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研究生: 劉凱博
Liu, Kai-Po
論文名稱: 溶血素蛋白之疫苗佐劑開發
Development of suilysin as vaccine adjuvant
指導教授: 朱純燕
Chu, Chun-Yen
鄭力廷
Cheng, Li-Ting
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: 107
語文別: 中文
論文頁數: 62
中文關鍵詞: 豬鏈球菌溶血素佐劑
外文關鍵詞: Streptococcus suis, suilysin, adjuvant
DOI URL: http://doi.org/10.6346/NPUST201900303
相關次數: 點閱:50下載:0
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  • 豬鏈球菌(Streptococcus suis, S. suis)主要引起豬的腦膜炎、敗血症,亦為一種人畜共通傳染病。溶血素(suilysin , Sly)是豬鏈球菌關鍵的毒力因子,會與細胞膜上膽固醇結合進而穿孔,再與其他毒力因子合作造成細胞裂解。研究指出,Sly在豬鏈球菌感染與炎症反應中起重要作用,低濃度Sly會刺激細胞激素分泌使嗜中性球和巨噬細胞,自然殺手細胞和細胞毒性T細胞的活化,引發TH1和TH2免疫反應,可以作為一種候選佐劑。本研究已成功使用原核系統表現重組Sly蛋白,確認蛋白質產物大小及抗原性,並完成Sly重組蛋白純化,搭配本實驗室已構築重組巴氏桿菌抗原製成疫苗免疫仔豬,利用流式細胞儀測定CD4+與CD8+ T細胞數量、酵素連結免疫分析抗體、Real-time PCR測定細胞激素表現量及攻毒試驗,藉此評估佐劑效力。

    Streptococcus suis (S. suis) causes meningitis septicemia of swine and it is also a zoonotic disease. Suilysin (Sly) is an essential virulence factors of streptococcus suis and creates transmembrane pores in cholesterol-containing membranes thereby causing cell lysis. Sly plays an important role in S. suis infection and inflammation. Low concentration sly can stimulate cytokine secretion, neutrophil, macrophage, natural killer cell and cytotoxic T cell, causing TH1 and TH2 immune response as a candidate adjuvant. We have constructed a gene fragment that removes toxicity and confirmed the size and antigenicity of the recombinant sly protein product. The sly recombinant protein has been purified successfully, combined with constructed rPMT-NC, and prepared as vaccine. Piglet were then immunized with the vaccine. We will evaluate adjuvant efficacy by ELISA analysis, and cytokine production by Real-time PCR. We will measure CD4+ and CD8+ T cells percentages by flow cytometry. We will perform challenge tests to evaluate vaccine protection rate.

