| Summary: | 博士 === 國立臺灣海洋大學 === 生物科技研究所 === 98 === The grouper iridovirus (GIV) belonged to the genus Ranavirus, family Iridoviridae, is one of the major viral pathogens that seriously damage aquaculture and wild fish worldwide. The function of two GIV immediate early genes 078R and 030L are presumed to be B-cell lymphoma (Bcl)-2-like gene and tumor necrosis factor receptor (TNFR)-like gene, respectively, by homology comparison analysis. The expression of 078R and 030L can be detected by Northern blot hybridization in GK (grouper kidney) cells as early as 2 hours post-GIV infection. Their significant expression can still be observed even in the presence of cycloheximide, a translation inhibitor, indicating that the 078R and 030L are viral immediate early genes. The UV irradiation induced GK cell cellular intrinsic apoptosis can be inhibited by the immediate GIV infection as early as 5 minutes post-GIV infection, suggesting that the inhibition of apoptosis is an early event upon GIV infection. Western blot showed that the 078R was translated in GK cells 3 hours post GIV infection the 078R protein and is carried by viral particles. To investigate its subcellular localization, the 078R was fused with green fluorescence protein and the expressed fusion protein was clearly observed on the mitochondria and other defined intracellular areas. This was consistent to the intracellular distribution of Bcl-2. In 078R-transfected HeLa cells, the high expression of 078R can effectively inhibit cellular apoptosis triggered by UV. These results suggested that the immediately early gene 078R might be exploited to inhibit GK cells apoptosis upon GIV infection. NF-κB is an important transcription factor involved in immune regulation. The NF-κB subunit c-rel gene from Epinephelus coioides was cloned. c-Rel has 640 amino acids containing a Rel homology domain (RHD), immunoglobulin-like folds (TIG), nuclear translocation signal (NLS). This gene was expressed mainly in the grouper tissue such as spleen, kidney, brain, gill, liver and heart. Study of intraperitoneal injection of lipopolysaccharide (LPS) and poly I:C into the grouper showed that the c-rel gene expression was induced in the tissue of heart and intestine. The grouper c-rel gene also involve the expression and regulation of 030L gene. The co-transfection of c-rel expression vector and 030L promoter analysis vector into grouper cells could induce the expression of 030L promoter as high as 35 folds compared to the control. Further analysis of the c-Rel binding site on the 030L promoter indicated that this was a novel NF-κB binding nucleotide sequence.
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