Abstract
BACKGROUND: Detection of hepatitis C virus specific antibodies is the initial step in chronic HCV diagnosis. HCV NS4B is among the most immunogenic HCV antigens and has been widely used in commercial Enzyme Immunoassays (EIA). Additionally, NS4B, a key protein in the virus replication, can be an alternative target for antiviral therapy. OBJECTIVES: Development of a new method for high-level expression and purification of NS4B coding region was the aim of the report. MATERIALS AND METHODS: 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 1 mM isopropyl-β-D-thiogalactopyranoside (IPTG) to the culture medium. Immunoreactivity of the purified recombinant proteins was evaluated by immunoblotting and indirect enzymelinked immunosorbent assay (ELISA). RESULTS: The recombinant NS4B protein was expressed and its immunoreactivity was confirmed by ELISA and western blotting. CONCLUSIONS: The directional TOPO cloning provides an efficient and easy platform for heterologous expression of immunoreactive HCV NS4B.