Expression, Purification, And Applications Of Hepatitis C Virus (HCV) Envelope Proteins

Hepatitis C virus (HCV) infection could lead to chronic hepatitis, liver cirrhosis, and even hepatocellular carcinoma, constituting a major threat to human health. There is a pressing need to develop effective anti-HCV vaccines. Different lines of evidence suggest that the envelope proteins of HCV, E1 (aa 192-383) and E2 (384-746), may play important and vital roles in its infection of target cells, thus becoming the main targets of HCV vaccine research. Research on envelope protein-based HCV vaccine demands a prompt solution to the problems of detection of antigen and evaluation of post-vaccination immune responses, which requires large amounts of highly purified envelope proteins and corresponding antibody or antiserum. Therefore, the expression and purification of subtype 1b envelope proteins by using the Escherichia coli system were studied.Four E2 fragments, aa 385-565, aa 450-565, aa 567-730 and aa 385-730, were expressed as 6xHis fusion proteins in E. coli to a high level and purified to high purity. In immuno-blotting, these fragments reacted specifically with hepatitis C patients' sera, suggesting that E. coli-derived E2 proteins carried HCV E2-specific, glycosylation-and-conformation-independent epitopes. ELISA system for the detection of anti-E2 antibodies in patients' sera was established using aa 385-730 fragment, which detected anti-E2 in 56% of hepatitis C patients and demonstrated a correlation between anti-E2 and HCV viraemia. By using aa 385-365 fragment, an ELISA system for the evaluation of post-E2-vaccination humoral immune responses was also established, and was successfully applied to recombinant vaccinia virus- and DNA-based vaccine research. By immunizing rabbits with aa 450-565 or aa 385-730 fragment, polyclonal sera were obtained that were able to recognize E2 proteins expressed in both E. coli and mammalian cells, and cross-react between certain HCV subtypes. Two E1 fragments, aa 192-340 and aa 192-326, were expressed as 6xHis fusion proteins in E. coli to a high level and purified to high purity. By immunizing rabbits with aa 192-326 fragment, polyclonal sera were obtained that were able to recognize E1 proteins expressed in both E. coli and mammalian cells, suggesting that E. coli-derived E2 proteins carried HCV E1-specific, glycosylation-and-conformation-independent epitopes.This work produced large amounts of high-purity envelope protein antigens and envelope-specific antisera, and established detection systems for envelope antigens as well as antibodies. Meanwhile, in the process of expressing envelope proteins, some useful insights into the mechanisms governing HCV envelope protein expression in E. coli were obtained.