水稻黑条矮缩病毒p8蛋白的原核表达、抗血清制备及其特性

Prekaryotic expression antiserum preparation and some properties of p8 protein of Rice black-streaked dwarf Fijivirus

水稻黑条矮缩病毒(RBSDV)p8蛋白由其基因组片段S8编码,根据RBSDV浙江分离物S8序列(AJ297431)设计特异性引物扩增编码p8蛋白N端部分的片段,并亚克隆至原核表达载体pET-32a+,然后以大肠杆菌BL21plysS为宿主菌进行高水平地表达,利用纯化的p8蛋白免疫小鼠,制备了p8蛋白的特异性抗血清。Western blot分析表明,p8蛋白在水稻病株中含量较为丰富,易于检测,而且p8蛋白参与病毒粒子的组成,表明p8是一个病毒结构蛋白。ELISA检测显示p8蛋白的抗血清可与不同来源的水稻、小麦、玉米病汁液发生强烈的血清学反应,表明该抗血清适用于RBSDV田间病株的快速诊断。

Protein p8 is encoded by the genome segment S8 of Rice black-streaked dwarf virus （RBSDV）. Based on the sequence of S8 （AJ297431）, a pair of S8-specific primers were designed and used to amplify a fragment encoding the N-part of p8 protein. The expected product was subcloned into the expression vector pET-32a^＋ , transformed into E. coli BL21plysS and expressed at high level. Antiserum specifically against p8 protein was prepared using the purified protein to immunize mouse. Western blotting revealed that the p8 protein was abundant and easily detected in the infected plants and that the p8 protein was involved in the particles, indicating that p8 is a structural protein of RBSDV. ELISA tests showed that the antiserum against p8 protein could react strongly with the saps of infected rice, maize and wheat from different sources, indicating that the antiserum should be used to rapidly diagnose the RBSDV infection in fields.