碩士 / 國立臺灣大學 / 動物學研究所 / 88 / WSSV genomic library of Hind III was constructed in pUC19, and contained a 4.4 kb genomic fragment named pmh15. The pmh15 nucleotide sequences have been identified and sequenced from the viral genome. The pmh15 was composed of six open reading frames (ORFs), including the longest ORF─pmh15 ORF1 with 2535 nucleotides. And the pmh15-ORF1 encodes 845 amino acid polypeptide of a theoretical size of 92 kDa from computer predictions. The sequence surrounding the methionine start codon (AGTATGG) conformed with the Kozak rule for efficient eukaryotic translation initiation and a polyadenylation consensus (polyA) signal is present 46 nt downstream of the translational stop codon. RT-PCR was used to investigate the temporal expression of the pmh15-ORF1, the results show that the pmh15-ORF1 gene may express early in the course of WSSV infection.
A DNA fragment containing the partial pmh15-ORF1 was amplified by PCR reaction for cloning from the start codon to amino acid 258, then ligated into pQE for in vivo expression. The partial pmh15-ORF1 was expressed in E. coli, which produced a 40 kDa protein product. And the N terminal amino acids of the expressed protein were sequenced as prediction. The purified protein was injected into a New Zealand white rabbit to produce antibodies for subsequent analysis.
The full-length pmh15-ORF1 was constructed in vector pcDNA3 for in vitro transcription/translation (TNT), and a major product was 100 kDa. The antibody against the N terminus of the WSSV 100 kDa protein could recognize the TNT product and a 100 kDa protein in WSSV-infected shrimp tissues.
The 100 kDa protein contains three possible transmembrane domains, 8 putative N-glycosylation sites and 31 possible phosphorylation sites. This viral protein showed no significant homology to any published envelope or transmembrane proteins of baculovirus and other large DNA viruses, that suggests WSSV could be a representative of a new baculovirus genus (whispovirus) or even a new virus family (Whispoviridae).
| Identifer | oai:union.ndltd.org:TW/088NTU00312015 |
| Date | January 2000 |
| Creators | Shih-Yin Yang, 楊仕音 |
| Contributors | Guang-Hsiung Kou, Chu-Fang Lo, 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 | 85 |
Page generated in 0.0013 seconds