Structural characteristics of flagellin FliC from Escherichia coli Nissle 1917 and its stimulating effect on Caco-2 cells
The purpose of the study was to analyze the structural characteristics of flagellin FliC(FliCEcN)of Escherichia coli Nissle 1917 and its stimulating effect on Caco-2 cells.FliCEcN protein was expressed by Escherichia coli BL21(DE3),purified by NTA column and verified by SDS-PAGE and Western-blot.The structure model of FliCEcN protein was predicted by SOPMA and Alphold2,and the structure of FliCEcN protein was analyzed by surface enhanced Raman spectroscopy and circular dichroism spectroscopy.The secretion level of immune related factors was detected after stimulating Caco-2 cells with FliCEcN protein.The results showed that the size of FliCEcN protein was 64 kDa,which could be specifically recognized by anti-His monoclonal antibody.The amino and carboxyl terminals of FliCEcN protein were mainly composed of α-helix,and the intermediate domain is mainly composed of β-fold.The maximum absorption peaks of amide Ⅰ region of FliCEcN protein were all located at 1 656 cm-1,and the main secondary structures were α-helix andβ-fold.FliCEcN's α-helix accounts for 44.4％,β-fold accounts for 23.4％,β-rotation angle accounts for 13.5％,and irregular curl accounts for 19.0％.The FliCEcN protein could effectively stimulate the secretion of interleukin-6(IL-6),tumor necrosis factor-α(TNF-α),and interleukin-10(IL-10)in Caco-2 cells.With the increase of stimulation time,the secretion level of IL-6 decreased,while the secretion level of IL-10 and TNF-α increased.The results show that α-helix and β-folding are the main structures of FliCEeN,which can promote the correct folding of its conformation and maintain its three-dimensional structure stability.The FliCEcN protein stimulated Caco-2 cells to secrete IL-6,IL-10,and TNF-αlevels is different.
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维普
