人肝金属硫蛋白-I_A基因在鱼腥藻中的克隆与表达  

The Cloning and Expression of Human Liver MetallothioneinIAgene in Anabaena sp.PCC7120

作　　者：任黎[1,2,3] 邵强[1,2,3] 施定基 戴继勋[1,2,3] 茹炳根 

机构地区：[1]北京大学蛋白质工程及植物基因工程国家重点实验室 [2]青岛海洋大学海洋生命学院 [3]中国科学院植物研究所

出　　处：《中国生物化学与分子生物学报》1998年第4期365-371,共7页Chinese Journal of Biochemistry and Molecular Biology

基　　金：国家"九五"攻关项目

摘　　要：将人工合成的人肝金属硫蛋白（ｍｅｔａｌｏｔｈｉｏｎｅｉｎ，简称ＭＴ）－ＩＡ基因插入至中间载体ｐＲＬ－４３９上强启动子ｐｓｂＡ后，再将其与穿梭载体ｐＫＴ－２１０相连，得到大肠杆菌－蓝藻穿梭表达载体ｐＫＴ－ＭＴ，用三亲接合转移法将ｐＫＴ－ＭＴ转入丝状体蓝藻－鱼腥藻７１２０，经链霉素筛选，得到了稳定的转人肝ＭＴ－ＩＡ基因鱼腥藻．纯化单藻落，液体扩大培养．从鱼腥藻中提取的质粒经Ｓｏｕｔｈｅｒｎ印迹分析，确定人肝ＭＴ－ＩＡ基因已转入鱼腥藻７１２０中，Ｗｅｓｔｅｒｎ印迹分析表明，金属硫蛋白在转人肝ＭＴ－ＩＡ基因鱼腥藻中得到了表达．经原子吸收光谱法测定表达量约为７００μｇＭＴ／ｇ鲜藻，重金属耐受性实验表明，得到了能耐受重金属－镉的转人肝ＭＴ－ＩＡ基因鱼腥藻，它将在清除水域中重金属污染和医药研究方面发挥重要作用．Human liver metallothioneinIA gene chemically synthesized was inserted following the strong promoter psbA on the intermediary vector pRL439,then the plasmid pRLMT was ligated with shuttle vector pKT210 on the site of EcoRⅠ to construct the shuttle expression plasmid pKTMT.The shuttle expression plasmid pKTMT was introduced into filamentous cyanobacteriumAnabaena sp.PCC 7120 by triparental conjugative transfer.After the selection of streptomycin,the stable transgenic Anabaena was obtained,and the purified monoclonal cyanobacterium cultured expandedly in BG11 liquid medium.The existence of human liver metallothioneinIA gene in transgenic Anabaena was confirmed by Southern blotting of plasmids isolated from Anabaena.The expression of metallothionein in transgenic Anabaena was demonstrated by Western blotting and the expression content was about 700 μg/g of fresh Anabaena.The metal tolerance experiments showed that transgenic Anabaena conferring heavy metalresistance was obtained,which would play important roles in clearing off the heavy metal pollution in waters and in some medical research.

关 键 词：转基因蓝藻 鱼腥藻7120 转移 表达 MT-IA基因 

分 类 号：Q789[生物学—分子生物学]