Construction and identification of human HCN4 gene recombinant adenovirus

AIM: To construct the recombinant adenovirus of human HCN4 gene. METHODS: HCN4 gene fragment was cloned into the shuttle plasmid pAdTrack-CMV to form the transfer vector by the methods of homogeneous recombination in bacteria and the recombinant adenovirus was then transfected into 293 cells using Lipofectamine 2000. The target gene was detected by polymerase chain reaction (PCR) and the titer and its infection rate were determined using the green fluorescent protein (GFP) expression in the shuttle plasmid. RESULTS: Restriction enzyme digestion analysis and the sequence analysis confirmed that the HCN4 gene was successfully inserted into the adenovirus vector. The titer of the recombinant adenovirus was 2.0×10 15 pfu/L. GFP was observed in the transfected 293 cells under a fluorescent microscope. CONCLUSION: The recombinant adenovirus containing human HCN4 gene is successfully constructed by the method of homogeneous recombination in bacteria.