拟态弧菌OmpU蛋白在草鱼肠组织中互作蛋白的筛选鉴定

Screening and identification of interacting intestinal proteins of Ctenopharyngodon idella with OmpU of Vibrio mimicus

摘要:

【目的】为了确定草鱼肠组织蛋白中能与拟态弧菌OmpU黏附素蛋白发生互作的蛋白。【方法】在前期构建重组表达质粒pET-32a-OmpU基础上，通过IPTG诱导表达及亲和层析纯化获得可溶性His-OmpU融合蛋白，经Western blot分析显示该重组融合蛋白与OmpU多克隆抗体具有良好的反应原性。以His-OmpU融合蛋白为诱饵蛋白，利用His-tag pull down试验筛选草鱼肠组织蛋白中与OmpU黏附素蛋白结合的蛋白，并经SDS-PAGE分离、液相色谱-串联质谱(LC-MS/MS)鉴定以及Mascot与蛋白数据库检索。【结果】共鉴定出5个体外互作蛋白，分别为α-肌动蛋白2 (α-actin 2)、细丝蛋白A (Filamin A)、α-辅肌动蛋白4 (α-actinin 4)、转胶蛋白2 (又称肌动蛋白结合蛋白2，Transgelin-2)和调宁蛋白2 (Calponin-2)。【结论】GO功能注释分析显示，这些互作蛋白属于细胞骨架蛋白和细胞骨架调节蛋白，在病原菌的黏附、内化和致病中起着重要作用。研究结果为后期研制黏附拮抗剂和探究拟态弧菌分子致病机理奠定了基础。

Abstract:

[Objective] In order to determine interaction between outer membrane protein U (OmpU) of Vibrio mimicus and the proteins of intestinal tissue of grass carp. [Methods] By construction of recombinant expression plasmid pET-32a-OmpU, we obtained the soluble fusion His-OmpU protein through IPTG induction and affinity purification. Western blot analysis showed the expressed His-OmpU protein could be combined by polyclonal antibody of OmpU. The proteins of intestinal tissue of grass carp interacting with OmpU was screened by His-tag pull down assay and identified by LC-MS/MS followed by SDS-PAGE. Based on searches of bioinformatics by Mascot software and protein database. [Results] Five proteins were verified which can interact with the OmpU in vitro, including α-actin 2, Filamin A, α-actinin 4, Transgelin-2, Calponin-2. [Conclusion] GO annotation indicated that these proteins are cytoskeletal protein and regulatory protein of cytoskeletal which play an important role in adhesion of pathogens, internalization and pathogenicity. These results provided a basis for studies on future development of adhesion antagonist and exploration the molecular pathogenesis of V. mimicus.