本文關(guān)鍵詞：腸道病毒分離鑒定及EV71快速高通量中和實(shí)驗(yàn)方法的建立　出處：《廈門大學(xué)》2009年碩士論文　論文類型：學(xué)位論文

  更多相關(guān)文章： 人腸道病毒71型 中和實(shí)驗(yàn) 酶聯(lián)免疫斑點(diǎn)法 動(dòng)物模型

【摘要】： 手足口病是兒童常見病,多種腸道病毒均可引起手足口病,其中以腸道病毒71型(EV71)為我國(guó)近幾年手足口病的主要病原體。EV71導(dǎo)致的手足口病較常引起中樞神經(jīng)系統(tǒng)損傷,重癥患者比例以及病死率均明顯高于其他腸道病毒,具有很重要的公共衛(wèi)生意義。目前尚未有成熟的EV71快速診斷試劑和預(yù)防疫苗,開發(fā)相關(guān)診斷試劑與疫苗對(duì)于手足口病的防治具有重要意義。本研究目的是建立腸道病毒的分離鑒定技術(shù)平臺(tái),獲得較為完整的EV71病毒庫;建立新型的EV71中和抗體快速高通量檢測(cè)方法:建立EV71動(dòng)物感染模型。本研究結(jié)果將為EV71的防治研究提供重要的基礎(chǔ)和工具。 本研究通過病毒分離鑒定建立了一個(gè)較為完整的腸道病毒庫,并且初步建立了EV71病毒的大規(guī)模生產(chǎn),濃縮和純化的工藝流程,已獲得了具有較高滴度和純度的EV71病毒顆粒。 本研究建立了一系列EV71鑒定的方法,包括EV71滴度檢測(cè)方法,免疫熒光方法,EV71中和抗體檢測(cè)方法等。傳統(tǒng)的TCID_50方法是目前常用的EV71中和抗體的檢測(cè)方法,該方法的實(shí)驗(yàn)周期長(zhǎng),工作量大,肉眼判斷結(jié)果存在主觀誤差,難以滿足大量標(biāo)本的檢測(cè)要求。鑒于傳統(tǒng)TCID_50方法所存在的問題,我們建立了快速、高通量的新型EV71中和抗體檢測(cè)方法。本方法是應(yīng)用酶標(biāo)記的EV71單克隆抗體與EV71病毒蛋白結(jié)合,通過TMB顯色使得被EV71感染的細(xì)胞顯示出區(qū)別顏色標(biāo)記,應(yīng)用斑點(diǎn)自動(dòng)掃描儀進(jìn)行掃描和計(jì)數(shù)。本研究對(duì)該方法的敏感性,特異性,交叉反應(yīng)性和重復(fù)性進(jìn)行了考察,結(jié)果表明該方法具有較好的性能。同時(shí)我們應(yīng)用該方法檢測(cè)了大量的單抗和多抗的EV71中和抗體滴度,結(jié)果顯示了該方法具有較好的應(yīng)用前景。 本論文也初步研究EV71的乳鼠感染模型,已經(jīng)通過顱腔注射的方式將EV71病毒接種到乳鼠體內(nèi),并觀察到乳鼠出現(xiàn)體重下降,顫抖,癱瘓甚至死亡等現(xiàn)象,在乳鼠的腦組織中檢測(cè)到EV71病毒,為后期的疫苗研發(fā)等工作提供了基礎(chǔ)。
[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.
【學(xué)位授予單位】：廈門大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2009
【分類號(hào)】：R725.1;R373

【引證文獻(xiàn)】

相關(guān)期刊論文 前1條

1 董彩文;張勝楠;王云龍;李玉林;孫新城;王國(guó)強(qiáng);李恒思;;腸道病毒71型的診斷方法及疫苗研究述評(píng)[J];鄭州輕工業(yè)學(xué)院學(xué)報(bào)(自然科學(xué)版);2012年05期

相關(guān)碩士學(xué)位論文 前1條

1 于賀娟;腸道病毒71型熒光RT-PCR檢測(cè)方法的建立及評(píng)價(jià)[D];鄭州大學(xué);2010年

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本文編號(hào)：1411849