Research On Construction And Immunogenicity Of Bp26 Gene Mutant Strains Of Brucella Melitensis M5-90

Brucellosis is zoonoses in the world caused by organisms belonging to the genus Brucella, the small parasitic bacteria gram-negative, facultative, intracellular bacteria. It is fever, abortion as the main symptoms of a serious threat to human and animal health. In recent years the morbidity was ascending. "Detect, kill, and immune " are the primary means for the current prevention and control of animal brucellosis. M5-90 vaccine has good immunogenicity, and can be effective in preventing brucellosis of sheep and goats. However, attenuated vaccines also have a common disadvantage, cannot differentiate the disease from the vaccinated animals after the current vaccine used, and some attenuated vaccine has atavism. Therefore the development of a new vaccine of weak virulence, but also has good immunogenicity, and differential diagnosis could be necessary.Suicide plasmid pGB261and pGB262 were constructed by traditional molecular biology technology. Through the method of homologous recombination, we screened Brucella gene deletion mutant by 100 mg/L ampicillin resistance screening and 5% sugar sensitivity screening after electroporation. The M5-90Abp261and M5-90Δbp262 mutants obtained were indistinguishable from the parental M5-90 strain by conventional bacteriological and PCR tests. Their stability were detected by continuous bacteria culture. In this study, SAT and RBPT tested antibody levels in SPF BALB/c mice. Determined the total IgG in mice and cytokine levels of IFN-γby iELISA. By flow cytometry detected CD4+ and CD8+ content of mice. Determined the virulence of gene mutants through the indicator of spleen index. Evaluation of protective immunity of mutants after challenge with wild-type Brucella melitensis 16M. Construct the prokaryotic expression vector pet-32a-BP261/BP262. Purified BP261 and BP262 protein.Western blot analysis specific antibodies in mice immunized for differential diagnosis.The results of this study:1. The M5-90 Abp261 and M5-90 Abp262 deletion mutant strain was successfully constructed. The reverse mutation did not occur within 15 passages. Gram staining showed that the physiological characteristics did not change of the mutants.2. Immunogenicity results showed the mutant strains can induce specific humoral and cellular immunity. And the antibody levels of the mutants close to or slightly higher than the parent strains.3. Two mutants virulence had been reduced, and the virulence has been significantly decreased of M5-90Abp262 mutants.4. The mutant strains with a certain degree of protective on the body.5. Purified to high expression protein of BP261 and BP2626. BP261 and BP262 in the WB test with the serum of immunized mice showed with the mutants immuned the mice did not produced antibodies against missing gene. BP261 and BP262 can be used as the detection protein for the differential diagnosis of Brucella M5-90 mutant strains and naturally infected with brucellosis.These results showed that the mutant strains of Brucella M5-90Abp261 and M5-90Abp262 has a good prospect as vaccine, but also to establish the differential diagnosis for serological detection methods to lay the foundation.