【作者】 胡萍萍；

【导师】 黄承志； 罗凌飞；

【作者基本信息】 西南大学 ， 生物化学与分子生物学， 2014， 博士

【摘要】 近年来随着生命科学的飞速发展及其与数学、物理学、化学等多种学科的交叉与融合,逐渐产生了一门新的学科-定量生物学。自此,科学家的目光从定性的描述性研究逐渐向定量的整体化方向转变,同时注重寻找生物系统的整体规律。定量生物学的发展对生命科学中的飞跃发展起着至关重要的作用。朊病毒病(Prion diseases)又称为传染性海绵状脑病(TSE),是一类人畜共患的神经退行性疾病。20世纪80年代欧洲爆发的“疯牛病”以及由此产生的人新型克雅氏病(nv-CJD),引发了世界范围的空前恐慌,给人类健康造成巨大的威胁。这类疾病具有相似的临床症状和组织病理学改变,都是由一种具有自我复制能力的蛋白感染粒子-朊病毒引起的。目前人们普遍认为朊病毒蛋白具有两种构象：细胞型朊蛋白(Cellular prion protein,PrPc)和致病型朊蛋白(scrapie of pathogenic isoform, Prp.sc)。朊病毒病的发生是由于PrPc发生错误折叠产生PrPSc,同时prpSc可以诱导更多的’PrPc发生构象转变,prpSc成指数型增加形成聚集体进而引发病变。但是TSE在宿主内的潜伏期很长,潜伏期内患病动物无明显临床症状,PrPsc含量极少；同时,不同毒株中朊病毒理化性质的差别非常大,导致TSEs的早期临床诊断和治疗都面临着巨大的挑战。本论文利用量子点(QDs)独特的发光性质及金银纳米颗粒的强等离子共振性质通过建立能量转移体系,从定量生物学的角度,将化学与生物学进行有机融合,实现了对朊蛋白的简单、快速、灵敏的定量检测,并根据朊蛋白的结构特点筛选出一种小分子多肽,对稳定朊蛋白的构象具有非常重要的作用。主要研究内容如下：1.基于朊蛋白-适配子相互作用实现朊病毒蛋白的灵敏检测SERS用于双核酸适配子与朊蛋白相互作用研究表面增强拉曼散射由于在分子水平上提供了比传统拉曼散射更丰富的物质结构信息,近年来得到了广大科学工作者的亲睐。有文献报道朊蛋白有两段DNA适配子,可以分别与其N末端23-90以及90-231两个表位结合,对朊蛋白具有更好地选择性。我们用考马斯亮蓝染料在对蛋白质-PrPc进行标记的同时作为拉曼信号分子,用于PrPc-aptamer之间的相互作用研究。实验过程中,我们将朊蛋白的适配子功能化修饰在Ag@Si NPs表面,因此,当PrPc与其适配子结合时,体系中形成Ag@Si聚集体,可以大大增强R-250的拉曼信号。该方法证明我们选用的两段朊蛋白适配子确实可以同时识别靶物的不同位点,形成PrPc-aptamer复合物,并结合纳米银增强的R-250拉曼信号用于朊蛋白的检测研究。对于进一步发展基于双核酸适配子策略的朊蛋白检测方法具有非常重要的作用。基于双适配子的金属增强荧光策略用于检测朊蛋白我们合成了一种表面易于生物功能化的硅包银纳米颗粒,利用该纳米银与荧光染料之间的金属增强荧光建立了可在溶液中进行的简单、快速的朊蛋白的分析方法。首先在硅包银纳米颗粒表面修饰抗朊蛋白核酸适配子(Apt1),另一段核酸适配子(Apt2)修饰Cy3染料。Apt1和Apt2可以同时识别PrPc的两个不同的结合位点,因此,当PrPc存在时适配子发生构象转变,分别与PrPc上相应的靶点进行结合,从而形成Ag@SiNPs-PrPc-Cy3三明治结构。通过控制核壳型纳米粒子硅壳的厚度调整该纳米探针与染料分子之间的距离。由于纳米银的强等离子共振性质,当纳米探针与染料分子之间的距离足够近时就会发生金属增强荧光,Cy3荧光强度的增加与PrPc浓度呈很好的线性关系,可用于灵敏检测PrPc。同时该方法也适用于复杂样品如小鼠脑组.织匀浆中PrPc的检测,及细胞内朊蛋白的实时成像分析。长程共振能量转移实现朊蛋白超灵敏检测我们进一步利用量子点和纳米金分别作为能量的供体和受体,量子点-金纳米粒子表面能量转移发展了一种长距离能量转移体系,实现了对朊蛋白的超灵敏检测。当体系中存在朊蛋白时,Ni2+与朊蛋白组氨酸标签的相互作用使得NTA-Ni2+修饰量子点和核酸适配子修饰的金纳米粒子因为距离拉近而发生表面能量转移,量子点的荧光被金纳米粒子猝灭,猝灭效率高达88.7%,能量转移距离9-22nm,据此建立的朊蛋白检测方法检测限可达33aM。由于量子点以及金纳米颗粒具有很好的生物稳定性,该能量转移体系不但可以用于复杂生物样品中PrPc的灵敏检测。同时,该长距离能量转移体系还可以用于标记活细胞内内源性朊蛋白,实时追踪其与细胞内物质相互作用及代谢反应途径,对于朊病毒病的致病机理及其临床监测都具有非常重要的作用。2.多肽与朊蛋白相互作用研究朊病毒病至今尚无有效的治疗方法,目前其首选治疗策略主要是稳定朊病毒蛋白中α-螺旋构象,抑制其向β-折叠的转化。朊蛋白180位的缬氨酸突变为异亮氨酸的180I突变蛋白的突变位点与其181位的糖基化位点非常接近,其生物化学性质对朊病毒病的影响非常重要。本文针对180I突变蛋白的182-190段序列设计了KNFTK、KTDVE、EMMKE和EVVKK等四种axyzβ型多肽。其中xyz由PrP序列中相关的疏水氨基酸组成,并嵌入碱性(α)和酸性(β)氨基酸之间,因此它可以通过静电作用力与PrP中相应的酸性和碱性氨基酸之间形成盐桥。盐桥的形成使得蛋白质的相对长度变短,对蛋白水解酶的敏感性降低,从而起到稳定α-螺旋的作用。研究发现,EVVKK能稳定朊病毒蛋白的构象,同时诱导p-折叠向α-螺旋的转变,因此这种多肽有望作为一种新的药物,用于朊病毒病的临床治疗。更多还原

【Abstract】 During the last decade, with the rapid development of life sciences, and the integration of which with a variety of subjects such as mathematics, physics, chemistry, etc., a new discipline-termed "Quantitative Biology" generated, in which scientists gradually focused on the a precise, quantitative study, seeking the universal law of biological systems. And the development of quantitative biology is of vital importance on the rapid development of the life sciences.Transmissible