Stable co-expression of human kappa opioid receptor and PKAcat-EGFP in CHO cells and functional identification
10.3969/j.issn.1001-1978.2018.09.025
- VernacularTitle: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
- Author:
Yu-Lei LI
1
;
Long LONG
;
Quan WEN
;
Li-Li WANG
;
Ze-Hui GONG
;
Rui-Bin SU
Author Information
1. 军事科学院军事医学研究院毒物药物研究所抗毒药物与毒理学国家重点实验室
- Keywords:
kappa opioid receptor;
PKA;
stable transfection;
high-content analysis;
signal transduction;
Chinese hamster ova-ry cell (CHO cell)
- From:
Chinese Pharmacological Bulletin
2018;34(9):1321-1326
- CountryChina
- Language:Chinese
-
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.
