Please use this identifier to cite or link to this item: http://hdl.handle.net/11455/13602
標題: 以真核及原核系統表現水禽環狀病毒外殼蛋白並應用於血清診斷學之研究
Eukaryotic and Prokaryotic Expression of Waterfowl Circovirus Capsid Protein Particles and Its Usage in Serological Diagnosis
作者: 辛岱倫
Shin, Dai-Len
關鍵字: Circovirus;環狀病毒;protein expression;蛋白質表現
出版社: 獸醫學系暨研究所
引用: 參考文獻 王芃宣。台灣地區鵝環狀病毒與鴨環狀病毒之PCR檢測、基因型分析與宿主特異性研究。國立中興大學碩士論文。2005。 李洸鉑。以大腸桿菌表現水禽環狀病毒外殼蛋白及複製蛋白並應用於血清抗體之調查。國立中興大學碩士論文。2006。 陳建宏。鵝環狀病毒封套蛋白與複製蛋白在大腸桿菌之表現及其在血清學診斷上之應用。國立中興大學碩士論文。2003。 陳秋麟。台灣鵝環狀病毒感染及其病原之研究。國立中興大學博士論文。2004。 黃士至。以桿狀病毒表現系統表現禽流感病毒之NA蛋白,並應用於NA亞型抗體之快速區分診斷。國立中興大學碩士論文。2004。 Acharya A, Gopinathan KP. Indentification of an enhancer-like element in the polyhedron gene upstream region of Bombyx mori nucleopolyhedrovirus. Journal of General Virology 82: 2811-19, 2001. Adair BM. Immunopathogenesis of chicken anemia virus infection. Developmental and Comparative Immunology 24:247-255, 2000. Allan GM, Ellis JA. Porcine circoviruses: a review. Journal of Veterinary Diagnostic Investigation 12: 3-14, 2000a. Allan GM, McNeilly F, Meehan BM. A sequential study of experimental infection of pigs with porcine circovirus and porcine parvovirus: Immunostaining of cryostat sections and virus isolation. Journal of Veterinary Medicine. B, Infectious Diseases and Veterinary Public Health 47:81-94, 2000b. Allan GM, McNeilly F, Meehan BM, Kennedy S, Mackie DP, Ellis JA, Clark EG, Espuna E, Saubi N, Riera P, Botner A, Charreyre C. Isolation and characterization of circoviruses from pigs with wasting syndromes in Spin, Denmark and Northern Ireland. Veterinary Microbiology 66:115-123, 1999. Allan GM, Meehan BM, Todd D, Kennedy S, McNeilly F, Ellis JA, Clark EG, Harding J, Espuna E, Botner A, Charreyre C. Novel porcine circoviruses from pig with wasting disease syndromes. Veterinary Record 142:467-468, 1998. Allan GM, Phenix KV, Todd D, McNulty MS. Some biological and physico-chemical properties of porcine circovirus. Zentralblatt fur Veterinarmedizin. Reihe B 41:17-26, 1994. Ball NW, Smyth JA, Weston JH, Borghmans BJ, Palya V, Glavits R, Ivanics E, Dan A, Todd D. Diagnosis of goose circovirus infection in Hungarian geese samples using polymerase chain reaction and dot blot hybridization tests. Avian Pathology 33: 51-58, 2004. Bassami MR, Berryman D, Wilcox GE, Raidal SR. Psittacine beak and feather disease virus nucleotide sequence analysis and its relationship to porcine circovirus, plant circoviruses, chicken anaemia virus. Virology 249:453-459, 1998. Belak S, Ballagi-Prodany A. Application of the polymerase chain reaction (PCR) in veterinary diagnostic virology. Veterinary Research Communications 17:55-72, 1993. Biagini P, Todd D, Bendinelli M, 8 other authors. Anellovirus. In Virus Taxonomy. Eighth Report of the International Committee on Taxonomy of Viruses, Edited by Fauquet CM, Mayo MA, Maniloff J, Desselberger U, Ball LA. London: Academic Press pp.335–341, 2005. Bougiouklis PA. Avian circoviruses of the genus Circovirus: A potential trigger in Pigeon breeder’s lung (PBL)/Bird fancier’s lung (BFL). Medical Hypotheses 68: 320–323, 2007. Chen CL, Chang PC, Lee MS, Shien JH, Ou SJ, Shieh HK. Nucleotide sequences of goose circovirus isolated in Taiwan. Avian Pathology 32:165-171, 2003. Chen CL, Wang PX, Lee MS, Shien JH, Shieh HK, Ou SJ, Chen CH, Chang PC. Development of a polymerase chain reaction procedure for detection and differentiation of duck and goose circovirus. Avian Diseases 50: 92-95, 2006. Cheung AK. Homologous recombination plays minor role in excision of unit-length viral