ES2360808T3 - GENOMIC RNA OF THE HUMAN VIRUS MOFIFIED FROM HEPATITIS C WITH AUTONOMOUS REPLICATIVE CAPACITY. - Google Patents

Abstract

Genomic RNA of the modified hepatitis C virus comprising parts of the genomic RNA of two or more hepatitis C virus strains, which comprises a 5 'untranslated region, a nucleus protein coding sequence, a coding sequence of the E1 protein, a coding sequence of the E2 protein, a coding sequence of the p7 protein, a coding sequence of the NS2 protein, a partial RNA sequence encoding the NS3, NS4, NS5A and NS5B proteins of a strain JFH1 shown in SEQ ID NO: 1 and a 3 'untranslated region, and which can be replicated autonomously, where one of the hepatitis C virus strains is a genotype 1b viral strain.

Description

Technical Field

The present invention relates to: a method for autonomously replicating the human hepatitis C virus (HCV) with various genotypes in a cultured cell system; to the modified HCV genomic RNA used for it; and to cells that replicate the HCV genomic RNA described above.

Background of the Technique

As a result of recent studies, it has become clear that the hepatitis C virus is classified into a large number of types, depending on the genotype or serotype. According to the phyloanalysis method of Simmonds et al., Which uses nucleotide sequences of HCV strains, a method that is currently being used as the main method for the classification of HCV genotypes, HCV is classified into the following 6 types : genotype 1a, genotype 1b, genotype 2a, genotype 2b, genotype 3a and genotype 3b (Non-Patent Document 1). These types are further classified into several subtypes. The nucleotide sequences of the full-length genomes of numerous HCV genotypes have also been determined (Patent Document 1 and Non-Patent Documents 2 to 4).

HCV causes chronic hepatitis as a result of persistent infection. A leading cause of chronic hepatitis, recognized on a global scale, is persistent HCV infection. In fact, approximately 50% of infected patients persistently develop chronic hepatitis and approximately 20% of patients progress to hepatocirrosis from 10 to 20 years. In addition, some of these patients develop fatal pathological conditions, for example liver cancer.

Currently, the main treatments against hepatitis C include the use of interferon-interfer or interferon-comb and the combined use of interferon- with ribavirin, which is a derivative of the purine nucleoside. However, although these treatments are applied in patients, only their therapeutic effects are observed in approximately 60% of such patients. When the treatments are finished after obtaining said therapeutic effects, more than half of the patients develop the recurrent disease. It has been known that the therapeutic effects of interferon depend on the HCV genotype. That is, it is said that the effects of interferon are scarce in genotype 1b and that its effects are high in genotype 2a (Non-Patent Document 5). In addition, the substrate specificity of the HCV protease varies depending on the genotype. The inhibitory activity of an inhibitor developed by the NS3 protease of genotype 1b is 50 times lower or more than that developed by the NS3 proteases of other genotypes (Non-Patent Document 6). Consequently, in order to develop an effective HCV therapeutic agent, it is required to develop the agent while confirming the reactivity of each of the HCV genotypes.

Recently, a HCV subgenomic RNA replicon has been obtained as an HCV-derived RNA that can be replicated autonomously (Patent Documents 2 and 3 and Non-Patent Documents 7 to 9). By this means, it is already possible to analyze the mechanisms of HCV replication using cultured cells. Said HCV subgenomic RNA replicon is obtained by replacing a structural protein present downstream of the HCV IRES, in the 5 'untranslated region of the HCV genomic RNA, with a neomycin resistance gene, and the EMCV- IRES that is linked downstream to it. This RNA replicon was introduced into human Huh7 liver cancer cells and then the cells were cultured in the presence of neomycin. As a result, it was shown that RNA replicon replicated autonomously in Huh7 cells. In addition, it was also demonstrated that several HCV subgenomic RNA replicons replicated autonomously in cells other than Huh7, such as human HeLa cervical cancer cells or human HepG2 liver cancer cells (Patent Document 3).

However, said HCV intracellular RNA replication systems have been obtained for limited genotypes, or rather, such systems have been obtained only by means of genomic RNA from a limited number of HCV strains. Therefore, with respect to HCV with a large number of genotypes, it is extremely difficult to analyze the differences in the therapeutic effects of the therapeutic agents developed against HCV that are caused by differences in the genotypes of the agents mentioned above. Said RNA replicon is an experimental system that only serves to evaluate the replication of the RNA of the virus during the HCV virus growth and replication process. Therefore, it is impossible for said RNA replicon to evaluate processes such as the formation of HCV virus particles in an infected cell, the release thereof outside the cell or the infection of a new cell.

Currently, the application of a method to evaluate processes such as the formation of HCV particles, the release thereof outside the cell and the infection of new cells is limited to an experimental system using animals, for example chimpanzees (Document No -Patent 10). However, this experimental system in which live animal bodies are used directly involves complicated operations and, therefore, it is extremely difficult to carry out analyzes with said experimental system. Consequently, in order to analyze processes such as the formation of HCV particles, the release thereof outside the cell and the infection of new cells, or in order to develop an anti-HCV agent that uses the inhibition of These processes as a mechanism of action, it is necessary to develop an extremely simplified experimental system capable of replicating these processes; namely, a HCV virus particle replication system that uses a cultured cell system.

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If it were possible to stably deliver HCV virus particles from said cultured cell system, the virus could be attenuated, or a non-infectious HCV virus could be produced by means based on molecular biology, thus using such viruses as vaccines. However, because the HCV protein sequences are different depending on the genotype, the antigenicity of HCV is also different depending on the genotype. In fact, the presence of several genotypes constitutes a significant impediment to the production of HCV vaccines (Non-Patent Document 11). Therefore, in order to effectively produce HCV vaccines it is also desired that HCV virus particles with various genotypes be produced stably in a cultured cell system.

It is known that HCV is a spherical particle with a size between 55 and 65 nm that exists in the blood of a patient infected with HVC. As a method 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, by these methods, only less than 1 ml of virus can be purified at a concentration of approximately 1M copies / ml. Therefore, these methods are not industrially applicable.

Various methods for purifying viral particles other than HCV have been designed to date (Patent Documents 4, 5, and 6, for example). However, as is clear from these publications, the viral particles have different properties and, therefore, the particles do not give useful information on what would be an optimal method to purify the human hepatitis C virus. The Patent Document 7 describes that the human hepatitis A virus, which is also a hepatitis virus, can be purified by removing DNA according to ion exchange chromatography. However, although the hepatitis A virus is also a hepatitis virus, it is a virus that has DNA as a gene. As is clear from the fact that the hepatitis C virus has RNA as a gene, there are no relevant similarities between the hepatitis A virus and the hepatitis C virus and, therefore, no information is given regarding to relevant purification methods. In order to use human hepatitis C viral particles as vaccines or the like in the industrial field in the future, it is required to purify such particles at a high level in a large volume. In these circumstances, the development of a purification method is anticipated.

Patent Document 1: Patent Publication JP (Kokai) No. 2002-171978 A

Patent Document 2: Patent Publication JP (Kokai) No. 2001-17187 A

Patent Document 3: WO2004 / 104198A1

Patent Document 4: Japanese Patent No. 3313117

Patent Document 5: JP Patent Publication (Kohyo) No. 2002-503484 A

Patent Document 6: Patent Publication JP (Kohyo) No. 2000-510682 A

Patent Document 7: Patent Publication JP (Kokoku) No. 6-48980 B (1994)

Non-Patent Document 1: Simmonds P. et al., Hepatology, 10 (1994) pp. 1321-1324

Non-Patent Document 2: Choo Q. L. et al., Science, 244 (1989) pp. 359-362

Non-Patent Document 3: Okamoto H. et al., J. Gen. Virol., 73 (1992) pp. 673-679

Non-Patent Document 4: Mori S. et al., Biochem. Biophis Res. Commun. 183 (1992) pp. 334-342

Non-Patent Document 5: Yoshioka K. et al., Hepatology, 16 (1992) pp. 293-299

Non-Patent Document 6: Thibeault D. et al., J. Virol., 78 (2004) pp. 7352-7359

Non-Patent Document 7: Blight et al., Science, 290 (2000) pp. 1972-1974

Non-Patent Document 8: Friebe et al., J. Virol., 75 (2001) pp. 12047-12057

Non-Patent Document 9: Kato T. et al., Gastroenterology, 125 (2003) pp. 1808-1817 Non-Patent Document 10: Kolykhalov et al., Science, 277 (1997) pp. 570-574

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Non-Patent Document 11: Farci P. et al., Semin Liver Dis 20 (2000) pp. 103-126

Non-Patent Document 12: Virology, 196 (1993) pp. 354-357

Non-Patent Document 13: Journal of General Virology 86 (2005) pp. 677-685

Description of the Invention

It is an object of the present invention to provide a method for replicating and amplifying hepatitis C virus with various genotypes in a cultured cell system.

As a result of intensive studies aimed at achieving the aforementioned object, the present inventors have produced genomic RNA from the modified hepatitis C virus by combining genomic RNA from a JFH1 strain of HCV, which can be replicated autonomously with the Genomic RNA from an HCV strain, which cannot be replicated autonomously in vitro. The inventors have discovered that the genomic RNA thus produced can be replicated autonomously in a cultured cell system. Specifically, with respect to the invention mentioned above, the present inventors have discovered that the introduction of a genomic portion comprised between the coding sequence of the NS3 protein of strain JFH1 and the 3 'terminal thereof allows the modification of the genomic RNA of the HCV 1b, which cannot replicate autonomously in vitro, to result in an RNA that can replicate autonomously in a cultured cell system.

That is, the present invention relates to genomic RNA of the modified hepatitis C virus comprising nucleotide sequences of portions of genomic RNA from two or more types of hepatitis C virus, comprising a 5 'untranslated region, a coding sequence of the core protein, a coding sequence of the E1 protein, a coding sequence of the E2 protein, a coding sequence of the p7 protein, a coding sequence of the NS2 protein, coding sequences of the NS3, NS4A, NS4B, NS5A proteins and NS5B of a JFH1 strain and a 3 'untranslated region, and which can be replicated autonomously, where one of the hepatitis C virus strains is a genotype 1b virus.

Specifically, in one embodiment, the present invention provides the genomic RNA of the modified hepatitis C virus, which is produced by replacing a portion of the genomic RNA of hepatitis C virus 1b comprised between a coding sequence of the NS3 protein and an NS5B protein coding sequence, which is a 3 'terminal genomic sequence, with a partial RNA sequence encoding the NS3, NS4, NS5A and NS5B proteins of a JFH1 strain shown in SEQ ID NO: 1 ( RNA sequence obtained by replacing T with U in a sequence corresponding to 3867-9678 of the DNA sequence deposited under GenBank Accession No. AB047639), and which can be replicated autonomously.

In another embodiment, the present invention provides genomic RNA of the modified hepatitis C virus, which is produced by the substitution of the coding sequence of the NS5B protein of the genomic RNA of the hepatitis C virus by the coding sequence of the NS5B protein of strain JFH1 shown in SEQ ID NO: 2 and which can be replicated autonomously, where one of the hepatitis C virus strains is a genotype 1b virus.

Strains of the two or more types of hepatitis C virus used here include a hepatitis C virus with genotype 1b and a hepatitis C virus with genotype 2a. Examples of viral strains with genotype 1b may include a strain with HCV1, a strain HCV TH, a strain J HCV, a strain JT HCV and a strain BK HCV. The viral strain with genotype 2a is a JFH1 strain of HCV.

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

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

By way of example of genomic RNA of the modified hepatitis C virus mentioned above, the present specification describes a genomic RNA of the modified hepatitis C virus, comprising:

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(to)

an RNA with the nucleotide sequence shown in SEQ ID NO: 11; or

(b)

an RNA with a nucleotide sequence comprising a deletion, substitution or addition of one or more, preferably 100, in particular 50 and especially 10 nucleotides, with respect to the nucleotide sequence shown in SEQ ID NO: 11 and which can be Autonomously replicate and generate viral hepatitis C particles.

In addition, the present invention also provides a cell into which the genomic RNA of the modified hepatitis C virus of the present invention is introduced and which replicates the genomic RNA of the hepatitis C virus described above and can generate viral particles. Here, a proliferative cell is preferably used as the host cell. Particularly preferred examples of such host cells may include eukaryotic cells, including cells from the human liver such as Huh7 cells, HepG2 cells, IMY-N9 cells, HeLa cells or 293 cells, human cervical cells and cells from the human fetal kidney.

Furthermore, the present invention also provides: a method for producing viral hepatitis C particles characterized in that it comprises the cell culture mentioned above and the recovery of the viral particles from the culture; and viral hepatitis C particles produced by the previous method.

In addition, the present invention also provides: a method for producing a cell infected by the hepatitis C virus that is characterized in that the method comprises the culture of the cell mentioned above and the infection of another cell by viral particles contained in the culture; and a cell infected by the hepatitis C virus produced by the previous method. In the present invention, said HCV particles are purified by column chromatography and / or density gradient centrifugation, to obtain HCV particles with a purity that allows their industrial use in pharmaceutical products. The chromatography used here consists of one or more types of chromatography selected from ion exchange chromatography, gel filtration chromatography and affinity chromatography. Density gradient centrifugation is carried out with one or more solutes selected from cesium chloride, sucrose and sugar polymers, to purify HCV.

