Adjuvant Effects of Deleting Hypervariable Domains of FliC of Escherichia coli Nissle 1917

Bacterial flagellin (FliC) can be used as a TLR5 ligand-like adjuvant. However, the sequences of hypervariable regions(HVR) of FliC from different bacteria vary, and their effects on adjuvants remain unclear. In this study, FliCΔ274–406 (deleting of D3 domain) and FliCΔ174–506 (deleting of D2-D3 domain) from Escherichia coli Nissle 1917 FliC (FliCEcN) were constructed and expressed in host bacteria, BL21. Purification was conducted using affinity chromatography on a Ni-NTA column, validation was done using SDS-PAGE and western blotting, the antigenicity and immunogenicity were detected using ELISA, and adjuvant effects were evaluated in Caco-2 cells and mice. The results showed that FliCEcN was mainly expressed in the bacterial supernatant, and the two truncated flagellins were expressed as inclusion bodies. Compared with FliCEcN, both FliCΔ274–406 and FliCΔ174–506 had considerable decreased antigenicity and immunogenicity. In Caco-2 cells, FliCΔ174–506 had a higher ability to promote the secretion of IL-8 than FliCEcN and FliCΔ274–406. In mice, FliCΔ174–506 showed a comparable adjuvant level to FliCEcN, while FliCΔ274–406 was less effective. Our data shows that adjuvant effects of FliCEcN with deletion of different regions of its HVR are inconsistent, and deleting its entire D2–D3 domain is better.