...
  • 主题
高级搜索  >
历史搜索 清空历史
首页> 外文期刊>Infection and immunity >Identification of surface-exposed linear B-cell epitopes of the nonfimbrial adhesin CS31A of Escherichia coli by using overlapping peptides and antipeptide antibodies.
【24h】

Identification of surface-exposed linear B-cell epitopes of the nonfimbrial adhesin CS31A of Escherichia coli by using overlapping peptides and antipeptide antibodies.

机译：使用重叠的肽段和抗肽抗体鉴定大肠杆菌的非膜粘附素CS31A的表面暴露的线性B细胞表位。

获取原文
           
页面导航
  • 摘要
  • 著录项
  • 相似文献
  • 相关主题

摘要

As a first step toward the design of an epitope vaccine, by using the nonfimbrial adhesin CS31A of Escherichia coli as a carrier, a low-resolution topological and epitope map of the CS31A subunit was developed by using solid-phase peptide synthesis and polyclonal rabbit antibodies raised against both native and denatured proteins. Peptides constituting antigenic epitopes on the major subunit (ClpG) of the multimeric CS31A antigen were identified by examining the binding of the antibodies to 249 overlapping nonapeptides covering the amino acid sequence of ClpG. With antibodies raised against denatured ClpG subunit, seven major epitope regions, corresponding to residues 10 to 18, 45 to 58, 88 to 107, 148 to 172, 187 to 196, 212 to 219, and 235 to 241, were located. Most of the epitopes were hydrophilic and were located in variable regions, residing largely in loop regions at the boundaries of secondary structural elements of ClpG. In contrast, antibodies raised against native CS31A antigen reacted only with the peptide AVNPNA (positions 179 to 184), demonstrating that this peptide was the only linear B-cell epitope of the native protein. The different immunogenic profiles of native CS31A antigen and denatured ClpG indicated that the denaturation process resulted in marked conformational changes in the protein, which could expose epitopes hidden or absent in native CS31A. To identify the surface-exposed epitopes, nine peptides covering the dominant antigenic regions of ClpG were synthesized and used to prepare site-specific antibodies. Antipeptide antibodies were tested, in a competitive enzyme-linked immunosorbent assay (ELISA), for cross-reactivity with native CS31A and denatured ClpG subunit. Four of these antipeptide antibodies bound to the native protein in an accessibility ELISA, indicating that residues 44 to 56, 174 to 190, 185 to 199, and 235 to 249 were surface exposed on CS31A. These data indicate that an immunodominant surface-exposed linear epitope was present in the region from positions 179 to 184 of ClpG in the native CS31A antigen on intact bacterial cells and suggest that the four surface-exposed epitopes constitute potential sites for insertions or substitutions with heterologous peptides.
机译：作为设计表位疫苗的第一步，通过使用大肠杆菌的非纤维粘附素CS31A作为载体，通过使用固相肽合成和多克隆兔抗体开发了CS31A亚基的低分辨率拓扑和表位图可以抵抗天然和变性蛋白质。通过检查抗体与覆盖ClpG氨基酸序列的249个重叠非肽的结合，鉴定了多聚CS31A抗原主要亚基（ClpG）上构成抗原表位的肽。用针对变性的ClpG亚基的抗体，定位了七个主要表位区域，分别对应于残基10至18、45至58、88至107、148至172、187至196、212至219和235至241。大多数表位是亲水的，位于可变区，主要位于ClpG二级结构元件边界的环区。相反，针对天然CS31A抗原产生的抗体仅与肽AVNPNA（位置179至184）反应，表明该肽是天然蛋白的唯一线性B细胞表位。天然CS31A抗原和变性的ClpG的不同免疫原性谱表明，变性过程导致蛋白质显着的构象变化，从而可能暴露天然CS31A中隐藏或不存在的表位。为了鉴定表面暴露的表位，合成了覆盖ClpG优势抗原区域的九种肽，并用于制备位点特异性抗体。在竞争性酶联免疫吸附测定（ELISA）中测试了抗肽抗体与天然CS31A和变性ClpG亚基的交叉反应性。在可访问性ELISA中，这些抗肽抗体中有四种与天然蛋白结合，表明残基44至56、174至190、185至199和235至249在CS31A上表面暴露。这些数据表明免疫原性表面暴露的线性表位存在于完整细菌细胞上天然CS31A抗原中ClpG 179位至184位的区域中，并且表明这四个表面暴露的表位构成了异源插入或取代的潜在位点肽。

著录项

相似文献

  • 外文文献
  • 中文文献
  • 专利
获取原文

客服邮箱：kefu@zhangqiaokeyan.com

京公网安备：11010802029741号 ICP备案号：京ICP备15016152号-6 六维联合信息科技 (北京) 有限公司©版权所有

  • 客服微信

  • 服务号