梅毒螺旋体硫氧还蛋白的原核表达、纯化及抗氧化活性分析  

Prokaryotic Expression, Purification and Anti-Oxidative Activity Analysis of Treponema pallidum Thioredoxin

作　　者：李冉辉[1,2] 杨杰 陈飞[1,3] 胡楚晖 赵飞骏[1,2] 

机构地区：[1]南华大学病原生物学研究所,特殊病原体放空湖南省重点实验室,衡阳421001 [2]湖南省分子靶标新药研究协同创新中心,衡阳421001 [3]南华大学医学院卓越医师班,衡阳421001

出　　处：《基因组学与应用生物学》2018年第1期280-285,共6页Genomics and Applied Biology

基　　金：国家自然科学基金项目(81373230);湖南省高校创新平台开放基金(No.13K081);湖南省科技计划项目(No.2015JC3087);南华大学大学生创新课题(2015-28)共同资助

摘　　要：本研究利用生物信息学方法预测梅毒螺旋体（Treponema pallidum,Tp）Trx蛋白的生物学性质。Trx蛋白无信号肽,定位在细胞质。二级结构无规卷曲占41.9%,β片层为55%,使用枯草芽胞杆菌Trx蛋白结构（PDB：2gzz.1.A）进行同源建模,获得Trx蛋白三级结构。根据TpTrx基因序列设计引物,以TpDNA为模板,PCR扩增得到大小为318 bp的Trx基因,然后将其连接到pET30a构建重组质粒pET30-Trx,将该重组质粒转化大肠杆菌BL21（DE3）,得到重组菌大肠杆菌。利用IPTG诱导目的蛋白表达,该质粒编码的重组Trx蛋白分子量约为16 kDa。使用Ni^（2＋）亲和层析纯化获得重组Trx蛋白,发现Trx蛋白具有二硫键还原酶活性。利用菌落计数法观察重组大肠杆菌在H_2O_2胁迫下的存活率,发现能表达Trx蛋白的大肠杆菌存活率高于对照。本研究利用生物信息学方法分析了TpTrx蛋白的性质,并发现它具有抗氧化功能,本研究为解析Tp的抗氧化机制提供实验依据。In this study, the biological characters of Trx protein from Treponema pallidum were predicted by bioinformatics. TpTrx protein had no signal peptides and was located in the cytosol. The percentages of random coil was 41.9% and beta sheet was 55%, respectively. Three dimensional structure of Trx was built by homology modeling using the structure of Bacillus subtilis Trx protein（PDB： 2 gzz.1.A） as template. The specific primers were designed according to the sequence of TpTrx gene. 318 bp TpTrx gene was obtained by PCR using TpDNA as template, which was connected to pET30 a to generate the recombinant plasmid pET30 a-Trx. The pET30 a-Trx was transformed into E. coli BL21（DE3） to create recombinant E. coli BL/pET30-Trx. The expression of Trx protein was induced by IPTG, and the molecular mass of recombinant was 16 kDa. Then it was purified by Ni^（2＋）-affinity chromatography, and the Trx protein had activity of disulfide reductase. This research observed survival ratios of the E. coli under H_2O_2 stress by colony counting method, which indicated that the survival ratios of the E. coli in Trx protein were higher than the contrast. This study analyzed the property of TpTrx protein by bioinformatic method to find the antioxidan function and and it might provide experimental evidence for exploring antioxidant mechanisms of Tp.

关 键 词：梅毒螺旋体 生物信息学 硫氧还蛋白 基因克隆 

分 类 号：R377.1[医药卫生—病原生物学]