豬環狀病毒相關疾病(Porcine circovirus associated disease, PCVAD)為病毒感染所引發的豬隻重要疾病，普遍存在於畜養豬隻國家的豬群中。而第二型豬環狀病毒(Porcine circovirus type II, PCV2)是主要具有致病性的病原，當爆發大流行時皆對養殖業造成重大的經濟損失。施打疫苗為最有效之防疫措施，因此我們希望針對此病毒發展高品質且低成本的疫苗，提供豬隻抵抗病毒感染的免疫力。過去我們實驗室已成功利用桿狀病毒/昆蟲細胞表現系統大量生產PCV2外殼蛋白(capsid protein, Cap)做為疫苗抗原，並且經由豬隻的動物實驗證實的確能夠誘發豬隻對第二型豬環狀病毒的免疫力。本研究藉由發展一套簡單並且可使用於大量生產的程序，針對Cap蛋白進行萃取及純化，並於此程序中保有類病毒顆粒(virus-like particle, VLP)的結構。我們於萃取步驟中先嘗試使用多種清潔劑及鹽類溶液作為萃取液，但在後續發現並無法有效萃取Cap蛋白形成之類病毒顆粒。因此我們改以加入物理性破壞方式於萃取程序中，並且改善物理性破壞方式，使此流程可被使用於大量生產。最後我們確定最佳之萃取程序為使用PBS搭配三次冷凍解凍去除雜蛋白質後，收取細胞碎片沉澱物加入鹽類溶液(20 mM sodium phosphate)並以高壓均質機進行均質。均質後之上清液內Cap蛋白純度由Image J軟體分析純度為38%，並且以此方式可萃取出70%以上的Cap蛋白質。萃取出之Cap蛋白質已可在豬隻的免疫實驗中達到明顯的免疫效果。而在後續純化的部分我們採用工業上常使用的陽離子交換樹脂，藉由改變沖提液鹽類濃度及pH值等條件，已確立可將Cap蛋白純度提升至90%以上之純化流程。最重要的是，我們在穿透式電子顯微鏡分析下，證實此萃取及純化程序可保留PCV2類病毒顆粒的構型。

Porcine circovirus associated disease (PCVAD) is a prevalent porcine disease. Porcine circovirus type II (PCV2) is the major etiologic pathogen and causes tremendous economic loss. Although the capsid (Cap) protein-based subunit vaccines have been commercialized, the high price of vaccination entails the need for effective and inexpensive vaccines. We have produced PCV2 Cap protein as the vaccine antigen using baculovirus/insect cell expression system and proved the antigen could elicit protective immune response against PCV2 infection in swine. Here we aimed to develop a cost-effective process to extract and purify PCV2 Cap-based vaccine. First, we attempted to use various detergents and salt solutions as extraction buffers, which however resulted in poor extraction efficiency. Therefore, we employed physical methods including sonication and high pressure homogenization in the process and elevated the extraction efficiency significantly. The optimal extraction process involved the use of phosphate-buffered saline and 3 cycles of freeze and thaw, followed by extraction of the Cap protein into 20 mM sodium phosphate with high pressure homogenizer. This process enabled the extraction of Cap protein at an efficiency exceeding 70% and a purity of 38%. Immunization of porcine with the extracted Cap proteins were able to elicit significant immune effects. We further developed a purification process based on cation exchange chromatography. By changing the salt concentrations and pH values of binding and eluting solutions, the purity of Cap protein was increased to higher than 90%. The purified Cap protein self-assembled into virus-like particles, as confirmed by electron microscopy. In summary, we developed an efficient and cost-effective process for the extraction and purification of Cap proteins as the potential PCV2 vaccine.

