Molecular cloning of the survivin gene promoter and its specific expression in the human laryngeal cancer Hep-2 cell line / 医学研究生学报

ABSTRACT

Objective: To construct the eukaryotic expression vector pSurp-EGFP regulated by the survivin gene promoter and to detect the specific expression of the promoter in human laryngeal cancer Hep-2 cells by green fluorescent protein assay.Methods: Thesurvivin gene promoter was generated by polymerase chain reaction(PCR) and the CMV promoter of the pShuttle vector replaced by the survivin gene promoter to generate the plasmid pSurp.The three plasmids pShuttle,pSurp and pEGFP-C1 were respectively double-enzyme digested so as to produce the plasmids pCMV-EGFP and pSurp-EGFP carrying the CMV or survivin promoter.The purified pCMV-EGFP and pSurp-EGFP were transfected into Hep-2 cell and vascular endothelial cell ECV304 using liposome transfection reagent and the expressions of EGFP detected by the fluorescent microscope.Results: Thesurvivin gene promoter was successfully cloned by PCR,and thesurvivin gene promoter-regulated pSurp-EGFP was constructed.Green fluorescence was observed in Hep-2 cells but not in ECV304. Conclusion: The high specific activity of the survivin gene promoter in Hep-2 cells that we successfully constructed attributes to the studies of tumor specific gene therapy.