人p16基因的重组腺病毒构建及鉴定

Construction and identification of human p16 gene recombinant adenovirus

目的构建含有人p16基因的重组腺病毒载体,并对其相关指标进行鉴定。方法用基因工程技术将人p16基因cDNA亚克隆至穿梭质粒pAdTrack-CMV上,利用PAdEasy系统进行细菌内同源重组,然后通过脂质体将正确重组体包裹并转染293T细胞,并在其中包装扩增病毒。采用PCR方法对重组腺病毒进行鉴定,利用穿梭质粒中所带绿色荧光蛋白GFP报告基因,对病毒滴度和感染效率进行检测。结果酶切鉴定及PCR结果证明p16基因重组腺病毒载体构建成功,病毒滴度达6.1×1010pfu/m l,对人成纤维细胞有较强感染能力。结论应用细菌内同源重组方法成功构建了含人p16基因的重组腺病毒载体。

Objective To construct the recombinant adenovirus encoding human p16 gene for future gene therapy. Methods The human p16 gene fragment was cloned into the shuttle plasmid pAdTrack-CMV to form the transfer vector by the method of homogenous recombination in bacteria. Then the recombinant adenovirus was transfected into 293T cells using Lipofectine DOTAP. The target gene was detected by polymerase chain reaction （PCR）. 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 p16 gene was successfully inserted into the adenovirus vector. The titer of the recombinant adenovirus was 6.1 × 10^10 pfu/ml. The adenovirus has a strong effect on human fibroblast cells. Conclusion The recombinant adenovirus containing human p16 gene was successfully constructed by the method of homogenous recombination in bacteria.