本文選題：基因 + 克隆　； 參考：《第三軍醫(yī)大學(xué)》2006年碩士論文

【摘要】： 研究背景 調(diào)節(jié)性T細(xì)胞(Treg)是機(jī)體免疫中非常重要的T細(xì)胞亞群,與機(jī)體的免疫系統(tǒng)自穩(wěn)狀態(tài)、自身免疫性疾病、移植物免疫排斥反應(yīng)及腫瘤發(fā)生有極其密切的聯(lián)系。但有關(guān)Treg特征性標(biāo)志物、細(xì)胞接觸性抑制和無能的分子基礎(chǔ)等根本問題尚未闡明。FOXP3是近年發(fā)現(xiàn)的一個轉(zhuǎn)錄因子,是目前公認(rèn)Treg細(xì)胞的一個特征性標(biāo)志。目前研究表明FOXP3特異表達(dá)于機(jī)體的CD4+CD25+Treg,介導(dǎo)CD4+CD25+Treg細(xì)胞在胸腺的發(fā)育,外周表達(dá)及功能維持。FOXP3基因和Treg細(xì)胞的關(guān)系首先是在Scurfy小鼠上發(fā)現(xiàn)的,Scurfy突變小鼠中此基因發(fā)生了突變,可引起致命性的淋巴細(xì)胞增殖性疾病,伴隨著多器官組織浸潤,在人類,與小鼠同源的Foxp3基因突變可有多種突變,可引起系統(tǒng)性自身免疫病IPEX (新生兒糖尿病、炎癥性腸炎,內(nèi)分泌綜合征等)綜合征。 我們移植免疫小組長期致力于調(diào)節(jié)性T細(xì)胞(Treg)的研究,FOXP3基因的發(fā)現(xiàn)極大的促進(jìn)了在分子水平上對Treg細(xì)胞的認(rèn)識,為我們進(jìn)一步研究Treg細(xì)胞功能提供了新的思路。然而對于FOXP3通過何種機(jī)制影響Treg發(fā)揮作用及其通過何種下游靶基因發(fā)生作用,這些問題鮮見文獻(xiàn)報道。闡明這些問題,將有助于Treg細(xì)胞的發(fā)育和其功能的研究,并對于自身免疫性疾病,腫瘤的治療及移植免疫耐受的誘導(dǎo)等臨床應(yīng)用具有非常重要的意義。蛋白質(zhì)相互作用研究是認(rèn)識基因功能的一個重要途徑,生物體系的運(yùn)作與蛋白質(zhì)之間的相互作用密不可分。發(fā)現(xiàn)和驗(yàn)證在生物體中相互作用的蛋白質(zhì)與核酸、蛋白質(zhì)與蛋白質(zhì)是認(rèn)識它們生物學(xué)功能的第一步。通過分析一個蛋白質(zhì)是否與已知功能的蛋白質(zhì)相互作用可得到揭示其功能的線索,如果兩個蛋白質(zhì)相互作用,它們一般參與相同或相關(guān)的細(xì)胞活動,相互作用的伴侶可以直接與生物學(xué)事件聯(lián)系起來。蛋白相互作用研究技術(shù)中,用于發(fā)現(xiàn)和研究在活細(xì)胞體內(nèi)的蛋白質(zhì)與蛋白質(zhì)之間相互作用的酵母雙雜交技術(shù)(yeast two-hybrid system)自建立以來已經(jīng)成為一種強(qiáng)有力的研究方法。 我們的研究將FOXP3列為Treg功能相關(guān)的候選蛋白,希望能從蛋白質(zhì)組學(xué)方面對Treg提出新的認(rèn)識。為此我們利用酵母雙雜交技術(shù)篩選FOXP3的相互作用蛋白。本研究從獲得FOXP3基因全長cDNA序列著手,構(gòu)建FOXP3誘餌融合蛋白表達(dá)載體,用酵母交配法釣取與FOXP3相互作用蛋白,以得到與其相互作用的陽性克隆,旨在對FOXP3基因的功能有較深入了解,為進(jìn)一步研究Treg細(xì)胞接觸抑制分子機(jī)制的物質(zhì)基礎(chǔ),并為我們能主動干預(yù)免疫反應(yīng)的進(jìn)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.
【學(xué)位授予單位】：第三軍醫(yī)大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2006
【分類號】：R392

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