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, FliC Δ274–406 (deleting of D3 domain) and FliC Δ174–506 (deleting of D2-D3 domain) from Escherichia coli Nissle 1917 FliC (FliC EcN ) 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 FliC EcN was mainly expressed in the bacterial supernatant, and the two truncated flagellins were expressed as inclusion bodies. Compared with FliC EcN , both FliC Δ274–406 and FliC Δ174–506 had considerable decreased antigenicity and immunogenicity. In Caco-2 cells, FliC Δ174–506 had a higher ability to promote the secretion of IL-8 than FliC EcN and FliC Δ274–406 . In mice, FliC Δ174–506 showed a comparable adjuvant level to FliC EcN , while FliC Δ274–406 was less effective. Our data shows that adjuvant effects of FliC EcN with deletion of different regions of its HVR are inconsistent, and deleting its entire D2–D3 domain is better.