β-淀粉样蛋白寡聚体的制备及其表征

发布时间：2018-03-03 16:26

本文选题：阿尔茨海默症　切入点：β-淀粉样蛋白　出处：《兰州大学》2017年硕士论文　论文类型：学位论文

【摘要】：阿尔茨海默症(Alzheimer Disease,AD)是一种中枢神经系统退行性疾病,又名老年痴呆。它的病理学特征分别是淀粉样蛋白斑和神经纤维结。近些年来,随着β-淀粉样蛋白质(β-amyloid,Aβ)假说的不断更新,越来越多的实验证据表明淀粉样蛋白质沉积只是AD致病的结果而不是导致其致病的关键因素,其寡聚体形式的淀粉样蛋白才是致病的罪魁祸首。为了从分子水平更深层次地理解淀粉样蛋白质错误折叠和聚集的分子机制,解析淀粉样蛋白质寡聚体的分子结构迫在眉睫。固体核磁共振技术是研究这类体系最有力的工具。但是,固体核磁共振技术解析蛋白结构对样品要求较高,需要大量的、均一性较好的样品。因此在体外制备符合其要求的寡聚体样品尤为重要。本论文主要研究内容包括以下三个部分:(1)Aβ1-40蛋白的获取运用分子克隆、蛋白表达纯化技术在大肠杆菌细胞中成功制备出高含量、高纯度的Aβ1-40蛋白。(2)Aβ1-40寡聚体样品的制备、表征在溶液环境中采用低温低盐以及添加抑制剂、金属离子等几个方面制备Aβ1-40蛋白的寡聚体,并结合电泳、ThT荧光光谱、ANS荧光光谱、圆二色光谱和透射电子显微镜技术等对寡聚体进行初步表征。我们发现Aβ1-40寡聚体呈圆饼状,直径为15nm-35nm左右。(3)抑制剂、金属离子对Aβ1-40蛋白聚集机制的研究在课题2的基础上,通过液体核磁共振技术,我们研究了抑制剂、金属离子与Aβ1-40蛋白相互作用的分子机制,进一步阐明Aβ蛋白聚集化过程的复杂性,对后续设计药物来抑制Aβ蛋白聚集化进程的发展,提供了一定的理论基础,对疾病的治疗和预防具有非常重要的价值和意义。
[Abstract]:Alzheimer's Disease (AD) is a degenerative disease of the central nervous system, also known as Alzheimer's disease. Its pathological features are amyloid plaques and neurofibril knots. In recent years, the 尾 -amyloid A 尾 hypothesis has been updated. A growing body of experimental evidence suggests that amyloid deposition is the result of AD rather than a key cause. In order to understand the molecular mechanism of misfolding and aggregation of amyloid proteins at the molecular level, It is urgent to analyze the molecular structure of amyloid protein oligomers. Solid nuclear magnetic resonance (SNMR) is the most powerful tool to study this kind of system. Therefore, it is very important to prepare in vitro oligomer samples that meet its requirements. The main contents of this thesis include the following three parts: molecular cloning of the extraction and application of 尾 1-40 protein. High content and high purity A 尾 1-40 protein A 尾 1-40 oligomer samples were successfully prepared by protein expression and purification technique. The samples were characterized by low temperature and low salt and adding inhibitors in solution environment. The oligomers of A 尾 1-40 protein were prepared from metal ions and characterized by electrophoretic fluorescence spectra (ans), circular dichroism (CD) and transmission electron microscopy (TEM). Study on the aggregation Mechanism of A 尾 1-40 protein by Metal Ions. On the basis of topic 2, we studied the molecular mechanism of the interaction between inhibitor, metal ion and A 尾 1-40 protein by liquid nuclear magnetic resonance (LNMR). Further elucidation of the complexity of A 尾 protein aggregation process provides a theoretical basis for the subsequent design of drugs to inhibit the development of A 尾 protein aggregation process, and is of great value and significance for the treatment and prevention of diseases.
【学位授予单位】：兰州大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R749.16

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