侵染浙贝母3种病毒CP基因的原核表达、抗血清制备及其病毒检测  

Prokaryotic Expression of TFMV,FVY and LMoV CP Gene,Antiserum Preparation and Virus Detection

作　　者：吴琪瑶[1] 韦传宝[2] 李进华[1] 

机构地区：[1]安徽大学资源与环境工程学院,合肥230601 [2]皖西学院安徽省植物生物技术实训中心,六安237012

出　　处：《植物研究》2013年第1期73-79,共7页Bulletin of Botanical Research

基　　金：安徽省教育厅重点项目基金(KJ2010A328;KJ2011A272)资助

摘　　要：根据GenBank报道的浙贝母花叶病毒(Thunberg fritillary mosaic virus,TFMV)、浙贝母Y病毒(Fritillary virus Y,FVY)和百合斑驳病毒(Lily mottle virus,LMoV)序列设计引物,扩增其CP基因。将CP基因插入表达载体pSBET,转化大肠杆菌BL21(DE3)Plys E菌株,IPTG诱导表达。经12%SDS-PAGE和5%～20%梯度SDS-PAGE两次纯化CP,分别免疫小鼠获得抗CP血清。采用Western blot分析确定抗体的特异性及其之间的血亲学关系;采用ELISA分析确定抗体是否能与天然病毒粒子结合。采用Western blot、间接ELISA法和Dot-ELISA法检测侵染浙贝母的3种病毒。结果表明,制备的抗体对CP有高度特异性,相互之间无交叉反应,且能与天然病毒离子结合。制备的抗血清可以用于检测3种病毒,其中间接ELISA法和Dot-ELISA法检测效果较好。According to Genbank,specific primers were designed to amplify the CP genes of TFMV,FVY and LMoV infecting Fritillaria thunbergii Miq.and the CP sequences were analyzed.Then the CP genes were inserted into pSBET vector and expressed in Escherichia coli BL21（DE3）plys E strain.The object proteins were purified by 12% SDS-PAGE firstly and subsequently 5%-20% gradient SDS-PAGE.The antiserum against the CPs was raised in mouse.The specificity and serological relationship were confirmed by Western blot analysis and the ability to combine with nature virus particles was confirmed by ELISA analysis.Western blot,indirect ELISA and Dot-ELISA techniques were used for the detection of viruses infecting F.thunbergii.The results indicated that the antiserum is specific to its CP and no acrossing-reaction with others.It could combine with nature virus particles.Indirect ELISA and Dot-ELISA techniques are suitable for detecting three kinds of viruses.

关 键 词：浙贝母 病毒 原核表达 抗血清制备 病毒检测 

分 类 号：Q785[生物学—分子生物学] S436.421[农业科学—农业昆虫与害虫防治]