Construction of Recombinant Adenovirus Vector Carrying the Vascular Endothelial Growth Factor 165 Using the AdEasy System

Objective To construct recombinant adenovirus vector carrying vascular endothelial growth factor 165 (VEGF165) and amplify the adenovirus vector in 293T cells.Methods VEGF165 obtained by PCR was digested and inserted into adenovirus shuttle plasmid pAdTraek-CMV to generate recombinant plasmid pAdTrack-VEGF165,and then the Pme I-linearized plasmid pAdTrack-VEGF165 was electroporated into E.coli BJ5183 cells that had been electroporated adenovirus backbone plasmid pAdEasy-1.The identified recombinant plasmid pAdEasy-VEGF165 DNA was digested with Pac I and transfered into 293T cells to package adenovirus,followed by identification of the recombinant adenovirus by means of observation of the enhanced green fluorescence protein (EGFP) expression under fluorescent microscope.After amplified in 293T cells,the obtained adenovirus was transfered into 293T cells again,and EGFP expression was detected.Results Recombinant adenoviral VEGF165 was constructed successfully and confirmed by restriction enzyme digestion, gene sequence and EGFP expression. Conclusion The recombinant adenoviral vector carrying VEGF165 was successfully constructed,and provided the basis of VEGF165 gene therapy.