Please use this identifier to cite or link to this item: http://hdl.handle.net/11455/35782
標題: Hi-5細胞在磁攪拌瓶之通氣培養條件及組胺酸取代對傳染性華氏囊病毒之次病毒顆粒純化的影響
Aeration of Hi-5 cell culture in Spinner and the Effect of Histidine Substitution on the Purification of Infectious Bursal Disease Virus Subviral Particles
作者: 張群岳
Chang, Cyun-Yue
關鍵字: 昆蟲細胞
insect cell
通氣培養
傳染性華氏囊病毒
固定化金屬離子親和性層析法
aeration culture
infectious bursal disease virus
immobilized-metal ion affinity chromatography
出版社: 生物科技學研究所
引用: O''REilly, D.R., L.K. Miller, and V.A. Luckow, Baculovirus expression vectors: a laboratory manual. 1992, New York: W.H. Freeman and Company. Possee, R.D. and S.C. Howard, Analysis of the polyhedrin gene promoter of the Autographa californica nuclear polyhedrosis virus. Nucleic Acids Res, 1987. 15(24): p. 10233-48. Palomares, L.A., J.C. Pedroza, and O.T. Ram´ırez, Cell size as a tool to predict protein productivity of the insect cell-baculovirus expression system. Biotechnol, 2001. 23: p. 359–364. Ramirez, O.T. and R. Mutharasan, Cell cycle- and growth phase-dependent variations in size distribution, antibody productivity, and oxygen demand in hybridoma cultures. Biotechnol Bioeng, 1990. 36(8): p. 839-48. Pijlman, G.P., et al., Autographa californica baculoviruses with large genomic deletions are rapidly generated in infected insect cells. Virology, 2001. 283(1): p. 132-8. WICKHAM, T.J., et al., Baculovirus defective interfering particles are responsible for variations in recombinant protein production as a function of multiplicity of infection Biotechnology Letters 1991. 13(7): p. 483-488. Hink, W.F. and P.V. Vail, Aplaqueassay for titration of alfalfaloopernuclearpolyhedrosisvirus in acabbagelooper (TN-368) cellline. Invertebrate Pathology, 1973. 22: p. 168-174. Lynn, D.E., Improved efficiency in determining the titer of the Autographa californica baculovirus nonoccluded virus. BioTechniques, 1992. 13(2): p. 282-5. Yahata, T., et al., Estimation of baculovirus titer by beta-galactosidase activity assay of virus preparations. BioTechniques, 2000. 29(2): p. 214-5. Kwon, M.S., et al., Development of an Antibody-Based Assay for Determination of Baculovirus Titers in 10 Hours. Biotechnology Progress, 2002. 18(3): p. 647-651. Janakiraman, V., et al., A rapid method for estimation of baculovirus titer based on viable cell size. J Virol Methods, 2006. 132(1-2): p. 48-58. Shen, C.F., J. Meghrous, and A. Kamen, Quantitation of baculovirus particles by flow cytometry. J Virol Methods, 2002. 105(2): p. 321-30. Lo, H.-R. and Y.-C. Chao, Rapid titre determination of baculovirus by quantitative real-time polymerase chain reaction. Biotechnology Progress, 2004. 20(1): p. 354-360. Lynn, D.E., Novel techniques to establish new insect cell lines. In Vitro Cell Dev Biol Anim, 2001. 37(6): p. 319-21. Vaughn, J.L., et al., The establishment of two cell lines from the insect Spodoptera frugiperda (Lepidoptera; Noctuidae). In Vitro, 1977. 13(4): p. 213-7. Smith, G.E., M.D. Summers, and M.J. Fraser, Production of human beta interferon in insect cells infected with a baculovirus expression vector. 1983. Biotechnology, 1992. 24: p. 434-43. Hink, W.F., Established insect cell line from the cabbage looper, trichoplusia ni. Nature, 1970. 226(5244): p. 466-7. Saarinen, M.A., et al., Recombinant protein synthesis in Trichoplusia ni BTI-Tn-5B1-4 insect cell aggregates. Biotechnol Bioeng, 1999. 63(5): p. 612-7. P. C. KULAKOSKY, M.L.S., AND H. A. WOOD, N-glycosylation of a baculovirus-expressed recombinant glycoprotein in three insect cell lines In Vitro Cellular & Developmental Biology - Animal 1998. 34: p. 101-108. Rhiel, M., C.M. Mitchell-Logean, and D.W. Murhammer, Comparison of Trichoplusia ni BTI-Tn-5B1–4 (High FiveTM) and Spodoptera frugiperda Sf-9 insect cell line metabolism in suspension cultures. Biotechnol. Prog, 1997. 55: p. 909-920. Kioukia, N., et al., Physiological and environmental factors affecting the growth of insect cells and infection with baculovirus. J Biotechnol, 1995. 