在大肠杆菌中表达的重组1型登革病毒NS5蛋白具有RNA依赖性RNA聚合酶活性。
Recombinant dengue type 1 virus NS5 protein expressed in Escherichia coli exhibits RNA-dependent RNA polymerase activity.
作者信息
Tan B H, Fu J, Sugrue R J, Yap E H, Chan Y C, Tan Y H
机构信息
Institute of Molecular and Cell Biology, National University of Singapore.
出版信息
Virology. 1996 Feb 15;216(2):317-25. doi: 10.1006/viro.1996.0067.
The complete nonstructural NS5 gene of dengue type 1 virus, Singapore strain S275/90 (D1-S275/90) was expressed in Escherichia coli as a glutathione S-transferase (GST) fusion protein (126 kDa). The GST-NS5 fusion protein was purified and the recombinant NS5 protein released from the fusion protein by thrombin cleavage. The recombinant NS5 had a predicted molecular weight of 100 kDa and reacted with antiserum against D1-S275/90 virus in Western blot analysis. The purified recombinant NS5 protein possessed RNA-dependent RNA polymerase activity which was inhibited (>99%) by antibodies against the recombinant NS5 protein. The polymerase product was shown to be a negative-stranded RNA molecule, of template size, which forms a double-stranded complex with the template RNA.
1型登革病毒新加坡株S275/90（D1-S275/90）的完整非结构NS5基因在大肠杆菌中作为谷胱甘肽S-转移酶（GST）融合蛋白（126 kDa）表达。纯化GST-NS5融合蛋白，并通过凝血酶切割从融合蛋白中释放重组NS5蛋白。重组NS5的预测分子量为100 kDa，在蛋白质免疫印迹分析中与抗D1-S275/90病毒的抗血清发生反应。纯化的重组NS5蛋白具有RNA依赖性RNA聚合酶活性，该活性被抗重组NS5蛋白的抗体抑制（>99%）。聚合酶产物显示为模板大小的负链RNA分子，它与模板RNA形成双链复合物。
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