本文選題：豬IRF1 + 組織分布��； 參考：《河南農(nóng)業(yè)大學(xué)》2015年碩士論文

【摘要】：干擾素調(diào)節(jié)因子(IFN regulatory factors,IRFs)是一類重要的轉(zhuǎn)錄因子,參與機(jī)體免疫應(yīng)答、炎癥反應(yīng)、腫瘤發(fā)生以及細(xì)胞凋亡等多種生物學(xué)過程。其中,IRF3和IRF7在干擾素的表達(dá)調(diào)控中起重要作用,參與機(jī)體抗病毒免疫應(yīng)答。近年來(lái)研究發(fā)現(xiàn)除IRF3和IRF7之外,IRF1在抗病毒免疫應(yīng)答中也發(fā)揮著重要作用。IRF1可能是IRF3和IRF7之外的另一種調(diào)節(jié)抗病毒免疫的轉(zhuǎn)錄因子,而且當(dāng)IRF3/7或IFNs的功能被抑制時(shí),IRF1可能為調(diào)節(jié)干擾素(IFNs)和干擾素激活基因(ISGs)的表達(dá)提供了備選機(jī)制。對(duì)于控制那些能夠拮抗IRF3和IRF7以及IFNs表達(dá)的病毒而言,IRF1介導(dǎo)的抗病毒應(yīng)答可能發(fā)揮著極其重要的作用。目前,豬IRF1的研究相對(duì)較少,其具有怎樣的功能特點(diǎn),特別是在豬抗病毒先天免疫過程中的作用如何尚不清楚。因此,本文開展了以下研究,以期更多了解豬IRF1在機(jī)體先天性抗病毒免疫機(jī)制中的作用。1.豬IRF1基因的體內(nèi)表達(dá)分析及病毒感染對(duì)其表達(dá)的影響為研究豬IRF1基因的組織分布,采集健康成年豬的心臟、肝臟、脾臟、肺臟、腎臟、胃、腦等組織,通過熒光定量PCR檢測(cè)各組織中IRF1基因的表達(dá)量。結(jié)果表明IRF1在所有檢測(cè)組織中均有表達(dá),其中脾臟中表達(dá)量最高,其次是淋巴結(jié)和肝臟,而在心臟、腦和膀胱等其他組織中表達(dá)量較低。IRF1在脾臟和淋巴結(jié)等免疫相關(guān)組織中表達(dá)量較高,提示其可能參與機(jī)體的免疫應(yīng)答。為進(jìn)一步研究病毒感染狀態(tài)下IRF1基因表達(dá)的變化,分別選取兩種DNA病毒(豬偽狂犬病毒,PRV;豬細(xì)小病毒,PPV)和兩種RNA病毒(豬傳染性胃腸炎病毒,TGEV;豬水泡性口炎病毒,VSV)為代表,分別檢測(cè)了PK-15細(xì)胞在不同病毒感染后IRF1的表達(dá)情況。結(jié)果表明除PRV流行毒株(QXX株)外,其他參試病毒感染均可上調(diào)IRF1基因的表達(dá),表明豬IRF1參與了機(jī)體對(duì)病毒感染的應(yīng)答反應(yīng)。2.豬IRF1對(duì)干擾素表達(dá)和病毒復(fù)制的影響為研究豬IRF1對(duì)干擾素表達(dá)的影響,從豬外周血淋巴細(xì)胞中擴(kuò)增豬IRF1基因,定向克隆到PCAGGS-HA載體中,構(gòu)建真核表達(dá)質(zhì)粒PCAGGS-HA-pIRF1,轉(zhuǎn)染PK-15細(xì)胞,過表達(dá)豬IRF1基因。利用雙熒光素酶報(bào)告系統(tǒng)檢測(cè)無(wú)刺激狀態(tài)和poly(I:C)刺激下豬IFN-β的啟動(dòng)子活性,并通過熒光定量PCR檢測(cè)IFN-β的轉(zhuǎn)錄水平。結(jié)果表明過表達(dá)豬IRF1在無(wú)刺激的狀態(tài)下即可激活豬IFN-β的啟動(dòng)子,并上調(diào)IFN-β的轉(zhuǎn)錄水平。在poly(I:C)誘導(dǎo)下,過表達(dá)IRF1能夠更為顯著的激活I(lǐng)FN-β的啟動(dòng)子,而且這種激活能力比豬IRF3和豬IRF7的激活能力更強(qiáng)。然而,當(dāng)通過RNA干擾技術(shù)敲減PK-15細(xì)胞中IRF1的表達(dá)后,在poly(I:C)誘導(dǎo)下,IFN-β的表達(dá)水平并無(wú)顯著變化,說明豬IRF1能促進(jìn)干擾素的表達(dá),但不是誘導(dǎo)干擾素表達(dá)的必要因子。為探索豬IRF1對(duì)病毒復(fù)制的影響,將PCAGGS-HA-pIRF1轉(zhuǎn)染PK-15細(xì)胞,分別檢測(cè)過表達(dá)IRF1對(duì)PRV和TGEV復(fù)制的影響,結(jié)果顯示過表達(dá)IRF1可顯著抑制PRV和TGEV的復(fù)制,其中PRV的病毒滴度降低了近10倍,TGEV的病毒滴度降低了20倍以上,表明豬IRF1能夠促進(jìn)體外培養(yǎng)細(xì)胞的抗病毒感染應(yīng)答反應(yīng),具有較強(qiáng)的抗病毒活性。3.穩(wěn)定表達(dá)豬IRF1基因PK-15細(xì)胞系的建立為構(gòu)建穩(wěn)定表達(dá)豬IRF1基因的PK-15細(xì)胞系,利用PiggyBac真核轉(zhuǎn)座子系統(tǒng)將豬源IRF1基因轉(zhuǎn)染入PK-15細(xì)胞中,通過嘌呤霉素和綠色熒光進(jìn)行雙重篩選獲得陽(yáng)性轉(zhuǎn)化細(xì)胞,再經(jīng)克隆純化得到單個(gè)陽(yáng)性細(xì)胞克隆株。通過觀察熒光和熒光定量PCR鑒定,該細(xì)胞系連續(xù)傳代20代后其IRF1表達(dá)量未出現(xiàn)衰減,表明穩(wěn)定表達(dá)IRF1基因的PK-15細(xì)胞系構(gòu)建成功,為進(jìn)一步研究豬IRF1基因的功能以及作用機(jī)制提供了有效工具。
[Abstract]:IFN regulatory factors (IRFs) is an important class of transcription factors that participate in many biological processes, such as immune response, inflammatory response, tumorigenesis, and cell apoptosis. Among them, IRF3 and IRF7 play an important role in the regulation of interferon expression and participate in the immune response of the body. In recent years, the research has found that IRF has been found to be the exception of IRF. In addition to 3 and IRF7, IRF1 plays an important role in the antiviral immune response,.IRF1 may be another transcription factor that regulates antiviral immunity beyond IRF3 and IRF7, and when the function of IRF3/7 or IFNs is suppressed, IRF1 may provide a alternative mechanism for regulating the expression of interferon (IFNs) and interferon stimulated genes (ISGs). IRF1 mediated antiviral responses may play an extremely important role in making the viruses that can antagonize the expression of IRF3 and IRF7 and IFNs. At present, the study of porcine IRF1 is relatively less, and how its functional characteristics, especially in the process of swine virus innate immunity, are not clear. In order to know more about the role of pig IRF1 in the mechanism of innate antiviral immunity, the expression of.1. IRF1 gene in vivo and the influence of virus infection on the expression of swine IRF1 gene are studied in order to study the tissue distribution of porcine IRF1 gene and collect the heart, liver, spleen, lung, kidney, stomach and brain of healthy