臺灣博碩士論文加值系統

本研究目的在於希望藉由單株抗體技術及植物的遺傳工程技術，建立一病毒防治新策略之抗體媒介的保護方法 ( Antibody-mediated protection ) 應用於植物的病毒病害防治。目前實驗所獲得的喜姆比蘭嵌紋病毒 ( cymbidium mosaic virus , 簡稱 CyMV ) 的抗體基因cDNA ( CyMV-H 10-1、CyMV-L 23 ) 及木瓜輪點病毒 ( papaya ringspot virus , 簡稱 PRV ) 的抗體基因cDNA ( PRV-H 10-9、PRV-L 3-8 ) 已經由南方墨點分析法 ( Southern blot analysis ) 證明構築在pBluescript II SK ( + ) 增殖質體中，分別形成 pBs-10-9 ( PRV-H )、 pBs-3-8 ( PRV-L )、 pBs-10-1 ( CyMV-H )、 pBs-23 ( CyMV-L ) 重組質體。分別將其進行核酸序列定序工作，核酸序列之譯讀結果， PRV-H 10-9及 PRV-L 3-8的核甘酸序列數目分別為1377及714，其相對應轉譯所得氨基酸數目為459及238，polyadenylation signals ( AATAAA ) 分別位於1446到1451及903到908，其3端均具有poly A的尾部，進行網路比對及電腦分析預測signal peptide時，顯示PRV-H 10-9的signal peptide位置由1到19氨基酸，而PRV-L 3-8的位置是由1到20氨基酸，CyMV-H 10-1及CyMV-L 23的核甘酸序列數目分別為1383及717，其相對應轉譯所得氨基酸數目分別為461及239 ， polyadenylation signals ( AATAAA ) 分別位於1455到1459及906到910，其3端均具有poly A的尾部，進行網路比對及電腦分析預測signal peptide時，顯示CyMV-H 10-1的signal peptide位置由1到19氨基酸，而CyMV-L 23的位置是由1到20氨基酸。為了進行抗體基因轉殖植物和抗體基因蛋白質表現，完成 CyMV 抗體基因轉殖植物重組質體 pBin-10-1 ( CyMV-H )、 pBin-23 ( CyMV-L ) 及 PRV抗體基因轉殖植物重組質體 pBin-10-9 ( PRV-H )、 pBin-3-8 ( PRV-L ) 與 CyMV抗體基因蛋白質表現重組質體 pET-10-1 ( CyMV-H )、 pET-23 ( CyMV-L )及 PRV抗體基因蛋白質表現重組質體 pET-10-9 ( PRV-H )、 pET-3-8 ( PRV-L )之構築，而構築好的 pET-23 ( CyMV-L )、pET-3-8 ( PRV-L )抗體基因蛋白質表現重組質體結果經SDS-PAGE電泳分析及西方墨點分析法偵測證明，分別在約31 KDa ( 資料未示 )和33 KDa處出現蛋白大量表現之蛋白帶，以西方墨點分析法偵測，亦有約31 KDa和33 KDa 反應訊號，顯示可在大腸桿菌系統內表現兩病毒輕鏈抗體蛋白。利用金鋼砂與木瓜擬胚化組織 ( 日昇與泰國品種 ) 在水中強力混合一分鐘，製造微小傷口，再以農桿菌 ( Agrobacterium tumefaciens ) 媒介感染轉移PRV抗體基因轉殖植物重組質體 pBin-10-9 ( PRV-H )、 pBin-3-8 ( PRV-L )。轉型後之擬胚化組織經培養於1/2 MS 添加20 g / l 蔗糖、0.001 ppm BA及8 % 洋菜 ( pH 5.8 ) 之培養基上共同培養48小時，之後移至含500 ppm Cefoxitin之相同培養基培養，3個星期後再移至含500 ppm Cefoxitin及100ppm Kanamycin 之培養基培養進一步篩選轉殖基因胚。

With the advance in monoclonal antibody technology and genetic engineering, the study aimed to come up with an effective method in antibody-mediated protection, which could be applied in the control of virus disease in plant. The cDNAs of antibody genes of cymbidium mosaic virus ( CyMV ) and papaya ringspot virus ( PRV ) were obtained and cloned in pBluescript II SK ( + ) plasmid, including recombinant plasmids pBs-10-9 ( PRV-H ), pBs-3-8 ( PRV-L ), pBs-10-1 ( CyMV-H ) and pBs-23 ( CyMV-L ), respectively. Results of nucleotide sequences analysis revealed that there were 1377 and 714 nucleotides in PRV-H 10-9 and PRV-L 3-8, respectively, and numbers of deduced amino acids were 459 and 238, respectively. Poly A signals ( AATAAA ) were found at the position from 1446 to 1451 for PRV-H 10-9 and from 903 to 908 for PRV-L 3-8. Results also showed that there were 1383 and 717 nucleotides in CyMV-H 10-1 and CyMV-L 23, respectively, and numbers of deduced amino acids were 461 and 239 amino acids, respectively. Poly A signals ( AATAAA ) were found at the position from 1455 to 1459 for CyMV-H 10-1 and from 906 to 910 for CyMV-L 23. The nucleotide sequences computed through internet and Hitachi software of DNASIS indicated that all of sequences had a signal peptide and a poly (A) tail. In order