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研究生:陳詠宜
研究生(外文):Yung-Yi Chen
論文名稱:重組豬瘟病毒封套醣蛋白E2與巴氏桿菌毒素次單位Tox1之選殖表現
論文名稱(外文):Expression of recombinant classical swine fever virus glycoprotein E2 and subunit pasteurella multocida toxin Tox1
指導教授:簡茂盛簡茂盛引用關係
學位類別:碩士
校院名稱:國立中興大學
系所名稱:獸醫病理學研究所
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:2002
畢業學年度:90
語文別:中文
論文頁數:85
中文關鍵詞:豬瘟病毒封套醣蛋白E2巴氏桿菌毒素桿狀病毒表現系統疫苗
外文關鍵詞:CSFV glycoprotein E2Pasteurella multocida Toxinbaculovirus expression vector systemvaccine
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摘要
豬瘟(classical swine fever; CSF)為豬隻之高度傳染性及高致死性的病毒性疾病,而E2為豬瘟病毒顆粒上三種封套醣蛋白之一,亦為豬瘟病毒結構中最主要引發豬隻產生中和抗體的抗原。為開發E2次單位標誌疫苗藉以區分施打疫苗與野外感染的豬隻,本實驗以桿狀病毒表現系統(baculovirus expression vector system; BEVS)表現重組之豬瘟病毒E2封套醣蛋白。而為使次單位疫苗更具有應用性,本實驗另外嘗試構築結合E2與其他重要疾病主要抗原之融合蛋白,期望在不影響彼此蛋白質摺疊與免疫原性的情況下,開發雙價次單位疫苗。Tox1為豬萎縮性鼻炎主要致病因子Pasteurella multocida產毒株所產生的毒素(Pasteurella multocida toxin; PMT)於大腸桿菌表現系統之N端重組次單位選殖株,並已證實此重組次單位抗原蛋白在豬隻具有良好之免疫原性與保護性。本實驗以桿狀病毒表現系統表現重組蛋白E2、Tox1與E2Tox1,步驟須先將其基因構築於轉置載體,利用轉置載體與桿狀病毒基因的同源性重組,將欲嵌入之基因置換至桿狀病毒的基因體內。並經過多次挑選藍綠色病毒斑之方式篩選並純化重組病毒後,再以具重組基因之桿狀病毒感染昆蟲細胞株,利用昆蟲細胞合成重組蛋白E2、Tox1與E2Tox1。經由重組病毒基因的定序與西方墨點法的確認,各重組蛋白均能在細胞株中正確表現,其分子量各約為39 kDa、60 kDa與95 kDa。再以此三種重組蛋白分別進行小鼠與豬隻之動物免疫實驗後,並證實重組蛋白E2、Tox1與E2Tox1亦均具有免疫原性,可引發對PMT毒素與豬瘟病毒之中和抗體的產生。初步證實本實驗所構築之蛋白質次單位疫苗具有免疫效力,未來可進行較大規模的動物免疫實驗並進行豬瘟病毒與PMT的攻毒試驗,以評估蛋白質次單位疫苗的保護效力。
Abstract
Classical swine fever (CSF) is a highly contagious and fatal viral disease of swine. Glycoprotein E2 is one of the three glycoproteins on the envelope of classical swine fever virus (CSFV) and is also the most immunogenic antigen of CSFV. Because the marker vaccine can be used for discriminating vaccinated from infected pigs, the recombinant glycoprotein E2 has been constructed in the baculovirus expression vector system (BEVS) and tested for its immunogenicity. In order to make the subunit vaccine more proficient, we also tried to fuse the E2 glycoprotein gene to another pathogenic antigen gene for developing a bivalent subunit vaccine based on the finding that the folding and immunogenicity of the fusion protein could be still recognized correctly. Tox1 is the N-terminal subunit of Pasteurella multocida toxin (PMT), and is the major virulence factor associated with progressive atrophic rhinitis (PAR). The N-terminal of recombinant subunit PMT showed highly protection in pigs for vaccine efficacy from prior trials. In this study, transfer vectors that were inserted the genes of E2, Tox1, and E2Tox1 respectively were first applied to obtain recombinant baculoviruses with linearlized baculovirus DNA by homologous recombination in Spodoptera frugiperda (Sf9) cells. After repeat screening with blue plaque assays and confirming the inserted genes by DNA sequencing, the recombinant baculoviruses were purified and successfully expressed recombinant proteins in Sf9 cells. The calculated molecular weights of expressed proteins were 39 kDa (E2), 60 kDa (Tox1), and 95 kDa (E2Tox1) and all could be recognized by the Western blotting method. In addition, these recombinant proteins were utilized for immunization in mice and swine and both could induce neutralizing antibodies against CSFV and PMT as well. The large scales of animal trails will still be needed to apply for further evaluation the efficacy of these recombinant subunit vaccines in the near future.
