Construction of recombinant adenoviral vector carrying LEDGFp52 gene by homologous recombination in bacteria and its expression in vitro

Objective To construct recombinant adenovirus vector carrying LEDGFp52 gene by homologous recombination in bacteria and to detect its expression in vitro.Methods LEDGFp52 gene was cloned to adenoviral shuttle plasmid pAdTrack-CMV.Then the resultant pAdTrack-CMV-LEDGFp52 was cotransfected into BJ5183 bacteria with the adenovirus backbone plasmid pAdEasy-1.The adenovirus plasmid carrying LEDGFp52 was generated with homologous recombination in bacteria and the adenoviruses were produced in 293 cells.293 cells were infected with adenoviruses and the expression of LEDGFp52 was detected by CPE(cytopathic effect) and westernblot.Results The titer of Ad-LEDGFp52 adenoviruses was up to 5×10 12 PFU·L -1 after proliferation in 293 cells.LEDGFp52 was expressed efficiently in 293 cells after infection.Conclusion The recombinant adenoviruses expressing LEDGFp52 was constructed successfully and could be used further in gene transfection experiments.