本文選題：禽流感病毒　切入點(diǎn)：血凝素　出處：《西北大學(xué)》2015年碩士論文

【摘要】：研究背景：禽流感病毒(avian influenza virus, AIV)可以致使許多禽類發(fā)生急性傳染病,一旦感染人類,往往會(huì)引起嚴(yán)重的呼吸道感染甚至導(dǎo)致宿主死亡。流感病毒表面的血凝素(hemagglutinin, HA)與其感染宿主細(xì)胞表面末端為唾液酸(SA)α2-3半乳糖(Gal)或SAa2-6Gal的糖鏈?zhǔn)荏w相結(jié)合,這一生物學(xué)過程標(biāo)志著流感病毒侵染宿主機(jī)體的開始。HA是流感病毒識(shí)別以及結(jié)合宿主細(xì)胞受體,介導(dǎo)病毒和宿主細(xì)胞膜融合進(jìn)而侵入宿主細(xì)胞的關(guān)鍵分子,也是流感病毒中和抗體以及疫苗研制的重要靶標(biāo)。HA既是糖蛋白,又是一種糖結(jié)合蛋白,其糖基化不僅能夠影響流感病毒與宿主受體之間的結(jié)合活性,還能改變病毒的毒力,也能參與流感病毒的逐步演化。HA糖鏈在流感病毒的整個(gè)生命周期中都發(fā)揮著極其重要的作用,因此準(zhǔn)確掌握流感病毒感染宿主時(shí)的HA糖鏈結(jié)構(gòu)及結(jié)構(gòu)變化帶來的影響,有助于進(jìn)一步理解HA糖鏈在流感病毒感染中的重要生物學(xué)功能。然而,目前HA上糖鏈結(jié)構(gòu)的表達(dá)情況知之甚少。因此,有必要對HA的糖鏈結(jié)構(gòu)進(jìn)行解析,希望能夠從HA糖鏈水平上為流感的預(yù)防和疫苗設(shè)計(jì)提供新的靶標(biāo)。實(shí)驗(yàn)方法：應(yīng)用特異性糖磁性微粒(SAa2-3Gal和SAa2-6Gal糖鏈-磁性微粒復(fù)合物)分別對三株鴨源禽流感病毒HA蛋白進(jìn)行分離純化,獲得各毒株的兩套HA(分別定義為HA2,3和HA2,6),并應(yīng)用凝集素芯片分析其糖鏈表達(dá)譜。隨后用肽苷酶PNGaseF酶將糖鏈從各樣本的HA2,3和HA2,6上釋放下來,并借助濾膜和親水樹脂分離純化HA糖鏈,再用基質(zhì)輔助激光解吸飛行時(shí)間質(zhì)譜(MALDI-TOF/TOF-MS)鑒定其糖鏈結(jié)構(gòu)。兩種技術(shù)互相補(bǔ)充,共同解析鴨源禽流感病毒HA的N-糖鏈譜。結(jié)果及討論：1.SDS-PAGE顯示應(yīng)用SAa2-3Gal和SAa2-6Gal糖鏈-磁性微粒復(fù)合物均能從三種鴨源禽流感病毒株A/Duck/Guangdong/17/2008 (H5N1)、A/Mallard/Jiangxi/16/2005(H5N2)和A/Duck/Guangdong/S-7-134/2004(H9N2)中分離純化出HA2,3和HA2,6。2.應(yīng)用凝集素芯片分析HA糖鏈表達(dá)情況,發(fā)現(xiàn)：(1)源自A/Duck/Guangdong/17/2008(H5N1)毒株的HA2,3和HA2,6共有5種凝集素DSA、RCA120、ACA、PHA-E和SNA顯示較明顯的陽性信號(hào),對應(yīng)高表達(dá)糖鏈為β-D-GlcNAc,(GlcNAcβ1-4)n, Galβ1-4GlcNAc; β-Gal, Galβ-1,4GlcNAc (type Ⅱ), Galβ1-3GlcNAc (type Ⅰ); biantennary N-glycans and Bisecting GlcNAc; Galβ1-3GalNAca-Ser/Thr(T-antigen);Sia2-6Galβ1-4Glc(NAc)等,提示這些糖鏈可能與該毒株HA的基本結(jié)構(gòu)功能相關(guān)。此外,識(shí)別糖鏈Galβ1-3GalNAc,Terminal GalNAc的BPL在H5N1HA2,3顯示明顯陽性信號(hào),而識(shí)別糖鏈Terminala-1,3 mannose的GNA和識(shí)別糖鏈High-Mannose, Manα1-6(Manα1-3)Man,terminal GlcNAc的ConA在H5N1HA2,6上顯示明顯陽性。這些特異高表達(dá)糖鏈可能與HA的受體結(jié)合活性相關(guān)。(2)源白毒株A/Mallard/Jiangxi/16/2005(H5N2)的HA2,3和HA2,6共有6種凝集素RCA-120、DSA、PHA-E、ACA、SNA和 ConA顯示明顯陽性信號(hào),對應(yīng)高表達(dá)糖鏈為β-D-GlcNAc, (GlcNAcβ1-4)n, Galβ1-4GlcNAc;β-Gal, Galβ-1,4GlcNAc (type Ⅱ), Galβ1-3GlcNAc (type Ⅰ); Galβ1-3GalNAca-Ser/Thr(T-antigen); biantennary N-glycansandBisecting GlcNAc; Sia2-6Galβ1-4Glc(NAc); High-Mannose, Mana 1-6(Mana 1-3)Man, terminal GlcNAc等,提示這些糖鏈可能與該毒株HA的基本結(jié)構(gòu)功能相關(guān)。另有識(shí)別糖鏈Terminala-1,3 mannose的凝集素GNA在H5N2 HA2,3上顯示陽性,而識(shí)別糖鏈trimers and tetramers of GlcNAc, core (GlcNAc) of N-glycan, oligosaccharide containing GlcNAc and MurNAc的凝集素STL在H5N2 HA2;6上顯示陽性,提示這些糖鏈可能與HA的受體結(jié)合活性相關(guān)。