一种简便的高通量细胞内GFP报告基因检测方法

doi:10.3969/j.issn.1004-616x.2015.05.014

癌变·畸变·突变 ›› 2015, Vol. 27 ›› Issue (5): 394-398,403.doi: 10.3969/j.issn.1004-616x.2015.05.014

一种简便的高通量细胞内GFP报告基因检测方法

杨少哲^1,2, 石海军^1,2, 褚欣然^1,2, 周小玲^1,2, 孙平楠^1,2

1. 汕头大学医学院干细胞P2实验室, 广东汕头 515041;
2. 广东省感染病与分子免疫病理重点实验室, 广东汕头 515041

收稿日期:2015-04-21 修回日期:2015-06-23 出版日期:2015-09-30 发布日期:2015-09-30
通讯作者: 孙平楠,E-mail:pingnan_sun@yahoo.com E-mail:pingnan_sun@yahoo.com
作者简介:杨少哲,E-mail:yangshaozhezhe@163.com。
基金资助:
国家自然科学基金(81571994,81570567);教育部留学回国人员科研启动基金(第49批);广东省自然科学基金(2014A030313483,2015A030313447);汕头大学医学院李嘉诚基金

A simple and high throughput screening method for GFP reporter assay in cells

YANG Shaozhe^1,2, SHI Haijun^1,2, CHU Xinran^1,2, ZHOU Xiaoling^1,2, SUN Pingnan^1,2

1. Stem Cell P2 Laboratory, Shantou University Medical College, Shantou 515041;
2. Guangdong Provincial Key Laboratory of Infectious Diseases and Molecular Immunopathology, Shantou 515041, Guangdong, China

Received:2015-04-21 Revised:2015-06-23 Online:2015-09-30 Published:2015-09-30

摘要/Abstract

摘要：

目的:建立一种简单、快速、高通量的检测GFP蛋白的方法。方法:用TECAN多功能酶标仪Infinite M200pro检测GFP蛋白在不同缓冲液中的荧光强度;用酶标仪检测含GFP的蛋白样品梯度稀释后的荧光强度,建立荧光强度与GFP相对含量之间的标准曲线;分别比较荧光酶标仪的检测结果与流式细胞术和Western-blotting检测结果的相关性。结果:使用50 mmol/L Tris-HCl(pH=10)的缓冲液稀释GFP蛋白更有利于检测低浓度的GFP;荧光酶标仪检测GFP的荧光读数同GFP相对含量之间的回归系数R²>0.99。荧光酶标仪检测GFP蛋白的结果与流式细胞术、Western-blotting的检测结果之间均呈现明显的相关性。结论:建立的利用多功能酶标仪检测GFP蛋白方法方便快捷,可以实现短时间对大样本进行检测。

关键词: 绿色荧光蛋白, 报告基因, 高通量检测, 多功能酶标仪

Abstract:

OBJECTIVE: To establish a simple, fast and high throughput screening method for green fluorescent protein (GFP) assay. METHODS:We applied TECAN multi function measuring instrument infinite M200pro to examine the fluorescence intensity of GFP sample in different dilution buffers, and established the standard curve between fluorescence intensity and GFP relative content. Compared the results obtained from multi function measuring instrument, fluorescence-activated cell sorting (FACS) and Western-blotting. RESULTS:50 mmol/L Tris-HCl (pH=10) was more suitable for examining GFP samples with low content than other dilution buffers. The regression coefficient R2 between fluorescent value and GFP content was above 0.99 by using multi function measuring instrument. The results obtained from multi function measuring instrument correlated significantly with those obtained from FACS and Western-blotting. CONCLUSION:The established method of GFP assay using multi function measuring instrument was convenient, fast and could be used to screen large samples in a short time.

Key words: green fluorescent protein, reporter gene, high throughput screening, multi function measuring instrument

中图分类号:

Q291

杨少哲, 石海军, 褚欣然, 周小玲, 孙平楠. 一种简便的高通量细胞内GFP报告基因检测方法[J]. 癌变·畸变·突变, 2015, 27(5): 394-398,403.

YANG Shaozhe, SHI Haijun, CHU Xinran, ZHOU Xiaoling, SUN Pingnan. A simple and high throughput screening method for GFP reporter assay in cells[J]. Carcinogenesis, Teratogenesis & Mutagenesis, 2015, 27(5): 394-398,403.

参考文献

[1] Giepmans BN,Adams SR,Ellisman MH,et al. The fluorescent toolbox for assessing protein location and function[J]. Science,2006,312(5771):217-224.

[2] Bell P,Vandenberghe LH,Wu D,et al. A comparative analysis of novel fluorescent proteins as reporters for gene transfer studies[J]. J Histochem Cytochem,2007,55(9): 931-939.

[3] Ni Q,Zhang J. Dynamic visualization of cellular signaling[J]. Adv Biochem Eng/Biot,2010, 119:79-97.

[4] Gerdes HH,Kaether C. Green fluorescent protein: applications in cell biology[J]. FEBS Letters,1996,389(1): 44-47.

[5] Ward WW,Bokman SH. Reversible denaturation of aequorea green-fluorescent protein:physical separation and characterization of the renatured protein[J]. Biochemistry,1982,21(19): 4535-4540.

[6] Sheen J,Hwang S,Niwa Y,et al. Green-fluorescent protein as a new vital marker in plant cells[J]. Plant J,1995,8(5): 777-784.

[7] Labas YA,Gurskaya NG,Yanushevich YG,et al. Diversity and evolution of the green fluorescent protein family[J]. PNAS USA,2002,99(7):4256-4261.

[8] Shaner NC,Patterson GH,Davidson MW. Advances in fluorescent protein technology[J]. Cell Sci, 2007,120(Pt 24):4247-4260.

[9] Crameri A,Whitehorn EA,Tate E,et al. Improved green fluorescent protein by molecular evolution using DNA shuffling[J]. Nat Biotechnol,1996,14(3):315-319.

[10] Heim R,Tsien RY. Engineering green fluorescent protein for improved brightness,longer wavelengths and fluorescence resonance energy transfer[J]. Curr Biol:CB,1996, 6(2): 178-182.

[11] Tamura K,Shimada T,Ono E,et al. Why green fluorescent fusion proteins have not been observed in the vacuoles of higher plants[J]. Plant J, 2003,35(4):545-555.

[12] Zheng H,Kunst L,Hawes C,et al. A GFP-based assay reveals a role for RHD3 in transport between the endoplasmic reticulum and Golgi apparatus[J]. Plant J,2004,37(3):398-414.

[13] 苟吉庆,李敏,张锐,等. 利用荧光分光光度计定量分析GFP基因的表达水平[J]. 生物技术通报,2001,(5): 27-29.

[14] Kutner RH,Zhang XY,Reiser J. Production,concentration and titration of pseudotyped HIV-1-based lentiviral vectors[J]. Nat Protoc,2009,4(4):495-505.

[15] Wu JJ,Liu YW,Sun MX. Improved and high throughput quantitative measurements of weak GFP expression in transgenic plant materials[J]. Plant Cell Rep,2011,30(7):1253-1260.

一种简便的高通量细胞内GFP报告基因检测方法

A simple and high throughput screening method for GFP reporter assay in cells

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