外膜蛋白Omp25在布鲁氏菌毒力及免疫保护中的作用研究
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摘要
布鲁氏菌病是一种危害严重的人兽共患病,在世界范围内有广泛流行,给人类健康和经济发展带来巨大损失。布鲁氏菌是一种胞内寄生菌,具有比较独特的胞内生存机制和免疫机制。它的致病机制以胞内生存为主要特征,而免疫机制则是在与宿主免疫系统斗争中逃避免疫反应而得以生存繁殖。针对布鲁氏菌的有效的免疫反应以细胞免疫为主,并且是多种抗原综合作用的结果。深入探讨布鲁氏菌的胞内生存机制和免疫保护机制,对于布鲁氏菌致病机制的理解、疫苗的保护机制以及新型疫苗的研发等具有重要意义。
外膜蛋白在稳定细菌外膜的结构、适应胞内外环境和抵抗胞内杀菌机制等方面起着重要作用,与细菌的毒力有密切关系。外膜蛋白位于细菌的表面,容易被免疫系统识别,在与宿主的相互作用中发挥作用。另一方面,很多外膜蛋白都具有免疫原性,其中很大一部分是免疫保护抗原。因此,很多有关细菌致病机制和免疫反应机制的研究都集中在外膜蛋白的研究上。
布鲁氏菌病的控制重在预防,大规模接种疫苗是有效控制疾病的重要措施。事实证明,尽管经历了很多对其它疫苗形式的尝试,如亚单位疫苗、基因工程疫苗和核酸疫苗等,但是减毒活疫苗仍是目前预防布鲁氏菌病最为有效的疫苗形式。减毒活疫苗之所以能够发挥有效的免疫保护作用,是否与其外膜的成分,特别是外膜蛋白有关?或者说外膜蛋白在减毒活疫苗中发挥了什么样的作用?这些问题的回答,有利于解释布鲁氏菌疫苗的作用机制和保护机制,也有利于进一步开发新型的疫苗。
为研究布鲁氏菌主要外膜蛋白在布鲁氏菌毒力和免疫保护中的作用,本研究中,我们首先利用蛋白质组技术,分离鉴定布鲁氏菌的外膜蛋白,明确外膜蛋白的组成。然后,选取主要的外膜蛋白,构建其缺失突变株,并比较分析这些突变株的胞内外的毒力表型,探讨其与毒力的关系。用M5及缺失突变菌株免疫小鼠,然后用M5本身和羊种布鲁氏菌强毒株16M进行攻击,观察M5及突变菌株的免疫保护性,探讨主要外膜蛋白在疫苗株免疫保护中的作用及其机制。最后,体外克隆表达主要外膜蛋白,分析其免疫原性,探讨其作为诊断抗原的可行性。通过以上研究,将初步了解羊布鲁氏菌外膜蛋白的组成,认识主要外膜蛋白在M5的毒力及免疫保护中的作用,并探索了主要外膜蛋白作为诊断抗原的可行性。
Brucellosis, which caused by Brucella, is one of the most important bacterial zoonoses endemic in the world, especially in developing countries. These pathogens can affect a broad range of mammals and cause serious economic losses. Three Brucella species, B. melitensis, B. abortus and B.suis, are virulent for humans, causing a chronic, debilitating disease with severe and sometimes fatal outcomes. Moreover, as Brucella spp. can be easily aerosolized, this pathogen could be used to develop biological weapons.
Brucella is an intracellular bacterium. Research on Brucella and its interaction between host reveals that Brucella activate reduced immune response. The virulence of Brucella depends upon its ability to survive and replicate in host cells. They replicate in endoplasmic reticulum (ER) and escape from the immune response. Effective immune response is mainly mediated by cellular immunity. Outer membrane proteins (OMPs) play important roles in stabilizing the structure of the outer membrane, adapting to external and intracellular environments, and Brucella virulence. OMPs are located on surface of bacteria and can be easily recognized by host immune system, therefore, some of them represent important immunoantigen.
Although many attepts have been tried for development of different vaccine types, live attenuated vaccine is the most efficient vaccine for Brucellosis for the present. In the present study, to analyze the roles of major OMPs in Brucella virulence and immunological protection, outer membrane proteome was separated by 2D eletrophoresis and the proteins were identified by MS. A total of 67 proteins were identified to be possibly membrane proteins. Nine of these protein spots are products of Omp25 and seven were those of Omp31. Omp25 is a member of the OmpA protein family, which have immunogenicity. Outer membranes proteins play important roles in stabilizing the structure of the outer membrane and are relative to the virulence. To predict its function, the sequence of omp25 was analyzed by bioinformatics methods. In the genome of Brucella, there are four omp25 genes(BMEI1249(omp25)、 BMEI1007(omp25b)、BMEI1829(omp25c) and BMEI1830(omp25d). Among the four genes, only omp25 (BMEI1249) have been shown to be related with brucella virulence .
To further investigate the function of omp25, the mutants of the four omp25 genes were constructed by homologous recombination, and then their virulence phenotypes were tested. Firstly, the kanamycin gene of pBBR1MCS-2 was PCR amplified and cloned at the multicloning site of pUC19 to generate a new suicide plasmid pUC19K, which was then used to construct the mutant strains. By using resistance replacement method, mutants of the four omp25 genes were successfully constructed, named BMΔ1007, BMΔ1249, BMΔ1829 and BMΔ1830.
In vitro growth curve assays showed that the four mutants and BM have similar growth curves. However, the mutants showed higher growth rate than the wild type strain at logarithmic phase. At stationary phase, BMΔ1007 showed the highest cell density. This implyied that omp25 negatively regulate growth of Brucella under the assaying conditions. When grow into stationary phase, BMΔ1249 autoaggregated to form clamps in culture, which were not observed in the other three mutant strains. Survival under stress conditions showed that the four mutants showed different sensitivity to the stress simulating intracellular environments, including high salt, high osmosis, low pH, heat shock and oxidative stress. Sensitivity of these mutants to polymyxin B and sodium deoxycholate were also increased. These data indicated that omp25 plays important roles in Brucella adaptation to hostile environments and maintaining integrity of membrane structure. Intracellular survival showed that the omp25 mutants could invade macrophage, but were rapidly cleared by host cell, implying that omp25 is important for Brucella intracellular replication and chonic infection. Balb/c mice was infected with M5 and omp25 mutants, and then challenged with BM and 16M to test their protection capability. When challenged with M5, no great differences were observed in protection efficiency between three mutants and M5, but the BMΔ1829 showed better protection. Then, BMΔ1249, BMΔ1829 and M5 was used to immunize mice and challenged with 16M. The mutant showed reduced protection than M5. The mice immunized with omp25 mutants showed lower antibody titer and IFN-v those infected with M5, implying that omp25 plays important roles in protection efficency of M5. At last, the four omp25 proteins were expressed in E. coli. Western blot result showed that all the proteins could react with brucellosis sera. Only omp25 protein could differentiate sera of M5 from that of the mutant strain.
The results above showed that the omp25 play important roles in brucella adaptation to hostile environments, intracellular survival and maintaining integrity of outer membrane. These data also showed that omp25 proteins play important roles in protection of live attenuated strain. These findings expand our knowledge of the function of Omp25 in Brucella survival both in vivo and in vitro, virulence phenotypes and immuno-protection, providing important clues for the understanding of the molecular mechanism of intracellular survival and protection mechanism of Brucella.
外膜蛋白在稳定细菌外膜的结构、适应胞内外环境和抵抗胞内杀菌机制等方面起着重要作用,与细菌的毒力有密切关系。外膜蛋白位于细菌的表面,容易被免疫系统识别,在与宿主的相互作用中发挥作用。另一方面,很多外膜蛋白都具有免疫原性,其中很大一部分是免疫保护抗原。因此,很多有关细菌致病机制和免疫反应机制的研究都集中在外膜蛋白的研究上。
布鲁氏菌病的控制重在预防,大规模接种疫苗是有效控制疾病的重要措施。事实证明,尽管经历了很多对其它疫苗形式的尝试,如亚单位疫苗、基因工程疫苗和核酸疫苗等,但是减毒活疫苗仍是目前预防布鲁氏菌病最为有效的疫苗形式。减毒活疫苗之所以能够发挥有效的免疫保护作用,是否与其外膜的成分,特别是外膜蛋白有关?或者说外膜蛋白在减毒活疫苗中发挥了什么样的作用?这些问题的回答,有利于解释布鲁氏菌疫苗的作用机制和保护机制,也有利于进一步开发新型的疫苗。
为研究布鲁氏菌主要外膜蛋白在布鲁氏菌毒力和免疫保护中的作用,本研究中,我们首先利用蛋白质组技术,分离鉴定布鲁氏菌的外膜蛋白,明确外膜蛋白的组成。然后,选取主要的外膜蛋白,构建其缺失突变株,并比较分析这些突变株的胞内外的毒力表型,探讨其与毒力的关系。用M5及缺失突变菌株免疫小鼠,然后用M5本身和羊种布鲁氏菌强毒株16M进行攻击,观察M5及突变菌株的免疫保护性,探讨主要外膜蛋白在疫苗株免疫保护中的作用及其机制。最后,体外克隆表达主要外膜蛋白,分析其免疫原性,探讨其作为诊断抗原的可行性。通过以上研究,将初步了解羊布鲁氏菌外膜蛋白的组成,认识主要外膜蛋白在M5的毒力及免疫保护中的作用,并探索了主要外膜蛋白作为诊断抗原的可行性。
Brucellosis, which caused by Brucella, is one of the most important bacterial zoonoses endemic in the world, especially in developing countries. These pathogens can affect a broad range of mammals and cause serious economic losses. Three Brucella species, B. melitensis, B. abortus and B.suis, are virulent for humans, causing a chronic, debilitating disease with severe and sometimes fatal outcomes. Moreover, as Brucella spp. can be easily aerosolized, this pathogen could be used to develop biological weapons.
