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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 the recombinant plasmid pGEX-Eg95-EgA31 of Echinococcus granulosus and study its expression in Escherichia coli BL21 (DE3). Methods Echinococcus granulosus was sonicated to extract total RNA. Eg95 and EgA31 antigen-encoding genes were amplified by RT-PCR. Then Eg95 and EgA31 were spliced by gene SOEing to obtain Eg95-EgA31 fusion gene. Clones The recombinant plasmid pGEX-Eg95-EgA31 was constructed and transformed into Escherichia coli BL21. The recombinant plasmid pGEX-Eg95-EgA31 was transformed into E. coli BL21 and induced by IPTG. SDS-PAGE and Westernblot The expression product was analyzed and identified. Results The Eg95-EgA31 fusion gene was amplified approximately 1 016 bp by gene splicing. Double-digestion confirmed that the Eg95-EgA31 fusion gene was successfully inserted into pGEX-1λT. SDS-PAGE analysis showed that the expressed product was about 62.5 ku Recombinant protein, consistent with the expected results, the expression of protein accounted for about 18% of total bacterial protein; Western blot identification of recombinant protein can be echinococcus granulosus infected rat serum identification. Conclusion The recombinant plasmid pGEX-Eg95-EgA31 was successfully constructed. The fusion protein was highly expressed in Escherichia coli BL21 and the expressed fusion protein had specific antigenicity.