Please wait a minute...

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  2010, Vol. 30 Issue (05): 81-86    
技术与方法     
代谢型谷氨酸受体5亚型高通量筛选模型的建立
石榴1,2*,蒋亚琴1,2,白艳秋1,2
1. 中国科学院北京基因组研究所 北京 101300
2.中国科学院研究生院 北京 100039
Establishment of High Throughput Screening System of mGluR5
SHI Liu1,2,JIANG Ya-qin1,2,BAI Yan-qiu1,2
1.Beijing Genomics Institute, Chinese Academy of Sciences, Beijing 101300, China
2.Graduate University of the Chinese Academy of Sciences, Beijing 100039, China
 全文: PDF(904 KB)   HTML
摘要：

目的: 利用荧光测钙的方法，建立了人源代谢型谷氨酸受体5亚型(mGluR5)的高通量筛选模型。方法: 通过基因合成得到mGluR5的ORF区域，并将其构建到pCNDA3.1真核表达载体上。将此重组质粒转染HEK293细胞，利用抗生素筛选和钙流检测方法得到稳定表达mGluR5的重组细胞株。结果: 对试验条件:接种密度,孵育时间,溶剂DMSO浓度进行了优化，建立了稳定可靠的实验系统。并使用该受体特异激动剂和拮抗剂进行了功能验证,其中激动剂EC50 L-Quisqualic acid(67.8nmol/L) > L-Glu(2.73μmol/L)，抑制剂IC50 MTEP(3.3nmol/L) > Fenobam(23.5nmol/L)系统Z因子为0.68,证明建立了一个人源mGluR5抑制剂的细胞筛选模型。利用此重组细胞株对360种化合物进行了筛选，找到了若干对mGluR5有抑制效果的化合物。结论: 此细胞模型适用于mGluR5抑制剂的高通量筛选。
关键词： 代谢型谷氨酸受体5亚型（mGluR5）;  高通量筛选（HTS）;  激动剂/拮抗剂    
Abstract:

Objective: A high throughput screening system of mGluR5 was established by Ca2+ mobilization detecting method. Methods: The human mGluR5 cDNA was cloned using gene synthesis technique and subcloned into the pCNDA3.1 mammalian expression vector. The recombinant plasmid was then transfected into HEK293 cells. The transfected cells were stably selected by G418. Single cell clones with high Ca2+ inducibility and low background were isolated. Results: Assay conditions were optimized, under which more robust signals were observed. Further validation results indicated that in this mGluR5 cell line, the potencies of mGluR5 agonists, antagonists were comparable with the published data. The rank order of agonists potency for mGluR5 was L-Quisqualic acid (EC50=67.8nM) > L-Glu (EC50=2.73μM). The rank order of antagonists potency for mGluR5 was MTEP (IC50=3.3nM) >Fenobam (IC50=23.5nM). The cell line was stable for at least 15 passages. The results of functional assay indicated that a HTS system for mGluR5 antagonist screening had been established successfully with a Z’ Prime of 0.68.By using this assay system, several positive compounds were identified which represented strong inhibition effect. Conclusion: The recombinant cell model is suitable for mGluR5 receptor targeted high throughput screening.
Key words: Metabotropic Glutamate Receptor 5 (mGluR5)    high throughput screening (HTS)    agonist/antagonist
收稿日期: 2009-12-23 出版日期: 2010-05-25
通讯作者: 石榴     E-mail: shiliu@hdbiosciences.com
服务　　
把本文推荐给朋友
加入引用管理器
E-mail Alert
RSS
作者相关文章　　
石榴
蒋亚琴
白艳秋

引用本文:

石榴 蒋亚琴 白艳秋. 代谢型谷氨酸受体5亚型高通量筛选模型的建立[J]. 中国生物工程杂志, 2010, 30(05): 81-86.

DAN Liu, JIANG E-Qin, BAI Yan-Qiu. Establishment of High Throughput Screening System of mGluR5. China Biotechnology, 2010, 30(05): 81-86.

