J4 ›› 2012, Vol. 38 ›› Issue (5): 943-947.

• 基础研究 • 上一篇    下一篇

巴斯德毕赤酵母表达的人抑瘤素M发酵条件及发酵产物分析

孔宁1|高海成2|刘国民3|陈月1|周余来1|颜炜群1   

  1. 1. 吉林大学药学院医用生物材料学教研室|吉林 长春130021;2. 吉林大学药学院临床药学与药事管理学教研室|吉林 长春130021;3.吉林大学第二医院骨科|吉林 长春130041
  • 收稿日期:2012-04-10 出版日期:2012-09-28 发布日期:2012-09-28
  • 通讯作者: 颜炜群(Tel:0431-85619715,E-mail: weiqunyan@jlu.edu.cn) E-mail: weiqunyan@jlu.edu.cn
  • 作者简介:孔 宁(1979-)|女|吉林省吉林市人|讲师|医学博士|主要从事医学生物工程研究。
  • 基金资助:

    吉林省卫生厅科研基金资助课题(3D511AJ83432)

Fermentation conditions of human oncostatin M expressed in Pichia pastoris and analysis of fermentation products

KONG Ning1, GAO Hai-cheng2, LIU Guo-min3,CHEN Yue1, ZHOU Yu-lai1|YAN Wei-qun1   

  1. 1. Department of Biomedical Materials, School of Pharmacy, Jilin University, Changchun 130021, China|2. Department of Clinical Pharmacy and Pharmacy Administration,School of Pharmacy, Jilin University, Changchun 130021, China|3. Department of Orthopedics, Second Hospital, Jilin University, Changchun 130021, China
  • Received:2012-04-10 Online:2012-09-28 Published:2012-09-28

摘要:

目的:研究巴斯德毕赤酵母表达的人抑瘤素M(hOSM)大规模发酵条件。方法:利用hOSM毕赤酵母高表达菌株,于试管水平进行发酵pH条件的摸索,以补料分批培养方式在 80 L发酵罐中对hOSM工程菌进行3个批次的高密度发酵,控制和优化各种发酵条件,通过SDS-PAGE对发酵产物进行分析。结果:最终确定在pH 5.0的FM21培养基中扩增菌体,待菌体密度达190 g/L 时添加甲醇诱导,在发酵液pH 5.5的条件下,发酵温度稳定在28℃,通入空气的流量为2 vvm, 转速为650 r/min,罐内压力为10 P,溶解氧保持在25%左右,甲醇流加速度在9.3 mL/h/L 初始发酵液体积,诱导36 h时结束发酵,hOSM占分泌总蛋白的28%,产量达到280 mg?L-1。结论:应用hOSM工程菌能够进行稳定的发酵,为进行下一步生物学功能测定提供充足的物质基础。

关键词: 抑瘤素M, 毕赤酵母, 发酵

Abstract:

Abstract:Objective  To optimize the fermentation condition of large scale fermentation of human oncostatin M(hOSM)  with engineering strain of Pichia pastoris. Methods The optimal pH condition of hOSM expressed in the methylotrophic yeast Pichia pastoris X-33 in tube-scale was determined and fermentation of hOSM was performed in an 80 L fermentor in a fed-batch mode for three times.The pH value, culture medium, dissolved oxygen, temperature, methanol feeding speed, initial biomass and etc were focused mainly.hOSM was analyzed by SDS-PAGE.Results  In the FM21 medium of pH 5.0, when a cell yield of 190 g/L wet weight was achieved, methanol induction phase began.In the fermentation broth of pH 5.5, the temperature was controlled at 28 ℃, a stirring speed of up to 650 r/min, the pressure of fermentor was 10 P, dissolved oxygen above 25% and the supply speed of methanol was 9.3 mL?h-1?L-1 initial fermentation volume, after methanol induction for about 36 h, the expression level of hOSM reached 280 mg/L. Conclusion  OSM is successfully obtained through genetic engineering for large scale production of hOSM for following biological functions.

Key words:  oncostatin M, Pichia pastoris;fermentation

中图分类号: 

  • Q78