马Ⅰ型RNA聚合酶启动子在流感研究中的应用及马IFITM蛋白抗流感病毒的研究

发布时间：2018-08-25 19:04

【摘要】：流感病毒反向遗传系统能够在体外对流感病毒的基因组进行操作,可以极大的方便对流感病毒的研究。流感病毒最小的复制单位由核糖核蛋白复合物(ribonucleoprotein complex,RNP)组成。在流感病毒反向系统中,PB1、PB2、PA、NP蛋白可以识别由Ⅰ型RNA聚合酶启动子驱动生成的负链病毒RNA,并以此为模板转录生成正链互补RNA及m RNA。真核生物Ⅰ型RNA聚合酶启动子具有种属特异性。一种生物的Ⅰ型RNA聚合酶不能识别另一种生物的Ⅰ型RNA聚合酶启动子。研究者已经克隆了人源、犬源、鸡源、鼠源Ⅰ型RNA聚合酶启动子,并将其用于流感病毒反向遗传系统的构建。但是,目前为止,还没有马源Ⅰ型RNA聚合酶启动子的研究报道。因此,对于流感病毒的很多研究不能在马源细胞上进行。基于此,在本研究中,利用真核生物Ⅰ型RNA聚合酶启动子的位置特点,在Gen Bank数据库和ensembl数据库中的马基因组中,检索并确定了马Ⅰ型RNA聚合酶启动子转录起始位点。以马肺细胞基因组DNA为模板,通过PCR,扩增得到了马Ⅰ型RNA聚合酶启动子,并构建了不同长度的马Ⅰ型RNA聚合酶启动子驱动的马流感病毒小基因组复制系统。结果表明马Ⅰ型RNA聚合酶启动子为500bp时,可以发挥最大转录活性。用建立的马流感病毒小基因组复制系统,对比了两株马流感病毒聚合酶活性的差异,发现PA蛋白可能是决定两者聚合酶活性差异的关键蛋白。在本研究中,还使用该系统证实了马Mx A蛋白会影响流感病毒聚合酶活性。最后,利用马Ⅰ型RNA聚合酶启动子构建了马流感病毒反向遗传系统,成功拯救出一株野生型马流感病毒和一株重组型马流感病毒。马Ⅰ型RNA聚合酶启动子的成功克隆及应用,丰富了我们对真核生物Ⅰ型RNA聚合酶启动子的认识。利用马Ⅰ型RNA聚合酶启动子构建的流感病毒小基因组复制系统是在马源细胞上研究流感病毒聚合酶活性的有效工具,而在此基础上构建的马流感病毒反向系统将方便研究者在马源细胞上对流感病毒进行操作。最近的研究表明,人源、猪源、鸡源、鸭源、鼠源IFITM蛋白都具有抗流感病毒活性。马属动物是流感病毒的重要宿主,但对于马源IFITM蛋白是否也具有抗流感病毒作用却未见报道。在本研究中,通过在Gen Bank数据库中检索马IFITM基因,设计了靶向马IFITM基因的特异性引物。通过RT-PCR,扩增得到了6个马IFITM基因。首先,我们描述了马IFITM m RNA的表达特性:通过荧光定量PCR,检测了马源细胞及组织中的IFITM m RNA表达水平,发现不同IFITM的m RNA表达水平有所差异;经干扰素处理和流感病毒感染后,马源细胞中IFITM m RNA表达水平会上调;但是感染马疱疹病毒后,马源细胞中IFITM m RNA表达水平未发生变化。然后,为了明确马IFITM蛋白的细胞定位,构建了表达马IFITM蛋白的真核表达载体,间接免疫荧光和Western blot实验都表明马IFITM蛋白成功表达;激光共聚焦实验的结果表明不同马IFITM蛋白的细胞定位并不相同;其中,马IFITM3蛋白与LAMP1蛋白呈现共定位现象,而其它马IFITM蛋白不与LAMP1蛋白共定位。最后,通过瞬时转染外源表达质粒和构建稳定表达IFITM蛋白细胞系这两种策略,利于基于Ⅰ型RNA聚合酶启动子的双荧光素酶报告系统,检测了马IFITM蛋白的抗流感活性:通过慢病毒感染实验,建立了稳定表达马IFITM蛋白的MDCK细胞系;间接免疫荧光试验的结果表明,在MDCK细胞系中,马IFITM蛋白都成功表达;利用基于马或犬Ⅰ型RNA聚合酶启动子的双荧光素酶报告系统,证实在细胞中过表达或者稳定表达马IFITM蛋白都会限制流感病毒复制。对马源IFITM蛋白的抗流感病毒活性研究,将加深我们对IFITM蛋白抗流感病毒作用的理解,也为相应的抗流感药物的研发提供了理论基础。2012年以来,在马群中发现了三种新的黄病毒科病毒:马丙型肝炎病毒(equine hepatitis C virus,EHCV)、EPg V(equine pegivirus)、泰勒病相关病毒(Theiler’s disease-associated virus,TDAV)。目前,在世界范围内,关于这三种新发马病病毒的报道较少。在国内的马群中,也没有这三种病毒的流行病学报道。在2014年和2015年,我们从国内的马群中采集了177份血清样品。通过PCR,在血清样品中,检测到了6份EHCV阳性样品和2份EPg V阳性样品。在检测过程中,没有发现这两种病毒共感染的证据。在所有样品中没有检测到TDAV核酸。对EHCV的NS5B基因、NS3基因、5’非转录区序列以及EPg V的NS3基因序列进行进化树分析。结果表明,EHCV和EPg V已经进化为两个分支,而且这两支病毒都在国内马群中流行。本研究丰富了我们对这三种新发黄病毒科马病病毒的流行地区以及基因进化的认识。
[Abstract]:Influenza virus reverse genetic system can manipulate the genome of influenza virus in vitro, so it is very convenient to study influenza virus. The smallest replication unit of influenza virus is composed of ribonucleoprotein complex (RNP). In influenza virus reverse system, PB1, PB2, PA, NP proteins can be recognized by I. Eukaryotic type I RNA polymerase promoters are species specific. One organism's type I RNA polymerase does not recognize another organism's type I RNA polymerase promoter. Chicken-derived, mouse-derived RNA polymerase promoter and its application in the construction of reverse genetic system of influenza viruses have not been reported so far. Therefore, many studies on influenza viruses can not be carried out on horse-derived cells. Location characteristics of polymerase promoter were studied. The transcription initiation sites of horse type I RNA polymerase promoter were searched and identified in the horse genome of Gen Bank and Ensembl databases. The horse type I RNA polymerase promoter was amplified by PCR using the genomic DNA of horse lung cells as template, and the horse type I RNA polymerase promoter with different lengths was constructed. The results showed that the promoter of equine influenza virus type I RNA polymerase could maximize the transcriptional activity when it was 500 bp. The difference of polymerase activity between the two equine influenza viruses was compared with the established system of equine influenza virus small