目錄
誌謝 I
摘要 II
Abstract III
目錄 VI
圖表目錄 V
第一章 文獻回顧 1
1-1 第二型豬環狀病毒(Porcine Circovirus Type 2, PCV2) 1
1-1-1 病毒結構與基因特性 2
1-1-2 感染與複製週期 3
1-2 豬環狀病毒相關疾病 3
1-2-1 診斷方式 5
1-3 第二型豬環狀病毒疫苗的發展 6
1-3-1 原型疫苗的發展 6
1-3-2 商業化的疫苗 8
1-3-3 PCV2類病毒顆粒疫苗 9
1-4 桿狀病毒/昆蟲細胞表現系統 9
1-4-1 桿狀病毒與昆蟲細胞簡介 9
1-4-2 桿狀病毒表現系統的發展與應用 11
1-4-3 flashBACTM桿狀病毒表現系統 12
1-5萃取與純化Cap蛋白質方式 14
1-5-1 Cap蛋白質萃取 14
1-5-2 Cap蛋白質純化 15
1-6研究動機 18
第二章 實驗材料及方法 24
2-1昆蟲細胞培養 24
2-2 重組桿狀病毒建構 24
2-2-1 聚合酶鏈鎖反應 24
2-2-2 flashBAC ULTRA™重組桿狀病毒的建構 25
2-2-3 重組桿狀病毒放大 25
2-2-4 終端稀釋法定量病毒效價 25
2-2-5第二型豬環狀病毒外殼蛋白生產 26
2-3 蛋白質的萃取程序建立 26
2-3-1蛋白質的萃取 26
2-3-2氯化銫連續密度梯度離心 29
2-4 蛋白質純化程序建立 29
2-5 實驗分析方法 30
2-5-1蛋白質膠體電泳法 30
2-5-2西方點墨法(Western blot) 31
2-5-3酵素連結免疫分析法 31
2-5-4 負染色穿透式電子顯微鏡 32
2-6 動物實驗 32
2-6-1豬隻來源 32
2-6-2 PCV2疫苗配製 33
2-6-3 PCV2來源與病毒力價測定 33
2-6-4 動物實驗設計 33
2-6-5 病材採樣 34
2-6-6 病理組織切片檢查 34
2-6-7 PCV2病毒以Real-time PCR定量分析 34
2-6-8 酵素免疫分析法(ELISA)分析血清中PCV2抗體力價 35
第三章 實驗結果與討論 42
3-1 Cap蛋白質萃取與分析 42
3-1-1 兩步驟萃取程序初步測試 42
3-1-2 萃取步驟加入醇類保護類病毒顆粒結構 45
3-1-3 參考相關文獻使用非物理性破壞方式萃取 46
3-1-4 物理性破壞方式 48
3-2 蛋白質的純化與分析 51
3-2-1蛋白質的純化 51
3-2-2穿透式電子顯微鏡分析 55
3-3動物實驗結果 55
3-3-1 日增重及體溫變化 55
3-3-2 病理組織檢查 56
3-3-3 以Real-time PCR分析血清內PCV2病毒量 56
3-3-4 酵素免疫分析法(ELISA)分析血清中PCV2抗體力價 56
3-4結果與討論 57
第四章 未來展望 81
4-1 生產條件最適化 81
4-2 使用生物反應器進行大量生產 81
4-3 純化程序部分 81
第五章 參考文獻 82

Airenne KJ, Hu YC, Kost TA, Smith RH, Kotin RM, Ono C, Matsuura Y, Wang S, Yla-Herttuala S. 2013. Baculovirus: an insect-derived vector for diverse gene transfer applications. Mol Ther 21(4):739-49.
Allan G, Krakowka S, Ellis J, Charreyre C. 2012. Discovery and evolving history of two genetically related but phenotypically different viruses, porcine circoviruses 1 and 2. Virus Res 164(1-2):4-9.
Allan GM, Phenix KV, Todd D, McNulty MS. 1994. Some biological and physico-chemical properties of porcine circovirus. Zentralbl Veterinarmed B 41(1):17-26.
Badgett MR, Auer A, Carmichael LE, Parrish CR, Bull JJ. 2002. Evolutionary dynamics of viral attenuation. J Virol 76(20):10524-9.
Biosciences A. 2002. Affinity Chromatography; Principles and Methods. Uppsala, Sweden: Amersham Biosciences.