    目錄
    摘 要 I
    Abstract II
    謝 誌 III
    目錄 IV
    圖表目錄 VIII
    第1章 緒言 1
    第2章 文獻回顧 3
    2.1 豬鏈球菌 3
    2.1.1豬鏈球菌概況 3
    2.1.2豬鏈球菌之微生物特徵 3
    2.1.3豬鏈球菌之流行病學 3
    2.1.4豬鏈球菌臨床症狀及致病機轉 4
    2.1.5豬鏈球菌主要毒力因子 5
    2.1.6如何預防及治療豬鏈球菌 5
    2.2豬鏈球菌溶血素 6
    2.2.1溶血素簡介 6
    2.2.2 溶血素功能 6
    2.3佐劑 7
    2.3.1佐劑簡介 7
    2.3.2抗原呈現(Presentation) 7
    2.3.3免疫調控(Immunomodulation) 7
    2.3.4引導抗原到目標免疫細胞 (Targeting) 8
    2.3.5抗原堆積作用 (Depot generation) 8
    2.3.6促進 T 淋巴細胞免疫反應 (Cytotoxin T lymphocyte, CTL) 8
    2.3.7 生物性佐劑 8
    2.3.8雙相油質佐劑 9
    2.4巴氏桿菌 (Pasteurella multocida ; Pm) 9
    2.4.1巴氏桿菌生物特性 9
    2.4.2巴氏桿菌流行病學 10
    2.4.3巴氏桿菌毒力因子 10
    2.5巴氏桿菌毒素(Pasteurella multocidatoxin) 10
    2.5.1巴氏桿菌毒素 10
    2.5.2巴氏桿菌毒素結構及功能 11
    2.5.3巴氏桿菌毒素導致豬萎縮性鼻炎之致病機轉 11
    第3章 材料與方法 12
    3.1 實驗所使用之菌株 12
    3.1.1 Escherichia coli DH5α 12
    3.1.2 Escherichia coli BL21 12
    3.1.3 pET-32a(+) (Novagen, Darmstadt, Germany) 12
    3.2溶血素蛋白(rsly)確認 12
    3.2.1使用特異性引子鑑定溶血素(rsly)基因 12
    3.2.2 使用特異性引子突變溶血素(rsly)基因 13
    3.2.3 DNA膠體電泳 13
    3.3以原核表現系統構築溶血素(msly) 13
    3.3.1 pET-32a(+)表現載體之製備 13
    3.3.2 PCR產物之純化 14
    3.3.3勝任細胞之製備 14
    3.3.4使用限制酶切除重組溶血素(msly)與pET-32a(+) 14
    3.3.5重組溶血素(msly)與pET-32a(+)之接合反應 15
    3.3.6以轉形作用 (transformation) 至E.coli DH5α 15
    3.3.7質體DNA之萃取 15
    3.3.8 pET-32a/ msly轉形作用 (transformation) 至E.coli BL21 16
    3.4重組蛋白之表現 16
    3.4.1重組溶血素蛋白(msly)之表現 16
    3.4.2 蛋白質電泳(SDS-PAGE) 16
    3.4.2.1重組溶血素(msly)製備 16
    3.4.2.2聚丙烯醯胺膠體製備 17
    3.4.2.3重組溶血素蛋白(msly)之定量 17
    3.4.3重組溶血素蛋白(msly)西方墨點法(Western Blot) 17
    3.4.4重組溶血素蛋白純化 18
    3.4.5重組溶血素蛋白透析 18
    3.5重組巴氏桿菌毒素蛋白之表現 19
    3.5.1 重組巴氏桿菌毒素蛋白之純化 19
    3.5.2 重組巴氏桿菌毒素蛋白之透析 19
    3.6疫苗之製備 19
    3.6.1豬隻實驗重組疫苗製備 19
    3.6.2 試製疫苗之特性及無菌試驗 19
    3.7豬巴氏桿菌 (P. multocida) 之培養 20
    3.7.1豬巴氏桿菌培養 20
    3.7.2巴氏桿菌生長曲線 20
    3.8動物試驗 20
    3.8.1巴氏桿菌之攻毒用培養 20
    3.8.2豬效力試驗 20
    3.9豬隻免疫反應分析 20
    3.9.1 蛋白質定量 20
    3.9.2酵素連結免疫吸附法 (Enzyme-linked immunosorbent assay, ELISA) 21
    3.9.3即時定量聚合酶連鎖反應(Real Time PCR, Q-PCR)進行細胞激素之定量 21
    3.9.3.1周邊血液淋巴球細胞(Peripheral blood mononuclear cell ;PBMC)之抽取 21
    3.9.3.2周邊血液淋巴球之細胞激素試驗 22
    3.9.3.3周邊血液淋巴球刺激抗原後細胞RNA萃取 22
    3.9.3.4反轉錄聚合酶連鎖反應(reverse transcription polymerase chain reaction;RT-PCR) 22
    3.9.3.5即時聚合酶連鎖反應(Real-time polyrmerase chain reaction:Real-time PCR) 22
    3.9.3.6樣本細胞激素mRNA基因量倍數計算 23
    3.9.4流式細胞儀(Flow cytometry)分析免疫細胞 24
    3.9.4.1周邊血液淋巴球細胞(Peripheral blood mononuclear cell ;PBMC)之抽取 24
    3.9.4.2抗體染色 24
    3.10統計分析 24
    第4章 結果 25
    4.1突變溶血素( msly)之構築 25
    4.1.1 溶血素(rsly)基因之構築與突變 25
    4.1.2突變溶血素( msly)確認與接合 25
    4.1.3轉型進入DH5α之確認 25
    4.1.4轉型進入BL-21之確認 25
    4.2突變溶血素( msly)蛋白表現 25
    4.2.1突變溶血素( msly)抗原性分析 26
    4.2.2突變溶血素( msly)純化與定量 26
    4.3重組巴氏桿菌毒素( rPMT-NC)蛋白表現 26
    4.4豬隻效力試驗 27
    4.4.1豬隻臨床症狀 27
    4.4.2豬隻體重 27
    4.4.3解剖病變評分 27
    4.4.4流式細胞儀分析CD4+及CD8+免疫細胞 27
    4.4.5免疫反應之分析血清中IgG表現量 27
    4.4.6細胞激素mRNA的表現量 28
    第5章 討論 50
    5.1構築突變溶血素 (msly) 探討 50
    5.2 豬鏈球菌溶血素與豬巴氏桿菌毒素重組蛋白分段探討 50
    5.3純化與透析探討 51
    5.4豬隻效力試驗探討 52
    5.5佐劑之探討 52
    第6章 參考文獻 53
    作者簡介 62

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