羊种布鲁氏菌M5-90疫苗株铁应答调控因子irr和rirA基因缺失株的构建与鉴定

为研究铁调控因子irr和rirA在羊种布鲁氏菌（Brucella melitensis）感染过程中的作用，本研究通过卡那替换的方法构建两个缺失株M5-90Δirr和M5-90ΔrirA，分别将亲本株和缺失株在相同营养条件下培养36 h，观察其振荡培养时的生长变化趋势。分别将1×10⁸ CFU M5-90Δirr、M5-90ΔrirA和M5-90接种到含1.5 mol/L NaCl、pH 2.5、pH 11.5、10 mmol/L H₂O₂的1 mL布鲁氏菌液体培养基，比较缺失株和亲本株在不同条件下的生长特性；分别以1×10⁶ CFU M5-90Δirr、M5-90ΔrirA和M5-90感染小鼠巨噬细胞RAW264.7检测缺失株的黏附、侵袭和胞内生存能力。结果显示，在相同体外培养条件下缺失株M5-90Δirr和M5-90ΔrirA生长速度明显低于亲本株；M5-90Δirr、M5-90ΔrirA在高盐、强酸和强碱环境下生存率均显著或极显著低于亲本株（P<0.05；P<0.01），在H₂O₂条件下这两个缺失株的生存率却显著高于亲本株（P<0.05）。与亲本株相比，缺失株在小鼠巨噬细胞RAW264.7内的侵袭和黏附能力均减弱，在感染后45 min缺失株M5-90Δirr和M5-90ΔrirA的黏附和侵袭能力均极显著低于亲本株（P<0.01），但在感染后24 h，这两个缺失株在细胞内繁殖能力与亲本株相比有所增强。本研究报道了irr和rirA基因不仅调控了羊种布鲁氏菌的生长，同时对细菌黏附和侵袭能力也产生一定的影响，M5-90Δirr和M5-90ΔrirA是两株具有潜力的布鲁氏菌候选疫苗株。

Construction and Identification of Iron Responsive Regulator irr and rirA Genes Deletion Strain of Brucella melitensis M5-90 Vaccine Strain

In order to explore the role of iron response regulator (irr) and rhizobial iron regulator (rirA) during Brucella melitensis infection,in this research,two mutants M5-90Δirr and M5-90ΔrirA were constructed through the kanamycin gene replacement method.These two mutants and their parental strain M5-90 were cultured for 36 h in the same condition,the growth ability were measured.1×10⁸ CFU M5-90Δirr,M5-90ΔrirA and M5-90 were inoculated into the 1 mL Brucella broth supplemented with 1.5 mmol/L NaCl,pH 2.5,pH 11.5 and 10 mmol/L H₂O₂ to detect the growth ability of these two mutants.The adhesion,invasion and intracellular survival ability of these two mutants were evaluated in RAW264.7 cells infected with 1×10⁶ CFU either M5-90Δirr,M5-90ΔrirA or M5-90.The results showed that under the same culture condition,the growth rates of two mutants M5-90Δirr and M5-90ΔrirA were significantly lower than that of their parental strain.The survival rates of M5-90Δirr and M5-90ΔrirA in high salt,strong acid and alkali environment were significantly lower than those in parental strains (P<0.05).The survival rates of these two mutants were significantly higher than the parental strain under hydrogen peroxide conditions (P<0.05).The invasion and adhesion capability of these two mutants inside the murine macrophages RAW264.7 were attenuated,especially at 45 min post infection,the invasion and adhesion capability of these two mutants were extremely significantly lower than M5-90 (P<0.01),however,24 h after infection,the ability of these two mutants to propagate in cells was enhanced compared with that of their parental strain.This was the first report that irr and rirA genes of B.melitensis not only associated with the virulence but also with the growth ability.To sum up,M5-90Δirr and M5-90ΔrirA were two promising Brucella vaccine candidates.