鲍曼不动杆菌铁蛋白的抗氧化功能研究
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摘要
【目的】克隆鲍曼不动杆菌铁蛋白(Abferritin)基因,并研究其抗氧化功能。【方法】荧光定量PCR检测氧化应激下Abferritin基因的表达量,并将其基因克隆到表达载体p ET28a以构建重组质粒p ET28a-Abferritin,转化大肠杆菌BL21(DE3)得到重组菌BL/p ET28aAbferritin,IPTG诱导目的蛋白表达并利用镍柱亲和层析纯化该蛋白。比色法测定Abferritin蛋白的Fe2+氧化酶活性,自由基清除实验测定其抗氧化功能。菌落计数法观察重组大肠杆菌在H2O2应激条件下的存活率。【结果】Abferritin基因在氧化应激下表达增高。重组质粒在大肠杆菌BL21(DE3)中高效表达,通过Ni2+亲和层析纯化获得了Abferritin蛋白。该蛋白具有Fe2+氧化酶活性,能有效减少氧自由基的形成及提高大肠杆菌抵抗氧化应激的能力。【结论】氧化应激能诱导Abferritin基因表达上调,且该蛋白具有亚铁氧化酶活性和抗氧化功能。
[Objective] This study aimed to clone the ferritin gene Abferritin from Acinetobacter baumannii and identify its anti-oxidative activity. [Methods] Relative expression of Abferritin under oxidative stress was analyzed by real-time PCR. The gene encoding sequence of Abferritin was inserted into the p ET28 a vector to generate the p ET28a-Abferritin recombinant plasmid. This plasmid was transformed into the E. coli BL21(DE3) to create transformed strain of BL/p ET28a-Abferritin. The Abferritin protein was expressed by IPTG induction and was purified by Ni2+-affinity chromatography. Kinetics of Fe2+ oxidation which catalyzed by Abferritin protein was determined by spectrophotometric analysis. Anti-oxidative activity of Abferritin was examined by the radical scavenging assay. Survival rations of the recombinant and control E. coli under the oxidative stress of H2O2 were also measured. [Results] Expression level of Abferritin was up-regulated in A. baumannii under oxidative stress. Abferritin protein was expressed in E. coli BL21(DE3) and was purified successfully. Ferroxidase activity assay demonstrated that the Abferritin protein could convert Fe2+ to Fe3+. The hydroxyl radicals were scavenged by the Abferritin protein in vitro and the ectopic expression of Abferritin could increase the survival ratios of E. coli cells under the oxidative stress. [Conclusion] Abferritin was strongly up regulated under oxidative stress and the Abferritin protein exhibited ferroxidase and anti-oxidative activity.
[Objective] This study aimed to clone the ferritin gene Abferritin from Acinetobacter baumannii and identify its anti-oxidative activity. [Methods] Relative expression of Abferritin under oxidative stress was analyzed by real-time PCR. The gene encoding sequence of Abferritin was inserted into the p ET28 a vector to generate the p ET28a-Abferritin recombinant plasmid. This plasmid was transformed into the E. coli BL21(DE3) to create transformed strain of BL/p ET28a-Abferritin. The Abferritin protein was expressed by IPTG induction and was purified by Ni2+-affinity chromatography. Kinetics of Fe2+ oxidation which catalyzed by Abferritin protein was determined by spectrophotometric analysis. Anti-oxidative activity of Abferritin was examined by the radical scavenging assay. Survival rations of the recombinant and control E. coli under the oxidative stress of H2O2 were also measured. [Results] Expression level of Abferritin was up-regulated in A. baumannii under oxidative stress. Abferritin protein was expressed in E. coli BL21(DE3) and was purified successfully. Ferroxidase activity assay demonstrated that the Abferritin protein could convert Fe2+ to Fe3+. The hydroxyl radicals were scavenged by the Abferritin protein in vitro and the ectopic expression of Abferritin could increase the survival ratios of E. coli cells under the oxidative stress. [Conclusion] Abferritin was strongly up regulated under oxidative stress and the Abferritin protein exhibited ferroxidase and anti-oxidative activity.
引文
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[14]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].Infection and Immunity,2012,80(10):3650-3659.
[15]Ni W,Cui J,Liang B,et al.In vitro effects of tigecycline in combination with colistin(polymyxin E)and sulbactam against multidrug-resistant Acinetobacter baumannii[J].The Journal of Antibiotics,2013,66(12):705-708.
[2]严卓彦,许镇坚,许光治,等.耐辐射奇球菌dps突变株的构建和蛋白功能初步研究[J].微生物学报,2014,47(5):610-615.
[3]Lu J,Holmgren A.The thioredoxin antioxidant system[J].Free Radical Biology and Medicine,2014,66:75-87.
[4]Zhao G,Ceci P,Ilari A,et al.Iron and hydrogen peroxide detoxification properties of DNA-binding protein from starved cells.A ferritin-like DNA-binding protein of Escherichia coli[J].The Journal of Biological Chemistry,2002,277(31):27689-27696.
[5]Lima AL,Oliveira PR,Paula AP.Acinetobacter infection[J].The New England Journal of Medicine,2008,358(26):1271-1281.
[6]Dijkshoorn L,Nemec A,Seifert H.An increasing threat in hospitals:multidrug-resistant Acinetobacter baumannii[J].Nature Reviews Microbiology,2007,5(12):939-951.
[7]Dy ME,Nord JA,La Bombardi VJ,et al.The emergence of resistant strains of Acinetobacter baumannii:clinical and infection control implications[J].Infection Control and Hospital Epidemiology,1999,20(8):565-567.
[8]Hirai Y.Survival of bacteria under dry conditions:from a viewpoint of nosocomial infection[J].The Journal of Hospital Infection,1991,19(3):191-200.
[9]Sampson TR,Liu X,Schroeder MR,et al.Rapid killing of Acinetobacter baumannii by polymyxins is mediated by a hydroxyl radical death pathway[J].Antimicrobial Agents and Chemotherapy,2012,56(11):5642-5649.
[10]Smith MG,Gianoulis TA,Pukatzki S,et al.New insights into Acinetobacter baumannii pathogenesis revealed by high-density pyrosequencing and transposon mutagenesis[J].Genes&Development,2007,21(5):601-614.
[11]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 Science,2003,12(8):1663-1674.
[12]Calhoun LN,Kwon YM.The ferritin-like protein Dps protects Salmonella enterica serotype Enteritidis from the Fenton-mediated killing mechanism of bactericidal antibiotics[J].International Journal of Antimicrobial Agents,2011,37(3):261-265.
[13]Krawczyk-Balska A,Lipiak M.Critical role of a ferritin-like protein in the control of Listeria monocytogenes cell envelope structure and stability under beta-lactam pressure[J].PLo S One,2013,8(10):e77808.
[14]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].Infection and Immunity,2012,80(10):3650-3659.
[15]Ni W,Cui J,Liang B,et al.In vitro effects of tigecycline in combination with colistin(polymyxin E)and sulbactam against multidrug-resistant Acinetobacter baumannii[J].The Journal of Antibiotics,2013,66(12):705-708.