Blanchard P, Mahe D, Cariolet R, Keranflec'h A, Baudouard MA, Cordioli P, Albina E, Jestin A. 2003a. Protection of swine against post-weaning multisystemic wasting syndrome (PMWS) by porcine circovirus type 2 (PCV2) proteins. Vaccine 21(31):4565-75.
Blanchard P, Mahe D, Cariolet R, Truong C, Le Dimna M, Arnauld C, Rose N, Eveno E, Albina E, Madec F and others. 2003b. An ORF2 protein-based ELISA for porcine circovirus type 2 antibodies in post-weaning multisystemic wasting syndrome. Vet Microbiol 94(3):183-94.
Boyce FM, Bucher NL. 1996. Baculovirus-mediated gene transfer into mammalian cells. Proc Natl Acad Sci U S A 93(6):2348-52.
Bucarey SA, Noriega J, Reyes P, Tapia C, Saenz L, Zuniga A, Tobar JA. 2009. The optimized capsid gene of porcine circovirus type 2 expressed in yeast forms virus-like particles and elicits antibody responses in mice fed with recombinant yeast extracts. Vaccine 27(42):5781-90.
Chaabihi H, Ogliastro MH, Martin M, Giraud C, Devauchelle G, Cerutti M. 1993. Competition between baculovirus polyhedrin and p10 gene expression during infection of insect cells. J Virol 67(5):2664-71.
Choi J, Stevenson GW, Kiupel M, Harrach B, Anothayanontha L, Kanitz CL, Mittal SK. 2002. Sequence analysis of old and new strains of porcine circovirus associated with congenital tremors in pigs and their comparison with strains involved with postweaning multisystemic wasting syndrome. Can J Vet Res 66(4):217-24.
Cox MM. 2012. Recombinant protein vaccines produced in insect cells. Vaccine 30(10):1759-66.
Crowther RA, Berriman JA, Curran WL, Allan GM, Todd D. 2003. Comparison of the structures of three circoviruses: chicken anemia virus, porcine circovirus type 2, and beak and feather disease virus. J Virol 77(24):13036-41.
Cseke LJ. 2004. Handbook of Molecular and Cellular Methods in Biology and Medicine. P. B. Kaufman GKP, C. J. Tsai, editor. Boca Raton, Fla, USA: CRC Press.
Darwich L, Segales J, Mateu E. 2004. Pathogenesis of postweaning multisystemic wasting syndrome caused by Porcine circovirus 2: An immune riddle. Arch Virol 149(5):857-74.
Davis TR, Trotter KM, Granados RR, Wood HA. 1992. Baculovirus expression of alkaline phosphatase as a reporter gene for evaluation of production, glycosylation and secretion. Biotechnology (N Y) 10(10):1148-50.
Drugmand JC, Schneider YJ, Agathos SN. 2012. Insect cells as factories for biomanufacturing. Biotechnol Adv 30(5):1140-57.
Ellis J, Hassard L, Clark E, Harding J, Allan G, Willson P, Strokappe J, Martin K, McNeilly F, Meehan B and others. 1998. Isolation of circovirus from lesions of pigs with postweaning multisystemic wasting syndrome. Can Vet J 39(1):44-51.
Fan H, Ju C, Tong T, Huang H, Lv J, Chen H. 2007. Immunogenicity of empty capsids of porcine circovius type 2 produced in insect cells. Vet Res Commun 31(4):487-96.
Fan H, Pan Y, Fang L, Wang D, Wang S, Jiang Y, Chen H, Xiao S. 2008. Construction and immunogenicity of recombinant pseudotype baculovirus expressing the capsid protein of porcine circovirus type 2 in mice. J Virol Methods 150(1-2):21-6.
Fenaux M, Opriessnig T, Halbur PG, Elvinger F, Meng XJ. 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 PG, Meng XJ. 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.
Finsterbusch T, Mankertz A. 2009. Porcine circoviruses--small but powerful. Virus Res 143(2):177-83.
