人穿孔素在sf9昆虫细胞中的表达和纯化

目的建立重组人穿孔素的表达和快速纯化方法。方法在sf9昆虫细胞内表达带His标签人穿孔素。使用人工制作简易的镍(Ni)柱装置进行蛋白质亲和层析。采用考马斯亮蓝染色、银染法及流式细胞计量术纯化的蛋白分析其理化性质;用人乳腺癌细胞MCF-7的PI染色法鉴定其生物学活性。结果纯化出的穿孔素具有在细胞膜打孔的生物学活性。与常规蛋白纯化方法如离子交换色谱、分子筛和疏水作用层析等方法相比,该方法操作简便、成本低,为人穿孔素接下来的研究以及其他蛋白质的纯化提供一定参考。结论以一种简单的方法在sf9昆虫细胞内表达并纯化出了有活性的人穿孔素。

Expression and purification of human perforin by sf9 insect cells

Objective To optimize the purification method of human perforin.Methods Perforin with His tag was expressed by sf9 insect cells, and then protein affinity chromatography was performed by Ni column device. The physicochemical property and biological activity of the purified protein was analyzed by coomassie brilliant blue staining, silver staining and flow cytometry. Results This method was more convenient and economical than traditional protein purification methods like ion exchange chromatography, molecular sieve and hydrophobic interaction chromatography, which providing new ideas for further research of perforin and purification of other protein. Conclusions Perforin can be expressed by sf9 insect cells and is purified by a simple method with biological activity.