猪传染性胃肠炎病毒S基因重组腺病毒的构建及特性研究

将克隆的猪传染性胃肠炎病毒(TGEV)全长S基因插入pAdTrack-CMV穿梭质粒中,形成的转移质粒pAdTrack-CMV/S线性化后,用pAdEasyTM系统电转化含有腺病毒骨架载体pAdEasy-1的感受态大肠埃希氏菌BJ5183,经同源重组,构建了含有全S基因的重组腺病毒载体,经酶切充分暴露反向末端重复序列后,与脂质体混合转染AD-293细胞,成功获得了AD-S复制缺陷型腺病毒,经检测证实,AD-S稳定性好,安全性高。转录水平检测证实,S基因得到了转录;荧光检测发现,外源蛋白已得到表达。

Construction of recombinant adenovirus with transmissible gastroenteritis virus spike gene and study on its characterizations

The spike(S) gene of transmissible gastroenteritis virus(TGEV) of swine was cloned and then subcloned into a shuttle vector pAdTrack-CMV,and the transfer vector pAdTrack-CMV/S was constructed.After being linearized,the pAdTrack-CMV/S was transformed into the E.coli strain BJ5183 cells and the recombinant supercoiled adenoviral vector pAdEasy-1 was constructed in the cells.(After) the inverted terminal repeat(ITR) sequence of pAdEay-1 was exposed fully by restriction enzyme(digestion), the resultant recombinant was transfected into AD-293 with lipoplast.Results showed that a stable and safety replication defective recombinant adenovirus AD-S was produced successfully.The TGEV S gene was transcribed and expressed,which was confirmed by a fluorescence microscope.