辣椒轻斑驳病毒cp基因的原核表达及抗血清制备  

Prokaryotic expression and antiserum preparation of the coat protein of Pepper mild mottle virus

作　　者：洪鲲[1,2] 郑兴华 李欲轲[2] 龚记熠 乙引 万晴姣 

机构地区：[1]贵州师范大学生命科学学院,贵阳550001 [2]贵州省植物生理与发育调控重点实验室,贵阳550001

出　　处：《植物病理学报》2017年第5期598-604,共7页Acta Phytopathologica Sinica

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]

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。Pepper mild mottle virus （PMMoV）, which belongs to the genus Tobamovirus, is one of the major viruses in pepper. In this study, coat protein （cp） gene of PMMoV was cloned into a prokaryotic expression vector pET32a（＋）, and transformed to the Escherichia coli strain BL21 （DE3）. The recombinant CP protein fused with Trx tag （35 kDa） was expressed after induction with IPTG, and the New Zealand rabbits were used to prepare the antiserum by immunization with the recombinant CP protein. Western blot results showed that the prepared antiserum strongly reacted with recombinant CP. Indirect enzyme linked immunosorbent assay （IDELISA） results showed that the titer of antiserum against PMMoV leaf sap reached 1： 4 000 and the detection sensitivity for the sap was 1 ： 3 200. Furthermore, the antiserum didn＇t react with samples infected by other six viruses, suggesting the high specificity of the prepared antiserum against PMMoV. The positive rate of IDELISA based on the prepared antiserum was 38%, which was comparable to the commercialized ELISA kits. These results indicated that the antiserum could be applied to ELISA diagnosis of PMMoV.

关 键 词：辣椒轻斑驳病毒 外壳蛋白 原核表达 抗血清 

分 类 号：S432.41[农业科学—植物病理学]