m_4AChR-Gilα融合蛋白的建立、表达及几种配体对其机能的影响  

The preparation,expression and effect of ligands on the function of m_4AChR-Gilα fusion protein

作　　者：王海波[1] 郭政东[2] 张海鹏[1] 赵立斌[3] 郭政礼[3] 

机构地区：[1]中国医科大学病理生理学教研室,辽宁沈阳110001 [2]中国医科大学药理学教研室,辽宁沈阳110001 [3]沈阳医学院附属中心医院药剂科,辽宁沈阳110024

出　　处：《中国生化药物杂志》2004年第5期257-260,264,共5页Chinese Journal of Biochemical Pharmaceutics

基　　金：国家自然科学基金资助项目 (No .3 0 1710 77);辽宁省自然科学基金资助项目 (No .0 0 2 0 3 9)

摘　　要：目的通过Baculovirus Sf9细胞系统制备并表达m4AChR Gilα融合蛋白 ,检测几种配体对融合蛋白质中m4AChR与Gilα间相互作用 ,并以该融合蛋白质为工具 ,筛选m4AChR的特异性配体。方法两步聚合酶链反应建立m4AChR Gilα融合cDNAs,并在Sf9细胞内表达。通过 [3 H]QNB和 [3 5S]GTPγS替代结合实验研究几种配体乙酰胆碱、卡巴胆碱、AF 10 2B、prifinium、AF DX116和阿托品对m4AChR Gilα融合蛋白的作用。结果m4AChR Gilα融合蛋白表达水平为 (4 7.0 4± 1.5 8)pmol/g蛋白 ,不同配体使融合蛋白质中Gilα与二磷酸鸟苷的亲和力发生变化。结论杆状病毒表达的m4AChR Gilα融合蛋白具备m受体配体结合特性和组分间的偶联功能。配体通过改变融合蛋白质中Gilα与二磷酸鸟苷的亲和力 ,活化该融合蛋白质。PurposeTo prepare m 4AChR-Gilα fusion protein in Baculovirus-Sf 9 cells system and to detect the effects of various muscarinic ligands on the interaction between m 4AChR and Gilα in the to fusion protein, then to screen ligands specific for m 4AChR.MethodsTo generate fused m 4AChR-Gilα DNA by two-step PCR, then to express in Sf9 cells. And to detect the pharmacological function of m 4AChR-Gilα fusion protein by [3H]QNB and [ 35S]GTPγS binding experiments. To explore the way of the activation of m 4AChR-Gilα fusion protein by various ligands including acetylcholine, carbamylcholine, AF-102B, prifinium, AF-DX116 and atropine.ResultsThe expression level of m 4AChR-Gilα fusion protein is (47.04±1.58) pmol/g protein. The affinity of guanosine diphosphate(GDP) to Gilα partner changed in the presence of different muscarinic ligands.ConclusionLigands of m 4AChR mediate different signal transductions by changing the affinity of Gilα and GDP. It was taken that m 4AChR-Gilα fusion protein as a model to screen ligands specific for mAChRs.

关 键 词：融合蛋白质 G-蛋白 配体 二磷酸鸟苷 

分 类 号：Q813[生物学—生物工程]