EV71和人类共同抗原的鉴定及其作为重症HFMD潜在致病机理的研究和高敏核酸酶联免疫荧光试验的建立

发布时间：2018-08-22 12:51

【摘要】：第一篇：EV71与人类共同抗原的鉴定及其作为HFMD重症潜在病因的研究手足口病主要是由肠道病毒71型和柯萨奇病毒16型两种病原引起的以感染5岁以下儿童，手足口部出现疱疹溃疡以及发热等为主要症状的疾病。由于EV71的感染，少数病例可出现严重的神经症状，如无菌性脑干脑炎、急性致死性神经性肺水肿、肺出血、急性呼吸衰竭等严重中枢神经系统症状而导致死亡。因此，澄清EV71的致病机理对重症的控制、及时制定正确的治疗方案减少死亡率具有重要的意义。此前，尽管对EV71致死性神经性症状进行了大量研究，但其真正致病机理尚不明确。目前，EV71对中枢神经系统的致病机理研究主要包括病毒感染对神经元的直接损伤、致病性细胞因子（IP-10, MCP-1, IL-6, IL-8, and G-CSF等）在神经系统中急剧升高导致的神经组织损伤以及EV71与人脑蛋白共同抗体的自身反应性而引发的自身免疫对神经系统的损伤三方面原因的内容。本文鉴定了EV71病毒与人类共同抗原，并主要以机体针对共同抗原产生的具有自身反应性的抗体为出发点，论述了共同抗体在HFMD重症病因中潜在的作用。本研究通过对病毒蛋白质组与人类蛋白质组的比对分析，首次鉴定出在人类脑干部高表达的RNA聚合酶的转录调节复合物的亚基25（mediator complexsubunit25，MED25）与EV71的病毒蛋白1（VP1）存在共同表位PPGAPKP，并将两种共同抗原进行了体外表达，制备了针对该表位的单克隆抗体2H2，假病毒中和试验检测显示该抗体无中和活性。通过Western blot和ELISA试验证实了2H2与MED25，VP1以及EV71病毒样颗粒的结合活性。使用测定蛋白间相互作用的方法测定了共同抗体与自身抗原的结合活性及亲和常数（KD），KD=8.0×10-7，为中等亲和力强度。动物免疫实验证明了两种共同抗原均能够刺激机体产生高滴度的针对共同表位的共同抗体。同时对患者血清进行共同抗体检测，结果显示EV71感染者的血清中同样存在高滴度的共同抗体。应用小动物活体成像技术监测共同抗体在体内的分部情况来反映该抗体对自身抗原的反应性。结果显示，除主要分布在肝脏外，在脑干及延髓处亦有分布，表明共同抗体在活体内可能与自身抗原结合进而引发免疫反应。本研究首次鉴定了共同抗原并系统地证实了自身反应性抗体在体内的大量存在并与自身抗原在体内的结合反应，为EV71的重症致病机理在有关自身免疫反应性方面的进一步研究提供了确实而充分的实验和理论依据。同时，对EV71新型疫苗的开发与应用也有重要的指导意义。第二篇高灵敏性核酸酶联免疫荧光试验的建立酶联免疫吸附试验（ELISA）在传染性病原检测中着实是一种非常有效的方法。但是在应用中仍然存在不足，如在某些领域里其检测灵敏度很难达到要求。为了进一步提高该方法的灵敏性，几十年来，科学工作者做了很多努力。虽然已有很多改善和提高，但是目前该方法的灵敏度及实用性仍不能适应现代医学某些领域的发展和少数传染病复杂化的程度。为了进一步提高该方法的灵敏性，本研究首次将核酸酶和荧光探针用于免疫吸附试验，被称为核酸酶联荧光寡核苷酸试验（Nuclease-linked Fluorescence Oligonucleotide Assay，NLFOA）。在该方法中，具有高酶活性的核酸酶TurboNuclease用作标记酶，水解荧光探针中链接荧光素和淬灭基团的寡核苷酸链，致使淬灭基团对荧光素的淬灭作用消失而发出可检测的荧光。由于核酸酶的高效性和荧光探针的高荧光强度而使源于被检分子的检测信号得到一定程度的放大，从而提高灵敏度。同时，具有信号放大效应的表面偶联有大量链霉亲和素分子的金纳米颗粒也被用于此方法中，能够使被捕获的单个被检抗原分子通过纳米颗粒-亲和素复合物结合大量生物素化的标记核酸酶，使被检测信号进一步放大再次提高灵敏度。本研究在纳米颗粒直径、标记核酸酶使用浓度、纳米颗粒-亲和素复合物使用浓度和荧光探针使用浓度等方面对NLFOA进行了条件的优化。通过检测人类艾滋病毒重要的标志性蛋白分子p24，，对NLFOA的检测灵敏性、特异性和重复性进行了评价。结果显示，NLFOA的最低检测限度为1.0pg/mL，低于传统ELISA的最低检测限度（10.0pg/mL）10倍。特异性在两次评价中均达到了100%，重复性在p24的高（100.0pg/mL）、中（50.0pg/mL）、低（1.5pg/mL）三个浓度水平的变异系数（CV）分别为7.8%、9.05%、8.4%，均低于被接受阈值10%。NLFOA是本实验室建立的一种高灵敏性检测抗原的方法，改变相应的抗体后可用与各种传染病抗原的检测。本方法首次把核酸酶和荧光探针应用与酶联免疫试验中，开拓了在免疫试验中使用核酸酶和荧光探针为底物新的思路，显著地提高了检测方法的灵敏性，为疾病的早期诊断避免病原的传播以及及时制定合理的治疗方案提供了强有力的技术手段。
[Abstract]:Part one: identification of EV71 and human common antigens and their potential role as a potential cause of HFMD
Hand-foot-mouth disease is mainly caused by enterovirus 71 and Coxsackievirus 16 in children under 5 years of age, herpes ulcer and fever in hand, foot and mouth. Severe neurological symptoms such as aseptic brainstem encephalitis and acute lethal neuropulmonary fluid can occur in a few cases due to EV71 infection. Severe central nervous system symptoms such as swelling, pulmonary hemorrhage, and acute respiratory failure lead to death. Therefore, it is important to clarify the pathogenesis of EV71 to control the severity of the disease and to formulate correct treatment plans to reduce mortality. At present, the pathogenesis of EV71 in the central nervous system mainly includes the direct injury of neurons caused by virus infection, the nerve tissue injury caused by the sharp increase of pathogenic cytokines (IP-10, MCP-1, IL-6, IL-8, and G-CSF, etc.) in the nervous system, and the self-reactivity of EV71 and human brain protein co-antibodies. This paper identifies the common antigen of EV71 virus and human beings, and discusses the potential role of common antibodies in the pathogenesis of HFMD. By comparative proteomic analysis, we identified for the first time that the transcriptional regulatory complex subunit 25 (MED25) of RNA polymerase overexpressed in human brain cadres shared a common epitope with the virus protein 1 (VP1) of EV71. Two common antigens were expressed in vitro and the monoclonal antibody 2 against this epitope was prepared. The binding activity of the antibody to MED25, VP1 and EV71 