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目的确定荧光蛋白标记是否影响GB1、GB1asa和GB2亚基在CHO细胞中的表达、亚细胞定位及功能。方法构建GB1-DsRed2和GB1asa-DsRed2真核表达载体,利用ELISA和IP3积累法检测融合蛋白在CHO细胞中的表达情况及功能,在激光扫描共聚焦显微镜下观测融合蛋白的亚细胞定位。结果荧光蛋白标记既不影响GB1、GB1asa和GB2亚基的表达,也不影响GB1和GB2在CHO细胞上的共定位过程,同时对二聚体的功能不存在显著的影响。结论GB1-DsRed2、GB1asa-DsRed2和GB2-EGFP在CHO细胞中具有正常的生理功能和分布,为进一步研究GABAB受体在细胞膜上的运动及分布以及各亚基之间的相互作用奠定了基础。
Objective To determine if the fluorescent protein marker affects the expression, subcellular localization and function of GB1, GB1asA and GB2 subunits in CHO cells. Methods The eukaryotic expression vectors GB1-DsRed2 and GB1asa-DsRed2 were constructed. The expression and function of fusion protein in CHO cells were detected by ELISA and IP3 accumulation. The subcellular localization of fusion protein was observed under laser scanning confocal microscopy. Results Fluorescent protein labeling did not affect the expression of GB1, GB1asA and GB2 subunits, nor did it affect the colocalization of GB1 and GB2 on CHO cells, but did not affect the function of dimers significantly. Conclusion GB1-DsRed2, GB1asas-DsRed2 and GB2-EGFP have normal physiological functions and distribution in CHO cells, which lays the foundation for further study on the movement and distribution of GABAB receptors on the cell membrane and the interaction between each subunit.