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

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.