請用此 Handle URI 來引用此文件：
http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/23975完整後設資料紀錄
| DC 欄位 | 值 | 語言 |
|---|---|---|
| dc.contributor.advisor | 郭應誠(Ing-Cherng Guo) | |
| dc.contributor.author | Jui-Yu Chiu | en |
| dc.contributor.author | 邱芮瑜 | zh_TW |
| dc.date.accessioned | 2021-06-08T05:13:23Z | - |
| dc.date.copyright | 2006-07-21 | |
| dc.date.issued | 2006 | |
| dc.date.submitted | 2006-07-14 | |
| dc.identifier.citation | Allan, G.M., McNeilly, F., Cassidy, J.P., Reilly, G.A., Adair, B., Ellis, W.A. and McNulty, M.S. (1995) Pathogenesis of porcine circovirus; experimental infections of colostrum deprived piglets and examination of pig foetal material. Vet Microbiol 44(1), 49-64.
Belyavskyi, M., Braunagel, S.C. and Summers, M.D. (1998) The structural protein ODV-EC27 of Autographa californica nucleopolyhedrovirus is a multifunctional viral cyclin. Proc Natl Acad Sci U S A 95(19), 11205-10. Biagini, P. (2004) Human circoviruses. Vet Microbiol 98(2), 95-101. Blanchard, P., Mahe, D., Cariolet, R., Keranflec'h, A., Baudouard, M.A., Cordioli, P., Albina, E. and Jestin, A. (2003) Protection of swine against post-weaning multisystemic wasting syndrome (PMWS) by porcine circovirus type 2 (PCV2) proteins. Vaccine 21(31), 4565-75. Breitling, R., Klingner, S., Callewaert, N., Pietrucha, R., Geyer, A., Ehrlich, G., Hartung, R., Muller, A., Contreras, R., Beverley, S.M. and Alexandrov, K. (2002) Non-pathogenic trypanosomatid protozoa as a platform for protein research and production. Protein Expr Purif 25(2), 209-18. Cardin, A.D. and Weintraub, H.J. (1989) Molecular modeling of protein-glycosaminoglycan interactions. Arteriosclerosis 9(1), 21-32. Chae, C. (2004) Postweaning multisystemic wasting syndrome: a review of aetiology, diagnosis and pathology. Vet J 168(1), 41-9. Chae, C. (2005) A review of porcine circovirus 2-associated syndromes and diseases. Vet J 169(3), 326-36. Cheung, A.K. (2003) Transcriptional analysis of porcine circovirus type 2. Virology 305(1), 168-80. Cheung, A.K. and Bolin, S.R. (2002) Kinetics of porcine circovirus type 2 replication. Arch Virol 147(1), 43-58. Darwich, L., Segales, J. and Mateu, E. (2004) Pathogenesis of postweaning multisystemic wasting syndrome caused by Porcine circovirus 2: An immune riddle. Arch Virol 149(5), 857-74. Ellis, J., Hassard, L., Clark, E., Harding, J., Allan, G., Willson, P., Strokappe, J., Martin, K., McNeilly, F., Meehan, B., Todd, D. and Haines, D. (1998) Isolation of circovirus from lesions of pigs with postweaning multisystemic wasting syndrome. Can Vet J 39(1), 44-51. Fan, H.Y., Chen, H.C., Tong, T.Z., Ju, C.M., Lu, J.Q. and Huang, H.L. (2005) [The expression of porcine circovirus type 2 ORF2 gene in insect cells and its character]. Sheng Wu Gong Cheng Xue Bao 21(6), 975-8. Fenaux, M., Halbur, P.G., Gill, M., Toth, T.E. and Meng, X.J. (2000) Genetic characterization of type 2 porcine circovirus (PCV-2) from pigs with postweaning multisystemic wasting syndrome in different geographic regions of North America and development of a differential PCR-restriction fragment length polymorphism assay to detect and differentiate between infections with PCV-1 and PCV-2. J Clin Microbiol 38(7), 