miR-145真核表达载体的构建及表达

目的构建miR-145的真核表达载体,为研究miR-145在结肠癌中的生物学功能奠定基础。方法设计并应用PCR扩增miR-145基因片段,将其导入真核表达载体pCMV-myc中构建重组质粒pCMV-miR-145后,将重组质粒转染入结肠癌细胞系HCT116中,运用RT-PCR检测miR-145的表达情况。结果酶切及DNA测序证实miR-145被正确克隆入真核表达载体pCMV-myc中,该重组质粒能在HCT-116细胞中高效表达miR-145。结论成功构建了miR-145的真核表达载体pCMV-miR-145,该载体能有效高表达miR-145。

Construction and Expression of a Eukaryotic Vector for miR- 145

Objective To construct a eukaryotic expression vector for miR-145,so as to lay a foundation for exploring its biological function in colon cancer.Methods The gene fragment of miR-145 was obtained by PCR and inserted into a eukaryotic expression plasmid,pCMV-myc.The constructed plasmid,pCMV-miR-145,was transfected into the colon cancer cell line HCT116.The expression level of miR-145 was detected by real-time PCR.Results The successful cloning of miR-145 into the eukaryotic expression vector pCMV-myc was confirmed by enzyme digestion and DNA sequencing analysis.The reconstructed plasmid could significantly increase the expression of miR-145 in HCT116 cells.Conclusions A eukaryotic expression vector for miR-145,pCMV-miR-145,is successfully constructed.It can highly express miR-145.