羊细粒棘球蚴病抗体间接ELISA检测方法的建立

Development of an Indirect ELISA for Detection of Sheep Antibodies against Echinococcosis granulosa

本研究旨在通过建立检测羊细粒棘球蚴病抗体的间接酶联免疫吸附试验(ELISA)方法,为羊细粒棘球蚴病的检测提供快速、简便的手段。作者利用DNAStar软件对GenBank上发表的细粒棘球蚴EG95蛋白的氨基酸序列进行分析,筛选出高度亲水的优势表位区EG95s,对该区域编码基因进行克隆并表达。以纯化的重组融合蛋白为包被抗原,按常规方法建立检测羊细粒棘球蚴病抗体的间接ELISA,并对各种条件进行优化。SDS-PAGE结果表明成功获得了可溶性好、表达效率高、纯化简便的重组融合蛋白GST-1EG95s和HIS-1EG95s,Western blot检测结果表明表达产物具有较好的反应原性。间接ELISA方法优化结果显示:HIS-1EG95s作为包被抗原效果优于GST-1EG95s。经统计学分析,确定间接ELISA方法的判定标准:OD450nm值≥0.235时判定为阳性,OD450nm值≤0.191时判定为阴性,介于二者之间则为可疑。分别对采自新疆的70份羊包虫阳性血清和70份阴性血清进行检测,结果表明该方法与新西兰Wallaceville动物研究中心提供的间接ELISA方法符合率为100%,阻断试验结果显示与其他蛋白无交叉反应,批内变异系数介于3.8%～5.6%,批间变异系数介于5.7%～8.5%,结果表明该方法特异性强、敏感性高、重复性好。作者所建立的羊细粒棘球蚴病抗体的间接ELISA检测方法有望为羊细粒棘球蚴病的检测提供快速、简便的手段。

This study was designed to develop an indirect ELISA for detection of sheep antibodies against Echinococcosis granulosa,which will provide a fast and simple means for detection Echinococcosis granulosa of sheep.The amino acid sequences of EG95 published in GenBank were analyzed using DNAStar software,and the hydrophilic epitopes were selected,named EG95s.Then the gene of EG95s were cloned and expressed.As a result,an indirect ELISA for detection of sheep antibodies against Echinococcosis granulosa were developed using the purified recombinant fusion protein as the coating antigen,therefore a variety of conditions were optimized.The results of SDS-PAGE showed that the recombinant fusion protein,GST-1EG95s and HIS-1EG95s,were efficiently expressed,which were soluble and purified easily.Then the results of Western blot showed that the expression products were reacted with positive serum of sheep with Echinococcosis granulosa,which indicated they were good immunogenicity.The optimized conditions of indirect ELISA showed that： HIS-1EG95s was better than GST-1EG95s as the coating antigen.The determine criteria of the indirect ELISA were： Samples with an OD450 nm value ≥ 0.235 were judged as positive,and with an OD450 nm value≤ 0.191 were judged as negative,while with an OD450 nm value ranging between them was suspicious.70 positive and negative serum of sheep collected in Xinjiang with or without Echinococcosis granulosa were detected by the indirect ELISA respectively,and the results showed that this indirect ELISA method was completely consistent with the indirect ELISA provided by Wallaceville Animal Research Centre of New Zealand.There were no cross reactivity detected with other proteins.The variant coefficient in batch and between batches varied from 3.8% to 5.6% and from 5.7% to 8.5%,respectively.All these results showed that the indirect ELISA possessed specificity,sensitivity and good reproducibility.The indirect ELISA for detection of sheep antibodies against Echinococcosis granulosa were successfully developed,which provide a fast and simple means,and can be used for clinical detection.