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

作　　者：卢训[1] 方琦[1] 尹跃艳[1] 秦西云[2] 丁铭[1] 

机构地区：[1]云南省农业科学院生物技术与种质资源研究所,云南省农业生物技术重点实验室,农业部西南农业基因资源与种质创制重点实验室,云南昆明650223 [2]云南省烟草农业科学研究院,云南昆明650031

出　　处：《西南农业学报》2014年第4期1547-1550,共4页Southwest China Journal of Agricultural Sciences

基　　金：中国烟草总公司重点科技项目(110200902065);云南省烟草公司科技计划项目(2010YN19)

摘　　要：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.

关 键 词：烟草花叶病毒 原核表达 多克隆抗体 DOT-ELISA 

分 类 号：S572[农业科学—烟草工业]