抗IgE单链抗体在大肠杆菌中可溶性高效表达条件的研究

中国生物工程杂志

2012, Vol. 32

Issue (11): 23-28

研究报告

抗IgE单链抗体在大肠杆菌中可溶性高效表达条件的研究

刘启刚^1,2, 代云见², 张勇侠², 王保成², 王明蓉²

1. 中国医药集团总公司四川抗菌素工业研究所成都 610052;
2. 成都生物制品研究所有限责任公司成都 610023

Efficient Soluble Expression of Anti-IgE scFv in E.coli and Optimization of Expression Conditions

LIU Qi-gang^1,2, DAI Yun-jian², ZHANG Yong-xia², WANG Bao-cheng², WANG Ming-rong²

1. Sichuan Industrial Institute of Antibiotics, Chengdu 610052, China;
2. Chengdu Institute of Biological Products Co., Ltd., Chengdu 610023, China

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摘要： 目的:以抗IgE单链抗体(anti-IgE scFv)为研究对象,以大肠杆菌周质高效表达可溶性单链抗体为目标,研究不同宿主细胞、培养基及培养条件对可溶性单链抗体表达产量的影响。方法:构建Rosetta(DE3)、BL21(DE3)和SoluBL21(DE3)三种大肠杆菌工程菌,研究不同碳源、氮源、培养基配方和培养条件对可溶性抗IgE单链抗体产量的影响。结果:携带抗IgE单链抗体基因的pET-IgE26质粒在新型宿主SoluBL21(DE3)中的表达产量明显优于传统的Rosetta(DE3)和BL21(DE3)宿主菌。经碳氮源筛选、培养基和培养条件的优化,确定SoluBL21(DE3)工程菌高效表达可溶性重组抗体的最佳培养基为:以M9培养基为基础,添加0.5%葡萄糖、0.6%酪蛋白胨和0.02%微量元素。最佳的培养条件为:以LB为种子培养基,37℃过夜培养至OD₆₀₀为3.0左右,以5%接种量接种于最佳发酵培养基中;接种后细胞生长条件:37℃,260r/min,培养3.5h;细胞诱导培养条件:当OD₆₀₀为1.5～1.8时,降温至25℃,添加0.1mmol/L IPTG,220r/min继续培养16h。经抗原ELISA检测,抗IgE单链抗体的可溶性表达量比原始对照组提高了5倍。结论:通过对宿主细胞类型的筛选、培养基及培养条件的优化,可以显著提高重组单链抗体在大肠杆菌周质空间内的表达产量。该研究结果不仅为规模化制备抗IgE单链抗体提供了技术支持,同时为大肠杆菌生产重组小分子抗体提供了有价值的参考。

关键词： 可溶性抗体产量; IgE单链抗体; 大肠杆菌; 表达条件优化

Abstract: Objective: An anti-IgE scFv was used to investigate the influence of different host strains, growth media, and culture conditions on the high-level expression of soluble single-chain antibody fragment in E. coli periplasm. Method: Three engineered bacterial strains, Rosetta (DE3), BL21(DE3), and SoluBL21(DE3), were constructed and effects of different carbon sources, nitrogen sources, growth media, and culture conditions on the amount of expression of soluble anti-IgE scFv were evaluated. Results: The expression level of pET-IgE26, a plasmid that carried anti-IgE scFv sequence, was significantly enhanced in the novel host strain SoluBL21(DE3), compared to that in traditional Rosetta (DE3) and BL21(DE3) host strains. After optimization of carbon and nitrogen sources, growth media and culture conditions, the optimal growth media for high-level expression of soluble recombinant antibodies in SoluBL21(DE3) engineered strain was found to be M9 medium with 0.5% glucose, 0.6% bacto casitone, and 0.02% trace elements. The best culture condition was defined as growing overnight culture in LB medium at 37℃, until OD₆₀₀reached approximately 3.0, then inoculate the optimal growth media with the overnight culture at 5% inoculum concentration, shake at 37℃, 260r/min for 3.5h. For induction, lower the temperature to 25℃ when OD₆₀₀ is between 1.5～1.8, add 0.1mmol/L IPTG, and incubate at 220r/min for 16h. ELISA detected that the expression of soluble anti-IgE scFv increased by 5-fold after optimization. Conclusion: Expression of recombinant scFv in E coli periplasm can be significantly enhanced through optimization of host strain types, growth media, and culture conditions. The study provides technical support for scale-up production of anti-IgE scFv and insights into the production of recombinant micromolecular antibody by E. coli.

Key words: Production of soluble antibodies IgE scFv E.coli Optimization of expression conditions

收稿日期: 2012-07-19 出版日期: 2012-11-25

ZTFLH:

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基金资助: 国家"十二五""重大新药创制"科技重大专项资助项目(2011ZX09506-005)

通讯作者: 王明蓉,电子信箱:mignrongw2007@163.com E-mail: mignrongw2007@163.com

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引用本文:

刘启刚, 代云见, 张勇侠, 王保成, 王明蓉. 抗IgE单链抗体在大肠杆菌中可溶性高效表达条件的研究[J]. 中国生物工程杂志, 2012, 32(11): 23-28.

LIU Qi-gang, DAI Yun-jian, ZHANG Yong-xia, WANG Bao-cheng, WANG Ming-rong. Efficient Soluble Expression of Anti-IgE scFv in E.coli and Optimization of Expression Conditions. China Biotechnology, 2012, 32(11): 23-28.

链接本文:

https://manu60.magtech.com.cn/biotech/CN/ 或 https://manu60.magtech.com.cn/biotech/CN/Y2012/V32/I11/23

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