激活NF-κB信号通路的TGEV蛋白筛选及功能域确定

发布时间：2018-05-18 02:04

本文选题：猪传染性胃肠炎病毒 + NF-κB　；参考：《东北农业大学》2017年硕士论文

【摘要】：猪传染性胃肠炎(Swine transmissible gastroenteritis,TGE)病原为猪传染性胃肠炎病毒(Transmissible gastroenteritis virus of swine,TGEV),临床上以发热、呕吐、严重腹泻、脱水和2周龄以内仔猪高死亡率为特征。目前,该病在多个国家均有报道,已成为大型养猪场仔猪腹泻性死亡的主要原因之一,极大地限制了我国养猪业的发展。已有研究表明,TGEV感染可导致小肠上皮严重病变,空肠回肠绒毛严重萎缩,同时可以促进肠系膜淋巴结细胞和树突状细胞产生IL-6、IL-8、TNF-α、IFN-α等大量的炎症因子。由此可见,TGEV感染机体后诱导宿主产生的炎症反应可能与其致病机制密切相关。NF-κB(Nuclear factor-kappa B)是一类在促炎症基因表达过程中起到关键作用的核蛋白因子,具有多重转录调节作用,在病毒诱导的炎症反应中发挥着关键作用。有研究表明TGEV感染能激活NF-κB信号通路,但TGEV诱导炎症反应的分子机制尚未确定。因此,本研究以确定TGEV感染细胞对NF-κB信号通路的调控作用为切入点,筛选TGEV激活NF-κB信号通路的关键蛋白,并进一步确定其激活NF-κB信号通路的关键功能域,以期为阐明TGEV诱导机体产生炎症反应的信号转导机制奠定基础。本实验利用双荧光素酶报告系统检测TGEV感染猪睾丸细胞(Swine testicle,ST)对NF-κB信号通路的影响,结果表明,接种TGEV滴度的升高和TGEV感染时间的增加均可导致双荧光素酶活性比值的显著升高,而紫外灭活的TGEV同对照组相比没有明显的变化;间接免疫荧光实验(Indirect immunofluorescence assay,IFA)结果表明,TGEV感染可以促进p65发生核转移。以上研究结果表明TGEV可以显著激活NF-κB信号通路并且其激活作用与TGEV的滴度和复制密切相关。为了进一步确定激活NF-κB信号通路的TGEV的关键蛋白,本研究通过RT-PCR的方法扩增了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各基因片段并构建各段基因的真核表达载体,Western Blot和IFA实验的结果表明TGEV各蛋白均在细胞中成功表达。利用荧光素酶报告系统检测TGEV各个蛋白对NF-κB信号通路有影响,结果表明Nsp1、Nsp2、Nsp3、Nsp6、Nsp14、ORF7均可激活NF-κB信号通路,其中,Nsp2的激活作用最为显著。利用Western Blot和IFA检测Nsp2对NF-κB信号通路经典途径中的关键因子IκBα、p65的影响,结果表明Nsp2可促进IκBα的降解及p65的磷酸化、核转移,由此可见,Nsp2可通过经典途径激活NF-κB信号通路。为确定TGEV Nsp2激活NF-κB信号通路的关键序列,本研究通过构建包含TGEV Nsp2各个关键功能域的基因片段的真核表达载体,利用荧光素酶报告系统确定Nsp2 1-120位氨基酸是激活NF-κB信号通路的关键序列。综上所述,本研究证实了TGEV感染可以显著激活NF-κB信号通路,并且其激活作用与TGEV的滴度和复制密切相关;TGEV Nsp2激活NF-κB信号通路的关键蛋白,其可通过诱导IκBα的降解、p65的磷酸化和核转移来激活NF-κB信号通路;其中,1-120位氨基酸是Nsp2激活NF-κB信号通路的关键序列。本研究成果为阐明TGEV感染引起的炎症反应的机制及TGEV的致病机理奠定基础。进一步全面深入地研究NF-κB信号通路在TGEV致病过程中的作用,将为靶向药物的研发提供线索。
[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.
【学位授予单位】：东北农业大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S852.65

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