Construction of a tumor specific expression vector under the control of survivin gene promoter

Objective:By amplification of survivin gene promoter and construction of eukaryotic expression vector under the control of survivin promoter to prove its tumor specific expression.Methods:Using Hep-2 cell genomic DNA as template,the survivin gene promoter region was amplified by polymerase chain reaction.The amplified survivin gene fragment and pShuttle vector were double-enzyme digested to construct vector pSurp bearing survivin gene promoter,which was further confirmed by restriction analysis and DNA sequencing.Afterwards,plasmids pSurp and pEGFP-C1 were respectively double-enzyme digested to produce the vector pSurp-EGFP contolled by survivin promoter.The plasmid pSurp-EGFP was transfected into Hep-2 cell and vessel endothelial cell ECV304 using liposome reagent and expression of EGFP was detected by fluorescent microscope and Western blot.Results:Survivin gene promoter was cloned successfully by PCR method and eukaryotic expression vector pSurp-EGFP controlled by survivin gene promoter was constructed.Green fluorescence was observed in Hep-2 cells while not in ECV304 after transfected by pSurp-EGFP.Western blot analysis results showed that pSurp-EGFP expressed EGFP protein only in Hep-2 cells.Conclusion:Successfull survivin gene promoter cloning and construction of its expression vector might be the initial step of tumor specific gene therapy.