表达兔瘟病毒VP60基因的重组病毒BHV-1的构建与鉴定

Abstract

为了构建表达兔瘟病毒VP60基因的牛疱疹病毒I型，首先构建含有兔瘟病毒VP60基因和CMV启动子的转移载体，然后将构建移载体与gE基因缺失的牛疱疹病毒I型基因组共转染牛肺细胞后收获增殖的病毒。通过蓝白斑筛选不含有LacZ基因的白色病毒蚀斑，反复纯化获得重组病毒BHV-1-VP60。PCR方法验证VP60基因的成功插入，间接免疫荧光试验和Western blot，结果证明了BHV-1-VP60中的VP60基因在易感细胞中获得了表达。本研究成功地构建了兔瘟病毒VP60基因的重组病毒BHV-1-VP60，为研制新型兔瘟活载体疫苗奠定了基础。 In order to construct the recombinant bovine heper virus I which expressed RHDV VP60 gene, first, we constructed a transfer vector containing RHDV VP60 gene and CMV promoter, and then the mixtures of parental virus DNA and transfer vector were transfected into bovine lung cells. Then the propagated viruses were harvested. The recombinant virus was obtained by selection for white virus plaques which did not contain LacZ gene. PCR assay showed that VP60 gene had been inserted into the recombinant virus genome; the expression of VP60 in infected cells was proved by indirect immunofluorescence assay and Western blot. This study successfully constructed the recombinant virus BHV-1-VP60, which provided a basis for the development of a new type of RHDV vector vaccine.