggcu 2580

gcuuucugcu ccgcuaugua cgugggggau cucugcggau cuguuuuccu cgucucccag 2640

uuguucaccu ucucgccucg ccggcaugag acagugcagg acugcaauug uucaaucuau 2700

cccggccacg uaucagguca ccgcauggcu ugggauauga ugaugaacug gucaccuaca 2760

acagcccuac ugguaucgca guuacuccgg aucccacaag ccgucgugga caugguggcg 2820

ggggcccacu ggggaguccu ggcgggccuu gccuacuauu ccauggcggg gaacugggcu 2880

aagguuuuga uugugcugcu acucuuugcc ggcguugaug gggcgaccua cgugacgggg 2940

gggucggaag ccagaggggc cucuggcuua gcaaaccucu uuucauuugg ggcgucucag 3000

aagauccagc ucauaaauac caacggcagu uggcacauca auagaacugc ccugaacugc 3060

aaugacuccc uccacacugg guuucuugcc gcgcuauucu acacacacaa auucaacgcg 3120

uccggauguc cagagcgcau ggccagcugc cgccccauug aagaguucgc ucagggguau 3180

ggucccauca cuuaugcuga gcccuccccc ucggaccaga ggcccuauug cuggcacuac 3240

gcgccucgac cgugugguau cauacccgcg ucgcaggugu gugguccagu guacugcuuc 3300

accccaagcc cuguuguggu ggggacgacc gaucgcuccg gugcccccac guauaauugg 3360

ggggcgaaug agacggacgu gcuguaucuc aacaacacgc ggccgccgca aggcaacugg 3420

uucggcugca cauggaugaa uggcaccggg uucaccaaga cgugcggggg ccccccgugc 3480

aacaucgggg ggggcggcaa caacaacacc uugaccugcc ccacggacug uuuccggaaa 3540

caccccgagg ccaccuacac caaauguggu ucgggaccuu gguugacacc uaggugcaug 3600

gucgacuacc cauacaggcu cuggcacuac cccugcaccg uuaacuuuac caucuuuaag 3660

guuaggaugu acgugggagg uguggagcac aggcucaacg ccgcaugcaa uuggacccga 3720

ggagagcguu guaacuuaga ggacagggau agaucagagc uuagcccgcu gcugcuguca 3780

acaacagagu ggcaggugcu accuuguucc uucaccaccc uaccggcucu guccacuggu 3840

uugauccauc uccaccagaa caucguggac gugcaauacc uguacgguau agggucggcg 3900

guugucuccu augcaaucaa augggaauau gucuuguugc ucuuccuccu ccuggcagac 3960

gcgcgcgucu gcgccugcuu guggaugaug cugcugauag cucaagcuga ggccgccuua 4020

gagaaccugg ugguccucaa ugcggcgucc cuggcuggag cgcauggccu ucucucuuuc 4080

cuuguguucu ucugugccgc uugguacauc aagggcaggu ugauccccgg ggcggcguau 4140

gcuuuuuacg gcguauggcc gcugcuccua cuccugcugg cguuaccacc acgagcauac 4200

gccuaugacg caccugugca cggacagaua ggcguggguu uguugauauu gaucacccuc 4260

uucacacuca ccccggggua uaagacccuc cucggccagu gucuguggug guugugcuau 4320

cuccugaccc ugggggaagc caugauucag gaguggguac cacccaugca ggugcgcggc 4380

ggccgcgaug gcaucgcgug ggccgucacu auauucugcc cggguguggu guuugacauu 4440

accaaauggc uuuuggcguu gcuugggccu gcuuaccucu uaagggccgc uuugacacau 4500

gugccguacu ucgucagagc ucacgcucug auaaggguau gcgcuuuggu gaagcagcuc 4560

gcggggggua gguauguuca gguggcgcua uuggcccuug gcagguggac uggcaccuac 4620

aucuaugacc accucacacc uaugucggac ugggccgcua gcggccugcg cgacuuagcg 4680

gucgccgugg aacccaucau cuucaguccg auggagaaga aggucaucgu cuggggagcg 4740

gagacggcug caugugggga cauucuacau ggacuucccg uguccgcccg acucggccag 4800

gagauccucc ucggcccagc ugauggcuac accuccaagg gguggaagcu ccuugcuccc 4860

aucacugcuu augcccagca aacacgaggc cuccugggcg ccauaguggu gaguaugacg 4920

gggcgugaca ggacagaaca ggccggggaa guccaaaucc uguccacagu cucucagucc 4980

