Establishment Of SAV Stablely Expressed Cell Pool And Puriifcation Of SAV Interacting Protein Complex

OBJECTIVE To Establish HEK293cell model in which SAV isstably expressing and purification of SAV interacting protein complex. METHODSRT-PCR was used to amplify SAV gene segment in HEK293cells and then the PCRproduct was cloned into pBabe-SBP-FLAG vector with BamHâ… andEcoRâ… restriction enzyme cutting sites. Co-transfection of pBabe-SBP-FLAG-SAVand the packing plasmids into HEK293T cells to produce retroviruses which will beused for infection of HEK293cells. Stable cell pool was selected by puromycine for2weeks and SAV expression was detected by Western-blot. SAV interacting proteincomplex was purified by streptavidin beads from the stable cell pool and virulized bysilver staining. RESULTS pBabe-SBP-FLAG-SAV expression vector wassuccessfully constructed. FLAG-SBP tagged SAV in HEK293stable cell pool wasdetected by straight Western-blot and IP-Western-blot. SAV interacting proteincomplex was captured by streptavidin beads and we found some specific bandspurified from SAV stable cell pool was visualized compare to control in silverstainning. CONCLUSION pBabe-SBP-FLAG-SAV eukaryotic expression plasmidand the stable cell pool is successfully constructed and the interacting protein complexis purified by streptavidin beads, which provide a foundation for further investigationof SAV.