Construction of a recombinant adenovirus containing the human vascular endothelial growth factor 165 gene

Objective To construct a recombinant adenovirus containing the human vascular endothelial growth factor 165(VEGF165)gene.Methods Plasmid pDC315 and plasmid pDC-VEGF were digested with EcoRⅠ and XbaⅠ, ligated a target fragment of DNA, then transformed into DH5α competent cells to construct recombinant plasmid pDC315-VEGF.The plasmid was analyzed with restriction enzyme digestion and identified by sequencing.The identified plasmid was then cotransfected with pBHGE3 into 293 cells to generate a recombinant adenovirus.The constructed adenovirus was identified by PCR and then amplified and purified.PFU titers were determined by plaque assays.Results Plasmid pDC315-VEGF was identified by restriction enzyme analysis and DNA sequencing to have a full length of human VEGF165 cDNA fragment.The adenovirus vector was correctly constructed by homologous recombination in 293 cells and confirmed by PCR, with a virus concentration of 5.0 × 109 pfu / mL.Conclusion A recombinant adenovirus vector containing human VEGF165 gene has been successfully constructed via homologous recombination with double plasmids.