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研究生:林伯澤
研究生(外文):Po-Tse Lin
論文名稱:桿狀病毒表面呈現流感血球凝集素用以改善單株抗體之生產與篩選流程
論文名稱(外文):Surface-Display of Hemagglutinin from Influenza Virus Using Baculovirus for Efficient Monoclonal Antibody Production and Screening
指導教授:趙裕展
指導教授(外文):Yu-Chan Chao
學位類別:碩士
校院名稱:國立陽明大學
系所名稱:生命科學系暨基因體科學研究所
學門:生命科學學門
學類:生物訊息學類
論文種類:學術論文
論文出版年:2015
畢業學年度:103
語文別:中文
論文頁數:101
中文關鍵詞:桿狀病毒表面呈現A 型流感病毒血球凝集素單株抗體
外文關鍵詞:Baculovirus surface displayInfluenza A virusHemagglutininMonoclonal antibody
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流感病毒為一重要的人畜共通病原,每年約有三百到五百萬人遭到重症感染,並有25至50萬人因而死亡。若能即時篩檢及監測不同流感病毒亞型的流行概況,對於防疫及基礎研究皆有重要的意義,但流感病毒容易產生突變,篩選不易。目前臨床檢測流感常用快篩試劑,以單株抗體進行免疫呈色反應,檢體注入後可快速判讀結果,然而此法的靈敏度參差不齊,約40-60% 的篩檢率,且通常無法判讀不同的 HA 亞型。本實驗已將流感病毒表面膜蛋白 (血球凝集素, Hemagglutinin, HA) 呈現於桿狀病毒外膜表面 (HA-Bac) 的技術,做為免疫原施打老鼠產生單株抗體 (monoclonal antibody),與傳統施打流感病毒相比,可避免操作時感染問題,同時小鼠也不會因病毒感染而死亡。此外,HA-Bac 的 HA 為三聚合體,與純化的單體蛋白相比,有較正確的構形。更進一步以 HA-Bac 轉導哺乳動物細胞表現 HA 做為篩選單株抗體的抗原盤,再將挑選出的單株抗體,進行專一性及親合性的測試,測定單株抗體對不同 HA 亞型的專一性及靈敏度,以達到篩選出高親合性之單株抗體,提升對流感病毒的檢測能力之目的。以濃縮之 HA-Bac 和純化 HA 膜蛋白個別施打於小鼠後,取出小鼠血清進行血球凝集抑制試驗,結果發現兩者抑制凝血效價相近,顯示兩者都可產生抑制 HA 功能型抗體。利用哺乳動物細胞表現 HA 後以篩選出來之單株抗體進行 ELISA 與抗原盤測試,取出五隻單株抗體進行專一性及親合力測試,驗證此新型抗原盤較傳統 ELISA 法更加準確。使用 HA-Bac 進行單株抗體篩選,與傳統施打純化高濃度蛋白相比,前者濃縮後即可達到後者相同的效價,省去純化的步驟,且和流感病毒相比,在操作上安全性佳,不會有人畜感染的問題,可取代流感病毒進行血球凝集測試。另外,我們也建立出新型單株抗體篩選之抗原盤,改善了傳統抗原盤篩選時偽陽性偏高的問題,在未來可進一步以流感病毒進行實測。以桿狀病毒系統改善單株抗體生產流程,既安全也簡易,對於流感的監測及防疫將有所貢獻。
Influenza virus is an important cause of diseases circulating between human and animals. These annual epidemics are responsible for approximately 3 to 5 million cases of severe illnesses, and about 250,000 to 500,000 deaths worldwide. Therefore, the establishment of a system that can screen, monitor and subtyping influenza viruses will bring tremendous value to both disease control and basic scientific research. Yet, the complex strains of influenza viruses and their high pathogenicity, make the virus manipulation and screening process difficult. Currently, the commercially available quick screening kit can only detect influenza virus with 40-60% accuracy, in addition, they cannot distinguish the subtypes of different influenza viruses. To meet these needs, our laboratory has developed pseudotyped viruses that present hemagglutinin (HA), the glycoprotein of influenza virus, on the baculovirus surface (HA-Bac) for antibody production and screening. Compared with traditional antibody production methods, which use influenza viruses as the immunogens, HA-Bac may be a better choice to prevent viral contamination upon mouse injection. HA can retain native trimer conformation on the HA-Bac for antibody stimulation. In this project, we successfully constructed the HA-Bac (from H1N1) and used it to produce the HA protein. We have vaccinated mice separately by either HA-Bac or purified HA. Then, we have collected the sera of mice at several time points for western blotting and hemagglutination inhibition assays. Results showed that both HA-Bac and purified HA elicited HA inhibiting antibodies. It also indicated HA-Bac are capable of agglutinating red blood cells, which serves as a convenient and safe tool to assay HA inhibitingantibodies. We further expressed HA on the surface of cells and performed cell-based immunofluorescence assay with 18 selected monoclonal antibodies and compared those with traditional ELISA method. We chose 5 antibodies for further assays. The results suggested that our system may be an easier antibody screening platform and possible to replace ELISA. Our work proposes a convenient and safer strategy in generating
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antibodies utilizing HA-Bac. This novel platform can replace three major components in traditional methods, including the use of HA-Bac as an immunogen to replace the influenza virus or purified HA protein, HA-bac instead of the influenza virus for performing hemagglutination inhibition assay, and the mammalian cell-based immunofluorescence staining instead the using of conventional ELISA. HA-Bac as a safety antigen, induction of high quality antibody by native trimmer form of HA on HA-Bac, and easy to condense HA-Bac in one step without the need of further purification, are multiple features that make HA-Bac a promising tool for antibody production.
