【摘要】：傳染性法氏囊病(Infectiou Bursal Disease, IBD)由傳染性法氏囊病病毒(Infectious Bursal Disease Virus, IBDV)引起,主要侵害雛雞淋巴組織,特別是法氏囊。該病不僅導致患病動物死亡,而且還可導致機體免疫抑制。microRNA (miRNA)是一種非編碼小分子RNA,長度約21-25nt,能在轉錄后降解靶基因mRNA或抑制基因的翻譯,在轉錄后和翻譯水平上對生理和病理過程起重要的調(diào)控作用。gga-miRNA-155在傳染性法氏囊病毒(IBDV)感染雞胚成纖維細胞CEF和SPF雞的病料被檢測到表達差異顯著。本文運用雙熒光素酶報告系統(tǒng)、Western Blo、qRT-PCR等分子生物學技術以及生物信息學方法,探究gga-miR-155對IBDV的復制的影響,并探究其作用的分子機理。研究包括兩個部分：1、gga-miR-155對IBDV復制和細胞IFN表達的影響首先合成gga-miR-155的模擬物gga-miR-155 mimics,轉染DF-1細胞后接種IBDV,分別在24h、48h收集細胞培養(yǎng)上清液,測定IBDV的病毒滴度,結果顯示,gga-miR-155 mimics能夠顯著抑制IBDV的復制。為進一步探究其抑制IBDV復制的分子機理,對病毒感染后的I型干擾素(IFN)檢測分析,發(fā)現(xiàn)Ⅰ型IFN的表達上調(diào)。由此表明gga-miR-155抑制IBDV復制的分子機理是通過增強細胞天然免疫功能而發(fā)生的。2、gga-miR-155抑制IBDV復制的分子機理生物信息學方法預測gga-miR-155的靶點,并在miRDB中gga-miR-155的眾多靶基因中選取SOCS1基因。SOCS1是一種信號轉導過程中的負性調(diào)節(jié)因子,對機體固有免疫以及適應性免疫起到重要的調(diào)控作用,可能與miR-155對IBDV的抑制作用有關,分別在mRNA水平和蛋白水平進行驗證。將SOCS1 mRNA3'UTR克隆至熒光素酶報告載體,雙熒光素酶報告系統(tǒng)中實驗組熒光素酶活性降低。qRT-PCR分析gga-miR-155 mimics轉染的DF-1細胞中SOCS1 mRNA水平比對照組顯著降低。將SOCS1 mRNA編碼區(qū)及其3’UTR克隆至pEGFP-N1-Flag標簽載體中,構建Flag與SOCS1共表達真核質(zhì)粒,將其與gga-miR-155 mimics共轉染DF1細胞并接毒IBDV, Western Blot檢測結果表明Flag蛋白表達下調(diào),也即SOCS1蛋白表達被抑制。以上結果表明,gga-miR-155抑制IBDV復制的分子機理是通過靶向調(diào)控SOCS1基因轉錄后表達從而增強細胞天然免疫功能而發(fā)生的。本實驗的研究結果可以為IBDV防控發(fā)掘新型抗病毒藥物與免疫分子靶標探索了新的技術路徑。
[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.
【學位授予單位】：南京師范大學
【學位級別】：碩士
【學位授予年份】：2015
【分類號】：S852.65

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1 杜希寧;gga-miR-155抑制傳染性法氏囊病病毒復制的分子機理[D];南京師范大學;2015年

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本文編號：2471897