与阿尔茨海默症相关的γ-分泌酶复合物的表达与纯化研究

发布时间：2018-08-29 17:02

【摘要】：阿尔茨海默症(Alzheimer’s disease,AD)是一种慢性致死性的神经退行性疾病,多发生在老年人群,但也有早发性AD,与遗传因素有关。其主要病理特征是β-淀粉样蛋白(amyloid-βprotein,Aβ)堆积形成的老年斑和神经纤维状缠结,造成神经元损伤。其中,Aβ的形成与三种分泌酶相关,γ-分泌酶被认为是其限速酶,β-淀粉样前体蛋白(amyloid-βprecursor protein,APP)经过γ-分泌酶剪切生成Aβ40/Aβ42。所以,研究γ-分泌酶的结构对于揭示γ-分泌酶的工作机制和治疗AD药物分子设计具有重要意义。γ-分泌酶是一个具有19次跨膜区域的膜内切割蛋白酶,由四个亚基presenilin(PS),nicastrin(NCT),PEN2和Aph1a在空间极为严密复杂排列组成。本研究中,利用昆虫杆状病毒表达系统、哺乳动物表达系统等成功表达四种组分和全酶复合物。由于膜蛋白疏水性,筛选合适的去垢剂溶解膜组分也是本研究中的重点,经过筛选,选择最佳的去垢剂,进行膜蛋白的提取和纯化。实验结果,得到了纯度较高的nicastrin蛋白,并进行体外去糖基化实验,其他组分和全酶复合物经过分步纯化后含量较低。这些结果为下一步各亚基间相互作用研究,全酶复合物的结构研究奠定了基础。
[Abstract]:Alzheimer's disease (Alzheimer's disease,AD) is a chronic and fatal neurodegenerative disease, which mostly occurs in the elderly, but early onset AD, is also associated with genetic factors. The main pathological features of amyloid- 尾 -amyloid protein (amyloid- 尾 protein,A 尾) are senile plaques and neurofibrillar tangles, resulting in neuronal damage. The formation of A 尾 is related to three secretory enzymes, 纬 -secretase is considered to be its rate-limiting enzyme, and 尾 -amyloid precursor protein (amyloid- 尾 precursor protein,APP) is shredded by 纬 -secretase to produce A 尾 40 / A 尾 42. Therefore, it is important to study the structure of 纬 -secretase, which is an intramembrane cleavage protease with 19 transmembrane regions, to reveal the working mechanism of 纬 -secretase and to design drug molecules for AD. It consists of four subunits, presenilin (PS) nicastrin (NCT), PEN2 and Aph1a. In this study, the insect baculovirus expression system and mammalian expression system were used to express the four components and the whole enzyme complex. Because of the hydrophobicity of membrane protein, it is also the key point of this study to select suitable dissolving membrane components of scale removal agent. After screening, the best scale removal agent is selected to extract and purify membrane protein. The results showed that the high purity nicastrin protein was obtained and the content of the other components and the whole enzyme complex was lower after purification in vitro. These results lay a foundation for the further study of the interaction between subunits and the structure of the whole enzyme complex.
【学位授予单位】：上海交通大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：R749.16

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