Anti-radiation damage effect of polyethylenimine as a toll-like receptor 5 targeted agonist

Hu, Zhiqiang; Xing, Yaling; Qian, Yuanyu; Chen, Xiaojuan; Tu, Jian; Ren, Lening; Wang, Kai; Chen, Zhongbin

A cell-based model for screening TLR5 agonists targeting TLR5-mediated NF-κB pathway Following the cloning of human TLR5 gene into pcDNA3.1-V5/HisB (BamHI/ApaI site) (Fig. 1A and B) , the stable expression of pcDNA3.1-V5/HisB-hTLR5 was confirmed in HEK 293T cell by western blot analysis, indicating that the molecular weight of the expressed protein was 96 kD (Fig. 1C) . Twenty-four hours after transfection of pcDNA3.1-V5/HisB-hTLR5 and NF-κB-luciferase into HEK 293T cells, these cells were treated with flagellin ranging from 0.1 to 10 μg/ml for 6 h to examine hTLR5 gene activation. Our results showed that NF-κB-luciferase activity was significantly activated by flagellin, especially at concentrations ranging from 1.0 to 10.0 μg/ml (Fig. 1D) , indicating that a cell-based model was established with the potential for screening of TLR5 agonists targeting the TLR5-mediated NF-κB pathway. Our data demonstrated that the TLR5 receptor-mediated NF-κB signaling pathway was specifically activated with the positive TLR5 ligand of flagellin in a dose-dependent manner, and the cell-based system has potential application for screening of new TLR5 agonists that target the TLR5-mediated NF-κB pathway.