摘要
布鲁氏菌病(Brucellosis,简称布病)是由布鲁氏菌(Brucella)引起的动物源性传染病,在我国被列为二类传染病,主要引起人的波状热和慢性感染以及反刍动物的不育和流产。尽管布病是一种古老的传染病,但人们对它的认识还很肤浅,很多问题还没有得到解决,如布鲁氏菌的致病机制、细胞内的寄生机制、免疫机制,不同种宿主寄生的差异,以及布病的免疫预防问题等。随着细菌蛋白质组学的发展,通过研究分析细菌蛋白质的组成分布及功能,给揭示上述问题带来了福音。布鲁氏菌的膜蛋白具有促进对表面的黏附、转运溶质和营养进入细胞、输出蛋白和大分子、允许细胞间信号交换、感受外部环境的变化、保持膜的稳定和维持细胞内相对于外周环境的高渗透压等功能。利用蛋白质组学技术研究分析布鲁氏菌膜蛋白,比较不同种间膜蛋白的差异、筛选鉴定免疫原性蛋白,对于研究布鲁氏菌的生物学分类、不同种间的关系,解释不同种宿主寄生的差异,从而挖掘布鲁氏菌毒力、免疫相关分子,阐明致病机制与免疫机制,进一步研发新型疫苗均具有重要意义。
本研究用碳酸盐和新兼性离子去污剂方法成功地提取了布鲁氏菌的膜蛋白,进行2-DE分离,获得了很好的蛋白分离效果。在对布鲁氏菌不同种的强毒株544A和16M进行了比较蛋白质组学研究后发现,在pI 4.0~7.0,MW 6.5~100kDa的2-DE图谱中共发现130个差别表达的蛋白质点,液相串联质谱(LC-MS/MS)鉴定了31个蛋白点,获得23个ORF,经生物信息学分析,这些蛋白涉及能量转化生成、代谢相关酶、信号传导、细胞膜合成等。比较结果显示544A和16M虽同为布鲁氏菌的强毒株,但各自的膜蛋白表达显示其在宿主环境中的代谢机制有明显不同:1)两者通过不同的能量生成与转化功能蛋白表达上调,以供在恶劣的宿主细胞环境中逃逸生存,如544A的gabD、gltA、BruAb2_0325,16M的BMEI0386、BMEⅡ0394;2)通过氨基酸代谢途径544A有ureC在极酸环境的保护机制,16M有BMEⅠ1635在缺氮环境中的保护途径;3)在544A中发现甲基转移酶家族ubiG、假想ABC输送胞质糖结合蛋白、Omp31、过氧化氢环境中保护性酶ahpC、通用压力蛋白BruAb1_1942等蛋白上调,为其适应胞内生存环境提供了生存条件,其中ahpC、ureC为已知的布鲁氏菌毒力因子;在16M中上调的Dps已有研究证明其在16M、M5和Rev1不同的布鲁氏菌强弱毒株中表达差异显著,可能为潜在的毒力因子。4)另在两种菌中均发现有上调的假定蛋白,如544A的BruAb1_1052、BruAb2_0291,16M的BMEI1092,在其他发表的差异蛋白质组学文献中也发现了不同的假定蛋白,这些蛋白没有生物信息学的支持对其功能无法预测,但暗示着布鲁氏菌中有很大一部分假定蛋白在它的胞内生存中发挥着重要作用。因此对标准强毒株不同的致病分子机制进行深入研究,对于理解布鲁氏菌的致病机制有重要的价值。
本研究同时对544A的膜蛋白用布鲁氏菌临床阳性牛、羊血清进行了免疫蛋白质组学研究。在pI 4.0~7.0,MW 6.5~100kDa的2-DE窗口上,共发现19个共同具有免疫原性的蛋白点,通过LC-MS/MS质谱分析和Mascot检索,获得19个ORF。19个蛋白中验证了一部分已知的免疫原性蛋白,也新发现了一部分免疫原性蛋白的,如gltA、ubiG、fabA、gap等,其中ubiG、BruAb1_1617、dadA为本研究中544A和16M的表达差异蛋白,猜测是可引发免疫应答的毒力因子。优选了5个蛋白基因进行PCR引物设计和扩增,在544A、16M和各自疫苗株S19、M5中均有这5个免疫相关蛋白基因的扩增,高的种间同源性和免疫原性提示这些蛋白可用于诊断和多肽疫苗研究。
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 4.0~7.0, MW 6.5~100kDa 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, BruAb2_0325, 16M of BMEI0386, BMEⅡ0394; 2) through amino acid metabolism pathway 544A was protected by ureC in acid environment, 16M was protected by BMEⅠ1 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 BruAb1_1942 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 asBruAb1_1052, BruAb2_0291 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, BruAb1_1617, 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
引文
[1] Pappas, G., Papadimitriou, P.. Challenges in Brucella bacteraemia, Int. J. Antimicrob., 2007:S29–31.
