本文選題：豬IRF1 + 組織分布��； 參考：《河南農(nóng)業(yè)大學》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,參與機體免疫應答、炎癥反應、腫瘤發(fā)生以及細胞凋亡等多種生物學過程。其中,IRF3和IRF7在干擾素的表達調(diào)控中起重要作用,參與機體抗病毒免疫應答。近年來研究發(fā)現(xiàn)除IRF3和IRF7之外,IRF1在抗病毒免疫應答中也發(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IRF3/7或IFNs的功能被抑制時,IRF1可能為調(diào)節(jié)干擾素(IFNs)和干擾素激活基因(ISGs)的表達提供了備選機制。對于控制那些能夠拮抗IRF3和IRF7以及IFNs表達的病毒而言,IRF1介導的抗病毒應答可能發(fā)揮著極其重要的作用。目前,豬IRF1的研究相對較少,其具有怎樣的功能特點,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在機體先天性抗病毒免疫機制中的作用。1.豬IRF1基因的體內(nèi)表達分析及病毒感染對其表達的影響為研究豬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檢測各組織中IRF1基因的表達量。結(jié)果表明IRF1在所有檢測組織中均有表達,其中脾臟中表達量最高,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量較低。IRF1在脾臟和淋巴結(jié)等免疫相關(guān)組織中表達量較高,提示其可能參與機體的免疫應答。為進一步研究病毒感染狀態(tài)下IRF1基因表達的變化,分別選取兩種DNA病毒(豬偽狂犬病毒,PRV;豬細小病毒,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,分別檢測了PK-15細胞在不同病毒感染后IRF1的表達情況。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,表明豬IRF1參與了機體對病毒感染的應答反應。2.豬IRF1對干擾素表達和病毒復制的影響為研究豬IRF1對干擾素表達的影響,從豬外周血淋巴細胞中擴增豬IRF1基因,定向克隆到PCAGGS-HA載體中,構(gòu)建真核表達質(zhì)粒PCAGGS-HA-pIRF1,轉(zhuǎn)染PK-15細胞,過表達豬IRF1基因。利用雙熒光素酶報告系統(tǒng)檢測無刺激狀態(tài)和poly(I:C)刺激下豬IFN-β的啟動子活性,并通過熒光定量PCR檢測IFN-β的轉(zhuǎn)錄水平。結(jié)果表明過表達豬IRF1在無刺激的狀態(tài)下即可激活豬IFN-β的啟動子,并上調(diào)IFN-β的轉(zhuǎn)錄水平。在poly(I:C)誘導下,過表達IRF1能夠更為顯著的激活I(lǐng)FN-β的啟動子,而且這種激活能力比豬IRF3和豬IRF7的激活能力更強。然而,當通過RNA干擾技術(shù)敲減PK-15細胞中IRF1的表達后,在poly(I:C)誘導下,IFN-β的表達水平并無顯著變化,說明豬IRF1能促進干擾素的表達,但不是誘導干擾素表達的必要因子。為探索豬IRF1對病毒復制的影響,將PCAGGS-HA-pIRF1轉(zhuǎn)染PK-15細胞,分別檢測過表達IRF1對PRV和TGEV復制的影響,結(jié)果顯示過表達IRF1可顯著抑制PRV和TGEV的復制,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能夠促進體外培養(yǎng)細胞的抗病毒感染應答反應,具有較強的抗病毒活性。3.穩(wěn)定表達豬IRF1基因PK-15細胞系的建立為構(gòu)建穩(wěn)定表達豬IRF1基因的PK-15細胞系,利用PiggyBac真核轉(zhuǎn)座子系統(tǒng)將豬源IRF1基因轉(zhuǎn)染入PK-15細胞中,通過嘌呤霉素和綠色熒光進行雙重篩選獲得陽性轉(zhuǎn)化細胞,再經(jīng)克隆純化得到單個陽性細胞克隆株。通過觀察熒光和熒光定量PCR鑒定,該細胞系連續(xù)傳代20代后其IRF1表達量未出現(xiàn)衰減,表明穩(wěn)定表達IRF1基因的PK-15細胞系構(gòu)建成功,為進一步研究豬IRF1基因的功能以及作用機制提供了有效工具。
[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.

