大肠杆菌Nissle 1917鞭毛蛋白FliC的结构特性及刺激Caco-2细胞作用的研究  

Structural characteristics of flagellin FliC from Escherichia coli Nissle 1917 and its stimulating effect on Caco-2 cell

作　　者：李双 杨泽敏 廖义潇 杨颖[1,2] LI Shuang;YANG Ze-min;LIAO Yi-xiao;YANG Ying

机构地区：[1]贵州大学动物科学学院,贵州贵阳550025 [2]贵州大学动物疫病研究所,贵州贵阳550025

出　　处：《饲料研究》2024年第6期95-99,共5页Feed Research

基　　金：2023年度贵州省基础研究计划项目(项目编号:黔科合基础-ZK[2023]一般105)。

摘　　要：试验旨在分析大肠杆菌Nissle 1917鞭毛蛋白FliC(FliC_(EcN))的结构特征及其对Caco-2细胞的刺激作用。试验通过大肠杆菌BL21(DE3)表达FliCEcN蛋白、经NTA柱纯化FliC_(EcN)蛋白并通过SDS-PAGE和Westernblot验证,利用SOPMA和Alphofold2预测FliC_(EcN)蛋白的结构模型、表面增强拉曼光谱和圆二色谱解析FliC_(EcN)蛋白的结构,使用FliC_(EcN)蛋白刺激Caco-2细胞后,检测免疫相关因子的分泌水平。结果显示,FliC_(EcN)蛋白的大小为64kDa,能够被抗His单克隆抗体特异识别;FliC_(EcN)蛋白的氨基和羧基末端主要由α-螺旋组成,中间结构域主要由β-折叠组成。FliC_(EcN)蛋白酰胺Ⅰ区最大吸收峰均位于1656 cm^(-1)处,主要二级结构为α-螺旋和β-折叠;FliC_(EcN)的α-螺旋占比44.4%,β-折叠占比23.4%,β-转角占比13.5%,无规则卷曲占比19.0%;FliC_(EcN)蛋白能够有效刺激Caco-2细胞分泌白细胞介素-6(IL-6)、肿瘤坏死因子-α(TNF-α)和白细胞介素-10(IL-10);随着刺激时间延长,IL-6的分泌水平呈下降趋势,IL-10和TNF-α的分泌水平呈增加趋势。研究表明,α-螺旋和β-折叠是构成FliC_(EcN)的主要结构,可促进其构象的正确折叠和维持其三维结构稳定,FliC_(EcN)蛋白刺激Caco-2细胞分泌IL-6、IL-10、TNF-α的水平存在差异。The purpose of the study was to analyze the structural characteristics of flagellin FliC(FliC_(EcN))of Escherichia coli Nissle 1917 and its stimulating effect on Caco-2 cells.FliC_(EcN) protein was expressed by Escherichia coli BL21(DE3),purified by NTA column and verified by SDS-PAGE and Western-blot.The structure model of FliC_(EcN) protein was predicted by SOPMA and Alphold2,and the structure of FliC_(EcN) protein was analyzed by surface enhanced Raman spectroscopy and circular dichroism spectroscopy.The secretion level of immune related factors was detected after stimulating Caco-2 cells with FliC_(EcN) protein.The results showed that the size of FliC_(EcN) protein was 64 kDa,which could be specifically recognized by anti-His monoclonal antibody.The amino and carboxyl terminals of FliC_(EcN) protein were mainly composed of α-helix,and the intermediate domain is mainly composed of β-fold.The maximum absorption peaks of amideⅠregion of FliC_(EcN) protein were all located at 1656 cm^(-1),and the main secondary structures were α-helix and β-fold.FliC_(EcN)'s α-helix accounts for 44.4%,β-fold accounts for 23.4%,β-rotation angle accounts for 13.5%,and irregular curl accounts for 19.0%.The FliC_(EcN) protein could effectively stimulate the secretion of interleukin-6(IL-6),tumor necrosis factor-α(TNF-α),and interleukin-10(IL-10)in Caco-2 cells.With the increase of stimulation time,the secretion level of IL-6 decreased,while the secretion level of IL-10 and TNF-αincreased.The results show thatα-helix andβ-folding are the main structures of FliC_(EcN),which can promote the correct folding of its conformation and maintain its three-dimensional structure stability.The FliCEcN protein stimulated Caco-2 cells to secrete IL-6,IL-10,and TNF-αlevels is different.

关 键 词：大肠杆菌Nissle1917 鞭毛蛋白 表面增强拉曼光谱 圆二色谱 生物学活性 

分 类 号：S816.7[农业科学—饲料科学]