臺灣博碩士論文加值系統

豬環狀病毒 (Porcine circovirus, PCV) 為小顆粒且不具有封套的DNA病毒，核酸為單股環狀的DNA。豬環狀病毒分為兩型，分別為不具致病性的豬環狀病毒第一型 (porcine circovirus type 1, PCV1)，及具致病性的豬環狀病毒第二型(porcine circovirus type 2, PCV2)。豬環狀病毒第二型 (PCV2) 易與其他病原體混合感染造成離乳多系統耗弱症候群 (post-weaning multisystemic wasting syndrome, PMWS)，造成嚴重的經濟損失。為了增加病毒力價，以做為效果較好的疫苗研究使用，因此本論文將進行下列試驗。將豬腎細胞株 (PK15 cell line)經過單株化之後，經過初步形態篩選，得到10G、C10、2CA、9BB等四株細胞，單一化之10G則較其他細胞為細長，9BB與2CA則較圓，其中C10形態差異較大，為不規則有觸角狀，且細胞較大。將形態迥異的細胞10G和C10，分別進行生長速率測試及對PCV2的感受性試驗，結果顯示：與原始PK15相比，10G的生長速率及病毒感受性較高，而C10則生長速率和感受性都明顯較低。其病毒感受性試驗中，於攻毒後24小時，PCV 2b及PCV 2d對10G與PK15的感受性並無差異；但攻毒後48小時，細胞被感染率開始上升，且10G較PK15高，攻毒96小時後，PCV 2b對10G之感染率達17.87%，但在PK15僅有3.18%。而PCV 2d感染48小時之結果與PCV 2b相仿，亦是10G較PK15高，於96小時結果中，10G細胞被感染率為18.03%，PK15則為10.5%；C10的感染率始終為0%。以PK15、10G、C10分別進行病毒增殖試驗，檢測細胞病毒外力價與real-time PCR測定之病毒核酸分子數，顯示病毒在感染PK15及10G細胞48小時後，開始大量產製並釋放於細胞外，攻毒後之力價可達106.25 TCID50/ml，而PK15、10G產製的病毒力價與real-time PCR測定之結果無顯著差異。C10的力價為0 TCID50/ml，real-time PCR之結果遠低於10G和PK15。本文為了測試豬干擾素γ對PCV2增殖的影響，將豬干擾素-γ之基因選殖入pET 24a表現載體中，並於大腸桿菌中表現，豬干擾素-γ全長基因表現之重組蛋白會沉澱，因此將序列中含有兩個胱胺酸 (cysteine) 之片段經由次選殖方式去除，IPTG誘導表現並純化後得到之pIFNγ-447重組蛋白，並沒有抗病毒活性，因此並未測試其對PCV2增殖的影響。本文選殖之10G細胞對PCV 2b及PCV 2d皆較PK15細胞感受性高，日後可進行培養工藝的改進，以利病毒之大量生產。

Porcine circovirus (PCV) is a small, circular, single stranded DNA virus. Currently, there are two types of PCV, the nonpathogenic PCV1 and the pathogenic PCV2. PCV2, along with other pathogenic agents, cause a severe disease in pigs called postweaning multiple wasting syndrome (PMWS), causing substantial economical losses. In order to increase PCV2 viral titer for the facilitation of PCV2 vaccine research, the following experiments were performed. Homogenous cells, 10G, C10, 2CA, and 9BB were screened and derived from limiting dilution and cell cloning of parental PK15 cells. 10G cells appeared thinner and longer, while 9BB and 2CA were more round. C10 cells were bigger in size and had irregular and branch-like shapes similar to dendrite cells. Since 10G and C10 were vastly different than parental PK15 in morphology, their permissivity to PCV2 infection was investigated. Results showed that 10G grew faster and had higher permissivity to PCV2 than parental PK15, while C10 grew slower and had lower permissivity. At 24 hr post-infection, 10G and PK15 were not different in susceptibility to PCV2 infection. However, at 48 hr post-infection, increasingly PCV2 infection was detected, where 10G was observed to be more permissive than PK15. At 96 hr post-infection, the infection rate of 10G with PCV 2b was 17.87%, compared to only 3.18% of PK15. Similar to PCV 2b infection of 10G, PCV 2d infection rate of 10G at 96 hr post-infection was 18.03%, which was again higher than the infectivity of parental PK15 at 10.5%. To evaluate PCV2 propagation