一种新的肠道病毒71型和柯萨奇A16型中和抗体检测技术

发布时间：2018-06-09 12:35

本文选题：肠道病毒71型 + 柯萨奇A16型　；参考：《吉林大学》2012年硕士论文

【摘要】：目的为了避免传统的EV71和CVA16病毒中和抗体检测需要操作活病毒且通过观察细胞病变（CPE）等缺点，本研究建立了基于假病毒系统的EV71和CVA16中和抗体检测方法，具有操作安全、快速、有效、客观、高通量等优点。方法本研究构建了高效表达病毒结构蛋白P1的真核质粒(VR1012-EGFP-2A-P1和pcDNA3.1-5’NTR-P1)，T7RNA聚合酶真核质粒（pcDNA3.1-T7RNA Pol）及携带有荧光素酶基因嵌合的基因组质粒(pT7-CVA16-luc和pT7-EV71-luc，荧光素酶基因luciferase替换病毒结构蛋白编码区)共3种质粒，并将这3种质粒共同转染了人胚肾细胞293T，形成了具有单轮感染复制能力的EV71或CVA16假病毒；该假病毒能够感染Vero或RD细胞并在细胞中表达了报告基因luciferase。将假病毒与病毒感染的病人血清孵育后感染靶细胞，，根据Luciferase表达量确定了血清中和抗体的强弱。用该技术对不同免疫血清进行了中和抗体分析，并比较了基于假病毒系统和基于活病毒系统的中和试验效果。结果成功包装了能够感染Vero或RD细胞并带有荧光素酶基因的EV71和CVA16假病毒，且荧光素酶基因可在靶细胞中表达；通过与传统方法比较，基于假病毒和活病毒两个中和试验系统获得的中和抗体滴度趋势一致。结论本研究建立了新的EV71和CVA16中和抗体检测技术，此技术可进一步应用于其它小RNA病毒科的中和抗体检测，如，HAV等；同时为确定不同基因型EV71和CVA16间是否有交叉免疫保护提供了可能。
[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.
【学位授予单位】：吉林大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R373.2

【参考文献】