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研究生:吳忠晉
研究生(外文):Jhong-Jin Wu
論文名稱:豬干擾素α基因重組蛋白誘發細胞抗PRRSV效力之探討
論文名稱(外文):Study on the effect of porcine interferon α induced Anti-PRRSV activity
指導教授:郭村勇
指導教授(外文):Tsun-Yung Kuo
學位類別:碩士
校院名稱:國立宜蘭大學
系所名稱:生物技術研究所碩士班
學門:農業科學學門
學類:畜牧學類
論文種類:學術論文
論文出版年:2007
畢業學年度:95
語文別:中文
論文頁數:64
中文關鍵詞:干擾素豬生殖與呼吸綜合症
外文關鍵詞:InterferonPRRS
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在動物體內,干擾素是屬於誘發性蛋白,當細胞受到病毒感染,會誘發宿主細胞表現干擾素,干擾素可誘發未受病毒感染之細胞表現抗病毒蛋白,進而保護宿主不受病毒感染。
豬生殖與呼吸綜合症(Porcine reproductive and respiratory syndrome ; PRRS) 是全世界豬隻重要傳染病之ㄧ,該病會造成仔豬發育不良、間質性肺炎,鼻炎等症狀以及懷孕母豬會有食慾不振、精神倦怠、發熱、流產、胎兒木乃伊化等現象。PRRSV雖已有商品化疫苗上市,但其成效不如預期,所以目前全世界仍遭受此病的危害,且造成重大的經濟損失。
在本研究中,以RT-PCR技術選殖豬干擾素α基因,經序列比對確認無誤後,分別選殖入pET 20b、pET 44a、pGEX 6p-1原核表現載體,進行不同條件誘導下探討各基因重組蛋白質的表現、純化及抗PRRSV病毒活性測試。結果顯示,各重組蛋白若是在37℃下進行誘導表現,皆會產生不可溶態的包涵體,當溫度下降到20℃進行誘導,三種表現載體皆會產生少部份的可溶態重組蛋白,特別是在20℃下又添加終濃度5 mM的Betaine時,其可溶態蛋白表現之比例可明顯增加。
分別將各基因重組蛋白分別處理MARC 145細胞後,以PRRSV進行攻毒,分別測定其病毒力價變化和以抑制半數細胞受感染為基礎推算干擾素之活性。結果顯示,包涵體形式的干擾素,經由變性、重摺疊(Refolding)及透析後的活性為6193 Unit/ml,而可溶態的干擾素活性為39173 Unit/ml,本論文提供了以E. coli為表現系統,產製具活性豬干擾素α基因重組蛋白的雛形資訊,為將來量產奠下深厚基石。
Interferon is an inducible protein. Upon viral infection of the animal body, the host cells express interferons which induce the expression of anti-viral proteins of non-infected cells to protect the host from further viral infection.
Porcine reproductive and respiratory syndrome (PRRS) is one of the most important infectious disease of swine in the world. This viral disease causes retarded growth, interstitial pneumonia, and rhinitis among the symptoms caused in swine, as well as loss of appetite, lethargy, fever, abortion, and mummified fetus in pregnant sows. Although PRRSV commercial vaccines are on the market, they are not as effective as expected. Therefore, PRRSV continues to plague many countries and causes devastating economic impact throughout the world.
In this research, the porcine interferon-a gene was cloned by RT-PCR technique and sequence analyzed for correctness. The gene was inserted into vectors pET 20b, pET 44a, and pGEX 6p-1 prokaryotic expression systems. Different induction conditions for interferon-α recombinant proteins expression were tested. The proteins were purified and assayed for their activities. Results showed that proteins expressed at 37°C were in the inclusion body state regardless of the expression vector used. Under lower temperature of 20℃, slight increase of soluble proteins were expressed in 3 prokaryotic expression systems. When expressed at 20℃ with the addition of 5 mM betaine, soluble proteins increased significantly.
In the anti-PRRSV cell viability assay, MARC 145 cells were treated with the various recombinant proteins and challenged with PRRSV. Suppression of PRRSV proliferation was measured with viral titration. The anti-viral activity of interferon-α recombinant proteins was assayed by the suppression of infection to half of the cells. Results showed the inclusion body interferon-a, after denaturing, refolding, and dialysis, its activity was 6193 Unit/ml. The activity of soluble interferon-αwas 39173 Unit/ml. This research study successfully produced in E. coli interferon-α recombinant proteins with anti-PRRSV activities and serves as the basis for large-scale production in the future.
目錄


中文摘要…………………………………………………………………………………i
英文摘要 ………………………………………………………………………………ii
目錄……………………………………………………………………………………iii
表目錄…………………………………………………………………………………vi
圖目錄…………………………………………………………………………………vii

第一章 緒言……………………………………………………………………………1

第二章 文獻探討………………………………………………………………………3

一、 干擾素的發現………………………………………………………………3
二、 誘發干擾素之因素…………………………………………………………3
三、 第一型干擾素抗病毒機制…………………………………………………3
四、 干擾素的應用………………………………………………………………4
五、 豬生殖與呼吸綜合症………………………………………………………5
六、 原核表現系統………………………………………………………………6

