m_1AChR-G_(11)融合蛋白的表达和功能的检测  

Detection of the expression and function of m_1AChR-G_(11) fusion protein

作　　者：郭政东[1] 王海波[2] 李智[1] 刘洪瑞[1] 于秀华[1] 郭政礼[3] 赵立斌[3] 

机构地区：[1]中国医科大学基础医学院药理学教研室,沈阳110001 [2]中国医科大学基础医学院病生教研室,沈阳110001 [3]沈阳医学院附属中心医院药剂科,沈阳110024

出　　处：《中国药理学通报》2003年第11期1294-1298,共5页Chinese Pharmacological Bulletin

基　　金：国家自然科学基金资助项目;No 3 0 1710 77

摘　　要：目的　表达m1AChR G11融合蛋白 ,检测不同配体对m1AChR与G11间相互作用的影响。方法　两步PCR建立m1AChR G11重组cDNAs ,并在Baculovirus InfectedInsectcells(Sf9)细胞内表达。 [3 H]QNB和 [3 5S]GTPγS结合实验检测m1AChR G11融合蛋白功能。结果　m1AChR G11融合蛋白的表达水平为 4 5 39± 2 2 6pmol·mg-1protein。不同配体的存在使融合蛋白中G11与GDP的亲和力发生变化。乙酰胆碱 ,卡巴胆碱 ,AF 10 2B ,Isopropamide ,R (+) hyoscyamine和阿托品存在以及无配体存在时 ,GDP的IC50 值分别是 131 82、6 1 6 6、2 8 18、3 2 4、6 0 2、5 2 5和 16 6 μmol·L-1。结论　Sf9细胞系统表达的m1AChR G11融合蛋白具备m受体配体结合的特性和组分间偶联功能。m1AChR G11融合蛋白对GDP的亲和性决定于m受体配体的性质 。AIM To generate m 1AChR-G 11 fusion protein in baculovirus-Sf9 cells system and detect the effects of various muscarinic ligands. METHODS m 1AChR-G 11 fused DNA was generated through a two-step PCR and then expressed in Sf9 cells to produce fusion protein. QNB and GTPγS binding experiment as performed to study the function of m 1AChR-G 11 fusion protein. RESULTS The expression level of m 1AChR-G 11 was (45.38±2.26) pmol·mg -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 ACh, CCh, AF-102B, Isopropamide, R-(+)-hyoscyamine and Atropine were 131.82?61.66?28.18?3.24?6.02 and 5.25 μmol·L -1 respectively,and that in the absence of muscarinic ligand was 16.60 μmol·L -1. CONCLUSION The m 1AChR-G 11 fusion protein has the pharmacological specificity of m 1 receptor and the efficient coupling interaction of the two partner. Affinity of GDP to ligand-bound fusion protein represents the species of muscarinic ligands.This is helpful in screening and detecting the new specific ligands to muscarinic receptors.

关 键 词：融合蛋白 毒簟碱乙酰胆碱受体 G蛋白 放射配体 激动剂 拮抗剂 

分 类 号：R329.25[医药卫生—人体解剖和组织胚胎学] R341.3[医药卫生—基础医学]