hPPARα真核表达载体的构建及鉴定

CONSTRUTION AND IDENTIFICATION OF HPPARα EUKARYOTIC EXPRESSING VECTOR

目的:构建pSG5/hPPARα真核表达载体,为进一步探讨在治疗糖尿病的过程中,传统中草药对hPPARα信号通路的分子调节机制奠定了基础。方法:从人类外周血中抽提总RNA,利用RT-PCR的方法进行扩增得到hPPARα基因片段。纯化后将其连接至克隆载体pGEM-Teasy中,并对重组载体pGEM-T/hPPARα进行基因测序鉴定。之后将hPPARα基因片段连接至瞬时真核表达载体pSG5中。经BamHⅠ酶切鉴定插入方向后,再次进行测序鉴定。利用瞬时转染技术,将pSG5/hPPARα真核表达载体转染至HepG2细胞中,随后通过western blot检测技术,检测hPPARα的瞬时表达情况。结果:菌落PCR、酶切电泳及测序结果证实,瞬时表达载体pSG5/hPPARα构建成功。western blot结果证明,转染了pSG5/hPPARα的HepG2细胞中,hPPARα基因得到了有效表达。结论:本实验成功构建了pSG5/hPPARα瞬时表达载体,可用于后续实验。

Objective：Construct eukaryotic expression vector pSG5/hPPARα,so as to establish basis for further exploration in the molecular mechanism of hPPARα signaling pathways,which are regulated by chinese herbal medicine during the treatment of diabetes.Methods： Total RNA was extracted from human peripheral blood and hPPARα fragments were amplified by RT-PCR.After being purified,the product of RT-PCR was inserted into a clone vector pGEM-Teasy.And the recombinant plasmids pGEM-T/hPPARα was identified by DNA sequencing.The obtained hPPARα fragment was inserted into transient eukaryotic expression vector pSG5.The direction of the insert was confirmed by restriction enzyme BamHⅠ digestion and DNA sequencing.After pSG5/hPPARα was transfected into HepG2 cells by transient transfection,the expression levels of hPPARα were measured by western blot.Results： The recombinant plasmid pSG5/hPPARα was constructed successfully and identified by colony PCR,enzymatic digestion and sequencing.And effective expression of hPPARα was also testified by western blot in HepG2 cell.Conclusion： The eukaryotic expression vector pSG5/hPPARα is constructed successfully and can be used for follow-up experiments.