碩士 / 國立臺灣大學 / 漁業科學研究所 / 99 / Grouper (Epinephelus spp.) is an important aquaculture fish species in Taiwan, but it is highly susceptible to iridovirus which often cause significant economic losses to grouper aquaculture. Accordingly, it is imperative to investigate the mechanisms of iridovirus infection and pathogenesis. The grouper iridovirus (GIV) genes can be classed into immediate early (IE), early (E) and late (L) genes according to their temporal synthesis upon infection. IE genes are regard as major roles in virus life cycle, because the transcripts of viral IE genes manipulate essential functions to benefit viral replication including controlling itself gene expression and altering host cell physiological status, such as cell cycle control, apoptosis and immune response. ORF108L is one of the immediate-early genes which our laboratory had identified from GIV. It contains 1,149 nucleotide and is composed of 382 amino acids which encode a 44.1 kDa protein. By comparative sequence analysis, 108L encodes infected cell polypeptide (ICP) 46 homolog which is highly conserved among the Iridoviridae family. However, there are no putative conserved domains have been found in ICP46 protein, so its actual function remains unknown.
The prokaryotic expression plasmid, pET-28a-CBP-Factor Xa-108L, was constructed and transformed into the E.coli strain BL-21 (DE3) for expression. Besides, the best conditions of expression soluble 108L-his recombinant protein and purification by Ni2+ affinity column are well-established. The RT-PCR data confirmed that GIV108L is an immediate early gene of GIV, because the transcript of GIV108L was firstly detected at 2 hours post infection and still expressed after cycloheximide treatment. By immunocytochemistry assay, GIV108L protein was predominantly distributed at the nucleus both in GK and HeLa cells. Finally, the 108L gene knockout virus is generated by homologous recombination. Comparing wild-type virus with recombinant virus, the virus titeration is lower and the presence of cytopathic effect (CPE) obscured by infecting GK cells with recombinant virus than wild-type virus. In summary, this study demonstrated that GIV108L may function in nucleus and involve in the propagation and replication of grouper iridovirus.
| Identifer | oai:union.ndltd.org:TW/099NTU05451023 |
| Date | January 2011 |
| Creators | Ching-Hui Hsu, 徐靖惠 |
| 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 | 73 |
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