| 毕赤酵母表达铜锌超氧化物歧化酶中试工艺研究 |
| |
| 引用本文： | 迟春萍,陈子杨,徐军,时成波,曹玉峰,李铮,贾媛,李雨桐,郭立君,田海山,王小杰,孙彪,翟东,冀长发,李校堃.毕赤酵母表达铜锌超氧化物歧化酶中试工艺研究[J].兽医大学学报,2012(2):267-271. |
| |
| 作者姓名： | 迟春萍 陈子杨 徐军 时成波 曹玉峰 李铮 贾媛 李雨桐 郭立君 田海山 王小杰 孙彪 翟东 冀长发 李校堃 |
| |
| 作者单位： | [1]吉林农业大学生命科学学院,吉林长春130118 [2]长春生物制品研究所,吉林长春130062 [3]四平华科生物技术有限责任公司,吉林四平136000 |
| |
| 基金项目： | 吉林省科技发展计划项目（20070924-2） |
| |
| 摘 要： | 正交设计优化铜锌超氧化物歧化酶毕赤酵母在摇瓶及30L发酵罐的诱导表达条件,用两步层析法纯化表达的目的蛋白,在不同温度下进行原液稳定性试验。用考马斯亮蓝检测蛋白含量,活性检测试剂盒检测目的蛋白活性。纯化后原液参照中华人民共和国药典三部2005版进行检测,用SDS-PAGE、HPLC检测纯度,免疫印迹法检测rhCu,Zn-SOD特异性,等点聚焦测定该酶等电点,地高辛法检测残余DNA含量,酶联免疫法测定残余蛋白含量。结果显示正交试验的大罐高密度发酵最适条件比摇瓶的最适条所获得的目的蛋白活性高8倍,纯化后,HPLC纯度为99.7%,原液比活性大于6.0×103 U/mg,各项检测指标达到生物制品检定标准。该酶在45、65、85、100℃的半衰期分别为240、120、30、15。具有良好的酶稳定性。本研究建立了发酵、纯化的中试工艺,获得高表达、高纯度及高比活的铜锌超氧化物歧化酶,为规模化生产奠定了基础。
|
| 关 键 词： | 铜锌超氧化物歧化酶 毕赤酵母 高密度发酵 纯化 |
Study on a process for production of rhCu,Zn-SOD in Pichia pastoris |
| |
| Authors: | CHI Chun-ping CHEN Zi-yang XU Jun SHI Cheng-bo CAO Yu-feng LI Zheng JIA Yuan LI Yu-tong GUO Li-jun TIAN Hai-shan WANG Xiao-jie SUN Biao ZHAI Dong JI Chang-fa LI Xiao- |
| |
| Institution: | kun(1.Jilin Agricultural University,Changchun 130118,China;2.Changchun Institute of Biological Products,Changchun 130062,China;3.Siping Huake Biotechnology Limited Company,Siping,Jilin 136000,China) |
| |
| Abstract: | To establish a method of high-density fermentation to express Cu,Zn-SOD with Pichia pastoris.With Orthogonal Design rhCu,Zn-SOD Pichia pastoris induced expression conditions in shake flask and 30L fermenter and then the technology of purification,to detected protein content with Coomassie blue detection,rhCu,Zn-SOD activity with activity assay kit.The purified bulk was detected follow The People＇s Republic of China Pharmacopoeia 2005 edition with SDS-PAGE and HPLC Western blot for rhCu,Zn-SOD specific,determination of isoelectric focusing the enzyme isoelectric point,digoxin detection for residual DNA content,Compared with the shake flask,the HCDC fermentation orthogonal optimal conditions can offer the target protein activity in Article 8 times higher,HPLC purity was 99.7%;SOD activity was greater than 6.0×103 U/mg.The detection index can meet the standards of Biological Products.The enzyme has good stability,the half-life at 45,65,85,100℃ was 240,120,30,15 min.Initially established HCDC fermentation,purification technology for the expression of rhCu,Zn-SOD with high purity and high specific activity,to lay the foundation for large-scale production. |
| |
| Keywords: | Cu Zn-SOD Pichia pastoris HCDC purification |
| 本文献已被 维普 等数据库收录！ |
|
