碩士 / 國立海洋大學 / 水產生物技術研究所 / 87 / Infectious pancreatic necrosis virus (IPNV) is one kind of the contagious and widespread fish diseases. IPNV belongs to the Birnaviridae family of virus, which has bisegmented ds-RNA genomes and often causes sever damage to the aquaculture industry in Taiwan.
The purpose of this study was to investigate whether the expression of hammerhead ribozyme in persistent Tilapia ovary-2 (TO-2) cells can protect the cells from viral replication. Seven target sites of hammerhead ribozyme were selected according to secondary structure of VP2 cDNA sequence predicted by GCG foldRNA program. Their target sites were 56, 78, 114, 187, 209, 227 and 255 respectively. The dsDNA of hammerhead ribozyme was synthesized by PCR and constructed into pcDNA3 and pTracer-CMV2 under cytomegalovirus and T7 promoter driving. VP2 cDNA was constructed into pCMV-luc upstream luciferase gene. When ribozyme and VP2-luciferase fusion gene co-expressed in transcription /translation coupled system, the decrease of luciferase activity was used to determine the inhibition activity of ribozyme. The result showed that the ribozyme RZ209 could inhibit the expression of VP2-luciferase fusion protein up to 35%. The plasmids p56, p78, p114, p187, p209, p227 and p255 were introduced into persistent TO-2 cells by lipofection. Five stable transfected subclones were selected for each ribozyme, respectively. After the northern blotting, most transfected cells could express hammerhead ribozyme especially subclones of RZ209. By using IPNV-E1S polyclonal antibody in the western blotting, the viral protein decreased in some ribozyme-expressing clones comparing to control clone after 24hr incubation. The ribozyme-expressing clones 56C, 56A, 78C, 78D, 114F, 114C, 209A, 209B, 209C, 209E, 209F, 227A, 227C, 227E, 255E and 255F had the inhibition activity of viral protein expression. All subclones of ribozyme RZ209 had inhibition activity of viral protein. In addition, all subclones of ribozyme RZ209 had less the viral cytopathic effect (CPE) compared to the control group. These results indicate that the expression of ribozyme RZ209 can inhibit viral protein expression in vitro and ex vivo.
| Identifer | oai:union.ndltd.org:TW/087NTOU0613009 |
| Date | January 1999 |
| Creators | Yieh-Her Hsiao, 蕭亦何 |
| Contributors | Jen-Leih Wu, 吳金洌 |
| Source Sets | National Digital Library of Theses and Dissertations in Taiwan |
| Language | zh-TW |
| Detected Language | English |
| Type | 學位論文 ; thesis |
| Format | 88 |
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