[2] Mohamed N. Seleem, Stephen M. Boyle, Nammalwar Sriranganathan, Brucellosis: A re-emerging zoonosis. Veterinary Microbiology, 2010, 140: 392–398.
[3] Deqiu S, Donglou X, Jiming Y, Epidemiology and control of brucellosis in China, Vet Microbiol, 2002: 165-182.
[4]尚德秋,布鲁氏菌病再度肆虐及其原因,中国地方病防治杂志, 2001, 16: 29-34.
[5]王玉飞, IV型分泌系统调控布鲁氏菌胞内生存的分子机制研究,博士学位论文, 2008.
[6]齐景文,布鲁氏菌病概述,中国兽医杂志, 2004, 40: 50-52.
[7] Scholz H.C., Hubalek Z., Sedlacek I., et al, Brucella microti sp. nov., isolated from the common vole Microtus arvalis. Int. J. Syst. Evol. Microbiol. 2008, 58: 375-382.
[8] Verger J.M., Grimont F., Grimont P.A., Grayon M., Taxonomy of the genus Brucella. Ann. Inst. Pasteur Microbiol, 1987, 138: 235–238.
[9] Foster G., Osterman B.S., Godfroid J., et al, Brucella ceti sp. nov. and Brucella pinnipedialis sp. nov. for Brucella strains with cetaceans and seals as their preferred hosts. Int. J. Syst. Evol. Microbiol., 2007, 57: 2688-2693.
[10] DelVecchio V.G., Kapatral V., Redkar R.J., et al, The genome sequence of the facultative intracellular pathogen Brucella melitensis, Proc. Natl. Acad. Sci. USA, 2002, 99: 443-448.
[11] Halling S.M., Peterson-Burch B.D., Bricker B.J., et al, Completion of the genome sequence of Brucella abortus and comparison to the highly similar genomes of Brucella melitensis and Brucella suis, J. Bacteriol., 2005, 187: 2715–2726.
[12] Paulsen, I.T., Seshadri, R., Nelson, K.E., et al, The Brucella suis genome reveals fundamental similarities between animal and plant pathogens and symbionts. Proc. Natl. Acad. Sci. U.S.A. 2002, 99: 13148–13153.
[13] Wattam, A.R., Williams, K.P., Snyder, E.E., et al, Analysis of ten Brucella genomes reveals evidence for horizontal gene transfer despite a preferred intracellular lifestyle, J. Bacteriol. 2009, 191: 3569–3579.
[14] Kohler S., Teyssier J., Cloeckaert A., et al, Participation of the molecular chaperone DnaK in intracellular growth of Brucella suis with in U9372 derived phagocytes, Mol Microbiol, 1996, 20: 701-712.
[15] Kim, S., Watarai, M., Makino, S., et al. Membrane sorting during swimming internalization of Brucella is required for phagosome trafficking decisions. Microb. Pathog., 2002, 33: 225–237.
[16] Watarai, M., Makino S, Fujii Y, et al, Modulation of Brucella-induced macropinocytosis by lipid rafts mediates intracellular replication. Cell Microbiol, 2002, 4: 341–355.
