Construction and identification of human ribonuclease gene recombinant adenovirus

Objective To construct the recombinant adenovirus vector containing human RI for future gene therapy.Methods The RI fragment was amplified by PCR according to the pT7-RI,then the fragment was cloned to T vector and subcloned to pAdTrack-CMV.The linearized shuttle plasmid was homogenously recombined with pAdEasy-1 in BJ5183.The candidate clone was further analyzed by restriction endonuclease digestion and PCR.Then the recombined adenovirus was transfected into 293 cells for packaging and amplifying.The titer and its infection rate were determined using the green fluorescent protein(GFP) expression in the shuttle plasmid.Results Restriction endonuclease and PCR analysis confirmed that the human RI gene was successfully inserted into the adenovirus vector.The titer of the recombinant adenovirus was 1.5×1010pfu/ml.Conclusion The recombinant adenovirus containing human RI gene was successfully constructed by the method of homogenous recombination in bacteria.