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研究生:賴怡君
研究生(外文):Lai, Yi-Chun
論文名稱:開發口蹄疫重組VP1蛋白次單位疫苗
論文名稱(外文):Immune responses of recombinant VP1 antigens of serotype O Foot-and-Mouth Disease Virus
指導教授:鍾曜吉   
指導教授(外文):Chung, Yao chi
口試委員:鍾曜吉 鄭力廷張雋曦
口試委員(外文):Chung, Yao chiCheng, Li TingCheung, Chun Hei
口試日期:2017-06-28
學位類別:碩士
校院名稱:國立屏東科技大學
系所名稱:動物疫苗科技研究所
學門:獸醫學門
學類:獸醫學類
論文種類:學術論文
論文出版年:2017
畢業學年度:105
語文別:中文
論文頁數:59
中文關鍵詞:口蹄疫結構蛋白次單位疫苗
外文關鍵詞:Foot and mouth disease (FMD)Structural protein (SP)Subunit vaccine
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口蹄疫(Foot and mouth disease, FMD)為高度傳染性之病毒性疾病,造成偶蹄類動物之水疱性疾病,對農畜業經濟影響重大。口蹄疫病毒是屬於小核醣核酸病毒科中的口瘡病毒屬,共有七個血清型,而台灣地區流行的病毒株以O型為主。口蹄疫病毒由VP1,VP2,VP3,VP4 四個結構蛋白組成,其中 VP1具有最多抗原決定位,可以誘導專一性免疫反應。
本研究的目的是以重組FMDV之結構蛋白基因裝載入pET24a載體以大腸桿菌表現系統表現此重組蛋白,將蛋白質純化後,再利用市售抗His抗體及抗FMDV VP1多株抗體進行西方點墨法和ELISA,以確認重組蛋白抗原性。
以重組VP1蛋白與ISA206佐劑混合製成次單位疫苗免疫小鼠,經由抗體力價試驗,確定疫苗對於小鼠會產生效力。未來期望可以在豬隻身上進行更多的測試,以證明重組VP1蛋白可具有良好保護效力。
Foot and mouth disease (FMD) is a highly contagious viral disease of cloven-hoofed animals, and causing very huge economic losses. The infection of the virus induces vesicular. FMDV belongs to genus Aphthovirus within the family Piconaviridae. There are seven serotypes of FMDV. The O type virus is the main strain in Taiwan. There are VP1-VP4 which are the four structural proteins of FMDV. The VP1 carries important epitopes against FMDV inducing specific immune responses.
The goal of this study was to recombinant FMDV VP1 gene into the pET24a vector, and used the Escherichia coli expression system to express this recombinant protein. After purified, the protein antigenicity was characterized by western blot and ELISA with anti-His and anti-FMDV VP1 to confirm the antigenic of recombinant protein.
The VP1 protein was mixed with ISA206 adjuvant to produce subunit vaccine. The efficacy of recombinant protein vaccine was determined by antibody titer test. The results showed that the group of vaccine antibody titer higher than negative control. The immne result proved that the recombinant protein had immunogenic for FMDV, which suggest that the subunit vaccne may be a valuable vaccine candidate to prevent FMD on swine.
摘 要 I
Abstract II
謝誌 ………………………………………………………………………….IV
目錄 …………………………………………………………………………..V
圖表目錄 VII
第1章 緒言 1
第2章 文獻回顧 3
2.3口蹄疫病毒分類與病理特性 (Foot-and-mouth disease virus, FMDV) 4
2.4 口蹄疫病毒基因組成 5
2.5 口蹄疫疫苗簡介 6
2.6 疫苗對口蹄疫疫情控制 7
第3章 材料與方法 13
3.1口蹄疫病毒基因片段來源 13
3.1.1口蹄疫O/TWN/99之P12A基因片段 13
3.1.2 pUC57-P12A 質體抽取 13
3.2.1 設計O99-VP1引子基因序列(Table 1) 13
3.2.2 聚合酶連鎖反應 (Polymerase chain reaction, PCR) 14
3.2.3膠體電泳法確認PCR產物大小 14
3.2.4 O99-VP1基因片段純化 14
3.3.1 限制酶酵素切割 (Enzyme digestion) 15
3.3.3 pET24a-O99-VP1轉型至勝任細胞DH5α (Transformation) 15
3.3.4 確認pET24a-O99-VP1基因 15
3.3.5 抽取pET24a-O99-VP1質體 16
3.5.4純化重組O99-VP1蛋白 19
3.5.5重組O99-VP1蛋白透析 19
3.5.6重組O99-VP1蛋白定量 20
3.6 疫苗製作 20
3.7 動物試驗 20
3.7.1 小鼠免疫 20
3.7.2 檢測小鼠血清Total IgG 21
3.7.3 檢測小鼠血清IgG1 21
3.7.4 檢測小鼠血清 IgG2a 22
3.7.5 小鼠脾臟細胞製備 23
3.7.6 淋巴細胞之計數 23
3.7.6 小鼠淋巴細胞增殖反應 24
3.7.7 計算細胞增生刺激指數 (Stimulation index, SI) 24
3.7.8 萃取小鼠脾臟細胞RNA 24
3.7.9 反轉錄聚合酶連鎖反應 (Reverse transcription PCR, RT-PCR) 25
3.7.10 即時聚合酶連鎖反應 (Real-time Quantitative PCR, Q-PCR) 25
第4章 結果 26
4.1 pET24a-O99-VP1之質體建構 26
4.2 大腸桿菌表現pET24a-O99-VP1蛋白之條件及抗原分析 26
4.3重組O99-VP1蛋白純化 26
4.4 重組O99-VP1蛋白透析 27
4.5 第一次動物實驗免疫後小鼠血液中的Total IgG抗體分析 27
4.6 第一次ICR小鼠試驗結果分析: IgG1 and IgG2a 28
4.7 第二次動物實驗免疫後小鼠血液中的Total IgG抗體分析 28
4.8 第二次BALB/c小鼠試驗結果分析: IgG1 and IgG2a 29
4.9 第二次BALB/c小鼠試驗結果分析:細胞激素( Cytokine) 29
第6章 參考文獻 51
作者簡介 59
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