碩士 / 國立宜蘭大學 / 生物技術與動物科學系生物技術碩士班 / 101 / Groupers are considered to be the most important fish species of aquaculture with high economic value in Taiwan. But the most severe impact in groupers rasing course is significant mortality resulted from Iridovirus and leaded to groupers in low survival state. Now how to improve immunity of groupers to anti-viral infection is an urgent issue.
This thesis mainly focused on the antibodies production and genes expression of GIV (grouper iridovirus)ORF059L, ORF061L, ORF064L. The ORF059L, ORF061 and ORF064L are 760、1122、1218 in length, with the predicted protein masses of 27.5、43.4、47.5 kDa respectively. First, the ORF059L (760-1521 nt)、ORF061L、ORF064L grouper genes were amplified by PCR and then the PCR products were cloned into the expression vectors following transform to E.coli BL21 (DE3) hosts. After IPTG induction, the recombinant proteins were purified by Ni-affinity columns and using as immunogenes to immunize mice. Finally, three specific monoclonal antibodies against ORF059L, ORF061L、ORF064L were produced.
The expression pattern of ORF059L, ORF061L、ORF064L in GIV-infected GK cells were first analyzed by RT-PCR and western blotting. Our data indicated that, the transcript of ORF061L and ORF064L were detected at 18 h p.i. whereas ORF059L was not detected until 24 h p.i. At the protein level data show same as RT-PCR. To further verify the nature of these three viral proteins, the expression of ORF059L, ORF061L、ORF064L were assayed in GIV-infected cells at the presence of cycloheximide (CHX) or cytosine arabinoside (AraC). RT-PCR and western blot analyses of GIV-infected GK cells revealed that ORF059L, ORF061L and ORF064L belong to late genes. The localization of ORF059L, ORF061L and ORF064L in GIV-infected GK cells were further characterized by immunocytochemistry with specific antibodies. ORF059L and ORF064L mainly aggregated in the nucleus whereas ORF061L aggregated in the cytoplasm around nucleus.
These studies demonstrate the validity of the three specific antibodies as immunodiagnostic and research reagents. We hope there antibodies can be widely used in iridovirus infection researching futurity.
| Identifer | oai:union.ndltd.org:TW/101NIU00108016 |
| Date | January 2013 |
| Creators | Zhi -Yu Chen, 陳緻宇 |
| Contributors | Yu-Shen Lai, 賴裕順 |
| Source Sets | National Digital Library of Theses and Dissertations in Taiwan |
| Language | zh-TW |
| Detected Language | English |
| Type | 學位論文 ; thesis |
| Format | 81 |
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