【摘要】 目的了解艰难梭菌新型氧化还原酶Fnr的性质和催化功能。方法通过PCR扩增艰难梭菌的fnr基因片段,利用限制性内切酶NheⅠ、XhoⅠ对PCR产物进行双酶切,酶切片段插入经相同酶切处理的pET28b(+)质粒中,构建pET28b(+)-fnr重组表达载体,测序验证正确后转化至E.coli C41(DE3)感受态细胞中,用IPTG诱导表达目的蛋白,Ni-NTA柱亲和层析纯化后进行纯度及酶活性测定。结果成功构建了pET28b(+)-fnr重组表达载体,转化E.coli C41(DE3)高效表达重组蛋白Fnr。柱层析纯化后进行SDS-PAGE分析,该蛋白由两个大小分别约为51×103和33×103的亚基组成,具有NAD+依赖的NADPH还原TTC的活性。其酶所展现的性质一致,表明它们具有相同的生化功能。结论成功表达和纯化了艰难梭菌Fnr重组蛋白,该蛋白具有与其他专性厌氧菌对应的氧化还原酶活性,为其生理功能奠定研究了基础。更多还原

【Abstract】 Objective To ascertain the properties and function of a novel oxidoreductase(Fnr)fromClostridium difficile. Methods The fnr gene of C.difficile was amplified using PCR.The product of PCR was digested with the restriction endonucleases NheI and XhoI and ligated into pET28 b(+),which was used to construct the recombinant expression vector pET28 b(+)-fnr.The recombinant expression vector was verified using sequencing and then transformed into E.coli C41(DE3)competent cells,and expression of the target protein was induced with IPTG.Recombinant Fnr protein was purified with affinity chromatography using an Ni-NTA column,and its purity and enzyme activity were determined. Results The recombinant expression vector pET28 b(+)-fnr was successfully constructed and transformed into E.coli C41(DE3)to efficiently express recombinant Fnr.The recombinant protein was purified with affinity chromatography and analyzed with SDS-PAGE.Results indicated that the obtained protein was highly pure and consisted of two subunits 51×103 and 33×103 in size.The purified protein displayed characteristic absorption at 390 nm and 430 nm and had specific activity of 0.55 U/mg at 30℃in the presence of NAD+according to a TTC reduction assay.In the absence of NAD+,only 30% was noted.These enzymes exhibit the same properties,indicating that they perform the same biochemical function. Conclusion Recombinant Fnr from C.difficile was successfully expressed and purified.The protein displays the same oxidoreductase activity as other obligate anaerobic bacteria.These findings have laid the foundation for study of its physiological function.更多还原