碩士 / 國立臺灣海洋大學 / 水產養殖學系 / 101 / Small interfering RNAs (siRNAs), mediators of a process of sequence-specific gene silencing called RNA interference (RNAi). In recent years, it have been studied in detail and applied as a viral disease control strategy. Here, we describe an antiviral activity of a siRNA that targets the major capsid protein (MCP) gene of grouper iridovirus (GIV) of Ranavirus, and according to various designed criterias, synthesized five siRNAs which were siR376, siR588, siR1133, siR807 and siR1270. We assessed the effect of GIV inhibition by these 5 siRNAs through in vitro and in vivo test. First we tested two types of grouper cell lines, grouper fin (GF-1) and swim bladder (SB) transfection efficiency with fluorescently labled siRNA, and the results displayed that transfection efficiency at the final siRNA concentration 100 nM were 95.3% and 75.5%, respectively. Following, GF-1 cell line was co-transfected with siRNAs and plasmid of pcDNA3-EGFP-stopMCP to test five siRNAs inhibited the expression of MCP gene by plasmid, in which the inhibitory effect of siR376 50% for the best. Following to analyze five siRNAs inhibition of viral replication by GIV infected two cell lines, that product cytopathic effect and expression of MCP, and siR376 that reduced cytopathic effect and expression of MCP gene was best than others, following was siR807. Due to siRNA had no significant effect with PNP gene, confirmed that siRNA targeted MCP has sequence-specific gene silencing. Finally, we used DOTAP for siRNA delivery to test antivral effect in vivo. The result indicated that both negative control groups and group of virus were death in full at the 7 days post challenge, and the cumulative mortality of siR376 is 66.7% and siR807 is 60.0%. Although these siRNAs were protective, but no significance was found. Follow up we should tyr to use of different doses and re-assessment of siRNA antiviral effect in vivo.
| Identifer | oai:union.ndltd.org:TW/101NTOU5086024 |
| Date | January 2013 |
| Creators | Wei-Ren Lin, 林韋任 |
| Contributors | Hsin-Yiu Chou, 周信佑 |
| Source Sets | National Digital Library of Theses and Dissertations in Taiwan |
| Language | zh-TW |
| Detected Language | English |
| Type | 學位論文 ; thesis |
| Format | 51 |
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