Résumé
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-beta-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