碩士 / 國立成功大學 / 生物科技研究所碩博士班 / 93 / The defense system of interferon(IFN) in vertebrate provides an initial line of defense against viral infection by inducing the synthesis of proteins that inhibit virus replication. At least three different proteins mediate the antiviral response and one of them, Mx protein, specifically inhibits the replication of wide range of RNA virus has been well established. Grouper(Epinephelus coioides)is one of the economical fish in Taiwan. Because the virus infection in adult red-spotted grouper is often light and unrecognized, grouper should have an efficient defense system against to nodaviruses. In the recent work, a homolog Mx (“OsgMx”) gene from orange-spotted grouper (Epinephelus coioides) had been cloned and sequenced. It contains an 1881 bp open reading frame region and encodes a protein with 626 amino acids. On protein structure, the grouper Mx protein is similar to the superfamily of large GTP-binding proteins. In this study, we found that the gMx could be expressed in grouper cell lines, GF-1(grouper fin cell) and GL-a(grouper liver cell), after treated with interferon-inducer poly [I:C] and it could prevent cell from death. The localization of gMx protein was determined by transient expression of gMx-GFP in GF1 cell line and detection endogenous gMx protein in GLa cell by immunofluorescence. The gMx protein was located in cytoplasm and assembles in endoplasmic reticulum after nodavirus infected. A cell transfection assay was used to examine the protection of the gMx cDNA clones against infection of NNV. The gMx would be a part of innat immunoresponses defence NNV. Furthermore, GST-Mx fusion proteins, which contain the major domain of GTP binding domain, central interactive domain (CID; 335-503 aa) and Leucine zipper domain(L/Z; 556-626 aa) were expressed to demonstrate that gMx protein could bind to nodavirus coat protein by GST pull-down and Far western blots. The CID and L/Z domain were the majar domains to associate with NNV coat protein. In conclusion, gMx protein in grouper maybe function as a part of protection system, which was related to nodavirus coat protein and to interfere with virus assembling processes in the cellular context against nodavirus, and prevents cell from death. In the future, gMx protein will be applied as a grouper antivirus agent on young grouper.
| Identifer | oai:union.ndltd.org:TW/093NCKU5111004 |
| Date | January 2005 |
| Creators | Yong-Lin Su, 蘇永霖 |
| Contributors | Tzong-Yueh Chen, 陳宗嶽 |
| Source Sets | National Digital Library of Theses and Dissertations in Taiwan |
| Language | zh-TW |
| Detected Language | English |
| Type | 學位論文 ; thesis |
| Format | 80 |
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