表达西尼罗病毒E蛋白重组腺病毒的构建及鉴定

Construction of the recombinant adenovirus expressing E protein of West Nile virus

为构建表达西尼罗病毒(WNV)E蛋白的重组腺病毒,本实验将WNV的E基因克隆至腺病毒穿梭载体pShuttle-CMV中,经PmeⅠ酶切线性化后电转化于含有人5型缺陷腺病毒的骨架的BJ5183感受态大肠杆菌中进行同源重组,构建重组质粒pAd-WNV-E;将其经PacⅠ酶切线性化后通过脂质体介导转染于人胚肾细胞Ad-293细胞,制备重组腺病毒rAd-WNV-E;经western blot以及间接免疫荧光鉴定表明WNV E蛋白在Ad-293细胞中获得有效表达。病毒滴度测定试验结果显示,该重组腺病毒与野毒型腺病毒复制能力相近。该重组腺病毒的构建为进一步研制表达WNV E蛋白的重组腺病毒的疫苗奠定了基础。

To construct recombinant adenovirus expressing E protein of West Nile virus （WNV）, the WNV E gene was cloned into the shuttle plasmid vector pshuttle-CMV. The resultant recombinant shuttle plasmid was linearized by Pme I and transformed into E.coli B J5183 to construct the recombinant adenovirus plasmid of pAd-WNV-E by homologous recombination. The recombinant virus of rAd-WNV-E was generated by transfecting the Pac I linearized pAd-WNV-E into Ad-293 cells. The WNV E protein was efficiently expressed from the recombinant virus detected by western blot and immunofluorescence assay, and the viral titration results showed that the replication ability of rAd-WNV-E was the similar to the wild type recombinant virus. These results provided the basis for further study on the recombinant virus vaccine against WNV.