China Animal Husbandry and Veterinary Medicine ›› 2020, Vol. 47 ›› Issue (1): 8-17.doi: 10.16431/j.cnki.1671-7236.2020.01.002

• Biotechnology • Previous Articles     Next Articles

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

ZHAO Xiaoli1, WANG Benben2, ZHANG Huan1, YANG Yajun2, YANG Ningning1, WANG Zhen1, YANG Qin1, WANG Yong1, CHEN Chuangfu1, CAO Xudong3,4   

  1. 1. College of Animal Science and Technology, Shihezi University, Shihezi 832000, China;
    2. College of Life Sciences, Shihezi University, Shihezi 832000, China;
    3. Department of Pathogen Physiology and Immunology, School of Medicine, Shihezi University, Shihezi 832000, China;
    4. Medical College, Hexi University, Zhangye 734000, China
  • Received:2019-07-08 Online:2020-01-20 Published:2020-01-17

Abstract: 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.

Key words: Brucella melitensis; iron response regulator; irr gene; rirA gene; virulence

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