genomes from head-to-tail direct tandem repeats of porcine circovirus during DNA replication in Escherichia coli. Archives of Virology 152: 1531-1539, 2007. Cheung AK. Transcriptional analysis of porcine circovirus type 2. Virology 305:168-180, 2003. Cheung AK, Bolin SR. Kinetics of porcine circovirus type 2 replication. Archives of Virology 147:43-58, 2002. Cho HS, Kim TJ, Lee JI, Park NJ. Serodiagnostic comparison of enzyme-linked immunosorbent assay and surface plasmon resonance for the detection of antibody to Porcine circovirus type 2. The Canadian Journal of Veterinary Research 70: 263–268, 2006. Claessens JA, Schrier CC, Mockett AP, Jagt EH, Sondermeijer PJ. Molecular cloning and sequence analysis of the genome of chicken anaemia agent. The Journal of General Virology. 72: 2003-2006, 1991. Clark EG. Post-weaning multisystemic wasting syndrome. Proceedings of the American Association for Swine Practices 28:499-501, 1997. Crowther RA, Berriman JA, Curran WL, Allan GM, Todd D. Comparison of the structures of three circoviruses: chicken anemia virus, porcine circovirus type2, beak feather disease virus. Journal of Virology 77:13036-13041, 2003. De Bore GF, van Roozelaar DJ, Moormann RJ, Jeurissen SH, van den Wijngaard JC, Hilbink F, Koch G. Interaction between chicken anaemia virus and live Newcastle disease vaccine. Avian Pathology 23:263-275, 1994. Eills J, Hassard L, Clark E, Harding J, Allan G, Wilson P, Strokappe J, Martin K, McNeilly F, Meehan B, Todd D, Haines D. Isolation of circovirus from lesions of pigs with postweaning multisystemic wasting syndrome. The Canadian Veterinary Journal 39:44-51, 1998. Gelderblom H, Kling S, Lurz R, Tischer I, von Bulow V. Morphological characterization of chicken anaemia agent (CAA). Archives of Virology 109:115-120, 1989. Goldsmith TL. Documentation of passerine circoviral infection. Proceedings of Association for Avian Veterinary 16:349, 1995. Gough RE, Drury SE. Circovirus-like particles in the bursae of young racing pigeons. The Veterinary Record 138:167, 1996. Hamel AL, Lin LL, Nayar GP. Nucleotide sequence of porcine circovirus associated with postweaning multisystemic wasting syndrome in pigs. Journal of Virology 72: 5262-5267, 1998. Harding JC. Postweaning multisystemic wasting syndrome (PMWS): preliminary epidemiology and clinical presentation. Pro. Am. Assoc. Sw. Prac. 28th Ann. Meet. Quebec Canada 28:503, 1997. Hattermann K, Schmitt C, Soike D, Mankertz A. Cloning and sequencing of Duck circovirus (DuCV). Archives of Virology 148:2471-2480, 2003. Heath L, Martin DP, Warburton L, Perrin M, Horsfield W, Kingsley C, Rybicki EP, Williamson AL. Evidence of unique genotypes of beak and feather disease virus in southern Africa. Journal of Virology 78:9277-9284, 2004. Heath L, Williamson AL, Rybicki EP. The capsid protein of Beak and Feather Disease Virus binds to the viral DNA and is responsible for transporting the replication-associated protein into the nucleus. Journal of Virology 80: 7219-7225, 2006. Higgins, Chung DA, Chung SH. Duck Lymphocytes: I. Purification and preliminary observations on surface markers. Journal of Immunological Methods 86:231-238, 1986. Islam MR, Johne R, Raue R, Todd D, Muller H. Sequence analysis of the full-length cloned DNA of a chicken anaemia virus (CAV) strain from Bangladesh: evidence for genetic grouping of CAV strains based on the deduced VP1 amino acid sequences. Journal of Veterinary Medicine. B, Infectious Diseases and Veterinary Public Health 49: 332-337, 2002. Kiupel M, Stevenson GW, Mittal SK, Clark