人重组白细胞介素11在毕氏酵母系统中的表达及纯化  

Expression and Purification of Recombinant Human Interleukin- 11 in Pichia pastoris

作　　者：朱剑昆[1] 许志祥[1] 黄伟达[2] 闵太善[2] 谢炜[1] 徐颖[1] 张学光[1] 

机构地区：[1]苏州大学免疫教研室,苏州215007 [2]Department of Biochemistry, Fudan University

出　　处：《中国医学科学院学报》2001年第2期127-131,共5页Acta Academiae Medicinae Sinicae

基　　金：国防科工委科学基金! (Y5573162);国际原子能机构 (IAEA)基金! (CPR/9/025)&&

摘　　要：目的 在甲醇营养型毕氏酵母蛋白质表达系统中高效表达人白细胞介素-11（ rhIL-11），便于进一步开发。方法 以人工设计合成的 rhIL-11基因，构建表达载体 pPICZα A-IL-11，经线性化后电转化导入毕氏酵母菌株 KM71，甲醇诱导表达，用 ELISA和 SDS-PAGE检测发酵上清中 IL-11的抗原性和表达量，用 IL-11依赖的 B9-11细胞株分析其生物学活性，采用疏水层析、离子交换和凝胶过滤纯化发酵上清中的 IL-11。 结果 序列分析表明，克隆载体中 IL-11人工基因序列与设计相符；基因工程菌株 KM71-2424在摇瓶培养上清中 IL-11的表达量超过 60 mg/L，生物学活性测定显示其比活性为 5.5× 107 U/mg，而标准品的生物学活性为 2.2× 107 U/mg。经过三步层析纯化得到电泳纯的 rhIL-11蛋白质。结论 成功获得 IL-11人工基因和稳定分泌重组蛋白的基因工程菌株 KM71-2424，该重组蛋白的生物学活性显著高于大肠杆菌表达的标准品，并获得较高纯度的重组蛋白。Objective To express recombinant human interleukin- 11 (rhIL- 11) in methylotropic yeast Pichia pastoris. Methods By designing and synthesizing an artificial gene for IL- 11, the expression vector pPICZα A- IL- 11 was constructed and introduced into Pichia pastoris by linearized electroporation. The rhIL- 11 protein was identified by ELISA and SDS- PAGE analysis. The bioactivity was analyzed by B9- 11 cell line. A combination of liquid chromatography was developed to purify the rhIL- 11 from ferment supernatant. Results The nucleotide sequence analysis indicated that the sequence of cloned artificial IL- 11 gene accorded with that of designed; the secreted yield of rhIL- 11 by yeast Pichia pastoris KM71- 2424 in flask reached 60 mg/L. The biological activity of IL- 11 in yeast supernatant and E.coli standard determined by B9- 11 was 5.5× 107 U/mg and 2.2× 107 U/mg respectively. The rhIL- 11 was purified to electrophoretic purity by a combination of liquid chromatography. Conclusion The human IL- 11 artificial gene was obtained and successfully expressed in the Pichia pastoris(KM71- 2424). The biological activity of IL- 11 in yeast supernatant was significantly higher than that of E.coli standard. The rhIL- 11 was purified to electrophoretic purity.

关 键 词：重组人白细胞介素-11 毕氏酵母 表达 分离 纯化 

分 类 号：R392－33[医药卫生—免疫学]