人PAX8与PPARγ的融合基因(PPFP)的克隆及其重组质粒的构建
作者:
作者单位:

作者简介:

李新营

通信作者:

基金项目:

国家自然科学基金资助项目(30600601)。


Cloning of PPFP fusion gene (composed of PAX8 and PPARγ) and construction of the recombinant vector containing human PPFP gene
Author:
Affiliation:

Fund Project:

  • 摘要
  • |
  • 图/表
  • |
  • 访问统计
  • |
  • 参考文献
  • |
  • 相似文献
  • |
  • 引证文献
  • |
  • 资源附件
  • |
  • 音频文件
  • |
  • 视频文件
    摘要:

    目的:探讨人PAX8/PPARγ融合基因(PPFP)表达质粒在甲状腺上皮细胞中的表达及作用。
    方法:从含有PAX8基因和PPARγ基因的质粒克隆模板PAX8-pOTB7及PPARγ-pCMV-SPORT6中,利用PCR方法调取目的基因PAX8和PPARγ,将PAX8和PPARγ定向连接后克隆到pEGFP-C1载体上,构建融合基因的真核表达质粒pEGFP-C1-PAX8/PPARγ,在大肠杆菌E.coli DH5α中转化并提取质粒,通过PCR和测序、分析比对验证PPFP融合基因后,将真核表达载体pEGFP-C1-PAX8/PPARγ的质粒用脂质体包合并转染人正常甲状腺上皮细胞Nthy ori 3-1,通过RT-PCR和Western blot鉴定PPFP融合基因于靶细胞内在mRNA和蛋白水平上的表达。
    结果:构建的质粒通过鉴定证实正确;该质粒转染人正常甲状腺上皮细胞Nthy ori 3-1后,经验证显示PPFP融合基因在mRNA和蛋白水平上顺利表达。
    结论:成功克隆了PPFP融合基因并构建其重组质粒pEGFP-C1-PAX8/PPARγ;该质粒转染人正常甲状腺上皮细胞Nthy ori 3-1后可顺利表达PPFP蛋白,为进一步研究PPFP基因致瘤作用的分子机制提供了实验基础。

    Abstract:

    Objective:To explore the expression and effect of the recombinant vector of human PAX8/ PPARγ fusion gene (PPFP) in human thyroid follicular epithelial cell Nthy ori 3-1.
    Methods:The PAX8 gene was isolated and amplified by PCR technique from PAX8- pOTB7 plasmid. The PPARγ gene was isolated and amplified by PCR technique from PPARγ- pCMV-SPORT6 plasmid. Then the two genes were directionally united and this united gene was subcloned into the eukaryotic expression plasmid of pEGFP-C1 vector to generate the eukaryotic expression recombinant vector, pEGFP-C1-PAX8/ PPARγ.The correct PPFP gene was confirmed by PCR, edoenzyme digestion, sequencing, analysis, and contrast. After the co-transfection of pEGFP-C1-PAX8/ PPARγ and lipofectamine 2000 into human thyroid follicular epithelial cell Nthy ori 3-1,the expressions of PPFP fusion gene on the levels of mRNA and protein were detected by RT-PCR and Western blot.
    Results:The correct eukaryotic expression recombinant vector, pEGFP-C1-PAX8/ PPARγ,was confirmed by verification.After the pEGFP-C1-PAX8/ PPARγ and Lipofectamine 2000 were transfected into Nthy ori 3-1 cells, the expressions of PPFP fusion gene on the levels of mRNA and protein were successfully detected.
    Conclusions:The PPFP fusion gene and the eukaryotic expression recombinant vector pEGFP-C1-PAX8/ PPARγ have been constructed successfully. After this recombinant vector is transfected into Nthy ori 3-1 cells,it could successfully express PPFP protein,and provides an experimental basis for further exploring the molecular mechanisms of PPFP′s tumorigenesis.

    参考文献
    相似文献
    引证文献
引用本文

刘剑鸣, 王志明, 李新营, 李劲东, 李萃, 段朝军, 汤参娥.人PAX8与PPARγ的融合基因(PPFP)的克隆及其重组质粒的构建[J].中国普通外科杂志,2009,18(11):1149-1155.
DOI:10.7659/j. issn.1005-6947.2009.11.012

复制
分享
文章指标
  • 点击次数:
  • 下载次数:
历史
  • 收稿日期:2009-08-12
  • 最后修改日期:2009-09-12
  • 录用日期:
  • 在线发布日期: 2009-11-15