Amplification and Cloning of Entire Structural Genome (Core- E2) of Hepatitis C virus

Abstract

HCV is the leading cause of liver related morbidity and mortality around the world. Chronic infection usually leads to serious outcomes like cirrhosis, hepatocellular carcinoma and metabolic abnormalities.  Inability of HCV to replicate in cell culture and absence of efficient and cost effective animal models are the major hurdles in developing therpeutic strategies against this virus. Cloning and expression of HCV entire structural genome in eukaryotic expression system was observed in the current study. Structural genes of HCV are important in mediating viral entry in the host cell and pathogenesis, most notably hepatocellular carcinoma. HCV 3a is the most prevalent genotype in Pakistan. RNA was extracted from HCV positive serum infected with 3a genotype, and entire structural genome (C-E2) was reverse transcribed. HCV (C-E2) region was amplified using PCR with gene specific primers having restriction sites. Digested product of this amplicon was cloned in mammalian expression vector pcDNA3.1+. Positive clones were confirmed after double restriction digetsion and sequencing PCR. This successful clone of (C-E2) would be a useful tool for transfection to particular cell lines and further investigation on stable cell lines using this clone may help in designing new therapies and studying interaction of viral proteins with host cells.