目 錄
頁次
中文摘要 ------------------------------------------------------------------- I
英文摘要 ------------------------------------------------------------------- Π
目錄 ------------------------------------------------------------------------- Ⅳ
圖次 ------------------------------------------------------------------------- Ⅵ
第一章 前言 ------------------------------------------------------------- 1
第二章 文獻探討 ------------------------------------------------------- 3
第ㄧ節 桿狀病毒表現系統 ---------------------------------- 3
一、桿狀病毒之介紹 ------------------------------- 3
二、桿狀病毒表現系統之發展 ------------------- 4
三、桿狀病毒表現系統之原理 ------------------- 5
四、桿狀病毒表現系統之特性 ------------------- 7
五、桿狀病毒表現系統之應用 ------------------- 7
第二節 豬瘟病毒與其封套醣蛋白 ------------------------- 9
一、豬瘟病毒 ---------------------------------------- 9
二、豬瘟病毒封套醣蛋白之研究 ---------------- 11
三、豬瘟病毒所引發之免疫反應 ---------------- 14
四、豬瘟疫苗之研發 ------------------------------- 16
第三節 豬萎縮性鼻炎與巴氏桿菌毒素 ------------------- 17
ㄧ、豬萎縮性鼻炎 ---------------------------------- 17
二、巴氏桿菌毒素 ---------------------------------- 18
第三章 材料與方法 ----------------------------------------------------- 26
第一節 重組桿狀病毒之構築與製備 ---------------------- 26
一、轉置載體之構築 ------------------------------- 25
二、共轉染作用 ------------------------------------- 31
三、病毒斑篩選 ------------------------------------- 33
四、重組桿狀病毒之確認 ------------------------- 34
五、重組病毒之增殖與力價測定 ---------------- 37
第二節 重組蛋白之表現與萃取 ---------------------------- 37
一、重組蛋白質最高表現量之時間點測定 ------ 37
二、重組蛋白之大量表現 ------------------------- 38
三、重組蛋白之萃取 ------------------------------- 38
四、重組蛋白濃度之測定 ------------------------- 39
第三節 重組蛋白之免疫試驗 ------------------------------- 39
一、BALB/c小鼠之免疫與攻毒試驗 ------------- 39
二、豬隻之免疫試驗 ------------------------------- 43
第四章 結果 ------------------------------------------------------------- 45
第一節 重組桿狀病毒之構築 ------------------------------- 45
一、轉置載體之構築 ------------------------------- 45
二、共轉染作用 ------------------------------------- 47
三、分離與純化重組桿狀病毒 ------------------- 48
四、重組桿狀病毒之確認 ------------------------- 49
五、重組桿狀病毒之力價 ------------------------- 50
第二節 重組蛋白之表現 ------------------------------------- 51
一、最高重組蛋白質表現量之測定 ------------- 51
二、重組蛋白之濃度 ------------------------------- 51
第三節 免疫試驗 ---------------------------------------------- 52
一、BALB/c小鼠之免疫攻毒試驗 -------------- 52
二、豬隻之免疫試驗 ------------------------------- 53
第五章 討論 ------------------------------------------------------------- 67
參考文獻 ------------------------------------------------------------------- 75
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