to obtain antibody genes transgenic plants and antibody genes expressed protein, we constructed several recombinant plasmids. The recombinant plasmids of antibody genes of CyMV and PRV were pBin-10-1 ( CyMV-H ), pBin-23 ( CyMV-L ), pBin-10-9 ( PRV-H ) and pBin-3-8 ( PRV-L ) for transformation to plants. The recombinant plasmids of antibody genes of CyMV and PRV were pET-10-1 ( CyMV-H ), pET-23 ( CyMV-L ), pET-10-9 ( PRV-H ) and pET-3-8 ( PRV-L ) for proteins expression in E. coli. SDS-PAGE and Western blot analysis showed that proteins were expressed in E. coli transformed with pET-23 ( CyMV-L ) and pET-3-8 ( PRV-L ) with molecular weights of 31 KDa ( SDS-PAGE data not shown ) and 33 KDa, respectively. By means of the triparental mating, the coding sequence for the heavy and light chains of monoclonal antibody ( mAb ) to PRV were cloned into the A. tumefaciens through Ti plasmid transformation system into papaya embryos of Sunrise and Tailand cultivars, respectively. The embryogenic tissues were vortexed with 600 mesh carborundum in sterilized water for 1 min before treating with A. tumefaciens containing pBin-10-9 ( PRV-H ) and pBin-3-8 ( PRV-L ). Transformed tissue were cultured on 1/2 MS medium for 2 days and then transfered to Cefoxitin ( 500 ppm ) selection medium for 3 weeks. The developed somatic embryos were transferred to 1/2 MS medium containing Kanamycin ( 100 ppm ).

中文摘要…………………………………………………………………I
英文摘要………………………………………………………………III
壹、前言…………………………………………………………………1
貳、材料與方法…………………………………………………………13
一、實驗材料…………………………………………………………13
二、質體DNA的大量抽取…………………………………………14
三、質體DNA的小量抽取…………………………………………14
四、DIG核酸探針的標識……………………………………………15
五、DNA洋菜膠體電泳分析………………………………………16
六、南方墨點分析法 ( Southern blot analysis) ……………………16
七、自動核酸序列分析 ( Autosequencing ) ………………………18
八、核甘酸及氨基酸序列的電腦分析………………………………18
九、DNA片段的回收與純化………………………………………19
十DNA的黏接反應 ( Ligation ) …………………………………20
十一、勝任細胞 ( Competent cell ) 的製備………………………21
十二、轉化作用 ( Transformation ) ………………………………22
十三、三親交配 ( Triparental mating ) ……………………………22
十四、植物轉殖 ( Plant transformation ) …………………………23
十五、聚合酵素鏈連鎖反應 ( Polymerase chain reaction ) ………24
十六、蛋白質的大量表現……………………………………………25
十七、SDS-PAGE分析蛋白質………………………………………26
十八、西方墨點分析法 ( Western blot analysis )…………………27
參、結果…………………………………………………………………29
一、 南方墨點分析法 ( Southern blot analysis ) …………………29
二、 核甘酸及氨基酸序列的分析…………………………………29
三、 DNA的黏接反應及轉化作用………………………………42
四、 植物轉殖………………………………………………………54
五、 轉殖植物的聚合酵素鏈連鎖反應偵測………………………54
六、蛋白質表現……………………………………………………60
肆、討論…………………………………………………………………65
伍、參考文獻……………………………………………………………75
陸、附錄…………………………………………………………………85

王嬿婷。1996。蘭花喜姆比蘭嵌紋病毒偵測技術之比較與應用。國立高雄師範大學科學教育研究所碩士論文。
位國慶。1995。台灣蝴蝶蘭病毒之發生與防治。農業世界 137:84-88。
李惠鈴。1990。蝴蝶蘭病毒之特性及其防治。國立中興大學植物病理研究所碩士論文。
張清安。1996。蘭花病毒病之特性及其防治。農業世界 137:14-20。
賴本智。1996。台灣蘭花產業的過去、現在與未來。農業世界 137:7-10。
Abel, P. P., Nelson, R. S., De, B., Hoffmann, N., Rogers, S. G., Fraley, R. T., and Beachy, R. N. 1986. Delay of disease development in transgenic plant that express the tobacco mosaic virus coat protein gene. Science 232: 738-743.
