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为获得特异性抗血清用于批量检测葡萄苗木中的葡萄卷叶伴随病毒3号(Grapevine leafroll associatedvirus 3,GLRaV-3),本研究用含有GLRaV-3CP蛋白基因的载体pMD18-CP为模板,PCR扩增CP蛋白基因。将PCR产物定向克隆到原核表达载体pET-28a(+)上,转化大肠杆菌BL21(DE3),筛选得到阳性克隆pET-G3CP。用终浓度为1mmol/L的IPTG进行诱导表达,SDS-PAGE电泳分析显示,GLRaV-3CP在大肠杆菌中得到高效表达。纯化表达产物后免疫家兔制备抗血清。经分析,抗血清效价为1/214,试验结果显示,此抗血清能用于葡萄植株样品中GLRaV-3不同分离物检测。
In order to obtain specific antiserum for batch detection of Grapevine leafroll associated virus 3 (GLRaV-3), we used a vector pMD18-CP containing the GLRaV-3CP protein gene as a template and PCR Amplify CP protein gene. The PCR product was cloned into prokaryotic expression vector pET-28a (+) and transformed into E. coli BL21 (DE3). The positive clone pET-G3CP was obtained. The expression of GLRaV-3CP was induced by IPTG at a final concentration of 1 mmol / L. SDS-PAGE analysis showed that GLRaV-3CP was highly expressed in E.coli. After purification of the expressed product, rabbits were immunized to prepare antiserum. After analysis, the antiserum titer is 1/214, and the test result shows that the antiserum can be used for detecting different GLRaV-3 isolates in grapevine samples.