第三章 材料與方法……………………………………………………………………8

豬干擾素α基因選殖與定序

一、 豬週邊血液單核細胞分離…………………………………………………8
二、 豬干擾素αRNA萃取…………………………………………………………8
三、 特異性引子的設計…………………………………………………………8
四、 反轉錄聚合酶鏈反應增幅干擾素α基因……………………………………9
五、 PCR產物確認與純化…………………………………………………………9
六、 限制酶切割反應………………………………………………………………9
七、 接合反應………………………………………………………………………10
八、 勝任細胞的製備與轉型作用…………………………………………………10
九、 重組質體的挑選與確認………………………………………………………11
十、 重組質體的定序確認…………………………………………………………11

豬干擾素α基因重組蛋白之表現及分析

一、 豬干擾素α基因重組蛋白表現………………………………………………11
二、 以SDS-PAGE確認不同表現載體重組蛋白的表現…………………………12
三、 西方墨漬法確認重組蛋白表現………………………………………………12
四、 豬干擾素α基因重組蛋白的純化與定量……………………………………13

干擾素α基因重組蛋白的抗病毒活性測試

一、 細胞株培養、病毒增殖與病毒力價測定……………………………………14
二、 抗病毒力價測試法……………………………………………………………15
三、 干擾素效價測試法……………………………………………………………15

第四章 結果………………………………………………………………………………17

豬干擾素α基因選殖、確認與表現

一、 豬干擾素α基因之選殖與確認…………………………………………………17
二、 豬干擾素α基因重組蛋白表現確認……………………………………………17

豬干擾素α基因重組蛋白抗病毒活性測試

一、 抗病毒力價測試法………………………………………………………………18
二、 干擾素效價測試法………………………………………………………………19

第五章 討論………………………………………………………………………………20

附錄……………………………………………………………………………………………23

參考文獻………………………………………………………………………………………50


表目錄


表一、干擾素基因分型與生物活性…………………………………………………………23
表二、細胞表面的干擾素TLRs與其接受體…………………………………………………24
表三、豬干擾素α基因特異性引子的設計……………………………………………………25


圖目錄


圖一、誘發干擾素表現的路徑………………………………………………………………26
圖二、第一型干擾素的接受體………………………………………………………………27
圖三、干擾素抗病毒機制路徑………………………………………………………………28
圖四、干擾素誘發抗病毒蛋白路徑,及其抗病毒機制……………………………………29
圖五、PBMC分離法……………………………………………………………………………30
圖六、pET system 原核表現載體…………………………………………………………31
圖七、pGEX system 原核表現載體………………………………………………………32
圖八、以RT-PCR反應增幅豬干擾素α基因電泳確認圖……………………………………33
圖九、豬干擾素α基因片段選殖入原核表現載體
pET 20b、pET 44a、pGEX 6p-1進行DNA定序及比對之結果…………………34
圖十、pET 20b/pIFNα/BL21 Gold以不同溫度誘導
干擾素重組蛋白表現之SDS-PAGE分析圖……………………………………………36
圖十一、pET 20b/pIFNα/BL21 Codon以不同溫度誘導
干擾素重組蛋白表現之SDS-PAGE分析圖……………………………………………37
圖十二、pGEX 6p-1/pIFNα/BL21 DE3以不同溫度誘導
干擾素重組蛋白表現之SDS-PAGE分析圖……………………………………………38
圖十三、pGEX 6p-1/pIFNα/BL21 Gold以不同溫度誘導
干擾素重組蛋白表現之SDS-PAGE分析圖……………………………………………39
圖十四、pGEX 6p-1/pIFNα/BL21 Codon以不同溫度誘導
干擾素重組蛋白表現之SDS-PAGE分析圖……………………………………………40
圖十五、pET 44a/pIFNα/BL21 DE3以不同溫度誘導
干擾素重組蛋白表現之SDS-PAGE分析圖……………………………………………41
圖十六、pET 44a/pIFNα/BL21 Gold以不同溫度誘導
干擾素重組蛋白表現之SDS-PAGE分析圖……………………………………………42
圖十七、pET 44a/pIFNα/BL21 Codon以不同溫度誘導
干擾素重組蛋白表現之SDS-PAGE分析圖……………………………………………43
圖十八、以Western法確認基因不可溶態豬干擾素α重組蛋白表現………………………44
圖十九、以Western法確認基因不可溶態豬干擾素α重組蛋白表現………………………45
圖二十、可溶態干擾素α基因重組蛋白抗病毒活性之病毒力價測試
細胞病變效應觀察結果…………………………………………………………………46
圖二十一、不可溶態干擾素α基因重組蛋白抗病毒活性之病毒力價測試
細胞病變效應觀察結果…………………………………………………………………47
圖二十二、可溶態豬干擾素α基因重組蛋白抗病毒活性之病毒
力價變化測試結果………………………………………………………………………48
圖二十三、不可溶態豬干擾素α基因重組蛋白抗病毒活性之病毒
力價變化測試結果………………………………………………………………………49
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