布鲁氏菌膜蛋白组学研究及免疫蛋白的筛选鉴定
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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
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
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