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运用点突变试剂盒构建pcDNA3.1-YFP-H148Q/V163真核表达载体,脂质体介导YFP-H148Q/V163S转染稳定表达Ano1的FRT细胞,荧光淬灭动力学试验检测YFP-H148Q/V163S对其氯离子的敏感性。测序结果显示,YFP(yellow fluorescent protein)特异编码序列上第508-510位碱基CAC突变为CAG,148位组氨酸H突变为谷氨酰胺Q;第553-555位碱基GTG突变为AGC,163位缬氨酸V突变为丝氨酸S。荧光淬灭动力学试验结果显示,向表达YFP-H148Q/V163S的FRT细胞加入Anol激活剂ionomycin和Cl~-,其相对荧光强度明显下降。pcDNA3.1-YFP-H148Q/V163真核表达载体成功构建,并证实表达于FRT胞浆中的YFP-H148Q/V163S具有对氯离子敏感的特性。
The eukaryotic expression vector pcDNA3.1-YFP-H148Q / V163 was constructed by point mutation kit. The FRT cells stably expressing Ano1 were transfected with YFP-H148Q / V163S by lipofectamine. Fluorescence quenching kinetics assay was used to detect YFP-H148Q / V163S its chloride ion sensitivity. The sequencing results showed that the base CAC at position 508-510 of YFP (yellow fluorescent protein) coding sequence was mutated to CAG and histidine H at position 148 was mutated to glutamine Q. The base GTG at position 553-555 was mutated to AGC , Valine V at position 163 is mutated to serine S Fluorescence quenching kinetics test results showed that the relative fluorescence intensity of Antim activators such as ionomycin and Cl ~ - decreased to FRT cells expressing YFP-H148Q / V163S. The pcDNA3.1-YFP-H148Q / V163 eukaryotic expression vector was successfully constructed and confirmed that the YFP-H148Q / V163S expressed in the cytoplasm of FRT has a chloride ion-sensitive character.