Human HCN4 gene and identification of recombinant adenovirus vector.docVIP

 PAGE \* MERGEFORMAT 15 Human HCN4 gene and identification of recombinant adenovirus vector Author: Cai Jun, Lin-sheng, Jiang Hong, Wang Teng, Zeng Bin, Luo Hao, Guo, Li Jun, Wang Lei [Keywords:] Adenoviridae Construction and identification of human HCN4 gene recombinant adenovirus [Abstract] AIM: To construct the recombinant adenovirus of human HCN4 gene. METHODS: HCN4 gene fragment was cloned into the shuttle plasmid pAdTrackCMV 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 * 1015 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. [Keywords] HCN gene; adenoviridae; recombination, genetic [Abstract] Objective: To construct hyperpolarization-activated cyclic nucleotide-gated channel gene (HCN4) recombinant adenovirus vector. Methods: The genetic engineering technology, HCN4 cDNA was cloned into shuttle vector pAdTrackCMV directed, using pAdeasy1 system for bacterial homologous recombination, after transfection of 293T cells liposome packaging, amplified, CsC1 density gradient ultracentrifugation purification. adenovirus using PCR method for identification, using the shuttle plasmid with a green fluorescent protein, GFP reporter gene, the virus drops degree and infection efficiency of monitoring. Results: The sequencing results show that HCN4 gene was suc