表达牛冠状病毒S1基因的重组人腺病毒的构建及鉴定

Construction and identification of recombinant adenovirus expressing S1 gene of bovine coronavirus

为构建表达牛冠状病毒(BCV)S1基因的重组人腺病毒疫苗,本实验通过人工合成密码子优化的截短BCV S1的基因片段(55 bp～1 650 bp)。将其克隆到重组腺病毒穿梭载体pShuttle-CMV中,并以PmeⅠ线性化后转化含有人5型腺病毒骨架的BJ5183感受态细胞,通过细菌内同源重组的方式获得重组腺病毒质粒pAdV-BCV-S1,经PacⅠ线性化后转染AD-293细胞获得重组腺病毒rAdV-BCV-S1。利用间接免疫荧光法和western blot法检测该重组腺病毒的S1基因在AD-293细胞中的表达情况,同时用一步生长曲线法,测定该重组腺病毒的复制能力。结果表明:BCV S1基因在感染细胞中获得了高效表达:子代重组腺病毒在感染细胞后60 h滴度最高,为6.8×108 pfu/mL。该重组腺病毒的构建为BCV重组疫苗的研究奠定了基础。

To construct recombinant adenovirus expressing bovine coronavirus（BCV）,a truncated BCV S1 gene fragment（55 bp to 1,650 bp） with optimized codons was chemically synthesized and cloned into the shuttle plasmid pShuttle-CMV.Recombinant adenovirus plasmid pAdV-BCV-S1 was generated by homologous recombination with bone plasmid pAdEasy-1 into E.coli BJ5183 cells.The pAdV-BCV-S1 was linearized and transfected into AD-293 cells for package of rAdV-BCV-S1.The S1 protein expressed in AD-293 cells was identified by indirected immunoflourescent assay and western blot assay.The results showed that the S1 gene of BCV was successfully inserted into adenovirus DNA with high level of expression.The highest titer of rAdV-BCV-S1 reached 6.8×108 pfu/mL at 60 h in infected cells.The construction of recombinant adenovirus carrying S1 gene of BCV provided alternative approach for developing novel vaccine against BCV.