[17] Rajashekara G, Eskra L, Mathison A, et al. Brucella: functional genomics and host pathogen interactions. Anim. Health Res. Rev. 2006, 7:1–11.
[18] Vergunst A.C., Lier M. C. M., Dulk-Ras A., et al, Positive charge is an important feature of the C-terminal transport signal of the VirB/D4-translocated proteins of Agrobacterium, Proc. Natl. Acad. Sci. U. S. A., 2005, 102: 832–837.
[19] Choi S.H., Greenberg E.P., Genetic dissection of DNA binding and luminescence gene activation by the Vibrio fischeri LuxR protein, J. Bacteriol., 1992, 174: 4064–4069.
[20] Starr T., Ng TW., Wehrly TD., Knodler LA., et al, Brucella intracellular replication requirestrafficking through the late endosomal / lysosomal compartment, Traffic., 2008, 9: 678–694.
[21] Harms J.S., Durward M. A., Magnani D. M., Evaluation of recombinant invasive, non-pathogenic Eschericia coli as a vaccine vector against the intracellular pathogen, Brucella, J. Immune Based Ther. Vaccines, 2009, 7:1-14.
[22] Rambow-Larsen A. A., Petersen E. M., Gourley C. R., et al, Brucella regulators self-control in a hostile environment, cell, 2009,10: 371-377.
[23] Yu DH, Hu XD, Cai H, A combined DNA vaccine encoding BCSP31, SOD, and L7/L12 confers high protection against Brucella abortus 2308 by inducing specific CTL responses, DNA Cell Biol, 2007, 26: 435-443.
[24] Munoz-Montesino C, Andrews E, Rivers R, Gonzalez-Smith A, Moraga-Cid G, Folch H, Cespedes S, Onate AA, Intraspleen delivery of a DNA vaccine coding for superoxide dismutase (SOD) of Brucella abortus induces SOD-specific CD4+ and CD8+ T cells, Infect Immun, 2004, 72: 2081-2087.
[25] Caron E, Cellier M, Liautard JP, et al, Complementation of a DnaK-deficient Escherichia coli strain with the dnaK/dnaJ operon of Brucella ovis reduces the rate of initial intracellular killing within the monocytic cell line U937, FEMS Microbiol Lett, 1994, 120: 335-340.
[26] Cellier MF, Teyssier J, Nicolas M, et al, Cloning and characterization of the Brucella ovis heat shock protein DnaK functionally expressed in Escherichia coli, J Bacteriol, 1992, 174: 8036-8042.
[27] Phillips RW, Roop RM, 2nd: Brucella abortus HtrA functions as an authentic stress response protease but is not required for wild-type virulence in BALB/c mice. Infect Immun 2001, 69:5911-5913.
[28] Phillips RW, Elzer PH, Robertson GT, et al, A Brucella melitensis high-temperature-requirement A (htrA) deletion mutant is attenuated in goats and protects against abortion, Res Vet Sci, 1997, 63: 165-167.
[29] Sola-Landa A, Pizarro-Cerda J, Grillo MJ, et al, A two-component regulatory system playing a critical role in plant pathogens and endosymbionts is present in Brucella abortus and controls cell invasion and virulence, Mol Microbiol, 1998, 29: 125-138.
[30] Lopez-Goni I, Guzman-Verri C, Manterola L, et al, Regulation of Brucella virulence by the two-component system BvrR/BvrS, Vet Microbiol, 2002, 90: 329-339.
[31] Guzman-Verri C, Manterola L, Sola-Landa A, et al, The two-component system BvrR/BvrS essential for Brucella abortus virulence regulates the expression of outer membrane proteins with counterparts in members of the Rhizobiaceae, Proc Natl Acad Sci U S A, 2002, 99: 12375-12380.
[32] Cordwell, S.J., Nouwens, A.S.,Walsh, B.J., Comparative proteomics of bacterial pathogens. Proteomics, 2001, 1:461–472.
