痢疾杆菌免疫及温度比较蛋白质组学研究
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摘要
痢疾杆菌是一类通过侵袭入肠道上皮细胞并进行复制从而导致人类出现腹泻的革兰氏阴性细菌。主要传播途径为粪口途径,由于感染剂量极低(10-100个菌),所以最长见的扩散形式是人与人之间的传播。据保守估计,全世界每年的感染人数约1.647亿,发展中国家有约1.632亿人次。每年死亡人数有110万。在发展中国家中福氏痢疾2a血清型占大多数。由于人们对痢疾杆菌的致病机理和宿主的免疫保护机制还不十分清楚,所以迄今为止仍未研究出能有效控制痢疾较为理想的疫苗。
本文首先使用免疫蛋白质组学手段对痢疾杆菌福氏2a 2457T株可能存在的抗原进行筛选和鉴定。分别提取了全菌体蛋白、膜蛋白、外膜蛋白以及胞外分泌蛋白,进行了不同pH梯度的双向电泳。对于痢疾杆菌的外膜蛋白建立了双向电泳参考图谱,通过软件分析在pH4-7 18厘米胶上计算出共126个点,其中109个进行胶内酶切并MALDI-TOF鉴定出87个,代表55种蛋白。结合根据基因组预测的外膜蛋白对鉴定的所有蛋白质点的等电点/分子量吻合程度、分布以及功能分类等进行了初步的分析。使用免疫家兔的血清,对不同组分的蛋白样品进行免疫印迹的研究,共找到23种蛋白质抗原,其中8种是已知抗原,15种为新抗原。有一个假想蛋白YaeT,功能未知,序列与Haemophilus influenzae的表面抗原D15以及Pasteurella multocida的oma87基因同源,免疫反应很强。有报道该基因在模拟的体内环境中转录水平提高,推测可能与痢疾杆菌的侵袭能力有关,值得进一步深入研究。
痢疾杆菌毒力的表达是温度相关的。当在37℃培养时痢疾杆菌表现出完整的毒性以及对Henle细胞的侵袭力,但是在30℃培养时则明显减毒及丧失侵袭力。本实验在以前工作的基础上改进方法,对30℃及37℃培养时菌体蛋白进行了双向电泳的比较,找到59个差异蛋白点,对应于53种蛋白质。结果显示,在30℃培养时检测到42种蛋白上调,12种蛋白下调(TufB既上调又下调)。通过对结果的分析,推测庄30℃培养时细菌的乙醛酸循环增强,蛋白质合成加强以及嘌呤合成的增加。其中庄免疫蛋白质组学工作中检测到的假想蛋白YaeT表达量也出现增加。
由于在电泳图谱上ArgT的变化十分显著,而在大肠杆菌中人工强制表达时不论在30℃还是37℃培养时均可以获得高表达,但是在痢疾杆菌中表达时则只有在30℃时可获得高表达。因此我们使用Red重组系统构建了argT失活的突变体,在30℃和37℃培养条件下分别与原始的2457T株进行双向电泳图谱的比较并得到了一些差异
Shigella is a genus of gram-negative bacilli that causes bacillary dysentery in humans by invading and replicating in epithelial cells lining the lower gut. Shigella infection spreads by the fecal-oral route. Because of the low infectious dose (10 to 100 organisms), person-to-person transmission probably is the most common. Worldwide there are approximately 164.7 million cases, of which 163.2 million in developing countries. Each year 1.1 million people are estimated to die from Shigella infection. In developing countries, S. flexneri 2a is most common. Because of the unknown mechanism of pathogenicity and immunoprotection, no ideal and effective vaccine has been developed till now.In this article immunoproteomic techniques were used to screen antigens of S. flexneri 2a 2457T. Whole cell proteins, membrane proteins, outer membrane proteins and extracellular proteins were extracted and performed 2-DE of different pH gradient. Reference map of OMPs were established and analyzed by software. Total 126 spots were calculated on 18cm pH 4-7 gel, 109 spots were cut and in-gel digested. Eighty-seven spots were identified successfully by MALDI-TOF/MS which represent 55 protein entries. The comparison between experimental and theoretical pI/MW distribution and their cellular role were analyzed. By using the sera of immunized rabbits, Western Blotting were performed with proteins of different components. Total of 23 antigens were found, 8 were known and 15 were unknown. One hypothetical protein-YaeT, with unknown function and strong immunoreactivity, was homologous with surface antigen D15 of Haemophilus influenzae and Oma87 of Pasteurella multocida. The transcription of yaeT was reported highly increased in a murine model and this gene was deduced associated with Shigella invasion.The expression of virulence genes of Shigella is temperature-dependent. Availability of invasion into Henle cells was reported when cultivated at 37℃ but not at 30℃. By using our improved methods, the whole cell proteins of bacteria cultivated at 30 and 37℃ were performed 2-DE. Fifty-nine different spots were found, which representing 53 protein entries by comparison of the 2-D gels. Forty-two proteins were up-regulated when cultivated at 30℃ and 12 were down-regulated at 37℃ (TufB was observed both of up-regulated and down-regulated). From the results we can deduce that the glyoxylate cycle pathway was
