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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
aeration culture
infectious bursal disease virus
immobilized-metal ion affinity chromatography
出版社: 生物科技學研究所
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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。
其他識別: U0005-1508201211200700
Appears in Collections:生物科技學研究所



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