大肠杆菌Nissle 1917 FliC高变区缺失的构建、原核表达及体外TLR5活性检测  

Construction,prokaryotic expression and in vitro TLR5 activity assay of Escherichia coli Nissle 1917 flagellin's hypervariable region deletion

作　　者：周桂仙 吴诗卉 王敏乐 廖义潇 李双 杨泽敏 杨颖[1,2] ZHOU Guixian;WU Shihui;WANG Minle;LIAO Yixiao;LI Shuang;YANG Zemin;YANG Ying(School of Animal Science,Guizhou University,Guiyang 550025,China;Guizhou Province Animal Biological Products Engineering Technology Research Center,Guiyang 550025,China)

机构地区：[1]贵州大学动物科学学院,贵州贵阳550025 [2]贵州省动物生物制品工程技术研究中心,贵州贵阳550025

出　　处：《中国兽医学报》2025年第3期449-457,共9页Chinese Journal of Veterinary Science

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

摘　　要：运用生物信息技术分析FliCEcN的结构和潜在的抗原表位;借助ClonExpress?同源重组技术设计引物,缺失FliCEcN高变区不同结构域,并克隆至pET-28a(+)表达载体中表达,经SDS-PAGE和Western blot鉴定鞭毛蛋白变体;采用鲎试剂显色法检测鞭毛蛋白变体中内毒素残留,并使用不同质量浓度的鞭毛蛋白变体刺激Caco-2细胞,通过检测IL-8的分泌水平来评估各鞭毛蛋白变体的生物学活性。生物信息分析结果显示,FliCEcN高变区的多数结构域被预测为含有潜在的抗原表位;PCR结果显示,fliC_(△820~1518)、fliC_(△736~963)、fliC_(△985~1200)、fliC_(△748~828)、fliC_(△1114~1191)和fliC_(△1225~1311)分别约为1095、1566、1578、1713、1716、1707 bp;SDS-PAGE结果显示,经镍柱纯化处理和透析复性的鞭毛蛋白变体分别约为41.36、57.06、57.50、61.97、61.95、61.56 kDa;Western blot结果显示,6个鞭毛蛋白变体均能与抗His单克隆抗体和大肠杆菌H7抗原诊断血清发生特异性反应;TLR5活性检测结果显示,缺失不同结构域的鞭毛蛋白变体保留了其TLR5激动剂功能。结果表明,本试验成功构建了6种FliC_(EcN)缺失高变区不同结构域的鞭毛蛋白变体,且各鞭毛蛋白变体均保留了其TLR5激动剂功能,展现了良好的生物学特性,为进一步研究鞭毛蛋白在缺失不同结构域后的佐剂效应以及鞭毛蛋白抗体滴度对其佐剂效应影响提供了参考依据。The structure and potential antigenic epitopes of FliC_(EcN)were analysed using bioinformatics technology.With the help of ClonExpress~?homologous recombination technology,primers were designed to deletion of different structural domains in the highly variable region of FliC_(EcN)and cloned into pET-28a(+)expression vector for expression.The expressed flagellin variants were identified by SDS-PAGE and Western blot.Endotoxin residues in the flagellin variants were detected by horseshoe crab reagent chromatography,and Caco-2 cells were stimulated with different concentrations of flagellin variants.The biological activity of each flagellin variant was assessed by detecting the secretion level of IL-8.Bioinformatic analysis showed that most of the structural domains in the highly variable region of FliC_(EcN)were predicted to contain potential antigenic epitopes.PCR results showed that fliC_(Δ820-1518),fliC_(Δ736-963),fliC_(Δ985-200),fliC_(Δ748-828),fliC_(Δ1114-1191),and flic_(Δ1225-1311)were approximately 1094,1566,1578,1713,1716 and 1707 bp,respectively.SDS-PAGE results showed that the sizes of the flagellin variants treated with nickel column purification and dialysis replication were about 41.36,57.06,57.50,61.97,61.95 and 61.56 kDa,respectively.Western blot results showed that all six flagellin variants reacted specifically with anti-His monoclonal antibody and E.coli H7 antigen diagnostic serum.The results of TLR5 activity assay showed that the flagellin variants missing different structural domains retained their TLR5 agonist function.In this study,six flagellin variants with different structural domains of FliC_(EcN)deletion hypervariable region were successfully constructed,and all of them retained their TLR5 agonist function and showed good biological properties.This provides a reference for further research on the adjuvant effect of flagellin after deletion of different structural domains and the effect of flagellin antibody titer on its adjuvant effect.

关 键 词：大肠杆菌Nissle 1917 鞭毛蛋白 佐剂 原核表达 TLR5 

分 类 号：S852.61[农业科学—基础兽医学] S852.2[农业科学—兽医学]