Expression, Purification Of HCV NS5B And Establishment Of A Novel Method For Measuring Its Enzyme Activity In Vitro

Hepatitis C virus (HCV) is the major etiologic agent responsible for non-A, non-B hepatitis. More than 170 million people, or 3% of the entire world population, are infected with HCV. Viral infection frequently leads to chronic liver diseases, including cir-rhosis and hepatocellular carcinoma. Although much research has been focused on the development of anti-HCV agents, no vaccine is available to date and there is no effective therapy for all genotypes of HCV. The current therapy against chronic infection of HCV is a combination of interferon-αand ribavirin. However, this is often accompanied by severe side effects, and the response rate of approximately 50% is not satisfactory. Therefore, more potent and broad-spectrum inhibitors are needed.HCV is a member of the Hepacivirus family and has a positive single-strand RNA genome composed of approximately 9600 nucleotides. The viral genome contains a single open reading frame translated into one polyprotein that is subsequently cleaved by both host and viral proteases. The polyprotein is composed of 3010-3033 amino acids. It is processed by a host cell signal peptidase and two virally encoded proteases into four structural proteins (C, El, E2, and p7) and six nonstructural proteins (NS2 to NS5B). The order of these proteins is NH2-C-E1-E2-p7-NS2-NS3-NS4A-NS4B-NS5A-NS5BCOOH.NS5B has been characterized as an RNA-dependent RNA polymerase (RdRp) based upon in vitro experiments. Close structural homologues of this enzyme do not exist within the uninfected host cell. Thus, it represents a good target for antiviral investigations.To construct the engineering E.coli. expressing full-length HCV NS5B and NS5B with C-terminal 21 aa and 51 aa truncated and to acquire the protein for screening the anti-HCV drugs targeting the NS5B. The coding regions of the three different length of HCV NS5B gene were amplified by PCR and were digested by BamH I and Xho I . The gene fragments were cloned into plasmid pET-28a(+) and the recombinant plasmids were transformed into E. coli BL21 (DE3). The positive recombinant plasmids were obtained by identification of PCR amplification,they were named pET-28a(+)-NS5B-FL,pET-28a(+)-NS5B-C21, pET-28a(+)-NS5B-C51 respectively. The engineering E.coli. harboring the recombinant pasmids were induced by IPTG and analysed by SDS-PAGE. NS5B-FL, NS5B-C21 and NS5B-C51 proteins were efficiently expressed, expressed NS5B-FL is in the form of inclusion and the solubility of expressed NS5B-C21 and NS5B-C51 proteins were increased obviously. The three expressed proteins were purified with Ni-Affinity column successfully, which establishing foundation for screening the anti-HCV drugs targeting the NS5B.The research successfully modified magnetic nanoparticle. RNA was fixed in the magnetic nanoparticle in the reaction system,and then added in primer,RDRP,NTP,Bio-11-UTP and so on. In the activity of RDRP, the other chain was extended.And then Avidin-Peroxidase added affinity Bio-11-UTP fixed. After scrubbing,added in TMB,judged the activity of RDRP through coloration or not.