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

Effect of enhanced green fluorescent protein fusion on Ano1 physiological feature

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Ca^(2+)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。

The aim of the present study was to investigate whether the physiological features of Anol were affected by enhanced green fluorescent protein （EGFP） fusing at Anol C-terminal. The eukaryotic expression vectors of Anol and EGFP-Anol were constructed, and these plasmids were transsfected into Fischer rat thyroid follicular epithelial （FRT） cells using liposome. The expression and location of Anol were examined by using inverted fluorescence microscope. The ability of Anol to transport iodide was detected by kinetics experiment of fluorescence quenching. The results showed that both Ano 1 and EGFP-Anol were expressed on FRT cell membrane and could be activated by Ca〉. There was no significant difference of the ability to transport iodide between Anol and EGFP-Anol. These results suggest Anol and EGFP-Anol have similar physiological feature.