食品科学2012，Vol.33Issue(5)：218-223,6.
构建人Cu，Zn-SOD毕赤酵母转化子及其高表达研究
Construction and High Expression of Pichia pastoris Transformants Carrying Human Cu/Zn Superoxide Dismutase Gene
摘要
Abstract
Artificial rhCu,Zn-SOD gene was synthesized according to the amino acid sequence of human SOD with yeast preferential codons, then cloned into the eukaryotic expression vector pPIC9K, named pPIC9K/Cu,Zn-SOD. The plasmid was transformed into Pichia pastoris GS 115 by electroporation and screened for high-copy transformants under the selective pressure. Three recombinant yeast strains were obtained with high expression. Gene copy number increased 2--8 folds by Southern blot identification. The expression activity increased 2- 4 fold. The recombinant gene copy number was positively correlated with the SOD protein yield. The Expressed protein was secreted to the supernatant as dimmer with low degree of glycosylation. The molecular weight was about 40 kD. The product can specificity bind to the antibody of Cu,Zn-SOD. Transformants entered the logarithmic growth phase after 16 h and the stable growth phase after 24 h in culture. After 50 passages, 3 recombinant strains remained stable, indicating it could be used for industrialization production of Cu,Zn-SOD. Cu,Zn-SOD activity was determined as 600U/mL in supematant. The high expression strain was build after the optimal shake flask culture conditions of pH 6.0, temperature 30 ℃, 1.5%（V/V） ethanol, and 72h induced.关键词
毕赤酵母GS115/Cu/Zn-SOD/电转化法/高拷贝/真核表达Key words
Pichia pastoris GS115/Cu,Zn-SOD/electrotransformation/high gene copy number/ereukaryofic expression分类
生物科学引用本文复制引用
迟春萍,时成波,曹玉锋,陈子杨,徐军,张健锋,贾媛,李铮,王小杰,牛灵,田海山,孙彪,李校堃..构建人Cu，Zn-SOD毕赤酵母转化子及其高表达研究[J].食品科学,2012,33(5):218-223,6.基金项目
吉林省科学技术发展重点项目 （）
