细粒棘球绦虫重组质粒pGEX-Eg95-EgA31的构建及其在大肠埃希菌BL21(DE3)中的表达

Construction and expression of the recombinant plasmid pGEX-Eg95-EgA31 of Echinococcus granulosus in Escherichia coli BL21(DE3)

目的构建细粒棘球绦虫重组质粒pGEX-Eg95-EgA31,研究该质粒在大肠埃希菌BL21(DE3)中的表达。方法超声粉碎细粒棘球蚴组织,提取总RNA,通过RT-PCR扩增获得Eg95和EgA31抗原编码基因,然后采用基因拼接法(gene SOEing)剪接Eg95和EgA31,得到Eg95-EgA31融合基因,克隆至原核表达载体pGEX-1λT,构建重组质粒pGEX-Eg95-EgA31,转化大肠埃希菌BL21,经异丙基硫代--βD-半乳糖苷(IPTG)诱导表达后用SDS-PAGE和Westernblot对表达产物进行分析和鉴定。结果基因拼接法扩增出约1 016 bp的Eg95-EgA31融合基因;双酶切证实Eg95-EgA31融合基因成功插入pGEX-1λT中,SDS-PAGE分析显示表达产物为分子质量单位约为62.5 ku的重组蛋白,与预期结果一致,表达的蛋白约占菌体总蛋白的18%;Western blot鉴定重组蛋白能被细粒棘球蚴感染鼠血清识别。结论成功构建了细粒棘球绦虫重组质粒pGEX-Eg95-EgA31,该质粒在大肠埃希菌BL21中获得了高效融合表达,表达的融合蛋白具有特异的抗原性。

Objective To construct and express the recombinant plasmid pGEX Eg95-EgA31 of Echinococcus granulosus in Escherichia coli BL21 （DE3）. Methods Total RNA was extracted from hydatid cysts by ultrasound-breaking, and the Eg95 and EgA31 antigen genes were amplified by RT PCR of the total RNA. The Eg95-EgA31 fusion gene ob- tained with gene SOEing was cloned into the prokaryotic expression plasmid pGEX-1λT and transformed into E. coli BL2 （DE3） to construct pGEX Eg95-EgA31. BL21（pGEX-Eg95-EgA31） was induced with isopropyl-β-D-thiogalactopyranosid （IPTG）, and the expressed products were analyzed and identified by SDS-PAGE and Western hlot. Results The 1 016 bp Eg95-EgA31 fusion gene was successfully amplified by gene SOEing and cloned into pGEX-1λT by restriction analysis, the recombinant plasmid pGEX-Eg95-EgA31 was successfully constructed. The relative molecular mass of the expressed recombinant protein was approximately 62.5 ku by SDS-PAGE, and the amount of the expressed protein was 18% of the total bacterial proteins. The fusion protein could be recognized by sera from mice infected with E. granulosus by Western blot. Conclusion The recombinant plasmid pGEX-Eg95 EgA31 of E. granulosus is successfully constructed and highly expressed in E. coli in the fused form with GST, and the expressed fusion protein shows specific antigenicity.