炭疽芽胞杆菌mntA基因缺失突变株的构建及鉴定

目的:通过同源重组的方法敲除炭疽芽胞杆菌减毒AP422株的mntA基因,使菌株进一步减毒,用于构建新的疫苗候选株。方法:利用PCR方法扩增mntA基因上下游同源臂后与温敏质粒连接,构建打靶载体,并转化炭疽芽胞杆菌减毒AP422株;利用抗生素和温度2种选择压力实现同源重组,敲除目标基因mntA,然后利用Cre-LoxP系统去除抗性筛选标记,得到无抗性标记的缺失突变株,并利用PCR和Western印迹等方法对重组菌进行系统鉴定,最后分析突变株的生物学性状。结果:敲除了AP422株的mntA基因,获得了无抗性标记的缺失突变株,突变株的生存竞争能力比原始菌株明显减弱。结论:突变株获得了进一步减毒,可用于构建新的疫苗候选株。

Construction and Identification of mntA Deletion Mutant of Bacillus anthracis AP422

Objective： To construct mntA gene deletion mutant of attenuated Bacillus anthracis AP422 strain. Methods： The upstream and downstream homologous arms of mntA gene were amplified by PCR, then they were linked to the thermosensitive plasmid to construct the targeting vector, which was transformed to the attenuated B. anthracis AP422. Under the pressure of antibiotics and temperature, mntA gene of AP422 was knocked out by homologous recombination, and then the resistance selection nant strain was identified by PCR and Western blotting marker was deleted using Cre-LoxP system. The recombi- and its biological character was also analyzed. Results： The mntA gene was successfully knocked out and the resistance selection marker was also deleted. Compared to the AP422 strain, the fitness of the mutant strain decreased significantly. Conclusion： The mntA gene deletion mutant strain is more attenuated and can potentially serve as an live attenuated vaccine candidate.