碩士 / 國立成功大學 / 生物科技研究所碩博士班 / 98 / Epinephelus coioides (E. coioides, orange-spotted grouper) is an important cultivated fish that has value of economy in Taiwan. Infecting with diseases was causing huge loss in aquatic industry. To uncover the response of fish that have encountered pathogens is necessary and urgent.
Many cytokines involved in innate immunity and work as the communication and regulation of the immune system. Tumor necrosis factor alpha (TNF-α) is an important molecule, which was secreted in primary inflammatory reaction. TNF-α can activate the immunocytes and the innate immune network system. There are more than one of TNF-α molecular were found in some fish, it is quite different with human and vertebrate animal we usually known. In this study, a novel TNF-α (TNF-α2) gene of E. coioides has been cloned. The length of mRNA is 1510 bp and the predicted protein has 240 amino acids. The identity between the ORF region of TNF-α1 and TNF-α2 was 52 % in uncleartide and 35 % in amino acid sequence. In order to identify the mRNA expression in various organs, the groupers inject intraperitoneally with LPS (40 mg / kg of body weight). Then organs are collected after 24 h and evaluated the expression of TNF-α mRNA. The expressional pattern of TNF-α1 and TNF-α2 are dissimilar. In larval stage, all two TNF-α perhaps are activatable by raise of Vibrio. Also these two TNF-α have different expression pattern under stimulation of bacteria. These results show that two TNF-α molecules may have different functions. However, the functions, distribution in different tissues, and regulation by different pathogens or stimulators of these two TNF molecules were not sufficient researched. In order to understand the difference between TNF-α1 and TNF-α2 in regulation mechanism, the promoter region of TNF-α genes have been cloned and analyzed. The transcription factor binding sites of TNF-α1 and TNF-α2 are dissimilar. In the future, the deletion assay of promoter region will proceed in cell line and the polyclonal antibody of TNF-α will be prepared to detect TNF-α expression in protein level. These can help us understand the roles of TNF-α1 and TNF-α2 in immune response.
| Identifer | oai:union.ndltd.org:TW/098NCKU5111119 |
| Date | January 2010 |
| Creators | Szu-YinWu, 吳思穎 |
| Contributors | Han-You Lin, 林翰佑 |
| Source Sets | National Digital Library of Theses and Dissertations in Taiwan |
| Language | zh-TW |
| Detected Language | English |
| Type | 學位論文 ; thesis |
| Format | 92 |
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