[Cloning of hepatitis C virus open reading frame sequence and non structural protein expressions of the virus]

To induce the expressions of the constituent proteins of hepatitis C virus (HCV) using a vaccinia virus expression system.

The open reading frame (ORF) sequence encoding HCV large polyprotein precursor was cloned into a vaccinia virus promoter to construct the recombinant plasmid pVHCV, which was subsequently used, along with a control plasmid (pSC59) in a parallel experiment, to transfect BHK21 cells via Lipofectamine 2000 reagent, followed by infection of the cells with vTF7-3 vaccinia viruses. After a 48-h culture, the expressions of HCV nonstructural proteins NS3 and NS5a in the cells were detected using Western blotting and immunofluorescence assay.

Western blotting analysis presented the nonstructural proteins NS3 and NS5a detected in pVHCV-transfected BKH21 cells as two bands with molecular weights of 70,000 and 56,000, respectively. In the immunofluorescence assay, intense granular fluorescence signal was detected in the cytoplasm of pVHCV-transfected cells, irrespective of the use of either NS3- or NS5a- specific mouse monoclonal antibody.

HCV nonstructural proteins NS3 and NS5a can be expressed in BKH21 cells.