M_2-G_(i1α)融合蛋白在Sf9细胞中的表达及M_2AChR特异性药物的鉴别  

The Specific Ligand Screening for M_2-G_(i1α) Fusion Protein Expressed in Sf9 Cells

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

机构地区：[1]中国医科大学药学院天然药物学研究室,沈阳110001

出　　处：《中国医科大学学报》2010年第6期428-431,共4页Journal of China Medical University

基　　金：国家自然科学基金资助项目(30171077)

摘　　要：目的通过杆状病毒-Sf9细胞系统表达M2-Gi1α融合蛋白,并利用M2-Gi1α融合蛋白为工具筛选毒蕈碱性乙酰胆碱受体亚型2(M2AChR)特异性配体,探讨M2-Gi1α融合蛋白中两组分相互作用的机制。方法通过两步PCR反应建立M2AChR与Gi1α融合cDNAs,并在Sf9昆虫细胞中表达M2-Gi1α融合蛋白。通过[3H]羟苯二乙酸奎宁酯([3H]QNB)配体饱和结合实验及[35S]GTPγS竞争性替代结合实验,检测M2-Gi1α融合蛋白表达水平,并筛选M2AChR的特异性配体。结果 M2-Gi1α融合蛋白的表达水平为(8.44±0.39)nmol·g-1膜蛋白。不同配体使M2-Gi1α融合蛋白中Gi1α与GDP的亲和力发生变化,乙酰胆碱、氧化震颤素、槟榔碱、阿托品、fangchinoline、levitimide的IC50值分别为:21.35,23.86,11.91,0.13,1.05,1.75μmol·L-1,无配体存在时为2.50μmol·L-1。结论杆状病毒-Sf9细胞表达系统表达的M2-Gi1α融合蛋白具备M受体与配体结合的特性和组分间的耦联功能,通过检测GDP亲和力的大小有利于筛选和鉴别M2受体亚型特异性药物。Objective Using M2-Gi1α fusion protein expressed by baculovirus-Sf9 cell system to find the specific ligand for M2 receptor and detect the interaction of the two parts of the fusion protein.Methods The fused M2-Gi1α cDNAs were generated in a two-step PCR and then expressed in Sf9 cells.[3H]QNB and[35S]GTPγS binding experiments were employed to study the function of M2-Gi1α fusion protein.Results The expression level of M2-Gi1α fusion protein was 8.44±0.39 nmol·g-1 protein.The affinity of GDP to the Gi1α part changed under the affection of different ligands.The IC50 value in the appearance of acetylcholine,oxotremorine,arecoline,atropine,fangchinoline,levitimide were 21.35 μmol·L-1,23.86 μmol·L-1,11.91 μmol·L-1,0.13 μmol·L-1,1.05 μmol·L-1,1.75 μmol·L-1,and 2.5 μmol·L-1 when there was no ligand.Conclusion The M2-Gi1α fusion protein expressed in baculovirus-Sf9 cell system has pharmacological specificity for M2 receptor and the efficient coupling function between the two parts.The M2-Gi1αfusion protein is a helpful tool for detecting the new specific ligands of the M2 receptor.

关 键 词：融合蛋白 毒蕈碱型乙酰胆碱受体 G蛋白耦联受体 兴奋剂 拮抗剂 

分 类 号：R962[医药卫生—药理学]