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作 者 ：吴琪瑶;韦传宝*;李进华

期 刊 ：植物研究 2013年 33卷 1期 页码：73-79

关键词：浙贝母；病毒；原核表达；抗血清制备；

Keywords:Fritillaria thumbergii Miq., virus, prokaryotic expression, antiserum preparation, virus detection,

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。

Abstract: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.

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