Additionally, the present invention also provides a method for screening ("screening") an anti-hepatitis C viral substance using the cell of the present invention or a cell infected with the hepatitis C virus. This method is characterized in that it comprises the culture. of the cell of the present invention, or a cell infected by the hepatitis C virus, in the presence of a test substance and the detection of viral hepatitis C virus or viral particles in the culture, thus assessing the effects of anti-hepatitis C virus in the test substance described above.

Also, the present invention also provides a method for producing a hepatitis C vaccine by the viral hepatitis C particles of the present invention, or a portion thereof, as antigens.

In addition, the present invention also provides: a method for replicating and / or expressing a foreign gene in a cell, characterized in that the method comprises the insertion of the RNA encoding the foreign gene into the genomic RNA of the modified hepatitis C virus. of the present invention and the introduction of genomic RNA into a cell of interest, to replicate or express the foreign gene within; and a viral vector directed to the liver cell, which comprises the genomic RNA of the modified hepatitis C virus of the present invention.

In accordance with the present invention, infectious HCV viral particles can be produced using a cultured cell system. In addition, even in the case of an HCV strain that cannot be replicated autonomously and that is isolated from patients, a region thereof corresponding to the region from the NS3 region to the 3 'terminal is replaced by genomic RNA from the virus. of JFH1, or the NS5B region is replaced by NS5B of JFH1, so that the above HCV strain can be autonomously replicated in vitro. Consequently, HCV viral particles with various genotypes can be produced in a cultured cell system and these viral particles are effectively used for studies related to the HCV infection process or for the production of a screening system for various substances that affect to said HCV infection process and a vaccine against HCV.

Brief Description of the Figures

Figure 1: Schematic view showing the procedures for constructing the DNA template used to produce the HCV genomic RNA of the present invention. The figure shows the structure of a plasmid clone pJFH1 obtained by inserting the full length HCV genome downstream of a T7 promoter. The symbols shown in the figure have the following meanings: T7: T7 RNA promoter; 5’-UTR: untranslated region 5 ’; C: core protein; E1, E2: envelope proteins; NS2, NS3, NS4A NS4B, NS5A, NA5B: non-structural proteins; 3’-UTR: 3 ’untranslated region; Agel, Pmel, XbaI: cleavage sites of the restriction enzymes of Agel, Pmel and Xbal; and GDD: the position of a GDD motif of amino acids that corresponds to the active center of an NS5B protein.

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Figure 2: photograph showing the results of Northern blot analysis indicating the replication of rJFH1 in Huh7 cells in which rJFH1 has been introduced, that is, HCV genomic RNA.

Figure 3: Results of the detection of a HCV core protein, an NS3 protein, an NS5A protein and an E2 protein, in a medium.

Figure 4: Results of the temporal course of changes in the release of a core protein from the cells into which HCV genomic RNA has been introduced, in a medium.

Figure 5: Includes graphs, each showing the amount of HCV core protein and the amount of HCV genomic RNA in each fraction obtained by sucrose density gradient fractionation of the Huh7 cell culture supernatant into which rJFH1 has been introduced . The filled circle represents a HCV nucleus protein and the empty circle represents HCV genomic RNA. Figure 5A shows the results for untreated Huh7 cells with rJFH1 introduced. Figure 5B shows the results of Huh7 cells with rJFH1 introduced treated with RNAse. Figure 5C shows the results of Huh7 cells with introduced rJFH1 treated with NP40. Figure 5D shows the results of Huh7 cells with introduced rJFH1 treated with NP40 + RNAse.

Figure 6: shows the infectious capacity of the secreted viral particles of the Huh7 cell culture solution with rJFH1 introduced. Figure 6A includes photographs showing the results of an immunostaining with an anti-core antibody (left) and with an anti-NS5A antibody (right). Figure 6B is a graph showing the number of positive cells stained with an anti-core antibody. Figure 6C includes graphs showing the change over time of the level of HCV RNA in the cells (left) and in the supernatant (right).

Figure 7: shows the capacity of infection of the secreted viral particles of the culture solution of Huh7 cells with rJCH1 / NS5B (jfh 1) introduced. Figure 7A is a graph showing the increase in HCV RNA of the viral particles secreted in the culture solution of Huh7 cells with rJCH1 / NS5B (jfh 1) introduced, in native Huh7 cells. Figure 7B is a graph showing the number of positive cells stained with an anti-core antibody.

Figure 8: shows the structure of a chimeric TH / JFH replicon.

Figure 9: shows the results concerning colony formation by transfection of rTH / JFH1 chimeric replicon RNA.

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

Figure 11: shows the elution profiles of gel filtration chromatography. The vertical axis represents the absorbance at a wavelength of 490 nm. S-300, S-400 and S-500 represent Sephacryl (R) S-300, S-400 and S-500 respectively. The horizontal axis represents the amount of elution eluted from the column.

Figure 12: shows the elution profiles of ion exchange chromatography. The vertical axis represents the amount of core protein in HCV particles.

Figure 13: shows the elution profiles of lectin affinity chromatography. The vertical axis represents the amount of core protein in HCV particles.

Figure 14: shows elution profiles in two types of affinity chromatography using heparin and sulfated cellulophin. The vertical axis represents the absorbance at a wavelength of 490 nm.

Figure 15: shows the elution profile of affinity chromatography with blue staining. The vertical axis represents the amount of core protein in HCV particles.

Figure 16: shows the purification profiles that involve the combined use of column chromatography with gradient centrifugation of sucrose density. The vertical axis represents the amount of core protein in HCV particles. With respect to sucrose density gradient centrifugation, the density of each solution fraction as well as the amount of core protein in HCV are shown on the vertical axis.

This specification includes the content as described in the specification and / or figures of Japanese Patent Applications No. 2004-243975, 2004-290801, 2005-69527 and 2005-69725, which are priority documents of the present application.

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Invention Embodiment

The present invention is described in detail below.

1. Chimeric modified viral genomic hepatitis C hepatitis

The genome of a hepatitis C virus (HCV) is a single-stranded RNA, that is strand (+), which is made up of approximately 9,600 nucleotides. This genomic RNA comprises a 5 'untranslated region (also called 5'-NTR or 5'-UTR), a translated region composed of a structural region and a non-structural region and a 3' untranslated region (also called 3'- NTR or 3'UTR). The structural region encodes HCV structural proteins and the non-structural region encodes various non-structural proteins.

Said HCV structural proteins (nucleus, E1 and E2) and non-structural HCV proteins (NS2, NS3, NS4A, NS4B, NS5A and NS5B) are translated as a continuous polyprotein from the translated region. The polyprotein is then subjected to limited digestion with protease so that the proteins can be released and generated. Among these structural and non-structural proteins (namely HCV virus proteins), the nucleus is a core protein and E1 and E2 are envelope proteins. The non-structural protein is a protein associated with the replication of a virus per se. It is known that NS2 has metalloprotease activity and that NS3 has serine protease activity (one third of the N-terminal side) as well as helicase activity (two thirds of the C-terminal side). In addition, it has also been reported that NS4A is a cofactor of NS3 protease activity and that NS5B has RNA-dependent RNA polymerase activity.

Currently, it is known that HCV genotypes are classified at least in type 1 to type

6. HCV is classified into several genotypes (HCV1a, HCV1b, HCV2a, HCV2b, etc.) depending on its sequence, according to the international classification of Simmonds et al. (see Simmonds P. et al., Hepatology, (1994) 10, pp. 1321-1324). In the present invention, HCV genomic RNA that cannot replicate autonomously is not limited to the types of known viruses mentioned above, but includes all types of HCV genomic RNA that cannot replicate autonomously, that is, with the ability to release infectious particles out of the cell. In the present invention, the expression "RNA that can be replicated autonomously" or "that replicates autonomously" is intended to mean that when HCV genomic RNA is introduced into a cell, HCV genomic RNA replicates autonomously , that is, it can release infectious particles out of the cell.

In the present specification, the RNA that includes the above-mentioned HCV genomic RNA that can be autonomously replicated in a cultured cell system is called "replicon RNA" or "RNA replicon." In the present specification, the replicon RNA of the present invention, which comprises the full length 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 generate viral particles. In addition, the genomic RNA of the modified hepatitis C virus of the present invention is full length HCV replicon RNA.

The genomic RNA of the modified hepatitis C virus of the present invention includes a genomic modified hepatitis C RNA that has the nucleotide sequences of genomic RNA portions of two or more types of hepatitis C viruses, comprising a non-region translated 5 ', a coding sequence of the core protein, a coding sequence of the E1 protein, a coding sequence of the E2 protein, a coding sequence of the p7 protein, a coding sequence of the NS2 protein, the coding sequence of the each protein of NS3, NS4A, NS4B, NS5A and NS5B of a JFH1 strain and a 3 'untranslated region, and can be replicated autonomously. Specifically, in one embodiment, the present invention includes genomic hepatitis of the modified hepatitis C virus that is produced by replacing a portion of the genomic RNA of the hepatitis C virus comprising from the coding sequence of the NS3 protein to the sequence coding for the NS5B protein, that is a genome sequence at the 3 'terminal, by a partial RNA sequence encoding the NS3, NS4, NS5A and NS5B proteins of strain JFH1 shown in SEQ ID NO: 1 (sequence of RNA obtained by substituting T for U in a sequence corresponding to 3867-9678 of the DNA sequence deposited under GenBank Accession No. AB047639), and which can be replicated autonomously.

In another embodiment, the present invention provides genomic RNA of the modified hepatitis C virus produced by replacing the coding sequence of the NS5B protein of the genomic RNA of the hepatitis C virus with the coding sequence of the NS5B protein of strain JFH1 shown in SEQ ID NO: 2, and that can be replicated autonomously.

Preferably, the present invention includes genomic RNA of the modified hepatitis C virus obtained by hepatitis C virus with genotypes 1b and 2a, having a nucleotide sequence comprising a 5 'untranslated region, a nucleus protein coding sequence , a coding sequence of the E1 protein, a coding sequence of the E2 protein, a coding sequence of the p7 protein, a coding sequence of the NS2 protein, the coding sequence of the each protein of NS3, NS4A, NS4B, NS5A and NS5B of strain JFH1 and a 3 'untranslated region, and which can be replicated autonomously.

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The genomic RNA of the modified hepatitis C virus described above may further comprise at least one selective marker gene and / or at least one reporter gene, and at least one IRES sequence.

In the present invention, using an HCV strain that can be replicated autonomously in a cultured cell system with the combination of an HCV strain that cannot replicate autonomously in said cultured cell system, due to two or More types of hepatitis C virus, the strain of HCV that cannot be replicated autonomously, can be modified to become replicated autonomously. Otherwise, a viral strain that replicates effectively autonomously can be modified to become replicated autonomously very effectively.

Specific examples of known strains of HCV type 1a may include a strain HCV-1, a strain HCV-H and a strain HCV-J1. Specific examples of known strains of HCV type 1b may include a strain HCV-con1, a strain HCV-TH, a strain HCV-J, a strain HCV-JT and a strain HCV-BK. Specific examples of known strains of HCV type 2a may include a strain HCV-J6, a strain JFH-1 and a strain JCH1. An example of a known strain of HCV type 2b may be an HC-J8 strain. An example of a known strain of HCV type 3a may be an E-b1 strain. The structure of these viruses is basically composed of 5’-UTR, core, E1, E2, p7, NS2, NS3, NS4a, NS4b, NS5a, NS5b and 3’-UTR (as described above). The nucleotide sequence of each region of each HCV strain mentioned above has been determined. For example, the nucleotide sequences of the regions corresponding to the nucleus, E1, E2, p7 and NS2 in the full length sequence of strain TH have been determined. In addition, in the sequence of the strain HCV-JT, the regions corresponding to the nucleus, E1, E2, p7 and NS2 have been determined. An example of the replicon RNA of the present invention may be chimeric HCV replicon RNA, which is obtained by a JFH1 strain of HCV type 2a and other strains other than the JFH1 strain of HCV type 2a, such as an HCV strain , an HCV-H strain, an HCV-J1 strain, an HCV-con1 strain, a HCV-TH strain (Wakita et al., J. Biol. Chem. (1994), 269, pp. 14205-14210; and Moradpour et al., Biochem. Biophys. Res. Commun., (1998) 246, pp. 920-924), an HCV-J strain, an HCV-JT strain, an HCV-BK strain, a HCV-J6 strain, a strain JCH1, strain HCJ8 or strain E-b1.

