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中国生物工程杂志

China Biotechnology
China Biotechnology
China Biotechnology  2010, Vol. 30 Issue (05): 81-86    DOI:
    
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
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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     
Received: 23 December 2009      Published: 25 May 2010
Corresponding Authors: Liu Shi     E-mail: shiliu@hdbiosciences.com
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DAN Liu
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Cite this article:

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

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

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