Construction and identification of lentiviral expression vector hsa-miR-150

Objective To construct lentiviral expression vector hsa-miR-150 and determine the expression of miR-150 in infected hepatocellular carcinoma cell line Hep3B.Methods The hsa-miR-150 gene fragment was synthesized chemically and cloned into lentiviral vector pGIPZ.The constructed recombinant plasmid pGIPZ-hsa-miR-150 was transfected to packaging cell line 293T,and the packaged virus was determined for titer.Recombinant lentivirus particles were harvested and concentrated,then stably transfected to Hep3B cells and determined for expression rate of GFP by flow cytometry,and for expression level of miR-150 by fluorescent quantitative PCR.Results PCR amplification of bacterial colony and sequencing proved that recombinant lentiviral expression vector pGIPZ-hsa-miR-150 was constructed correctly.The titer of recombinant lentivirus was 5.7 × 108/ml.The expression rate of GFP in Hep3B cells stably transfected with pGIPZ-hsa-miR-150 was(97.78 ± 1.14)%,while the expression level of miR-150 increased by 6-folds as compared with those in blank control group(P 0.05).Conclusion The lentiviral expression vector hsa-miR-150 was constructed successfully and highly expressed in Hep3B,which laid a foundation of further study on biological function of miR-150.