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目的利用氨基酸定点突变技术以大鼠野生型α7烟碱型乙酰胆碱受体(nAChR)为模板制备α7nAChR突变体。方法采用体外转录、定点突变、凝胶电泳、双电极电压钳等技术对α7nAChR进行点突变,在非洲爪蟾卵母细胞上表达α7nAChR及其突变体,并研究其受体活性功能。结果将α7nAChR第111位的丙氨酸突变为丝氨酸,制备了α7nAChR的点突变体,测定了突变体对激动剂乙酰胆碱(ACh)的半数效应浓度(EC_(50))为165.6μmol/L。结论该结果不仅为受体定点突变提供了1种方法,同时为药物筛选和研究α7nAChR结构与功能关系提供了模型。
Objective To prepare the α7nAChR mutant using the wild-type α7 nicotinic acetylcholine receptor (nAChR) as a template by using amino acid site-directed mutagenesis. Methods The α7 nAChR gene was mutated in vitro by site - directed mutagenesis, site - directed mutagenesis, gel electrophoresis and bipolar voltage clamp. The α7 nAChR and its mutants were expressed on Xenopus laevis oocytes and their receptor activity was studied. Results The a7nAChR point mutant was prepared by mutating alanine at position 111 of α7 nAChR to serine. The half - effect concentration (EC50) of the mutant on acetylcholine (ACh) was determined to be 165.6 μmol / L. Conclusion The results not only provide a method for site-directed mutagenesis of receptors, but also provide a model for drug screening and studying the relationship between structure and function of α7 nAChR.