碩士 / 國立宜蘭大學 / 生物技術與動物科學系動物科學碩士班 / 106 / Grouper iridovirus (GIV) belongs to the genus Ranavirus of the family Iridoviridae, is a kind of large icosahedral viruses containing a single molecule of linear double-stranded DNA packed in a viral particle with diameter ranging from 120 to 350 nm, and viral genome is predicted to encode 120 open reading frames (ORFs). Grouper (Epinephelus spp.) is an economically important fish species worldwide and GIV is a major viral pathogens resulting in heavy economic losses in grouper aquaculture. In order to prevent virus infection, it is necessary to understand genes function of GIV. The aim of this study is to investigate the genes expression and subcellular proteins localization of GIV-48R, GIV-50L and GIV-107L during GIV infection in vitro.
The complete ORFs of GIV-48R, GIV-50L and GIV-107L were amplified by PCR from GIV genomic DNA and the amplified fragments were cloned into the pET23a expression vector, respectively. After transformation into BL21 (DE3) cells, the estimated size of recombinant GIV-48R, GIV-50L and GIV-107L fusion proteins were 22 kDa, 35 kDa and 21 kDa, respectively. These recombinant proteins were purified by Ni-affinity column and were used as immunogen to immunize mice and rabbits.
RT-PCR analyses revealed that during GIV infection in grouper kidney cells, GIV-107L was detected as early as 3 hours post infection (h p.i.), GIV-48R at 9 h p.i., but GIV-50L was not detected during the infection process. Analyses with cycloheximide (a protein synthesis inhibitor) or cytosine arabinoside (a DNA synthesis inhibitor) revealed that GIV-107 was expressed immediately after infection, whereas GIV-48R was expressed at an early stage. The localization of GIV-48R gene in GIV-infected cells was further characterized by immunofluorescence microscopy with the polyclonal antibody. The GIV-48R protein mainly aggregated in the virus assembly site of cytoplasm of the infected cells and suggesting GIV-48R was associated with virus assembly.
| Identifer | oai:union.ndltd.org:TW/106NIU00289003 |
| Date | January 2018 |
| Creators | YANG, PO-CHENG, 楊博証 |
| Contributors | LAI, YU-SHEN, LIN, YU-AN, 賴裕順, 林育安 |
| Source Sets | National Digital Library of Theses and Dissertations in Taiwan |
| Language | zh-TW |
| Detected Language | English |
| Type | 學位論文 ; thesis |
| Format | 54 |
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