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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
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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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