烟草花叶病毒原核表达外壳蛋白抗体制备及快速检测方法建立

Preparation of Polyclonal Antibody and Establishment of Rapid Detection Method for Tobacco Mosaic virus

用RT-PCR从感染烟草花叶病毒(Tobacco mosaic virus,TMV)的烟草样品中克隆该病毒的外壳蛋白基因,病毒外壳蛋白基因再亚克隆到原核表达载体pET-30a(+)中,构建成重组原核表达载体,转化大肠杆菌BL21,经IPTG诱导、Ni+NTA亲和柱纯化获重组蛋白。以纯化的重组蛋白为抗原免疫家兔,制备TMV外壳蛋白多克隆抗体,并用制备的多克隆抗体建立了更简便、经济、特异性更强的DOT-ELISA检测法。

The full length eDNA of tobacco mosaic virus （TMV） which encoded coat protein was cloned from the virus infected tobacco asmpies by RT-PCR, and subcloned into a prokaryotic expression vector pET-30a（ ＋ ）. The recombinant prokaryotic expression vector was used to transform Eschedch/a co//B[21. 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 TMV. Using polyclonal antibodies and DOT- ELISA were established for reliable, sensitive and specific detection of TMV.