38(3): p. 243-51. Donaldson, M.S. and M.L. Shuler, Effects of long-term passaging of BTI-Tn5B1-4 insect cells on growth and recombinant protein production. Biotechnol Prog, 1998. 14(4): p. 543-7. Reuveny, S., et al., Effect of temperature and oxygen on cell growth and recombinant protein production in insect cell cultures. Appl Microbiol Biotechnol, 1993. 38(5): p. 619-23. Ikonomou, L., et al., Effect of partial medium replacement on cell growth and protein production in the insect cell-baculovirus system. Cytotechnology, 2004. 44: p. 67-76. Wilkie, G.E., H. Stockdale, and S.V. Pirt, Chemically-defined media for production of insect cells and viruses in vitro. Dev Biol Stand, 1980. 46: p. 29-37. Donaldson, M.S. and M.L. Shuler, Low-cost serum-free medium for the BTI-Tn5B1-4 insect cell line. Biotechnol Prog, 1998. 14(4): p. 573-9. Palomares, L.A., S. López, and O.T. Ramı́rez, Utilization of oxygenuptakerate to assess the role of glucose and glutamine in the metabolism of infected insectcellcultures. Biochemical Engineering Journal, 2004. 19(1): p. 87-93. Kunas, K.T. and E.T. Papoutsakis, Damage mechanisms of suspended animal cells in agitated bioreactors with and without bubble entrainment. Biotechnol Bioeng, 1990. 36(5): p. 476-83. Scott, R.I., J.H. Blanchard, and C.H.R. Ferguson, Effects of oxygen on recombinantproteinproduction by suspension cultures of Spodoptera frugiperda (Sf-9) insect cells. Enzyme and Microbial Technology, 1992. 14(10): p. 798-804. Gotoh, T., et al., Significant increase in recombinant protein production of a virus-infected Sf-9 insect cell culture of low MOI under low dissolved oxygen conditions. J Biosci Bioeng, 2002. 94(5): p. 426-33. Zhang, F., et al., The effect of dissolved oxygen (DO) concentration on the glycosylation of recombinant protein produced by the insect cell–baculovirus expression system. Biotechnology and Bioengineering, 2002. 77(2): p. 219-224. Wu, J., Mechanisms of animal cell damage associated with gas bubbles and cell protection by medium additives. J Biotechnol, 1995. 43(2): p. 81-94. Murhammer, D.W. and C.F. Goochee, Sparged animal cell bioreactors: mechanism of cell damage and Pluronic F-68 protection. Biotechnol Prog, 1990. 6(5): p. 391-7. King, G.A., et al., Recombinant B-galactosidase production in serum-free medium by insect cells in a 14-liter airlift bioreactor. Biotechnol. Prog, 1992. 8(6): p. 567-571. Weber, W., et al., Optimisation of protein expression and establishment of the Wave Bioreactor for Baculovirus/insect cell culture. Cytotechnology, 2002. 38(1-3): p. 77-85. van den Berg, T.P., et al., Infectious bursal disease (Gumboro disease). Rev Sci Tech, 2000. 19(2): p. 509-43. Rodriguez-Lecompte, J.C., et al., Infectious bursal disease virus (IBDV) induces apoptosis in chicken B cells. Comp Immunol Microbiol Infect Dis, 2005. 28(4): p. 321-37. Saif, Y.M., Immunosuppression induced by infectious bursal disease virus. Vet Immunol Immunopathol, 1991. 30(1): p. 45-50. Muller, H., C. Scholtissek, and H. Becht, The genome of infectious bursal disease virus consists of two segments of double-stranded RNA. J Virol, 1979. 31(3): p. 584-9. Lee, L.H., Characterization of nonradioactive hybridization probes for detecting infectious bursal disease virus. J Virol Methods, 1992. 38(1): p. 81-92. Castón, J.R., et al., C Terminus of Infectious Bursal Disease Virus Major Capsid Protein VP2 Is Involved in Definition of the T Number for Capsid Assembly. journal of virology, 2001. 75(22): p. 10815-10828. Irigoyen, N., J.R. Castón, and J.F. Rodríguez, Host Proteolytic Activity Is Necessary for Infectious Bursal Disease Virus Capsid Protein Assembly. Journal of Biological Chemistry, 2012. 287: p. 24473-24482. Lee, C.-C., et al., Crystal structure of infectious bursal disease virus VP2 subviral particle at 2.6Å resolution: Implications in virion assembly and immunogenicity. Journal of Structural Biology, 2006. 