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绿色荧光蛋白作为基因表达和蛋白质定位的报告分子。

Green fluorescent protein as a reporter of gene expression and protein localization.

作者信息

Kain S R, Adams M, Kondepudi A, Yang T T, Ward W W, Kitts P

机构信息

CLONTECH Laboratories, Inc., Palo Alto, CA, USA.

出版信息

Biotechniques. 1995 Oct;19(4):650-5.

PMID:8777060
Abstract

The green fluorescent protein (GFP) from the jellyfish Aequorea victoria is rapidly becoming an important reporter molecule for monitoring gene expression and protein localization in vivo, in situ and in real time. GFP emits bright green light (lambda max = 509 nm) when excited with UV or blue light (lambda max = 395 nm, minor peak at 470 nm). The fluorescence excitation and emission spectra of GFP are similar to those of fluorescein, and the conditions used to visualize this fluorophore are also suitable for GFP. Unlike other bioluminescent reporters, the chromophore in GFP is intrinsic to the primary structure of the protein, and GFP fluorescence does not require a substrate or cofactor. GFP fluorescence is stable, species-independent and can be monitored non-invasively in living cells and, in the case of transparent organisms, whole animals. Here we demonstrate GFP fluorescence in bacterial and mammalian cells and introduce our Living Colors line of GFP reporter vectors, GFP protein and anti-GFP antiserum. The reporter vectors for GFP include a promoterless GFP vector for monitoring the expression of cloned promoters/enhancers in mammalian cells and a series of six vectors for creating fusion protein to either the N or C terminus of GFP.

摘要

来自维多利亚多管水母的绿色荧光蛋白(GFP)正迅速成为一种重要的报告分子,用于在体内、原位和实时监测基因表达及蛋白质定位。当用紫外线或蓝光(λmax = 395 nm,次要峰值在470 nm)激发时,GFP会发出明亮的绿光(λmax = 509 nm)。GFP的荧光激发和发射光谱与荧光素相似,用于观察这种荧光团的条件也适用于GFP。与其他生物发光报告分子不同,GFP中的发色团是蛋白质一级结构所固有的,并且GFP荧光不需要底物或辅因子。GFP荧光稳定、不依赖物种,并且可以在活细胞中进行非侵入性监测,对于透明生物体而言,还可以对整个动物进行监测。在这里,我们展示了细菌和哺乳动物细胞中的GFP荧光,并介绍了我们的GFP报告载体、GFP蛋白和抗GFP抗血清的“活色生香”系列。用于GFP的报告载体包括一个无启动子的GFP载体,用于监测哺乳动物细胞中克隆启动子/增强子的表达,以及一系列六个载体,用于创建与GFP的N端或C端融合的蛋白质。

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