[33] Fuqua WC, Winans SC, Greenberg EP, Quorum sensing in bacteria:the LuxR-LuxI family of cell density-responsive transcriptional regulators,J Bacteriol,1994,176:269-275。
[34] Delrue RM, Deschamps C, Leonard S, et al, A quorum-sensing regulator controls expression of both the type IV secretion system and the flagellar apparatus of Brucella melitensis,Cell Microbiol, 2005, 7:1151-1161.
[35] Rambow-Larsen AA, Rajashekara G, Petersen E, Splitter G, Putative quorum-sensing regulator BlxR of Brucella melitensis regulates virulence factors including the type IV secretion system and flagella,J Bacteriol,2008,190:3274-3282。
[36] Phillips RW, Elzer PH, Roop RM, II, A Brucella melitensis high temperature requirementA(htrA) deletion mutant demonstrates a stress response defective phenotype in vitro and transient attenuation in the BALB/c mouse model,Microb Pathog,1995,19:227-284。
[37] Zhan Y., Yang J.I., Cheers C., Cytokines response of T-cell subsets from Brucella abortus infected mice to soluble Brucella proteins, Infect. Immun., 1993, 61: 2841–2847.
[38] Huang L.Y., Reise Sousa C., Itoh Y., et al, IL-12 induction by a TH1-inducing adjuvant in vivo: dendritic cell subsets and regulation by IL-10, J. Immunol., 2001, 167:1423–1430.
[39] Murphy E.A., Sathiyaseelan J., Parent M.A., et al, Interferon-gamma is crucial for surviving a Brucella abortus infection in both resistant C57BL/6 and susceptible BALB/c mice, Immunology, 2001, 103: 511–518.
[40] Oliveira S.C., Splitter G.A., CD8+ type 1 CD44hi CD45RBlo T lymphocytes control intracellular Brucella abortus infection as demonstrated in major histocompatibility complex class I- and Class II-deficient mice, Eur. J. Immunol., 1995, 25: 2551–2557.
[41] Sergio C. Oliveira, Nancy Soeurt, Gary Splitter, Molecular and cellular interactions between Brucella abortus antigens and host immune responses, Veterinary Microbiology, 2002, 90: 417–424.
[42] Canning P C,Roth J A,and Deyoe B L, Release of 5'-guanosine monophosphate and adenine by Brucella abortus and their role in the intracellular survival of the bacteria, J. Infect. Dis.,1986, 154:464-470.
[43] Liautard J P, Dornand A. G. J., Kohler S., Interactions between professional phagocytes and Brucella spp, Microbiologia, 1996.12:197-206.
[44] Kohler S, Layssac M, Naroeni A, et al, Secretion of listeriolysin by Brucella suis inhibits its intramacrophagic replication, Infect.Immun.2001.69:2753-2756.
[45] Cloeckaert A, Vizcaino N, Paquet JY, et al, Major outer membrane proteins of Brucella spp.:past, present and future,Vet Microbiol, 2002, 90:229-247.
[46] Tibor A, Wansard V, Bielartz V, et al, Effect of omp10 or omp19 deletion on Brucella abortus outer membrane properties and virulence in mice, Infect. Immun., 2002, 70:5540-5546.
[47] Salhi I, Boigegrain RA, Machold J, et al,Characterization of new members of the group 3 outer membrane protein family of Brucella spp.,Infect Immun,2003,71: 4326-4332.
[48] Eschenbrenner M., Wagner M.A., Horn T.A., Comparative proteome analysis of Brucella melitensis vaccine strain Rev 1 and a virulent strain 16M, 2002, 184: 4962–4970.
[49] Joseph P. Connolly, Diego Comerci, Timothy G. Alefantis, et al, Proteomic analysis of Brucella abortus cell envelope and identification of immunogenic candidate proteins for vaccine development, Proteomics 2006, 6:3767–3780.
[50] Henningsen R, Gale BL, Straub KM, et al, Application of zwitterionic detergents to the solubilization of integral membrane proteins for two-dimensional gel electrophoresis and mass spectrometry, Proteomics, 2002, 2: 1479-1488.
