Key words: HCV, cloning, structural gene, core, E1, E2
By: Afiono Agung Prasetyo
The main purpose of this research is to get replicon and infectious system of Hepatitis C virus from locale isolate (Indonesia). The HCV replicon and infectious system are going to be used for replication strategy, diagnosis, vaccine, and therapy study of HCV. The present purpose of this study is to clone the HCV’s structural genes for diagnosis and vaccine development study. Previously, the HCV RNA was extracted from all plasma with anti-HCV positive followed with cDNA synthesis and nested RT-PCR addressed the part of HCV NS5B and E1-E2 region. The positive PCR products were directly sequenced and phylogenetic analysis. The predominant HCV genotype was HCV 1a, and the best isolate for cloning was 09IDSKAC-20. To clone of its structural genes, primers were designed with FastPCR. The HCV RNA was re-extracted from the plasma aliquot of 09IDSKAC-20. Cloning was performed via RT-PCR using Accuprime Pfx Polymerase, followed with PCR products purification. The PCR products were then subcloned into pETBlue-1 vector. The recombinant plasmis were transformed into competent cells. Competent cells were propagated, harvested, and the plasmid DNA was extracted and sequenced using universal primers for pETBlue-1: pETBlueUP primer #70604-3 for forward primer and pETBlueDOWN primer #70603-3 for reverse primer. The sequencing results were analysed using Sequence Viewer and MEGA4.