布鲁菌铁转录调控因子rirA基因突变株的构建与鉴定

发布时间：2018-04-13 22:26

本文选题：布鲁菌 + 铁转录调控因子　；参考：《动物医学进展》2017年10期

【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。
[Abstract]:In order to construct the mutant of iron transcription regulator rirA gene of Brucella bovis S2308 螖 RIA, the effect of this gene on its own growth and its effects on the adhesion invasion and intracellular reproduction of different types of cells were investigated.By means of homologous recombination and resistance substitution, the rirA gene was replaced by kana-resistant gene, and the mutant S2308 螖 rirA was obtained.The parent strain S2308, vaccine strain RB51 and mutant strain S2308 螖 rirA were cultured under the same nutritional conditions.Human embryonic trophoblastic cells (HPT-8) and mouse macrophages (RAW264.7) were infected with each strain.The results showed that the mutant of Brucella rirA gene was obtained successfully and there was no reverse mutation within 10 generations, and the growth trend of S2308 螖 rirA was not changed obviously under the same culture condition, and its agglutination state was similar to that of parent strain.The adhesion and invasion test showed that S2308 螖 rirA had significantly stronger adhesion and invasion ability to HPT-8 cells than its parent strain, but its adhesion and invasion ability to RAW264.7 was weaker than that of parent strain to some extent, and Brucella spp. After 24 h invasion of HPT-8 cells, Brucella spp.The number of intracellular bacteria increased significantly, and the reproductive ability of S2308 螖 rirA was significantly lower than that of parent strain after 24 h infection of macrophages.These results suggest that the iron transcription regulator rirA gene is involved in the intracellular parasitism of Brucella and is closely related to the virulence of the strain.
【作者单位】：石河子大学兵团动物疾病防控重点实验室;石河子大学新疆地方与民族高发病教育部重点实验室;新疆畜牧科学院兽医研究所;
【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)
【分类号】：S852.614

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