| Objective Through constructing two HCV full-length plasmid with chimeric HCV en-velope protein genes of 1b or 1a sub-type,to develop cell culture system for chimeric HCV, and further provide effective model for screening anti-virus drugs and evaluation of immune effects upon major genotype from china. Methods 1.Overlapping PCR was used to clone HCV envelope gene (E1/E2) of Hebei isolates (1b) in china and H77 strain(1a)which were inserted into JFH1(2a) backbone to construct chimeric virus (1b/2a and 1a/2a) full-length plasmid with regions of envelope protein gene replaced reciprocally.2. Then the 1b/2a and 1a/2a full-length plasmid were digested by Xbal single enzyme and transcribed in vitro to ob-tain full-length virus RNA. After the RNA were transfected into Huh7.5.1 cell strain,indi-rect immunofluorescence method and western blot assay were employed to determine the posi-tive cell number of transfection and expression of the protein. Results 1. This study success-fully constructed chimeric virus (1b/2a and 1a/2a)full-length plasmid pHeBei (E1E2)/ JFH1 and pH77(E1E2)/JFH1 with regions of envelope protein gene replaced reciprocally.2. Respectively using pHeBei (E1E2)/JFH1 and pH77 (E1E2)/JFH1 as template to tran-scribe in vitro, then HCV RNA was transfected into Huh7.5.1 hepatocarcinoma cells.72h af-ter transfection, The results of indirect immunofluorescence method showed that transfection efficiency was between 1% to 3%,and from 20% to 50% after transfection for 5 days;The re-sults of western blot showed that there were expressions of HCV nonstructural protein NS3 and structural protein Core. Conclusions It is the first time in domestic that HCV cell culture sys-tem for chimeric envelope gene of HCV strain isolated from HeBei province china. Virus RNA can replicate and express in cultured cells and produce chimeric HCV, and can transmit among co-cultured cells. |
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