(3)大多凝集素在A/Duck/Guangdong/S-7-134/2004 (H9N2)的HA2,3和HA2,6上都顯示陽性的熒光信號(hào),其中AAL、RCA120、GNA和SNA以及DBA這5種凝集素表達(dá)熒光信號(hào)呈明顯陽性,而BPL、ACA、PWM等凝集素顯示較強(qiáng)陽性信號(hào),指示高表達(dá)的糖鏈為：Fucal-6 GlcNAc(core fucose),Fucα1-3(Galβ1-4)GlcNAc; β-Gal, Galβ-1,4GlcNAc (type Ⅱ), Galβ1-3GlcNAc (type Ⅰ); Terminala-1,3 mannose; Sia2-6Galβ1-4Glc(NAc)以及αGalNAc, Tn antigen, GalNAcαl-3((Fucal-2))Gal (blood group A antigen)等。與H5N1和H5N2相比,H9N2 HA表達(dá)陽性信號(hào)的凝集素較多,且H9N2的HA2,3和HA2,6糖鏈表達(dá)譜差異不是很明顯,這可能與其較低的致病性有關(guān)。不過,H9N2的HA2,3和HA2,6在糖鏈表達(dá)量上還是有差異的。(4)凝集素RCA120、SNA和ACA在三株流感病毒的HA2,3和HA2,6上都顯示陽性信號(hào),對應(yīng)高表達(dá)糖鏈為β-Gal, Galp-1,4GlcNAc (type Ⅱ), Galβ1-3GlcNAc (type Ⅰ); Sia2-6Galβ1-4Glc(NAc)和Galβ1-3GalNAca-Ser/Thr(Tn)等,提示這些糖鏈可能與流感病毒HA的基本結(jié)構(gòu)或功能相關(guān)。此外,H5N1和H5N2的HA所表達(dá)的糖鏈譜具有一部分相似的高表達(dá)糖鏈,而H9N2與它們的差異還是比較明顯的,這可能是因?yàn)榍皟煞N病毒都屬于H5亞型,同源性較大。3.質(zhì)譜技術(shù)解析流感病毒HA糖鏈,發(fā)現(xiàn)：(1)①H5N1的HA2,3顯示18個(gè)糖鏈峰,HA2,6顯示16個(gè)糖鏈峰,其中共同存在15個(gè)糖鏈峰,HA2,3特異存在3個(gè)糖鏈峰,HA2,6特異存在1個(gè)糖鏈峰；②H5N2的HA2,3顯示17個(gè)糖鏈峰,HA2,6顯示20個(gè)糖鏈峰,共同存在15個(gè)糖鏈峰,HA2,3特異存在2個(gè)糖鏈峰,HA2,6特異存在5個(gè)糖鏈峰；③H9N2的HA2,3顯示20個(gè)糖鏈峰,HA2,6顯示21個(gè)糖鏈峰,共同存在18個(gè)糖鏈峰,HA2,3特異存在2個(gè)糖鏈峰,HA2,6特異存在3個(gè)糖鏈峰。這些糖鏈結(jié)構(gòu)涉及的單糖有甘露糖,半乳糖,巖藻糖,N-乙酰葡糖糖胺,N.乙酰半乳糖胺等,具體糖鏈結(jié)構(gòu)見文中圖譜。這些共同存在的糖鏈結(jié)構(gòu)可能與該毒株HA的基本結(jié)構(gòu)功能相關(guān),而HA2,3和HA2,6特異存在的糖鏈結(jié)構(gòu)可能與HA的受體結(jié)合活性相關(guān)。(2)三個(gè)毒株的6種HA共同存在12個(gè)糖鏈峰,涉及的糖鏈結(jié)構(gòu)可能有Mannose,Galactose,Gal-GlcNAc,GalNAc-GlcNAc,fucose-GlcNAc等,提示這些糖鏈結(jié)構(gòu)可能是維持HA基本結(jié)構(gòu)和功能所需的。此外,即使在同一糖鏈峰其糖鏈表達(dá)強(qiáng)度也是不同的,整體上HA2,3要比HA2,6的表達(dá)強(qiáng)度高一些,這可能與樣本都來自禽源有關(guān)系。(3)凝集素芯片顯示SNA在此三株病毒中均表達(dá),同時(shí)質(zhì)譜鑒定也發(fā)現(xiàn)唾液酸糖鏈結(jié)構(gòu)Sia2-6Galβ1-4Glc(NAc),推測該糖鏈結(jié)構(gòu)可以使病毒之間相互結(jié)合,發(fā)生團(tuán)聚,從而降低病毒的致病力。這可能是許多流感病毒可以長期潛伏于鴨類卻表現(xiàn)低致病力的原因。4.綜合分析,推測流感病毒HA上存在兩套糖鏈系統(tǒng),一套糖鏈系統(tǒng)(命名為HA2,3)可能用于控制流感病毒感染禽類,另一套糖鏈系統(tǒng)(命名為HA2,6)可能用于控制流感病毒感染人類。
[Abstract]:Research background: avian influenza virus (avian influenza, virus, AIV) can cause many avian acute infectious diseases, once infected humans, and even lead to death of the host will often cause severe respiratory infection. The influenza virus hemagglutinin (hemagglutinin, HA) and the infection of the host cell surface terminal sialic acid (SA) - alpha 2-3 lactose (Gal) sugar chain SAa2-6Gal receptor or the combination of the biological process marks the influenza virus infected host organism.HA is influenza virus host cell receptor recognition and binding, mediated by the viral and host cell membrane fusion of key molecules and invade host cells, and is also the influenza