恶性疟原虫配子体期特异性蛋白Pfgdv1克隆表达及鉴定

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。

Cloning,expression and identification of gametocyte specific protein Pfgdv1 of Plasmodium falciparum

Objective Objective To clone a gametocyte specific protein Pfgdv1 of Plasmodium falciparum，express and identify re?combinant Pfgdv1 protein in vitro. Methods Methods PCR was performed to amplify Pfgdv1 from P. falciparum DNA which was gotfrom the patient who was infected with P. falciparum，and the PCR product was inserted into pET28a（+）vector. pET28a?Pfg?dv1 recombinant plasmid was constructed and transformed into E. coli host BL21（DE3+） . IPTG was used to induce the recombi?nant Pfgdv1 protein fused with His tag，and the protein was purified by His?NTA affinity chromatography. The recombinant pro?tein was identified by SDS?PAGE and Western blotting. Results Results The PCR product of Pfgdv1 gene was about 1.65 kb，meetingthe expectation of predicted fragment size. The recombinant protein was about 67 kDa，which could be recognized by His?Tagmonoclonal antibody. Conclusion Conclusion The Pfgdv1 gene of P. falciparum is successfully cloned，and the recombinant Pfgdv1 pro?tein is expressed， thereby providing an opportunity for further study on transmission blocking vaccine.