Expression,purification And Research Of Structure Of Two Proteins Related To Human Infectious Diseases

Both HIV and EV68 are RNA Virus which have serious threat to public health.However,the specificity and effective antiviral vaccines or drugs are unavailable to date.So to explore the virus replication mechanism and new drug targets have become the hotspot in research of virology.The aim of this thesis is to provide a solid basis for the research for pathogenic mechanism of virus as well as the development of the antiviral drug.HIV is a retrovirus infecting human immune system cells,and classified as HIV-1 and HIV-2 type.HIV-1 type is the main pathogens of the global AIDS epidemic.To inhibit viral replication,host cells express APOBEC3 H as a kind of HIV restriction factor,and different APOBEC3 H haplotypes have diverse anti-viral activity.A3 H HapII as the most stable haplotype has strong ability of antiviral activity and retrotransposon inhibition,and also can be efficiently degraded by HIV-1 Vif.Thus,the research of A3 H Hap II has a profound significance.We use the baculovirus-insect expression system to express large quatities soluble A3 H hap II proteins,and also prove that it can specific interact with HIV-1 Vif.First,We construct the A3 H hapII-His recombinant bacmid,and then the recombinant bacmid transfects into insect sf9 cells to generate the third generation of recombinant baculovirus through amplification.Then the baculovirus infect sf9 cells to express the targer protein.We can use Western Blot to detect the expression of the target protein.After purified by Ni-NAT,A3 H hapII has specific interaction with HIV-1 Vif is confirmed by co-immunoprecipitation assay.In this thesis,we successfully established baculovirus-insect expression system of A3 H hapII,and those results will laid a foundation for the further study of the interaction mechanism between A3 H hapII and HIV-1 Vif as well as anti-hiv drug targets.EV68 is a RNA virus mainly causing respiratory disease.In recent years,different regions and countries have different levels outbreaks of EV68 infection,and EV68 has raised widespread concerns.The EV68 genome encodes seven nonstructural and four structure viral proteins.The nonstructural protein 3A plays an important role in the replication complex.The soluble N-terminal(1-61)of 3A protein has important significance to biological function,but the structure of EV68 3A-N has not yet reported.This thesis expresses the soluble region of EV-D68 3A protein through the prokaryotic expression system and also has a preliminary research on the target protein.First,we construct the EV68 3A-N prokaryotic expression plasmid and E.coli BL21(DE3)is transformed with the recombinant plasmid to express the the fusion protein His-SUMO-3A(1-61).Afer primary purified by by the Ni-NTA and ULP Protease,using multiple methods including Ni-NTA,anion exchange chromatography and gel filtration chromato-graphy to further purify the target protein.And then we use chemical cross-linking reaction assay to indentify the multiple polymerization state of the 3A-N region.Finally we can obtain about 5 mg target protein with purity > 95% from 1L Escherichia coli BL21 and demonstrate the multiple polymerization form of the target protein.Thus,this thesis provides a solid basis for the research for the 3A crystal structure and the development of the 3A-based antiviral drugs.