virus antibody and vaccine development target.HA is a glycoprotein, is a carbohydrate binding protein, its glycosylation can not only combine active influence between influenza virus and host receptor, can change the disease Virus virulence of influenza virus can also participate in the gradual evolution of the.HA sugar chain in the whole life cycle of influenza virus play an extremely important role, therefore accurately grasp the impact of HA sugar chain structure and structural changes of the host influenza virus infection, have important biological functions help to further understand the HA sugar chain in the influenza virus infection. However, little is the expression of sugar chain structure of HA is known. Therefore, it is necessary to sugar chain structure of HA was analyzed, according to the HA sugar chain level for the prevention of influenza vaccine design and provide a new target. Method: application specific carbohydrate magnetic particles (SAa2-3Gal and SAa2-6Gal sugar chain - magnetic particles) of HA avian influenza virus from duck protein three strains were isolated and purified to obtain the strain of the two sets of HA (defined as HA2,3 and HA2,6), and analyze the application of lectin chip The sugar chain expression. Then release the sugar chain down from the sample HA2,3 and HA2,6 peptide glycoside enzyme PNGaseF, and the separation and purification of HA sugar chain and hydrophilic resin membrane, and matrix assisted laser desorption time-of-flight mass spectrometry (MALDI-TOF/TOF-MS) identification of the sugar chain structure. Two technologies complement each other, HA bird analysis of common influenza virus of duck origin N- sugar chain spectrum. Results and conclusion: 1.SDS-PAGE display using SAa2-3Gal and SAa2-6Gal sugar chain - magnetic particles are from three kinds of duck derived avian influenza virus strain A/Duck/Guangdong/17/2008 (H5N1), A/Mallard/Jiangxi/16/ 2005 (H5N2) and A/Duck/Guangdong/S-7-134/2004 (H9N2) HA2,3 and HA2,6.2. were isolated and purified by lectin microarray analysis of HA sugar chain expression, found that: (1) from A/Duck/Guangdong/17/2008 (H5N1) strains HA2,3 and HA2,6 were 5 RCA120, lectin DSA, ACA, PHA-E and SNA showed that The positive signal significantly, corresponding to the high expression of -D-GlcNAc beta sugar chain (GlcNAc beta 1-4) n, Gal beta 1-4GlcNAc; beta -Gal, Gal beta -1,4GlcNAc (type II), Gal beta 1-3GlcNAc (type 1); biantennary N-glycans and Bisecting GlcNAc Gal 1-3GalNAca-Ser/Thr (T-antigen); beta; beta Sia2-6Gal 1-4Glc (NAc) etc. these