重组西尼罗病毒NS5融合蛋白依赖RNA的RNA聚合酶特性鉴定

西尼罗病毒(West Nile virus, WNV)非结构蛋白NS5是病毒基因组复制的关键蛋白.以病毒全长cDNA克隆为模板，PCR扩增获得NS5的RNA依赖的RNA聚合酶(RdRp)活性区（NS5pol）及该蛋白完整的编码序列（NS5F），分别克隆于原核表达载体pET-28a 并转化至大肠杆菌E.coliBL21（DE3）中诱导表达.表达的可溶性重组蛋白经Ni柱亲和层析纯化后进行SDS-PAGE和Western印迹鉴定.结果显示,二者均为病毒特异蛋白,且纯度均在90%以上.进一步的体外RdRp分析及EMSA的结果表明，NS5pol和NSF5均有较高的RdRp活性，且该活性具有RNA模板序列和二级结构的特异性.获得的具有RdRp活性的NS5pol和NS5F为西尼罗病毒基因组复制相关元件的研究奠定了基础.

Identification of RNA-dependent RNA Polymerase Activity of Recombinant West Nile Virus NS5 Protein

The NS5 protein of West Nile virus (WNV) is critically involved in the replication of viral RNAs. The full-length NS5 (NS5F) and the NS5 fragment encoding the RdRp, RNA-dependent RNA polymerase, domain (NS5pol) were PCR amplified from a full-length WNV cDNA clone and cloned into the prokaryotic expression vectors pET-28a, respectively. The NS5pol and NS5F recombinant plasmids were introduced into E.coli BL21(DE3) and induced with 0.2 mmol/L IPTG at 15 ℃ for transgene expression. SDS-PAGE analysis demonstrated that both proteins were produced in soluble forms and were able to be purified with nickel affinity chromatography at greater than 90% purities. Purified NS5pol and NS5F were reactive to WNV and 6×His tag rabbit polyclonal antibodies as determined by Western blotting. EMSA and in vitro RdRp assay have shown that both NS5pol and NS5F exhibited potent RdRp activities, which was dependent on the RNA sequences and secondary structures. Our discoveries may serve as a foundation to further identification of critical elements in WNV genome that are essential for viral RNA synthesis.