目錄
致謝............................... i
摘要 ............................... ii
Abstract ........................ iii
目錄 .............................. v
表目錄......................... x
圖目錄 ......................... xi
壹、引言 ................... 1
一、流感病毒歷史背景介紹 ...................................................................................................... 1
二、流感病毒的生物特性 .......................................................................................................... 3
三、流感病毒的防疫及監測 ...................................................................................................... 4
四、Hemagglutinin 血球凝集素 ................................................................................................ 6
五、桿狀病毒表現系統基本背景介紹 ...................................................................................... 7
六、桿狀病毒表現系統表現原理 .............................................................................................. 9
七、桿狀病毒表面抗原呈現技術之原理及應用 .................................................................... 10
貳、研究目的 .................................................................................................................................... 13
参、材料與方法 ................................................................................................................................ 15
一.實驗材料 ............................................................................................................................... 15
(一)桿狀病毒表現系統 ..................................................................................................... 15
1-1. 線狀同源重組系統 ........................................................................................... 15
1-2. 質體 ................................................................................................................... 16
1-3. 細胞培養 ........................................................................................................... 18
(二)藥品與試劑 ................................................................................................................. 19 2-1. 一般藥品與耗材 ............................................................................................... 19 2-2. 市售套組 ........................................................................................................... 22
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2-3. 培養基 ............................................................................................................... 23
(三)器材設備 ..................................................................................................................... 23 二、實驗方法 ............................................................................................................................ 24
(一)病毒重組轉移質體建構 ............................................................................................. 24
1-1. PCR 反應 ........................................................................................................... 25
1-2. DNA 瓊脂膠體 (agarose gel) 電泳 ................................................................. 26
1-3. 回收電泳膠體之 DNA ..................................................................................... 26
1-4. 質體接合 ........................................................................................................... 27
1-5. 質體轉形 ........................................................................................................... 27 1-6. Colony PCR: ....................................................................................................... 28 1-7. 抽取質體 ........................................................................................................... 29 1-8. 核酸定序 ........................................................................................................... 29
(二)生產 HA-bac 重組桿狀病毒 .................................................................................... 30
2-1. 共轉染實驗 ...................................................................................................... 30
2-2. 終點稀釋法 (End-point dilution) ..................................................................... 30
