Please use this identifier to cite or link to this item:
Production and Purification of Recombinant Infectious Bursal Disease Viruses D78 by Using Microcarrier Culture DF-1 Cell Line
|關鍵字:||傳染性華氏囊病病毒;微載體;DF-1;磁攪拌瓶;IBDV;microcarrier;DF-1;spinner||引用:|| A.L. van Wezel, Growth of cell-strains and primary cells on micro-carriers in homogeneous culture, Nature, 216(5110) (1967), 64-5.  A.K. Chen, S. Reuveny, S.K. Oh, Application of human mesenchymal and pluripotent stem cell microcarrier cultures in cellular therapy: achievements and future direction, Biotechnol Adv., 31(7) (2013), 1032-46.  A.S. Cosgrove, AN APPARENTLY NEW DISEASE OF CHICKEN-AVIAN NEPHROSIS, 6(Avian Diseases), (1962) 5.  H. Muller, M.R. Islam, R. Raue, Research on infectious bursal disease--the past, the present and the future, Vet Microbiol, 97(1-2) (2003), 153-65.  I. Eldaghayes, L. Rothwell, A. Williams, D. Withers, S. Balu, F. Davison, P. Kaiser, Infectious bursal disease virus: strains that differ in virulence differentially modulate the innate immune response to infection in the chicken bursa, Viral Immunol, 19(1) (2006), 83-91.  M. Brandt, K. Yao, M. Liu, R.A. Heckert, V.N. Vakharia, Molecular determinants of virulence, cell tropism, and pathogenic phenotype of infectious bursal disease virus, J Virol, 75(24) (2001), 11974-82.  B. Da Costa, C. Chevalier, C. Henry, J.C. Huet, S. Petit, J. Lepault, H. Boot, B. Delmas, The capsid of infectious bursal disease virus contains several small peptides arising from the maturation process of pVP2, J Virol, 76(5) (2002), 2393-402.  E. Lombardo, A. Maraver, I. Espinosa, A. Fernandez-Arias, J.F. Rodriguez, VP5, the nonstructural polypeptide of infectious bursal disease virus, accumulates within the host plasma membrane and induces cell lysis, Virology, 277(2) (2000), 345-57.  E. Lombardo, A. Maraver, J.R. Caston, J. Rivera, A. Fernandez-Arias, A. Serrano, J.L. Carrascosa, J.F. Rodriguez, VP1, the putative RNA-dependent RNA polymerase of infectious bursal disease virus, forms complexes with the capsid protein VP3, leading to efficient encapsidation into virus-like particles, J Virol, 73(8) (1999), 6973-83.  E.V. Grgacic, D.A. Anderson, Virus-like particles: passport to immune recognition, Methods, 40(1) (2006), 60-5.  D.L. Caspar, A. Klug, Physical principles in the construction of regular viruses, Cold Spring Harb Symp Quant Biol, 27 (1962), 1-24.  C. Troupin, A. Dehee, A. Schnuriger, P. Vende, D. Poncet, A. Garbarg-Chenon, Rearranged genomic RNA segments offer a new approach to the reverse genetics of rotaviruses, J Virol, 84(13) (2010), 6711-9.  X. Qi, Y. Gao, H. Gao, X. Deng, Z. Bu, X. Wang, C. Fu, X. Wang, An improved method for infectious bursal disease virus rescue using RNA polymerase II system, J Virol Methods, 142(1-2) (2007), 81-8.  M.Y. Wang, Y.Y. Kuo, M.S. Lee, S.R. Doong, J.Y. Ho, L.H. Lee, Self-assembly of the infectious bursal disease virus capsid protein, rVP2, expressed in insect cells and purification of immunogenic chimeric rVP2H particles by immobilized metal-ion affinity chromatography, Biotechnol Bioeng, 67(1) (2000), 104-11.  C.S. Chen, S.Y. Suen, S.Y. Lai, G.R. Chang, T.C. Lu, M.S. Lee, M.Y. Wang, Purification of capsid-like particles of infectious bursal disease virus (IBDV) VP2 expressed in E. coli with a metal-ion affinity membrane system, J Virol Methods, 130(1-2) (2005), 51-8.  S.Y. Lai, G.R. Chang, H.J. Yang, C.C. Lee, L.H. Lee, V.N. Vakharia, M.Y. Wang, A single amino acid in VP2 is critical for the attachment of infectious bursal disease subviral particles to immobilized metal ions and DF-1 cells, Biochim Biophys Acta, 1844(7) (2014), 1173-82.  