苹果茎痘病毒外壳蛋白的原核表达及抗血清制备

Prokaryotic expression of Apple stem pitting virus coat protein and antiserum preparation

利用基因重组技术将苹果茎痘病毒Applestempittingvirus(ASPV)完整cp基因在大肠杆菌中表达,以纯化蛋白为抗原免疫新西兰兔制备其抗血清,并采用ACP-ELISA法测定其效价和应用PAS-ELISA法对其抗血清的有效性进行初步鉴定。结果显示,cp基因获得了高效表达,表达产物与Anti-His抗体产生特异性免疫反应。制备的抗血清具有较强的特异性,效价为1:800。PAS-ELISA检测结果显示,9个苹果样品中有8个样品与RT-PCR检测结果一致,仅1个样品RT-PCR检测为阳性,而PAS-ELISA检测为阴性。表明所制备的抗血清具有一定的应用潜力。

The complete cp gene of Apple stem pitting virus （ASPV）was expressed in Escherichia coli using gene recombinant method, and the antiserum was produced after the rabbit was immunized with purified protein. The titer was determined by antigen coating plate-ELISA （ ACP-ELISA）, and validity of the antiserum was preliminarily identified by protein A sandwich ELISA （PAS-ELISA）. As a result, the cp gene was highly expressed, and the expression product could react with Anti-His antibody. Antiserum with high specificity was produced, and the titer was determined to be 1 ： 800. The detection results of PAS-ELISA showed, among 9 apple samples infected by ASPV, eight of them could be detected by both methods, the rest one, however, could not be detected by PAS-ELISA, but could by RT-PCR. It suggested the antiserum produced possesses applied potential to a certain extent.