in PK15 and homogeneous 10G and C10 cells, viral titer and real-time PCR DNA quantification were measured post PCV2 infection. Results showed that PCV2 increasingly released from PK15 and 10G cells at 48 hr post-infection, with viral titers reaching 106.25 TCID50/ml. From the results of virus titration and real-time PCR quantification, 10G and PK15 did not show significant differences in supporting PCV2 propagation, albeit 10G gave slightly higher results than PK15. The viral titer of C10 was 0 TCID50/ml and its real-time PCR results were also far lower than 10G and PK15. Moreover, in order to test the effect of porcine INF-γ on PCV2 replication, porcine INF-γ was cloned into pET 24a for recombinant protein expression in E. coli. Initially, the expressed porcine INF-γ recombinant proteins precipitated during refolding. Thus, two cysteine amino acids were removed with PCR mutation and after IPTG induction, pIFNγ-447 recombinant protein was expressed and subsequently purified. Since it did not display anti-viral activity, further trials on its effect on PCV2 propagation were not conducted. In this study, 10G homogenous cells were cloned and were found to be more permissive to both PCV 2b and PCV 2d infection compared to PK15. From the results, indicated that 10G cells can provide the production of large quantities of PCV2.

中文摘要 …………………………………………………………………………… i
英文摘要 …………………………………………………………………………… ii
目錄 ……………………………………………………………………………… iv
表目錄 …………………………………………………………………………… vii
圖目錄 …………………………………………………………………………… viii

第一章、前言 …………………………………………………………………………1
第二章、文獻探討 ……………………………………………………………………2
一、豬環狀病毒的歷史背景 ………………………………………………………2
二、猪環狀病毒的構造 ……………………………………………………………2
三、猪環狀病毒二型臨床症狀和病理變化 ………………………………………2
四、猪環狀病毒二型間的基因分型分析 …………………………………………3
五、細胞株分離或增殖猪環狀病毒二型之現況 …………………………………3
六、猪干擾素γ (pIFN-γ) 介紹…………………………………………………… 4
七、同源分株PK15細胞系具有對PCV2增殖的效果 …………………………4
第三章、實驗策略 ……………………………………………………………………5
第四章、材料與方法 …………………………………………………………………6
第一節、PK15細胞株的培養、PCV2病毒增殖與病毒力價測定 ………………6
一、PK15細胞株的培養……………………………………………………………6
二、PK15細胞之單一化分離培養…………………………………………………6
三、不同單一化PK15之細胞增殖試驗……………………………………………6
四、PCV 2b與PCV 2d病毒來源 …………………………………………………7
五、不同單一化PK15細胞對PCV2之感受性試驗 ……………………………7
六、PCV 2b及PCV 2d之病毒增殖試驗 …………………………………………7
七、以間接螢光抗體染色法
測定PCV2之病毒力價(IFA; indirect fluorescence assay) …………………8
八、即時定量聚合酵素連鎖反應（Real-time PCR）
測PCV 2b及PCV 2d之病毒核酸變化量…………………………………8
第二節、猪干擾素γ 基因選殖與蛋白質表現 ……………………………………9
一、豬周邊血液單核細胞（PBMC）的分離………………………………………9
二、豬干擾素γ之誘導與RNA萃取………………………………………………9
三、特異性引子的設計 ……………………………………………………………9
四、反轉錄聚合酶鏈反應增幅豬干擾素γ基因 ………………………………10
五、PCR產物確認與純化 ……………………………………………………… 10
六、限制酵素切割反應……………………………………………………………10
七、接合反應………………………………………………………………………11
八、勝任細胞的製備與轉型作用…………………………………………………11
九、重組質體的挑選與確認………………………………………………………11
十、重組質體的定序確認…………………………………………………………12
