利用T载体克隆快速构建布鲁氏菌缺失突变株

Construction of Brucella Deletion Mutant by Using T Cloning Vector

目的:建立一种基于T载体快速克隆构建布鲁氏菌突变株的方法,提高布鲁氏菌突变株构建的效率。方法:采用融合PCR的方法,将待缺失基因上下游的同源臂与卡那霉素抗性基因融合起来,构建突变盒,然后将突变盒直接与T载体连接,构建突变载体,将载体转入布鲁氏菌感受态细胞并筛选抗性克隆,进而获得布鲁氏菌的缺失突变株。结果:结合融合PCR和T载体快速克隆,能够在48h之内构建好突变载体,与传统的酶切连接相比,效率高、周期短。结论:基于T载体快速克隆是一种非常高效的构建突变株的方法,为布鲁氏菌突变株的构建提供了一种新方法。

Construction of mutant strain is an essential method in pathogenesis researches.In the present study,the Brucella deletion mutant was constructed by using T cloning vector.At first,the homology arms of upstream and downstream of the target gene were fused with kanamycin resistance gene by fusion PCR.Then,the mutant cassette was ligated with T vector,resulted in the mutant plasmid.And the mutant plasmid was transformed into recombination competent cells and recombinants were selected by kanamycin resistance.As the results showed,the mutant construction was convenient and the mutant could be generated by only one round of selection with high efficiency.Therefore,this recombinational cloning strategy provides a new method to construct the Brucella mutant and would accelerate gene function analysis of Brucella.