论文部分内容阅读
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。
The gene was cloned into the prokaryotic expression vector pET-28a and transformed into E. coli BL21 (DE3) according to the sequence of the coat protein gene of Cherry virus Aisolate Beijing (CVA-BJ) obtained in the previous study. IPTG at a concentration of 1 mmol / L was induced to express. Ni beads adsorption method to purify the expression product after immunization of rabbits to prepare antiserum. The titer of antiserum measured by indirect ELISA was 1:2048. The purified protein and total protein of cherry leaf were used as the test material, and Western blot analysis showed that the antiserum was highly specific. Indirect ELISA method for detecting cherry disease leaves, the results were positive, consistent with the RT-PCR test results. The results showed that the prepared antiserum could be used for the detection of CVA in susceptible cherry samples.