Figure 1.

Expression and characterization of recombinant DIII and FliC-DIII proteins. (A) DIII, FliC, and recombinant protein FliC and DIII, which were linked by GS₄ linker, were constructed into the pET-22b(+) vector with a C-terminal His-tag for protein expression proteins. (B) The DIII proteins (D1DIII, D2DIII, D3DIII, D4DIII, ZDIII) and the FliC-DIII fusion proteins (FliC-D1DIII, FliC-D2DIII, FliC-D3DIII, FliC-D4DIII, and FliC-ZDIII) were expressed by E.coli BL21 (DE3) and purified using nickel-chelated affinity chromatography. Purified proteins were verified by SDS-PAGE staining with Coomassie blue. (C) TLR5 signal functional assay was performed by co-culturing the10-fold serial dilutions of FliC or recombinant proteins with HEK 293A expressing hTLR5 receptor and NF-κB reporter vector. The cells were disrupted and treated with Neolite luciferase substrate. The TLR5 activity was measured by luciferase activity. (D) BALB/c mice were immunized two doses of 20 μg FliC-D2DIII or a mixture of 20 μg FliC and 20 μg D2DIII in a 3-week interval. Sera were collected 2 weeks after the second-dose immunizations and analyzed by FRNT assay. FRNT₅₀ values were calculated by GraphPad Prism 6. (**, p <0.01).