博士 / 國立臺灣大學 / 動物學研究所 / 100 / A shrimp cDNA microarray and immunoblotting were used to confirm that white sopt syndrome virus (WSSV) infection up-regulated expression of the important redox regulator thioredoxin (Trx). WSSV immediate early gene #1 (IE1) was identified as a possible target protein of Trx and selected for further study. In a pull down assay, we found that recombinant Trx bound to IE1 under oxidizing conditions, and a co-immunoprecipitation assay showed that Trx bound to WSSV IE1 when the transfected cells were subjected to oxidative stress. A pull down assay with Trx mutants showed that no IE1 binding occurred when cysteine 62 was replaced by serine. EMSA showed that the DNA binding activity of WSSV IE1 was down-regulated under oxidative conditions, and that PmTrx restored the DNA binding activity of the inactivated, oxidized WSSV IE1. Another EMSA experiment showed that IE1’s Cys-X-X-Cys motif and cysteine residue 55 were necessary for Trx binding. Measurement of the ratio of reduced glutathione to oxidized glutathione (GSH/GSSG) in WSSV-infected shrimp showed that oxidative stress was significantly increased at 48 hpi. The biological significance of Trx was also demonstrated in a dsRNA Trx knockdown experiment where suppression of shrimp Trx led to significant decreases in mortality and viral copy numbers. Based on all of these results, we hypothesize that Trx uses the same binding sites and redox control mechanism in vitro and in vivo, and that Trx increase WSSV’s pathogenicity by rescuing IE1’s DNA binding ability in vivo.
| Identifer | oai:union.ndltd.org:TW/100NTU05312009 |
| Date | January 2012 |
| Creators | Jiun-Yan Huang, 黃俊諺 |
| Contributors | , 郭光雄, 羅竹芳 |
| Source Sets | National Digital Library of Theses and Dissertations in Taiwan |
| Language | zh-TW |
| Detected Language | English |
| Type | 學位論文 ; thesis |
| Format | 102 |
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