5株H9N2亞型AIV血凝素結(jié)構(gòu)分析及感染A549細胞的蛋白質(zhì)組學研究
發(fā)布時間：2018-07-28 21:18
【摘要】：H9N2亞型禽流感病毒自1994年以來在中國大陸地區(qū)的家禽中廣泛流行,雖然該亞型屬于低致病性,但當繼發(fā)其它病原感染時,能導致很高的發(fā)病率和死亡率,給家禽養(yǎng)殖造成巨大損失。不僅如此,該亞型病毒血凝素的氨基酸序列的變化,使更多的流行株變得能夠與哺乳動物細胞的受體結(jié)合,能夠傳染給人,引起角膜炎或輕度類似感冒癥狀,引起公眾對該病原的關(guān)注。近幾年的研究表明H9N2亞型禽流感病毒可以作為新型流感病毒的來源。H7N9和H10N8亞型流感病毒的內(nèi)部基因均是來自H9N2亞型流感病毒。了解H9N2亞型禽流感病毒感染哺乳動物的適應(yīng)過程顯得更加迫切。為篩選出對人肺臟上皮細胞敏感性較強的毒株,本研究通過間接免疫熒光的結(jié)果判斷敏感性的高低,通過比較本實驗室保存的5株H9N2亞型禽流感病毒對人肺臟上皮細胞的敏感性發(fā)現(xiàn)A/Chicken/Shandong/ch/2011敏感性最高。本研究從蛋白高級結(jié)構(gòu)角度出發(fā),采用同源建模的方法,比較了易于和不易感染人肺臟上皮細胞的毒株血凝素蛋白差異氨基酸所造成的結(jié)構(gòu)差異。血凝素蛋白在一級結(jié)構(gòu)上受體結(jié)合位點的第198和234位氨基酸有差別,但這兩處在高級結(jié)構(gòu)上沒有差異。第336-342位氨基酸雖然一級結(jié)構(gòu)相同,但是高級結(jié)構(gòu)有差異,A/Chicken/Shandong/ch/2011的高級結(jié)構(gòu)在這個位置形成了α-螺旋,但A/Chicken/Shandong/W4/2012等其他4株病毒在這個位置卻是無規(guī)則卷曲。通過比較5株病毒血凝素蛋白糖基化位點的差異,發(fā)現(xiàn)在218位,298位和313位糖基化位點有差異。為揭示這些糖基化位點在大量毒株中的變化趨勢,選取了NCBI上華東地區(qū)包含血凝素序列全長的雞源H9N2亞型流感病毒病毒序列,進行糖基化位點進化情況的統(tǒng)計。發(fā)現(xiàn)H9N2亞型流感病毒血凝素片段中,保守的糖基化位點有29位NST,141位NVS,298位NTT,305位NVS,492位NGT。2006年之后的毒株,絕大多數(shù)新增兩個糖基化位點,218位NRT,313位NCS。為更好地了解H9N2亞型禽流感病毒跨種感染哺乳動物的分子機制,本研究采用雙向電泳技術(shù)對H9N2亞型禽流感病毒感染人肺臟上皮細胞后不同時間點差異表達的蛋白質(zhì)進行了探索。結(jié)合質(zhì)譜鑒定技術(shù),共有17個差異表達的蛋白質(zhì)被成功鑒定。位于位于NF-κB和IFN調(diào)節(jié)因子通路的蛋白被鑒定,表明NF-κB和IFN調(diào)節(jié)因子通路在H9N2亞型禽流感病毒感染哺乳動物細胞過程起到了作用。肌動蛋白和角蛋白被鑒定,表明細胞骨架在H9N2亞型禽流感病毒感染哺乳動物細胞過程中發(fā)揮了重要作用。
[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.
【學位授予單位】：山東農(nóng)業(yè)大學
【學位級別】：碩士
【學位授予年份】：2015
【分類號】：S852.65
本文編號：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.
【學位授予單位】：山東農(nóng)業(yè)大學
【學位級別】：碩士
【學位授予年份】：2015
【分類號】：S852.65
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相關(guān)碩士學位論文 前1條
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，本文編號：2151605
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