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標題: 以微載體懸浮培養DF-1細胞生產重組傳染性華氏囊病病毒D78及純化
Production and Purification of Recombinant Infectious Bursal Disease Viruses D78 by Using Microcarrier Culture DF-1 Cell Line
作者: 謝羽璿
Yu-Hsuan Hsieh
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
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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.
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