天津医药

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人nanog-delta48基因的克隆及其真核表达载体的构建

邓璐林1,章诺贝1,张吉翔2   

  1. 1. 南昌大学第二附属医院
    2. 南昌大学第二附医院消化内科
  • 收稿日期:2011-11-04 修回日期:2012-03-19 出版日期:2012-09-15 发布日期:2012-09-15
  • 通讯作者: 张吉翔

Cloing human nanog-delt48 gene and constructing its eukryotic expression vector

1, 1,   

  • Received:2011-11-04 Revised:2012-03-19 Published:2012-09-15 Online:2012-09-15

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摘要： 摘要：目的：构建pcDNA5/FRT-nanog-delta48真核表达载体，检测其在肝癌细胞7721中的表达。方法：通过RT-PCR的方法克隆nanog基因的剪切变异体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转染到肝癌细胞7721中，通过RT-PCR初步鉴定其在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重组真核表达载体成功，使其在7721细胞中过表达，MTT检测nanog-delta48转染入7721细胞后，增值能力增强。结论：剪接变异体nanog-delta48基因可能具有与nanog基因类似的活性，将为进一步研究nanog-delta48基因的功能以及与nanog基因的关系及奠定基础。

关键词: 人, nanog, 剪接变异体, 基因克隆, 真核表达, 瞬时转染

Abstract: Abstract: 【Objective】: Construct the eukaryotic expression vector of pcDNA5/ FRT-nanog-delta48 and detect its expression in liver cancer cells 7721.【Method】: Clone the alternatively-spliced variant of nanog (nanog-delta48 )full-length coding sequence by RT-PCR, then insert the right PCR product into pMD18-T vector, after DNA analysis, subclone the correct identification into pcDNA5/FRT. The eukaryotic expression vector of pcDNA5/FRT-nanog-delta48 was transiently transfected into human liver cancer cell 7721 by liposome mediated, then identify its expression by RT-PCR and assess its proliferation by MTT.【Result】:The full-length coding sequence of human nanog-delta48 was cloned succefully.The eukaryotic expression vector of pcDNA5/ FRT-nanog-delta48 was proved to be constructed successful by DNA analysis, and it overexpressed in 7721 cells. MTT result showed cell proliferation increased after transfection.【Conclusion】:The alternatively-spliced variant of nanog(nanog-delta48) has similar activity to that of the full length version. It will lay the foundation for the further study of its own functions and the relationship with nanog of full-length

Key words: Human, nanog gene, alternatively-spliced variant, gene cloning, eukaryotic expression, transient transfection