Establishment of IL-1β Antisense RNA Expression Vector and Its Effect on Expression Level of IL-1β in Murine Hepatoma Cells

Objective To construct murine interleukin 1β(IL-1β) antisense RNA expression vectors and analysis its effect on expression level of IL-1β in murine hepatoma cells.Methods Murine IL-1β gene segments of IL-1β1(264bp) and IL-1β2(284bp) was amplified through RT-PCR,purified PCR products were inserted inversely into PafpIRES2-EGFP to construct PafpIRES2-antiIL-1β1 and PafpIRES2-antiIL-1β2 antisense RNA expression vectors.Recombinant plasmid was verified through clolony PCR screening and DNA sequencing.The established vectors were transfected into H22 hepatoma cells by jetPEI.Specific expression the vectors were observed under fluorescence microscope and the level of the IL-1β were assessed through RT-PCR.Results Two gene segments of 264b and 284bp were prepared through RT-PCR as expected.After insertion into PafpIRES2-EGFP,repeated PCR screening identified several positive clones,as proved by DNA sequencing that the two gene fragments inserted were identical to the reported murine IL-1β in GenBank(M15131),and then the recombinant vectors were named as PafpIRES2-antiIL-1β1 and PafpIRES2-antiIL-1β2.Following transfection into H22 and YAC-1 cells,48h later bright green fluorescence could be seen only on the cell surface of H22 but not YAC-1 cells when observed under fluorescent microscope,indicating that both vectors could specifically express in hepatoma cells.RT-PCR indicated that the expression level of IL-1β was markedly inhibited by the antisense RNA compare with cells transfected with PafpIRES2-EGFP,significant inhibition was observed in cells transfected with PafpIRES2-antiIL-1β2.Conclusion Two murine IL-1β antisense RNA expression vectors PafpIRES2-antiIL-1β1 and PafpIRES2-antiIL-1β2 were successfully constructed,and the recombinant vectors could decrease the expression level of IL-1β effectively in murine hepatoma cells.