DNA重组酶Cre介导载体间基因的重组转移

Recombinase Cre Mediated DNA Recombination and Gene's Transferring Between Vectors

DNA重组酶Cre可以识别LoxP位点,使含有LoxP位点的DNA分子发生重组:2个同向LoxP之间的DNA片段被删除,2个环状DNA分子被整合为一个大分子.基于Cre酶的这些作用特性,构建了一套载体间基因的重组转移体系,在Cre酶的作用下,gfp基因被从基因供体pTLG上切除下来,然后转移到基因受体pET-LoxP上,从而快速、简便地完成了gfp基因高效表达载体pET-gfp的构建.gfp基因在大肠杆菌BL21(DE3)中被诱导表达,使菌落产生了可视的绿色荧光.通过对荧光菌落的计数分析,比较了环状基因供体pTLG和线性基因供体pTLG对有效重组率的影响.使繁琐的传统载体构建变为简单的酶促反应,极大地简化了载体构建步骤,为Cre酶在基因克隆和亚克隆中的应用提供了很好的研究基础.

DNA recombinase Cre can recognize LoxP sites and result in recombination of DNA molecules. Recombination between two directly oriented LoxP sites excises the inserted DNA. And recombination between two circular DNA molecules, which contains a directly oriented LoxP site each one, generates a cointegrate. Based on these traits of Cre recombinase, a gene-transfer and gene sub-cloning system was constructed. gfp gene was excised from gene-donor vector pTLG and transferred directly to gene-receiving vector pET-LoxP mediated by Cre recombinase, completing the construction of pET-gfp very quickly and simply. Afterwards, gfp gene was expressed in Escherichia coli BL21： （DE3） and generated visible green fluorescence colonies. It extremely simplified traditional courses of vector construction to only a reaction mediated by recombinase. Recombination ratios affected by circular gene-donor vector pTLG and linear pTLG were quantified and compared with. A good reference was provided for gene easy cloning or sub-cloning mediated by Cre recombinase.