Identification And Characterization Of The Antigens Induced During Infection Of Brucella Abortus A19

Brucellosis is one of the most important zoonotic diseases worldwide, brucella spp., the causative agent of brucellosis, has not only caused substantial economic losses in animal industry, but also threatened greatly to human health. Brucella, replicating within host cells, have no classical toxins and fail to induce strong innate immunity. It is very difficult to control the brucella due to the lack of its pathogenesis information. In this study, In Vivo-Induced Antigen Technology (IVIAT) has been used to identify the antigens induced specifically during the infection of brucella. The results will be of great significance for further studies on its molecular pathogenesis, diagnostic markers and vaccine development. This study mainly includes four parts as described below:1. Construction of the genomic expression library of Brucella abortus-A19In this study, the genomic DNA of B. abortus A19was isolated and partly digested by Mbo I successfully. The digestion conditions of the genomic DNA, such as the concentrate of the enzyme, reaction temperature and time were optimized. The optical condition was determined as0.07u Mbo I μg DNA at37℃for45min. The DNA fragments ranging from500to3000bp were recovered and cloned at a ratio of1:1(quantity) into the expression vector pET28abc which have been digested by40u BamHI for6h and20u dephosphorylated by SAP for4h. The ligation products were electroporated into E.coli DH5a and BL21(DE3) to construct the expression library. Fifty colonies were detected by polymerase chain reaction (PCR) using primers specific for the vectors to determine the insertion ratio. By this strategy, the genomic expression library of B. abortus has been successfully constructed for further study.2. Identification of the antigens induced during the infection of B. abortus using IVIATB. abortus bovine positive sera were collected from several farms. Eight sera samples were pooled equally and absorbed with bacterial whole cells and cell lysates of in vitro grown B, abortus A19and E. coli BL-21(DE3). The sera were determined by ELISA for the adsorption efficiency after each step and used for the screening of the expression library. In total,55positive clones were obtained and sequenced. Bioinformatics analysis revealed that33antigens induced during the infection of B, abortus were identified. These antigens belong to categories of cell surface structure, molecule synthesis, energy metabolism, regulation, transpot systems and some other unknown functions.3. Cloning, expression and antigenecity analysis of the selected antigens and their sero-reactivities to33B. abortus bovine positive seraThe genes encoding Fumly, SDR, MDH, Nirase, Fumhy, EnvZ, AfuA and D15were selected for cloning and expression. The antigenicities of the recombinant proteins were also determined. It showed that all of the recombinant proteins exibited antigenic response against B, abortus bovine positive sera by using Western blot analysis. Moreover, We detected the sero-reactivities of these proteins with33convalescent bovine sera that were naturally infected with B, abortus by Western blot. The results showed that the positive rates for Fumly, SDR, MDH, Nirase, Fumhy, EnvZ, D15, and AfuA were29/33,27/33,25/33,24/33,18/33,13/33,8/33and6/33, respectively. Rabbit antisera against the above eight recombinant prteins were generated, and the immunofluorescence assay was then employed to probe the bacteria in different culture conditions, like in TSB in tubes, in infected Raw267.4cells in vitro and in infected mouse spleen in vivo. It revealed that the rabbit antiserum against SDR can stain the bacteria in both infected cells and mouse spleen, but not in TSB in tubes, indicating that the SDR was an antigen induced specifically during the infection of B. abortus, which may help to find a potential diagnostic target in brucellosis.4. Biological characterization and monoclonal antibody preparation of Bacterial surface antigen (D15) in B. abortusA19In this study, the biological characteristics and subcellular localization of Brucella abortus A19bacterial surface antigen (D15) of were also determined. D15showed the binding activity to plasminogen, but not to fibronectin in immunoblotting assay. Localization experiments showed that the D15is a membrane protein. In addition, three monoclonal antibodies agaist D15were generated for further study.