为构建表达Ⅰ型鸭病毒性肝炎病毒(DHV-I)VP1蛋白的重组腺病毒,本实验采用RT-PCR方法扩增DHV-Ⅰ HP-1株VP1基因,克隆于腺病毒穿梭质粒pShuttle-CMV中,并将其电转化于含腺病毒pAdeasy骨架质粒的大肠杆菌BJ5183中,通过同源重组制备含有重组腺病毒的质粒.重组质粒经PacⅠ酶切线性化后转染AD293细胞,获得重组腺病毒(rAd-VP1).间接免疫荧光和western blot鉴定结果显示,DHV-Ⅰ的VP1蛋白在重组腺病毒感染的AD293中获得表达.rAd-VP1的制备为鸭免疫实验效果评价奠定了基础.%In order to construct recombinant adenovirus expressing the VP1 protein of duck hepatitis virus type Ⅰ (DHV-Ⅰ),the VP1 gene was amplified by RT-PCR and cloned into pShuttle-CMV vector.The recombinant plasmid was transferred into E.coli B J5183 competent cells containing adenovirus backbone vector to produce recombinant adenovirus plasmid by homologous recombination.The positive recombinant plasmid was linearized by Pac Ⅰ and transfected into AD293 cells.The recombinant adenovirus (rAd-VP1) was rescued and identified by indirect immunofluorescence assay and western blot.The results showed that rAd-VP1 expressing VP1 protein of DHV-Ⅰ was constructed successfully which laid the foundation for the evaluation of immune efficacy in ducks.
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