張群岳, 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, 國立中興大學生物科技學研究所, 2012.  J. Porath, J. Carlsson, I. Olsson, G. Belfrage, Metal chelate affinity chromatography, a new approach to protein fractionation, Nature, 258(5536) (1975), 598-9.  E. Hochuli, H. Dobeli, A. Schacher, New metal chelate adsorbent selective for proteins and peptides containing neighbouring histidine residues, J Chromatogr, 411 (1987), 177-84.  J. Crowe, H. Dobeli, R. Gentz, E. Hochuli, D. Stuber, K. Henco, 6xHis-Ni-NTA chromatography as a superior technique in recombinant protein expression/purification, Methods Mol Biol, 31 (1994), 371-87.  K. Rekha, C. Sivasubramanian, I.M. Chung, M. Thiruvengadam, Growth and replication of infectious bursal disease virus in the DF-1 cell line and chicken embryo fibroblasts, Biomed Res. Int., 2014 (2014), 494835.  S.A. Christman, B.W. Kong, M.M. Landry, D.N. Foster, Chicken embryo extract mitigates growth and morphological changes in a spontaneously immortalized chicken embryo fibroblast cell line, Poultry Sci., 84(9) (2005), 1423-31.  Y. Wang, X. Qi, H. Gao, Y. Gao, H. Lin, X. Song, L. Pei, X. Wang, Comparative study of the replication of infectious bursal disease virus in DF-1 cell line and chicken embryo fibroblasts evaluated by a new real-time RT-PCR, J Virol Methods, 157(2) (2009), 205-10.  D.H. Jackwood, Y.M. Saif, J.H. Hughes, Replication of infectious bursal disease virus in continuous cell lines, Avian Dis, 31(2) (1987), 370-5.  劉聖哲, 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, 國立中興大學生物科技學研究所, 2018.  G.H.L. Sciences, Microcarrier Cell Culture Principles and Methods, 2013.  M.A. Arifin, M. Mel, R. Ahmad Raus, S. S. H., A. Ideris, Growth studt of DF-1 cell line in microcarrier bioreactor, 2nd International Conference on Science and Technology, (2008).  L. Ikonomou, J.C. Drugmand, G. Bastin, Y.J. Schneider, S.N. Agathos, Microcarrier culture of lepidopteran cell lines: implications for growth and recombinant protein production, Biotechnology Prog, 18(6) (2002), 1345-55.  S. Rourou, A. van der Ark, T. van der Velden, H. Kallel, 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) (2007) 3879-89.  L.J. Reed, H. Muench, A simple method of estimating fifty percent endpoints, The American Journal of Hygiene, 27 (1938), 493-497.  S.C. Arya, E. Ghosh, P.L. Banerjee, Effect of rust on haemagglutination test, J Clin Pathol, 22(2) (1969), 246.  M. Mel, M. Arifin, H. Sohif, S. Hassan, Optimization of Process Conditions for High Cell Density Proliferation of DF-1 Cells in Bioreactor, Med J Malaysia, 65 (2010).  M.Y.C. He, S.A. Stacker, R. Rossi, M.M. Halford, Counting nuclei released from microcarrier-based cultures using pro-fluorescent nucleic acid stains and volumetric flow cytometry, Biotechniques, 63(1) (2017), 34-36.  C.M. Brown, K.D. Bidle, Attenuation of virus production at high multiplicities of infection in Aureococcus anophagefferens, Virology, 466-467 (2014), 71-81.  K. Trabelsi, S. Majoul, S. Rourou, H. Kallel, Development of a measles vaccine production process in MRC-5 cells grown on Cytodex1 microcarriers and in a stirred bioreactor, Appl Microbiol Biotechnol, 93(3) (2012), 1031-40.  I. Jordan, K. John, K. Howing, V. Lohr, Z. Penzes, E. Gubucz-Sombor, Y. Fu, P. Gao, T. Harder, Z. Zadori, V. Sandig, Continuous cell lines from the Muscovy duck as potential replacement for primary cells in the production of avian vaccines, Avian Pathol, 45(2) (2016), 137-55.  