Gillespie J, Juhan NM, DiCristina J, Key KF, Ramamoorthy S, Meng XJ. 2008. A genetically engineered chimeric vaccine against porcine circovirus type 2 (PCV2) is genetically stable in vitro and in vivo. Vaccine 26(33):4231-6.
Gillespie J, Opriessnig T, Meng XJ, Pelzer K, Buechner-Maxwell V. 2009. Porcine circovirus type 2 and porcine circovirus-associated disease. J Vet Intern Med 23(6):1151-63.
Hamel AL, Lin LL, Nayar GP. 1998. Nucleotide sequence of porcine circovirus associated with postweaning multisystemic wasting syndrome in pigs. J Virol 72(6):5262-7.
Hawtin RE, Zarkowska T, Arnold K, Thomas CJ, Gooday GW, King LA, Kuzio JA, Possee RD. 1997. Liquefaction of Autographa californica nucleopolyhedrovirus-infected insects is dependent on the integrity of virus-encoded chitinase and cathepsin genes. Virology 238(2):243-53.
Hitchman RB, Locanto E, Possee RD, King LA. 2011. Optimizing the baculovirus expression vector system. Methods 55(1):52-7.
Hodgson JJ, Arif BM, Krell PJ. 2011. Interaction of Autographa californica multiple nucleopolyhedrovirus cathepsin protease progenitor (proV-CATH) with insect baculovirus chitinase as a mechanism for proV-CATH cellular retention. J Virol 85(8):3918-29.
Hofmann C, Sandig V, Jennings G, Rudolph M, Schlag P, Strauss M. 1995. Efficient gene transfer into human hepatocytes by baculovirus vectors. Proc Natl Acad Sci U S A 92(22):10099-103.
Horlen KP, Dritz SS, Nietfeld JC, Henry SC, Hesse RA, Oberst R, Hays M, Anderson J, Rowland RR. 2008. A field evaluation of mortality rate and growth performance in pigs vaccinated against porcine circovirus type 2. J Am Vet Med Assoc 232(6):906-12.
Hung C-L. 2012. Production of porcine circovirus type 2 capsid protein by using baculovirus-insect cell s expression system. Hsinchu, Taiwan: National Tsing Hua university. 62 p.
Johnson CS, Joo HS, Direksin K, Yoon KJ, Choi YK. 2002. Experimental in utero inoculation of late-term swine fetuses with porcine circovirus type 2. J Vet Diagn Invest 14(6):507-12.
Ju C, Fan H, Tan Y, Liu Z, Xi X, Cao S, Wu B, 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.
Kao Y-C. 2013. Recombinant Protein Vaccine Production using the Baculovirus Expression System: Development and Optimization of Production Process. Hsinchu, Taiwan: National Tsing Hua university. 88 p.
Karp G. 2008. Cell andMolecular Biology: Concepts and Equipments. London, UK: John Wiley & Sons.
Karuppannan AK, Kwang J. 2011. ORF3 of porcine circovirus 2 enhances the in vitro and in vivo spread of the of the virus. Virology 410(1):248-56.
Keller SA, Bauer M, Manolova V, Muntwiler S, Saudan P, Bachmann MF. 2010. Cutting edge: limited specialization of dendritic cell subsets for MHC class II-associated presentation of viral particles. J Immunol 184(1):26-9.
Kirnbauer R, Booy F, Cheng N, Lowy DR, Schiller JT. 1992. Papillomavirus L1 major capsid protein self-assembles into virus-like particles that are highly immunogenic. Proc Natl Acad Sci U S A 89(24):12180-4.
Kuzio J, Jaques R, Faulkner P. 1989. Identification of p74, a gene essential for virulence of baculovirus occlusion bodies. Virology 173(2):759-63.
Lin L-L. 2014. Development of Bivalent Vaccine against Swine Diseases using Baculovirus/Insect Cells Expression System. Hsinchu, Taiwan: National Tsing Hua university. 78 p.
Liu F, Wu X, Li L, Liu Z, Wang Z. 2013. Use of baculovirus expression system for generation of virus-like particles: successes and challenges. Protein Expr Purif 90(2):104-16.
