酵母双杂交系统筛选FOXP3相互作用蛋白

发布时间：2018-05-12 05:54

本文选题：基因 + 克隆　；参考：《第三军医大学》2006年硕士论文

【摘要】： 研究背景调节性T细胞(Treg)是机体免疫中非常重要的T细胞亚群,与机体的免疫系统自稳状态、自身免疫性疾病、移植物免疫排斥反应及肿瘤发生有极其密切的联系。但有关Treg特征性标志物、细胞接触性抑制和无能的分子基础等根本问题尚未阐明。FOXP3是近年发现的一个转录因子,是目前公认Treg细胞的一个特征性标志。目前研究表明FOXP3特异表达于机体的CD4+CD25+Treg,介导CD4+CD25+Treg细胞在胸腺的发育,外周表达及功能维持。FOXP3基因和Treg细胞的关系首先是在Scurfy小鼠上发现的,Scurfy突变小鼠中此基因发生了突变,可引起致命性的淋巴细胞增殖性疾病,伴随着多器官组织浸润,在人类,与小鼠同源的Foxp3基因突变可有多种突变,可引起系统性自身免疫病IPEX (新生儿糖尿病、炎症性肠炎,内分泌综合征等)综合征。我们移植免疫小组长期致力于调节性T细胞(Treg)的研究,FOXP3基因的发现极大的促进了在分子水平上对Treg细胞的认识,为我们进一步研究Treg细胞功能提供了新的思路。然而对于FOXP3通过何种机制影响Treg发挥作用及其通过何种下游靶基因发生作用,这些问题鲜见文献报道。阐明这些问题,将有助于Treg细胞的发育和其功能的研究,并对于自身免疫性疾病,肿瘤的治疗及移植免疫耐受的诱导等临床应用具有非常重要的意义。蛋白质相互作用研究是认识基因功能的一个重要途径,生物体系的运作与蛋白质之间的相互作用密不可分。发现和验证在生物体中相互作用的蛋白质与核酸、蛋白质与蛋白质是认识它们生物学功能的第一步。通过分析一个蛋白质是否与已知功能的蛋白质相互作用可得到揭示其功能的线索,如果两个蛋白质相互作用,它们一般参与相同或相关的细胞活动,相互作用的伴侣可以直接与生物学事件联系起来。蛋白相互作用研究技术中,用于发现和研究在活细胞体内的蛋白质与蛋白质之间相互作用的酵母双杂交技术(yeast two-hybrid system)自建立以来已经成为一种强有力的研究方法。我们的研究将FOXP3列为Treg功能相关的候选蛋白,希望能从蛋白质组学方面对Treg提出新的认识。为此我们利用酵母双杂交技术筛选FOXP3的相互作用蛋白。本研究从获得FOXP3基因全长cDNA序列着手,构建FOXP3诱饵融合蛋白表达载体,用酵母交配法钓取与FOXP3相互作用蛋白,以得到与其相互作用的阳性克隆,旨在对FOXP3基因的功能有较深入了解,为进一步研究Treg细胞接触抑制分子机制的物质基础,并为我们能主动干预免疫反应的进程、方向提供新的思路。
[Abstract]:Research background Regulatory T cell (T cell) is a very important T cell subgroup in immune system, which is closely related to autostable state of immune system, autoimmune disease, graft immune rejection and tumorigenesis. However, some fundamental problems, such as the characteristic marker of Treg, the molecular basis of cell contact inhibition and incompetence, have not been clarified. FOXP3 is a transcription factor discovered in recent years and is a characteristic marker of Treg cells. Current studies have shown that FOXP3 is specifically expressed in CD4 CD25 tregs of the body, mediating the development of CD4 CD25 Treg cells in the thymus, peripheral expression and functional maintenance. The relationship between FOXP3 gene and Treg cells is first found in Scurfy mice. It can cause fatal lymphocytic proliferative disease, accompanied by multiple organ tissue infiltration. In humans, Foxp3 gene mutations homologous to mice can be mutated, which can cause systemic autoimmune disease IPEX (neonatal diabetes, inflammatory enteritis, neonatal diabetes, inflammatory enteritis). Endocrine syndrome, etc. The discovery of FOXP3 gene has greatly promoted the understanding of Treg cells at the molecular level and provided a new way for us to further study the function of Treg cells. However, there are few reports on the mechanism by which FOXP3 affects the role of Treg and the downstream target genes. To elucidate these problems will be helpful to the study of the development and function of Treg cells, and will be of great significance in the clinical application of autoimmune diseases, tumor therapy and the induction of transplantation immune tolerance. Protein interaction is an important way to understand gene function. The interaction between protein and biological system is closely related. The discovery and verification of proteins and nucleic acids, proteins and proteins interacting in organisms is the first step in understanding their biological functions. Analyzing whether a protein interacts with a protein with a known function leads to clues to its function. If two proteins interact, they are generally involved in the same or related cellular activity. Interacting partners can be directly associated with biological events. Yeast two-hybrid system, which is used to find and study the interaction between protein and protein in living cells, has become a powerful research method since its establishment. Our study listed FOXP3 as a candidate protein related to the function of Treg, hoping to provide a new understanding of Treg in proteomics. Therefore, we used yeast two-hybrid technique to screen the interacting protein of FOXP3. In this study, the full-length cDNA sequence of FOXP3 gene was obtained, the expression vector of FOXP3 bait fusion protein was constructed, and the interacting protein with FOXP3 was isolated by yeast mating method. The aim of this study is to understand the function of FOXP3 gene, to further study the material basis of the mechanism of contact inhibition in Treg cells, and to provide a new way of thinking for us to intervene in the process of immune response.
【学位授予单位】：第三军医大学
【学位级别】：硕士
【学位授予年份】：2006
【分类号】：R392

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