Brucella is an intracellular bacterium. Research on Brucella and its interaction between host reveals that Brucella activate reduced immune response. The virulence of Brucella depends upon its ability to survive and replicate in host cells. They replicate in endoplasmic reticulum (ER) and escape from the immune response. Effective immune response is mainly mediated by cellular immunity. Outer membrane proteins (OMPs) play important roles in stabilizing the structure of the outer membrane, adapting to external and intracellular environments, and Brucella virulence. OMPs are located on surface of bacteria and can be easily recognized by host immune system, therefore, some of them represent important immunoantigen.
Although many attepts have been tried for development of different vaccine types, live attenuated vaccine is the most efficient vaccine for Brucellosis for the present. In the present study, to analyze the roles of major OMPs in Brucella virulence and immunological protection, outer membrane proteome was separated by 2D eletrophoresis and the proteins were identified by MS. A total of 67 proteins were identified to be possibly membrane proteins. Nine of these protein spots are products of Omp25 and seven were those of Omp31. Omp25 is a member of the OmpA protein family, which have immunogenicity. Outer membranes proteins play important roles in stabilizing the structure of the outer membrane and are relative to the virulence. To predict its function, the sequence of omp25 was analyzed by bioinformatics methods. In the genome of Brucella, there are four omp25 genes(BMEI1249(omp25)、 BMEI1007(omp25b)、BMEI1829(omp25c) and BMEI1830(omp25d). Among the four genes, only omp25 (BMEI1249) have been shown to be related with brucella virulence .
To further investigate the function of omp25, the mutants of the four omp25 genes were constructed by homologous recombination, and then their virulence phenotypes were tested. Firstly, the kanamycin gene of pBBR1MCS-2 was PCR amplified and cloned at the multicloning site of pUC19 to generate a new suicide plasmid pUC19K, which was then used to construct the mutant strains. By using resistance replacement method, mutants of the four omp25 genes were successfully constructed, named BMΔ1007, BMΔ1249, BMΔ1829 and BMΔ1830.
In vitro growth curve assays showed that the four mutants and BM have similar growth curves. However, the mutants showed higher growth rate than the wild type strain at logarithmic phase. At stationary phase, BMΔ1007 showed the highest cell density. This implyied that omp25 negatively regulate growth of Brucella under the assaying conditions. When grow into stationary phase, BMΔ1249 autoaggregated to form clamps in culture, which were not observed in the other three mutant strains. Survival under stress conditions showed that the four mutants showed different sensitivity to the stress simulating intracellular environments, including high salt, high osmosis, low pH, heat shock and oxidative stress. Sensitivity of these mutants to polymyxin B and sodium deoxycholate were also increased. These data indicated that omp25 plays important roles in Brucella adaptation to hostile environments and maintaining integrity of membrane structure. Intracellular survival showed that the omp25 mutants could invade macrophage, but were rapidly cleared by host cell, implying that omp25 is important for Brucella intracellular replication and chonic infection. Balb/c mice was infected with M5 and omp25 mutants, and then challenged with BM and 16M to test their protection capability. When challenged with M5, no great differences were observed in protection efficiency between three mutants and M5, but the BMΔ1829 showed better protection. Then, BMΔ1249, BMΔ1829 and M5 was used to immunize mice and challenged with 16M. The mutant showed reduced protection than M5. The mice immunized with omp25 mutants showed lower antibody titer and IFN-v those infected with M5, implying that omp25 plays important roles in protection efficency of M5. At last, the four omp25 proteins were expressed in E. coli. Western blot result showed that all the proteins could react with brucellosis sera. Only omp25 protein could differentiate sera of M5 from that of the mutant strain.
The results above showed that the omp25 play important roles in brucella adaptation to hostile environments, intracellular survival and maintaining integrity of outer membrane. These data also showed that omp25 proteins play important roles in protection of live attenuated strain. These findings expand our knowledge of the function of Omp25 in Brucella survival both in vivo and in vitro, virulence phenotypes and immuno-protection, providing important clues for the understanding of the molecular mechanism of intracellular survival and protection mechanism of Brucella.
引文
[1]Boschiroli M L, Foulongne V, O'Callaghan D. Brucellosis: a worldwide zoonosis [J]. Curr Opin Microbiol, 2001, 4: 58-64.
[2]Young E J. An overview of human brucellosis [J]. Clin Infect Dis, 1995, 21: 283-289.
[3]Schurig G G, Sriranganathan N, Corbel M J. Brucellosis vaccines: past, present and future [J]. Vet Microbiol, 2002, 90: 479-496.
[4]Bowden R A, Estein S M, Zygmunt M S, et al. Identification of protective outer membrane antigens of Brucella ovis by passive immunization of mice with monoclonal antibodies [J]. Microbes Infect, 2000, 2(5): 481-488.
[5]Edgardo Moreno, Cloeckaert A, Ignacio Moriyon. Brucella evolutin and taxonomy [J]. Vet Micro, 2002, 90: 209-227.
[6]Franco M P, Mulder, M, Gilman, R H and Smits H L. Human brucellosis [J]. Lancet Infect Dis, 2007, 7: 775-786.
[7]尚德秋.布氏菌病研究进展[J].中国地方病防治杂志, 2004, 19: 204-212.
[8]Jarvis B W. Rough lipopoly saccharide from Brucella abortus and Esc herichia coli differentially activates the same mitoge activated protei nkinase signaling pathways for tumornecrosis factor alphain RAW264.7 macrophage-likecell [J]. Infe Immun, 2002, 70(12): 7165-7168.
[9]Corbel M J. Brucellosis: an overview [J]. Emerg Infect Dis, 1997, 3: 213-221.
[10]Verger J M, Grayon M, Tibor A, Wansard V, Letesson J J, Cloeckaert A. Differentiation of Brucella melitensis, B.ovis and B.suis biovar 2 strains by use of membrane protein-or cytoplasmic protein-specific gene probes [J]. Res Microbiol, 1998, 149: 509-517.
[11]Foster G, Jahans K L, Reid R J, et al. Isolation of Brucella species from cetaceans, seals and an otter [J]. Vet Rec, 1996, 138: 583-6.
[12]Ross H M, Foster G, Reid R J, et al. Brucella species infection in sea-mammals [J]. Vet Rec, 1994, 134: 359.
[13]Ross H M, Jahans K L, Mac Millan A P, et al. Brucella species infection in North Sea seal and cetacean populations [J]. Vet Rec, 1996, 138: 647-8.
[14]齐景文.布鲁氏菌病概述[J].中国兽医杂志, 2004, 40: 50-52.
[15]Brinley Morgan W J and Corbel M J. Brucella Infections in Man and Animals: Contagious Equine Metritis. 547-570, 1990 [C]. In M. T. Parker and L. H. Collier(ed.), Topley and Wilson's principles of bacteriology, virology and immunology, 8th ed. Edward Arnold, London, England.
[16]Ko J, Splitter G A. Molecular host-pathogen interaction in brucellosis: currentunderstanding and future approaches to vaccine development for mice and humans [J]. Clin Microbiol Rev, 2003, 16: 65-78.
[17]Chevalier P E, Bonnefoy G and Touboul P. Brucella pancarditis with fatal outcome [J]. Presse Med, 1996, 25: 628-630.
[18]Mc Quiston J R, Vemulapalli T J, Inzana G G, et al. Genetic characterization of a Tn 5-disrupted glycosyltransferase gene homolog in Brucella abortus and its effect on lipopolysaccharide composition and virulence [J]. Infect. Immun, 1999, 67: 3830-3835.
[19]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.
[20]Riley L K and Robertson. D C. Ingestion and intracellular survival of Brucella abortus in human and bovine polymorphonuclear leukocytes [J]. Infect Immun, 1984, 46: 224-230.
[21]Kohler S, Layssac M, Naroeni A, et al. Secretion of listeriolysin by Brucella suis inhibits its intramacrophagic replication [J]. Infect Immun, 2001, 69: 2753-2756.
