孕酮诱导的蜕膜蛋白Depp在哺乳细胞株的转染表达与定位

Human Depp was transfected for expression and localization in mammalian cell lines

目的将孕酮诱导的蜕膜蛋白Depp基因构建到pC-DGFP表达载体中,分析其在哺乳细胞株中的表达和定位情况。方法用PCR方法扩增Depp全长序列,插入真核表达载体pCDGFP中,将构建好的质粒转染至COS7细胞,在荧光显微镜下观察其细胞定位;转染至HEK 293T细胞,提取总蛋白,利用GFP抗体进行Western blot检测。结果荧光显微镜观察表明,Depp蛋白主要分布在COS7细胞的胞质内,在细胞核周呈斑块状分布,在细胞核未见到明显表达,在HEK293T细胞中能有效表达。结论成功构建pCDGFP-Depp表达载体并确定Depp蛋白在哺乳细胞株中能有效表达。

Objective To construct with pCDGFP expression vector for human wild Depp and investigate the expression and localization of the preliminary study in mammalian cell lines.Methods The full length fragment of Depp was used to amplify by PCR,then this fragment inserted into the eukaryotic expression vector pCDGFP to construct expression vector.Transfected into COS7 cells to observe localization under fluorescence microscope;transfected into HEK 293T cells,extracted total protein for Western blot to test GFP antibody.Results Fluorescence microscopy showed that Depp protein localized mainly in the cytoplasm,not in the nucleus with patchy distribution,The expression vector could express efficiently in HEK 293T cell line.Conclusion pCDGFP-Depp expression vector is successfully constructed,and identified Depp protein can be effectively expressed in mammalian cells lines.