His-N2蛋白体外排除抑制大肠杆菌DH5α黏附猪肠黏液蛋白的研究

in vitro Exclusive Inhibition of E. coli DH5α Adhesion to Porcine Intestinal Mucus by His-N2 Protein

以植物乳杆菌KLDS1.0320候选表面黏附蛋白NP_785232 N端前两个结构域组成的融合表达蛋白为研究对象,采用体外模拟肠道环境的方法研究其对大肠杆菌DH5α黏附猪肠黏液的排除抑制作用。对猪肠黏液进行固定,加入His-N2融合表达蛋白37℃孵育1h,再加入大肠杆菌DH5α菌悬液37℃孵育1h,洗去未黏附的菌体,之后用1%Triton?X-100进行裂解,将裂解液涂布LB琼脂平板以进行菌落计数。结果表明:pH值为6.6时,相对于缓冲液阴性对照组,His-N2蛋白对大肠杆菌DH5α黏附猪肠黏液的抑制率为(50.37±3.66)%,相对于BSA蛋白对照组的抑制率为(38.33±4.55)%;pH值为7.5时,相对于缓冲液阴性对照组,His-N2蛋白对大肠杆菌DH5α黏附猪肠黏液的抑制率为(42.18±3.44)%,相对于BSA蛋白对照组的抑制率为(34.48±3.90)%。在体外模拟条件下,由植物乳杆菌KLDS1.0320的候选表面黏附蛋白NP_785232 N端前两个结构域组成的His-N2蛋白能排除抑制大肠杆菌DH5α对猪肠黏液蛋白的黏附。

His-N2 protein, consisting of two domains at the N terminus of Lactobacillus plantarum KLDS 1.0320 adhesion surface protein NP_785232, was selected as the candidate in this work. This protein was used to explore the exclusive inhibition against E. coli DH5α adhesion to porcine intestinal mucus in vitro. First, porcine mucus was fixed on 96-well cell plates and incubated at 37 ℃ for 1 h after adding His-N2 protein, and then the solution containing E. coli DH5α was added.After co-incubation at 37 ℃ for 1 h, the mucus was washed and disrupted with 1% Triton X-100. Ten-fold dilutions of the cell lysates were prepared and spread on LB agar plates for counting of E.coli DH5α. Results indicated that His-N2 protein could inhibit the adhension of E. coli DH5α to porcine intestinal mucus by (50.37 ± 3.66)% compared with PBS and by (38.33% ± 4.55)% compared with BSA protein at pH 6.6, while the inhibitory rates were (42.18 ± 3.44)% and (34.48 ± 3.90)% at pH 7.5, respectively. Therefore, His-N2 protein is involved in the exclusive inhibition of E. coli DH5α adhersion to porcine intestinal mucus in vitro.