pEGFP-N1-PGC-1α重组质粒的构建及体外表达

Construction and Expression of pEGFP-N1-PGC-1α Recombinant Plasmid in Vitro

目的构建PGC—1α真核表达载体pEGFP—N1-PGC-1α重组质粒,鉴定及体外表达。方法通过RT-PCR方法从组织中克隆PGC-1α基因片段,酶切后将其定向插入真核表达载体pEGFP—N1中,构建pEGFP—N1-PGC-1α重组质粒。结果双酶切法、测序法鉴定表明目的基因正确插入质粒。结论成功构建了pEGFP—N1-PGC-1α真核表达载体,转染huh-7细胞后,可瞬时、有效表达,为进一步研究PGC-1α调节HBV、HPV生物合成奠定了基础。

Objective To construct eukaryotic expression plasmid carrying PGC-1α gene, and observe its expression in huh-7 cells. Methods PGC-1α gene was obtained from tissue through RT-PCR method and inserted eukaryotic expression vector pEGFP-N1. Thus pEGFP-N1-PGC-1α recombinant plasmid was constructed and transfected into huh-7 cells. Results The inserted fragment was identified by double enzyme digestion and DNA sequencing. Conclusion pEGFP-N1-PGC-1α recombinant plasmid was constructed and expressed successfully in huh-7 cells, which lays the foundation for future studies on the regulation of PGC-1α on HBV and HPV biosynthesis.