5株H9N2亚型AIV血凝素结构分析及感染A549细胞的蛋白质组学研究
发布时间：2018-07-28 21:18
【摘要】：H9N2亚型禽流感病毒自1994年以来在中国大陆地区的家禽中广泛流行,虽然该亚型属于低致病性,但当继发其它病原感染时,能导致很高的发病率和死亡率,给家禽养殖造成巨大损失。不仅如此,该亚型病毒血凝素的氨基酸序列的变化,使更多的流行株变得能够与哺乳动物细胞的受体结合,能够传染给人,引起角膜炎或轻度类似感冒症状,引起公众对该病原的关注。近几年的研究表明H9N2亚型禽流感病毒可以作为新型流感病毒的来源。H7N9和H10N8亚型流感病毒的内部基因均是来自H9N2亚型流感病毒。了解H9N2亚型禽流感病毒感染哺乳动物的适应过程显得更加迫切。为筛选出对人肺脏上皮细胞敏感性较强的毒株,本研究通过间接免疫荧光的结果判断敏感性的高低,通过比较本实验室保存的5株H9N2亚型禽流感病毒对人肺脏上皮细胞的敏感性发现A/Chicken/Shandong/ch/2011敏感性最高。本研究从蛋白高级结构角度出发,采用同源建模的方法,比较了易于和不易感染人肺脏上皮细胞的毒株血凝素蛋白差异氨基酸所造成的结构差异。血凝素蛋白在一级结构上受体结合位点的第198和234位氨基酸有差别,但这两处在高级结构上没有差异。第336-342位氨基酸虽然一级结构相同,但是高级结构有差异,A/Chicken/Shandong/ch/2011的高级结构在这个位置形成了α-螺旋,但A/Chicken/Shandong/W4/2012等其他4株病毒在这个位置却是无规则卷曲。通过比较5株病毒血凝素蛋白糖基化位点的差异,发现在218位,298位和313位糖基化位点有差异。为揭示这些糖基化位点在大量毒株中的变化趋势,选取了NCBI上华东地区包含血凝素序列全长的鸡源H9N2亚型流感病毒病毒序列,进行糖基化位点进化情况的统计。发现H9N2亚型流感病毒血凝素片段中,保守的糖基化位点有29位NST,141位NVS,298位NTT,305位NVS,492位NGT。2006年之后的毒株,绝大多数新增两个糖基化位点,218位NRT,313位NCS。为更好地了解H9N2亚型禽流感病毒跨种感染哺乳动物的分子机制,本研究采用双向电泳技术对H9N2亚型禽流感病毒感染人肺脏上皮细胞后不同时间点差异表达的蛋白质进行了探索。结合质谱鉴定技术,共有17个差异表达的蛋白质被成功鉴定。位于位于NF-κB和IFN调节因子通路的蛋白被鉴定,表明NF-κB和IFN调节因子通路在H9N2亚型禽流感病毒感染哺乳动物细胞过程起到了作用。肌动蛋白和角蛋白被鉴定,表明细胞骨架在H9N2亚型禽流感病毒感染哺乳动物细胞过程中发挥了重要作用。
[Abstract]:H9N2 subtype avian influenza virus has been widely prevalent in domestic poultry since 1994. Although this subtype belongs to low pathogenicity, it can lead to high morbidity and mortality when secondary pathogens are infected and cause huge loss to poultry breeding. More popular strains become able to bind to the receptors of mammalian cells, be able to infect humans, cause keratitis or slightly similar cold symptoms, and cause public concern for the pathogen. In recent years, studies have shown that the H9N2 subtype avian influenza virus can be used as the internal gene of the.H7N9 and H10N8 subtype influenza viruses of the new influenza virus. It is all from the H9N2 subtype influenza virus. It is more urgent to understand the adaptation process of the H9N2 subtype avian influenza virus infection in mammals. In order to screen out the strains with strong sensitivity to human lung epithelial cells, the sensitivity of this study is judged by indirect immunofluorescence, and 5 H9N2 subtypes of avian influenza kept in our laboratory have been compared. The sensitivity of the virus to human lung epithelial cells was found to be the highest in A/Chicken/Shandong/ch/2011 sensitivity. From the point of view of the protein advanced structure, the structural difference between the hemagglutinin protein difference in the virus strain of human lung epithelial cells was compared with the homologous modeling method. There is a difference in the 198th and 234Th - bit amino acid of the receptor binding site on the first order structure, but there is no difference in the advanced structure. The 336-342 - position amino acid, although the same structure is the same, but the advanced structure is different. The advanced structure of A/Chicken/Shandong/ch/2011 is formed in this position as alpha helix, but A/Chicken/Shandong/W4/2012 and so on. By comparing the glycosylation sites of the 5 virus hemagglutinin proteins, the 4 strains of the virus were found to be different in the 218, 298 and 313 glycosylation sites. To reveal the trend of these glycosylation sites in a large number of virulent strains, the chicken source H9N containing the full length of the hemagglutinin sequence on the East China region of NCBI was selected. The sequence of the 2 subtype influenza virus virus was used to carry out the evolution of glycosylation sites. In the H9N2 subtype influenza virus hemagglutinin fragment, the conservative glycosylation sites have 29 NST, 141 NVS, 298 NTT, 305 NVS, 492 NGT.2006 years later, and the vast majority of the new glycosylation sites, 218 NRT, and 313 bit NCS. are better understood. The molecular mechanism of H9N2 subtype avian influenza virus infection in mammals. This study explored proteins expressed differently at different time points after H9N2 subtype avian influenza virus infection in human lung epithelial cells. A total of 17 differentially expressed proteins were successfully identified. The proteins of the NF- kappa B and IFN regulatory factor pathway were identified, indicating that the NF- kappa B and IFN regulatory factor pathway played a role in the mammalian cell process of the H9N2 subtype avian influenza virus infection. The actin and keratin were identified, indicating that the cytoskeleton played an important role in the process of the H9N2 subtype avian influenza virus infection of mammalian cells.
