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【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。
【Objective】 In order to confirm the interaction between grass carp intestinal protein and OmpU adhesin protein. 【Method】 The recombinant plasmid pET-32a-OmpU was constructed in the early stage. The soluble His-OmpU fusion protein was obtained by IPTG induction and purification by affinity chromatography. Western blot analysis showed that the recombinant fusion protein and OmpU polyclonal antibody had Good reaction. His-OmpU fusion protein was used as the bait protein. His-tag pull down assay was used to screen the proteins that bind OmpU adhesin in grass carp intestinal tissues. The proteins were separated by SDS-PAGE and analyzed by liquid chromatography-tandem mass spectrometry (LC-MS / MS) and Mascot and protein database search. 【Result】 Five in vitro interacting proteins were identified, which were α-Actin 2, Filamin A, α-Actin 4, , Transgenic protein 2 (also called actin binding protein 2, Transgelin-2) and Calponin-2. 【Conclusion】 GO functional annotation analysis showed that these interacting proteins belong to cytoskeleton proteins and cytoskeleton regulatory proteins, which play an important role in the adhesion, internalization and pathogenicity of pathogenic bacteria. The results laid the foundation for the later development of adhesion antagonists and to explore the molecular mechanism of mimicry Vibrio.