Coexpression of human IL-12 in insect cells transfected by double promoter baculovirus expression vector

Objective Interleukin 12 (IL 12) is a heterodimeric cytokine composed of two covalently linked chains, P40 and P35. It is necessary to express recombinant human IL 12 for studying the biological effects of this cytokine in vivo and in vitro. Methods Peripheral blood mononuclear cells (PBMC), adherent cells and KB cell line were treated with Staphylococcus aureus CowanⅠ strain (SAC), IFN γ+LPS and PDBu, respectively. Total RNA was extracted from these cells by the guanidine isothiocyanate method. cDNA for both P40 and P35 subunits of IL 12 were cloned by RT PCR. Double promoter eukaryotic expression vectors, pAcUW51/IL 12 containing IL 12 P35 and P40 cDNA, were constructed. The vectors were used to co transfect the insect cells (Sf9) with linearized polyhedrosis virus genomic DNA. The cell clones expressed the rhIL 12 products were analyzed by SDS PAGE, Western blot, ELISA and biological assays. Results The molecular weight of recombinant human IL 12 products is about 67kD on SDS PAGE and Western blot in non reducing conditions. The expression level of recombinant human IL 12 was about 15.4μg 15.7μg/10 6 cells. The biological activities identified by two biological assays showed that the expressed products increased the proliferation of human PHA activated PBMC and also the human NK cell mediated cytotoxicity. Conclusion Double promoter baculovirus expression vector pAcUW51/IL 12 was successfully constructed and both P40 and P35 subunits of IL 12 were correctly coexpressed in insect cells.