基础医学与临床 ›› 2017, Vol. 37 ›› Issue (1): 56-61.

• 研究论文 • 上一篇    下一篇

EGS-C67抑制HCV基因表达及其抗病毒活性的测定

张成成1,李小英2,伍婧茹1,张文军1   

  1. 1. 广东药学院
    2. 广东药科大学病原生物学与免疫学系
  • 收稿日期:2016-02-18 修回日期:2016-06-13 出版日期:2017-01-05 发布日期:2016-12-30
  • 通讯作者: 张文军 E-mail:tzhangwj@126.com
  • 基金资助:
    中国博士后科研基金资助项目

Inhibiting effect of EGS-C67 on HCV gene expression and determination of its antiviral activity

  • Received:2016-02-18 Revised:2016-06-13 Online:2017-01-05 Published:2016-12-30
  • Contact: Wen-Jun ZHANG E-mail:tzhangwj@126.com

摘要: 目的 基于丙肝病毒(HCV)基因组保守区5’UTR的序列,设计一段相应的外部引导序列(EGS),鉴定其体外靶向切割活性及胞内抗病毒作用。方法 用计算机软件对HCV 基因保守区的5’非翻译区(5’UTR)序列与结构进行分析,选择针对HCV第67位胞嘧啶C与第68位鸟嘌呤G之间的位点,作为设计的靶位,设计相应的EGS(EGS-C67), 将该EGS导入HCV感染的Huh7.5.1细胞,通过Northern blot及Western blot试验检测HCV基因的表达,通过荧光定量PCR检测EGS-C67的抗病毒活性。结果 体外切割试验表明EGS-C67对HCV RNA片段具有明显的靶向引导活性。在宿主细胞内EGS-C67可显著抑制HCV基因的表达,可使Huh7.5.1细胞培养上清中HCV的滴度降低约150倍(p<0.01)。结论 EGS-C67作为一种新型抗HCV核酸分子,其成功构建为抑制HCV增殖提供了一种新的抗病毒策略。

关键词: 丙型肝炎病毒, 外部引导序列 , 抗病毒

Abstract: Objective To explore advantage of RNA guiding activity in vitro cleavage assay and intracellular antiviral effects for hepatitis C, basing on one External Guide Sequence (EGS)sequence of the conserved region of 5’UTR of HCV genome. Methods We analyzed the sequence and structure of the conserved region of 5’UTR of HCV genome with computer software and designed one External Guide Sequence (EGS) which was direct at the potential targeting site (C67-G68). We tested the gene expression of HCV in Northern blot and Western blot and its antiviral activity by fluorescence quantitative PCR in the Huh7.5.1 cell which was transfected by EGS-C67. Results EGS-C67 had obvious advantage of RNA guiding activity in vitro cleavage assay. Our results indicated that EGS-C67could obviously inhibit the gene expression of HCV and inhibit the proliferation of HCV and down-regulate the titer of HCV about 150 times(p<0.01). Conclusions EGS-C67 , the new antiviral RNA has notably antiviral activity in cultured cells , thus could provide a new promising approach for development of anti-HCV strategy.

Key words: HCV, external guide sequence , antiviral

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