[22]Anderson T D and Cheville N F. Ultrastructural morphometric analysis of Brucella abortus-infected trophoblasts in experimental placentitis. Bacterial replication occurs in rough endoplasmic reticulum. Am [J]. Pathol, 1986, 124: 226-237.
[23]Detilleux P G, Deyoe B L and Cheville N F. Penetration and intracellular growth of Brucella abortus in nonphagocytic cells in vitro [J]. Infect Immun, 1990,
[24]Pizarro-Cerda J S, Meresse R G, Parton G, et al. Brucella abortus transits through the autophagic pathway and replicates in the endoplasmic reticulum of nonprofessional phagocytes [J]. Infect Immun, 1998, 112: 212-216.
[25]Dornand J, Gross A, Lafont V, Liautard J, Oliaro J and Liautard J P. The innate immune response against Brucella in humans [J]. Vet Microbiol, 2002, 90: 383-394.
[26]Gross A, Terraza A, Ouahrani-Bettache S, Liautard J P and Dornand,J. In vitro Brucella suis infection prevents the programmed cell death of human monocytic cells [J]. Infect Immun , 2000, 68: 342-351.
[27]Arenas G N, Staskevich A S, Aballay A and Mayorga L S. Intracellular trafficking of Brucella abortus in J774 macrophages [J]. Infect Immun, 2000, 68: 4255-4263.
[28]Pizarro-Cerda J, Moreno E and Gorvel J P. Invasion and intracellular trafficking of Brucella abortus in nonphagocytic cells [J]. Microbes Infect, 2000, 2: 829-835.
[29]Guzman-Verri C, Manterola L, Sola-Landa A, Parra A, Cloeckaert A, Garin J,Gorvel J P, Moriyon I, Moreno E and Lopez-Goni I. The two-component system BvrR/BvrS essential for Brucella abortus virulence regulates theexpression of outer membrane proteins with counterparts in members of the Rhizobiaceae [J]. Proc Natl Acad Sci USA, 2002, 99: 12375-12380.
[30]Celli J, Chastellier C, Franchini D M, Pizarro-Cerda J, Moreno E and Gorvel J P. Brucella evades macrophage killing via VirB-dependent sustained interactions with the endoplasmic reticulum [J]. Exp Med, 2003, 198: 545-556.
[31]Foulongne V, Bourg G, Cazevieille C, Michaux-Charachon S, O'Callaghan D. Identification of Brucella suis genes affecting intracellular survival in an in vitro human macrophage infection model by signature-tagged transposon mutagenesis [J]. Infect Immun, 2000, 68: 1297-1303.
[32]Qiu M, Wang J, Wang H, Chen Z, Dai E, Guo Z, Wang X, Pang X, Fan B, Wen J, et al. Use of the COOH Portion of the Nucleocapsid Protein in an Antigen-Capturing Enzyme-Linked Immunosorbent Assay for Specific and Sensitive Detection of Severe Acute Respiratory Syndrome Coronavirus [J]. Clin Diagn Lab Immunol, 2005, 12: 474-476.
[33]Lin J, Ficht T A. Protein synthesis in Brucella abortus induced during macrophage infection [J]. Infect Immun, 1995, 63: 1409-1414.
[34]Rafie-Kolpin M, Essenberg R C, Wyckoff J H. Identification and comparison of macrophage-induced proteins and proteins induced under various stress conditions in Brucella abortus [J]. Infect Immun, 1996, 64: 5274-5283.
[35]Teixeira-Gomes A P, Cloeckaert A, Zygmunt M S. Characterization of heat, oxidative, and acid stress responses in Brucella melitensis [J]. Infect Immun, 2000, 68: 2954-2961.
[36]Eskra L, Canavessi A, Carey M, Splitter G. Brucella abortus genes identified following constitutive growth and macrophage infection [J]. Infect Immun, 2001, 69: 7736-7742.
[37]Lillo AM, et al. Functional expression and characterization of eryA, the erythriol kinase of Brucella abortus, and enzymetic Synthesis of L-erythritol-4-phosphate [J]. Vet Bull, 2003, 73(9): 1249-
[38]Robertson G T, et al. The Brucella abortus Lon functions as a qeneralized streis respinse protease and is required for wildtype virulence in BALB/C mice [J]. Mol Micro Immun, 2000, 35(3): 577-588.
[39]Roop R M, et al. Rexamination of the role of the Brucella melitensis Htr A stress response protease in virurence in pregnant goats [J]. Vet Microb, 2001, 82(1): 91-95.
[40]Baldwin C L and Winter A J. Macrophages and Brucella Immunol[M]. Ser, 1994, 60: 363-380.
[41]Rasool O, Freer E, Moreno E, Jarstrand C. Effect of Brucella abortus lipopolysaccharide on oxidative metabolism and lysozyme release by human neutrophils [J]. Infect Immun, 1992, 60: 1699-1702.
[42]Allen C A, Adams L G, Ficht T A. Transposon-derived Brucella abortus roughmutants are attenuated and exhibit reduced intracellular survival [J]. Infect Immun, 1998, 66: 1008-1016.
[43]Pei J and Ficht T A. Brucella abortus rough mutants are cytopathic for macrophages in culture[J]. Infection and immunity, 2004, 72: 440-450.
[44]Sola-Landa A, Pizarro-Cerda J, Grillo M J, Moreno E, Moriyon I, Blasco J M, Gorvel J P, Lopez-Goni I. 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 [J]. Mol Microbiol, 1998, 29: 125-138.
[45]Lopez-Goni I, Guzman-Verri C, Manterola L, Sola-Landa A, Moriyon I, Moreno E. Regulation of Brucella virulence by the two-component system BvrR/BvrS [J]. Vet Microbiol, 2002, 90: 329-339.
[46]Ujmam-Verri C, Manterola C, Sola-landa A, et al. Two-component system BvrR/Bvrs esseatial for Brucella abortus virulence requlates the espression of outer membrane proteins with counter derts in members of rhizobioceae [J]. Vet Bull, 2003, 73(4): 520.
[47]Boschiroli M L, Ouahrani-Bettache S, Foulongne V, Michaux-Charachon S, Bourg G, Allardet-Servent A, Cazevieille C, Liautard J P, O'Callaghan D. The Brucella suis virB operon is induced intracellularly in macrophages [J]. Proc Natl Acad Sci USA, 2002, 99: 1544-1549.
[48]Boschiroli M L, Ouahrani-Bettache S, Foulongne V, Michaux-Charachon S, Bourg G, Allardet-Servent A, Cazevieille C, Lavigne J P, Liautard J P, Ramuz M, O'Callaghan D. Type IV secretion and Brucella virulence [J]. Vet Microbiol, 2002, 90: 341-348.
[49]O'Callaghan D, Cazevieille C, Allardet-Servent A, Boschiroli M L, Bourg G, Foulongne V, Frutos P, Kulakov Y, Ramuz M. A homologue of the Agrobacterium tumefaciens VirB and Bordetella pertussis Ptl type IV secretion systems is essential for intracellular survival of Brucella suis [J]. Mol Microbiol, 1999, 33: 1210-1220.
[50]Sieira R, Comerci D J, Sanchez D O, et al. A homoloque of an operon requred for DNA tranger in agrobacterium is required in Brucelluar multipeation [J]. J Bact, 2000, 182(17): 4849-4855.
[51]Rouot B, Alvarez-Martinez M T, Marius C, Menanteau P, Guilloteau L, Boigegrain R A, Zumbihl R, O'Callaghan D, Domke N, Baron C. Production of the type IV secretion system differs among Brucella species as revealed with VirB5- and VirB8-specific antisera [J]. Infect Immun, 2003, 71: 1075-1082.
[52]Comerci D J, Martinez-Lorenzo M J, Sieira R, Gorvel J P, Ugalde R A. Essential role of the VirB machinery in the maturation of the Brucella abortus-containing vacuole [J]. Cell Microbiol, 2001, 3: 159-168.
[53]Delory M, Hallez R, Letesson J J, De Bolle X. An RpoH-like heat shock sigma factor is involved in stress response and virulence in Brucella melitensis 16M [J].J Bacteriol, 2006, 188: 7707-7710.
[54]Yu D H, Hu X D, Cai H. A combined DNA vaccine encoding BCSP31, SOD, and L7/L12 confers high protection against Brucella abortus 2308 by inducing specific CTL responses [J]. DNA Cell Biol, 2007, 26: 435-443.
[55]Munoz-Montesino C, Andrews E, Rivers R, Gonzalez-Smith A, Moraga-Cid G, Folch H, Cespedes S, Onate A A. Intraspleen delivery of a DNA vaccine coding for superoxide dismutase (SOD) of Brucella abortus induces SOD-specific CD4+ and CD8+ T cells [J]. Infect Immun, 2004, 72: 2081-2087.
[56]Caron E, Cellier M, Liautard J P, Kohler S. 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 [J]. FEMS Microbiol Lett, 1994, 120: 335-340.
