Proteomics Of Membrane Proteins Of Macrophage Infected By Brucella.melitensis 16M

Brucellosis is the zoonotic diseases caused by the brucella, serious harm to livestock development and human health. Brucellosis is a worldwide public health problem, and there are many countries around the world involved in brucellosis. Recent years, the monitoring results showed that brucellosis was more serious in China. Sheep and cattle brucellosis infection rates rose faster, and became one of the fastest growing diseases that increased number of notifiable and reported infectious diseases.In China B. melitensis,B. abortus,B. suis are mainly prevalent, among them, B. melitensis are more common species of brucella and virulence is the strongest, virulent strain of brucella is a typical intracellular parasites. But its pathogenesis is still unclear, such as adhesion of cells, phagosome forming, living and replication in the cell, immune evasion, etc, it remains to be further study the related pathogenesis. Membrane proteins is the sum of all membrane proteins of cell in a specific time and space, Membrane proteomics'research contributes to research of cell signal transduction, cell-cell interactions and interaction between cells and pathogens. Differential proteomics is to find and screen the significant proteins of various samples caused by certain factors which is to get some key proteins'analysis of properties and functions. Therefor, membrane protein differential proteomics'development and technology application will bring new ideas to the pathogenesis of brucellosis.This research, differential proteomics to research membrane protein changes of B. melitensis virulent strain 16M infected macrophage, in order to find anti-Brucella related host cell membrane proteins. First, the brucella and macrophages were cultured, and then the the cells RAW264.7 were infected with B. melitensis 16M: 500:1, the first antibody and second antibody was 1:80 / 1:80 for indirect immunofluorescence at 4.5h, and observed by transmission electron microscopy, then the infected cells were broken and cultured in TSB solid medium, to further verify the infection of cells. Results established an infection model of bacteria infected cells by optimizing a variety of conditions of infections. Then membrane proteins were extracted using the homemade extraction lysate and kit lysate to extract the membrane protein of infected macrophage, this research used the kit to extract membrane proteins by BCA method quantitatived the proteins and by SDS-PAGE comparised experiment, which separation is good, protein concentration is high, operation is easy. Then the two-dimensional gel electrophoresis did the first was isoelectric focusing,pH range in the 3-10 with a large range of solid dry strip pH was choiced, after two equilibrium, the second was the SDS-PAGE, stained, choiced rapid ammoniacal silver stain by comparison, high sensitivity, option of the first solid dry strip pH range was appropriate, protein separation was effective. And then repeated the experiment samples. Results got the number of protein spots were more scattered clear, clean background of the gel patterns.The image analysised by Image-master2D found that different protein spots of ratio≥1.5 had 147 and the different protein spots of ratio≥2.0 had 54 at infection group and the control group. From the large difference in the selection of 31 protein spots, 22 protein spots were detected by MALDI-TOF-MS mass spectrometry identified method. Western Blotting experiment analysised the changes of the expression in the infected group with the control group, which experimental results and 2D gel electrophoresis analysis results were consistent. Most of these proteins located on the biofilm searched by NCBI database, and included Heat shock protein1,Fructose-bisphosphate aldolase C,Superoxide dismutase,Peptidyl-prolyl cis-trans isomerase A,Protein disulfide-isomeraseA3,ATP synthase subunit,alpha-enolase[Mus musculus] , etc. there also were some cells tubulin protein, scaffold protein, binding protein. Most protein involved in the cell's life activities, such as ATP synthesis and transportion, protein folding processing, signal transduction, carbohydrate metabolism, and they may be involved in cellular immunity of anti-infection Brucella process.This study firstly used differential proteomics to analysis of the infected and control groups of host cell membrane proteins and firstly screened the protein related brucella infection and parasitic interaction from the host cells. All those would give us a new direction to reveal the pathogenesis of brucella, and also provided a scientific basis to find new drug targets,nurture brucellosis resistance of new varieties of genetically modified.