小鼠金属硫蛋白在聚胞藻中的金属诱导表达与纯化  

Metal induced Expression of Mouse Metallothionein 1 cDNAin Synechocystis sp.PCC 6803

作　　者：郭祥学[1,2,3] 赵晖 施定基 徐旭东[1,2,3] 茹炳根 

机构地区：[1]北京大学生命科学学院生物化学及分子生物学系蛋白质工程及植物基因工程国家重点实验室 [2]中国科学院植物研究所光合作用研究室 [3]美国密执安州立大学植物学国家实验室

出　　处：《生物工程学报》1998年第4期405-411,共7页Chinese Journal of Biotechnology

基　　金：国家863生物高技术项目基金

摘　　要：应用蓝藻类金属硫蛋白基因启动子（ｓｍｔＯＰ）的金属诱导性，在单细胞的聚胞藻ＰＣＣ６８０３中表达小鼠金属硫蛋白结构基因（ｍＭＴ１ｃＤＮＡ）。在大肠杆菌ＨＢ１０１中构建含有ｓｍｔＯＰ和ｍＭＴ１ｃＤＮＡ的穿梭表达载体ｐＫＴＭＲＥ，经质粒转移，链霉素筛选，Ｓｏｕｔｈｅｒｎ和Ｗｅｓｔｅｒｎ杂交分析鉴定得稳定的转基因工程藻落。同时，做小批量锌诱导表达，并纯化了外源蛋白，５Ｌ培养液含鲜藻重５０ｇ，得到３５ｍｇｍＭＴ１；转基因藻在高金属浓度下的耐受性测定表明，外源基因的表达提高了蓝藻对金属离子的抗性，约为野生藻的２倍。Cyanobacterium MT like promoter( smt O P) was used in expression of mMT 1 cDNA in Synechocystis sp.PCC 6803 to enhance metalbinding ability and specialty.Shuttle expression vector pKT MRE was constructed in E.coli (HB101),and through triparental conjugated transfer,Streptomycin screening,Southern and Western blotting analysis,transgenic cyanobacterium clone was acquired.The cultivation and Zn 2+ inducing of transgenic cyanobacterium were carried on ,and the foreign mMT 1 was purified through the improved gel filtration and ion exchange chromatography.About 3.5mg mMT 1 were obtained from 5g wet cyanobacterial cells in 5L culture scale.Cyanobacterial metal resistance measurement indicated that the expression of foreign mMT 1 enhanced the transgenic cyanobacterial metalresistance to two times than the wild Synechocystis sp.PCC 6803

关 键 词：聚胞藻PCC6803 smtO-P区域 金属硫蛋白 蓝藻 

分 类 号：Q78[生物学—分子生物学] Q513