[51]刘秉阳,布鲁氏菌病学,人民卫生出版社, 1989,1:40-41.
[52] Verger J. M.; Grimont F.; Grimont P. A.; Grayon M., Brucella, a monospecific genus as shown by deoxyribonucleic acid hybridization, Int. J. Syst. Bacteriol., 1985, 35: 292-295.
[53] Rosemarie B. Alcantara, Richard D. A. Read, Michelle Wright Valderas, et al, Intact Purine Biosynthesis Pathways Are Required for Wild-Type Virulence of Brucella abortus 2308 in the BALB/c Mouse Model, Infection and immunity, 2004, 72: 4911–4917.
[54] Mohamed N. Seleem, Stephen M. Boyle, Nammalwar Sriranganathan, Brucella: A pathogen without classic virulence genes, Veterinary Microbiology, 2008, 129: 1–14.
[55] JoséLuis Lavín, Tim T. Binnewies, Antonio G. Pisabarro, et al, Differences in two-component signal transduction proteins among the genus Brucella: Implications for host preference and pathogenesis, Veterinary Microbiology, 2010, 1:1-10.
[56] Chung-Dar Lu. Pathways and regulation of bacterial arginine metabolism and perspectives for obtaining arginine overproducing strains, Appl Microbiol Biotechnol, 2006, 70: 261-272.
[57] Saier M.H. Jr. Families of transmembrane sugar transport proteins, Mol. Microbiol., 2000, 35:99-710.
[58] Koning S.M., Albers S. V., Konings W.N., et al, Sugar transport in (hyper) thermophilic archaea, Res. Microbiol., 2002, 153: 61-67.
[59] Horlacher R., Xavier K.B., Santos H., Di, et al, Archaeal binding protein-dependent ABC transporter: molecular and biochemical analysis of the trehalose/maltose transport system of the hyperthermophilic archaeon Thermococcus litoralis, J. Bacteriol., 1998, 180: 680-689.
[60] Al-Mariri A, Tibor A, Mertens P, et al, Induction of immune response in BALB/c mice with a DNA vaccine encoding bacterioferritin or P39 of Brucella spp, Infect Immun, 2001, 69: 6264-6270.
[61] Delpino MV, Cassataro J, Fossati CA, et al, Brucella outer membrane protein Omp31 is a haemin-binding protein, Microbes Infect, 2006, 8: 1203-1208.
[62] Sherman D.R., Mdluli K., Hickey M.J., et al, Compensatory ahpC gene expression in isoniazid-resistant mycobacterium tuberculosis. Science, 1996, 272: 1641–1643.
[63] Chen L., Xie Q.W., Nathan C., Alkyl hydroperoxide reductase subunit C (AhpC) protects bacterial and human cells against reactive nitrogen intermediates, Mol. Cell, 1998, 1: 795–805.
[64] Chae H.Z., Robison K., Poole L.B., Church G., et al. Cloning and sequencing of thiol-specific antioxidant from mammalian brain: alkyl hydroperoxide reductase and thiol-specific antioxidant define a large family of antioxidant enzymes, Proc. Natl. Acad. Sci. U.S.A., 1994, 91: 7017–7021.
[65] Eschenbrenner M, Horn TA, Wagner MA,et al, Comparative Proteome Analysis of Laboratory Grown Brucella abortus 2308nd Brucella melitensis 16M, J Proteome Res, 2006, 5(7) :731 1740.
[66]赵忠鹏,吴朔,罗德炎,等,羊种布鲁氏菌疫苗株M5和强毒株16M的比较蛋白质组学研究,中国人兽共患病学报, 2007,23(12):1172-1175.
[67] Jonathan Pevsner,孙之荣译,生物信息学与功能基因组学,化学工业出版社,2006, 1: 231-232.
[68] Gygi S.P., Corthals G.L., Zhang Y., et al, Evaluation of two-dimensional gel electrophoresis-based proteome analysis technology, Proc. Natl. Acad. Sci., 2000, 97: 9390–9395.