spongiform encephalopathies (TSE), or prion diseases, is a group of infectious neurodegenerative disorders accompanied by cognitive impairments, extensive brain damage and neuronal dysfunction. Although these diseases are rare, their unique mechanism of transmission and the concerns generated by the recent appearance of a new variant form of CJD, which has been linked to consumption of meat contaminated with BSE, have put prions in the spotlight. Increasing evidence now supports the idea that the central event is the conformational change of PrP through a posttranslational process, from cellular form (PrPc) to disease-causing conformation (PrPSc). Prion diseases always have a long incubation period before an extremely rapid clinical stage, which makes erlay diagnosis of the diseases is far to achieve. Simple, fast, and sesitive detection strategy of the diseases related protein could be helpful for rational design of novel therapeutic and diagnostic methods.In this contribution, seval novel prion protein detection strategys are proposed based on prion-aptamer interaction, which is simple, fast, sensitive. Additionally, we designed a axyzβ type peptides, which show great potential for the stability of a-helix. The mainly issues of this contribution are as follows: 1. New methods for prion protein detection based on PrP-aptamer interactionSurface-Enhanced Raman Scattering Investigations on Prion Protein-dual Aptamer Interaction Surface-enhanced Raman scattering (SERS) spectra, which can provide large information on the chemical structure of the probed substances, have been successfully performed as a well-established tool in complex biological system. PrP is reported possessing two different distinct binding epitopes for two aptamers. One aptamer (Aptl) recognizes epitope23-90of the N-termina. while the other one (Apt2) specifically binds with the90-231of prion, which is corresponded to the β-sheet structure of PrP. Considering the strong Raman activity, Coomassie brilliant blue (R-250) was employed to label target proteins and probe protein-aptmer interactions, and an original quantitative study to investigate the interaction between PrPc and its two aptamers was develped based on SERS spectroscopy. Experimently, Aptl and Apt2are conjugated to silica coated silver (Ag@Si) NPs, followed by the addition of R-250labeled PrP. After well mixed with the target proteins, the aptamers undergo a conformational transition from an unfolded structure to a folded conformation, which possesses high affinity with the target, leading to the nanoparticles aggregation, sequentially inducing enhanced raman scattering. This strategy, which is addressing two aptamers recognizing distinct epitopes of the target, is simple, rapid, and high specificity to probe protein-aptamer recognition in the solution.Metal-enhanced fluorescence