【摘要】 目的　制备毒蕈碱乙酰胆碱受体m5亚型与G 蛋白亚单位G1 1 (m5AChR G1 1 )融合蛋白 ,探索m5AChR与G1 1 之间的耦联功能、相互作用及其影响因素。方法　两步PCR法建立m5AChR与G1 1 融合cDNAs,并在Sf9细胞内表达。 [3 H]QNB和 [3 5S]GTPγS结合实验检测m5AChR与G1 1 融合蛋白的功能。结果m5AChR G1 1 融合蛋白的表达水平为 (60 .4± 2 .0 )nmol·g-1 膜蛋白。不同配体存在使融合蛋白中G1 1 与GDP的亲和力发生变化。乙酰胆碱(ACh)、卡巴胆碱、毛果芸香碱、异丙铵、异丙嗪及阿托品存在时 ,GDP的IC50值分别为 1 35,63 ,1 82 ,4.1 ,8.9和 5.9μmol·L-1 ,无配体存在时 ,GDP的IC50 值为 2 3.4μmol·L-1 。结论　杆状病毒Sf9细胞系统表达的m5AChR G1 1 融合蛋白具备m受体配体结合特性和组分间耦联的功能。m5AChR G1 1 融合蛋白对GDP亲和力取决于m受体配体的性质 ,分析GDP的亲和力的变化有利于筛选和鉴别m5受体亚型特异性配体更多还原

【Abstract】 AIM To test the couping function, the interreaction and the influence factors between m 5 muscarinic acetylcholine receptor(m 5AChR) and G 11 protein by generating m 5AChR and G 11 fusion protein(m 5AChR G 11 ) in baculovirus Sf9 cells system. METHODS m 5AChR G 11 Fused DNA was generated in a two step PCR and then expressed in Sf9 cells to produce fusion protein. QNB and [γ 35 S]GTP binding experiments were used to study the function of m 5AChR G 11 fusion protein. RESULTS The expression level of m 5AChR G 11 was (60.4± 2.0) nmol·g -1 protein. The affinity of GDP to G 11 partner changed in the presence of different muscarinic ligands. IC 50 values of GDP in the presence of acetylcholine(ACh), carbachol, pilocarpine, isopropamide, promethazine and atropine were 135, 63, 182, 4.1, 8.9 and 5.9 μmol·L -1 , respectively, and that in the absence of muscarinic ligand was 23.4 μmol·L -1 . CONCLUSION The m 5AChR G 11 fusion protein expressed in baculovirus sf9 cells system has pharmacological specificity for m 5 receptor and the efficient coupling function between the two partners. Affinity of GDP to ligand bound fusion protein represents the species of muscarinic ligands. This is helpful to screen and detect new specific ligands of the muscarinic receptors subtype.更多还原