臺灣博碩士論文加值系統

山羊關節腦炎病毒（CAEV）屬於反轉錄病毒科之慢病毒，此病毒感染特徵為潛伏期長，會引發多器官疾病的症狀，且症狀都屬於一些慢性消耗性疾病。主要的傳染途徑為小羊吸吮到受感染的初乳或是藉著羊隻與羊隻間的接觸而造成傳染。CAEV已廣泛流行於世界各地，尤其是在集約式乳羊養殖盛行的國家，已造成養羊業者重大的損失，特別是在乳汁的產量嚴重受到的影響和羊隻的利用率也大幅下降。目前針對CAEV並無有效藥物及疫苗可供預防和治療。因此，本實驗選殖CAEV上保守性高且具抗原決定位的gag基因，作為次單位疫苗研發的題材。利用Nested RT-PCR的方式由羊奶中擴增出gag基因片段約996 bp，並構築到表現載體pQE-Trisystem中，利用M15大腸桿菌系統在37℃下以0.5mM的IPTG誘導4小時後，可大量表現重組蛋白，經SDS-PAGE與western blotting分析後証實為目標蛋白，再利用親和性管柱進行目標蛋白的純化與分離。為了測試重組蛋白是否有誘導抗體產生之效力，我們利用BALB/c小鼠進行免疫注射產生多株抗體，經ELISA分析免疫注射後2週的小鼠血清，皆可明顯偵測到高濃度的IgG產生，未來將更進一步進行田間試驗，研究重組蛋白是否對山羊產生有效的CAEV IgG進而預防山羊關節炎腦炎病毒的感染。

Caprine arthritis-encephalitis virus (CAEV) is a retrovirus belonging to the Lentivirus subfamily. CAE is a chronic goat disease characterized by progressive inflammation in multiple organs or tissue systems. CAEV’s transmission pathways can be either from dam to kid through colostrums or through prolonged contact between infected and healthy animals. The prevalence of CAEV infection, especially in zones of production, has significantly reduced farmers’ profits due to fall in production of goat milk, early slaughter of undesirable stock, and delay of exports. Currently, there is no commercially available vaccine to control or prevent CAEV infection. The CAEV’s gag gene has been shown to possess good antigenic epitopes and high conservative amino acid sequence in relative CAEV strains. Therefore, the gag gene product may serve well as subunit vaccine against CAEV infection in goat. In this report, CAEV’s gag gene was first amplified from infected goat milk by nested RT-PCR. The product of nested RT-PCR was confirmed by DNA sequencing analysis and found to encode a 996 bp open reading frame of CAEV’s gag gene. This cloned gag gene was applied in a M15 E. coli expression system via the vector of pQE-trisystem. By western blotting assay, the gag gene product is demonstrated to express in M15 after IPTG’s induction. The gene product of cloned gag gene was further purified by nickel-affinity chromatography to homogeneity. These results lay the foundation of developing subunit vaccine against CAEV in the near future.

