基于卵粘蛋白糖基化结构解析的抗病毒活性分子机制研究

It has been emphasized to research on the biological function of natural active protein in food field. In this project, the anti-virus activity of ovomucin will be studied based on the glycosylation analysis. Firstly, the glycosylation information of composition, content, structure and sequence of monosaccharide in N-linked and O-linked carbohydrate chain will be analyzed based on the Glycoproteomics and lectin microarrays. Before and after the cut off of glycan, the α, β-subunit molecular weight will be gained by MODAL-TOF-MS. The N-glycosylation and O-glycosylation sites will be identified by fast atom bombardment mass spectrometry, HPLC-MS, electron spray ionization mass spectrometry and Infrared multiple photon dissociation mass spectrometry. Then, the interaction and interaction force between ovomucin active fragments and viral surface antigen will be detected by atomic force microscope single-molecule force spectroscopy. The association, dissociation process will be analyzed by surface plasmon resonance. The number of binding sites and thermodynamic characteristics as changes of entropy and enthalpy during the interaction between both will be analyzed by isothermal titration calorimetry. The dual polarisation interferometry will be used for the binding mode and conformational changes. Moreover, the active mode and site will be found out , the anti-virus molecular mechanism in will be revealed. The study will not only provide insight into developing avian natural active proteins but also offer novel ideas and theoretical basis about the design and screen of antivirus natural substances.

天然活性蛋白质功能的研究已成为食品领域的研究重点。本项目以鸡蛋卵粘蛋白为对象，采用糖蛋白组学技术、凝集素芯片解析卵粘蛋白N-linked和O-linked糖链的单糖组成、含量、结构序列等糖基化信息；运用MODAL-TOF-MS质谱仪得到脱糖前后α、β亚基分子量；利用快原子轰击质谱、HPLC-MS、电喷雾电离质谱、红外多光子断裂串级质谱等，确定N-、O-糖基化位点；应用原子力显微镜单分子力谱技术测定病毒表面抗原与卵粘蛋白活性片段的相互作用及作用力；表面等离子体共振分析其结合、解离过程；通过等温滴定量热分析结合的焓变、熵变等热力学特征及结合位点数；利用双偏振极化干涉研究其结合方式与构象变化，揭示作用方式与活性位点，阐明糖链抗病毒作用与构效关系，解析卵粘蛋白抗病毒分子机制，为禽蛋天然活性蛋白的应用及抗病毒活性物质的设计与筛选提供新思路与理论基础。

β-消去反应得出O型糖基化的潜在位点Thr和Ser在β-消去反应发生后显著减少，其中Thr含量降低57.6%，Ser含量降低53.5%，这证明超过半数的Thr/Ser发生了O糖基化修饰。OVM结构与功能分析发现，含有两个亚基，其中α亚基mucin5B分子量230KDa；β亚基mucin6分子量132KDa，有1条信号肽，为分泌蛋白。在mucin5B的 N末端没有发现乙酰化位点，mucin6在第二位S和第三位T上共有2个位点。糖基化方面mucin5B上存在75个N糖基化潜在位点，58个O糖基化潜在位点；mucin6上存在52个N糖基化潜在位点，54个O糖基化潜在位点。.排阻色谱测定OVM表观分子量为633 kDa，糖链部分的分子量为267.9 kDa，占总蛋白的42.29%。将OVM酶解并去糖基化处理，利用HPLC-ESI-MS/MS技术鉴定出α亚基糖基化位点存在N型21个，O型230个；鉴定β亚基糖基化位点，N型位点8个，O型位点88个。在糖链组成上，mucin5B中共鉴定到的1427条糖链，其中有509条含有神经氨酸，占总糖链的35.67%；mucin6中共鉴定到的262条的糖链，其中有78条含有神经氨酸，占总糖链的29.80%。.研究OVM含有59.1%的α2-6键和40.9%的α2-3键。ELISA实验证明H5N1与H1N1的血凝素皆可与OVM结合，而去除神经氨酸之后，OVM与血凝素的结合能力显著下降，其中对H5N1血凝素的结合能力下降了71.5%，而与H1N1血凝素的结合能力也减弱64.25%。竞争实验表明，OVM其他糖链组分如半乳糖，岩藻糖，甘露糖等不参与同血凝素的结合。研究发现游离神经氨酸与OVM混合后，使得OVM与血凝素的相互作用显著的增强，其响应值超过5倍以上（P<0.001），提高了OVM与流感病毒血凝素结合的能力。去除神经氨酸后，OVM的α螺旋减少1.7%，而无规卷曲增加1.2%。荧光表明去除神经氨酸导致OVM中Trp和Tyr暴露，内源荧光增强。.鉴定了神经氨酸亚型为NeuAc。OVM显著抑制病毒基因在A549细胞中的表达降84.9%，去糖链后的作用减弱甚至消失，与病毒组无显著性差异。WB实验显示，受OVM抑制病毒NP蛋白的表达量显著下降。免疫荧光OVM与血凝素发生特异性相互作用，导致病毒不能侵染细胞。去糖基化OVM与血凝素的结合减弱。透射