[57]Cellier M F, Teyssier J, Nicolas M, Liautard J P, Marti J, Sri Widada J. Cloning and characterization of the Brucella ovis heat shock protein DnaK functionally expressed in Escherichia coli [J]. J Bacteriol, 1992, 174: 8036-8042.
[58]Phillips R W, Roop R M, 2nd. Brucella abortus HtrA functions as an authentic stress response protease but is not required for wild-type virulence in BALB/c mice [J]. Infect Immun, 2001, 69: 5911-5913.
[59]Elzer P H, Phillips R W, Kovach M E, Peterson K M, Roop R M, 2nd. Characterization and genetic complementation of a Brucella abortus high-temperature-requirement A (htrA) deletion mutant [J]. Infect Immun, 1994, 62: 4135-4139.
[60]Phillips R W, Elzer P H, Roop R M, II. A Brucella melitensis high temperature requirement A (htrA) deletion mutant demonstrates a stress response defective phenotype in vitro and transient attenuation in the BALB/c mouse model [J]. Microb Pathog, 1995, 19: 227-284.
[61]Phillips R W, Elzer P H, Robertson G T, Hagius S D, Walker J V, Fatemi M B, Enright F M, Roop R M, 2nd. A Brucella melitensis high- temperature- requirement A (htrA) deletion mutant is attenuated in goats and protects against abortion [J]. Res Vet Sci, 1997, 63: 165-167.
[62]Taminiau B, Daykin M, Swift S, Boschiroli M L, Tibor A, Lestrate P, De Bolle X, O'Callaghan D, Williams P, Letesson J J. Identification of a quorum-sensing signal molecule in the facultative intracellular pathogen Brucella melitensis [J]. Infect Immun, 2002, 70: 3004-3011.
[63]Davies D G, Parsek M R, Pearson J P, et al. The involvement of cell-to-cell signals in the development of a bacterial biofilm [J]. Science, 1998: 295-298.
[64]Rambow-Larsen A A, 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]. J Bacteriol, 2008, 190: 3274-3282.
[65]Edmonds M P, Cloeckaert A, Booth N J, et al. Attenuation of a Brucella abortus mutant lacking a major 25 KDa outer membrane protein in cattle [J]. Am J Vet Res, 2001, 62: 1461-1466.
[66]Sha Z, et al. Brucella abortus is a periplasmic protein lacking a standard signal Sequence [J]. J Bact, 1994, 176(23): 7375.
[67]Ewalt D R, Bricker B J. Validation of the abbreviated Brucella AMOS PCR as a rapid screening method for differentiation of Brucella abortus Field strain isolates and Vaccine Strains S19 and RB51 [J]. J Clin Microb, 2000, 38(8): 3085-3086.
[68]Cloeckaert A, Kerkhofs P, Limet J N. Antibody response to Brucella outer membrane proteins in bovine brucellosis: Immunoblot analysis and competitive enzyme-linked immunosorbent assay using monoclonal antibodies [J]. J Clin Microbiol, 1992, 30: 3168-3174.
[69]Cloeckaert A, Vizcaíno N, paquet J Y, et al. Major outer membrane proteins of Brucella spp. past, present and future [J]. Veterinary Microbiology, 2002, 90: 229-247.
[70]Cloeckaert A, Zygmunt M S, de werglfosse P, et al. Demonst ration of peptidoglycan-associ-ated Brucella outer membrane proteins by use of monoclonac antibodies [J]. J Gen Microbiol, 1992, 138: 1543-1550.
[71]Tibor A, Wansard V, Bielartz V, Delrue R M, Danese I, Michel P, Walravens K, Godfroid J, Letesson J J. Effect of omp10 or omp19 deletion on Brucella abortus outer membrane properties and virulence in mice [J]. Infect Immun, 2002, 70: 5540-5546.
[72]Cloeckaert A, et al. Classifcation of Brucella spp. isolated from marine mammals by DNA polymorphism at the OMP2 locus [J]. Vet Bull, 2002, 72(1): 20-21.
[73]Cloeckaert A, Verger J M, Grayon M, et al. Rest riction site polymorphism of the genes encoding the major 25 and 36kDa outer membrane proteins of Brucella [J]. Microbiology, 1995, 141: 2111-2121.
[74]Cloeckaert A, Verger J M, Grayon M, et al. Classification of Brucella spp. isolated from marine mammals by DNA polymorphism at the Omp2 locus [J]. Microbes Infect, 2001, (3): 729-738.
[75]Ficht T A, Husseinen H S, Derr J, et al. Species-specific sequences at the omp2 locus of Brucella type strains[J]. Int J Syst Bacteriol, 1996, 46: 329-331.
[76]Mobasheri H, Ficht T A, Marquis H, Lea E J, Lakey J H. Brucella Omp2a and Omp2b porins: single channel measurements and topology prediction [J].FEMS Microbiol Lett, 1997, 155: 23-30.
[77]Paquet J Y, Daz M A, Genevrois S, et al. Molecular, antigentic, and functional analyses of Omp2b porin size variants of Brucella spp [J]. J Bacteriol, 2001, 183: 4839-4847.
[78]Oliveira S C, Splitter G A. Immunization of mice with recombinant L7/L12ribosomal protein confers protection against Brucella abortus infection [J]. Vaccine, 1996, 14(10): 959-962.
[79]Jubier-Maurin V, Boigegrain R A, Cloeckaert A, et al. Maior outer membrane protein Omp25 of Brucella suis is involved in inhibition of tumor necrosis factor alpha production during infection of human macrophages [J]. Infect Immun, 2001, 69(8): 4823-4830.
[80]Matthew D, Edmonds, Cloeckaert A, Philip H E. Brucella species lacking the major outer membrane protein Omp25 are attenuated in mice and protect against Brucella melitensis and Brucella ovis [J]. Vet Microbiol, 2002, 88: 205-221.
[81]Boigegrain R A, Salhi I, Alvarez-Martinez M T, Machold J, Fedon Y, Arpagaus M, Weise C, Rittig M, Rouot B. Release of periplasmic proteins of Brucella suis upon acidic shock involves the outer membrane protein Omp25 [J]. Infect Immun, 2004, 72: 5693-5703.
[82]Viza no N, Cloeckaert A, Zygmunt M S, et al. Characterisation of a Brucella species 25-kb DNA fragment deleted from Brucella abortus reveals a large gene cluster related to the synthesis of a polysaccharide [J]. Infect Immun, 2001, 69: 6738-6748.
[83]Cassataro J, Pasquevich K, Bruno L, Wallach J C, Fossati C A, Baldi P C. Antibody reactivity to Omp31 from Brucella melitensis in human and animal infections by smooth and rough Brucellae [J]. Clin Diagn Lab Immunol, 2004, 11: 111-114.
[84]Salhi I, Boigegrain R A, Machold J, Weise C, Cloeckaert A, Rouot B. Characterization of new members of the group 3 outer membrane protein family of Brucella spp [J]. Infect Immun, 2003, 71: 4326-4332.
[85]Dubray G, Charriaut C. Evidence of three major polypeptide species and two major polysaccharide species in the Brucella outer membrane [J]. Ann Rech Vet, 1983, 14: 311-318.
[86]Dubray G. Protectives antigens in brucellosis [J]. Ann Inst Pasteur/Microbiol (Paris), 1987, 138: 84-87.
[87]Cloeckaert A, Jacques I, Limet J N, et al. Immunogenic properties of Brucella melitensis cell-wall fractions in BALB/C mice [J]. J Med Microbiol, 1995, 42: 200-208.
[88]Guiuoteau L A, Lavoucan K, Vizano N, et al. Immunogenicity of recombinant Escherichia coli expressing the omp31 gene of Brucella melitensis in BALB/C mice [J]. Vaccine, 1999, 17: 353-361.
[89]Jimnezde Bags M P, Elzer P H, Blasco J M, et al. Protective immunity to Brucella ovis in BALB/C mice following from primary infection or immuniation with subcellular vaccines [J]. Infect Immun, 1994, 62: 632-638.
[90]Kittelberge R, Diack D S, Vizca no N, et al. Characterisation of an immunodominant antigen in Brucella ovis and evaluation of it s use in anenzyme2linked immunosorbent assay [J]. Vet Microbiol, 1998, 59: 213-227.
[91]Betsy J, Bricker S, Halling M. Enhancement of the Brucella AMOS PCR Assay for Differentiation of Brucella abortus Vaccine Strain S19 and RB51 [J]. Clin Microbiol, 1995, 33: 1640-1642.
[92]Jiang X, Leonard B, Benson R, et al. Macrophage control of Brucella abortus: role of reactive oxygen intermediates and nitric oxide [J]. Cell Immunol, 1993, 151: 309-319.
[93]Gay B, Sanchez-Teff S and Caravano R. Ultrastructural localization of NADPH-oxidase activity in murine peritoneal macrophages during phagocytosis of Brucella. Correlation with the production of superoxide anions [J]. Virchows Arch B, 1984,45: 147-155.
[94]Baldwin C L, Jiang X and Fernandes D M. Macrophage control of Brucella abortus: influence of cytokines and iron [J]. Trends Microbiol, 1993, 1: 99-104.
