Please use this identifier to cite or link to this item: http://hdl.handle.net/11455/31194
標題: 蝴蝶蘭黃化斑點病毒之單元抗體及康乃馨斑駁病毒蝴蝶蘭分離株之多元抗體之製備及應用
Generation and application of monoclonal antibody against Phalaenopsis chlorotic spot virus and polyclonal antibody against Carnation mottle virus Phalaenopsis orchid isolate
作者: 陳呈權
Chen, Cheng-quan
關鍵字: Phalaenopsis chlorotic spot virus
蝴蝶蘭黃化斑點病毒
Carnation mottle virus
monoclonal antibody
polyclonal antibody
康乃馨斑駁病毒
單元抗體
多元抗體
出版社: 植物病理學系所
引用: 伍、 引用文獻 行政院農業委員會。2008。96 年農業統計年報。行政院農業委員會編印。台北。108頁。 行政院農業委員會動植物防疫檢疫局。2001。植物保護圖鑑系列 6-洋蘭保護。行政院農業委員會動植物防疫檢疫局編印。台北。34頁。 安寶貞。1995。臺灣的蘭花疫病。植物病理學會刊 4:152-162 頁。 宋一鑫、吳雅芳、陳燿煌、陳紹崇、林明瑩、陳昇寬、鄭安秀。2006。環控設施 栽培之蝴蝶蘭落蕾現象。植保會刊 48:65-70 頁。 位國慶。1994。臺灣蝴蝶蘭真菌及細菌病害之發生與防治。中華植物保護學會特刊新二號。中華植物保護學會編印。119-131 頁。 許秀惠。1994。臺灣花卉之細菌性病害。中華植物保護學會特刊新二號。中華民國植物保護學會編印。63-75 頁。 張清安。1995。花卉病害 (1) 蘭花病害。臺灣農家要覽。農作篇 (三) 豐年社。臺北。500頁。 張清安。2005。蘭花病毒病。植物保護技術專刊系列1。行政院農業委員會動植物防疫檢疫局。台北。62頁。 張清安。2007。蘭花種苗病毒檢測與相關產業發展趨勢。農業生技產業季刊第九期。42-48頁。 曾素玲、童伯開、蔡竹固。1992。石斛蘭葉斑病之發生與藥劑防治。植保會刊。 34:8-16頁。 楊秀珠。1994。觀賞植物灰黴病之發生及防治。臺灣花卉病蟲害研討會專刊。中華植保學會編印。167-176 頁。 鄭尤琇。2007。新蝴蝶蘭病毒之鑑定及其特性之研究暨以可產生短片段干擾RNA之人工合成核苷酸片段發展新式可廣泛性抗多種病毒之轉基因抗病策略.國立中興大學植物病理研究所博士論文。124 頁。 謝廷芳。1995。花卉病害 (1) 蘭花病害。臺灣農家要覽。農作篇 (三) 豐年社。臺北。243頁。 謝廷芳、張義璋、杜金池。2000。由Bipolaris zizaniae引起的文心蘭花瓣斑點病 .植物病理學會刊9卷2期。75-78頁。 Anandalakshmi, R., Pruss, G. J., Ge, X., Marathe, R. , Mallory, A. C., Smith, T. H., and Vance V. B. 1998. A viral suppressor of gene silencing in plants. Proc. Natl. Acad. Sci. U.S.A. 95: 13079-13084. Baker, C. A., Davsion, D., and Jones, L. 2007. Impatiens necrotic spot virus and Tomato spotted wilt virus Diagnosed in Phalaenopsis Orchids from Two Florida Nurseries. Plant Dis. 91:1515. Blanco-Urgoiti, B., Sanchez, F., Perez, de san Roman, C., Dopazo, J., and Ponz, F. 1998. Potato virus Y group C isolate are homogeneous pathotype but two different genetic strains. J. Gen. Virol. 79: 2037-2042. Brigneti1, G., Voinnet1, O., 3, Li, W.-X., Ji, L.-H., Ding, S.-W., and Baulcombe1, D. C. 1998. Viral pathogenicity determinants are suppressors of transgene silencing in Nicotiana benthamiana. EMBO J. 17: 6739-6746. Brodeur, B. R., and Tsang, P. S. 1986. High yield monoclonal antibody production in ascites. J. Immunol. Meth. 86: 239-241. Carrington, J. C., Freed, D. D., Sanders, T. C. 1989. Autocatalytic processing of potyvirus helper component proteinase in Escherichia coli and in vitro. J. Gen. Virol. 63:4459-4463. Chang, M.-U., Doi, Y., Yora, K., and Arai, K. 1976. Morphology and intracellular appearance of Orchid fleck virus. Ann. Phytopathol. Soc. Japan 42:156-157. Chang, M.-U., Chun, H.-H., Baek, D.-H., and Chung, J.-D. 1991. Studies on the viruses in orchids in Korea. 2. Dendrobium mosaic virus, Odontoglossum ringspot virus, Orchid fleck virus, and unidentified potyvirus. Korean J. Plant Pathol. 7:118-129. Chen, C.-C., and Ko, W.-F., Lin, C.-Y., Jan, F.-J., and Hsu, H. T. 2003. First Report of Carnation mottle virus in Calla lily (Zantedeschia spp.) Plant Dis. 87:1539. Clark, M. F., and Adams, A. N. 1977. Characteristics of the microplate method of enzyme-linked immunosorbent assay for the detection of plant viruses. J. Gen. Virol. 