A Research On The Establishment Of A New Cell Culture Model Of Hepatitis C Virus

[Objective]:This project aims to build a new plasmid containing the gene of the full-length of HCV genome and the luciferase gene, which can be directly transfected into cells so that the expression of luciferase can reflect the HCV RNA expression in cells and to some extent quantitate the HCV RNA level. To preliminarily establish a new kind of practical and convenience cell culture research model for HCV infection in vitro.[Methods]:1. Use the gene cloning method to insert the firefly luciferase gene fragment into the HCV full-length gene replicon, construct a kind of dicistronic HCV replicon eukaryote expression plasmid which contains HCV full-length gene and firefly luciferase gene in vitro, and then excise the18nucleotides including RNA polymerase active site in NS5B end to construct a kind of replication-defective recombinant plasmid. The recombinant plasmid and the corresponding replication-defective recombinant plasmid are identified by restriction enzyme digestion and sequencing analysis.2. Make use of calcium phosphate to mediate the plasmid transfection to HEK-293T cells.3. Detect firefly luciferase values in transfected HEK-293T cells with a luminometer.4. Semi-quantitative detect HCV RNA transcription levels with plus and minus strand primers respectively with RT-PCR.5. Detect HCV RNA in the cell culture medium with qPCR.[Results]:1. Plasmid restriction enzyme digestion and sequencing analysis proved the correct construction of the recombinant eukaryotic expression plasmid and the replication-defective recombinant plasmid.2. With plus and minus strand primers as a RT primer, we can both amplify the target segment,which implies that recombinant eukaryotic expression plasmid can correctly transcript HCV RNA and it’s efficiency replication in HEK-293T cells is permitted. We found out that the HCV RNA level in the cell culture medium is up to10IU/ml.3. Only can plus strand primer be used to amplify the target segment, shows that replication-defective recombinant plasmid can take the correct transcription in HEK-293T cells, but can not replicate, the HCV RNA levels in the cell culture medium is less than500IU/ml.[Conclusions]:A success in construction of the recombinant eukary-otic expression plasmid and the corresponding replication-defective reco-mbinant plasmid and preliminary verification of its expression in HEK-293T cells will provide an effective technology platform in HCV cell culture model for further research of HCV pathogenesis and drug screening.