共表达人Kappa阿片受体与Gq嵌合体GqG66Di5的稳定细胞株构建与鉴定  

Construction and Identification of a Stable Cell Line Co-expressing Human Kappa Opioid Receptor and Gq Chimera GqG66Di5

作　　者：石晶晶 张毅 陈学军 靳倩 朱思庆 李丽琴 王陈 SHI Jingjing;ZHANG Yi;CHEN Xuejun;JIN Qian;ZHU Siqing;LI Liqin;WANG Chen(State Key Laboratory of NBC Protection for Civilian,Beijing 102205,China)

机构地区：[1]国民核生化灾害防护国家重点实验室,北京102205

出　　处：《防化研究》2023年第2期25-32,共8页CBRN DEFENSE

摘　　要：Kappa阿片受体(Kappa Opioid Receptor,KOR)是新药研究的重要靶标之一,建立稳定表达相应受体的细胞株是实现快速高效筛选靶向化合物的前提。通过构建人KOR过表达质粒,将其分别与5种不同的Gq嵌合体质粒共转染至CHO-FlpIn细胞,筛选出最佳Gq嵌合体;通过抗生素加压筛选以及单克隆化获得稳转细胞株;采用Western Blot检测KOR表达,并进一步通过FLIPR钙信号对细胞株的药理学功能进行鉴定。基于以上途径,本研究构建了稳定共表达人KOR与Gq嵌合体GqG66Di5的稳定细胞株CHO-OPRK1-Gq。该细胞株高表达KOR,具有对特异性激动剂U-69593(EC50=0.039μmol/L±0.006μmol/L)和抑制剂Naloxone(IC50=0.43μmol/L±0.28μmol/L)响应的药理学功能,实现了基于Gq途径介导信号转导的KOR功能检测。在CHOOPRK1-Gq细胞株的基础上,建立了体外高通量筛选靶向KOR配体的新方法,为研发靶向KOR的药物奠定了基础。Kappa opioid receptor(KOR)is one of the important targets for the research of new drugs,and the establishment of cell lines that stably express KOR is a prerequisite for rapid and efficient screening of targeted compounds.The human KOR overexpression plasmid was constructed and co-transfected into CHO-FlpIn cells with five different Gq chimeric plasmids to screen the best Gq chimera.Stable cell lines were obtained through antibiotic pressure screening and monoclonalization.KOR expression was detected by Western Blot,and the pharmacological function of the cell lines was further identified by FLIPR calcium signaling.In this study,a stable cell line CHO-OPRK1-Gq that stably co-expressed human KOR and Gq chimera GqG66Di5 was constructed,which had high expression of KOR,and had a pharmacological function that responded to the specific agonist U-69593(EC50=0.039μmol/L±0.006μmol/L)and inhibitor Naloxone(IC50=0.43μmol/L±0.28μmol/L),enabling the detection of KOR function based on Gq pathway-mediated signal transduction.Based on the CHO-OPRK1-Gq cell line,a new method is established for high-throughput screening of KOR targeting ligands in vitro,which lays the foundation for the development of KOR targeting drugs.

关 键 词：Kappa阿片受体 Gq嵌合体 钙离子 高通量筛选 

分 类 号：Q786[生物学—分子生物学]