【作者】 迟春萍； 隋红； 刘畅； 时成波； 陈子杨； 徐军； 曹玉峰； 贾媛； 郭立君； 王艳芳； 牛灵； 王小杰； 田海山； 孙彪； 冀长发； 李校堃；

【Author】 CHI Chun-ping(1,2),SUI Hong1,LIU Chang1,SHI Cheng-bo1,CHEN Zi-yang1,XU Jun1, CAO Yu-feng1,JIA Yuan1,GUO Li-jun1 TIAN Hai-shan2,NIU Ling4,SUN Biao3, JI Chang-fa3,LI Xiao-kun2 (1.Changchun Institute of Biological Products,Changchun 130062,China;2.Jilin Agricultural University, Changchun 130118,China;3.Siping HuakeBiotechnologyLimited Company,Siping 136000,China; 4.Institute of Microbiology,Chinese Academy of Sciences,Beijing 100080,China)

【机构】 长春生物制品研究所； 吉林农业大学； 中国科学院微生物研究所； 四平华科生物技术有限责任公司；

【摘要】 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,获得的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,其分子量为40 kDa左右,低糖基化,均为分泌表达。Western blot法分析,对Cu,Zn-SOOD抗体具有特异性反应。转化子在培养16 h后进入对数生长期,24 h后进入生长稳定期;转化子培养20 h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600 U/mL。确立最适摇瓶培养条件为pH 6.0,30℃,1.5甲醇诱导浓度诱导72 h上清的目的蛋白表达最好,构建的高表达菌株为中试工艺研究奠定了基础。更多还原

【Abstract】 To constructeeukaryotic expression vector and transform into Pichiapastoris forhigh 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 expression of 4 - 8 times.The recombinant gene copy number was positively correlated with the expression product;expression product is a dimer,its molecular weight was about 40 kDa,low degree of glycosylation,rhCu,Zn-SOD are secretory expression of soluble form. Transformants entered the logarithmic growth phase after 16 h in culture,and the stable growth phase after 24 hours;Cu, Zn-SOD activity was induced to 600 U/mL in supernatant.After 50 passages,3 recombinant strainremained stable;It laid the foundation for the industrialization model.更多还原