In this study,a unique antigenic epitope 87YAEYI91 on E protein of duck Tembusu virus (DTMUV) was identified by phage display technology with purified monoclonal antibodies (MAb) 1A5 against DTMUV E protein.To determine whether the 87YAEYI91 sequence is conserved among the E proteins of flaviviruses,we aligned the E protein amino acid sequences of flaviviruses.The results revealed that YAEYI was highly conservative in DTMUV strain,but the epitope sequence wasn't conserved among the corresponding amino acid sequence of the E protein in other strains of flavivirus.Dot blotting assay showed that epitope 87 YAEYI91 had a good affinity with positive sera to DTMUV,and there was not cross-reacting with JEV-,DENV-,WNV-positive sera,suggesting that 87YAEYI91 was DTMUV unique epitope.An Epitope-ELISA assay was established by using unique epitope (87YAEYI91) fused protein and used to test duck serum samples which were also detected by the Neutralization Test as the standard method.According to results,the specificity of this test was 100％.The sensitivity of this epitope-based peptide serologic test for DTMUV infection was 96％.So,Epitope-ELISA was a specific method for detecting antibodies against DTMUV.%旨在建立鸭坦布苏病毒(DTMUV)的特异性血清学诊断方法.本研究以纯化的单克隆抗体1A5为固相筛选分子,利用噬菌体展示技术鉴定DTMUV囊膜(E)蛋白的特异性抗原表位,并利用DNAStar分析抗原表位序列在DTMUV中保守性和与其他黄病毒的E蛋白相应位点的氨基酸序列相似性.结果成功筛选到DTMUV E蛋白的一个线性抗原表位s7YAEYI91,该抗原表位在DTMUV中具有高度的保守性,无氨基酸位点差异,而与其他黄病毒E蛋白相应位点的氨基酸序列不具有相似性.Dot-Blotting结果表明表位87YAEYI91与DTMUV阳性血清具有良好的亲和性,而与JEV、DENV、WNV等黄病毒阳性血清间不出现交叉反应,可见抗原表位87YAEYI91为DTMUV所特有的抗原表位.利用DTMUV特异性抗原表位.7YAEYI91建立Epitope-ELISA血清学诊断方法并对126份鸭血清样品进行检测,以中和试验作为标准进行比较,结果显示Epitope-ELISA检测方法的相对特异性为100％,相对敏感度为96％,并且Epitope-ELISA与其他黄病毒血清间不出现交叉反应性,表明本研究建立的Epitope-ELISA检测方法是一种特异的、灵敏的ELISA血清学诊断方法.
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