RNA Interference For Inhibition Of EBV Latent Membrane Gene LMP1 Expression

Objective To construct a recombinant retroviral vector pSUPER-LMPl that target EB V latent membrane protein gene 1(LMP1) and select a stable virus-producing cell clone, to explore the apoptosis of EBV positive Raji cell (LMP1~+) after the target gene was silenced by RNAi.Methods The 60nt sequences encoded for transcribing LMP1 shRNA were cloned into a retrovirual vector pSUPER.retro with DNA recombinant technique. The recombinant vector was identified by the electrophoresis analysis of restriction enzyme digestion and DNA sequencing. The packaging cell PA317 was transfected with recombinant plasmid using liposome-based transfection method and the stable virus-producing cell clone was selected by using G-418 medium. The viral supernatant was used to infect target cells, the expression of LMPl was tested by RT-PCR and the apoptosis of Raji cells was tested by flow cytometry.Results The result of DNA sequencing demonstrated that 60bp had been inserted in the expected site and the insertion sequence was exactly correct. When the recombinant vector was transfected into the packaging cell, green fluorescent protein (GFP) was expressed. The anti-G418 positive clones were also selected and they could excrete recombinant retrovirus. RT-PCR result showed that high-efficiency inhibition of LMPl mRNA was achieved by retrovirus vector mediated RNAi in Raji cells, and the LMPl silencing induced Raji cells apoptosis by flow cytometry.Conclusion We succeeded in constructing a recombinant retroviral vector pSUPER-LMPl and selecting a stable virus-producing packaging cell line. LMPl expression in Raji cells was specifically silenced by recombinant retrovirus pSUPER-LMPl, and the effect of specific silencing of LMPl can induce the apoptosis of EBV positive Raji cells.