Generation And Evaluation Of Brucella Melitensis Vaccine M5-90 Strain With VirB12 Gene Deletion

Brucellosis is one of the five most common bacterial zoonoses in the world caused by organisms belonging to the genus Brucella, gram-negative, non-spore-forming, facultative, intracellular bacteria. The genus Brucella consist of seven species: Brucella melitensis B. suis (hogs), B. abortus (cattle), B. ovis(sheep), B. canis (dogs), B. neotomae (wood rats), and B. maris(marine mammals). In China, Brucella melitensis, B. suis (hogs) and B. abortus (cattle) are the most harmful to public health, especially B. melitensis .The methods of serological detection of Brucellosis are significant to the control and prevention of this disease. Currently, M5-90, A19 and S2 are the main vaccines for preventing brucella in China, however, these vaccines all share the common problem in that they can not be differentiated from the natural infection. This hinders the prevention and control of Brucellosis. Therefore, new brucellar marker vaccines are needed.VirB12 gene exists in type IV secretion system which is a virulence factor of brucella. VirB12 protein resides on the surface of cell membranes and plays a key role in the host cell evading mechanism of brucella. According to related reports, VirB12 protein can induce host cells to produce certain antibodies, which we can use to detect the bacteria. M5-90 originated from M5 and its biological safety and effectiveness have been tested by practice, and so we may get a new marker vaccine through deleting virB12 gene of M5-90.In this research we cloned the virB12 gene of B. melitensis and expressed it in E. coli. An indirect ELISA was set up using purified VirB12 as acoating antigen. 60 clinical B. melitensis positive sera and 30 B. melitensis negative sera were examined then the coincidence is 91.7%, comparing with commercial C-ELISA Kit and the rose bengal plate test, indicating that VirB12 recombinant protein could be applied for the detection of B. melitensis infection.Further in this research, we deleted virB12 gene in M5-90 by homologous recombination and got M5-90-virB12. Then we firstly estimated the biosafety of M5-90-virB12. Results show that spleen CFU in mice injected by M5-90-virB12 or M5-90 is continuously reduced, they are basally same, which proves that M5-90-virB12 possesses the quality of low pathogenicity of its parent strain M5-90. Secondly, we compare immunal efficiacy of recombinant strain M5-90-virB12 with its parent strain M5-90. The results show that two groups immunized with M5-90-virB12 and M5-90 can induce high-level immune response against the attack of virulent strain M28. Finally, the detection levels of antibody anti-sLPS in serum from goats injected by M5-90-virB12 and M5-90 are nearly the same.This research proves that VirB12 protein can be a serological marker of brucella and provides a new candidate gene for construction of brucella marker vaccine.