人nanog-delta48基因的克隆及其真核表达载体的构建

目的:构建pcDNA5/FRT-nanog-delta48真核表达载体,检测其在肝癌SMMC-7721细胞中的表达。方法:通过RT-PCR的方法克隆nanog基因的选择性剪接变异体nanog-delta48全长编码序列,连接入pMD18-T载体,经鉴定正确后亚克隆入pcDNA5/FRT,构建pcDNA5/FRT-nanog-delta48真核表达载体,测序无误后经脂质体介导转染到SMMC-7721细胞中,通过RT-PCR初步鉴定其在SMMC-7721细胞中的表达,并通过四甲基偶氮唑盐(MTT)法检测转染前后的细胞增殖活力。结果:成功克隆nanog-delta48基因全长编码区,测序证明pcDNA5/FRT-nanog-delta48重组真核表达载体构建成功,使其在SMMC-7721细胞中过表达,MTT检测nanog-delta48转染入SMMC-7721细胞后,增殖能力增强。结论:剪接变异体nanog-delta48基因可能具有与nanog基因类似的活性。

Cloning Human Nanog-delta48 Gene and Constructing Its Eukryotic Expression Vector

Objective:To construct the eukaryotic expression vector of pcDNA5/FRT-nanog-delta48 and detect its expression in human hepatoma cell line SMMC-7721.Methods:The alternatirely-spliced variant of nanog(nanog-delta48) full-length coding sequence was cloned by RT-PCR,and then inserted into pMD18-T vector.After DNA analysis,the correct identification was sub-cloned into pcDNA5/FRT.The eukaryotic expression vector of pcDNA5/FRT-nanog-delta48 was transiently transfected into human liver cancer SMMC-7721 cells by liposome-mediated transfection,then its expression was identified by RT-PCR and its proliferation was assessed by MTT.Results:The full-length coding sequence of human nanog-delta48 was cloned successfully.The eukaryotic expression vector of pcDNA5/FRT-nanog-delta48 was proved to be constructed successfully by DNA analysis,which was over-expressed in SMMC-7721 cells.MTT result showed that the cell proliferation increased after transfection.Conclusion:The alternatively-spliced variant of nanog(nanog-delta48) has similar activity to that of the full length version.