Analysis of immumoreactivity of heterologously expressed non-structural protein 4B (NS4B) from Hepatitis C Virus (HCV) genotype 1a

Detection of antibodies against HCV is the initial step in chronic HCV diagnosis. HCV NS4B is among the most immunogenic HCV antigens and it has been widely used in commercial Enzyme Immunoassays (EIA) for HCV diagnosis. Furthermore, since NS4B is a key protein in the virus replication, it is an alternative target for antiviral therapy. Hence, rapid, high yield, and economical production of recombinant HCV NS4B is an obligation for producing HCV diagnostic kits and developing anti-HCV drugs. In the current study, we aimed to develop a new method for high-level expression and purification of NS4B coding region. Viral RNA was purified from the serum of an HCV positive patient and NS4B coding region was amplified using nested RT-PCR. PCR products were cloned into pET102/D-TOPO expression vector and transformed into E. Coli BL21. Induction was performed by adding 1mM isopropyl-β-D-thiogalactopyranoside (IPTG) to the culture medium. Immunogenicity of the purified recombinant proteins was evaluated by immunoblotting and indirect enzyme-linked immunosorbent assay (ELISA). The accuracy of the construct was confirmed by colony PCR and sequencing. SDS-PAGE analysis showed successful expression of the recombinant protein. ELISA and western blotting confirmed the immunoreactivity of the recombinant NS4B. The directional TOPO cloning provides an efficient and easy method for cloning and expression of recombinant HCV NS4B. The directional TOPO cloning should be evaluated for production of other viral proteins.