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

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.