34:475-483. Cohen, Y., Gisel, A., and Zambrysky, P. C. 2000. Cell-to-cell and systemic movement of recombinant green fluorescent protein-tagged Turnip crinkle virus. Virology 273:258–266. Cronin, S., Verchot, J., Haldeman-Cahill, R., Schaad, M. C., and Carrington, J. C. 1998. Long-distance movement factor: a transport function of the potyvirus helper component proteinase. Plant Cell 7: 549-559. Dallot, S., Quiot-Douine, L., Saenz, P., Cervera, M. T., Garcia, J.-A., and Quiot, J.-B. 2001. Identification of Plum pox virus Determinants Implicated in Specific Interactions with Different Prunus spp. Phytopathology 91: 159-164. Doi, Y., Chang, M. U., and Yora, K. 1977. Orchid fleck virus. CMI/ABB Descriptions of Plant Viruses No. 183. Doi, Y., and Yora, K. 1978. Cymbidium mild mosaic virus, isolation of cymbidium mild mosaic virus. Korean J. Plant Prot. 17:131-138. Dougherty, W. G., Carrington, J. C., Cary, S. M., and Parks, T. D. 1998. Biochemical and mutational analysis of a plant virus polyprotein cleavage site. EMBO J. 7: 1281–1287. Farreyrol, K., Pearson, M. N., Grisoni, M., Cohen, D., and Beck, D. 2006. Vanilla mosaic virus isolates from French Polynesia and the Cook Islands are Dasheen mosaic virus strains that exclusively infect Vanilla. Arch. Virol. 151, 905–919. Farreyrol, K., Pearson, M. N., Grisoni, M., and Quillec, L. L. F., 2001. Severe stunting of Vanilla tahitensis in French Polynesia caused by Cucumber mosaic virus (CMV), and the detection of the virus in V. fragrans in Reunion Island. Plant Pathol. 50:414. Fauquet, C. M., Mayo, M. A., Maniloff, J., Desselberger, U., and Ball, L. A. 2005. Virus Taxonomy: The Eight Report of the International Committee on Taxonomy of Viruses. Elsvier/Academic Press, U.S.A. 1162 pp. Francki, R. I. B. 1970. Cymbidium mosaic virus. CMI/AAB Descriptions of Plant Viruses No. 27. Fry, C. R., Zimmerman, M. T., and Scott, S. W. 2004. Occurrence of Colombian datura virus in the Terrestrial Orchid, Spiranthes cernua. J. Phytopathol. 152:200-203. Gara, I. W., Kondo, H., Maeda, T., Inouye, N., and Tamada, T. 1998. Calanthe mild mosaic virus, a new potyvirus causing a mild mosaic disease of Calanthe orchid in Japan. J. Phytopathol. 146:357-363. Gibbs, A., Mackenzie, A., Blanchfield, A., Cross, P., Wilson, C., Kitajima, E., Nightingale, M., and Clements, M. 2000. Virus of orchids in Australia: their identification, biology and control. Aust. Orchid Rev. 65:10-21. Goff, L. M., and Corbett, M. K. 1977. Association of Tomato ringspot virus with chlorotic leaf streak of Cymbidium orchids. Phytopathology 67:1096-1100. Gonsalves, D., and Ishii, M. 1980. Purification and serology of Papaya ringspot virus. Phytopathology 70:1027-1032. Gooderham, K. 1984. Transfer techniques in protein blotting. Pages 165-178 in: Molecular Biology. Vol. 1 Proteins. J. M. Walk (ed.), Humana Press, Clifton, NJ. Guaragna, M. A., Odum, O., and Jordan, R. 2006. Detection and characterization of two previously undescribed potyvirus in the terrestrial orchid Spiranthes cernua. Acta Hortic. 