Hainan Key Lab of Tropical Animal Reproduction & Breeding and Epidemic Disease Research (Construction Period), Animal Genetic Engineering Key Lab of Haikou, College of Agriculture, Hainan University, Haidian Island, Haikou 570228, People's Republic of China.
Curr Microbiol. 2011 May;62(5):1355-62. doi: 10.1007/s00284-010-9863-3. Epub 2011 Jan 12.
RNA interference (RNAi) provides a powerful promising approach to inhibit viral infection specifically. To explore the possibility of using RNAi as a strategy against HEV infection, which is a serious public health problem in developing countries of Asia, Middle East, Africa, and in Mexico, after the fusion protein expression plasmids pEGFP-ORF3 which contain the EGFP reporter gene and SHEV ORF3 as silencing target, were constructed; EGFP-ORF3 fusion protein expressing HEK 293 cell lines were established; and four siRNAs targeting ORF3 gene were designed, synthesized, and used to transfect the stable cell lines. At 24, 48, and 72 h after transfection, flow cytometry, real-time quantitative PCR, and Western blot were used to assess the expression level of ORF3. The results demonstrated that specific siRNAs which are sequence dependant effectively inhibited mRNA accumulation and protein expression of SHEV ORF3 in HEK 293 cells. These findings provide useful information for the development of RNAi-based prophylaxis and therapy for SHEV infection.
RNA 干扰（RNAi）为特异性抑制病毒感染提供了一种强大而有前途的方法。为了探索 RNAi 作为一种策略来对抗戊型肝炎病毒（HEV）感染的可能性，HEV 是亚洲、中东、非洲和墨西哥等发展中国家的一个严重公共卫生问题。在构建含有 EGFP 报告基因和 SHEV ORF3 的融合蛋白表达质粒 pEGFP-ORF3 之后；建立了表达 EGFP-ORF3 融合蛋白的 HEK 293 细胞系；并设计、合成了针对 ORF3 基因的四个 siRNA 用于转染稳定细胞系。转染后 24、48 和 72 小时，通过流式细胞术、实时定量 PCR 和 Western blot 评估 ORF3 的表达水平。结果表明，序列依赖性特异性 siRNA 有效抑制了 HEK 293 细胞中 SHEV ORF3 的 mRNA 积累和蛋白表达。这些发现为基于 RNAi 的预防和治疗 SHEV 感染提供了有用的信息。
