葡萄卷叶伴随病毒3号抗血清的制备及应用

Preparation and application of antiserum against Grapevine leafroll associated virus 3

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。

The pMD18-CP containing CP gene of Grapevine leafroll associated virus 3 （GLRaV-3） was used for PCR amplification in order to obtain a specific antiserum for reliable GLRaV-3 diagnosis in routine tests. The PCR products of GLRaV-3 CP gene were cloned into the expression vector pET-28a. The resulted recombinant plasmid pET-G3CP was transformed into E. coil BL21 （DE3）. SDS-PAGE analysis showed that the recombinant GLRaV-3 CP was expressed as a 40 ku inclusion body protein at high level induced by IPTG at final concentration of 1 mmol/L. Antiserum was prepared after the rabbit was immunized with the purified recombinant CP. The antiserum titer was determined to be 1/2TM ,and the antiserum was specific and sensitive for different GLRaV-3 isolates detection.