The Study Of Interaction Between Influenza A Virus Protein And Host Protein

Influenza A viruses are enveloped viruses within the family Orthomyxoviridae.They contain a single-stranded, negative sense, segmented RNA genome whichconsists of eight segments of viral RNA (vRNA). NS1protein is encoded on acollinear mRNA derived from vRNA segment eight. It is a multiple-function proteinthat plays a significant role in the restraining the synthesis of host proteins, and alsohas impact on the cell apoptosis and regulation of viral pathogenicity and virulence.In this study, we synthesized the NS1gene of1918influenza A virus and clonedit into prokaryotic expression vector pET28a. The soluble recombinant proteinsHis-NS1was expressed in E.coli BL21(DE3) under the optimal condition. The NS1protein was purified by nickel chelate affinity chromatography method and then wasinjected into immune mouse to prepare the polyclonal antibody. ELISA assay wereused to analysis the titer of the serum. The results showed that the titer of antiserumwas1:1.6105.pCTAP-NS1was constructed and was transfected in HEK cells by Lipofectamine2000. The expression level of NS1was then deternmined by Western Blot assay at48h posttransfection. Futhermore, HEK cells were transfected with pCTAP-NS1, andanti-G418cell clones were screened using G418. The results showed that HEK cellclones expressing NS1were obtained by G418pressure selection after contimuouspassage for40days by using RT-PCR and Western Blot anaysis.Tandem affinity purification was used in our study to purify cellular interactors ofNS1protein, one of which was identified as HSP70by two-dimensionalelectrophoresis and mass spectrum. The interaction between HSP70and NS1wasfurther conformed by GST Pull Down assay and confocal immunofluorescence.GST-RBD (RNA Binding Domain), GST-ED (Effect Domain) and GST-NS1werepurified to search for the interaction interface. Results of GST Pull Down indicatedthat HSP70interacted with NS1both in RNA Binding Domain and Effect Domain.Confocal immunofluorescence assay also demonstrated that they were well co-locatedin the cytoplasm. Summing up the above, our study preliminary indicated that HSP70 interacted with NS1in the cytoplasm and made a foundation for NS1protein functionresearch in cell cycle regulation and induction of apoptosis.