碩士 / 國立宜蘭大學 / 園藝學系碩士班 / 101 / Epinephelus spp. (i.e., groupers) are aquaculture products that are critical to the Taiwanese economy. Recently, the grouper iridovirus (GIV) infection has caused considerable fatality rates in groupers. A commercially available inactive vaccine administered through intraperitoneal injection currently provides effective protection to groupers; however, to combat frequent occurrences of the GIV epidemic, a more convenient and economical vaccine strategy is required. Producing animal therapeutic proteins in plants can minimize the risk of latent pathogenic contamination in animals. The major capsid protein (MCP) composing the GIV capsid accounts for approximately 40% of the total virus protein. The MCP is a potential antigen candidate because of its high conservation among various species.
This study cloned the MCP gene of the GIV onto plant expression vectors activated by the CaMV 35S promoter. The Agrobacterium tumefaciens species of LAB4404 and EHA105 were transfected to leaf lettuce for expression. In the experiment, shoot regeneration was induced using the lettuce cotyledon as explants. The plants transformed from LAB4404 for transfection were selected using kanamycin and possessed a regeneration rate of approximately 3.5%. Polymerase chain reaction analysis was conducted on the DNA extracted from the plants that were regenerated using the transgenic pKcMCP plasmid. Testing on 35 plants showed that 24 exhibited positive reactions, verifying that the MCP gene could be inserted into the lettuce genome. The Western blot test results showed that 21 plants expressed the target protein at the 50 kDa position, showing that transgenic plants can translate the MCP protein with a successful transformation rate of approximately 2.1%. In addition, the regenerated plants grew normally and were fertile. The NPTII selectable marker gene was detected in the T1 plant, indicating that the gene was passed on to the offspring. The test results showed that lettuce is a potential bioreactor for expressing the MCP. Future studies can apply the MCP to feed additives and conduct oral animal experiments to validate its protection effectiveness.
| Identifer | oai:union.ndltd.org:TW/101NIU00378004 |
| Date | January 2013 |
| Creators | Wei-Ting Guo, 郭瑋婷 |
| Contributors | Jinn-Chin Yiu, 尤進欽 |
| Source Sets | National Digital Library of Theses and Dissertations in Taiwan |
| Language | zh-TW |
| Detected Language | English |
| Type | 學位論文 ; thesis |
| Format | 71 |
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