臺灣博碩士論文加值系統

　　利用植物細胞核基因轉殖技術，發展家禽里奧病毒(avian reovirus；ARV)的可食性疫苗。隨著植物生物技術的發展，以植物生產動物疫苗可降低成本、可口服、較為安全，無人畜共通疾病、可表現於貯存組織運送方便等優點，許多以植物生產疫苗的研究，在動物試驗中證實是可引發動物的免疫反應，並可以對抗病原菌的感染。經由前人的研究，家禽里奧病毒之外鞘膜蛋白(outer capsid protein) σC可誘發中和抗體的產生，因此，我利用植物細胞核基因轉殖技術將S1基因分別轉殖至苜蓿與菸草之中，利用不同的轉殖載體使σC蛋白分別表現於苜蓿與菸草。在PCR的測試及GUS的測試，可證實家禽里奧病毒S1基因已融合於苜蓿及菸草的基因體之中；以西方墨點法除了得知σC蛋白質的確可表現在已轉殖的植株外，且可篩選出表現最高的轉殖植株，以此做為雞隻的可食性疫苗，希望可誘發雞隻產生中和抗體，並可抵抗家禽里奧病毒的感染。

　Several animal feeding experiments have shown that transgenic plant with over expression the surface antigen of pathogen can cause animal to induce immunity to against pathogen attack. In order to develop plant-based vaccine for poultry against avian reovirus, I have cloned S1 gene into plant nuclear expression vector pCAMBIA1301 and pCAMIBA2301. I also have transformed the expression constructs into both alfalfa and tobacco tissues by Agrobacteria-mediated transformation method. The alfalfa and tobacco transformants were selected with media containing antibiotics (hygomycin or kanamycin). PCR analysis and GUS histochemical assay have confirmed that the S1 gene have integrated into the chromosome of alfalfa and tobacco . transformantsI selected the transgenic lines with highest σC protein expression level by western blot analysis, and tested the immune response by injecting the plant–expressed σC protein to mice.

第一章、前言..............................................1
第二章、文獻探討..........................................3
1.疫苗的重要性及其優缺點.............................3
2.以植物生產動物疫苗的優點...........................5
3.如何利用植物來生產動物疫苗.........................6
3.1細胞核基因轉殖.................................6
3.2葉綠體基因轉殖.................................7
3.3病毒表現系統...................................8
4. 利用植物來做為動物疫苗的發展現況 .................9
4.1以植物開發人體疫苗.............................9
4.2以植物開發畜用體疫苗......................... 13
5. 以植物生產疫苗有待解的問題....................... 14
6. 研究目標-里奧病毒.................................16
第三章、材料與方法 ..................................... 18
1. 實驗材料......................................... 18
1.1家禽里奧病毒S1基因的來源.................... . 18
1.2轉殖材料之培養............................... 18
2. 轉殖表現載體的構築............................... 19
2.1 p35S-S1-1301和p35S-S1-2301表現載體的構築......19
2.2 pAct1-S1-1301及pAct1-S1-2301表現載體的構築....20
3. 表現載體轉入農桿菌中............................. 21
4. 農桿菌植物基因轉殖............................... 22
4.1苜蓿農桿菌基因轉殖............................ 22
4.2菸草農桿菌基因轉殖............................ 23
5. 抽取植物基因體................................... 23
6. GUS組織染色分析...................................24
7. PCR 測試......................................... .....24
8. 抽取植物蛋白質................................... 24
8.1 小量抽取植物蛋白質........................... 24
8.2 大量抽取植物蛋白質........................... 25
9. 免疫老鼠......................................... 25
10. 西方墨點法...................................... .....26
11. 製備兔子抗ARV S1133 σC蛋白之免疫血清.............28
