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中国生物工程杂志

CHINA BIOTECHNOLOGY
中国生物工程杂志
中国生物工程杂志  2015, Vol. 35 Issue (2): 78-83    DOI: 10.13523/j.cb.20150212
技术与方法     
重组大肠杆菌不耐热肠毒素B亚单位的中试发酵及纯化工艺
刘亚龙, 闫东明, 翁樑, 邹雪, 刘丹, 彭超, 苏亚南, 闫锦锦, 张静, 郭志燕
芜湖康卫生物科技有限公司 芜湖 241000
Pilot-scale Fermentation and Purification of a Recombinant E. coli Heat-labile Enterotoxin B Subunit
LIU Ya-long, YAN Dong-ming, WENG Liang, ZOU Xue, LIU Dan, PENG Chao, SU Ya-nan, YAN Jin-jin, ZHANG Jing, GUO Zhi-yan
Wuhu Kangwei Biontechnology Co., Ltd., Wuhu 241000, China
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摘要：

重组基因工程菌经中试发酵后,诱导表达出以包涵体形式存在的重组大肠杆菌不耐热肠毒素B亚单位(LTB)蛋白。采用Q Sepharose High Perfomance 阴离子交换层析,将洗涤、裂解后得到的可溶性蛋白进行纯化,得到纯度高达98%的重组LTB蛋白。利用Sephadex G-25分子筛层析法将重组LTB蛋白中的盐和尿素脱去,使蛋白复性,最终获得目的蛋白纯度为95%。将得到的重组LTB蛋白进行免疫双扩散实验,结果表明此蛋白具有良好的生物学活性。通过高效表达重组LTB蛋白的大肠杆菌发酵,确定了其中试发酵工艺,并建立了Q柱一步纯化即可获得目的蛋白的方法,该工艺简捷、高效、易于工业化生产,为重组LTB蛋白的生产和应用奠定了基础。
关键词： LTB;  包涵体;  纯化    
Abstract:

The recombinant bacteria were induced to express Escherichia coli heat labile enterotoxin B subunit (LTB) in the form of inclsion body after pilot-scale fermentation. Q Sepharose High Perfomance Anion exchange column chromatography were used to purify the soluble protein after processing of washing and lysing, resulting to obtain the recombinant protein LTB with a high purify of 98%. Renaturation the protain through Sephadex G-25 molecular sieve chromatography to remove the salts and urea, and ultimately obtain the target protein of 95% putify. The immune double diffusion test was performed to indicate that the target protein has a good biological activity. This was the first time to determine the optimal pilot-scale fermentation conditions, the production process of E. coli LTB recombinant proteins, and also to establish the method in the use of Q column to obtain the target protein in only one step. The process was more convenient and efficient for industrial production which laid a solid foundation for the production and application of recombinant LTB protein.
Key words: LTB    Inclusion bodies    Purification
收稿日期: 2014-10-28 出版日期: 2015-02-25
ZTFLH:  Q789  
基金资助:

国家科技重大专项子课题(2014ZX09102042-002)、安徽省科技攻关计划(1301041020)资助项目
通讯作者: 翁樑     E-mail: wengliang1603@sina.com
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引用本文:

刘亚龙, 闫东明, 翁樑, 邹雪, 刘丹, 彭超, 苏亚南, 闫锦锦, 张静, 郭志燕. 重组大肠杆菌不耐热肠毒素B亚单位的中试发酵及纯化工艺[J]. 中国生物工程杂志, 2015, 35(2): 78-83.

LIU Ya-long, YAN Dong-ming, WENG Liang, ZOU Xue, LIU Dan, PENG Chao, SU Ya-nan, YAN Jin-jin, ZHANG Jing, GUO Zhi-yan. Pilot-scale Fermentation and Purification of a Recombinant E. coli Heat-labile Enterotoxin B Subunit. China Biotechnology, 2015, 35(2): 78-83.

链接本文:

https://manu60.magtech.com.cn/biotech/CN/10.13523/j.cb.20150212    或    https://manu60.magtech.com.cn/biotech/CN/Y2015/V35/I2/78


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