| Brucellosis is a extensively zoonotic and chronic infectious disease caused by Brucella. It caused serious calamity to animals and serious harm to human. Fortunately, Vaccination is the most suitable approach to control the disease. The live attenuated B.melitensis M5-90 strain is considered as the best vaccine available for the prophylaxis of brucellosis in sheep and goats. However, the vaccine could easily lead to abortion of the pregnancy with the possibility of virlence recovery, while it can not distinguish between natural infection and vaccination which hindering the clinical brucellosis serological diagnosis and epidemiological investigation, so the application of the vaccine has been limited.Therefore, we develop the gene deletion vaccines that can effectively distinguish natural infection and vaccination, which was proved one way to prevent brucellosis.The upstream and downstream homologous sequences of WboA gene and pgm gene as also as gfp gene and SacB gene were amplified by PCR and cloned into the pMD18-T simple vector. The amplified sequences of upstream and downstream homologous arm sequences and gfp gene were connected to the vector pGEM-7Zf after double digestion, respectively, named pGEM-7zf-△WboA and pGEM-7zf-△pgm-gfp, and then SacB gene was connected to two vectors, respectively. The object vectors were transformed into competent strain M5-90 cells, and The M5-90△WboA and M5-90 Apgm gene deletion strains were screened; 3.0×106 CFU dose of△WboA, Apgm and M5-90 vaccine strains were inoculated Balb/c mice.The virulence of different strains was evaluated by spleen index. The IgG antibodies and Cytokine response (IL-2, IFN-γ) of peripheral blood of mice were tested by ELISA.Using flow cytometry to analyze CD4+ and CD8+ T lymphocytes of peripheral blood and evaluating the immune protection by inoculating Brucella 16M to mices immuned after 30 days; A pair of primers specific to the WboA gene and pgm gene of B.melitensis was designed and synthesized according to the nucleotide sequences of 16M strain published in GenBank.WboA gene and pgm gene was amplified by PCR. The PCR products of WboA gene and pgm gene was cloned into pET-28a (+). The recombinant bacteria were transformed into E.coli BL21 cells and induced by IPTG to express protein. The expressed proteins were purified with Ni-NTA agaros, and identified by Western blot. The anti-WboA protein antibody and anti-pgm protein antibody were detected by Western blot analysis using the purified WboA protein and pgm protein as the first antibody and serum of mice immunizd with M5-90,△WboA and Apgm as the second antibody.The M5-90△WboA and M5-90 Apgm gene deletion strains were successfully obtaind. The virulence of M5-90AWboA was significantly reduced than the M5-90, and the immune protection was lower than the parent strain, in addition, the virulence of Apgm gene deletion strains was as same as the parent strain, but the immune protection was better than the parent strain; the expressed WboA protein and pgm protein showed good immunogenicity, Western blot pilot that both△WboA and Apgm can not induce Balb/c mice to produce the corresponding anti-protein antibodies. Meanwhile, the mice serum of M5-90,△WboA and Apgm were test by the SAT and RBPT, the results showed that the△WboA group and Apgm group did not produce agglutinating antibody, but M5-90 group produce agglutinating antibody.It proved that the WboA gene and pgm gene can be used in differential diagnosis between vaccination and natural infection. |
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