11.1構築表現載體..................................28
11.2 蛋白質表現及純化............................ 28
11.3 免疫兔子.................................... 31
12. 酵素免疫吸附分析(ELISA) .........................32
第四章、結果..............................................33
1. 獲得兔子抗σC之免疫血清...........................33
2. 獲得持續性表現家禽里奧病毒S1基因轉殖苜蓿及菸草品系
2.1在轉殖苜蓿的部份..................................34
2.2在轉殖菸草的部份..............................34
3. GUS histochemical的分析...........................35
3.1在轉殖苜蓿的部份..............................35
3.2在轉殖菸草的部份..............................35
4. 轉殖植株苜蓿和菸草外源蛋白表現量之測量........... 35
4.1 在苜蓿方面...................................35
4.2在菸草方面................................... 36
4.3 σC重組蛋白的定量.............................36
5. 動物實驗......................................... 37

第五章、討論..............................................38
1. 於苜蓿和菸草中持續性表現σC之重組蛋白..............38
2. 苜蓿和菸草的農桿菌轉殖效率差異................... 38
3. GUS的表現量與σC重組蛋白的表現並無直接關聯...... ..39
4. 35S啟動子和 Act1啟動子蛋白質表達量的差異......... 39
5. 動物實驗的結果....................................40
6. 以植物生產家禽里奧病毒疫苗....................... ....40
第六章、參考文獻......................................... 41
圖目錄
圖一、大腸桿菌表現載體結構圖pET42a-S1.....................46
圖二、不同的誘導時間下σC-GST重組蛋白的表現量..............47
圖三、重組蛋白σC-GST經由硫酸氨沉澱和8% SDS-PAGE純化的結果............. 48
圖四、以西方墨點法分析兔子的免疫血清抗σC-GST之效價...... .49
圖五、以西方墨點法分析兔子的免疫前血清抗σC-GST之效價......50
圖六、農桿菌雙偶型載體pAct1-S1-1301與p35S-S1-1301.........51
圖七、農桿菌雙偶型載體pAct1-S1-1301與p35S-S1-1301.........52
圖八、以PCR檢測苜蓿轉殖植株的基因體中是否含有S1基因.......53
圖九、以PCR檢測苜蓿轉殖植株的基因體中是否含有S1基因.......54
圖十、以PCR檢測苜蓿轉殖植株的基因體中是否含有S1基因.......55
圖十一、以PCR檢測苜蓿轉殖植株的基因體中是否含有S1基因.....56
圖十二、以PCR檢測菸草轉殖植株的基因體中是否含有S1基因.....57
圖十三、組織GUS組織結果................................ ..58
圖十四、組織GUS組織結果................................ ..59
圖十五、以西方墨點法分析苜蓿轉殖植株中σC的表現量(p35S-S1-1301轉殖載體組) ..........................................60
圖十六、以西方墨點法分析苜蓿轉殖植株中σC的表現量(p35S-S1-1301轉殖載體組) ......................................... 61
圖十七、以西方墨點法分析苜蓿轉殖植株中σC的表現量(pAct1-S1-1301轉殖載體組) .................... .....................62
圖十八、以西方墨點法分析菸草轉殖植株中σC的表現量.........63
圖十九、以西方墨點法分析苜蓿與菸草轉殖植株中σC的表現量....64
圖二十、以ELISA法分析老鼠血清anti-σC的力價...............65
圖二十一、以ELISA法分析老鼠血清在免疫後第35天時anti-σC抗體的力價...................................................66
圖二十二、以ELISA法比較老鼠血清在免疫後第35天時anti-σC抗體的力價...................................................67

表目錄
表一、比較利用各種表現系統來生產疫苗的優缺點..............68
表二、以植物生產疫苗的優點................................69
表三、各種植物表現系統的比較..............................71
表四、以發展的動物疫苗....................................72
表五、分析各轉殖載體組所得的轉殖數目......................77

郭良嘉. (2001). 家禽里奧病毒基因演化研究及ELISA診斷試劑之研製. 國立屏東科技大學獸醫系碩士論文.
張清俊. (2002). 高等植物葉綠體的基因轉殖與植物生物技術. 科學農業 50, 243-248.
Anna Modelska, B.D., N. Sleysh, Zhen Fang Fu, Klaudla Steplewski, D.Craig Hooper, Hilary Koprowski, and Vidadi Yusibov. (1998). Immunization against rabies with plant-derived antigen. Proc Natl Acad Sci U S A 95, 2481-2485.
