Taura syndrome為一種極高死亡率（60 - >90%）的蝦病，引發此病症的病毒為Taura syndrome virus（TSV），TSV已被暫時歸類為Dicistroviridae一科。經比對分析後推測包含有proteinase、 RNA-dependent RNA polymerase(RdRp) 和helicase等氨基酸序列motifs，此特性相似於許多picornavirus，推測TSV coat-polyprotein會經由蛋白質水解酵素(protease)剪切出具有功能蛋白質產物。
目前TSV病毒3C protease和polyportein間的作用機制仍不清楚，本研究將TSV非結構性蛋白氨機酸序列與其他已知功能的picornavirus 蛋白質作比對分析，比對出一保守區間，再利用選殖的方式，構築TSV VP-polyprotein（pTSVVP）、 3C protease（pTSV3C）、 protease＋RdRp-polyprotein(pET3CD) 和同時表現TSV protease及病毒外殼蛋白單一載體(pTSVVP3C)等質體，利用大腸桿菌表現推測之TSV protease precursor及病毒外殼聚蛋白(VP-polyprotein)，並利用eletroelution 的方式純化VP-polyprotein並利用免疫小鼠產生多株抗體（Anti-VP-polyprotein polyclonal antibody），在原核細胞外系統轉錄轉譯表現此二蛋白並利用Anti-VP-polyprotein polyclonal antibody做西方點墨法研究後發現有大小近似於VP2+VP1及VP1+VP3 precursor protein之條帶，可能為VP-polyprotein之次蛋白。

Taura syndrome virus (TSV), the epidemic agent of Taura syndrome (TS), was tentatively classified in the family Dicistroviridae, caused disastrous losses with high mortality rates from 60 to >90% of affected pond-cultured Litopenaeus vannamei. The amino acid sequence alignments of ORF1 revealed sequence motifs characteristic of a helicase, a protease and an RNA-dependent RNA polymerase, similar to the non-structural proteins of picornavirus, suggesting perhaps a similar to picornavirus polyprotein processing. Proteolytic processing of the TSV polyprotein and protease is still unclear. In order to further understand the role of protease interaction with polyprotein, several clones were constructed. In this study, we constructed several clones that expressed the TSV virus coat protein(VP-polyprotein)（pTSVVP）, putative 3C protease（pTSV3C）, protease＋RdRp-polyprotein(pET3CD) and both VP+3C proteins(pTSVVP3C) in E. coli. The VP-polyprotein band was excised from SDS-polyacrylamide gels and electroeluted for antibody production in mice. Processing of the VP-polyprotein by 3C protease was carried out in the in vitro co-transcription/translation system and detected by Western blot. The results showed there were protein bands corresponding to the sizes of VP2+VP1 and VP1+VP3 indicating that the processing might have been partial or incomplete.

摘要
序論
文獻探討
材料與方法
結果
討論

丁雲源， 2004，水產養殖手冊，水產試驗所

Altschul, S. F., Madden, T. L., Schaffer, A. A., Zhang, J., Zhang, Z., Miller, W. & Lipman, D. J. (1997). Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Research 25, 3389-3402.

Anonymous. (1994). The Taura syndrome: EPA mission arrived in Ecuador. Acuacultura del Ecuador 2, 4-10.

Bedard, K M. ＆ Selmler, B. L. (2004). Regulation of picornavirus gene expression. Microbes and Infection 6, 702-713.

Bergmann, E. M., Mosimann, S. C., Chernaia, M. M., Malcolm, B. A. & James, M. N. G. (1997). The refined crystal structure of the 3C gene product from hepatitis A virus : specific proteinase activity and RNA recognition. Journal of Virology 71, 2436-2448.

Blom, N., Hansen, J., Blaas, D. ＆ Brunak, S. (1996) Cleavage site analysis in picornaviral polyproteins: Discovering cellular targets by neural networks. Protein Science 5, 2203-2216

Bonami, J.-R., Hasson, K. W., Mari, J., Poulos, B. T. & Lightner, D. V. (1997). Taura syndrome of marine penaeid shrimp: characterization of the viral agent. Journal of General Virology 78, 313-319.

Bradford, M. M. (1976). A rapid and sensitive method for the quantification of microgram quantities of protein utilizing the principle of protein-dye binding. Analytical Biochemistry 72, 248-254.

Bragard, C., Duncan, G. H., Wesley, S. V., Naidu, R. A. & Mayo, M. A. (2000). Virus-like particles assemble in plants and bacteria expressing the coat protein gene of Indian peanut clump virus. Journal of General Virology 81, 267–272.

