唾液和血清中抗流感病毒保护性蛋白质的筛选及鉴定

发布时间：2018-06-09 14:28

本文选题：唾液 + 血清　；参考：《西北大学》2013年硕士论文

【摘要】：流行性感冒(Influenza)是一种由流感病毒侵染机体而引发的急性呼吸道传染病。已有研究证实,甲型流感病毒血凝素(HA)分子与流感病毒宿主细胞表面SAα2-3Gal或SAα2-6Gal糖链末端受体的结合,是流感病毒侵染机体的开始。不同流感病毒宿主细胞表面上的糖链受体结构是不同的,禽流感病毒血凝素(HA)主要识别和结合末端为SAα2-3Gal的糖链受体,而人流感病毒血凝素(HA)主要识别和结合末端为SAα2-6Gal的糖链受体。研究报道指出SAα2-6Gal糖链末端结构主要存在于人上呼吸道细胞表面,在人下呼吸道细胞表面仅有少量SAα2-3Gal糖链末端结构的存在。这些发现为抗流感病毒的相关研究提供了全新的思路,提示SAα2-3Gal和SAα2-6Gal糖链结构可以作为分离纯化血凝素,以及能够与血凝素竞争性结合该糖链受体糖结合蛋白的工具。本研究以此为思路,利用SAα2-3Gal和SAα2-6Gal糖链结构分别从健康人唾液和血清中分离及鉴定抗流感病毒保护性蛋白,并结合生物信息学研究手段对这些蛋白质的相关信息进行深入分析,寻找这些蛋白质与糖链SAα2-3Gal和SAα2-6Gal结合的结构域。主要参考本实验室已经建立的羟基化磁性微粒分离纯化糖结合蛋白的方法,利用SAα2-3Gal和SAα2-6Gal糖链修饰的功能化磁性微粒,从健康人唾液和血清中分别分离纯化糖链SAα2-3Gal和SAα2-6Gal结合蛋白(抗流感病毒保护性),并对这些蛋白质进行质谱鉴定；通过质谱数据分析唾液和血清中SAα2-3Gal和SAα2-6Gal结合蛋白的异同,为进一步研究人体内抗流感病毒保护性蛋白质在预防流感传播中的作用提供理论依据；最后利用motif-x基序分析模型,预测了特异性结合SAα2-3Gal和SAα2-6Gal糖链蛋白质的糖结合域。对获得的质谱数据进行整理、筛选、统计和分析,得到以下实验结果：1)从健康人唾液中共鉴定到116个SAα2-3Gal和SAα2-6Gal糖结合蛋白,其中94个SAα2-3Gal糖结合蛋白,83个SAα2-6Gal糖结合蛋白,与这两种糖链都结合的蛋白质有61个,与SAα2-3Gal糖链特异性结合蛋白质有33个,与SAα2-6Gal糖链特异性结合蛋白质有22个；两组唾液糖结合蛋白质的相对分子质量和等电点分布较为类似；由emPAI分析结果可知,Ratio Value(即mol%(SA2-6Gal):mol%(SAα2-3Gal)大于1.5的蛋白质有35个,小于0.66的蛋白质有9个；2)从健康人血清中共鉴定到144个SAα2-3Gal和SAα2-6Gal糖结合蛋白,其中117个SAα2-3Gal糖结合蛋白,115个SAα2-6Gal糖结合蛋白,与这两种糖链都结合的蛋白质有88个,与SAα2-3Gal糖链特异性结合蛋白质有29个,与SAa2-6Gal糖链特异性结合蛋白质有27个；两组血清糖结合蛋白的相对分子质量和等电点分布较为类似；由emPAI分析结果可知,Ratio Value(即mol%(SAα2-6Gal): mol%(SAα2-3Gal))大于1.5的蛋白质有52个,小于0.66的蛋白质有23个；3)统计分析可知,唾液和血清中与这两种糖链同时结合的蛋白质有13种,与SAα2-6Gal糖链同时结合的蛋白质仅有2种,分别为免疫球蛋白G-λ2链C区和免疫球蛋白G-κ链Ⅴ-Ⅳ区：与SAα2-3Gal糖链结合的蛋白质未出现重叠。根据motif-x基序分析可知,SAα2-3Gal糖链特异性结合蛋白质的糖结合潜在特征性糖结合域有44种,而SAα2-6Gal糖链特异性结合蛋白的糖结合潜在特征性糖结合域只有1种。
[Abstract]:Influenza virus (Influenza) is an acute respiratory infection caused by influenza virus infection. It has been proved that the binding of HA molecules with SA alpha 2-3Gal or SA alpha 2-6Gal sugar chain terminal receptor on the surface of the host cell of influenza virus is the beginning of influenza virus infection. The structure of the sugar chain receptor on the surface of the main cell is different, and avian influenza virus hemagglutinin (HA) mainly identifies and combines the sugar chain receptor at the end of SA alpha 2-3Gal, while human influenza virus hemagglutinin (HA) mainly identifies and combines the end of the glucose chain receptor with SA alpha 2-6Gal. The study shows that the terminal structure of the SA alpha 2-6Gal chain is mainly in the human upper respiratory tract. On the surface of the cell, only a small amount of SA alpha 2-3Gal sugar chain terminal structure exists on the surface of the human lower respiratory tract. These findings provide a new idea for the related research of anti influenza virus, suggesting that SA alpha 2-3Gal and SA alpha 2-6Gal sugar chain structure can be used to separate and purify hemagglutinin, and to combine with hemagglutinin to combine the sugar chain receptor with the hemagglutinin. In this study, we use SA alpha 2-3Gal and SA alpha 2-6Gal sugar chain structure to separate and identify anti influenza virus protective proteins from healthy human saliva and serum, and analyze the related information of these proteins in depth with bioinformatics research methods, and find these proteins and sugar chain SA alpha 2-3Gal and S. The domain of A alpha 2-6Gal binding.
