钙库调控的钙通道Orai1体外研究方法的建立

Establishment of an in Vitro Method to Study Store-Operated Calcium Channel Orai1

目的：建立钙通道Orai1的体外研究方法。方法：利用脂质体重组技术,将体外纯化的Orai1蛋白重组到脂质体膜上,利用蔗糖密度梯度离心来检测其重组效率及Orai1蛋白在脂质体膜上的结构,并利用钙染料Fura-2检测脂质体内钙离子的释放。结果：成功制备了脂质体及体外纯化了GST-Orai1融合蛋白,蔗糖密度梯度离心结果证明GST-Orai1蛋白成功重组到脂质体上,以及Orai1蛋白以多聚体的形式定位在脂质体膜上。钙离子释放实验证明脂质体内钙离子包装完好,可用于后续Orai1钙通道的功能研究。结论：利用脂质体重组技术建立了一种新的Orai1的研究方法,能够更直接有效地研究其功能及其活化机制。

Objective： To establish an in vitro method of calcium channel Orai1. Methods： The purified orai1 protein was reconstituted into liposome membrane by the liposome reconstitution technology,and the recombinant efficiency and the structure of Orai1 protein on the liposome membrane were detected by sucrose density gradient centrifugation,and the release of calcium in the liposome was detected by calcium dye Fura-2.Results： The liposomes were successfully prepared and the GST-Orai1 fusion protein was purified in vitro. The sucrose density gradient centrifugation showed that the GST-Orai1 protein was successfully reconstituted into liposomes,and the Orai1 protein was located on the liposome membrane in the form of polymer. Calcium release experiments showed that calcium ions were loaded into the liposomes and could be used for functional study of Orai1 calcium channels. Conclusion： A new method of Orai1 was established to study its function and activation mechanism more directly and effectively.