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目的构建日本血吸虫重组质粒pET28α-Sj26GST,研究该质粒在大肠埃希菌BL21(DE3)中的表达。方法超声粉碎日本血吸虫成虫,提取总RNA,通过RT-PCR扩增获得Sj26GST抗原编码基因,克隆至原核表达载体pET28α,构建重组质粒pET28α-Sj26GST,转化大肠埃希菌BL21(DE3),经异丙基硫代-β-D-半乳糖苷(IPTG)诱导表达后用SDS-PAGE和Western blot对表达产物进行分析和鉴定。结果 RT-PCR扩增出676 bp的Sj26GST基因;双酶切和PCR鉴定证实Sj26GST基因成功插入pET28α中,SDS-PAGE分析显示表达产物为相对分子质量约为36×103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26%;Western blot鉴定重组蛋白能被日本血吸虫感染的兔血清识别。结论成功构建了日本血吸虫重组质粒pET28α-Sj26GST,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 pET28α-Sj26GST of Schistosoma japonicum and study its expression in Escherichia coli BL21 (DE3). Methods Sj26GST antigen encoding gene was amplified by RT-PCR and then cloned into prokaryotic expression vector pET28α. The recombinant plasmid pET28α-Sj26GST was constructed and transformed into Escherichia coli BL21 (DE3) After induction with IPTG, the expressed products were analyzed and identified by SDS-PAGE and Western blot. Results The Sj26GST gene of 676 bp was amplified by RT-PCR. The double-digestion and PCR proved that the Sj26GST gene was successfully inserted into pET28α. SDS-PAGE analysis showed that the recombinant protein was expressed as a recombinant protein with molecular weight of 36 × 103, Consistent with the expression of protein accounted for about 26% of total bacterial protein; Western blot identification of recombinant proteins can be infected with Schistosoma japonicum rabbit serum identification. Conclusion The recombinant plasmid pET28α-Sj26GST of Schistosoma japonicum was constructed successfully. The fusion protein was highly expressed in Escherichia coli BL21, and the expressed fusion protein has specific antigenicity.