Screening Brucella Diagnostic Antigen And The Protection Assessment And Construction Of Prpa Genetic Marker Vaccine Strain

Brucellosis is a brucella infection by the extremely serious human zoonoses and Brucellosis is widely popular in the many countries and regions of world, prevention and control of brucellosis depend on accurate diagnosis and vaccination. Although the researches on the diagnosis of brucellosis and the detection of Brucella are having been on constantly, but they mainly focus on a few known immunogens, and so the research progress is slow. Therefore, to find a new candidated antigens of diagnostic value has become an important content in the brucella of study. Vaccine is the most effective and economical method in prevention and control of brucellosis, and the live attenuated vaccine is most widely used. However, the traditional live attenuated vaccine can not distinguish between natural infection and artificial immune, so seriously affects on the diagnosis of brucellosis. Knocking out the antigen gene to build Brucella strains with the molecular marker, and evaluating the feasibility of attenuated live vaccine as a candidate antigen.it has become an important strategy to find the ideal live attenuated vaccine.In this study, a total of32brucella proteins were selected, expressed and purified and finally18proteins were obtained according to the early study of our laboratory on the brucella outer membrane proteome and secretion proteome and the analyzed results of protein chip antibody spectrum which have been reported abroad. These18proteins were used to detect brucella immune response against mouse serum, rabbit serum and patient serum by Western blot and the proteins with good immunogenicity which can be as candidated diagnostic antigens were filtered out. As a result,8proteins with immunogenicity against the rabbit serum were obtained,9proteins with immunogenicity against the patients serum were obtained, and only1protein against the mouse serum was obtained. However, part of the proteins have cross-immunogenicity, and finally, we obtained a total of13proteins with immunogenicity.When we analyzed8proteins with immunogenicity against the patients serum, we found a protein PrpA with immunomodulatory effects. Previous studies have shown that the Brucella PrpA can secrete IL-10and inhibit the host immunization, so we inferred PrpA protein a ideal target to construct a brucella vaccine strain with the molecular marker. Therefore, in this study, we constructed prpA deletion mutant and evaluated its feasibility as a molecular marker vaccine. Since most of the foreign plasmid can not replicate in Brucella, we used a method which can quickly build the brucella deletion mutant strain with no trace. First, N-terminal, C-terminal homologous arms of prpA gene and the kanamycin resistance gene were fused by PCR, and the mutation box with N-terminal, C-terminal homologous arms and the kanamycin resistance gene was obtained. Then, the mutation box was cloned into T vector, and then the recombined plasmid vector was transformed into S19live attenuated vaccine strains, and the prpA deletion mutant strain S19△prpA by the resistance gene replacement was successfully constructed. Finally, the virulence and protection of the marked strain were analyzed through a mouse model. The experimental results showed that the absence of the immune regulatory protein gene prpA is harmful for brucella to establish a chronic infection in the host and the virulence of S19was significantly reduced, however, the protection was not affected after prpA gene was deleted. Thus, compared with the traditional attenuated live vaccine strains, not only the security of S19A prpA was significantly improved, but also PrpA can be as a diagnostic antigen to distinguish between vaccine immunity and natural infection. Therefore, S19△prpA is a good candidated attenuated live vaccine strain and can be used in further study.