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

Total RNA was extracted from CaCo-2 cells using TRIZOL reagent. DNA sub-cloning technique was utilized to clone the hTLR5 gene (Fig. 1A) . The F0 fragment (254 bp) was synthesized. Fragments of the F1 (1126 bp), F2 (869 bp) and F3 (633 bp) were all obtained using the reverse transcriptase polymerase chain reaction (RT-PCR) method. . F3 upstream and downstream primers were P6 and P2, respectively, and their sequences are the following: P6: 5′GTC ACT CTG ACT CTG TTC CTC ATG 3′ (upstream), P2: 5′ CCG CGG GCC CTC GGA GAT GGT TGC TAC AGT TTG CAA C 3′ (downstream). The fragment F0 was fused with the fragments of F1, F2 and F3 using PCR methods, respectively, resulting in Fa and Fb fragments. These two fragments were then ligased with pcDNA3.1-V5/HisB vector. The sequence of hTLR5 in pcDNA3.1-V5/HisB recombinant was confirmed by restriction digestion and sequencing (Fig. 1B) . The pcDNA3.1-V5/HisB-hTLR5 construct was transfected into HEK293T cells using Lipofectamine TM 2000 reagent, and the expressed target protein of hTLR5 was verified by western blotting.