m_3AChR-G_(11α)融合蛋白表达及功能检测  

Detection of the expression and function of m3AChR-G11 fusion protein

作　　者：白立川[1] 郭政东[1] 张雪薇[1] 孙科[1] 姜爱民[1] 

机构地区：[1]中国医科大学药物毒理学教研室,辽宁沈阳110001

出　　处：《中国药理学通报》2010年第8期1017-1020,共4页Chinese Pharmacological Bulletin

基　　金：国家自然科学基金资助项目(No30171077)

摘　　要：目的制备m3AChR-G11α融合蛋白,探索m3AChR与G11间的偶联功能、相互作用及其影响因素。方法通过应用两步PCR方法构建m3AChR-G11α融重组的cDNAs,并在Baculovirus-infected Insect cells(Sf9)细胞内表达m3AChR-G11α融合蛋白。BCA法测定Sf9细胞表达的膜蛋白总含量,[3H]QNB和[35S]GTPγS放射性配体结合实验检测融合蛋白的偶联功能。结果 BCA法测定Sf9细胞膜蛋白的总浓度为2.86g·L-1,M3-G11α融合蛋白的表达水平为7.76×10-9mol·g-1protein。不同配体存在时使融合蛋白中的G11α与GDP的亲和力发生变化。ACh,Pilo,CCh,MCN-A-343,Atro,4-Damp,Dafi及无配体存在时,GDP的IC50值分别为:82.21,93.70,12.10,14.30,1.93,1.37,0.72和1.99×10-6mol·L-1。结论 Sf9细胞表达的m3AChR-G11α融合蛋白具有m受体配体结合的特性及两组分间偶联的功能,m3AChR-G11α融合蛋白中的G11亚基对GDP的亲和性取决于m受体配体的性质,分析GDP的亲和性变化有利于筛选和鉴别m3受体亚型特异性配体。Aim To generate m3AChR-G11 fusion protein in baculovirus-Sf9 cells and test the couping function,the interation and the influence factors be-tween m3AChR and G11 protein,as well as screen the specific ligands for m3AChR. Methods m3AChR-G11 fused DNA was generated through a two-step PCR and then expressed in Sf9 cells to produce fusion protein. The total concentration for membrane protein was de-tected by BCA method,[3H]QNB and [35 S]GTPγS binding experiment as perfomed to study the function of m3AChR-G11 fusion protein. Results The expression level of m3AChR-G11 was 7. 76 × 10 -9 mol·g -1. The affinity of GDP to G11 partner changed in the presence of different muscarinic ligands. IC50 values of GDP in the presence of ACh,Pilo,CCh,MCN-A-343,Atro,4-Damp and Dafi were 82. 2,93. 70,12. 10,14. 30, 1. 93,1. 37,0. 72 × 10 -6 mol · L -1 respectively,and that in the absence of muscarinic was 1. 99 × 10 -6 mol ·L-1.Concluslons The m3AChR-G11 fusion protein has the pharmacological specificity of m3 receptor and the efficient coupling interaction of the two partners. 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：M受体 G蛋白 融合蛋白 激动剂 拮抗剂 放射性配体 

分 类 号：R329.2[医药卫生—人体解剖和组织胚胎学] R341.3[医药卫生—基础医学]