of nano-core-shell structure used for sensitive detection of prion protein with a dual-aptamer strategy. We developed a novel metal-enhanced fluorescence (MEF) and dual-aptamer-based strategy to achieve the prion detection in solution and intracellular protein imaging simultaneously, which shows high promise for nanostructure-based biosensing. In the prescence of prion protein, core-shell Ag@SiO2. which are functionalized covalently by single stranded aptamer (Aptl) of prions and Cyanine3(Cy3) decorated the other apater (Apt2) were coupled together by the specific interaction between prions and the anti-prion aptamers in solution. By adjusting shell thickness of particles, a dual-aptamer strategy combined MEF can be realized by the excitation and/or emission retes of Cy3. It was found that the enhanced fluorescence intensities followed a linear relationship in the range of50-300μM, which is successfully applied to the detection of PrP in the mice brain homegenates. as well as in the live cells. Ultra-sensitive detection of prion protein with a long range resonance energy transfer strategy. We developed an aptamer-mediated Long range Resonance Energy Transfer (LrRET) strategy to achieve the detection of cellular prion protein (PrPc) at attomolar level, which could be extended to the detection of other analogous proteins. Under optimal conditions,0.82-3.30fM of PrPccan be quantitatively detected with the limit of determination of33aM (3a). The strong fluorescence signals of QDs could be distinguished byconfocal imaging in vivo, and efficiently quenched with the addition of AuNPs-Apt conjugate. The visual LrRET system is promising to real-time trace the endogenous cellular prion protein.2. Interaction studies of the peptides and prion proteinUntil recently, no effective treatment strategy is avaliable for prion diseases, while the widely accepted therapautic method is. based on the stability of a-helical conformation, and the inhibition of transformation between a-helical and β-sheet Herein, we designed four axyzβ type peptides for sequence range of182-190of1801prion protein (a valine to isoleucine mutation), which plays an important role in the pathological prion disease, xyz is composed of hydrophobic amino acids (aa) that are found in the prion protein, while a/β stands for basic or acidic amino acids, which can interact with the relevant acidic or basic aa in prion protein to form salt briges. And the existence of salt briges lead to the shortening of protein length, and make it more stable.The results showed that only the sequence of EVVKK can bring a very good stability, and induce the conformation changes from β-sheet to a-helix, while the effect of other three peptides can be neglected, which can act as a reference for the development of peptide drugs, and further for the clinical treatment.更多还原