代谢型谷氨酸受体5亚型高通量筛选模型的建立

Establishment of High Throughput Screening System of mGluR5

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EC50L-Quisqualic acid(67.8nmol/L)>L-Glu(2.73μmol/L),抑制剂IC50MTEP(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。

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=67.8nmol/L） L-Glu（EC50=2.73μmol/L）.The rank order of antagonists potency for mGluR5 was MTEP（IC50=3.3nmol/L）Fenobam（IC50=23.5nmol/L）.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.