【作者】 彭丹；

【导师】 谭宇蓉；

【作者基本信息】 中南大学 ， 生物学， 2013， 硕士

【摘要】 目的：人呼吸道合胞病毒(RSV)是引起严重性呼吸道疾病的主要原因之一,是引起后继哮喘发生的重要危险因素。筛选RSV的2个非结构蛋白NS1和NS2在支气管上皮中的相互作用蛋白,检测其诱发支气管上皮细胞异常信号转导,从而探究其驱动T淋巴细胞亚群漂移机制。方法：构建含NS1或NS2基因的过表达慢病毒载体感染支气管上皮细胞,免疫共沉淀法和质谱法筛选并鉴定其相互作用蛋白；Western blot法鉴定相互作用蛋白泛素化修饰；亚硫酸盐测序检测Notch1基因甲基化；支气管上皮细胞与CD4+T淋巴细胞共培养,流式细胞术检测CD4+T淋巴细胞亚群分化,ELISA法检测共培养体系上清中细胞因子IFN-γ、IL-4、IL-17表达。结果：(1)NSl与组蛋白H2BD相互作用,NS1与组蛋白H2BD相互作用的关键区域是NS1延伸蛋白C区；(2)NS1与组蛋白H2BD相互作用诱导组蛋白H2BD单泛素化修饰；(3)NS1诱导Notch1去甲基化改变；(4)NS1对支气管上皮细胞驱动的T淋巴细胞亚群分化无影响,NS2可抑制Th17的分化,该效应可被泛素化酶抑制剂所逆转。(5)病毒非结构蛋白对共培养体系上清中细胞因子分泌无影响。结论：NS1与H2BD特异性结合并诱导H2BD单泛素化修饰,并进一步诱导下游靶基因Notch1的激活。NS2抑制Th17的分化,该效应可被泛素化酶抑制剂所逆转。更多还原

【Abstract】 Objectives:Human respiratory syncytial virus (RSV), a major cause of severe respiratory diseases, constitutes an important risk factor for the development of subsequent asthma. The present study was designed to screen the interacting proteins of two nonstructural(NS1and NS2)proteins and test abnormal signal transduction of bronchial epithelial in searching for mechanism of the subsets drift of CD4+T lymphocytes.Methods:Constructing lentiviral vectors of NS1or NS2to infect bronchial epithelial cells, immune coprecipitation and mass spectrometry technologies were used to screen and identify their interacted proteins; Western blotting technology was used to identify the ubiquitination modification of interacting proteins; bisulfite sequencing was used to detect the methylation of Notch1gene. Co-culturing bronchial epithelial cells and CD4+T lymphocytes, flow cytometry was used to detect the subsets differentiation of CD4+T lymphocyte, ELISA assay was used to detect the secretions of cytokine IFN-gamma, IL-4and IL-17in the supernatant.Results:(1) NS1can interact with histone H2BD with its elongin C binding region;(2) NS1can induce monoubiquitination of histone H2BD interaction;(3) NS1can induce the demethylation of Notch1gene;(4) NS1had no effects on bronchial epithelial cell-driven subsets differentiation of T lymphocyte, while NS2inhibited the differentiation of Th17, which can be reversed by inhibitors of ubiquitination.(5) Viral non-structural proteins had no effects on the secretions of cytokines in co-cultured supernatant. Conclusion:NS1interacted with H2BD specifically and induced H2BD monoubiquitination and subsequent activation of downstream Notch1. NS2inhibited Th17’s differentiation, which was related with degradation activity of ubiquitination.更多还原