EG, Haines DM. Circovirus-like viral associated disease in weaned pigs in Indiana. Veterinary Pathology 5:303-307, 1998. Kothlowa S, Mannesb NK, Schaerera B, Rebeskic DE, Kaspersa B, Schultzb U. Characterization of duck leucocytes by monoclonal antibodies. Developmental and Comparative Immunology 29: 733-748, 2005. Latimer KS, Rakich PM, Niagro FD, Ritchie BW, Steffens WL, Campagnoli RP, Pesti DA, Lukert PD. An updated review of psittacine beak and feather disease. J Assoc avian Vet 5:211-220, 1991. Leisy DJ, Rohrmann GF. Characterization of the replication of plasmids containing hr sequences in baculovirus-infected Spodoptera frugiperda cells. Virology 196: 722–730, 1993. Liu CM, Ihara T, Nunoya T, Ueda S. Development of an ELISA based on the baculovirus- expressed capsid protein of porcine circovirus type2 as antigen. The Journal of Veterinary Medical Science. 66: 237-242, 2004. Liu Q, Tikoo SK, Babiuk LA. Nuclear localization of the ORF2 protein encoded by porcine circovirus type 2. Virology. 285: 91-99, 2001a. Liu Q, Willson P, Attoh-Poku S, Babiuk LA. Bacterial expression of an immunologically reactive PCV2 ORF2 fusion protein. Protein Expression and Purification. 21: 115-120, 2001b. Lukert PD, de Boer GF, Dale JL, Keese P, McNulty MS, Randles JW, Tischer I. The Circoviridae. In Virus Taxonomy. Classification and Nomenclature of Viruses. Sixth Report of the International Committee of Taxonomy of Viruses. F. A. Murphy, C. M. Fauquet, D. H. L. Bishop, S. A. Ghabrial, A. W. Jasivs, G. P. Martelli, M. A. Mayo, M. D. Summers (eds.). New York: Springer. pp. 166-168, 1995. Majima K, Kobara R, Maeda S. Divergence and evolution of homologous regions of Bombyx mori nuclear polyhedrosis virus. Journal of Virology 67: 7513-21, 1993. Mankertz A, Domingo M, Folch JM, LeCann P, Jestin A, Segales J, Chmielewicz B, Plana-Duran J, Soike D. Characterisation of PCV-2 isolates from Spain, Germany and France. Virus Research 66:65-77, 2000a. Mankertz A, Hattermann K, Ehlers B, Soike D. Cloning and sequencing of columbid circovirus (CoCV), a new circovirus from pigeons. Archives of Virology 145:2469-2479, 2000b. Mankertz A, Hillenbrand B. Replication of porcine circovirus type1 requires two proteins encoded by the viral rep gene. Virology 279:429-438, 2001. Mankertz A, Mueller B, Steinfeldt T, Schmitt C, Finsterbusch T. New reporter gene-based replication assay reveals exchangeability of replication factors of Porcine Circovirus type1 and type2. Journal of Virology 77:9885-9893, 2003. Mankertz A, Persson F, Mankertz J, Blaess G, Buhk HJ. Mapping and characterization of the origin of DNA replication of porcine circovirus. Journal of Virology 71: 2562-2566, 1997. Marchal ID, Jarvis L, Cacan R, Verbert A. Glycoproteins from insect cells: sialylated or not? Biological Chemistry 382: 151-159. 2001. McFerran JB, McNulty MS, Curran WL. Diagnosis of avian viral diseases by electron microscopy. American Journal of Veterinary Research 39:505-508, 1978. McNulty MS. Chicken anaemia agent: a review. Avian Pathology 20:187-203, 1991. Meehan BM, Creelan JL, McNulty MS, Todd D. Sequence of porcine circovirus DNA: affinities with plant circoviruses. The Journal of General Virology 78:221-227, 1997. Meehan BM, McNeilly F, Todd D, Kennedy S, Jewhurst VA, Ellis JA, Hassard LE, Clark EG, Haines DM, Allan GM. Characterization of novel circovirus DNAs associated with wasting syndromes in pigs. Journal of General Virology 79: 2171–2179, 1998. Miyata H, Tsunoda H, Kazi A, Yamada A, Khan MA, Murakami J, Kamahora T, Shiraki K, Hino