2-3. 病毒效價測定 .................................................................................................... 31
2-3-1. TCID50 (Tissue culture infectious dose 50) ............................................. 31
2-3-2. qPCR ....................................................................................................... 32
2-4. 病毒增殖 ............................................................................................................ 34
2-5. 重組病毒純化 ................................................................................................... 34
2-5-1. 蔗糖濃度梯度離心純化重組病毒 ........................................................ 34
2-5-2. FPLC 病毒純化 ..................................................................................... 35
2-5-3. TFF-Hollow fiber 病毒純化 .................................................................. 36
2-6. 血球凝集試驗 ................................................................................................... 37
2-7. 電子顯微鏡負染色觀察 ................................................................................... 37
(三) HA 膜蛋白萃取與純化 ............................................................................................ 37
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3-1. 病毒感染、細胞收取及破碎 ........................................................................... 38
3-2. 膜蛋白重力純化 ............................................................................................... 38
3-3. 蛋白濃縮與定量 ............................................................................................... 39
3-4. 聚丙烯醯胺膠體電泳 (Poly-Acrylamide Gel Electrophoresis, PAGE) .......... 40
(四)抗原盤製作 ................................................................................................................. 41
4-1. 昆蟲細胞抗原盤製作 ....................................................................................... 42
4-2. 哺乳動物細胞抗原盤製作 ............................................................................... 42
4-2-1. VERO E6 細胞轉導條件測試 ............................................................... 42
4-2-2. 抗原盤條件測試 .................................................................................... 43
(五)老鼠免疫試驗 ............................................................................................................. 43
5-1. 抗原接種 ........................................................................................................... 43
5-2. 綜合血清效價測試 ........................................................................................... 43
5-2-1. 西方墨點法 ............................................................................................ 43 5-2-2. 酵素免疫分析法 .................................................................................... 44 5-2-3. 血球凝集抑制試驗 ................................................................................ 44 5-2-4. 抗原盤測試 ............................................................................................ 44 (六)單株抗體篩選及測試 ................................................................................................. 45 6-1. 單株抗體篩選 ................................................................................................... 45 6-1-1. 酵素免疫分析法 .................................................................................... 45 6-2-2. 抗原盤測試 ............................................................................................ 45 6-2. 流式細胞儀測定單株抗體結合效力 ............................................................... 45 (七)西方墨點法 ................................................................................................................. 46 7-1. 蛋白質轉漬 ....................................................................................................... 47 7-2. 抗體使用 ........................................................................................................... 48 7-3. 底片呈色 ........................................................................................................... 48
肆、結果 ............................................................................................................................................ 49
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一、小鼠免疫抗原製備 ............................................................................................................ 49
