辣椒轻斑驳病毒cp基因的原核表达及抗血清制备

发布时间：2018-11-11 17:52

【摘要】：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。
[Abstract]:Capsicum light mottle virus (Pepper mild mottle virus,PMMoV) belongs to the genus Tobacco Mosaic virus (TMV) and is one of the most important viruses in capsicum. The coat protein (cp) gene of PMMoV was cloned into prokaryotic expression vector pET32a () and transformed into Escherichia coli BL21 (DE3). After IPTG induction, the recombinant PMMoV CP protein was expressed in soluble form. After purification, the fusion CP protein with molecular weight of about 35 kDa was obtained. New Zealand rabbits were immunized with the recombinant protein as antigen. The results of. Western blot detection showed that the antiserum reacted strongly with the recombinant CP protein. The results of indirect enzyme-linked immunosorbent assay (ID-ELISA) showed that the titer of the antiserum against recombinant CP protein reached 1:25 600, and for PMMoV disease juice was 1:4 000. The detection sensitivity was 1:3 2002#. The antiserum did not react with the other 6 viruses of the same genus or different genus of PMMoV and had good specificity. The antiserum was used to detect 42 field pepper samples, and the positive rate was 38%, which was consistent with the results of commercial kit, which indicated that the antiserum could be used for the ELISA diagnosis of PMMoV.
【作者单位】：贵州师范大学生命科学学院;贵州省植物生理与发育调控重点实验室;
【基金】：教育部长江学者和创新团队发展计划资助(PCSIRT1227) 贵州省重点实验室项目[黔科合计Z字(2011)4005号] 贵州省农业攻关项目[黔科合NY字(2014)3036]
【分类号】：S436.418

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