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  

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

作　　者：江阳[1] 时宏珍[1] 黄伟达[2] 谢炜[1] 赵文宝[1] 张学光[1] 

机构地区：[1]苏州大学生物技术研究所,苏州215007 [2]复旦大学生物化学系,上海200433

出　　处：《中国免疫学杂志》2003年第2期82-87,共6页Chinese Journal of Immunology

基　　金：江苏省自然科学重点项目资助(批准号 BI98100)

摘　　要：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-13基因工程菌株,用rhIL-13来替代IL-4体外诱导DC。结果:得到表达量达25 mg/L且分泌稳定的rhIL-13基因工程菌株;rhIL-13具有rhIL-4同样的体外诱导外周血单核细胞向DC增殖分化的作用,MHCII、CD1α、CD80、CD83、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,其所诱导的未成熟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-13基因工程菌株,rhIL-13在体外可有效地诱导外周血单核细胞向DC分化。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.

关 键 词：人重组白细胞介素-13 毕氏酵母 外周血单核细胞 DC分化 基因表达 基因工程 

分 类 号：Q78[生物学—分子生物学]