adult pigs by fluorescence quantitative PCR detection The expression of IRF1 gene in all the tissues showed that IRF1 was expressed in all the detected tissues, the highest expression in the spleen, followed by lymph nodes and liver, and the lower expression of.IRF1 in other tissues such as the heart, brain and bladder and other immune related tissues such as the spleen and lymph nodes, suggesting that it may participate in the body. In order to further study the changes in IRF1 gene expression in the virus infection state, two kinds of DNA viruses (porcine pseudorabies virus, PRV, porcine parvovirus, PPV) and two RNA viruses (porcine infectious gastroenteritis virus, TGEV, porcine vesicular stomatitis virus, VSV) were selected respectively as the representative, and the PK-15 cells were detected respectively in IRF1 after different virus infection. The results showed that in addition to the PRV strain (QXX strain), the expression of IRF1 gene was up-regulated by other test virus infection, indicating that porcine IRF1 was involved in the response of the organism to the virus infection, and the effect of.2. pig IRF1 on the expression of interferon and virus replication was the study of the effect of pig IRF1 on the expression of interferon, from the peripheral blood lymphocytes of pigs. The porcine IRF1 gene was amplified and cloned into the PCAGGS-HA vector. The eukaryotic expression plasmid PCAGGS-HA-pIRF1 was constructed, PK-15 cells were transfected and porcine IRF1 gene was overexpressed. The promoter activity of the pig IFN- beta was detected by the double luciferase reporter system and the poly (I:C) stimulated the promoter activity of the IFN- beta, and the transcriptional level of IFN- beta was detected by the fluorescence quantitative PCR. The overexpression of porcine IRF1 activates the promoter of porcine IFN- beta and up-regulated the transcriptional level of IFN- beta. Under the induction of poly (I:C), overexpression of IRF1 can more significantly activate the IFN- beta promoter, and this activation ability is stronger than the activation energy of pig IRF3 and pig IRF7. However, when RNA interference technology knocks down PK-15. After the expression of IRF1 in the cells, the expression level of IFN- beta was not significantly changed under the induction of poly (I:C), indicating that pig IRF1 could promote the expression of interferon, but not a necessary factor to induce the expression of interferon. In order to explore the effect of IRF1 on the replication of the virus, PCAGGS-HA-pIRF1 transfected to PK-15 cells, and the expression of IRF1 to PRV and TGEV replication was detected respectively. The results showed that overexpression of IRF1 could significantly inhibit the replication of PRV and TGEV, in which the virus titer of PRV decreased by nearly 10 times, and the virus titer of TGEV decreased by more than 20 times. It indicated that pig IRF1 could promote the response to antiviral infection of cultured cells in vitro, and had strong anti disease activity.3. to express the PK-15 cell line of pig IRF1 gene. To construct a PK-15 cell line that stably expressed IRF1 gene, the porcine IRF1 gene was transfected into PK-15 cells by PiggyBac eukaryotic transposon system. The positive transformed cells were screened by double screening of purinomycin and green fluorescence, and the single positive cell clones were cloned and purified by cloning. The fluorescence and fluorescence quantitative PC were observed. R showed that the IRF1 expression of the cell line was not attenuated after 20 generations of continuous passage, indicating that the PK-15 cell line that stably expressed the IRF1 gene was constructed successfully, which provided an effective tool for further study of the function and mechanism of the pig IRF1 gene.

【學(xué)位授予單位】：河南農(nóng)業(yè)大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2015
【分類號(hào)】：S852.65

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