Construction of eukarytic expression vector of enhanced green fluorescence protein driven by telomerase catalytic subunit promoter and its expression targeted in human lung cancer cells / 医学研究生学报

ABSTRACT

Objective:To construct an eukaryotic expression vector of enhanced green fluorescence protein(EGFP) gene driven by telomerase catalytic subunit(hTERT) gene promoter and observe the specific expression of EGFP in lung cancer cell lines.Methods:The 1100bp promoter fragment was obtained by enzyme digestion from a recombinant plasmid of pGL3-hTERTp containing the hTERT promoter.The hTERT promoter was then subcloned into the upstream of the report gene EGFP of pEGFP-1 without promoter.The expression vector pEGFP-hTERTp was successfully constructed.The vector pEGFP-N1 containing cytomegalovirus(CMV) promoter was used as a positive control.The vector pEGFP-1 without promoter was used as a negative control.The vectors were transfected into human lung cancer cell lines 95D,NCI-H446,A2,A549,LTEP-a-2,YTMLC and normal MRC-5 through lipofectamine respectively.EGFP expression was detected under the fluorescence microscope.Results:pEGFP-hTERTp was confirmed by enzyme digestion with correct result.That the EGFP expression was detected in all of eight lung cancer cells transfected with pEGFP-hTERTp,but not in MRC cells.By contrast,high intensity EGFP expression was observed in both lung tumor cells and normal cells,which were transfected with pEGFP-N1.Conclusion:The EGFP controlled by hTERT promoter can be expressed specifically in lung cancer cell lines.hTERT promoter may be used as an excellent regulation element in tumor-targeting gene therapy.