烟草丛顶病毒ORF1蛋白的多克隆抗体制备及应用  

Polyclonal antibody preparation and application of ORF1-encoded protein of Tobacco bushy top virus

作　　者：王国鲁 王德亚 于成明[1] 原雪峰[1] 

机构地区：[1]山东农业大学植物保护学院山东省农业微生物重点实验室,泰安271018

出　　处：《植物病理学报》2016年第1期63-71,共9页Acta Phytopathologica Sinica

基　　金：国家自然科学基金资助项目(31370179);山东省自然科学基金资助项目(ZR2013CM015)

摘　　要：PCR扩增烟草丛顶病毒(Tobacco bushy top virus,TBTV)的ORF1序列并克隆到原核表达载体pEHISTEV中,转化大肠杆菌Rosetta菌株经IPTG诱导表达TBTV ORF1蛋白。利用切胶纯化的ORF1蛋白免疫新西兰大耳白兔制备并获得抗血清,间接ELISA检测效价为1:24 3000。经抗原亲和纯化从抗血清中得到特异性和灵敏度俱佳的ORF1多克隆抗体。Western blot分析显示,TBTV ORF1多克隆抗体既可以检测田间发病的烟草丛顶病样品中ORF1蛋白,也可检测在体内和体外翻译体系中的TBTV ORF1蛋白的表达。另外发现ORF2蛋白以ORF1延长蛋白的形式存在,根据ORF1和ORF2的重叠情况及潜在的七核苷酸滑动序列和下游的稳定二级结构,推测此ORF1延长蛋白是移码翻译产物。ORF1 sequence of Tobacco bushy top virus （TBTV） was amplified and cloned into prokaryotic ex- pression vector pEHISTEV. The recombination plasmid containing ORF1 of TBTV was transformed into E. coli Rosetta and performed to express ORF1 protein under the induction of IPTG. The purified ORF1 protein from SDS-PAGE gel was used to inject the rabbits as antigens to get antiserum with high titer at 1 ： 24 3000 by indi- rect ELISA.Through the antigen affinity purification, the polyclonal antibody against ORF1 protein with higher sensitivity and specificity Was purified from whole antiserum. The polyclonal antibody could be used to detect the ORF1 of the TBTV-infected tobaccos in the field as well as in vivo （refer to Arabidopsis protoplasts） and in vitro translation system （refer to WGE）, in which TBTV full-length RNA was incubated. In addition, ORF2 was identified to be expressed as ORFl-extended protein, possibly through translational frameshift inechanism based on the over-lapping status of ORF1 and ORF2, potential hepta-nucleotide slippery sequence and down- stream stable stem-loop structure, This project provided substantial serological support for the consequent research on the detailed mechanism of the multi-levels of translation of ORF1 and ORF2 in Tobacco bushy top virus.

关 键 词：烟草丛顶病毒 ORF1基因 原核表达 抗血清 移码翻译 

分 类 号：S432.41[农业科学—植物病理学]