鞭毛蛋白对丁酸梭菌黏附肠上皮细胞的影响

Effect of flagellin on the adhesion of Clostridium butyricum to intestinal epithelial cells

目的研究鞭毛蛋白对丁酸梭菌黏附肠上皮细胞的影响,为阐释丁酸梭菌作为益生菌发挥有益作用提供实验基础。方法提取纯化丁酸梭菌DSM 10702鞭毛蛋白,并通过质谱对蛋白进行鉴定;将鞭毛蛋白作为抗原免疫小鼠获得抗血清,并通过酶联免疫吸附试验(ELISA)测定抗血清效价;最后用人结肠腺癌细胞系HT-29和Caco-2构建肠上皮细胞黏附模型,利用鞭毛蛋白处理细胞和抗鞭毛蛋白抗血清预处理细菌,两种细胞黏附模型均采用t检验将两个处理组的黏附水平分别与对照组进行比较。结果丁酸梭菌DSM 10702表达的鞭毛蛋白由FF104_14415基因编码,其分子量为29.56kDa,获得的抗血清效价为1∶16000。在HT-29细胞黏附模型中,与对照组相比,鞭毛蛋白预处理组和抗鞭毛蛋白抗血清预处理组丁酸梭菌的黏附显著降低,差异有统计学意义(t=2.870、6.135,P<0.05);在Caco-2细胞黏附模型中,两处理组与对照组相比,丁酸梭菌黏附均显著降低,差异有统计学意义(t=6.778、18.020,P<0.05)。结论鞭毛蛋白有助于丁酸梭菌黏附肠上皮细胞,本研究为深入了解丁酸梭菌黏附机制提供了实验依据。

Objective To study the effect of flagellin on the adhesion of Clostridium butyricum to intestinal epithelial cells,and to provide an experimental basis for explaining the beneficial role of Clostridium butyricum as a probiotic.Methods The flagellin of Clostridium butyricum DSM 10702 was extracted and purified,and the protein was identified by mass spectrometry.The antiserum was obtained by immunizing mice with flagellin as an antigen,and the antiserum titer was determined by enzyme-linked immunosorbent assay(ELISA).Finally,human colon adenocarcinoma cell lines HT-29 and Caco-2 were used to construct an intestinal epithelial cell adhesion model,and flagellin was used to treat the cells and anti-flagellin and antiserum pretreatment bacteria.The t-test was used to compare the adhesion levels of the two treatment groups with that of the control group.Results The flagellin expressed by Clostridium butyricum DSM 10702 was encoded by FF104_14415 genes with a molecular weight of 29.56kDa,and the obtained antiserum titer was 1∶16000.In the HT-29 cell adhesion model,compared with the control group,the adhesion of Clostridium butyricum in the flagellin pretreatment group and the anti-flagellin antiserum pretreatment group was significantly reduced,and the difference was statistically significant(t=2.870,6.135,P<0.05).In the Caco-2 cell adhesion model,Clostridium butyricum adhesion was significantly reduced in the two treatment groups compared with the control group,and the difference was statistically significant(t=6.778,18.020,P<0.05).Conclusion Flagellin contributes to Clostridium butyricum adhering to intestinal epithelial cells,and this study provides an experimental basis for further understanding of the adhesion mechanism of Clostridium butyricum.