长双歧杆菌NCC2705纯化蛋白烯醇化酶、Tuf蛋白的黏附作用研究

目的：验证功能蛋白烯醇化酶（enolase）和Tuf蛋白是否为长双歧杆菌（Bifidobacterium longum）NCC2705的黏附因子。方法将长双歧杆菌NCC2705与Caco-2细胞共培养，显微镜观察加入纯化蛋白烯醇化酶、Tuf蛋白前后细胞黏附细菌数的变化并进行统计学分析。将Caco-2细胞、长双歧杆菌NCC2705、致病菌（弗氏志贺菌或伤寒沙门菌）共培养，检测加入纯化蛋白烯醇化酶、Tuf蛋白前后上清液中乳酸脱氢酶（ LDH）和肿瘤坏死因子-α（ TNF-α）的水平。结果加入纯化蛋白烯醇化酶、Tuf蛋白之后，Caco-2细胞黏附细菌数显著降低（P＜0．05）。 Caco-2细胞与致病菌共培养时，LDH和TNF-α的水平显著增高（P＜0．05）；Caco-2细胞与长双歧杆菌NCC2705和致病菌共培养时，LDH和TNF-α的水平显著降低（P＜0．05），然而加入纯化蛋白烯醇化酶和Tuf蛋白后，LDH和TNF-α的水平又出现显著增高（P＜0．05）。结论长双歧杆菌NCC2705可竞争性抑制致病菌对肠上皮细胞的黏附，而烯醇化酶和Tuf蛋白可竞争性抑制长双歧杆菌对肠上皮细胞黏附作用，是双歧杆菌的黏附因子。

Adhesion of enolase and Tuf from B.longum NCC2705

Objective To demonstrate that enolase and Tuf are direct adhesions of Bifidobacterium longum（ B.longum） NCC2705.Methods B.lougum NCC2705 was co-cultured with Caco-2.The change of adhesion was tested by microscopy after the purified proteins（GST-Eno and GST-Tuf） were added into the co-culture, and then statistical analysis was conduc-ted.Caco-2 cells were incubated with B.lougum NCC2705 and pathogenic bacteria （Shigella flexneri 2a or Salmonella）. The level ofcytokine LDH and TNF-αfor supernatant was tested after the purified proteins （ GST-Eno and GST-Tuf） were added.Results The number of bacteria adherent to Caco-2 was reduced significantly （ P 〈0.05 ） after the purified proteins （ GST-Eno/GST-Tuf） were added .While Caco-2 was co-cultured with pathogens , the level of LDH and TNF-αwas significantly increased （P〈0.05）.When Caco-2 was incubated with B.lougum NCC2705 and pathogenic bacteria , the level of LDH and TNF-αwas significantly reduced （P〈0.05）, but the level of LDH and TNF-αincreased once more （P〈0.05） after the purified protein （GST-Eno/GST-Tuf） was added.Conclusion B.longum NCC2705 can competitive-ly inhibit production of pathogenic bacteria in intestinal epithelial cell adhesion , while the enolization enzyme and Tuf proteins can competitively inhibit long bifidobacteria in intestinal epithelial cell adhesion effect .They are adhesion factors of bifidobacteria .