肠道病毒71型外壳蛋白VP1全长在大肠杆菌中的高效表达及初步活性测定

目的:重组表达肠道病毒71型(EV71)外壳蛋白VP1全长,用于研制血清学检测试剂和疫苗研发。方法:在获得EV71全长基因并测序正确的基础上,将外壳蛋白VP1全长基因克隆到表达载体pET28a(+)上,构建重组表达质粒pET28a(+)/VP1,转化大肠杆菌BL21,IPTG诱导表达,利用Ni2+亲和层析柱对重组蛋白进行纯化,采用双抗原夹心检测技术评价重组抗原与27份EV71抗体阳性血清和18份阴性血清的反应情况。结果:重组EV71-VP1蛋白在大肠杆菌中诱导6 h后可获得高效表达,能与27份EV71抗体阳性血清中的21份发生阳性反应,EV71双抗原夹心检测与中和血清测试结果具有很好的一致性(P0.05)。结论:实现了肠道病毒71型外壳蛋白VP1的高效表达,为肠道病毒71型诊断试剂和疫苗的研究奠定了基础。

Over Expression of the Whole Capsid Protein VP1 of Enterovirus 71 in E.coli and the Preliminary Activity Determination

Objective： To clone,express the recombinant capsid protein VP1 of enterovirus 71（EV71）,and make preparations for the detection and vaccine.Methods： Based on the full length clone of the capsid protein VP1 of EV71,VP1 gene was cloned into pET28a（＋） vector to construct the recombinant plasmid pET28a（＋） / VP1.Re-combinant VP1 protein was expressed in E.coli and purified by metal（Ni2＋） chelating affinity chromatography.Evaluate the reaction between the recombination antigen and the 27 portions of anti-EV71 positive and 18 portions negative by direct sandwich of ELISA.Results： The recombinant capsid protein can be over experssed in E.coli and has well antigenicity.21 from 27 have been detected.The results are concordance between the direct sand-wich of ELISA and the neutralization test（P0.05）.Conclusion： The capsid protein VP1 of EV71 was expressed successfully,which could be useful for developing diagnose reagent or vaccine of EV71.