阴离子交换蛋白1在HEK293t细胞中的高效表达与鉴定

Expression and identification of anion exchanger 1 in HEK293t cells

目的 在人胚肾HEK2 93t(2 93t)细胞高效表达具有阴离子转运活性的阴离子交换蛋白1(AE1)。方法 用传统磷酸钙转染法将质粒pRBG4- AE1转入2 93t细胞,免疫荧光及Westernblotting验证蛋白表达及正常的细胞膜定位后,应用6 - 甲氧基- N- (3- 磺丙基)喹啉铵(SPQ)观察表达的AE1是否具有阴离子转运活性。结果 在2 93t细胞中,显示AE1高效表达。转染pRBG4 - AE1后2 93t细胞相对于正常以及转染空载体对照组的阴离子交换功能明显增强。结论 利用传统磷酸钙转染法,在2 93t可高效表达具有正常跨膜拓扑结构及阴离子转运活性的AE1,为进一步研究其相关功能奠定了基础。

Objective To express the functional anion exchanger 1(AE1) in HEK293t (293t) cells.Methods The cDNA encoding full length AE1 was transfected into 293t cells with calcium phosphate.Expression of AE1 in 293t cells was detected by western-blotting and fluorescence microscopy.6-methoxy-N-(3-sulfopropyl)quinolinium(SPQ)was used to detect the anion exchange activity of AE1.Results AE1 was efficiently expressed in the transfected cells and the anion exchange activity of these cells was obviously increased compared with the activity of the control.Conclusion AE1 could be abundantly expressed on the membrane of 293t cells with calcium phosphate and has normal transmembrane-topology and anion exchange activity.A useful cell model is provided for the further functional investigation of AE1.