Study On Molecular Marker And Virulence Deleted Vaccine Strain △YZ-2 Of B.abortus

Brucellosis is a contagious disease of human and animal,which outbreak in many areas of the world and cause a loss in productivity for a considerable time.In these years the morbidity was ascending and some terrorist organizations or rogue states might target livestock industry by employing the etiologic agent of Brucellosis.Vaccination is the only best way to prevent the disease, but we cannot differen tiate the disease from the vaccinated animals after the current vaccine used, and some attenuated vaccine has atavism.So it recommended the development of modern diagnostic methods to rapidly detect an outbreak, as well as the investment of additional funds in animal disease research to develop marker vaccines that would allow easy diagnostic distinction of vaccinated from infected or convalescent animals by OIE.BP26 protein of Brucellosis is an immunodominance protein,which has high immunogenicity in animal and human.So it was used as a diagnosis antigen to detect Brucellosis.BMP18(Omp19) is one of the main virulence protein of Brucellosis.In this study,a mutanted gene of BP26 was inserted into pBK-CMV and sacB gene was inserted into pBK-CMV.And a Bp26 gene mutated suicide plasmid was constructed by replace part of Bp26 gene of B. abortus with Luc NF+ gene, a gene amplified from Luciferase gene.A second suicide plasmid was constructed based on pBluescript KS II+ by the same method.The two suicide plasmids have pUC ori and can stay and replicate in E.coli,but cannot stay and replicate in B. abortus,that is why we call them suicide plasmids.They all have sacB gene,which was cloned from Bacillus subtilis, and its product can hydrolysis sucrose to fruit sugar to form fructosan.Fructosan can cause cell to death.Two suicide plasmids was transformed into YZ strain of Brucellosis,respectively. Recombinated strain within which homologous recombination occurred was selected on 5% sucrose-liver-medium.BP26 gene of B. abortus was amplified and cloned into pET-28a,the recombinated plasmid named pETBp26.And then the recombinated plasmid was transformed into BL21 and induced by IPTG.The expressed fusion protein BP26-His was purified by Ni-NTA resin.Immunological activity of BP26 protein was identified by Western-blot.Furthermore,â–³YZ-2 and YZ was inoculated into baby mouse respectively, and its antibody was detected by ELISA based on BP26 as antigen which produced by pETBp26. The result showed that we can differentiate mouse inoculated by YZ from mouse inoculated byâ–³YZ-2,that is to say the disease can be differentiated from the vaccinated by ELISA afterâ–³YZ-2 used.Study on virulence ofâ–³YZ-2 and S29 showed thatâ–³YZ-2 ,from which a Bmp18 gene deleted,has lower virulence than YZ.All the results showed thatâ–³YZ-2 of B.abortus was constructed, with a molecular marker and virulence gene of Bmp18 deleted, successfully.