Viral hepatitis caused by the hepatitis C virus (HCV) affects millions of people worldwide. The non-structural protein 3 (NS3), one of the most conserved proteins in HCV, is the target of many therapeutic studies. The NS3 protease domain (NS3p) has a range of cytotoxic T lymphocyte (CTL) epitopes, and synthesizing the protein inside the cells is the most appropriate way to present it to the immune system. We developed a tool to study this kind of presentation, using two vectored particle (VP) systems, one based on the Semliki Forest virus (SFV) and the other on HCV pseudoparticles (HCVpp), both carrying the protease domain of the NS3 gene. In addition to producing the particles, we developed a method to quantify these VPs using qRT-PCR. We produced batches of approximately 2.4 × 104 SFV-NS3p/μL and 4.0 × 102 HCVpp-NS3p/μL. BHK-21 and HuH-7 cells treated with the VPs expressed the NS3 protein, thus showing the functionality of this system.
Keywords: Electroporation of HEK-293T cells; Hepatitis C; Transfection; Vectored particles (VP); qPCR titration.
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