【摘要】 目的采用Sf9细胞系统表达m5AChRG11α融合蛋白,通过检测GDP与m5AChRG11α融合蛋白的亲和力大小来鉴别m5AChR的特异性激动剂和拮抗剂。方法用两步PCR反应重组m5AChRG11α融合蛋白cDNAs,并插入到pBacPAK9病毒载体中,利用Sf9细胞表达m5AChR受体蛋白和m5AChRG11α融合蛋白,通过[3H]QNB结合饱和实验及[35S]GTPγS竞争性替代结合实验,检测受体表达水平及GDP与m5AChRG11α融合蛋白的亲和力。结果m5AChRG11α融合蛋白表达水平是(47.6±3.2)nmol·g-1膜蛋白。不同配体的存在使融合蛋白中G11α与GDP的亲和力发生变化。ACh、YM796、Oxotremorine、Methixene、Dextimide及atropine存在以及无配体存在时,GDP的IC50值分别是128.0,72.1,68.5,16.2,14.9,9.7和20.8μmol·L-1。结论Sf9细胞系统表达的m5AChRG11α融合蛋白具备M受体配体结合的特性和组分间偶联功能。m5AChRG11α融合蛋白对GDP的亲和性决定于M受体配体的性质。对于m5AChRG11α融合蛋白,ACh是m5AChR完全激动剂,YM796和Oxotremorine是部分激动剂;atropine,Methixene和Dextimide是拮抗剂。更多还原

【Abstract】 Aim The m₅AChR-G 11α fusion protein was expressed by baculovirus-Sf9 cells system, then using it to identify the specific agonists and antagonists for m₅AChR via detecting the affinity of GDP and m₅AChR-G 11α. Methods The m₅AChR-G 11α fused cDNAs were generated via a two-step PCR protocol and inserted into pBacPAK9 virus vector. We expressed m₅AChR-G 11α fusion protein and m₅AChR protein using baculovirus-Sf9 cell system. [ 3H]QNB and [ 35S]GTPγS binding tests were performed to detect the expressional level of receptor proteins and determine the affinity of GDP and m₅AChR-G 11α fusion protein. Results The expression level of m₅AChR-G 11α was （47.6±3.2） nmol·g -1 protein. The affinity of GDP to G 11α partner changed in the presence of different muscarinic ligands. IC 50 values of GDP in the presence of ACh, YM796, Oxotremorine, Methixene, Dextimide and atropine were 128.0, 72.1, 68.5, 16.2, 14.9 and 9.7 μmol·L -1 respectively, and that in the absence of muscarinic ligand was 20.8 μmol·L -1. Conclusion The m₅AChR-G 11α fusion protein has the pharmacological specificity of M₅ receptor and the efficient coupling interaction of the two partner. Affinity of GDP to ligand bound m₅AChR-G 11α fusion protein represents the species of muscarinic ligands. ACh is a full agonist for m₅AChR-G 11α fusion protein, YM796 and oxotremorine are partial agonists, while methixene, dextimide and atropine are antagonists.更多还原