【摘要】 目的对刚地弓形虫1-脱氧-D-木酮糖-5-磷酸还原异构化酶(TgDXR)基因进行克隆、表达、纯化和生物学特性分析。方法收集、纯化RH株弓形虫速殖子,提取cDNA和基因组DNA;PCR扩增TgDXR的基因片段;构建成熟TgDXR/pET-24b重组原核表达载体;经双酶切、PCR及测序鉴定阳性克隆;在大肠杆菌BL21中用IPTG诱导表达。亲和层析纯化重组TgDXR蛋白,并对该蛋白的生物学特性和酶的动力学活性进行分析。结果从弓形虫RH株的cDNA和基因组DNA中分别扩增出长度为1 542bp和5 464bp的TgDXR片段,成功构建重组质粒;SDS-PAGE结果表明,目的基因在大肠杆菌中高效表达。重组蛋白的相对分子量约50kDa。酶活性实验显示,Mg2+、Mn2+是最佳金属螯合离子,反应最佳pH值为7.5,该酶活性抑制剂膦胺霉素(Fosmidomycin)对该酶蛋白的IC50为0.52μmol/L。结论 RH株刚地弓形虫TgDXR可在原核表达系统中高效表达,该重组蛋白具有生物学活性,并有望作为弓形虫特异性药物靶标。更多还原

【Abstract】 To clone,express and purify of the 1-deoxy-D-xylulose-5-phosphate reductoisomerase(DXR)gene from Toxoplasma gondii RH strain and analyze its biological characters,the DXR of T.gondii(TgDXR)was amplified from cDNA and genomic DNA by PCR.The mature domain of TgDXR was cloned into the pET-24 bvector to construct the prokaryotic expression vector TgDXR/pET-24 b,which was efficiently expressed in E.coli BL21(RIL).Recombinant TgDXR was purified by Ni-NTA affinity chromatography.The biological properties of this enzyme were characterized and enzyme activity/fosmidomycin(Fos)inhibition assay was tested.Result showed that TgDXR in the non-mevalonate isoprene biosynthesis pathway was a potential target for developing Toxoplasma killing drugs.更多还原