王金和Wang, Ching-Ho臺灣大學:獸醫學研究所朱文玉Chu, Wen-YuWen-YuChu2010-05-042018-07-092010-05-042018-07-092008U0001-1607200815021700http://ntur.lib.ntu.edu.tw//handle/246246/178894本研究之目的為利用對抗H5N2 亞型家禽流行性感冒病毒 (avian influenza viruses，AIV) HA1區域之單株抗體 (monoclonal antibodies，mAbs) 分別發展抗原捕捉型酵素聯結免疫吸附法 (antigen-capture enzyme-linked-immunosorbent assay，AC-ELISA) 以及阻斷型酵素聯結免疫吸附法 (blocking enzyme-linked-immunosorbent assay，B-ELISA)，以期能早期檢測出H5亞型AIV及抗H5亞型AIV抗體，另外亦進一步分析本研究室之前開發塗鍍全病毒的B-ELISA (簡稱Virus-B-ELISA) 敏感性及特異性，並針對此Virus-B-ELISA塗鍍抗原步驟加以改良以減短操作時間。實驗方法為將mAbs做為捕捉與偵測抗體，以AC-ELISA的架構進行H5亞型病毒檢測，並將mAbs標示過氧化氫酶做為追蹤子 (tracer)，以B-ELISA的架構進行雞隻血清檢測。結果，發展出之AC-ELISA只能偵測到屬於歐亞世系之H5亞型毒株，而無法測得其他世系之AIV (cut-off value=0.1)，其最低辨認限制為4.2×104/0.1 mL EID50；至於塗鍍重組HA1蛋白的B-ELISA (簡稱rHA1-B-ELISA) 則因H5亞型AIV誘發的多株抗體無法與重組蛋白質有效結合，而使H5陰性或陽性血清的測定值無顯著差異；在Virus-B-ELISA方面，與血球凝集抑制試驗相較，其檢測雞隻血清之敏感性為95.76％ (113/118)、特異性為90.78％ (266/293)，且改良前後Virus-B-ELISA之Kappa值為0.9539，代表二者間之結果幾乎完全吻合，故以改良式Virus-B-ELISA能更快速地偵測出H5亞型AIV抗體。以上總結可得，本研究發展的AC-ELISA和Virus-B-ELISA可有效運用於H5亞型歐亞世系AIV抗原及H5亞型AIV抗體檢測。The purpose of this study is to use monoclonal antibodies (mAbs) against a H5N2 avian influenza virus (AIV) HA1 domain to develop an antigen-capture enzyme-linked-immunosorbent assay (AC-ELISA) and a blocking enzyme-linked-immunosorbent assay (B-ELISA) for early detection of H5 subtype AIV and anti-H5 AIV antibodies, respectively. Besides, we also evaluated the sensitivity and the specificity of a H5-subtype AIV B-ELISA developed in our laboratory by coating with whole virus as the antigen (called Virus-B-ELISA) and improved the virus coated step of the Virus-B-ELISA to save time. These mAbs were used as the capture antibodies and detector antibodies for the detection of H5 AIV by AC-ELISA. These mAbs were also labeled with horseradish peroxidase to become the tracers for the detection of H5 antibodies in chicken serum by B-ELISA. The result showed that the AC-ELISA only detected the H5 subtype Eurasia lineage strain and did not cross react with other lineage AIV (cut-off value=0.1). The detection limit was as little as 4.2×104/0.1 mL EID50. As to the B-ELISA coated with recombinant HA1 protein (rHA1) as the antigen (called rHA1-B-ELISA), the serum polyclonal antibodies induced by H5 AIV couldn’t effective binding with rHA1. The measurements of the H5-positive or negative serum are not significantly different. In Virus-B-ELISA, the sensitivity based on the hemagglutination inhibition test was 95.76％ (113/118) and the specificity was 90.78％ (266/293). The Kappa value between original Virus-B-ELISA and improved Virus-B-ELISA was 0.9539 meant an almost perfect consistency. So the improved Virus-B-ELISA can detect the H5 subtype AIV antibodies more rapidly than the original one. The AC-ELISA and the Virus-B-ELISA are useful to detect H5 subtype Eurasia lineage AIV antigen and H5 subtype AIV antibody, respectively.