The effects of lentiviral vector-mediated delivery of short hairpin RNA on CD28 expression in mouse T lymphocytes

Objective To explore the effects of lentiviral vector-mediated delivery of short hairpin RNA on CD28 expression in mouse T lymphocytes.Methods Three pairs of specific interference sequences targeting CD28 gene and one pair of non-specific RNA interference sequences were designed and synthesized,and were thereafter respectively inserted into lentiviral vector pLB downstream of U6 promoter,so as to construct four recombinant plasmids.293FT cells were co-transfected with CD28/shRNA transfer plasmid and two lentiviral packaging plasmids,and the lentivirus titer was determined according to the expression level of green fluorescent protein(GFP).Mouse spleen lymphocytes were infected by three specific or one control lentivirus,and then CD28 mRNA transcription and protein expression of these cells was detected by real-time RT-PCR and Western blot,respectively.Results PCR analysis and DNA sequencing confirmed that the four recombinant lentiviral transfer plasmids were successfully constructed.The titer of recombinant lentivirus was above 1×1011 U/L.After CD28/shRNA virus infection,CD28 expression in mouse T lymphocytes were significantly downregulated at both mRNA and protein levels.Conclusion The lentiviral RNAi vector of CD28 gene have been successfully constructed,and they can effectively inhibit the expression of CD28 gene in mouse T lymphocytes.Thus it provides a basis for further study of CD28 gene-targeted gene therapy on graft-versus-host disease.