重组人白介素18的发酵、纯化及功能鉴定  

Fermentation,Purification and Identification of Recombinant Human Interleukin-18

作　　者：安秀梅[1] 杨莉莉[1] 李慧[1] 曹水[1] 任秀宝[1] 

机构地区：[1]天津医科大学附属肿瘤医院研究所免疫室,天津市"肿瘤防治"重点实验室,300060

出　　处：《天津医药》2009年第7期559-561,I0004,共4页Tianjin Medical Journal

基　　金：天津市科技创新专项资金项目(项目编号.06FZZDSF01500)

摘　　要：目的:利用构建的重组菌株Pichia pastoris X-33/hIL-18,在3.7L发酵罐中表达重组人白介素18(hIL-18),探讨其大规模发酵及纯化工艺。方法:复苏工程菌X-33/hIL-18于BMGY培养基,在其A600达到6左右时转入发酵罐进行高密度发酵。发酵液经过滤浓缩柱、疏水层析和阴离子交换层析分离纯化。结果:工程菌在3.7L发酵罐采用甲醇诱导补料批式发酵,在pH6.0,溶氧值(DO)20%～30%,温度28℃,甲醇诱导48h重组hIL-18的表达产量为202mg/L,发酵液经纯化后纯度可达97%以上,并初步证明重组hIL-18能协同IL-2诱导PBMCs分泌IFN-γ和协同IL-2增强NK细胞的杀伤活性。结论:利用Pichia pastoris分泌型表达系统和建立的分离纯化方法,能获得大量较高纯度的重组hIL-18,为进一步研究其活性和功能奠定基础。Objective： To investigate the expression of recombinant human intedeukin-18 （hIL-18） in 3.7 L fermenter with the constructed engineer train Pichiapastoris X-33/hIL-18, and the procedures of expression and purification thereof. Methods： The train X-33/hlL- 18 was inoculated in BMGY medium and then inoculated into the fermenter until the A 600 of the culture reached about 6. The supernatant of fermentation was isolated and purified with centrifugal filter devices, hydrophobic chromatography column and anion exchange chromatography column. Results： The recombinant hIL-18 was expressed in 3.7 L fermenter with batch feed methanol and the concentration reached 202 mg/L. After the purification, the purity could be more than 97%. The recombinant hIL-18 was shown to induce interferon-gamma （IFN-3,） production by human peripheral blood mononuclear cells （PBMCs）, and enhanced NK cell cytotoxicity synergistically with IL-2. Conclusion： A great deal of the recombinant hlL-18 with higher purity could be harvested by Pichia pastoris expression system. This study showed a new potential for further study of its function and activities.

关 键 词：白细胞介素18 毕赤酵母 发酵 培养基 分离和提纯 

分 类 号：R979.14[医药卫生—药品] R284.2[医药卫生—药学]