College of Agriculture, Hainan Key Laboratory of Tropical Animal Reproduction & Breeding and Epidemic Disease Research, Animal Genetic Engineering Key Laboratory of Haikou, Hainan University, Haidian Island, Haikou, People's Republic of China.
Folia Microbiol (Praha). 2013 Jul;58(4):335-42. doi: 10.1007/s12223-012-0212-8. Epub 2012 Nov 29.
Avian influenza has emerged as a devastating disease and may cross species barrier and adapt to a new host, causing enormous economic loss and great public health threats, and non-structural protein 1 (NS1) is a multifunctional non-structural protein of avian influenza virus (AIV) that counters cellular antiviral activities and is a virulence factor. RNA interference (RNAi) provides a powerful promising approach to inhibit viral infection specifically. To explore the possibility of using RNAi as a strategy against AIV infection, after the fusion protein expression plasmids pNS1-enhanced green fluorescent protein (EGFP), which contain the EGFP reporter gene and AIV NS1 as silencing target, were constructed and NS1-EGFP fusion protein expressing HEK293 cell lines were established, four small interfering RNAs (siRNAs) targeting NS1 gene were designed, synthesized, and used to transfect the stable cell lines. Flow cytometry, real-time quantitative polymerase chain reaction, and Western blot were performed to assess the expression level of NS1. The results suggested that sequence-dependent specific siRNAs effectively inhibited mRNA accumulation and protein expression of AIV NS1 in vitro. These findings provide useful information for the development of RNAi-based prophylaxis and therapy for AIV infection.
禽流感已成为一种毁灭性疾病，可能跨越物种屏障并适应新宿主，造成巨大的经济损失和严重的公共卫生威胁，而非结构蛋白 1（NS1）是禽流感病毒（AIV）的一种多功能非结构蛋白，可对抗细胞抗病毒活性，是一种毒力因子。RNA 干扰（RNAi）为特异性抑制病毒感染提供了一种强大的有前途的方法。为了探索将 RNAi 用作抗 AIV 感染策略的可能性，构建了包含 EGFP 报告基因和 AIV NS1 作为沉默靶标的融合蛋白表达质粒 pNS1-增强型绿色荧光蛋白（EGFP），并建立了表达 NS1-EGFP 融合蛋白的 HEK293 细胞系，设计、合成了针对 NS1 基因的四个小干扰 RNA（siRNA），并用于转染稳定细胞系。通过流式细胞术、实时定量聚合酶链反应和 Western blot 评估 NS1 的表达水平。结果表明，序列依赖性特异性 siRNA 可有效抑制 AIV NS1 的体外 mRNA 积累和蛋白表达。这些发现为基于 RNAi 的 AIV 感染预防和治疗的发展提供了有用信息。