Arntzen, A.M.W.a.C.J. (2000). Plants for delivery of edible vaccines. Curr Opin Biotechnol 11, 126-129.
Carrillo, C., Wigdorovitz, A., Oliveros, J.C., Zamorano, P.I., Sadir, A.M., Gomez, N., Salinas, J., Escribano, J.M., and Borca, M.V. (1998). Protective immune response to foot-and-mouth disease virus with VP1 expressed in transgenic plants. J Virol 72, 1688-1690.
Castanon, S., Marin, M.S., Martin-Alonso, J.M., Boga, J.A., Casais, R., Humara, J.M., Ordas, R.J., and Parra, F. (1999). Immunization with potato plants expressing VP60 protein protects against rabbit hemorrhagic disease virus. J Virol 73, 4452-4455.
Chikwamba, R.K., Scott, M.P., Mejia, L.B., Mason, H.S., and Wang, K. (2003). Localization of a bacterial protein in starch granules of transgenic maize kernels. Proc Natl Acad Sci U S A 100, 11127-11132.
Daniell, H., Streatfield, S.J., and Wycoff, K. (2001a). Medical molecular farming: production of antibodies, biopharmaceuticals and edible vaccines in plants. Trends Plant Sci 6, 219-226.
Daniell, H., Lee, S.B., Panchal, T., and Wiebe, P.O. (2001b). Expression of the native cholera toxin B subunit gene and assembly as functional oligomers in transgenic tobacco chloroplasts. J Mol Biol 311, 1001-1009.
Dus Santos, M.J., Wigdorovitz, A., Trono, K., Rios, R.D., Franzone, P.M., Gil, F., Moreno, J., Carrillo, C., Escribano, J.M., and Borca, M.V. (2002). A novel methodology to develop a foot and mouth disease virus (FMDV) peptide-based vaccine in transgenic plants. Vaccine 20, 1141-1147.
Galun, E., and Galun, E. (2001). The manufacture of medical and health products by transgenic plants. Imperial college, 125-181.
Golosby, R.A., Kindt, T.J., and Osborne, B.A. (2001). Immunology. W.H. Freeman and company new york.
Hamilton, W.D. (2001). The green revolution:plants as heterologous expression vectors. Vaccine 19, 2735-2741.
Haq, T.A., Mason, H.S., Clements, J.D., and Arntzen, C.J. (1995). Oral immunization with a recombinant bacterial antigen produced in transgenic plants. Science 268, 714-716.
Haq, T.A.M., Hugh S.. (1995). Oral immunization with a recombinant bacterial antigen produced in transgenic plants. Science 268, 714-716.
Horn, M.E., Woodard, S.L., and Howard, J.A. (2004). Plant molecular farming: systems and products. Plant Cell Rep 22, 711-720.
Hu, Y.-C., Liu, H.-J., and Chung, Y.-C. (2002). High level expression of the key antigenic proein,from avian reovirus into insect cells and its purification by immobilized metal affinity chromatography. Biotechnology Letters, 1017-1022.
Huang, Z., Dry, I., Webster, D., Strugnell, R., and Wesselingh, S. (2001). Plant-derived measles virus hemagglutinin protein induces neutralizing antibodies in mice. Vaccine 19, 2163-2171.
Huang, Z., G. Elkin, et al. (2005). Virus-like particle expression and assembly in plants: hepatitis B and Norwalk viruses. Vaccine 23(15): 1851-8.
J. Erickson, W.-J.Y., J. Brandle and R. Rymerson. (2002). Molecualr farming of plants and animlas for human and veterinary medicine.
Jani, D., Singh, N.K., Bhattacharya, S., Meena, L.S., Singh, Y., Upadhyay, S.N., Sharma, A.K., and Tyagi, A.K. (2004). Studies on the immunogenic potential of plant-expressed cholera toxin B subunit. Plant Cell Rep 22, 471-477.
