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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 recombinant plasmid pGEX-Eg95 of Echinococcus granulosus and study its expression in E. coli BL21 (DE3). Methods Echinococcus granulosus was sonicated to extract total RNA. The Eg95 antigen gene was amplified by RT-PCR. The gene was cloned into prokaryotic expression vector pGEX-1λT to construct recombinant plasmid pGEX-Eg95. The recombinant plasmid was transformed into E. coli BL21. After induced by IPTG, the expressed products were analyzed and identified by SDS-PAGE and Western blotting. Results The 471 bp Eg95 antigen gene was amplified by RT-PCR. Double-digestion confirmed that the gene encoding Eg95 was inserted into pGEX-1λT successfully. SDS-PAGE analysis showed that the expressed product was a recombinant protein with a relative molecular mass of about 42 500, The results showed that the expressed protein accounted for about 21% of the total bacterial protein. Western blot showed that the recombinant protein could be recognized by the serum of mice infected with Echinococcus granulosus. Conclusion The recombinant plasmid pGEX-Eg95 of Echinococcus granulosus was constructed successfully. The fusion protein was highly expressed in E. coli BL21, and the expressed fusion protein has specific antigenicity.