布鲁氏菌Ⅳ型分泌系统效应蛋白BMEI1135基因缺失株的构建及鉴定

Construction and identification of Ⅳ secretion system effector protein BMEI1135 gene deletion mutant of Brucella

为了初步探讨BMEI1135基因在布鲁氏菌感染过程中的作用,以16M为模板利用同源重组和抗性替换的方法,构建羊种布鲁氏菌16M(简称16M)BMEI1135基因缺失株(16M△BMEI1135)。将亲本株16M、疫苗株M5-90、缺失株16M△BMEI1135在相同起始浓度下震荡培养,观察其生长变化趋势;将各菌株置于强酸、强碱、高盐、热休克应激条件下,观察其生存率;侵染小鼠巨噬细胞RAW264.7后,比较其在胞内的生存能力,并在不同时间点收集细胞提取RNA,通过qRT-PCR检测自噬相关基因的表达。研究结果显示:成功获得了布鲁氏菌BMEI1135基因缺失株且20代内未发生回复性突变现象。与亲本株相比,16M△BMEI1135在体外培养生长趋势与亲本株相似,只是细菌的浓度存在一定差异;突变株体在外界应激条件下生存能力低于亲本株;胞内CFU显示侵染4 h后缺失株胞内细菌数量明显下降(P<0.01);qRT-PCR检测到突变株的ULKI、Beclin1表达量均显著降低(P<0.01)。本实验的研究结果表明:布鲁氏菌Ⅳ型分泌系统效应蛋白与16M的胞内生存介导的细胞自噬密切相关,该研究为16M胞内寄生机制的研究奠定了基础。

To explore the role of BMEI1135 genes in Brucella infection process,we regarded 16 M as a template then appied homologous recombination and resistant alternative method to construct Brucella melitensis 16M(referred to 16M) BMEI1135 gene deletion strains(16MABMEI1135).Under the same conditions of shaking culture,the growth of the parental strain 16 M,vaccine strain M5-90,mutant 16M△BMEI1135 was observed in vitro,and then,each strains was placed in strong acid,alkali and salt,heat shock environment in vitro,the survival rate of which was observed.Mouse macrophage RAW264.7 were infected with parental and mutant strains,then their ability of surviving in host cells was compared,and cells were collected at different time points and extracted RNA,soon after we detected autophagy-related genes using qRT-PCR.The results showed that the 16MABMEI1135 mutant strains were successfully obtained.The mutants were genetic stability within 20 passages.Compared with the parental strain,the growing trend of mutant and parent strains were coincident in vitro,but there were some differences in the concentration of bacteria.The mutants had decreased survival rate under the stress conditions.After 4h post-infection,the numbers of the mutant strains in host cells significantly decreased(P<0.01).ULK1 and Beclinl expression levels of the mutants significantly reduced which were detected by qRT-PCR(P<0.01).The results of this study showed that Brucella type Ⅳ secretion system effector proteins associated with 16 M mediated-cell autophagy,the research laid the foundation for the study of 16 M intracellular parasitism mechanisms.