722:209-218. Hacker, D. L., Petty, I. T., Wei, N., and Morris, T. J., 1992. Turnip crinkle virus genes required for RNA replication and virus movement. Virology 186:1–8. Hajimord, M. R., Ding, X. S., Flasinski, S., Mahajan, S., Graff, E., Haldeman-Carhii, R., Carrington, J. C., and Cassidy, B. G. 1996. NIa and NIb of Peanut Stripe Potyvirus Are Present in the Nucleus of Infected Cells, but Do Not Form Inclusions. Virology. 244: 368-379. Hollings, M., Stone, O. M., and Barton, R. J. 1977. Pathology, soil transmission and characterization of Cymbidium ringspot, a virus from Cymbidium orchids and white clover (Trifolium repens). Ann. Appl. Biol. 85:233-248. Hoogenraada, N., Helmana, T., and Hoogenraad, J. 1983. The effect of pre-injection of mice with pristane on ascites tumour formation and monoclonal antibody production .J. Immunol. Meth. 61: 310-317. Huang, C.-H., and Chang, Y.-C. 2006. Basella rugose mosaic virus, a new potyvirus infecting Basella rubra. Plant Pathol. 55:819. Hu, J. S., Wang, M., Ferreira, S., and Ogata, D. 1992. Tomato spotted wilt virus on Oncidium orchids in Hawaii. Plant Dis. 76:426. Hu, J. S., Ferreria, S., Wang, M., Borth, W. B., Mink, G., and Jordan, R. L. 1995. Purification, host range, serology and partial sequencing of Dendrobium mosaic potyvirus, a new member of the Bean common mosaic virus subgroup. Phytopathology 85:542-546. Inouye, N. 1969. Cucumber mosaic virus isolated from Dendrobium. Agric. Res. 53:49-60. Inouye, N. 1973. A new virus isolated from Dendrobium. Ann. Phytopathol. Soc. Japan 39:367-368. Inouye, N., and Gara, I. W. 1996. Detection and identification of viruses of orchids in Indonesia. Bull. Res. Inst. Bioresourc. Okayama University 4:109-118. Inouye, N., Maeda, T., and Mistsuhata, K. 1988. A strain of Clover yellow vein virus isolated from Calanthe sp. Acta Hortic. 234:61-68. Inouye, N., Maeda, T., Mistusuhata, K., and Gara, I. W. 1998. Habenaria mosaic virus, a new member of potyvirus from Habenaria radiata orchid. Bull. Res. Inst. Bioresourc. Okayama University 5:155-168. Jensen, D. D., and Gold, H. A. 1951. A virus ring spot of Odontoglossum orchid: symptoms, transmission and electron microscopy. Phytopathology 41:648-653. Jordan, R., Guaragna, M. A., Kinard, G., and Lynn, S. 2002. Detection and first report of Dasheen mosaic virus and a second potyvirus infecting the terrestrial orchid Spiranthes cernua. Acta Hortic. 568:253-260. Kasschau, K. D., and Carrington, J.C. 1998. A counterdefensive strategy of plant ciruses: suppression of posttranscriptional gene silencing. Cell 95: 461-470. Kasiwazaki, S., Minobe, Y., Omura, T., Hibino, H. 1990. Nucleotide sequence of barley yellow mosaic virus RNA1: a close evolutionary relationship with potyviruses. J. Gen. Virol. 71: 2781-2790. Kasiwazaki, S., Minobe, Y., Hibino, H. 1991. Nucleotide sequence of barley yellow mosaic virus RNA2. J. Gen. Virol. 