Construction of lentiviral vector specific for mouse B7-1 gene interference and study on silencing effects induced by lentivirus-mediated B7-1 RNAi / 中国免疫学杂志

ABSTRACT

Objective:To construct lentiviral vector specific for mouse B7-1 RNA interference and study lentivirus-mediated B7-1 gene silencing effects in L929 fibroblast cells.Methods:Three candidate sequences for B7-1 RNAi selected from coding sequence of mouse B7-1 transcription were used to design short hairpin RNA ( shRNA ) templates and then cloned into lentiviral expression plasmid followed with correctness identification of inserted sequence by DNA sequencing.Recombinant lentivirus were prepared by co-transfecting lentiviral expression vector and packaging plasmids into 293T cells.Then the resulting culture supernatant containing infectious lentiviral particles was pooled and centrifuged via ultra-centrifugation.Infectious titer of the preparations was determined by detecting the expression of GFP in 293T cells after transfected by lentivirus.Cultured L929 cells were transfected with lentivirus to deter-mine transduction efficiency and silencing efficacy of B7-1 expression by flow cytometry.Transducted L929 cells were then screened using puromycin to generate stable cell clones followed by flow cytometry analysis of GFP and B7-1 expression.A mixed reaction system consisting of stable B7-1 silencing L929 cells and mouse splenic T cells was used to analyze ability of the established cell line to trigger T cells proliferation.Results: Lentiviral expression vector for mouse B7-1 RNAi was correctly constructed with inserted sequences as designed.Recombinant RNAi lentivirus were prepared with titers ranging (3-5) ×108 TU/ml and efficacy to mediate GFP transgene expression and B7-1 silencing.B7-1 expression and the ability to trigger T cells proliferation of stable L929 cells were suppressed significantly ( P<0.05 ).Conclusion: We generated lentiviral vector specific for mouse B7-1 RNAi with high performance of transduction efficiency as well as B7-1 silencing efficacy and the recombinant RNAi lentivirus can mediated stable B7-1 gene silencing in L929 cells and inhibition of T cells proliferation induced by B7-1/CD28 co-stimulatory signal.