【作者】 肖赛金；

【导师】 黄承志；

【作者基本信息】 西南大学 ， 分析化学， 2010， 博士

【摘要】 朊病毒病（prion diseases）是一类致死性神经退行性疾病,是细胞型朊蛋白（cellular prion protein, PrP^C）经构象转变形成致病型朊蛋白(scrapie prion protein, prp^Sc)的结果。上世纪末欧洲疯牛病的大范围爆发及其由牛传染给人的新型克雅氏病的发现使得人们“谈牛色变”,并迫使欧盟迅速建立朊病毒病检测的标准方法。朊病毒病的诊断尤其是早期诊断既能实现该疾病流行趋势的监控、确保肉类产品、输血、手术、血库和血浆产品安全,又使得该病在永久性脑损伤发生前即可进行有效治疗。基于朊病毒病抗体的传统检测方法因抗体制备复杂、与靶物的亲和力和特异性不高等缺点而无法应用于朊病毒病的早期诊断中。因此简单、快速、准确的朊病毒病早期诊断方法亟待建立。朊病毒蛋白配体因能够特异性的与朊蛋白相互作用而得到科学工作者的广泛关注。如适配子是一类新兴的与靶物具有很高的亲和力和选择性的核酸配体,自发现以来就因具抗体无法比拟的优势成为抗体替代物在各个领域得到广泛应用。本文将配体应用于朊病毒病分析中,建立了一系列基于配体的朊病毒蛋白分析方法。主要研究内容如下：1.单标记aptamer分子灯标的设计及朊蛋白的检测中。根据G碱基在距离适当时能猝灭染料如罗丹明、荧光素等的荧光的性质,本文设计了基于G碱基猝灭的单标记适配子分子灯标,成功解决了分子灯标双标记的操作复杂、成本高、适配子亲和力下降等问题。将所设计的适配子分子灯标应用于朊病毒蛋白检测时发现,在朊病毒蛋白浓度为1.1-44.7μg/mL时,荧光强度与蛋白浓度呈现很好的线性关系,检测限为0.3μg/mL （3σ）。与传统的抗体检测方法相比,本文所建立的方法简单、快速且选择性高。2.基于适配子和汞离子的朊蛋白免标记检测。根据T碱基能与Hg相互作用形成T-Hg²⁺-T结构的特点,本文建立了基于适配子和Hg²⁺的免标记的朊病毒蛋白检测方法。研究结果表明,适配子与Hg²⁺作用后形成链间双链结构,引起双链嵌入荧光染料Syber Green I的荧光显著增强；但当加入朊病毒蛋白后,朊病毒蛋白与适配子的特异性相互作用促使T-Hg-T结构打开从而双链结构被破坏,Syber Green I的荧光降。当朊蛋白浓度为13.0-156.0 nmol/L时,Syber GreenI的荧光强度与朊蛋白浓度呈现很好的线性关系,线性方程为I_F-F0= 58.33-8.42 c_PrP,相关系数R=0.992。3.双适配子策略在多靶物分析中的应用。与单适配子策略相比,双适配子策略具有更高的选择性和灵敏度,并能显著提高分析方法灵敏度。本研究将双适配子策略应用于多靶物分析中并选择凝血酶、腺苷、朊病毒蛋白（PrP）三种与疾病相关的生物分子作为研究对象。结果表明,双适配子策略能够实现同一样品中凝血酶、腺嘌呤核苷酸和朊蛋白的同时检测,且三种靶物的类似物如鸟嘌呤核苷酸、胞嘧啶核苷酸、尿嘧啶核苷酸、IgG、蜗牛酶、牛血清白蛋白等对多靶物测定不产生干扰,显示出双适配子策略在多靶物分析中的高选择性。4.双适配子分子逻辑门在朊病毒蛋白构象区分中的应用。作为逻辑门延伸的分子逻辑门以一个或多个复杂的生物或化学反应过程为输入信号,通过一定的逻辑运算简化为单一输出结果,例如疾病的诊断通过分子逻辑门可简化为“患病”或“正常”。本文借助分子逻辑门的优势并将双适配子策略应用其中,成功构建了能够进行“异或”（XOR）和“或”（OR）逻辑运算的双适配子分子逻辑门。结果表明,PrP^C符合“异或”逻辑运算而PrP^Res符合“或”逻辑运算。与传统的朊病毒蛋白区分方法相比,本文所构建的双适配子分子逻辑门具有四个优点：（1）双适配子分子逻辑门非常简单且能够应用于朊病毒蛋白不同构象的区分；（2）使用双适配子策略使得分子逻辑门具有极高选择性,无需分离和纯化即可实现血清中的朊病毒蛋白的区分；（3）双适配子分子逻辑门的输入信号（PrP和盐酸胍）和元件（MMPs-Apt1和QDs-Apt2）的成本较低,且都是化学稳定分子,而这是构建更快、更高效、更小型的计算器所必需的；（4）因Apt1只在磁微米粒子表面形成单层而不涉及固定构象,所以元件之一的MMPs-Apt1能够通过简单的方法进行循环使用。5.肝素钠诱导朊病毒蛋白构象变化的光谱分析。粘多糖是一种在细胞膜表面和溶酶体中均有表达的朊蛋白配体分子,其在朊病毒病中发生中所发挥的作用目前仍存在争议。本文以肝素钠作为粘多糖的代表,通过共振光散射光谱、荧光光谱和圆二色光谱的变化研究了肝素钠与人重组细胞型朊蛋白(rhPrP^C_23-231)的相互作用。结果表明,肝素钠与朊蛋白相互作用后光散射和荧光信号均得到增强,且当rhPrP^C_23-231浓度为0.41-16.46μg/mL时,rhPrP^C_23-231浓度与共振散射光强度呈现很好的线性关系,线性方程：I_RLS=183.84+272.40 c₍rhPrP^C_23-231,相关系数R=0.999。同时对朊蛋白和肝素钠相互作用体系的荧光寿命表征表明,肝素钠促使朊蛋白的荧光寿命有一定程度的延长,而圆二色光谱的表征则表明肝素钠能诱导朊蛋白从富含α-螺旋的构象向富含β-折叠的构象转变。本研究基于配体所建立的一系列朊病毒蛋白分析方法,方法简单、快速且灵敏,双适配子逻辑门方法更因具有超高的灵敏度和选择性而有望开发成朊病毒病的早期诊断方法。更多还原

【Abstract】 Prion diseases, a group of fatal neurodegerative disorders, arised when cellular prion protein （PrP^C） undergoes some conformation rearrangement and formes the pathological isoform (PrP^Sc). The huge epizootic of "mad cow" in european and the discovery of variant CJD （vCJD, the transmission form of "mad cow" in human） at the end of last centrury, created enormours concerns among consumers and forced the European Commission to develop new simpler and faster diagnositc tests for prion diseases. Especially pre-symptomatic diagnostical that will do favors in the following aspects:firstly, the extent of epizootic would under surveillance by the goverment; secondly, ensure the safety of meat products, blood transfission, surgery, blood banks and plasma products; thirdly, identify prion diseases in the early stage so that the treatment could be initiated before the appearance of permanent brain damage. So simpler, faster and