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完整後設資料紀錄
DC 欄位 | 值 | 語言 |
---|---|---|
dc.contributor.advisor | 闕玲玲(Ling-Ling Chueh) | |
dc.contributor.author | Ying-Ting Wang | en |
dc.contributor.author | 王映庭 | zh_TW |
dc.date.accessioned | 2021-06-15T04:12:28Z | - |
dc.date.available | 2021-12-31 | |
dc.date.copyright | 2011-08-19 | |
dc.date.issued | 2011 | |
dc.date.submitted | 2011-08-17 | |
dc.identifier.citation | Addie, D.D., Jarrett, O., 1992, A study of naturally occurring feline coronavirus infections in kittens. Vet Rec 130, 133-137.
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dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/45286 | - |
dc.description.abstract | 貓冠狀病毒 (Feline coronavirus, FCoV) 是致命性之貓傳染性腹膜炎之病原。FCoV可區分為第一型 (Type I) 及第二型 (Type II)。世界各地FCoV病毒分型調查結果皆以Type I感染為主,然Type II感染卻顯示與疾病較為相關。 冠狀病毒藉由病毒上的棘蛋白 (Spike) 辨識細胞上的特異性receptor進入標的細胞中,此蛋白除了造成病毒與細胞間融合,亦是誘導中和抗體產生的重要醣蛋白。由於棘蛋白在第一型與第二型FCoV中具相當大的差異,因此為目前用以區分此兩型病毒之主要標的。由於台灣不曾有過全省大規模FCoV血清學之調查,第一型與第二型FCoV之盛行率亦仍不明。本研究以桿狀病毒表現系統針對兩型FCoV棘蛋白上差異區間,進行型別特異性重組蛋白之表現,分析其特異性,並將之運用於兩型別病毒感染貓隻血清之檢測。首奶尷R兩型FCoV棘蛋白基因發現其receptor結合位之基因序列區間相似性極低 (19.2%),將此段約500 bp之基因序列以PCR進行增幅與純化,選殖入桿狀病毒表現載體,製作具表現兩型FCoV部分棘蛋白之重組桿狀病毒,並順利獲得力價達2 × 106 PFU/ ml之高效價重組病毒。以此重組病毒生產兩型FCoV之部分棘蛋白利用相關抗體,辨認出符合預期大小約為23-25 kDa之兩型重組蛋白質。進一步發現兩型重組病毒以MOI 0.1,分別感染Sf9細胞72小時以及96小時後可獲得最高蛋白質的表現量。利用免疫沈澱法確立第一型重組蛋白之特異性。運用兩重組抗原免疫螢光染色分析確定感染型別之貓隻臨床血清,發現各型別之抗血清可辨認其相對應重組蛋白,而隨著血清稀釋可見螢光訊號之遞減,表示所生產之兩型別重組蛋白具有特異性,同時具定量抗體力價之功能。未來可將此兩重組蛋白純化後進一步應用於ELISA檢測的發展,進行台灣兩型貓冠狀病毒盛行率之調查。 | zh_TW |
dc.description.abstract | Feline infectious peritonitis (FIP), is a fatal disease caused by feline coronavirus (FCoV). According to the serum neutralizing capacity and the antigenicity relation to canine coronavirus, FCoVs can be divided into two serotypes, namely, Type I and Type II. Type I FCoV is predominant in the field. Despite the higher prevalence of Type I FCoV infection, infection of Type II FCoV appears to be highly significantly correlated to FIP. Coronavirus infection is determined by the interaction between the spike protein and its corresponding receptors on the target cells. S gene has been used in the differentiation of different type(s) of FCoV infection due to the low similarity between the two viruses. The seroprevalence of different types of FCoV infection in Taiwan is unknown yet. This study aim to investigate the seroprevalence of different types of FCoV infection in Taiwan through the expression of type specific spike proteins from Type I and II FCoV. Spike gene of two types of FCoV were aligned, and a low similarity region corresponding to the receptor binding domain of spike protein from Type II virus and the related region of Type I virus were selected, amplified and cloned into a baculovirus expression system. Two recombinant viruses with the titer up to 2 × 106 PFU/ml have been obtained. Cells were infected with these recombinant viruses and total protein was harvested, the type-specific recombinant proteins could be recognized by anti-serum with the expected size of 23-25kDa. While Sf9 cells were infected with these two recombinant viruses at a multiplicities of infection 0.1, the highest protein yield can be obtained at 72 hour and 96 hour post infection for type I and II RBD recombinant viruses, respectively. The specificity of type I recombinant protein could be evaluated by co-immunoprecipitation. Sera from cats infected with different types of FCoV could specifically identify corresponding recombinant proteins by immunofluorescence assay. The intensity of fluorescence was declined through serial dilution of anti-serum, which indicates the proteins is type-specific and could be used for the titration of the specific antibody. These type-specific recombinant proteins will be purified and used for the establishment of ELISA for further serological surveillance. | en |
