人核糖核酸酶抑制因子基因重组腺病毒载体的构建及鉴定

Construction and identification of human ribonuclease gene recombinant adenovirus

目的构建含有人核糖核酸酶抑制因子(hRI)基因的重组腺病毒载体。方法以含有全长cDNA的pT7-RI为模板,PCR扩增hRI,经T载体克隆后,酶切亚克隆到穿梭质粒pAdTrack-CMV上,在BJ5183细菌内和pAdEasy-1同源重组。筛选阳性克隆,酶切、PCR及测序鉴定,线性化后脂质体法转染293细胞进行包装、扩增。通过观察绿色荧光蛋白(GFP)的表达及PCR扩增目的基因等方法鉴定重组的腺病毒。结果酶切鉴定及PCR结果证明hRI基因重组腺病毒载体构建成功,病毒滴度为1.5×1010pfu/ml。结论应用细菌内同源重组法成功构建了含hRI基因的重组腺病毒载体。

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 subeloned to pAdTrack-CMV. The linearized shuttle plasmid was homogenously reeombined with pAdEasy-1 in BJ5183. The candidate clone was further analyzed by restriction endonuelease 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 × 10^10pfu/ml. Conclusion The recombinant adenovirus containing human RI gene was successfully constructed by the method of homogenous recombination in bacteria.