Expression And Purification Of Avian Influenza Virus RNA Polymerase From Eukaryotic Cell And Preliminary Research Of The Host Factors Combining With It

In this study, the RNA-dependent RNA polymerase (RdRp) of avian influenza virus (AIV) was expressed and purified from eukaryotic cells in two ways.One is developing one hybridoma cell strain named 5G11 by confusing SP2/0 cells and spleen cells of mouse immunized with prokaryotic expressed PB2 protein . The results of Western-blot indicated that 5G11 can react with seavral H5 subtype strains'PB2 protein. The results of subtype analysis showed that the heavy chain of 5G11 belonged to IgG2a and the light chain was theκchain. two different H5 subtype AIV strains'RdRp can be purified from virus infected MDCK with 5G11 by co-immunoprecipitation assay,although these two stains possess different virulence to mouse.we found and identified several proteins that may sociate with virus RdRp by 2-D electrophoresis and mass spectrographic analysis. The other method is that we constructed eurokaryotic expression reconstructed plasmids of RdRp,with TAP-tag in the fused protein PB2 subunit.Then,by tandem affinity purification procedure (TAP), virus RdRp was pufied from plasmids cotransfected 293T cells.In summary, we have set up reliable method of expression and purification of AIV's RdRp in eurokaryotic cells and we purified virus RdRp from eurokaryotic cell.Based on this, the host factors combining with virus RdRp was researched preliminarily.All these work together provides an efficient avenue to study the host factors'function in AIV replicating in mammalian cells.This work helps us to understand better the pathogenicity machnism of influenza virus and to go a step further ,to develop new drugs with high efficiency in future.