Study on the expression of recombination of human interleukin-13 (rhIL-13) in methylotropic yeast Pichia pastoris and its effect on the induction of DC to differen- tiate hi vitro / 中国免疫学杂志

ABSTRACT

Objective:To express recombination human interleukin-13(rhIL-13)in methylotropic yeast Kchia pastoris and study its effect on the induction of DC from PBMC in vitro. Methods: An artificial gene with the optional condon usage of Kchia pastoris for IL-13 was designed and synthesized by T4 DNA ligase and PCR.The expression vector pPICZoA-IL-13 was constracted and introduced in Kchia pastoris GS115 by electroporation after linearized with Sacl.Recombinants were selected by plating cells on YPD/Zeocine plates.The recombinant protein secreted by the yeast was identified by 15%SDS-PAGE,ELISA and Western blot.The abilities to induce DC differentiation of IL-13 and IL-4 were compared. Results: Based on the result of the 15% SDS-PAGE and Western blot assay, an about 13 kD recommbinant protein expressed in the yeast supernatant was identified to be recombinant human IL-13. Adherent PBMC cultured in GM-CSF,TNFa and IL-13 displayed morphological characteristic of DC generated in media containing IL-4. They formed cellular clumps and had extensive dendrites sharp. Fluorescence-activated cell sorter analysis showed that they expressed a high level of MHC class II ,CDla,CD80,CD83,CD86. The purity and yield of both DC populations are not significantly different with IL-13 or IL-4. The phagocytosis of immature DC induced by IL-13 is potent. They were also equally potent stimulators of allogeneic lymphocytes in the mixed lymphocyte reaction. Conclusion: rhIL-13 can substitute for IL-4 in the proliferation and development of dendritic cells.