(一) 構築 GP64 表面抗原呈現之轉移質體 .................................................................. 49
(二) 終點稀釋法分離 HA-Bac單一重組病毒株 ........................................................... 50
(三) 重組病毒濃縮及純化結果 ....................................................................................... 50
3-1. FPLC 純化 HA-Bac 病毒 ............................................................................... 50
3-2. TFF-中空纖維膜濃縮 HA-Bac 病毒與 HA 活性測試 ................................. 51
(四) 膜蛋白表現與純化 ................................................................................................... 52
二、小鼠血清學測試結果 ........................................................................................................ 52
(一) 西方墨點法測試與 ELISA ...................................................................................... 53
(二) 血球凝集抑制試驗 ................................................................................................... 54
三、桿狀病毒轉導抗原盤建立與條件測試 ............................................................................ 55
(一) 昆蟲細胞感染性抗原盤測試 ................................................................................... 55
(二) 西方墨點法驗證 HA 蛋白於 VERO E6 表現之條件測試 ................................. 56
(三) 以小鼠血清測試於 VERO E6轉導條件之結果 .................................................... 57
(四) 測試單株抗體於 VERO E6抗原盤之結果 ............................................................ 58
(五) 測試單株抗體於 VERO E6 與 U2OS 抗原盤之結果 ......................................... 59
四、單株抗體功能性測試 ........................................................................................................ 60
(一) 單株抗體血球凝集抑制試驗 ................................................................................... 60
(二) 流式細胞儀分析單株抗體之特性 ........................................................................... 60
(三) 西方墨點法分析單株抗體之特性 ........................................................................... 60
伍、討論 ............................................................................................................................................ 62
一、流感疫情監控與快速篩檢之重要性 ................................................................................ 62
二、HA 表面呈現桿狀病毒之應用與純化技術改良 ............................................................ 63
三、哺乳動物轉導之抗原盤應用 ............................................................................................ 65
四、單株抗體改善與地區性流感資料庫之建立 .................................................................... 66
陸、結論 ............................................................................................................................................ 67
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柒、參考資料 .................................................................................................................................... 98
表目錄
表一、引子序列清單 68
表二、進行小鼠免疫之不同免疫原清單 69

圖目錄
圖一、單株抗體生產流程圖 70
圖二、生產H5表面呈現之桿狀病毒 71
圖三、建構 H1 基因與 GP64 基因重組之轉移質體 72
圖四、製造H1表面呈現病毒與西方墨點法確認外源蛋白表現 73
圖五、以 FPLC 進行重組桿狀病毒之純化結果 74
圖六、以中空纖維膜濃縮重組病毒與 HA 活性測試 75
圖七、HA 膜蛋白之純化結果 76
圖八、西方墨點法測試 77
圖九、ELISA 測試 78
圖十、血球凝集抑制試驗 80
圖十一、以桿狀病毒系統建立單株抗體篩選用抗原盤之概念圖 82
圖十二、以免疫螢光法在昆蟲細胞表現膜蛋白並以之篩選抗體 83
圖十三、增強 HA-Bac 轉導於哺乳類細胞表現 HA 蛋白之條件測試結果 84
圖十四、不同轉導條件於哺乳類細胞表現 HA 蛋白之條件測試結果 85
圖十五、細胞免疫螢光染色進行血清學測試之結果 86
圖十六、細胞免疫螢光染色進行血清學測試之結果 87
圖十七、ELISA 與細胞免疫螢光染色進行單株抗體測試之結果 89
圖十八、VERO E6 單株抗體測試 90
圖十九、U2OS 單株抗體測試 91
圖二十、測定單源抗體之血球凝集抑制效價 92
圖二十一、以流式細胞儀測定單源抗體與HA蛋白之結合能力 93
圖二十二、以西方墨點法測定單源抗體與HA蛋白之結合能力 94
圖二十三、不同單源抗體篩選方式之結果比較表 95
圖二十四、小量濃縮純化桿狀病毒之比較結果 96
圖二十五、以桿狀病毒生產不同流感病毒的 HA 以激發單株抗體 97


柒、參考資料
1. Bouvier, N. M., and P. Palese. 2008. The biology of influenza viruses. Vaccine 26 Suppl 4:D49-53.
2. CDC. 2012. Prevention and control of influenza with vaccines: recommendations of the advisory committee on immunization practices (ACIP)--United States, 2012-13 influenza season. Morbidity and mortality weekly report 61:613-618.
3. Cox, N. J., and K. Subbarao. 1999. Influenza. The lancet 354:1277-1282.
4. Cros, J. F., and P. Palese. 2003. Trafficking of viral genomic RNA into and out of the nucleus: influenza, thogoto and borna disease viruses. Virus research 95:3-12.
5. Drake, J. W. 1993. Rates of spontaneous mutation among RNA viruses. Proceedings of the national academy of sciences of the United States of America 90:4171-4175.
