共表达人μ阿片受体与Gq蛋白的稳定细胞株的建立及功能鉴定  

Establishment and identification of stable cell lines co-expressing hMOR and Gq protein

作　　者：石晶晶 张毅 陈学军 朱思庆 王陈 李丽琴 SHI Jingjing;ZHANG Yi;CHEN Xuejun;ZHU Siqing;WANG Chen;LI Liqin(State Key Laboratory of NBC Protection for Civilians,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：《实验技术与管理》2023年第5期24-30,共7页Experimental Technology and Management

基　　金：国民核生化灾害防护国家重点实验室科研基金(SKLNBC2020-15)。

摘　　要：建立共表达人μ阿片受体(humanμ-opiatereceptor,hMOR,OPRM)与Gq蛋白的CHO-Flp In稳定细胞模型,并鉴定其药理学功能,可为体外高通量筛选靶向OPRM的药物奠定基础。该研究首先构建重组表达质粒OPRM-pcDNA5/FRT,并进行鉴定;然后通过脂质体转染法将OPRM-pcDNA5/FRT、GqG66Di5-pIRES/puro3和FLP重组酶质粒POG44共转染CHO-Flp In细胞,经抗性加压和有限稀释法挑取耐药单克隆,采用FLIPR钙信号检测方法筛选阳性克隆株;最后,通过RT-q PCR对细胞中的OPRM和GqG66Di5m RNA表达水平进行检测,并选用OPRM激动剂DAMGO和抑制剂Naloxone对稳定细胞株的药理学功能进行鉴定。结果显示:经酶切确定了重组质粒的正确构建;通过重组质粒转染、抗生素加压筛选以及钙信号测定获得22个具有活性的克隆细胞株,其中15号细胞株的荧光信号值最高,命名为Gq-OPRM1-CHO;与对照组相比,Gq-OPRM1-CHO细胞组中OPRM与GqG66Di5基因m RNA水平分别升高约400倍和120倍;在Gq-OPRM1-CHO细胞中,FLIPR钙信号检测激动剂DAMGO的EC_(50)为0.02±0.002μmol/L,抑制剂Naloxone的IC_(50)为0.04±0.003μmol/L。该研究成功建立了OPRM与GqG66Di5蛋白稳定共表达的细胞模型Gq-OPRM1-CHO,该细胞株具有对OPRM激动剂和拮抗剂特异性反应的药理学功能。To establish a stable CHO-FlpIn cell model co-expressing humanμ-opiate receptor(hMOR,OPRM)and Gq protein and identify its pharmacological function,which may provide a basis for high-throughput screening of drugs targeting OPRM in vitro.Firstly,the recombinant expression plasmid OPRM-pcDNA5/FRT is constructed and identified.Then,OPRM-pcDNA5/FRT,GqG66Di5-pIRES/puro3 and FLP recombinase plasmid POG44 are co-transfected into CHO-FlpIn cells by liposome transfection method,and resistant monoclonals are selected by resistance pressure and the limiting dilution method,the FLIPR calcium signal assay is used to screen positive clones.Finally,the expression levels of OPRM and GqG66Di5 mRNA in the stable cells are detected by quantitative real-time PCR(RT-qPCR),and the pharmacological functions are identified by OPRM agonist DAMGO and antagonist Naloxone.The results show that the correct construction of the recombinant plasmid is confirmed through enzyme digestion.22 active clone cell lines are obtained by recombinant plasmid transfection,antibiotic pressure screening and calcium signal assay,among them,cell line No.15,named Gq-OPRM1-CHO,has the highest fluorescence signal value.Compared with the control group,the mRNA levels of OPRM and GqG66Di5 genes in Gq-OPRM1-CHO group are increased by 400 and 120 folds,respectively.The Gq-OPRM1-CHO cell lines are further used to detect the calcium signal of OPRM agonists and antagonists by FLIPR,where the EC_(50) of the agonist DAMGO is 0.02±0.002μmol/L and the IC_(50) of the antagonist naloxone is 0.04±0.003μmol/L.In this study,a cell model,Gq-OPRM1-CHO,with stable co-expression of OPRM and GqG66Di5 protein is successfully established,and the stable cell line has pharmacological functions that specifically respond to OPRM agonists and antagonists.

关 键 词：Μ阿片受体 G蛋白耦联受体 钙离子 实时荧光检测分析系统 DAMGO 

分 类 号：Q291[生物学—细胞生物学]