本文首先使用免疫蛋白质组学手段对痢疾杆菌福氏2a 2457T株可能存在的抗原进行筛选和鉴定。分别提取了全菌体蛋白、膜蛋白、外膜蛋白以及胞外分泌蛋白,进行了不同pH梯度的双向电泳。对于痢疾杆菌的外膜蛋白建立了双向电泳参考图谱,通过软件分析在pH4-7 18厘米胶上计算出共126个点,其中109个进行胶内酶切并MALDI-TOF鉴定出87个,代表55种蛋白。结合根据基因组预测的外膜蛋白对鉴定的所有蛋白质点的等电点/分子量吻合程度、分布以及功能分类等进行了初步的分析。使用免疫家兔的血清,对不同组分的蛋白样品进行免疫印迹的研究,共找到23种蛋白质抗原,其中8种是已知抗原,15种为新抗原。有一个假想蛋白YaeT,功能未知,序列与Haemophilus influenzae的表面抗原D15以及Pasteurella multocida的oma87基因同源,免疫反应很强。有报道该基因在模拟的体内环境中转录水平提高,推测可能与痢疾杆菌的侵袭能力有关,值得进一步深入研究。
痢疾杆菌毒力的表达是温度相关的。当在37℃培养时痢疾杆菌表现出完整的毒性以及对Henle细胞的侵袭力,但是在30℃培养时则明显减毒及丧失侵袭力。本实验在以前工作的基础上改进方法,对30℃及37℃培养时菌体蛋白进行了双向电泳的比较,找到59个差异蛋白点,对应于53种蛋白质。结果显示,在30℃培养时检测到42种蛋白上调,12种蛋白下调(TufB既上调又下调)。通过对结果的分析,推测庄30℃培养时细菌的乙醛酸循环增强,蛋白质合成加强以及嘌呤合成的增加。其中庄免疫蛋白质组学工作中检测到的假想蛋白YaeT表达量也出现增加。
由于在电泳图谱上ArgT的变化十分显著,而在大肠杆菌中人工强制表达时不论在30℃还是37℃培养时均可以获得高表达,但是在痢疾杆菌中表达时则只有在30℃时可获得高表达。因此我们使用Red重组系统构建了argT失活的突变体,在30℃和37℃培养条件下分别与原始的2457T株进行双向电泳图谱的比较并得到了一些差异
Shigella is a genus of gram-negative bacilli that causes bacillary dysentery in humans by invading and replicating in epithelial cells lining the lower gut. Shigella infection spreads by the fecal-oral route. Because of the low infectious dose (10 to 100 organisms), person-to-person transmission probably is the most common. Worldwide there are approximately 164.7 million cases, of which 163.2 million in developing countries. Each year 1.1 million people are estimated to die from Shigella infection. In developing countries, S. flexneri 2a is most common. Because of the unknown mechanism of pathogenicity and immunoprotection, no ideal and effective vaccine has been developed till now.In this article immunoproteomic techniques were used to screen antigens of S. flexneri 2a 2457T. Whole cell proteins, membrane proteins, outer membrane proteins and extracellular proteins were extracted and performed 2-DE of different pH gradient. Reference map of OMPs were established and analyzed by software. Total 126 spots were calculated on 18cm pH 4-7 gel, 109 spots were cut and in-gel digested. Eighty-seven spots were identified successfully by MALDI-TOF/MS which represent 55 protein entries. The comparison between experimental and theoretical pI/MW distribution and their cellular role were analyzed. By using the sera of immunized rabbits, Western Blotting were performed with proteins of different components. Total of 23 antigens were found, 8 were known and 15 were unknown. One hypothetical protein-YaeT, with unknown function and strong immunoreactivity, was homologous with surface antigen D15 of Haemophilus influenzae and Oma87 of Pasteurella multocida. The transcription of yaeT was reported highly increased in a murine model and this gene was deduced associated with Shigella invasion.The expression of virulence genes of Shigella is temperature-dependent. Availability of invasion into Henle cells was reported when cultivated at 37℃ but not at 30℃. By using our improved methods, the whole cell proteins of bacteria cultivated at 30 and 37℃ were performed 2-DE. Fifty-nine different spots were found, which representing 53 protein entries by comparison of the 2-D gels. Forty-two proteins were up-regulated when cultivated at 30℃ and 12 were down-regulated at 37℃ (TufB was observed both of up-regulated and down-regulated). From the results we can deduce that the glyoxylate cycle pathway was
引文
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[2] Ueberle B, Frank R, Herrmann R. The proteome of the bacterium Mycoplasma pneumoniae: Comparing predicted open reading frames to identified gene products. Proteomics 2002, 2, 754-764.
