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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: 黃士至
關鍵字: 桿狀病毒;禽流感
出版社: 獸醫學系
家禽流行性感冒病毒 (Avian influenza virus;AIV)屬於A型流行性感冒病毒。依據病毒表面的醣蛋白區分為15種HA (H1-H15) 及9種NA (N1-N9)亞型,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,由於義大利發生H7N1血清亞型之LPAI及HPAI相繼於養禽場爆發而造成經濟上的重大損失,該國政府便研發了DIVA(Differentiating Infected from Vaccinated Animals)檢測技術,藉以有效的控制了家禽流行性感冒。本研究亦依DIVA實驗架構,應用了Bac-N-BlueTM 昆蟲桿狀病毒表現系統及Bac-to-Bac® 昆蟲桿狀病毒表現系統,分別構築了N1、N3、N7重組桿狀病毒,並以High Five昆蟲細胞進行重組蛋白表現,製備了N1、N3、N7重組蛋白,而重組蛋白以SDS-PAGE分析結果可發現N3及N7之重組全長蛋白可能具有醣基化之現象。另一方面,以N3及N7重組蛋白為抗原,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N3及N7蛋白之辨識分析,發現N3重組蛋白可特異性的區分N3及其他各種NA血清亞型之高免血清,而N7重組蛋白可區別N7與N6、N8以外之NA血清亞型之高免血清。將N3及N7重組蛋白應用間接免疫螢光抗體染色法(iIFA),分析9種NA血清亞型之雞隻高免血清時,可發現N3、N7血清對於相同血清亞型的重組蛋白之辨識效果最佳,呈現強陽性的螢光,並且視野下大於80%以上之感染細胞的細胞膜上有強陽性反應,可與其它亞型血清做明顯區分,尤其以N1、N2、N5、N9之區分效果最佳,因此本研究所表現之N3與N7重組蛋白應可應用於DIVA技術,區分不同NA亞型之血清,進而可協助禽流感病毒之監控與防治。

Avian influenza virus is a type A influenza virus. According to virus surface glycoprotein, fifteen HA subtypes(H1-H15)and nine NA subtypes(N1-N9)have been identified. During 1999 and 2000, chickens in Italy were infected by H7N1 viruses of low or high pathogenicity, which resulted in a drastic economic loss. Because of this impact, the government of Italy developed an effective DIVA methodology as a tool for the eradication of AI in Italy. In this study, recombinant baculovirus containing the N1, N3 and N7 genes were constructed by Bac-N-BlueTM or Bac-to-Bac® expression system. High Five insect cells were used to produce recombinant NA(rNA) protein. Possible glycosylations of the full length N3 and N7 recombinant proteins were observed by SDS-PAGE. These two rNA proteins were used to detect antibodies of 9 NA subtypes by multi-screen channel Western blot analysis, and the result showed that N3 recombinant protein could discriminate the N3-specific sera from sera of other subtypes. Moreover, N7 recombinant protein could discriminate the N7-specific sera from sera of other subtypes, except for the N6- and N8-specific sera. 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 sera of the same subtype had strong positive fluorescence against the rNA proteins. Therefore, this iIFA can discriminate sera of N3 and N7 subtypes from sera of other subtypes, especially from N1, N2, N5 and N9 subtypes. The recombinant N3 and N7 proteins produced in this study could be used to establish a DIVA method that could help the surveillance and prevention of AIV.
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