Liu LJ, Suzuki T, Tsunemitsu H, Kataoka M, Ngata N, Takeda N, Wakita T, Miyamura T, Li TC. 2008. Efficient production of type 2 porcine circovirus-like particles by a recombinant baculovirus. Arch Virol 153(12):2291-5.
Liu Q, Tikoo SK, Babiuk LA. 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, Babiuk LA. 2001b. Bacterial expression of an immunologically reactive PCV2 ORF2 fusion protein. Protein Expr Purif 21(1):115-20.
Luckow VA, Lee SC, Barry GF, Olins PO. 1993. Efficient generation of infectious recombinant baculoviruses by site-specific transposon-mediated insertion of foreign genes into a baculovirus genome propagated in Escherichia coli. J Virol 67(8):4566-79.
Luckow VA, Summers MD. 1988. Signals important for high-level expression of foreign genes in Autographa californica nuclear polyhedrosis virus expression vectors. Virology 167(1):56-71.
M.Wink. 2006. An Introduction to Molecular Biotechnology: Molecular Fundamentals, Methods and Application in Modern Biotechnology. Weinheim, Germany: Wiley-VCH.
Mahe D, Blanchard P, Truong C, Arnauld C, Le Cann P, Cariolet R, Madec F, Albina E, 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, Hillenbrand B. 2001. Replication of porcine circovirus type 1 requires two proteins encoded by the viral rep gene. Virology 279(2):429-38.
Masumoto M, Ohde T, Shiomi K, Yaginuma T, Niimi T. 2012. A Baculovirus immediate-early gene, ie1, promoter drives efficient expression of a transgene in both Drosophila melanogaster and Bombyx mori. PLoS One 7(11):e49323.
McCullough KC, Summerfield A. 2009. Targeting the porcine immune system--particulate vaccines in the 21st century. Dev Comp Immunol 33(3):394-409.
Meehan BM, Creelan JL, McNulty MS, Todd D. 1997. Sequence of porcine circovirus DNA: affinities with plant circoviruses. J Gen Virol 78 ( Pt 1):221-7.
Merrington CL, Bailey MJ, Possee RD. 1997. Manipulation of baculovirus vectors. Mol Biotechnol 8(3):283-97.
Metz SW, Pijlman GP. 2011. Arbovirus vaccines; opportunities for the baculovirus-insect cell expression system. J Invertebr Pathol 107 Suppl:S16-30.
Monteiro F, Carinhas N, Carrondo MJ, Bernal V, Alves PM. 2012. Toward system-level understanding of baculovirus-host cell interactions: from molecular fundamental studies to large-scale proteomics approaches. Front Microbiol 3:391.
National Center for I, Respiratory D. 2011. General recommendations on immunization --- recommendations of the Advisory Committee on Immunization Practices (ACIP). MMWR Recomm Rep 60(2):1-64.
Nawagitgul P, Morozov I, Bolin SR, Harms PA, Sorden SD, Paul PS. 2000. Open reading frame 2 of porcine circovirus type 2 encodes a major capsid protein. J Gen Virol 81(Pt 9):2281-7.
Opriessnig T, Meng XJ, Halbur PG. 2007. Porcine circovirus type 2 associated disease: update on current terminology, clinical manifestations, pathogenesis, diagnosis, and intervention strategies. J Vet Diagn Invest 19(6):591-615.
Pan Q, He K, Huang K. 2008. Development of recombinant porcine parvovirus-like particles as an antigen carrier formed by the hybrid VP2 protein carrying immunoreactive epitope of porcine circovirus type 2. Vaccine 26(17):2119-26.
Perez-Martin E, Gomez-Sebastian S, Argilaguet JM, Sibila M, Fort M, Nofrarias M, Kurtz S, Escribano JM, Segales J, Rodriguez F. 2010. Immunity conferred by an experimental vaccine based on the recombinant PCV2 Cap protein expressed in Trichoplusia ni-larvae. Vaccine 28(11):2340-9.
