人类野生型及突变型PPARγ基因真核表达质粒的构建和鉴定

Construction and identification of eukaryotic expression plasmids of human wild-type and mutant PPARγ gene

目的构建人类野生型过氧化物酶增殖物激活受体γ(PPARγ)及其突变型PPARγMut基因的真核表达质粒。方法采用RT-PCR和重叠延伸PCR技术扩增人类野生型及功能区缺失的突变型PPARγ全长序列,通过DNA重组技术分别将其重组于pcDNA3.1-Myc载体,构建myc标记的PPARγWT及PPARγMut融合蛋白表达质粒,通过酶切电泳分析和DNA测序的方法对重组表达质粒进行鉴定。结果通过酶切电泳和测序结果证实,构建的重组表达质粒目的基因片段为人类野生型PPARγ及突变型PPARγMut的cDNA。结论成功构建野生型及其功能区缺失的突变型PPARγ质粒,为进一步探讨PPARγ基因在乳腺癌发生和发展中的作用奠定了基础。

Objective To construct the eukaryotic expression plasmids of human wild-type peroxidase proliferator activated receptor γ(PPARγ) and its mutant PPARγMut gene.Methods The full sequences of wild-type and mutant PPARγ were amplified by RT-PCR and overlap extension PCR technique,which were then reconstructed into the eukaryotic expression vector pcDNA3.1-Myc to form the myc-tagged fusion protein expression plasmids of PPARγWT and PPARγMut by DNA recombinant technology.The recombinant expression plasmids were identified by enzyme digestion and DNA sequencing analysis.Results The enzyme digestion and DNA sequencing analysis showed that the constructed gene fragments of recombinant expression plasmids were the cDNA of human wild-type PPARγ and its mutant PPARγMut.Conclusion The plasmids of wild-type and mutant PPARγ have been successfully constructed,which lays a foundation for further exploring the role of PPARγ gene in the development and progression of breast cancer.