本文關鍵詞：櫻桃谷肉鴨IRF4和IRF8基因克隆及其生物功能分析　出處：《山東農業(yè)大學》2017年碩士論文　論文類型：學位論文

  更多相關文章： 櫻桃谷肉鴨 先天性免疫 氨基酸對比分析 干擾素調節(jié)因子 生物學功能

【摘要】：干擾素(IFN)在天然的抗病毒免疫反應中具有重要的作用。哺乳動物物的干擾素根據基因結構、蛋白結構、生物學功能不同可分為Ⅰ型、Ⅱ型、Ⅲ型。干擾素調節(jié)因子(IRF)是一類轉錄因子,主要調節(jié)干擾素和干擾素刺激性應答基因(ISG)的表達,同時IRF家族本身也受IFN的調節(jié)。IRFs在病毒感染的早期先天免疫應答中起關鍵作用。IRF4是淋巴細胞特異性的轉錄調節(jié)因子,與其它轉錄因子形成復合體,通過調節(jié)IFN及一些淋巴因子的表達,參與抗病毒、Th細胞分化和B細胞成熟。IRF8也稱為干擾素保守序列結合蛋白(IFN consensus sequence binding protein,ICSBP),是一種核轉錄因子,激活后可刺激I型IFN及ISGs的轉錄。IRF8在調控細胞因子信號轉導、干擾素轉錄、細胞增殖、先天性免疫和適應性免疫等方面發(fā)揮重要的作用。干擾素調節(jié)因子的生物學功能的研究集中在哺乳動物上(大部分在鼠上),雞以及魚類也有部分報道,而在鴨子上關于干擾素調節(jié)因子的研究尚未見報道。基于上述原因,本課題在國內外率先開展了鴨子上干擾素調節(jié)因子的克隆及生物功能分析的研究。本研究以櫻桃谷肉鴨作為實驗動物,研究內容主要包括三部分:第一部分櫻桃谷肉鴨IRF4、IRF8的克隆鑒定與進化分析。根據預測的鴨IRF4、IRF8基因保守區(qū)域設計特異性引物,以健康櫻桃谷肉鴨脾臟反轉錄得到的cDNA為模板,經PCR擴增得到IRF4、IRF8片段,通過測序鑒定目的基因。其中IRF4的ORF序列長1341 bp,編碼446個氨基酸;IRF8的ORF序列長1290 bp,編碼429個氨基酸。根據IRF4和IRF8的進化樹顯示,櫻桃谷肉鴨與鴻雁的同源性最高,IRF4同源性達98.4%,IRF8同源性達96.8%,此外,櫻桃肉鴨IRF4和IRF8與土綏雞和原雞的同源性也非常高,但與魚類親緣關系較遠。第二部分櫻桃谷肉鴨上IRF4、IRF8與其他物種的氨基酸對比分析以及生物學功能分析。IRF4和IRF8與多種物種進行氨基酸序列比對,例如原雞、鴻雁、人、小鼠和牙鲆,結果發(fā)現IRF4和IRF8均可分為七個區(qū)域:N末端(N terminus),DNA結合結構域(DBD),推定的核定位信號(NLS),轉錄激活結構域(TD),外顯子6(exon 6),干擾素調節(jié)因子關聯域(IAD)和C末端(C terminus)。IRF4和IRF8蛋白均具有三個保守的結構域:N末端DBD結構域,C端IAD結構域和NLS結構域。第三部分櫻桃谷肉鴨IRF4、IRF8在健康鴨各個組織的分布情況以及感染病毒之后IRF4、IRF8的消長規(guī)律。通過熒光定量PCR檢測健康櫻桃谷肉鴨各組織中IRF4 m RNA和IRF8 mRNA在21種組織中的表達情況。檢測發(fā)現,櫻桃谷肉鴨IRF4在脾臟中表達量最高,其次是法氏囊,而在皮膚和大腦中表達量較低。櫻桃谷肉鴨IRF8在各組織中的表達量差異非常大,在肝臟中的表達量最高,為法氏囊的154倍;在心臟和盲腸中的表達量也非常高,均達到法氏囊的100倍左右,而在肌肉中表達量最低。為闡明櫻桃谷肉鴨IRF4和IRF8是否參與宿主的抗病毒免疫應答,本實驗使用鴨坦布蘇病毒、鴨新型呼腸弧病毒和鴨瘟病毒,通過腿部肌肉注射感染櫻桃谷肉鴨,檢測攻毒之后第1、3、5天在脾臟和大腦中IRF4、IRF8的表達變化情況。結果顯示。當被鴨坦布蘇病毒感染后,3天中兩組織中IRF4的表達差異均達到極顯著水平,在脾臟中的第3天上調達到最大值為96.5倍;腦中第5天上調達到1572.4倍。當被鴨瘟病毒感染后,脾臟中IRF4上調水平較低,均在10倍以下,但在檢測的3天中不斷升高;大腦在感染后的上調趨勢明顯,但在1、3、5天上調水平逐漸下降,其中第一天上調達到33.6倍。當被鴨新型呼腸弧病毒感染后,脾臟中IRF4在1、3、5天的含量逐漸升高,但總體含量較低;而在大腦中的上調趨勢非常明顯,在第1天上調就達到4388.3倍,隨后上調倍數逐漸下降。櫻桃谷肉鴨脾臟和大腦中的IRF8在感染3種病毒后的第1、3、5天表達量均呈現上調,感染鴨瘟病毒和鴨新型呼腸弧病毒后,脾臟中的上調倍數明顯高于大腦;而感染鴨坦布蘇病毒后,大腦中的上調倍數高于脾臟,被三種病毒感染后各組脾臟和大腦上調均達到極顯著水平。當被鴨坦布蘇病毒感染后,脾臟在第3天上調倍數達到最大值為342.2倍,但在第5天下降到201.8倍;大腦中上調非常明顯均達到1000倍左右,其中第3天上調倍數最大為1385.9倍。當被鴨瘟病毒感染后,脾臟在第1天上調倍數最高達到106.9倍;而腦中相對較低。當被鴨新型呼腸弧病毒感染后,脾臟和大腦在感染后的第1天上調均達到最高水平,其中脾臟達到10843.9倍,第3天和第5天上調倍數逐漸減小。本研究表明,櫻桃谷肉鴨中存在IRF4和IRF8這兩個轉錄調節(jié)因子,且在健康鴨的組織中廣泛分布。不同類型的病毒感染櫻桃谷肉鴨后,IRF4和IRF8在大腦和脾臟中的表達量均顯著上調,表明這兩個因子參與宿主的抗病毒反應。本研究為進一步探究鴨IRF4和IRF8的抗病毒作用機理以及其介導的信號通路奠定了基礎,有助于認識鴨的先天性免疫系統,豐富免疫學知識,為疾病防控奠定理論基礎。
[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.

【學位授予單位】：山東農業(yè)大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：S834

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