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

Construction and Expression of the Recombinant Plasmid pGEX-Eg95 in Escherichia coli BL21(DE3)

目的构建细粒棘球绦虫重组质粒pGEX-Eg95,并研究该质粒在大肠杆菌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Eg95抗原编码基因;克隆至原核表达载体pGEX-1λT,构建重组质粒pGEX-Eg95;转化大肠杆菌BL21,经异丙基硫代-β-D-半乳糖苷(IPTG)诱导表达后用SDS-PAGE和Western blotting对表达产物进行分析和鉴定。结果RT-PCR扩增出471 bp的Eg95抗原编码基因;双酶切证实Eg95抗原编码基因成功插入pGEX-1λT中;SDS-PAGE分析显示表达产物为相对分子质量约42 500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21%;Western blot鉴定显示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,该质粒在大肠杆菌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 in Escherichia coli BL21 （DE3）. Methods Total RNA was extracted from hydatid cyst of Echinococcus granulosus by ultrasonication method. Eg95 gene, amplified by RT-PCR from the total RNA, was cloned into prokaryotic expression plasmid pGEX- 1λT and transformed into E.coli BL2 （DE3） to form pGEX-Eg95. BL21 （pGEX-Eg95） was then induced with isopropyl-β-D-thiogalactopyranosid （IPTG）, and the expressed products were analyzed and identified by SDS-PAGE and Western blot. Results Eg95 gene with the size of 471 bp was successfully amplified by RT-PCR and cloned into pGEX-1λT, and the recombinant plasmid pGEX-Eg95 was successfully constructed. The molecular mass of the expressed recombinant protein was approximately 42 500 as determined by SDS-PAGE. Amount of the expressed protein was 21% of the total bacterial proteins. Western blot analysis showed that tThe expressed fusion protein could be recognized by the immune sera from mice infected with E. granulosus. Conclusion The plasmid pGEX-Eg95 was successfully constructed and the fusion protein was highly expressed in E.coli. The expressed recombinant protein was also immunogenic.