【學位授予單位】：河南農(nóng)業(yè)大學
【學位級別】：碩士
【學位授予年份】：2015
【分類號】：S852.65

【相似文獻】

相關(guān)期刊論文 前10條

1 唐學璽,楊震,劉成圣;小麥耐鹽細胞系的篩選及其抗氧化能力分析[J];青島海洋大學學報(自然科學版);2002年01期

2 甘煩遠;鄭光植;;植物培養(yǎng)細胞次級代謝產(chǎn)物高產(chǎn)細胞系的篩選[J];國外醫(yī)藥(植物藥分冊);1990年01期

3 沈超,鄭從義,李朝陽,屈三甫;離體培養(yǎng)細胞系冷休克敏感差異性的研究[J];氨基酸和生物資源;2003年04期

4 顏永杉,馮尚,劉連瑞,甄惠欣,馮之凡;大鼠非致瘤四倍體細胞系的建立及細胞遺傳學和酶學特征[J];遺傳學報;1987年04期

5 韓嶸,陳偉勝,童英,尚克剛,孟令國;兩個可進入種系的ES細胞系的建立[J];遺傳學報;1999年03期

6 ;為再生醫(yī)學提供高通量細胞系表征服務[J];辦公自動化;2014年05期

7 郭惠東;特日格樂;尚愛萍;鄭文靜;劉剛;道日娜;王蓉蓉;李瑤;李煜;;Hela細胞HGPRT缺陷型細胞系構(gòu)建[J];中國細胞生物學學報;2013年02期

8 張念慈;尹文林;沈錦玉;曹錚;顧志敏;;團頭魴尾鰭細胞系TQ-8801的建立[J];科技通報;1991年02期

9 馬端端，王曉民，王昕紅，萬有，陳光慧，，韓濟生;GDNF在多種細胞系中表達的研究[J];北京醫(yī)科大學學報;1995年04期

10 唐學璽,賈敬芬;小麥耐鹽細胞系對鹽脅迫的傷害性反應[J];植物學報;1999年07期

相關(guān)會議論文 前10條

1 王官清;;水痘-帶狀皰疹病毒報告細胞系的構(gòu)建及應用研究[A];中華醫(yī)學會第16次全國皮膚性病學術(shù)年會摘要集[C];2010年

2 張永忠;盛紅;李琳;安萍;陳佩蘭;鄭菁;;對兩株人細胞系的鑒定[A];中國細胞生物學學會第五次會議論文摘要匯編[C];1992年

3 劉永霞;朱小麗;陳玉華;郭飛;沈默;陳占國;徐威;陳德瑩;陶志華;;雄激素非依賴前列腺癌細胞系代謝組學的初步研究[A];中華醫(yī)學會第九次全國檢驗醫(yī)學學術(shù)會議暨中國醫(yī)院協(xié)會臨床檢驗管理專業(yè)委員會第六屆全國臨床檢驗實驗室管理學術(shù)會議論文匯編[C];2011年

4 張秀軍;;血源細胞系中P2Y受體的基因表達[A];中國細胞生物學學會2005年學術(shù)大會、青年學術(shù)研討會論文摘要集[C];2005年

5 張力;龔明玉;何瑞龍;張立廣;;X-射線對HepG2細胞系細胞周期進程的影響[A];第五屆全國中醫(yī)藥免疫學術(shù)研討會——暨環(huán)境·免疫與腫瘤防治綜合交叉會議論文匯編[C];2009年

6 張鳳蘊;樊擁軍;陳君平;張飚;王麗群;;3種T細胞系細胞毒作用的比較[A];慶祝黑龍江省免疫學會成立十周年（1993—2003）論文集[C];2003年

7 王浩浩;滕理送;曹江;;新型靶向性藥物BP3323對SW480和SW620細胞系的作用研究[A];中國細胞生物學學會醫(yī)學細胞生物學學術(shù)大會論文集[C];2006年

8 張建華;張彩;許曉群;王郡甫;魏海明;;IL-12和IL-15對人NK92細胞系體外細胞毒效應的影響[A];第九屆全國腫瘤生物治療學術(shù)會議論文集[C];2006年

9 張慧;范列英;盧天寶;宗明;;RNAi表達載體干擾四型肽精氨酸脫亞氨基酶基因在HL-60細胞系中的表達[A];中華醫(yī)學會第七次全國檢驗醫(yī)學學術(shù)會議資料匯編[C];2008年

10 周竹娟;鄭健;;轉(zhuǎn)核技術(shù)建立可增殖神經(jīng)細胞系的實驗研究[A];第十一屆全國神經(jīng)病學學術(shù)會議論文匯編[C];2008年

相關(guān)重要報紙文章 前5條

1 吳一福;我國建立首株HCC-7721/VCR細胞系[N];中國醫(yī)藥報;2006年

2 ;Ⅱ型膠原特異性T細胞系與關(guān)節(jié)炎發(fā)病機制的研究[N];中國醫(yī)藥報;2003年

3 張中橋;西京醫(yī)院建立穩(wěn)定表達MAGE-n細胞系[N];中國醫(yī)藥報;2006年

4 唐風;美建立前列腺癌新細胞系[N];中國醫(yī)藥報;2006年

5 記者 朱慶、蘇雁 通訊員 崔俊;白血病研究有重大突破[N];光明日報;2003年

本文編號：1805906