布鲁氏菌自杀载体的改建及其在突变株构建中的应用

Development of a new suicide plasmid and its application to Brucella mutant construction

目的:改建布鲁氏菌自杀载体.方法:用NdeⅠ将pKOBEG-SacB质粒中的反向筛选基因SacB切下来,与同样酶切处理的pUC19质粒连接,得到pUC19-SacB,经测序和蔗糖筛选实验证实.结果:成功将SacB从pKOBEG-SacB中切下来并插入到pUC19质粒中,构建得到了pUC19- SacB.测序和蔗糖筛选实验证实.插入的sacB基因具有较好的反筛功能.我们利用该质粒,成功的构建了布鲁氏菌Ⅳ型分泌系统的突变株,改建的载体能高效的应用于布鲁氏菌染色体的精确修饰.结论:构建了一个新的自杀质粒pUC19-SacB,该质粒能用于布鲁氏菌无痕缺失突变株的构建.

AIM： To developed a new suicide plasmid, pUC19-SacB, for Brucella mutant construction. METHODS： The counter-selection gene sacB, released from plasmid pKOBEG-SacB by digestion with Nde Ⅰ, was inserted into the same site of plasmid pUC19 to produce pUC19-SacB. This plasmid was then applied to the construction of a Brucella mutant. DNA sequencing and sucrose selection were used. RESULTS： The counter-selection gene sacB was successfully released from pKOBEG-SacB and cloned into pUC19 to produce pUC19-SacB. Using DNA sequencing and sucrose selection, the pUC19-SacB plasmid was shown to have counter-selection capability. Using pUC19-SacB, we successfully constructed an unmarked deletion mutant of the VirB operon, showing that this plasmid can be efficiently used to modify the Brucella chromosome.CONCLUSION： The successfully constructed new suicide plasmid pUC19-SacB can be used to construct a Brucella unmarked mutant.