Preliminary Study On Immuoproteomics Of Outer Membrane Proteins Of Brucella

Brucellosis is a zoonosis caused by Brucella species, occurs widly in the world, harms seriously to animal husbandry and human health, and causes serious economic losses. Brucellosis is prevails in 170 countries in the world and 28 provinces in our country, and is getting more and more serious in recent years.The genus Brucella consists currently of 7 nomenspecies 21 subgroups including marine mammal species. The classification is based on the microbiological features and host preference among the various nomenspecies, B.abotus, B.melitensis, B.suis, B.canis and B.maris have been reported to infect humans. Brucella is a Gram-negative, facultative intracellular bacterium that infects macrophages where the bacteria persist and effectively evade immune elimination. This pathogen infects ruminants, causing abortion and infertility, and in humans, leading to chronic stages of the disease known as undulant fever. Brucella has been postulates to be a potential agent of biowarfare and bioterrorism.Vaccine has been of great importance in the prophylaxis of brucellosis due to specific pathogenic mechanism and cruelessness of brucellosis. Up to now, the live attenuated vaccine still plays an important part in reducing the risk of transmission to human and livestocks in spite of its problems on safety and efficiency. With the complete sequencing of Brucella genome and the development of reverse vaccinology, it provides the necessary tools for a proteomic-based analysis of Brucella. At present, the study of new Brucella vaccines focuses on the genetic engineering vaccines and it could elicit a protective immune response, but the protection is incomplete. Therefore in this study, the immunoproteomics methods were successfully applied in identifying immunogenic proteins of Brucella vaccine strain M5, which helps to search for other immunogenic proteins and identification antigen candidates for use in vaccine development and as potential vaccine targets in future.(1) Rabbit-anti-Brucella serum was prepared, then two-dimension electrophoresis was used to separate the outer membrane proteins of Brucella vaccine strain M5, which were cultures under laboratory conditions. The Western blot was also used to screen immunogenic proteins. After in-gel digestion, twenty-one immunoreactive spots were identified by matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF-MS). All the peptide mass fingerprints (PMFs) were searched by the program (www.matrixscience). These proteins represented 12 discrete open reading frames (ORFs). These proteins were not only outer membrane proteins, but also cytoplasm prtoteins. Some of them were involoved in protein synthesis and protein fate, others were related to substance metabolism, and fuctions of one protein were unknown. The immunoproteomics methods were successfully applied in identifying immunogenic proteins of Brucella vaccine strain M5, and laid a fundation for vaccine candidate antigens in future.(2) According to the reports and bioinformatics, the five genes were chosen and cloned from B.melitenise vaccine strain M5 and inserted into prokaryotic expression vector pET32a expressed in E.coli BL21. At the same time, these 5 genes were inserted into the eukaryotic expression vector pVAX1, used to transfect the recombinant plasmids and identify expressed proteins using Western blot analyses and immunchemistry.Balb/c mice were immunized with these candidate vaccines three times every 10d. The DNA vaccine was used in first two immunization, and then the recombinant proteins were boosted in the third immunization. And it elicited both humoral and cellular immune responses. Experimental animals exhibited specific antibodies after the first immunization and increased steadily. The ELISA, ELISPOT, FCM and MTT assays were used to analyse the humoral and cellular immune responses. The results indicated that the candidate DNA vaccines could elicit higher humoral and cellular immune responses. This study laid the foundation for further research on developing effective prevention methods to infection of Brucella based on vaccine strategy.