more accurate methods for prion diseases are in the ugent need of development. The application of conventional methods that are based on antibodies to pre-symptomatic diagnostical are limited by many reasons, such as the complicated procedure of antibodies producing, insufficient affinity and selectivity to targets. Aptamers, new-born nucleic acids ligands that bind to targets with high affinity and selectivity, are superior to antibodies in many aspects and thus has been widely applied to a variety of fields. In this contribution, aptamers are introduced to the analysis of prion diseases and a serial of prion protein detection methods have been developed based on aptamers. The main researches of this contribution include.1. Guanine-based aptamer beacon design and its application for PrP analysis. An aptamer-participated haprin structure was designed by employing cellular prion protein （PrP^C） as a model protein, and thus an aptamer-mediated turn-on fluorescence assay for proteins was developed in this contribution. The designed aptamer-participatedhaprin structure consists of three segments.Namely, an aptamer sequence located in the loop, three guanine bases at 3’-terminal, and a fluophor modified at 5’-terminal. It was found that the guanine bases at the 3’-terminal could quench the fluorescence of the fluophor such as tetramethyl-6-carboxyrhodamine （TAMRA） at the 5’-terminal about 76.6% via electron transfer if the guanine bases are close enough to the fluophor, and the quenched fluorescence could get restored when the target protein is present since the interaction, which could be confirmed by measuring fluorescence lifetime, between TAMRA-aptamer and the target protein forces the guanines away from TAMRA so that TAMRA-modified aptamer changes into turn-on state. A linear relationship was then constructed between the turn-on fluorescence intensity and the concentration of PrPC in the range from 1.1 to 44.7μg/mL with a limit of detection of 0.3μg/mL （3a）.2. Label-free PrP detection based on aptamer and Hg²⁺. A new lable-free method for prion protein detection was developed based on the fact that T bases can interact with Hg²⁺ to form T-Hg²⁺-T structure. It was found that when aptamers were interact with Hg²⁺, intramolecular T-Hg²⁺-T structure was formed and thus the fluorescence of double-strand dyes such as Syber GreenⅠ（SGⅠ） enhanced; however, the T-Hg²⁺-T structure destroyed with the addition of prion protein since the specifical interaction between aptamer and its target protein force aptamer underwent conformation change, and thus the fluorescence of SGⅠdisappeared. The fluorescence intensities of SGⅠhave good linear relationship with the concenctration of prion protein ranging from 13.0 to 156.0 nmol/L, R= 0.992.3. Compared with single-aptamer strategy, dual-aptamer strategy has higher affinity and selectivity and the employment of dual-aptamer strategy will dramatically increase the sensitivity and selectivity of aptamer-assays. Herein, dual-aptamer strategy was introduced to multi-targets analysis to solve the problem that limited the application of multi-targets analysis. Three targets, including ATP, thrombin and prion protien, are all related to diseases were selected for multi-targets analysis in this contribution. The results show that the detection of thrombin, ATP and PrP could be achieved simultanously in the same sample, and the analoges of the three targets such as adenosine, guanine, thymidine, IgG, snailase, BSA have on cross-reaction on the detection of targets, indicating the high selectivity of dual-aptamer strategy for multi-target analysis. 