Department of Microbiology, University of São Paulo, Brazil.
J Virol Methods. 2010 Aug;167(2):186-92. doi: 10.1016/j.jviromet.2010.04.003. Epub 2010 Apr 23.
The dengue virus NS1 protein has been shown to be a protective antigen under different experimental conditions but the recombinant protein produced in bacterial expression systems is usually not soluble and loses structural and immunological features of the native viral protein. In the present study, experimental conditions leading to purification and refolding of the recombinant dengue virus type 2 (DENV-2) NS1 protein expressed in Escherichia coli are described. The refolded recombinant protein was recovered as heat-stable soluble dimers with preserved structural features, as demonstrated by spectroscopic methods. In addition, antibodies against epitopes of the NS1 protein expressed in eukaryotic cells recognized the refolded protein expressed in E. coli but not the denatured form or the same protein submitted to a different refolding condition. Collectively, the results demonstrate that the recombinant NS1 protein preserved important conformation and antigenic determinants of the native virus protein and represents a valuable reagent either for the development of vaccines or for diagnostic methods.
登革热病毒 NS1 蛋白已被证明在不同的实验条件下是一种保护性抗原，但在细菌表达系统中产生的重组蛋白通常不可溶，并失去了天然病毒蛋白的结构和免疫学特征。本研究描述了导致在大肠杆菌中表达的重组登革热病毒 2 型（DENV-2）NS1 蛋白的纯化和重折叠实验条件。通过光谱方法证明，重折叠的重组蛋白以热稳定的可溶性二聚体形式回收，保留了结构特征。此外，针对真核细胞中表达的 NS1 蛋白表位的抗体识别在大肠杆菌中表达的重折叠蛋白，但不识别变性形式或在不同重折叠条件下的相同蛋白。总的来说，这些结果表明，重组 NS1 蛋白保留了天然病毒蛋白的重要构象和抗原决定簇，是开发疫苗或诊断方法的有价值的试剂。
