Objective: To induce the expressions of the constituent proteins of hepatitis C virus (HCV) using a vaccinia virus expression system.
Methods: 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.
Results: 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.
Conclusion: HCV nonstructural proteins NS3 and NS5a can be expressed in BKH21 cells.
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