链接本文:

https://manu60.magtech.com.cn/biotech/CN/    或    https://manu60.magtech.com.cn/biotech/CN/Y2010/V30/I05/81

［1］ O’Brien J A, Lemaire W, Chen T B,et al.A family of highly selective allosteric modulators of the metabotropic glutamate receptor subtype 5. Mol Pharmacol, 2003, 64(3):731740. 
［2］ Lavreysen H,Poul E L,Gompel P V,et al.Supersensitivity of human metabotropic glutamatela receptor signaling in L929sA cell.Mol Pharmacol 2002,61(5):12441254. 
［3］ Kouhen O E,Lehto S J,Pan J B,et al.Blockade of mGluR1 receptor results in analgesia and disruption of motor and cognitive performances: effects of A841720, a novel noncompetitive mGluR1 receptor antagonist.British Journal of Pharmacology.2006,149:761774. 
［4］ Abe T,Sugihara H,Nawa H,et al.Molecular characterization of a novel metabotropic glutamate receptor mGluR5 coupled to inositol phosphate/Ca2+signal transduction.The Journal of Biological Chemistry,1992,267(19):1336113368. 
［5］ Watkins J C,Jane D E.The glutamate story.British Journal of Pharmacology,2006,147 Suppl 1:S100S108. 
［6］ Gass J T,Osborne M P,Watson N L,et al.mGluR5 antagonism attenuates methamphetamine renforcement and prevents reinstatement of methamphetamineseeking behavior in rat.Neuropsychopharmacology,2009,34(4):820833. 
［7］ Bckstrm P,Hyyti P.Ionotropic and metabotropic glutamate receptor antagonism attenuates cueinduced cocaine seeking.Neuropsychopharmacology,2006,31(4):778786. 
［8］ Ayala J E,Chen Y,Banko J L,et al. mGluR5 positive allosteric modulators facilitate both hippocampal LTP and LTD and enhance spatial learning. Neuropsychopharmacology,2009,34(9):20572071. 
［9］ 曹婧,张烨,彭志刚.组胺H3受体激活剂高通量筛选细胞模型的研究.中国生物工程杂志,2008,28(9):6167. Cao J, Zhang Y, Peng Z G. China Biotechnology,2008,28(9):6167. 
［10］ 张巨,程杰,陈静.HepG2/mdr1稳定细胞系的建立及特性研究.中国生物工程杂志,2009,29(5):1722. Zhang J, Cheng J, Chen J. China Biotechnology,2009,29(5):1722. 
［11］ 张娅玲,白艳秋.代谢性谷氨酸受体4亚型(mGluR4)高通量药物筛选模型的建立.生物工程学报,2009,25(3):457463. Zhang Y L, Bai Y Q. Chinese Journal of Biotechnology,2009,25(3):457463. 
［12］ Mutel V,Ellis G J,Adam G,etal.Characterization of ［3H］quisqualate binding to recombinant rat metabotropic glutamate la and 5a receptors and to rat and human brain sections.Journal of Neurochemistry,2000,75(6):25902601. 
［13］ Porter R H, Jaeschke G, Spooren W,et al. Fenoban:a clinically validated nonbenzodiazepine anxiolytic is a potent,selective,and noncompetitive mGlu5 receptor antagonist with inverse agonist activity.J Pharmacol,2005,315(2):711721. 
［14］ Zhang J H,Chuang T D,Oldenburg K R.A simple statistical parameter for use in evaluation and validation of high throughput screening assays. Journal of Biomolecular Screening,1999,4(2):6773. 
［15］ Hemstapat K,Paulis T D,Chen Y L,et al.A novel class of positive allosteric modulators of mGluR1 interact with a site distinct from that of negative allosteric modulators.Molecular Pharmacology. 2006 ,70(2):616626.
No related articles found!
主管：中国科学院 　主办：中国科学院文献情报中心 　中国生物技术发展中心 　中国生物工程学会