重组白细胞抑制因子和水蛭肽嵌合蛋白工程菌的高密度发酵  

High cell density fermentation of engineered E.coli expressingrecombinant neutrophil inhibitory factor and hirulog hybrid

作　　者：冀成法 马鲁南 柳常青 滕学东 邵明学 刘忠 张贵民 JI Chengfa;MA Lunan;LIU Changqing;TENG Xuedong;SHAO Mingxue;LIU Zhong;ZHANG Guimin(National Engineering Laboratory of High Expression in Mammalian Cells,Lunan Pharmaceutical Group Co.,Ltd.,Linyi 276006,China;Lunan New Time Bio-Tech Co.,Ltd.,Linyi 273400,China;Shandong Provincial Engineering Laboratory for Protein Drugs,Shandong New Time Pharmaceutical Co.,Ltd.,Linyi 273400,Chi)

机构地区：[1]鲁南制药集团股份有限公司哺乳动物细胞高效表达国家工程实验室,山东临沂276006 [2]鲁南新时代生物技术有限公司,山东临沂273400 [3]山东新时代药业有限公司山东省蛋白类药物工程实验室,山东临沂273400

出　　处：《生物加工过程》2021年第3期250-255,共6页Chinese Journal of Bioprocess Engineering

基　　金：山东省新旧动能转换重大工程课题(2020235)。

摘　　要：重组白细胞抑制因子和水蛭肽嵌合蛋白(TNHH)是治疗用生物制品Ⅰ类新药,具有多种功效,可用于治疗脑卒中。本文旨在优化TNHH工程菌发酵工艺,以实现该工程菌的高密度发酵和目的蛋白的高效表达。笔者采用摇瓶试验和发酵罐发酵,从培养基碳、氮源配比正交试验入手,对工程菌生长和目的蛋白表达影响较大的单因子条件进行重点考察,如温度、pH、诱导策略、诱导剂和抗生素替代及补料流加方式。通过优化,6批50 L发酵工艺稳定,菌体密度(OD 600)均能达79以上,目的蛋白表达量均在30%以上,质粒丢失率均低于10%。优化后的发酵工艺稳定,产量提高258%,质粒丢失率保持在10%以内,为TNHH项目进一步大规模生产奠定了基础。Recombinant neutrophil inhibitory factor and hirulog hybrid(TNHH)is class I new drugs for therapeutic biological products,which have multiple effects and can be used in the treatment of stroke.In this study,we aimed to optimize the fermentation process of E.coli producing recombinant targeted neutrophil inhibitory factor and hirulog hybrid(TNHH),so as to achieve the high-density fermentation and high expression of the target protein.Using shake flask test and tank fermentation,the ratio of carbon and nitrogen source from medium orthogonal experiment was researched firstly,then the factors that have a great effect on bacterial growth and protein expression were analyzed,such as temperature,pH,induction time,inducers and antibiotic replacement and filling material flow and method.Under the optimal conditions,6 repeated batches were fermented in a 50-L fermenter,and the bacterial density(OD 600)could reach more than 79,the expression level of target protein was higher than 30%,and the plasmid loss rate was less than 10%.The optimized fermentation process was stable,and the production of inclusion body increased by 258%.The plasmid loss rate could keep within 10%,laying a foundation for further large-scale production of TNHH.

关 键 词：脑卒中 TNHH 大肠杆菌 高密度发酵 质粒丢失率 水蛭肽 

分 类 号：TQ920.6[轻工技术与工程—发酵工程]