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中国生物工程杂志

CHINA BIOTECHNOLOGY
中国生物工程杂志
中国生物工程杂志  2022, Vol. 42 Issue (3): 38-46    DOI: 10.13523/j.cb.2108063
技术与方法     
稳定表达人源GABAAR-CHO细胞株的建立
张毅,王陈,石晶晶,陈学军,张瑞华,靳倩,石童*(),李丽琴*()
国民核生化灾害防护国家重点实验室 北京 102205
Establishment of Human GABAAR-CHO Cell Line Stable Expression
ZHANG Yi,WANG Chen,SHI Jing-jing,CHEN Xue-jun,ZHANG Rui-hua,JIN Qian,SHI Tong*(),LI Li-qin*()
State Key Laboratory of NBC Protection for Civilian, Beijing 102205, China
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摘要：

目的: 构建α1亚基诱导表达、β2和γ2L亚基稳定表达的人源α1β2γ2L-GABAAR-CHO(Chinese hamster ovary)细胞株。方法: 从人cDNA文库中扩增α1、β2、γ2L亚基编码基因,分别构建亚基表达载体;将三个亚基表达载体共转染CHO-K1细胞,通过抗性筛选、膜电位检测法进行稳定表达克隆筛选;通过qPCR、Western blot对亚基表达进行鉴定;以激动剂GABA、阳性变构调节剂地西泮(diazepam,Dia)、拮抗剂荷包牡丹碱(bicuculine)为工具药,采用全细胞膜片钳方法及膜电位检测法对稳定表达细胞的药理学功能进行鉴定。结果: 经克隆筛选获得表达量较高的α1β2γ2L-GABAAR-CHO并对其亚基表达鉴定,结果显示该细胞稳定表达α1、β2、γ2L亚基,构建的α1β2γ2L-GABAAR-CHO细胞仅在加入四环素(tetracyclin)诱导的情况下表达α1亚基并与β2、γ2L组装成具有功能活性的α1β2γ2L-GABAAR;对其进行全细胞膜片钳检测研究发现,GABA可对其产生激动效应,引起α1β2γ2L-GABAAR-CHO细胞产生氯离子通道特征性电流变化,Dia可剂量依赖性地增强GABA对α1β2γ2L-GABAAR的激动效应;在膜电位检测研究中,获得GABA激动效应EC50为(177.72 ± 15.92)nmol/L,Dia变构效应EC50为(3.63±0.52)μmol/L,拮抗剂Bicuculine拮抗效应IC50为(538.83±29.55)nmol/L。结论: 通过采用诱导表达策略,成功构建了α1β2γ2L-GABAAR-CHO稳定表达细胞株,该细胞株具有对激动剂、阳性变构剂、拮抗剂特异性检测的药理学功能。
关键词： α1β2γ2L-GABAAR-CHO;  诱导表达;  膜电位检测;  GABA;  地西泮    
Abstract:

Objective: A functional α1β2γ2L-GABAAR-CHO human cell line is constructed in which α1 subunit is inducd expression and β2 and γ2L subunits are stable expression. Methods: The coding genes of human subunit α1, β2 and γ2L were amplified from human cDNA library, and the subunit vectors were respectively constructed. The three subunit vectors were cotransfected into CHO-K1 cells, and the stable expression clones were screened by resistance screening and membrane potential detection. The expression of subunits were identified by qPCR and western blot;the pharmacological function of α1β2γ2L-GABAAR-CHO line were identified by whole-cell patch clamp detection and membrane potential detection method. Results: The α1β2γ2L-GABAAR-CHO with high expression level was obtained by screening the clones. The cells stably expressed α1, β2 and γ2L subunits. The constructed α1β2γ2L-GABAAR-CHO cells expressed α1 subunit in the presence of tetracycline, and assembled with β2 and γ2L subunits to form α1β2γ2L-GABAAR with functional activity. Whole-cell patch clamp detection of α1β2γ2L-GABAAR-CHO showed that GABA could stimulate it and cause the characteristic current change of chloride channel in α1β2γ2L-GABAAR-CHO, and diazepam could enhance the activation effect of GABA on α1β2γ2L-GABAAR. Membrane potential detection showed that EC50 of agonist GABA was (177.72±15.92) nmol/L, EC50 of allosteric agent diazepam was (3.63±0.52) μmol/L, and IC50 of antagonist bicuculine was (538.83±29.55) nmol/L, respectively. Conclusion: α1β2γ2L-GABAAR-CHO cell line is successfully constructed by the induced expression strategy, which has the pharmacological function of specific detection of agonists, positive allosteric agents and antagonists.
Key words: α1β2γ2L-GABAAR-CHO    Induced expression    Membrane potential detection    GABA    Diazepam
收稿日期: 2021-08-27 出版日期: 2022-04-07
ZTFLH:  Q819  
通讯作者: 石童,李丽琴     E-mail: tong198282@126.com;llq969696@163.com
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张毅
王陈
石晶晶
陈学军
张瑞华
靳倩
石童
李丽琴

引用本文:

张毅, 王陈, 石晶晶, 陈学军, 张瑞华, 靳倩, 石童, 李丽琴. 稳定表达人源GABAAR-CHO细胞株的建立[J]. 中国生物工程杂志, 2022, 42(3): 38-46.

ZHANG Yi, WANG Chen, SHI Jing-jing, CHEN Xue-jun, ZHANG Rui-hua, JIN Qian, SHI Tong, LI Li-qin. Establishment of Human GABAAR-CHO Cell Line Stable Expression. China Biotechnology, 2022, 42(3): 38-46.

链接本文:

https://manu60.magtech.com.cn/biotech/CN/10.13523/j.cb.2108063    或    https://manu60.magtech.com.cn/biotech/CN/Y2022/V42/I3/38

Gene Forward primer Reverse primer
α1亚基 ATGGATTGGTTTATT GCCGTG CTGTTGGAGCGTAGTGTTGTTT
β2亚基 TCCTCTCCTGG GTCTCCTTC GGTGTTGATTGTGGTCATTGTG
γ2L亚基 CATCTTTGTCTTCTCTGCTCTGG TTGTCCTTGCTTGGTTTCCG
GAPDH GTATTGGACGCCTGGTTACC CGCTCCTGGAAGATGGTGATGG
表1  α1、β2、γ2L、GAPDH亚基引物序列
图1  GABAAR各亚基表达载体图谱
图2  FLIPR膜电位检测α1β2γ2L-GABAAR的表达
图3  qPCR鉴定GABAAR细胞株α1、β2、γ2L亚基的表达
图4  Western blot鉴定α1、γ2L亚基蛋白表达
图5  全细胞膜片钳检测工具药GABA和Dia的激动效应
图6  膜电位检测工具药激动剂GABA、阳性变构剂Dia和拮抗剂Bicuculine剂量-效应关系
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