Research On Construction And Immunogenicity Of Δgmd, Δper And Δmanb Mutant Strains Of Brucella Melitensis 015

Brucellosis was a zoonotic disease,a great threat to human health and livestock,listed as class B infectious diseases. There has been no brucellosis vaccine, live attenuated vaccine were mainly used to animal to prevention and control of brucellosis. Most animal brucellosis vaccine were live attenuated vaccine of smooth type, the larger side effect was cann’t distinguish between natural infection and artificial immunity. Brucella pathogenesis is not entirely clear. Lipopolysaccharide(LPS) is a very important virulence factor of Brucella, with strong antigenicity.According to available studies show, LPS’s integrity are closely related to its cellular memory alive, multiplication and virulence. gmd, per and manb are associated with LPS biosynthetic processes of smooth Brucella. Deletion of these genes can lead to a smooth type Brucella becomes to a rough type Brucella.Objective: In the study, organisms type 3 epidemic strains 015 of B. melitensis gmd, per and manb gene was deleted by homologous recombination construct,to correspond rough type deletion strains; and used murine macrophage RAW264.7 and Kunming Preliminary mice to evaluat the virulence of attenuats, detected the immune mouse serum IgG and IFN-γ levels; respectively, while expressed gmd, per and manb protein, Western blot verify its reactogenicity; aims to develop B. melitensis vaccines,able to distinguish between vaccinated and naturally infected.Methods: In this study, using inactivated B.melitensis 015 as templateas,respectively amplified upstream and downstream of the gmd, per and manb genes homologous arm sequence,using Bacillus subtilis as a template, amplified SacB gene sequence., The cloned sequences were connected to the pMD19-T simple vector,using double disgestion method homologous arm sequences upstream and downstream connected to the suicide vector pGEM-7Zf for constructing the suicide vector in the pGEM-7Zf-gmd,pGEM-7Zf-manb and pGEM-7Zf-per,respectively;and then SacB gene was connected to pGEM-7Zf-gmd,pGEM-7Zf-manb and pGEM-7Zf-per for constructing the suicide vector in the pGEM-7Zf-gmd-SacB(pgs),pGEM-7Zf —manb-SacB(pms)and pGEM-7Zf-per-SacB(pps); The pgs,pms and pps vectors were electrically transferred to B.melitensis 015 strains of competent cells by homologous recombination were successfully constructed 015 gmd, manb and per gene deletion strains,the Brucella gene deletion mutants by 100 mg/L ampicillin resistance screening and 8% sugar sensitivity screening,named Δgmd,Δmanb and Δper.TheΔgmd,Δmanb and Δper mutants were identified by PCR passage for 20 generations of genetic stability. When Δgmd,Δmanb and Δper mutants infect murine macrophages cells RAW264.7 using deletion mutants immune and Kunming mice of 6-week-oldmice. The virulence of different strains was evaluated by CFU counts;preliminary validation mice antibodies by conventional serological testslevel, the IgG antibodies and Cytokine response(IFN-γ) of peripheral blood of mice were tested by ELISA;and evaluating the immune protection by inoculating B.melitensis 015 to mices immuned after 14 days.In addition, gmd,manb and per gene was amplified by PCR. The PCR products of gmd,manb and per gene was cloned into pET-28a(+). The recombinant bacteria were transformed into E.coli BL21 cells and induced by IPTG to express protein. The the gmd,manb and per protein were detected by Western blot analysis using Brucella naturally infected sheep serum as the first antibody.Results: We successfully constructed a genetically stable rough-type Brucella015Δgmd, 015Δper, 015Δmanb deletion strains, mutation did not respond within 20generations; 015Δgmd, 015Δper and 015Δmanb deletion strains at the cellular level and in-vivo in mice demonstrated toxic forces are significantly reduced and has good immunogenicity; naturally infected sheep sera confirmed gmd, manb and protein per reactogenicity well,the candidate vaccines has potential value.Conclusion: The constructed Brucella 015Δgmd, 015Δper and 015Δper deletion strains combined with the corresponding protein is expected to research and develop the candidate vaccine and diagnostic methods.