本文關(guān)鍵詞：L.brevis M8 S-層蛋白的黏附性及其引起Caco-2細胞蛋白質(zhì)差異表達研究　出處：《湖南農(nóng)業(yè)大學(xué)》2012年碩士論文　論文類型：學(xué)位論文

  更多相關(guān)文章： 乳酸菌 S-層蛋白 黏附性 蛋白質(zhì)

【摘要】：乳酸菌作為益生菌在功能性食品產(chǎn)業(yè)中具有很大的應(yīng)用前景,其發(fā)揮益生性的重要前提為黏附至胃腸道中。乳酸菌的S-層蛋白由于其特殊的結(jié)構(gòu)位置,一直被人們認為參與了黏附的過程,然而此方面的研究卻很少。目前,已有人成功的將S-層蛋白的編碼基因表達到不含S-層蛋白的乳酸球菌中,發(fā)現(xiàn)可以增強乳酸球菌的黏附力。這表明S-層蛋白在增強益生菌的黏附性方面具有一定的潛力。因此,對于S-層蛋白的黏附性及黏附機制的研究顯得尤為重要。 本論文主要研究L. brevis M8S-層蛋白對結(jié)腸癌細胞Caco-2的黏附性及其引起腸細胞蛋白變化情況,旨在進一步確定S-層蛋白的黏附性及其黏附機制。主要的研究內(nèi)容和結(jié)果如下： 1、采用5mol/L鹽酸胍提取L. brevis M8S-層蛋白,并采用SephadexG-75凝膠層析柱對提取的蛋白質(zhì)溶液進行分離純化。經(jīng)SDS-PAGE檢測得出：以Tris-HCL為洗脫液,SephadexG-75為層析柱,可以獲得電泳純度的、具生物活性的S-層蛋白。 2、運用免疫學(xué)原理,研究含S-層蛋白的L. brevis M8菌體抗體結(jié)合前后與Caco-2的黏附力變化情況；同時以細胞總蛋白為研究對象、S-層蛋白為第一抗體、膠體金標記的S-層蛋白抗體為第二抗體進行Western Blot,從而研究S-層蛋白對腸細胞蛋白黏附特性。試驗結(jié)果得出：含有S-層蛋白的菌體黏附力高于不含S-層蛋白的菌體；S-層蛋白可以與分子量在130kDa、63kDa、45kDa、0kDa及13kDa的腸細胞蛋白發(fā)生較好的黏附。 3、采用蛋白質(zhì)雙向電泳技術(shù)對S-層蛋白黏附腸細胞引起其變化的情況進行分析,分別設(shè)置正常組、試驗組1(0.2mg/mlS-層蛋白處理)和試驗組2(0.5mg/mlS-層蛋白處理)。用PDQuest軟件對圖片進行分析得出：試驗組1存在差異表達蛋白12個,上調(diào)蛋白2個,下調(diào)蛋白5個,未表達的蛋白有5個且無新蛋白表達；試驗組2中存在差異點17個,上調(diào)蛋白3個,下調(diào)蛋白8個,未表達蛋白5個且引起了1種新蛋白的表達。
[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.
【學(xué)位授予單位】：湖南農(nóng)業(yè)大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2012
【分類號】：R151

【參考文獻】

相關(guān)期刊論文 前1條

1 賈宇峰,林秋霞,郭堯君,郭鷂,劉少君;蛋白質(zhì)雙向電泳圖像分析[J];生物化學(xué)與生物物理進展;2001年02期

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