【作者】 窦敏；

【导师】 沈明山；

【作者基本信息】 厦门大学 ， 细胞生物学， 2007， 硕士

【摘要】 Armored RNA技术是近几年发展起来的一种新的RNA质控品的制备技术。该技术的原理就是利用基因工程方法将包含有大肠杆菌MS2噬菌体的外壳蛋白基因的序列及外源片段克隆到表达载体中,这一载体能够将外源克隆片段转录成重组RNA,并利用载体上MS2的外壳蛋白基因合成的外壳蛋白将其装配成球状RNA病毒结构的RNA-蛋白质复合体,我们称之为RNA病毒样颗粒。目前Armored RNA技术正逐渐应用于RNA病毒的检测方面。首先,根据Armored RNA技术的原理,构建了能表达耐核糖核酸酶病毒样颗粒的载体,选取口蹄疫病毒IRES RNA片断为内标检测基因,克隆到表达载体噬菌体基因的下游,最终得到能够表达病毒样颗粒的原核表达载体,转化大肠杆菌进行诱导表达。通过对表达产物的双酶消化实验进行初步确认。提取表达产物的RNA进行反转录PCR,可以确定表达产物是含口蹄疫病毒IRES RNA的病毒样颗粒。蔗糖密度梯度离心纯化表达产物,透射电镜观察其形态。一系列的实验结果表明,本研究制备得到的病毒样颗粒为含口蹄疫病毒IRES RNA,直径约为26nm的圆形颗粒。该病毒样颗粒具有耐核糖核酸酶的特性,稳定性良好,易保存。其次,通过设计荧光双链探针对病毒样颗粒RNA进行一步法反转录实时荧光PCR检测。我们以IRES为检测靶序列,系统优化了检测病毒样颗粒RNA的反应体系和反应条件。结果表明,在最优的检测条件下,荧光双链探针可以在2h内通过一步法反转录荧光PCR检测出病毒样颗粒外壳蛋白包裹的IRES RNA。最后本研究通过优化反应体系,以病毒样颗粒为内标,对禽流感病毒进行实时荧光PCR检测。实验结果说明,实时荧光PCR可以迅速准确的检测禽流感病毒,同时由于内标的存在,也避免了在整个病毒检测过程中抑制因子的存在或者人为误差等因素造成的假阴性结果。本研究制备的含有口蹄疫病毒IRES RNA的病毒样颗粒,通过对其进行的一系列鉴定实验和应用于禽流感病毒检测的实验,可以看出该病毒样颗粒具有与RNA病毒相似的结构,即都是蛋白外壳包裹核酸的蛋白-RNA复合体,所以可以把该病毒样颗粒与其它病毒一起经历核酸提取、反转录和PCR扩增检测,从而对RNA病毒的检测进行全程监控,避免了假阴性结果的出现。该病毒样颗粒具有耐核糖核酸酶的特性,并且稳定性好、易于储存、安全可靠。因此本研究所制备的病毒样颗粒为以后RNA病毒的检测提供了一个良好的平台。更多还原

【Abstract】 Armored RNA technology is a new technique of preparation for RNA quality control, which is developed in recent years. The principle is that E.coli bacteriophage MS2 coat protein gene and foreign fragment was cloned into the expression vector by genetic engineering methods, and the foreign fragment can be transcripted into recombinant RNA. The recombinant RNA is packaged by MS2 coat protein as RNA-protein complex. This spherical RNA-protein complex was named as virus-like particles. Now Armored RNA technology is gradually applied to the research of detection of RNA viruses.Firstly, according to the principle of Armored RNA technology, a vector that can express RNase-resistant virus-like particles was constructed. The Foot-and-mouth disease virus IRES RNA fragment was selected as the internal standard gene. The gene was cloned into the expression vector, then the prokaryotic expression vector expressing virus-like particles was obtained and was induced. The expression products was confirmed by the enzyme digestion assay. The virus-like particles RNA was extracted and was identified by RT-PCR. Then we confirmed that the expression products were virus-like particles containing foot-and-mouth disease virus IRES RNA. The expression products were purified by sucrose density gradient centrifugation and were observed by transmission electron microscope. A series of experimental results showed that the expression products were circular virus-like particles containing foot-and-mouth disease virus IRES RNA. The diameter of the virus-like particles is about 26nm. The prepared virus-like particles with good stability are easy to preserve because of the RNase - resistant characteristic.Secondly, the IRES gene was selected as the target, and we tested the virus-like particles RNA by double-stranded fluorescent probes in one-step reverse transcript real-time fluorescence PCR assay. Then we optimized the reaction systems and the reaction conditions. The results showed that double-stranded fluorescent probes could test the virus-like particles IRES RNA in the optimal one-step reverse transcript real-time fluorescence PCR assay. Finally, the prepared virus-like particles was selected as internal control, and avian influenza virus was tested through adjusting the reaction system in real-time fluorescence PCR assay. The experiment results showed that avian influenza virus could be quickly tested by real-time PCR. And because of the internal control, we can also avoid the false negative results caused by inhibitors and human error in the assay or other factors.The virus-like particles containing foot-and-mouth disease virus IRES RNA prepared in the study were tested by a series of experiments and applied to the detection assay of avian influenza virus. From these experiments, we can know that the structure of the virus-like particles is similar as RNA virus. Both of them are RNA-protein complexes with RNA wrapped by coat proteins. So we can put the virus-like particles together with other virus, then they experience RNA extraction, reverse transcription PCR amplification and detection in order to monitor the whole process and avoid false negative results. The virus-like particles with RNase - resistant characteristics have good stability. They are also secure and easy to store. Therefore, The virus-like particles prepared in the study provide a good platform for the detection of RNA viruses.更多还原