Please use this identifier to cite or link to this item: http://hdl.handle.net/11455/14016
DC FieldValueLanguage
dc.contributor.advisor謝快樂zh_TW
dc.contributor.advisor張伯俊zh_TW
dc.contributor.author何蓓音zh_TW
dc.date2003zh_TW
dc.date.accessioned2014-06-06T06:51:34Z-
dc.date.available2014-06-06T06:51:34Z-
dc.identifier.urihttp://hdl.handle.net/11455/14016-
dc.description.abstract1997年香港爆發高病原性H5N1血清亞型之家禽流行性感冒,造成6人不幸死亡,經由分析比對其序列後,推斷該病毒是經由已感染的禽鳥類直接傳染給人類,1999年與2001年H5N1亞型病毒又於中國大陸及香港之雞、鴨間流傳,造成嚴重之經濟損失,鑑於兩岸間之往來日益頻繁,此H5亞型病毒對本省養禽業具極大之潛在危險,另亦有人畜共通疾病之隱憂。為求有效地監控此病毒,本實驗室先前已成功建立一株H5亞型之HA(hemagglutinin)蛋白單源抗體(命名為MAb-H5),並開發以MAb-H5建立阻斷性ELISA(blocking ELISA)快速診斷H5亞型血清之技術,為了進一步瞭解此MAb-H5單源抗體之特性,本實驗將H5亞型之HA蛋白抗原決定區,即第108-299個胺基酸之基因共分成17個片段,再將這些不同片段以基因重組技術選殖至原核表現系統表現蛋白,並配合Western blot找出主要抗原決定區。結果發現單源抗體MAb-H5主要辨識區位於第200-231胺基酸序列中,此外將此區比對香港H5毒株序列,從中找出相對之核苷酸序列,以化學合成法合成此片段進行蛋白質表現,發現單源抗體MAb-H5亦能辨識香港H5毒株之HA蛋白,顯示以MAb-H5所建立之阻斷性ELISA應能偵測到香港1997年所分離H5毒株之抗血清。另一方面,阻斷性ELISA套組在H1∼H15標準血清3倍稀釋的條件下,H5亞型血清阻斷性可達O.D值0.208,其餘14亞型血清O.D值均在2.076以上,而陽性血清之檢出敏感性為93.3%,田間陰性血清之檢測特異性可達93.7%(競爭百分比30%訂為 cut-off value)。未來除了增加田間血清之檢測數量外,亦嘗試改變檢驗條件,以期能夠達到更高之敏感度與特異性,希望藉此檢驗試劑來快速診斷高病原性H5亞型血清,以預防病毒之入侵與蔓延。zh_TW
dc.description.abstractAn outbreak of highly pathogenic H5N1 avian influenza occurred in Hong Kong in 1997 and caused the death of 6 people. Nucleotide sequence analysis showed that this virus was transmitted directly from avian species to humans. In 1999 and 2001, the H5N1 virus circulated again in chickens and ducks at Hong Kong and China, and caused a severe economic loss. Because of the rapid and frequent communication between Taiwan and China, the H5N1 virus might invade Taiwan and impose a potential risk to the poultry industry of Taiwan. To develop a rapid method for the survey of this virus infection, we have developed a monoclonal antibody (MAb-H5) against the HA (hemagglutinin) protein of H5 virus, and developed a blocking ELISA for the detection of antibody against the H5 virus. To locate the binding site of MAb-H5, we divided the HA protein (residues 108-299) of the H5 subtype into 17 fragments, and expressed these fragments in E. coli. By Western blot, we were able to localize the binding site of MAb-H5 at residues 200-231 of the HA protein. We also synthesized a gene fragment encoding the residues 200-231 of the HA protein of the H5N1 virus isolated in Hong Kong in 1997. By E. coli expression and Western blot analysis we showed that MAb-H5 could recognized the HA protein of the H5N1 virus isolated in 1997 in Hong Kong. H1-H15 reference sera (1:3 dilution) was tested against this blocking ELISA, and the result showed that the H5 antiserum gave an absorbance 0.208 whereas sera of other subtypes gave absorbance higher than 2.076. The sensitivity of the blocking ELISA was 93.3 when tested against