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[Construction of Brucella abortus A19-delta VirB12 mutant and evaluation of its protective efficacy against 2308 strain challenge in BALB/c mice].

OBJECTIVE
A19-deltaVirB12 deletion mutant of Brucella abortus was constructed by using homologous recombination technology. BALB/c mice were vaccinated intraperitoneally with the mutant to evaluate protective efficacy against Brucella abortus 2308 challenge.


METHOD
A SacB gene was amplified by PCR from pIB279 plasmid. The sequences upstream and downstream of the VirB12 gene were amplified by PCR from Brucella abortus A19. These three PCR products were subsequently inserted into pBK-CMV vector, namely pBK-CMV-SacB-VirB12. This construct was transformed into Brucella abortus A19. The A19-delta VirB12 mutants were obtained by Kan(r) and 5% sucrose selection. Six-week-old female BALB/c mice were distributed into three treatment groups, including A19-delta VirB12 group, A19 group and PBS control group. BALB/c mice were vaccinated intraperitoneally at a dose of 5.0 x 10(4) CFU. At the 45-days post-immunization, all of mice were challenged with 2308 strain. Fifteen days after thechallenge, the levels of infection were expressed as means of the log10 CFU/spleen values. The histological changes were assessed among the groups.


RESULTS
Compared with PBS control group, the A19-delta VirB12 deleted mutant had astatistically significant protection against 2308 challengesimilar to A19 strain. Western blotting showed that A19-delta VirB12 mutant did not express VirB12 protein.


CONCLUSION
The A19-delta VirB12 deleted mutantelicits a strong protective immunity, and may becomea promising vaccine candidate.