第三節、豬干擾素γ基因重組蛋白的表現與確認 ………………………………12
一、重組蛋白表現…………………………………………………………………12
二、重組蛋白型態確認……………………………………………………………12
三、十二烷基磺酸鈉-聚丙烯醯胺膠電泳分析 …………………………………13
四、西方墨漬法確認重組蛋白表現………………………………………………13
五、包涵體態重組蛋白的清洗與溶解……………………………………………14
六、以親合性管柱純化豬干擾素重組蛋白………………………………………14
七、包涵體態重組蛋白的透析……………………………………………………14
八、蛋白質定量……………………………………………………………………15
九、猪干擾素γ基因重組蛋白的抗病毒活性測試………………………………15
第五章、結果…………………………………………………………………………16
第一節、PK15細胞株的單一化、PCV2病毒增殖與病毒力價測定 ……………16
一、P K15細胞單一化 ……………………………………………………………16
二、不同單一化PK15之細胞增殖 ………………………………………………16
三、不同單一化PK15細胞對PCV2之感受性試驗 ……………………………16
四、PCV 2b及PCV 2d之病毒增殖試驗 ………………………………………17
第二節、豬干擾素γ 基因選殖與重組蛋白質表現 ………………………………17
一、豬周邊血液單核細胞分離後誘導豬干擾素γ ………………………………17
二、豬干擾素γ (pIFN-γ)基因之選殖與確認 ……………………………………17
三、豬干擾素γ基因重組蛋白的表現與型態確認………………………………18
四、猪干擾素γ基因重組蛋白誘導細胞抗PRRSV之活性測試 ………………19
第六章、討論…………………………………………………………………………43
參考文獻……………………………………………………………………………47
附錄…………………………………………………………………………………53

Allan, G. M. and J. A. Ellis. 2000. Porcine circoviruses: A review. J. Vet. Diagn. Invest. 12: 3-14.
Allan, G. M., K. V. Phenix, D. Todd, and M. S. Mcnulty. 1994. Some biological and physico-chemical properties of porcine circovirus. J. Vet. Med. 41: 17-26.
Berridge, M. V. and A. S. Tan. 1993. Characterization of the cellular reduction of 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT): Subcellular localization, substrate dependence, and involvement of mitochondrial electron transport in MTT reduction. Arch. Biochem. Biophys. 303: 474-82.
Carman, S., H. Y. Cai, J. DeLay, S. A. Youssef, B. J. McEwen, C. A. Gagnon, D. Tremblay, M. Hazlett, P. Lusis, J. Fairles, H. S. Alexander, and T. van Dreumel. 2008. The emergence of a new strain of porcine circovirus-2 in Ontario and Quebec swine and its association with severe porcine circovirus associated disease - 2004-2006. Can. J. Vet. Res. 72: 259-268.
Chae, C. 2004. Postweaning multisystemic wasting syndrome: a review of aetiology, diagnosis and pathology. Vet. J. 168: 41-49.
Charley, B., K. McCullough, and S. Martinod. 1988. Antiviral and antigenic properties of recombinant porcine interferon gamma. Vet. Immunol. Immunopathol. 19: 95-103.
Cheung, A.K. 2003. Transcription analysis of porcine circovirus type 2. Virology 305: 168-180.
Cheung, A. K., K. M. Lager, O. I. Kohutyuk, A. L. Vincent, S. C. Henry, R. B. Baker, R. R. Rowland, and A. G. Dunham. 2007. Detection of two porcine circovirus type 2 genotypic groups in United States swine herds. Arch. Virol. 152: 1035-1044.
Clark, E.G. 1996. Pathology of the post-weaning multisystemic wasting syndrome of pigs. Proc. West Can. Assoc. Swine Pract. 1996: 22-25.
Chomczynski, P., and N. Sacchi. 1987. Single-step method of RNA isolation by acid guanidiniumthiocyanate-phenol-chloroform extraction. Anal. Biochem. 162: 156-159.

