长滩军团菌临床分离株与环境分离株全菌蛋白的比较与分析
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摘要
目的采用蛋白质组学方法,对长滩军团菌血清1型临床分离株(LL-1)与环境分离株(CD-1)全菌蛋白进行比较和分析,探索军团菌临床株与环境分离株是否存在蛋白表达差异,为进一步认识、研究军团菌提供依据。方法培养、收集CD-1与LL-1菌株,用超声兼尿素-3-[(3-胆酰胺丙基)-乙二胺]-1丙磺酸(CHAPS)-二硫苏糖醇(DTT)裂解法提取全菌蛋白,SDS-PAGE及Western blot对二者全菌蛋白进行初步比较,确定二者全菌蛋白表达存在差异后,采用双向电泳(2-DE)联合MALDI-TOF-TOF质谱深入研究,并对质谱鉴定的差异点进行Western blot验证。结果初步SDS-PAGE及Western blot试验表明,CD-1与LL-1存在蛋白表达差异。进一步的2-DE研究结果共获得142个蛋白。CD-1与LL-1相比,18个蛋白点表达上调、19个蛋白表达下调。对其中显著差异的四个点进行质谱分析、Mascot软件查询、蛋白数据库搜库后证实CD-1表达下调的为热休克蛋白70(Chaperone protein Dna K)和30 s核糖体蛋白S1(30S ribosomal protein S1);CD-1表达上调的蛋白为假想蛋白TP04_0359(hypothetical protein TP04_0359)和一个未知蛋白。对进行质谱分析的四个蛋白点进行Western blot验证确实是军团菌蛋白。结论 LL-1与CD-1蛋白表达存在差异,此试验为进一步研究军团菌毒力和致病及疫苗研制奠定了基础。
Objective Through proteomic methods to compare and analyze the total expressed proteins of clinical and environmental isolates of Legionella longbeachae sg1, and explore the differentially expressed protein between them. And the aim is to lay the foundation for further understanding and study on Legionella spp. Methods The total expressed protein from the strains of CD-1 and LL-1 was extracted by Sonication-Urea-3- [-(3-cholamidopropyl)-dimethylammonio ]-1-propanesulfonate(CHAPS)-DTT method, and preliminarily compared through SDS-PAGE and Western blot. After the differentially expressed proteins were found, 2-dimensionnal electrophoresis(2-DE) and MALDI-TOF-TOF were employed to obtain more information of the protein. And then Western blot was adopted to verify differentially expressed protein spots which identified by mass spectrometry. Results The preliminary data from SDS-PAGE and Western blot showed that the differentially expressed proteins exist between CD-1 and LL-1. The 2-DE revealed 142 protein spots. Compared with LL-1,CD-1 has 18 protein spots up-regulated and 19 down-regulated. Among the differentially expressed protein spots, 4 significant difference spots of them were identified as Heat shock protein 70, 30 S ribosomal protein S1,hypothetical protein TP04_0359 and the unknown protein by mass spectrometry, Mascot inquiring and database searching in CD-1. And the first two proteins were down-regulated while the following two were up-regulated. The results of Western blot demonstrated that the 4 differentially expressed protein spots were belong to Legionella. Conclusion There are differences between the total expressed protein of CD-1 and LL-1.The study may be helpful for virulence and pathogenic an alysis of Legionella, and stand as a valuable resource for vaccine development against Legionella.
Objective Through proteomic methods to compare and analyze the total expressed proteins of clinical and environmental isolates of Legionella longbeachae sg1, and explore the differentially expressed protein between them. And the aim is to lay the foundation for further understanding and study on Legionella spp. Methods The total expressed protein from the strains of CD-1 and LL-1 was extracted by Sonication-Urea-3- [-(3-cholamidopropyl)-dimethylammonio ]-1-propanesulfonate(CHAPS)-DTT method, and preliminarily compared through SDS-PAGE and Western blot. After the differentially expressed proteins were found, 2-dimensionnal electrophoresis(2-DE) and MALDI-TOF-TOF were employed to obtain more information of the protein. And then Western blot was adopted to verify differentially expressed protein spots which identified by mass spectrometry. Results The preliminary data from SDS-PAGE and Western blot showed that the differentially expressed proteins exist between CD-1 and LL-1. The 2-DE revealed 142 protein spots. Compared with LL-1,CD-1 has 18 protein spots up-regulated and 19 down-regulated. Among the differentially expressed protein spots, 4 significant difference spots of them were identified as Heat shock protein 70, 30 S ribosomal protein S1,hypothetical protein TP04_0359 and the unknown protein by mass spectrometry, Mascot inquiring and database searching in CD-1. And the first two proteins were down-regulated while the following two were up-regulated. The results of Western blot demonstrated that the 4 differentially expressed protein spots were belong to Legionella. Conclusion There are differences between the total expressed protein of CD-1 and LL-1.The study may be helpful for virulence and pathogenic an alysis of Legionella, and stand as a valuable resource for vaccine development against Legionella.
引文
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[17]Ericsson M,Tarnvik A,Kuoppa K,et al.Increased synthesis of Dna K,Gro EL,and Gro ES homologs by Francisella tularensis LVS in response to heat and hydrogen peroxide[J].Infect Immun,1994,62(1):178-183.
