CAJ | 학술논문

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、10 μg/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的金属离子螫和柱,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.
목적 분리장상피Caco-2세포중여IbeA상호작용적단백.방법 표체병순화중조IbeA,장1、5、10 μg/ml적His-IbeA급우혈청백단백여Caco-2단층세포4℃부육30min후.재37℃이용경대매소보호실험측정각조대장간균K1치병주E44침습솔적변화.렬해Caco-2득전세포단백,상양지결합유His-IbeA적금속리자석화주,His pull down순화특이성결합단백,전영분석,Far-Western blotting진행량자결합특이성적험증.병분석결합단백N말단안기산서렬.결과 중조IbeA조단E44침습Caco-2,병정일정제량의뢰관계,대조조BSA대침습몰유현저영향.His pull down적방법순화출량개결합조대,Far-Western blotting험증료결합적특이성.병측정강결합조대pI약5.0,N단서렬위MASITKLP.결론 분리득도량개여IbeA상호작용적단백,위연구IbeA분자치병궤제전정료기출.
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 rain at 4 ℃. Gentamicin protection assay was used to test the invasion or E. coli K1 pathogenic isolate E44 in Caco-2 cells. The binding proteins were purified from Caco-2 by IbeA-Cu~(2+) 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.