摘要
为了构建牛布鲁菌S2308(简称S2308)的铁转录调控因子rirA基因突变株(S2308ΔrirA),探讨该基因对自身生长的影响以及其对不同类型细胞黏附侵袭和胞内繁殖的作用。利用同源重组和抗性替换的方法,以卡那抗性基因替换rirA基因,获得突变株S2308ΔrirA。将亲本株S2308、疫苗株RB51和突变株S2308ΔrirA在相同营养条件下培养,观察其振荡培养时的生长变化趋势和静置培养时的聚集状态。将各菌株侵染人胚胎滋养层细胞(HPT-8)和小鼠巨噬细胞(RAW264.7),分别检测其黏附侵袭能力和胞内生存能力。结果显示,成功获得了布鲁菌rirA基因突变株且10代内未发生回复性突变;与亲本株相比,S2308ΔrirA在相同体外培养条件下其生长趋势未发生明显改变,且其凝集状态与亲本株类似;黏附侵袭试验显示,S2308ΔrirA对HPT-8细胞的黏附侵袭能力显著强于亲本株,而其对RAW264.7的黏附侵袭能力在一定程度上弱于亲本株;布鲁菌胞内生存试验发现,布鲁菌侵染HPT-8细胞24h后,其胞内细菌数量显著升高,而侵染巨噬细胞24h后,S2308ΔrirA的繁殖能力明显低于亲本株。这些结果表明,铁转录调控因子rirA基因参与了布鲁菌胞内寄生的过程,并与菌株的毒力强弱存在密切联系。
To construct the iron transcription factor rirA gene mutant （S2308△rirA） of B. abortus S2308 （S2308） ,and explore the impact of this gene on its growth and the function of cell invasion or intracellular survival in various cells,the methods of homologous recombination and replacement were applied, and the kanamycin gene was used to replace rirA gene obtaining mutant S2308△rirA. The parent strain S2308, vac- cine strain RB51 and mutant S2308△rirA were cultured in the same nutrient condition. The growth and aggregation status were recorded. Then human trophoblast cells （HPT-8） and macrophages （RAW264. 7） were infected with these strains. The invasive and survival abilities were detected. The results showed that the S2308△rirA mutant strain was successfully obtained, and was genetically stable within 10 passages. Compared with the parent strain, the growing trends and aggregation of mutant and parent strains were co- incident in vitro. The adhesion ability of S2308 rirA was significantly stronger than that of the parent strain to HPT-8 cells, but slightly weaker to RAW264.7 cells. After 24 h post-infection, the numbers of Brucella in HPT-8 cells were significantly increased, while the bacterial counts in RAW264.7 cells infected with S2308ArirA were significantly lower than that of the parent strain. All of these results indicated that the iron transcription factor rirA gene is involved in the process of intracellular parasitism ofBrucella, and closely related with the virulence.
作者
王震
王勇
张倩
陈创夫
郭飞
李天森
张辉
王远志
刘志强
WANG Zhen WANG Yong ZHANG Qian CHEN Chuang-fu GUO Fei LI Tian-sen ZHANG Hui WANG Yuan-zhi LIU Zhi-qiang(Key Laboratory of Prevention and Control of Animal Disease, Shihezi University, Shihezi, Xinj iang , 832000, China Education Key Laboratory of Xinjiang Endemic and Ethnic Disease, Shihezi University, Shihezi, Xinjiang, 832000 ,China Veterinary Research Institute of Xinjiang Academy of Animal Sciences ,Urumqi , Xinjiang , 830000, China)
出处
《动物医学进展》
北大核心
2017年第10期1-7,共7页
Progress In Veterinary Medicine
基金
新疆生产建设兵团动物疾病防控重点实验室开放课题(BTDJ06)
国家自然科学基金项目(31572491
31660705)