口試委員審定書.............................................i謝......................................................ii要.....................................................iiibstract..................................................iv錄.......................................................v目錄...................................................xii目錄..................................................xiii一章 序言...............................................1二章 文獻回顧...........................................3一節 家禽流行性感冒病毒之病原學.........................3-1.1 家禽流行性感冒病毒分類..............................3-1.2 家禽流行性感冒病毒命名..............................3-1.3 家禽流行性感冒病毒基因體結構........................4-1.4 家禽流行性感冒病毒型態構造..........................4-1.5 家禽流行性感冒病毒蛋白質功能........................5-1.5.1 聚合酶蛋白B2 (basic polymerase protein 2，PB2)....5-1.5.2 聚合酶蛋白B1 (basic polymerase protein 1，PB1)....5-1.5.3 聚合酶蛋白A (acidic polymerase protein，PA).......6-1.5.4 血球凝集素 (hemagglutinin，HA)....................6-1.5.5 核蛋白 (nucleoprotein，NP)........................8-1.5.6 神經胺酸酶 (neuraminidase，NA)....................8-1.5.7 基質蛋白 (matrix proteins，M).....................9-1.5.8 非結構蛋白 (nonstructural proteins，NS).........10-1.6 家禽流行性感冒病毒複製機制.........................10-1.7 流行性感冒病毒抗原之變異性.........................11-1.8 流行性感冒病毒物理化學特性.........................12二節 家禽流行性感冒病毒之病況..........................13-2.1 家禽流行性感冒病毒病原性之評估.....................13-2.2 家禽流行性感冒病毒感染自然宿主範圍.................14-2.3 家禽流行性感冒病毒臨床症狀.........................14-2.4 家禽流行性感冒病毒病理變化.........................15三節 家禽流行性感冒病毒之實驗室診斷....................16-3.1 AIV抗原之偵測......................................16-3.1.1 全病毒分離.......................................16-3.1.2 偵測病毒核酸.....................................17-3.1.3 偵測病毒蛋白.....................................17-3.2 AIV抗體之檢測......................................18-3.2.1 A型流感抗體檢測..................................18-3.2.2 亞型特異性流感抗體檢測...........................18四節 酵素聯結免疫吸附法................................19-4.1 ELISA偵測抗原......................................19-4.1.1 直接型三明治型ELISA (direct sandwich ELISA)......19-4.1.2 一步法直接型三明治型ELISA (one step direct sandwich ELISA)...........................................19-4.1.3 間接型三明治型ELISA (indirect sandwich ELISA)....20-4.2 ELISA檢測抗體......................................20-4.2.1 間接型ELISA (indirect ELISA).....................20-4.2.2 直接型競爭型ELISA (direct competitive ELISA).....20-4.2.3 阻斷型ELISA (blocking ELISA).....................21-4.2.4 間接型競爭型ELISA (indirect competitive ELISA)...21-4.2.5 IgM捕捉型ELISA (IgM-capture ELISA)...............21三章 材料與方法........................................22一節 家禽流行性感冒病毒製備............................223-1.1 家禽流行性感冒病毒毒株............................22-1.2 家禽流行性感冒病毒增殖.............................22-1.3 家禽流行性感冒病毒濃縮.............................23-1.4 家禽流行性感冒病毒純化.............................23二節 H5亞型家禽流行性感冒病毒單株抗體..................24-2.1 單株抗體製備.......................................24-2.2 單株抗體純化.......................................25-2.2.1 硫酸銨分劃法.....................................25-2.2.2 Protein A親和性管柱純化抗體......................25-2.3 蛋白質定量.........................................26三節 開發H5亞型AIV抗原捕捉型ELISA......................26-3.1 選擇適當之單株抗體發展AC-ELISA.....................26-3.1.1 檢測單株抗體與A/duck/Yunlin/04 (H5N2) 結合能力...26-3.1.2 單株抗體標示過氧化氫酶............................27-3.1.3 以棋盤方格法找出最佳化AC-ELISA...................28-3.1.4 以棋盤方格法檢測αH5-3 mAb發展AC-ELISA之條件.....28-3.2 最佳化AC-ELISA cut-off值計算.......................28-3.2.1 製作雞隻氣管乳劑.................................28-3.2.2 