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.

细菌鞭毛蛋白 (FliC) 可用作 TLR5 配体样佐剂。然而，不同细菌的FliC高变区（HVR）序列不同，其对佐剂的影响仍不清楚。在本研究中，构建了来自大肠杆菌Nissle 1917 FliC (FliC _{EcN ) 的 FliC Δ274–406}（删除 D3 结构域）和 FliC _Δ174–506（删除 D2-D3 结构域），并在宿主细菌BL21中表达。使用 Ni-NTA 柱上的亲和层析进行纯化，使用 SDS-PAGE 和蛋白质印迹进行验证，使用 ELISA 检测抗原性和免疫原性，并在 Caco-2 细胞和小鼠中评估佐剂效应。结果表明，FliC _EcN主要在细菌上清液中表达，两条截短的鞭毛蛋白以包涵体的形式表达。与FliC _EcN相比，FliC _Δ274-406和FliC _Δ174-506的抗原性和免疫原性均显着降低。在Caco-2细胞中，FliC _{Δ174-506比FliC EcN}和FliC _Δ274-406具有更高的促进IL-8分泌的能力。在小鼠中，FliC _{Δ174-506显示出与 FliC EcN}相当的佐剂水平，而 FliC _Δ274-406的效果较差。我们的数据显示，删除其HVR不同区域的FliC _{EcN的佐剂效果不一致，删除其整个D2-D3结构域效果更好。}