[95]Jiang X and Baldwin C L. Iron augments macrophage-mediated killing of Brucella abortus alone and in conjunction with interferon-gamma [J]. Cell Immunol, 1993, 148: 397-407.
[96]Young E J, Borchert F L and Musher D M. Phagocytosis and killing of Brucella by human polymorphonuclear leukocytes [J]. J Infect Dis, 1985, 151: 682-690.
[97]Nathan C and Shiloh M U. Reactive oxygen and nitrogen intermediates in the relationship between mammalian hosts and microbial pathogens [J]. Proc Natl Acad Sci USA, 2000, 97: 8841-8848.
[98]尚德秋.布鲁氏菌感染与免疫研究近况[J].中国地方病杂志, 2003, 18(3): 154-157.
[99]金山,乌尼,哈斯木伦.布鲁氏菌生物学特性研究进展[J].内蒙古畜牧科学, 2000, (08): 32~34.
[100]Corbeil L B, K Blau T J, Inzana K H, et al. Killing of Brucella abortus by bovine serum [J]. Infect Immun, 1988, 56: 3251-3261.
[101]Hoffmann E M and Houle J J. Failure of Brucella abortus lipopolysaccharide (LPS) to activate the alternative pathway of complement [J]. Vet Immunol Immunopathol, 1983, 5: 65-76.
[102]Fernandez-Prada C M, M Nikolich R, Vemulapalli N, et al. Deletion of wboA enhances activation of the lectin pathway of complement in Brucella abortus and Brucella melitensis [J]. Infect Immun, 2001, 69: 4407-4416.
[103]Bellamy R. The natural resistance-associated macrophage protein and susceptibility to intracellular pathogens [J]. Microbes Infect, 1999, 1: 23-27.
[104]Ho M and Cheers C. Resistance and susceptibility of mice to bacterial infection. IV. Genetic and cellular basis of resistance to chronic infection with Brucella abortus [J]. J Infect Dis, 1982, 146: 381-387.
[105]Barthel R, Feng J, Piedrahita J A et al. Stable transfection of the bovine NRAMP1 gene into murine RAW264.7 cells: effect on Brucella abortus survival[J]. Infect.Immun, 2001, 69: 3110-3119.
[106]Araya L N, Elzer P H, Rowe G E, et al.Temporal development of protective cell-mediated and humoral immunity in BALB/c mice infected with Brucella abortus [J]. J Immunol, 1989, 143: 3330-3337.
[107]Oliveira S C and Splitter G A. CD8+ type 1 CD44hi CD45 RBlo T lymphocytes control intracellular Brucella abortus infection as demonstrated in major histocompatibility complex class I and class II-deficient mice [J]. Eur J Immunol, 1995, 25: 2551-2557.
[108]Winter A J, Duncan J R, Santisteban C G, et al. Capacity of passively administered antibody to prevent establishment of Brucella abortus infection in mice [J]. Infect Immun, 1989, 57: 3438-3444.
[109]Montaraz J A and Winter A J. Comparison of living and nonliving vaccines for Brucella abortus in BALB/c mice [J]. Infect Immun, 1986, 53: 245-251.
[110]Michaux-Charachon S, Bourg G, Jumas-Bilak E, et al. Genome structure and phylogeny in the genus Brucella [J]. J Bacteriol, 1997, 179: 3244-3249.
[111]Montaraz J A, Winter A J, Hunter D M, et al. Protection against Brucella abortus in mice with O-polysaccharide-specific monoclonal antibodies [J]. Infect Immun, 1986, 51: 961-963.
[112]Schurig G G, Roop R M, Bagchi J T, et al. Biological properties of RB51 a stable rough strain of Brucella abortus [J]. Vet Microbiol, 1991, 28: 171-188.
[113] Ko J, Gendron-Fitzpatrick A and Splitter G A. Susceptibility of interferon regulatory factor (IRF-1) and interferon consensus sequence binding protein (ICSBP) deficient mice to brucellosis [J]. J Immunol, 2002, 168: 2433-2440.
[114]Zhan Y, Kelso A and Cheers C. Cytokine production in the murine response to Brucella infection or immunization with antigenic extracts [J]. Immunology, 1993, 80: 458-464.
[115]Zhan Y and Cheers C. Endogenous gamma interferon mediates resistance to Brucella abortus infection [J]. Infect Immun, 1993, 61: 4899-4901.
[116]Zhan Y, Z Liu and Cheers C. Tumor necrosis factor alpha and interleukin-12 contribute to resistance to the intracellular bacterium Brucella abortus by different mechanisms [J]. Infect Immun, 1996, 64: 2782-2786.
[117]Mingle C K, Manthei C A, Jasmin A M. The stability of reduced virulence exhibited by Brucella abortus [J]. J Am Vet MED Assoc, 1941, 99: 203.
[118]Philip H Elzer, Fred M Enright, L Colby, et al. Protection against infection and abortion induced by virulent Challenge exposure after oral vaccination of Cattle with Brucell abortus strain RB51 [J]. AJNR, 1998, 59(12): 1575-1578.
[119]Henningsen R G B, Straub K M, et al. Application of zwitterionic detergents to the solubilization of integral membrane proteins for two-dimensional gel electrophoresis and mass spectrometry [J]. Proteomics, 2002, 2: 1479-1488.
[120]Gorg A, Obermaier C, Boguth G, Harder A, Scheibe B, Wildgruber R, Weiss W.The current state of two-dimensional electrophoresis with immobilized pH gradients[J]. Electrophoresis, 2000, 21: 1037-1053.
[121]Delpino M V, Cassataro J, Fossati C A, Goldbaum F A, Baldi P C. Brucella outer membrane protein Omp31 is a haemin-binding protein[J]. Microbes Infect, 2006, 8: 1203-1208.
[122]Perucho M, Hanahan D, Lipsich L, et al. Isolation of the chicken thymidine kinase gene by plasmid rescue[J]. Nature, 1980, 285: 207-210.
[123]Lin J, Huang S, Zhang Q. Outer membrane proteins: key players for bacterial adaptation in host niches[J]. Microbes Infect, 2002, 4: 325-331.
[124]Weiser JN, Gotschlich E C. Outer membrane protein A (OmpA) contributes to serum resistance and pathogenicity of Escherichia coli K-1[J]. Infect Immun, 1991, 59: 2252-2258.
[125]Edmonds M D, Cloeckaert A, Elzer PH, Brucella species lacking the major outer membrane protein Omp25 are attenuated in mice and protect against Brucella melitensis and Brucella ovis[J], Vet Microbiol, 2002, 88: 205-221.
[126]Caro-Hernandez P, Fernandez-Laqo L, de Miquel M L, et al. Role of the Omp25/Omp31 family in outer membrance properties and virulence of Brucella. Ovis[J]. Infect Immun, 2007, 75(8): 4050-4061.
[127]Porte F, Liautard J P, Kohler S. Early acidification of phagosomes containing Brucella suis is essential for intracellular survival in murine macrophages[J]. Infect Immun, 1999, 67: 4041-4047. .
[2]Young E J. An overview of human brucellosis [J]. Clin Infect Dis, 1995, 21: 283-289.
[3]Schurig G G, Sriranganathan N, Corbel M J. Brucellosis vaccines: past, present and future [J]. Vet Microbiol, 2002, 90: 479-496.
[4]Bowden R A, Estein S M, Zygmunt M S, et al. Identification of protective outer membrane antigens of Brucella ovis by passive immunization of mice with monoclonal antibodies [J]. Microbes Infect, 2000, 2(5): 481-488.
[5]Edgardo Moreno, Cloeckaert A, Ignacio Moriyon. Brucella evolutin and taxonomy [J]. Vet Micro, 2002, 90: 209-227.
[6]Franco M P, Mulder, M, Gilman, R H and Smits H L. Human brucellosis [J]. Lancet Infect Dis, 2007, 7: 775-786.
[7]尚德秋.布氏菌病研究进展[J].中国地方病防治杂志, 2004, 19: 204-212.
[8]Jarvis B W. Rough lipopoly saccharide from Brucella abortus and Esc herichia coli differentially activates the same mitoge activated protei nkinase signaling pathways for tumornecrosis factor alphain RAW264.7 macrophage-likecell [J]. Infe Immun, 2002, 70(12): 7165-7168.
[9]Corbel M J. Brucellosis: an overview [J]. Emerg Infect Dis, 1997, 3: 213-221.
[10]Verger J M, Grayon M, Tibor A, Wansard V, Letesson J J, Cloeckaert A. Differentiation of Brucella melitensis, B.ovis and B.suis biovar 2 strains by use of membrane protein-or cytoplasmic protein-specific gene probes [J]. Res Microbiol, 1998, 149: 509-517.
[11]Foster G, Jahans K L, Reid R J, et al. Isolation of Brucella species from cetaceans, seals and an otter [J]. Vet Rec, 1996, 138: 583-6.