病毒RNA萃取......................................29-3.2.3 H5 subtyping RT-PCR..............................29-3.2.4 洋菜膠體電泳.....................................30-3.2.5 計算最佳化AC-ELISA cut-off值.....................31-3.3 最佳化AC-ELISA辨認病毒毒株分析.....................32-3.3.1 H5亞型家禽流行性感冒病毒HA基因序列親源性分析.....32-3.3.2 測試最佳化AC-ELISA辨認之病毒毒株.................32-3.4 最佳化AC-ELISA靈敏度分析...........................32-3.4.1 家禽流行性感冒病毒力價測定.......................32-3.4.1.1 雞隻紅血球懸浮液製備...........................32-3.4.1.2 血球凝集試驗 (hemagglutination test，HA test)..33-3.4.1.3 病毒50％雞胚胎蛋感染/致死劑量 (50％ embryo infectious/lethal dose，EID50/ELD50).....................33-3.4.2 測試最佳化AC-ELISA之檢測靈敏度...................34-3.4.3 比較最佳化AC-ELISA與RT-PCR之檢測效力.............34四節 Virus-B-ELISA雞隻田間試驗分析.....................35-4.1 雞隻田間血清樣本收集...............................35-4.2 Virus-B-ELISA操作流程..............................35-4.3 血球凝集抑制試驗 (hemagglutination inhibition test，HI test)..................................................36-4.4 Virus-B-ELISA敏感性及特異性分析....................36-4.5 改良之Virus-B-ELISA確效試驗........................37五節 開發H5亞型家禽流行性感冒病毒rHA1-B-ELISA..........37-5.1 A/duck/Yunlin/04病毒核酸HA1基因選殖................37-5.1.1 增幅A/duck/Yunlin/04 HA1基因為3段短基因片段......37-5.1.1.1 萃取pGEXH5-HA1質體.............................37-5.1.1.2 聚合酶鏈反應 (polymerase chain reaction，PCR)..38-5.1.1.3 DNA片段之分離與純化............................39-5.1.2 TA cloning kit基因選殖...........................40-5.1.2.1 TA cloning步驟.................................40-5.1.2.2 重組TA質體colony PCR...........................41-5.1.2.3 重組TA質體基因核酸定序.........................41-5.1.3 pET-21a表現載體基因選殖..........................42-5.1.3.1 限制酶酵素作用 (digestion).....................42-5.1.3.2 pET-21a表現載體接合反應 (ligation).............42-5.1.3.3 重組pET-21a表現載體轉形 (transformation).......43-5.1.3.4 重組pET-21a表現載體檢定........................43-5.2 重組蛋白質分析.....................................43-5.2.1 重組蛋白質表現...................................43-5.2.2 SDS-膠體電泳 (SDS-PAGE)..........................44-5.2.3 SDS-膠片染色法...................................45-5.2.4 西方墨點法確認重組蛋白質.........................45-5.2.5 檢測單株抗體與重組蛋白結合能力...................45-5.2.6 單株抗體標示過氧化氫酶...........................46-5.2.7 重組蛋白質純化...................................46-5.3 rHA1-B-ELISA條件測試...............................46-5.3.1 以棋盤方格法找出rHA1-B-ELISA條件.................46-5.3.2 rHA1-B-ELISA檢測雞隻血清.........................47四章 結果..............................................48一節 H5亞型家禽流行性感冒病毒單株抗體..................48-1.1 單株抗體製備.......................................48-1.2 單株抗體純化及定量.................................48二節 H5亞型AIV AC-ELISA之開發..........................48-2.1 AC-ELISA單株抗體之選擇.............................48-2.1.1 單株抗體與A/duck/Yunlin/04 (H5N2) 結合能力.......48-2.1.2 最佳化AC-ELISA條件...............................49-2.1.3 以棋盤方格法檢測αH5-3 mAb發展AC-ELISA之條件.....49-2.2 最佳化AC-ELISA之cut-off值..........................50-2.3 最佳化AC-ELISA辨認病毒毒株分析.....................50-2.3.1 H5亞型家禽流行性感冒病毒HA基因序列親源性分析.....51-2.3.2 最佳化AC-ELISA辨認之病毒毒株.....................51-2.4 最佳化AC-ELISA靈敏度分析...........................51-2.4.1 家禽流行性感冒病毒EID50/ELD50測定................51-2.4.2 最佳化AC-ELISA之檢測靈敏度.......................51-2.4.3 