摘要
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 construct the point mutant of ratα7nicotinic acetylcholine receptor(nAChR)and evaluate its function.Methods Site-directed mutagenesis was used to construct α7 nAChR point mutant,which was expressed in Xenopus oocytes and functional tested by electrophysiology with two-electrode voltage-clamp techniques.Results An α7 nAChR point mutant was constructed by mutating Alanine(Ala)at position 111of α7 nAChR to Serine(Ser)successfully.The EC(50) of the α7 nAChR mutant to the agonist acetylcholine(ACh)was 165.6μmol/L.Conclusion The results would provide an effective method for site-directed mutagenesis,and offer a realistic model for drug screening and characterization of structure-function relationships of α7 nAChR.
作者
朱晓鹏
于津鹏
雷宇苗
黄艺
长孙东亭
罗素兰
ZHU Xiao-peng YU Jin-peng LEI Yu-miao HUANG Yi ZHANGSUN Dong-ting LUO Su-lan(Key Laboratory of Tropical Biological Resources of Ministry of Education, Key Laboratory for Marine Drug of Haikou, College of Marine Science, Hainan University, Haikou 570228, Chin)
出处
《中国海洋药物》
CAS
CSCD
2017年第3期73-80,共8页
Chinese Journal of Marine Drugs
基金
国家自然科学基金重点国际合作项目(81420108028)
国家自然科学基金项目(41366002)
长江学者和创新团队发展计划(IRT-15R15)
海南省高等学校科学研究项目(Hnky2017-16)资助
关键词
α7乙酰胆碱受体
定点突变
表达与功能检测
双电极电压钳技术
α7 nAChR
site-directed mutagenesis
expression and functional test
two-electrode voltageclamp techniques