Recombinant Brucella Abortus S19 Expressing Mycobacterium Tuberculosis Ag85B Antigen

Brucellosis is a worldwide zoonosis caused by brucella which can infect more than sixty domestic and wild life species. The major signs of cattle infected by B.abortus are abortions, stillbirths and the birth of weak offspring. Bovine tuberculosis, resulting from Mycobacterium bovis, is another important worldwide zoonosis whose common symptoms include progressive emaciation, a low-grade fluctuating fever, weakness and inappetenc. Infected cattle are the major source of transmission and the most susceptible specie is the dairy cow. These two diseases not only are responsible for the great Economic loss but also bEcome the sources of transmission to human beings, which lead to serious public hazard.Vaccination is the major way of preventing brucellosis. B. abortus S19 have been used as a vaccine strain for a long time and its safety and efficiency have been fully proved. There is no efficient vaccine for preventing bovine tuberculosis all the time and its prevention primarily depends on passively stamping out. Recent studies show that Ag85B antigen secreted and retained in the cell wall of M. tuberculosis can induce protective immune response after injected into animals. Brucella and mycobacteria are all facultative intracellular pathogens, their mechanisms of pathogenesis and immune response are in common and they are also can infect cattle. Particularly in developing countries and rural poor areas, their infection in cattle is more serious.bp26 gene is a candidate target site for deletion vaccine of brucellosis and its deletion do not affect the immune efficiency of the vaccine. Moreover, the immuce response induced by bp26-deleted vaccine can be differentiated from that by nature infection by serum methology. So in this study, mycobacterium bovis ag85b gene ORF and kanamycin resistence gene were rEcombinated into the genome of S19 vaccine strain by taking replace of bp26 gene and sceened the double-crossover recombinant strain S19-ag85b. The result of Western blot shows that Ag85B antigen can be correctly expressed in recombinant strain S19-ag85b which was continuously cultured in TSA-YE plate containing kanamycin for 20 generations and ag85b gene is genetically stable in recombinant strain S19-ag85b.4-6 week old BALB/c mice were injected with 108 CFU of recombinant strain S19-ag85b or parent strain S19 intraperitoneally and these mice were killed after 1, 3, 6, 9, 12, 15 weeks, measuring the spleen weights and spleen CFU. Results show that spleen CFU in mice injected by S19-ag85b or S19 is continuously reduced, they are basally same. In 15th week, these two strains are almostly cleared, which proves that S19-ag85b possesses the quality of low pathogenicity of its parent strain S19.Further experiment was conducted to compare immunal efficiacy of recombinant strain S19-ag85b and its parent strain S19. In this experiment, 4-6 week old BALB/c mice were intraperitoneally immunized with 105CFU were chanllenged with 104CFU of B. melitensis M28 or 5×104CFU of B. abortus 544 intraperitoneally 45days post injection. Then these mice were killed after 15 days, measuring the spleen weights and spleen CFU. The results show that two groups immunized with S19-ag85b and S19 can induce high-level immune response against the attack of virulent strain either M28 or 544, because mice spleen weigh and CFU number isolated from spleens 45 days post vaccination in groups immunized with S19-ag85b and S19 are equal and much lower compared with the PBS control group.This study sets a fundamental foundation for the research of combined vaccine of bovine brucellosis and tuberculosis.