Construction,prokaryotic expression and in vitro TLR5 activity assay of Esche-richia coli Nissle 1917 flagellin's hypervariable region deletion
10.16303/j.cnki.1005-4545.2025.03.08
- VernacularTitle: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
- Author:
Guixian ZHOU
1
;
Shihui WU
1
;
Minle WANG
1
;
Yixiao LIAO
1
;
Shuang LI
1
;
Zemin YANG
1
;
Ying YANG
1
Author Information
1. 贵州大学动物科学学院,贵州 贵阳 550025;贵州省动物生物制品工程技术研究中心,贵州 贵阳 550025
- Publication Type:Journal Article
- Keywords:
Escherichia coli Nissle 1917;
flagellin;
adjuvant;
prokaryotic expression;
TLR5
- From:
Chinese Journal of Veterinary Science
2025;45(3):449-457
- CountryChina
- Language:Chinese
-
Abstract:
The structure and potential antigenic epitopes of FliCEcN were analysed using bioinformat-ics technology.With the help of ClonExpress? homologous recombination technology,primers were designed to deletion of different structural domains in the highly variable region of FliCEcN 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 differ-ent 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 FliCEcN were predicted to contain potential antigenic epitopes.PC R results showed that fliC△820-1 518,fliC△736-963,fliC△985-1 200,fliC △748-828,fliC△1 114-1 191,andfliC△1 225-1311 were approximately 1 095,1 566,1 578,1 713,1 716 and 1 707 bp,respectively.SDS-PAGE results showed that the sizes of the flagellin variants treated with nickel column purifi-cation and dialysis replication were about 41.36,57.06,57.50,61.97,61.95 and 61.56 kDa,respec-tively.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 FliCEcN 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 flagel-lin antibody titer on its adjuvant effect.
