重组人粒细胞-巨噬细胞集落刺激因子在毕赤酵母中的表达及活性分析  

Expression and Bioactivity Analysis of hGM-CSF in Pichia pastoris

作　　者：宋淼[1,2] 刘波[2] 王越[1,2] 刘党生[2] 吴军[1] 

机构地区：[1]军事医学科学院生物工程研究所,北京100071 [2]沈阳药科大学制药工程学院,辽宁沈阳110015

出　　处：《生物技术通讯》2008年第6期848-851,共4页Letters in Biotechnology

基　　金：国家高技术研究发展计划(2007AA02Z103)

摘　　要：目的:利用毕赤酵母野生型菌株GS115(his4)和突变型菌株GJK01(ura3,ade1,arg4,his4,och1)表达人粒细胞-巨噬细胞集落刺激因子(hGM-CSF),并对其活性及糖基修饰情况进行比较。方法:用PCR技术扩增目的基因,引入XhoⅠ、NotⅠ双酶切位点,构建pPIC9-hGM-CSF表达载体,电击转化毕赤酵母GS115和GJK01感受态细胞,得到工程菌;经甲醇诱导,对其表达产物进行糖基分析和生物学活性分析。结果:hGM-CSF在野生型菌株GS115和突变型菌株GJK01中均得到表达,其中野生菌表达的为过度糖基化的糖蛋白,而突变型菌株表达的为低糖化的糖蛋白,且二者均可刺激TF-1细胞的生长,比活性分别为9.79×105和2.21×105U/mL。结论:利用酵母工程菌表达了低糖化的重组hGM-CSF,为其药物研究提供了新的思路,也为酵母的糖基工程改造提供了良好的糖蛋白模型。Objective： To compare degree of N-glycosylation and bioactivity of the recombinant human granulocyte- macrophage colony-stimulating factor（hGM-CSF） expressed in wild-type and mutant type Pichia pastoris. Methods： Gene of interest was amplified by PCR, then the expression vector of pPIC9-hGM-CSF was constructed and transformed into wild type and mutant type P.pastoris by electroporation. N-glycan and bioactivity of the expressed products were analyzed after the expression strains were induced by methanol. Results： Different with the hyperglycosylated hGM-CSF expressed in wild type P.pastoris, hGM-CSF expressed in the ochl deletion strain contained smaller N-glycan, and their specific activities were 9.79x105 and 2.21x105 U/mL respectively. Conclusion： Low N-glycosylated hGM-CSF expressed in the ochl deletion strain makes possible for medical research of recombinant hGM-CSF, and for production of complex human glycoproteins in glyco-enginnering yeast strain.

关 键 词：人粒细胞-巨噬细胞集落刺激因子 毕赤酵母 N-糖基化 生物活性 

分 类 号：Q78[生物学—分子生物学]