【作者】 唐吉军；

【导师】 谢剑炜；

【作者基本信息】 中国人民解放军军事医学科学院 ， 药物分析学， 2011， 博士

【摘要】 2000年人类基因序列测定的提前完成,标志着后基因组时代的序幕随之升起。蛋白质组学研究即为其中的重要分支。蛋白质组学旨在发现具有诊断和/或治疗意义的蛋白质,以揭示疾病的发生机制,从而为早期诊断、分子分型、治疗效果以及预后判断提供依据,并寻找可能成为药物作用的新靶点。为了能在分子水平上阐述表达量极低的功能性蛋白质在生理、病理过程中所起到的作用,就必须建立相应的高灵敏度、高特异性的活性蛋白质检测方法。常规的蛋白质检测方法大都基于抗体的免疫分析法而开展,而作为一类新型识别元件,寡核苷酸适配体受到了广泛关注并且在很多应用方面替代免疫分析中的抗体。适配体是一类通过体外指数富集配基的系统进化技术(SELEX)获得的单链寡核苷酸序列(RNA或ssDNA),其可以特异性地结合目标分子,例如无机离子、有机小分子、氨基酸、多肽、蛋白质、整个细胞等。由于核酸适配体与靶分子的相互作用类似于抗原与抗体结合,因此它们又被称为“化学抗体”。而相比抗体,适配体具有一些自身独特的优势,例如易于化学合成与修饰、良好的重现性与稳定性、较好的组织渗透性和很小的免疫原性、以及可扩增性。本论文以我们课题组已筛选获得的特异识别重组人促红细胞生成素α(rHuEPO-α)的适配体807-39 nt作为研究对象,基于适配体的结构特征以及适配体可扩增性,设计了一类具有普适性的高灵敏检测方法。此外,生物毒素作为一类重要的高毒性生物源物质,可作为潜在的生物战剂而被恐怖分子作为实施恐怖活动的工具,同时,其还是一类重要的免疫毒素类抗癌药物。因此,生物毒素的快速检测在军事、反恐和药学领域都存在迫切需要,此亦是本课题组长期致力的研究方向之一。核糖体失活蛋白(RIP)是其中一类重要的生物毒素,其通过抑制蛋白质合成而导致细胞中毒死亡。本论文中,我们以II型核糖体失活蛋白--蓖麻毒素为例,基于其糖苷酶活性建立了高灵敏度的毒素蛋白毒性体外分析新方法。本研究论文分为以下四章。第一章为前言部分,本章节主要综述了适配体的筛选、特性及其在蛋白质分析检测中的应用,以及代表性RIP--蓖麻毒素的理化性质、中毒机理以及不同原理的检测技术的发展状况。本章的最后则归纳了本研究论文的意义、内容与创新点。在第二章中,我们基于适配体的识别能力和可扩增特性,构建了两种经磁珠分离后实时定量PCR检测rHuEPO-α的检测策略。策略A,称为“杂交后识别”:先将适配体与互补链杂交,并通过互补链末端的生物素结合到链霉亲和素偶联的磁珠上,当加入rHuEPO-α后,适配体与靶分子的相互作用导致杂交区的部分双螺旋结构被破坏,适配体序列进入溶液,最后通过测量溶液中适配体的量来推算靶分子的含量;而策略B,即“杂交前识别”,是先将适配体与rHuEPO-α在溶液中充分混合以形成复合物,然后加入较高浓度的已固定在磁珠上的互补链,以结合溶液中游离适配体,通过实时定量PCR技术,测定上清液中适配体的浓度,从而计算出rHuEPO-α的浓度。对两种策略进行了系统优化,经比较,策略B的灵敏度、线性范围、重现性等分析指标更佳,更适于定量测定缓冲液和人工尿液中的rHuEPO-α,在未经任何富集处理的情况下检测限分别为1 pmol/L和6 pmol/L,线性范围分别为6 pmol/L ~ 100 nmol/L和30 pmol/L ~ 100 nmol/L。然而,对于实际生物样品例如人血清中靶蛋白的检测,样品中高丰度的基质会严重干扰PCR扩增,而目前常用的核酸样品前处理技术又难以适用于本检测策略。我们利用碱基互补配对原理设计合成了分别与适配体两端引物区结合的互补链,通过凝胶阻滞实验(EMSA)筛选出最佳的互补链,并将优选的生物素化的互补链连接到链霉亲和素磁珠上,以此为探针捕获复杂基质中的PCR扩增模板。研究结果表明,尽管检测灵敏度略有降低,但是应用该前处理方法,可成功地将所建立的检测策略应用到正常人血清中的rHuEPO-α定量检测。第三章则进一步验证了基于磁分离技术的适配体实时定量PCR分析方法的普适性。基于适配体与靶分子之间高效、专一的结合特性,近来适配体在多个领域尤其是分析化学等领域得到了广泛应用,但大都针对单一靶分子而展开检测,普适性的检测方法仍然是基于适配体的分析检测技术所亟需解决的难点。根据第二章中成功建立的基于磁分离技术的适配体实时定量PCR检测策略,本章则分别选取了代表生物大分子的凝血酶和代表有机小分子的ATP作为检测对象,考察上述检测策略的普适性,为建立基于适配体的靶分子通用检测方法奠定基础。研究结果表明,仅需简单优化互补链序列,该基于磁分离技术的适配体实时定量PCR普适型检测策略可成功地对结合缓冲液中添加的凝血酶和ATP的浓度进行准确定量,尤其是针对分子量仅为一个碱基大小的ATP靶分子的检测。第四章建立了基于糖苷酶活性的RIP毒性分析新方法。RIP是一类专一性水解真核或原核生物的核糖体RNA中一个保守的腺苷酸的N-C糖苷键,释放出一个腺嘌呤碱基使核糖体失活,从而抑制蛋白质生物合成的生物毒素。该类蛋白具有极强的细胞毒性,且尚无特效解毒药,因此针对其建立包括定性、定量以及活性分析在内的一整套灵敏、简便的检测方法具有重要现实意义。II型RIP--蓖麻毒素亦是禁止化学武器公约(CWC)附表1清单化学品中唯一的蛋白类毒素,目前本课题组已针对蓖麻毒素已经建立了较为完整的定性、定量分析方法,但尚无法判定蓖麻毒素的活性(毒性)状态。为解决该问题,并进一步完善蓖麻毒素的系统分析,我们基于毒素的糖苷酶活性建立了体外毒性分析方法,以LC-TOF/MS测定脱嘌呤反应中释放出的腺嘌呤的量来确定毒素的活性。我们系统优化了该分析方法的条件,包括液相色谱-质谱条件、脱嘌呤反应中所用的内标、脱氧寡核苷酸底物、反应pH值、温度和时间、保护蛋白的浓度以及反应终止液等。基于上述优化的脱嘌呤反应体系,可准确测定了低至10 ng/mL的活性蓖麻毒素,而经免疫磁珠亲和捕获后检测限可降至1 ng/mL。在特异性考察中发现毒素糖苷酶的体外脱嘌呤反应具有很强的特异性,不仅常见的蛋白酶不影响糖苷键的稳定性,而且肽N-糖苷酶F和β-葡糖醛酸糖苷酶同样不干扰毒素体外脱嘌呤反应。进一步以体外脱嘌呤反应为基础,建立了RIP的毒性分析平台,并应用于包括蓖麻凝集素、蓖麻毒素A链及相思子毒素在内的II型RIP,结果显示该平台实现了毒素毒性的简便、快速、灵敏的分析检测,并且可以满足实际生物样品中毒素活性的快速侦检需求。更多还原