2494-503. Fenaux, M., Halbur, P.G., Haqshenas, G., Royer, R., Thomas, P., Nawagitgul, P., Gill, M., Toth, T.E. and Meng, X.J. (2002) Cloned genomic DNA of type 2 porcine circovirus is infectious when injected directly into the liver and lymph nodes of pigs: characterization of clinical disease, virus distribution, and pathologic lesions. J Virol 76(2), 541-51. Fenaux, M., Opriessnig, T., Halbur, P.G., Elvinger, F. and Meng, X.J. (2004) A chimeric porcine circovirus (PCV) with the immunogenic capsid gene of the pathogenic PCV type 2 (PCV2) cloned into the genomic backbone of the nonpathogenic PCV1 induces protective immunity against PCV2 infection in pigs. J Virol 78(12), 6297-303. Fenaux, M., Opriessnig, T., Halbur, P.G. and Meng, X.J. (2003) Immunogenicity and pathogenicity of chimeric infectious DNA clones of pathogenic porcine circovirus type 2 (PCV2) and nonpathogenic PCV1 in weanling pigs. J Virol 77(20), 11232-43. Grasland, B., Loizel, C., Blanchard, P., Oger, A., Nignol, A.C., Bigarre, L., Morvan, H., Cariolet, R. and Jestin, A. (2005) Reproduction of PMWS in immunostimulated SPF piglets transfected with infectious cloned genomic DNA of type 2 porcine circovirus. Vet Res 36(5-6), 685-97. Hamel, A.L., Lin, L.L. and Nayar, G.P. (1998) Nucleotide sequence of porcine circovirus associated with postweaning multisystemic wasting syndrome in pigs. J Virol 72(6), 5262-7. Harding, J.C. (2004) The clinical expression and emergence of porcine circovirus 2. Vet Microbiol 98(2), 131-5. Ju, C., Fan, H., Tan, Y., Liu, Z., Xi, X., Cao, S., Wu, B. and Chen, H. (2005) Immunogenicity of a recombinant pseudorabies virus expressing ORF1-ORF2 fusion protein of porcine circovirus type 2. Vet Microbiol 109(3-4), 179-90. Kamstrup, S., Barfoed, A.M., Frimann, T.H., Ladekjaer-Mikkelsen, A.S. and Botner, A. (2004) Immunisation against PCV2 structural protein by DNA vaccination of mice. Vaccine 22(11-12), 1358-61. Kushnir, S., Gase, K., Breitling, R. and Alexandrov, K. (2005) Development of an inducible protein expression system based on the protozoan host Leishmania tarentolae. Protein Expr Purif 42(1), 37-46. Lekcharoensuk, P., Morozov, I., Paul, P.S., Thangthumniyom, N., Wajjawalku, W. and Meng, X.J. (2004) Epitope mapping of the major capsid protein of type 2 porcine circovirus (PCV2) by using chimeric PCV1 and PCV2. J Virol 78(15), 8135-45. Liu, C., Ihara, T., Nunoya, T. and Ueda, S. (2004) Development of an ELISA based on the baculovirus-expressed capsid protein of porcine circovirus type 2 as antigen. J Vet Med Sci 66(3), 237-42. Liu, J., Chen, I., Du, Q., Chua, H. and Kwang, J. (2006) The ORF3 protein of porcine circovirus type 2 is involved in viral pathogenesis in vivo. J Virol 80(10), 5065-73. Liu, J., Chen, I. and Kwang, J. (2005) Characterization of a previously unidentified viral protein in porcine circovirus type 2-infected cells and its role in virus-induced apoptosis. J Virol 79(13), 8262-74. Liu, Q., Tikoo, S.K. and Babiuk, L.A. (2001a) Nuclear localization of the ORF2 protein encoded by porcine circovirus type 2. Virology 285(1), 91-9. Liu, Q., Willson, P., Attoh-Poku, S. and Babiuk, L.A. (2001b) Bacterial expression of an immunologically reactive PCV2 ORF2 fusion protein. Protein Expr Purif 21(1), 