Expression of the full-length HCV core subgenome from HCV gentoype-1a and genotype-3a and evaluation of the antigenicity of translational products

Abstract

Hepatitis C virus (HCV) infection is a major public health problem in India. Detection of HCV and its genotypes by simple and economic assays is a prime requirement in the planning of antiviral treatments for patients infected with this virus. Although commercial assays are available for the detection of both HCV RNA and genotypes, efforts aimed at the development of simple and economical systems for these measurements are still going on. The present study was designed to clone and express the HCV CORE gene from HCV genotype-1a and genotype-3a and use the peptides to develop immunoassays for the detection of genotype-specific antibodies in sera samples. One hundred and thirty-five serum samples from patients with liver and renal diseases were screened for HCV RNA by real-time PCR, followed by HCV genotyping in RNA-positive sera by restriction fragment length polymorphism, sequencing, and phylogenetic analysis. The HCV CORE gene was amplified from sera carrying HCV genotype-1a and genotype-3a and cloned and expressed in the pET19b vector. The translational products were used to develop a western blot assay for the detection of genotype-specific anti-HCV antibodies. The HCV CORE gene, from both genotypes, was cloned and expressed successfully, with production of a 26 kDa recombinant protein in either case. Using peptides in a western blot assay, 101 sera samples were tested for the anti-HCV CORE antibody. Each peptide showed a reaction with anti-HCV total antibody without showing any genotype-specific binding. This indicates that individual peptides obtained from different genotypes do not have a genotype-specific epitope to bind with antibodies. Cloning and expression of the HCV CORE gene from genotype-1a and genotype-3a was successful. However, the peptides formed did not show genotype-specific binding with anti-HCV.