目 錄
致 謝………………………………………………………………Ⅰ
目 錄………………………………………………………………Ⅲ
圖次索引………………………………………………………………Ⅶ
中文摘要………………………………………………………………Ⅸ
英文摘要………………………………………………………………XI
第一章 序論……………………………………………………………1
第一節 歷史背景………………………………………………………1
第二節 山羊關節炎腦炎病毒特………………………………………2
2-1 CAEV分類 ………………………………………………………2
2-2 CAEV之結構與物理化學特性 …………………………………4
2-3 CAEV基因結構 …………………………………………………5
2-4 CAEV基因結構及蛋白質功能 …………………………………6
第三節 病毒增殖 ………………………………………………………9
第四節 CAEV誘發之免疫反應…………………………………………10
第五節 山羊關節炎腦炎之臨床症狀與病理變化……………………12
第六節 流行病學………………………………………………………15
第七節 CAEV診斷方法…………………………………………………16
第八節 CAEV傳播方式…………………………………………………17
第九節 CAEV防治方法…………………………………………………18
第十節 研究目的………………………………………………………19
參考文獻 ………………………………………………………………20
第二章 山羊關節炎腦炎病毒之gag基因選殖及核酸序列分析……32
第一節 前言 …………………………………………………………32
第二節 材料與方法 …………………………………………………34
2-1 羊奶來源………………………………………………………34
2-2 病毒RNA萃取…………………………………………………34
2-3 反轉錄酵素反應（Reverse Transcription；RT）…………35
2-4 cDNA純化………………………………………………………36
2-5 巢式聚合酶連鎖反應（Nested-PCR）………………………37
2-6 DNA純化 ………………………………………………………41
2-7 質體與PCR產物之接合作用…………………………………42
2-8 轉型作用………………………………………………………43
2-9質體DNA純化 …………………………………………………44
2-10利用限制酶確認重組質體……………………………………45
2-11 DNA定序………………………………………………………46
第三節 結果……………………………………………………………49
3-1 利用第一組引子進行PCR反應之最適化條件………………49
3-2 利用第二組引子進行PCR反應之最適化條件……………50
3-3 CAEV-gag 基因選殖 ………………………………………50
3-4 序列分析……………………………………………………51
第四節 討論 ………………………………………………………52
參考文獻……………………………………………………………55
第三章 建構重組質體pQE-trisystem-gag以發展次單位疫苗 …72
第一節 前言………………………………………………………72
第二節 材料與方法………………………………………………73
2-1 表現載體之構築……………………………………………73
2-2 質體與載體之製備…………………………………………73
2-3 接合反應（Ligation）……………………………………75
2-4 勝任細胞（competent cell）之製備 ………………… 75
2-5 重組質體之轉型（transformation）……………………76
2-6 質體DNA純化 ………………………………………………77
2-7 利用限制酶確認重組質體…………………………………78
2-8 DNA定序（DNA sequencing） ……………………………79
2-9 重組蛋白之表現……………………………………………82第三節 結果………………………………………………………90
3-1 pQE-trisystem-gag之構築………………………………90
3-2重組質體之定序分析………………………………………90
3-3 重組蛋白之表現……………………………………………91第四節 討論 ………………………………………………………93
參考文獻 ………………………………………………………… 95
第四章 CAEV gag重組蛋白之免疫試驗 ……………………………105
第一節 前言 ………………………………………………………105
第二節 材料與方法 ………………………………………………106
2-1 實驗老鼠 ……………………………………………………106
2-2 重組蛋白之製備 ……………………………………………106
2-3免疫計畫………………………………………………………107
2-4 酵素結合免疫吸附法（ELISA） ……………………………108
2-5 點墨法分析（Dot blotting）………………………………109
2-6 蛋白質電泳分析 ……………………………………………111
2-7西方點墨法……………………………………………………113
第三節 結果 ………………………………………………………114
3-1 酵素結合免疫吸附法（ELISA）……………………………114
3-2 點墨法分析（Dot blotting）………………………………115
3-3 電泳分析與西方點墨法 ……………………………………115
第四節 討論 ………………………………………………………116
圖 次
第二章 山羊關節炎腦炎病毒之gag基因選殖及核酸序列分析
Fig.1 pGEM®-T Easy Vector圖譜 …………………………………56
Fig.2 利用第一組引子進行PCR反應之最佳鎂離子濃度…………57
Fig.3 利用第一組引子進行PCR反應之最佳黏合溫度……………58
Fig.4 利用第一組引子進行PCR反應之最佳黏合溫度……………59
Fig.5 利用第二組引子進行PCR反應之最佳鎂離子濃度…………60
Fig.6 利用第二組引子進行PCR反應之最佳黏合溫度……………61
Fig.7 利用第二組引子進行PCR反應之最佳黏合溫度……………62
Fig.8 利用第二組引子進行PCR反應之最佳黏合溫度……………63
Fig.9 pGEMT-CAEVgag 選殖株電泳確認圖…………………………64
Fig.10 定序後完整的CAEV-gag DNA序列…………………………65
Fig.11 選殖株與其他相鄰病毒株之gag基因序列比對 ……66、67
Fig.12 選殖株與其他相鄰病毒株之gag基因核酸相似性圖與親緣關係樹狀圖………………………………………………… 68
Fig.13 gag基因之胺基酸序列……………………………………… 69
Fig.14選殖株與其他相鄰病毒株之胺基酸比較圖…………………70
Fig.15選殖株與其他相鄰病毒株之GAG蛋白之胺基酸相似性圖與親緣關係樹狀圖 ………………………………………………71
第三章 建構重組質體pQE-trisystem-gag以發展次單位疫苗
Fig. 1 pQE-Trisystem質體構造……………………………………96
Fig. 2 將gag基因選殖於pQE-Trisystem之選殖株電泳分析圖.97
Fig. 3、4、5不同IPTG誘導劑量對XL-1-Blue與M15表現宿主之重組蛋白表現量之影響………………………………98、99、100
Fig.6 Dot blot分析圖…………………………………………… 101
Fig.7 於37℃下不同誘導時間（1、2、3、4小時）對E. coli（M15）表現重組蛋白之影響 ……………………………………… 102
Fig.8 利用western blotting分析重組蛋白在不同時間誘導下其蛋白質的表現情形 …………………………………………… 103
Fig. 9 重組蛋白經純化後其SDS-PAGE和Western blotting分析圖 …………………………………………………………… 104
第四章 CAEV gag重組蛋白之免疫試驗
Fig. 1 經ELISA分析結果………………………………………… 118
Fig. 2 經ELISA分析各組間其長條圖比較………………………118
Fig. 3 Dot blotting分析圖………………………………………119
Fig. 4 蛋白質電泳圖 …………………………………………… 120
Fig. 5 Western blotting分析……………………………………121

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