解脲支原体Ferritin基因的原核表达及抗氧化功能研究
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摘要
目的构建解脲支原体Ferritin蛋白的原核表达载体,纯化重组Ferritin蛋白并测定其抗氧化功能,为解析解脲支原体的抗氧化机制奠定基础。方法利用荧光定量PCR检测氧化胁迫下Ferritin基因的相对表达量。根据Ferritin基因序列,设计引物,PCR扩增Ferritin全长片段。将解脲支原体Ferritin基因中编码色氨酸的密码子TGA突变为TGG,将突变后的Ferritin基因连接到原核表达载pET28a得到重组质粒pET28a-Ferritin,转化大肠埃希菌BL21(DE3)得到重组菌BL/pET28a-Ferritin,IPTG诱导Ferritin蛋白表达并用镍柱亲和层析进行纯化。体外试验测定Ferritin蛋白的Fe2+结合特性,亚铁氧化酶活性和抗氧化能力。结果 Ferritin基因在H2O2或者CHP胁迫表达上调。成功构建能够表达全长Ferritin蛋白的重组质粒pET28a-Ferritin。诱导并纯化得到理论分子质量单位为22ku的重组Ferritin蛋白。结合Fe2+后,Ferritin蛋白的内源荧光值降低。Ferritin具有Fe2+氧化酶活性,能催化Fe2+生成Fe3+,并能够减少氧自由基的产生。结论解脲支原体Ferritin基因在氧化胁迫条件下表达上调,其表达产物具有亚铁氧化酶活性和抗氧化功能。
Objectives The aims of this study were to clone a ferritin gene fromUreaplasma urealyticum,to over-expresses and purify the recombinant ferritin protein in E.coli,and to evaluate the anti-oxidative activity of ferritin.The protective role of ferritin should provide a foundation for understanding the mechanisms by which U.urealyticumtolerates oxidative stress. Methods The relative level of ferritin expression under oxidative stresses was analyzed using realtime PCR.The ferritin gene fromU.urealyticum was cloned using PCR.The genetic codon TGA,which encodes the amino acid tryptophan in U.urealyticum,was replaced by TGG.The altered ferritin coding sequence was over-expressed in E.coli BL21(DE3)and purified using Ni 2+-affinity chromatography.The Fe2+binding ability and ferroxidase and antioxidant activity of the ferritin protein were studied via in vitro experiments. Results Ferritin mRNA was significantly up-regulated when U.urealyticum was subjected to H2O2 or CHP stress.The ferritin gene with the altered TGA codon was successfully over-expressed in E.coli and purified,producing a 22-kDa band in SDS-PAGE analysis.Addition of Fe2+dramatically quenched the intrinsic fluorescence of ferritin.The recombinant ferritin converted Fe2+into Fe3+and inhibited the Fenton reaction,indicating apparent ferroxidase activity. Conclusion Ferritin was up-regulated in U.urealyticumunder oxidative stress,and ferritin possesses ferroxidase activity and anti-oxidative activity.
Objectives The aims of this study were to clone a ferritin gene fromUreaplasma urealyticum,to over-expresses and purify the recombinant ferritin protein in E.coli,and to evaluate the anti-oxidative activity of ferritin.The protective role of ferritin should provide a foundation for understanding the mechanisms by which U.urealyticumtolerates oxidative stress. Methods The relative level of ferritin expression under oxidative stresses was analyzed using realtime PCR.The ferritin gene fromU.urealyticum was cloned using PCR.The genetic codon TGA,which encodes the amino acid tryptophan in U.urealyticum,was replaced by TGG.The altered ferritin coding sequence was over-expressed in E.coli BL21(DE3)and purified using Ni 2+-affinity chromatography.The Fe2+binding ability and ferroxidase and antioxidant activity of the ferritin protein were studied via in vitro experiments. Results Ferritin mRNA was significantly up-regulated when U.urealyticum was subjected to H2O2 or CHP stress.The ferritin gene with the altered TGA codon was successfully over-expressed in E.coli and purified,producing a 22-kDa band in SDS-PAGE analysis.Addition of Fe2+dramatically quenched the intrinsic fluorescence of ferritin.The recombinant ferritin converted Fe2+into Fe3+and inhibited the Fenton reaction,indicating apparent ferroxidase activity. Conclusion Ferritin was up-regulated in U.urealyticumunder oxidative stress,and ferritin possesses ferroxidase activity and anti-oxidative activity.