Pham DQ, Hice RH, Sivasubramanian N, Federici BA. 1993. The 1629-bp open reading frame of the Autographa californica multinucleocapsid nuclear polyhedrosis virus encodes a virion structural protein. Gene 137(2):275-80.
Possee RD, Hitchman RB, Richards KS, Mann SG, Siaterli E, Nixon CP, Irving H, Assenberg R, Alderton D, Owens RJ and others. 2008. Generation of baculovirus vectors for the high-throughput production of proteins in insect cells. Biotechnol Bioeng 101(6):1115-22.
Rodriguez-Arrioja GM, Segales J, Balasch M, Rosell C, Quintant J, Folch JM, Plana-Duran J, Mankertz A, Domingo M. 2000. Serum antibodies to porcine circovirus type 1 and type 2 in pigs with and without PMWS. Vet Rec 146(26):762-4.
Roques E, Girard A, Gagnon CA, Archambault D. 2013. Antibody responses induced in mice immunized with recombinant adenovectors expressing chimeric proteins of various porcine pathogens. Vaccine 31(24):2698-704.
Rosell C, Segales J, Ramos-Vara JA, Folch JM, Rodriguez-Arrioja GM, Duran CO, Balasch M, Plana-Duran J, Domingo M. 2000. Identification of porcine circovirus in tissues of pigs with porcine dermatitis and nephropathy syndrome. Vet Rec 146(2):40-3.
Roussel G, Tinti E, Perpete E, Michaux C. 2013. Refolding of SDS-denatured proteins using amphipathic cosolvents and osmolytes. Curr Protoc Protein Sci Chapter 28:Unit28 5.
Saarinen MA, Troutner KA, Gladden SG, Mitchell-Logean CM, Murhammer DW. 1999. Recombinant protein synthesis in Trichoplusia ni BTI-Tn-5B1-4 insect cell aggregates. Biotechnol Bioeng 63(5):612-7.
Seeliger FA, Brugmann ML, Kruger L, Greiser-Wilke I, Verspohl J, Segales J, Baumgartner W. 2007. Porcine circovirus type 2-associated cerebellar vasculitis in postweaning multisystemic wasting syndrome (PMWS)-affected pigs. Vet Pathol 44(5):621-34.
Segales J, Kekarainen T, Cortey M. 2013. The natural history of porcine circovirus type 2: from an inoffensive virus to a devastating swine disease? Vet Microbiol 165(1-2):13-20.
Shen HG, Zhou JY, Huang ZY, Guo JQ, Xing G, He JL, Yan Y, Gong LY. 2008. Protective immunity against porcine circovirus 2 by vaccination with ORF2-based DNA and subunit vaccines in mice. J Gen Virol 89(Pt 8):1857-65.
Shibata I, Okuda Y, Yazawa S, Ono M, Sasaki T, Itagaki M, Nakajima N, Okabe Y, Hidejima I. 2003. PCR detection of Porcine circovirus type 2 DNA in whole blood, serum, oropharyngeal swab, nasal swab, and feces from experimentally infected pigs and field cases. J Vet Med Sci 65(3):405-8.
Small EJ, Fratesi P, Reese DM, Strang G, Laus R, Peshwa MV, Valone FH. 2000. Immunotherapy of hormone-refractory prostate cancer with antigen-loaded dendritic cells. J Clin Oncol 18(23):3894-903.
Smith GE, Summers MD, Fraser MJ. 1983. Production of human beta interferon in insect cells infected with a baculovirus expression vector. Mol Cell Biol 3(12):2156-65.
Smith WJ, Thomson JR, Done S. 1993. Dermatitis/nephropathy syndrome of pigs. Vet Rec 132(2):47.
Tan SC, Yiap BC. 2009. DNA, RNA, and protein extraction: the past and the present. J Biomed Biotechnol 2009:574398.
Thermo. 2008. Pack Beaded Affinity Resin into Columns. New Hampshire, NH, USA: Thermo Scientific.
Thomas CJ, Brown HL, Hawes CR, Lee BY, Min MK, King LA, Possee RD. 1998. Localization of a baculovirus-induced chitinase in the insect cell endoplasmic reticulum. J Virol 72(12):10207-12.