genome replication. In this study, we also used this system to confirm that horse Mx A protein can affect the polymerase activity of influenza virus. Finally, a reverse genetic system of equine influenza virus was constructed using horse type I RNA polymerase promoter, and a wild equine influenza virus and a recombinant equine influenza virus were successfully rescued. The successful cloning and application of the promoter of NA polymerase enriched our understanding of the promoter of Eukaryotic type I RNA polymerase.The replication system of influenza virus small genome constructed by the promoter of horse type I RNA polymerase is an effective tool for studying the polymerase activity of influenza virus in horse-derived cells. The virus reverse system will facilitate researchers to operate on influenza virus on horse origin cells. Recent studies have shown that IFITM protein from human, pig, chicken, duck * and mouse sources has anti influenza activity. Equine is an important host of influenza virus, but it has not reported whether IFITM protein has anti influenza effect. In this study, six horse IFITM genes were amplified by RT-PCR. Firstly, we described the expression characteristics of horse IFITM RNA: IFITM RNA expression levels in horse-derived cells and tissues were detected by fluorescence quantitative PCR. It was found that the expression level of IFITM m RNA was different in different IFITM cells; IFITM m RNA expression level was up-regulated in horse-derived cells after treatment with interferon and infection with influenza virus; however, the expression level of IFITM m RNA in horse-derived cells did not change after infection with equine herpes virus. Eukaryotic expression vectors, indirect immunofluorescence and Western blot assays showed that equine IFITM protein was successfully expressed; confocal laser scanning showed that different equine IFITM proteins had different cellular localization; among them, equine IFITM3 protein and LAMP1 protein were co-localized, while other equine IFITM proteins were not co-localized with LAMP1 protein. Then, two strategies, transient transfection of exogenous expression plasmids and construction of stable IFITM expression cell lines, were used to detect the anti-influenza activity of equine IFITM proteins by using a dual luciferase reporting system based on type I RNA polymerase promoter. The results of epidemic fluorescence assay showed that equine IFITM protein was successfully expressed in MDCK cell lines, and the double luciferase reporting system based on horse or dog type I RNA polymerase promoter was used to confirm that overexpression or stable expression of equine IFITM protein in cells would restrict influenza virus replication. Since 2012, three new Flaviviridae viruses have been found in horses: equine hepatitis C virus (EHCV), EPg virus (equine pegivirus), and Theiler disease-related virus (Theiler). At present, there are few reports about these three new equine disease s viruses in the world. There is no epidemiological report of these three viruses in domestic horses. In 2014 and 2015, 177 serum samples were collected from domestic horses. TDAV nucleic acid was not detected in all samples. Evolutionary tree analysis was performed on the NS5B gene, NS3 gene, 5'non-transcriptional region sequence and NS3 gene sequence of EHCV. This study enriches our understanding of the epidemic areas and genetic evolution of the three new flavivirus equine diseases viruses.
【学位授予单位】：华南农业大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：S852.65

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