共表达人kappa阿片受体与PKAcat-EGFP的CHO稳定细胞株建立及功能鉴定

摘要

Aim To establish a cell model which stably co-ex-press human kappa opioid receptor (hKOR) and enhanced green fluorescent protein ( EGFP) labeled catalytic domain of cAMP-dependent protein kinase A(PKAcat) fusion protein (PKAcat-EGFP) in Chinese hamster ovary(CHO) cells, laying the foun-dation for the high-throughput screening of hKOR drugs and drug molecular mechanisms in vitro. Methods Hygromycin B resist-ant hKOR recombinant plasmid [ pcDNA3.1/Hygro ( + ) -hKOR] was transfected into CHO cells stably expressing PKA-cat-EGFP by a lipofectin based method. Transfected cells were selected in culture medium containing hygromycin B. The posi-tive clones were selected by PKA redistribution assay. Z’ factor was used for evaluation and validation the reliability of the cell model. PKA redistribution assay and LANCE cAMP 384 Kit were used to test the function of the receptors in selected clone. Results CHO-PKAcat-EGFP/hKOR-13 cell model exhibited stable response in PKA redistribution assay and LANCE cAMP 384 Kit. Treated with 100 nmol·L-1U-50488 for 30 min, the average value of Z’ factor was 0.596, proving the reliability of the cell model. The hKOR expression in cell model remained stable after a few generations. Conclusion The CHO-PKAcat-EGFP/hKOR-13 cell model with stable co-expression of hKOR and PKAcat-EGFP has been successfully established.%目的 在中国仓鼠卵巢( Chinese hamster ovary, CHO)细胞上建立人kappa阿片受体( human kappa opioid receptor, hKOR)及增强型绿色荧光蛋白( enhanced green fluorescent protein,EGFP)标记的蛋白激酶A催化亚基( catalytic domain of cAMP-dependent protein kinase A, PKAcat ) 融合蛋白(PKAcat-EGFP)稳定共表达的细胞模型,为体外高通量筛选作用于hKOR的药物及药物分子机制研究打下基础.方法通过脂质体介导法将潮霉素 B 抗性的 hKOR 重组质粒[pcDNA3.1/Hygro( +)-hKOR]转染入已稳定表达 PKAcat-EGFP的CHO细胞中,随后用含潮霉素B的选择性培养基培养细胞,有限稀释法挑取耐药单克隆,PKA重分布实验筛选阳性克隆,利用Z’因子对建立的细胞模型的可靠性进行评价,利用PKA重分布实验与LANCE cAMP 384 Kit检测受体功能.结果 PKA重分布实验与LANCE cAMP 384 Kit结果表明,CHO-PKAcat-EGFP/hKOR-13 号克隆反应性良好,100 nmol·L-1的U-50488作用时的Z’平均值为0.596,证明了该细胞模型的可靠性,且经多次传代后的受体表达也能保持稳定.结论 成功建立了hKOR与PKAcat-EGFP融合蛋白稳定共表达的细胞模型CHO-PKAcat-EGFP/hKOR-13.