樱桃谷肉鸭IRF4和IRF8基因克隆及其生物功能分析

发布时间：2018-01-06 05:19

本文关键词：樱桃谷肉鸭IRF4和IRF8基因克隆及其生物功能分析　出处：《山东农业大学》2017年硕士论文　论文类型：学位论文

【摘要】：干扰素(IFN)在天然的抗病毒免疫反应中具有重要的作用。哺乳动物物的干扰素根据基因结构、蛋白结构、生物学功能不同可分为Ⅰ型、Ⅱ型、Ⅲ型。干扰素调节因子(IRF)是一类转录因子,主要调节干扰素和干扰素刺激性应答基因(ISG)的表达,同时IRF家族本身也受IFN的调节。IRFs在病毒感染的早期先天免疫应答中起关键作用。IRF4是淋巴细胞特异性的转录调节因子,与其它转录因子形成复合体,通过调节IFN及一些淋巴因子的表达,参与抗病毒、Th细胞分化和B细胞成熟。IRF8也称为干扰素保守序列结合蛋白(IFN consensus sequence binding protein,ICSBP),是一种核转录因子,激活后可刺激I型IFN及ISGs的转录。IRF8在调控细胞因子信号转导、干扰素转录、细胞增殖、先天性免疫和适应性免疫等方面发挥重要的作用。干扰素调节因子的生物学功能的研究集中在哺乳动物上(大部分在鼠上),鸡以及鱼类也有部分报道,而在鸭子上关于干扰素调节因子的研究尚未见报道。基于上述原因,本课题在国内外率先开展了鸭子上干扰素调节因子的克隆及生物功能分析的研究。本研究以樱桃谷肉鸭作为实验动物,研究内容主要包括三部分:第一部分樱桃谷肉鸭IRF4、IRF8的克隆鉴定与进化分析。根据预测的鸭IRF4、IRF8基因保守区域设计特异性引物,以健康樱桃谷肉鸭脾脏反转录得到的cDNA为模板,经PCR扩增得到IRF4、IRF8片段,通过测序鉴定目的基因。其中IRF4的ORF序列长1341 bp,编码446个氨基酸;IRF8的ORF序列长1290 bp,编码429个氨基酸。根据IRF4和IRF8的进化树显示,樱桃谷肉鸭与鸿雁的同源性最高,IRF4同源性达98.4%,IRF8同源性达96.8%,此外,樱桃肉鸭IRF4和IRF8与土绥鸡和原鸡的同源性也非常高,但与鱼类亲缘关系较远。第二部分樱桃谷肉鸭上IRF4、IRF8与其他物种的氨基酸对比分析以及生物学功能分析。IRF4和IRF8与多种物种进行氨基酸序列比对,例如原鸡、鸿雁、人、小鼠和牙鲆,结果发现IRF4和IRF8均可分为七个区域:N末端(N terminus),DNA结合结构域(DBD),推定的核定位信号(NLS),转录激活结构域(TD),外显子6(exon 6),干扰素调节因子关联域(IAD)和C末端(C terminus)。IRF4和IRF8蛋白均具有三个保守的结构域:N末端DBD结构域,C端IAD结构域和NLS结构域。第三部分樱桃谷肉鸭IRF4、IRF8在健康鸭各个组织的分布情况以及感染病毒之后IRF4、IRF8的消长规律。通过荧光定量PCR检测健康樱桃谷肉鸭各组织中IRF4 m RNA和IRF8 mRNA在21种组织中的表达情况。检测发现,樱桃谷肉鸭IRF4在脾脏中表达量最高,其次是法氏囊,而在皮肤和大脑中表达量较低。樱桃谷肉鸭IRF8在各组织中的表达量差异非常大,在肝脏中的表达量最高,为法氏囊的154倍;在心脏和盲肠中的表达量也非常高,均达到法氏囊的100倍左右,而在肌肉中表达量最低。为阐明樱桃谷肉鸭IRF4和IRF8是否参与宿主的抗病毒免疫应答,本实验使用鸭坦布苏病毒、鸭新型呼肠弧病毒和鸭瘟病毒,通过腿部肌肉注射感染樱桃谷肉鸭,检测攻毒之后第1、3、5天在脾脏和大脑中IRF4、IRF8的表达变化情况。结果显示。当被鸭坦布苏病毒感染后,3天中两组织中IRF4的表达差异均达到极显著水平,在脾脏中的第3天上调达到最大值为96.5倍;脑中第5天上调达到1572.4倍。当被鸭瘟病毒感染后,脾脏中IRF4上调水平较低,均在10倍以下,但在检测的3天中不断升高;大脑在感染后的上调趋势明显,但在1、3、5天上调水平逐渐下降,其中第一天上调达到33.6倍。当被鸭新型呼肠弧病毒感染后,脾脏中IRF4在1、3、5天的含量逐渐升高,但总体含量较低;而在大脑中的上调趋势非常明显,在第1天上调就达到4388.3倍,随后上调倍数逐渐下降。樱桃谷肉鸭脾脏和大脑中的IRF8在感染3种病毒后的第1、3、5天表达量均呈现上调,感染鸭瘟病毒和鸭新型呼肠弧病毒后,脾脏中的上调倍数明显高于大脑;而感染鸭坦布苏病毒后,大脑中的上调倍数高于脾脏,被三种病毒感染后各组脾脏和大脑上调均达到极显著水平。当被鸭坦布苏病毒感染后,脾脏在第3天上调倍数达到最大值为342.2倍,但在第5天下降到201.8倍;大脑中上调非常明显均达到1000倍左右,其中第3天上调倍数最大为1385.9倍。当被鸭瘟病毒感染后,脾脏在第1天上调倍数最高达到106.9倍;而脑中相对较低。当被鸭新型呼肠弧病毒感染后,脾脏和大脑在感染后的第1天上调均达到最高水平,其中脾脏达到10843.9倍,第3天和第5天上调倍数逐渐减小。本研究表明,樱桃谷肉鸭中存在IRF4和IRF8这两个转录调节因子,且在健康鸭的组织中广泛分布。不同类型的病毒感染樱桃谷肉鸭后,IRF4和IRF8在大脑和脾脏中的表达量均显著上调,表明这两个因子参与宿主的抗病毒反应。本研究为进一步探究鸭IRF4和IRF8的抗病毒作用机理以及其介导的信号通路奠定了基础,有助于认识鸭的先天性免疫系统,丰富免疫学知识,为疾病防控奠定理论基础。
[Abstract]:Interferon (IFN) plays an important role in antiviral immune response in mammals. Natural objects according to the interferon gene structure, protein structure, biological function can be divided into type I, II, III. Interferon regulatory factor (IRF) is a transcription factor, regulating interferon and interferon stimulated genes (response the expression of IRF and ISG), the family itself is also regulated by IFN.IRFs in the early innate immune response to virus infection plays a key role in.IRF4 transcription, lymphocyte specific regulatory factor, form complexes with other transcription factors, involved in the regulation of IFN expression by antiviral, and some lymphokines, differentiation and B cell