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研究生: 林玟因
Lin, Wen-Yin
論文名稱: 開發牛病毒性下痢重組E2蛋白次單位疫苗
Development of recombinant E2 antigen subunit vaccine against bovine viral diarrhea virus
指導教授: 鍾曜吉
Chung, Yao-Chi
學位類別: 碩士
Master
系所名稱: 獸醫學院 - 動物疫苗科技研究所
Graduate Institute of Animal Vaccine Technology
畢業學年度: 106
語文別: 中文
論文頁數: 56
中文關鍵詞: 牛病毒性下痢病毒E2蛋白次單位疫苗
外文關鍵詞: Bovine viral diarrhea virus, E2 protein, subunit vaccine
DOI URL: http://doi.org/10.6346/THE.NPUST.AVT.008.2018.D03
相關次數: 點閱:26下載:2
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  • 牛病毒性下痢病毒(Bovine viral diarrhea virus, BVDV)是造成全世界牛群疾病的主要病原體,並對其對生產和一般健康狀況的不利影響造成重大經濟損失。 接種疫苗廣泛用於控制全世界BVDV感染的表現,但是BVDV野毒株的異質性和BVDV建立持續感染的獨特能力限制了疫苗的有效性。
    在這項研究中,我們為台灣BVDV野生型構建了一種新的重組pET24a-BVDV-E2,其表達BVDV具有高免疫原性E2蛋白。使用大腸桿菌表達系統(Escherichia coli expression system)來表達這種重組蛋白並 Western blotting驗證E2蛋白的抗原性,然後將E2蛋白與ISA 206佐劑混合製成疫苗。我們已經確認注射小鼠疫苗的功效,雖然E2亞單位疫苗沒有達到預期的效果,但E2蛋白與少量滅活疫苗混合可提高滅活疫苗的有效性。 因此,我們推測rE2蛋白可能可以增強滅活BVDV疫苗效力。

    Bovine viral diarrhea virus (BVDV) is a major pathogen responsible for diseases in cattle population worldwide and causes substantial economic losses for its negative effect on production and general health conditions. Vaccination is widely used to control manifestations of BVDV infections in the world, but in the heterogeneity of BVDV field strains and the unique ability of BVDV to establish persistent infections, limiting the effectiveness of the vaccine. In this study, we have constructed a novel recombinant pET24a-BVDV- E2 for Taiwan BVDV wild-type expressing the most immunogenic E2 antigen of BVDV. This recombinant protein was expressed by Escherichia coli expression system. The expression of E2 protein was verified by Western blotting. And then the E2 protein was mixed with ISA 206 adjuvent to produce subunit vaccine. We have confirmed the efficacy of the vaccine by Injected mice, the E2 subunit vaccine does not deliver on expectations, But the E2 protein mixed with a small amount of inactivated vaccine improve the effectiveness of inactivated vaccines. Therefore, we speculate that rE2 protein perhaps be an effective way to enhance the protective efficacy of inactivated BVDV vaccines.

    目錄
    第1章 緒言 1
    第2章 文獻探討 3
    2.1牛病毒性下痢病毒背景 3
    2.2牛病毒性下痢病毒結構和分型 4
    2.3傳播病毒 5
    2.4臨床症狀 6
    2.4.1急性感染 6
    2.4.2胎兒感染 7
    2.4.3持續感染 7
    2.4.4粘膜疾病 8
    2.5預防控制方法 8
    2.6使用疫苗接種控制BVDV的情況 8
    2.7 BVDV疫苗的發展 9
    2.8本研究的目標與方向 11
    第3章 材料與方法 13
    3.1基因片段來源 13
    3.2 E2基因片段 13
    3.2.1設計BVDV-E2引子基因序列(Table 1) 13
    3.2.2 聚合酶連鎖反應(Polymerase chain reaction, PCR) 13
    3.2.3 膠體電泳法確認 PCR 產物大小 13
    3.2.4 BVDV-E2基因片段純化 14
    3.3 建構大腸桿菌表現系統 14
    3.3.1 限制酶酵素切割 (Enzyme digestion) 14
    3.3.2 黏合pET24a vector和BVDV-E2 (Ligation) 14
    3.3.3 pET24a- BVDV-E2轉型至勝任細胞 DH5α (Transformation) 15
    3.3.5 抽取pET24a- BVDV-E2質體 15
    3.4 表現 BVDV-E2蛋白 15
    3.4.1 pET24a- BVDV-E2 轉型至勝任細胞 BL21 15
    3.4.2 BL21- pET24a- BVDV-E2 小量誘導蛋白測試 16
    3.4.3 BL21- pET24a- BVDV-E2 大量誘導蛋白 16
    3.5 蛋白質分析、純化與定量 17
    3.5.1十二烷基硫酸鈉聚丙烯醯胺膠體(SDS-PAGE)製備 17
    3.5.2重組 BVDV-E2蛋白進行SDS-PAGE電泳分析 17
    3.5.3 重組 BVDV-E2蛋白進行抗原分析-西方墨點方(Western blot) 18
    3.5.4 純化重組 BVDV-E2蛋白 18
    3.5.5 重組 BVDV-E2蛋白透析 19
    3.5.6 重組 BVDV-E2 蛋白定量 19
    3.6 疫苗製作 19
    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 20
    3.7.3 小鼠脾臟細胞製備 21
    3.7.4 淋巴細胞之計數 21
    3.7.5 萃取小鼠脾臟細胞 RNA 22
    3.7.8即時聚合酶連鎖反應(Quantitative real time polymerase chain reaction, Q-PCR) 22
    第4章 結果 24
    4.1 pET24a- BVDV-E2質體建構 24
    4.2大腸桿菌表現BVDV-E2條件測試及抗原分析 24
    4.3 BVDV-E2蛋白 24
    4.4 重組 BVDV-E2蛋白透析 25
    4.5重組 BVDV-E2蛋白純化回收率 25
    4.6 第一次動物實驗 25
    4.7 第二次動物實驗 26
    第5章 討論 45
    第6章 參考文獻 49
    作者簡介 56

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