蜜蜂蜂毒糖蛋白组分析

Honeybee venom is a complicated defensive toxin and has a wide range of pharmacologically active compounds that are useful for human therapeutics. Some compounds, however, can result in pain, local inflammation, irritation and death once the people stung by honeybees. To better utilize as medicinal ingredients, the key issues is that the allergenic reactions need to be reduced to a lowest extent or even completely removed. This demand a better understanding of the honeybee venomic toxicology. Proteomics is an efficiently approach that can investigation of the biological function of proteins on a systematic view. It is known that the protein glycosylation is a major driving force of the allergens in the insect venom. So far, the glycoproteomics analysis of the honeybee venom still remians lacking. In this project, to address the toxic mechanism of the bee venom and relationships between the allergen and pharmaceutical active of the bee venom and their glycosylation modification, complementary proteome strategies will be adopted by two-dimensional gel electrophoresis, shotgun analysis, Hydrazide and Lectin enrichment, follwed by high performance liquid chromatography tandem mass spectrometry and bioinformatics. The study will not only significantly extend the glycoproteome coverage of venom but also add vital new knowledge in understanding the innate biochemical nature of venom at the glycoproteome levels. The result will not only comprehend the honeybee venomic toxicology but also provide the theoretic base for the developing the anti-allergic serum and the efficiently develop pharmacological application as potential drug of the bee venom.

蜜蜂蜂毒（蜂毒）具广泛药理学活性，但粗制蜂毒含过敏原，易引起生物体过敏反应，甚至危及生命。因此理解蜂毒的毒性机理，减缓乃至去除其过敏成份是高效利用蜂毒中有效药用成份所急需解决的关键问题之一。蛋白组学可系统全面阐述蛋白质在生命活动过程中的功能和作用机制。蜂毒中蛋白糖基化修饰与其毒性机理（如过敏原的过敏性）密切相关。迄今为止，国内外利用糖蛋白组学方法对蜂毒的毒性机理研究还未见报道。本研究拟采用糖蛋白组学的研究手段，利用富集效率互补的糖蛋白/糖肽富集方法（酰肼法、凝集素法）和功能互补的高灵敏度高分辨率液质联用仪器，结合生物信息学分析，以及生物大分子互作、体外细胞实验等蛋白功能研究手段，深入研究蜂毒蛋白/多肽的糖基化修饰特点与其过敏性和药用活性。这将为探明蜂毒毒理学机理，研制抗蜂毒过敏血清和蜂毒蛋白有效药用成份的高效利用提供理论依据。

蜂毒主要是由蜜蜂毒腺分泌的蛋白和多肽类物质组成，具有多种药理活性，是开发临床药物的天然宝库。但是，蜂蜇后，轻则造成局部红肿、发热、疼痛，重则过敏性休克危及生命。因此如何合理利用蜂毒的有效药用成份，减缓乃至彻底去除其过敏成份对生物体的危害，是目前急需要解决的问题。蛋白质糖基化修饰与其生化特征和生物学活性和功能密切相关。对毒素而言，糖基化修饰与过敏原的过敏性密切相关。尽管意蜂是我国饲养的重要蜂种，但是从糖蛋白质组学的角度研究我国主要饲养蜂种蜂毒的毒性机理还未进行深入研究。本研究采用糖蛋白组学的研究手段，利用富集效率互补的糖蛋白/糖肽富集方法（IMAC法、凝集素法）和高灵敏度高分辨率液质联用仪器（Thermo Q-Exactive LC MS/MS），结合生物信息学分析，体外细胞实验等蛋白功能研究手段，深入研究蜂毒蛋白/多肽的糖基化修饰特点与其过敏性和药用活性，共从意蜂蜂毒样品中分析出19个糖基化修饰蛋白。其中有5个蛋白含有两个糖基化位点，其余蛋白均仅有一个糖基化位点被鉴定出来。并利用在线的motif分析软件WebLogo对蜂毒中N-糖基化位点的保守序列分析发现，蜂毒中约2/3的N-糖肽属于N-X-T motif，剩余的1/3属于N-X-S motif。这项研究为深入了解蜂毒的毒理学机理和研制相应抗过敏血清及合理利用蜂毒蛋白中有效药用成份，提供理论依据和实践基础，也对保护人类健康具有重要意义。