In addition, a preferred example of the modified HCV genomic RNA of the present invention may be a HCV genomic RNA obtained by substituting a region corresponding to the region from the NS3 region to the 3'-terminal side of the HCV genomic RNA. of the JFH1 strain of hepatitis C virus by JFH 1 viral genomic RNA, or by substitution of the NS5B protein coding sequence with the NS5B protein coding sequence of another HCV genomic RNA or by insertion here of the previous sequence. For example, in the case of JCH1 (ref) of the HCV genomic RNA, which is known to not be able to replicate in vitro, a region corresponding to the region from the NS3 region thereof to the 3'-terminal side it is replaced by JFH1 viral genomic RNA so that the HCV genomic RNA can be modified to result in an HCV genomic RNA that can replicate autonomously.

In addition, in the case of clone Con-1 (ref) of the HCV genomic RNA with HCV genotype 1b (Accession No. to EMBL AJ238799), a portion of the RNA sequence thereof encoding proteins NS3, NS4, NS5A and NS5B is replaced by the RNA sequence of a JFH1 strain that encodes the NS3, NS4, NS5A and NS5B proteins, or only the portion of the RNA sequence that encodes the NS5B protein of the Con-1 clone (ref) with HCV genotype 1b it is replaced by the RNA sequence encoding the NS5B protein of strain JFH1, so that the HCV genomic RNA can be modified to result in a HCV genomic RNA that can be replicated autonomously.

The full length replicon that uses a Con-1 clone gene can be replicated autonomously, but does not form HCV particles (see Pietschmann et al., Journal of Virology, (2002) 76, pp. 4008-4021). However, as described in the example of the present invention, said HCV particles can be formed by replacing a portion of the RNA sequence encoding the NS3, NS4, NS5A and NS5B proteins with the RNA sequence that encodes the NS3, NS4, NS5A and NS5B proteins of strain JFH1. That is, according to the method of the present invention, the genomic hepatitis C virus RNA that can replicate autonomously but that is unable to form HCV particles can be converted into genomic RNA from the modified hepatitis C virus capable of form particles

In addition, even in the case of an HCV that is unable to produce a replicon that can replicate autonomously, such as a TH strain or a JCH strain, HCV particles are formed by producing a chimeric gene thereof with the strain JFH-1, as described in the example of the present invention. Accordingly, the present invention allows the conversion of genomic HCV RNA that cannot autonomously replicate into a genomic hepatitis of the modified hepatitis C virus that can form HCV particles.

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In addition, by introducing a mutation in NS5B of the RNA sequence portion of strain JFH1, the growth of the HCV genomic RNA ends and so does the generation of HCV particles. Therefore, apparently, NS5B plays an important role in allowing HCV genomic RNA to replicate autonomously and generate particles.

Currently, HCV is classified into several genotypes (HCV1a, HCV1b, HCV2a, HCV2b, etc.) depending on its sequence, according to the international classification of Simmonds et al., (See Simmonds P. et al., Hepatology, ( 1994) 10, pp. 1321-1324). In the present invention, HCV genomic RNA that cannot be replicated autonomously is not limited to the types of known viruses mentioned above, but includes all types of HCV genomic RNA that cannot replicate autonomously.

In the present invention, the NS5B protein coding sequence is an NS5B protein coding sequence derived from strain JFH1 (SEQ ID NO: 3), but it has the nucleotide sequence shown in SEQ ID NO: 2. However, the NS5B protein coding sequence of the present invention also includes nucleotide sequences that can hybridize with the nucleotide sequence shown in SEQ ID NO: 2 under astringent conditions, provided said nucleotide sequences encode the amino acids that function. as an NS5B protein (for example, an NS5B protein comprising a conservative substitution).

The term "astringent conditions" is intended to indicate, for example, conditions consisting of a concentration of sodium between 300 and 2,000 mM and a temperature between 40 ° C and 75 ° C, preferably a concentration of sodium between 600 and 900 mM and a temperature of 65 ° C. Those skilled in the art can easily obtain the aforementioned NS5B homologue, with reference to molecular cloning (Sambrook J. et al., Molecular Cloning: A Laboratory Manual 2nd ed., Cold Spring Harbor Laboratory Press, 10 Skyline Drive Plainview, NY (1989)).

The HCV genomic RNA of the present invention has an RNA sequence portion that encodes the NS3, NS4, NS5A and NS5B proteins in the HCV genomic RNA with JFH1 or an NS5B protein coding sequence.

In one embodiment, the HCV genomic RNA of the present invention is an RNA having a nucleotide sequence that includes a 5 'untranslated region, a nucleus protein coding sequence, an E1 protein coding sequence, a sequence. encoding the E2 protein, a coding sequence for the NS2 protein, a coding sequence for the NS3 protein, a coding sequence for the NS4A protein, a coding sequence for the NS4B protein, a coding sequence for the NS5A protein, a coding sequence for the NS5B protein and a 3 'untranslated region in the genomic RNA of the hepatitis C viral strain. In addition, in the above RNA, the aforementioned RNA sequence portion encoding the NS3, NS4, NS5A and NS5B proteins is a portion of the RNA sequence that encodes the NS3, NS4, NS5A and NS5B proteins that comes from the HCV genomic RNA with JFH1 introduced strangely. Preferably, it is RNA in which the coding sequence of the NS5B protein thereof is a coding sequence of the NS5B protein derived from HCV genomic RNA with JFH1 introduced strangely.

In the specification of the present invention, "5 'untranslated region (5'-NTR or 5'-UTR)", "nucleus protein coding sequence (nucleus region or C region)", "coding sequence of the E1 protein (E1 region), "E2 protein coding sequence (E2 region)", "NS2 protein coding sequence (NS2 region)", "NS3 protein coding sequence (NS3 region)", "coding sequence of the NS4A protein (NS4A region) ”,“ NS4B protein coding sequence (NS4B region) ”,“ NS5A protein coding sequence (NS5A region) ”,“ NS5B protein coding sequence (NS5B region) ”,“ 3 '(3'-NTR or 3'-UTR) ”untranslated region and other regions or specific sites are already known in several genotypes. The aforementioned regions or sites of an unknown HCV strain can be readily determined by aligning the full length genomic RNA sequence of a known HCV with that of the previous HCV strain.

The term "selective marker gene" is used in the present invention in reference to a gene that can confer on selectivity cells to select only those cells in which the gene has been expressed. A common example of such a selective marker gene may be an antibiotic resistance gene. Examples of such a selective marker gene that can preferably be used in the present invention may include a neomycin resistance gene, a thymidine kinase gene, a kanamycin resistance gene, a pyriamine resistance gene, a adenylyl transferase gene, a zeocin resistance gene and a puromycin resistance gene. Among them, the neomycin resistance gene and the thymidine kinase gene are preferred, a neomycin resistant gene being especially preferred. However, the selective marker genes used in the present invention are not limited thereto.

The term "gene reporter" is used in the present invention in reference to a marker gene that encodes a genetic product that acts as an indicator of gene expression. A common example of such a reporter gene may be a structural gene of an enzyme that catalyzes a light reaction or a staining reaction. Examples of reporter genes that can preferably be used in the present invention may include a chloramphenicol acetyltransferase gene derived from the transposon Tn9, a -glucuronidase or -galactosidase gene derived from Escherichia coli, a luciferase gene, a Green fluorescent protein gene, a jellyfish-derived aequorin gene and a secreted form of the human placental alkaline phosphatase gene (SEAP). However, the reporter genes used in the present invention are not limited thereto.

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One of the selective marker and reporter genes mentioned above may be included in the replicon RNA, or both may also be included therein. With respect to said selective marker gene or reporter gene, the gene may be included in the genomic RNA of the modified hepatitis C virus or two or more genes may also be contained within it.

The HCV genomic RNA of the present invention may further comprise an RNA encoding any foreign gene that must be expressed in the cells into which the full length HCV genomic RNA is introduced. This RNA encoding a foreign gene may be linked downstream of the untranslated region 5 ’or may be linked upstream of the untranslated region 3’. Likewise, said RNA can be inserted in any space between a coding sequence of the core protein, a coding sequence of the E1 protein, a coding sequence of the E2 protein, a coding sequence of the NS2 protein, a coding sequence of the NS3 protein, a coding sequence of the NS4A protein, a coding sequence of the NS4B protein, a coding sequence of the NS5A protein and a coding sequence of the NS5B protein.

When the HCV genomic RNA comprising the RNA encoding a foreign gene is translated into the cells into which the RNA has been introduced, this allows a genetic product encoded by the foreign gene to be expressed. Accordingly, said HCV genomic RNA comprising an RNA encoding a foreign gene can preferably also be used for the purpose of generating the genetic product of the foreign gene in cells.

In the HCV genomic RNA of the present invention, the coding sequences of the viral proteins mentioned above, the foreign gene and others are linked together so that they can be translated from the HCV genomic RNA using a correct reading frame. The proteins encoded by the HCV genomic RNA are preferentially linked to each other through cleavage sites for protease or the like, so that the proteins are translated in the form of a continuous polypeptide and allowed to be expressed, and thereby that the polypeptide is then segmented with the protease within each protein and then released.

The HCV genomic RNA thus produced, which comprises a part of the RNA sequence encoding the NS3, NS4, NS5A and NS5B proteins of strain JFH1, is introduced into suitable host cells to obtain recombinant cells that can autonomously replicate. HCV genomic RNA, and preferably they can persistently replicate autonomously HCV genomic RNA (ie they can replicate HCV genomic RNA). In the following specification, said recombinant cells that can replicate the HCV genomic RNA comprising a part of the RNA sequence encoding the NS3, NS4, NS5A and NS5B proteins of strain JFH1 are called "replicating cells of the HCV genomic RNA. "

The type of host cells used for such "HCV genomic RNA replicating cells" is not particularly limited, provided they can be subcultured. Eukaryotic cells are preferred. Especially preferred are human cells and in particular human cells derived from the liver, human cervical cells and human cells derived from the fetal kidney. In addition, proliferative cells that include cancer cell strains or stem cell strains are preferred. Among others, Huh7 cells, HepG2 cells, IMY-N9 cells, HeLa cells, 293 cells and the like are particularly preferred. Commercial cells can be used as such cells or said cells can be obtained from depository institutions of cells. Otherwise, cells constituted from any cell (cancer cells or stem cells, for example) can be used.

HCV genomic RNA can be introduced into host cells by any known technique. Examples of such introduction methods may include electroporation, particle bombardment, lipofection, calcium phosphate, microinjection and DEAE-sepharose. Among them, a method involving electroporation is particularly preferred.

Genomic HCV RNA can be introduced separately or mixed with another nucleic acid and then introduced. In order to change the amount of HCV genomic RNA introduced while keeping the amount of RNA introduced constant, a certain amount of HCV genomic RNA can be mixed with total cellular RNA extracted from the cells into which genomic RNA should be introduced HCV, to prepare a certain total amount of RNA, the total amount of RNA can be introduced into cells. The amount of HCV genomic RNA introduced into the cells can be determined depending on the introduction method used. The amount of said HCV genomic RNA introduced preferably ranges from 1 picogram to 100 micrograms, in particular between 10 picograms and 10 micrograms.

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Replication of HCV genomic RNA in "HCV genomic RNA replicating cells" can be confirmed by any known RNA detection method. For example, the total RNA extracted from the cells is subjected to the Northern hybridization method by means of a specific DNA fragment of the HCV genomic RNA introduced as a probe, or to the RT-PCR method with specific primers of the HCV genomic RNA introduced.

In addition, when an HCV protein is detected in the proteins extracted from "HCV genomic RNA replicating cells", it can be determined which cells replicate the HCV genomic RNA. Said HCV protein can be detected by any known method to detect the protein. For example, said HCV protein can be detected by allowing an antibody to react with an HCV protein that must be expressed from the genomic HCV RNA introduced, so that it reacts with a protein extracted from the cells. More specifically, a sample of protein extracted from the cells is subjected to blotting on a nitrocellulose membrane, then an anti-HCV protein antibody (for example, an anti-NS3 specific antibody or a antiserum extracted from a patient with hepatitis C) react with it, and then, for example, the anti-HCV protein antibody is detected.

The fact that HCV genomic RNA can be replicated autonomously can be confirmed, for example, by transfecting Huh7 cells with RNA as a target, culturing Huh7 cells and subjecting the RNA extracted from the cells of the culture obtained to Northern Blot hybridization with a probe capable of specifically detecting the introduced RNA, but said confirmation method is not limited to this. Specific operations to confirm that the RNA can be replicated autonomously are found in descriptions concerning the confirmation of HCV protein expression or the detection of HCV genomic RNA in the example of the present specification.

2. HCV particle production

Genomic HCV RNA replicating cells produced as described above are capable of generating viral HCV particles in vitro. That is, the HCV genomic RNA replicating cells of the present invention are cultured in a suitable medium and the generated viral particles are then harvested from the culture (preferably, a culture solution), thus easily obtaining HCV particles.

