博士 / 國立臺灣大學 / 動物學研究研究所 / 94 / White spot syndrome virus (WSSV) virions were purified from the hemolymph of experimentally infected crayfish Procambarus clarkii, and their proteins were separated by 8 to 18% gradient sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) to give a protein profile. The visible bands were then excised from the gel, and following trypsin digestion of the reduced and alkylated WSSV proteins in the bands, the peptide sequence of each fragment was determined by liquid chromatography–nano-electrospray ionization tandem mass spectrometry (LC-nanoESI-MS/MS) using a quadrupole/time-of-flight mass spectrometer. Comparison of the resulting peptide sequence data against the nonredundant database at the National Center for Biotechnology Information identified 33 WSSV structural genes, 20 of which are reported here for the first time. Since there were six other known WSSV structural proteins that could not be identified from the SDS-PAGE bands, there must therefore be a total of at least 39 (33 ＋ 6) WSSV structural protein genes.
However, several details of the virus structure and assembly remain controversial, including the role of one of the major structural proteins, VP26. After isolating WSSV nucleocapsids by treatment with Triton X-100 and CsCl isopycnic equilibrium centrifugation, mass spectrometry identified only VP664 and four other minor structural proteins, VP160A, VP160B, VP60B, VP51C. Surprisingly, VP15 was not detected in this fraction.
To locate the other structural proteins, Triton X-100 was used in combination with various concentrations of NaCl to separate intact WSSV virions into distinct fractions such that each fraction contained: envelope and tegument proteins; tegument and nucleocapsid proteins; or nucleocapsid proteins only. From the protein profiles and Western blotting, VP26, VP36A, VP39A, and VP95 were all identified as tegument proteins as distinct from the envelope proteins (VP19, VP28, VP31, VP36B, VP38A, VP51B, VP53A) and nucleocapsid proteins (VP664, VP51C, VP60B, VP15). We also found that VP15 dissociated from the nucleocapsid at high salt concentrations even though DNA was still present. These results were confirmed by the above identified nucleocapsid component proteins using proteomic methods, by a trypsin sensitivity assay, and by an immunogold assay.
| Identifer | oai:union.ndltd.org:TW/094NTU05312006 |
| Date | January 2006 |
| Creators | Jyh-Ming Tsai, 蔡志明 |
| Contributors | , 郭光雄, 羅竹芳 |
| Source Sets | National Digital Library of Theses and Dissertations in Taiwan |
| Language | en_US |
| Detected Language | English |
| Type | 學位論文 ; thesis |
| Format | 78 |
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