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標題: 應用無血清Vero細胞培養技術生產本土型傳染性華氏囊病病毒
A serum-free Vero cell culture process for the production of a local infectious bursal disease virus isolate
作者: 吳愷軒
Kai-Syuan Wu
關鍵字: 本土型傳染性華氏囊炎病病毒P3009;無血清培養基;量產與純化;Infectious Bursal Disease Virus;Serum-free medium;Mass production and virus purification
引用: A MA. 2008. Growth study of DF-1 cell line in microcarrier bioreactor. 2nd International Conference on Science and Technology. Arya S, Ghosh E, Banerjee P. 1969. Effect of rust on haemagglutination test. Journal of clinical pathology 22(2):246. Brown CM, Bidle KD. 2014. Attenuation of virus production at high multiplicities of infection in Aureococcus anophagefferens. Virology 466:71-81. Caramelli M, Ru G, Acutis P, Forloni G. 2006. Prion Diseases. CNS drugs 20(1):15-28. Chen T, Chen K. 2009. Investigation and application progress of vero cell serum-free culture. International Journal of Biology 1(2):41. Cho B, Raymond R, Hill R. 1979. Growth of infectious bursal disease virus with plaque formation in chick embryo fibroblast cell culture. Avian diseases:209-218. Chun J. 1981. Serially subcultivated cells as substrates for poliovirus production for vaccine. Developments in biological standardization 47:25-33. Crespi CL, Thilly WG. 1981. Continuous cell propagation using low‐charge microcarriers. Biotechnology and Bioengineering 23(5):983-993. Crowe J, Dobeli H, Gentz R, Hochuli E, Stiiber D, Henco K. 1994. 6xffis-ni-nta chromatography as a superior technique in recombinant protein expression/purification. Protocols for gene analysis: Springer. p 371-387. Cubas-Gaona LL, Diaz-Beneitez E, Ciscar M, Rodríguez JF, Rodríguez D. 2018. Exacerbated apoptosis of cells infected with infectious bursal disease virus (IBDV) upon exposure to Interferon alpha (IFN-α). Journal of virology:JVI. 00364-18. Delgui L, Oña A, Gutiérrez S, Luque D, Navarro A, Castón JR, Rodríguez JF. 2009. The capsid protein of infectious bursal disease virus contains a functional α4β1 integrin ligand motif. Virology 386(2):360-372. Dias AD, Elicson JM, Murphy WL. 2017. Microcarriers with synthetic hydrogel surfaces for stem cell expansion. Advanced healthcare materials 6(16):1700072. Elsdale T, Bard J. 1972. Collagen substrata for studies on cell behavior. The Journal of cell biology 54(3):626-637. Giard DJ, Thilly W, Wang D, Levine D. 1977. Virus production with a newly developed microcarrier system. Applied and environmental microbiology 34(6):668-672. Griffiths B. 1990. Perfusion systems for cell cultivation. Bioprocess Technol 10:217. Grinnell F. 1978. Cellular adhesiveness and extracellular substrata. International review of cytology: Elsevier. p 65-144. Grinnell F, Hays DG, Minter D. 1977. Cell adhesion and spreading factor: partial purification and properties. Experimental cell research 110(1):175-190. Hannik C, Cohen H. 1978. New approach to the production of concentrated and purified inactivated polio and rabies tissue culture vaccines. Developments in biological standardization 41:159-168. Healthcare G, Biosciences A. 2005. Microcarrier cell culture: principles and methods. General Electric Company. Hitchman RB, Siaterli EA, Nixon CP, King LA. 2007. Quantitative real‐time PCR for rapid and accurate titration of recombinant baculovirus particles. Biotechnology and bioengineering 96(4):810-814. Hitchner S. 1970. Infectivity of infectious bursal disease virus for embryonating eggs. Poultry science 49(2):511-516. Hochuli E, Döbeli H, Schacher A. 1987. New metal chelate adsorbent selective for proteins and peptides containing neighbouring