人重组白细胞介素-13在毕氏酵母中的表达及其体外诱导DC分化作用的研究

目的：在毕氏酵母中表达重组人IL-13并探讨其对DC的诱导作用。方法：用基因工程的方法人工合成IL-13的全长片段，并转化至毕氏酵母菌株GS115中，筛选后获得表达量相对较高且分泌稳定的rhIL-l3基因工程菌株，用rhIL-l3来替代IL-4体外诱导DC。结果：得到表达量达25mg/L且分泌稳定的rhIL-l3基因工程菌株；rhIL-l3具有rhIL-4同样的体外诱导外周血单核细胞向比增殖分化的作用,MHCII、CDlα、CD80、CD80、CD86等DC表型分析和混合淋巴细胞反应刺激T细胞增殖的作用与IL-4诱导得到的成熟DC并无显著差别，其所诱导的末成熟DC具有很强的摄取抗原能力。结论：获得表达量相对较高且分泌稳定的rhIL-l3基因工程菌株，rhIL-l3在体外可有效地诱导外周血单核细胞向DC分化。

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 differentiate in vitro

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.