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

建立利用萤光素酶快速检测细胞活性的方法

1.
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

详细信息

中图分类号: Q814.9
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出版历程
- 收稿日期: 2011-06-01
- 修回日期: 2011-09-01
- 刊出日期: 2012-09-25

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

1.
Key Laboratory of Brain Functional Genomics, Ministry of Education, Shanghai Key Laboratory of Brain Functional Genomics, East China Normal University, Shanghai 200062, China

摘要

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.
- 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.
- firefly luciferase /
- protein expression /
- protein purification /
- cell viability assay

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参考文献(1)

[1]

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