Anderson, J. M., Palukaitis, P., and Zaitlin, M. 1992. A defective replicase gene induces resistance to cucumber mosaic virus in transgenic tobacco plants. Proc. Natl. Acad. Sci. USA 89:8759-8763.
Anamthawat, K., and Vajrabhaya, T. 1979. Survey of the occurrence of cymbidium mosaic virus in dendrobium orchids in Bankok. Aust. Orchid. Rev. 44 (2):79-81.
Baulcome, D. C., Saunders, G. R., Bevan, M. W., Mayo, M. A., and Harrison, B. D. 1986. Expression of biologically active viral satellite RNA from the nuclear genome of transformed plants. Nature 321:446-449.
Beachy, R. N. 1990. Coat protein mediated resistance against virus infection. Ann. Rev. Phytopathol. 28, 451-474.
Bednarek, S. Y. and Raikhel, N. V. 1992. Intracellular trafficking of secretory proteins. Plant Mol. Biol. 20:133-150.
Bidney, D., Scelonge, C., Martich, J., Burrus, M., Sims, L., and Huffman, G. 1992. Microprojectile bombardment of plant tissues increases transformation frequency by Agrobacterium tumefaciens. Plant Mol. Biol. 18:301-313.
Brown, P. 1985. Viruses-A serious problem for orchid growers. Green- Thumb Denver 42 (4):117-119.
Chen, M. H., and Chen, C. C. 1992. Plant regeneration from Carica protoplasts. Plant Cell Rep. 11:404-407.
Chen, M. H., Wang, D. J., and Maeda, E. 1987. Somatic embroyogenesis and plant regeneration in Carica papaya L. tissue culture derived from root explants. Plant Cell Rep. 6:348-351.
Cheung, S. C., Dietzschold, B., Koprowski, H. Notkins, A. L., and Rando, R. F. 1992. A recombinant human Fab expressed in E. coli neutralizes Rabies virus. J Virol. 11:6714-6720.
Chia, T. F., and Chan, Y. S. 1992. Chracterization of cymbidium mosaic virus coat protein gene and its expression in transgenic tobacco plant. Plant Mol. Biol. 18:1091-1099.
Chomczynski, P. and Sacchi, N. 1987. Single-step method of RNA isolation by acid-guanidium-thiocyante-phenol-chloroform extraction. Anal. Bio. 162:156-159.
Cuzzo, M., O''Connell, K. M., Kaniewsk, W., Fang, R. X., Chua, N. H., and Tumer, N. E. 1988. Viral protection in transgenic tobacco plants expressing the cucumber mosaic virus coat protein or its antisense RNA. Bio/Technology 6:549-557.
Darvean, R. P. 1992. Expression of antibody fragment in Escherichia coli. J. Clini. Immunol. 15:25-29.
David, J. R. 1996. Environmental risk assessment of releases of transgenic plants containing virus-derived inserts. Trangenic Research. 5:359-362.
De Neve, M., De, Losse, M., Jacobs, A., Van, H. H., Kaluza, B., Weidle, U., Van, M. M., and Depicker, A. 1993. Assembly of an antibody and its derived antibody fragment in Nicotiana and Arabidopsis. Transgen Res. 2:227-237.
De Bruijne, E., DeLangehe, E., and Van, R. R. 1974. Action of homones and embryoid formation in callus cultures of Carica papaya. Int. Symp. Fytofarm Fytiata. 26:637-645.
Duenas, M., and Borrebaeck. C. A. K. 1994. Clonal selection and amplification of phage displayed antibodies by linking antigen recognition and phage replication. Bio / Technol. 12:999-1002.
Faccioli, G., and Marani, F. 1979. Cymbidium mosaic virus associated with flower necrosis in Cattleya orchids. Phytopath. Medit. 18:21-25.
Schouten, A., Roosien, J., de Boer, J. M., Wilmink., A. Rosso, M-N., Bosch, D., Stiekema, W. J., Gommers, F. J., Bakker, J., and Schots, A. 1997. Improving scFv antibody expression levels in the plant cytosol. 415:235-241.