【Abstract】 In pace with the successful completion of the Human Genome Project (HGP) in 2000, proteomics raised the curtain on postgenomic era. The intense interests of proteomics are to find proteins with potential diagnostic and/or therapeutic significance, so as to foster a better understanding of disease processes, and to provide basis for early detection of disease, molecular typing, therapeutic efficacy and prognosis assessment. Besides, proteomics is also applied to discover new biological markers for drug action. To demonstrate the functions of extremely low expression of proteins during physiological and pathologic processes, there is an urgent need to establish methods for active proteins with high sensitivity and selectivity. The conventional assays are mainly antibodies-based on immunoassays. Nevertheless, as a new molecular recognition module, oligonucleotide aptamers are currently focused on and have shown their wide applications instead of antibodies in immunoassays.Aptamers are one type of single-stranded oligonucleotides (RNA or ssDNA) generated through an in vitro selection process termed SELEX (systematic evolution of ligands by exponential enrichment), and they can specifically bind to their target molecules, such as inorganic ions, organic small molecules, amino acids, polypeptides, proteins, and even whole cells. Nucleic acid aptamers have been named as“chemical antibodies”because they interact with their targets with high binding affinity and specificity comparable with the interaction between antibodies and antigens. Besides, aptamers have many unique advantages, such as ease of chemical synthesis and modification, superior reproducibility and stability, good tissue penetration and much low immunogenicity, and amplification capability. In this dissertation, based on aptamer 807-39 nt, which has been successfully in vitro isolated by our research group and can specifically recognize recombinant human erythropoietinα(rHuEPO-α), we describe a novel, universal and highly sensitive methodlogy for quantification of rHuEPO-α, which combined the structure characteristics with the amplification feature of aptamers.In addition, biotoxins are an important kind of poisonous substances produced by living organism. Besides being potential biological warfare agents for terrorist use, biotoxins could likewise be used as immunotoxins to treat cancer. Therefore, a rapid detection of biotoxins is an urgent need in military, counterterrorism and pharmaceutical fields, which is also a key research direction of our research group. Ribosome-inactivating protein (RIP) is a type of biotoxins that can inhibit protein synthesis and result in cell death. Taking ricin type II RIP for an example, we demonstrate an in vitro high sensitive assay for quantification of toxic RIP based on its N-glycosidase activity.This dissertation consists of four chapters.The first chapter is the introduction. In this chapter, we summarized the selection methods and features of aptamers and their applications in protein detection. The physical and chemical properties of ricin as a representative of RIP, mechanism of poisoning and different detection techniques were introduced as well. The objectives, contents and new insights of this dissertation were briefly outlined at the end of this chapter.In chapter two, benefited from