Joung, Y. H., J. W. Youm, et al. (2004). Expression of the hepatitis B surface S and preS2 antigens in tubers of Solanum tuberosum."Plant Cell Rep 22(12): 925-30.
Kang, T.J., Kim, Y.S., Jang, Y.S., and Yang, M.S. (2005a). Expression of the synthetic neutralizing epitope gene of porcine epidemic diarrhea virus in tobacco plants without nicotine. Vaccine 23, 2294-2297.
Kang, T.J., Han, S.C., Kim, M.Y., Kim, Y.S., and Yang, M.S. (2004). Expression of non-toxic mutant of Escherichia coli heat-labile enterotoxin in tobacco chloroplasts. Protein Expr Purif 38, 123-128.
Kang, T.J., Seo, J.E., Kim, D.H., Kim, T.G., Jang, Y.S., and Yang, M.S. (2005b). Cloning and sequence analysis of the Korean strain of spike gene of porcine epidemic diarrhea virus and expression of its neutralizing epitope in plants. Protein Expr Purif 41, 378-383.
Kapusta, J., Modelska, A., Figlerowicz, M., Pniewski, T., Letellier, M., Lisowa, O., Yusibov, V., Koprowski, H., Plucienniczak, A., and Legocki, A.B. (1999). A plant-derived edible vaccine against hepatitis B virus. Faseb J 13, 1796-1799.
Kong, Q., Richter, L., Yang, Y.F., Arntzen, C.J., Mason, H.S., and Thanavala, Y. (2001). Oral immunization with hepatitis B surface antigen expressed in transgenic plants. Proc Natl Acad Sci U S A 98, 11539-11544.
Liu, H.J., Lee, L.H., Hsu, H.W., Kuo, L.C., and Liao, M.H. (2003). Molecular evolution of avian reovirus: evidence for genetic diversity and reassortment of the S-class genome segments and multiple cocirculating lineages. Virology 314, 336-349.
Liz J. Richter, Y.T., Charies J.Arntzen, Hugh S. Mason. (2000). Production of hepatitis B surface antigen in trasgenic plants for oral immunization. Nat Biotechnol 18, 1167-1171.
Ma, J.K. (2000). Genes, greens, and vaccines. Nat Biotechnol 18, 1141-1142.
Ma, J.K., Drake, P.M., and Christou, P. (2003). The production of recombinant pharmaceutical proteins in plants. Nat Rev Genet 4, 794-805.
Martinez-Costas, J., A. Grande, et al. (1997). Protein architecture of avian reovirus S1133 and identification of the cell attachment protein. J Virol 71(1): 59-64.
Marusic, C., Rizza, P., Lattanzi, L., Mancini, C., Spada, M., Belardelli, F., Benvenuto, E., and Capone, I. (2001). Chimeric plant virus particles as immunogens for inducing murine and human immune responses against human immunodeficiency virus type 1. J Virol 75, 8434-8439.
Mason, H.S., Lam, D.M., and Arntzen, C.J. (1992). Expression of hepatitis B surface antigen in transgenic plants. Proc Natl Acad Sci U S A 89, 11745-11749.
Mason, H.S., Haq, T.A., Clements, J.D., and Arntzen, C.J. (1998). Edible vaccine protects mice against Escherichia coli heat-labile enterotoxin (LT): potatoes expressing a synthetic LT-B gene. Vaccine 16, 1336-1343.
Mason, H.S., Warzecha, H., Mor, T., and Arntzen, C.J. (2002). Edible plant vaccines: applications for prophylactic and therapeutic molecular medicine. Trends Mol Med 8, 324-329.
Mason, H.S., Ball, J.M., Shi, J.J., Jiang, X., Estes, M.K., and Arntzen, C.J. (1996). Expression of Norwalk virus capsid protein in transgenic tobacco and potato and its oral immunogenicity in mice. Proc Natl Acad Sci U S A 93, 5335-5340.
McElroy, D., Zhang, W., Cao, J., and Wu, R. (1990). Isolation of an efficient actin promoter for use in rice transformation. Plant Cell 2, 163-171.
