建立利用萤光素酶快速检测细胞活性的方法  

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

作　　者：祝静静[1] 鲍秋颖[1] 王宇萌[1] 夏钢[1] 

机构地区：[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

出　　处：《华东师范大学学报（自然科学版）》2012年第5期45-53,84,共10页Journal of East China Normal University(Natural Science)

基　　金：上海市浦江人才计划(08PJ1404100)

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.The firefly luciferase gene was PCR-amplified from plasmid pGL4.26 and subcloned into a bacterial overexpression vector pET24a. Expressed lueiferase fusion protein was purified using Ni-NTA affinity chromatograph and its activity was confirmed using Bright-GloTM 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.

关 键 词：萤火虫萤光素酶 蛋白表达 蛋白纯化 细胞活性 

分 类 号：Q814.9[生物学—生物工程]