细粒棘球绦虫重组EG95抗原在绵羊绦虫蚴病血清抗体检测中的应用

Application of recombinant EG95 antigen(rEG95) of Echinococcus granulosus for the detection of serum antibodies in sheep against infections of larval stage of taeniids

为了分析评价细粒棘球绦虫重组蛋白EG95(rEG95)在血清抗体检测上的效果,以细粒棘球绦虫EG95重组质粒pGEM-EG95为模板,经PCR扩增后将目的片段亚克隆至pET-28a(+),构建重组表达载体pET-EG95,在大肠杆菌BL21中表达,SDS-PAGE分离和电洗脱纯化重组表达产物rEG95。建立重组蛋白间接ELISA方法,检测绵羊带绦虫蚴病血清抗体水平。结果表明,表达产物经SDS-PAGE测定分子质量为21ku左右。该蛋白可被棘球蚴病阳性血清特异性识别,表明该表达产物具有反应原性。应用该重组蛋白间接ELISA检测18份脑多头蚴、15份细颈囊尾蚴、20份棘球蚴感染绵羊血清,24份疫苗抗原rEG95免疫羊血清和来自棘球蚴病疫区的179份绵羊血清,其阳性检出率分别是66.67%、80.00%、100%、91.67%和58.2%,而14份健康羊血清反应为阴性,组间差异有一定统计学意义(P<0.05)。表明EG95蛋白可作为绵羊带绦虫蚴病共同保护性抗原和诊断抗原而加以应用。

To evaluate the efficacy of recombinant protein on serum antibodies,EG95 gene was amplified from recombinant plasmid pGEM-EG95 by PCR,and was then subcloned into the pET-28a（＋） vector.The recombinant pET-EG95 plasmid was expressed in the Escherichia coli BL21.The expressed product of rEG95 gene was identified by SDS-PAGE and Western-blotting,and purified for further use by electro-eluting method.The level of serum antibodies was determined by indirect ELISA.Results indicated that the recombinant protein rEG95 with the molecular weight of approximate 21 ku had specifically reacted with the positive sera from sheep infected with the larval stage of Echinococcus granulosus,suggesting that the protein had a promising reactinogenicity.Indirect ELISA showed that by using the rEG95 protein,thepositive rate was 66.67%,80.00%,100%,91.67% and 58.2% in 18 cases of sheep with Taenia multiceps,15 cases of Cysticercus tenuicollis,20 cases of E.granulosus,24 cases of the immunized group with rEG95 and 179 cases of an epidemic area of echinococcosis,respectively, with statistically significant diffe-rences in the positive rates among the different groups（P0.05）.There was positive reactivity with ovine sera from the control healthy group. In conclusion,rEG95 protein was used as a promising antigen for the co-protective vaccine and immunodiagnostic agent of sheep with larval taeniid infections.