S. Identification of a novel GC-rich 113-nucleotide region to complete the circular, single-stranded DNA genome of TT virus, the first human circovirus. Journal of Virology 73:3582-3586, 1999. Morozov I, Sirinarumitr T, Sorden SD, Halbur PG, Morgan MK, Yoon KJ, Paul PS. Detection of a novel strain of porcine circovirus in pigs with postweaning multisystemic wasting syndrome. Journal of Clinical Microbiology 36:2535-2541, 1998. Mushahwar IK, Erker JC, Muerhoff AS, Leary TP, Simons JN, Birkenmeyer LG, Chalmers ML, Pilot-Matias TJ, Dexai SM. Molecular and biophysical characterization of TT virus: evidence for a new virus family infecting humans. Proceedings of the National Academy of Sciences of the United States of America 96: 3177-3182, 1999. Nawagitul P, Harms PA, Morozov I, Thacker BJ, Sorden SD, Lekcharoensuk C, Paul PS. Modified indirect porcine circovirus (PCV) type 2-based and recombinant capsid protein (ORF2)-based Enzyme-Linked Immunosorbent Assays for detection of antibodies to PCV. Clinical and Diagnostic Laboratory Immunology 9: 33-40, 2002. Nawagitul P, Morozov I, Bolin SR, Harms PA, Sorden SD, Paul PS. Open reading frame 2 of porcine circovirus type 2 encodes a major capsid protein. Journal of General Virology. 81: 2281-2287, 2000. Nawagitul P, Morozov I, Paul PS, Thangthumniyom N, Wajjawalku W, Meng XJ. Epitope mapping of the major capsid protein of type 2 porcine circovirus (PCV2) by using chimeric PCV1 and PCV2. Journal of Virology 78: 8135-8145, 2004. Niagro FD, Forsthoefel AN, Lawther RP, Kamalanathan L, Ritchie BW, Latimer KS, Lukert PD. Beak and feather disease virus and porcine circovirus genomes: intermediates between the geminiviruses and plant circoviruses. Archives of Virology 143: 1723-1744, 1998. Nishizawa T, Okamoto H, Nishizawa T, Ukita M. A novel DNA virus (TTV) associated with elevated transaminase levels in posttransfusion hepatitis of unknown etiology. Biochemical and Biophysical Research Communications 1241:92-97, 1997. Noteborn MH, de Boer GF, van Roozelaar DJ, Karreman C, Kranenburg O, Vos JG, Jeurissen SH, Hoeben RC, Zantema A, Koch G, et al. Characterization of cloned chicken anemia virus DNA that contains all elements for the infectious replication cycle. Journal of Virology 65:3131-3139, 1991. Okamoto H, Akahane Y, Ukita M, Fukuda M, Tsuda F, Miyakawa Y and Mayumi M. Fecal excretion of a nonenveloped DNA virus (TTV) associated with posttransfusion non-A-G hepatitis. Journal of Medical Virology 56: 128-132, 1998. Phenix KV, Weston JH, Ypelaar I, Lavazza A, Smyth JA, Todd D, Wilcox GE, Raidal SR. Nucleotide sequence analysis of a novel circovirus of canaries and its relationship to other members of the genus Circovirus of the family Circoviridae. The Journal of General Virology 82:2805-2809, 2001. Racine S, Kheyar A, Gagnon CA, Charbonneau B, Dea S. Eucaryotic expression of the nucleocapsid protein gene of porcine circovirus type 2 and use of the protein in an indirect immunofluorescence assay for serological diagnosis of postweaning multisystemic wasting syndrome in pigs. Clinical and Diagnostic Laboratory Immunology 11: 736-741, 2004. Raidal SR, Cross GM. The haemagglutination spectum of psittacine beak and feather disease virus. Avian Pathology 23:621-630, 1994. Repnik U, Knezevic M, Jeras M. Simple and cost-effective isolation of monocytes from buffy coats. Journal of Immunological Methods 278: 283– 292, 2003. Ritchie BW, Niagro FD, Lukert PD, Steffens WL, 3rd, Latimer KS. Characterization of a new virus from cockatoos with psittacine beak and feather