细粒棘球蚴Eg95s基因的串联表达及表达系统优化  

Tandem Expression of Eg95s Gene of Echinococcus granulosus and Optimization of the Expression System

作　　者：刘丹[1] 贾红[1] 侯绍华[1] 丁敏[1] 朱鸿飞[1] 

机构地区：[1]中国农业科学院北京畜牧兽医研究所动物医学实验室,北京100081

出　　处：《中国畜牧兽医》2009年第8期33-37,共5页China Animal Husbandry & Veterinary Medicine

基　　金：国家科技支撑计划(2008BAK51B08)

摘　　要：为研制新型棘球蚴病基因工程疫苗,根据GenBank公布的细粒棘球蚴Eg95基因序列,针对其中一段348 bp高度亲水的优势表位序列设计引物,扩增Eg95s基因,利用同尾酶法基因同向串联方案,获得3拷贝Eg95s串联体。分别用两种表达载体pGEX-6p-1和pET-32a构建重组质粒,转化入3种大肠杆菌表达宿主BL21(DE3)、Rosseta(DE3)和Origami(DE3),对其进行IPTG诱导表达,经免疫印迹分析结果表明,均能正确表达具有免疫原性的Eg95s蛋白。通过重组蛋白的表达量、可溶性、纯化方法等方面的比较,对Eg95s重组表达系统的载体及宿主菌进行了优化,结果表明,3拷贝的串联Eg95s在以pET-32a为表达载体,Rosseta(DE3)为宿主菌的系统中表达最佳。最终获得了表达量高、可溶性好、纯化方便的Eg95s重组蛋白表达系统,为大规模生产棘球蚴病基因工程疫苗提供了科学依据。According to the sequence of Eg95 gene published on GenBank, primers were designed from a segment about 348 bp sequences encoding highly hydrophilic dominate epitopes, designating as Eg95s. Three repeats of EG95s were obtained by isoschizomer construction method. Three Eg95s were cloned into two expression vectors, pGEX-6p-1 and pET-32a, then transformed into three host bacterias, Escherichia coli BL21 （DE3）, Rosseta （DE3） and origami （DE3） for expression, respectively. The expression products were identified by Western blotting, the expression systems were optimizated by detecting the expression level, solubility of the products and their purify methods. The results showed that the best quality of three EG95s was obtained in the expression system of pET 32a and Rosseta （DE3）. The Eg95s fusion protein highly expressed by the optimized expression system was with good solubility, and easy to purify, which is benefit for the further preparation of Echinococcus granulosus gene engineering vaccine on a large scale.

关 键 词：Eg95s 串联表达 表达系统 优化 

分 类 号：S852.7[农业科学—基础兽医学]