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目的原核表达、纯化肺炎链球菌(Streptococcus pneumoniae)Tpx蛋白并测定其活性。方法利用生物信息学方法分析Tpx蛋白的生物学特性。设计特异性引物,PCR扩增Tpx基因并对进行双酶切,酶切片段连接到表达质粒pET28a。将重组质粒转化大肠埃希菌BL21(DE3),利用IPTG诱导重组Tpx蛋白表达,使用Ni 2+亲和层析进行纯化,采用氧化铁二甲酚橙法(FOX)测定其活性。结果生物信息学分析Tpx蛋白无信号肽,无跨膜结构域。α-螺旋、延伸主链、β-转角和无规卷曲的含量分别为30%、27.6%、11.6和30.7%。晶体结构在蛋白结构数据库(PDB)中的登陆号为1psq.1.A。PCR扩增Tpx基因片段大小为492bp,连接到pET28a后获得重组质粒pET28a-Tpx。重组质粒转化DE3后经IPTG诱导表达分子质量单位约为22ku的重组蛋白,经亲和层析得到高纯度的重组Tpx蛋白,该蛋白能分解H2O2和叔丁基过氧化氢(t-BHP)。结论成功构建pET28a-Tpx质粒,表达的重组Tpx蛋白具有氧化酶活性,为研究肺炎链球菌的抗氧化机制奠定了实验基础。
Objective To prokaryotic express and purify Tpx protein of Streptococcus pneumoniae and determine its activity. Methods Bioinformatics methods were used to analyze the biological characteristics of Tpx protein. Specific primers were designed to amplify the Tpx gene by PCR and double digestion. The digested fragment was ligated into the expression plasmid pET28a. The recombinant plasmid was transformed into Escherichia coli BL21 (DE3), the recombinant Tpx protein was induced by IPTG, purified by Ni2 + affinity chromatography, and its activity was determined by the method of iron oxide xylenol orange (FOX). Results Bioinformatics analysis of Tpx protein without signal peptide, no transmembrane domain. The contents of α-helix, extended backbone, β-turn and random coil were 30%, 27.6%, 11.6 and 30.7%, respectively. The crystal structure has a login number of 1 psq.1.A in the Protein Structure Database (PDB). The size of the fragment of Tpx gene amplified by PCR was 492bp, and the recombinant plasmid pET28a-Tpx was obtained after ligated to pET28a. Recombinant plasmids were transformed into DE3 and induced by IPTG to express recombinant protein with a molecular weight of about 22ku. The recombinant Tpx protein was purified by affinity chromatography, which can decompose H2O2 and tert-butyl hydroperoxide (t-BHP). Conclusion The plasmid pET28a-Tpx was successfully constructed and the expressed recombinant Tpx protein possessed the activity of oxidase, which laid the experimental foundation for studying the mechanism of anti-oxidation of Streptococcus pneumoniae.