the positive sera, and the specificity was 93.7 when tested against the negative field sera (30% competition was used as the cut-off value). The further work of this study will include the test of more field sera, and modification of the test condition to increase the sensitivity and specificity of this test. We believe that the blocking ELISA could provide a rapid tool for the detection of antibody of the H5 virus, and will contribute to the prevention of the invasion and spreading of H5 virus.en_US
dc.description.tableofcontents中文摘要…………………………………………………………………. Ⅰ 英文摘要…………………………………………………………………. Ⅱ 目次………………………………………………………………………. Ⅲ 表次………………………………………………………………………. Ⅴ 圖次………………………………………………………………………. Ⅵ 第一章 緒言……………………………………………………………. 1 第二章 文獻探討………………………………………………………. 2 2-1 歷史背景及現況…………………………………………... 2 2-2 病毒特徵…………………………………………………... 5 2-3 病毒型態、結構與功能…………………………………… 6 2-3.1 血球凝集素………………………………………… 6 2-3.2 神經胺酸酶………………………………………… 7 2-3.3 基質蛋白…………………………………………… 8 2-3.4 聚合酶蛋白………………………………………… 8 2-3.5 核蛋白……………………………………………… 8 2-3.6 非結構蛋白………………………………………… 9 2-4 病毒複製過程……………………………………………... 9 2-5 病毒物理化學特性………………………………………... 10 2-6 病毒抗原特性……………………………………………... 10 2-7 宿主範圍…………………………………………………... 11 2-8 診斷方法…………………………………………………... 12 2-8.1 偵測抗原………………………………………….... 12 2-8.2 偵測抗體…………………………………………… 13 2-9 酵素連結免疫吸附測定之應用原理……………………... 14 2-10 單源抗體……………………………………………….….. 15 2-10.1單源抗體之歷史背景…………………………….… 15 2-10.2融合瘤細胞篩選之原理………………………….… 15 2-6.3 單株抗體之特性與應用…………………………….. 16 第三章 材料與方法……………………………………………………. 17 3-1 重組表現載體之構築………………………………….….. 17 3-1.1 增幅基因………………………………………........ 17 3.1.2 重組基因之選殖………………………………........ 18 3.1.3 重組基因之表現與確認………………………........ 20 3-2 化學合成法合成核酸片段之分析……………………....... 23 3-2.1 以化學合成法合成核酸片段……………………… 23 3-2.2 合成片段之分析…………………………………… 23 3-3 AI-H5 ELISA之試驗……………….……………………... 23 3-3.1 利用以H5重組蛋白為抗原之ELISA kit檢測血清……………………………….………………….. 24 3-3.2 血清之前處理……………………….……………... 24 3-4 以血球凝集抑制試驗確認血清中之抗體力價…………… 24 3-4.1 偵測抗原力價…………………………….………... 24 3-4.2 偵測抗體力價……………………………………… 25 第四章 結果………………………………………................................. 27 4-1 MAb-H5之特性分析……………………………………… 27 4-1.2 確認MAb-H5對15亞型AIV之辨識特異性……. 27 4-1.2 確認MAb-H5對重組蛋白之辨識特異性………… 27 4-1.3 分析MAb-H5辨識之抗原決定位………………… 27 4-1.4 MAb-H5對Ck/HK/97之特異性分析……………... 29 4-1.4 MAb-H5對pET32a-H5-Dk/HYK/80與pET32a-H5-Ck/HK/97重組蛋白之結合活性比較... 29 4-2 AI-H5 blocking ELISA之分析……………………………. 30 第五章 討論……………………………………………………………. 51 參考文獻…………………………………………………………………. 56zh_TW
dc.language.isoen_USzh_TW
dc.publisher獸醫學系zh_TW
dc.subjectH5亞型家禽流行性感冒zh_TW
dc.titleH5亞型高病原性家禽流行性感冒之單源抗體特性分析及其在以阻斷性ELISA快速診斷H5亞型血清技術之應用zh_TW
dc.titleCharacterization of monoclonal antibody against H5 subtype of highly pathogenic avian influenza virus and its application to rapid detection of H5-specific antibody by blocking ELISAen_US
dc.typeThesis and Dissertationzh_TW
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