uuccucggaa caaccaucuc ggggguuuug uggacuguuu accacggagc uggcaacaag 5040

acucuagccg gcuuacgggg uccggucacg cagauguacu cgagugcuga gggggacuug 5100

guaggcuggc ccagcccccc ugggaccaag ucuuuggagc cgugcaagug uggagccguc 5160

gaccuauauc uggucacgcg gaacgcugau gucaucccgg cucggagacg cggggacaag 5220

cggggagcau ugcucucccc gagacccauu ucgaccuuga agggguccuc gggggggccg 5280

gugcucugcc cuaggggcca cgucguuggg cucuuccgag cagcugugug cucucggggc 5340

guggccaaau ccaucgauuu cauccccguu gagacacucg acguuguuac aaggucuccc 5400

acuuucagug acaacagcac gccaccggcu gugccccaga ccuaucaggu cggguacuug 5460

caugcuccaa cuggcagugg aaagagcacc aaggucccug ucgcguaugc cgcccagggg 5520

uacaaaguac uagugcuuaa ccccucggua gcugccaccc ugggguuugg ggcguaccua 5580

uccaaggcac auggcaucaa ucccaacauu aggacuggag ucaggaccgu gaugaccggg 5640

gaggccauca cguacuccac auauggcaaa uuucucgccg augggggcug cgcuagcggc 5700

gccuaugaca ucaucauaug cgaugaaugc cacgcugugg augcuaccuc cauucucggc 5760

aucggaacgg uccuugauca agcagagaca gccgggguca gacuaacugu gcuggcuacg 5820

gccacacccc ccgggucagu gacaaccccc caucccgaua uagaagaggu aggccucggg 5880

cgggagggug agauccccuu cuaugggagg gcgauucccc uauccugcau caagggaggg 5940

agacaccuga uuuucugcca cucaaagaaa aagugugacg agcucgcggc ggcccuucgg 6000

ggcaugggcu ugaaugccgu ggcauacuau agaggguugg acgucuccau aauaccagcu 6060

cagggagaug ugguggucgu cgccaccgac gcccucauga cgggguacac uggagacuuu 6120

gacuccguga ucgacugcaa uguagcgguc acccaagcug ucgacuucag ccuggacccc 6180

accuucacua uaaccacaca gacuguccca caagacgcug ucucacgcag ucagcgccgc 6240

gggcgcacag guagaggaag acagggcacu uauagguaug uuuccacugg ugaacgagcc 6300

ucaggaaugu uugacagugu agugcuuugu gagugcuacg acgcaggggc ugcgugguac 6360

gaucucacac cagcggagac caccgucagg cuuagagcgu auuucaacac gcccggccua 6420

cccguguguc aagaccaucu ugaauuuugg gaggcaguuu ucaccggccu cacacacaua 6480

gacgcccacu uccucuccca aacaaagcaa gcgggggaga acuucgcgua ccuaguagcc 6540

uaccaagcua cggugugcgc cagagccaag gccccucccc cguccuggga cgccaugugg 6600

aagugccugg cccgacucaa gccuacgcuu gcgggcccca caccucuccu guaccguuug 6660

ggcccuauua ccaaugaggu cacccucaca cacccuggga cgaaguacau cgccacaugc 6720

augcaagcug accuugaggu caugaccagc acgugggucc uagcuggagg aguccuggca 6780

gccgucgccg cauauugccu ggcgacugga ugcguuucca ucaucggccg cuugcacguc 6840

aaccagcgag ucgucguugc gccggauaag gagguccugu augaggcuuu ugaugagaug 6900

gaggaaugcg ccucuagggc ggcucucauc gaagaggggc agcggauagc cgagauguug 6960

aaguccaaga uccaaggcuu gcugcagcag gccucuaagc aggcccagga cauacaaccc 7020

gcuaugcagg cuucauggcc caaaguggaa caauuuuggg ccagacacau guggaacuuc 7080

auuagcggca uccaauaccu cgcaggauug ucaacacugc cagggaaccc cgcgguggcu 7140

uccaugaugg cauucagugc cgcccucacc aguccguugu cgaccaguac caccauccuu 7200

cucaacauca ugggaggcug guuagcgucc cagaucgcac cacccgcggg ggccaccggc 7260

uuugucguca guggccuggu gggggcugcc gugggcagca uaggccuggg uaaggugcug 7320

guggacaucc uggcaggaua uggugcgggc auuucggggg cccucgucgc auucaagauc 7380

