臺灣博碩士論文加值系統

羊傳染性化膿性病毒(Orf virus，ORFV) 隸屬於痘病毒科的副痘病毒屬，發生於世界各地的山羊與綿羊，其症狀為造成口腔潰瘍和膿皰等，對於成羊來說發生率和死亡率不高，但對於年幼動物ORFV的感染具有致死性，ORFV為具有封套，含雙股DNA的基因體，由於其基因體較大，具有非必要基因可與外源基因置換，基於其限制性的宿主、局部性的皮膚感染、可重複接種等特性，ORFV作為載體之發展具有相當高的潛力，而前人的研究中也有以ORFV為載體表達外源性蛋白，例如:狂犬病的醣蛋白質基因、禽流感的HA、NA、豬瘟病毒E2 蛋白等，台灣於2008年山羊痘疫情爆發，對飼養的經濟動物產業造成嚴重的經濟損失，而為了控制其疫情皆有賴於國外疫苗之進口，台灣目前並無研發和自製之羊痘疫苗，有鑑於此本研究試圖以ORFV為載體，用以架構可以表達羊痘病毒的主要免疫源蛋白(P32)的重組病毒。製備重組病毒所需的transfer vector中含有自ORFV基因體中增幅出的open reading frame 127以及128核酸序列；以此作為flanking sequence，透過homologous recombination將羊痘病毒P32 基因重組入ORFV基因體中。經由PCR、南方墨點法檢測基因型，並以西方墨點法、免疫螢光偵測P32蛋白質的表現，確認已成功製備P32-ORFV重組病毒，未來將藉由動物實驗以證實其所產生之免疫反應。

Orf virus (ORFV), belonging to parapoxvirus, causes skin lesions in infected sheep and goats. Due to the restricted host range, the skin tropism, the absence of systemic virus spread, and the short-lived ORFV vector-specific immunity allowing repeated immunizations, it has been successfully used as a novel viral vector system for expressing foreign antigens to prevent infectious diseases of swine, rabbit, and avian. Hence, study herein aimed to establish ORFV as a bivalent vaccine platform for goat. In 2008, goatpox (GP) outbreaks caused substantial loss in the production and productivity of goats in Taiwan. Considering that control of goatpox virus (GPV) infection relies on foreign vaccine, the goal of current study is to construct a recombinant ORFV that would express goatpox virus P32, the major immunogenic protein. To do so, coding regions of GPV P32 and eGFP that serves as a selection marker were inserted between the flanking sequences, i.e. open reading frame 127 and 128 of the ORFV genome. By homologous recombination, recombinant ORFV with GPV p32 gene (GPV P32-ORFV) was generated. At the present, the presence and expression of GPV P32 gene were confirmed by polymerase chain reaction and western blot analysis, respectively. The immune characteristics of GPV P32-ORFV will be further tested in animal models.

目錄
中文摘要 i
Abstract ii
第一章 序言 1
第二章 文獻探討 2
第一節 羊傳染性化膿性病毒 (orf virus) 2
第二節 流行病學與傳播 3
第三節 臨床症狀 4
第四節 組織病理學 5
第五節 人類感染羊傳染性化膿性病毒 5
第六節 羊傳染性化膿性病毒之診斷 6
第七節 疫苗 7
一、不活化疫苗(inactivated vaccine) 7
二、減毒疫苗(attenuated vaccine) 8
三、載體疫苗(vector vaccine) 8
四、病毒載體疫苗 9
第八節、羊傳染性化膿性病毒作為溶瘤病毒 11
第九節、山羊痘病毒簡介 11
第十節、山羊痘病毒臨床症狀 12
第十一節、山羊痘病毒病變 12
第十二節、山羊痘病毒之診斷 13
第十三節、山羊痘病毒與羊傳染性化膿性病毒之區別 13
第十四節、山羊痘病毒蛋白質抗原P32蛋白 14
第十五節、山羊痘病毒之防治 14
第十六節研究目的 14
第三章 材料與方法 16
第一節 P32蛋白基因原核表現載體之構築 16
1 聚合酶鏈鎖反應 (polymerase chain reaction, PCR) 16
2 瓊脂凝膠製作 16
3 DNA瓊脂電泳分析(agarose gel electrophoresis) 17
4 DNA片段純化 17
5 限制酵素切割 18
6 接合反應(ligation) 18
7 勝任細胞製備 (Competent cells) 18
8 轉型作用(transformation) 19
9 菌落聚合酶連鎖反應 (Colony polymerase chain reaction, colony PCR) 19
10 菌株之挑選和質體抽取 20
11 限制酵素切割確認和定序 20
第二節 原核表現載體之建構 21
1 pUC19-orf128 21
2 pUC19-ORF128- ORF127 21
3 pUC19-ORF128- ORF127-eGFP 22
4 pUC19-ORF128- ORF127-eGFP-Oligo 22
5 pET Duet-GPP32 23
6 pUC19-ORF128- ORF127-eGFP-Oligo-GPP32 23
第三節 蛋白質膠體電泳分析 24
1 膠台的組裝 24
2 蛋白質膠體的配方 24
3蛋白電泳膠片的製作 24
4電泳分析 25
5西方墨點法(Western blotting) 25
第四節 蛋白質交互作用 26
1 細胞培養 26
2 冷凍細胞 27
3 病毒培養 27
4 轉染(transfection) 27
5 免疫螢光分析immunofluorescence assay (IFA) 28
第五節 重組病毒之建立 29
一 病毒斑挑選與PCR基因型鑑定 29
二 南方墨點法 29
第四章 結果 39
第一節原核表現載體之構築及確認 39
第二節 羊傳染性化膿性重組病毒之建立 42
第三節 PCR確認重組病毒DNA片段 42
第四節南方墨點法確認基因嵌入之位置 43
第五節 西方墨點法檢測重組病毒F1L、P32、eGFP蛋白表現 45
第六節 利用西方墨點法以野外株山羊痘抗體偵測P32蛋白的表現 46
第七節 免疫螢光染色 46
第八節 病毒生長曲線 47
第五章 討論 60

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