臺灣博碩士論文加值系統

摘 要

家禽流行性感冒病毒（AI）擁有八段單股RNA基因，其中第八片段即NS（nonstructural） gene。NS gene可以轉譯出兩個非結構性蛋白，NS1與NS2。其中NS1是最主要之非結構性蛋白，具有高度保留性，抗原性也具有交叉反應性，與病毒的複製和抑制宿主干擾素反應有關。因此在細胞被感染的早期，會被大量合成且累積於細胞核中，並誘發針對NS1之抗體。若是以不活化疫苗免疫動物則只能產生針對結構性蛋白之抗體，而無針對非結構性蛋白之抗體，故利用NS1之特性來作為感染的指標。本研究中，利用大腸桿菌表現AI之NS1，並結合western blot assay與ELISA系統進行檢測三組經人工接種而獲得的血清，分別為接種live AI virus、免疫inactivated AI virus與inactivated semi-purified AI virus，每組四隻SPF雞，於接種後每隔7天收集血清，並以the standard agar gel precipitin（AGP）test作為此次研究之黃金標準進行檢測。於第35天時，此三組於AGP皆呈現陽性反應，但是接種活毒之組別於western blot assay與ELISA皆呈現陽性反應，以不活化病毒免疫之組別呈現微弱之反應，以不活化之部分純化病毒免疫之雞隻呈現低程度之陽性反應。由以上結果證實，此次建立之診斷系統具有敏感性。有鑑於傳統之AGP、Hemagglutination inhibition test及市面上的檢測試劑皆是以檢測流感病毒的結構性蛋白為主，但無法區分被免疫與被感染之家禽。本研究利用NS1之特性，以western blot assay與ELISA系統進行檢測三組之血清，以感染活毒時誘發之抗體最高，以不活化之部分純化病毒進行免疫其抗體量次之，以不活化病毒進行免疫其抗體最微弱，且抗體的高低與NS1含量成正相關，由此證實可用於區分被免疫與被感染之家禽。雖然western blot assay與ELISA系統皆能進行偵測NS1 antibody，但是ELISA相較於western blot assay其所需之抗原量較少、具有高度的敏感性、能大量且快速地篩檢檢體，並以數字之方式來呈現，因此用重組的NS1蛋白來建立ELISA系統已經蔚為趨勢，也期望本實驗能有助於AI的診斷。

Abstract

Avian influenza (AI) is a serious infectious disease caused by avian influenza virus (AIV) belonging to type A Orthomyxovirus. Vaccination programs for controlling avian influenza (AI) in poultry have limitations because of differentiating between vaccinated and virus-infected birds. Nonstructural (NS) proteins can be used as markers because viral infection can induce antibodies against both structural and nonstructural antigens. While immunized with inactivated viral vaccines, animals elicit antibodies from only structural proteins. Two nonstructural proteins (NS1 and NS2) are present in cells when infected with influenza virus. The NS1 protein of AIV is the major nonstructural protein and is highly conserved among AIV subtypes. The antigenic analyses of the purified NSl protein from several subtypes have indicated cross-reactivity among all influenza A virus strains. The presence of an anti-NSl response provides a useful measure in influenza viral infection, and highly circulating anti-HA antibodies. In order to use the immune response of the NSl protein in a routine diagnostic setting, it is necessary to develop an enzyme-linked immunosorbent assay (ELISA) with recombinant NS1. We have used NS1, the conserved nonstructural protein of influenza A virus, as a differential diagnostic marker for influenza virus infection. Experimentally infected poultry were evaluated for the ability to induce antibodies reactive to NS1 recombinant protein produced in Escherichia coli, which was cloned from H6N1 AIV A / Chicken / Taiwan / NCHU-0507 / 99 strain. Immune sera were obtained from SPF chickens inoculated with live AI virus, inactivated semi-purified AI virus, or inactivated AI virus. Animals that received live AI virus, inactivated purified AI virus, or inactivated AI virus were found to possess antibodies against AI virus, as measured by the standard agar gel precipitin (AGP) test. Seroconversion to positive for antibodies to the NS1 protein was achieved in birds experimentally infected with live AI virus, as determined by enzyme-linked immunosorbent assay (ELISA) and Western blot analysis. In contrast, animals inoculated with inactivated AI virus had faint seroconversion to positive for antibodies to the NS1 protein, and animals vaccinated with inactivated semi-purified AI virus had low, but detectable, levels of NS1 antibodies. These results demonstrate the potential benefit of a simple, specific ELISA for anti-NS1 antibodies that may have diagnostic value for the poultry industry, this ELISA is useful for serological diagnosis to distinguish poultry infected with influenza viruses from those immunized with inactivated vaccine.