augucuggcg agaagcccuc uauggaagau gucaucaauc uacugccugg gauccugucu 7440

ccgggagccc uggugguggg ggucaucugc gcggccauuc ugcgccgcca cgugggaccg 7500

ggggagggcg cgguccaaug gaugaacagg cuuauugccu uugcuuccag aggaaaccac 7560

gucgccccua cucacuacgu gacggagucg gaugcgucgc agcgugugac ccaacuacuu 7620

ggcucucuua cuauaaccag ccuacucaga agacuccaca auuggauaac ugaggacugc 7680

cccaucccau gcuccggauc cuggcuccgc gacguguggg acuggguuug caccaucuug 7740

acagacuuca aaaauuggcu gaccucuaaa uuguucccca agcugcccgg ccuccccuuc 7800

aucucuuguc aaaaggggua caagggugug ugggccggca cuggcaucau gaccacgcgc 7860

ugcccuugcg gcgccaacau cucuggcaau guccgccugg gcucuaugag gaucacaggg 7920

ccuaaaaccu gcaugaacac cuggcagggg accuuuccua ucaauugcua cacggagggc 7980

cagugcgcgc cgaaaccccc cacgaacuac aagaccgcca ucuggagggu ggcggccucg 8040

gaguacgcgg aggugacgca gcaugggucg uacuccuaug uaacaggacu gaccacugac 8100

aaucugaaaa uuccuugcca acuaccuucu ccagaguuuu ucuccugggu ggacggugug 8160

cagauccaua gguuugcacc cacaccaaag ccguuuuucc gggaugaggu cucguucugc 8220

guugggcuua auuccuaugc ugucgggucc cagcuucccu gugaaccuga gcccgacgca 8280

gacguauuga gguccaugcu aacagauccg ccccacauca cggcggagac ugcggcgcgg 8340

cgcuuggcac ggggaucacc uccaucugag gcgagcuccu cagugagcca gcuaucagca 8400

ccgucgcugc gggccaccug caccacccac agcaacaccu augacgugga cauggucgau 8460

gccaaccugc ucauggaggg cgguguggcu cagacagagc cugaguccag ggugcccguu 8520

cuggacuuuc ucgagccaau ggccgaggaa gagagcgacc uugagcccuc aauaccaucg 8580

gagugcaugc uccccaggag cggguuucca cgggccuuac cggcuugggc acggccugac 8640

uacaacccgc cgcucgugga aucguggagg aggccagauu accaaccgcc caccguugcu 8700

gguugugcuc uccccccccc caagaaggcc ccgacgccuc ccccaaggag acgccggaca 8760

gugggucuga gcgagagcac cauaucagaa gcccuccagc aacuggccau caagaccuuu 8820

ggccagcccc ccucgagcgg ugaugcaggc ucguccacgg gggcgggcgc cgccgaaucc 8880

ggcgguccga cguccccugg ugagccggcc cccucagaga cagguuccgc cuccucuaug 8940

cccccccucg agggggagcc uggagauccg gaccuggagu cugaucaggu agagcuucaa 9000

ccuccccccc aggggggggg gguagcuccc gguucgggcu cggggucuug gucuacuugc 9060

uccgaggagg acgauaccac cgugugcugc uccaugucau acuccuggac cggggcucua 9120

auaacucccu guagccccga agaggaaaag uugccaauca acccuuugag uaacucgcug 9180

uugcgauacc auaacaaggu guacuguaca acaucaaaga gcgccucaca gagggcuaaa 9240

aagguaacuu uugacaggac gcaagugcuc gacgcccauu augacucagu cuuaaaggac 9300

aucaagcuag cggcuuccaa ggucagcgca aggcuccuca ccuuggagga ggcgugccag 9360

uugacuccac cccauucugc aagauccaag uauggauucg gggccaagga gguccgcagc 9420

uuguccggga gggccguuaa ccacaucaag uccgugugga aggaccuccu ggaagaccca 9480

caaacaccaa uucccacaac caucauggcc aaaaaugagg uguucugcgu ggaccccgcc 9540

aaggggggua agaaaccagc ucgccucauc guuuacccug accucggcgu ccgggucugc 9600

gagaaaaugg cccucuauga cauuacacaa aagcuuccuc aggcgguaau gggagcuucc 9660

uauggcuucc aguacucccc ugcccaacgg guggaguauc ucuugaaagc augggcggaa 9720

aagaaggacc ccauggguuu uucguaugau acccgaugcu ucgacucaac cgucacugag 9780

agagacauca ggaccgagga guccauauac caggccugcu cccugcccga ggaggcccgc 9840

