Construction of eukaryotic expression vector of fusion protein pEGFP/hVEGF165 and its expression in vascular endothelial cells / 中南大学学报(医学版)

ABSTRACT

OBJECTIVE@#To construct the plasmid of human vascular endothelial cell growth factor165 and green fluorescence protein report gene eukaryotic expression vector of fusion protein pEGFP /hVEGF165, and to detect its expression in vascular endothelial cells.@*METHODS@#We amplified full-length of gene VEGF165 by PCR, cloned in direction in multiple clone sites of pEGFP-N1, constructed recombinant plasmid of pEGFP/hVEGF165. Through enzyme digestion, PCR, and sequencing analysis, we also performed liposome-mediated transfection of vascular endothelial cells of in vitro cultivation, and detected the expression of fusion protein pEGFP/hVEGF165 using fluorescence microscope, RT-PCR, and Western blot.@*RESULTS@#Both gene VEGF165 and multiple clone site of pEGFP-N1 confirmed by PCR, enzyme digestion, and sequence analysis. EGFP/VEGF protein was expressed in vascular endothelial cells after pEGFP/VEGF165 recombinant plasmid transfected vascular endothelial cells.@*CONCLUSION@#Fusion protein eukaryotic plasmid of report gene EGFP and VEGF165 is successfully constructed, and EGFP/VEGF can be expressed in vascular endothelial cells, which lays a foundation for the application of VEGF gene in treating ischemia vascular diseases.