Genetical Engineering Of Hepatitis C Virus Rna Dependent Rna Polymerase And Establishment Of Anovel Method For Its Enzyme Activity Assay And Preliminary Application Of The Method

Hepatitis C virus (HCV) infection remains a global problem, which is the major cause of severe chornic hepatitis that progresses to liver cihrrosis and hepatocellular carcinoma. Chronic infection with HCV represents a serious threat to human health and it shows an upward trend in infection rates. Since the research of cell and animal models are not progressing smoothly, there is no effective vaccine to prevent HCV infection presently. Therefore, there is an obvious and urgent need to develop more effective and tolerated treatments. HCV RNA-dependent RNA polymerase (RdRp) is a nonstructural protein of HCV which has been characterized as the catalytic core of viral replicase. There is no close structural homologues of this enzyme existing within the uninfected host cell. Thus, it represents a good target for antiviral investigations and HCV RdRp inhibitor screening.The key point of HCV RdRp inhibitors screening in vitro is to obtain HCV RdRp protein with good solubility and high enzyme activity, while the C terminal of the protein has some effects on its solubility and enzyme activity. Different expression systems have significant differences in pritein expression levels as well as its biological activity. It is very important to select an appropriate expression system for improving the expression level. Currently available are a variety of different expression systems, such as E. coli, insect cells, yeast and mammalian cells. In this study, E.coli, Pichia pastoris and Chinese hamster ovary (CHO) cells were applied to the research. The positive RdRp recombinant plasmids with C-terminal 21 aa truncated were obtained by identification of PCR amplification, they were named pet28a(+)-HCV-RdRp, pPICZα-HCV-RdRp and pcDNA3.1-HCV-RdRp respectively. The three recombinant plasmids were expressed and identified in E.coli, Pichia pastoris and CHO cells.Several factors influencing RdRp expression level and solubility were optimized by using single factor tests. The expressed RdRp was detected by SDS-PAGE, the results showed that amplification cultural time, IPTG concentration, induction temperature and induction time have significant effects on solubility of the expressed RdRp. The best condition for soluble expression of HCV RdRp was obtained. The expressed RdRp protein were purified with Ni-Affinity column and then detected by Western blot, the HCV RdRp fusion protein was efficiently soluble expressed in E.coli.To optimize the sequence of pcDNA3.1 vector, transcriptional regulatory sequences, signal peptide of human IgG light chain in variable region and 8×his tag were introduced into the vector, which expected to make HCV RdRp protein secreted into the cell supernatant. After transfection of CHO cells with the recombinant plasmid, the expressed protein was detected by Competitive ELISA and was purified through Ni affinity chromatography. HCV RdRp protein was expressed in the supernatant of CHO cells. However , it showed a low expression.The recombinant plasmid pPICZα-HCV-RdRp was transfected into Pichia pastoris GS115 with electric shock. A single colony was inoculated in the medium at 30℃, and then induced by methanol continuously for 72 hours. The results of Western blot showed that the HCV RdRp protein was not secretively expressed in Pichia pastoris yeast. The study combined magnetic nanoparticle enrichment and separation as well as ELISA technologies, successfully established a novel method for measuring the enzyme activity of HCV RdRp in vitro. The RNA template was fixed in the magnetic nanoparticle, then rNTP, purified HCV RdRp, Bio-16-UTP and buffer were added into the reaction system. In the activity of RdRp, another chain was extended. By the separation of external magnetic, non-specific adsorption fragments were removed. Then HRP labeled Avidin–Peroxidase was added to affinity Bio-16-UTP. After scrubbing, added TMB, judged the activity of RdRp through coloration or not. The concentration and conditions of factors involved in replication in vitro were optimized, and the best reaction conditions were obtained. The novel activity assay method established the foundation for HCV RdRp inhibitor high flux screening in vitro.