带3蛋白C末端与血型糖蛋白A在哺乳动物细胞内相互作用的研究

Interaction between band 3 c-end and glycophorin A in mammalian cells

目的 研究人类红细胞带 3蛋白C末端与血型糖蛋白A(GPA)之间在哺乳动物细胞内的相互作用。方法 利用穿梭位点 ,将AE1 c end和GPA的基因从pGADT7 AE1 c end和pGBKT7 GPA中分别亚克隆至pM和pVP16载体上 ,将pVP16 AE1 c end、pM GPA及报告基因质粒pG5CAT共同转染HeLa细胞 ,采用ELISA检测报告基因CAT蛋白 (氯霉素乙酰转移酶 )的活性。结果 转染后 4 8～ 72h检测到CAT蛋白的表达 ,其表达水平和活性明显高于阴性对照。结论 本实验在哺乳动物细胞内证明了带 3蛋白C末端与GPA之间有直接的相互作用。

Objective To identify the interaction between band 3 c end and glycophorin A(GPA) in mammalian cells.Methods Using the shuttle sites of EcoR Ⅰ and Pst Ⅰ, the cDNA fragment of AE1 c end and GPA were subcloned into the vector pVP16 and pM from the pGADT 7 AE1 c end and pGBKT 7 GPA plasmids, respectively. The two recombinant plasmids were cotransfected along with a chloramphenicol acetyltransferase (CAT) reporter vector into HeLa cells.The activity of CAT protein was measured by ELISA. Results The level of CAT expression and activity were detected 48～72h after transfection.The expression level of CAT protein and activity of CAT protein were markedly increased than those of negative control.Conclusion There is a direct interaction between band 3 c end and GPA in mammalian cells.