目次
中文摘要.................................................I
英文摘要.................................................II
目次.....................................................IV
表次.....................................................VI
圖次.....................................................VII
第一章 緒言..............................................1
第二章 文獻探討..........................................2
第一節 家禽流行性感冒病毒歷史背景........................2
第二節 家禽流行性感冒病毒性狀............................4
2-2.1 病毒特性...........................................5
2-2.2 病毒結構及基因體...................................5
2-2.3 病毒的複製.........................................9
2-2.4 病毒物理化學特性...................................10
2-2.5 病毒抗原性改變特性.................................10
第三節 宿主範圍..........................................11
第四節 病毒實驗室診斷....................................12
2-4.1 病原直接分離與同定.................................13
2-4.2 直接證實病毒之存在.................................14
2-4.3 免疫螢光試驗病毒蛋白...............................14
2-4.4 證實病毒核酸之存在.................................14
2-4.5 由血清抗體證實感染.................................15
第五節 酵素連結免疫吸附分析法(ELISA)之應用原理...........16
第三章 材料與方法........................................18
第一節 病毒增殖、純化及其力價測定........................18
3-1.1 病毒來源...........................................18
3-1.2 實驗動物...........................................18
3-1.3 病毒增殖...........................................18
3-1.4 病毒純化...........................................18
3-1.5 病毒力價測定.......................................19
第二節 病毒之核酸序列分析................................19
3-2.1 病毒核酸的萃取.....................................19
3-2.2 反轉錄聚合酶鏈反應人工引子與反應條件...............19
3-2.3 反轉錄聚合酶鏈反應產物的確認.......................20
3-2.4 反轉錄聚合酶鏈反應產物的純化.......................21
3-2.5 反轉錄聚合酶鏈反應產物選殖.........................21
3-2.6 反轉錄聚合酶鏈反應產物之定序反應...................22
3-2.7 定序結果之序列分析.................................22
第三節 病毒重組蛋白之製備及確認..........................23
3-3.1 基因之增幅.........................................23
3-3.2 基因之選殖.........................................24
3-3.3重組質體之表現、純化及確認..........................26
第四節 血清..............................................29
3-4.1 實驗血清...........................................29
3-4.2 田間血清...........................................30
第五節 血清抗體檢測......................................30
3-5.1 膠體免疫擴散試驗...................................30
3-5.2 血球凝集抑制試驗...................................31
3-5.3 Indirect NS1-ELISA.................................31
第四章 結果..............................................33
第一節 病毒增殖、純化及其力價測定........................33
4-1.1 病毒增殖...........................................33
4-1.2 病毒之部分純化.....................................33
4-1.3 病毒力價測定.......................................33
第二節 病毒之核酸序列分析................................33
4-2.1 RT-PCR產物之確認...................................33
4-2.2 NS基因序列分析與親緣樹圖...........................34
4-2.3 NS1基因序列分析與親緣樹圖..........................34
第三節 病毒重組蛋白之製備及確認..........................34
4-3.1 NS1基因之選殖......................................34
4-3.2 重組質體之表現、純化及確認.........................35
4-3.3 重組蛋白特異性分析與確認...........................35
第四節 Indirect NS1-ELISA之建立與應用....................36
4-4.1 建立原核表現系統之Indirect NS1-ELISA之檢測方法.....36
4-4.2 評估indirect NS1-ELISA應用於檢測實驗血清...........37
4-4.3 評估indirect NS1-ELISA應用於檢測田間血清...........37
第五章 討論..............................................59
參考文獻.................................................66

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