重组质粒Eg.EF-1/pGEX-6P-1的构建及原核诱导表达

将细粒棘球蚴（Echinococcus granulosus,Eg.）延伸因子（Elongation factor 1,EF-1）与pGEX-6P-1构建成重组质粒并进行表达、纯化及对其特性进行鉴定.将构建好的EF-1/pGEM-T经双酶切后获取目的基因片段,定向重组于表达载体pGEX-6P-1并转化大肠杆菌（Escherichia coli）E.coli BL21,IPTG诱导表达,用蛋白亲和层析柱GSTrap FF对表达产物进行纯化,特异性蛋白解离酶PreScission^TM Protease酶切融合蛋白从中获取重组Eg.EF-1;SDS-PAGE和Western blot方法鉴定表达产物.获得的Eg.EF-1/pGEX-6P-1/E.coli BL21菌株,诱导表达融合蛋白和纯化分离得到的31 ku Eg.EF-1均能被细粒棘球蚴天然抗原免疫的兔多克隆抗血清识别.证明成功构建出具有有效表达的Eg.EF-1/pGEX-6P-1菌株,初步证实重组蛋白具有较好的抗原性.

Construction of Recombinant Eg. EFY DGEX-6p-1 and Expresssion in E. coli

To obtain the recombinant protein Eg. EF -1 by gene engineering, the gene fragment of Eg. EF 1 was obtained by restrietedly digesting the Eg. EF-1/pGEM T, and subelonding into expression vector pGEX-6P-1. The Eg. EF- 1/pGDX-6P-1 was constructed and the fusion protein Eg. EF-1/GST expressed in E. coil BL21 induced by IPTG. The Eg. EF-1/GST was purified by chromatography using GSTrap FF and the isolated 31ku Eg. EF-1 has been obtained after the fusion protein digested by the PreScission^TM Protease. Using Western blotting the gg. EF- 1 and Eg. EF 1/GST can be identified by the sera of rabbits immunized by hydatid cyst fluid from CE patients. The recombinant fusion protein Eg. EF 1/GST has a same epitope as the nature antigen.