Modelska, A., Dietzschold, B., Sleysh, N., Fu, Z.F., Steplewski, K., Hooper, D.C., Koprowski, H., and Yusibov, V. (1998). Immunization against rabies with plant-derived antigen. Proc Natl Acad Sci U S A 95, 2481-2485.
Netherwood, T., Martin-Orue, S.M., O'Donnell, A.G., Gockling, S., Graham, J., Mathers, J.C., and Gilbert, H.J. (2004). Assessing the survival of transgenic plant DNA in the human gastrointestinal tract. Nat Biotechnol 22, 204-209.
Richter, L.J., Thanavala, Y., Arntzen, C.J., and Mason, H.S. (2000). Production of hepatitis B surface antigen in transgenic plants for oral immunization. Nat Biotechnol 18, 1167-1171.
Shih, W. L., H. W. Hsu, et al. (2004). Avian reovirus sigmaC protein induces apoptosis in cultured cells. Virology 321(1): 65-74.
Streatfield, S.J., and Howard, J.A. (2003). Plant-based vaccines. Int J Parasitol 33, 479-493.
Streatfield, S.J., Lane, J.R., Brooks, C.A., Barker, D.K., Poage, M.L., Mayor, J.M., Lamphear, B.J., Drees, C.F., Jilka, J.M., Hood, E.E., and Howard, J.A. (2003). Corn as a production system for human and animal vaccines. Vaccine 21, 812-815.
Thanavala, Y., Yang, Y.F., Lyons, P., Mason, H.S., and Arntzen, C. (1995). Immunogenicity of transgenic plant-derived hepatitis B surface antigen. Proc Natl Acad Sci U S A 92, 3358-3361.
Thanavala, Y., Mahoney, M., Pal, S., Scott, A., Richter, L., Natarajan, N., Goodwin, P., Arntzen, C.J., and Mason, H.S. (2005). Immunogenicity in humans of an edible vaccine for hepatitis B. Proc Natl Acad Sci U S A 102, 3378-3382.
Tregoning, J.S., Nixon, P., Kuroda, H., Svab, Z., Clare, S., Bowe, F., Fairweather, N., Ytterberg, J., van Wijk, K.J., Dougan, G., and Maliga, P. (2003). Expression of tetanus toxin Fragment C in tobacco chloroplasts. Nucleic Acids Res 31, 1174-1179.
Tuboly, T., Yu, W., Bailey, A., Degrandis, S., Du, S., Erickson, L., and Nagy, E. (2000). Immunogenicity of porcine transmissible gastroenteritis virus spike protein expressed in plants. Vaccine 18, 2023-2028.
Walmsley, A.M., and Arntzen, C.J. (2003). Plant cell factories and mucosal vaccines. Curr Opin Biotechnol 14, 145-150.
Walsh, G. (2002). Proteins Biochemistry and Biotechnology, 234-247.
Wang, Y., Zhang, W., Cao, J., McElroy, D., and Wu, R. (1992). Characterization of cis-acting elements regulating transcription from the promoter of a constitutively active rice actin gene. Mol Cell Biol 12, 3399-3406.
Wickramasinghe, R., J. Meanger, et al. (1993). Avian reovirus proteins associated with neutralization of virus infectivity. Virology 194(2): 688-96.
Wigdorovitz, A., Mozgovoj, M., Santos, M.J., Parreno, V., Gomez, C., Perez-Filgueira, D.M., Trono, K.G., Rios, R.D., Franzone, P.M., Fernandez, F., Carrillo, C., Babiuk, L.A., Escribano, J.M., and Borca, M.V. (2004). Protective lactogenic immunity conferred by an edible peptide vaccine to bovine rotavirus produced in transgenic plants. J Gen Virol 85, 1825-1832.
Wu, H., N. K. Singh, et al. (2004). Immunization of chickens with VP2 protein of infectious bursal disease virus expressed in Arabidopsis thaliana. Avian Dis 48(3): 663-8.
Yu, J., and Langridge, W.H. (2001). A plant-based multicomponent vaccine protects mice from enteric diseases. Nat Biotechnol 19, 548-552.