The ability to generate viral particles from the HCV genomic RNA replicating cells can be confirmed by any known viral detection method. For example, a culture solution containing cells that presumably generate viral particles is fractionated according to sucrose density gradient and then both the density, concentration of HCV core proteins, and the amount of HCV genomic RNA of each fraction are measured. . As a consequence, when the peak of the HCV core protein corresponds to that of the HCV genomic RNA and when the density of a fraction in which the peak is detected is lower than the density of the same fraction, which is fractionated after treatment the culture supernatant with 0.25% NP40 (polyoxyethylene (9) octylphenyl ether) (for example between 1.15 mg and 1.22 mg), it can be confirmed that the cells have the capacity to generate viral particles.

The HCV viral particles released in the culture solution can also be detected by reacting an antibody with a core protein, an E1 protein or an E2 protein. In addition, it is also possible to indirectly detect the existence of HCV viral particles by increasing the HCV genomic RNA contained in the HCV viral particles in the culture solution and then detecting the amplified product following the RT-PCR method with specific primers.

3. Infection of other cells with the HCV particles of the present invention

The HCV viral particles generated by the method of the present invention have the capacity to infect cells (preferably HCV sensitive cells). The present invention also provides a method for producing a hepatitis C virus infected cell comprising the culture of HCV genomic RNA replicating cells and then the infection of other cells (preferably HCV sensitive cells) with viral particles contained in the culture obtained (preferably a culture solution). The term "HCV sensitive cells" is used herein with reference to cells that are capable of being infected by HCV. Said HCV-sensitive cells are preferably liver cells or lymphocyte cells, without being limited thereto. Specific examples of such liver cells may include primary liver cells, Huh7 cells, HepG2 cells, IMY-N9 cells, HeLa cells and 293. Specific examples of lymphocyte cells may include Molt4 cells, HPB-Ma cells and Daudi cells. However, not limited to them.

When the cells (for example HCV-sensitive cells) are infected by HCV particles generated in the HCV genomic RNA replicating cells of the present invention, the HCV genomic RNA is replicated in the infected cells and then viral particles are formed. Next, by allowing the cells to become infected with viral particles generated in the HCV genomic RNA replicating cells of the present invention, the HCV genomic RNA can be replicated in the cells, and then viral particles can be produced.

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When animals that can be infected with the HCV virus, for example chimpanzees, are infected by viral HCV particles generated in the HCV genomic RNA replicating cells of the present invention, the particles can cause HCV-derived hepatitis in the animals.

4. HCV particle purification

A solution containing HCV virus used in the purification of HCV particles from one or more selected from blood from a patient infected with HCV, cultured cells infected with HCV, a cell culture medium containing cells that generate cells can be derived HCV particles as a result of genetic recombination and a solution obtained from cell homogenate.

The solution containing HCV virus is subjected to centrifugation and / or filtration by a filter to remove cells and cell debris. The solution obtained by removing these residues can be concentrated at a magnification of 10 to 100 times using an ultrafiltration membrane with a molecular weight limit between 100,000 and 500,000.

The solution containing the HCV from which the residues have been removed can be purified by chromatography or density gradient centrifugation as described below, or by the combined use of density gradient centrifugation chromatography, in any order. Representative methods of chromatography and density gradient centrifugation will be described below, but the present invention is not limited thereto.

A gel filtration chromatography can be used to purify the HCV particles, preferably with a chromatographic support having, as a gel matrix, a crosslinked polymer composed of allyldextran and N, N'-methylene-bisacrylamide, and in particular with Sephacryl (R) S-300, S-400 and S-500.

Ion exchange chromatography can be used to purify HCV particles, preferably by Q-Sepharose (R) as ion exchange resin and preferably with SP Sepharose (R) as cation exchange resin.

An affinity chromatography can be used to purify HCV particles using preferably as a ligand-like resin that is allowed to bind to a substrate selected from heparin, sulfated cellulophin, lectin and various pigments. This affinity chromatography can be used to purify HCV particles preferably with HP (R) HiTtrap heparin, HiTrap blue HP (R), Benzamidine FF (R) HiTrap, sulfated cellulophin or supports to which LCA, ConA, RCA- bind 120 and WGA. Said affinity chromatography can be used to purify HCV particles preferably with sulfated cellulophin as support. Unexpectedly, HCV particles are purified with a 30-fold increase, relative to the total protein mass coefficient in solution, relative to the number of HCV RNA copies before and after purification.

In purification by density gradient centrifugation, as a solute forming a density gradient, cesium chloride, sucrose, Nycodenz (R) or a sugar polymer such as Ficoll (R) or Percoll (R) is preferably used. In particular, sucrose is used. In addition, as the solvent to be used herein, water or a buffer solution such as a phosphate buffer, a Tris buffer, an acetate buffer or a glycine buffer is preferred.

The temperature applied to the purification preferably ranges between 0 ° C and 40 ° C, in particular between 0 ° C and 25 ° C and especially between 0 ° C and 10 ° C.

In a purification method involving density gradient centrifugation, the centrifugal force applied to the purification preferably ranges between 1x104 and 1x109 g, in particular between 5x104 and 1x107 g and especially between 5x104 and 5x105 g.

With respect to the combined use of the purification methods, density gradient centrifugation and column chromatography can be combined in any order. Preferably, after having purified the HCV particles by multiple types of column chromatography, the result is subjected to density gradient centrifugation. In particular, a column ion exchange chromatography is carried out and then an affinity chromatography to obtain a fraction containing HCV particles and then the fraction obtained is purified by density gradient centrifugation. Especially, a fraction containing HCV particles obtained by column chromatography using Q-Sepharose (R) is further purified on a column with sulphated cellulophin and then the fraction obtained containing HCV particles is purified by gradient centrifugation of density. In addition, dialysis or ultrafiltration can be carried out between the column chromatography process and the density gradient centrifugation process to replace a solute in the solution containing HCV particles and / or to concentrate the HCV particles.

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5. Other embodiments of the present invention

HCV genomic RNA replicates with great efficiency in the HCV genomic RNA replicating cells of the present invention. Consequently, using the HCV genomic RNA replicating cells of the present invention, a high performance HCV genomic RNA can be produced.

In the present invention, HCV genomic RNA replicating cells are cultured and RNA is extracted from the culture (cultured cells and / or culture medium). Then, the extracted RNA is electrophoresed to isolate and purify the separated HCV genomic RNA, thus obtaining HCV genomic RNA. The RNA thus produced comprises a genomic HCV sequence. By providing such a method to produce the RNA comprising the HCV genomic sequence, more detailed analysis of the HCV genome is possible.

In addition, the HCV genomic RNA replicating cells of the present invention can preferably be used to produce an HCV protein. Said HCV protein can be obtained by any known method. For example, HCV genomic RNA is introduced into cells to produce recombinant cells. The recombinant cells are then cultured and the protein obtained from the culture obtained (cultured cells and / or culture medium) is recovered by the usual methods.

HCV viral particles may be directed to liver cells. Thus, a viral vector directed to liver cells can be produced by means of the HCV genomic RNA of the present invention. This viral vector is preferably used for genetic therapy. In the present invention, the RNA encoding a foreign gene is incorporated into the HCV genomic RNA and then the RNA is introduced into the cells, to introduce the above-mentioned foreign gene into the cells. The foreign gene can then be replicated and then expressed in the cells.

In addition, RNA is produced by exchanging the coding sequence of the E1 protein and / or E2 protein in the HCV genomic RNA with the envelope protein of a virus from other living species. The RNA produced is then introduced into the cells, to produce viral particles. Thus, it is possible that the cells of several living species can be infected by RNA. In this case, a foreign gene is also incorporated into the HCV genomic RNA and the RNA obtained can be used as a viral vector directed to the cells, so that the foreign gene can be expressed in several types of cells, depending on the directivity of a cell. recombinant virus envelope protein.

The present invention also relates to a method for producing a viral vector containing a foreign gene, which comprises the insertion of the RNA encoding the foreign gene into the HCV genomic RNA, the introduction of the genomic RNA into the cells and the culture. thereof so that cells can generate viral particles.

The present invention also provides a method for producing a hepatitis C vaccine or a virus vaccine used for the genetic recombination of a envelope protein using the HCV particles of the present invention or a portion thereof, as an antigen, or using particles produced by genetic recombination of the virus envelope protein to alter the directivity of the cells or a part thereof, as an antigen. In addition, a HCV infection neutralizing antibody can also be produced using the HCV particles of the present invention or a portion thereof, as an antigen, or using particles produced by genetic recombination of the virus envelope protein to alter the virus. directivity of the cells or a part thereof, as an antigen.

The HCV genomic RNA replicating cells of the present invention, or HCV infected cells that are infected by viral particles generated in the HCV genomic RNA replicating cells, can be used, for example, for HCV replication or for reconstruction of viral particles, or as a test system for screening a substance that favors or inhibits the release of viral particles (hepatitis C antiviral substance). Specifically, for example, said cells are cultured in the presence of a test substance and genomic HCV RNA or viral particles are detected in the culture obtained. Next, it is determined whether the test substance mentioned above favors or not, or inhibits or not, the replication of the replicon RNA or the HCV genomic RNA, the formation of said viral particles or the release thereof, thus seeking a substance that favors or inhibits the development of hepatitis C viruses. In this case, the HCV genomic RNA contained in the culture can be detected by measuring the amount of HCV genomic RNA in the RNA extracted from the aforementioned cells, the proportion thereof or its presence or absence. Viral particles contained in the culture (mainly, a culture solution) can be detected by measuring the amount of HCV protein contained in the culture solution, the proportion thereof or its presence or absence.

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The HCV particles generated in the HCV genomic RNA replicating cells of the present invention, as well as HCV-sensitive cells, can be used as assay systems for screening a substance that favors or inhibits the binding of HCV to cells. Specifically, for example, HCV-sensitive cells are cultured together with HCV particles generated in the HCV genomic RNA replicating cells of the present invention in the presence of a test substance. Next, HCV genomic RNA or viral particles are detected in the culture obtained. It is determined whether the test substance mentioned above favors or not, or inhibits or not, the replication of HCV genomic RNA or the formation of viral particles, thus investigating a substance that favors or inhibits the development of hepatitis C virus.

Said HCV genomic RNA or viral particles can be detected according to the means mentioned above or the examples described below. The test system described above can be used for the production or evaluation of a preventive agent, a therapeutic agent, or a diagnostic agent for hepatitis C virus infection.

Specific examples of using the test system of the present invention mentioned above are presented below.

(1) Screening of a substance that inhibits the development of HCV and the infection that accompanies it

Examples of substances that inhibit the development of HCV and the accompanying infection may include: an organic compound that directly or indirectly affects the development of HCV and the infection that accompanies it; and an antisense oligonucleotide that hybridizes with the HCV genome target sequence

or a complementary strand thereof, to directly or indirectly affect the development of HCV or the translation of an HCV protein.

(2) Evaluation of various substances with antiviral activity in cell culture

An example of the different substances mentioned above may be a substance obtained by the rational design of drugs or high capacity screening (for example, isolated and purified enzyme).

(3) Identification of a new target to be attacked, used for the treatment of patients infected with HCV

In order to identify a host cell protein that plays an important role in the replication of an HCV virus, for example the HCV genomic RNA replicating cells of the present invention can be used.

(4)

Evaluation of the ability of the HCV virus to acquire resistance to agents or the like, and identification of the mutation associated with said resistance

(5)

Production of the viral protein used as an antigen that can be used for the development, production and evaluation of a diagnostic agent or therapeutic agent for hepatitis C virus infection

(6)

Production of viral protein and attenuated HCV used as antigens that can be used for the development, production and evaluation of a vaccine against hepatitis C virus infection

Examples

The present invention is described more specifically based on the following examples and figures. However, these examples are not intended to limit the technical scope of the present invention.

Example 1: Production of HCV genomic RNA

1. Construction of the expression vector

The DNA corresponding to the total genomic region of the virus of a JFH1 strain of hepatitis C virus (genotype 2a) isolated from patients suffering from fulminant hepatitis was obtained from a clone of JFH1 comprising the full length genomic cDNA of the cited viral strain (Kato T. et al., J. Med. Virol. 64 (2001) pp. 334-339). The DNA obtained was then inserted downstream of a T7 RNA promoter sequence that had been inserted into a pUC19 plasmid. Specifically, an RT-PCR fragment obtained by amplifying the viral RNA of strain JFH1 was cloned into a pGEM-T EASY vector (Promega), to obtain various plasmid DNAs such as pGEM1-258, pGEM44-486, pGEM317-849 , pGEM617-1323, pGEM1141-2367, pGEM2285-3509, pGEM3471-4665, pGEM4547-5970, pGEM5883-7003, pGEM6950-8035, pGEM7984-8892, pGEM8680-9283, pGEM92319634, and pG967959, and pG967959, and pG96794, and pG96719434, and pG96719834, and pG96719934, and pG967196, , Gastroenterology, 125 (2003) pp. 1808-1817). The genomic cDNA of the virus contained in each plasmid was ligated with each other by the PCR method and using restriction enzymes and thus the full length genomic cDNA was cloned. A T7 RNA promoter sequence was inserted upstream thereof, to obtain a JFH1 clone (pJFH1) (Figure 1). It should be noted that the full length cDNA sequence of the pJFH has been registered in the International DNA Databank (DDBJ / EMBL / GenBank) under accession No. AB047639.