Fitch, M. M. M., and Manshardt, R. M. 1990. Somatic embryogenesis and plant regeration from immature zygotic embryos of papaya (Carica papaya L.). Plant Cell Rep. 9:320-324.
Frowd, J. A., and Tremaine, J. H. 1977. Physical , chemical , and serological properties of cymbidium mosaic virus. Phytopathology 67:43-49.
Golemboski, D. B., Lomonossoff, G. P., and Zaitlin, M. 1990. Plants transformed with a tobacco mosaic virus monstructural gene sequence are resistant to the virus. Proc. Natl. Acad. Sci. USA 87:6311-6315.
Harrison., B. D., Mayo., M. A., and Baulcome, D. C. 1987. Virus resistance in transgenic plants that express cucumber mosaic virus satellite RNA. Nature 328:799-802.
Hayashimoto, A., Li, Z., and Murai, N. 1990. A polyethylene glycol-mediated protoplast transformation system for production of fertile transgenic rice plants. Plant Physiol. 93:857-863.
Hiatt, A., Cafferkey, R., and Bowdish, K. 1989. Production of antibodies in transgenic plants. Nature 342:76-78.
Hiatt, A. 1990. Antibodies produced in plants. Nature 344:469-470.
Hiatt, A., and Ma, J. K. 1992. Monoclonal antibody engineering in plants. FEBS Lett. 307:71-75.
Hiruki, C., Chen, M. H., and Rao, D. V. 1980. Ultrastructure of Cattleya leaf cells infected with cymbidium mosaic virus. Acta. Hortic. 110:273-279.
Huston, J. S., Levinson, D., Mudgett-Hunter, M. Tai, M. -S., Novotny, J., Margolies, M. N., Ridge, R. J., Bruccoleri, R. E., Haber, E., Crea, R., and Oppermann, H. 1988. Protein engineering of antibody binding sites: recovery of specific activity in an anti-digoxin single-chain Fv analogue produced in Escherichia coli. Proc. Natl. Acad. Sci. USA. 85:5879-5883.
Jensen, D. D. 1951. Mosaic or black streak disease of cymbidium orchids. Phytopathology 41:401-414.
Johnson, G., and Bird, R. E. 1991. Construction of single-chain Fv derivatives of monoclonal antibodies and their production in Escherichia coli. Methods Enzymol. 203:88-98.
Lawson, R. H., and Hearon, S. S. 1973. Symptomatology of cattleya mericlones infected with cymbidium mosaic virus. Amer. Orchid Soc. Bull. 42:1071-1074.
Lim, S. T., Wong, S. M., Yeong, C. Y., Lee, S. C., and Goh, C. J. 1993. Rapid detection of cymbidium mosaic virus by the polymerase chain reaction. J. Virol. Methods 41:37-46.
Lin, C. C. 1980. Strains of papaya ringspot virus and their cross protection. Ph. D. thesis. National Taiwan University, Taipei.
Litz, R. E., and Conover, R. A. 1982. In vitro somatic embryogenesis and plant regeneration from Carica papaya L. ovular callus. Plant Sci. Lett. 26: 153-158
Litz, R. E., Ohair, S. K., and Conover, R. A. 1983. In vitro of Carica papaya L. cotyledons. Scientia Hort. 19: 287-293.
Lothar, F. F., Andrea, K., Ulrich, C., and Judith, C. 1996. Expression of single-chain antibody fragments (scFv) specific for beet necrotic yellow vein virus coat protein or 25KDa protein in Escherichia coli and Nicotiana benthamiana. Plant Mol. Biol. 32:979-986.
Luzzago, A., Felici, F., Tramontano, A., Pessi, and Cortese, R. 1993. Mimicking of discontinuous epitopes by phage-displayed peptides. I. Epitope mapping of human H ferritin using a phage library of constrained peptides. Gene. 128: 51-57.
Morrison, S. L., Johnson, M. J., Herzenberg, L. A.,and Oi, V. T. 1984. Chimeric human antibody molecules:mouse antigen-binding domains with human constant region domains. Proc. Natl. Acad. Sci. USA. 81:6851-6857.
Michael, T., and Wilson, A., 1993. Strategies to protect crop plants against viruses: Pathogen-derived resistance blossoms. Proc. Natl. Acad. Sci. USA. Vol. 90, pp. 3134-3141.
