樱桃病毒A北京分离物外壳蛋白的原核表达及抗血清制备

根据前期研究获得的樱桃病毒A北京分离物(Cherry virus Aisolate Beijing,CVA-BJ)外壳蛋白基因序列设计引物,将此基因克隆到原核表达载体pET-28a后转化大肠杆菌BL21(DE3),以终浓度为1 mmol/L的IPTG进行诱导表达。Ni珠吸附法纯化表达产物后免疫家兔制备抗血清。间接ELISA测得抗血清效价为1∶2048。以纯化后的蛋白和樱桃叶片总蛋白为检测材料,Western blot分析表明抗血清具有高度特异性。间接ELISA方法检测樱桃病叶,结果呈阳性,与RT-PCR检测结果一致。表明制备的抗血清可用于感病樱桃样品中CVA的检测。

Prokaryotic expression of coat protein of Cherry virus A isolate Beijing and preparation of the polyclonal antiserum

According to previous work, Cherry virus A Beijing isolate(CVA-BJ) cp gene was cloned into pET-28a vector and fusion protein was expressed in Escherichia coli strain BL21(DE3).After purification through affinity Ni column, the fusion protein was used to immunize the white rabbit to produce antiserum.The titer of the antiserum was 1:2 048 tested by indirect ELISA.Western blot analysis indicated that the antiserum was specific to CVA.With diseased cherry leaves confirmed by RT-PCR as test material, positive reactions occurred in indirect ELISA test.The results indicated that the antiserum could be applied in detecting CVA in the field.