肠道病毒分离鉴定及EV71快速高通量中和实验方法的建立

发布时间：2018-01-12 00:15

本文关键词：肠道病毒分离鉴定及EV71快速高通量中和实验方法的建立　出处：《厦门大学》2009年硕士论文　论文类型：学位论文

【摘要】： 手足口病是儿童常见病,多种肠道病毒均可引起手足口病,其中以肠道病毒71型(EV71)为我国近几年手足口病的主要病原体。EV71导致的手足口病较常引起中枢神经系统损伤,重症患者比例以及病死率均明显高于其他肠道病毒,具有很重要的公共卫生意义。目前尚未有成熟的EV71快速诊断试剂和预防疫苗,开发相关诊断试剂与疫苗对于手足口病的防治具有重要意义。本研究目的是建立肠道病毒的分离鉴定技术平台,获得较为完整的EV71病毒库;建立新型的EV71中和抗体快速高通量检测方法:建立EV71动物感染模型。本研究结果将为EV71的防治研究提供重要的基础和工具。本研究通过病毒分离鉴定建立了一个较为完整的肠道病毒库,并且初步建立了EV71病毒的大规模生产,浓缩和纯化的工艺流程,已获得了具有较高滴度和纯度的EV71病毒颗粒。本研究建立了一系列EV71鉴定的方法,包括EV71滴度检测方法,免疫荧光方法,EV71中和抗体检测方法等。传统的TCID_50方法是目前常用的EV71中和抗体的检测方法,该方法的实验周期长,工作量大,肉眼判断结果存在主观误差,难以满足大量标本的检测要求。鉴于传统TCID_50方法所存在的问题,我们建立了快速、高通量的新型EV71中和抗体检测方法。本方法是应用酶标记的EV71单克隆抗体与EV71病毒蛋白结合,通过TMB显色使得被EV71感染的细胞显示出区别颜色标记,应用斑点自动扫描仪进行扫描和计数。本研究对该方法的敏感性,特异性,交叉反应性和重复性进行了考察,结果表明该方法具有较好的性能。同时我们应用该方法检测了大量的单抗和多抗的EV71中和抗体滴度,结果显示了该方法具有较好的应用前景。本论文也初步研究EV71的乳鼠感染模型,已经通过颅腔注射的方式将EV71病毒接种到乳鼠体内,并观察到乳鼠出现体重下降,颤抖,瘫痪甚至死亡等现象,在乳鼠的脑组织中检测到EV71病毒,为后期的疫苗研发等工作提供了基础。
[Abstract]:Hand, foot and mouth disease is a common disease in children. A variety of enterovirus can cause hand, foot and mouth disease. Enterovirus 71 (EV71) is the main pathogen of hand-foot-mouth disease in China in recent years. The proportion of severe patients and mortality are significantly higher than other enterovirus, which has important public health significance. There is no mature rapid EV71 diagnostic reagent and prophylactic vaccine. It is of great significance to develop diagnostic reagents and vaccines for the prevention and treatment of HFMD. The purpose of this study was to establish a platform for isolation and identification of enterovirus and to obtain a complete EV71 virus library. To establish a new rapid high-throughput detection method for EV71 neutralizing antibody: to establish an animal model of EV71 infection. The results of this study will provide an important foundation and tool for the prevention and treatment of EV71. In this study, a complete enterovirus library was established by virus isolation and identification, and the process of large-scale production, concentration and purification of EV71 virus was preliminarily established. EV71 virus particles with high titer and purity were obtained. In this study, a series of EV71 identification methods were established, including EV71 titer detection method, immunofluorescence method. EV71 neutralizing antibody detection method. The traditional TCID_50 method is the commonly used detection method of EV71 neutralizing antibody. This method has a long experimental period and a large workload. There is subjective error in the result of naked eye judgment and it is difficult to meet the detection requirements of a large number of specimens. In view of the problems existing in the traditional TCID_50 method, we have established a fast method. High throughput new EV71 neutralizing antibody detection method. This method is using enzyme labeled EV71 monoclonal antibody to bind to EV71 virus protein. By TMB, the cells infected with EV71 showed different color markers, and were scanned and counted by dot scanner. This study was sensitive and specific to this method. The cross-reactivity and repeatability were investigated and the results showed that the method had good performance. At the same time, we used the method to detect a large number of McAb and multi-antibody EV71 neutralizing antibody titers. The results show that this method has a good application prospect. This paper also preliminarily studies the infection model of EV71 in neonatal mice. EV71 virus has been inoculated into the body by cranial injection, and the body weight loss and tremor were observed in the newborn mice. The detection of EV71 virus in the brain tissue of the newborn rat provides the basis for the later research and development of vaccine.
【学位授予单位】：厦门大学
【学位级别】：硕士
【学位授予年份】：2009
【分类号】：R725.1;R373

【引证文献】