Th cell maturation.IRF8 is also known as interferon conserved sequence binding protein (IFN consensus sequence binding, protein, ICSBP) is a nuclear transcription factor,.IRF8 transcription activation after stimulation of I type IFN and ISGs In the regulation of cytokine signal transduction, interferon transcription, cell proliferation, play an important role in innate immunity and adaptive immunity. Study on biological function of interferon regulatory factor concentration in mammals (mostly in mice), chicken and fish also have some of the reports, and in the research on the duck interferon regulatory factor is has not been reported. Based on the above reasons, this paper first carried out research and Analysis on the duck interferon regulatory factor gene cloning and biological function at home and abroad. In this study, the ducks as the experimental animal, the research content mainly includes three parts: the first part of the ducks IRF4, molecular cloning and phylogenetic analysis of IRF8. According to the prediction of duck IRF4 IRF8 gene, conserved region specific primers were designed to get healthy ducks spleen reverse transcription cDNA as template, amplified by PCR IRF4, IRF8 Fragments were confirmed by DNA sequencing. The ORF gene sequence of IRF4 1341 BP in length, encoding 446 amino acids; ORF IRF8 sequence length of 1290 BP, encoding 429 amino acids. According to the phylogenetic tree of IRF4 and IRF8 showed that the ducks and geese of the highest homology of IRF4 homology 98.4%, IRF8 homology is 96.8% in addition, IRF4 and IRF8, cherry duck and chicken gallus and soil Sui homology is very high, but the fish and distantly related. The second part ducks on IRF4, IRF8 and other species of amino acid comparative analysis and biological function analysis of.IRF4 and IRF8 with various species of amino acid sequence, such as gallus, Hongyan, human, mice and flounder, the results showed that IRF4 and IRF8 can be divided into seven regions: the end of the N (N terminus), DNA binding domain (DBD), the putative nuclear localization signal (NLS), transcription activation domain (TD), exon 6 (exon 6), interferon Regulator associated domain (IAD) and C (C terminus) at the end of.IRF4 and IRF8 proteins have three conserved domains: N terminal DBD domain, C terminal IAD domain and NLS domain. The third part of the ducks in IRF4, the distribution of IRF8 in various tissues and healthy duck virus infection after IRF4. The dynamic law of IRF8. The expression of the fluorescent quantitative PCR detection in IRF4 m RNA healthy ducks and IRF8 mRNA in 21 tissues. Detection of IRF4 in the spleen of ducks in the highest expression level, followed by the bursa, and in skin and brain in low expression. Expression difference the ducks IRF8 in different tissues is very large, the expression in liver is highest, 154 times of bursa; expression in the heart and the cecum is also very high, reached about 100 times of the bursa, and the lowest expression in muscle. In