virus-like particles was confirmed by Western blot and ELISA. The binding activity and affinity constant (KD), KD=8.0 *10-7, were determined by the method of protein-protein interaction. Animal immunoassay showed that both common antigens could stimulate the body to produce high titers of common antibodies against common epitopes. At the same time, common antibodies were detected in the sera of patients with EV71 infection. The results showed that there were high titers of common antibodies in the sera of EV71 infected persons. The results showed that the antibody was mainly distributed in the liver, but also in the brain stem and medulla oblongata, suggesting that the common antibody might bind to the autoantigen in vivo and induce an immune response. This study identified the common antigen for the first time and systematically confirmed its autoreactivity. A large number of antibodies exist in vivo and react with autoantigens in vivo, which provides a solid and sufficient experimental and theoretical basis for the further study of the pathogenesis of EV71 and the development and application of new EV71 vaccine.
Establishment of second highly sensitive nucleic acid ELISA
Enzyme-linked immunosorbent assay (ELISA) is a very effective method in the detection of infectious pathogens. However, there are still some shortcomings in the application, such as the detection sensitivity in some areas is difficult to meet the requirements. In order to further improve the sensitivity of this method, scientists have made a lot of efforts for decades. In order to further improve the sensitivity of this method, nuclease and fluorescent probe were first used in immunosorbent assay, called nuclease-linked fluorescent oligonucleotide assay. Nuclease-linked fluorescence Oligonucleotide Assay (NLFOA). In this method, a nuclease with high enzyme activity, Turbo Nuclease, is used as a labeling enzyme to hydrolyze the oligonucleotide chains linking fluorescein and quenching groups in fluorescent probes, resulting in the disappearance of quenching effect of quenching groups on fluorescein and the detection of fluorescence. The high efficiency of the enzyme and the high fluorescence intensity of the fluorescent probe enlarge the detection signal from the detected molecule to a certain extent, so as to improve the sensitivity. NLFOA was characterized by nanoparticle diameter, labeled nuclease concentration, nanoparticle-avidin complex concentration and fluorescent probe concentration. The sensitivity, specificity and repeatability of NLFOA were evaluated by detecting p24, an important marker of human HIV. The results showed that the minimum detection limit of NLFOA was 1.0 pg/mL, 10 times lower than the minimum detection limit of traditional ELISA (10.0 pg/mL). The specificity of NLFOA was 100% and the repeatability was 100% in both evaluations. The coefficient of variation (CV) of high (100.0 pg/mL), medium (50.0 pg/mL) and low (1.5 pg/mL) concentrations of p24 were 7.8%, 9.05% and 8.4% respectively, which were lower than the accepted threshold of 10%. NLFOA is a highly sensitive method established in our laboratory for detecting antigens, and can be used to detect various infectious disease antigens after changing the corresponding antibodies. The application of acid enzyme and fluorescent probe and enzyme-linked immunoassay (ELISA) have opened up a new way of using nuclease and fluorescent probe as substrate in immunoassay, which has greatly improved the sensitivity of detection methods, and provided a powerful technical means for early diagnosis of diseases to avoid the spread of pathogens and formulate reasonable treatment plans in time.
【学位授予单位】：吉林大学
【学位级别】：博士
【学位授予年份】：2015
【分类号】：R725.1

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