CLONING, PHYLOGENETIC AND EXPRESSION ANALYSIS OF NS2 GENE FROM HCV INFECTED PATIENTS IN PESHAWAR

ABSTRACT
Hepatitis is liver-specific blood borne disease, which may be acute and chronic. Hepatitis C virus is one of the fundamental
cause of liver inflammation and could result in cirrhosis and hepatocellular carcinoma. The prevalence of HCV
in Pakistan has been reported to be 4.7% and genotype 3a has been found to be the leading cause of infection in
Pakistan. HCV is RNA virus, single stranded and belongs to the family flaviviridae. The RNA genome is 9.6kb in length.
NS2, one of the important antiviral drug targets is a transmembrane protein. It contains an autoprotease mechanism
causing cleavage at NS2/NS3 junction. NS2 interacts with E1, E2, P7, NS3-4A, NS5A and NS5B thus maintaining
co-ordination between different kinds of proteins. In this study a total of 50 patients having positive HCV RNA in their
serum were selected from Peshawar district. Extraction of viral RNA followed by cDNA synthesis was performed and
then we PCR amplify NS2 gene and was confirmed through agarose gel electrophoresis and subsequently sequenced
through Sanger sequencing. Next NS2 gene was cloned into TA vector and sequenced. Phylogenetic analysis was
performed on the sequences reported in this study with sequences reported from different regions of the world. NS2
gene was cloned into expression vector and expression analysis was performed in Huh-7 cell line. Cell culture model
system developed in this study could be used in future for screening antiviral drugs, as alternative treatment options
are needed. In Khyber Pakhtunkhwa few large scale studies have been conducted in different districts and cities but
no detailed data is available concerning NS2 gene. This study is a starting step towards the development