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辣椒轻斑驳病毒(Pepper mild mottle virus,PMMoV)属于烟草花叶病毒属,是辣椒的重要病毒种类之一。将PMMoV的外壳蛋白(cp)基因克隆至原核表达载体pET32a(+)中,转化大肠杆菌BL21(DE3),经IPTG诱导,重组PMMoV CP蛋白以可溶形式表达,纯化后获得分子量约35 kDa的融合CP蛋白。以该重组蛋白为抗原免疫新西兰兔制备了PMMoV CP特异性抗血清。Western blot检测结果表明该抗血清与重组CP蛋白发生强烈的免疫反应,间接酶联免疫吸附测定(ID-ELISA)结果显示该抗血清针对重组CP蛋白的效价达1∶25 600,针对PMMoV病汁液的效价达1∶4 000,检测灵敏度为1∶3 200,且该抗血清不与PMMoV同属或不同属的其它6种病毒汁液发生免疫反应,具有较好的特异性。利用该抗血清对42份田间辣椒病样进行检测,阳性率达38%,与进口商品化试剂盒检测结果一致,说明该抗血清可用于PMMoV的ELISA诊断。
Pepper mild mottle virus (PMMoV) belongs to the genus Tobacco mosaic virus and is one of the important virus types in pepper. The coat protein (cp) gene of PMMoV was cloned into prokaryotic expression vector pET32a (+) and transformed into E.coli BL21 (DE3). After induced by IPTG, the recombinant PMMoV CP protein was expressed in soluble form and purified to obtain a molecular weight of about 35 kDa Fusion protein CP. PMMoV CP specific antiserum was prepared by immunizing New Zealand rabbits with the recombinant protein as antigen. The results of Western blot showed that the antiserum reacted strongly with recombinant CP protein. The indirect ELISA (ID-ELISA) showed that the antiserum against recombinant CP protein had a titer of 1:25 600. For the detection of PMMoV disease The titer of the juice reached 1: 4000, the detection sensitivity was 1: 3 200, and the antiserum did not immunoreact with the other 6 virus juices belonging to the same or different genus PMMoV and had good specificity. The antisera was used to detect 42 samples of pepper in the field with a positive rate of 38%. The results were in good agreement with the results of the imported commercial kit, indicating that the antiserum can be used in the ELISA diagnosis of PMMoV.