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[0010] In view of the above situation, there is a need to develop a production method for HCV particles that can produce high-yield infectious HCV particles with a genotype 1b structure that is less effective against existing therapies and that can be cultured in a persistent infection system
Method used
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Embodiment 1
[0154] Embodiment 1: Construction of TH / JFH-1 plasmid
[0155] As the cDNA of HCV genomic RNA, a TH / JFH-1 chimeric cDNA was prepared, wherein the 5' UTR was the JFH-1 strain of genotype 2a (GenBank Accession No. AB047639, Kato, T. et al., Gastroenterology, 125: 1808 -1817, 2003), the N-terminal 33 amino acid residues of core protein to NS2 protein are TH strains of genotype 1b (Wakita, T. et al., J.Biol.Chem., 269:14205-14210, 1994, Moradpour, D. et al., Biochem.Biophys.Res.Commun., 246:920-924, 1998 and International Publication WO2006 / 022422), the N-terminal 34 amino acid residues ~ 3'UTR of NS2 are JFH- 1 plant.
[0156] The amino acid sequence of the protein encoded by TH / JFH-1 is shown in SEQ ID NO:5. For the preparation of these plasmids, see figure 1 .
[0157] Specifically, pJFH1 (Wakita, T. et al., Nat. Med., 11:791-796, 2005, International Publication WO2004 / 104198) and pTH ( International Publication WO2006 / 022422), the pJFH1 is a plasmidDNA constructed by cl...
Embodiment 2
[0163] Example 2: In vitroRNA synthesis and its introduction into cells
[0164] pTH / JFH1 was cleaved with XbaI, extracted with phenol / chloroform, and precipitated with ethanol. Next, this XbaI cleavage fragment was treated with mung beannuclease to remove the remaining nucleotide sequence derived from the 3' end of the XbaI recognition sequence. Then proteinase K treatment, phenol / chloroform extraction, and ethanolprecipitation were performed to purify the DNA fragment. Using the cutplasmid as a template, the MEGAscriptT7 kit (Ambion) was used to react at 37°C for 3 hours to synthesize HCV RNA. After the reaction, the synthesized RNA was treated with DNaseI, extracted with acid phenol, and precipitated with ethanol to purify it.
[0165] will be 3×10 6 Huh7 cells and 10 μg HCV RNA were suspended in 400 μl Cytomix solution (120 mM KCl, 0.15 mM CaCl 2 , 10mM K 2 HPO 4 / KH 2 PO 4 , 25mM Hepes, 2mM EGTA, 5mM MgCl 2 , 20 mM ATP, 50 mM glutathione), transferred to 4 mm...
Embodiment 3
[0166] Example 3: HCV particles produced by cells introduced with TH / JFH-1 RNA
[0167] When the TH / JFH-1RNA-introduced cells were subcultured, the HCV core protein contained in the culture supernatant was quantified using the HCV antigenELISA kit (Oiso Co., Ltd.) to confirm the production of HCV particles. As a result, the HCV core amount in the culture supernatant decreased over time until the 23rd day after the introduction, but the HCV core amount began to increase after 26 days from the introduction, and showed a constant high yield after 34 days ( figure 2 ). Therefore, it is believed that: TH / JFH-1RNA did not have high virus production ability at the beginning when it was introduced into Huh7 cells, but then the adaptive mutation necessary for virus production was introduced into the virus genome, so that TH / JFH-1RNA had high virus production ability. Virus production capacity.
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Abstract
Disclosed are: an infectious chimeric HCV particle which can be used as a vaccine; Core protein, E1 protein, E2 protein and p7 protein, all of which are derived from a hepatitis type-C virus strain other than JFH-1 strain; NS2 protein derived from hepatitis type-C virus JFH-1 strain or a hepatitis type-C virus strain other than JFH-1 strain, or a chimeric NS2 protein composed of NS2 protein derived from hepatitis type-C virus JFH-1 strain and NS2 protein derived from a hepatitis type-C virus strain other than JFH-1 strain; a nucleic acid containing a chimeric gene derived from a hepatitis type-C virus, which comprises domains respectively encoding NS3 protein, NS4A protein, NS4B protein, NS5A protein and NS5B protein all derived from JFH-1 strain in this order from the 5'-side toward the 3'-side, wherein a proline residue at position-328 from the amino acid residue located at the N-terminal of the Core protein is substituted by an amino acid residue other than a proline residue; a chimeric HCV particle containing the nucleic acid; and use of the HCV particle as a vaccine.
Description
technical field [0001] The present invention relates to nucleic acids containing chimeric genes derived from hepatitis C virus, chimeric hepatitis C virus particles of JFH-1 strains and strains other than JFH-1 strains (preferably strains belonging to genotype 1a, 1b or 2a), A vector for producing the virus particle, and a cell for producing the virus particle. [0002] The present invention also relates to a method for screening anti-HCV drugs using the virus particle, a vaccine obtained by inactivating or attenuating the virus particle, and an anti-HCV antibody that recognizes the virus particle as an antigen. Background technique [0003] Hepatitis C virus (hereinafter abbreviated as "HCV") was discovered by Choo et al. in 1989 and identified as the causative virus of non-A non-B hepatitis (Non-Patent Document 1). HCV infection is the main cause of persistent infection after chronic hepatitis and transformation to liver cirrhosis and liver cancer. According to reports, t...
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