Objective To construct a short hairpin shRNA recombinant lentiviral vector targeting mouse VASP gene to interfere with the expression of VASP in mouse alveolar macrophage line(MH-S)cells.Methods shRNA targeting mouse VASP gene were de-signed,inserted into the shuttle plasmid GV644-EGFP vector,and identified by polymerization chain reaction(PCR)and sequencing.The recombinant plasmids GV644-VASP-shRNA-EGFP,auxiliary packaging pHelper 1.0 and pHelper 2.0 were co-transfected into 293T cells for packaging of recombinant lentivirus lenti-GV644-VASP-shRNA-EGFP,and titers were determined by fluorescent la-beling.MH-S cells were transfected with lenti-VASP-shRNA-EGFP at different multiple of infection(MOI)values,and the optimal MOI value was obtained according to the infection efficiency,and the expression of the target gene was detected by real-time fluorescence quantitative polymerase chain reaction(RT-qPCR)and Western blot.Results The shuttle plasmid GV644-VASP-shRNA-EGFP was successfully constructed by PCR and sequencing identification,and the recombinant lenti-VASP-shRNA-EGFP was further pack-aged with a viral titer of 2 × 109/(TU·ml),and the optimal MOI value of MH-S cells was 50,which significantly inhibited the expres-sion of VASP after infection with MH-S cells(P＜0.05).Conclusion In this study,the shRNA recombinant lentiviral vector targeting mouse VASP gene was successfully constructed,which significantly inhibited the expression of VASP in mouse MH-S cells,which laid a foundation for further exploring the role of VASP gene in macrophage phenotype regulation.
维普
万方数据