PDF(Pubmed)
Abstract：
Since 2010, the duck Tembusu virus (DTMUV) has caused a severe outbreak of egg drop syndrome in laying ducks in China, which has resulted in substantial financial losses in the poultry industry. DTMUV nonstructural protein 1 (NS1), as the only secreted protein, could aid in the development of therapeutic antibodies and diagnostic techniques; however, there are few studies on the preparation and epitope identification of monoclonal antibodies (mAbs) against DTMUV NS1. In this study, by indirect enzyme-linked immunosorbent assay (ELISA), Western blotting, and indirect immunofluorescence assay, we screened 6 mAbs (8A4, 8E6, 10F12, 1H11, 3D5, 5C11) that could specifically recognize DTMUV NS1. For epitope mapping of mAbs, a series of GST-tagged truncated fusion proteins of DTMUV NS1 were constructed by prokaryotic expression. Finally, the 4 shortest linear epitopes were identified by indirect ELISA and Western blotting. The epitope 133FVIDGPK139 was recognized by 8A4, the epitope 243IPKTLGGP250 was recognized by 8E6, the epitope 267PWDEK271 was recognized by 10F12, and 156EDFGFGVL163 was recognized by 1H11, 3D5, and 5C11. By sequence alignment and cross-reaction tests, we found that 8A4 and 8E6 had high specificity for DTMUV NS1 compared with that of other mAbs, but 10F12, 1H11, 3D5, and 5C11 exhibited a clear degree of cross-reaction with dengue virus (DENV), Japanese encephalitis virus (JEV), West Nile virus (WNV), and Zika virus (ZIKV) NS1. Finally, the predicted crystal structure analysis showed the approximate spatial positions of the 4 epitopes on the NS1 dimer. In summary, our study revealed 2 specific mAbs for DTMUV NS1 recognition and 4 multiflavivirus mAbs for DENV, JEV, WNV, and ZIKV NS1 recognition.
摘要：
自2010年以来,鸭Tembusu病毒(DTMUV)在我国蛋鸡中引起严重的掉蛋综合征,这给家禽业造成了巨大的经济损失。DTMUV非结构蛋白1(NS1),作为唯一的分泌蛋白，可以帮助开发治疗性抗体和诊断技术；然而，关于针对DTMUVNS1的单克隆抗体（mAb）的制备和表位鉴定的研究很少。在这项研究中,通过间接酶联免疫吸附测定(ELISA)，西方印迹,和间接免疫荧光分析，我们筛选了6种可以特异性识别DTMUVNS1的单克隆抗体（8A4，8E6，10F12，1H11，3D5，5C11）。对于单克隆抗体的表位定位，通过原核表达构建了一系列带有GST标签的DTMUVNS1截短融合蛋白。最后,通过间接ELISA和蛋白质印迹法鉴定了4个最短的线性表位。表位133FVIDGPK139被8A4识别,表位243IPKTLGGP250被8E6识别,表位267PWDEK271被10F12识别,156EDFGFGVL163被1H11、3D5和5C11识别。通过序列比对和交叉反应测试,我们发现8A4和8E6对DTMUVNS1的特异性高于其他单克隆抗体，但10F12、1H11、3D5和5C11与登革病毒（DENV）表现出明显的交叉反应，日本脑炎病毒（JEV），西尼罗河病毒(WNV)和寨卡病毒（ZIKV）NS1。最后,预测的晶体结构分析显示了NS1二聚体上4个表位的近似空间位置。总之,我们的研究揭示了2个特异性单克隆抗体的DTMUVNS1识别和4个多黄病毒单克隆抗体的DENV，JEV,WNV,和ZIKVNS1识别。
