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增强型绿色荧光蛋白融合对Ano1钙激活Cl−通道生理特性的影响

郑锴, 许会静, 藏雨轩, 侯毅鞠, 张丽, 杨海鸥, 朱洁, 方芳, 郝峰

吉林医药学院1生物化学检验教研室；临床血液学检验教研室，吉林 132013

摘要

本研究旨在探讨融合在Ano1钙激活Cl−通道(anoctamin1) C末端的增强型绿色荧光蛋白(EGFP)对Ano1生理特性的影响。分别构建融合和未融合EGFP的Ano1真核表达载体，通过脂质体介导分别将这两类真核表达载体转染至Fischer大鼠甲状腺滤泡上皮(FRT)细胞，倒置荧光显微镜下观察融合和未融合EGFP的Ano1在FRT细胞中的表达与定位；荧光淬灭动力学检测Ano1转运I−的能力。结果显示，融合和未融合EGFP的Ano1均表达于FRT细胞膜上，且均能被Ca2+激活，融合和未融合EGFP的Ano1转运I−的能力无明显差异。以上结果提示，Ano1和EGFP-Ano1具有相似的生理特性。

关键词： 融合蛋白; 增强型绿色荧光蛋白; Ano1；生理特性；钙激活Cl−; 通道

分类号：R3

[Effect of enhanced green fluorescent protein fusion on Ano1 physiological feature.] [Article in Chinese]

ZHENG Kai, XU Hui-Jing, ZANG Yu-Xuan, HOU Yi-Ju, ZHANG Li, YANG Hai-Ou, ZHU Jie, FANG Fang, HAO Feng

Department of Biochemistry Laboratory； Department of Hematology, Jilin Medical College, Jilin 132013, China

Abstract

The aim of the present study was to investigate whether the physiological features of Ano1 were affected by enhanced green fluorescent protein (EGFP) fusing at Ano1 C-terminal. The eukaryotic expression vectors of Ano1 and EGFP-Ano1 were constructed, and these plasmids were transfected into Fischer rat thyroid follicular epithelial (FRT) cells using liposome. The expression and location of Ano1 were examined by using inverted fluorescence microscope. The ability of Ano1 to transport iodide was detected by kinetics experiment of fluorescence quenching. The results showed that both Ano1 and EGFP-Ano1 were expressed on FRT cell membrane and could be activated by Ca2+. There was no significant difference of the ability to transport iodide between Ano1 and EGFP-Ano1. These results suggest Ano1 and EGFP-Ano1 have similar physiological feature.

Key words: fusion protein; enhanced green fluorescent protein; Ano1; physiological feature

收稿日期：2015-03-30　　录用日期：2015-09-14

通讯作者：郝峰　　E-mail: haof863@126.com

引用本文：

郑锴, 许会静, 藏雨轩, 侯毅鞠, 张丽, 杨海鸥, 朱洁, 方芳, 郝峰. 增强型绿色荧光蛋白融合对Ano1钙激活Cl−通道生理特性的影响[J]. 生理学报 2015; 67 (6): 623-628.

ZHENG Kai, XU Hui-Jing, ZANG Yu-Xuan, HOU Yi-Ju, ZHANG Li, YANG Hai-Ou, ZHU Jie, FANG Fang, HAO Feng. [Effect of enhanced green fluorescent protein fusion on Ano1 physiological feature.] [Article in Chinese]. Acta Physiol Sin 2015; 67 (6): 623-628 (in Chinese with English abstract).