Transient expression of the hepatitis C virus core and NS3 proteins in mouse cell line BNL CL.2

Abstract

HCV infections may cause, self-limited disease as well as more commonly chronic infections that result in chronic liver disease, cirrhosis, and hepatocellular carcinoma. Therfore, there is an important need for a vaccine to protect against infectin by this virus. The induction of neutralizing antibodies of broad reactivity was hampered by several reasons; heterogeneous nature of HCV genome, high nutation rate, and hypervariable region in envelope proteins. As a preliminary step of induction of HCV-specific cytotoxic T-lymphocytes (CTL), we transfered MFG recombinant retroviral vectors, which contained the genes encoding HCV core protein and NS3 protein, into mouse cells, and then investigated the expression of two proteins. Cationic lipid-mediated gene transfer (lipofection) was employed as a transfection method. For the high-efficiency transfection, we optimized several factors which are known to be critical. The ratio of lipid to DNA, the length of time the cells are exposed to the lipid/DNA complexes, and the seeding density were determined on the basis of -galactosidase activity exhibited by a control reporter plasmid RSV- Gal. The synthesis of a core protein product approximately 21 kDa (P21) was confirmed, and a processed form of P21 was also detected. HCV NS3 protein of about 65 kDa was expressed in MFG recombinant retroviral vector DNA-transfected mouse cells. Bicistronic recombinant plasmids successfully led to synthesis of the two proteins.