de Boisseson, C., V. Beven, L. Bigarre, R. Thiery, N. Rose, E. Eveno, F. Madec, A. Jestin. 2004. Molecular characterization of Porcine circovirus type 2 isolates from post-weaning multisystemicwasting syndrome-affected and non-affected pigs. J. Gen. Virol. 85: 293-304.
Devos, R., H. Cheroutre, Y. Taya, and W. Fiers. 1982. Isolation and characterization of IFN-gamma mRNA derived from mitogen-induced human splenocytes. J. Interferon Res. 2: 409-420.
Dianzani, F., O. Scheglovitova, M. Gentile, V. Scanio, C. Barresi, B. Ficociello, F. Bianchi, D. Fiumara, and M. R. Capobianchi. 1996. Interferon gamma stimulates cell-mediated transmission of HIV Type 1 from abortively infected endothelial cells. AIDS Res. Hum. Retrovir. 12: 621-627.
Dupont, K., E. O. Nielsen, P. Baekbo, and L. E. Larsen. 2008. Genomic analysis of PCV2 isolates from Danish archives and a current PMWS case–control study supports a shift in genotypes with time. Vet. Microbiol. 128: 56-64.
Ellis, J. A., A. Bratanich, E.G. Clark, G. Allan, B. Meehan, D.M. Haines, J. Harding, K.H. West, S. Krakowka, C. Konoby, L. Hassard, K. Martin and F. McNeilly. 2000. Coinfection by porcine circoviruses and porcine parvovirus in pigs with naturally acquired postweaning multisystemic wasting syndrome. J. Vet. Diagn. Invest. 12: 21-27.
Fan, Y. H., K. C. Chow, S. Y. Huang, L. M. Chi, C. J. Huang, and S. H. Chiou. 2007. A missense polymorphism in porcine interferon-γ cDNA affects antiviral activity of the protein variant. Mol. Immunol. 44: 3297-3304.
Gilpin, D.F., K. McCullough, B. M. Meehan, F. McNeilly, I. McNair, L. S. Stevenson, J. C. Foster, J. A. Ellis, S. Krakowka, B. M. Adair, and G. M. Allan. 2003. In vitro studies on the infection and replication of porcine circovirus type 2 in cells of the porcine immune system. Vet. Immunol. Immunopathol. 94: 149-161.
Grau-Roma, L., E. Crisci, M. Sibila, S. Lopez-Soria, M. Nofrarias, M. Cortey, L. Fraile, A. Olvera, and J. Segales. 2008. A proposal on porcine circovirus type 2 (PCV2) genotype definition and their relation with postweaning multisystemicwasting syndrome (PMWS) occurrence. Vet. Microbiol. 128: 23-35.
Harding, J. H. S. 1996. Post-weaning multisystemic wasting syndrome (PMWS): preliminary epidemiology and clinical findings. In: Proceedings of the West. Can. Assoc. Swine Prac. 21.
Horner, G. 1991. Pig circovirus antibodies present in New Zealand pigs. Surveil Wellington 18: 23.
Hu, V. W., G. E. Black, A. Torres-Duarte, and F. P. Abramson. 2002. 3H-thymidine is a defective tool with which to measure rates of DNA synthesis. FASEB Jour. 16: 1456-1457
Katze, M. G., Y. He, and M. Jr. Gale. 2002. Viruses and interferon: a fight for supremacy. Nat. Rev. Immunol. 2: 675-687.
Ladekjær-Mikkelsen, A. S., J. Nielsen, T. Stadejek, T. Storgaard, S. Krakowka, J. Ellis, F. McNeilly, G. Allan and A. Bøtner. 2002. Reproduction of postweaning multisystemic wasting syndrome (PMWS) in immunostimulated and non-immunostimulated 3-week-old piglets experimentally infected with porcine circovirus type 2 (PCV2). Vet. Microbiol. 89: 97-114.
Larochelle, R., R. Magar, S. D’Allaire. 2002. Genetic characterization and phylogenetic analysis of porcine circovirus type 2 (PCV2) strains from cases presenting various clinical conditions. Virus Res. 90: 101-112.
Liu, J., I. Chen, and J. Kwang. 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: 8262-8274.
