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標題: 利用毛細管帶域電泳法分離及檢測傳染性華氏囊病毒顆粒
Separation and identification of infectious bursal disease virus particles by capillary zone electrophoresis
作者: 謝昕翰
Hsieh, Hsin-Han
關鍵字: capillary zone electrophoresis;毛細管帶域電泳;infectious bursal disease virus particles;傳染性華氏囊病病毒
出版社: 微生物暨公共衛生學研究所
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傳統上蛋白質組成之分析,通常都是藉由高效能液相層析(HPLC),但是HPLC的動相溶劑常會被微粒分子阻礙,進而影響其再現性及解析度,且會產生大量的有機溶劑廢液。毛細管帶域電泳(Capillary zone electrophoresis,CZE) 是另一種有效的分析方法,分析樣品所需時間短,且兼顧效能的穩定性及準確性,而且只需要微量的樣品量即可進行分析。CZE是根據物質其荷質比之不同來進行分離,且最重要的是CZE可以使病毒保持在完整的顆粒下進行分析,並不會在操作過程中造成病毒顆粒的損壞。在CZE分離中最重要的就是其緩衝液和pH值,不同的緩衝液及酸鹼值所適用的對象皆不相同。CZE至今已經成功應用在許多病毒的分析研究上,包括菸草鑲嵌病毒、人類鼻病毒、重組腺病毒和輪狀病毒等等,但尚未有文獻提到使用CZE來分析傳染性華氏囊病病毒 (Infectious bursal disease virus,IBDV)。傳染性華氏囊病又稱作甘保羅病,由IBDV所引起,是一種具有高度傳染性的免疫抑制疾病,好發於年幼的雞隻,在全球家禽業已造成重大的經濟損失。完整的IBDV病毒顆粒和其免疫性具有高度相關性,並可誘導產生具保護性之免疫反應。因此,深入了解IBDV病毒顆粒之特性並將其應用在疫苗之製備,已成為當前重要之課題。本實驗之目的,是希望建立一個毛細管電泳分析系統,用來分析並檢測傳染性華氏囊病毒顆粒。首先,將經IBDV感染三天後之華氏囊和磷酸緩衝液混合,進行均質形成病毒原液。接著利用30 % 蔗糖進行初步的純化,緊接著進行氯化銫等密度梯度離心,完成病毒的分離與純化。純化之病毒經穿透式電子顯微鏡的觀察,以及反轉錄聚合

Traditional methods for the analysis of protein components, such as high performance liquid chromatograph (HPLC), are either time-consuming or inefficient. Instead, capillary zone electrophoresis(CZE)is a rapid, accurate and efficient technique used to separate mixtures of charged molecules/macromolecules. The most important feature of CZE is that intact shape of protein can be maintained during the operation. The separation occurs in a buffer solution where analytes separate and move according to their electrophoretic mobility past a detector. The CZE has been employed to study diverse viruses including tobacco mosaic virus, human rhinovirus, recombinant adenovirus and recombinant rotavirus etc, but no attempts have been made to analyze infectious bursal disease virus(IBDV). Infectious bursal disease(IBD)is a highly contagious and immunosuppressive disease caused by IBDV in young chickens. IBDV is responsible for major economic losses in the poultry industry worldwide. The integrity of IBDV particles is highly associated with its immunogenicity and the ability to induce protective immune responses. Therefore, characterization of IBDV particle is important to demonstrate a consistent vaccine manufacturing process. The purpose of this study is to setup a CZE to separate and identify IBDV particles prepared by sucrose and cesium chloride(CsCl) centrifugation from IBDV infected bursae. Bursae were collected from chickens infected with IBDV three days after challenge and homogenized in 1x PBS. IBDV were purified from bursal homogenate by ultracentrifugation with 30 % sucrose first and followed with CsCl solution. The purified IBDV were confirmed by transmission electron microscopy (TEM)and RT-PCR and applied into CZE with 1x PBS or BGE buffer (pH = 7 or 9) as running buffer using uncoated fused-silica capillaries. However, no peak was observed. A boric acid(pH = 9)running buffer was used and successfully form a single peak with electrophoretic mobility of 10 minutes indicating the IBDV particles. Furthermore, the single peak of IBDV particle was confirmed by binding IBDV with monoclonal anti-VP2 antibody and the virus-antibody complex was also analyzed by CZE. The signal of the previous peak of IBDV reduced and the virus-antibody complex formed an extra peak with electrophoretic mobility of 30 minutes. The results demonstrated that CZE is a capable method to separate and identify IBDV particles instead of time-consuming methods such as TEM and RT-PCR.
其他識別: U0005-1708201117051000
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