6. Edwin, D. K. 2006. Influenza pandemics of the 20th century. Emerging infectious disease journal 12:9.
7. Ernst, W., R. Grabherr, D. Wegner, N. Borth, A. Grassauer, and H. Katinger. 1998. Baculovirus surface display: construction and screening of a eukaryotic epitope library. Nucleic acids research 26:1718-1723.
8. Gerster, P., E. M. Kopecky, N. Hammerschmidt, M. Klausberger, F. Krammer, R. Grabherr, C. Mersich, L. Urbas, P. Kramberger, T. Paril, M. Schreiner, K. Nobauer, E. Razzazi-Fazeli, and A. Jungbauer. 2013. Purification of infective baculoviruses by monoliths. Journal of chromatography. A 1290:36-45.
9. Ghedin, E., N. A. Sengamalay, M. Shumway, J. Zaborsky, T. Feldblyum, V. Subbu, D. J. Spiro, J. Sitz, H. Koo, P. Bolotov, D. Dernovoy, T. Tatusova, Y. Bao, K. St George, J. Taylor, D. J. Lipman, C. M. Fraser, J. K. Taubenberger, and S. L. Salzberg. 2005. Large-scale sequencing of human influenza reveals the dynamic nature of viral genome evolution. Nature 437:1162-1166.
99
10. Haase, S., L. Ferrelli, M. L. Pidre, and V. Romanowski. 2013. Genetic engineering of baculoviruses.
11. Hilleman, M. R. 2002. Realities and enigmas of human viral influenza: pathogenesis, epidemiology and control. Vaccine 20:3068-3087.
12. Jeffery, T., and M. David. 2006. 1918 Influenza: the mother of all pandemics. Emerging infectious disease journal 12:15.
13. Kaikkonen, M. U., J. K. Raty, K. J. Airenne, T. Wirth, T. Heikura, and S. Yla-Herttuala. 2006. Truncated vesicular stomatitis virus G protein improves baculovirus transduction efficiency in vitro and in vivo. Gene therapy 13:304-312.
14. Kash, J. C., A. G. Goodman, M. J. Korth, and M. G. Katze. 2006. Hijacking of the host-cell response and translational control during influenza virus infection. Virus research 119:111-120.
15. Kataoka, C., Y. Kaname, S. Taguwa, T. Abe, T. Fukuhara, H. Tani, K. Moriishi, and Y. Matsuura. 2012. Baculovirus GP64-mediated entry into mammalian cells. Journal of virology 86:2610-2620.
16. Kobasa, D., S. M. Jones, K. Shinya, J. C. Kash, J. Copps, H. Ebihara, Y. Hatta, J. Hyun Kim, P. Halfmann, M. Hatta, F. Feldmann, J. B. Alimonti, L. Fernando, Y. Li, M. G. Katze, H. Feldmann, and Y. Kawaoka. 2007. Aberrant innate immune response in lethal infection of macaques with the 1918 influenza virus. Nature 445:319-323.
17. Kost, T. A., J. P. Condreay, and D. L. Jarvis. 2005. Baculovirus as versatile vectors for protein expression in insect and mammalian cells. Nature biotechnology 23:567-575.
18. Krammer, F., and P. Palese. 2013. Influenza virus hemagglutinin stalk-based antibodies and vaccines. Current opinion in virology 3:521-530.
19. Kumar, A., R. Zarychanski, R. Pinto, and et al. 2009. Critically ill patients with 2009 influenza a (H1N1) infection in canada. The journal of the American medical association 302:1872-1879.
100
20. Lakadamyali, M., M. J. Rust, H. P. Babcock, and X. Zhuang. 2003. Visualizing infection of individual influenza viruses. Proceedings of the national academy of sciences 100:9280-9285.
21. Lin, W., H. Fan, X. Cheng, Y. Ye, X. Chen, T. Ren, W. Qi, and M. Liao. 2011. A baculovirus dual expression system-based vaccine confers complete protection against lethal challenge with H9N2 avian influenza virus in mice. Virology journal 8:273.
