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目的克隆恶性疟原虫配子体期特异性基因(Plasmodium falciparum gametocyte development 1 gene,Pfgdv1),体外表达和鉴定重组Pfgdv1蛋白。方法通过PCR法从恶性疟原虫感染病人血液DNA样本中扩增Pfgdv1基因,插入到原核表达载体p ET28a(+),构建p ET28a-Pfgdv1重组表达质粒,转化至大肠埃希菌(E.coli)BL21(DE3+),通过异丙基-β-D-硫代吡喃半乳糖苷诱导表达重组蛋白,经Ni+-亲和层析柱纯化重组蛋白,纯化产物经十二烷基磺酸钠-聚丙烯酰胺凝胶电泳(SDS-PAGE)和Western blotting鉴定。结果 PCR扩增的Pfgdv1基因长度约为1.65 kb,重组p ET28a-Pfgdv1质粒构建成功,插入方向正确无框移,转化至E.coli BL21(DE3+)所表达的重组蛋白分子量约为67 k Da,且能被抗His标签单克隆抗体识别。结论成功克隆了Pfgdv1基因,表达并纯化了重组Pfgdv1蛋白,为进一步研究恶性疟原虫配子体期传播阻断疫苗奠定了基础。
Objective To clone Plasmodium falciparum gametocyte development 1 gene (Pfgdv1) and express and identify the recombinant Pfgdv1 protein in vitro. Methods The Pfgdv1 gene was amplified by PCR from blood DNA samples of P. falciparum infected patients and inserted into the prokaryotic expression vector p ET28a (+) to construct the recombinant plasmid pET28a-Pfgdv1. The recombinant plasmid was transformed into E.coli The recombinant protein was induced by isopropyl-β-D-thiogalactopyranoside and the recombinant protein was purified by Ni + - affinity chromatography. The purified product was purified by sodium dodecyl sulfate - poly Acrylamide gel electrophoresis (SDS-PAGE) and Western blotting identification. Results The length of Pfgdv1 gene amplified by PCR was about 1.65 kb. The recombinant plasmid pET28a-Pfgdv1 was constructed successfully and its insertion direction was correct without frame shift. The molecular weight of the recombinant protein transformed into E. coli BL21 (DE3 +) was about 67 kDa, And can be recognized by anti-His-tag monoclonal antibodies. Conclusion The Pfgdv1 gene was successfully cloned and the recombinant Pfgdv1 protein was expressed and purified, which laid the foundation for the further study of the phage vaccine against P. falciparum.