猪血凝性脑脊髓炎病毒S1蛋白基因在毕赤酵母中的表达  

Expression of S1 gene of hemagglutinating encephalomyelitis virus in Pichia pastoris yeast

作　　者：陆慧君[1] 贺文琦[1] 高丰[1] 常灵竹[1] 赵魁[1] 盖显英[2] 王龙涛[1] 宋德光[1] 

机构地区：[1]吉林大学畜牧兽医学院,吉林长春130062 [2]吉林大学第一医院,吉林长春130021

出　　处：《中国兽医学报》2008年第1期1-4,共4页Chinese Journal of Veterinary Science

基　　金：国家自然科学基金资助项目(30671551);吉林省科技发展计划重点资助项目(20060206-2)

摘　　要：应用特异性引物从猪血凝性脑脊髓炎病毒(HEV)中扩增出S1蛋白基因,PCR产物纯化后克隆入pGEM-T载体中,得到重组质粒pTS1。用EcoRI和Not I双酶切pTS1,回收目的基因S1片段将其定向克隆到pPICZαA中,构建重组质粒pPICZαAS1。用BstXI酶切pPICZαAS1使其线性化,并电转至感受态毕赤酵母细胞GS115。PCR法鉴定阳性重组子,用1%甲醇诱导表达后,进行SDS-PAGE及Western blot分析。结果显示,在酵母菌培养基上清中检测到相对分子质量为73000的重组蛋白,且该重组蛋白可与HEV多克隆抗体发生特异性血清学反应,表明HEV的S1蛋白片段在毕赤酵母中获得成功表达。To express SI protein of HEV in Pichia pastoris yeast, SI gene was amplified by PCR from HEV with a pair of specific primers. Then PCR products were purified and cloned into pGEM-T vector to obtain the pasmid pTSI. The intersting gene frag-ment was recovered after the double enzyme difestion of EcoR I/Not I,then subcloned into pPICZαA for secretory expression. The recombinant pPICZaASI was linealized with BstX I and then tmsformed into GS115 yeast cells by electroporation for expression under the induction of 1% methanol. The recombinated strains were screened by PCR technique. The expression products were identi-fied by SDS-PAGE and Western-blot. The results showed that there was a molecular weight of 73 000, which could be specifically recongnized by polyclonic antibody against HEV. It suggested that the SI protein of HEV was obtained with Pichia pastoris expression system.

关 键 词：猪 血凝性脑脊髓炎病毒 S1蛋白 毕赤酵母 表达 

分 类 号：S858.9[农业科学—临床兽医学]