[12]Ross H M, Foster G, Reid R J, et al. Brucella species infection in sea-mammals [J]. Vet Rec, 1994, 134: 359.
[13]Ross H M, Jahans K L, Mac Millan A P, et al. Brucella species infection in North Sea seal and cetacean populations [J]. Vet Rec, 1996, 138: 647-8.
[14]齐景文.布鲁氏菌病概述[J].中国兽医杂志, 2004, 40: 50-52.
[15]Brinley Morgan W J and Corbel M J. Brucella Infections in Man and Animals: Contagious Equine Metritis. 547-570, 1990 [C]. In M. T. Parker and L. H. Collier(ed.), Topley and Wilson's principles of bacteriology, virology and immunology, 8th ed. Edward Arnold, London, England.
[16]Ko J, Splitter G A. Molecular host-pathogen interaction in brucellosis: currentunderstanding and future approaches to vaccine development for mice and humans [J]. Clin Microbiol Rev, 2003, 16: 65-78.
[17]Chevalier P E, Bonnefoy G and Touboul P. Brucella pancarditis with fatal outcome [J]. Presse Med, 1996, 25: 628-630.
[18]Mc Quiston J R, Vemulapalli T J, Inzana G G, et al. Genetic characterization of a Tn 5-disrupted glycosyltransferase gene homolog in Brucella abortus and its effect on lipopolysaccharide composition and virulence [J]. Infect. Immun, 1999, 67: 3830-3835.
[19]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.
[20]Riley L K and Robertson. D C. Ingestion and intracellular survival of Brucella abortus in human and bovine polymorphonuclear leukocytes [J]. Infect Immun, 1984, 46: 224-230.
[21]Kohler S, Layssac M, Naroeni A, et al. Secretion of listeriolysin by Brucella suis inhibits its intramacrophagic replication [J]. Infect Immun, 2001, 69: 2753-2756.
[22]Anderson T D and Cheville N F. Ultrastructural morphometric analysis of Brucella abortus-infected trophoblasts in experimental placentitis. Bacterial replication occurs in rough endoplasmic reticulum. Am [J]. Pathol, 1986, 124: 226-237.
[23]Detilleux P G, Deyoe B L and Cheville N F. Penetration and intracellular growth of Brucella abortus in nonphagocytic cells in vitro [J]. Infect Immun, 1990,
[24]Pizarro-Cerda J S, Meresse R G, Parton G, et al. Brucella abortus transits through the autophagic pathway and replicates in the endoplasmic reticulum of nonprofessional phagocytes [J]. Infect Immun, 1998, 112: 212-216.
[25]Dornand J, Gross A, Lafont V, Liautard J, Oliaro J and Liautard J P. The innate immune response against Brucella in humans [J]. Vet Microbiol, 2002, 90: 383-394.
[26]Gross A, Terraza A, Ouahrani-Bettache S, Liautard J P and Dornand,J. In vitro Brucella suis infection prevents the programmed cell death of human monocytic cells [J]. Infect Immun , 2000, 68: 342-351.
[27]Arenas G N, Staskevich A S, Aballay A and Mayorga L S. Intracellular trafficking of Brucella abortus in J774 macrophages [J]. Infect Immun, 2000, 68: 4255-4263.
[28]Pizarro-Cerda J, Moreno E and Gorvel J P. Invasion and intracellular trafficking of Brucella abortus in nonphagocytic cells [J]. Microbes Infect, 2000, 2: 829-835.
[29]Guzman-Verri C, Manterola L, Sola-Landa A, Parra A, Cloeckaert A, Garin J,Gorvel J P, Moriyon I, Moreno E and Lopez-Goni I. The two-component system BvrR/BvrS essential for Brucella abortus virulence regulates theexpression of outer membrane proteins with counterparts in members of the Rhizobiaceae [J]. Proc Natl Acad Sci USA, 2002, 99: 12375-12380.
[30]Celli J, Chastellier C, Franchini D M, Pizarro-Cerda J, Moreno E and Gorvel J P. Brucella evades macrophage killing via VirB-dependent sustained interactions with the endoplasmic reticulum [J]. Exp Med, 2003, 198: 545-556.
[31]Foulongne V, Bourg G, Cazevieille C, Michaux-Charachon S, O'Callaghan D. Identification of Brucella suis genes affecting intracellular survival in an in vitro human macrophage infection model by signature-tagged transposon mutagenesis [J]. Infect Immun, 2000, 68: 1297-1303.
[32]Qiu M, Wang J, Wang H, Chen Z, Dai E, Guo Z, Wang X, Pang X, Fan B, Wen J, et al. Use of the COOH Portion of the Nucleocapsid Protein in an Antigen-Capturing Enzyme-Linked Immunosorbent Assay for Specific and Sensitive Detection of Severe Acute Respiratory Syndrome Coronavirus [J]. Clin Diagn Lab Immunol, 2005, 12: 474-476.
[33]Lin J, Ficht T A. Protein synthesis in Brucella abortus induced during macrophage infection [J]. Infect Immun, 1995, 63: 1409-1414.
[34]Rafie-Kolpin M, Essenberg R C, Wyckoff J H. Identification and comparison of macrophage-induced proteins and proteins induced under various stress conditions in Brucella abortus [J]. Infect Immun, 1996, 64: 5274-5283.
[35]Teixeira-Gomes A P, Cloeckaert A, Zygmunt M S. Characterization of heat, oxidative, and acid stress responses in Brucella melitensis [J]. Infect Immun, 2000, 68: 2954-2961.
[36]Eskra L, Canavessi A, Carey M, Splitter G. Brucella abortus genes identified following constitutive growth and macrophage infection [J]. Infect Immun, 2001, 69: 7736-7742.
[37]Lillo AM, et al. Functional expression and characterization of eryA, the erythriol kinase of Brucella abortus, and enzymetic Synthesis of L-erythritol-4-phosphate [J]. Vet Bull, 2003, 73(9): 1249-
[38]Robertson G T, et al. The Brucella abortus Lon functions as a qeneralized streis respinse protease and is required for wildtype virulence in BALB/C mice [J]. Mol Micro Immun, 2000, 35(3): 577-588.
[39]Roop R M, et al. Rexamination of the role of the Brucella melitensis Htr A stress response protease in virurence in pregnant goats [J]. Vet Microb, 2001, 82(1): 91-95.
[40]Baldwin C L and Winter A J. Macrophages and Brucella Immunol[M]. Ser, 1994, 60: 363-380.
[41]Rasool O, Freer E, Moreno E, Jarstrand C. Effect of Brucella abortus lipopolysaccharide on oxidative metabolism and lysozyme release by human neutrophils [J]. Infect Immun, 1992, 60: 1699-1702.
[42]Allen C A, Adams L G, Ficht T A. Transposon-derived Brucella abortus roughmutants are attenuated and exhibit reduced intracellular survival [J]. Infect Immun, 1998, 66: 1008-1016.
[43]Pei J and Ficht T A. Brucella abortus rough mutants are cytopathic for macrophages in culture[J]. Infection and immunity, 2004, 72: 440-450.
[44]Sola-Landa A, Pizarro-Cerda J, Grillo M J, Moreno E, Moriyon I, Blasco J M, Gorvel J P, Lopez-Goni I. 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 [J]. Mol Microbiol, 1998, 29: 125-138.
[45]Lopez-Goni I, Guzman-Verri C, Manterola L, Sola-Landa A, Moriyon I, Moreno E. Regulation of Brucella virulence by the two-component system BvrR/BvrS [J]. Vet Microbiol, 2002, 90: 329-339.
[46]Ujmam-Verri C, Manterola C, Sola-landa A, et al. Two-component system BvrR/Bvrs esseatial for Brucella abortus virulence requlates the espression of outer membrane proteins with counter derts in members of rhizobioceae [J]. Vet Bull, 2003, 73(4): 520.
[47]Boschiroli M L, Ouahrani-Bettache S, Foulongne V, Michaux-Charachon S, Bourg G, Allardet-Servent A, Cazevieille C, Liautard J P, O'Callaghan D. The Brucella suis virB operon is induced intracellularly in macrophages [J]. Proc Natl Acad Sci USA, 2002, 99: 1544-1549.
[48]Boschiroli M L, Ouahrani-Bettache S, Foulongne V, Michaux-Charachon S, Bourg G, Allardet-Servent A, Cazevieille C, Lavigne J P, Liautard J P, Ramuz M, O'Callaghan D. Type IV secretion and Brucella virulence [J]. Vet Microbiol, 2002, 90: 341-348.
[49]O'Callaghan D, Cazevieille C, Allardet-Servent A, Boschiroli M L, Bourg G, Foulongne V, Frutos P, Kulakov Y, Ramuz M. A homologue of the Agrobacterium tumefaciens VirB and Bordetella pertussis Ptl type IV secretion systems is essential for intracellular survival of Brucella suis [J]. Mol Microbiol, 1999, 33: 1210-1220.
