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豫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.