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Subsequently, with respect to a region of NS5B in pJFH1 (nucleotide sequence: SEQ ID NO: 2; amino acid sequence: SEQ ID NO: 3), a GDD motif of the amino acids corresponding to the active center of RNA polymerase encoded by the anterior region was mutated to GND, to produce a mutant plasmid clone pJFH1 / GND. Since the amino acid sequence of the active center of an NS5B protein encoded by the mutant plasmid clone pJFH1 / GND is mutated, this clone cannot express an active NS5B protein, necessary for HCV RNA replication.

Subsequently, an E1 region and an E2 region of JFH1 were deleted to produce pJFH1 / E1E2. In addition, the total length HCV cDNA of a J6CF strain (GenBank Accession No. AF177036) different from the JFH1 strain and that of a JCH1 strain (Kato T., et al., J. Med. Virol. 64 (2001 ) pp. 334-339) were inserted downstream of a T7 RNA promoter sequence that had been inserted into a plasmid pUC19, to produce pJ6CF and pJCH1, respectively. In addition, the NS5B coding region of pJCH1 was replaced by NS5B of JFH1, to produce pJCH1 / NS5B (jfh1).

2. Production of HCV genomic RNA

In order to produce a template DNA used for RNA synthesis, each of pJFH1, pJFH1 / GND, pJFH1 / E1-E2, pJ6CF, pJCH1 and pJCH1 / NS5B (jfh1) was segmented with the restriction enzyme Xbal. Next, 10 to 20 g of each of these Xbal cleavage fragments was incubated with 20 U of Mung Bean Nuclease (total amount of reaction solution: 50 ) at 30 ° C for 30 minutes. Mung Bean Nuclease is an enzyme that catalyzes a selective digestion reaction of a single-stranded part of a two-stranded DNA. In general, when the RNA is directly synthesized using the aforementioned Xbal cleavage fragment as a template, the replicon RNA is synthesized, to whose terminal 3 '4 CUGA nucleotides are redundantly added which constitute a part of a recognition sequence from Xbal. Thus, in the present example, said Xbal cleavage fragment was treated with Mung Bean Nuclease to remove the 4 CUGA nucleotides from the Xbal cleavage fragment. Next, the solution thus treated with Mung Bean Nuclease containing a cleavage fragment with Xbal was subjected to a protein removal treatment according to common methods, so that the cleavage fragment with Xbal, from which the 4 had been removed CUGA nucleotides, could be purified. The purified fragment was used as template DNA.

Subsequently, the RNA was synthesized in vitro from the previous template DNA. Said RNA was synthesized by reacting 20 µl of a reaction solution containing 0.5 to 1.0 µg of the template DNA at 37 ° C for 3 to 16 hours, using MEGAscript manufactured by Ambion.

At the end of RNA synthesis, DNAse (2U) was added to the reaction solution and the mixture was allowed to react at 37 ° C for 15 minutes. Next, the RNA was extracted with phenolic acid and the template DNA was removed. Thus, the various types of HCV RNA synthesized from the above-mentioned template DNA from pJFH1 and pJFH1 / GND were designated rJFH1, rJFH1 / GND, rJFH1 / E1-E2, rJ6CF, rJCH1 and rJCH1 / NS5B (jfh1).

With respect to the HCV RNA thus obtained, rJFH1 is RNA produced by DNA under GenBank Accession Number AB047639 as a template; JFH1 / GND is RNA produced using, as a template, DNA obtained by replacing G in nucleotide 8618 with A, with respect to DNA under GenBank Accession Number AB047639; rJFH1 / E1-E2 is RNA produced using, as a template, DNA comprising a deletion of part 989-2041 of the DNA sequence, with respect to DNA under GenBank Accession Number AB047639; rJ6CF is RNA produced by means of DNA under GenBank Accession Number AF177036 as a template; rJCH1 is RNA produced by means of DNA under GenBank Accession Number AB047640 as a template; and rJCH1 / NS5B (jfh1) is RNA produced using, as a template, DNA obtained by linking part 1-3866 of the DNA sequence of the DNA under GenBank Accession No. AB047640, to part 3867-9678 of the DNA sequence of DNA under GenBank Accession Number AB047639, through the AvrII site of the restriction enzyme. The nucleotide sequences of these RNAs can be confirmed. This latter genomic RNA of the modified hepatitis C virus is an embodiment of the present invention.

Example 2: Generation of HCV genomic RNA replicating cells and viral particles in cells

1. HCV genome replication and generation of viral particles in cells

Each of the full length HCV genomic RNAs synthesized above (rJFH1 and rJFH1 / GND) was adjusted so that the total RNA level reached 10 g. Subsequently, the RNAs mixed in Huh7 cells were introduced by the electroporation method. Huh7 cells treated by electroporation were inoculated in a culture plate and then cultured for 12 hours, 24 hours, 48 hours, and 72 hours. The cells were then recovered and then the RNA was extracted from them. The extracted RNA was analyzed by the Northern blot method. Said Northern blot analysis was performed according to Molecular Cloning, A Laboratory Manual, 2nd edition, J. Sambrook, E.F. Fritsch, T. Maniatis, Cold Spring Harbor Laboratory Press (1989). The RNA extracted from the cells was subjected to denatured agarose electrophoresis. Upon completion of the electrophoresis, the RNA was transcribed into a positively charged nylon membrane. A 32P-labeled RNA or DNA probe produced from pJFH1 was allowed to hybridize with the RNA transcribed in the membrane, as described above. The membrane was then washed and then exposed to a film, thus detecting a specific RNA band of the HCV genome.

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As shown in Figure 2, when the cells were transfected with JFH1 / GND, the introduced RNA band was confirmed as a weak signal 4 hours after transfection. However, this signal was a time-dependent attenuation and, 24 hours later, almost no signal band was confirmed.

On the other hand, when the cells were transfected with rJFH1, 4 to 12 hours after transfection, the signal intensity of the introduced RNA band was almost the same as in the case of the introduction of JFH1 / GND. Then, the signal was attenuated once, but a clear signal from the RNA band was confirmed from 24 hours onwards. This signal was HCV specific. In other words, it was considered that a part of the introduced rJFH1 RNA was replicated and developed. Such replication was not observed in rJFH1 / GND obtained by mutation of the active motif of NS5B, which was an RNA replicating enzyme. Thus, it was confirmed that NS5B activity was important for the replication of full-length HCV RNA. The same experiment was carried out using strain JCH1 (Kato T. et al., J. Med. Virol. 69 (2001) pp. 334-339), which had been isolated from patients with chronic hepatitis by the present inventors. In the case of this strain, the replication of HCV RNA was not confirmed at all.

2. HCV protein detection

A protein was extracted in a time-dependent manner from rJFH1 or rJFH1 / GND RNA transfected cells according to the usual methods and then analyzed by SDSPAGE and Western blot. For said analysis, Huh7 cells were transiently transfected with expression plasmid DNA that included an NS3, NS5A, nucleus or E2 gene, and the cell extract obtained was used as a positive control (NS3 protein). In addition, a protein extracted from untransfected Huh7 cells was used as a negative control. A sample of protein extracted from each cell clone was blotted onto a PVDF membrane (Immobilon-P, manufactured by Millipore). Next, an anti-NS3 specific antibody (supplied by Dr. Moradpour; Wolk B. et al., J. Virology. 2000;

74: 2293-2304), an anti-NS5A specific antibody (produced by inserting the NS5A region of JFH1 into an expression vector and using it with a mouse according to DNA immunization procedures), a anti-core specific antibody (clone 2H9 antibody) and an anti-E2 specific antibody (produced by synthesis of the GTTTVGGAVARSTN peptide (SEQ ID NO: 4) in the E2 region of JFH1 and the CDLEDRDRSQLSPL peptide (SEQ ID NO: 5) inside, and then by immunization of a rabbit with the two synthetic peptides), were used to detect the NS3, NS5A, nucleus and E2 proteins encoded by the JFH1 RNA. In addition, as an intrinsic control, an actin protein was detected by an anti-actin antibody.

As shown in Figure 3, in the cells transfected with rJFH1, from 24 hours after transfection the NS3, NS5A, nucleus and E2 proteins were detected, and it was confirmed that the increase in the level of expression depended on time. In contrast, in cells transfected with rJFH1 / GND, or in non-transfected Huh7 cells, none of said NS3, NS5A, nucleus, and E2 proteins were detected. It was found that these proteins were expressed within as a result of autonomous replication of the transfected rJFH1.

From the results obtained in points 1 and 2 above, it was confirmed that rJFH1 is replicated in cells constituted by transfection with rJFH1.

3. Detection of HCV core protein in a culture medium of transfected cells

Huh7 cells, in which rJFH1, rJFH1 / GND, rJFH1 / E1-E2, rJ6CF and rJCH1, were inoculated into a culture plate. The cells were then grown inside for 2 hours, 12 hours, 24 hours, 48 hours and 72 hours. Next, the HCV core protein contained in the culture medium was measured. This measurement was carried out by the IRMA test with HCV Ortho antigen (Aoyagi et al., J. Clin. Microbiol., 37 (1999) pp. 1802-1808).

As shown in Figure 4, a core protein was detected in the culture medium 48 to 72 hours after transfection with rJFH1. On the other hand, in the culture medium of cells transfected with rJFH1 / GND, rJ6CF and rJCH1 no HCV core proteins were detected. A small amount of HCV core protein was detected in the culture medium of cells transfected with rJFH1 / E1-E2. rJFH1 / GND, rJ6CF, and rJCH1 cannot replicate autonomously in Huh7 cells, while rJFH1 and rJFH1 / E1-E2 can replicate autonomously. Thus, it was discovered that autonomous replication of the HCV RNA introduced is essential for the release of said core protein and, in addition, that E1 and E2 are necessary so that a large amount of core protein can be stably released out of the cells.

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4. Detection of HCV particles in culture medium of transfected cells

In order to analyze whether the core protein released into the culture medium in the above-mentioned example was or was not secreted in the form of viral particles, the culture medium obtained 6 days after transfection with rFJH1 was fractionated in density gradient of saccharose. That is, 2 ml of a 60% sucrose solution (weight / weight) (dissolved in 50 mM Tris, pH 7.5 / 0.1M NaCl / 1 mM EDTA), 1 ml of a 50% sucrose solution , 1 ml of a 40% sucrose solution, 1 ml of a 30% sucrose solution, 1 ml of a 20% sucrose solution and 1 ml of a 10% sucrose solution were laminated in a centrifugal tube and then 4 ml of the culture supernatant of a sample was laminated on top. This tube was then centrifuged at 400,000 rpm. at 4 ° C for 16 hours using a Beckmann SW41Ti rotor. At the end of centrifugation, 0.5 ml of each of the fractions was recovered from the bottom of the centrifuge tube. The density, the concentration of HCV core protein and the number of HCV RNA copies were tested for each fraction. Replicon RNA detection by quantitative RT-PCR was carried out by RNA detection 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, the RNA of the replicon contained in the RNA extracted from the cells was amplified by PCR using the following synthetic primers and the EZ rTth RNA PCR kit (Applied Biosystems), and was then detected using the ABI Prism 7700 sequence detector system (Applied Biosystems).

R6-130-S17: 5’-CGGGAGAGCCATAGTGG-3 ’(SEQ ID NO: 6)

R6-290-R19: 5’-AGTACCACAAGGCCTTTCG-3 ’(SEQ ID NO: 7)

TaqMan probe, R6-148-S21FT: 5’-CTGCGGAACCGGTGAGTACAC-3 ’(SEQ ID NO: 8)

As shown in Figure 5A, the peak of the core protein corresponded to that of HCV RNA at a fraction of 1.17 mg / ml. The density of this fraction was found to be approximately 1.17 mg / ml. It was a lighter specific gravity than that of a bound product consisting of a core protein and nucleic acid, which had been previously reported. If the core protein and HCV RNA existing in the 1.17 mg / ml fraction form a structure of HCV particles, this fraction is considered to be nuclease resistant. Therefore, a culture solution obtained 6 days after transfection with JFH1 was treated with 10 µg / ml RNase A for 20 minutes and then fractionated in a sucrose density gradient.

As a consequence, as shown in Figure 5B, the HCV RNA was broken down and the core protein peak and that of the HCV RNA were detected at a fraction of 1.17 mg / ml, as in the case of no be treated with RNAse A. That is, it was confirmed that the core protein and HCV RNA existing in the 1.17 mg / ml fraction formed a HCV particle type structure.

Next, the culture solution was subjected to the same fractionation as described above, after being treated with 0.25% NP40. As a consequence, the peak of the core protein and that of HCV RNA changed to 1.28 mg / ml (Figure 5C). Then, when the culture solution was treated simultaneously with 0.25% NP40 as well as with RNAse A, the HCV RNA peak disappeared (Figure 5D). Therefore, it was considered that a surface membrane with low specific gravity containing lipids had been exfoliated from the viral particles as a result of the NP40 treatment, so that the particles became core particles consisting only of nucleic acid and a core protein that they did not have a viral type structure, resulting in an increase in specific gravity.

