EV71和人類共同抗原的鑒定及其作為重癥HFMD潛在致病機理的研究和高敏核酸酶聯(lián)免疫熒光試驗的建立
發(fā)布時間：2018-08-22 12:51
【摘要】：第一篇：EV71與人類共同抗原的鑒定及其作為HFMD重癥潛在病因的研究 手足口病主要是由腸道病毒71型和柯薩奇病毒16型兩種病原引起的以感染5歲以下兒童，手足口部出現(xiàn)皰疹潰瘍以及發(fā)熱等為主要癥狀的疾病。由于EV71的感染，少數(shù)病例可出現(xiàn)嚴重的神經(jīng)癥狀，如無菌性腦干腦炎、急性致死性神經(jīng)性肺水腫、肺出血、急性呼吸衰竭等嚴重中樞神經(jīng)系統(tǒng)癥狀而導(dǎo)致死亡。因此，澄清EV71的致病機理對重癥的控制、及時制定正確的治療方案減少死亡率具有重要的意義。此前，盡管對EV71致死性神經(jīng)性癥狀進行了大量研究，但其真正致病機理尚不明確。目前，EV71對中樞神經(jīng)系統(tǒng)的致病機理研究主要包括病毒感染對神經(jīng)元的直接損傷、致病性細胞因子（IP-10, MCP-1, IL-6, IL-8, and G-CSF等）在神經(jīng)系統(tǒng)中急劇升高導(dǎo)致的神經(jīng)組織損傷以及EV71與人腦蛋白共同抗體的自身反應(yīng)性而引發(fā)的自身免疫對神經(jīng)系統(tǒng)的損傷三方面原因的內(nèi)容。本文鑒定了EV71病毒與人類共同抗原，并主要以機體針對共同抗原產(chǎn)生的具有自身反應(yīng)性的抗體為出發(fā)點，論述了共同抗體在HFMD重癥病因中潛在的作用。本研究通過對病毒蛋白質(zhì)組與人類蛋白質(zhì)組的比對分析，首次鑒定出在人類腦干部高表達的RNA聚合酶的轉(zhuǎn)錄調(diào)節(jié)復(fù)合物的亞基25（mediator complexsubunit25，MED25）與EV71的病毒蛋白1（VP1）存在共同表位PPGAPKP，并將兩種共同抗原進行了體外表達，制備了針對該表位的單克隆抗體2H2，假病毒中和試驗檢測顯示該抗體無中和活性。通過Western blot和ELISA試驗證實了2H2與MED25，VP1以及EV71病毒樣顆粒的結(jié)合活性。使用測定蛋白間相互作用的方法測定了共同抗體與自身抗原的結(jié)合活性及親和常數(shù)（KD），KD=8.0×10-7，為中等親和力強度。動物免疫實驗證明了兩種共同抗原均能夠刺激機體產(chǎn)生高滴度的針對共同表位的共同抗體。同時對患者血清進行共同抗體檢測，結(jié)果顯示EV71感染者的血清中同樣存在高滴度的共同抗體。應(yīng)用小動物活體成像技術(shù)監(jiān)測共同抗體在體內(nèi)的分部情況來反映該抗體對自身抗原的反應(yīng)性。結(jié)果顯示，除主要分布在肝臟外，在腦干及延髓處亦有分布，表明共同抗體在活體內(nèi)可能與自身抗原結(jié)合進而引發(fā)免疫反應(yīng)。本研究首次鑒定了共同抗原并系統(tǒng)地證實了自身反應(yīng)性抗體在體內(nèi)的大量存在并與自身抗原在體內(nèi)的結(jié)合反應(yīng)，為EV71的重癥致病機理在有關(guān)自身免疫反應(yīng)性方面的進一步研究提供了確實而充分的實驗和理論依據(jù)。同時，對EV71新型疫苗的開發(fā)與應(yīng)用也有重要的指導(dǎo)意義。 第二篇高靈敏性核酸酶聯(lián)免疫熒光試驗的建立 酶聯(lián)免疫吸附試驗（ELISA）在傳染性病原檢測中著實是一種非常有效的方法。但是在應(yīng)用中仍然存在不足，如在某些領(lǐng)域里其檢測靈敏度很難達到要求。為了進一步提高該方法的靈敏性，幾十年來，科學(xué)工作者做了很多努力。雖然已有很多改善和提高，但是目前該方法的靈敏度及實用性仍不能適應(yīng)現(xiàn)代醫(yī)學(xué)某些領(lǐng)域的發(fā)展和少數(shù)傳染病復(fù)雜化的程度。為了進一步提高該方法的靈敏性，本研究首次將核酸酶和熒光探針用于免疫吸附試驗，被稱為核酸酶聯(lián)熒光寡核苷酸試驗（Nuclease-linked Fluorescence Oligonucleotide Assay，NLFOA）。在該方法中，具有高酶活性的核酸酶TurboNuclease用作標記酶，水解熒光探針中鏈接熒光素和淬滅基團的寡核苷酸鏈，致使淬滅基團對熒光素的淬滅作用消失而發(fā)出可檢測的熒光。由于核酸酶的高效性和熒光探針的高熒光強度而使源于被檢分子的檢測信號得到一定程度的放大，從而提高靈敏度。同時，具有信號放大效應(yīng)的表面偶聯(lián)有大量鏈霉親和素分子的金納米顆粒也被用于此方法中，能夠使被捕獲的單個被檢抗原分子通過納米顆粒-親和素復(fù)合物結(jié)合大量生物素化的標記核酸酶，使被檢測信號進一步放大再次提高靈敏度。本研究在納米顆粒直徑、標記核酸酶使用濃度、納米顆粒-親和素復(fù)合物使用濃度和熒光探針使用濃度等方面對NLFOA進行了條件的優(yōu)化。通過檢測人類艾滋病毒重要的標志性蛋白分子p24，，對NLFOA的檢測靈敏性、特異性和重復(fù)性進行了評價。結(jié)果顯示，NLFOA的最低檢測限度為1.0pg/mL，低于傳統(tǒng)ELISA的最低檢測限度（10.0pg/mL）10倍。特異性在兩次評價中均達到了100%，重復(fù)性在p24的高（100.0pg/mL）、中（50.0pg/mL）、低（1.5pg/mL）三個濃度水平的變異系數(shù)（CV）分別為7.8%、9.05%、8.4%，均低于被接受閾值10%。NLFOA是本實驗室建立的一種高靈敏性檢測抗原的方法，改變相應(yīng)的抗體后可用與各種傳染病抗原的檢測。本方法首次把核酸酶和熒光探針應(yīng)用與酶聯(lián)免疫試驗中，開拓了在免疫試驗中使用核酸酶和熒光探針為底物新的思路，顯著地提高了檢測方法的靈敏性，為疾病的早期診斷避免病原的傳播以及及時制定合理的治療方案提供了強有力的技術(shù)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.
【學(xué)位授予單位】：吉林大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2015
【分類號】：R725.1
本文編號：2197149
[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.
【學(xué)位授予單位】：吉林大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2015
【分類號】：R725.1
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