小麦矮缩病毒外壳蛋白基因的原核表达、抗体制备及应用

 小麦矮缩病毒(Wheat dwarf virus,WDV)引起的小麦矮缩病是近年来我国小麦生产中的一种重要病毒病害,急需研发快速精准的检测技术用于预测预报和病毒-介体相互作用的研究。本研究应用Gateway重组技术构建了外壳蛋白基因(Coat protein, CP)的原核表达载体,将重组表达载体转化大肠杆菌Rosetta,经IPTG诱导获得CP基因原核表达蛋白。以重组蛋白为抗原免疫新西兰大白兔制备得到了相应的抗体,Western blot检测表明制备的抗体能与CP重组蛋白、感病小麦和带毒叶蝉特异性结合,说明获得的抗体特异性高。用获得的抗体进行免疫荧光标记,观察到病毒分布在介体叶蝉的前中肠和中中肠部位,为WDV的预测预报和介体条沙叶蝉传毒机制的研究奠定了基础。

Antiserum preparation of Wheat dwarf virus using coat protein expressed in Escherichia coli and its application

Wheat dwarf virus (WDV) has severely affected wheat production in China in recently years. It is urgent to develop accurate detection technologies for disease forcast and researches on interaction between virus and vector. In this study, the expression plasmids of WDV CP gene were constructed by the technology of Gateway and then transformed into E. coli strain Rosetta. Recombinant protein was obtained through IPTG induction. The recombinant protein was used to immunize rabbits for preparation of polyclonal antibodies. The analysis of Western blotting indicated that the prepared antibodies could specially binding with recombined CP protein, wheat plant and leafhopper infected by WDV respectively. It was observed that WDV distributed in the anterior and middle midgut of the leafhopper vector by immunofluorescence. The result can provide support for the study of prediction and the mechanism of transmission.Key words:Wheat dwarf virus; prokaryotic expression; polyclonal antibody; Western blot; immunofluorescence