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  本文選題：腸道病毒71型 + 柯薩奇A16型��； 參考：《吉林大學》2012年碩士論文

【摘要】：目的為了避免傳統(tǒng)的EV71和CVA16病毒中和抗體檢測需要操作活病毒且通過觀察細胞病變（CPE）等缺點，本研究建立了基于假病毒系統(tǒng)的EV71和CVA16中和抗體檢測方法，具有操作安全、快速、有效、客觀、高通量等優(yōu)點。方法本研究構(gòu)建了高效表達病毒結(jié)構(gòu)蛋白P1的真核質(zhì)粒(VR1012-EGFP-2A-P1和pcDNA3.1-5’NTR-P1)，T7RNA聚合酶真核質(zhì)粒（pcDNA3.1-T7RNA Pol）及攜帶有熒光素酶基因嵌合的基因組質(zhì)粒(pT7-CVA16-luc和pT7-EV71-luc，熒光素酶基因luciferase替換病毒結(jié)構(gòu)蛋白編碼區(qū))共3種質(zhì)粒，并將這3種質(zhì)粒共同轉(zhuǎn)染了人胚腎細胞293T，形成了具有單輪感染復制能力的EV71或CVA16假病毒；該假病毒能夠感染Vero或RD細胞并在細胞中表達了報告基因luciferase。將假病毒與病毒感染的病人血清孵育后感染靶細胞，，根據(jù)Luciferase表達量確定了血清中和抗體的強弱。用該技術(shù)對不同免疫血清進行了中和抗體分析，并比較了基于假病毒系統(tǒng)和基于活病毒系統(tǒng)的中和試驗效果。結(jié)果成功包裝了能夠感染Vero或RD細胞并帶有熒光素酶基因的EV71和CVA16假病毒，且熒光素酶基因可在靶細胞中表達；通過與傳統(tǒng)方法比較，基于假病毒和活病毒兩個中和試驗系統(tǒng)獲得的中和抗體滴度趨勢一致。結(jié)論本研究建立了新的EV71和CVA16中和抗體檢測技術(shù)，此技術(shù)可進一步應用于其它小RNA病毒科的中和抗體檢測，如，HAV等；同時為確定不同基因型EV71和CVA16間是否有交叉免疫保護提供了可能。
[Abstract]:Objective to avoid the disadvantages of the traditional neutralizing antibody detection of EV71 and CVA16 virus which requires the operation of live virus and observe the cytopathic effect of CPE), a method of neutralizing antibody detection of EV71 and CVA16 based on pseudovirus system was established in this study, which is safe and fast in operation. Effective, objective, high throughput and other advantages. Methods in this study, the eukaryotic plasmids VR1012-EGFP-2A-P1 and pcDNA3.1-5 NTR-P1T7RNA polymerase were constructed, and the genomic plasmids pT7-CVA16-luc and pT7-EV71-lucone carrying luciferase gene chimeric were constructed. The luciferase gene luciferase was used to replace the virus. There are three kinds of plasmids, These three plasmids were co-transfected into human embryonic kidney cells 293T to form EV71 or CVA16 pseudoviruses which could infect Vero or Rd cells and express the reporter gene luciferase. After the pseudovirus was incubated with the infected patient's serum, the target cells were infected, and the level of neutralizing antibody was determined according to the expression of Luciferase. Neutralization antibodies of different immune sera were analyzed by this technique, and the neutralization test results based on pseudovirus system and live virus system were compared. Results EV71 and CVA16 pseudoviruses, which could infect Vero or Rd cells and contain luciferase gene, were successfully packaged, and luciferase gene could be expressed in target cells. The neutralizing antibody titers obtained from two neutralization test systems, pseudovirus and live virus, showed the same trend. Conclusion A new neutralizing antibody detection technique for EV71 and CVA16 has been established in this study, which can be further applied to the detection of neutralizing antibodies in other small RNA virology, such as HAV. At the same time, it is possible to determine whether different genotypes EV71 and CVA16 have cross immune protection.
【學位授予單位】：吉林大學
【學位級別】：碩士
【學位授予年份】：2012
【分類號】：R373.2

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本文編號：1999747