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目的表达纯化肠道病毒71型(EV71)VP1蛋白,初步建立人血清中抗EV71 IgG间接ELISA检测方法。方法通过化学法合成肠道病毒71型VP1全基因序列,构建重组质粒pET-43.1a(+)/VP1,转化大肠杆菌BL21(DE)3,IPTG诱导表达,镍金属亲和层析纯化目的蛋白,产物经SDS-PAGE和Western blot鉴定。以目的蛋白作为包被抗原,初步建立间接ELISA检测方法,并对77份6～10岁无手足口病症状儿童血清及手足口病现症患儿血清进行检测。结果成功构建重组质粒pET-43.1a(+)/VP1,VP1融合蛋白分子量约为43 kD,纯度可达95%;初步建立了人血清中抗EV71 IgG间接ELISA检测方法,36例HFMD现症患儿中EV71 IgG阳性22例(61.11%);41例无手足口症状儿童血清中EV71 IgG阳性21例(51.22%)。结论表达产物具有较好的免疫原性,可用于肠道病毒71抗体检测。
Objective To express and purify the VP1 protein of enterovirus 71 (EV71) and establish an indirect ELISA method for the detection of anti-EV71 IgG in human serum. Methods The full-length VP1 gene of enterovirus 71 was synthesized by chemical method. The recombinant plasmid pET-43.1a (+) / VP1 was constructed and transformed into E.coli BL21 (DE) 3. The recombinant protein was induced by IPTG and purified by nickel metal affinity chromatography , The product was identified by SDS-PAGE and Western blot. The target protein was used as coating antigen, and the indirect ELISA method was established. The sera from 77 children aged 6-10 years with no symptoms of hand-foot-mouth disease and children with HFMD were detected. Results The recombinant plasmid pET-43.1a (+) / VP1 was successfully constructed. The molecular weight of the VP1 fusion protein was about 43 kD with a purity of 95%. The indirect ELISA method was established for the detection of anti-EV71 IgG in human serum and 36 cases of HFMD There were 22 cases (61.11%) of EV71 IgG positive in children, and 21 cases (51.22%) of EV71 IgG positive in 41 cases without hand-foot mouth symptoms. Conclusion The expressed product has good immunogenicity and can be used for the detection of enterovirus 71 antibody.