解脲支原体Ferritin基因的原核表达及抗氧化功能研究

发布时间：2018-10-29 16:54

【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。
[Abstract]:Objective to construct the prokaryotic expression vector of Ferritin protein of Ureaplasma Urealyticum, purify the recombinant Ferritin protein and determine its antioxidant function, so as to lay a foundation for analyzing the antioxidant mechanism of Ureaplasma Urealyticum. Methods fluorescence quantitative PCR was used to detect the relative expression of Ferritin gene under oxidative stress. According to the sequence of Ferritin gene, primers were designed and the full length Ferritin fragment was amplified by PCR. The TGA encoding tryptophan in Ureaplasma Urealyticum Ferritin gene was mutated into TGG,. The mutated Ferritin gene was linked to the prokaryotic expression pET28a to obtain the recombinant plasmid pET28a-Ferritin, transformed into Escherichia coli BL21 (DE3) to obtain the recombinant strain BL/pET28a-Ferritin,. IPTG induced the expression of Ferritin protein and purified it by nickel affinity chromatography. The Fe2 binding characteristics, the activity of ferrous oxidase and the antioxidant ability of Ferritin protein were measured in vitro. Results the expression of Ferritin gene was up-regulated under H2O2 or CHP stress. Construction of Recombinant plasmid pET28a-Ferritin. capable of expressing Full-length Ferritin protein The recombinant Ferritin protein with theoretical molecular weight unit (22ku) was induced and purified. After binding to Fe2, the endogenous fluorescence value of Ferritin protein decreased. Ferritin had the activity of Fe2 oxidase, which could catalyze the production of Fe3 from Fe2 and reduce the production of oxygen free radical. Conclusion Ureaplasma Urealyticum Ferritin gene is up-regulated under oxidative stress, and its expression product has the activity of ferrous oxidase and antioxidant function.
【作者单位】：南华大学病原生物学研究所特殊病原体防控湖南省重点实验室;郴州市第一人民医院检验科;
【基金】：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)
【分类号】：R375

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