[3] Vandahl B, Birkelund S, Demol H, et al. Proteome analysis of the Chlamydia pneumoniae elementary body. Electrophoresis 2001, 22, 1204-1223.
[4] Langen H, Gray C, Roder D, et al. Front genome to proteome: Protein map of Haemophilus influenzae. Electrophoresis 1997, 18, 1184-1192.
[5] Bumann D, Meyer TF, Jungblut PR. Proteome analysis of the common human pathogen Helicobacter pylori. Proteomics 2001, 1, 473-479.
[6] Liao X, Ying TY, Wang HL, et al. A two-dimensional proteome map of Shigella flexneri. Electrophoresis 2003, 24, 2864-2882.
[7] Guillot A, Gitton C, Anglade P, et al. Proteomics analysis of Lactococcus lactis, a lactic acid bacterium. Proteomics 2003, 3, 337-354.
[8] Coelho A, de Otiveira Santos E, Faria ML, et al. A proteome reference map for Vibrio cholerae EI Tor. Proteomics 2004, 4, 1491-1504.
[9] Bini L, Sanchez-Campillo M, Santucci A, et al. Mapping of Chlamydia trachomatis proteins by Immobiline-polyacrylamide two-dimensional electrophoresis: Spot identification by N-terminal sequencing and immunoblotting. Electrophoresis 1996, 17, 185-190.
[10] McAtee CP, LIM MY, Fung K, et al. Identification of potential diagnostic and vaccine candidates of Helicobacter pylori by two-dimensional gel electrophoresis, sequence analysis, and serum profiling. Clin Diagn Lab Immunol 1998, 5, 537-542.
[11] Haas G, Karaali G, Ebermayer K, et al. Immunoproteomics of Helicobacter pylori infection and relation to gastric disease. Proteomics 2002, 2, 313-324.
[12] Kimmel B, Bosserhoff A, Frank R, et al. Identification of immunodominant antigens from Helicobacter pylori and evaluation of their reactivities with sera from patients with different gastroduodenal pathologies. Infect Immun 2000, 68, 915-920.
[13] Nilsson I. Utt M, Nillson HO, et al. Two-dimensional electrophoretic and immunoblot analysis of cell surface proteins of spiral-shaped and coccoid forms of Helicobacter pylori. Electrophoresis 2000, 21, 2670-2677.
[14] Ariel N, Zvi A, Makarova KS, et al. Genome-based bioinformatic selection of chromosomal Bacillus anthracis putative vaccine candidates coupled with proteomic identification of surface-associated antigens, Infect Immun 2003, 71, 4563-4579.
[15] Pardo M, Ward M, Pitarch A, et al. Cross-species identification of novel Candida albicans immunogenic proteins by combination of two-dimensional polyacrylamide gel electrophoresis and mass spectrometry. Electrophoresis 2000, 21, 2651-2659.
[16] Pitarch A. Pardo M, Jimenez A, et al. Two-dimensional gel electrophoresis as analytical tool for identifying Candida albicans immunogenic proteins. Electrophoresis 1999, 20, 1001-1010.
[17] Jennison AV, Verma NK. Shigella flexneri infection: pathogenesis and vaccine development. FEMS Micro Rev 2004, 28: 43-58.
[18] Maurelli AT, Blackmon B, Curtiss Ⅲ R. Temperature-dependent expression of virulence genes in Shigella species, Infect Immun 1984, 43,195-201
[19] 张延龄,张晖.疫苗学.科学出版社,北京,2004.
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