人穿孔素在sf9昆虫细胞中的表达和纯化

基础医学与临床 ›› 2020, Vol. 40 ›› Issue (1): 54-59.

人穿孔素在sf9昆虫细胞中的表达和纯化

陈风晔, 刘孟雨, 周雅博, 梁晓雨, 黄波, 解静^*

中国医学科学院基础医学研究所北京协和医学院基础学院医学分子生物学国家重点实验室免疫学系, 北京 100005

收稿日期:2019-10-12 修回日期:2019-11-22 出版日期:2020-01-05 发布日期:2019-12-27
通讯作者: ^*xiejing@ibms.pumc.edu.cn
基金资助:
国家自然科学基金(81802847,81702825)

Expression and purification of human perforin by sf9 insect cells

CHEN Feng-ye, LIU Meng-yu, ZHOU Ya-bo, LIANG Xiao-yu, HUANG Bo, XIE Jing^*

National Key Laboratory of Medical Molecular Biology, Department of Immolunology, Institute of Basic Medical Sciences CAMS & School of Basic Medicine PUMC, Beijing 100005, China

Received:2019-10-12 Revised:2019-11-22 Online:2020-01-05 Published:2019-12-27
Contact: ^*xiejing@ibms.pumc.edu.cn

摘要/Abstract

摘要： 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。

关键词: 蛋白纯化, 人穿孔素, 蛋白活性, 打孔

Abstract: 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.

Key words: protein purification, perforin, protein activity, pore-formation

中图分类号:

陈风晔, 刘孟雨, 周雅博, 梁晓雨, 黄波, 解静. 人穿孔素在sf9昆虫细胞中的表达和纯化[J]. 基础医学与临床, 2020, 40(1): 54-59.

CHEN Feng-ye, LIU Meng-yu, ZHOU Ya-bo, LIANG Xiao-yu, HUANG Bo, XIE Jing. Expression and purification of human perforin by sf9 insect cells[J]. Basic & Clinical Medicine, 2020, 40(1): 54-59.

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