人FOXP3 cDNA的克隆及在原核细胞中的表达

发布时间：2018-03-12 14:28

本文选题：FOXP3　切入点：cDNA　出处：《第四军医大学》2007年硕士论文　论文类型：学位论文

【摘要】： 乙型肝炎病毒(hepatitis B virus,HBV)慢性感染是非常严重的人类健康问题,对HBV感染慢性化发生机制的研究是寻找新的有效的治疗方法的基础。几十年来世界各国的科学家就此进行了大量的研究,发现特异性免疫功能低下是HBV感染慢性化的关键,但是未能阐明免疫功能低下发生的机制。目前认为维持机体免疫耐受的主要机制是中枢耐受和外周耐受,在外周耐受中调节性T细胞(Treg细胞)具有非常重要的作用,已成为当前免疫学研究的热点。国内外多项研究结果显示调节性T细胞与慢性HBV感染者特异性免疫反应低下有一定的关联。国外多个研究小组已证明FOXP3(称叉头状/翼状螺旋转录因子)在Treg细胞上特异性表达,且对Treg细胞的发育和功能是必需的。这极大地促进了在分子水平上对Treg细胞的认识,但当前对于FOXP3是如何调控Treg细胞的发育及其通过何种下游靶基因发生作用的机制仍知之甚少。阐明这些问题,对寻找以调节性T细胞为靶点的新的治疗自身免疫性疾病、肿瘤和包括慢性乙型病毒性肝炎在内的慢性感染性疾病具有重要意义。本实验在成功克隆人FOXP3 cDNA的基础上,构建了人FOXP3蛋白的原核表达载体并在大肠杆菌中进行了表达,为进一步完成对该蛋白的纯化及免疫学功能研究奠定了基础。方法:从脐带血分离的单个核细胞中提取总RNA,巢式RT-PCR技术扩增FOXP3 cDNA,产物纯化后T-A克隆连接至中间载体pMD18-T,利用测序正确质粒,构建其原核表达载体pRSET-A-FOXP3,转化大肠杆菌BL-2(1DE3)pLysS,IPTG诱导表达6×His融合蛋白,表达产物经SDS-PAGE及Western blot检测及鉴定。结果与结论:经序列测定、SDS-PAGE及Western blot等方法证实成功克隆了人FOXP3 cDNA,构建了其原核表达载体,并在大肠杆菌中得到有效表达。
[Abstract]:Chronic hepatitis B virus infection is a very serious human health problem. The study on the mechanism of chronic HBV infection is the basis of finding new and effective treatment methods. In recent decades, scientists all over the world have done a lot of research, and found that the low specific immune function is the key to chronic HBV infection. At present, it is considered that the main mechanism of maintaining immune tolerance is central tolerance and peripheral tolerance, and the regulatory T cells (Treg cells) play a very important role in peripheral tolerance. Many domestic and foreign studies have shown that regulatory T cells are associated with the low specific immune response of chronic HBV infections. Several foreign research groups have demonstrated that FOXP3 (forkhead / winglike helical transcription factor) is specifically expressed on Treg cells and is necessary for the development and function of Treg cells. This has greatly promoted the understanding of Treg cells at the molecular level. However, little is known about how FOXP3 regulates the development of Treg cells and the mechanism by which downstream target genes play a role. To elucidate these problems, we can find a new treatment for autoimmune diseases targeting regulatory T cells. Tumor and chronic infectious diseases including chronic hepatitis B are of great significance. Based on the successful cloning of human FOXP3 cDNA, the prokaryotic expression vector of human FOXP3 protein was constructed and expressed in Escherichia coli. It lays a foundation for the further study of the purification and immunological function of the protein. Methods: total RNAs were extracted from mononuclear cells isolated from umbilical cord blood. FOXP3 cDNA was amplified by nested RT-PCR. T-A clone was purified and ligated to the intermediate vector pMD18-T, and the correct plasmid was sequenced. The prokaryotic expression vector pRSET-A-FOXP3 was constructed and transformed into Escherichia coli BL-2DE1DE1DE3DE-pLysSS-IPTG to express 6 脳 His fusion protein. The expression product was detected and identified by SDS-PAGE and Western blot. Results and conclusion: the human FOXP3 cDNAs were cloned successfully by sequencing SDS-PAGE and Western blot, and the prokaryotic expression vector was constructed and expressed effectively in Escherichia coli.
【学位授予单位】：第四军医大学
【学位级别】：硕士
【学位授予年份】：2007
【分类号】：R346

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