The Construction of Recombinant Adenoviral Plasmid by Homologous Recombination in Bacteria and the Preparation of Recombinant Adenovirus Expressing EGFP and Human eNOS

Objective To construct recombinant adenoviral plasmid containing human endothelial nitric oxide synthase (heNOS) cDNA using homologous recombination in bacteria and to prepare recombinant adenovirus expressing enhanced green fluorescent protein (EGFP) and heNOS. Methods HeNOS cDNA was digested from plasmid pHCMV SP1A-heNOS with EcoR I and subcloned into the shuttle vector to generate the transfer plasmid pShuttle-heNOS-EGFP. The vector pShuttle-heNOS-EGFP was linearized with Pme I and transformed into ultracompetent BJ5183 bacteria containing pAdEasy-1. The positive clone of homologous recombination (pAdEasy-heNOS-EGFP) was identified by PCR, enzyme digestion, and DNA sequencing. The positive recombinant adenoviral plasmid was digested with Pac I and transfected AD293 cells with Lipofectamine 2000 to package recombinant adenovirus particles. The recombinant adenovirus was purified with density gradient centrifugation of cesium chloride. PCR was used to characterize the recombinant adenovirus expressing heNOS. The titer and purity of the recombinant adenovirus was determined by ultraviolet spectrometry. Results HeNOS cDNA was successfully inserted into the shuttle vector. Homologous recombination occurred between pShuttle-heNOS-EGFP and pAdEasy-1 within BJ5183 to generate pAdEasy-heNOS-EGFP. The recombinant adenovirus was confirmed to be successfully packaged within AD293 cells with PCR. The titer of the purified Ad-heNOS-EGFP was 6.5 ×1015 pfu/L. Conclusion The construction of adenovirus plasmid by homologous recombination in bacteria can be quickly and easily performed. The preparation of recombinant adenovirus Ad-heNOS-EGFP with high titer provides a basis for gene therapy of erectile dysfunction. EGFP expression is a useful tool for directly monitoring the infection of target cells and the expression of gene of interest.