鸭源禽流感病毒血凝素N-糖链谱的研究

发布时间：2018-03-25 22:48

  本文选题：禽流感病毒　切入点：血凝素　出处：《西北大学》2015年硕士论文

【摘要】：研究背景：禽流感病毒(avian influenza virus, AIV)可以致使许多禽类发生急性传染病,一旦感染人类,往往会引起严重的呼吸道感染甚至导致宿主死亡。流感病毒表面的血凝素(hemagglutinin, HA)与其感染宿主细胞表面末端为唾液酸(SA)α2-3半乳糖(Gal)或SAa2-6Gal的糖链受体相结合,这一生物学过程标志着流感病毒侵染宿主机体的开始。HA是流感病毒识别以及结合宿主细胞受体,介导病毒和宿主细胞膜融合进而侵入宿主细胞的关键分子,也是流感病毒中和抗体以及疫苗研制的重要靶标。HA既是糖蛋白,又是一种糖结合蛋白,其糖基化不仅能够影响流感病毒与宿主受体之间的结合活性,还能改变病毒的毒力,也能参与流感病毒的逐步演化。HA糖链在流感病毒的整个生命周期中都发挥着极其重要的作用,因此准确掌握流感病毒感染宿主时的HA糖链结构及结构变化带来的影响,有助于进一步理解HA糖链在流感病毒感染中的重要生物学功能。然而,目前HA上糖链结构的表达情况知之甚少。因此,有必要对HA的糖链结构进行解析,希望能够从HA糖链水平上为流感的预防和疫苗设计提供新的靶标。实验方法：应用特异性糖磁性微粒(SAa2-3Gal和SAa2-6Gal糖链-磁性微粒复合物)分别对三株鸭源禽流感病毒HA蛋白进行分离纯化,获得各毒株的两套HA(分别定义为HA2,3和HA2,6),并应用凝集素芯片分析其糖链表达谱。随后用肽苷酶PNGaseF酶将糖链从各样本的HA2,3和HA2,6上释放下来,并借助滤膜和亲水树脂分离纯化HA糖链,再用基质辅助激光解吸飞行时间质谱(MALDI-TOF/TOF-MS)鉴定其糖链结构。两种技术互相补充,共同解析鸭源禽流感病毒HA的N-糖链谱。结果及讨论：1.SDS-PAGE显示应用SAa2-3Gal和SAa2-6Gal糖链-磁性微粒复合物均能从三种鸭源禽流感病毒株A/Duck/Guangdong/17/2008 (H5N1)、A/Mallard/Jiangxi/16/2005(H5N2)和A/Duck/Guangdong/S-7-134/2004(H9N2)中分离纯化出HA2,3和HA2,6。2.应用凝集素芯片分析HA糖链表达情况,发现：(1)源自A/Duck/Guangdong/17/2008(H5N1)毒株的HA2,3和HA2,6共有5种凝集素DSA、RCA120、ACA、PHA-E和SNA显示较明显的阳性信号,对应高表达糖链为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,提示这些糖链可能与该毒株HA的基本结构功能相关。此外,识别糖链Galβ1-3GalNAc,Terminal GalNAc的BPL在H5N1HA2,3显示明显阳性信号,而识别糖链Terminala-1,3 mannose的GNA和识别糖链High-Mannose, Manα1-6(Manα1-3)Man,terminal GlcNAc的ConA在H5N1HA2,6上显示明显阳性。这些特异高表达糖链可能与HA的受体结合活性相关。(2)源白毒株A/Mallard/Jiangxi/16/2005(H5N2)的HA2,3和HA2,6共有6种凝集素RCA-120、DSA、PHA-E、ACA、SNA和 ConA显示明显阳性信号,对应高表达糖链为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,提示这些糖链可能与该毒株HA的基本结构功能相关。另有识别糖链Terminala-1,3 mannose的凝集素GNA在H5N2 HA2,3上显示阳性,而识别糖链trimers and tetramers of GlcNAc, core (GlcNAc) of N-glycan, oligosaccharide containing GlcNAc and MurNAc的凝集素STL在H5N2 HA2;6上显示阳性,提示这些糖链可能与HA的受体结合活性相关。(3)大多凝集素在A/Duck/Guangdong/S-7-134/2004 (H9N2)的HA2,3和HA2,6上都显示阳性的荧光信号,其中AAL、RCA120、GNA和SNA以及DBA这5种凝集素表达荧光信号呈明显阳性,而BPL、ACA、PWM等凝集素显示较强阳性信号,指示高表达的糖链为：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。与H5N1和H5N2相比,H9N2 HA表达阳性信号的凝集素较多,且H9N2的HA2,3和HA2,6糖链表达谱差异不是很明显,这可能与其较低的致病性有关。不过,H9N2的HA2,3和HA2,6在糖链表达量上还是有差异的。(4)凝集素RCA120、SNA和ACA在三株流感病毒的HA2,3和HA2,6上都显示阳性信号,对应高表达糖链为β-Gal, Galp-1,4GlcNAc (type Ⅱ), Galβ1-3GlcNAc (type Ⅰ); Sia2-6Galβ1-4Glc(NAc)和Galβ1-3GalNAca-Ser/Thr(Tn)等,提示这些糖链可能与流感病毒HA的基本结构或功能相关。此外,H5N1和H5N2的HA所表达的糖链谱具有一部分相似的高表达糖链,而H9N2与它们的差异还是比较明显的,这可能是因为前两种病毒都属于H5亚型,同源性较大。3.质谱技术解析流感病毒HA糖链,发现：(1)①H5N1的HA2,3显示18个糖链峰,HA2,6显示16个糖链峰,其中共同存在15个糖链峰,HA2,3特异存在3个糖链峰,HA2,6特异存在1个糖链峰；②H5N2的HA2,3显示17个糖链峰,HA2,6显示20个糖链峰,共同存在15个糖链峰,HA2,3特异存在2个糖链峰,HA2,6特异存在5个糖链峰；③H9N2的HA2,3显示20个糖链峰,HA2,6显示21个糖链峰,共同存在18个糖链峰,HA2,3特异存在2个糖链峰,HA2,6特异存在3个糖链峰。这些糖链结构涉及的单糖有甘露糖,半乳糖,岩藻糖,N-乙酰葡糖糖胺,N.乙酰半乳糖胺等,具体糖链结构见文中图谱。这些共同存在的糖链结构可能与该毒株HA的基本结构功能相关,而HA2,3和HA2,6特异存在的糖链结构可能与HA的受体结合活性相关。(2)三个毒株的6种HA共同存在12个糖链峰,涉及的糖链结构可能有Mannose,Galactose,Gal-GlcNAc,GalNAc-GlcNAc,fucose-GlcNAc等,提示这些糖链结构可能是维持HA基本结构和功能所需的。此外,即使在同一糖链峰其糖链表达强度也是不同的,整体上HA2,3要比HA2,6的表达强度高一些,这可能与样本都来自禽源有关系。(3)凝集素芯片显示SNA在此三株病毒中均表达,同时质谱鉴定也发现唾液酸糖链结构Sia2-6Galβ1-4Glc(NAc),推测该糖链结构可以使病毒之间相互结合,发生团聚,从而降低病毒的致病力。这可能是许多流感病毒可以长期潜伏于鸭类却表现低致病力的原因。4.综合分析,推测流感病毒HA上存在两套糖链系统,一套糖链系统(命名为HA2,3)可能用于控制流感病毒感染禽类,另一套糖链系统(命名为HA2,6)可能用于控制流感病毒感染人类。
[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 鏄剧ず闃虫，

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