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目的 克隆并表达恶性疟原虫Pf12基因 ,为进一步研究其抗原表位奠定基础。 方法 将恶性疟原虫Pf12基因克隆入高效融合表达载体 pGEX 4T 1,转化大肠杆菌BL2 1(DE3 ) ,2 8℃、IPTG诱导表达 ,SDS PAGE和Western blot免疫印迹分析表达产物。 结果 成功构建重组质粒 pGEX Pf12 ,经诱导后表达出含外源基因的融合蛋白 ,SDS PAGE分析表达产物分子质量约 66ku ,Western blot免疫印迹表明 ,表达的产物能特异地被抗GST多抗 (1∶5 0 0稀释 )识别 ,亦能较特异地与抗疟原虫鼠免疫血清结合。 结论 恶性疟原虫Pf12在原核表达系统 pGEX 4T 1/BL2 1(DE3 )中获得成功表达 ,为下一步表达蛋白纯化 ,以及研究Pf12基因包含的抗原表位提供试验依据。
Objective To clone and express Pf12 gene of Plasmodium falciparum and lay foundation for further study of its antigenic epitope. Methods Pf12 gene of Plasmodium falciparum was cloned into pGEX 4T 1, which was transformed into E. coli BL21 (DE3) and induced by IPTG at 28 ℃. The expressed product was analyzed by SDS PAGE and Western blot. Results The recombinant plasmid pGEX Pf12 was successfully constructed and the fusion protein containing exogenous gene was expressed after induction. The molecular weight of the expressed product was about 66ku by SDS PAGE. Western blotting showed that the expressed product was specifically blocked by anti-GST polyclonal antibody : 500 dilutions), but also more specifically binds to anti-Plasmodium immune sera. Conclusion Plasmodium falciparum Pf12 was successfully expressed in prokaryotic expression system pGEX 4T 1 / BL2 1 (DE3), which provided the experimental basis for the further purification of the expressed protein and the study of the epitopes contained in Pf12 gene.