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为探讨嗜酸乳杆菌(Lactobacillus acidophilus)分泌的相关蛋白质及其促进肠道稳态的功能作用,本研究以L.acidophilus作为研究对象,固体培养获得胞外蛋白粗提液,采用Sephadex G-100凝胶层析纯化,收集胞外蛋白各组分,用考马斯亮蓝法和SDS-PAGE方法分别测定各蛋白组分的含量及分子量,然后采用四氮唑蓝比色分析法(MTT法)鉴定各蛋白组分抑制HT-29细胞增殖的能力。研究显示,分离L.acidophilus胞外蛋白获得两个蛋白峰,分子量分别为67 ku和37 ku,含量分别为0.066 mg/m L和0.021mg/m L,不同浓度(0.001μg/m L、0.01μg/m L、0.1μg/m L和1μg/m L)的67 ku和37 ku胞外蛋白分别对HT-29细胞作用12 h、24 h、48 h和72 h,均显示抑制细胞增殖的作用,且呈现明显的量效关系和时间依赖效应,其中1μg/m L的67 ku和37 ku胞外蛋白对HT-29细胞作用48 h的抑制率分别为(38.41±1.94)%和(45.06±1.58)%。综上所述,固体培养L.acidophilus可获得两种胞外蛋白(即37 ku和67 ku),其对HT-29细胞增殖具有不同程度的抑制作用,其中37 ku胞外蛋白的抑制作用较强。
In order to explore the related proteins secreted by Lactobacillus acidophilus and its function of promoting intestinal homeostasis, L.acidophilus was taken as the research object. The crude extract of extracellular protein was obtained by solid culture. Sephadex G-100 Gel filtration chromatography, the components of extracellular proteins were collected, and the content and molecular weight of each protein component were determined by Coomassie brilliant blue method and SDS-PAGE method, and then identified by MTT method The ability of each protein component to inhibit HT-29 cell proliferation. Studies have shown that the two protein peaks were obtained from the extracellular protein of L. acidophilus, with molecular weights of 67 ku and 37 ku, respectively, at concentrations of 0.066 mg / ml and 0.021 mg / ml, respectively, at different concentrations of 0.001 μg / ml μg / mL, 0.1μg / mL and 1μg / mL respectively) inhibited the proliferation of HT-29 cells after treated with 67 ku and 37 ku extracellular proteins for 12 h, 24 h, 48 h and 72 h, respectively (38.41 ± 1.94)% and (45.06)%, respectively. The inhibitory rates of 67 μg and 37 ku extracellular proteins at 1 μg / mL on HT-29 cells for 48 h were ± 1.58)%. In conclusion, two kinds of extracellular proteins (37 ku and 67 ku) were obtained from solid culture L. acidophilus, which inhibited the proliferation of HT-29 cells to a certain extent. The inhibitory effect of 37 ku extracellular protein Strong.