豬環狀病毒相關疾病、豬瘟、豬生殖與呼吸綜合症皆為病毒感染所引發的豬隻重要疾病，存在於全世界各國家的豬群中，每當爆發大流行皆造成養殖業的重大經濟損失，而施打對應疫苗為最有效的防疫策略，因此我們希望針對這些病毒發展品質高且成本低的疫苗，提供豬隻抵抗病毒感染的免疫力。先前實驗室已成功利用昆蟲細胞/桿狀病毒表現系統大量生產豬環狀病毒的外殼蛋白(Cap protein)做為疫苗抗原，並且經由豬隻動物實驗證實能夠誘發豬隻對豬環狀病毒的免疫力。後續我們也藉由去除天然的核停留訊號使Cap蛋白不會累積於細胞核中限制產量及增加純化困難，然而進一步藉由加入額外的胞外分泌訊號仍舊無法使Cap蛋白由細胞質分泌至胞外，藉由共同感染帶有功能性蛋白Vankyrin以及Heat Shock Protein基因的重組桿狀病毒，也無法增加Cap蛋白產量及提升溶解度。研究過程中我們也觀察到去除核停留訊號的Cap蛋白結構不穩定，有蛋白質容易降解的情形，為了解決以上的問題，我們設計新的重組桿狀病毒建構，將具有分泌訊號的豬瘟病毒以及豬生殖與呼吸綜合症病毒的結構蛋白融合於Cap蛋白的N端成為分泌型融合蛋白，成功的改變Cap蛋白在細胞內的分布位置，且此融合蛋白含有兩個病毒抗原，因此具有發展為二價疫苗的潛力。

摘要 I
Abstract II
目錄 III
圖表目錄 VI
第一章 文獻回顧 1
1-1 第二型豬環狀病毒 1
1-1-1 病毒結構與基因特性 2
1-1-2 第二型豬環狀病毒疫苗的發展 3
1-1-3 商業化疫苗 4
1-2 豬瘟病毒 5
1-2-1 病毒結構與基因特性 5
1-2-2 豬瘟疫苗的發展 6
1-2-3 商業化的疫苗 8
1-3 豬生殖與呼吸綜合症病毒 8
1-3-1 病毒結構與基因特性 9
1-3-2 豬生殖與呼吸綜合症病毒疫苗發展 10
1-3-3 商業化疫苗 11
1-4 桿狀病毒/昆蟲細胞表現系統 12
1-4-1桿狀病毒與昆蟲細胞簡介 12
1-4-2桿狀病毒表現系統的發展與應用 13
1-4-3 flashBACTM桿狀病毒表現系統 13
1-5功能性蛋白 15
1-5-1伴侶蛋白Heat shock protein 70、Heat shock protein 40 15
1-5-2 Vankyrin protein 15
1-6研究動機 16
第二章 實驗材料及方法 21
2-1昆蟲細胞培養 21
2-2 重組桿狀病毒建構 21
2-2-1 聚合酶鏈鎖反應 21
2-2-2 flashBACULTRA™重組桿狀病毒的建構 22
2-2-3 重組桿狀病毒放大 24
2-2-4 終端稀釋法定量病毒效價 24
2-2-4第二型豬環狀病毒外殼蛋白生產 24
2-2-5多支病毒共感染以生產第二型豬環狀病毒外殼蛋白 25
2-3 蛋白質的萃取與純化 25
2-3-1蛋白質的萃取 25
2-3-2蔗糖密度梯度離心 25
2-3固定化金屬離子親和性層析法 26
2-4 實驗分析方法 26
2-4-1膠體電泳法 26
2-4-2西方點墨法 27
2-4-3免疫螢光染色 28
2-4-4酵素連結免疫分析法 29
2-5穿透式電子顯微鏡觀察 29
第三章 實驗結果與討論 35
3-1 輔助性蛋白對於Cap蛋白產量與性質的影響 35
3-1-1 Chaperone protein：Hsp70 and Hsp40 35
3-1-2 Vankyrin protein：P-vank-1 and I2-vank3 36
3-2 Cap蛋白的純化與結構分析 37
3-2-1 Cap蛋白萃取 38
3-2-2 Cap蛋白純化 39
3-2-3 Cap蛋白結構分析 40
3-3 新建構分泌型融合蛋白 40
3-3-1 建構新型重組桿狀病毒 41
3-3-2 分泌型融合蛋白特性分析 42
3-3-3 融合蛋白萃取與純化 43
3-3-4融合蛋白結構分析 44
3-4 討論 45
第四章 未來展望 62
4-1 探討融合蛋白在細胞內分布位置 62
4-2 生產程序最適化 63
4-3 融合其他病毒抗原 63
第五章 參考文獻 64

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