关键词: 毕赤酵母GS115, Cu, Zn-SOD, 电转化法, 高拷贝, 真核表达

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 GS115 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 supernatant. 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.

Key words: Pichia pastoris GS115, Cu,Zn-SOD, electrotransformation, high gene copy number, ereukaryotic expression