肠道病毒71型重组VP1蛋白的表达和EV71型手足口病血清学诊断方法的建立

目的 获得纯化的具有免疫活性的肠道病毒71型VP1蛋白,建立EV71感染早期、快速和准确的ELISA血清学诊断方法.方法 通过PCR方法扩增出VP1基因,定向克隆到原核表达载体pET-21b(+),阳性质粒转化入E.coli B121(DE3)感受态细胞经IPTG诱导,SDS-PAGE电泳和蛋白免疫印迹分析目的 蛋白的表达水平.纯化的VPI蛋白用作包被抗原,建立手足口病(HFMD)患者抗-EV71-IgM和IgG的血清学诊断方法.结果 成功表达和纯化了VP1重组蛋白,所表达的蛋白能被EV71型手足口病患者血清所识别.调查发现,与正常人和EV71阴性手足口病患儿比较,EV71阳性手足口病血清中抗-EV71-IgM和IgG中A值显著升高.差异具有统计学意义(P<0.05).与RT-PCR结果比较,发现该方法IgM的诊断敏感性和特异性分别为73%和77%;该IgG诊断方法的敏感性和特异性分别为82%和83%.完成该试验仪需4 h.结论 利用pET原核表达系统成功克隆、表达和纯化了肠道病毒71型重组外壳蛋白VPI,且具有良好的抗原性.该抗原可用于研制EV71血清学诊断试剂盒.

Expression of recombinant VP1 protein of Enterovirus 71 and development of serological assay for detection of EV71 infection

Objective To obtain a recombinant purified Enterovims 71 VP1 protein and establishment of an early,rapid and accurate serolngieal ELISA (enzyme-linked immunesorbent assay)for detection of EV71 infection. Methods VPI gene was amplified by PCR and cloned into pET-21b ( + ) vector,the positive recombinant plasmid were transformed into E. coil BI21 ( DE3 ),and was induced with IPTG,the recombinant protein by SDS-PAGE and Western Blot assays. Finally,the recombinant purified VP1 protein was used as a coated antigen for detection of serum anti-lgM and lgG against EV71 by ELISA. Results The purified VP1 was obtained,and it can be recognized by sera of patients with EV71 infection associated with hand-foot-mouth disease. The A values of anti-EV71 lgM and IgG were significantly elevated as compared to healthy objects and HFMD patients without EV71 infection ( P < 0.05) .The sensitivity and specificity of IgM to EV71 were 73% and 77% compared with the RT-PCR results,respectively; and those of IgG being 82% and 83%,respectively. Conclusions The recombinant protein VP1 was produced and purified,and it was proved to have a good antigenicity and eould be used to develop a serological diagnosis kit for EV71 infection in the future.