中国药理学通报2018，Vol.34Issue(9)：1321-1326,6.DOI:10.3969/j.issn.1001-1978.2018.09.025
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
Stable co-expression of human kappa opioid receptor and PKAcat-EGFP in CHO cells and functional identification
摘要
Abstract
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.关键词
kappa阿片受体/蛋白激酶A/稳定转染/高内涵分析/信号转导/中国仓鼠卵巢细胞Key words
kappa opioid receptor/PKA/stable transfection/high-content analysis/signal transduction/Chinese hamster ova-ry cell (CHO cell)分类
医药卫生引用本文复制引用
李玉蕾,龙隆,温泉,王莉莉,宫泽辉,苏瑞斌..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[J].中国药理学通报,2018,34(9):1321-1326,6.基金项目
国家科技部"重大新药创制"科技重大专项( No 2015ZX09501003) （ No 2015ZX09501003）
