基于微流控芯片的Cx43巨型脂蛋白体的制备及分析方法研究

In order to overcome the shortcomings of the in vivo protein expression strategy, an artificial method is used to integrate membrane proteins onto the giant liposome membrane, which can construct a giant proteoliposome model to simulate the biological membrane and membrane protein structure. This method has important value in some basic studies on cells, organelles, membrane proteins, etc. as well as some application studies such as targeted delivery, target identification, and so on. However, the existing method is low efficiency, poor controllability, wide particle size distribution, as well as difficult for analysis and evaluation, which limit its extensive application. In this project, a novel microfluidic method is proposed for high-efficiency construction and evaluation of giant proteoliposomes. Precise microfluidic way is used to realize fast, efficient, controllable construction of giant proteoliposomes. Several microfluidic modules for collection, purification, evaluation, and so on, are integrated to improve efficiency and avoid pollution. The gap junction protein Connexin 43 (Cx43) is selected as the model for membrane protein research. It is the membrane channel structure for direct cell-cell communication, and it is closely related to many pathological and physiological processes. However, the detailed mechanism of action is still not clear, so the study on Cx43 is of significant importance. Through efficient and controllable construction of the Cx43 giant proteoliposomes, dielectrophoretic collection and purification, as well as on-chip analysis and evaluation, a routinely available microfluidic method can be finally established for the preparation and analysis of giant proteoliposomes.

为弥补膜蛋白相关研究中体内蛋白表达策略的不足，用人工方法将膜蛋白整合到巨型脂质体膜上可以构建出能仿真生物膜及膜上蛋白结构的巨型脂蛋白体模型。该方法在细胞、细胞器、膜蛋白等基础研究，靶向传输、靶点识别等应用研究中均有重要价值。但是，现有方法效率低、可控性差、粒径分布广、分析评价难，限制其广泛应用。本课题拟建立一种新颖的微流控巨型脂蛋白体高效构建和分析评价方法。利用精确的微流控手段实现巨型脂蛋白体的快速、高效、可控制备，并集成收集、纯化、分析评价等多个微流控模块，提高效率、避免污染。选用间隙连接蛋白Cx43为膜蛋白研究模型，它是细胞间直接进行物质交流的膜通道，与许多病理生理过程密切相关。但具体作用机制仍不清晰，对其研究具有重要意义。通过Cx43巨型脂蛋白体的高效、可控制备，介电电泳收集、纯化，在片分析和评价研究，最终建立一种可广泛应用的巨型脂蛋白体制备、分析的微流控方法。

为弥补膜蛋白相关研究中体内蛋白表达策略的不足，用人工方法将膜蛋白整合到巨型脂质体膜上可以构建出能仿真生物膜及膜上蛋白结构的巨型脂蛋白体模型。该方法在细胞、细胞器、膜蛋白等基础研究，靶向传输、靶点识别等应用研究中均有重要价值。但是，现有方法效率低、可控性差、粒径分布广、分析评价难，限制其广泛应用。. 通过三年的研究工作，我们完成了微流控巨型脂蛋白体高效构建和分析评价方法的建立。利用构建的多种微结构阵列和微通道结构，在芯片上实现巨型脂质体的有效制备。利用无细胞蛋白合成法合成Cx43蛋白完成相关验证，并开展了Cx43蛋白与巨型脂质体整合的多种方法研究，实现了Cx43巨型脂蛋白体的制备。在芯片上制备两类含有收集和纯化的结构，在芯片上初步实现Cx43巨型脂蛋白体的收集和纯化。采用多种微电极阵列对Cx43巨型脂蛋白体开展分析和评价研究。研究工作实现了预期目标，巨型脂蛋白体制备、分析的微流控方法的建立，为巨型脂蛋白体模型的广泛应用奠定了坚实的基础。