115-20. Mahe, D., Blanchard, P., Truong, C., Arnauld, C., Le Cann, P., Cariolet, R., Madec, F., Albina, E. and Jestin, A. (2000) Differential recognition of ORF2 protein from type 1 and type 2 porcine circoviruses and identification of immunorelevant epitopes. J Gen Virol 81(Pt 7), 1815-24. Mankertz, A., Caliskan, R., Hattermann, K., Hillenbrand, B., Kurzendoerfer, P., Mueller, B., Schmitt, C., Steinfeldt, T. and Finsterbusch, T. (2004) Molecular biology of Porcine circovirus: analyses of gene expression and viral replication. Vet Microbiol 98(2), 81-8. Mankertz, A. and Hillenbrand, B. (2001) Replication of porcine circovirus type 1 requires two proteins encoded by the viral rep gene. Virology 279(2), 429-38. Mankertz, A. and Hillenbrand, B. (2002) Analysis of transcription of Porcine circovirus type 1. J Gen Virol 83(Pt 11), 2743-51. Mankertz, A., Persson, F., Mankertz, J., Blaess, G. and Buhk, H.J. (1997) Mapping and characterization of the origin of DNA replication of porcine circovirus. J Virol 71(3), 2562-6. McKeown, N.E., Opriessnig, T., Thomas, P., Guenette, D.K., Elvinger, F., Fenaux, M., Halbur, P.G. and Meng, X.J. (2005) Effects of porcine circovirus type 2 (PCV2) maternal antibodies on experimental infection of piglets with PCV2. Clin Diagn Lab Immunol 12(11), 1347-51. McNeilly, F., McNair, I., Mackie, D.P., Meehan, B.M., Kennedy, S., Moffett, D., Ellis, J., Krakowka, S. and Allan, G.M. (2001) Production, characterisation and applications of monoclonal antibodies to porcine circovirus 2. Arch Virol 146(5), 909-22. Meehan, B.M., Creelan, J.L., McNulty, M.S. and Todd, D. (1997) Sequence of porcine circovirus DNA: affinities with plant circoviruses. J Gen Virol 78 ( Pt 1), 221-7. Meehan, B.M., McNeilly, F., Todd, D., Kennedy, S., Jewhurst, V.A., Ellis, J.A., Hassard, L.E., Clark, E.G., Haines, D.M. and Allan, G.M. (1998) Characterization of novel circovirus DNAs associated with wasting syndromes in pigs. J Gen Virol 79 ( Pt 9), 2171-9. Misinzo, G., Delputte, P.L., Meerts, P., Lefebvre, D.J. and Nauwynck, H.J. (2006) Porcine circovirus 2 uses heparan sulfate and chondroitin sulfate B glycosaminoglycans as receptors for its attachment to host cells. J Virol 80(7), 3487-94. Misinzo, G., Meerts, P., Bublot, M., Mast, J., Weingartl, H.M. and Nauwynck, H.J. (2005) Binding and entry characteristics of porcine circovirus 2 in cells of the porcine monocytic line 3D4/31. J Gen Virol 86(Pt 7), 2057-68. Nawagitgul, P., Morozov, I., Bolin, S.R., Harms, P.A., Sorden, S.D. and Paul, P.S. (2000) Open reading frame 2 of porcine circovirus type 2 encodes a major capsid protein. J Gen Virol 81(Pt 9), 2281-7. Roca, M., Balasch, M., Segales, J., Calsamiglia, M., Viaplana, E., Urniza, A., Hattermann, K., Mankertz, A., Plana-Duran, J. and Domingo, M. (2004) In vitro and in vivo characterization of an infectious clone of a European strain of porcine circovirus type 2. J Gen Virol 85(Pt 5), 1259-66. Segales, J., Rosell, C. and Domingo, M. (2004) Pathological findings associated with naturally acquired porcine circovirus type 2 associated disease. Vet Microbiol 98(2), 137-49. Steinfeldt, T., Finsterbusch, T. and Mankertz, A. (2001) Rep and Rep' protein of porcine circovirus type 1 bind to the origin of replication