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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。
Objective To clone and express the nitroreductase (Nrd) gene in Mycobacterium abscess and analyze its corresponding protein bioinformatics. Methods The whole genome sequence of Nrd was amplified from Mycobacterium abscess by PCR and expressed in vitro using prokaryotic expression system. Bioinformatics methods were used to predict and analyze the important parameters such as physicochemical properties, structure and function of Nrd protein. The homology modeling of its tertiary structure was carried out. Results The target fragment of 660 bp was amplified from Mycobacterium abscess and the recombinant protein with His tag of about 30 kDa was obtained in vitro. Bioinformatics analysis showed that the Nrd protein has 219 amino acids with theoretical molecular weight and isoelectric point of 24.38 kDa and 6.52, respectively. It is an unstable hydrophilic protein (instability coefficient is 54.52, total average hydrophilicity is - 0.244). The secondary structure consisted of α-helix, β-sheet and random coil. The amino acid sequence of the secondary structure was 60% similarity to the 3gr3 A chain in the protein structure database, The template constructed a three-dimensional structural molecular model of Nrd protein. Conclusion The Nrd protein in Mycobacterium abscess was successfully expressed and purified. The prediction shows that the protein is a member of the nitro-reductase family, suggesting that the Nrd protein may perform the function of reducing p-nitrobenzoate in M. abscess.