[50]Sieira R, Comerci D J, Sanchez D O, et al. A homoloque of an operon requred for DNA tranger in agrobacterium is required in Brucelluar multipeation [J]. J Bact, 2000, 182(17): 4849-4855.
[51]Rouot B, Alvarez-Martinez M T, Marius C, Menanteau P, Guilloteau L, Boigegrain R A, Zumbihl R, O'Callaghan D, Domke N, Baron C. Production of the type IV secretion system differs among Brucella species as revealed with VirB5- and VirB8-specific antisera [J]. Infect Immun, 2003, 71: 1075-1082.
[52]Comerci D J, Martinez-Lorenzo M J, Sieira R, Gorvel J P, Ugalde R A. Essential role of the VirB machinery in the maturation of the Brucella abortus-containing vacuole [J]. Cell Microbiol, 2001, 3: 159-168.
[53]Delory M, Hallez R, Letesson J J, De Bolle X. An RpoH-like heat shock sigma factor is involved in stress response and virulence in Brucella melitensis 16M [J].J Bacteriol, 2006, 188: 7707-7710.
[54]Yu D H, Hu X D, Cai H. A combined DNA vaccine encoding BCSP31, SOD, and L7/L12 confers high protection against Brucella abortus 2308 by inducing specific CTL responses [J]. DNA Cell Biol, 2007, 26: 435-443.
[55]Munoz-Montesino C, Andrews E, Rivers R, Gonzalez-Smith A, Moraga-Cid G, Folch H, Cespedes S, Onate A A. Intraspleen delivery of a DNA vaccine coding for superoxide dismutase (SOD) of Brucella abortus induces SOD-specific CD4+ and CD8+ T cells [J]. Infect Immun, 2004, 72: 2081-2087.
[56]Caron E, Cellier M, Liautard J P, Kohler S. 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 [J]. FEMS Microbiol Lett, 1994, 120: 335-340.
[57]Cellier M F, Teyssier J, Nicolas M, Liautard J P, Marti J, Sri Widada J. Cloning and characterization of the Brucella ovis heat shock protein DnaK functionally expressed in Escherichia coli [J]. J Bacteriol, 1992, 174: 8036-8042.
[58]Phillips R W, Roop R M, 2nd. Brucella abortus HtrA functions as an authentic stress response protease but is not required for wild-type virulence in BALB/c mice [J]. Infect Immun, 2001, 69: 5911-5913.
[59]Elzer P H, Phillips R W, Kovach M E, Peterson K M, Roop R M, 2nd. Characterization and genetic complementation of a Brucella abortus high-temperature-requirement A (htrA) deletion mutant [J]. Infect Immun, 1994, 62: 4135-4139.
[60]Phillips R W, Elzer P H, Roop R M, II. A Brucella melitensis high temperature requirement A (htrA) deletion mutant demonstrates a stress response defective phenotype in vitro and transient attenuation in the BALB/c mouse model [J]. Microb Pathog, 1995, 19: 227-284.
[61]Phillips R W, Elzer P H, Robertson G T, Hagius S D, Walker J V, Fatemi M B, Enright F M, Roop R M, 2nd. A Brucella melitensis high- temperature- requirement A (htrA) deletion mutant is attenuated in goats and protects against abortion [J]. Res Vet Sci, 1997, 63: 165-167.
[62]Taminiau B, Daykin M, Swift S, Boschiroli M L, Tibor A, Lestrate P, De Bolle X, O'Callaghan D, Williams P, Letesson J J. Identification of a quorum-sensing signal molecule in the facultative intracellular pathogen Brucella melitensis [J]. Infect Immun, 2002, 70: 3004-3011.
[63]Davies D G, Parsek M R, Pearson J P, et al. The involvement of cell-to-cell signals in the development of a bacterial biofilm [J]. Science, 1998: 295-298.
[64]Rambow-Larsen A A, 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]. J Bacteriol, 2008, 190: 3274-3282.
[65]Edmonds M P, Cloeckaert A, Booth N J, et al. Attenuation of a Brucella abortus mutant lacking a major 25 KDa outer membrane protein in cattle [J]. Am J Vet Res, 2001, 62: 1461-1466.
[66]Sha Z, et al. Brucella abortus is a periplasmic protein lacking a standard signal Sequence [J]. J Bact, 1994, 176(23): 7375.
[67]Ewalt D R, Bricker B J. Validation of the abbreviated Brucella AMOS PCR as a rapid screening method for differentiation of Brucella abortus Field strain isolates and Vaccine Strains S19 and RB51 [J]. J Clin Microb, 2000, 38(8): 3085-3086.
[68]Cloeckaert A, Kerkhofs P, Limet J N. Antibody response to Brucella outer membrane proteins in bovine brucellosis: Immunoblot analysis and competitive enzyme-linked immunosorbent assay using monoclonal antibodies [J]. J Clin Microbiol, 1992, 30: 3168-3174.
[69]Cloeckaert A, Vizcaíno N, paquet J Y, et al. Major outer membrane proteins of Brucella spp. past, present and future [J]. Veterinary Microbiology, 2002, 90: 229-247.
[70]Cloeckaert A, Zygmunt M S, de werglfosse P, et al. Demonst ration of peptidoglycan-associ-ated Brucella outer membrane proteins by use of monoclonac antibodies [J]. J Gen Microbiol, 1992, 138: 1543-1550.
[71]Tibor A, Wansard V, Bielartz V, Delrue R M, Danese I, Michel P, Walravens K, Godfroid J, Letesson J J. Effect of omp10 or omp19 deletion on Brucella abortus outer membrane properties and virulence in mice [J]. Infect Immun, 2002, 70: 5540-5546.
[72]Cloeckaert A, et al. Classifcation of Brucella spp. isolated from marine mammals by DNA polymorphism at the OMP2 locus [J]. Vet Bull, 2002, 72(1): 20-21.
[73]Cloeckaert A, Verger J M, Grayon M, et al. Rest riction site polymorphism of the genes encoding the major 25 and 36kDa outer membrane proteins of Brucella [J]. Microbiology, 1995, 141: 2111-2121.
[74]Cloeckaert A, Verger J M, Grayon M, et al. Classification of Brucella spp. isolated from marine mammals by DNA polymorphism at the Omp2 locus [J]. Microbes Infect, 2001, (3): 729-738.
[75]Ficht T A, Husseinen H S, Derr J, et al. Species-specific sequences at the omp2 locus of Brucella type strains[J]. Int J Syst Bacteriol, 1996, 46: 329-331.
[76]Mobasheri H, Ficht T A, Marquis H, Lea E J, Lakey J H. Brucella Omp2a and Omp2b porins: single channel measurements and topology prediction [J].FEMS Microbiol Lett, 1997, 155: 23-30.
[77]Paquet J Y, Daz M A, Genevrois S, et al. Molecular, antigentic, and functional analyses of Omp2b porin size variants of Brucella spp [J]. J Bacteriol, 2001, 183: 4839-4847.
[78]Oliveira S C, Splitter G A. Immunization of mice with recombinant L7/L12ribosomal protein confers protection against Brucella abortus infection [J]. Vaccine, 1996, 14(10): 959-962.
[79]Jubier-Maurin V, Boigegrain R A, Cloeckaert A, et al. Maior outer membrane protein Omp25 of Brucella suis is involved in inhibition of tumor necrosis factor alpha production during infection of human macrophages [J]. Infect Immun, 2001, 69(8): 4823-4830.
[80]Matthew D, Edmonds, Cloeckaert A, Philip H E. Brucella species lacking the major outer membrane protein Omp25 are attenuated in mice and protect against Brucella melitensis and Brucella ovis [J]. Vet Microbiol, 2002, 88: 205-221.
[81]Boigegrain R A, Salhi I, Alvarez-Martinez M T, Machold J, Fedon Y, Arpagaus M, Weise C, Rittig M, Rouot B. Release of periplasmic proteins of Brucella suis upon acidic shock involves the outer membrane protein Omp25 [J]. Infect Immun, 2004, 72: 5693-5703.
[82]Viza no N, Cloeckaert A, Zygmunt M S, et al. Characterisation of a Brucella species 25-kb DNA fragment deleted from Brucella abortus reveals a large gene cluster related to the synthesis of a polysaccharide [J]. Infect Immun, 2001, 69: 6738-6748.
[83]Cassataro J, Pasquevich K, Bruno L, Wallach J C, Fossati C A, Baldi P C. Antibody reactivity to Omp31 from Brucella melitensis in human and animal infections by smooth and rough Brucellae [J]. Clin Diagn Lab Immunol, 2004, 11: 111-114.
[84]Salhi I, Boigegrain R A, Machold J, Weise C, Cloeckaert A, Rouot B. Characterization of new members of the group 3 outer membrane protein family of Brucella spp [J]. Infect Immun, 2003, 71: 4326-4332.
[85]Dubray G, Charriaut C. Evidence of three major polypeptide species and two major polysaccharide species in the Brucella outer membrane [J]. Ann Rech Vet, 1983, 14: 311-318.