in vitro. Virology 291(1), 152-60. Tischer, I., Mields, W., Wolff, D., Vagt, M. and Griem, W. (1986) Studies on epidemiology and pathogenicity of porcine circovirus. Arch Virol 91(3-4), 271-6. Tischer, I., Peters, D., Rasch, R. and Pociuli, S. (1987) Replication of porcine circovirus: induction by glucosamine and cell cycle dependence. Arch Virol 96(1-2), 39-57. Tischer, I., Rasch, R., Tochtermann, G. (1974) Characterization of papovavirus- and picornavirus-like particles in permanent pig kidney cell lines. Zentralbl. Bakteriol. [Orig A] 226(2), 153-67. Todd, D. (2004) Avian circovirus diseases: lessons for the study of PMWS. Vet Microbiol 98(2), 169-74. Todd, D., Creelan, J.L. and McNulty, M.S. (1991) Dot blot hybridization assay for chicken anemia agent using a cloned DNA probe. J Clin Microbiol 29(5), 933-9. Wang, C., Huang, T.S., Huang, C.C., Tu, C., Jong, M.H., Lin, S.Y. and Lai, S.S. (2004) Characterization of porcine circovirus type 2 in Taiwan. J Vet Med Sci 66(5), 469-75. Wang, X., Jiang, W., Jiang, P., Li, Y., Feng, Z. and Xu, J. (2006) Construction and immunogenicity of recombinant adenovirus expressing the capsid protein of porcine circovirus 2 (PCV2) in mice. Vaccine 24(16), 3374-80. Zhou, J.Y., Shang, S.B., Gong, H., Chen, Q.X., Wu, J.X., Shen, H.G., Chen, T.F. and Guo, J.Q. (2005) In vitro expression, monoclonal antibody and bioactivity for capsid protein of porcine circovirus type II without nuclear localization signal. J Biotechnol 118(2), 201-11. | |
| dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/23975 | - |
| dc.description.abstract | 豬離乳後多消耗性症候群 (PMWS)的發生雖然是多因子性，但第二型豬環狀病毒 (PCV2)為主要元兇。PCV2的感染是世界性的，在台灣PCV2抗體盛行率高達八成以上。PCV2的ORF2為外鞘蛋白 (capsid protein)，含有中和抗原決定位而能引起中和抗體。全長ORF2的次單位疫苗與DNA疫苗在實驗感染的豬隻上測試顯示，次單位疫苗比DNA疫苗有較好的保護效力，迄今卻未見有疫苗上市，推測可能是ORF2的重組蛋白質量產與純化上的困難所致。利用原核系統成功表現全長ORF2並不容易，本論文將全長ORF2 (ORF2F)與含較高抗原性區域片段分前、中、後縮短的ORF2片段，稱為ORF2N、ORF2M與ORF2C於大腸桿菌中表現，只有ORF2M、ORF2C能成功表現並純化出重組蛋白，且純化量高達約20 mg/L，所製備的小鼠抗血清及單株抗體，不但能辨識病毒抗原，也具中和抗體力價，其中，ORF2M血清可達到32倍。據此，本論文所製備的ORF2M有潛力作為PCV2次單位疫苗之素材。本論文也嘗試利用真核原蟲表現系統表現ORF2，卻無法大量表現。
PCV2常伴隨其他病原存在動物體中，增加單獨分離此病毒的困難度，再者分離的PCV2病毒在細胞培養中並不易增殖純化。本論文構築具感染性PCV2質體，利用細胞培養建立PCV2量產模式，作為穩定單純的病毒來源。同時觀察病毒在PK-15細胞中增殖時，幾種主要病毒蛋白的表現情況，以及他們對病毒複製能力的影響。結果顯示，病毒感染PK-15細胞後12小時，以西方墨點法偵測細胞質或細胞核都能發現ORF2蛋白的存在，但此時ORF1蛋白則尚未表現，直到感染後24小時才稍見出現，ORF2似乎較ORF1早被表現。另外，ORF1、ORF2及ORF3蛋白的過度表現不顯著影響每一代病毒的增殖，然而ORF2的過多表現可能促成CPE的提早出現；反之，ORF1過多表現時卻延後CPE的出現，因而讓細胞能多繼代2代，生產更多的病毒液。本結果可作為量產PCV2病毒細胞模式的參考條件。 | zh_TW |
| dc.description.abstract | Post-weaning multisystemic wasting syndrome (PMWS) becomes a major problem in many pig-producing countries throughout the world. Porcine circovirus type 2 (PCV2) has been identified as an essential component of PMWS. Approximately 83.5% of the pigs in Taiwan were seropositive to PCV2. The open reading frame 2 (ORF2) encoding the major capsid protein with epitopes to induce neutralizing antibodies is the potential target gene for vaccine development in against PCV2 infection. However the difficulty to effectively express ORF2 seems to limit the development of PCV2 vaccine. In this study, we expressed full-length, N-terminal, middle and C-terminal ORF2 covering high antigenic epitopes by E. coli. Only ORF2M and ORF2C could be overexpressed and purified. The