Proteomics Of Membrane Proteins Of Macrophage Infected By Brucella.Abortus544A

Research on proteomics analysis of membrane proteins of macrophage infected by Brucella abortus544A. Brucellosis is a highly contagious zoonotic diseases in human and animals caused by the brucella. It has severely affected the development of animal husbandry and public health security. Now brucellosis is spreading in the world and the onset number is increasing year by year. In the recent years, the epidemic and explosion of brucellosis in human and animals are very serious in China and the Brucella infection in sheep and cattle are increasing and a new high infection rate was found in human. The brucellosis has become one of the fastest growing diseases that increased number of notifiable and reported infectious diseases.Brucellae are typical intracellular bacteria and can escape the phagocytosis in host. However, the interaction mechanism of Brucella and macrophage is still unknown and which proteins in host cells that help Brucella adhere macrophages and invadate into macrophages and the intracellular traffic and replication are still unknown. The macrophages constitute the first line ofdefense of the innate immune response against invading microorganisms and the relative proteins expressed in cell membrane are playing key role for interaction Brucella and macrophages.Therefore, the comparative proteomics mathods was used for screening and identificating these preoteins,the difference proteins were identified by MALDITOF-MS,which will provide scientific evidence for interaction mechanism of brucella and host cell.In this study,the infection model of Brucella infected-macrophages was first constructed in order to analyze difference proteins of macrophage before infection and post infection. We optimized the infection time and the proportion of primary antibody and secondary antibody. The results revealed that the MOI of200:1, infection time of2h, and the antibody dilution of1:80was the optimization for infecting between Brucella and macrophages. The transmission electron microscope was used to observe the Brucella in macrophages and the bacterial culturing in TSA was used to detect the intracellular Brucella in macrophages.Then we extracted the membrane proteins from infected-macrophages and noninfected-macrophages by the membrane protein extracting kit and the proteins were quantitated by BCA methods.The proteins concentration were12.5mg/ml and10.98mg/ml.In the end, the membrane proteins were separated by two-dimensional gel electrophoresis in range of pH4to7and molecule weight7kDa to175kDa. The defference proteins were screened by the software, Image-master2D and the results revealed that16defference protein spots were screened.The defference proteins were identified by MALDITOF-MS and the results revealed that13defference proteins were identified successfully, they represented12proteins, respectively. The bioinformation function of12proteins were retrieved in www.expasy.org and the results revealed that these proteins participated glycolysis, energy metabolism, ion transportation, cytoskeleton synthesis, protein injury and reparation and gene transcriptional control in macrophage life process,respectively.Therefore,the study would provide the scientific evidence for understanding the interaction mechamnism between Brucella and macrophage.