Brock, J. A., Gose, R., Lightner, D. V. & Hasson, K. W. (1995). An overview of Taura syndrome, an important disease of farmed Penaeus vannamei. In swimming through troubled water. Proceedings of the Special Session on Shrimp Farming, pp. 84-94. Edited by C. L. Browdy & J. S. Hopkins. Baton Rouge, LA: World Aquaculture Society.

Bujard, H., Gentz, R., Lanzer, M., Stuber, D., Muller, M., Ibrahimi, I., Hauptle, M. T. ＆ Dobberstein, B. (1987). A T5 promoter based transcription-translation system for the analysis of proteins in vivo and in vitro. Methods in Enzymology 155, 416-433.

Cohen, J. I., Ticehurst, J. R., Purcell, R. H., Buckler-White, A. & Baroudy, B. M. (1987). Complete nucleotide sequence of wild-type hepatitis A virus : comparison with different strains of hepatitis A virus and other picornaviruses. Journal of Virology 61, 50-59.

Culley A. I., Lang, A. S. ＆ Suttle, C. A. (2003). High diversity of unknown picorna-like viruses in the sea. Nature 424, 1054-1057.

Hunke, S. ＆ Betton, J. M. (2003). Temperature effect on inclusion body formation and stress response in the periplasm of Escherichia coli. Molecular Microbiology 50, 1579-1589.

Harris, K. S., Xiang, W., Alexander, L., Lane, W. S., Paul, A. V. & Wimmer, E. (1994). Interaction of poliovirus polypeptide 3CDpro with the 5’ and 3’ termini of the poliovirus genome. Identification of viral and cellular cofactors needed for efficient binding. Journal of Biological Chemistry 269, 27004-27014.

Hasson, K. W., Lightner, D. V., Poulos, B. T., Redman, R. M., White, B. L., Brock, J. A. & Bonami, J. R. (1995). Taura syndrome in Penaeus vannamei: demonstration of a viral etiology. Diseases of Aquatic Organisms 23, 115-126.

Hasson, K. W., Lightner, D. V., Mari, J., Bonami, J.-R., Poulos, B. T., Mohney, L. L., Redman, R. M. & Brock, J. A. (1999). The geographic distribution of Taura syndrome virus (TSV) in the Americas: determination by histopathology and in situ hybridization using TSV specific cDNA probes. Aquaculture 171, 13-26.

Johnson, K. N. & Christian, P. D. (1998). The novel genome organization of the insect picorna-like virus Drosophila C virus suggests this virus belongs to a previously undescribed virus family. Journal of General Virology 79, 191-203.

Lee, C., Levin, A. ＆ Branton. D. (1987) Copper staining: a five-minute protein stain for sodium dodecyl sulfate-polyacrylamide gels. Analytical Biochemistry 308, 308-312.

Lightner, D. V., Redman, R. M., Hasson, K. W. & Pantoja, C. R. (1995). Taura syndrome in Penaeus vannamei (Crustacea: Decapoda): gross signs, histopathology and ultrastructure. Diseases of Aquatic Organisms 21, 53–59.

Lightner, D. V. (1996). Epizootiology, distribution and the impact on international trade of two penaeid shrimp viruses in the Americas. Revue Scientfique et Technique Office International des Epizooties 15, 579-601.

Lightner, D. V. & Redman, R. M. (1998). Shrimp diseases and current diagnostic methods. Aquaculture 164, 201-220

Lightner, D. V. & Redman, R. M. (1998). Strategies for the control of viral diseases of shrimp in the Americas. Fish Pathology 33, 165-180.

Lo, C. F. ＆ Kou, G. H. (1998). Virus-associated white spot syndrome of shrimp in Taiwan: A review. Fish Pathology 33, 365-371

Lutsyk, M. D., Oleshko, P. S. ＆ Vovkanych, A. S. (1990). Electroelution of protein fractions from a polyacrylamide gel. Ukr Biokhim Zh 62, 308-312.

Mari, J., Bonami, J.-R. & Lightner, D. V. (1998). Taura syndrome of penaeid shrimp: cloning of viral genome fragments and development of specific gene probes. Diseases of Aquatic Organisms 33, 11-17.

Nevin, D. E.＆ Pratt, J. M. (1991). A coupled in vitro transcription-translation system for the exclusive synthesis of polypeptides expressed from the T7 promoter . FEBS Letters 291, 259-263.

Nunan, L. M., Poulos, B. T. ＆ Lightner, D. V. (1998). Reverse transcription polymerase chain reaction (RT-PCR) used for the detection of Taura syndrome virus (TSV) in experimentally infected shrimp. Disease of Aquatic Organisms 34, 87-91.

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