人HCN4基因重组腺病毒载体的构建和鉴定

Construction and identification of human HCN4 gene recombinant adenovirus

目的:构建超极化激活环核苷酸门控通道基因(HCN4)重组腺病毒载体.方法:采用基因工程技术,将HCN4cDNA定向克隆至穿梭载体pAdTrackCMV中,利用胞包装、扩增,CsC1密度梯度超速离心纯化.采用PCR方法对重组腺病毒进行鉴定,利用穿梭质粒中带有绿色荧光蛋白GFP报告基因,对病毒滴度和感染效率进行监测.结果:测序结果证明成功构建了HCN4基因重组腺病毒载体,病毒滴度达2.0×1015pfu/L.结论:应用细菌内同源重组法成功构建了含人HCN4基因的重组腺病毒载体.

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.