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/mL和0.021 mg/mL，不同浓度（0.001 μg/mL、0.01 μg/mL、0.1 μg/mL和1 μg/mL）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1 μg/mL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。

To explore the related proteins secreted by Lactobacillus acidophilus and their functional roles in promoting gut homeostasis, the extracellular protein crude extract was obtained from L. acidophilus solid culture, and each fraction of the extracellular proteins was collected after purification and isolation using Sephadex G-100 gel chromatography. The amount of protein and molecular mass of each protein fraction were determined by Coomassie brilliant blue method and SDS-PAGE electrophoresis, respectively. Then, the inhibitory effect of each L. acidophilus extracellular protein fraction on the proliferation of HT-29 cells was investigated by 3-(4,5-dimethylthiazol-2-yl)-2,5- diphenyltetrazolium bromide (MTT) assay. Two protein peaks were obtained after purification, their molecular weights were 67 ku and 37 ku, respectively, and their protein contents were 0.066 mg/mL and 0.021 mg/mL, respectively. HT-29 cells were treated with both 67 ku and 37 ku extracellular proteins at concentrations of 0.001 μg/mL, 0.01 μg/mL, 0.1 μg/mL, and 1 μg/mL for 12 h, 24 h, 48 h, or 72 h, respectively. Both proteins showed an inhibitory effect on the proliferation of HT-29 cells in a time- and dose-dependent manner. Among the tested concentrations, the inhibition rates of the 67 ku and 37 ku extracellular proteins at μg/mL on HT-29 cells were (38.41±1.94)% and (45.06±1.58)%, respectively. In conclusion, L. acidophilus cultured on solid medium produced two types of extracellular proteins (37 ku and 67 ku), which inhibited the proliferation of HT-29 cells to different degrees, and the 37 ku extracellular protein exhibited a relatively strong inhibitory effect.

国家自然科学基金资助项目（31071522）