| 代海丽,王艳,孙权,时连瑞.靶向柯萨奇病毒B3 siRNA的设计与筛选[J].济宁医学院学报,2020,43(1):14-18 |
| 靶向柯萨奇病毒B3 siRNA的设计与筛选 |
| Design and screening of small interfering RNA targetting to coxsackievirus B3 |
| 投稿时间：2020-01-09 |
| DOI：10.3969/j.issn.1000-9760.2020.01.004 |
| 中文关键词: 柯萨奇B3病毒;RNA干扰;siRNA |
| 英文关键词: Coxsackievirus B3;RNA interference;Small interfering RNA |
| 基金项目:山东省医药卫生科技发展计划项目（2018WS210） |
| 作者 | 单位 | | 代海丽 | 山东英才学院医学院, 济南 250101 | | 王艳 | 山东英才学院医学院, 济南 250101 | | 孙权 | 山东英才学院医学院, 济南 250101 | | 时连瑞 | 山东英才学院医学院, 济南 250101 |
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| 中文摘要: |
| 目的 针对柯萨奇病毒B3（CVB3）基因筛选抑制效率较高的siRNA新序列，同时探究顺式作用复制元件（CRE）以外的2C区是否是设计siRNA序列的有效靶点。方法 在CVB3的基因组中以3D、3C、2C、2A、VP3、VP2和VP1区为靶点设计新的siRNA序列，在HeLa细胞中转染siRNA序列，转染12h后，进行CVB3感染。感染病毒36h后收毒，用TCID50、MTT、Western blot和real-time RT-PCR 4种方法从不同水平上检测本研究设计筛选的siRNA序列在CVB3复制方面的抑制效果。结果 设计的siRNA序列共11条，其中5条对CVB3的复制在细胞的死亡率、病毒的相关致病性、RNA分子水平和蛋白表达水平4个方面均表现出了较好抑制效果。其中siRNA-5对CVB3复制的抑制率达到了84%，本序列是针对2C区CRE以外的部分设计的。结论 本文发现了5条新的抑制率较高的siRNA靶序列，同时首次证明CRE以外2C区也是设计siRNA序列的有效靶点。 |
| 英文摘要: |
| Objective To investigate several effective siRNAs against different regions of the CVB3 genome for CVB3 silencing in vitro and explore whether the sequences except CRE within the 2C region was potential targets for CVB3-specific siRNAs design.Methods In this study,eleven siRNAs were designed to target seven distinct regions of the CVB3 genome including 3D,3C,2C,2A,VP3,VP2 and VP1.All of the siRNAs were individually transfected into HeLa cells,which were subsequently infected with CVB3 12h later.After 36h the virus were harvested and the impacts of RNA interference (RNAi) on viral replication were evaluated using five measures:50% tissue culture infectious dose (TCID50),3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide (MTT) assay,Western blot,and real-time RT-PCR.Results Five of the eleven siRNAs were highly efficient at inhibiting viral replication in the levels of cell viability,cytopathic effect,infectious viral particles,viral protein expression and viral RNA.This was especially true for siRNA-5,which targeted the ATPase 2C.The viral RNA recovered from samples with siRNA-5 treatment was decreased by 84%.Conclusion Our results revealed five effective siRNAs for CVB3 silencing and provided evidence that the sequences except CRE within the 2C region may also be potential targets for CVB3-specific siRNAs design. |
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