肠上皮细胞中大肠杆菌侵袭素IbeA结合蛋白的分离

Purification of E. coli invasin IbeA-binding protein in intestinal epithelial cells

目的分离肠上皮Caco-2细胞中与IbeA相互作用的蛋白。方法表达并纯化重组IbeA,将1、5、10μg/ml的His-IbeA及牛血清白蛋白与Caco-2单层细胞4℃孵育30min后,在37℃利用庆大霉素保护实验测定各组大肠杆菌K1致病株E44侵袭率的变化。裂解Caco-2得全细胞蛋白,上样至结合有His-IbeA的金属离子螯和柱,His pull down纯化特异性结合蛋白,电泳分析,Far-Western blotting进行两者结合特异性的验证,并分析结合蛋白N末端氨基酸序列。结果重组IbeA阻断E44侵袭Caco-2,并呈一定剂量依赖关系,对照组BSA对侵袭没有显著影响。His pull down的方法纯化出两个结合条带,Far-Western blotting验证了结合的特异性。并测定强结合条带pI约5.0,N端序列为MASITKLP。结论分离得到两个与IbeA相互作用的蛋白,为研究IbeA分子致病机制奠定了基础。

Objective To purify IbeA-binding protein from intestinal epithelial Caco-2 cells. Methods Recombinant IbeA was purified, and 1, 5, and 10 μg/ml His-IbeA and bovine serum albumin （control） were preincubated with confluent Caco-2 monolayer for 30 min at 4 ℃. Gentamicin protection assay was used to test the invasion of E. coli K1 pathogenic isolate E44 in Caco-2 cells. The binding proteins were purified from Caco-2 by IbeA-Cu2＋ sepharose affinity chromatography, and validated by Far-Western blotting. The N-terminal amino acid sequence of the binding protein was determined using Edman assay. Results E44 invasion in Caco-2 cells was blocked by the recombinant IbeA in a dose-dependent manner. Two binding bands were obtained with His pull-down, and the binding specificity was demonstrated by Far-Western blotting. The N-terminal amino acid sequence of IBP200 was MASITKLP with an isoelectric point of about 5.0. Conclusion Two novel Caco-2 proteins interacting with IbeA of E. coli have been purified and identified.