碩士 / 國立臺灣大學 / 漁業科學研究所 / 101 / Grouper (Epinephelus spp.) are highly economic value farmed fish in Taiwan. In addition to the farming competition from China and Southeast Asia countries, the high mortality caused by iridovirus infection became one of the thorniest issues in grouper aquaculture. Therefore, the study of mechanism involving in the iridovirus infection is the most important current events. Using cDNA microarray assay, our laboratory had identified 21 immediate early genes from grouper iridovirus including GIV030L. The GIV030L contains 696 nucleotides and is composed of 231 amino acids which encode a 25.9 kDa protein. According NCBI database comparative sequence analysis, GIV030L represents a tumor necrosis factor receptor (TNFR) homolog with a TNF ligand binding extracellular domain and a transmembrane domain, but lacking a intracellular domain. This incomplete TNFR-like viral protein may serve as a decoy and play a role to help iridovirus against host TNF attacking system.
In this study, GIV030L was constructed into pcDNA3CF eukaryotic expression vector and was transfected into HeLa cells. However, the overexpressed GIV030L can not prevent the HeLa cells from apoptosis trigged by UV-irradiation mediated intrinsic apoptosis pathway in the immunocytochemistry and Terminal deoxynucleotidyl Transferase mediated dUTP Nick End Labeling (TUNEL) assays. To establish the grouper kidney (GK) cells apoptosis through TNF and TNFR extrinsic pathway, the TNF-α gene was cloned from orange-spotted grouper and was constructed into pET-43.1a(+)-Factor Xa prokaryotic expression vector. The E. coli expressed recombinant TNF-α contains a NusA protein at N-terminal to help TNF-α under correct folding. After Ni2+ affinity column purification and Factor Xa protease digestion, the soluble recombinant grouper TNF-α was subjected to apoptosis experiment. With no harmful concentration of Actinomycin D, the GK cells were gradually deteriorated fallowing the increase of recombinant TNF-α addition.
| Identifer | oai:union.ndltd.org:TW/101NTU05451012 |
| Date | January 2013 |
| Creators | Yi-Sen Wang, 王奕森 |
| Contributors | Chi-Yao Chang, 張繼堯 |
| Source Sets | National Digital Library of Theses and Dissertations in Taiwan |
| Language | zh-TW |
| Detected Language | English |
| Type | 學位論文 ; thesis |
| Format | 50 |
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