We mainly refer to the method of separating and purifying the sugar binding protein by the hydroxy magnetic particles established in our laboratory, and using the functional magnetic particles modified by SA alpha 2-3Gal and SA alpha 2-6Gal sugar chain to separate and purify the sugar chain SA alpha 2-3Gal and SA alpha 2-6Gal in combination with the egg white (anti influenza virus protection) from the healthy human saliva and serum. Mass spectrometric identification and analysis of the similarities and differences of SA alpha 2-3Gal and SA alpha 2-6Gal binding protein in saliva and serum by mass spectrometry, provide a theoretical basis for further research on the role of anti influenza virus protective proteins in the prevention of influenza transmission. Finally, the motif-x based sequence analysis model is used to predict the specific binding of SA alpha 2-3Gal. The sugar binding domain of SA alpha 2-6Gal sugar chain protein.
The obtained mass spectrometry data were collated, screened, statistics and analyzed. 1) 116 SA alpha 2-3Gal and SA alpha 2-6Gal binding proteins were identified from healthy human saliva, of which 94 SA alpha 2-3Gal sugar binding proteins, 83 SA alpha 2-6Gal sugar binding proteins, and 61 proteins combined with these two sugars, and SA alpha 2-3Gal sugar. There are 33 chain specific protein binding proteins and 22 proteins associated with SA alpha 2-6Gal sugar chain specific binding proteins; the relative molecular mass and isoelectric point distribution of the two groups of saliva sugar binding proteins are similar; the results of emPAI analysis show that there are 35 proteins of Ratio Value (mol% (SA2-6Gal): mol% (SA alpha 2-3Gal) greater than 1.5, less than 0.66 of protein 9; 2) 144 SA alpha 2-3Gal and SA alpha 2-6Gal binding proteins were identified from healthy human serum, of which 117 SA alpha 2-3Gal sugar binding proteins, 115 SA alpha 2-6Gal sugar binding proteins, 88 proteins combined with these two sugar chains, 29 with SA alpha 2-3Gal sugar chain specific binding egg white, and specific binding protein of SAa2-6Gal sugar chain. The relative molecular mass and isoelectric point distribution of serum sugar binding proteins in the two groups were similar, and the results of emPAI analysis showed that Ratio Value (mol% (SA alpha 2-6Gal): mol% (SA a 2-3Gal)) had 52 proteins greater than 1.5, 23 protein less than 0.66; 3) statistical analysis showed that saliva and serum were associated with the two sugar chains. There are 13 kinds of proteins combined with the protein of SA alpha 2-6Gal sugar chain. There are only 2 proteins, which are the immunoglobulin G- lambda 2 chain C region and the immunoglobulin G- kappa chain V - IV region: the protein binding to the SA alpha 2-3Gal chain is not overlapped. According to the motif-x motif analysis, the sugar chain specific binding protein of SA alpha 2-3Gal is potential for sugar binding. There are 44 kinds of characteristic sugar binding domains, while the sugar binding potential characteristic sugar binding domain of SA alpha 2-6Gal sugar chain binding protein is only 1.
【学位授予单位】：西北大学
【学位级别】：硕士
【学位授予年份】：2013
【分类号】：Q51;R511.7

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