恶性疟原虫网状细胞结合蛋白同源体5F1段的克隆、表达及初步鉴定

Cloning,expression and identification of reticulocyte-binding protein homologue 5 F1 fragment in Plasmodium falciparum

目的克隆表达恶性疟原虫网状细胞结合蛋白同源体5(PfRh5)F1片段,并评价其抗原性。方法 PCR扩增目的基因片段,克隆到表达载体pET28a(+)中,构建PfRh5F1/pET28a原核表达载体。IPTG诱导表达目的基因,SDS-PAGE电泳分析表达产物,并用WesternBlot检测其抗原性。结果成功构建了PfRh5F1/pET28a原核表达系统,并在大肠杆菌中以包涵体形式高效表达,表达产物能被恶性疟原虫感染患者血清识别,而不能被间日疟原虫感染患者及正常人血清识别。结论恶性疟原虫PfRh5F1段在大肠杆菌中获得高效表达且表达产物具有良好的抗原性。

In order to clone and express reticulocyte-binding protein homologue 5（PfRh5）F1 fragment gene of Plasmodium falciparum,the 429bp PfRh5 gene（91-519）was specifically amplified by polymerase chain reaction and cloned into pET28a（＋）vector.The recombinant plasmid was then transformed and induced to express in E.coli Rosetta.The expressed product was analyzed by SDS-PAGE and Western Blot respectively.And the expressed protein was insoluble with a size of about 21.9 kDa as predicted.Also,it exhibited a specific reaction with immune sera obtained from patients with Pf maleria.These results demonstrate that the PfRh5 F1 fragment has been successfully expressed and the expressed protein has certain antigenicity.