order to clarify the Sakura Whether the antiviral immune response Momodani duck IRF4 and IRF8 in the host, duck Tembusu virus used in this experiment, the new duck reovirus and duck plague virus infection, by intramuscular injection of ducks challenged, detection after 1,3,5 days in spleen and IRF4 in the brain, the expression of IRF8. The results showed that when. Is duck Tembusu virus after infection, expression of IRF4 two in 3 days in the organization reached significant level in the spleen third days up to a maximum of 96.5 times; in the brain fifth to 1572.4 times. When the sky up by duck plague virus after infection in the spleen, the up regulation of IRF4 level is low, are in the following 10 times, but in the detection of 3 days increased; in brain after infection was significantly up-regulated, but decreased in 1,3,5 days by the first level, up to 33.6 times. When the sky was new duck reovirus infection after IRF4 in spleen The content of 1,3,5 days gradually increased, but the overall content is low; and the upward trend in the brain is very obvious, on the first day increase reached 4388.3 times, then up-regulated gradually decreased. The ducks in the spleen and brain IRF8 in 3 kinds of viral infection after day 1,3,5 expression were increased, infection of duck new duck plague virus and reovirus, up-regulated in spleen was significantly higher than that of the brain; and the infection of duck Tembusu virus, up-regulated in the brain was three higher than that in the spleen, spleen and brain after infection were up-regulated reached significant level. When the duck Tembusu virus after infection, the spleen reached the maximum value is 342.2 times in the third heaven adjustable multiples, but in fifth fell to 201.8 times; in the brain increased obviously reached about 1000 times, third of which is 1385.9 times the maximum adjustment ratio of heaven. When duck plague virus after infection in the spleen The first day adjustment ratio up to 106.9 times; and the brain is relatively low. When the new duck reovirus infection, spleen and brain have reached the highest level in the first day after infection, the spleen reached 10843.9 times, third days and fifth days up-regulated gradually decreases. This study shows that IRF4 and IRF8 these two transcription factors are widely distributed and the ducks, duck in healthy tissues. Different types of virus infection in ducks, the expression of IRF4 and IRF8 in the brain and spleen weight were significantly up-regulated, indicating that these two factors involved in host antiviral responses. This research laid the foundation for the to further explore the duck IRF4 and IRF8 antiviral mechanism and signaling pathways mediated, contribute to the innate immune system of duck, rich knowledge and lay the theoretical foundation for immunology, disease prevention and control.

【学位授予单位】：山东农业大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S834

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