可结合Caco-2细胞表面蛋白的副溶血弧菌外膜蛋白的筛选与鉴定

目的]筛选可结合Caco-2细胞表面蛋白的副溶血弧菌外膜蛋白。方法]筛选副溶血弧菌外膜中的黏附蛋白,将Caco-2细胞表面蛋白生物素化并固定于中性卵白素树脂上,进一步利用亲和层析技术来筛选副溶血弧菌的外膜蛋白。结果]通过LC-MS/MS质谱技术鉴定出3个候选蛋白:ATP synthase subunit alpha、ATP synthase subunit beta和outer membrane protein U。通过对这3个蛋白进行克隆基因和原核表达,成功纯化得到相应重组蛋白。通过进一步的间接免疫荧光试验,发现3种蛋白对于Caco-2细胞均有黏附作用。结论]推测ATP synthase subunit alpha、ATP synthase subunit beta和outer membrane protein U这3种蛋白可能是潜在的黏附因子。

Screening and Identification of Outer Membrane Protein of Vibrio parahaemolyticus Binding to Caco-2 Cell Surface Proteins

Objective]To screen the outer membrane protein of Vibrio parahaemolyticus(VP)binding to Caco-2 cell surface proteins.Meth-od]In order to screen adhesins of VP,the cell surface proteins of Caco-2 cells were biotinylated and then employed as the baits to capture host binding outer membrane proteins of VP by using affinity chromatography.Result]ATP synthase subunit alpha,ATP synthase subunit beta and outer membrane protein U were identified by LC-MS/MS approach.The three recombinant candidate proteins were expressed and purified,fur-therly their binding activity to Caco-2 cells were evaluated via indirect immune-flourescence assays.Conclusion]The three outer membrane proteins of VP which are capable of binding to Caco-2 cell surface proteins are candidates of VP adhesion.