荧光标记法初探植物乳杆菌ST-Ⅲ对Caco-2细胞的粘附机理

A Fluorescence Labeling Method for the Study on Mechanism of Adhesion of Lactobacillus plantarum ST-Ⅲ on Caco-2 Cells

摘要:

利用荧光探针CFDA-SE标记植物乳杆菌ST-Ⅲ, 测定其对Caco-2细胞粘附能力的变化, 提取相关物质, 探讨其粘附机理。化学和酶处理ST-Ⅲ菌悬液发现, 经胃蛋白酶、胰蛋白酶、氯化锂、苯酚、盐酸胍和热处理能显著降低ST-Ⅲ的粘附性, 表明表面蛋白或脂磷壁酸(Lipoteichoic acid)可能参与了ST-Ⅲ对Caco-2细胞的粘附。粘附抑制试验和可逆性结合实验证实表面蛋白而非脂磷壁酸参与了ST-Ⅲ对Caco-2细胞的粘附。结果表明ST-Ⅲ的表面蛋白粗提物可能含有粘附素类的S-层蛋白(S-layer protein), 经SDS-PAGE电泳分析, 此粗提物主要成分的分子量分别为72.7、34.1和24.3 kD。

Abstract:

To study the mechanism of adhesion of Lactobacillus plantarum ST-Ⅲ on Caco-2 cells, the adhesive ability of the bacterial cell to the mammal cells was investigated by fluorescence labeling with CFDA-SE and the components on the cell surface involved in the adhesion were extracted. After treatments by chemicals and enzymes, it was shown that pepsin, trypsin, LiCl, phenol, guanidine hydrochloride and heat treatment could signigfantly decrease the adhesive ability of ST-Ⅲ, indicating that the S-layer protein or lipoteichoic acid may be related to the adhesion of ST-Ⅲ to Caco-2 cells. The suppressive test and reversible binding test confirmed that the S-layer protein mediated the adhesion process, whereas the lipoteichoic acid on the bacterial cell surface did not participated in the adhesion. The results showed that the crude surface protein extracts of ST-Ⅲ might contain S-layer protein which participated in the adhesion. The SDS-PAGE assay showed that the crude extracts was composed of three proteins with molecular weight of 72.7 kD, 34.1 kD and 24.3 kD respectively.