[69]赵忠鹏,布鲁氏菌毒力、免疫相关分子的筛选研究,博士学位论文,军事医学科学院,2008.
[70] Sládek NE., Human aldehyde dehydrogenases: potential pathological, pharmacological, and toxicological impact, J. Biochem Mol. Toxicol., 2003, 17(1):7-23.
[71] Vasiliou V, Pappa A, Petersen DR. Role of aldehyde dehydrogenases in endogenous and xenobiotic metabolism. Chem Biol Interact. 2000 Dec 1: 129(1-2):1-19.
[72] Marchitti SA, Deitrich RA, Vasiliou V. Neurotoxicity and metabolism of the catecholamine-derived 3,4-dihydroxyphenylacetaldehyde and 3,4-dihydroxyphenylglycolaldehyde: the role of aldehyde dehydrogenase. Pharmacol Rev. 2007 59(2):125-50.
[73] Tong L, Acetyl-coenzyme A carboxylase: crucial metabolic enzyme and attractive target for drug discovery, Cell. Mol. Life Sci., 2005, 62 (16): 1784–803.
[74] Corbett JW, Harwood JH, Inhibitors of mammalian acetyl-CoA carboxylase, Recent Patents Cardiovasc Drug Discov, 2007, 2 (3): 162–180.
[75] Teixeira-Gomes A.P., Characterization of heat oxidative and acid stress responses in Brucella melitensis. Infect. Immun., 2000, 68:2954–2961.
[76] Oddo M., Renno T., Attinger A., et al, Fas ligand-induced apoptosis of infected human macrophages reduces the viability of intracellular Mycobacterium tuberculosis, J. Immunol., 1998, 160(1):5448-5454.
[77] Connolly J P, Comerci D, Alefantis T G, et al, Proteomic analysis of Brucella abortus cell envelope and identification of immunogenic candidate proteins for vaccine development, Proteomics, 2006,6:3767-3780.
[78] Molloy MP, Isolation of Bacterial Cell Membranes Proteins Using Carbonate Extraction. Methods in Molecular Biology, 2008, 424: 397-402.
[1] Scholz, H.C., Hubalek, Z., Sedlacek, I., et al, Brucella microti sp. nov., isolated from thecommon vole Microtus arvalis. Int. J. Syst. Evol. Microbiol. 2008, 58: 375-382.
[2] Verger, J.M., Grimont, F., Grimont, P.A., Grayon,M., Taxonomy of the genus Brucella. Ann. Inst. Pasteur Microbiol, 1987,138: 235–238.
[3] Foster, G., Osterman, B.S., Godfroid, J., et al, Brucella ceti sp. nov. and Brucella pinnipedialis sp. nov. for Brucella strains with cetaceans and seals as their preferred hosts. Int. J. Syst. Evol. Microbiol. 2007, 57: 2688-2693.
[4] Ugalde, Rodolfo A., Intracellular lifestyle of Brucella spp. common genes with other animal pathogens, plant pathogens, and endosymbionts, Microb. Infect., 1999, 1: 1211–1219.
[5] O’Callaghan, D., Cazevieille, C., Allardet-Servent, et al, A homologue of the Agrobacterium tumefaciens VirB and Bordatella pertussis Ptl type IV secretion systems is essential for intracellular survival of Brucella suis, Mol. Microbiol., 1999, 33: 1210–1220.
[6] Corbel, M.J., 1997. Brucellosis: an overview. Emerg. Infect. Dis. 2, 213–221.
[7] Morris, J.A., The use of polyacrylamide gel electrophoresis in taxonomy of Brucella, J. Gen. Microbiol., 1973, 76: 231–237.
[8] Santos, J.M., Verstreate, D.R., Perera, V.Y., Winter, A.J., Outer membrane proteins from rough strains of four Brucella species, Infect. Immun., 1984, 46: 188–194.
[9] Gamazo, C., Winter, A.J., Moriyo′n, I., et al, Comparative analyses ofproteins extracted by hot saline or released spontaneously into outer membrane blebs from field strains of Brucella ovis and Brucella melitensis, Infect. Immun., 1989, 57: 1419–1426.
