【摘要】：目的構(gòu)建解脲支原體Ferritin蛋白的原核表達(dá)載體,純化重組Ferritin蛋白并測定其抗氧化功能,為解析解脲支原體的抗氧化機制奠定基礎(chǔ)。方法利用熒光定量PCR檢測氧化脅迫下Ferritin基因的相對表達(dá)量。根據(jù)Ferritin基因序列,設(shè)計引物,PCR擴增Ferritin全長片段。將解脲支原體Ferritin基因中編碼色氨酸的密碼子TGA突變?yōu)門GG,將突變后的Ferritin基因連接到原核表達(dá)載pET28a得到重組質(zhì)粒pET28a-Ferritin,轉(zhuǎn)化大腸埃希菌BL21(DE3)得到重組菌BL/pET28a-Ferritin,IPTG誘導(dǎo)Ferritin蛋白表達(dá)并用鎳柱親和層析進行純化。體外試驗測定Ferritin蛋白的Fe2+結(jié)合特性,亞鐵氧化酶活性和抗氧化能力。結(jié)果 Ferritin基因在H2O2或者CHP脅迫表達(dá)上調(diào)。成功構(gòu)建能夠表達(dá)全長Ferritin蛋白的重組質(zhì)粒pET28a-Ferritin。誘導(dǎo)并純化得到理論分子質(zhì)量單位為22ku的重組Ferritin蛋白。結(jié)合Fe2+后,Ferritin蛋白的內(nèi)源熒光值降低。Ferritin具有Fe2+氧化酶活性,能催化Fe2+生成Fe3+,并能夠減少氧自由基的產(chǎn)生。結(jié)論解脲支原體Ferritin基因在氧化脅迫條件下表達(dá)上調(diào),其表達(dá)產(chǎn)物具有亞鐵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.
【作者單位】： 南華大學(xué)病原生物學(xué)研究所特殊病原體防控湖南省重點實驗室;郴州市第一人民醫(yī)院檢驗科;
【基金】：特殊病原體防控湖南省重點實驗室資助項目(湘科計字[2014]5號,湘教通〔2012〕312號)
【分類號】：R375

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