From these results, it was confirmed that the viral RNA had been replicated by transfection of the Huh7 cells with rJFH1 and that the viral particles are formed by this means and released into the culture solution.

5. Experiment regarding the infectivity of viral particles in the culture medium

Huh7 cells were transfected with rJFH1 and the infectivity of the HCV particles secreted in a culture medium was examined. The culture supernatant was recovered 3 days after transfection of Huh7 cells with rJFH1 or rJFH1 / E1-E2. The recovered culture medium was centrifuged and the centrifuged supernatant was recovered, followed by filtration by a 0.45 µm filter. In the presence of this culture medium, Huh7 cells that had not been transfected with RNA were cultured. 48 hours later, the cells were fluorescently immunostained with an anti-core antibody or an anti-NS5A antibody. As shown in Figure 6A, in the case of cultured cells in the presence of a culture medium obtained by transfection of Huh7 cells with rJFH1, the expression of a core protein and an NS5A protein was observed in the cells. On the other hand, in the case of cultured cells in the presence of a culture medium obtained by transfection of Huh7 cells with rJFH1 / E1-E2, said expression of a core protein and an NS5A protein was not observed in the cells ( data not revealed).

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Subsequently, a culture supernatant was recovered 3 days after transfection of rHuh7 cells with JFH1 and then concentrated to a 30-fold magnification using an ultrafilter (limit: 1 x 105 Da). Huh7 cells that had not been transfected with RNA were cultured in 100 µl of a culture medium containing HCV particles concentrated on a 15 mm coverslip. 48 hours later, the cells were immunostained with an anti-core antibody and then the number of positive cells stained with the anti-core antibody was counted, namely infected cells. As a consequence, as shown in Figure 6B, 394.0 ± 26.5 infected cells (approximately 0.51% of the total cells) were confirmed. It was then confirmed if this infection was due

or not to HCV particles that had been secreted in the culture medium as a result of the transfection of Huh7 cells with rJFH1. That is, using a culture medium prepared by subjecting a culture solution used for the treatment of UV infection and another culture medium prepared without the RNA transfection step, Huh7 cells that had not been transfected with RNA were cultured in a 15 mm coverslip. 48 hours later, the cells were immunostained with an anti-core antibody and then the number of infected cells was counted. As a result, in the case where the cells were treated with UV, the number of infected cells was drastically reduced. In the case of the culture medium prepared without the RNA transfection step, no infected cells were observed.

In addition, it was examined whether HCV infectious particles amplified or not the RNA in the cells and then released new HCV particles in the culture medium. Huh7 cells that had not been transfected with RNA were cultured in 100 µl of a culture medium containing HCV particles prepared by concentrating a culture medium obtained 48 hours after transfection of Huh7 cells with rJFH1. Then, the cells and the culture medium were recovered every day and the RNA was recovered from it. The amount of HCV RNA was tested by the method mentioned above. As a consequence, as shown in Figure 6C, HCV RNA was amplified to a certain amount in the cells and the amount of HCV RNA increased time-dependent in the supernatant. On the other hand, the same test was carried out using a culture solution obtained by transfecting Huh7 cells with rJFH1 / E1-E2. However, HCV RNA was not detected in the cells or in the culture solution.

From these results, it was confirmed that HCV particles secreted in the culture medium have infectivity as a result of the transfection of Huh7 cells with rJFH1 and also have the ability to amplify HCV RNA in infected cells and produce new ones. HCV particles.

6. Production of HCV viral particles by rJCH1 / NS5B (jfh1)

It was examined whether or not HCV particles are secreted in a culture medium as a result of the transfection of Huh7 cells with rJCH1 / NS5B (jfh1), or if the secreted HCV particles exhibit or not infectivity. A culture solution obtained 6 days after transfection of Huh7 cells with rJCH1 / NS5B (jfh1) was concentrated by the method described in section 5 above. In the presence of this culture medium, Huh7 cells that had not been transfected with RNA were cultured and time-dependent changes in the amount of HCV RNA in the cells were tested. At 12 hours after the start of the culture, the amount of HCV RNA in the cells increased in a time-dependent manner (Figure 7A). In addition, Huh7 cells that had not been transfected with RNA were grown on a 15 mm coverslip, and then the cells were cultured in the presence of the concentrated culture medium. 48 hours later, the cells were immunostained with an anti-core antibody and then the number of positive cells stained with the anti-core antibody was counted, namely infected cells. As a consequence, as shown in Figure 7B, infected cells were observed. From these results, it was discovered that HCV particles secreted in a culture medium acquire infectivity as a result of the transfection of Huh7 cells with rJCH1 / NS5B (jfh1) and that they also have the ability to amplify HCV RNA in Infected cells and produce new HCV particles.

Consequently, even in the case of a strain that cannot replicate autonomously in vitro, such as an HCV strain isolated from patients, the replacement of the HS5B region of the same with NS5B of rJFH1 allows the autonomous replication thereof in a cell culture system as well as the generation of HCV particles.

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Example 3

1. Production of HCV viral particles by ConI / C-NS2 / JFH-1

Huh7 cells were transfected with chimeric HCV RNA comprising the NS5B part of a Con-1 strain with genotype 1b of HCV and JFH-1 and then investigated whether or not HCV particles were secreted in a culture solution and if the secreted HCV particles present or not infectivity.

The sequence of a Con-1 strain with HCV genotype 1b corresponding to 1 to 1,026 (core regions, E1, E2, p7 and NS2 of the ConI strain) was ligated downstream of 5’-UTR from a JFH strain

1. Next, region 1.031-3.030 of strain JFH-1 (from NS3 to NS5b) was also ligated downstream thereof. Then, the 3’-UTR of strain JFH-1 was also linked downstream of it, to produce a construct. Using this construct, chimeric HCV RNA of rCon1 / CNS2 / JFH-1 was produced by the method described in Example 1-2 above. Next, Huh7 cells were transfected with the previous RNA by the method described in Example 2-1 above. Huh7 cells were transfected with HCV RNA and the core protein contained in the supernatant was measured over time. From about 48 hours onwards, said core protein was detected in the supernatant and, therefore, it could be confirmed that HCV particles were generated in the cell supernatant. Subsequently, the supernatant was concentrated at a 20-fold increase by ultrafiltration and then the concentrate was added to Huh7 cells. 48 hours after culture, the cells were stained with an anti-NS3 rabbit antibody.

As a consequence, no positive cells of the antiNS3 antibody or rJFH1 / E1-E2 were observed in the simulation, but said positive cells of the anti-NS3 antibody were detected in rJFH-1 and rCon1 / C-NS2 / JFH-1. From these results, it was confirmed that rCon1 / C-NS2 / JFH-1 can generate HCV infectious particles, as with JFH-1.

Example 4: Production of full length chimeric HCV replicon RNA derived from full length chimeric HCV genomic RNA

1. Construction of the expression vector

The DNA (clone JFH-1: SEQ ID NO: 9) that contained the full length genomic cDNA of a JFH-1 strain (genotype 2a), which is a hepatitis C virus isolated from patients suffering from fulminant hepatitis, is inserted downstream of a T7 RNA promoter sequence into a plasmid pUC19, to produce plasmid DNA.

Specifically, an RT-PCR fragment obtained by amplifying the viral RNA of strain JFH-1 was cloned into a pGEM-T EASY vector (Promega), to obtain various plasmid DNAs such as pGEM1-258, pGEM44-486, pGEM317 -849, pGEM617-1323, pGEM1141-2367, pGEM2285-3509, pGEM3471-4665, pGEM4547-5970, pGEM5883-7003, pGEM6950-8035, pGEM7984-8892, pGEM86809283, pGEM9231-934 and p9EM9-934 and p9EM9-934 and p. Gastroenterology, (2003) 125: pp. 1808-1817). The cDNA derived from the genomic RNA of the virus contained in each plasmid was ligated with each other by PCR and using restriction enzymes, and thus the full length genomic cDNA was cloned. A T7 RNA promoter sequence was inserted upstream of the full length virus genome. Hereinafter, the plasmid DNA thus constructed is called pJFH1. It should be noted that the production of the JFH-1 clone mentioned above is described in Japanese Patent Publication (Kokai) No. 2002-171978 A and the document by Kato et al. (Kato et al., J. Med. Virol., (2001) 64 (3): pp. 334-339). In addition, the nucleotide sequence of the full-length cDNA of clone JFH-1 has been registered in the International DNA Databank (DDBJ / EMBL / GenBank) under Accession No. AB047639.

Subsequently, EMCV-IRES (internal ribosome entry site for the encephalomyocarditis virus) and a neomycin resistance gene (neo; also called the neomycin phosphotransferase gene) were inserted between the 5 'untranslated region and the core region of pJFH1, which was plasmid DNA, to construct pFGREP-JFH1, which was plasmid DNA. Said construction was carried out in accordance with the procedures of Ikeda et al. (Ikeda et al., J. Virol., (2002) 76 (6): pp. 2997-3006).

2. Construction of the chimeric expression vector

The JFH1 strain is HCV derived from type 2a HCV. A TH strain derived from HCV type 1b (Wakita et al., J. Biol. Chem., (1994) 269, pp. 14205-14210; and Moradpour et al., Biochem. Biophys. Res. Commun., (1998 ) 246, pp. 920-924) was used to produce a chimeric HCV vector. The core parts, E1, E2, and p7 of pFGREP-JFH1 as produced above were replaced by those of strain TH, to produce chimeric HCV, pFGREP-TH / JFH1.

In the present specification, the full-length RNA sequence of strain JFH-1 mentioned above (derived from a JFH-1 clone) and the partial RNA sequence of strain TH used to produce the anterior chimeric body (partial genomic RNA (1-3748) comprising a part corresponding to the region from the 5 'untranslated region of the HCV strain TH to the NS3 region thereof), are shown in SEQ ID NO: 9 and 10, respectively. In the full-length genomic RNA sequence of strain JFH-1 (SEQ ID NO: 9) mentioned above, the "untranslated region 5 '" corresponds to 1-340, the "core protein coding sequence" corresponds to 341913, the "coding sequence of the E1 protein" corresponds to 914-1489, the "coding sequence of the E2 protein" corresponds to 1490-2590, the "coding sequence of the NS2 protein" corresponds to 27803430, the "coding sequence of the NS3 protein ”corresponds to 3431-5323, the“ NS4A protein coding sequence ”corresponds to 5324-5486, the“ NS4B protein coding sequence ”corresponds to 5487-6268 the“ NS5A protein coding sequence ”corresponds to 6269-7663 and the "NS5B protein coding sequence" corresponds to 7664-9442.

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3. Production of chimeric HCV replicon full length

In order to produce template DNA used for the synthesis of full-length chimeric HCV replicon RNA, the expression vector pFGREP-TH / JFH1 as constructed above was cloned with the restriction enzyme Xbal. Next, 10 to 20 µg of the Xbal cleavage fragment was mixed in 50 µl of a reaction solution and the obtained mixture was incubated with 20 U of Mung Bean Nuclease at 30 ° C for 30 minutes. Mung Bean Nuclease is an enzyme that catalyzes a selective digestion reaction of a single-stranded part in two-stranded DNA. In general, when the RNA is directly synthesized using the aforementioned Xbal cleavage fragment as a template, the replicon RNA is synthesized, to whose terminal 3 '4 CUGA nucleotides are redundantly added which constitute a part of a recognition sequence from Xbal. Thus, in the present example, said Xbal cleavage fragment was treated with Mung Bean Nuclease, to remove the 4 CUGA nucleotides from the Xbal fragment. Next, the solution thus treated with Mung Bean Nuclease containing the Xbal cleavage fragment was subjected to a protein removal treatment according to the usual methods, so that the Xbal cleavage fragment, from which the 4 CUGA nucleotides will be purified. The purified fragment was used as template DNA.

Subsequently, RNA was synthesized in vitro from template DNA using T7 RNA polymerase. MEGAscript manufactured by Ambion was used for said RNA synthesis. 20 µl of a reaction solution containing 0.5 to 1.0 µg of the template DNA was allowed to react according to the instructions provided by the manufacturer.

At the end of RNA synthesis, DNAse (2U) was added to the reaction solution and the obtained mixture was reacted at 37 ° C for 15 minutes. Next, the RNA was extracted with phenolic acid and the template DNA was removed. Thus, the RNA synthesized from the template DNA mentioned above from pFGREP-TH / JFH1 was called rFGREP-TH / JFH1. The nucleotide sequence of the chimeric HCV genomic RNA in rFGREP-TH / JFH is shown in SEQ ID NO: 11. Said rFGREP-TH / JFH is an example of the full length chimeric HCV replicon RNA of the present invention.

