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Thesis Advisor徐霞
Degree Grantor中国科学院研究生院
Place of Conferral北京
Degree Discipline生物工程
Keyword阿尔兹海默症 Ad7c-ntp 多肽配体

由于阿尔兹海默疾病的治疗局限性,因此其早期诊断是预防与治疗的关键。AD7c-NTP作为早期诊断阿尔兹海默症的一个生化指标,具有无创性的优势。目前针对该蛋白检测的试剂盒主要以多克隆抗体检测为主,而多克隆抗体检测存在易发生交叉反应、批间重复性差等缺点。因此本研究采用分子模拟的方法设计和AD7c-NTP相互作用的小分子多肽;通过基因克隆、原核表达等方法合成AD7c-NTP蛋白;利用液相色谱法检测小分子多肽与AD7c-NTP的相互作用,进而实现对模拟尿液中的AD7c-NTP的检测。本研究得到的主要结果如下:(1) 在pUC57载体上构建AD7c-NTP基因,将基因转化于BL21进行蛋白表达,在1.1 mmol/L IPTG时获得最高表达量,通过蛋白纯化复性,获得了具生物活性的AD7c-NTP。(2) 利用Autodock Vina分子对接软件,设计筛选出12种与AD7c-NTP相互作用高的小分子配体,进一步通过GROMACS在水环境下进行进行动力学模拟,确定WRDW、DEWH以及RWDW作为肽筛选的候选序列。(3) 液相色谱检测结果显示WRDW、DEWH以及RWDW对AD7c-NTP的动态吸附率分别为90%、85%和86%,且WRDW对AD7c-NTP的吸附率与分离体系的pH值、离子强度和缓冲液组成无关。(4) WRDW和DEWH对IgG和HSA的非特异性吸附在pH 9的条件下即可洗脱,相反,WRDW和DEWH对AD7c-NTP的吸附只能在pH 2的条件下洗脱。(5) WRDW和DEWH对AD7c-NTP的检测范围范围为100~900 ng/mL,检测限都达到了100 ng/mL,同时成功地实现了对模拟尿液中的AD7c-NTP的检测。

Other Abstract

Early diagnosis is critical for treating Alzheimer’s diease due to no effective methods for preventing and curing Alzheimer’s disease has been found. Alzheimer-associated neuronal thread protein (AD7c-NTP) as a sensitive, easily obtained and noninvasive biomarker for AD disease detection has attracted more and more attentions in recent years. However, the current ELISA method using polyclonal antibody to detect AD7c-NTP leads to the difficulty in the comparison between different batches. Hence, in this study, a novel peptide for detecting AD7c-NTP was developed using molecular simulations. The binding capacity of the designed peptides to AD7c-NTP was determined using chromatography. The main conclusions are as the followings:(1) The recombinant AD7c-NTP was expressed using BL21 containing AD7c-NTP gene synthesized in pUC57. The optimal protein expression is achieved using 1.1 mmol/L IPTG.(2) 12 peptides were developed based on the binding free energy between the peptide and AD7c-NTP using Autodock Vina. Further dynamic molecular simulations in the water environment at room temperature using the GROMACS indicate that three peptides of WRDW, DEWH and RWDW have the strongest interactions with AD7c-NTP.(3) The binding capacity of WRDW, DEWH and RWDW to AD7c-NTP is 90%、85% and 86%, respectively, determined using chromatography. There is little effect of pH, NaCl concentration and buffer type on the binding capacity of WRDW to AD7c-NTP. (4) The bound HSA and IgG can be differentiated using different elution conditions. The bound AD7c-NTP can only be eluted at pH 2 while more than 90% of the bound HSA and IgG can be eluted at pH 9.(5) The detection linearity range of the peptide of WRDW and DEWH is 100-900 ng/mL,and the detection limit is 100 ng/mL. The designed peptide can be successfully used to detect AD7c-NTP in the mimic urine samples.

Document Type学位论文
Recommended Citation
GB/T 7714
张燕. 一种用于检测AD7c-NTP的多肽配体设计及应用研究[D]. 北京. 中国科学院研究生院,2016.
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