dc.description.provenance | Made available in DSpace on 2021-06-15T04:12:28Z (GMT). No. of bitstreams: 1 ntu-100-R98629024-1.pdf: 23283109 bytes, checksum: dcfd79ad69364840f76eee60535bd73e (MD5) Previous issue date: 2011 | en |
dc.description.tableofcontents | 摘要............................................................................................................................ VI
英文摘要................................................................................................................... VII 第一章、序言........................................................................................................... 1 第二章、文獻回顧................................................................................................... 3 第一節、研究背景.......................................................................................... 3 第二節、病毒特徵.......................................................................................... 3 2.1 病毒分類........................................................................................... 4 2.2 基因體特性....................................................................................... 4 2.3 冠狀病毒S protein之差異與宿主之關連性.................................... 7 2.4 貓冠狀病毒之分型........................................................................... 8 第三節、貓冠狀病毒與疾病.......................................................................... 10 3.1 宿主與流行病學............................................................................... 11 3.2 致病機制與免疫反應....................................................................... 12 3.3 臨床症狀與診斷方法....................................................................... 14 3.3.1 臨床症狀.............................................................................. 14 3.3.2 臨床上的診斷...................................................................... 15 3.4 治療與預防控制............................................................................... 16 3.4.1 臨床上的治療...................................................................... 16 3.4.2 疫苗預防與控制.................................................................. 17 第三章、材料與方法............................................................................................... 19 第一節、病毒與細胞...................................................................................... 19 第二節、型別特異性基因序列之建構.......................................................... 20 2.1 型別特異性基因序列之選取........................................................... 20 2.2 型別特異性基因產物之製備........................................................... 20 2.2.1 病毒RNA萃取...................................................................... 20 2.2.2 反轉錄反應.......................................................................... 21 2.2.3 質體DNA之萃取................................................................. 22 2.2.4 聚合酶鏈鎖反應.................................................................. 22 2.2.5 型別特異性基因產物純化.................................................. 24 2.3 型別特異性基因產物之基因選殖................................................... 24 2.3.1 TA接合反應....... .................................................................. 24 2.3.2 勝任細胞之準備.................................................................. 25 2.3.3 質體DNA做細菌轉型.......................................................... 25 第三節、重組病毒建構與重組蛋白質表現.................................................. 26 3.1 建構重組pFastBacHT-RBD的質體................................................. 26 3.2 重組NTU 156 RBD-bacmid 、NTU 2 RBD-bacmid的 生成................................................................................................... 27 3.3 萃取NTU156 RBD-bacmid、NTU 2 RBD-bacmid......................... 28 3.4 NTU 156 RBD、NTU 2 RBD-baculovirus重組病毒製備.............. 29 3.5 重組蛋白質表現條件最佳化與蛋白質純化................................... 30 3.5.1 重組蛋白質表現條件最佳化.............................................. 