Example 5: Production of chimeric HCV replicon RNA replicating cells of full length and constitution of the cell clone

1. Introduction of full length chimeric HCV genomic RNA in cells

Different amounts of the full length chimeric HCV genomic RNA (rFGREP-TH / JFH1) as synthesized above were mixed with total cellular RNA extracted from Huh7 cells, resulting in the total amount of RNA at 10 g. Subsequently, RNA mixed into Huh7 cells was introduced by electroporation method. After the cells were cultured for 16 to 24 hours, G418 was added thereto in different amounts. The culture continued while the culture solution was changed to a fresh solution twice a week. At the end of the culture for 21 days, the surviving cells were stained with crystal violet. The number of dyed colonies was counted and then the number of colonies obtained by weight of RNA used for transfection was calculated. In addition, in several culture plates, the colonies of surviving cells were cloned and the culture continued. RNA, genomic DNA and a protein were extracted from the cloned cells, and then the detection of full length chimeric HCV replicon RNA, the presence or absence of incorporation of a neomycin resistance gene into the DNA was investigated. Genomic and expression of an HCV protein. The results are shown in detail below.

2. Colony formation capacity

As a consequence of the aforementioned transfection, colony formation by cells was still observed in a case where the concentration of G418 was 1.0 mg / ml. The rFGREP-TH / JFH1 replicon RNA was considered to replicate autonomously in Huh7 cells transfected with the rFGREP-TH / JFH1 replicon RNA, and that a neomycin resistance gene was persistently expressed to maintain resistance to G418. Thus, the cells could develop and Huh7 cells acquire colony formation capacity.

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Example 6: Infectivity of the HCV chimeric virus in the culture supernatant

Experiment concerning the infectivity of chimeric HCV viral particles in culture supernatant

Huh7 cells were transfected with rFGREP-TH / JFH1 and then the culture supernatant containing clones of replicating cells of the chimeric HCV replicon RNA was recovered. The culture supernatant was added to Huh7 cells that had not been infected, such that Huh7 cells were infected with viral particles in the culture supernatant. The day after infection, 0.3 mg / ml of G418 was added to the culture medium containing the infected Huh7 cells and then the mixture was cultured for 21 days. At the end of the culture, the cells were fixed and then stained with crystal violet. As a result, colony formation was observed in cells infected with the culture supernatant containing full length chimeric HCV replicon RNA replicating cells obtained by transfection with rFGREP-TH / JFH1. This shows that the full length chimeric HCV replicon RNA replicating cell clones obtained by transfection with rFGREP-TH / JFH1 generate infectious HCV and also that HCV exhibits infectivity to new cells.

Example 7: HCV particle purification

1. Gel filtration

Figure 11 shows the distribution of HCV particles in each fraction by gel filtration chromatography. The gel supports used were Sephacryl (R) S300, S400 and S500. A solution containing HCV particles used for column chromatography was purified by column chromatography containing each of the above gel supports. The buffer used for purification comprised 10 mM Tris-hydrochloride, 1 mM ethylenediaminetetraacetic acid and 100 mM sodium chloride (pH 8.0). As a consequence, in the case of using Sephacryl (R) S-300, HCV particles were obtained at a passing fraction called a vacuum fraction. Thus, using Sephacryl (R) S-300, the HCV particles were separated from the small molecular weight proteins, so that the salt concentration of the solution could be changed. The ratio between HCV core protein and total protein mass was 3.78 when compared to HCV particles before column purification and, therefore, the ratio between HCV particles and total protein increased. . On the other hand, in the case of using Sephacryl (R) S-400 and S-500, as the HCV particles were obtained at an eluted fraction depending on the molecular weight, the particles can be separated from other proteins with different molecular weights.

2. Ion exchange chromatography

Figure 12 shows the distribution of HCV particles in each fraction by ion exchange chromatography. The gel supports used were SP Sefarosa HP (R) and Q Sefarosa HP (R).

In the case of a column using SP Sepharose HP (R), the column was equilibrated with 50 mM of a citric acid buffer (pH 6.2). A solution containing HCV particles was added to the column that had been concentrated by an ultrafilter with a fractional molecular weight between 100,000 and 500,000 and then diluted with 50 mM of a citric acid buffer (pH 6.2). Next, 50 mM of a citric acid buffer (pH 6.2) was passed through the column at a flow rate approximately 10 times higher than that of the column. Subsequently, 50 mM citric acid buffer (pH 6.2) to which 0.1M NaCl, 0.3M NaCl and 1M NaCl had been added, were successively passed through the column at a volume approximately 3 times greater than that of the spine. Next, 50 mM of a citric acid buffer (pH 6.2) to which 1M NaCl had been added passed through the column at a volume approximately 5 times greater than that of the column (1M fraction of NaClW). As a result, HCV particles were eluted in the 50 mM fraction of citric acid buffer (pH 6.2) to which 0.1M NaCl had been added.

In the case of a column using Q Sepharose HP (R), the column was equilibrated with 50 mM of a Tris-HCl buffer (pH 8.0). A solution containing HCV particles was added to the column that had been concentrated by an ultrafilter with a fractional molecular weight between 100,000 and 500,000 and then diluted with 50 mM Tris-HCl buffer (pH 8.0). Next, 50 mM of a Tris-HCl buffer (pH 8.0) passed through the column, at a volume approximately 10 times greater than that of the column. Subsequently, 50 mM Tris-HCl buffers (pH 8.0) to which 0.1M NaCl, 0.3M NaCl and 1M NaCl had been added successively passed through the column, at a volume approximately 3 times greater than that of the column. Then, 50 mM of a Tris-HCl buffer (pH 8.0) to which 1M NaCl had been added, passed through the column at a volume approximately 5 times greater than that of the column (1M fraction of NaClW). As a result, HCV particles were eluted in the 50 mM fraction of Tris-HCl buffer (pH 8.0), to which 0.3M NaCl had been added. The ratio between HCV core protein and total protein mass was 2.32 when compared to HCV particles before column purification and, therefore, the ratio between HCV particles and total protein increased. .

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3. Affinity chromatography

Figure 13 shows the distribution of HCV particles in each fraction by lectin affinity chromatography. In affinity chromatography supports were used to which each of RCA-120, ConA, LCA and WGA are attached.

In the case of affinity chromatography with ConA, LCA and WGA, the column was equilibrated with a phosphate buffered saline. A solution containing HCV particles that had been concentrated by an ultrafilter with a molecular weight limit between

100,000 and 500,000 and then diluted with a phosphate buffered saline solution. Next, the phosphate buffered saline solution passed through the column at a volume approximately 10 times greater than that of the column. Subsequently, a phosphate buffered saline solution to which 0.35M lactose had been added passed through the column, at a volume approximately 3 times greater than that of the column. Next, a phosphate buffered saline solution to which 0.5M lactose had been added passed through the column, at a volume approximately 5 times greater than that of the column. As a result, in the case of affinity chromatography with LCA and ConA, no specific binding to the support was observed. In the case of WGA affinity chromatography, the HCV particles were eluted in the phosphate buffered saline fraction to which 0.35M lactose had been added.

In the case of RCA-120 affinity chromatography, the column was equilibrated with a phosphate buffered saline solution. A solution containing HCV particles that had been concentrated by an ultrafilter with a fractional molecular weight between 100,000 and 500,000 was added to the column and then diluted with a phosphate buffered saline solution. Next, the phosphate buffered saline solution passed through the column at a volume approximately 10 times greater than that of the column. Subsequently, the phosphate buffered saline solution to which 0.38M lactose had been added passed through the column, at a volume approximately 3 times greater than that of the column. Next, a phosphate buffered saline solution to which 0.38M lactose had been added passed through the column, at a volume approximately 5 times greater than that of the column. As a result, in the case of RCA-120 affinity chromatography, HCV particles were eluted in the phosphate buffered saline fraction to which 0.38M lactose had been added.

Figure 14 shows the distribution of HCV particles in each fraction by affinity chromatography with heparin and sulfated cellulophin.

In each affinity chromatography, the column was equilibrated with 20 mM of a phosphate buffer (pH 7.0). A solution containing HCV particles was added to the column that had been concentrated by an ultrafilter with a molecular weight limit between 100,000 and 500,000 and then diluted with 20 mM phosphate buffer (pH 7.0). Next, the phosphate buffer (pH 7.0) passed through the column at a volume approximately 10 times higher than that of the column. Subsequently, phosphate buffers (pH 7.0) to which any one of 0.1M, 0.3M, 0.5M and 1M NaCl had been added successively passed through the column at a volume approximately 3 times greater than that of the column . Then, 20 mM of a phosphate buffer (pH 7.0) to which 1M NaCl had been added passed through the column at a volume approximately 5 times greater than that of the column. As a result, in the case of heparin affinity chromatography, HCV particles were eluted in the 20 mM fraction of phosphate buffer (pH 7.0) to which 0.3M NaCl had been added. The ratio between HCV core protein and total protein mass was 0.36 when compared to HCV particles before column purification and, therefore, the ratio between HCV particles and total protein was reduced. In the case of affinity chromatography with sulfated cellulophin, HCV particles were eluted in the 20 mM fraction of phosphate buffer (pH 7.0) to which 0.1M NaCl had been added.

Figure 15 shows the distribution of HCV particles in each fraction by affinity chromatography by blue staining.

In affinity chromatography by blue staining, a support obtained by binding Blue Cibacron F3G-A to agarose particles was used for the column. The column was equilibrated with 20 mM phosphate buffer (pH 7.0). A solution containing HCV particles was added to the column that had been concentrated by an ultrafilter with a molecular weight limit between 100,000 and

500,000 and then diluted with 20 mM of a phosphate buffer (pH 7.0). Next, a phosphate buffered saline solution passed through the column at a volume approximately 10 times greater than that of the column. Subsequently, 20 mM phosphate buffers (pH 7.0) to which 1M or 2M NaCl had been added passed successively through the column, at a volume approximately 3 times greater than that of the column. Then, 20 mM of a phosphate buffer (pH 7.0) to which 2M NaCl had been added passed through the column, at a volume approximately 5 times greater than that of the column. As a result, HCV particles were eluted in a non-binding fraction of the column. The ratio between HCV core protein and total protein mass was 3.33 when compared to HCV particles before column purification and, therefore, the ratio between HCV particles and total protein increased. .

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4. Centrifugation in sucrose density gradient

The HCV particles were purified by the combined use of column chromatography and sucrose density gradient centrifugation with reference to the examples mentioned above.

First, the HCV particles were purified using Q Sepharose HP (R). The column was equilibrated with 50 mM of a Tris-HCl buffer (pH 8.0). A solution containing HCV particles was added to the column that had been concentrated by an ultrafilter with a fractional molecular weight between 100,000 and 500,000 and then diluted with 50 mM of a Tris-HCl buffer (pH 8.0). Next, 50 mM of a Tris-HCl buffer (pH 8.0) passed through the column at a volume approximately 10 times greater than that of the column. Subsequently, 50 mM of the Tris-HCl buffer (pH 8.0) to which each of 0.1M NaCl, 0.3M NaCl and 1M NaCl had been added successively passed through the column, at a volume approximately 3 times greater than of the column. Next, 50 mM of a Tris-HCl buffer (pH 8.0) to which 1M NaCl had been added passed through the column, at a volume approximately 5 times greater than that of the column (1M fraction of NaClW). As a result, as shown in Figure 16A, HCV particles were eluted in the 50 mM fraction of the Tris-HCl buffer (pH 8.0) to which 0.3M NaCl had been added; the 50 mM fraction of the Tris-HCl buffer (pH 8.0) to which 1M NaCl had been added; and the 1M fraction of NaClW. Fractions containing HCV particles were collected. The ratio between HCV core protein and total protein mass was 2.29 when compared with HCV particles before column purification and, therefore, the ratio between HCV particles and total protein increased. .

Second, HCV particles were purified by sulfated cellulophin chromatography. In each chromatography, the column was equilibrated with 20 mM of a phosphate buffer (pH 7.0). A solution containing HCV particles obtained by concentration by an ultrafilter with a molecular weight limit between 100,000 and 500,000 containing the HCV particle fractions purified with Q Sepharose HP (R) was added to the column, and then dilution of the result with 20 mM phosphate buffer (pH 7.0). Next, the phosphate buffer (pH 7.0) passed through the column at a volume approximately 10 times higher than that of the column. Subsequently, 20 mM phosphate buffers (pH 7.0) to which 0.25M or 1M NaCl had been added successively passed through the column, at a volume approximately 3 times greater than that of the column. Then, 20 mM of a phosphate buffer (pH 7.0) to which 1M NaCl had been added passed through the column, at a volume approximately 5 times greater than that of the column. As a result, as shown in Figure 16B, HCV particles were eluted primarily in 20 mM phosphate buffer (pH 7.0) to which 1M NaCl had been added. The ratio between HCV core protein and the total protein mass in 20 mM phosphate buffer (pH 7.0) to which 1M NaCl had been added was 31.4 when compared to HCV particles before purification in column Therefore, the ratio between HCV particles and total protein increased.