Lukert, P., G. F. de Boer, and J. L. Dale. 1995. The Circoviridae. In: Virus taxonomy. Sixth Report of the International Committee on Taxonomy of Viruses, Edited by Murphy F. A., Fauquet C. M., and Bishop D. H. L. Springer-Verlag, Vienna and New York. pp. 166-168.
Mankertz, A., J. Mankertz, K. Wolf, and H. J. Buhk. 1998. Identification of a protein essential for replication of porcine circovirus. J. Gen. Virol. 79: 381-384.
Martins Gomes de Castro, A. M., A. Cortez, M. B. Heinemann, P. E. Brandao, and L. J. Richtzenhain. 2007. Genetic diversity of Brazilian strains of porcine circovirus type 2 (PCV-2) revealed by analysis of the cap gene (ORF-2). Arch.Virol. 152: 1435-1445.
Meerts, P., G. Misinzo, and H. J. Nauwynck. 2005a. Enhancement of porcine circovirus 2 replication in porcine cell lines by IFN-γ before and after treatment and by IFN-α after treatment. J. Interferon Cytokine Res. 25: 684-693.
Meerts, P., G. Misinzo, and F. McNeilly, and H. J Nauwynck. 2005b. Replication kinetics of different porcine circovirus 2 strains in PK-15 cells, fetal cardiomyocytes and macrophages. Arch. Virol. 150: 427-441
Misinzo, G., P. L. Delputte, D. J. Lefebvre, and H. J. Nauwynck. 2008a. Increased yield of porcine circovirus-2 by a combined treatment of PK-15 cells with interferon-gamma and inhibitors of endosomal-lysosomal system acidification. Arch. Viorl. 153: 337-342.
Misinzo, G., P. L. Delputte, and H. J. Nauwynck. 2008b. Inhibition of endosome- lysosome system acidification enhances porcine circovirus 2 infection of porcine epithelial cells. J. Virol. 82: 1128-1136.
Misinzo, G., P. Meerts, M. Bublot, J. Mast, H. M. Weingartl, and H. J. Nauwynck. 2005. Binding and entry characteristics of porcine circovirus 2 in cells of the porcine monocytic line 3D4/31. J. Gen. Virol. 86: 2057-2068.
Nawagitgul, P., I. Morozov, S. R. Bolin, P. A. Harms, S. D. Sorden, and P. S. Paul. 2000. Open reading frame 2 of porcine circovirus type 2 encodes a major capsid protein. J. Gen. Virol. 81: 2281-2287.
Nawagitgul P., P. A. Harms, I. Morozov, B.J. Thacker, S.D. Sorden, C. Lekcharoensuk, and P. S. Paul. 2002. Modified indirect porcine circovirus (PCV) type 2-based and recombinant capsid protein (ORF2)-based enzyme-linked immunosorbent assays for detection of antibodies to PCV. Clin. Diagn. Lab Immunol. 9: 33-40.
Olvera, A., M. Cortey, and J. Segales. 2007. Molecular evolution of porcine circovirus type 2 genomes: phylogeny and clonality. Virology 357: 175-185.
Reed, L. J. and H. Muench. 1938. A simple method of estimating fifty percent endpoints. Amer. J. Hyg. 27: 493-497.
Rubinstein, S., Familletti, P. C. and S. Pestka. 1981. Convenient assay for interferons. J. Virol. 37: 755-758.
Samuel, C.E. 1991. Antiviral actions of interferon. Interferon-regulated cellular proteins and their surprisingly selective antiviral activities. Virology 183: 1-11.
Sanchez, R. E. Jr., Meerts P., Nauwynck H. J. and Pensaert M. B. 2003. Change of porcine circovirus 2 target cells in pigs during development from fetal to early postnatal life. Vet. Microbiol. 95: 15-25.
