重组慢病毒表达载体mpCDH-CMV-RFP的构建及鉴定+

Objective] To construct the recombinant lentivirus plasmid mpCDH-CMV-RFP that can be used to stably express miRNAs. [Methods] The red fluorescent protein (RFP), miR-30 loop and flanking sequence amplified from expression vector pTRIPZ was cloned into the lentivirus vector pCDH-CMV-MCS-EF1-copGFP. The recombinant plasmid mpCDH, packaging plasmid psPAX2 and envelope plasmid pMD2.G were co-transfected into the 293T cells. The transfection efficiency was determined by observing the expression of RFP. Gradient dilution method was used to detect the virus titer after harvest the filtered culture medium. To determine the expression of target miRNA, the precursor sequence of miR-134-3p was inserted into the constructed lentivirus vector mpCDH and served as a positive control. The obtained recombinant lentiviruses Lv-mpCDH and Lv-miR-134-3p were used to infect HeLa cells and the relative expression level of miR-134-3p was detected using fluorescence quantitative PCR(RT-PCR). [Results] The construction of recombinant plasmid mpCDH was confirmed by restriction endonuclease and sequencing. The titer of Lv-mpCDH was 8×105 pfμ/mL. With the Lv-mpCDH and Lv-miR-134-3p infection in HeLa cells, RT- PCR results showed that the mpCDH mediated miR-134-3p expression was significantly increased after Lv-miR-134-3p infection . [Conclusion] We successfully constructed a recombinant lentivirus vector mpCDH which can be used for stable expression of mature miRNAs.