gga-miR-155抑制传染性法氏囊病病毒复制的分子机理
发布时间：2019-05-08 12:04
【摘要】：传染性法氏囊病(Infectiou Bursal Disease, IBD)由传染性法氏囊病病毒(Infectious Bursal Disease Virus, IBDV)引起,主要侵害雏鸡淋巴组织,特别是法氏囊。该病不仅导致患病动物死亡,而且还可导致机体免疫抑制。microRNA (miRNA)是一种非编码小分子RNA,长度约21-25nt,能在转录后降解靶基因mRNA或抑制基因的翻译,在转录后和翻译水平上对生理和病理过程起重要的调控作用。gga-miRNA-155在传染性法氏囊病毒(IBDV)感染鸡胚成纤维细胞CEF和SPF鸡的病料被检测到表达差异显著。本文运用双荧光素酶报告系统、Western Blo、qRT-PCR等分子生物学技术以及生物信息学方法,探究gga-miR-155对IBDV的复制的影响,并探究其作用的分子机理。研究包括两个部分：1、gga-miR-155对IBDV复制和细胞IFN表达的影响首先合成gga-miR-155的模拟物gga-miR-155 mimics,转染DF-1细胞后接种IBDV,分别在24h、48h收集细胞培养上清液,测定IBDV的病毒滴度,结果显示,gga-miR-155 mimics能够显著抑制IBDV的复制。为进一步探究其抑制IBDV复制的分子机理,对病毒感染后的I型干扰素(IFN)检测分析,发现Ⅰ型IFN的表达上调。由此表明gga-miR-155抑制IBDV复制的分子机理是通过增强细胞天然免疫功能而发生的。2、gga-miR-155抑制IBDV复制的分子机理生物信息学方法预测gga-miR-155的靶点,并在miRDB中gga-miR-155的众多靶基因中选取SOCS1基因。SOCS1是一种信号转导过程中的负性调节因子,对机体固有免疫以及适应性免疫起到重要的调控作用,可能与miR-155对IBDV的抑制作用有关,分别在mRNA水平和蛋白水平进行验证。将SOCS1 mRNA3'UTR克隆至荧光素酶报告载体,双荧光素酶报告系统中实验组荧光素酶活性降低。qRT-PCR分析gga-miR-155 mimics转染的DF-1细胞中SOCS1 mRNA水平比对照组显著降低。将SOCS1 mRNA编码区及其3’UTR克隆至pEGFP-N1-Flag标签载体中,构建Flag与SOCS1共表达真核质粒,将其与gga-miR-155 mimics共转染DF1细胞并接毒IBDV, Western Blot检测结果表明Flag蛋白表达下调,也即SOCS1蛋白表达被抑制。以上结果表明,gga-miR-155抑制IBDV复制的分子机理是通过靶向调控SOCS1基因转录后表达从而增强细胞天然免疫功能而发生的。本实验的研究结果可以为IBDV防控发掘新型抗病毒药物与免疫分子靶标探索了新的技术路径。
[Abstract]:Infectious bursal disease (Infectiou Bursal Disease, IBD) is caused by infectious bursal disease virus (Infectious Bursal Disease Virus, IBDV), which mainly infects the lymphoid tissue of chicks, especially the bursa of Fabricius. MicroRNA (miRNA) is a non-coding small molecule RNA, with a length of about 21 脳 25 NT, which can degrade the target gene mRNA or the translation of the inhibitory gene after transcription, and it can not only lead to the death of diseased animals, but also lead to immunosuppression. GCA-miRNA-155 plays an important role in the regulation of physiological and pathological processes at post-transcriptional and translational levels. Significant differences were detected in the expression of GGA-SPF in chicken embryo fibroblast CEF and SPF chickens infected with infectious bursal virus (IBDV). In this paper, the effects of gga-miR-155 on the replication of IBDV and the molecular mechanism of IBDV replication were investigated by using double luciferase reporting system, Western Blo,qRT-PCR and other molecular biological techniques and bioinformatics methods. The study consists of two parts: 1. The effects of Gaga mir _ (155) on IBDV replication and IFN expression in DF-1 cells were first transfected with gga-miR-155 mimics, an analogue of gga-miR-155 synthesis, and then inoculated with IBDV, for 24 h, and the supernatant of cell culture was collected 48 h later. The viral titers of IBDV were determined and the results showed that gga-miR-155 mimics could significantly inhibit the replication of IBDV. In order to explore the molecular mechanism of its inhibition of IBDV replication, the expression of type I IFN was up-regulated by detection and analysis of type I interferon (IFN) after virus infection. These results suggest that the molecular mechanism of gga-miR-155 inhibiting IBDV replication is through enhancing the innate immune function of cells. 2. The molecular mechanism of Gaga mir 155 inhibiting IBDV replication is to predict the target of gga-miR-155 by bioinformatics method. SOCS1 gene was selected from many target genes of gga-miR-155 in miRDB. SOCS1 is a negative regulatory factor in signal transduction process, which plays an important role in regulating innate immunity and adaptive immunity of the body. It may be related to the inhibitory effect of miR-155 on IBDV, which was verified at mRNA level and protein level respectively. SOCS1 mRNA3'UTR was cloned into luciferase reporter vector and the luciferase activity of the experimental group was decreased in the double luciferase reporting system. The level of SOCS1 mRNA in the DF-1 cells transfected with gga-miR-155 mimics was significantly lower than that in the control group by qRT-PCR analysis. The coding region of SOCS1 mRNA and its 3'UTR were cloned into pEGFP-N1-Flag tag vector to construct eukaryotic plasmid co-expressed by Flag and SOCS1, and co-transfected with gga-miR-155 mimics into DF1 cells. The results of IBDV, Western Blot assay showed that the expression of Flag protein was down-regulated. That is, the expression of SOCS1 protein was inhibited. These results suggest that the molecular mechanism of gga-miR-155 inhibiting IBDV replication is through targeted regulation of the post-transcriptional expression of SOCS1 gene to enhance the innate immune function of cells. The results of this study can be used to explore new antiviral drugs and immune molecular targets for IBDV prevention and control.
