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建立以哺乳动物α3β4乙酰胆碱受体(nAChR)为分子靶标的药物筛选模型。将大鼠乙酰胆碱受体的α3与β4亚基基因分别进行体外转录,获得α3与β4亚基基因的cRNA,按1∶1的比例将适量α3与β4的cRNA混合后,显微注射到新鲜分离的非洲爪蟾卵母细胞中,在ND96溶液中、17℃下培养24 h后,通过双电极电压钳技术(TEVC)检测受体α3β4 nAChR的乙酰胆碱(Ach)门控电流。同时注入α3、β4 cRNA的非洲爪蟾卵母细胞孵育24h后,可记录到明显的Ach门控内向电流,未注射或只注射ddH2O的对照组细胞则没有任何电流产生。α3β4乙酰胆碱受体在非洲爪蟾卵母细胞膜上成功表达,以此为药物作用靶点,可用于大量生物毒素等物质的活性筛选实验模型。
A drug screening model using mammalian α3β4 acetylcholine receptor (nAChR) as a molecular target was established. The α3 and β4 subunit genes of rat acetylcholine receptor were respectively transcribed in vitro to obtain the cRNA of the α3 and β4 subunit genes. The appropriate amount of α3 and β4 cRNA were mixed in a ratio of 1: 1 and then microinjected into freshly isolated Of Xenopus laevis oocytes were incubated in ND96 solution at 17 ℃ for 24 h. The acetylcholine (Ach) gated currents of receptor α3β4 nAChR were detected by two-electrode voltage clamp technique (TEVC). At 24 h after incubation with α3 and β4 cRNA, a significant Ach-gated inward current was recorded, whereas none of the control cells injected with ddH2O did not produce any current. α3β4 acetylcholine receptor in Xenopus oocytes membrane successfully expressed as a drug target, can be used for a large number of biological toxins and other substances in the activity of screening experimental model.