臺灣博碩士論文加值系統

豬生殖與呼吸綜合症(Porcine reproductive and respiratory syndrome；PRRS)的爆發造成世界經濟嚴重受挫。而此疾病的發生是由PRRS病毒(PRRSV)所造成，PRRSV為具封套的RNA病毒，屬於動脈病毒屬家族 (Arteriviridae)。高致病性的PRRSV在中國大陸盛行，造成豬隻持續性發燒，甚至死亡。因此開發有效抵抗高致病性的PRRSV疫苗是重要的議題。其主要的結構蛋白被認為能引發免疫反應抵抗PRRSV的感染，其中醣蛋白GP5與基質蛋白M為病毒的主要結構蛋白，也是重要的免疫原蛋白，而GP5與M蛋白能以雙硫鍵連結方式形成GP5/M 異源二聚體於病毒表面上，並且能夠誘導中和抗體，因此為開發疫苗的關鍵性目標。本實驗以發展具安全性且更具效力之次單位疫苗為目標，利用桿狀病毒表現系統 (BEVS) 表現修飾的GP5與M蛋白，將GP5與M之基因選殖於pFastBacTM載體，轉型於DH10Bac™利用抗生素及藍白篩選重組bacmid並且轉染至Sf9昆蟲細胞。以利用SDS-PAGE及西方墨點法評估此重組蛋白之抗原性以及其於疫苗開發之潛力。

Porcine reproductive and respiratory syndrome (PRRS) is the most important infectious disease in pigs, caused huge economic losses in the world. This infectious disease is caused by PRRS virus (PRRSV), an enveloped RNA virus of the Arterivirdae family. A highly pathogenic PRRSV, which causes continuous high fever and a high proportion of deaths in pigs, has emerged and prevailed in Mainland China. Huge efforts should be made towards the development of an efficient vaccine against the highly pathogenic PRRSV. PRRSV has major structural proteins which those have been considered very important to arouse the immune responses against PRRSV infection. Glycoprotein 5 (GP5) and non-glycosylated matrix protein (M) are important major structural proteins and immunogenic protein of PRRSV. GP5 and M proteins form disulfide-linked heterodimer(GP5/M) in the viral surface. It might induce neutralizing antibodies, and thus is a primary target for developing a subunit vaccine. The objective of this study is to develop a more efficient subunit vaccine against PRRS by baculovivrus expression vector system. Modified GP5 and M proteins were expressed in baculovirus expression vector system (BEVS). The recombinant plasmids containing modified GP5 and M genes have been cloned into pFastBacTM vector and were transformed into DH10BacTM. Antibiotic selection and blue/white screening were used to select recombinant bacmids and insect cells were transfected with recombinant bacmids. These recombinant proteins was analyzed by SDS-PAGE and western blot. The potential use of the GP5 amd M protein as a subunit vaccine will be further evaluated.