tips, and the basic structure of sugar chain may strain HA function. In addition, the identification of sugar chain Gal beta 1-3GalNAc, GalNAc Terminal BPL in H5N1HA2,3 showed significant positive signal, GNA and identification of High-Mannose sugar chain and the identification of the sugar chain Terminala-1,3 mannose, Man 1-6 alpha (Man alpha 1-3) Man, terminal GlcNAc ConA in the H5N1HA2,6 display was positive. High expression of these specific sugar chains may be related with the HA receptor binding activity. (2) the source of white strain A/Mallard/Jiangxi/16/2005 (H5N2) HA2,3 and HA2,6 6 in RCA-120 DSA, coagulation, PHA-E, ACA, SNA and ConA display Positive signal, corresponding to the high expression of -D-GlcNAc beta sugar chain (GlcNAc beta 1-4) n, Gal beta 1-4GlcNAc; beta -Gal, Gal beta -1,4GlcNAc (type II), Gal beta 1-3GlcNAc (type 1); Gal beta 1-3GalNAca-Ser/Thr (T-antigen); biantennary N-glycansandBisecting GlcNAc; Sia2-6Gal beta 1-4Glc (NAc); High-Mannose, Mana 1-6 (Mana 1-3) Man, terminal GlcNAc, suggesting that these sugar chains may the basic structure and function of HA. The strain GNA and Terminala-1,3 lectin sugar chain recognizing mannose in H5N2 HA2,3 positive, and trimers and tetramers of sugar chain recognizing GlcNAc, core (GlcNAc) of N-glycan, oligosaccharide containing GlcNAc and MurNAc the lectin STL in H5N2 HA2; 6 showed positive, suggesting that these sugar chains may activity associated with HA receptors. (3) most of lectin in A/Duck/Guangdong/S-7-134/2004 (H9N2) HA2,3 and HA2, 6 showed positive fluorescence signal, including AAL, RCA120, GNA and SNA and DBA these 5 kinds of lectin fluorescence signal was significantly positive, while BPL, ACA, PWM and other lectins showed strong positive signal, indicating the high expression of sugar chain: Fucal-6 GlcNAc (core fucose), Fuc (alpha Gal beta 1-3 1-4) GlcNAc; beta -Gal, Gal beta -1,4GlcNAc (type II), Gal beta 1-3GlcNAc (type 1); Terminala-1,3 mannose; Sia2-6Gal 1-4Glc (NAc) and alpha beta GalNAc, Tn antigen, GalNAc alpha L-3 ((Fucal-2)) Gal (blood group A antigen). Compared with H5N1 and H5N2, H9N2 HA the expression of lectin more positive signals, and the H9N2 HA2,3 and HA2,6 sugar chain expression difference is not very obvious, which may be related to the low pathogenicity. However, the expression of H9N2 HA2,3 and HA2,6 in the sugar chain still have differences on the amount. (4) SNA lectin RCA120, and ACA in three strains of influenza virus HA2,3 and HA2,6 are 鏄劇ず闃蟲，

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