發(fā)布時(shí)間：2018-05-18 02:04

  本文選題：豬傳染性胃腸炎病毒 + NF-κB　； 參考：《東北農(nóng)業(yè)大學(xué)》2017年碩士論文

【摘要】：豬傳染性胃腸炎(Swine transmissible gastroenteritis,TGE)病原為豬傳染性胃腸炎病毒(Transmissible gastroenteritis virus of swine,TGEV),臨床上以發(fā)熱、嘔吐、嚴(yán)重腹瀉、脫水和2周齡以內(nèi)仔豬高死亡率為特征。目前,該病在多個(gè)國(guó)家均有報(bào)道,已成為大型養(yǎng)豬場(chǎng)仔豬腹瀉性死亡的主要原因之一,極大地限制了我國(guó)養(yǎng)豬業(yè)的發(fā)展。已有研究表明,TGEV感染可導(dǎo)致小腸上皮嚴(yán)重病變,空腸回腸絨毛嚴(yán)重萎縮,同時(shí)可以促進(jìn)腸系膜淋巴結(jié)細(xì)胞和樹(shù)突狀細(xì)胞產(chǎn)生IL-6、IL-8、TNF-α、IFN-α等大量的炎癥因子。由此可見(jiàn),TGEV感染機(jī)體后誘導(dǎo)宿主產(chǎn)生的炎癥反應(yīng)可能與其致病機(jī)制密切相關(guān)。NF-κB(Nuclear factor-kappa B)是一類在促炎癥基因表達(dá)過(guò)程中起到關(guān)鍵作用的核蛋白因子,具有多重轉(zhuǎn)錄調(diào)節(jié)作用,在病毒誘導(dǎo)的炎癥反應(yīng)中發(fā)揮著關(guān)鍵作用。有研究表明TGEV感染能激活NF-κB信號(hào)通路,但TGEV誘導(dǎo)炎癥反應(yīng)的分子機(jī)制尚未確定。因此,本研究以確定TGEV感染細(xì)胞對(duì)NF-κB信號(hào)通路的調(diào)控作用為切入點(diǎn),篩選TGEV激活NF-κB信號(hào)通路的關(guān)鍵蛋白,并進(jìn)一步確定其激活NF-κB信號(hào)通路的關(guān)鍵功能域,以期為闡明TGEV誘導(dǎo)機(jī)體產(chǎn)生炎癥反應(yīng)的信號(hào)轉(zhuǎn)導(dǎo)機(jī)制奠定基礎(chǔ)。本實(shí)驗(yàn)利用雙熒光素酶報(bào)告系統(tǒng)檢測(cè)TGEV感染豬睪丸細(xì)胞(Swine testicle,ST)對(duì)NF-κB信號(hào)通路的影響,結(jié)果表明,接種TGEV滴度的升高和TGEV感染時(shí)間的增加均可導(dǎo)致雙熒光素酶活性比值的顯著升高,而紫外滅活的TGEV同對(duì)照組相比沒(méi)有明顯的變化;間接免疫熒光實(shí)驗(yàn)(Indirect immunofluorescence assay,IFA)結(jié)果表明,TGEV感染可以促進(jìn)p65發(fā)生核轉(zhuǎn)移。以上研究結(jié)果表明TGEV可以顯著激活NF-κB信號(hào)通路并且其激活作用與TGEV的滴度和復(fù)制密切相關(guān)。為了進(jìn)一步確定激活NF-κB信號(hào)通路的TGEV的關(guān)鍵蛋白,本研究通過(guò)RT-PCR的方法擴(kuò)增了TH-98株TGEV的Nsp1、Nsp2、Nsp3、Nsp4、Nsp5、Nsp6、Nsp7、Nsp8、Nsp9、Nsp10、Nsp11+Nsp12、Nsp13、Nsp14、Nsp15、Nsp16、S基因、ORF3a、ORF3b、E基因、M基因、N基因、ORF7各基因片段并構(gòu)建各段基因的真核表達(dá)載體,Western Blot和IFA實(shí)驗(yàn)的結(jié)果表明TGEV各蛋白均在細(xì)胞中成功表達(dá)。利用熒光素酶報(bào)告系統(tǒng)檢測(cè)TGEV各個(gè)蛋白對(duì)NF-κB信號(hào)通路有影響,結(jié)果表明Nsp1、Nsp2、Nsp3、Nsp6、Nsp14、ORF7均可激活NF-κB信號(hào)通路,其中,Nsp2的激活作用最為顯著。利用Western Blot和IFA檢測(cè)Nsp2對(duì)NF-κB信號(hào)通路經(jīng)典途徑中的關(guān)鍵因子IκBα、p65的影響,結(jié)果表明Nsp2可促進(jìn)IκBα的降解及p65的磷酸化、核轉(zhuǎn)移,由此可見(jiàn),Nsp2可通過(guò)經(jīng)典途徑激活NF-κB信號(hào)通路。為確定TGEV Nsp2激活NF-κB信號(hào)通路的關(guān)鍵序列,本研究通過(guò)構(gòu)建包含TGEV Nsp2各個(gè)關(guān)鍵功能域的基因片段的真核表達(dá)載體,利用熒光素酶報(bào)告系統(tǒng)確定Nsp2 1-120位氨基酸是激活NF-κB信號(hào)通路的關(guān)鍵序列。綜上所述,本研究證實(shí)了TGEV感染可以顯著激活NF-κB信號(hào)通路,并且其激活作用與TGEV的滴度和復(fù)制密切相關(guān);TGEV Nsp2激活NF-κB信號(hào)通路的關(guān)鍵蛋白,其可通過(guò)誘導(dǎo)IκBα的降解、p65的磷酸化和核轉(zhuǎn)移來(lái)激活NF-κB信號(hào)通路;其中,1-120位氨基酸是Nsp2激活NF-κB信號(hào)通路的關(guān)鍵序列。本研究成果為闡明TGEV感染引起的炎癥反應(yīng)的機(jī)制及TGEV的致病機(jī)理奠定基礎(chǔ)。進(jìn)一步全面深入地研究NF-κB信號(hào)通路在TGEV致病過(guò)程中的作用,將為靶向藥物的研發(fā)提供線索。
[Abstract]:The pathogen of Swine transmissible gastroenteritis (TGE) is porcine infectious gastroenteritis virus (Transmissible gastroenteritis virus of swine, TGEV). It is clinically characterized by fever, vomiting, severe diarrhea, dehydration and high mortality of piglets within 2 weeks of age. One of the main causes of piglet diarrhoea death in piggery has greatly restricted the development of the pig industry in China. It has been shown that TGEV infection can lead to severe intestinal epithelial lesions, severe atrophy of the jejunum ileum villi, and can promote mesenteric lymph node cells and dendritic cells to produce a large number of inflammatory factors, such as IL-6, IL-8, TNF- a, IFN- A and so on. It can be seen that the inflammatory response induced by TGEV infected host may be closely related to the pathogenesis of.NF- kappa B (Nuclear factor-kappa B), a class of nuclear protein factors that play a key role in the expression of pro-inflammatory genes, with multiple transcriptional regulation, and plays a key role in the virus induced inflammatory response. Some studies have shown that TGEV infection can activate the NF- kappa B signaling pathway, but