| Brucellosis, a disease caused by Brucella, which are Gram-negative coccobaccilli that are nonspore-forming, nonmotile, and nonencapsulated, continues to be a worldwide zoonotic disease. At the same time, Brucella is also considered to be one of the most important biological warfare agents and bioterrorism agents. It is subdivided, on the basis of its pathogenicity and host preference, into seven nomen species. Human can become infected with B.melitensis, B.abortus, B.suis, rarely B.canis and B.maris. Infection results in a chronic disease in human known as undulant fever. In livestock the disease is characterized by abortion and sterility. Recently the incidence of human and livestock brucellosis increases rapidly.Vaccination and eradication of infected hosts have been key factors in the control of brucellosis in livestock. Vaccines provide the most important mechanism for protecting human populations against brucellosis. Live, attenuated vaccines have been used to control the spread of the disease in animals, however, they are considered unsafe for human use and they induce abortion in pregnant livestock. To date, no safe human vaccine is available against Brucella infection. Many of molecular mechanisms on virulence and immunity are far from clear, which block developing new-type vaccines against Brucella.Brucella are one of the organisms whose genome has been completely sequenced, closed, and annotated. In post-genomic era, it is anticipated that many interesting aspects of Brucella biology including virulence factors, protective antigens will be elucidated, which will be useful for developing a safer and more efficacious vaccine. Reverse vaccinology, has been utilized to identify novel candidate proteins for vaccine development. In this study, comparative proteomics and immunoproteomics were used to screen virulence associated and immunogenic proteins, which will provide useful knowledge for finding new drug targets and designing new-type vaccines against Brucella.Firstly, whole cell proteins, outer membrane proteins were extracted and performed 2-DE. Reference maps were established perfectly.Secondly, comparative proteomics was used to compare the total proteins of B.melitensis M5 with that of B.melitensis 16M. 14 different spots were found, which represent 8 discrete ORFs. These proteins were functioned in energy metabolism, protein synthese, molecular chaperones, and so on. From the results we can deduce that attenuation mechanisms of M5 differs from B.melitensis Rev 1. Thirdly, immunoproteomic techniques were used to screen antigens of M5. Whole cell proteins, outer membrane proteins were extracted and separated by 2-DE and analyzed by WB assay. 21 immunogenic protein spots from outer membrane proteins were found with sera collected from rabbits infected with Brucella and identified by MALDI-TOF/MS, which represent 12 discrete ORFs. The total protein of Brucella was screened the same as outer membrane proteins. 67 immunogenic protein spots were found , which represent 57 discrete ORFs. At the same time, immunogenic protein spots were also found with sera collected from clinical patients and infected animals. The elucidation of the immunome of Brucella provided a number of candidate proteins for developing vaccines. The results might be helpful in the prophylaxis and therapy of Brucella.Finally, According bioinformatics, five genes were cloned, expressed in prokaryotic expression vector and eukaryotic expression vector, purified. BALB/c mice were immunized with these candidate vaccines. ELISA,WB,ELISPOT,FCM and MTT assays were used to analyse the humoral and cellular immune responses.This study paved the way for further reverse vaccinology research on developing a safer, more effective new-type vaccine against Brucella.
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