链霉菌噬菌体1010和P13启动子在大肠杆菌中的克隆和表达  

CLONING AND EXPRESSION OF STREPTOMYCES PHAGE 1010 AND P13 PROMOTERS IN ESCHERICHIA COLI

作　　者：王筠[1] 陈孝康[1] 周健明[1] 朱逸忻 沈仁权[1] 盛祖嘉[1] 

机构地区：[1]复旦大学遗传学研究所

出　　处：《中国抗生素杂志》1990年第1期14-19,共6页Chinese Journal of Antibiotics

摘　　要：将螺旋链霉菌噬菌体1010和红霉菌噬菌体P13的San3A和HindⅢ酶切片段分别插入启动子探测质粒pGA46的BgIⅡ和HindⅢ位点,在大肠杆菌中获得了氯霉素抗性、四环素抗性的重组质粒。限制性内切酶酶切产物的电泳分析和DNA分子杂交分析结果表明重组质粒确由载体pGA46和噬菌体酶切片段连接而成。以限制性内切酶HindⅢ、BamHⅠ、EcoRⅠ、PstⅠ、SolⅠ等对重组质粒进行酶切分析,并应用限制性内切酶和核酸酶BAl-31酶切重组质粒pSS31中的噬菌体DNA插入顺序。得到分子量由1.2Kb缩短至0.25Kb而仍保留启动子功能的片段,用大肠杆菌的启动子探测质粒pGA46作为载体分别克隆于San3A和HindⅢ酶切的大肠杆菌的启动子和探测质粒,在含有抗生素的培养基上分别选择T_c^r和C_m^r转化子。Using E. coli promoter-probe plasmid pGA46 as the vector, Sau 3 A and HindⅢ restriction fragments from Streptomyces erythyreus phage p13 DNA and Streptotnyces spiramyceticus phage 1010 DNA have been cloned into the Bg1Ⅱ and Hind Ⅲ sites of pGA46, respectively. Transformants were selected on medium that allowed the selection of T_e^n and C_m^2 recombinant plasmids. Southern blot hybridization was used to as demonstrate that the insertion in the recombinant plasmids were in fact derived from Streptomyces phages restriction endonucleases and nuclease BAL31 were used for further reducing the size of the inserted fragment to 250 bp in recombinant plasmid pSS31 still with full promoter activity.

关 键 词：链霉菌噬菌体 启动子 克隆 表达 

分 类 号：R394.8[医药卫生—医学遗传学]