Nucleic acid construct containing fulllength genome of human hepatitis C virus, recombinant fulllength virus genome-replicating cells having the nucleic acid construct transferred thereinto and method of producing hepatitis C virus Particle
View Patent ↗The present invention provides a method for replicating efficiently an RNA containing fulllength HCV genomic sequence and a method for producing HCV virus particles containing fulllength HCV replicon RNA or fulllength HCV genomic RNA by using a cell culture system. Further, the present invention relates to a method for producing hepatitis C virus particles which comprises culturing a cell, into which a replicon RNA comprising a nucleotide sequence comprising a fulllength genomic RNA sequence of hepatitis C virus of the genotype 2 a , at least one selectable marker gene and/or at least one reporter gene and at least one IRES sequence or the fulllength genomic RNA of hepatitis C virus of the genotype 2 a is introduced, and generating virus particles in the culture medium. Still further the present invention relates also to a hepatitis C vaccine and an antibody against hepatitis C virus particles.
1. A replicon RNA, comprising a nucleotide sequence comprising a 5′ untranslated region comprising the nucleotide sequence of SEQ ID NO: 1, a core protein coding sequence comprising the nucleotide sequence of SEQ ID NO: 2, an E1 protein coding sequence comprising the nucleotide sequence of SEQ ID NO: 3, an E2 protein coding sequence comprising the nucleotide sequence of SEQ ID NO: 4, an NS2 protein coding sequence comprising the nucleotide sequence of SEQ ID NO: 5, an NS3 protein coding sequence comprising the nucleotide sequence of SEQ ID NO: 6, an NS4A protein coding sequence comprising the nucleotide sequence of SEQ ID NO: 7, an NS4B protein coding sequence comprising the nucleotide sequence of SEQ ID NO: 8, an NS5A protein coding sequence comprising the nucleotide sequence of SEQ ID NO: 9, an NS5B protein coding sequence comprising the nucleotide sequence of SEQ ID NO: 10, and a 3′ untranslated region of genomic RNA comprising the nucleotide sequence of SEQ ID NO: 11, at least one selectable marker gene and/or at least one reporter gene, and at least one IRES sequence.
2. The replicon RNA according to claim 1 , wherein said nucleotide sequence comprises the 5′ untranslated region, the at least one selectable marker gene and/or the at least one reporter gene, and the at least one IRES sequence, and the core protein coding sequence, the E1 protein coding sequence, the E2 protein coding sequence, the NS2 protein coding sequence, the NS3 protein coding sequence, the NS4A protein coding sequence, the NS4B protein coding sequence, the NS5A protein coding sequence, the NS5B protein coding sequence, and the 3′ untranslated region, in this order in the 5′ to 3′ direction.
3. A replicon RNA, comprising a 5′ untranslated region, a core protein coding sequence, an E1 protein coding sequence, an E2 protein coding sequence, an NS2 protein coding sequence, an NS3 protein coding sequence, an NS4A protein coding sequence, an NS4B protein coding sequence, an NS5A protein coding sequence, an NS5B protein coding sequence, and a 3′ untranslated region, at least one selectable marker gene and/or at least one reporter gene, and at least one IRES sequence, wherein said replicon comprises an RNA comprising the nucleotide sequence shown in SEQ ID NO: 13; which has autonomous replication ability and virus particle production ability.
4. A method for producing a cell which replicates a replicon RNA and produces a virus particle, comprising introducing the replicon RNA according to claim 1 or 3 into a cell.
5. The method according to claim 4 , wherein the cell is a proliferative cell.
6. The method according to claim 4 , wherein the cell is a eukaryotic cell.
7. The method according to claim 6 , wherein the eukaryotic cell is a human liver-derived cell, a human uterine cervix-derived cell or a human fetal kidney-derived cell.
8. The method according to claim 6 , wherein the eukaryotic cell is a Huh7 cell, a HepG2 cell, an IMY-N9 cell, a HeLa cell or a 293 cell.
9. An isolated cell obtainable by the method according to claim 4 , which replicates the replicon RNA and produces the virus particle.
10. A method for producing a hepatitis C virus particle, comprising culturing the cell according to claim 9 to allow the cell to produce the virus particle.
11. An isolated hepatitis C virus particle obtainable by the method according to claim 10 .
12. A method for producing a hepatitis C virus-infected cell, comprising culturing the cell according to claim 9 and infecting other cells with the virus particle in the culture.
13. An isolated hepatitis C virus-infected cell obtainable by the method according to claim 12 .
14. A method for screening an anti-hepatitis C virus substance, comprising culturing, in the presence of a test substance, at lease one selected from the group consisting of following (a), (b) and (c):
(a) the cell according to claim 9 ,
(b) an isolated cell infected with the hepatitis C virus particle produced by the cell of (a), and
(c) the hepatitis C virus particle and a hepatitis C virus permissive cell;
and detecting the replicon RNA or the virus particles in the resulting culture.
15. A hepatitis C immunogenic composition, comprising the hepatitis C virus particle according to claim 11 .
16. A method for producing a hepatitis C immunogenic composition, comprising mixing the particles according to claim 11 with a pharmaceutically acceptable excipient.
17. A method for producing a hepatotropic virus vector for gene therapy by integrating an RNA encoding a foreign gene into the replicon RNA according to claim 1 .
18. A hepatotropic virus vector obtainable by the method according to claim 17 .
19. A method for replicating and/or expressing a foreign gene in a cell, comprising inserting an RNA encoding the foreign gene to the replicon RNA according to claim 1 and introducing it into said cell and culturing said cell.
20. The method according to claim 19 , wherein the cell is a proliferative cell.