disease. Virology 171:83-88, 1989. Scriba A, Luciano L, Steiniger. High-yield purification of rat monocytes by combined density gradient and immunomagnetic separation. Journal of Immunological Methods 189: 203-216, 1996. Scott ANJ, Beckett A, Smyth JA, Ball NW, Palya V, Todd D. Serological diagnosis of goose circovirus infections. Avian Pathology 35: 495-499, 2006. Smith GE, Summers MD, Fraser MJ. Production of human beta interferon in insect cells infected with a baculovirus expresssion vector. Molecular and Cellular Biology 3: 2156-65, 1983. Smyth JA and Carroll BP. Circovirus infection in European racing pigeons. The Veterinary Record 136:173-174, 1995. Smyth JA, Soike D, Moffett D, Weston JH, Todd D. Circovirus-infected geese studied by in situ hybridization. Avian Pathology 34(3):227-232, 2005. Soike D. Circovirus infections in pigeons. Tierarztliche Praxis 25:52-54, 1997. Soike D, Albrecht K, Hattermann K, Schmitt C, Mankertz A. Novel circovirus in mulard ducks with developmental and feathering disorders. The Veterinary Record 154:792-793, 2004. Soike D, Hattermann K, Albrecht K, Segales J, Domingo M, Schmitt C, Mankertz A. A diagnostic study on columbid circovirus infection. Avian Pathology 30:605-611, 2001. Soike D, Kohler B and Albrecht K. A circovirus-like infection of geese related to a runting syndrome. Avian Pathology 28:199-202, 1999. Takahashi K, Iwasa Y, Hijikata M, Mishiro S. Identification of a new human DNA virus (TTV-like mini virus, TLMV) intermediately related to TT virus and chicken anemia virus. Archives of Virology 145(5):979-993, 2000. Tischer I, Peters D, Rasch R, Pociuli S. Replication of porcine circovirus: indu ction by glucosamine and cell cycle dependence. Archives of Virology 96(1-2):39-57. 1987. Tischer I, Rasch R, Tochtermann G. Characterization of papovavirus-and picornavirus-like partickes in permanent pig kidney cell lines. Zentralblatt fur Bakteriologie, Parasitenkunde, Infektionskrankheiten und Hygiene. Erste Abteilung Originale. Reihe A 226:153-167, 1974. Todd D, McNulty MS, Adair BM, Allan GM. Animal circoviruses. Advances in Virus Research 57:1-70, 2001a. Todd D, Niagro FD, Ritchie BW, Curran W, Allan GA, Lukert PD, Latimer KS, Steffens III WL, McNulty MS. Comparison of three animal viruses with circular single-stranded DNA genomes. Archives of Virology 117:129-135, 1991. Todd D, Weston JH, Soike D, Smyth JA. Genome sequence determinations and analyses of novel circoviruses from goose and pigeon. Virology 286:354-362, 2001b. Trundova M, Celer V. Expression of porcine circovirus 2 ORF2 gene requires codon optimized E.coli cells. Virus Genes. 34: 199-204, 2007. Woods LW, Latimer KS, Barr BC, Niagro FD, Campagnoli RP, Nordhausen RW, Castro AE. Circovirus-like infection in a pigeon. Journal of Veterinary Diagnostic Investigation 5: 609-612, 1993. Woods LW, Latimer KS, Niagro FD, Riddell C, Crowley AM, erson ML, Daft BM, Moore JD, Campagnoli RP, Nordhausen RW. A retrospective study of circovirus infection in pigeons: nine cases (1986-1993). Journal of Veterinary Diagnostic Investigation 6: 156-164, 1994. Yu S, Vincent A, Opriessnig T,Carpenter S,Kitikoon P, Halbur PG and Thacker E. Quantification of PCV2 capsid transcript in peripheral blood mononuclear cells (PBMCs) in vitro. Veterinary Microbiology 123: 34–42, 2007. Yuasa N. Effect of chemicals on the infectivity of chicken anaemia virus.Avian Pathology 21: 315-319, 1992. Yuasa N, Taniguchi T, Yoshida I. Isolation and some characteristics of an agent inducing anemia in chicks. Avian diseases 23: 366-385, 1979.