4. Dual-aptamer logic gate for the discrimination of prion-diseases-associated isoform. As the extension of logic gate, molecular logic gate that performs one or more inputs resulting from complex biological or chemical processes and produces a single output through logic operation, leaving the diagnosis of disease either "yes" or "no". Combining the advanteges of logic gate and dual-aptamer strategy, a dual-aptamer logic gate that is capable of OR and XOR logic operations (PrPc behaviors XOR logic operation while PrP^Res represents OR logic operation) has been designed. Compared with convential discrimination assays, the present dual-aptamer logic gate possesses the following advantages. Firstly, the dual-aptamer strategy could achieve highly selective discrimination of PrPRes in serum without isolation of target proteins prior to assay. Secondly, the dual-aptamer logic gate is simple and could be applied for prion diseases-associated isoform discrimination. Thirdly, the inputs （PrP and Gdn-HCl） and gates （MMPs-Apt1 and QDs-Apt2） used here are all cost-saving and chemical stable molecules, which are crucial for the development of smaller, more effective molecular-scaled computers. Fourthly, the gate （MMPs-Apt1） could be reused without loss of sensitivity since it only includes a surface-tethered monolayer of Apt1 without preferred conformation.5. Spectra characterization of the conformational changes of human cellular prion protein induced by heparin. Glycosaminoglycans（GAGs） is a ligand that distributes both in cell membrane surface and in endosomes. Up to now, the role of GAGs played in prions is still in controversy. By using heparin as an example, we investigated the interaction of GAGs and recombinant human cellular prion protein (rhPrP～C_23-231) by measuring the spectral features of the resonance light scattering （RLS）, fluorescence and circular dichroism （CD）. It was found that the intensity of both RLS and fluorescence get increased when the interaction of heparin with rhPrP^C_23-231 occurred and RLS intensities have good relationship with the concentration of rhPrP^C_23-231 ranging from 0.41 to 16.46μg/mL, with R=0.999. Meanwhile, the interaction between rhPrP^C_23-231and heparin make the fluorescence lifetime of rhPrP^C_23-231 extend, and the CD spectra indicate that heparin could induce the conformation rich in alfa-helix change to conformation rich in beta-sheet. In summary, we have developed a serial of prion protein detection methods based on aptamers. All of these methods are simple, fast, cost-saving and sensitivity, especially the dual-aptamer logic gate which are ultra-sensitive and selective for prion protein discrimination might further be applied to prion diseases pre-symptomatic diagnostical. Also, the researches present here extend the application of aptamers in diseases diagnosis.更多还原