histidine residues. Journal of Chromatography A 411:177-184. Horng C-bC. 1976. PRIMARY CULTURE OF MAMMALIAN CELLS ON MICROCARRIER SURFACE. Horng CB, McLimans W. 1975. Primary suspension culture of calf anterior pituitary cells on a microcarrier surface. Biotechnology and Bioengineering 17(5):713-731. Hussain I, Rasool M. 2005. Adaptation of an indigenous very virulent infectious bursal disease virus on Vero cell line. Pakistan Veterinary Journal 25(3):103-106. Jackwood DH, Saif Y, Hughes J. 1987. Replication of infectious bursal disease virus in continuous cell lines. Avian Diseases:370-375. Jordan I, John K, Höwing K, Lohr V, Penzes Z, Gubucz-Sombor E, Fu Y, Gao P, Harder T, Zádori Z. 2016. Continuous cell lines from the Muscovy duck as potential replacement for primary cells in the production of avian vaccines. Avian Pathology 45(2):137-155. Jorio H, Tran R, Kamen A. 2006. Stability of serum‐free and purified baculovirus stocks under various storage conditions. Biotechnology progress 22(1):319-325. Kibenge FS, Dhillon A, Russell R. 1988. Growth of serotypes I and II and variant strains of infectious bursal disease virus in Vero cells. Avian Diseases:298-303. Lee L, Lu J, Lii N. 1988. Characterization of infectious bursal disease virus isolated in Taiwan. J. Chin. Soc. Vet. Sci 14:89-100. Lin T-W, Lo C-W, Lai S-Y, Fan R-J, Lo C-J, Chou Y-m, Thiruvengadam R, Wang AH-J, Wang M-Y. 2007. Chicken heat shock protein 90 is a component of the putative cellular receptor complex of infectious bursal disease virus. Journal of virology 81(16):8730-8741. Liu C-C, Wu S-C, Wu S-R, Lin H-Y, Guo M-S, Hu AY-C, Chow Y-H, Chiang J-R, Shieh D-B, Chong P. 2018. Enhancing enterovirus A71 vaccine production yield by microcarrier profusion bioreactor culture. Vaccine 36(22):3134-3139. Lonsdale H. 1982. The growth of membrane technology. Journal of membrane science 10(2-3):81-181. Lukert P, Leonard J, Davis R. 1975. Infectious bursal disease virus: antigen production and immunity. American journal of veterinary research 36(4 Pt 2):539. Markvicheva E, Grandfils C. 2004. Microcarriers for animal cell culture. Fundamentals of Cell Immobilisation Biotechnology: Springer. p 141-161. Maroudas N. 1975. Adhesion and spreading of cells on charged surfaces. Journal of theoretical Biology 49(2):417-424. Meignier B. 1979. Cell culture on beads used for the industrial production of foot-and-mouth disease virus. Developments in biological standardization 42:141-145. Meignier B, Mougeot H, Favre H. 1980. Foot and mouth disease virus production on microcarrier-grown cells. Developments in biological standardization 46:249-256. Mered B, Albrecht P, Hopps HE. 1980. Cell growth optimization in microcarrier culture. In vitro 16(10):859-865. Montagnon B, Fanget B, Nicolas A. 1981. The large-scale cultivation of VERO cells in micro-carrier culture for virus vaccine production. Preliminary results for killed poliovirus vaccine. Developments in biological standardization 47:55-64. Ng YC, Berry J, Butler M. 1996. Optimization of physical parameters for cell attachment and growth on macroporous microcarriers. Biotechnology and bioengineering 50(6):627-635. Ou C, Wang Q, Yu Y, Zhang Y, Ma J, Kong X, Liu X. 2017. Chemokine receptor CCR5 and CXCR4 might influence virus replication during IBDV infection. Microbial pathogenesis 107:122-128. Pittelkow MR, SCOTT RE. New techniques for the in vitro culture of human skin keratinocytes and perspectives on their use for grafting of patients with extensive burns; 1986. Elsevier. p 771-777. Porath J, Carlsson J, Olsson I, Belfrage G. 1975. Metal chelate affinity