脓肿分枝杆菌硝基还原酶基因的克隆表达及生物信息学分析(英文)

Cloning,expression,and bioinformatics analysis of the Mycobacterium abscessus nitroreductase gene

目的克隆表达脓肿分枝杆菌中的硝基还原酶(nitroreductase,Nrd)基因,并对其对应的蛋白质进行生物信息学分析。方法利用PCR技术从脓肿分枝杆菌中扩增Nrd的全基因序列,采用原核表达系统进行体外表达。采用生物信息学方法对Nrd蛋白的理化性质、结构和功能等重要参数进行了预测和分析,对其三级结构进行了同源建模。结果从脓肿分枝杆菌中扩增出660bp的目的片段,体外表达获得了大小约为30kDa的带有His标签的重组蛋白。生物信息学分析显示Nrd蛋白有219个氨基酸组成,理论分子量和等电点分别为24.38kDa和6.52,是一种不稳定的亲水性蛋白质(不稳定系数为54.52,总平均亲水性为-0.244);二级结构以α-螺旋、β-折叠和无规卷曲为主要构件;该蛋白的氨基酸序列与蛋白质结构数据库中3gr3蛋白A链的相似性最高为60%,以3gr3蛋白A链为模板构建了Nrd蛋白的三维结构分子模型。结论成功表达并纯化了脓肿分枝杆菌中的Nrd蛋白。预测结果显示该蛋白是硝基还原酶家族成员之一,这提示Nrd蛋白在脓肿分枝杆菌中可能执行还原对硝基苯甲酸的功能。

The purpose of this study was to clone and express the nitroreductase （Nrd） gene of Mycobacteriurn abscessus （M. abscessus） and perform bioinformatics analysis of its corresponding protein. The fragment of the Nrd gene of M. abscessus was amplified using PCR and expressed in vitro using the prokaryotic expression system. The bioinformatics analysis was used to predict and analyze the important parameters of the Nrd protein, such as the physical and chemical properties, protein struc- ture, and its functions. The tertiary structure of the Nrd protein was also investigated. A 660 bp target fragment was amplified from M. abscessus, and a His-tagged recombinant protein with a molecular weight of about 30 kDa was obtained after in vitro expression. The bioinformatics analysis revealed that the Nrd protein was composed of 219 amino acids, and that its theoretical molecular weight was 24.38 kDa and its isoelectric point was 6.52. The Nrd protein was an unstable hydrophilic protein （the instability coefficient was 54.52, the grand average of hydropathicity was -0. 244）. The secondary structure of the Nrd protein was mainly composed of a-helixes, l]-sheets, and random coils. The amino acid sequence of the protein had the highest similari- ty （60%） with the A chain of the 3gr3 protein in the Protein Data Bank. Therefore, the molecular model of the three-dimen- sional structure of the Nrd protein was constructed using the 3gr3 protein A chain as a template. The Nrd protein of M. ab scessus was successfully expressed and purified. The bioinformatics analysis predicted that the Nrd protein is a member of the Nrd family and should have nitrobenzoic acid-reducing functions.