Identification of one B-cell epitope from NS1 protein of duck Tembusu virus with monoclonal antibodies.
Identification of one B-cell epitope from NS1 protein of duck Tembusu virus with monoclonal antibodies.
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DOI：
10.1371/journal.pone.0181177
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发表时间：
2017
期刊：
影响因子：
3.7
通讯作者：
Li F
中科院分区：
文献类型：
--
作者：
Ti J;Li Z;Li X;Lu Y;Diao Y;Li F
本研究描述了通过间接酶联免疫吸附测定 (ELISA) 使用单克隆抗体 (mAb) 3G2 鉴定 TMUV NS1 蛋白上的一个线性 B 细胞表位。本研究将NS1蛋白在原核表达系统中表达并纯化。一种针对 NS1 蛋白的 mAb 是从用重组蛋白 NS1 免疫的 Balb/c 小鼠中产生的。使用 PGEX-6P-1 载体表达一组覆盖整个 NS1 蛋白的 35 个部分重叠的多肽，并使用 mAb 3G2 进行筛选。通过间接 ELISA 和蛋白质印迹获得并鉴定了一种针对 mAb 的多肽。为了准确定位表位，从多肽的羧基端和氨基端依次去除一或两个氨基酸残基。表达并纯化了一系列截短的寡肽。使用 mAb 3G2 识别并鉴定了线性 B 细胞表位的最小决定簇。准确的线性 B 细胞表位是位于 NS1 蛋白中的 269DEKEIV274。此外，序列比对表明该表位在TMUV毒株和其他黄病毒中分别具有高度保守性和特异性。 TMUV NS1 蛋白的线性 B 细胞表位可能有利于新疫苗和诊断检测的开发。
This study describes the identification of one linear B-cell epitope on TMUV NS1 protein with monoclonal antibody (mAb) 3G2 by indirect enzyme-linked immunosorbent assay (ELISA). In this study, NS1 protein was expressed in prokaryotic expression system and purified. One mAb against NS1 protein was generated from Balb/c mice immunized with recombinant protein NS1. A set of 35 partially-overlapping polypeptides covering the entire NS1 protein was expressed with PGEX-6P-1 vector and screened with mAb 3G2. One polypeptide against the mAb was acquired and identified by indirect ELISA and western-blot. To map the epitope accurately, one or two amino acid residues were removed from the carboxy and amino terminal of polypeptide sequentially. A series of truncated oligopeptides were expressed and purified. The minimal determinant of the linear B cell epitope was recognized and identified with mAb 3G2. The accurate linear B-cell epitope was 269DEKEIV274 located in NS1 protein. Furthermore, sequence alignment showed that the epitope was highly conserved and specific among TMUV strains and other flavivirus respectively. The linear B-cell epitope of TMUV NS1 protein could benefit the development of new vaccines and diagnostic assays.
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