chromatography, a new approach to protein fractionation. Nature 258(5536):598. Price PJ. 2017. Best practices for media selection for mammalian cells. In Vitro Cellular & Developmental Biology-Animal 53(8):673-681. Rasool MH, Hussain I. 2006. Preparation and evaluation of Vero-cell infectious bursal disease vaccine in Pakistan. Vaccine 24(15):2810-2814. Reed LJ, Muench H. 1938. A simple method of estimating fifty per cent endpoints. American journal of epidemiology 27(3):493-497. Rekha K, Sivasubramanian C, Chung I-M, Thiruvengadam M. 2014. Growth and replication of infectious bursal disease virus in the DF-1 cell line and chicken embryo fibroblasts. BioMed research international 2014. Reuveny S, Thoma R. 1986. Apparatus and methodology for microcarrier cell culture. Advances in applied microbiology: Elsevier. p 139-179. Roldão A, Oliveira R, Carrondo MJ, Alves PM. 2009. Error assessment in recombinant baculovirus titration: evaluation of different methods. Journal of virological methods 159(1):69-80. Rourou S, van der Ark A, van der Velden T, Kallel H. 2007. A microcarrier cell culture process for propagating rabies virus in Vero cells grown in a stirred bioreactor under fully animal component free conditions. Vaccine 25(19):3879-89. Sheets R. 2000. History and characterization of the vero cell line. US Food and Drug Administration CfBEaR, ed. US Food and Drug Administration, Silver Spring, MD. Spier RE, Griffiths JB. 1985. Animal cell biotechnology: Academic Press. Tierney M, Lamour K. 2005. An introduction to reverse genetic tools for investigating gene function. The Plant Health Instructor. Van Hemert P, Kilburn D, Van Wezel A. 1969. Homogeneous cultivation of animal cells for the production of virus and virus products. Biotechnology and Bioengineering 11(5):875-885. Van Wezel A. 1967. Growth of cell-strains and primary cells on micro-carriers in homogeneous culture. Nature 216(5110):64. Van Wezel A. 1971. New trends in the preparation of cell substrates for the production of virus vaccines. Progress in immunobiological standardization 5:187. Van Wezel A. 1973. Microcarrier cultures of animal cells. Tissue Culture: Elsevier. p 372-377. Van Wezel A, Van Der Velden-de Groot C, Van Herwaarden J. 1980. The production of inactivated poliovaccine on serially cultivated kidney cells from captive-bred monkeys. Developments in biological standardization 46:151. Van Wezel A, Van Herwaarden J, Van de Heuvel-de Rijk E. 1979. Large-scale concentration and purification of virus suspension from microcarrier culture for the preparation of inactivated virus vaccines. Developments in biological standardization 42:65. Van Wezel A, Van Steenis G. 1978. Production of an inactivated rabies vaccine in primary dog kidney cells. Developments in biological standardization 40:69. Widdel F. 2007. Theory and measurement of bacterial growth. Di dalam Grundpraktikum Mikrobiologie 4(11):1-11. Wu S-C, Liu C-C, Lian W-C. 2004. Optimization of microcarrier cell culture process for the inactivated enterovirus type 71 vaccine development. Vaccine 22(29-30):3858-3864. Zielke HR, Ozand PT, Tildon JT, Sevdalian DA, Cornblath M. 1978. Reciprocal regulation of glucose and glutamine utilization by cultured human diploid fibroblasts. Journal of cellular physiology 95(1):41-48. 張程凱. 2008. 利用固定化金屬親和性管柱純化傳染性華氏囊病毒之研究. 中興大學生物科技學研究所學位論文:1-40. 郭權輝. 1998. 生物分子固定化技術在微載體細胞培養之應用: National Taiwan University Graduate Institute of Chemical Engineering. 黃齡玉. 2012. 傳染性華氏囊炎病毒於 DF-1 細胞的增殖及以膠體過濾層析法純化病毒顆粒. 中興大學生物科技學研究所學位論文:1-48. 劉聖哲. 2018. 三個重組 D78 傳染性華氏囊病病毒之生產與固定化金屬離子親和性層析純化方法. 中興大學生物科技學研究所學位論文:1-49.
