人源CDC50基因的分子克隆及在E.coli中的表达

Molecular Cloning and Expression of Human CDC50 Gene in E.coli

糖皮质激素受体 (GR)为一种特异性配体诱导的转录因子在机体发育、生理及行为等过程中发挥重要作用 .这一过程往往受到转录协同调节因子的控制 ,包括协同抑制因子或协同激活因子的作用 .克隆了人源双跨膜受体分子 ,并命名为hCDC5 0 .该分子编码区有 10 5 6bp ,与鼠源基因mCDC5 0在核苷酸水平有 90 %一致性 .将hCDC5 0蛋白的 16 4～ 317编码序列与GST融合构建原核表达质粒进行重组蛋白的表达与纯化并制备多抗 .用二维双向扩散检测抗体为阳性 ,抗原的滴度为12 5 μg/ml.体外翻译研究显示该基因编码蛋白约为 4 0kD ,与Western检测在动物细胞中的表达一致 .用hCDC5 0真核表达质粒与GR萤光报告系统共转染COS 7细胞显示 ,hCDC5 0对GR信号转导有明显的抑制作用 ,其对GR信号通路的抑制作用达 3～ 4倍以上 .hCDC5 0可能为一个新的协同抑制因子 。

Glucocorticoid receptor (GR) is a specific ligand\|inducing transcription factor which plays important roles in diverse cellular and physiological processes such as development and behaviors,and is affected by other non\|ligand molecules called coregulators,such as corepressors or coactivators.The transmembrane protein human CDC50 (hCDC50) was cloned.The complete coding sequence is 1056 bp long and contains two putative transmembrane helices in either N\| or C\| terminus.The nucleotide sequences of human and mouse CDC 50 have 90% identity.The GST\|hCDC50 fusion protein was expressed with pGEX\|4T\|2 system in E.coli, and purified by glutathione\|Sepharose 4B chromatography.Polyclonal antibody against the fusion protein was produced by inoculation of the fusion protein into rabbit.The concentration of the antibody to the recombinant protein was 125 μg/ml.Using luciferase reporter system,a significant reduction on gene expression was observed as cotransfecting hCDC50 and GR into COS\|7 cells,indicating that hCDC50 might be an important novel co\|regulator to repress GR signaling.