the molecular mechanism of TGEV induced inflammatory response has not been determined. Therefore, this study aims to identify the key proteins that regulate the NF- kappa B signaling pathway by determining the regulatory role of TGEV infected cells, and to further determine its activation of the NF- kappa B signal pathway. The key functional domain is expected to lay the foundation for clarifying the signal transduction mechanism of TGEV induced inflammatory response in the body. This experiment uses a dual luciferase reporter system to detect the effect of TGEV infection on the NF- kappa B signaling pathway in swine testis cells (Swine testicle, ST). The results show that the increase of TGEV titer and the increase of TGEV infection time can lead to the increase of the time of TGEV infection. The activity ratio of the double luciferase increased significantly, while the TGEV inactivated by ultraviolet (Indirect immunofluorescence assay, IFA) showed that TGEV infection could promote the nuclear transfer of p65. The results showed that TGEV could significantly activate the NF- kappa B signaling pathway and its excitation. In order to further determine the key proteins that activate the NF- kappa B signaling pathway, this study amplified the Nsp1 of the TH-98, TGEV, TGEV, Nsp1, Nsp3, Nsp4, genes, genes, genes, genes, genes, genes, genes, genes, genes, genes, genes, genes, genes, genes, genes, genes, genes, genes, genes, genes, genes, genes, genes, genes, genes, genes, genes, genes, genes, genes, genes, genes, genes, genes, genes, genes, genes, genes, genes, genes, genes, genes, genes, genes, genes, genes, genes, genes, genes, genes, genes, genes, genes, genes, TGEV genes were amplified by RT-PCR. The ORF7 gene fragment and the construction of the eukaryotic expression vector of each segment gene, the results of the Western Blot and IFA experiments showed that all the TGEV proteins were expressed successfully in the cells. The luciferase reporter system was used to detect the effects of each protein on the NF- kappa B signaling pathway. The results showed that Nsp1, Nsp2, Nsp3, Nsp6, and the TGEV pathway could be activated. Among them, the activation of Nsp2 is the most significant. Western Blot and IFA are used to detect the effect of Nsp2 on the key factor of NF- kappa B signaling pathway, I kappa B alpha, p65. The results show that Nsp2 can promote the degradation of I kappa alpha and the phosphorylation and nuclear transfer. The key sequence of the NF- kappa B signaling pathway is made by constructing the eukaryotic expression vector containing the gene fragments of the key functional domains of TGEV Nsp2, and using the luciferase reporter system to determine the key sequence of the Nsp2 1-120 amino acids to activate the NF- kappa B signaling pathway. To sum up, this study confirms that TGEV infection can significantly activate the NF- kappa B. The signaling pathway is closely related to the titer and replication of TGEV; TGEV Nsp2 activates the key protein of the NF- kappa B signaling pathway, which can activate the NF- kappa B signaling pathway by inducing the degradation of I kappa B alpha, the phosphorylation and nuclear transfer of p65, in which the 1-120 amino acid is the key sequence of Nsp2 activation of the NF- kappa signaling pathway. The results of this study are To elucidate the mechanism of TGEV infection and the pathogenesis of TGEV, further study the role of NF- kappa B signaling pathway in the pathogenesis of TGEV, and will provide clues for the research and development of targeted drugs.
【學(xué)位授予單位】：東北農(nóng)業(yè)大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：S852.65

