Screening And Identification Of Brucella Relative Virulent Genes

Brucellosis is one anthropozoonosis with serious hazard. Pathogenic Brucella spp. is intracellular parasites and has no classical virulence factors, so the pathogenesis of which is still not clear. In this study, we deeply explored the pathogenesis of Brucella spp. in molecular level by screening Brucella virulent relative genes or metabolic pathway relative genes.This study was based on the bank of B.melitensis 16M transposon mutagenized mutants constructed previously by our laboratory, to observe growth characteristics by screening the differences of mutant strain plates of 6336 mutants were screened, and 1127 mutants were screened out by the first time. After repeated three times, we found that screening results were not consistent to some mutants. Then we streak cultured each CFU of 170 mutants of the second screening time, and screened again by picking single colony and finally screened out 27 mutants with the same results, which including 25 auxotroph mutants and 2 mutants which sensitive to spleen infusion. After observed the vitro culture characteristic of 27 mutants, we found that the CFU in the complete medium had more 101 than that in the minimal medium to the same mutant, and the diameter of the former CFU was enlarged obviously. In which, V-8 strain had much deposition in liquid culture and was identified to be a rough mutant through crystal violet staining, the others were all smooth strain; V-24 and V-26 had different culture time from differential with other mutants, after Brucella spp. specific type PCR identification, we found that V-24 was not Brucella, but V-26 was Brucella with specificity strap. Afterward, we used murine macrophage J744.A1 model to evaluate 23 mutants virulence by conducting cellular infection test. The results showed that the abilities of multiplication and duplication of V-5, V-8 and V-14 mutants in macrophage were all decreased compared to B. melitensis 16M. Finally,10 strains of screened 27 mutant strains were sequenced successfully by genome macrorestriction, connection and inverse PCR.This study established the foundation of seeking Brucella virulent relative genes or metabolic relative genes by screening out 27 mutant strains and identifing the transposon interrupted gene.