重组靶向性抗肿瘤融合蛋白EGF-E4orf4毕赤酵母工程菌中试发酵工艺的建立  

Development of Pilot Fermentation Procedure for Recombinant Pichia pastoris for Tumor-targeting Fusion Protein EGF-E4orf4

作　　者：孙成金[1] 王健[1] 崔春青[1] 郭鹏[1] 陈祥鹏[1] 张振龙[1] 

机构地区：[1]北京生物制品研究所第四研究室,北京100024

出　　处：《中国生物制品学杂志》2011年第6期711-714,共4页Chinese Journal of Biologicals

基　　金：国家863"基因操作与蛋白质技术"重大专项资助(2008AA02Z118)

摘　　要：目的建立重组靶向性抗肿瘤融合蛋白EGF-E4orf4毕赤酵母工程菌的中试发酵工艺。方法优化EGF-E4orf4工程菌二级种子培养时间,按5%的比例接种于20 L发酵培养基,设定培养阶段温度为30℃,诱导表达阶段温度为28℃,在发酵过程中通过调节搅拌速度、通气量和罐压等措施使DO维持在20%～35%,采用三步发酵法进行发酵,优化诱导pH值及诱导时间,并在已优化的工艺条件下,稳定发酵3批。结果确定二级种子的培养时间为15 h,最适诱导pH值为6.0,最适诱导时间为48 h;中试发酵3批,菌体A600均值达(327.67±17.96),菌体湿重均值达(308.33±9.07)g/L,EGF-E4orf4表达量均值为(200.00±5.57)mg/L。结论已初步建立了重组EGF-E4orf4毕赤酵母工程菌的中试发酵工艺,为EGF-E4orf4的产业化奠定了基础。Objective To develop a pilot fermentation procedure for recombinant Pichia pastoris for tumor-targeting fusion protein EGF-E4orf4（adenovirus early region 4 open reading frame 4 protein）.Methods The time for culture of secondary seeds of recombinant P.pastoris for EGF-E4orf4 was optimized.The seeds were inoculated into 20 L of fermentation medium at a volume ratio of 5%,and the temperatures at culture and induction stages were set as 30 and 28℃ respectively.The DO during fermentation was maintained at 20% ~ 35% by the measures such as regulating stirring rate,aeration and pressure in fermenter.The recombinant P.pastoris was cultured by three-step fermentation,and the pH value and time for induction were optimized.Three batches of recombinant P.pastoris were fermented under the optimal condition.Results The optimal time for culture of secondary seeds was 15 h,while the optimal pH value and time for induction were 6.0 and 48 h respectively.The mean A600 value and wet weight of 3 batches of recombinant P.pastoris cultured by fermentation were（327.67 ± 17.96） and（308.33 ± 9.07） g/L respectively,while the mean expression level of EgF-E4orf4 was（200.00 ± 5.57） mg/L.Conclusion The pilot fermentation procedure for recombinant P.pastoris for EGF-E4orf4 was preliminarily developed,which laid a foundation of industrialization of EGF-E4orf4.

关 键 词：毕赤酵母 腺病毒早期区4第4编码蛋白 发酵 

分 类 号：Q78[生物学—分子生物学] R392-33[医药卫生—免疫学]