全部 标题 作者
关键词 摘要

OALib Journal期刊
ISSN: 2333-9721
费用:99美元

查看量下载量

相关文章

更多...

肺炎链球菌三磷酸甘油醛脱氢酶GAPDH与热休克蛋白DnaJ的相互作用

, PP. 1629-1635

Keywords: 肺炎链球菌,三磷酸甘油醛脱氢酶,热休克蛋白DnaJ,蛋白相互作用

Full-Text   Cite this paper   Add to My Lib

Abstract:

目的验证肺炎链球菌(Streptococcuspneumoniae,S.pn)糖酵解关键酶三磷酸甘油醛脱氢酶(glyceraldehyde-3-phosphatedehydrogenase,GAPDH)和热休克蛋白DnaJ的相互作用。方法PCR扩增S.pnD39GAPDH蛋白编码基因gap全长,将其克隆至pET-28a(+),重组质粒经PCR、双酶切及测序鉴定正确后转化大肠杆菌BL21(DE3),IPTG诱导GAPDH-6His的表达,经Ni-NTA亲和层析柱纯化后,用SDS-PAGE鉴定其纯度。重组GAPDH蛋白经腹部皮下免疫昆明小鼠获得多克隆抗体。Westernblot鉴定GAPDH的保守性。采用大肠杆菌双杂交系统、直接结合实验、生物膜层干涉技术(Bio-layerInterferometry,BLI)验证GAPDH和DnaJ的相互作用。结果获得了纯度达90%的GAPDH重组蛋白,并获得了效价达107的抗GAPDH多克隆抗体,Westernblot检测显示在7株不同血清型S.pn的全菌裂解液和培养上清中均存在和抗GAPDH抗体反应的特异性条带。大肠杆菌双杂交系统显示GAPDH和DnaJ共转化菌株能够在3-AT平板及双选择培养平板上生长,提示二者在细菌胞内有相互作用。直接结合实验和BLI显示随着加入GAPDH蛋白浓度的增加,二者的结合量也增加,提示GAPDH和DnaJ的结合具有浓度依赖性。BLI实验也显示GAPDH和DnaJ的亲和常数为1.12×10-7mol/L。结论糖酵解关键酶GAPDH在肺炎链球菌中保守表达,且为分泌性蛋白;该蛋白和热休克蛋白DnaJ在细胞内存在相互作用,且为直接相互作用。

References

[1]  Jedrzejas M J. Pneumococcal virulence factors: structure and function[J]. Microbiol Mol Biol Rev, 2001, 65(2): 187-207. [2]O’Brien K L, Wolfson L J, Watt J P, et al. Burden of disease caused by Streptococcus pneumoniae in children younger than 5 years: global estimates[J]. Lancet, 2009, 374(9693): 893-902. [3]Jin H, Agarwal S, Agarwal S, et al. Surface export of GAPDH/SDH, a glycolytic enzyme, is essential for Streptococcus pyogenes virulence[J]. MBio, 2011, 2(3): e00068-11. [4]Wang G, Xia Y, Cui J, et al. The roles of moonlighting proteins in bacteria[J]. Curr Issues Mol Biol, 2013, 16(1): 15-22. [5]Boel G, Jin H, Pancholi V. Inhibition of cell surface export of group A streptococcal anchorless surface dehydrogenase affects bacterial adherence and antiphagocytic properties[J]. Infect Immun, 2005, 73(10): 6237-6248. [6]Terao Y, Yamaguchi M, Hamada S, et al. Multifunctional glyceraldehyde-3-phosphate dehydrogenase of Streptococcus pyogenes is essential for evasion from neutrophils[J]. J Biol Chem, 2006, 281(20): 14215-14223. [7]Bergmann S, Rohde M, Hammerschmidt S. Glyceraldehyde-3-phosphate dehydrogenase of Streptococcus pneumoniae is a surface-displayed plasminogen-binding protein[J]. Infect Immun, 2004, 72(4): 2416-2419. [8]Attali C, Durmort C, Vernet T, et al. The interaction of Streptococcus pneumoniae with plasmin mediates transmigration across endothelial and epithelial monolayers by intercellular junction cleavage[J]. Infect Immun, 2008, 76(11): 5350-5356. [9]Terrasse R, Tacnet-Delorme P, Moriscot C, et al. Human and pneumococcal cell surface glyceraldehyde-3-phosphate dehydrogenase (GAPDH) proteins are both ligands of human C1q protein[J]. J Biol Chem, 2012, 287(51): 42620-42633. [10]Jin H, Song Y P, Boel G, et al. Group A streptococcal surface GAPDH, SDH, recognizes uPAR/CD87 as its receptor on the human pharyngeal cell and mediates bacterial adherence to host cells[J]. J Mol Biol, 2005, 350(1): 27-41. [11]Agarwal V, Kuchipudi A, Fulde M, et al. Streptococcus pneumoniae endopeptidase O (PepO) is a multifunctional plasminogen- and fibronectin-binding protein, facilitating evasion of innate immunity and invasion of host cells[J]. J Biol Chem, 2013, 288(10): 6849-6863. [12]Price K E, Greene N G, Camilli A. Export requirements of pneumolysin in Streptococcus pneumoniae[J]. J Bacteriol, 2012, 194(14): 3651-3660. [13]Schneewind O, Missiakas D M. Protein secretion and surface display in Gram-positive bacteria[J]. Philos Trans R Soc Lond B Biol Sci, 2012, 367(1592): 1123-1139. [14]Chatzi K E, Sardis M F, Karamanou S, et al. Breaking on through to the other side: protein export through the bacterial Sec system[J]. Biochem J, 2013, 449(1): 25-37. [15]Cai Y, Yan W, Xu W, et al. Screening and identification of DnaJ interaction proteins in Streptococcus pneumoniae[J]. Curr Microbiol, 2013, 67(6): 732-741. [16]王哲, 王建敏, 马峰, 等. 肺炎链球菌SpxB蛋白的细胞表达定位、保守性分析及其生物学功能初探[J]. 第三军医大学学报, 2014, 36(14): 1486-1491. [17]蔡莺莺. 肺炎链球菌热休克蛋白DnaJ相互作用蛋白的筛选及鉴定[D]. 重庆: 重庆医科大学, 2013. [18]龚燕华, 彭小忠. 蛋白质相互作用及亚细胞定位原理与技术[M]. 北京: 中国协和医科大学出版社, 2010: 35-71. ?

Full-Text

Contact Us

service@oalib.com

QQ:3279437679

WhatsApp +8615387084133