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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。
Objective To construct a prokaryotic expression vector for Ferritin protein of Ureaplasma urealyticum, purify the recombinant Ferritin protein and determine its anti-oxidant function, and lay a foundation for analyzing the anti-oxidation mechanism of Ureaplasma urealyticum. Methods Fluorescence quantitative PCR was used to detect the relative expression of Ferritin gene under oxidative stress. According to the Ferritin gene sequence, primers were designed to amplify the full-length Ferritin fragment by PCR. The codon TGA encoding tryptophan in Ferritin gene of Ureaplasma urealyticum was mutated to TGG, and the mutated Ferritin gene was ligated into prokaryotic expression vector pET28a to obtain recombinant plasmid pET28a-Ferritin, which was transformed into Escherichia coli BL21 (DE3) BL / pET28a-Ferritin, IPTG induced Ferritin protein expression and purification by nickel column affinity chromatography. Ferritin protein’s Fe2 + binding properties, ferrous oxidase activity and antioxidant capacity were determined in vitro. Results Ferritin gene was up-regulated by H2O2 or CHP stress. The recombinant plasmid pET28a-Ferritin was successfully constructed to express the full-length Ferritin protein. The recombinant Ferritin protein with the theoretical molecular mass unit of 22 ku was induced and purified. When combined with Fe2 +, the endogenous fluorescence of Ferritin decreased. Ferritin has Fe2 + oxidase activity that catalyzes the formation of Fe3 + from Fe2 + and reduces the production of oxygen free radicals. Conclusion Ferritin gene of Ureaplasma urealyticum is up-regulated under oxidative stress, and its expression product has ferrooxidase activity and anti-oxidant function.