比較最佳化AC-ELISA與RT-PCR之檢測效力.............52三節 Virus-B-ELISA雞隻田間試驗分析.....................52-3.1 雞隻田間血清樣本Virus-B-ELISA及HI test試驗.........52-3.2 Virus-B-ELISA敏感性及特異性分析....................52-3.3 改良之Virus-B-ELISA確效試驗........................53四節 rHA1-B-ELISA之開發................................53-4.1 A/duck/Yunlin/04病毒核酸HA1基因選殖................53-4.1.1 PCR增幅A/duck/Yunlin/04 HA1基因為3片段...........53-4.1.2 TA重組質體之確認.................................54-4.1.3 限制酶酵素作用...................................54-4.1.4 pET-21a重組質體之確認檢定........................54-4.2 重組蛋白質分析.....................................55-4.2.1 重組蛋白質表現...................................55-4.2.2 西方墨點法確認重組蛋白質.........................55-4.2.3 檢測單株抗體與重組蛋白結合能力...................56-4.2.4 重組蛋白質純化...................................56-4.3 rHA1-B-ELISA條件測試...............................57-4.3.1 以棋盤方格法找出rHA1-B-ELISA條件.................57-4.3.2 rHA1-B-ELISA檢測雞隻血清.........................57五章 討論..............................................58一節 H5亞型AIV單株抗體.................................58二節 H5亞型AIV AC-ELISA之開發..........................59三節 Virus-B-ELISA雞隻田間試驗分析.....................61四節 rHA1-B-ELISA之開發................................64考文獻..................................................66 目 錄able 1、百分飽和濃度之硫酸銨添加量.......................77able 2、病毒株分型及病毒尿囊液EID50與ELD50定量...........78able 3、HI test及Virus-B-ELISA偵測雞隻血清H5亞型AIV抗體..79able 4、Virus-B-ELISA確效試驗............................80 目 錄igure 1、A型流行性感冒病毒型態構造模式圖.................81igure 2、H3亞型毒株HA1頭區5個抗原決定位之自然變異情形....82igure 3、A/duck/Yunlin/04 HA1基因選殖模式圖..............83igure 4、pET-21a vector結構與構築模式圖..................84igure 5、Indirect ELISA測定單株抗體和A/duck/Yunlin/04病毒之結合能力..................................................85igure 6、以棋盤方格法配對不同濃度之αH5-3、αH5-6單株抗體以找出最佳化之AC-ELISA組合..................................85igure 7、H5亞型家禽流行性感冒病毒HA基因序列親源樹........86igure 8、檢測最佳化AC-ELISA可辨認之病毒毒株..............87igure 9、最佳化AC-ELISA之檢測靈敏度......................88igure 10、比較最佳化AC-ELISA與subtyping RT-PCR之檢測效力.88igure 11、HI test H5抗體陽性血清檢測Virus-B-ELISA之敏感性89igure 12、HI test H5抗體陰性血清檢測Virus-B-ELISA之特異性89igure 13、增幅A/duck/Yunlin/04 HA1基因為3片段之電泳圖....90igure 14、TA重組質體轉型於JM109後之colony PCR電泳圖......91igure 15、pET-21a vector及重組TA質體BamHI/XhoI限制酶酵素切割純化....................................................92igure 16、重組pET-21a表現載體轉型至BL21 (DE3) 之colony PCR電泳圖....................................................93igure 17、SDS-PAGE確認重組蛋白質表現.....................94igure 18、Western blotting分析重組蛋白質之His-tag片段抗原性........................................................95igure 19、Western blotting分析重組蛋白質帶有的短片段A/duck/Yunlin/04 HA1蛋白抗原性............................96igure 20、Indirect ELISA測定各重組蛋白質與4株單株抗體的結合能力......................................................97igure 21、SDS-PAGE分析rHA1-C-pET-21a重組蛋白之純化透析...98igure 22、以棋盤方格法找出rHA1-B-ELISA條件...............99igure 23、rHA1-B-ELISA檢測H5亞型AIV抗體陰性/陽性雞隻血清.99application/pdf20509382 bytesapplication/pdfen-US家禽流行性感冒病毒H5亞型單株抗體抗原捕捉型酵素聯結免疫吸附法阻斷型酵素聯結免疫吸附法血球凝集素Avian influenza virusH5 subtype monoclonal antibodyAntigen-capture enzyme-linked-immunosorbent assayBlocking enzyme-linked-immunosorbent assayHemagglutininthesishttp://ntur.lib.ntu.edu.tw/bitstream/246246/178894/1/ntu-97-R95629004-1.pdf