重组人小分子抗体ScFv-Fc发酵条件的优化及纯化  

An optimized Fermentation and Purification Process for a Recombinant Human Antibody ScFv-Fc Fragment Expressed in Pichia Pastoris

作　　者：王丁丁[1] 苏曼曼[2] 胡丽莉[1] 袁丽颖[3] 颜炜群[2] 

机构地区：[1]广东药学院生命科学与生物制药学院,广东广州510006 [2]吉林大学药学院,吉林长春130021 [3]吉林大学附属第三幼儿园,吉林长春130021

出　　处：《现代生物医学进展》2011年第16期3048-3051,共4页Progress in Modern Biomedicine

基　　金：国家自然科学基金青年基金项目(81000923);吉林大学科学前沿与交叉学科创新创新项目(200903339)

摘　　要：目的:研究重组人小分子抗体ScFv-Fc在毕赤酵母中分泌表达的最佳条件,以及ScFv-Fc的纯化方法。方法:分别从甲醇浓度、pH、诱导时间等方面对毕赤酵母重组菌株产生ScFv-Fc的发酵过程进行了优化;通过硫酸铵沉淀结合protein A亲和层析柱,对ScFv-Fc的纯化方法进行了研究。结果:确定ScFv-Fc在毕赤酵母中分泌表达的最佳条件为:在pH5.2的条件下,以0.5%甲醇诱导72 h。经过protein A亲和层析柱纯化后,ScFv-Fc纯度可达94%以上。结论:确定了ScFv-Fc在毕赤酵母中分泌表达的最佳条件以及纯化方法,为重组抗体分子诊断、治疗试剂的开发以及抗体的人源化奠定了物质基础。Objective： To obtain an optimized fermentation and purification process for high-level production of a recombinant human antibody ScFv-Fc fragment expression secreted in Pichia pastoris. Methods： The growth conditions of the transformant strain were optimized in 50 ml conical tubes including pH, methanol concentration and inducing time. The ScFv-Fe was purified using a two-step scheme： ammonium aulfate fractionation, protein A Sepharose. Results： SeFv-Fc production was found to increase with 0.5% （v/v） methanol concentration after 72 b induction. Protein production was also greatly affected by pH, resulting in higher yields at 5.2 pH value. The ScFv-Fc was purified more than 94% purity by using protein A Sepharose. Conclusions： The results provided a best process for expression and purification of functional recombinant human monoclonal antibody ScFv-Fc. It suggests a potential use of this antibody generating method by Pichia pastoris and indicates the potential of scFv-Fc fusion proteins as therapeutic candidates.

关 键 词：ScFv—Fc 表达 毕赤酵母 发酵优化 纯化 

分 类 号：Q75[生物学—分子生物学] Q78