| Brucellosis, which is caused by Brucella, is one of the most important bacterial zoonoses endemic in many countries, especially in developing countries. Brucellosis has been an emerging disease since the discovery of Brucella melitensis by Bruce in 1887. The geographical distribution of brucellosis is constantly changing, with new foci emerging or re-emerging. All data suggest that worldwide economic losses due to brucellosis are extensive not only in animal production but also in public health.The most critical problem that controlling and eradicating brucellosis in animals and human beings is that the investigation in its pathogenic mechanism is not fully clear and have no safe and effective brucellosis vaccines. Brucella is an intracellular bacterium.and the intracellular survival mechanism of the virulent strains are completely different from the vaccine ones, which maybe caused by the different structure in their outer membranes.Therefore, in the present study,the outer membrane proteins of the two strains were analyzed by comparative proteomitics, and these different expressed proteins were further analyzed in transcriptional level by the semiquantitative RT-PCR, and the related outer membranes were identified, which provide new clues for the understanding on the role of outer membrane proteins in Brucella virulence and intracellular survival.In regions with high prevalence of the disease, the only way of controlling and eradicating this zoonosis is by vaccination of all susceptible hosts and elimination of infected animals. They remains used to vaccinate the animals, although live, attenuated vaccine, such as S19,Rev.1,M5,S2 have some disadvantages:they can be infectious for humans; they can interfere with diagnosis; they may result in abortions when administered to pregnant animals; and the vaccine strain can spread in the region. Howerver, have no a safe and effective vaccine against human brucellosis. It will be favor that a new vaccine overcoming the above-mentioned drawback,such as subunit vaccine, which will be urgent needs for controlling the brucellosis in animals and humans.In the present study, the immunogenic candidate antigens were identified by immoproteomics from B.melitensis 16M soluble proteins. And tne immunologic function were further evaluated by combining the molecular biology and vaccinology with the aim to design new-type vaccines against Brucella.First, the immoproteomics strategy was used to screen novel immunogenic candidate proteins for the development of brucellosis subunit vaccine.The soluble proteins of Brucella melitensis were extracted and separated by two-dimensional electrophoresis (2-DE), and the Western blotting analysis were performed probing with the Brucella-infected serm from humans, cattles and goats.The immunodominant proteins were identified by Liquid chromatography tandem mass spectrometry (LC-MS/MS).Some common immunodominant proteins recognized by the three different serum will be new immunogenic candidate proteins for the development of brucellosis subunit vaccine.Then, these immunogenic candidate proteins were expressed in E.coli for evaluating their immunogenicity and protective ability in the mice model, which laid a foundation for obtaining really effective vaccine antigens candidates.The corresponding results were obtained based on the above studies:1,33 differentially expressed proteins from Brucella melitensis virulent strain 16M and vaccine strain M5 outer membrane proteins were found.The results of LC-MS/MS and bioinfermatics indicated that they represented 26 open reading frames. And 22 and 5 proteins were up-regulated in 16M and M5, respectively. The data of bioinfermation retrieval and related references showed that the up-regulated proteins in 16M mainly participated in energy metabolism, fat metabolism, protein, amino acid and polysaccharides biosynthesis and the one in M5 were important immunogenic proteins.The results of semi-quantitative RT-PCR showed that 5 Brucella memebrane proteins, which are wbkC (piont16), UTP-glucose-1-phosphate-uridylyltransferase (piont 35), purH (piont 20), Omp10 (piont 33) and sucB (piont 27), are also difference in transcription level, which basically participate in bacteria cell membrane biosynthesis.2,The 56 immunodominant proteins were successfully identified by LC-MS/MS. Among them,11 common antigens were recognized by the serum from Brucella-infected humans, cattles and goats serum. 3,The results from the evaluation of immunogenicity and protective ablity in mice model showed that 4 of 11 immunodominant proteins, they are ahcY(piont 5), RSβ(piont 6), RSα(piont 12), Isovaleryl-CoA dehydrogenase (piont 17), could significantly induce the Th1 cell immnune response characterized by high level of IFN-γand IL-2. And they provided partially protective ability against B.melitensis 16M chanllenge, They provided the log unit protection of 2.13,1.12,1.12 and 1.24, respectively.And the protein ahcY can confer the similar profective ability(2.13) to the vaccine strain M5 (2.34).Finally, our study provided the basis to reveal that the role of Brucella membrane protein in Brucella virulence and intracellular survival and our approaches may provide a new strategy for screening vaccine candidates in combination with immunoproteomics and analysis of the immunogenicity and protective ability in a mouse model.
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