acugccauac acucgcugac ugagagacuu uacguaggag ggcccauguu caacagcaag 9900

ggucaaaccu gcgguuacag acguugccgc gccagcgggg ugcuaaccac uagcaugggu 9960

aacaccauca caugcuaugu gaaagcccua gcggccugca aggcugcggg gauaguugcg 10020

cccacaaugc ugguaugcgg cgaugaccua guagucaucu cagaaagcca ggggacugag 10080

gaggacgagc ggaaccugag agccuucacg gaggccauga ccagguacuc ugccccuccu 10140

ggugaucccc ccagaccgga auaugaccug gagcuaauaa cauccuguuc cucaaaugug 10200

ucuguggcgu ugggcccgcg gggccgccgc agauacuacc ugaccagaga cccaaccacu 10260

ccacucgccc gggcugccug ggaaacaguu agacacuccc cuaucaauuc auggcuggga 10320

aacaucaucc aguaugcucc aaccauaugg guucgcaugg uccuaaugac acacuucuuc 10380

uccauucuca ugguccaaga cacccuggac cagaaccuca acuuugagau guauggauca 10440

guauacuccg ugaauccuuu ggaccuucca gccauaauug agagguuaca cgggcuugac 10500

gccuuuucua ugcacacaua cucucaccac gaacugacgc ggguggcuuc agcccucaga 10560

aaacuugggg cgccaccccu cagggugugg aagagucggg cucgcgcagu cagggcgucc 10620

cucaucuccc guggagggaa agcggccguu ugcggccgau aucucuucaa uugggcggug 10680

aagaccaagc ucaaacucac uccauugccg gaggcgcgcc uacuggacuu auccaguugg 10740

uucaccgucg gcgccggcgg gggcgacauu uuucacagcg ugucgcgcgc ccgaccccgc 10800

ucauuacucu ucggccuacu ccuacuuuuc guagggguag gccucuuccu acuccccgcu 10860

cgguagagcg gcacacacua gguacacucc auagcuaacu guuccuuuuu uuuuuuuuuu 10920

uuuuuuuuuu uuuuuuuuuu uuuuuuuucu uuuuuuuuuu uuucccucuu ucuucccuuc 10980

ucaucuuauu cuacuuucuu ucuugguggc uccaucuuag cccuagucac ggcuagcugu 11040

gaaagguccg ugagccgcau gacugcagag agugccguaa cuggucucuc ugcagaucau 11100

gu 11102

Claims (23)

1. A modified hepatitis C virus genomic RNA which is composed of two or more hepatitis C virus genomic RNAs and comprises a 5 'untranslated region of a hepatitis C virus strain having a genotype of 1b or 2a, a core protein coding sequence, an E1 protein coding sequence, a p7 protein coding sequence, an E2 protein coding sequence, an NS2 protein coding sequence, a partial RNA sequence coding for the proteins NS3, NS4, NS5A and NS5B of the JFH1 strain shown in SEQ ID NO. 1, and a 3' untranslated region of a hepatitis C virus strain having a genotype of 1b or 2a, and which has an autonomous replication ability.

2. A modified hepatitis C virus genomic RNA which is composed of two or more hepatitis C virus genomic RNAs and which comprises a 5 'untranslated region of a hepatitis C virus strain having a genotype of 1b or 2a, a core protein coding sequence, an E1 protein coding sequence, a p7 protein coding sequence, an E2 protein coding sequence, an NS2 protein coding sequence, an NS3 protein coding sequence, an NS4A protein coding sequence, an NS4B protein coding sequence, an NS5A protein coding sequence, an NS5B protein coding sequence of the JFH1 strain shown in SEQ ID No. 2, and a 3' untranslated region of a hepatitis C virus strain having a genotype of 1b or 2a, and which has an autonomous replication ability.

