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| 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 | |
| 引用本文： | 迟春萍,隋红,刘畅,时成波,王艳芳,郭立君,李校堃.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[J].中国兽医学报,2011,31(12). |
| 作者姓名： | 迟春萍 隋红 刘畅 时成波 王艳芳 郭立君 李校堃 |
| 作者单位： | 1. 长春生物制品研究所,吉林长春130062／吉林农业大学,吉林长春130118 2. 长春生物制品研究所,吉林长春,130062 3. 吉林农业大学,吉林长春,130118 |
| 基金项目： | 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。 |
| 关 键 词： | 毕赤酵母GS115 Cu Zn-SOD 电转化法 高拷贝 真核表达 |
High expression of rhCu/Zn superoxide dismutase in Pichiapastoris |
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| CHI Chun-ping,SUI Hong,LIU Chang,SHI Cheng-bo,WANG Yan-fang,GUO Li-jun,LI Xiao-kun.High expression of rhCu/Zn superoxide dismutase in Pichiapastoris[J].Chinese Journal of Veterinary Science,2011,31(12). | |
| Authors: | CHI Chun-ping SUI Hong LIU Chang SHI Cheng-bo WANG Yan-fang GUO Li-jun LI Xiao-kun |
| Institution: | CHI Chun-ping1,2,SUI Hong1,LIU Chang1,SHI Cheng-bo1,WANG Yan-fang2,GUO Li-jun1,LI Xiao-kun1(1.Changchun Institute of Biological Products,Changchun 130062,China,2.Jilin Agricultural University,Changchun 130118,China) |
| Abstract: | 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 d... |
| Keywords: | PichiapastorisGS115 Cu Zn-SOD electrotransformation high gene copy number eukaryotic expression |
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