Construction of M5-ΔznuA and M5-Δbp26-ΔznuA mutant strains of Brucella melitensis and assessment of their virulence and immunogenicity

Objective To harvest a kind of live vaccine of Brucella melitensis with less virulence, the novel B.melitensis live vaccine was constructed through homologous recombination.

Method M5 and M5-Δbp26 were used as the parental strains.znuA gene was knocked out through the recombined suicide plasmid.The biological characteristic of mutant strains was examined by inspecting and annlyzing the growth curve, stability of generating the virulence in Balb/c mice.

Result and conclusion The results of PCR amplification and sequencing showed that two types of genic deleted mutant strains were constructed successfully, which were designated as M5-ΔznuA and M5-Δbp26-ΔznuA.Compared with the parental strain M5, these two mutant stains had the growing characteristics similar to M5 and possessed a stable genic generation.Based on the mass and counts of spleen of Balb/c, the results demonstrated that M5-Δbp26-ΔznuA exhibited the lowest virulence among all strains, followed by M5-ΔznuA and M5-Δbp26.The quantity of specific antibody of serum revealed that the ability to induce humoral immune reaction was not affected by znuA deletion.In this study, two gene deletion mutants of B.melitensis live vaccine have been yielded, and they might be a new candidate vaccine.