G-CSF受体和EPO受体胞内区蛋白在酵母细胞中的转录激活功能的比较研究

Transcriptional activation function of intracellular domain of EPOR and G-CSFR protein in yeast: a comparative study

目的 探索应用酵母双杂交系统筛选造血因子受体胞内区结合蛋白的可行性。方法 利用RT PCR从小鼠NFS 6 0和BET 2细胞中扩增G CSF受体和EPO受体胞内区全长序列 ,并连接至酵母表达载体pGBKT7,转化酵母菌株AH10 9,提取蛋白进行West ernblotting分析 ,进一步用β 半乳糖苷酶方法检测蛋白转录激活活性。结果 Westernblotting结果显示 ,G CSF受体和EPO受体在酵母细胞中均可以正常表达。β 半乳糖苷酶检测结果发现 ,G CSF受体在酵母细胞中不呈现转录激活功能 ,然而EPO受体则出现转录激活功能。结论 应用酵母双杂交系统筛选造血因子受体胞内区结合蛋白可适用G CSF受体研究 ,但不适用EPO受体 ,该筛选蛋白的方法并非适用于所有的造血因子受体。

Objective To investigate the feasibility of searching for proteins which interact with intracellular domain of hematopoietic growth factor receptors using yeast two-hybrid system. Methods RT-PCR method was performed to amplify the genes of intracellular domains of G-CSF receptor and EPO receptor in NFS-60 and BET-2 cells of mice. The genes were cloned into yeast expression plasmid pGBKT7 vector,and then transformed into yeast AH109. The yeast proteins were isolated and analyzed with Western blotting. Transcriptional activation was analyzed by the β-galactosidase colony-lift filter assay. Results The intracellular domains of G-CSF receptor and EPO receptor genes were successfully cloned into pGBKT7 vector. The results of Western blotting assay showed that both proteins were expressed in the yeast cells. The β-galactosidase colony-lift filter assay demonstrated that G-CSF receptor alone had no activity of transcriptional activation,while the EPO receptor alone could activate transcription. Conclusion The findings suggested that intracellular domain of G-CSF receptor could be used as a bait to find interacting proteins using yeast two-hybrid system,while that of the EPO receptor could not. Therefore the system could not be applied to all hematopoietic factor receptor.