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.
出处
《微生物学通报》
CAS
CSCD
北大核心
2014年第12期2474-2480,共7页
Microbiology China
基金
国家自然科学基金项目(No.31000091)
特殊病原体防控湖南省重点实验室资助项目(湘科计字[2014]5号
湘教通[2012]312号)
关键词
鲍曼不动杆菌
铁蛋白
抗氧化活性
Acinetobacter baumannii,Ferritin protein,Anti-oxidative activity