A.D. Dias, J.M. Elicson, W.L. Murphy, Microcarriers with Synthetic Hydrogel Surfaces for Stem Cell Expansion, Adv Healthc Mater, 6(16) (2017).  E.S. Giotis, C.S. Ross, R.C. Robey, A. Nohturfft, S. Goodbourn, M.A. Skinner, Constitutively elevated levels of SOCS1 suppress innate responses in DF-1 immortalised chicken fibroblast cells, Sci Rep, 7(1) (2017), 17485.  C. Ou, Q. Wang, Y. Yu, Y. Zhang, J. Ma, X. Kong, X. Liu, Chemokine receptor CCR5 and CXCR4 might influence virus replication during IBDV infection, Microbial Pathogenesis, 107 (2017), 122-128.  M.S. Lee, S.R. Doong, S.Y. Lai, J.Y. Ho, M.Y. Wang, Processing of infectious bursal disease virus (IBDV) polyprotein and self-assembly of IBDV-like particles in Hi-5 cells, Biotechnology Prog, 22(3) (2006), 763-9.  L.L. Cubas-Gaona, E. Diaz-Beneitez, M. Ciscar, J.F. Rodriguez, D. Rodriguez, Exacerbated apoptosis of cells infected with infectious bursal disease virus (IBDV) upon exposure to Interferon alpha (IFN-alpha), J Virol, 92(11) (2018), e00364-18.||摘要:||
傳染性華氏囊病病毒 (IBDV) 主要感染3~6週齡幼雞的華氏囊，破壞其B淋巴細胞，因抑制了雞隻的免疫系統，使其容易因感染其他疾病造成死亡，對養雞場造成嚴重的經濟損失。先前研究指出，IBDV的外殼蛋白VP2與其致病性有關，且暴露在其外側的VP2 His253是能與Ni-NTA吸附結合的胺基酸殘基，因此可利用固定化金屬離子親和性層析管柱來純化病毒顆粒，根據軟體計算將IBDV rD78外殼VP2上暴露面積較大數個胺基酸位置，利用反向遺傳學進行點突變得到重組病毒rD78-P222H、rD78-Q324H。本實驗先以不同磁攪拌瓶轉速以及初始DF-1細胞密度找出最適合磁攪拌瓶-微載體系統的培養條件，結果為轉速30 rpm，細胞起始密度為5 x 105 cells/mL。再來以IBDV rD78-WT以不同病毒感染劑量(MOI) 1、0.1、0.01、0.001感染磁攪拌瓶培養的DF-1細胞，其中以MOI 0.1可以得到較高的病毒力價。之後將所得之重組病毒分別用MOI 0.1感染磁攪拌瓶-微載體系統培養之DF-1細胞並觀察細胞病變效應及測量病毒力價，結果顯示以磁攪拌瓶-微載體系統生產之病毒力價皆高於以T175培養盒生產的病毒力價，rD78-WT約提高1.2倍、rD78-P222H約2.6倍、rD78-Q324H約1.2倍。而利用IMAC純化則於pH 4時得到最多的病毒。
Microcarrier is a small carrier that is harmless to cells. It is added to the culture medium to make the adherent cells attach and grow. It used in spinner flask or fermentor to bring about target product. Infectious bursal disease virus (IBDV) mainly affects 3 to 6 weeks old chicks and destroys their B lymphocytes. The chicken immune system is suppressed, and dies due to other disease. Previous studies have indicated that IBDV capsid protein VP2 is related to its pathogenicity, and the VP2 His253 residue can bind to Ni-NTA that can purify by immobilized metal-ion affinity chromatography (IMAC). Using Discovery studio 4.1 calculates the large exposed area of amino acid on IBDV rD78 capsid VP2. Using reverse genetics to get mutant recombinant virus rD78-P222H and rD78-Q324H. We determine the optimization stirring speed and inoculation cell density for the spinner-microcarrier system. The result was that the stirred speed was 30 rpm and initial cell density was 5 x 105 cells/mL. Then the multiplicity of infection (MOI) of IBDV rD78-WT was determined to be 0.1 for the highest virus titer. Therefore, we infected DF-1 cell cultured in spinner-microcarrier system by using recombinant IBDV D78-P222H、Q324H, observed cytopathic effect (CPE) and measured virus titer. The result showed that all of the virus titer from spinner-microcarrier system was higher than produced in T175 culture flask. The rD78-WT increased about 1.2 times, rD78-P222H about 2.6 times, and rD78-Q324H about 1.2 times. In the virus production, spinner-microcarrier system can save one day compared with using T flask. The virus was purified by IMAC and most viruses were eluted at pH 4.0.
|Appears in Collections:||生物科技學研究所|
Show full item record
Files in This Item:
TAIR Related Article
Items in DSpace are protected by copyright, with all rights reserved, unless otherwise indicated.