Please use this identifier to cite or link to this item: http://hdl.handle.net/11455/63266
標題: Production and Application of the Ha Antigen of Avian Influenza Virus H5 and H7 Subtypes in Yeast and Baculovirus Expression System
禽流感H5及H7亞型病毒之HA抗原在酵母菌和桿狀病毒表現系統的生產及應用
作者: 王敏盈
賴素媛
關鍵字: 畜牧獸醫類, 生物技術;應用研究
摘要: 
自從1997 年香港爆發禽流感以來,全世界近幾年又開始籠罩在禽流感的威脅下,每年因為禽流感所造成的經濟損失相當慘重。禽流感病毒屬於A 型流感病毒,只有少數HA 亞型(H1, H2, H3)及NA 亞型(N1, N2)確定會感染人類並造成呼吸道的疾病。儘管如此,由於流感病毒變異快速,在最近H5N1 的衝擊下,造成越來越多人被感染及死亡的病例出現;WHO 更發出全球警訊說禽流感轉變為人傳人乃遲早之事,屆時全球死亡人數將急遽攀升。要預防禽流感的爆發,除了良好的監控系統外,就是以施打疫苗為主。本實驗室目前已能在昆蟲細胞中成功表現H7N1 亞型病毒的HA 蛋白,每ml 可達40μg之產量,並且可以固定化金屬親合性管柱純化得到高純度的蛋白,此蛋白具備醣基化及trimer 的功能性構型,目前正在進行雞隻的免疫試驗。本計劃除利用昆蟲細胞表現H7N1的HA 蛋白外,我們也把H5N2 亞型的HA 及酵母菌的生產方式納入研究中,以發展一個利用酵母菌表現系統及桿狀病毒表現系統生產及純化H5N2 及H7N1 亞型病毒的HA之高效率流程,此一個製造技術平台之建立,不僅可提供日後緊急疫苗製造所需之技術,亦可快速生產高純度、高安全性的HA 以供將來開發新型之檢驗試劑或進行蛋白結晶以瞭解不同亞型之HA 蛋白的三度立體空間結構。所以預期兩年內完成以下目標:第一年:1. 完成H5N2 及H7N1 全長及分泌型HA 基因在桿狀病毒表現載體上的構築。2. 攜帶H5N2 及H7N1 全長及分泌型HA 基因之重組桿狀病毒的獲得。3. 完成HA 蛋白在昆蟲細胞的表現、純化及特性分析。4. 雞隻免疫試驗及HI 抗體力價分析。第二年:1. 完成H5N2 及H7N1 全長及分泌型HA 基因在酵母菌表現載體上的構築。2. 完成HA 蛋白在酵母菌的表現、純化及特性分析。3. 完成10 公升級量產條件最適化。4. 雞隻免疫試驗及HI 抗體力價分析。

Since the outbreak of avian influenza in Hong Kong in 1997, people worldwide begin tolive under the threat of avian influenza. The avian influenza results in huge economic loss inpoultry industry every year. The infectious virus belongs to the type A influenza virus, onlysome HA subtypes (H1, H2, H3) and NA subtypes (N1, N2) can infect and cause respiratorydiseases in human. However, due to the highly antigenic variation and under the impact ofH5N1 circulation, increasing cases of infection and death in human were reported. The WHOeven warn that the avian influenza will become human transmissible sooner and later andthen the mortality worldwide will increase sharply. Besides the well surveillance system, theadministration of vaccine is the best way to prevent the outbreak of avian influenza. For thispurpose, we have already expressed the HA protein of the H7 subtype successfully usingbaculovirus expression system. The expressed HA protein was glycosylated and with a nativetrimer conformation. The yield achieves 40 μg per ml of culture, and they can be purifiedusing the immobilized metal-ion chromatography. The vaccination trials in chickens areprogressing currently. In the future, we plan to produce the H5 and H7 HA subunit vaccinesusing baculovirus and yeast expression system, if these vaccines were effective, the resultswill provide a technical platform to meet the emergency need of vaccine manufacture. Weanticipate the research results in this plan can achieve the demands of fast producing, highpurity, and safety concern in HA subunit vaccine producing.To achieve our goal, the following specific aims will be finished in two years.First year:1. To construct transfer vectors carrying the genes of the full length HA andthe secreted form of HA (for either H5N2 or H7N1 subtype of AVI).2. To obtain recombinant baculoviruses expressing the full length HA and thesecredted form of HA (for either H5N2 or H7N1 subtype of AVI).3. To perform the expression of the recombinant HA proteins in insect cellsand purification of proteins.4. To carry out the chicken vaccination experiments and HI analysis.Second year:1. To construct the yeast expression vectors carrying the genes of the fulllength HA and the secreted form of HA (for either H5N2 or H7N1subtype of AVI).2. To express the recombinant HA proteins in Pichia and to purify therecombinant HA proteins.3. To perform a 10-liter bench-scale production and purification of therecombinant HA proteins.4. To carry out the chicken vaccination experiments and HI analysis.
URI: http://hdl.handle.net/11455/63266
其他識別: NSC95-2313-B005-036-MY2
Appears in Collections:生物科技學研究所

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