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[21]Delvillani F,Papiani G,Deho G,et al.S1 ribosomal protein and the interplay between translation and m RNA decay[J].Nucleic Acids Res,2011,39(17):7702-7715.
[22]Twine S,Shen H,Harris G,et al.BALB/c mice,but not C57BL/6 mice immunized with a Deltaclp B mutant of Francisella tularensis subspecies tularensis are protected against respiratory challenge with wild-type bacteria:association of protection with post-vaccination and post-challenge immune responses[J].Vaccine,2012,30(24):3634-3645.
[2]Laura GV,Christophe R,Christel C,et al.Comparative and functional genomics of legionella identified eukaryotic like proteins as key players in host-pathogen interactions[J].Front Microbiol,2011,2:208.
[3]Wang G,Ying X,Da LC,et al.Application of multilocus sequence analysis(MLSA)for accurate identification of Legionella spp.Isolated from municipal fountains in Chengdu,China,based on 16S r RNA,mip,and rpo B genes[J].J Microbiol,2012,50(1):127-136.
[4]Richards AM,Von Dwingelo JE,Price CT,et al.Cellular microbiology and molecular ecology of Legionella-amoeba interaction[J].Virulence,2013,4(4):307-314.
[5]Tiaden A,Hilbi H.alpha-Hydroxyketone synthesis and sensing by Legionella and Vibrio[J].Sensors(Basel),2012,12(3):2899-2919.
[6]Newton HJ,Ang DKY,van Driel IR,et al.Molecular pathogenesis of infections caused by Legionella pneumophila[J].Clin Microbiol Rev,2010,23(2):274-298.
[7]Amodeo MR,Murdoch DR,Pithie AD.Legionnaires'disease caused by Legionella longbeachae and Legionella pneumophila:comparison of clinical features,host-related risk factors,and outcomes[J].Clin Microbiol Infect,2010,16(9):1405-1407.
[8]Abu Kwaik Y,Gao LY,Stone BJ,et al.Invasion of protozoa by Legionella pneumophila and its role in bacterial ecology and pathogenesis[J].Appl Environ Microbiol,1998,64(9):3127-3133.
[9]Atlas RM.Legionella:from environmental habitats to disease pathology,detection and control[J].Environ Microbiol,1999,1(4):283-293.
[10]Aurell H,Farge P,Meugnier H,et al.Clinical and environmental isolates of Legionella pneumophila serogroup 1cannot be distinguished by sequence analysis of two surface protein genes and three housekeeping genes[J].Appl Environ Microbiol,2005,71(1):282-289.
[11]Kilic NK,Stensballe A,Otzen DE,et al.Proteomic changes in response to chromium(VI)toxicity in Pseudomonas aeruginosa[J].Bioresour Technol,2010,101(7):2134-2140.
[12]Liang S,Fu A,Zhang Q,et al.Honokiol inhibits Hep G2migration via down-regulation of IQGAP1 expression discovered by a quantitative pharmaceutical proteomic analysis[J].Proteomics,2010,10(7):1474-1483.
[13]Zeng X,Yang P,Chen B,et al.Quantitative secretome analysis reveals the interactions between epithelia and tumor cells by in vitro modulating colon cancer microenvironmen[J].J Proteomics,2013,89:51-70.
[14]Retzlaff C,Yamamoto Y,Hoffman PS,et al.Bacterial heat shock proteins directly induce cytokine m RNA and interleukin-1 secretion in macrophage cultures[J].Infect Immun,1994,62(12):5689-5693.
[15]Skar CK,Kruger PG,Bakken V.Characterisation and subcellular localisation of the Gro EL-like and Dna K-like proteins isolated from Fusobacterium nucleatum ATCC 10953[J].Anaerobe,2003,9(6):305-312.
[16]Eremeeva ME,Ching WM,Wu Y,et al.Western blotting analysis of heat shock proteins of Rickettsiales and other eubacteria[J].FEMS Microbiol Lett,1998,167(2):229-237.
[17]Ericsson M,Tarnvik A,Kuoppa K,et al.Increased synthesis of Dna K,Gro EL,and Gro ES homologs by Francisella tularensis LVS in response to heat and hydrogen peroxide[J].Infect Immun,1994,62(1):178-183.
[18]Amemura-Maekawa J,Watanabe H.Cloning and sequencing of the dna K and grp E genes of Legionella pneumophila[J].Gene,1997,197(1-2):165-168.
[19]Hoffman PS,Garduno RA.Surface-associated heat shock proteins of Legionella pneumophila and Helicobacter pylori:roles in pathogenesis and immunity[J].Infect Dis Obstet Gynecol,1999,7(1-2):58-63.
[20]Macellaro A,Tujulin E,Hjalmarsson K,et al.Identification of a 71-kilodalton surface-associated Hsp70 homologue in Coxiella burnetii[J].Infect Immun,1998,66(12):5882-5888.
[21]Delvillani F,Papiani G,Deho G,et al.S1 ribosomal protein and the interplay between translation and m RNA decay[J].Nucleic Acids Res,2011,39(17):7702-7715.
[22]Twine S,Shen H,Harris G,et al.BALB/c mice,but not C57BL/6 mice immunized with a Deltaclp B mutant of Francisella tularensis subspecies tularensis are protected against respiratory challenge with wild-type bacteria:association of protection with post-vaccination and post-challenge immune responses[J].Vaccine,2012,30(24):3634-3645.