the specific recognition and the amplification capability of aptamers, we constructed two detection strategies for quantification of rHuEPO-αby magnetic beads-based aptameric real-time PCR assay. Strategy A is termed as“recognition-after-hybridization”, in which a partial duplex hybridization of aptamer and biotinylated complementary sequence (CS) is immobilized onto Streptavidin MagneSphere? Paramagnetic Particles (SA-PMPs) via the biotin-streptavidin interaction. With the addition of rHuEPO-α, aptamer will switch its conformation to bind targets while the duplex will disassemble. As a result, aptamer will dissociate from PMPs and form a complex with target in aqueous solution. Upon magnetic separation, the dissociated aptamer can be collected for real-time PCR analysis. Strategy B is termed as“recognition-before-hybridization”, in which aptamer is firstly incubated with rHuEPO-αto form a stable complex in an aqueous homogeneous solution. Biotinylated CS linked SA-PMPs is then used to hybridize the redundant unbound aptamer, acting as a“removing-the-background”module. After being separated with a magnet, a series of aptamers corresponding to various concentrations of rHuEPO-αare collected. The parameters including complementary sequences, the composition of binding buffer and ratio of CS and recognition aptamer sequence are optimized. After comparing two strategies on their sensitivity and feasibility, Strategy B was finally adopted as a preferred one to measure rHuEPO-α. The limit of detections (LODs) of 1 pmol/L and 6 pmol/L rHuEPO-α, and wide dynamic ranges from 6 pmol/L to 100 nmol/L and 30 pmol/L to 100 nmol/L were obtained for spiked binding buffer and matrix-half diluted artifical urine, respectively.If the target proteins exist in the real biological samples such as human serum, the high abundant matrix in biosamples would seriously block the amplification of real-time PCR. Otherwise, commonly used pretreatment techniques for nucleic acid samples are not suitable for above-mentioned strategy A or B. Following base-pairing principle, we designed and synthesized two complementary strands which could respectively bind primer regions at both ends of aptamer. The most suitable complementary strand was screened by electrophoretic mobility shift assay (EMSA), and linked to SA-PMPs via the biotin-streptavidin interaction, thus the formed probes could efficiently capture amplification templates from complex matrix. The results showed that this established pretreatment strategy could successfully help to determine the concentration of rHuEPO-αin human serum, in spite of a slightly lowered sensitivity.In chapter three, the universality of magnetic-based aptameric real-time PCR assay was validated. Based on high effeciency and specificity of binding characteristics between aptamer and target, aptamer were widely applied to various fields, e.g. analytical chemistry, but most aptameric methods were adopted to detect a single target molecule. There is still an urgent need to develop a universal aptameric