摘 要 I
Abstract II
謝誌 IV
目錄 V
圖表目錄 IX
第1章 緒言 1
第2章 文獻回顧 4
2.1 豬繁殖與呼吸道綜合症 4
2.1.1 豬繁殖與呼吸道綜合症病原之背景 4
2.1.2 PRRSV之分類 4
2.1.3 PRRSV之結構及特性 5
2.1.3 PRRSV之感染 6
2.1.4 PRRSV Life cycle 7
2.1.5 PRRSV之傳播途徑 7
2.1.6 PRRSV臨床症狀 8
2.1.7 PRRSV之診斷 8
2.1.8 PRRSV治療及預防 9
2.1.9 PRRSV結構蛋白GP5 9
2.1.10 PRRSV結構蛋白M 10
2.1.11中國高致病性豬生殖與呼吸綜合症 10
2.2 桿狀病毒表現系統 (Baculovirus expression system) 11
2.2.1 桿狀病毒之分類 11
2.2.2 桿狀病毒之結構 12
2.2.3 桿狀病毒之生活史 12
2.2.4 桿狀病毒之基因表現 13
2.2.5桿狀病毒基因操作流程 13
第3章 材料與方法 15
3.1 本實驗使用菌株與細胞種類 15
3.1.1 菌株種類 15
3.1.2 細胞株種類 15
3.1.3 載體 15
3.1.4 儀器設備 15
3.2豬生殖與呼吸綜合症之ORF5蛋白GP5之選殖 16
3.2.1 PRRSV cDNA之萃取 16
3.2.1.1 以PRRSV進行總量cDNA之萃取 16
3.2.1.2 核糖核酸濃度的定量與計算 16
3.2.2 yT&;A-GP5、yT&;A-dNGP5、yT&;A-M 質體之構築 17
3.2.2.1引子(Primer)之設計 17
3.2.2.2 聚合酶連鎖反應 (Polymerase chain reaction, PCR) 17
3.2.2.3 DNA瓊脂糖凝膠電泳 18
3.2.2.4 DNA膠體純化 (Gel purification) 18
3.2.2.5 TA clone 接合反應 19
3.2.2.6 勝任細胞(Competent cell)之製備 19
3.2.2.7 轉型作用(Transformation) 20
3.2.2.8 藍白篩選 (Blue/White screeing) 20
3.2.2.9 以PCR方式確定選殖基因GP5、dNGP5、M(PH)以及M(P10)片段 21
3.2.2.10 yT&;A-GP5、yT&;A-dNGP5與yT&;A-M質體之萃取 22
3.2.2.11 以限制酵素處理重組質體帶有選殖GP5、NGP5及M基因片段 22
3.2.2.12定序 (Sequencing) 24
3.3 桿狀病毒表現系統Baculovirus expression vector system (BEVS) 24
3.3.1 pDual-GP5與pDual-dNGP5轉型載體之構築 24
3.3.1.1 yT&;A-GP5及yT&;A-dNGP5質體抽取 24
3.3.1.2 GP5、dNGP5與pDual以電泳法回收DNA 24
3.3.1.3 DNA瓊脂糖凝膠電泳 25
3.3.1.4 DNA膠體純化 (Gel purification) 25
3.3.1.5接合反應 (Ligation) 25
3.3.1.6轉型作用(Transformation) 26
3.3.1.7以PCR方式確定選殖基因GP5以及dNGP5片段 26
3.3.1.8 pDual-GP5及pDual-dNGP5質體抽取 26
3.3.1.9以限制酵素處理重組質體帶有選殖GP5以及dNGP5基因片段 26
3.3.2 pDual-M-GP5與pDual-M-dNGP5轉型載體之構築 26
3.3.2.1 yT&;A-M、pDual-GP5及pDual-dNGP5質體抽取 27
3.3.2.2 pDual-GP5、pDual-dNGP5與M以電泳法回收DNA 27
3.3.2.3 DNA瓊脂糖凝膠電泳 27
3.3.2.4 DNA膠體純化 (Gel purification) 27
3.3.2.5接合反應 (Ligation) 28
3.3.2.6轉型作用(Transformation) 28
3.3.2.7以PCR方式確定選殖基因M-GP5以及M-dNGP5片段 28
3.3.2.8 pDual-M-GP5及pDual-M-dNGP5質體抽取 28
3.3.2.9以限制酵素處理重組質體帶有選殖M-GP5以及M-dNGP5基因片段 29
3.3.3 Bacmid-GP5、Bacmid-dNGP5、Bacmid-M-GP5與Bacmid-M-dNGP5之構築 29
3.3.3.1轉型作用(Transformation) 30
3.3.3.2以PCR方式確定選殖基因GP5、dNGP5、M-GP5及M-dNGP5片段 30
3.3.3.3 Bacmid-GP5、Bacmid-dNGP5、Bacmid-M-GP5與Bacmid-M-dNGP5質體抽取 31
3.3.3.4以PCR方式確定重組Bacmid之基因GP5、dNGP5、M-GP5及M-dNGP5片段 32
3.3.4細胞株之培養 33
3.3.4.1昆蟲細胞Sf-9細胞株之培養 33
3.3.4.2 Sf9細胞繼代培養 33
3.3.4.3 Sf9細胞計數 34
3.3.4.4 Sf-9細胞冷凍保存 34
3.3.5昆蟲細胞Sf-9之轉染作用(Transfection) 34
3.3.5.1細胞之轉染作用(Transfection) 34
3.3.6重組桿狀病毒 35
3.3.6.1分離初代桿狀病毒(Isolating P1 Viral Stock) 35
3.3.6.2放大桿狀病毒(Amplifying P2 Viral Stock) 35
3.3.6.3抽取重組病毒之DNA 36
3.3.6.3利用PCR 確認重組病毒 36
3.3.7表現重組蛋白GP5、dNGP5、M-GP5和M-dNGP5 36
3.3.7.1 生產重組蛋白GP5、dNGP5、M-GP5和M-dNGP5 36
3.3.7.2蛋白質定量 37
3.3.7.3聚丙烯醯胺膠體電泳(SDS-PAGE) 37
3.3.7.4蛋白質轉漬 (Transfer) 38
3.3.7.5西方墨點法呈色分析 39
第4章 結果 40
4.1 豬呼吸道綜合症病毒之GP5、dNGP5及ORF5 蛋白之選殖 40
4.1.1yT&;A-GP5、yT&;A-dNGP5以及yT&;A-M載體之構築 40
4.1.2 基因之序列比對 40
4.2桿狀病毒表現系統 (Baculovirus expression system) 40
4.2.1 pDual-GP5以及pDual-dNGP5載體之構築 40
4.2.2 pDual-M-GP5以及pDual-M-dNGP5載體之構築 41
4.2.3 Bacmid-GP5、Bacmid-dNGP5、Bacmid-M-GP5與Bacmid-M-dNGP5之構築 41
4.2.4增幅可表現GP5、dNGP5、M-GP5及M-dNGP5之重組Baculovirus 42
4.2.5以SDS-PAGE分析重組蛋白GP5、dNGP5、M-GP5和M-dNGP之表現 42
4.2.6 測定重組蛋白GP5、dNGP5、M-GP5和M-dNGP之抗原性 43
4.2.7 SDS-PAGE及西方墨點法分析重組蛋白dNGP5之最佳表現時間點及蛋白定量 43
第5章 討論 67
參考文獻 71
附錄 78
作者簡介 85

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