【学位授予单位】：南京师范大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S852.65
本文编号：2471897
[Abstract]:Infectious bursal disease (Infectiou Bursal Disease, IBD) is caused by infectious bursal disease virus (Infectious Bursal Disease Virus, IBDV), which mainly infects the lymphoid tissue of chicks, especially the bursa of Fabricius. MicroRNA (miRNA) is a non-coding small molecule RNA, with a length of about 21 脳 25 NT, which can degrade the target gene mRNA or the translation of the inhibitory gene after transcription, and it can not only lead to the death of diseased animals, but also lead to immunosuppression. GCA-miRNA-155 plays an important role in the regulation of physiological and pathological processes at post-transcriptional and translational levels. Significant differences were detected in the expression of GGA-SPF in chicken embryo fibroblast CEF and SPF chickens infected with infectious bursal virus (IBDV). In this paper, the effects of gga-miR-155 on the replication of IBDV and the molecular mechanism of IBDV replication were investigated by using double luciferase reporting system, Western Blo,qRT-PCR and other molecular biological techniques and bioinformatics methods. The study consists of two parts: 1. The effects of Gaga mir _ (155) on IBDV replication and IFN expression in DF-1 cells were first transfected with gga-miR-155 mimics, an analogue of gga-miR-155 synthesis, and then inoculated with IBDV, for 24 h, and the supernatant of cell culture was collected 48 h later. The viral titers of IBDV were determined and the results showed that gga-miR-155 mimics could significantly inhibit the replication of IBDV. In order to explore the molecular mechanism of its inhibition of IBDV replication, the expression of type I IFN was up-regulated by detection and analysis of type I interferon (IFN) after virus infection. These results suggest that the molecular mechanism of gga-miR-155 inhibiting IBDV replication is through enhancing the innate immune function of cells. 2. The molecular mechanism of Gaga mir 155 inhibiting IBDV replication is to predict the target of gga-miR-155 by bioinformatics method. SOCS1 gene was selected from many target genes of gga-miR-155 in miRDB. SOCS1 is a negative regulatory factor in signal transduction process, which plays an important role in regulating innate immunity and adaptive immunity of the body. It may be related to the inhibitory effect of miR-155 on IBDV, which was verified at mRNA level and protein level respectively. SOCS1 mRNA3'UTR was cloned into luciferase reporter vector and the luciferase activity of the experimental group was decreased in the double luciferase reporting system. The level of SOCS1 mRNA in the DF-1 cells transfected with gga-miR-155 mimics was significantly lower than that in the control group by qRT-PCR analysis. The coding region of SOCS1 mRNA and its 3'UTR were cloned into pEGFP-N1-Flag tag vector to construct eukaryotic plasmid co-expressed by Flag and SOCS1, and co-transfected with gga-miR-155 mimics into DF1 cells. The results of IBDV, Western Blot assay showed that the expression of Flag protein was down-regulated. That is, the expression of SOCS1 protein was inhibited. These results suggest that the molecular mechanism of gga-miR-155 inhibiting IBDV replication is through targeted regulation of the post-transcriptional expression of SOCS1 gene to enhance the innate immune function of cells. The results of this study can be used to explore new antiviral drugs and immune molecular targets for IBDV prevention and control.
【学位授予单位】：南京师范大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S852.65
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1 杜希宁;gga-miR-155抑制传染性法氏囊病病毒复制的分子机理[D];南京师范大学;2015年
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