摘要: 
鵝環狀病毒(GoCV)與鴨環狀病毒(DuCV)為目前新興之水禽傳染病原體。此兩種病毒於台灣近年造成大量水禽的感染,會以羽毛脫落、體重減輕、生長遲緩等臨床症狀出現並導致水禽養殖業者的嚴重損失。在近年的研究中多以聚合脢連鎖反應(polymerase chain reaction;PCR)作為水禽環狀病毒的主要的檢測方法。但由於缺乏一有效之體外培養環境,致使於血清學方面的研究較少。本研究主題為發展抗體檢測技術並且應用於抗體的調查,並以人工感染DuCV的方式研究病毒於感染後抗原與抗體之關係。實驗分為三部份,第一部份,以原核pET 表現系統及真核Bac-to-Bac表現系統以表現水禽環狀病毒之外殼蛋白與核定向基因缺損之部份外殼蛋白。並以原核表現之重組蛋白為抗原,建立以multiscreen之western blot檢測水禽環狀病毒抗體;以真核表現之蛋白建立indirect immunofluorescence assay(iIFA),並評估兩者對田間血清檢測效力之優劣。結果顯示,原核系統之western blot assy具有良好之敏感性,而以真核所製備iIFA具有良好之特異性,且無論對鴨或鵝之田間血清皆有90%以上之一致性。第二部份,併用抗原及抗體檢測技術,以人工感染方式經由點眼點鼻途徑接種DuCV病毒於無抗原抗體反應之雛鴨,自血清學及病毒學上判定水禽環狀病毒於感染後之情況。DuCV於感染實驗鴨隻後始可自共泄腔中檢出抗原;於人工感染後第三週始出現抗體陽性,第五週為具有最高之抗體陽性率,而感染七週後抗體陽性率開始下降;病毒血症則是於感染後持續至感染後八週實驗結束。第三部份之研究目標為DuCV感染後之鴨隻,週邊血液單核細胞(PBMC)與病毒間的關係。利用兩種分離PBMC方式及利用PCR及iIFA檢驗結果皆間接證明DuCV可能位於單核球內,且可在其中進行外殼蛋白表現,利用iIFA更可於聚集之單核球及類巨噬細胞中發現病毒蛋白之呈現。因此我們推測,DuCV感染後所生成之抗體無法完全中和病毒,進而導致持續性病毒血症之現象。

Goose Circovirus (GoCV) and Duck Circovirus (DuCV) are two new emerging infectious agents of waterfowls. Recently, waterfowls infected with these virus exhibited clinical signs with feather disorder, poor body condition and low weight, which subsequently causes economic loss in Taiwan. Polymerase chain reaction (PCR) is the most important method for the diagnosis of these viruses. Lacking of an in vitro propagation system, the development of serological tests was limited. Our work was divided into three parts; the first part was to present the capsid protein of GoCV and DuCV by prokaryotic and eukaryotic expression system to establish further serological diagnosis and epidemiology study. With the antigens prepared by prokaryotic expression system, we sated up a multiscreen western blotting assay for serological diagnosis, while indirect immunofluorescence assay (iIFA) was developed by eukaryotic expressed antigen. The results showed that multiscreen western blotting assay had a better sensitivity while the iIFA had great specificity. The consistence between these two assays was more than 90%. In the second part of this study, we combined both antibody and antigen diagnosis for experimental inoculation of 14-day-old ducklings with plasma, cloacal swab and peripheral blood mononuclear cells (PBMC) containing DuCV. Virus-specific antibody was detected in plasma samples collected at 3 weeks post inoculation (wpi) and stayed at 100% positive at 5 wpi, fell down at 7 wpi. Viremia and virus shedding were persisted from the first week after inoculation till the end of experiment. The final part of this study was to find out the relationship between PBMC and virus after DuCV infection. Results of iIFA showed the virus protein particles were existed in monocytes/macrophage-like cells. With two separating methods for PBMC cytotaxonomy could also proof indirectly that DuCV may exist in monocytes with capsid protein expression. With all of the tests in this study, we presumed that the antibodies of DuCV could not neutralize the virus completely, while the virus persisted in monocytes could lead to long term viremia.
URI: http://hdl.handle.net/11455/13602
其他識別: U0005-2108200822112400
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