Construction Of Deletion Mutant Of Brucella S2308and Study On Its Biological Characteristics

Brucellosis is a world-wide popular zoonotic infectious disease caused by Brucella, which seriously harm to human and livestock’s health.Because in vivo induced genes influence to survival and pathogenesis of pathogen in host, the research of effect in vivo inducted gene in the pathogenesis of Brucella has an important value on clarifying the development and pathogenesis of brucellosis. Argininosuccinate lyase (Asly) was an in vivo induced antigen of Brucella, which screened and identified by the laboratory. To further explore the role of Asly in the process of Brucella infection in vivo, the study prepared Asly monoclonal antibody, built Asly mutant and carried out a study on biological functions of Asly. The experiments include the following:The full-length sequence of Asly was amplified and cloned into the prokaryotic expression vector pET-28a and then the recombinant expression plasmid pET28a-Asly was built. pET28a-Asly was induced to express and purified by column chromatography after transformed into E. coli BL21. SDS-PAGE analysis showed that the His-Asly was expressed as soluble fusion protein. The western blot (WB) showed that the His-Asly has a immunogenicity.BALB/c mice were immuned by purified His-Asly, and the spleen cells were fused with myeloma cells. The positive clones were selected with an indirect ELISA method by Asly-coated enzyme label plate. We finally obtained one strain cells stably secreting antibody against Asly, named as A5F2. BALB/c mice were inoculated intraperitoneally with hybridoma to produce ascites antibodies,the results revealed that the titers of McAbs, A5F2in ascites by ELISA was1:5.12×106respectively. WB identification results showed that the hybridoma monoclonal antibody had high specificity.Recombinant plasmid pUC19-U-C-D was constructed and further electronically transformed into S2308component cells. For the positive recombinant strains, Asly gene was completely replaced by chloramphenicol through homologous recombination. Western blot analysis showed that the protein of S2308could be recognized by Asly monoclonal antibody ascites, but not by Asly gene deletion strains. The results above suggested that S2308ΔAsly deletion mutant was successfully constructed.The RNA of macrophage RAW264.7was isolated and reversed after the macrophage were infected. Established real-time PCR method was used to detect and analyse differences in the level of transcription of TNF-a, IFN-γ, IL-2, IL-4, IL-6and II-10cytokines in RAW264.7cells.The results showed that only IL-4was differentially expressed at Oh; expression levels of all cytokines were significant differences between S2308and S2308ΔAsly invading to the macrophagesat different time points after2h and4h; TNF-a, IFN-γ, IL-2,IL-10were significant difference at8h; and TNF-a, IL-6at36h.In order to understand the impact of S2308ΔAsly for virulence factors, the transcription level of per, pmm, sodC, virB4, virB5, virB8, BvrR, BvrS and omp25were analysed by the establish real-time PCR method. The results showed that the transcription level of sodC、per、virB4、virB5and virB8were upregulated,pmm、BvrR omp25and BvrS were downregulated.The growth characteristics, adhesion, invasion of macrophages and proliferation in macrophages of S2308and S2308ΔAsly were studied, the results showed that the capacity of S2308ΔAslys adhesion and invasion of macrophages did not have significant change. Intracellular survival experiment showed that although S2308ΔAsly could survive and proliferate in macrophages, the activity was significantly reduced.In summary, high specificity Asly monoclonal antibody was successfully prepared in this study; S2308ΔAsly deletion mutant was successfully constructed. The real-time PCR results displayed that the deletion of Asly led to different expression of cytokines. Down regulated virB5, BvrR and BvrS hindered the immune response of macrophages; the capacity of S2308ΔAslys adhesion and invasion of macrophages had no significant change, but proliferation was reduced. All the results above provided reference for further study of Asly.