yield of purified ORF2M or ORF2C was about 20 mg/L. Mouse antisera and monoclonal antibodies produced by ORF2M or ORF2C recognized PCV2 under Western blotting and immunofluorescence assay. The neutralization titer of ORF2M-induced serum was about 32X, suggesting that ORF2M is a potential candidate of PCV2 subunit vaccine. We also tried but failed to express ORF2 in protozoa expression system. It is hard to isolate PCV2 from field sample and to propagate it in cell culture. We constructed the infectious clone of PCV2 to generate a biologically pure and homogeneous infectious virus stock to study viral behaviors. In PCV2-infected PK-15 cells ORF2’ was detected at 12 hours post inoculation (h.p.i.) by Western blotting, but ORF1 was detected at 24 h.p.i.. The expression of ORF2 seemed to be earlier than that of ORF1. On the other hand, overexpression of ORF1, ORF2 or ORF3 had no effect on viral propagation. However, overexpression of ORF2 advanced, but ORF1 delayed the appearanceof cytopathic effect (CPE). The ORF1-delayed CPE increased the life span of PCV2-infected cells and then viral propagation. The results provide useful information for efficiently producing PCV2 virus stock in PK-15 cell model. | en |
| dc.description.provenance | Made available in DSpace on 2021-06-08T05:13:23Z (GMT). No. of bitstreams: 1 ntu-95-R93629003-1.pdf: 8403410 bytes, checksum: 1370914931271dd5c60c4d97a4cebf29 (MD5) Previous issue date: 2006 | en |
| dc.description.tableofcontents | 目錄
中文摘要..................................................I 英文摘要.................................................II 目錄....................................................III 表次......................................................V 圖次.....................................................VI 第一章 緒言............................................1 第二章 文獻探討........................................3 第一節 豬環狀病毒......................................3 1-1 豬環狀病毒之構造................................3 1-2 豬環狀病毒之複製................................4 1-3 PCV1 Rep protein與Rep’ protein之功能...........4 1-4 PCV2 ORF1之研究.................................5 1-5 PCV2 ORF2之研究.................................6 1-6 PCV2 ORF3之研究.................................6 1-7 PCV2病毒蛋白抗原性位置分析......................7 1-8 PCV2吸附宿主細胞之研究..........................8 第二節 豬環狀病毒疫苗之開發............................8 2-1 PCV2 DNA疫苗與次單位疫苗........................8 2-2 PCV2重組病毒液苗...............................10 2-3 移行抗體對PCV2實驗感染之影響...................11 第三章 第二型豬環狀病毒ORF2蛋白的表現與應用...........13 第一節 緒言...........................................13 第二節 材料與方法.....................................14 2-1 利用E. coli表現ORF2重組蛋白....................14 2-1-1 ORF2重組蛋白E. coli表現質體之構築..............14 2-1-2 重組蛋白之誘導表現.............................14 2-1-3 重組蛋白之純化.................................14 2-1-4 蛋白質定量.....................................15 2-1-5 SDS-PAGE蛋白質膠片電泳.........................15 2-1-6 西方墨點法.....................................16 2-2 利用利什曼原蟲表現ORF2重組蛋白.................16 2-2-1 ORF2重組蛋白利什曼原蟲表現質體之構築...........16 2-2-2 利什曼原蟲Leishmania tarentolae的培養、保存....17 2-2-3 ORF2重組蛋白原蟲表現質體之轉染.................17 2-2-4 利什曼原蟲的篩選...............................18 2-2-4-1 抗生素篩選.....................................18 