22. Lucas, P. M., O. W. Morgan, T. F. Gibbons, A. C. Guerrero, G. M. Maupin, J. L. Butler, L. C. Canas, V. P. Fonseca, S. J. Olsen, and V. H. MacIntosh. 2011. Diagnosis of 2009 Pandemic influenza A (pH1N1) and seasonal influenza using rapid influenza antigen tests, San Antonio, Texas, april–june 2009. Clinical infectious diseases 52:S116-S122.
23. Miller, L. K. 1988. Baculoviruses as gene expression vectors. Annual review of microbiology 42:177-199.
24. Morens, D. M., J. K. Taubenberger, and A. S. Fauci. 2008. Predominant role of bacterial Pneumonia as a cause of death in pandemic influenza: implications for pandemic influenza preparedness. Journal of infectious diseases 198:962-970.
25. Murphy, B. R., and M. L. Clements. 1989. The systemic and mucosal immune response of humans to influenza A virus. New Strategies for Oral Immunization 146: 107-116.
26. Nayak, D. P., E. K. Hui, and S. Barman. 2004. Assembly and budding of influenza virus. Virus research 106:147-165.
27. Neumann, G., T. Noda, and Y. Kawaoka. 2009. Emergence and pandemic potential of swine-origin H1N1 influenza virus. Nature 459:931-939.
28. Pidre, M. L., M. L. Ferrelli, S. Haase, and V. Romanowski. 2013. Baculovirus display: a novel tool for vaccination.
29. Prabakaran, M., S. R. Kumar, K. V. Raj, X. Wu, F. He, J. Zhou, and J. Kwang. 2014. Cross-protective efficacy of baculovirus displayed hemagglutinin against highly pathogenic influenza H7 subtypes. Antiviral research 109:149-159.
101
30. Sakurai, A., and F. Shibasaki. 2012. Updated values for molecular diagnosis for highly pathogenic avian influenza virus. Viruses 4:1235-1257.
31. Steinhauer, D. A. 1999. Role of hemagglutinin cleavage for the pathogenicity of influenza virus. Virology 258:1-20.
32. Suzuki, Y. 2005. Sialobiology of influenza: molecular mechanism of host range variation of influenza viruses. Biological &; pharmaceutical bulletin 28:399-408.
33. Tamura, S., T. Tanimoto, and T. Kurata. 2005. Mechanisms of broad cross-protection provided by influenza virus infection and their application to vaccines. Japanese journal of infectious diseases 58:195-207.
34. Tang, X. C., H. R. Lu, and T. M. Ross. 2010. Hemagglutinin displayed baculovirus protects against highly pathogenic influenza. Vaccine 28:6821-6831.
35. van Oers, M. M., and J. M. Vlak. 2007. Baculovirus genomics. Current drug targets 8:1051-1068.
36. Wagner, R., M. Matrosovich, and H. D. Klenk. 2002. Functional balance between haemagglutinin and neuraminidase in influenza virus infections. Reviews in medical virology 12:159-166.
37. Wu, Q., L. Fang, X. Wu, B. Li, R. Luo, Z. Yu, M. Jin, H. Chen, and S. Xiao. 2009. A pseudotype baculovirus-mediated vaccine confers protective immunity against lethal challenge with H5N1 avian influenza virus in mice and chickens. Molecular immunology 13:2210 - 2217.
38. Yang, D.-G., Y.-C. Chung, Y.-K. Lai, C.-W. Lai, H.-J. Liu, and Y.-C. Hu. 2007. Avian influenza virus hemagglutinin display on baculovirus envelope: cytoplasmic domain affects virus properties and vaccine potential. Molecular therapy 15:989-996.
39. 林佳蓓, 高明輝, 方怡雅, 王德原, 羅吉方. 2011. A型流行性感冒病毒快速診斷試劑效能評估調查研究. 食品藥物研究年報 2:7.
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