新型磁性分子印迹复合颗粒可控制备方法的建立及其对活性蛋白的富集性能与识别机理研究

Active protein was a kind of protein with particular functional activities which has great values for the investigation and application. However, the operations of traditional extracting processes of active proteins were complicated and the efficiency, specificity and activity preservation rate were not good enough. In order to solve these problems, magnetic nanoparticles and molecularly imprinted technology were combined together in this study. Magnetic molecularly imprinted composite particles with core-shell structures were produced for the target active protein. The enrichment performances and recognition mechanisms of composite particles to the target active proteins were investigated particularly. The absorptive dynamic and thermodynamic properties and selectivities of composite particles to the target active proteins were defined. The variations of configurations and activities of active proteins during the enrichment procedure were clarified in this investigation. The binding sites and recognition driving forces of composite particles to the target active proteins were also confirmed. On this basis, a prediction model for the enrichment performances of composite particles to the target active protein was constructed. The novel magnetic molecularly imprinted composite particles with excellent enrichment performance to the target active proteins were controllable prepared aiming at the configurations of target proteins according to this prediction model. The target active proteins in actual complicated system could be purified rapidly and efficiently using the controllable prepared composite particles. Meanwhile, the controllable prepared composited particles could also have a great potential in the enrichment and purification of other biological macromolecules in actual complicated system.

活性蛋白是一大类具有独特功能活性的蛋白质，具有重要的研究和实用价值。目前活性蛋白的分离提取存在操作复杂、效率低、选择性差、活性保留率低等问题。针对这些问题，本项目将磁性纳米颗粒与分子印迹技术相结合，制备针对目标活性蛋白的具有核-壳结构的磁性分子印迹复合颗粒，对复合颗粒对目标活性蛋白的富集性能和识别机理进行详细研究，明确复合颗粒对目标活性蛋白的吸附动力学、吸附热力学特性和吸附选择性，确定目标活性蛋白在富集纯化过程中的构象与活性变化规律，明确目标活性蛋白与复合颗粒的结合位点与识别驱动力，构建复合颗粒对目标活性蛋白富集性能的预测模型，依据模型的指导实现对目标活性蛋白具有优良富集性能的新型磁性分子印迹复合颗粒的可控制备。通过本项目的研究可根据目标蛋白的结构可控制备磁性分子印迹复合颗粒，实现复杂体系中目标活性蛋白的快速高效富集纯化，同时为复杂体系中其它生物大分子的快速高效富集纯化提供新的研究思路。

蛋白质是食品中的重要组成成分之一，通常具有较高的营养价值和优良的功能特性，蛋白质的分离纯化是蛋白质研究的基础和前提。针对传统蛋白质分离提取过程中存在的操作复杂、效率低、选择性差、活性保留率低等问题，本项目将磁性纳米颗粒与分子印迹技术相结合，选择适宜的制备条件，成功制备了针对目标蛋白的具有核-壳结构的磁性分子印迹复合颗粒；采用多种方法对制备的磁性分子印迹复合颗粒进行了表征，验证了官能团在颗粒上的成功修饰；研究了环境条件对磁性分子印迹复合颗粒对目标蛋白富集性能的影响规律，探讨了复合颗粒对目标蛋白的识别机理和识别驱动力；构建了复合颗粒对目标蛋白的吸附动力学和等温吸附模型；研究了环境条件对实际蛋白体系理化与功能特性的影响规律，为复合颗粒对实际体系中目标蛋白的富集性能及蛋白功能性预测提供理论指导。通过本项目研究制备的磁性分子印迹复合颗粒结合了磁性纳米颗粒分离简单快速和分子印迹技术选择性高的优点，对目标蛋白富集具有较高的特异性，并且具有良好的重复使用性，通过选择适宜的颗粒制备及富集纯化条件，可以用于实际复杂体系中目标蛋白的快速高效富集纯化，同时也为复杂体系中其它生物大分子的快速高效富集纯化提供了新的研究思路。研究具有较好的理论研究意义和实际应用价值。