155(1): p. 74-86. Doong, S.R., Strong and Heterogeneous Adsorption of Infectious Bursal Disease VP2 Subviral Particle with Immobilized Metal Ions Dependent on Two Surface Histidine Residues. Anal. Chem., 2007. Ho, J.-Y., et al., Vaccine development through terminal deletions of an infectious bursal disease virus protein 2 precursor variant. Process Biochemistry, 2010. 45(5): p. 786-793. Wyeth, P.J., J.D. O''Brien, and G.A. Cullen, Improved performance of progeny of broiler parent chickens vaccinated with infectious bursal disease oil-emulsion vaccine. Avian Dis, 1981. 25(1): p. 228-41. Maas, R.A., et al., Efficacy of inactivated infectious bursal disease (IBD) vaccines: comparison of serology with protection of progeny chickens against IBD virus strains of varying virulence. Avian Pathol, 2001. 30(4): p. 345-54. Busso, D., R. Kim, and S.-H. Kim, Using an Escherichia coli cell-free extract to screen for soluble expression of recombinant proteins Journal of Structural and Functional Genomics, 2004. 5: p. 69-74. DeJong, J. and R.G. Roeder, A single cDNA, hTFIIA/alpha, encodes both the p35 and p19 subunits of human TFIIA. Genes Dev, 1993. 7(11): p. 2220-34. Shi, Y., et al., Transcriptional repression by YY1, a human GLI-Krüippel-related protein, and relief of repression by adenovirus E1A protein. cell 1991. 67(2): p. 377-388. Pohl, E., et al., Architecture of a protein central to iron homeostasis: crystal structure and spectroscopic analysis of the ferric uptake regulator. Mol Microbiol, 2003. 47(4): p. 903-15. Pesce, A., et al., Unique structural features of the monomeric Cu,Zn superoxide dismutase from Escherichia coli, revealed by X-ray crystallography. J Mol Biol, 1997. 274(3): p. 408-20. Widdel, F., Theory and Measurement of Bacterial Growth. basic practical microbiology, 2010. 楊函蓁, 研究傳染性華氏囊病毒次病毒顆粒VP2蛋白表面胺基酸His249與His253對鎳離子之親合性及免疫原性的影響. 碩士論文, 2008. 卓子暄, 傳染性華氏囊炎病毒VP2次病毒粒子與固定化鎳離子吸附之點突變分析. 碩士論文, 2011. A.Taticek, R. and M.L. Shuler, Effect of elevated oxygen and glutamine levels on foreign protein production at high cell densities using the insect cell-baculovirus expression system. Biotechnology and Bioengineering, 1996. 54(2): p. 142-152. Annathur, G.V., et al., Improvements in spinner-flask designs for insect-cell suspension culture. Biotechnol Appl Biochem, 2003. 38(Pt 1): p. 15-8. Mitchell-Logean, C. and D.W. Murhammer, Bioreactor Headspace Purging Reduces Dissolved Carbon Dioxide Accumulation in Insect Cell Cultures and Enhances Cell Growth. Biotechnol. Prog, 1997. 13: p. 875-877. Vallejos, J.R., et al., Integrating a 250 mL-spinner flask with other stirred bench-scale cell culture devices: a mass transfer perspective. Biotechnol Prog, 2011. 27(3): p. 803-810. Chen, C.-S., et al., Purification of capsid-like particles of infectious bursal disease virus (IBDV) VP2 expressed in E. coli with a metal-ion affinity membrane system. Journal of Virological Methods, 2005. 130(1-2): p. 51-58. pathange, L.P., et al., correlation between protein binding strength on immobilized metal affinity chromatography and the histidine-related protein surface structure. Anal. Chem., 2006. 78(13): p. 4443-4449. Niebaa, L., et al., biacore Analysis of Histidine-Tagged Proteins Using a Chelating NTA Sensor Chip. Anal. Biochem, 1997. 252(2): p. 217-228. Knecht, S., et al., Oligohis-tags: mechanisms of binding to Ni2+-NTA surfaces. Journal of Molecular Recognition, 2009. 22(4): p. 270-279. Roldão, A., et al., Error assessment in recombinant baculovirus titration: Evaluation of different methods. Journal of Virological Methods, 2009. 159(1): p. 69-80. Hitchman, R.B., et al., Quantitative real-time PCR for rapid and accurate titration of recombinant baculovirus particles. Biotechnology and Bioengineering, 2007. 96(4): p. 810-814. Jorio, H., R. Tran, and A. Kamen, Stability of serum-free and purified baculovirus stocks under various storage conditions. Biotechnol Prog, 2006. 22(1): p. 319-25. Block, H., et al., Chapter 27 Immobilized-Metal Affinity Chromatography (IMAC)A Review. 2009. 463: p. 439-473.
