基于磁性纳米生物共轭体分离纯化过敏原蛋白的研究

Allergen proteins are antigenic substances that cause allergic reactions and standardized allergens are dependent on the diagnosis and desensitization of allergic diseases. Therefore, the application of single antigen to clinic is of great significance for improving the diagnosis and treatment of allergic reactions in China. The antibodies, epitopes mimic peptides and specific sequences of DNA can recognize the allergen protein with high selectivity and affinity. Meanwhile, the outstanding properties of magnetic nanoparticles further endow them to be able to capture the targets with good stability. The combination of the two features will definitely open a new avenue for highly selective separation and purification of allergen proteins..The present project will focus on the following issues: to prepare and purify antibodies, peptides and nucleic acid by gene engineering technique; to combine the biomolecules and magnetic nanoparticles and to construct a magnetic nanobioconjugate by bioconjugate techniques; to explore the properties of magnetic nanobioconjugate for selective recognition of allergens by optimizing material synthesis and protein adsorption conditions; to study the interaction mechanism between the protein domain and the bioconjugate, and to establish a new method for highly selective separation and purification of allergens in dust mite samples.

过敏原蛋白是引起过敏反应的抗原性物质，过敏性疾病诊断及脱敏疗法都依赖于标准化过敏原，因此将单一抗原用于临床，对提高我国变态反应的诊疗水平有重要的意义。单克隆抗体、表位模拟多肽和特定序列DNA等生物分子对目标过敏原蛋白质具有超高特异性的识别能力，同时磁性纳米粒子以其优越的性能作为基底材料可提高他们捕获靶点的能力，因此将二者结合形成新型的磁性纳米生物共轭体，有望为过敏原蛋白质的高选择性分离纯化开辟新的途径。.本项目旨在运用基因工程等生物技术，将选定的抗体、多肽和核酸制备并纯化出来，通过生物偶联的方法与磁性纳米粒子相结合构建磁性纳米生物共轭体，通过优化材料合成和蛋白质吸附条件探究磁性纳米生物共轭体选择性识别过敏原蛋白性能，研究过敏原蛋白质结构域与生物共轭体之间的相互作用机理，并应用在实际尘螨样品中过敏原蛋白的分离纯化。

鸡蛋中的糖蛋白既是鸡蛋中的营养成分和抗菌活性成分,也是公认的食物过敏原。本项目通过N-(3-二甲基氨基丙基)-N′-乙基碳二亚胺盐酸盐(EDC)，将Aβ1-42嫁接到超支化聚乙烯亚胺(PEI)修饰的磁性纳米粒子上，制备出Aβ1-42偶联的磁性纳米粒子，Aβ1-42@MNP。利用Aβ1-42与糖蛋白潜在的识别作用，将Aβ1-42@MNP作为吸附剂对糖蛋白进行分离纯化的研究。建立了Aβ1-42@MNP在鸡蛋清溶液中高丰度卵清蛋白去除和低丰度糖蛋白分离鉴定的新方法。使用Aβ1-42@MNP对蛋清进行两步提取后，共鉴定出81种蛋白质，而未经任何处理的蛋清中只能鉴定出46种蛋白质。值得关注的是DH结构域蛋白被大量纯化出来，占纯化出蛋白含量的60.38%，说明Aβ1-42与DH结构域蛋白具有潜在的相互作用。该方法的有效性将为扩大低丰度蛋白的鉴定范围提供坚实的基础，并将有助于AD生物标志物的发现。该实验对促进糖蛋白组学的基础研究有重要意义，拓展基于多肽生物共轭体在蛋白质分离纯化领域的应用研究。