傳染性華氏囊炎病毒 (Infectious Bursal Disease Virus, IBDV) 為Birnaviridae科Avibirnavirus屬的病毒,主要侵害雞隻華氏囊之淋巴細胞,引發嚴重的免疫抑制作用,繼而感染其他疾病 (二次感染) 造成死亡。根據先前文獻指出,使用非洲綠獼猴腎細胞 (Vero cell) 進行傳染性華氏囊炎病毒強毒株 (vvIBDV) 感染可生產高力價減毒疫苗,為建立無血清Vero細胞培養技術生產本土型傳染性華氏囊病病毒之產程以利後續疫苗開發,本研究先以實驗用角瓶,於無血清培養基中小量培養Vero細胞,並持續以IBDV P3009本土病毒株進行感染,待病毒適應於細胞複製生產後,觀察到病毒力價明顯提升。之後分別以微載體 (Microcarrier) 與滾瓶 (roller bottle) 進行細胞放大培養,並感染病毒。微載體系統主要搭配磁攪拌瓶 (spinner flask) 或發酵槽 (fermenter) 所建構之細胞懸浮培養系統,培養可於無血清狀態下生長的Vero細胞,調整培養基種類及體積、攪拌轉速、通氣等因子以建立細胞生長最佳條件。滾瓶主要搭配滾輪式混合器使細胞能大面積貼附於滾瓶壁生長。待兩者系統細胞培養穩定,再以不同病毒感染劑量(multiplicity of infection, MOI) 如10-1、10-2、10-3比例感染IBDV P3009病毒株,於感染後不同時間點觀察細胞病變效應 (cytopathic effect, CPE) 與病毒增生情形,並以TCID50 (median tissue culture infectious dose ) 測定病毒力價。結果顯示,Vero細胞於磁攪拌瓶中攪拌轉速30 rpm培養五天後可獲得細胞密度2.1×106 cells/mL;而以不同病毒感染劑量感染病毒時病毒力價皆只可達到105pfu/mL,而使用滾瓶培養Vero細胞,可達到107pfu/ml高病毒力價。進一步利用濃縮(Concentrate)、超高速離心(Ultracentrifugation) 、38%蔗糖沉降(38% sucrose cushion)三種不同純化方式沉降病毒,濃縮可以獲得39%的病毒回收率。實驗最後探討以固定化金屬離子親和性層析(immobilized-metal ion affinity chromatography, IMAC)將生產的病毒液進行純化,再透過電子顯微鏡觀察,已確認能生產並純化得到完整的IBDV 病毒顆粒。未來將持續研究以提高spinner flask生產的病毒力價,並以發酵槽操作達到量產之目的,做為日後生產本土型IBDV病毒疫苗的基礎。

Infectious bursal disease virus (IBDV) belongs to the genus Avibirnavirus of family Birnaviridae, and mainly infects chicken fibroblast lymphocytes resulting in a death by severe immunosuppression and secondary infection. According to the previous literature, the very virulent infectious bursal disease virus (vvIBDV) was adapted to grow in Vero-cell line after ninth serial passages. This ninth passage virus was evaluated as live attenuated Vero-cell adapted vaccine (Rasool and Hussain 2006). To produce a local infection for the establishment of serum-free Vero cell culture technology .The labor of the diseased bursal disease virus is developed for the follow-up vaccine. In this study, the Vero cells were cultured in a small amount in a serum-free medium, and the infection was continued with the IBDV P3009 native virus strain. The virus was adapted to the cell replication production. After that, it was observed that the viral titer was significantly improved. Afterwards, the cells were cultured in a Microcarrier and a roller bottle, respectively, and infected with a virus. A serum-free Vero cell suspension culture system was constructed by microcarriers combined with spinner flask .Vero cells which can be grown in serum-free state, and adjust the type and volume of the medium, stirring speed, ventilation and other factors. To establish optimal conditions for cell growth. Roller bottles are mainly used with roller mixers to allow cells to grow on a large area of the roller wall. The system culture of both systems is stable. Vero cells were infected with IBDV P3009 at different multiplicity of infection (MOI) of 10-1, 10-2 or 10-3, respectively, and the virus titer was determined with TCID50 (Median tissue culture infectious dose) assay. Analysis of progeny virus production in cell culture revealed a maximum titer of 107 pfu/ml after 96 hours post infection MOI of 10-2. The produced virus solution was subjected to immobilized-metal ion affinity chromatography (IMAC) for virion purification and the complete IBDV particles were confirmed after transmission electron microscopy examination. In the future, continuous research will be conducted to increase the value of the virus produced by spinner and to achieve the purpose of mass production with fermenter operation.
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