Nelson, R. S., McCormick, S. M., Delannay, X., Dube, P., Layton, J., Aderson, E. J., Kaniewska, M., Porksch, R., Horsch, R. B., Rogers, S. G., Fraley, R. T., and Beachy, R. N. 1988. Virus tolerance, plant growth, and field performance of transgenic tomato plants expressing coat protein from tobacco virus. Bio/Technology 6:403-490.
Neo, K. K., Wong, S. M., and Wu, M. 1993. Nucleotide sequence and in vitro translation of the coat protein gene of cymbidium mosaic virus. Virus Genes. 7(2):157-170.
Neuhaus, G., and Spangenberg, G. 1990. Plant transformation by microinjection technique. Physiol. Plant. 79: 213-217.
Okemura, A. K., Kamemoto, H., and Ishii, M. 1984. Incidence and Expression of Cymbidium Mosaic Virus in Dendrobium Hybrid. HITAHR, College of tropical agriculture and human resources. University of Hawii.
Paul, J. J., and Rob, A. S. 1992. Agrobacterium and plant genetic engineering. Plant Mol. Biol. 19:15-38.
Pisi, A., Marani, F., and Bertaccini, A. 1982. Intracellular inclusions in host plants infected with cymbidium mosaic virus. Phytopath. Medit. 21:27-30.
Pluckthun, A. 1990. Antibodies from Escherichia coli. Nature. 347:497-498.
Purcifull, D. E., Edwardson, J. R., Hiebert, E., and Gonsalves, D. 1984. Papaya ringspot virus. CMI/AAB Descriptions of plant Viruses. No.84
Rezaian, M. A., Skene, K. G. M., and Ellis, J. G. 1988. Anti-sense RNAs of cucumer mosaic virus in transgenic plants assessed for control of the virus. Plant Mol. Biol. 11:463-471.
Rhodes, C. A., Pierce, D. A., Mettler, I. J., Mascarenhas, D., and Detmer, J. J. 1988. Genetically transformed maize plants from protoplasts. Science. 240:204-207.
Stachel, S. E., Messens, E., Montagu, M. V., and Zambryski, P. 1985. Identification of the signal molecules by wounded plant cells that activate T-DNA transfer in Agrobacterium tumefaciens. Nature. 318:624-629.
Udo, C., and Ulrike, F. 1994. Expression of engineered antibodies in plant cells. Plant Mol. Biol. 26:1023-1030.
Tavladoraki, P., Benvenuto, E.,Trinca,S., Martinis, D., and Galeffi, P. 1993. Transgenic plants expressing a functional single-chain Fv antibody are specifically protected from virus attack. Nature 366:469-472.
Tsay, H. S., and Su, C. Y. 1985. Anther culture of papaya (Carica papaya L.) Plant Cell Rep. 4:28-30.
Tien, R., and Wu, G. -S. 1991. Satellite RNA for the biocontrol of plant disease. Advances in Virus Research 39,321-339.
Tyutyulkova, S., and Paul., S. 1994. Selection of functional human immunoglobulin light chains from a phage-display library. Appl. Biochem. Biotechnol. 47:191-198.
Wang, H. L., Yeh, S. D., Chiu, R. J., and Gonsalves, D. 1987. Effectivess of cross-protection by mild mutants of papaya ringspot virus for control of ringspot disease of papaya in Taiwan. Plant Dis. 71:491-497.
Wang, H. Y., and Tadayuki, I. 1995. Antibody expression and engineering. Chapter 5-6.
Yeh, S. -D., and Gonsalves, D. 1984. Evaluation of induced mutants of papaya ringspot virus for control by cross protection. Phytopathology. 74. 1086-1091.
Yeh, S. -D., and Gonsalves, D. 1994. Practies and perspective of control of papaya ringspot virus by cross protection. Adv. Dis. Vector. Res. 10:237-257.
Yeh, S. -D., Gonsalves, D., Wang, H. -L., Namba, R., and Chiu, R. -J. 1988. Control of papaya ringspot virus by cross protection. Plant Dis. 72. 375-380.
Yie, S. T., and Liaw, S. I. 1977. Plant regeneration from shoot tips and callus of papaya. In Vitro. 13:564-567.
Yuen, C. K. K. H., Kamemoto, H., and Ishii, M. 1979. Transmission of cymbidium mosaic virus through seed propagation in Dendrobium. Amer. Orchid Soc. Bull. 48(12):1245-1248.
Zettler, F. W., Ko, N. J., Wisler, G. C., Elliott, M. S., and Wong, S. M. 1990. Viruses of orchids and their control. Plant Dis. 74(9):621-626.