Tischer I, Gelderblom H, Vettermann W, Koch MA. 1982. A very small porcine virus with circular single-stranded DNA. Nature 295(5844):64-6.
Trible BR, Suddith AW, Kerrigan MA, Cino-Ozuna AG, Hesse RA, Rowland RR. 2012. Recognition of the different structural forms of the capsid protein determines the outcome following infection with porcine circovirus type 2. J Virol 86(24):13508-14.
Trundova M, Celer V. 2007. Expression of porcine circovirus 2 ORF2 gene requires codon optimized E. coli cells. Virus Genes 34(2):199-204.
van Oers MM. 2011. Opportunities and challenges for the baculovirus expression system. J Invertebr Pathol 107 Suppl:S3-15.
Vialard JE, Richardson CD. 1993. The 1,629-nucleotide open reading frame located downstream of the Autographa californica nuclear polyhedrosis virus polyhedrin gene encodes a nucleocapsid-associated phosphoprotein. J Virol 67(10):5859-66.
Vlak JM, Klinkenberg FA, Zaal KJ, Usmany M, Klinge-Roode EC, Geervliet JB, Roosien J, van Lent JW. 1988. Functional studies on the p10 gene of Autographa californica nuclear polyhedrosis virus using a recombinant expressing a p10-beta-galactosidase fusion gene. J Gen Virol 69 ( Pt 4):765-76.
Wang K, Huang L, Kong J, Zhang X. 2008. Expression of the capsid protein of porcine circovirus type 2 in Lactococcus lactis for oral vaccination. J Virol Methods 150(1-2):1-6.
Wang X, Jiang P, Li Y, Jiang W, Dong X. 2007. Protection of pigs against post-weaning multisystemic wasting syndrome by a recombinant adenovirus expressing the capsid protein of porcine circovirus type 2. Vet Microbiol 121(3-4):215-24.
Watson JD. 2004. Molecular Biology of the Gene. T. A. Baker SPB, A. Gann, M. Lecine, R. Losick, editor. San Francisco, Calif, USA: Benjamin Cummings.
Welch J, Bienek C, Gomperts E, Simmonds P. 2006. Resistance of porcine circovirus and chicken anemia virus to virus inactivation procedures used for blood products. Transfusion 46(11):1951-8.
Wheelwright SM. 1991. Protein Purification: Design and Scale up of Downstream Processing. New York, NY, USA: Carl Hanser.
Wickham TJ, Nemerow GR. 1993. Optimization of growth methods and recombinant protein production in BTI-Tn-5B1-4 insect cells using the baculovirus expression system. Biotechnol Prog 9(1):25-30.
Williams GV, Rohel DZ, Kuzio J, Faulkner P. 1989. A cytopathological investigation of Autographa californica nuclear polyhedrosis virus p10 gene function using insertion/deletion mutants. J Gen Virol 70 ( Pt 1):187-202.
Wu PC, Lin WL, Wu CM, Chi JN, Chien MS, Huang C. 2012. Characterization of porcine circovirus type 2 (PCV2) capsid particle assembly and its application to virus-like particle vaccine development. Appl Microbiol Biotechnol 95(6):1501-7.
Yang K, Li W, Niu H, Yan W, Liu X, Wang Y, Cheng S, Ku X, He Q. 2012. Efficacy of single dose of an inactivated porcine circovirus type 2 (PCV2) whole-virus vaccine with oil adjuvant in piglets. Acta Vet Scand 54:67.
Yin S, Sun S, Yang S, Shang Y, Cai X, Liu X. 2010. Self-assembly of virus-like particles of porcine circovirus type 2 capsid protein expressed from Escherichia coli. Virol J 7:166.
Zhang X, Zhou J, Wu Y, Zheng X, Ma G, Wang Z, Jin Y, He J, Yan Y. 2009. Differential proteome analysis of host cells infected with porcine circovirus type 2. J Proteome Res 8(11):5111-9.