Protemomic Analysis Of Membrane Proteins And Identification Of Immunogenic Proteins On Brucella

Brucellosis, which is caused by brucella, is one of the most important bacterial zoonoses endemic and classified as second level infectious diseases in China, mainly causes human abortus fever,chronic infections and the infertility and miscarriage for ruminants. Although Brucellosis is an ancient disease, there are many problems still unresolved for it, such as pathogenesis, parasitism within the cell, immune system, disparity of the different species hosts parasitic, and the prevention of brucella. With the development of bacterial proteomics, analysis of bacteria by studying the distribution and function of protein composition has brought these issues to reveal the gospel. Brucella membrane proteins can promote the adhesion of surface, transport solute and nutrient into the cell, output protein and macromolecule, allow the exchange of signals between cells, feel the changes in the external environment, and maintain membrane stability and high osmotic pressure of cells relative to peripheral environment. Proteomics technology used to analysis brucella membrane protein, compare membrane proteins from different species, screening and identification the immunogenic proteins of brucella, is beneficial for the research of its biological classification and the relationship between different species, interpreting differences of the different species to host parasitism, screening brucella virulences and immune-related molecules, and has great significance to clarify the pathogenesis and the immune system for further development of new vaccines.In our research, carbonate and the new ion detergent were successfully used to extract the membrane proteins of brucella, and 2-DE got good proteins separation. The proteomics research was used to compare different species of brucella virulent strain, brucella menlitensis 16M and brucella abortus 544A. In the pI 4.0⁷.0, MW 6.5¹00kDa of the 2-DE gel 130 differential spots were screening, 31 spots were identified by LC-MS/MS and 23 ORFs were gained in the final. By bioinformatic analysis, these proteins involved in energy conversion generation, metabolic enzymes, signal transduction, cell membrane synthesis. 544A and 16M comparison results show that although both are the wild virulence strain of brucella, the different expression of membrane proteins show the significantly different metabolic mechanisms in host environment: 1) the two strains escaped and survived in the poor host cell environment by the different energy production and conversion functions protein expression, such as 544A of gabD, gltA, BruAb2₀325, 16M of BMEI0386, BMEâ…¡0394; 2) through amino acid metabolism pathway 544A was protected by ureC in acid environment, 16M was protected by BMEâ… 1 635 in nitrogen deficiency environment; 3) in 544A strain, methyltransferase family ubiG, hypothetical ABC transport cytoplasmic sugar-binding protein, Omp31, hydrogen peroxide environment protective enzyme ahpC, general stress protein BruAb1₁942 overexpressed, adapted to the intracellular survival, and ahpC,ureC were known as virulence factors of brucella; in 16M Dps overexpressed has been discovered by the studies that comparison proteomics of brucella menlitensis 16M, M5 and Rev1, and regards as a potential virulence factors. 4) hypothetical protein were found in two strains, such asBruAb1₁052, BruAb2₀291 in 544A, BMEI1092 in 16M. In other published comparison proteomics research were also found several hypothetical proteins with no biological information offered its function prediction, it suggests that there are a large part of hypothetical proteins play an important role on brucella survives in intracellular protein. Therefore, it suggested that there is significant value for understanding the pathogenesis by study the pathogenic molecular mechanisms of different virulent brucella strain.In this study, immunoproteomics was utilized to identify novel immunogenic proteins of the brucella abortus 544A membrane proteins responded to positive clinical serum from cattle, sheep. In the pI 4.0 7.0, MW 6.5 100kDa of 2-DE window, there were 19 common immunogenic spots identified by LC-MS/MS and Mascot search, gained 19 ORF. Among of them, known immune molecules was found, and there were also some new discovered in this study, such as gltA, ubiG, fabA, gap, etc.. UbiG, BruAb1₁617, dadA identified as the different proteins between 544A and 16M in this study, were guessed to be potential virulence factors that can introduced the immune response. Five immunogenic proteins were selected to amplificate in 544A, 16M, and their respective vaccine strain S19, M5 by PCR. Result showed the whole genes of the five immune-related all existed in the four strains brucella, high homology and imunogenicity of them suggested these proteins can be used for diagnosis and further research as immune dominant protein peptide vaccine...