L.brevis M8 S-层蛋白的黏附性及其引起Caco-2细胞蛋白质差异表达研究

发布时间：2018-01-14 21:19

本文关键词：L.brevis M8 S-层蛋白的黏附性及其引起Caco-2细胞蛋白质差异表达研究　出处：《湖南农业大学》2012年硕士论文　论文类型：学位论文

更多相关文章： 乳酸菌 S-层蛋白 黏附性 蛋白质

【摘要】：乳酸菌作为益生菌在功能性食品产业中具有很大的应用前景,其发挥益生性的重要前提为黏附至胃肠道中。乳酸菌的S-层蛋白由于其特殊的结构位置,一直被人们认为参与了黏附的过程,然而此方面的研究却很少。目前,已有人成功的将S-层蛋白的编码基因表达到不含S-层蛋白的乳酸球菌中,发现可以增强乳酸球菌的黏附力。这表明S-层蛋白在增强益生菌的黏附性方面具有一定的潜力。因此,对于S-层蛋白的黏附性及黏附机制的研究显得尤为重要。本论文主要研究L. brevis M8S-层蛋白对结肠癌细胞Caco-2的黏附性及其引起肠细胞蛋白变化情况,旨在进一步确定S-层蛋白的黏附性及其黏附机制。主要的研究内容和结果如下： 1、采用5mol/L盐酸胍提取L. brevis M8S-层蛋白,并采用SephadexG-75凝胶层析柱对提取的蛋白质溶液进行分离纯化。经SDS-PAGE检测得出：以Tris-HCL为洗脱液,SephadexG-75为层析柱,可以获得电泳纯度的、具生物活性的S-层蛋白。 2、运用免疫学原理,研究含S-层蛋白的L. brevis M8菌体抗体结合前后与Caco-2的黏附力变化情况；同时以细胞总蛋白为研究对象、S-层蛋白为第一抗体、胶体金标记的S-层蛋白抗体为第二抗体进行Western Blot,从而研究S-层蛋白对肠细胞蛋白黏附特性。试验结果得出：含有S-层蛋白的菌体黏附力高于不含S-层蛋白的菌体；S-层蛋白可以与分子量在130kDa、63kDa、45kDa、0kDa及13kDa的肠细胞蛋白发生较好的黏附。 3、采用蛋白质双向电泳技术对S-层蛋白黏附肠细胞引起其变化的情况进行分析,分别设置正常组、试验组1(0.2mg/mlS-层蛋白处理)和试验组2(0.5mg/mlS-层蛋白处理)。用PDQuest软件对图片进行分析得出：试验组1存在差异表达蛋白12个,上调蛋白2个,下调蛋白5个,未表达的蛋白有5个且无新蛋白表达；试验组2中存在差异点17个,上调蛋白3个,下调蛋白8个,未表达蛋白5个且引起了1种新蛋白的表达。
[Abstract]:Lactic acid bacteria (Lactic acid bacteria) as probiotics have a great application prospect in the functional food industry. The important premise to play the probiotics is to adhere to the gastrointestinal tract. The S- layer protein of lactic acid bacteria due to its special structural position. It has been thought to be involved in the adhesion process, but little research has been done in this field. At present, Slaminin coding genes have been successfully expressed in Lactococcus lactis without Slaminin. It was found that the adhesion of Lactococcus lactis could be enhanced, which indicated that S- laminin had some potential in enhancing the adhesion of probiotics. It is very important to study the adhesion and adhesion mechanism of S-laminin. In this paper, we studied the adhesion of L. brevis M8S- layer protein to Caco-2 of colon cancer cells and the changes of intestinal cell proteins. The aim of this study is to further determine the adhesion of S- laminin and its adhesion mechanism. The main contents and results are as follows: 1. 5 mol / L guanidine hydrochloride was used to extract L. brevis M8S- layer protein. The extracted protein solution was separated and purified by SephadexG-75 gel chromatography column. SDS-PAGE detection showed that Tris-HCL was used as eluant. SephadexG-75 was used as an chromatographic column to obtain purified and bioactive S- layer proteins. 2. Using immunological principle, the adhesion force of L. brevis M8 antibody containing S- layer protein to Caco-2 was studied before and after binding. At the same time, the total cell protein was used as the first antibody, and the colloidal gold labeled S- layer protein antibody was used as the second antibody to carry out Western Blot. The adhesion of S- layer protein to intestinal cell protein was studied. The results showed that the adhesion of S- layer protein to intestinal cell protein was higher than that of cell without S- layer protein. S- laminin could be well adhered to intestinal cell proteins with molecular weight of 130kDa, 63kDa, 45kDa0kDa and 13kDa. 3. Protein two-dimensional electrophoresis was used to analyze the changes caused by Slaminin adhesion to intestinal cells and set up normal group respectively. Test group (1: 0. 2 mg / ml S- layer protein treatment) and test group (2 0. 5 mg / ml S- layer protein treatment). The results showed that there were 12 differentially expressed proteins in group 1 by PDQuest software. There were 2 up-regulated proteins, 5 down-regulated proteins, 5 unexpressed proteins and no new proteins. In group 2, there were 17 points of difference, 3 up-regulated proteins, 8 down-regulated proteins, and 5 unexpressed proteins, which resulted in the expression of a new protein.
【学位授予单位】：湖南农业大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R151

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