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標題: A型流感病毒RNA聚合酶抗體之製備與應用
Preparation and Application of the Antibodies against the Influenza A Virus RNA Polymerase
作者: 孫惠芬
Sun, Hui-Fen
Contributors: 黃千衿;林春福
徐維莉
Wei-Li Hsu
中興大學
關鍵字: influenza virus;RNA polymerase;antibody
RNA聚合酶抗體
日期: 2010
Issue Date: 2012-09-04 16:45:25 (UTC+8)
Publisher: 獸醫公共衛生學研究所
摘要: 流感病毒RNA聚合酶(簡稱RdRp)由三種病毒次單位聚合酶蛋白組成:PA、PB1、PB2,為轉錄及複製所必須,突顯出在抗病毒機制上的重要性。為了了解流感病毒聚合酶的相關機制,抗體是最常用來分析的重要工具。本實驗利用原核系統表現重組流感病毒(A/ Puerto Rico/ 8/ 34)RdRp親水性氨基酸,重組蛋白質以鎳離子螯合管柱純化,經定量後進行動物接種,最後獲得不同動物來源的抗體: rabbit anti-PA、 chicken anti-PB1、 mouse anti-PB2。使用免疫轉漬法確認抗體對流感病毒H1N1、H6N1,聚合酶具有專一性外,也可辨識桿狀病毒系統(baculovirus system)表現的流感病毒聚合酶次單位蛋白,相同的結果亦在間接免疫螢光分析中可見。另外使用protein A agarose成功地純化不同抗體樣本中的IgG分子。後續運用多種免疫分析方法檢視抗流感病毒藥物:薑黃素(curcumin)與薑辣素([6]-gingerol),在間接免疫螢光分析觀察到薑黃素的作用在阻礙了流感病毒對細胞的感染,而薑辣素則無此作用,但對病毒蛋白質的合成具有降低的趨勢,因此兩者藥物是以不同方式達到抑制流感病毒的增殖。最後藉由商品化試劑包覆rabbit anti-PA IgG分子形成脂質複合體,運送抗體進入細胞質,以間接免疫螢光分析比較細胞對病毒感染情形,在帶有抗體分子的細胞中,病毒蛋白質的生成減少,初步證實以抑制流感病毒聚合酶功能的抗病毒策略具有可行性。未來以人類與動物體應用原則下,期許以流感病毒為主體的virosome形式包覆有效抑制流感病毒聚合酶的物質,直接在呼吸道對病毒發揮屏障作用,以這樣的方式達到預防與治療的雙重效果。
Influenza virus RNA polymerase (RdRP) containing three subunits: PA, PB1, PB2, is required for transcription and replication of viral RNA that has become a promising target for developing an alternative anti-viral agent. The antibodies were the most useful tools for study the influenza RdRp interaction. Recombinant three subunits RdRP of influenza virus (A/Puerto Rico/8/34) were expressed in prokaryotic system and were then used as antigens to produce antibodies from different animals: rabbit anti-PA, chicken anti-PB1, mouse anti-PB2. Consistent results were obtained from the immunoblot and indirected immunofluorescence assays: these antibodies were able to specifically recognize both the influenza A viruses polymerase from H1N1 or H6N1 infected and baculovirus system expressed. The IgG molecule was successfully purified through protein A agarose. Subsequently, application of such antibodies in study of the mechanism of anti-viral agents, curcumin and [6]-gingerol, the indirected immunofluorescence assays revealed that both chemicals reduced the viral replication via different mechanisms: curcumin interferenced the infection rate of influenza virus but [6]-gingerol didn't. Finally, the intracellular delivered with lipoplex, the complex of commercial reagent and rabbit anti-PA IgG, that appeared to reduce influenza viral protein synthesis. Hence, antibodies against viral RdRp may serve as an alternative therapeutic target for influenza virus infection. We expected that extend combination the influenza virosome and RdRp antagonist to set up the immuno-barrier of the respiratory tract by nasal admission. By this rule for human and animal to achieve prevent and therapeutic effect.
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