[86]Dubray G. Protectives antigens in brucellosis [J]. Ann Inst Pasteur/Microbiol (Paris), 1987, 138: 84-87.
[87]Cloeckaert A, Jacques I, Limet J N, et al. Immunogenic properties of Brucella melitensis cell-wall fractions in BALB/C mice [J]. J Med Microbiol, 1995, 42: 200-208.
[88]Guiuoteau L A, Lavoucan K, Vizano N, et al. Immunogenicity of recombinant Escherichia coli expressing the omp31 gene of Brucella melitensis in BALB/C mice [J]. Vaccine, 1999, 17: 353-361.
[89]Jimnezde Bags M P, Elzer P H, Blasco J M, et al. Protective immunity to Brucella ovis in BALB/C mice following from primary infection or immuniation with subcellular vaccines [J]. Infect Immun, 1994, 62: 632-638.
[90]Kittelberge R, Diack D S, Vizca no N, et al. Characterisation of an immunodominant antigen in Brucella ovis and evaluation of it s use in anenzyme2linked immunosorbent assay [J]. Vet Microbiol, 1998, 59: 213-227.
[91]Betsy J, Bricker S, Halling M. Enhancement of the Brucella AMOS PCR Assay for Differentiation of Brucella abortus Vaccine Strain S19 and RB51 [J]. Clin Microbiol, 1995, 33: 1640-1642.
[92]Jiang X, Leonard B, Benson R, et al. Macrophage control of Brucella abortus: role of reactive oxygen intermediates and nitric oxide [J]. Cell Immunol, 1993, 151: 309-319.
[93]Gay B, Sanchez-Teff S and Caravano R. Ultrastructural localization of NADPH-oxidase activity in murine peritoneal macrophages during phagocytosis of Brucella. Correlation with the production of superoxide anions [J]. Virchows Arch B, 1984,45: 147-155.
[94]Baldwin C L, Jiang X and Fernandes D M. Macrophage control of Brucella abortus: influence of cytokines and iron [J]. Trends Microbiol, 1993, 1: 99-104.
[95]Jiang X and Baldwin C L. Iron augments macrophage-mediated killing of Brucella abortus alone and in conjunction with interferon-gamma [J]. Cell Immunol, 1993, 148: 397-407.
[96]Young E J, Borchert F L and Musher D M. Phagocytosis and killing of Brucella by human polymorphonuclear leukocytes [J]. J Infect Dis, 1985, 151: 682-690.
[97]Nathan C and Shiloh M U. Reactive oxygen and nitrogen intermediates in the relationship between mammalian hosts and microbial pathogens [J]. Proc Natl Acad Sci USA, 2000, 97: 8841-8848.
[98]尚德秋.布鲁氏菌感染与免疫研究近况[J].中国地方病杂志, 2003, 18(3): 154-157.
[99]金山,乌尼,哈斯木伦.布鲁氏菌生物学特性研究进展[J].内蒙古畜牧科学, 2000, (08): 32~34.
[100]Corbeil L B, K Blau T J, Inzana K H, et al. Killing of Brucella abortus by bovine serum [J]. Infect Immun, 1988, 56: 3251-3261.
[101]Hoffmann E M and Houle J J. Failure of Brucella abortus lipopolysaccharide (LPS) to activate the alternative pathway of complement [J]. Vet Immunol Immunopathol, 1983, 5: 65-76.
[102]Fernandez-Prada C M, M Nikolich R, Vemulapalli N, et al. Deletion of wboA enhances activation of the lectin pathway of complement in Brucella abortus and Brucella melitensis [J]. Infect Immun, 2001, 69: 4407-4416.
[103]Bellamy R. The natural resistance-associated macrophage protein and susceptibility to intracellular pathogens [J]. Microbes Infect, 1999, 1: 23-27.
[104]Ho M and Cheers C. Resistance and susceptibility of mice to bacterial infection. IV. Genetic and cellular basis of resistance to chronic infection with Brucella abortus [J]. J Infect Dis, 1982, 146: 381-387.
[105]Barthel R, Feng J, Piedrahita J A et al. Stable transfection of the bovine NRAMP1 gene into murine RAW264.7 cells: effect on Brucella abortus survival[J]. Infect.Immun, 2001, 69: 3110-3119.
[106]Araya L N, Elzer P H, Rowe G E, et al.Temporal development of protective cell-mediated and humoral immunity in BALB/c mice infected with Brucella abortus [J]. J Immunol, 1989, 143: 3330-3337.
[107]Oliveira S C and Splitter G A. CD8+ type 1 CD44hi CD45 RBlo T lymphocytes control intracellular Brucella abortus infection as demonstrated in major histocompatibility complex class I and class II-deficient mice [J]. Eur J Immunol, 1995, 25: 2551-2557.
[108]Winter A J, Duncan J R, Santisteban C G, et al. Capacity of passively administered antibody to prevent establishment of Brucella abortus infection in mice [J]. Infect Immun, 1989, 57: 3438-3444.
[109]Montaraz J A and Winter A J. Comparison of living and nonliving vaccines for Brucella abortus in BALB/c mice [J]. Infect Immun, 1986, 53: 245-251.
[110]Michaux-Charachon S, Bourg G, Jumas-Bilak E, et al. Genome structure and phylogeny in the genus Brucella [J]. J Bacteriol, 1997, 179: 3244-3249.
[111]Montaraz J A, Winter A J, Hunter D M, et al. Protection against Brucella abortus in mice with O-polysaccharide-specific monoclonal antibodies [J]. Infect Immun, 1986, 51: 961-963.
[112]Schurig G G, Roop R M, Bagchi J T, et al. Biological properties of RB51 a stable rough strain of Brucella abortus [J]. Vet Microbiol, 1991, 28: 171-188.
[113] Ko J, Gendron-Fitzpatrick A and Splitter G A. Susceptibility of interferon regulatory factor (IRF-1) and interferon consensus sequence binding protein (ICSBP) deficient mice to brucellosis [J]. J Immunol, 2002, 168: 2433-2440.
[114]Zhan Y, Kelso A and Cheers C. Cytokine production in the murine response to Brucella infection or immunization with antigenic extracts [J]. Immunology, 1993, 80: 458-464.
[115]Zhan Y and Cheers C. Endogenous gamma interferon mediates resistance to Brucella abortus infection [J]. Infect Immun, 1993, 61: 4899-4901.
[116]Zhan Y, Z Liu and Cheers C. Tumor necrosis factor alpha and interleukin-12 contribute to resistance to the intracellular bacterium Brucella abortus by different mechanisms [J]. Infect Immun, 1996, 64: 2782-2786.
[117]Mingle C K, Manthei C A, Jasmin A M. The stability of reduced virulence exhibited by Brucella abortus [J]. J Am Vet MED Assoc, 1941, 99: 203.
[118]Philip H Elzer, Fred M Enright, L Colby, et al. Protection against infection and abortion induced by virulent Challenge exposure after oral vaccination of Cattle with Brucell abortus strain RB51 [J]. AJNR, 1998, 59(12): 1575-1578.
[119]Henningsen R G B, Straub K M, et al. Application of zwitterionic detergents to the solubilization of integral membrane proteins for two-dimensional gel electrophoresis and mass spectrometry [J]. Proteomics, 2002, 2: 1479-1488.
[120]Gorg A, Obermaier C, Boguth G, Harder A, Scheibe B, Wildgruber R, Weiss W.The current state of two-dimensional electrophoresis with immobilized pH gradients[J]. Electrophoresis, 2000, 21: 1037-1053.
[121]Delpino M V, Cassataro J, Fossati C A, Goldbaum F A, Baldi P C. Brucella outer membrane protein Omp31 is a haemin-binding protein[J]. Microbes Infect, 2006, 8: 1203-1208.
[122]Perucho M, Hanahan D, Lipsich L, et al. Isolation of the chicken thymidine kinase gene by plasmid rescue[J]. Nature, 1980, 285: 207-210.
[123]Lin J, Huang S, Zhang Q. Outer membrane proteins: key players for bacterial adaptation in host niches[J]. Microbes Infect, 2002, 4: 325-331.
[124]Weiser JN, Gotschlich E C. Outer membrane protein A (OmpA) contributes to serum resistance and pathogenicity of Escherichia coli K-1[J]. Infect Immun, 1991, 59: 2252-2258.
[125]Edmonds M D, Cloeckaert A, Elzer PH, Brucella species lacking the major outer membrane protein Omp25 are attenuated in mice and protect against Brucella melitensis and Brucella ovis[J], Vet Microbiol, 2002, 88: 205-221.
[126]Caro-Hernandez P, Fernandez-Laqo L, de Miquel M L, et al. Role of the Omp25/Omp31 family in outer membrance properties and virulence of Brucella. Ovis[J]. Infect Immun, 2007, 75(8): 4050-4061.
[127]Porte F, Liautard J P, Kohler S. Early acidification of phagosomes containing Brucella suis is essential for intracellular survival in murine macrophages[J]. Infect Immun, 1999, 67: 4041-4047. .