3. The modified hepatitis c virus genomic RNA as claimed in claim 1 or 2, wherein the virus strain of genotype 1b is selected from the group consisting of HCV-con1 strain and HCV-TH strain.

4. The modified hepatitis C virus genomic RNA as claimed in claim 1 or 2, wherein the virus strain of genotype 2a is selected from the HCV-JFH1 strain and the HCV-JCH1 strain.

5. A hepatocyte-targeted viral vector comprising the modified hepatitis c virus genomic RNA of claim 1 or 2.

6. A cell into which the modified hepatitis C virus genomic RNA according to claim 1 or 2 has been introduced, which is capable of replicating the hepatitis C virus genomic RNA and producing viral particles.

7. A hepatitis C virus particle obtained from a culture by culturing the cell of claim 6.

8. A method for purifying HCV viral particles by subjecting a liquid or cell debris containing the HCV viral particles according to claim 7 to combined column chromatography and/or density gradient centrifugation.

9. The method of claim 8, wherein the column chromatography is one or more selected from the group consisting of ion exchange chromatography, gel filtration chromatography, and affinity chromatography.

10. The method of claim 9, wherein the ion exchange chromatography is one or more chromatography selected from the group consisting of anion exchange chromatography and cation exchange chromatography; the gel filtration chromatography is a chromatography using one or more resins selected from the group consisting of Sephacryl-S300, Sephacryl-S400, and Sephacryl-S500; the affinity chromatography is a chromatography using one or more resins selected from the group consisting of Sulfated cellulofine, heparin, and lectin.

11. The method of claim 9, wherein the chromatography is sulfocelllulofine chromatography.

12. The method of claim 8, wherein the density gradient centrifugation is performed using one or more solutes selected from cesium chloride, sucrose, and sugar polymers.

13. The method of claim 8, wherein in the purification method, anion exchange chromatography, surface cellulose chromatography, and sucrose density gradient centrifugation are used at least once and in any order in combination.

An HCV viral particle obtained by a method for purifying an HCV viral particle by subjecting a liquid or cell debris containing the HCV viral particle according to claim 7 to a combined column chromatography and density gradient centrifugation.

15. The HCV viral particle according to claim 14, which is purified by column chromatography, wherein the column chromatography is one or more selected from the group consisting of ion exchange chromatography, gel filtration chromatography, and affinity chromatography.

16. The HCV viral particle according to claim 14, which is purified by column chromatography, wherein the ion exchange chromatography is one or more selected from the group consisting of anion exchange chromatography and cation exchange chromatography; the gel filtration chromatography is a chromatography using one or more resins selected from the group consisting of Sephacryl-S300, Sephacryl-S400, and Sephacryl-S500; the affinity chromatography is a chromatography using one or more resins selected from the group consisting of sulfated celllulofine, heparin, and lectin.

17. The HCV viral particle according to claim 14, which is purified by column chromatography, wherein said chromatography is a sulfonated cellulose chromatography.

18. The HCV viral particles of claim 14, which are purified by density gradient centrifugation, wherein said density gradient centrifugation is performed using one or more solutes selected from cesium chloride, sucrose, and sugar polymers.

19. The HCV viral particle according to claim 14, wherein said purification method comprises a combination of anion exchange chromatography, sulfonated cellulose chromatography, and sucrose density gradient centrifugation.

20. A hepatitis C virus-infected cell infected with the hepatitis C virus particle according to claim 7.

21. A method for producing a hepatitis C virus-infected cell, comprising culturing the cell according to claim 6, and recovering the virus particle from the culture medium.

22. A method for producing a hepatitis C virus-infected cell, comprising culturing the cell according to claim 6 to allow another cell to be infected with the virus particle in the culture medium.

23. A method for replicating and/or expressing a foreign gene in a cell in vitro, comprising:

inserting RNA encoding a foreign gene into the modified hepatitis C virus genomic RNA of claim 1 or 2, and introducing the modified hepatitis C virus genomic RNA into a target cell to replicate or express the foreign gene.