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目的诱导表达并纯化细粒棘球绦虫EgAgB8/1重组抗原和EgAgB8/1-EgAgB8/2重组嵌合抗原,比较两个重组蛋白对囊型包虫病(CE)的血清学诊断价值。方法 IPTG诱导转染至E.coliBL21(DE3)LysS的pET32a-EgAgB8/1和pET32a-EgAgB8/1-EgAgB8/2重组原核表达质粒,表达和纯化EgAgB8/1重组蛋白和EgAgB8/1-EgAgB8/2重组嵌合蛋白,用SDS-PAGE电泳分析鉴定重组蛋白,并通过超声裂解法进行纯化,以CE病人及囊虫病人血清为一抗,Western blot法检测两个重组蛋白免疫反应性。结果细粒棘球绦虫重组抗原pET32a-EgAgB8/1和pET32a-EgAgB8/1-EgAgB8/2重组原核表达质粒得到成功诱导表达,经SDS-PAGE电泳分析,重组蛋白分子质量单位为28 ku和38 ku;Western blot结果表明,EgAgB8/1重组蛋白和EgAgB8/1-EgAgB8/2重组嵌合蛋白均能被CE病人血清特异性识别,16份CE病人血清中,以EgAgB8/1重组蛋白为抗原时11份阳性,以EgAgB8/1-EgAgB8/2重组蛋白为抗原时13份阳性;用EgAgB8/1和EgAgB8/1-EgAgB8/2重组蛋白检测12份囊虫病人血清,分别有5份和3份阳性。结论 EgAgB8/1-EgAgB8/2重组嵌合蛋白具有抗原特异性,对囊型包虫病的血清学诊断价值优于EgAgB8/1重组蛋白。
Objective To induce the expression and purification of EgAgB8 / 1 recombinant antigen and EgAgB8 / 1-EgAgB8 / 2 recombinant chimeric antigen, and to evaluate the serological diagnostic value of two recombinant proteins for cystic echinococcosis (CE). Methods Recombinant plasmid pET32a-EgAgB8 / 1 and pET32a-EgAgB8 / 1-EgAgB8 / 2 transfected into E.coli BL21 (DE3) LysS were induced by IPTG to express and purify EgAgB8 / 1 recombinant protein and EgAgB8 / 1-EgAgB8 / 2 The recombinant chimeric protein was identified by SDS-PAGE. The recombinant protein was identified by SDS-PAGE and purified by sonication. Serum of CE patients and cysticercosis patients was used as primary antibody. Western blot was used to detect the immunoreactivity of the two recombinant proteins. Results The recombinant eukaryotic expression plasmid pET32a-EgAgB8 / 1 and pET32a-EgAgB8 / 1-EgAgB8 / 2 were induced successfully. SDS-PAGE analysis showed that the molecular weight of the recombinant protein was 28 ku and 38 ku ; Western blot results showed that both EgAgB8 / 1 recombinant protein and EgAgB8 / 1-EgAgB8 / 2 recombinant chimeric protein could be specifically recognized by the serum of CE patients. In the serum of 16 patients with CE and EgAgB8 / 1 recombinant protein as antigen 13 were positive with EgAgB8 / 1-EgAgB8 / 2 recombinant protein as antigen and 12 with cysticercosis by EgAgB8 / 1 and EgAgB8 / 1-EgAgB8 / 2 recombinant proteins respectively, 5 and 3 Positive. Conclusion The recombinant chimeric protein of EgAgB8 / 1-EgAgB8 / 2 is antigen-specific and its serological diagnostic value for cystic echinococcosis is better than that of EgAgB8 / 1 recombinant protein.