[10] Brooks-Worrell, B.M., Splitter, G.A., Sodium dodecyl sulfate- and salt-extracted antigens from various Brucella species induce proliferation of bovine lymphocytes. Infect. Immun., 1992, 60: 2136–2138.
[12] Hill, R.A., Cook, D.R., Protein profiles of Brucella suis and Brucella abortus in isoelectric focusing and sodium dodecyl sulphate-polyacrylamide gel electrophoresis. Vet. Microbiol., 1994, 39:25–32.
[13] Verstreate D.R., Creasy M.T., Caveney N.T., et al, Outermembrane proteins of Brucella abortus: isolation and characterization, Infect. Immun., 1982, 35: 979–989.
[14] Verstreate, D.R., Winter, A.J., Comparison of sodium dodecyl sulfate-polyacrylamide gel electrophoresis profiles and antigenic relatedness among outer membrane proteins of 49 Brucella abortus strains, Infect. Immun., 1984, 46: 182–187.
[15] Tabatabai, L.B., Deyoe, B.L., Characterization of salt-extractable protein antigens from Brucella abortus by crossed immunoelectrophoresis and isoelectricfocusing, Vet. Microbiol., 1984, 9: 549–560.
[16] Teixeira-Gomes A.P., Cloeckaert A., Be′zard G., et al, Mapping and identification of Brucella melitensis proteins by two-dimensional electrophoresis and microsequencing, Electrophoresis, 1997, 18:156–162.
[17] Teixeira-Gomes A.P., Cloeckaert A., Be′zard G., Identification and characterization of Brucella ovis immunogenic proteins using two-dimensional electrophoresis and immunoblotting, Electrophoresis, 1997,18:1491–1497.
[18] Teixeira-Gomes, A.P., Cloeckaert, A., Zygmunt, M.S., Characterization of heat, oxidative, and acid stress responses in Brucella melitensis, Infect. Immun., 2000, 68: 2954–2961.
[19] Sowa, B.A., Kelly, K.A., Ficht, T.A., Adams, L.G., Virulence associated proteins of Brucella abortus identified by paired two-dimensional gel electrophoretic comparisons of virulent, vaccine, and LPS deficient strains, Appl. Theor. Electrophor., 1992, 3: 33–40.
[20] Rafie-Kolpin, M., Essenberg, R.C., Wyckoff III, J.H., Identification and comparison of macrophageinduced proteins and proteins induced under various stress conditions in Brucella abortus. Infect. Immun. 1996, 64, 5274–5283.
[21] Lopez, M.F., Berggren, K., Chernokalskaya, E., et al, A comparison of silver stain and SYPRO Ruby protein gel stain with respect to protein detection in two-dimensional gels and identification by peptide mass profiling, Electrophoresis, 2000, 21: 3673–3683.
[22] Wildgruber, R., Harder, A., Obermaier, C., Boguth, G. et al., Towards higher resolution: two-dimensional electrophoresis of Saccharomyces cerevisiae proteins using overlapping narrow immobilized pH gradients, Electrophoresis, 2000, 21: 2610–2616.
[23] Wagner, M.A., Eschenbrenner, M., Horn, T.A., Kraycer, J.A., et al, Global analysis of the Brucella melitensis proteome: identification of proteins expressed in laboratory-grown culture. Proteomics, 2002, 2: 1047–1060.
[24] Gygi, S.P., Corthals, G.L., Zhang, Y. et al., Evaluation of two-dimensional gelelectrophoresis-based proteome analysis technology. Proc. Natl. Acad. Sci. U.S.A., 2000, 97, 9390–9395.
[25] Verger, J.-M., Grimont, F., Grimont, P.A.D., Grayon, M., Brucella, a monospecific genus as shown by deoxyribonucleic acid hybridization, Int. J. Sys. Bact., 1985, 35: 292–295.
[26] Michaux-Charachon S., Bourg G., Jumas-Bilak E., et al, Genome structure and phylogeny in the genus Brucella, J. Bacteriol., 1997, 179: 3244–3249.
