带绿色荧光蛋白标签的人细胞分裂周期蛋白25同源蛋白的真核表达及其生物学功能研究

Eukaryotic Expression of EGFP-Tagged Human Cell Division Cy clin 25 Homolog C and its Biological Function Detection

目的:构建带增强型绿色荧光蛋白(EGFP)标签的人细胞分裂周期蛋白25同源蛋白C(cdc25c)基因的真核表达载体pEGFP-cdc25c,并检测其在人胚肾293T细胞中的表达定位情况及生物学功能。方法:采用PCR技术从实验室已有质粒中扩增人cdc25c基因,并将其克隆到pEGFP-C1载体中;将重组质粒转染人胚肾293T细胞,Western印迹检测转染细胞的表达情况,荧光显微镜观察cdc25c蛋白在细胞中的定位,并利用cdc2 Tyr15位特异性抗体验证EGFP-cdc25c作为磷酸酯酶的生物学功能。结果:双酶切和测序鉴定表明,pEGFP-cdc25c真核表达质粒构建成功;转染293T细胞后获得表达,在荧光显微镜下,表达阳性的细胞呈绿色,并定位于细胞质;Western印迹结果表明,EGFP-cdc25c能增加cdc2 Tyr15位的磷酸化水平,起到拮抗内源性cdc25c的功能。结论:构建了带EGFP标签的人cdc25c基因真核表达载体,该载体能够在哺乳动物细胞293T中表达,表达产物定位于细胞质;EGFP-cdc25c能够发挥显性负性作用,为深入研究cdc25c的生物学功能奠定了重要基础。

Objective： To construct the eukaryotic expression vector of human cell division cyclin 25 homolog C （cdc25c） fused with EGFP, and to detect its expression, subcellular location and biological function in the human kidney 293T cells. Methods： Human cdc25c gene was obtained from myccdc25c plasmid by PCR and cloned in to the pEGFPC1 vector. Human 293T cells were transfected with the recombinant plasmid pEGFPcdc25c and the expression of EGFPcdc25c was detected by Western blot and fluorescence microscope. The biological function of EGFPcdc25c was examined with antiphosphocdc2（Tyr15）. Results： cdc25c eukaryotic expression vector fused with EGFP was successfully constructed and the expression of EGFPcdc25c was verified by Western blot. Fluores cence microscope analysis showed that the positive expression cells were green and EGFPcdc25c were located in the cytoplasm. EGFPcdc25c could enhance the phosphorylation level of cdc2 Tyr^5, which may antagonize the function of endogenous cdc25c. Conclusion： The eukaryotic expression vector of pEGFPcdc25c was successfully constructed and EGFPcdc25c was expressed in the cytoplasm of 293T cells. EGFPcdc25c may have dominant negative activity, which has laid foundation for the further study of the function of cdc25c.