番茄斑萎病毒运动蛋白NSm的原核表达及抗体制备

 将番茄斑萎病毒(Tomato spotted wilt virus,TSWV)运动蛋白NSm基因克隆到原核表达载体pET-32a (+) 中,利用大肠杆菌系统表达NSm蛋白,以Ni^2＋-NTA agarose亲和柱纯化该蛋白与His组氨酸的融合蛋白,免疫家兔制备融合蛋白多克隆抗体。Western blot检测显示,该抗体与NSm重组蛋白以及普通烟病叶中的NSm蛋白具有特异性反应。采用该抗体对TSWV感染的普通烟叶片低温超薄切片进行免疫胶体金标记,透射电镜观察发现特异性金颗粒主要定位在病叶细胞的胞间连丝中,而健康烟草叶片组织中没有金颗粒特异性标记。结果表明,该抗体可用于研究NSm在细胞中的定位。

Expression and antiserum preparation of the NSm of Tomato spotted wilt virus

The nonstructural(NSm)protein gene of Tomato spotted wilt virus(TSWV)was cloned into pET-32a (+) vector and transformed into E.coli strain BL21 (DE3) pLys S for protein expression. After purification by Ni²⁺-NTA agarose affinity chromatography, the expressed protein was used as antigen to prepare polyclonal antiserum in rabbit. Western blot analysis of the produced antiserum indicated that it can specifically react with either the expressed NSm protein or that derived from the naturally TSWV-infected tobacco. Based upon this polyclonal antiserum, immuno-gold labeling revealed that special colloid gold particles could be observed at the plasmodesmata of TSWV-infected cells, whereas no gold particle could be obaserved at the plasmodesmata of the healthy control. Collectively, the polyclonal antiserum against NSm prepared in our study could be used to detect the distribution of NSm protein in the infected leaf cells.