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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 coat protein gene S10 of the virus was cloned from rice infected with rice black-streaked dwarf virus (RBSDV) by RT-PCR, and then the coat protein gene was subcloned into prokaryotic expression vector PET-32a In which a recombinant prokaryotic expression vector pET32a-CP was constructed. The recombinant expression vector was transformed into E. coli BL21 (DE3), induced by IPTG and purified by Ni + NTA affinity column to obtain a thioredoxin-containing fusion protein with a molecular weight of about 76 kD. The purified recombinant protein was used as the antigen to immunize rabbits to prepare the polyclonal antibody of RBSDV coat protein. The polyclonal antibody of RBSDV coat protein was prepared and the reliable, sensitive and specific immunoprecipitation RT-PCR and Dot-blot ELISA to detect RBSDV were established. Virus disease diagnosis to provide technical support.