Brucella Building, Vjbr Genetic Marker Vaccine Strain Analysis And Differential Diagnosis Of Research On Protective Immunity

Brucellosis is a worldwide zoonotic disease caused by Brucella pathogens, which lead to serious economic loss and public health risk. Live attenuated vaccines have been proven to be the best vaccines and are used worldwide for prevention against animal brucellosis. However, its use is limited due to its residual virulence and identifical immune responses to those induced by infection. To resovle these shortcomings, numerous efforts have been made to develop new vaccines by targeted deletion of virulence genes or antigenic ones, being an important strategy for ideal live attenuated vaccine development.According to the reports on marked vaccine strains and results from our previous study, the feasibility of 16Mâ–³vjbR as a vaccine candidate was evaluated in this study. First, a mutant 16Mâ–³vjbR was reconstrcted using resistance replacement method. Then, the attenuation phenotypes are confirmed, and the protection and immunogenicity was analyzed. And to establish a diagnosis method, the vjbR was cloned and expressed, and used to differentiate immunization and infection.Brucella do not have plasmid, and many exogeneous plasmids do not replicate in it. Using this characteristic, we developed a new method to construct vjbR mutant. The homologous arms of vjbR were fused with kanamycin gene and generate the targeting cassette, which was cloned into T-cloning plasmid. The recombinant plasmid was transformed into Brucella and resistant colonies were selected, generating the vjbR targeted mutant 16Mâ–³vjbR.Balb/c mice were immuned with 16Mâ–³vjbR, and serum were collected at 2,4,6 weeks for monitoring antibody titers. Spleens were isolated and stimulated with Brucella whole cell proteins to analyze cellular immunity. The Rev.1 was used as positive control, and PBS, the negative control. The results showed that immunization with 16Mâ–³vjbR could provide protection against 16M challenges. The mutant induced secretion of IgG, IFN-y and IL-10, indicating that 16Mâ–³vjbR could induced both humoral and cellular immune response.In order to test whether the vjbR protein could be used as diagnosis antigen, the vjbR gene was cloned and expressed in E. coli. This protein was used to react with sera from 16Mâ–³vjbR or 16M immunized mice. Both western blot and indirect ELISA showed that vjbR could react with sera from 16M immunized mice but not with that from 16Mâ–³vjbR immunized ones, indicating that vjbR is antigenic and could be used as diagnosis antigen for differentiation of immunization and infection.In conclusion,16Mâ–³vjbR is an ideal live attenuated vaccine candidate, which deserve further evaluation as vaccine strains.