引文
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[16]Dussurget O,Dumas E,Archambaud C,et al.Listeria monocytogenes ferritin protects against multiple stresses and is required for virulence[J].FEMS Microbiol Lett,2005,250(2):253-61.
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[18]Stain B,Giudice DG,Bianca DV,et al.The neutrophil-activating protein(NAP)of Helicobacter pylori is a protective antigen and a major virulence factor[J].Exp Med,2000,191(9):1467-75
[19]Fu HW.Helicobacter pylori neutrophil-activating protein:from molecular pathogenesis to clinical applications[J].World J Gastroenterol,2014,20(18):5294-301.
[2]Lu J,Holmgren A.The thioredoxin antioxidant system[J].Free Radic Biol Med,2014(66):75-87.
[3]Chiancone E,Ceci P,Ilari A,et al.Iron and proteins for iron storage and detoxification[J].Biometals,2004,17(3):197-202.
[4]Andrews SC,Robinson AK,Rodriguez-Quinones F.Bacterial iron homeostasis[J].FEMS Microbiol Rev,2003,27(2-3):215-37.
[5]Khare G,Gupta V,Nangpal P,et al.Ferritin structure from Mycobacterium tuberculosis:comparative study with homologues identifies extended C-terminus involved in ferroxidase activity[J].PloS one,2011,6(4):e18570.
[6]Su M,Cavallo S,Stefanini S,et al.The so-called Listeria innocua ferritin is a Dps protein.Iron incorporation,detoxification,and DNA protection properties[J].Biochemistry,2005,44(15):5572-8.
[7]Shimada Y,Ito S,Mizutani K,et al.Bacterial loads of Ureaplasma urealyticum contribute to development of urethritis in men[J].Int J STD AIDS,2014,25(4):294-8.
[8]Larsen B,Hwang J.Mycoplasma,Ureaplasma,and adverse pregnancy outcomes:a fresh look[J].Infect Dis Obstet Gynecol,2010(2010):521921.
[9]Radonic A,Kovacevic V,Markotic A,et al.The clinical significance of Ureaplasma urealyticum in chronic prostatitis[J].J Chemother,2009,21(4):465-6.
[10]Waites KB,Schelonka RL,Xiao L,et al.Congenital and opportunistic infections:Ureaplasma species and Mycoplasma hominis[J].Semin Fetal Neonatal Med,2009,14(4):190-9.
[11]Haas A,Goebel W.Microbial strategies to prevent oxygen-dependent killing by phagocytes[J].Free Radic Res Commun,1992,16(3):137-57.
[12]Ko SH,Choi SW,Ye SK,et al.Comparison of anti-oxidant activities of seventy herbs that have been used in Korean traditional medicine[J].Nutr Res Pract,2008,2(3):143-51.
[13]Glass JI,Lefkowitz EJ,Glass JS,et al.The complete sequence of the mucosal pathogen Ureaplasma urealyticum[J].Nature,2000,407(6805):757-62.
[14]Kilic MA,Spiro S,Moore GR.Stability of a 24-meric homopolymer:comparative studies of assembly-defective mutants of Rhodobacter capsulatus bacterioferritin and the native protein[J].Protein Sci,2003,12(8):1663-74.
[15]Calhoun LN,Kwon YM.The ferritin-like protein Dps protects Salmonella enterica serotype Enteritidis from the Fenton-mediated killing mechanism of bactericidal antibiotics[J].Int J Antimicrob Agents,2011,37(3):261-5.
[16]Dussurget O,Dumas E,Archambaud C,et al.Listeria monocytogenes ferritin protects against multiple stresses and is required for virulence[J].FEMS Microbiol Lett,2005,250(2):253-61.
[17]Pandey R,Rodriguez GM.A ferritin mutant of Mycobacterium tuberculosis is highly susceptible to killing by antibiotics and is unable to establish a chronic infection in mice[J].Infect Immun,2012,80(10):3650-9.
[18]Stain B,Giudice DG,Bianca DV,et al.The neutrophil-activating protein(NAP)of Helicobacter pylori is a protective antigen and a major virulence factor[J].Exp Med,2000,191(9):1467-75
[19]Fu HW.Helicobacter pylori neutrophil-activating protein:from molecular pathogenesis to clinical applications[J].World J Gastroenterol,2014,20(18):5294-301.