Cloning and expression of NS۳ helicase fragment of hepatitis C virus and the study of its immunoreactivity in HCV infected patients

Objective(s): Hepatitis C is a major cause of liver failure worldwide. Current therapies applied for this disease are not fully effective and produce side effects in most cases. Non-structural protein ۳ helicase (NS۳) of HCV is one of the key enzymes in viral replication and infection. Therefore, this region is a promising target to design new drugs and therapies against HCV infection. The aim of this study was cloning and expression of HCV NS۳ helicase fragment in Escherichia coli BL۲۱ (DE۳) using pET۱۰۲/D-TOPO expression vector and studying immunoreactivity of the expressed antigen in Iranian infected with hepatitis C. Materials and Methods: The viral RNA was extracted from the serum of HCV infected patient. The NS۳ helicase region was amplified by RT-PCR. The PCR product was directionally cloned into the expression vector pET۱۰۲/D-TOPO and transformed into the BL۲۱ strain of E. coli (DE۳). The transformed bacteria were then induced by adding ۱mM isopropyl-β-D-thiogalactopyranoside (IPTG) into the culture medium to enhance the protein expression. SDS-PAGE and western blotting were carried out to identify the protein under investigation, and finally purified recombinant fusion protein was used as the antigen for ELISA method. Results: Theinsertion of theDNA fragment of the NS۳ regioninto the expression vectorwas further confirmed by PCR and sequencing. SDS-PAGE analysis showed the successful expression of the recombinant protein of interest. Furthermore, immunoreactivity of fusion NS۳ helicase was confirmed by ELISA and western blotting. Conclusion: It seems that this recombinant protein could be a useful source of antigen for future studies on HCV diagnosis and therapy.

Keywords:

HCV , Molecular Cloning , NS۳ helicase , Recombinant protein