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標題: 以桿狀病毒表現系統表現禽流感病毒之NA蛋白,並應用於NA亞型抗體之快速區分診斷
Expression of the NA protein of avian influenza virus by baculovirus system and its application to rapid subtyping of NA-antibodies
作者: 鍾明卉
Gung, Ming-Huei
關鍵字: avian influenza;家禽流行性感冒;neuraminidase;baculovirus;insect cell;神經安酸酶桿狀病毒;昆蟲細胞
出版社: 獸醫微生物學研究所
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家禽流行性感冒病毒 (Avian influenza virus;AIV)屬於正黏液病毒科(Orthomyxoviridae)之A型流行性感冒病毒。其病毒表面具有兩種醣基化的蛋白可引發宿主之中和抗體,分別為血球凝集素(Hemagglutinin,簡稱HA)和神經胺酸酶(Neuramindase,簡稱NA)。目前在禽鳥類已證實HA可區分為16種血清亞型,而NA則有9種,其中引起高病原性家禽流行性感冒 (HPAI)主要為H5及H7血清亞型之病毒。在1999-2000年間,義大利的雞場曾相繼爆發家禽流行性感冒病毒 H7N1血清型之高病原性(HPAI)及低病原性(LPAI)感染,造成重大的經濟損失,因而開發DIVA(Differentiating Infected from Vaccinated Animals)之檢測技術,以控制雞隻的感染及減少經濟損失。本研究亦使用DIVA之架構,應用昆蟲桿狀病毒表現系統Bac-to-Bac®重組桿狀病毒,並以Sf9及High FiveTM昆蟲細胞進行重組蛋白表現,製備N1、N2重組蛋白,經由SDS-PAGE分析重組蛋白之分子量結果判定N1、N2之重組全長蛋白可能皆具有醣基化之情形。另一方面,應用multi-screen channel 西方轉漬反應(Western blot),進行9種NA亞型血清對重組N1及N2蛋白之辨識分析,以N1及N2重組蛋白為抗原,發現N1、N2重組蛋白皆可特異性的確認相同血清亞型及區別其他各種NA血清亞型之高免血清。而以間接免疫螢光抗體染色法 (iIFA) 分析結果9種NA血清亞型之雞隻高免血清,可發現N1重組蛋白可區別N2以外之NA血清亞型,但仍以N1重組蛋白對於N1之高免血清的反應最為顯著。本研究應用義大利所開發之DIVA技術之概念,預先製備N1與N2重組蛋白,以昆蟲細胞大量表現,希望可達到區分不同NA亞型之血清之目的,進而可協助台灣地區禽流感病毒之監控與防治。

Avian influenza viruses are members of the orthomyxoviridae family and are grouped into type A influenza virus according to antigenic characteristics of their core proteins. These viruses can be further classified by their surface proteins hemagglutinin (H1-H16) and neuraminidase (N1-N9). During 1999 to 2000, outbroke of low or high pathogenic H7N1 avian influenza occured in Italy and led to a sobering economic loss. In consequence, the government of Italy developed an effective DIVA methodology as a tool for the eradication of AI. The goal of this study is to establish a DIVA methodology for rapid subtyping of NA-antibodies. The method is to use Bac-to-Bac® baculovirus express system to construct recombinant baculovirus containing N1 and N2 genes. Sf9 and High FiveTM insect cells were then used to produce recombinant NA (rNA) protein by infection with recombinant baculovirus. Possible glycosylations of the full length N1 and N2 recombinant proteins were observed based on the molecular weight of SDS-PAGE. However, the amount of recombinant N1 and N2 proteins expressed in insect cells were low. These two rNA proteins were probed with antibodies of 9 NA subtypes by multi-screen channel Western blot analysis, and the result showed that both N1 and N2 recombinant protein could discriminate the N1 and N2 sera, respectively. Moreover, the two rNA proteins were used as antigens to detect antibodies of 9 NA subtypes by indirect immunofluorescence assay (iIFA) analysis, and the result showed that recombinant N1 can discriminate each subtype sera except for the N2. The recombinant N1 and N2 proteins produced in this study could be used to establish a DIVA method that could help the surveillance and prevention of AIV in Taiwan.
其他識別: U0005-1107200614340300
Appears in Collections:微生物暨公共衛生學研究所

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