30 3.5.2 重組蛋白質純化.................................................................. 31 第四節、型別特異性蛋白質確認及分析...................................................... 32 4.1 SDS-PAGE以及Western blot分析.................................................... 32 4.2 間接免疫螢光染色........................................................................... 33 4.2.1 貓隻血清中抗FCoV之抗體之檢測..................................... 33 4.2.2 檢測貓隻血清抗體與型別特異性蛋白質結合之結果........ 33 4.3 免疫沈澱法....................................................................................... 34 第四章、結果........................................................................................................... 36 第一節、型別特異性基因序列之建構.......................................................... 36 1.1 型別特異性基因序列之選取........................................................... 36 1.2 重組pFastBac HT-RBD真核表現質體之建構................................ 37 1.3 重組NTU 2 RBD、NTU 156 桿狀病毒製備.................................. 37 1.4 西方墨點法分析重組蛋白質之表現............................................... 38 1.5 重組蛋白質表現條件最佳化........................................................... 39 1.6 重組蛋白質純化............................................................................... 39 第二節、型別特異性重組蛋白質之確認...................................................... 40 2.1 間接免疫螢光染色........................................................................... 40 2.2 免疫螢光染色確認感染血清可辨認其特異性抗原....................... 40 2.3 免疫沈澱法....................................................................................... 41 第五章、討論........................................................................................................... 43 附圖........................................................................................................................... 50 附表........................................................................................................................... 60 參考文獻................................................................................................................... 63 圖次 圖一、 Type I與type II FCoV棘蛋白部分胺基酸序列比對................................... 50 圖二、引子煉合溫度選擇之條件最佳化............................................................... 51 圖三、NTU 2 RBD、NTU 156 RBD與pFastBac HT接合,形成為 pFastBac HT-RBD質體之確.......................................................................... 51 圖四、重組NTU 2RBD、NTU 156 RND bacmid菌株之確認............................... 52 圖五、偵測感染細胞中重組病毒複製情形,並利用病毒斑試驗 純化單一重組病毒......................................................................................................... 53 圖六、利用西方墨點法分析重組蛋白質表現情形............................................... 54 圖七、利用不同MOI與攻毒時間進行重組蛋白質表現最佳化條件之建立........ 55 圖八、利用His GraviTrapTM affinity column進行蛋白質純化,並進 行蛋白質電泳與西方墨點分析................................................................... 56 圖九、透過免疫螢光染色進行抗貓冠狀病毒陽性血清之篩選........................... 57 圖十、型別特異性免疫螢光染色之建立............................................................................ 58 圖十一、免疫沈澱法作為重組蛋白型別特異性區分之指標............................... 59 表次 表一、本研究中用以增幅特異性基因產物與重組病毒bacmid之引子對........... 60 表二、本研究所使用之19支血清其背景資料及IFA結果與力價判讀................. 61 | |
dc.language.iso | zh-TW | |
dc.title | 應用第一型與第二型貓冠狀病毒型別特異性棘蛋白於感染之區分 | zh_TW |
dc.title | Application of Type Specific Spike Protein in the Differentiation of Type I and II Feline Coronavirus Infection | en |
dc.type | Thesis | |
dc.date.schoolyear | 99-2 | |
dc.description.degree | 碩士 | |
dc.contributor.coadvisor | 萬灼華(Cho-Hua Wan) | |
dc.contributor.oralexamcommittee | 王金和,林昭男,陳?銘,林俊宏,萬灼華 | |
dc.subject.keyword | 貓冠狀病毒,棘蛋白,區分, | zh_TW |
dc.subject.keyword | feline coronavirus,spike protein,differentiation, | en |
dc.relation.page | 73 | |
dc.rights.note | 有償授權 | |
dc.date.accepted | 2011-08-17 | |
dc.contributor.author-college | 獸醫專業學院 | zh_TW |
dc.contributor.author-dept | 獸醫學研究所 | zh_TW |
顯示於系所單位: | 獸醫學系 |
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