Construction and characterization of replication-deficient adenoviral vector coding for human vascular endothelial growth factor 165

Objective To construct recombinant adenovirus vector carrying the gene of human vascular endothelial growth factor 165 for study of therapeutic angiogenesis in severe coronary artery disease.Methods The VEGF165 cDNA was extracted from pUCCAGGS/hVEGF165 with EcoRI and inserted into the E 1-deleted expression plasmid pHCMVSP1A shuttle vector,hamed Ad-hVEGF165.Ad-hVEGF165 was co-transfected with the plasmid pJM17 into 293 cells by liposome-maliated mehtad Ad-hVEGF165 expressing human VEGF165 cDNA was generated by homolgous recombination and confirmed by PCR.Ad-hVEGF165 was propagated in 293 cells and purified by CsC1 density pruification.The titer of each viral stock was determined by measuring the absorbance at 260 nm.Results VEGF165 cDNA was successfully inserted into the shuttle vector pHCMVSP1A.pAd-hVEGF165 was confirmed by NcoI and XhoI digestion.Ad-hVEGF165 was characterized by PCR.The virus titer was 1.94×10 12pfu/ml?Conclusions The recombinant replication-deficient adenovirus vector coding for human vascular endothelial growth factor 165 is successfully constructd.This investigation provides the basis for study of therapeutic angiogenesis of coronary artery disease.