纳米荧光素酶的表达、纯化及性质鉴定  

Expression,Purification and Characterization of Nano Luciferase in Escherichia coli

作　　者：侯家鹏 丁澦[1] 

机构地区：[1]复旦大学生命科学学院,上海200438

出　　处：《云南农业大学学报（自然科学版）》2017年第2期191-196,共6页Journal of Yunnan Agricultural University:Natural Science

基　　金：国家自然科学基金项目(31470764)

摘　　要：由荧光素酶(luciferase)催化的一大类发光反应被统称为生物发光(bioluminescence),该反应具有背景低、灵敏度高、线性范围广等特点,是高灵敏检测的发展方向之一。课题组在近期的研究中全基因合成了一种纳米荧光素酶(nano luciferase),研究清楚了其理化性质的基础上,将其作为一种新型的生物发光检测工具。将荧光素酶基因克隆至原核高表达载体上,通过自动诱导系统进行了表达。通过Ni-NTA亲和层析纯化和分子筛将重组纳米荧光素酶进行纯化,其产量可稳定达到每升菌25.3 mg,纯度超过98%;通过酶活实验鉴定了纳米荧光素酶的活性,在1.0×10^(-4)~1.0×10^(-7)mg/m L的范围内,该酶均具有良好的线性反应,其线性检测下限低至1.0×10^(-7)mg/m L(约5pmol)。纳米荧光素酶具有灵敏度高、数据重复性好、操作简便、稳定性高、易于保存等多重优点,可利用其作为基础平台,开发具有应用前景的超灵敏新型生物发光探针。A large group of luminescence reactions catalyzed by luciferase are called bioluminescence. Since bioluminescence has been characterized as low background, high sensitivity and wide linear dy- namic range. It is applied widely in sensitive molecular biology detection. In this research, we have synthesized a novel luciferase （nano lueiferase） by whole gene synthesis, to make it a novel biolumi- nescent detection tool. Nano luciferase was cloned into a prokaryotic expression vector and expressed in an auto-induced system. Recombinant nano luciferase was purified by Ni -NTA affinity chromatogra- phy and size exclusive chromatography. The typical yield of recombinant nano luciferase was 25.3 mg from a liter culture medium with purity above 98%. Then, we tested the activity of recombinant nano luciferase, from 1.0 ×10^-4 mg/mL to 1.0 ×10^-7 mg/mL, nano luciferase has good linear response, the detection limit was as low as 1.0 ×10^-7 mg/mL （ around 5 pmol）. The assay based on nano lucif- erase is uhrasensitive, good repeatable and easy operated. It would be applied as a basic tool to devel- op new sensitive bioluminescent probes with promising novel applications.

关 键 词：纳米荧光蛋白 原核表达 纯化 

分 类 号：Q617[生物学—生物物理学]