Establishment of the Recombinant Murine IL-1β Vector pLIVE-IL-1β and Its Expression in Hepa1-6 Cells

Objective To establish a murine IL-1β expression vector for in vivo assay and analysis its specific expression in Hepa1-6 hepatoma cells.Methods Total RNA was prepared from peripheral blood monocytes(PBMCs) of BALB/C mouse treated with LPS(5 g/ml),full length IL-1β gene(822bp) was obtained through RT-PCR,purified PCR product digested with Xho Ⅰ and Nhe Ⅰ was cloned into pLIVETM to construct the recombinant murine IL-1β expression vector for in vivo research.Positive recombinant vector was verified through colony PCR,restriction enzyme assay(Xho Ⅰ and Nhe Ⅰ) and DNA sequencing.Purified pLIVE-IL-1β plasmid was transfected into Hepa1-6 hepatoma cells using TransITTM.The expression level of IL-1β was detected by RT-PCR 48h after transfection and compared with cells with pLIVE.Results A 822bp gene segment were amplified from total RNA isolated from PBMCs treated with LPS which was compatible to the sequence expected.Purified PCR product and pLIVETM digested with Xho Ⅰ and Nhe Ⅰ were ligated together to create the recombinant IL-1β expression vector pLIVE-IL-1β.When screening by colony PCR,it was showed that we got several positive clones,further assay by restriction enzyme assay indicated that positive clones gave rise to a 822bp DNA fragment which was finally proved to be identical to the full length murine IL-1β reported by GeneBank.The recombinant vector were then transfected into Hepa1-6 hepatoma cells,48h after transfection,RT-PCR indicated that the expression level of IL-1β was significantly enhanced compare with cells transfected with pLIVETM.Conclusion The authors successfully constructed a murine IL-1β expression vector pLIVE-IL-1β for in vivo research,the recombinant vector could stably express high level of IL-1β in murine hepatoma cells.