Screening The Cellular Proteins Interaction With HCV NS2 Protein And Study On The Mechanism Of Diosicn To Inhibit Virus Infection

The mechanism of HCV infection and replication isn't clarified well now because of the absence of good replicons in vitro and small animal mode. The structure and function of respect HCV proteins and the interaction of viral and cellular proteins are needed to be further studied as well. The function of NS2, a nonstructural protein, in the replication of HCV is still unknown yet to date. In order to have a deep understanding of the structure and function of NS2, NS2 was expressed in prokaryotic and eukaryotic cells first and anti-NS2 antibody was generated by immunizing rabbits. The gene corresponding to the 66 amino acids of C-terminal of NS2 protein were successfully cloned and expressed in E.coli and the proteins were purified through affinity chromatography and immunized the rabbits. In addition to this, the full length of ns2 was successfully expressed in 293 and HepG2 cells by using adenovirus expression system.In chapter 3, we screened a possible host factor interacting with NS2. His-tag pull down in combination with silver stain was carried out and found a host protein interacting with the NS2 protein. Through Mass-spectromy, three candidated proteins: ZNF136,SMC2 and GOA4 were gained after compared the database.Finally, we found that a traditional Chinese medicin dioscin could inhibit adenovirus and vesicular stomatitis virus (VSV) infection in vitro. The possible mechanism is that dioscin may destroy viral surface structure and/or its receptors in cell surface. We also demonstrated that dioscin could decrease the secretion of both HbsAg and HbeAg. These experiments set a foundation for the application of antiviral function of dioscin.