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 peDNA5/ 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.%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.
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