In addition, HCV particles were purified by sucrose density gradient centrifugation. The 20 mM fraction of the phosphate buffer (pH 7.0) to which 1M NaCl had been added by sulfated cellulophin chromatography was concentrated by an ultrafilter with a molecular weight limit between 100,000 and 500,000 and then diluted with a buffer TEN (10 mM Tris-HCl buffer (pH 8.0), 0.1M sodium chloride and 1 mM ethylenediaminetetraacetic acid (pH 8.0)). A solution containing HCV particles was laminated on a solution obtained by lamination of 60%, 50%, 40%, 30%, 20% and 10% sucrose solutions, and the obtained solution was centrifuged at 390 kg for 18 hours. at 4 ° C. As the HCV particles were grouped into a fraction with a specific gravity of approximately 1.2, the fraction was collected. The ratio between HCV core protein and total protein mass in the collected fraction was 1.69 when compared to HCV particles before column purification. Therefore, the ratio between HCV particles and total protein increased.

In the fraction containing purified HCV particles by sucrose density gradient centrifugation, the ratio between the HCV core protein and the total protein mass was approximately 120 times that purified when compared to the ratio before the start of the column chromatography The final fraction contained 109 copies / ml of HCV particles.

All publications, patents and patent applications cited here are incorporated herein by reference in their entirety.

Industrial Application

The present invention allows the production of HCV viral particles with various genotypes in a cultured cell system. That is, even in the case of an HCV strain that cannot replicate autonomously in vitro, such as HCV strains isolated from patients, the part of the RNA sequence thereof that encodes the NS3, NS4 proteins, NS5A and NS5B is replaced by a part of the RNA sequence that encodes the NS3, NS4, NS5A and NS5B proteins of JFH1, so that the above HSV strain can replicate autonomously in a cultured cell system, thus producing particles HCV The HCV particles purified by the present invention can be used directly as a vaccine for medical use. The HCV genomic RNA or viral particles provided by the present invention can also be used as a viral vector for a gene.

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5 strange. In addition, the method of the present invention can also be used for studies concerning an HCV infection process or for the production of a screening system for various substances that affect said HCV infection process.

Free Text of the Sequence Listing SEQ ID NO: 1 sequence encoding the NS3 to NS5 proteins of JFH1 (cDNA sequence)

10 SEQ ID NO: 2 sequence encoding the NS3 to NS5 proteins of JFH1 (cDNA sequence) SEQ ID NO: 3 NS5B protein of JFH1 SEQ ID NO: 4 Synthetic peptide designed based on an E2 fragment of JFH1 SEQ ID NO: 5 Synthetic peptide designed based on an E2 fragment of JFH1 SEQ ID NO: 6 Primer (R6-130-S17)

15 SEQ ID NO: 7 Primer (R6-290-R19) SEQ ID NO: 8 TaqMan Probe (R6-148-S21FT) SEQ ID NO: 9 Genomic RNA of the full length Hepatitis C virus derived from the

strain JFH1 (clone JFH-1) SEQ ID NO: 10 Genomic RNA sequence comprising the 5 'UTR to NS3 region of strain TH1 SEQ ID NO: 11 chimeric Hepatitis C virus genomic RNA derived from strain JFH1 HCV (clone JFH-1) and strain HCV TH

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SEQUENCE LIST

<110> Tokyo Metropolitan Organization for Medical Research Toray Industries Inc.

<120> Hepatitis C virus genomic RNA that can replicate autonomously

<130> PH-2565-PCT

<140>

<141>

<150> JP 2004-290801

<151> 01.10.2004

<150> JP 2004-243975

<151> 24.08.2004

<150> JP 2005-069527

<151> 11.03.2005

<150> JP 2005-069725

<151> 11.03.2005

<160> 11

<170> Patentln 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> sequence encoding the NS3 to NS5 proteins of JFH1 (cDNA sequence)

<400> 1

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<210> 2

<211> 1773 5 <212> DNA

<213> Hepatitis C virus

<220>

<221> CDS

<222> (1) .. (1773) 10 <220>

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

<400> 2

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<210>

 3

<211>

 591

5

<212>  PRT

<213>

Hepatitis C virus

<220>

<223>

NS5B protein from JFH1

<400>

 3

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<210> 4

<211> 14

<212> PRT

<213> Artificial Sequence

<220>

<223> Description of the Artificial Sequence: Synthetic peptide designed on the basis of the E2 fragment of JFH1

<400> 4

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<210> 5

<211> 14

<212> PRT

<213> Artificial Sequence 15 <220>

<223> Description of the Artificial Sequence: Synthetic peptide designed based on the E2 fragment of JFH1

<400> 5

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20 <210> 6

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<211> 17

<212> DNA

<213> Artificial sequence

<220>

<223> Description of the Artificial Sequence: primer (R6-130-S17)

<400> 6 cgggagagcc atagtgg 17

<210> 7

<211> 19

<212> DNA

<213> Artificial Sequence

<220>

<223> Description of the Artificial Sequence: primer (R6-290-R19)

<400> 7 agtaccacaa ggcctttcg 19

<210> 8

<211> 21

<212> DNA

<213> Artificial Sequence

<220>

<223> Description of the Artificial Sequence: TaqMan probe (R6-148-S21FT)

<400> 8 ctgcggaacc ggtgagtaca c 21

<210> 9

<211> 9707

<212> RNA

<213> Homo sapiens

<220>

<223> Genomic RNA of the full-length hepatitis C virus derived from strain JFH1 (clone JFH-1)

<400> 9

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<210>

 10

<211>

 3748

<212>

 DNA

5

<213> Hepatitis C virus

<220>

<223>

Genomic RNA sequence comprising the 5 ’UTR to NS3 region of strain TH1

<400>

 10

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<210> 11

<211> 11102 5 <212> RNA

<213> Homo sapiens

<220>

<223> Chimeric hepatitis C virus genomic RNA derived from HCV strain JFH1 (clone JFH-1) and HCV strain TH

10 <400> 11

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Claims (30)

one.

Genomic RNA of the modified hepatitis C virus comprising parts of the genomic RNA of two or more hepatitis C virus strains, which comprises a 5 'untranslated region, a nucleus protein coding sequence, a coding sequence of the E1 protein, a coding sequence of the E2 protein, a coding sequence of the p7 protein, a coding sequence of the NS2 protein, a partial RNA sequence encoding the NS3, NS4, NS5A and NS5B proteins of a strain JFH1 shown in SEQ ID NO: 1 and a 3 'untranslated region, and which can be replicated autonomously, where one of the hepatitis C virus strains is a genotype 1b viral strain.

2.

Genomic RNA of the modified hepatitis C virus comprising parts of the genomic RNA of two or more hepatitis C virus strains, which comprises a 5 'untranslated region, a nucleus protein coding sequence, a coding sequence of the E1 protein, a coding sequence for the E2 protein, a coding sequence for the p7 protein, a coding sequence for the NS2 protein, a coding sequence for the NS3 protein, a coding sequence for the NS4A protein, a coding sequence for the protein NS4B, a coding sequence of the NS5A protein, a coding sequence of the NS5B protein of a strain JFH1 shown in SEQ ID NO: 2 and a non-translated region 3 ', and which can be replicated autonomously, where one of the Hepatitis C virus strains is a viral strain of genotype 1b.

3.

Hepatitis C modified genomic RNA according to claim 1 or 2, characterized in that the genotype 1b viral strain is selected from a strain HCV-con1, a strain HCVTH, a strain HCV-J, a strain HCV-JT and a strain HCV-BK.

Four.

Genomic RNA of the modified hepatitis C virus according to claim 1 or 2, characterized in that said one of the hepatitis C virus strains is a HCV-con1 strain of genotype 1b.

5.

Genomic RNA of the modified hepatitis C virus according to claim 4, characterized in that the sequence encoding the E1 protein, the E2 protein, the p7 protein and the NS2 protein of the HCV-con1 strain is ligated downstream of the untranslated region 5 'of strain JFH1 and, downstream thereof, the sequence encoding the NS3, NS4A, NS4B, NS5A and NS5B proteins of strain JFH1 and, downstream thereof, the 3' untranslated region of the strain JFH1.

6.

Genomic RNA of the modified hepatitis C virus according to claim 1 or 2, characterized in that said one of the hepatitis C virus strains is a genotype 1b HCV-TH strain.

7.

Genomic RNA of the modified hepatitis C virus according to claim 6, comprising the nucleotide sequence as shown in SEQ ID NO: 11.

8.

Genomic RNA of the modified hepatitis C virus comprising parts of the genomic RNA of two hepatitis C virus strains, characterized in that said hepatitis C virus strains are a JPH1 strain and the JCH1 strain, and said genomic virus RNA Hepatitis C modified comprises a sequence corresponding to 1 to 3866 of the GenBank Accession No. AB047640 sequence linked to a sequence corresponding to 3867 to 9678 of the GenBank Accession No. AB047639 sequence, and which can be replicated autonomously

9.

Viral vector directed to liver cells, comprising the genomic RNA of the modified hepatitis C virus according to any of claims 1 to 8.

10.

Cell in which the genomic RNA of the modified hepatitis C virus is introduced according to any one of claims 1 to 8, and which replicates the genomic RNA of the hepatitis C virus and can generate viral particles.

eleven.

Hepatitis C viral particles, which are obtained from a culture obtained by culturing the cell according to claim 10.

12.

Method for purifying HCV particles by subjecting a liquid or a product obtained from the cell homogenate containing HCV particles according to claim 11 to a column chromatography and / or density gradient centrifugation used in combination.

13.

Method according to claim 12, characterized in that the column chromatography is one

or various types of chromatographs selected from ion exchange chromatography, gel filtration chromatography and affinity chromatography.

14.

Method according to claim 13, characterized in that the ion exchange chromatography is one or more types of chromatographs selected from anionic chromatography and

cationic chromatography, gel filtration chromatography is one or more types of chromatography using a resin selected from Sephacryl-S300 (R), SephacrylS400 (R) and Sephacryl-S500 (R), and affinity chromatography is one or several types of chromatographs using a resin selected from sulfated cellulophin, heparin and lectin.

fifteen.

Method according to claim 13, characterized in that the chromatography is sulphated cellulophin chromatography.

16.

Method according to claim 12, characterized in that density gradient centrifugation is carried out using one or more solutes selected from cesium chloride, sucrose and sugar polymers.

17.

Method according to claim 12, characterized in that, in the purification method, anion exchange chromatography, sulphated cellulophin chromatography and sucrose density gradient centrifugation are carried out, at least once, respectively, and combined in any order.

18.

HCV particles, which are obtained by means of a method for purifying HCV particles, characterized in that a liquid or a solution obtained from the cell homogenate containing HCV particles according to claim 11 is subjected to column chromatography and centrifugation in density gradient used in combination.

19.

HCV particles according to claim 18, characterized in that the column chromatography is one or more types of chromatographs selected from ion exchange chromatography, gel filtration chromatography and affinity chromatography.

twenty.

HCV particles according to claim 18, characterized in that the ion exchange chromatography is one or more types of chromatographs selected from anionic chromatography and cationic chromatography, gel filtration chromatography is one

image 1 or various types of chromatographs using a resin selected from Sephacryl-S300 (R), Sephacryl-S400 (R), and Sephacryl-S500 (R), and affinity chromatography is one or more types of chromatography using a selected resin between sulfated cellulophin, heparin, and lectin.

twenty-one.

HCV particles according to claim 18, characterized in that the chromatography is sulfated cellulophin chromatography.

22

HCV particles according to claim 18, characterized in that density gradient centrifugation is carried out using one or more solutes selected from cesium chloride, sucrose and sugar polymers.

2. 3.

HCV particles according to claim 18, which are purified by means of the purification method, wherein anion exchange chromatography, sulphated cellulophin chromatography and gradient centrifugation of sucrose density are carried out in combination.

24.

Use of viral hepatitis C particles according to claim 11 or 18 as antigens to produce a hepatitis C vaccine and / or a neutralizing antibody.

25.

Cell infected by the hepatitis C virus, which is infected by viral hepatitis C particles according to claim 11 or 18.

26.

Method for producing a hepatitis C viral particle, characterized in that the method comprises culturing the cell according to claim 10 and recovering the viral particles from the culture.

27.

Method for producing a cell infected with the hepatitis C virus, characterized in that the method comprises culturing the cell according to claim 10, recovering a supernatant from the cell culture and applying the supernatant to another cell to infect said cell. another cell with viral particles contained in the culture supernatant.

28.

Method for screening a hepatitis C antivirus substance, characterized in that the method comprises culturing the cell according to claim 10 or 25 in the presence of a test substance and detecting hepatitis C virus RNA or viral particles in the culture, thus evaluating the effects of the hepatitis C virus on the test substance.

29.

Method for producing a vaccine against hepatitis C and / or neutralizing antibodies by using viral hepatitis C particles according to claim 11 or 18 as antigens.

30

Method for replicating and / or expressing a foreign gene in a cell, characterized in that the method comprises the insertion of the RNA encoding the foreign gene into the genomic RNA of the hepatitis C modified virus according to any one of claims 1 to 8, and the introduction of genomic RNA into a cell of interest, to replicate or express the foreign gene inside.