Establishment of SAV stablely expressed cell pool and purification of SAV interacting protein complex / 中国药学杂志
Chinese Pharmaceutical Journal
;
(24): 1716-1719, 2012.
Article
Dans Chinois
| WPRIM
| ID: wpr-860576
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.
Texte intégral:
Disponible
Indice:
WPRIM (Pacifique occidental)
langue:
Chinois
Texte intégral:
Chinese Pharmaceutical Journal
Année:
2012
Type:
Article
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