【參考文獻(xiàn)】

相關(guān)期刊論文 前7條

1 趙生軍;張勇;閻萍;郭憲;;PRDM家族蛋白結(jié)構(gòu)與功能研究進(jìn)展[J];中國(guó)畜牧獸醫(yī);2016年05期

2 鐘望;田野;牛曉蕓;王艷麗;張顯浩;陳瑞愛(ài);賀東生;;豬傳染性胃腸炎病毒NC株的分離鑒定及生物學(xué)特性試驗(yàn)[J];中國(guó)獸醫(yī)雜志;2012年11期

3 仝鋼;張彥明;唐青海;王津津;;豬傳染性胃腸炎病毒(TGEV)對(duì)豬小腸黏膜上皮細(xì)胞活性的影響[J];西北農(nóng)業(yè)學(xué)報(bào);2011年01期

4 王龍濤;葛晨霞;黃海龍;胡桂學(xué);高豐;;仔豬人工感染豬傳染性胃腸炎病毒的病理分析[J];安徽農(nóng)業(yè)科學(xué);2010年28期

5 黃小波;曹三杰;文心田;楊恒;秦學(xué)遠(yuǎn);;豬傳染性胃腸炎病毒SC-H株的分離與鑒定[J];黑龍江畜牧獸醫(yī);2008年08期

6 劉登存 ,趙華;豬傳染性胃腸炎的防治[J];山東畜牧獸醫(yī);2003年04期

7 吳國(guó)平,尹燕博,吳時(shí)友;豬傳染性胃腸炎病毒(TGEV)研究進(jìn)展[J];中國(guó)獸醫(yī)雜志;2003年02期

相關(guān)博士學(xué)位論文 前2條

1 方瑩;激活NF-κB信號(hào)通路的高致病性PRRSV非結(jié)構(gòu)蛋白的篩選及其作用機(jī)制研究[D];華中農(nóng)業(yè)大學(xué);2012年

2 史慧玲;冠狀病毒Nsp1蛋白生物學(xué)機(jī)理研究及HR2片段的抗體研發(fā)[D];華東師范大學(xué);2012年

相關(guān)碩士學(xué)位論文 前4條

1 吳蔚;豬傳染性胃腸炎病毒非結(jié)構(gòu)蛋白nsp14誘導(dǎo)IFN-β的分子機(jī)制[D];華中農(nóng)業(yè)大學(xué);2016年

2 王瑩瑩;嗜酸乳桿菌胞外多糖對(duì)TGEV感染ST細(xì)胞TLR3及部分細(xì)胞因子表達(dá)的影響[D];東北農(nóng)業(yè)大學(xué);2015年

3 劉寧;豬瘟病毒核衣殼蛋白C亞細(xì)胞定位信號(hào)的鑒定[D];吉林農(nóng)業(yè)大學(xué);2014年

4 田運(yùn)嬌;Gsα基因干擾對(duì)心力衰竭幼鼠心肌鈣調(diào)控蛋白的作用研究[D];重慶醫(yī)科大學(xué);2010年

，

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