Segales, J., A. Olvera, L. Grau-Roma, C. Charreyre, H. Nauwynck, L. Larsen, K. Dupont, K. McCullough, J. Ellis, S. Krakowka, A. Mankertz, M. Fredholm, C. Fossum, S. Timmusk, N. Stockhofe-Zurwieden, V. Beattie, D. Armstrong, B. Grassland, P. Baekbo, and G. Allan. 2008. PCV-2 genotype definition and nomenclature. Vet. Rec. 162: 867-868.
Schroder K., P.J. Hertzog, T. Ravasi, and D.A. Hume. 2004. Interferon-gamma: an overview of signals, mechanisms and functions. J. Leukoc. Biol. 75: 163-189.
Timmusk, S., P. Wallgren, I. M. Brunborg, F. H. Wikstrom, G. Allan, B. Meehan, M. McMenamy, F. McNeilly, L. Fuxler, K. Belak, D. Podersoo, T. Saar, M. Berg, and C. Fossum. 2008. Phylogenetic analysis of porcine circovirus type 2 (PCV2) pre- and post-epizootic postweaning multisystemicwasting syndrome(PMWS). Virus Genes. 36: 509-520.
Tischer I., D. Peters, R. Rasch, and S. Pociuli. 1987. Replication of porcine circovirus: induction by glucosamine and cell cycle dependence. Arch. Virol. 96: 36-57.
Tischer, I., H. Gelderblom, W. Vettermann, and M. A. Koch. 1982. A very small porcine virus with circular single-stranded DNA. Nature 295: 64-66.
Tischer, I., R. Rasch, and G. Tochtermann. 1974. Characterization of papovavirus- and picornavirus-like particles in permanent pig kidney cell lines. Zentralbe. Bakteriol. Org. 226: 153-167.
Tischer, I., W. Mields, and D. Wolff. 1986. Studies on the pathogenicity of porcine circovirus. Arch. Virol. 91: 271-276.
Truong, C., D. Mahe, P. Blanchard, M. Le Dimna, F. Madec, A. Jestin and E. Albina. 2001. Identification of an immunorelevant ORF2 epitope from porcine circovirus type 2 as a serological marker for experimental and natural infection. Arch. Virol. 146: 1197-1211.
Weining, K.C., U. Schultz, U. Munster, B. Kaspers, and P. Staeheli. 1996. Biological properties of recombinant chicken interferon-gamma. Eur. J. Immunol. 26: 2440-2447.
Wellenberg, G.J., S. Pesch, F. W. Berndsen, P. J. Steverink, W. Hunneman, T. J. Van der Vorst, N. H. Peperkamp, V. F. Ohlinger, R. Schippers, J. T. Van Oirschot, and M. F. de Jong. 2000. Isolation and characterization of porcine circovirus type 2 from pigs showing signs of post-weaning multisystemic wasting syndrome in The Netherlands. Vet. Q. 22: 167-172.
Wen, L. B., X. Guo, and H. C. Yang. 2005. Genotyping of porcine circovirus type 2 from a variety of clinical conditions in China. Vet. Microbiol. 110: 141-146.
Wang, Z. T., H. C. Yang, and X. Guo. 2002. Epidemiological investigation on infection of PCV2 in intensive swine farms. J. Chin. Vet. Sci. 38: 3-6.
Wang, F., X. Guo, X. Ge, Z. Wang, Y. Chen, Z. Cha, and H. Yang. 2009. Genetic variation analysis of Chinese strains of porcine circovirus type 2. Virus Res. 145: 151-156.
Wiederkehr, D. D., T. Sydler, E. Buergi, M. Haessig, D. Zimmermann, A. Pospischil, E. Brugnera, and X. Sidler. 2009. A new emerging genotype subgroup within PCV-2b dominates the PMWS epizooty in Switzerland. Vet. Microbiol. 136: 27-35.
Williams, J. G., G. J. Jurkovich, and R. V. Maier. 1993. Interferon-gamma: a key immunoregulatory lymphokine. J. Surg. Res. 54: 79-93.
Yu, Z., Adeline L., Jennifer L., Qiang J., Anbu K. K., and Jimmy K. 2007. Enhanced replication of porcine circovirus type 2 (PCV2) in a homogeneous subpopulation of PK15 cell line. Virology 369: 423-430.