Establishment of SAV stablely expressed cell pool and purification of SAV interacting protein complex / 中国药学杂志

ABSTRACT

OBJECTIVE: To Establish HEK293 cell model in which SAV is stably expressing and purification of SAV interacting protein complex. METHODS: RT-PCR was used to amplify SAV gene segment in HEK293 cells and then the PCR product was cloned into pBabe-SBP-FLAG vector with BamH I and EcoR I restriction enzyme cutting sites. Co-transfection of pBabe-SBP-FLAG-SAV and the packing plasmids into HEK293T cells to produce retroviruses which will be used for infection of HEK293 cells. Stable cell pool was selected by puromycine for 2 weeks and SAV expression was detected by Western-blot. SAV interacting protein complex was purified by streptavidin beads from the stable cell pool and virulized by silver staining. RESULTS: pBabe-SBP-FLAG-SAV expression vector was successfully constructed. FLAG-SBP tagged SAV in HEK293 stable cell pool was detected by straight Western-blot and IP-Western-blot. SAV interacting protein complex was captured by streptavidin beads and some specific bands purified from SAV stable cell pool was visualized compare to control in silver stainning. CONCLUSION: pBabe-SBP-FLAG-SAV eukaryotic expression plasmid and the stable cell pool is successfully constructed and the interacting protein complex is purified by streptavidin beads, which provide a foundation for further investigation of SAV.