assay. Based on the detection strategy described in chapter two, thrombin and ATP were chosen as detection targets for biomacromolecules and organic small molecules, respectively, to investigate the general applicability of such a strategy. The results demonstrated that previous described detection strategy could successfully determine the concentrations of thrombin and ATP spiked in binding buffer only after a simple optimization of hybridized sequences. Even for small organic molecule ATP, the size of that as same as a base, this strategy also has good feasibility.In the last chapter, a sensitive assay of bioactive RIPs was established on the basis of their N-glycosidase activity. RIPs are a kind of biotoxins that possess N-glycosidase activity and can specifically cleave nucleotide N-C glycosidic bonds at the general conserved residue in eukaryotic and prokaryotic rRNAs, blocking the synthesis of protein, and resulting in deactivation of ribosome. Since such proteins have extreme cytotoxicity and there is not yet any valid antitodes, it is of important realistic significance to establish sensitive and convenient assays for qualification, quantification and activity analysis of biotoxins. It should be mentioned that ricin (RCA60), type II RIP, is also the sole protein toxin listed in Schedule I of the Chemical Weapons Convention (CWC). A set of systemic qualitative and quantitative assays for ricin have been developed in our research group but lack of helpful tools to judge the activity (toxicity) of ricin. To overcome such a problem and further complete the systematic analysis of ricin, an in vitro analytical method of toxicity was established in virtue of its glycosidase activity, in which the toxicity of ricin was determined by measuring the release of adenine in depurination reaction by using LC-TOF/MS. Several parameters were systematically optimized, including conditions of LC and MS, internal standard, deoxyoligonucleotide substrate, pH, temperature, time, concentrations of protective protein and stop buffer of depurination reaction. Under the above optimized conditions, the active ricin at a concentration as low as 10 ng/mL was accurately determined, and the LOD could be reduced to 1 ng/mL with addition of immunocapture. We also found that ricin N-glycosidase in depurination reaction possessed high specificity, neither common proteases (including lysozyme, pronase and trypsin) or PNGase F andβ-Glucuronidase affected the stability of glycosidic bonds, and interfered with the depurination reaction of RIP toxins. Based on depurination reaction of ricin toxin in vitro, a rapid and sensitive analytical platform for toxicity analysis of RIP was developed. Several RIPs such as RCA60, ricin agglutinin (RCA120), ricin A chain (RTA) and abrin were tested in this platform. The results showed that this platform provided a convenient, rapid and sensitive assay for toxicity analysis of RIPs, and would meet the requirements for rapidly detecting activity of RIPs in the real biological samples.更多还原