2-2-4-2 PCR篩選........................................18 2-2-5 ORF2重組蛋白表現程度之評估.....................19 2-3 PCV2 ORF2抗體之製備............................19 2-3-1 小鼠抗血清之製備...............................19 2-3-2 單株抗體之製備.................................20 2-3-2-1 小鼠骨髓瘤細胞株NS-1 cell之培養................20 2-3-2-2 融合瘤細胞之產生...............................20 2-3-2-3 融合瘤細胞之篩選與單株化.......................20 2-3-3 酵素連結抗體吸附法.............................21 2-4 抗體中和試驗...................................21 第三節 結果...........................................23 3-1 利用E. coli表現ORF2重組蛋白....................23 3-2 利用利什曼原蟲表現ORF2重組蛋白.................24 3-3 PCV2 ORF2抗體之製備............................25 3-4 抗體中和試驗...................................25 第四節 討論...........................................27 第四章 第二型豬環狀病毒感染性質體之產生與應用.........29 第一節 緒言...........................................29 第二節 材料與方法.....................................30 2-1 利用感染性質體生產PCV2病毒.....................30 2-1-1 細胞培養.......................................30 2-1-2 第二型豬環狀病毒感染性質體.....................30 2-1-3 轉染...........................................30 2-1-4 PCV2病毒液之收集...............................31 2-1-5 PCV2病毒力價測定...............................31 2-1-6 間接免疫螢光法.................................31 2-2 PCV2 ORF1、ORF2或ORF3對PCV2複製能力之影響......32 2-2-1 PCV2 ORF1、ORF2或ORF3真核表現載體構築..........32 2-2-2 共同轉染.......................................32 2-3 PK-15細胞對PCV2感染不同時間點蛋白表現之輪廓....32 2-3-1 感染...........................................32 2-3-2 細胞質與細胞核蛋白之萃取.......................32 2-3-3 細胞質與細胞核蛋白之分析.......................33 第三節 結果...........................................34 3-1 利用感染性質體生產PCV2病毒.....................34 3-2 PCV2 ORF1、ORF2或ORF3對PCV2複製能力之影響......34 3-3 PK-15細胞對PCV2感染不同時間點蛋白表現之輪廓....35 第四節 討論...........................................37 參考文獻................................................66 | |
| dc.language.iso | zh-TW | |
| dc.subject | 複製相關蛋白 | zh_TW |
| dc.subject | 第二型豬環狀病毒 | zh_TW |
| dc.subject | 次單位疫苗 | zh_TW |
| dc.subject | 重組蛋白 | zh_TW |
| dc.subject | 感染性質體 | zh_TW |
| dc.subject | 外鞘蛋白 | zh_TW |
| dc.subject | recombinant protein | en |
| dc.subject | infectious clone | en |
| dc.subject | subunit vaccine | en |
| dc.subject | Rep protein | en |
| dc.subject | capsid protein | en |
| dc.subject | porcine circovirus type II | en |
| dc.title | 第二型豬環狀病毒重組ORF2與感染性質體的分子選殖 | zh_TW |
| dc.title | Molecular Cloning of Recombinant ORF2 and Infectious Clone of Porcine Circovirus Type II | en |
| dc.type | Thesis | |
| dc.date.schoolyear | 94-2 | |
| dc.description.degree | 碩士 | |
| dc.contributor.oralexamcommittee | 賴秀穗(Shiow-Suey Lai),龐飛(Victor Fei Pang),鄭謙仁(Chian-Ren Jeng),張繼堯(Chi-Yao Chang) | |
| dc.subject.keyword | 第二型豬環狀病毒,外鞘蛋白,複製相關蛋白,次單位疫苗,感染性質體,重組蛋白, | zh_TW |
| dc.subject.keyword | porcine circovirus type II,capsid protein,Rep protein,subunit vaccine,infectious clone,recombinant protein, | en |
| dc.relation.page | 70 | |
| dc.rights.note | 未授權 | |
| dc.date.accepted | 2006-07-16 | |
| dc.contributor.author-college | 生物資源暨農學院 | zh_TW |
| dc.contributor.author-dept | 獸醫學研究所 | zh_TW |
| 顯示於系所單位： | 獸醫學系 | |
文件中的檔案：
| 檔案 | 大小 | 格式 | |
|---|---|---|---|
| ntu-95-1.pdf 未授權公開取用 | 8.21 MB | Adobe PDF |
系統中的文件，除了特別指名其著作權條款之外，均受到著作權保護，並且保留所有的權利。