[27] Cordwell, S.J., Nouwens, A.S.,Walsh, B.J., Comparative proteomics of bacterial pathogens. Proteomics, 2001, 1:461–472.
[28] Michel Eschenbrenner, Troy A. Horn, et al, Comparative Proteome Analysis of Laboratory Grown Brucella abortus 2308 and Brucella melitensis 16M, J. Proteome Res., 2006, 5 (7):1731 -1740.
[29] Eschenbrenner M., Wagner M.A., Horn T.A., Comparative proteome analysis of Brucella melitensis vaccine strain Rev 1 and a virulent strain 16M, 2002, 184: 4962–4970.
[30]赵忠鹏,吴朔,罗德炎,等,羊种布鲁氏菌疫苗株M5和强毒株16M的比较蛋白质组学研究,中国人兽共患病学报, 2007, 23:1172-1175.
[31] Gao YH, et al, Comparative proteomics research on THP-1 cells infected with Brucella, 2006, 46(4):629-34.
[32] Connolly JP, Comerci D, Alefantis TG, et al, Proteomic analysis of Brucella abortus cell envelope and identification of immunogenic candidate proteins for vaccine development Proteomics, 2006, 6(13):3767-3780.
[33] Sascha Al Dahouk, Karsten N?ckler, Holger C, et al, ScholzImmunoproteomic characterization of Brucella abortus 1119-3 preparations used for the serodiagnosis of Brucella infections, Journal of Immunological Methods, 2006, 309: 34– 47.
[34] DelVecchio VG, Wagner MA, Eschenbrenner M, et al, Brucella proteomes - a review, Veterinary Microbiology, 2002, 90 (1 -4) : 593 - 603.
1.曾政.布鲁氏菌毒力、免疫相关分子的筛选研究[D].北京:中国人民解放军军事医学科学院,2008.
2. Henningsen R, Gale BL, Straub KM, et al. Application of zwitterionic detergents to the solubilization of integral menbrane proteins for two-dimensional gel electrophoresis and mass spectrometry[J]. Proteomics,2002, 2(11):1479-1488.
3. Axel C, Nieves V,Jean YP, et al.Major outer membrane proteins of Brucella spp past, present and future[J].veternaru Microbiology.2002,90:229-247.
4. Paola CH, Luis FL, Mar?′a-Jesu′s de Miguel, et al. Role of the Omp25/Omp31 Family in Outer Membrane Properties and Virulence of Brucella ovis[J]. Infection and immunity,2007, 75: 4050–4061.
5. Connolly J P,Comerci D,Alefantis T G,et al.Proteomic analysis of Brucella abortus cell envelope and identification of immunogenic candidate proteins for vaccine development [J].Proteomics,2006,6:3767-3780.
6. Contreras-Rodriguez A, Ramirez-Zavala B, Contreras A,et al. Purification and characterization of an immunogenic aminopeptidase of Brucella melitensis[J]. Infect Immun, 2003, 71: 5238-5244。
7. Jinkyung Ko, Gary A. Splitter. Molecular Host-Pathogen Interaction in Brucellosis: Current Understanding and Future Approaches to Vaccine Development for Mice and Humans[J]. Clinical microbiology review,2003,16:65–78.
8. Sascha AD, Karsten N, Holger CS,et al. Immunoproteomic characterization of Brucella abortus 1119-3 preparations used for the serodiagnosis of Brucella infections[J]. Journal of Immunological Methods.2006,309:34–47.
9. Isabelle J, Jean MV, Karine L, et al.Immunological responses and protective efficacy against Brucella melitensis induced by bp26 and omp31 B. melitensis Rev.1 deletion mutants in sheep[J]. Vaccine, 2007(25) :794–805.
10. Joseph PC, Diego C, Timothy GA, et al. Proteomic analysis of Brucella abortus cell envelope and identification of immunogenic candidate proteins for vaccine development[J]. Proteomics 2006(6): 3767–3780.