通过一种新方法使T7基因Ⅰ置换araBAD基因簇  

Utilization of a New Method for Replacement of araBAD Genes by T7 Gene Ⅰ

作　　者：沈林[1] 方宏清[2] 戴红梅[2] 李树龙[2] 周长林[1] 陈惠鹏[2] 

机构地区：[1]中国药科大学生命科学与技术学院,江苏南京210009 [2]军事医学科学院生物工程研究所,北京100071

出　　处：《生物技术通讯》2009年第5期643-646,共4页Letters in Biotechnology

摘　　要：目的:用T7RNA聚合酶基因T7基因Ⅰ置换大肠杆菌基因组中的araBAD基因簇。方法:通过分析大肠杆菌基因组中araBAD基因序列,设计两侧同源臂,构建高拷贝数的打靶质粒,并且在这个质粒上的打靶片段两侧各加上一个归巢内切酶位点;将辅助质粒和打靶质粒同时转化到宿主体内,在L-阿拉伯糖的诱导下表达归巢内切酶和Red重组酶,实现体内重组,使T7基因I置换araBAD基因簇。结果:在4株不同的大肠杆菌中用T7基因I置换了araBAD基因簇。结论:成功地将T7基因Ⅰ整合到araBAD基因位置,为构建PAra-PT7表达系统奠定了基础。Objective： To utilize a new method for araBAD genes substitution by T7 gene I. Methods： Carry out this gene substitution utilizing an assistant plasmid and a high-copy target plamid together. To design the homology flanking with analyzing araBAD genes sequence, and construct the high-copy target plasmid consist of two homing endonuclease sites at the side of homology flanking. Transform target plasmid and assistant plasmid into host cell together, and express homing endonuclease and Red recombinase under induction by L-arabinose, as a result the in vivo recombination and gene substitution are carried out. Results： The genes substitution is carried out by this new method in four different strains of E.coli. Conclusion： T7 gene I inserts into the genome and substitute araBAD locus successfully, which is the foundation of PAra-PT7 expression system.

关 键 词：大肠杆菌 T7基因Ⅰ araBAD基因簇 归巢内切酶 L-阿拉伯糖 

分 类 号：Q78[生物学—分子生物学]