烟草蚀纹病毒外壳蛋白基因的克隆及在毕赤酵母中的表达  

Cloning and expression of tobacco etch virus coat protein gene in Pichia pastoris

作　　者：王秀敏[1] 吴云锋[2] 韩烈保[1] 

机构地区：[1]北京林业大学草坪研究所,北京100083 [2]西北农林科技大学分子植物病毒研究室,杨凌712100

出　　处：《中国烟草学报》2009年第1期61-64,共4页Acta Tabacaria Sinica

基　　金：陕西省烟草病毒病监控技术研究项目(2001KN-4);西北农林科技大学创新团队项目资助;国家自然科学基金(30270876)

摘　　要：利用RT-PCR方法获得烟草蚀纹病毒(TEV)外壳蛋白(CP)基因,大小为789bp。经过EcoRI和Not Ⅰ双酶切、定向克隆到pPIC9K,构建了真核表达载体pPIC9K-TEVCP。将重组质粒用Sal I线性化后电转化导入毕赤酵母GS115中,经PCR鉴定为阳性的菌落,利用甲醇进行诱导表达,筛选出了高效表达菌株GS115-11和GS115-14,表达的蛋白大小约为37kD。表达产物经SDS-PAGE割胶纯化后,免疫家兔,制备了特异性抗血清,ELISA法检测效价为1:1500。Western blot结果显示,制备的抗血清可以用来检测田间的发病植株。A TEV CP gene was obtained by means of RT-PCR amplification. The product was digested with restrictive endonu- clease Eco RI/Not I, and inserted into Pichia pastoris expression vector pPIC9K. Recombinant plasmid was linearized with Sal I and then transferred into GS115 strain by electroporation. Whether the TEV CP gene was integrated into alcohol oxidase promoter（AOX）locus on yeast chromosome was verified by amplification with special primers. SDS-PAGE result showed that the TEV CP gene was expressed highly in GS115-11 and GS115-14, and the molecular weight of expression product induced with 1% methanol was about 37 kD. The product was used as antigen to immunize the rabbit and the titer of antiserum was 1 ： 1500. Result of Western blot showed that the antiserum can be used to detect diseased tobaccos in the field.

关 键 词：烟草蚀纹病毒 外壳蛋白 毕赤酵母:表达 

分 类 号：S435.72[农业科学—农业昆虫与害虫防治]