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PCR扩增烟草丛顶病毒(Tobacco bushy top virus,TBTV)的ORF1序列并克隆到原核表达载体pEHISTEV中,转化大肠杆菌Rosetta菌株经IPTG诱导表达TBTV ORF1蛋白。利用切胶纯化的ORF1蛋白免疫新西兰大耳白兔制备并获得抗血清,间接ELISA检测效价为1:24 3000。经抗原亲和纯化从抗血清中得到特异性和灵敏度俱佳的ORF1多克隆抗体。Western blot分析显示,TBTV ORF1多克隆抗体既可以检测田间发病的烟草丛顶病样品中ORF1蛋白,也可检测在体内和体外翻译体系中的TBTV ORF1蛋白的表达。另外发现ORF2蛋白以ORF1延长蛋白的形式存在,根据ORF1和ORF2的重叠情况及潜在的七核苷酸滑动序列和下游的稳定二级结构,推测此ORF1延长蛋白是移码翻译产物。
The ORF1 of Tobacco bushy top virus (TBTV) was amplified by PCR and cloned into the prokaryotic expression vector pEHISTEV. The E. coli Rosetta strain was induced to express TBTV ORF1 protein by IPTG. New Zealand white rabbits were immunized with gel-purified ORF1 protein to prepare and obtain antisera. The indirect ELISA detection titer was 1:24 3000. The affinity-purified ORF1 polyclonal antibody with high specificity and sensitivity was obtained from the antiserum by antigen affinity purification. Western blot analysis showed that the polyclonal antibody against TBTV ORF1 could detect the ORF1 protein in tobacco bushy disease samples in field and the TBTV ORF1 protein expression in vivo and in vitro. In addition, the ORF2 protein was found to exist as an ORF1 elongation protein. According to the overlap of ORF1 and ORF2, the potential heptad sequence and the stable secondary structure, the ORF1 elongation protein is presumed to be a frameshift translation product.