Abstract: To construct a classical pathway expressing secretory human interleukin 1 beta (shIL-1β) recombinant vector and to analyze its expression level in H7402 hepatoma cells. MethodsThe total RNA was isolated from LPS stimulated healthy donor PBMCs, and the full length human interleukin 1 beta(hIL-1β) gene was obtained by RTPCR. The human EPO signal peptide gene was prepared by primer overlapping extension, and fusion gene encoding both the EPO signal peptide and mature IL-1β protein was synthesized through gene splicing by over lap extension(SOE). The product was directly cloned into pIRES2EGFP to construct the recombinant eukaryotic expression vector pIRES2EGFPshIL-1β which was verified by PCR, restriction enzyme assay(BamH I and EcoR I) and DNA sequencing. Purified pIRES2EGFPshIL-1β was stably transfected into the H7402 hepatoma cells by jetPEI, then the expression of the recombinant vector was verified under fluorescence microscopy, and the level of fusion gene mRNA was determined by RTPCR and of hIL-1β in cellular supernatant was assessed by ELISA. ResultsThe expression vector pIRES2EGFPshIL-1β that could induce the expression of hIL-1β was successfully prepared through the conventional protein secretory pathway. After the recombinant vector was transfected and selected with G418, the human H7402／shIL-1β cells could effectively express the fusion gene mRNA, and the active hIL-1β in the cellular supernatant was also significantly increased compared with the H7402／mock cells. ConclusionThe recombinant pIRES2EGFPshIL-1β vector that could express hIL-1β was successfully established through the conventional protein secretory pathway.

Key words: Human interleukin 1 beta, Secretion expression, Hepatoma, Signal peptide