| Brucella are facultative intracellular parasites,belonging to the ?-2 branch of Proteobacteria,which infect a variety of land and aquatic mammals,including pigs,cattle,goats,sheep,dogs,dolphins,whales,seals,and desert wood mice.Animals infected with Brucella can cause miscarriage,infertility,low reproductive survival rate,reduced meat and milk production,decreased productivity.Invasion associated locus B(ialB)gene is located on chromosome ? of Brucella.At present,there are few reports about ialB,which is mainly related to Bartonella bacilli invasion of red blood cells.Although the ialB homologous genes are widely conserved,especially in rhizobidia,which include Brucella suis,their possible enzymatic reactions and/or interactions with the host remain unknown.Therefore,it is of great significance to construct an Brucella ialB deletion strain and study the possible functions of ialB in Brucella and whether it has effects on adhesion,invasion and intracellular survival for revealing the pathogenic mechanism of Brucella.In this study,we use the principle of homologous recombination,pUC18 plasmid was used to construct pUC18-ialB-Kan R vector,and then the constructed vector was transferred into competent cells of Brucella S2 strain by electric shock.After antibiotic screening,PCR and fluorescence quantitative PCR identification,the Brucella ialB deletion strain was obtained.Then,the complement vector PBBRori-AMP-ialB was constructed by seamless DNA cloning technology.The constructed vector was transferred into competent cells of the Brucella ialB deletion strain by electric shock,and the complement strain was obtained by antibiotic screening,PCR and Western Blot identification.The basic biological characteristics of Brucella S2 strain,Brucella ialB deletion strain and complement strain,and the ability of adhesion,invasion and proliferation of the above three strains in RAW264.7cell were further determined.The experimental results were as follows:1.The Brucella ialB deletion strain was constructed by homologous recombination technique,the complement strain was constructed by seamless DNA cloning technique.2.By observing the morphology of the colony on TSA plate,measuring the growth curve and detecting the bacterial activity,it was found that the growth of Brucella ialB deletion strain was inhibited,and the activity was lower than that of Brucella S2 strain and complement strain.The survival rates of Brucella S2 strain,Brucella ialB deletion strain and complement strain were tested in response to acid stress,high-osmolarity stress,low-osmolarity stress,oxidative stress and polymyxin B stress,and it was found that the survival rates of Brucella ialB deletion strain was lower than that of Brucella S2 strain and complement strain under acid stress,high-osmolarity stress,low-osmolarity stress and polymyxin B stress,and higher than that of Brucella S2 strain and complement strain under oxidative stress.The morphology of the above three strains was observed by scanning electron microscopy,and it was found that compared with Brucella S2 strain,the length of the Brucella ialB deletion strain became shorter,the diameter increased,and the morphology became rounded as a whole.3.Real-time fluorescent quantitative PCR detected the expression of polar elongation related gene,peptidoglycan synthesis related genes,OMPs related genes and lipopolysaccharide transporters related genes.The results showed that the expression of the above genes in the Brucella ialB deletion strain was down-regulated compared with that in the Brucella S2 strain.4.The proliferation of Brucella S2 strain,Brucella ialB deletion strain and complement strain in RAW264.7 cells was detected.It was found that the intracellular proliferation of Brucella ialB deletion strain was lower than that of Brucella S2 strain and complement strain.These results indicated that the activity of Brucella ialB deletion strains decreased;Polar growth may be affected and cell wall synthesis may be blocked;The proliferation ability in RAW264.7 cells was reduced. |
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