人乙醛脱氢酶2基因在毕赤酵母SMD1168中的表达研究  

Study on the expression system of human acetaldehyde dehydrogenase 2 gene in Pichia SMD1168

作　　者：黄锟[1] 赵玉凤[1] 赵锦[1] 雷明科[1] 林宏楠[1] 王存文[1] 吴元欣[1] 

机构地区：[1]武汉工程大学化工与制药学院绿色化工过程省部共建教育部重点实验室,430073

出　　处：《中国医药生物技术》2010年第1期44-48,共5页Chinese Medicinal Biotechnology

基　　金：湖北省国际科技合作重点项目计划(2006CA013);湖北省科技攻关计划(2007AA201C27;2007AA301B24)

摘　　要：目的得到一株产人乙醛脱氢酶2(acetaldehyde dehydrogenase-2,ALDH2)的重组毕赤酵母菌株,优化其发酵条件以满足生产和科研需求。方法将ALDH2基因整合到质粒pPIC9K,构建重组表达载体pPIC9K-ALDH2,将重组表达质粒pPIC9K-ALDH2经电转化到毕赤酵母SMD1168中,转化子经MD平板筛选后,用G418筛选多拷贝重组子,测序鉴定。通过改变诱导温度、pH、初始OD值、甲醇浓度等诱导条件,对SMD1168(pPIC9K-ALDH2)发酵的条件进行优化。结果ALDH2cDNA整合到毕赤酵母基因组中,得到SMD1168(pPIC9K-ALDH2);获得最佳的诱导表达优化条件:在28℃、pH=7.0、诱导初始OD600=12、每隔24h添加1.5%甲醇、转速为300r/min,发酵所产生的人ALDH2酶活可达到0.115U/ml,相对较高。结论以蛋白酶缺陷型毕赤酵母SMD1168为宿主,分泌表达质粒pPIC9K为载体,能够成功构建重组子SMD1168(pPIC9K-ALDH2)。Objective To obtain a recombinant strain of Pichia which can produce human ALDH2 and then prepare for a large number of human ALDH2 through optimization of fermentation conditions. Methods The pPIC9K-ALDH2 was linearized by SacI and transformed by electroporation into Pichia cells SMD1168（defective with histidine）.The transformants were selected with MD culture plate and identified by PCR.And then,the multicopy recombinant Pichia strain was selected by G418 resistance. Results ALDH2 cDNA which is integrated into the genome of Pichia named SMD1168（pPIC9K-ALDH2）;At the best optimization of induced expression conditions：28℃,pH=7.0,induced by an initial OD600=12,adding 1.5%methanol every 24 h, rotational speed at 300 r/min,the human ALDH2 activity could reach 0.115 U/ml by fermentation,which is relatively high. Conclusion This study used protease-deficient Pichia SMD1168 as a host,secretory expression plasmid pPIC9K as vector to construct a recombinant SMD1168（pPIC9K-ALDH2）.

关 键 词：毕赤酵母 醛脱氢酶 乙醛 基因 

分 类 号：Q78[生物学—分子生物学]