水稻黑条矮缩病病毒外壳蛋白基因S10的原核表达、多克隆抗体制备及应用  

Prokaryotic Expression of Coat Protein Gene S10 of Rice Black-Streaked Dwarf Virus,and Preparation and Application of Its Polyclonal Antibody

作　　者：欧阳元龙[1,2,3] 吴建祥[2] 熊如意[3] 周益军[3] 周雪平[2] 

机构地区：[1]南京师范大学生命科学学院,江苏南京210046 [2]浙江大学农业与生物技术学院生物技术研究所,浙江杭州310029 [3]江苏省农业科学院植物保护研究所,江苏南京210014

出　　处：《中国水稻科学》2010年第1期25-30,共6页Chinese Journal of Rice Science

基　　金：农业部公益性行业(农业)科研专项资助项目(nyhyzx07-051);现代农业产业技术体系建设专项资金资助项目;国家自然科学基金资助项目(30670087)

摘　　要：用RT-PCR方法从感染水稻黑条矮缩病毒(rice black-streaked dwarf virus,RBSDV)水稻中克隆该病毒的外壳蛋白基因S10,然后将此外壳蛋白基因再亚克隆到原核表达载体PET-32a中构建成重组原核表达载体pET32a-CP。将重组表达载体转化大肠杆菌BL21(DE3),经IPTG诱导,Ni+ NTA亲和柱纯化获得分子量约为76kD含硫氧还蛋白的融合蛋白。以纯化的重组蛋白为抗原免疫兔子制备RBSDV外壳蛋白的多克隆抗体,并用制备的多克隆抗体建立了可靠、灵敏、特异的检测RBSDV的免疫捕获RT-PCR及Dot-blot ELISA方法,为该水稻病毒病的诊断提供技术支持。The full length cDNA of rice black-streaked dwarf virus （RBSDV） segment 10 （S10） which encoded coat protein was cloned from the virus infected rice samples by RT-PCR,and subeloned into a prokaryotic expression vector pET 32a. The re combinant prokaryotic expression vector （pET-32a-CP） was used to transform Escherichia coli BL21 （DE3）. A 76 kD TrxA fusion protein was obtained with induction of IPTG and purification of Ni^＋ NTA affinity column. The purified recombinant protein was used to immunize rabbits for production of polyclonal antibodies against the coat protein of RBSDV. Using poly- clonal antibodies, immunoeapture RT-PCR and Dot-blot ELISA were established for reliable, sensitive and specific detection of RBSDV. The two detection methods utilized polyclonal antibodies provide technical support for the diagnosis of RBSDV disease.

关 键 词：水稻黑条矮缩病病毒 原核表达 多克隆抗体 免疫捕获反转录聚合酶链式反应 斑点酶联免疫吸附测定 

分 类 号：S435.111.4[农业科学—农业昆虫与害虫防治]