狂犬病毒糖蛋白单、双拷贝基因重组腺病毒的构建

Construction of recombinant adenoviruses expressing one and two copies of the rabies virus glycoprotein gene

将携带狂犬病毒糖蛋白单、双拷贝基因的穿梭载体pShuttle-G和pShuttle-dG分别经PmeⅠ线性化后电转化含E1和E3区缺失的人5型腺病毒骨架质粒pAdEasy-1的感受态菌株BJ5183-AD-1进行细菌内同源重组,经抗性筛选,PCR、PacⅠ酶切及测序鉴定,构建了狂犬病毒糖蛋白单、双拷贝基因重组腺病毒质粒pAdHu5-G和pAdHu5-dG,线性化后分别转染Ad-293细胞进行病毒包装。结果显示,在第4天发生细胞病变效应,且荧光显微镜下观察到绿色荧光蛋白的表达;Western-blot证实2株重组腺病毒表达的糖蛋白能被特异性单抗识别;遗传稳定性分析显示,病毒传至10代时单G和双G基因均稳定遗传,没有发生缺失和突变。为比较分析2株重组腺病毒糖蛋白的表达时效及免疫效果奠定了基础。

Shuttle vectors pShuttle-G and pShuttle-dG carrying one or two copies of the rabies virus glycoprotein gene were respectively linearized with PmeⅠ and electrotransformed competent cells BJ5183-AD-1 which were pre-transformed with the pAdEasy-1 plasmid to produce recombinant Ad plasmids pAdHu5-G and pAdHu5-dG.Then they were single digested with Pac Ⅰ and linearized,and transfected the AD-293 cells.After development of cell cytopathic effect（CPE）,infected cells showed green fluorescence by fluorescent microscopy.Western-blot analysis showed the glycoprotein protein expressed could be bound with mouse anti-rabies glycoprotein monoclonal antibody.Genetic stability analysis revealed that the single G and double G genes could inherit stably and no mutation or absence happened.This work lays a good foundation for research on the different immunogenicity of the two recombinant Ads rAdHu5-G and rAdHu5-dG.