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標題: 應用大腸桿菌表現家禽流行性感冒病毒血球凝集素以區別病毒血清亞型
Application of Escherichia coli-Expressed Hemagglutinin of Avian Influenza Virus in Viral Serotyping
作者: 吳靜如
Wu, JingRu
關鍵字: Avian influenza (AI)
Hemagglutinin (HA)
出版社: 獸醫學系
摘要: 家禽流行性感冒 (Avian Influenza, AI),係由A型流行性感冒病毒所引起全身性或呼吸道感染的疾病。在禽類已證實有15種HA血清亞型 (HA1-HA15) 及9種NA血清亞型 (NA1-NA9),且水禽類為所有HA1-HA15亞型AI病毒之貯留者 (reservoir)。1997年在香港爆發H5N1強毒型AI大流行,此H5N1病毒對雞群具有高致病性,並可直接感染人類造成死亡,此一發現更加突顯AI病毒可藉基因重組,而形成新的高致病性病毒株之重大隱憂。目前最常用以區分AI病毒血清亞型之方法為血球凝集抑制試驗 (hemagglutination inhibition test, HI),此方法缺乏敏感性及快速性。本研究利用反轉錄酶鏈反應 (Reverse transcription polymerase chain reaction, RT-PCR),增幅HA1~HA15亞型病毒之HA基因片段,並將這些片段分別嵌入pET-32a表現載體內,再轉型至大腸桿菌BL-21 (DE3)中,進行重組HA蛋白質表現。這些重組蛋白經純化後,以西方轉印分析法 (Western blot analysis) 證實,HA1~HA15亞型之重組蛋白,對於15種HA血清亞型之雞隻高免血清具有極高之特異性,即單一亞型之重組蛋白僅能被相同亞型之高免血清所辨識,因此利用這些重組蛋白進行Western blot,可快速區分病毒抗血清之亞型。此外,HA3、HA5、HA6、HA7、HA9亞型之重組蛋白,亦被應用於塗鍍 (coating) ELISA plate,製備ELISA套組作為診斷用途。結果發現,此ELISA套組則可區分出HA3、HA5、HA6、HA7、HA9抗血清之亞型。另外,以這些重組蛋白進行Western blot或ELISA分析,亦成功診斷出台灣田間雞隻被HA6亞型病毒感染之案例。本研究之結果證實以原核系統所表現之HA重組蛋白,可用於區分AI病毒抗血清之亞型,此為一新的發現,將可用於協助改進現有之診斷技術,以達快速診斷AI病毒並區分其亞型之需求。
Avian influenza (AI) virus, a type A influenza virus, which might cause from a local respiratory to a systemic fatal infection in the affected animals. Fifteen HA subtypes (HA1~HA15) and nine NA subtypes (NA1~NA9) of avian influenza virus (AIV) have been found in avian species, and waterfowls are the main reservoir of these viruses. An outbreak of H5N1 virus infection occurred in Hong Kong in 1997; the virus was found highly pathogenic to chickens, and caused a lethal infection in human. This finding protrudes the importance of the generation of highly pathogenic viruses by recombination between different AIVs. The conventional method for differentiating subtypes of AIV is hemagglutination inhibition (HI) test, which is both time-consuming and insensitive. In this study, the HA gene fragment of 15 subtypes of AIV were amplifies by RT-PCR, and cloned individually into the expression vector pET32a. The recombinant plasmids were transformed into E. coli host BL-21 (DE3) for expression of recombinant HA proteins. After purification, these recombinant proteins were found to be useful in Western blot analysis for the differentiation of HA1~HA15 antisera. Moreover, the recombinant HA3, HA5, HA6, HA7, and HA9 were used for the coating of ELISA plate for the diagnosis of AIV infection. We found that this recombinant HA-ELISA could differentiate the antisera of HA3, HA5, HA6, HA7, and HA9 subtypes. In addition, recombinant HA-Western blot and HA-ELISA successfully identify a field case in which chickens were infected by AIV of HA6 subtype. This study shows the first time that recombinant HA protein expressed by a prokaryotic system can be used in differentiation of subtypes of antisera of AIV. The application of this recombinant HA-ELISA assay might help to improve the conventional diagnosis method of AIV, and to provide a rapid and specific tool for the subtype-differentiation of AIV.
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