布鲁菌Ⅳ型分泌系统效应蛋白DK63-887基因缺失株的构建及鉴定  

Construction and Identification of Ⅳ Secretion System Effector Protein DK63-887 Gene Deletion Mutant of Brucella

作　　者：江雅丽[1] 陈创夫[2] 李志强[2] 李默[3] 李爽[2] 王震[2] 张欢[2] 张辉[2] 郭飞[3] 

机构地区：[1]新疆石河子大学生命科学学院,新疆石河子832003 [2]新疆石河子大学动物科技学院,新疆石河子832000 [3]新疆石河子大学医学院,新疆石河子832003

出　　处：《动物医学进展》2016年第5期30-37,共8页Progress In Veterinary Medicine

基　　金：国家国际科技合作项目(2013DFA32380);国家科技支撑计划子课题(2013BAI05B05);国家自然科学基金项目(31572491;31502067);自治区研究生教育创新计划科研创新项目(XJGRI2015040)

摘　　要：为了构建羊布鲁菌16M(简称16M)的DK63-887基因缺失株(16MΔDK63-887),探讨该基因与16M介导自噬的关系。利用同源重组和抗性替换的方法,以卡那基因替换DK63-887基因,获得突变株16MΔDK63-887。将亲本株16M、疫苗株M5-90、突变株16MΔDK63-887在相同条件下振荡培养,观察其生长趋势变化;将各菌株置于不同外界环境中,观察其生存率;将各菌株侵染小鼠巨噬细胞,比较它们在宿主细胞内的生存能力及RT-qPCR检测自噬相关基因的表达。成功获得了布鲁菌DK63-887基因缺失株且在20代内未发生回复性突变现象。与亲本株相比,16MΔDK63-887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;突变株在外界应激条件下生存能力低于亲本株;侵染4h后缺失株胞内细菌数量明显下降;RT-qPCR检测到突变株的ULKI、Beclin1表达量均显著降低(P<0.01),结果表明,布鲁菌Ⅳ型分泌系统效应蛋白与16M介导的细胞自噬密切相关,为16M胞内寄生机制的研究奠定了基础。To construct the DK63-887 gene deletion mutant （16MADK63-887） of Brucella melitensis 16M （referred to as 16M）, and explore the relationship between the gene and 16M-mediated autophagy, the methods of homologous recombination and replacement were applied, and the kanamycin gene was used to replace DK63-887 gene obtaining mutant 16MADK63-887. Under the same conditions of shaking culture, we observed growth of the parental strain 16M, vaccine strain M5-90, mutant 16MADK63-887, and then, each strain was placed in different environments, the survival rate of which was observed. Mouse macropha- ges were infected with parental and mutant strains, then we compared their ability of surviving in host cells and detected autophagy-related genes using RT-qPCR. The 16MADK63-887 mutant strain was successfully obtained. The mutants were genetically stable within 20 passages. Compared with the parental strain, the growing trends 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. ULK1 and beclinl expression levels of the mutants significantly reduced which were detected by RT-qPCR（P〈0.01）. The results of this study showed that Brucella type Ⅳ secretion system effector proteins associated with 16M mediated-cell autophagy, the research laid the foundation for the study on 16M intracellular parasitism mechamsms.

关 键 词：布鲁菌 DK63—887基因 缺失株 自噬相关基因 

分 类 号：S852.614[农业科学—基础兽医学]