建立利用萤光素酶快速检测细胞活性的方法

华东师范大学学报(自然科学版) ›› 2012, Vol. 2012 ›› Issue (5): 45-53,84.

建立利用萤光素酶快速检测细胞活性的方法

祝静静，鲍秋颖，王宇萌，夏钢

华东师范大学脑功能基因组学教育部重点实验室、上海市脑功能基因组学重点实验室，上海 200062

收稿日期:2011-06-01 修回日期:2011-09-01 出版日期:2012-09-25 发布日期:2012-09-29

Development of a luciferase-based method for rapid cell viability detection

ZHU Jing-jing, BAO Qiu-ying, WANG Yu-meng, XIA Gang

Key Laboratory of Brain Functional Genomics, Ministry of Education, Shanghai Key Laboratory of Brain Functional Genomics, East China Normal University, Shanghai 200062, China

Received:2011-06-01 Revised:2011-09-01 Online:2012-09-25 Published:2012-09-29

摘要/Abstract

摘要： 运用PCR的方法，从萤火虫萤光素酶基因载体 pGL4.26 扩增萤火虫萤光素酶基因片段，将其插入连接于原核表达载体pET24a 中，构建重组表达载体pET24a-Luc.经酶切鉴定及序列分析后，将重组载体转化到表达菌株大肠杆菌BL21（DE3）中，获得阳性重组菌BL21/pET24a-Luc.IPTG诱导蛋白高效表达并通过镍柱亲和层析纯化萤火虫萤光素酶.该目的蛋白活性用Bright-GloTM试剂进行验证并用于建立一种基于测量ATP含量的检测细胞生物活性的方法.与传统的细胞生物活性检测试剂盒MTT，CCK-8以及Alamar Blue比较，该方法具有反应迅速、活力高、灵敏度好、生产方便的优点，具有实际应用的潜力.

关键词: 萤火虫萤光素酶, 蛋白表达, 蛋白纯化, 细胞活性

Abstract: The firefly luciferase gene was PCR-amplified from plasmid pGL4.26 and subcloned into a bacterial overexpression vector pET24a. Expressed luciferase fusion protein was purified using Ni-NTA affinity chromatograph and its activity was confirmed using Bright-GloTM kit. Based on the ATP-dependency of luciferase reaction, we developed a cell viability assay, which is faster, more convenient, and more sensitive in detect cell viability than generally used MTT, CCK-8 and Alamar Blue methods.

Key words: firefly luciferase, protein expression, protein purification, cell viability assay

中图分类号:

Q814.9

祝静静，鲍秋颖，王宇萌，夏钢. 建立利用萤光素酶快速检测细胞活性的方法[J]. 华东师范大学学报(自然科学版), 2012, 2012(5): 45-53,84.

ZHU Jing-jing, BAO Qiu-ying, WANG Yu-meng, XIA Gang. Development of a luciferase-based method for rapid cell viability detection[J]. Journal of East China Normal University(Natural Sc, 2012, 2012(5): 45-53,84.

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