【学位授予单位】：山东农业大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S852.65
本文编号：2151605
[Abstract]:H9N2 subtype avian influenza virus has been widely prevalent in domestic poultry since 1994. Although this subtype belongs to low pathogenicity, it can lead to high morbidity and mortality when secondary pathogens are infected and cause huge loss to poultry breeding. More popular strains become able to bind to the receptors of mammalian cells, be able to infect humans, cause keratitis or slightly similar cold symptoms, and cause public concern for the pathogen. In recent years, studies have shown that the H9N2 subtype avian influenza virus can be used as the internal gene of the.H7N9 and H10N8 subtype influenza viruses of the new influenza virus. It is all from the H9N2 subtype influenza virus. It is more urgent to understand the adaptation process of the H9N2 subtype avian influenza virus infection in mammals. In order to screen out the strains with strong sensitivity to human lung epithelial cells, the sensitivity of this study is judged by indirect immunofluorescence, and 5 H9N2 subtypes of avian influenza kept in our laboratory have been compared. The sensitivity of the virus to human lung epithelial cells was found to be the highest in A/Chicken/Shandong/ch/2011 sensitivity. From the point of view of the protein advanced structure, the structural difference between the hemagglutinin protein difference in the virus strain of human lung epithelial cells was compared with the homologous modeling method. There is a difference in the 198th and 234Th - bit amino acid of the receptor binding site on the first order structure, but there is no difference in the advanced structure. The 336-342 - position amino acid, although the same structure is the same, but the advanced structure is different. The advanced structure of A/Chicken/Shandong/ch/2011 is formed in this position as alpha helix, but A/Chicken/Shandong/W4/2012 and so on. By comparing the glycosylation sites of the 5 virus hemagglutinin proteins, the 4 strains of the virus were found to be different in the 218, 298 and 313 glycosylation sites. To reveal the trend of these glycosylation sites in a large number of virulent strains, the chicken source H9N containing the full length of the hemagglutinin sequence on the East China region of NCBI was selected. The sequence of the 2 subtype influenza virus virus was used to carry out the evolution of glycosylation sites. In the H9N2 subtype influenza virus hemagglutinin fragment, the conservative glycosylation sites have 29 NST, 141 NVS, 298 NTT, 305 NVS, 492 NGT.2006 years later, and the vast majority of the new glycosylation sites, 218 NRT, and 313 bit NCS. are better understood. The molecular mechanism of H9N2 subtype avian influenza virus infection in mammals. This study explored proteins expressed differently at different time points after H9N2 subtype avian influenza virus infection in human lung epithelial cells. A total of 17 differentially expressed proteins were successfully identified. The proteins of the NF- kappa B and IFN regulatory factor pathway were identified, indicating that the NF- kappa B and IFN regulatory factor pathway played a role in the mammalian cell process of the H9N2 subtype avian influenza virus infection. The actin and keratin were identified, indicating that the cytoskeleton played an important role in the process of the H9N2 subtype avian influenza virus infection of mammalian cells.
【学位授予单位】：山东农业大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S852.65
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