柯萨奇病毒A组16型衣壳蛋白VP1的原核表达及初步活性测定

Expression of the Capsid Protein VP1 of Coxsackie Virus Group A 16 Strain in E.coli and the Preliminary Activity Determination

目的:原核表达柯萨奇病毒A组16型(CVA16)衣壳蛋白VP1,以便于研制血清学检测试剂。方法:在基因库中钓取CVA16-VP1的全长序列,采用PCR逐步合成法合成其全长基因,测序正确后克隆到表达载体pET28a(+)中,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;建立捕获免疫酶联法检测IgM抗体,检测20份手足口病阳性血清和30份阴性血清,评价重组抗原的灵敏度和特异性;采用CVA16全病毒免疫的抗小鼠血清进行Western印迹。结果:重组CVA16-VP1蛋白在大肠杆菌中获得高效表达;用重组蛋白抗原检测,20份手足口病患儿阳性血清中有4份阳性,其中1份同时为肠道病毒71型(EV71)VP1阳性,30份阴性血清无反应。结论:实现了CVA16-VP1的高效表达,初步结果显示重组蛋白具有较好的抗原性,为柯萨奇病毒A组16型诊断试剂的研究奠定了基础。

Objective： To prokaryotic express the recombinant capsid protein VP1 of Coxsackie virus group A 16 strain（CVA16） and make preparations for the detection. Methods： The gene of CVA16-VP1 was synthesised by bridging-PCR, then was cloned into pET28a（＋） vector to construct the recombinant plasmid pET28a（＋）/VPI. Recombinant CVA16-VP1 protein was expressed in E.coli BL21 and was purified by Ni^2＋ chelating affinity chromatography. The recombination CVA16-VP1 antigen was evaluated by detection the 20 portions of HFMD positive and 30 portions negative by IgM-ELISA. Results： The recombinant CVA16-VPl can be over experssed in E.coli. 4 from 20 portions of HFMD positive have been detected, 1 of them was both positive by CVAI6-VP1 and EV71-VP1. 30 negative do not respond. Conclusion： The recombination CVA16-VP1 was expressed, and has well antigenicity, which could be useful for developing diagnose reagent or vaccine of CVA16.