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

设计人Cu,Zn-SOD酵母偏爱密码子并化学合成,与pPIC9K连接,构建酵母偏爱密码子的人Cu,Zn-SOD基因真核表达载体,通过电转化和持续加压筛选毕赤酵母GS115高拷贝转化子,获得的重组高表达酵母菌株建立主种子批。经Southern blot鉴定,基因拷贝数提高2～8倍,活性检测显示提高2～4倍。重组菌的目的基因拷贝数与表达产物呈正相关;表达产物为二聚体,其分子质量为40kD左右,低糖基化,均为分泌表达。Western blot法分析,对Cu,Zn-SOD抗体具有特异性反应。转化子在培养16h后进入对数生长期,24h后进入生长稳定期;转化子培养20h左右进行诱导表达最为合适。高拷贝的3株重组菌经50次传代后插入的目的基因保持稳定;Cu,Zn-SOD转化子用正交试验筛选摇瓶的诱导表达条件,经诱导表达,Cu,Zn-SOD表达上清最高活性大于600U/mL。确立最适摇瓶培养条件为pH6.0、30℃、体积分数1.5%甲醇诱导72h测得上清的目的蛋白表达最好,表明构建了高表达菌株。

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.