猪IRF1抗病毒功能的初步研究及稳定表达IRF1基因PK-15细胞系的构建

发布时间：2018-04-26 11:52

本文选题：猪IRF1 + 组织分布　；参考：《河南农业大学》2015年硕士论文

【摘要】：干扰素调节因子(IFN regulatory factors,IRFs)是一类重要的转录因子,参与机体免疫应答、炎症反应、肿瘤发生以及细胞凋亡等多种生物学过程。其中,IRF3和IRF7在干扰素的表达调控中起重要作用,参与机体抗病毒免疫应答。近年来研究发现除IRF3和IRF7之外,IRF1在抗病毒免疫应答中也发挥着重要作用。IRF1可能是IRF3和IRF7之外的另一种调节抗病毒免疫的转录因子,而且当IRF3/7或IFNs的功能被抑制时,IRF1可能为调节干扰素(IFNs)和干扰素激活基因(ISGs)的表达提供了备选机制。对于控制那些能够拮抗IRF3和IRF7以及IFNs表达的病毒而言,IRF1介导的抗病毒应答可能发挥着极其重要的作用。目前,猪IRF1的研究相对较少,其具有怎样的功能特点,特别是在猪抗病毒先天免疫过程中的作用如何尚不清楚。因此,本文开展了以下研究,以期更多了解猪IRF1在机体先天性抗病毒免疫机制中的作用。1.猪IRF1基因的体内表达分析及病毒感染对其表达的影响为研究猪IRF1基因的组织分布,采集健康成年猪的心脏、肝脏、脾脏、肺脏、肾脏、胃、脑等组织,通过荧光定量PCR检测各组织中IRF1基因的表达量。结果表明IRF1在所有检测组织中均有表达,其中脾脏中表达量最高,其次是淋巴结和肝脏,而在心脏、脑和膀胱等其他组织中表达量较低。IRF1在脾脏和淋巴结等免疫相关组织中表达量较高,提示其可能参与机体的免疫应答。为进一步研究病毒感染状态下IRF1基因表达的变化,分别选取两种DNA病毒(猪伪狂犬病毒,PRV;猪细小病毒,PPV)和两种RNA病毒(猪传染性胃肠炎病毒,TGEV;猪水泡性口炎病毒,VSV)为代表,分别检测了PK-15细胞在不同病毒感染后IRF1的表达情况。结果表明除PRV流行毒株(QXX株)外,其他参试病毒感染均可上调IRF1基因的表达,表明猪IRF1参与了机体对病毒感染的应答反应。2.猪IRF1对干扰素表达和病毒复制的影响为研究猪IRF1对干扰素表达的影响,从猪外周血淋巴细胞中扩增猪IRF1基因,定向克隆到PCAGGS-HA载体中,构建真核表达质粒PCAGGS-HA-pIRF1,转染PK-15细胞,过表达猪IRF1基因。利用双荧光素酶报告系统检测无刺激状态和poly(I:C)刺激下猪IFN-β的启动子活性,并通过荧光定量PCR检测IFN-β的转录水平。结果表明过表达猪IRF1在无刺激的状态下即可激活猪IFN-β的启动子,并上调IFN-β的转录水平。在poly(I:C)诱导下,过表达IRF1能够更为显著的激活IFN-β的启动子,而且这种激活能力比猪IRF3和猪IRF7的激活能力更强。然而,当通过RNA干扰技术敲减PK-15细胞中IRF1的表达后,在poly(I:C)诱导下,IFN-β的表达水平并无显著变化,说明猪IRF1能促进干扰素的表达,但不是诱导干扰素表达的必要因子。为探索猪IRF1对病毒复制的影响,将PCAGGS-HA-pIRF1转染PK-15细胞,分别检测过表达IRF1对PRV和TGEV复制的影响,结果显示过表达IRF1可显著抑制PRV和TGEV的复制,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,表明猪IRF1能够促进体外培养细胞的抗病毒感染应答反应,具有较强的抗病毒活性。3.稳定表达猪IRF1基因PK-15细胞系的建立为构建稳定表达猪IRF1基因的PK-15细胞系,利用PiggyBac真核转座子系统将猪源IRF1基因转染入PK-15细胞中,通过嘌呤霉素和绿色荧光进行双重筛选获得阳性转化细胞,再经克隆纯化得到单个阳性细胞克隆株。通过观察荧光和荧光定量PCR鉴定,该细胞系连续传代20代后其IRF1表达量未出现衰减,表明稳定表达IRF1基因的PK-15细胞系构建成功,为进一步研究猪IRF1基因的功能以及作用机制提供了有效工具。
[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.

【学位授予单位】：河南农业大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S852.65

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