构建铜锌超氧化物歧化酶毕赤酵母转化子及其高表达分析  

High expression of rhCu/Zn superoxide dismutase in Pichiapastoris

作　　者：迟春萍[1,2] 隋红[1] 刘畅[1] 时成波[1] 王艳芳[2] 郭立君[1] 李校堃[1] 

机构地区：[1]长春生物制品研究所,吉林长春130062 [2]吉林农业大学,吉林长春130118

出　　处：《中国兽医学报》2011年第12期1763-1768,共6页Chinese Journal of Veterinary Science

基　　金：吉林省科学技术发展重点资助项目(20070924-02)

摘　　要：设计铜锌超氧化物歧化酶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铜锌超氧化物歧化酶基因真核表达载体;通过电转化和持续加压筛选毕赤酵母GS115高拷贝转化子,获得重组高表达酵母菌株建立主种子批。经Southern blot鉴定,高拷贝基因比低拷贝高2～8倍,表达活性高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,相对分子质量为40 000左右,低糖基化,均为分泌表达。West-ern 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。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℃,1.5%甲醇诱导浓度诱导72h上清的目的蛋白表达最好。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。本研究为中试工艺研究奠定了基础。To constructe eukaryotic expression vector and transform into Pichiapastorisforhigh expression of recombinant human Cu,Zn-SOD gene.By electrotransformation and continued pressure screening,access to 4 recombinant yeast strains protein expression was significantly increased.By Southern blot identification,gene high copy number increased 2-8 times,expression increased 2-4 times than low copy number.The recombinant gene copy number was positively correlated with the expression product;expression product is a dimer,its molecular weight was about 40 000,low degree of glycosylation,rhCu,Zn-SOD are secretory expression of soluble form.Transformants entered the logarithmic growth phase 16 h after culture,and the stable growth phase after 24 h;Cu,Zn-SOD activity was induced to 600 U/mL in supernatant with pH6.0,30℃,1.5% methanol for 72 h.After 50 passages,3 recombinant strainremained stable;It laid the foundation for the industrialization model.

关 键 词：毕赤酵母GS115 Cu Zn-SOD 电转化法 高拷贝 真核表达 

分 类 号：S852.2[农业科学—基础兽医学]