Effect of Dual Promoter on Expression of Enhancement Green Fluorescent Protein

Objective To investigate the effect of dual promoter on expression of enhancement green fluorescent protein(EGFP)as well as its promotion efficacy.Methods Promoters H1,U2,U3 and U6 were cloned to the MCS site of vector pEGFP-N1 containing CMV promoter respectively,and the constructed dual promoter expression vectors were transfected to 293T cells.The transfected cells were observed by fluorescent microscopy,and the transfection efficacy and EGFP gene expression efficacy(expressed as fluorescent index)were determined by flow cytometry.Results Both PCR and restriction analysis proved that the dual promoter expression vectors were constructed correctly.The transfection efficacies of pEGFP-N1 and dual promoter expression vectors inserted with H1,U2,U3 and U6 promoters were 26.57% ± 1.54%,28.57% ± 0.99%,16.14% ± 1.69%,22.63% ± 1.77% and 17.89% ± 1.84%,and the EGFP gene expression efficacies in the cells transfected with the recombinant plasmids were 142.79 ± 31.26,103.59 ± 25.90,19.67 ± 0.52,58.16 ± 14.58 and 15.40 ± 1.92,respectively.Conclusion The expression efficacy driven by CMV promoter combined with H1 promoter were similar to that by CMV promoter alone.However,the expression efficacies driven by CMV promoter combined with U2,U3 and U6 respectively were significantly lower than that by CMV promoter alone.