摘要: Spinner flask plays a key role in insect cells culture operations in laboratory-scale. With the culture volume increased in a spinner flask, gas-liquid mixing effect will decline. Because Oxygen is a key nutrient in insect cell culture bioprocesses, it’s not conducive for the cultivation in spinner in large-scale. In order to increase the dissolved oxygen in the media, sparging it with air, directly enters the culture medium. However, direct sparging can damage insect cells through bursting bubbles and bubble accumulation. To improve the bubble-associated damage, adding a protective agent and antifoam reduce the cell damage. Aeration of Hi-5 cell culture, an additional 0.2% (v/v) antifoam AF emulsion inhibits the bubble accumulation and 0.2% (w/v) Pluronic F-68 protects cells against shear stress. Maximum cell density was 5 x 106 cells/ml in aeration culture, and specific growth rate was 0.032h-1. Infectious bursal disease virus (IBDV) is a double stranded RNA virus and a highly contagious disease of young chickens at 3 to 6 weeks of age. VP2, one of IBDV’s capsid proteins, is achieved by the induction of neutralizing antibodies in the chicken. In order to mass produce and develop safer vaccines, we have expressed the VP2 protein using an insect cell-baculovirus expression vector system (IC-BEVS). The VP2 protein spontaneously forms a dodecahedral T = 1 subviral particle (SVP) and good protection is achieved for young chickens. In previous studies, VP2 SVPs without His-tag can be purified by IMAC. To further study the effect of the exposed surface of a histidine residue on a VP2 SVPs’ interaction with a metal ion, a series of VP2 SVPs variants were generated. We first calculated the exposed area of the residues on the VP2 SVPs loop. Then we selected the residues with an exposed area of more than 100Å, and they were substituted in histidine. VP2 SVPs variants were analyzed for the interaction between surface histidine and the immobilized nickel ion in an IMAC column. These results show that a direct correlation between protein binding affinity and exposed surface area of the histidine. The surface histidine is involved in intramolecular hydrogen bond, reduced the binding affinity, as compared with the variant containing a histidine residue with a similar exposed surface area.
實驗室規模的昆蟲細胞培養經常使用磁攪拌瓶(spinner flask)做為生物反應器。當磁攪拌瓶培養體積增加時,氣體交換率會下降,使培養基的溶氧減少不利細胞生長。尤其昆蟲細胞對氧氣的需求比其他哺乳類細胞來的高,更不利於在磁攪拌瓶培養。為了增加培養基的溶氧,本實驗利用直接通氣法,將空氣直接導入培養基中來增加溶氧量有利於細胞生長。然而,利用直接通氣法會產生泡沫的累積與破裂的氣泡會產生剪應力傷害細胞,所以本實驗藉由添加抗剪力劑與消泡劑減少細胞的傷害。實驗結果顯示通氣培養Hi-5細胞,需額外添加0.2%(v/v) antifoam AF emulsion抑制泡沫累積,以及0.2% (w/v) Pluronic F-68保護細胞抵抗剪應力,細胞生長最大細胞密度達5 x 106 cells/ml,比生長速率為0.032h-1,相較無供氣組有利細胞生長與更高細胞密度。 傳染性華氏囊病毒(infectious bursal disease virus, IBDV)是雙股RNA病毒,對三至六周齡的幼雞具有高度的傳染性。VP2為IBDV外殼主要結構蛋白之一,本實驗室利用桿狀病毒/昆蟲表現系統表達IBDV的VP2蛋白,可自行組裝成T=1的次病毒顆粒(Subviral particle, SVP),做為疫苗施打於幼雞有良好的免疫保護效果。SVP無融合His-tag就可利用固定化金屬離子親和性層析法(immobilized-metal ion affinity chromatography, IMAC)純化。本實驗室先前研究證實SVP表面胺基酸H253可與Ni-NTA結合。為了更進一步瞭解SVP與Ni-NTA的結合機制。本實驗先將H253突變為Alanine使SVP失去吸附能力,再利用結構軟體分析VP2三聚體的P domain的胺基酸暴露面積,選取高暴露面積的胺基酸位置以組胺酸取代後測試與固定化鎳離子結合能力。結果顯示暴露面積大於100Å的組胺酸: VP2-H253AQ221H、VP2-H253AS251H、VP2-H253AQ320H與VP2-H253AA321H皆可與固定化鎳離子結合純化,而VP2-H253AQ320H的側鏈因為參與分子間氫鍵而降低與鎳離子吸附能力。總合上述結果顯示SVP的表面組胺酸暴露面積與分子間氫鍵的參與會是影響與Ni-NTA的結合的關鍵因子。
URI: http://hdl.handle.net/11455/35782
其他識別: U0005-1508201211200700
文章連結: http://www.airitilibrary.com/Publication/alDetailedMesh1?DocID=U0005-1508201211200700
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