目的构建携带miR-214的重组慢病毒表达载体，为研究miR-214的功能和作用机制奠定基础。方法从小鼠基因组DNA扩增miR-214基因，测序鉴定后克隆至pCDH-CMV-MCS-EF1-copGFP慢病毒载体上，测序鉴定、病毒包装后转染HEK293细胞，测定病毒滴度。将重组慢病毒载体感染HEK293细胞，应用萤光定量PCR法检测miR-214表达。结果重组慢病毒载体pCDH-CMV-miR-214-EF1-copGFP测序完全正确，pCDH-CMV-miR-214-EF1-copGFP感染的HEK293细胞中miR-214表达显著增强。结论成功构建小鼠miR-214重组慢病毒载体，并可在HEK293细胞中稳定表达出miR-214，为进一步进行miR-214的功能和机制研究奠定了试验基础。% Objective To construct the lentiviral expression system carring miR-214. Methods MiR-214 was amplified from mouse genomic DNA by PCR,then was cloned into the lentiviral vector of pCDH-CMV-MCS-EF1-copGFP and sequenced. The HEK293 cells were transfected with recombinant lentiviral vector and the titer of virus was detected by green fluorescent protein determination. Finally the expression of miR-214 in HEK293 cells infected with recombinant lentiviral vector was analysed by using real-time quantitative PCR. Results PCR and DNA sequencing assays demonstrated that the recombinant lentiviral vector of pCDH-CMV-miR-214-EF1-copGFP was successfully constructed. The expression level of miR-214 in HEK293 was significantly increased after transfection with pCDH-CMV-miR-214-EF1-copGFP. Conclusion The recombinant vector of pCDH-CMV-miR-214-EF1-copGFP was successfully constructed and it can induced a high miR-214 expression in HEK293 cells.
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