72: 995-999. Keith, L. M., Sewake, K. T., and Zee, F. T. 2005. Isolation and characterization of Burkholderia gladioli from orchids in Hawaii. Plant Dis. 89:1273-1278. Kitajima, E. W., Blumenschein, A., and Costa, A. S. 1974. Rodlike particles associated with ringspot symptoms in several orchid species in Brazil. J. Phytopathol. 81:280-286. Klein, R. R., Rodriguez-Cerezo, E., Hunt, A. G., and Shaw, J. G.. 1994. Mutational analysis of the Tobacco vein mottling virus genome. Virology. 204: 759-769. Koh, D. C.-Y., Liu, D. X., and Wong, S.-M. 2002. A six-nucleotide segment within the 3’ untranslated region of Hibiscus chlorotic ringspot virus plays an essential role in translational enhancement. J. Virol. 76: 1144-1153. Kohler, G.., and Milstein, C. 1975. Continuous cultures of fused cells secreting antibody of predefine specificity. Nature 256:495-497. Ko, N. J. 1988. Cytological identification of Cucumber mosaic virus infecting Phalaenopsis. Proc. Natl. Acad. Sci. Counc. Repub. China B 12:46-51. Lain, S., Riechmann, J. L., and Garcia, J. A. 1990. RNA helicase: a novel activity associated with a protein encoded by a positive strand RNA virus. Nucleic Acid Res. 18: 7003-7006. Lain, N. S., Martin, M. T., Riechmann, J. L., and Garcia, J. A. 1991. Novel catalytic activity associated with positive-strand RNA virus infection: nucleic acid-stimulated ATPase activity of the plum pox potyvirus helicaselike protein. J. Virol. Methods 65: 1-6. Lawson, R. H., and Hsu, H. T. 1995. Orchid. P 409-420 in: Virus and virus-like diseases of bulb and flower crops. G. Loebenstein, R. H. Lawson, and A. A. Brunt (eds), John Wiley & Sons, West Sussex, United Kingdom. Lee, S.-C., Chang, H., and Chang, Y.-C. 2008. First record of Bean yellow mosaic virus infecting a member of the family Orchiaceae, Phaius tankervilliae. Plant Pathol. Bull. 17: 89. Lee, S.-C., and Chang, Y.-C. 2006. Multiplex RT-PCR detection of two orchid viruses with an internal control of plant nad5 mRNA. Plant Pathol. Bull. 15:187-196. Lesemann, D., and Doraiswamy, S. 1975. Bullet-shaped virus-like particles in chlorotic and necrotic leaf lesions of orchids. Phytopathol. Z. 83:27-39. Lesemann, D. E. 1977. Long, filamentous virus-like particles associated with vein necrosis of Dendrobium phalaenopsis. Phytopathol. Z. 89:330-339. Lesemann, D. E., and Begtrup, J. 1971. Electron microscopic demonstration of a bacilliform virus in Phalaenopsis. Phytopathol. Z. 71:257-269. Lesemann, D. E., and Koenig, R. 1985. Identification of Bean yellow mosaic virus in Masdevallia. Acta Hortic. 164: 347-354. Lesemann, D. E., and Vetten, H. J. 1985. The occurrence of Tobacco rattle and Turnip mosaic viruses in Orchis sp., and of an unidentified potyvirus in Cypripedium calceolus. Acta Hortic. 164:45-54. Liu, X.-H., Zheng, H.-W., Liu X., Liu, X.-J., Tan, Z.-B., and Rong, T.-Z. 2005. Isolation of the capsid protein gene of maize dwarf mosaic virus and its transformation in maize. Sheng Wu Gong Cheng Xue Bao 21: 144-148. Li, W.-Z., Qu, F., and Morris, T. J. 1998. Cell-to-cell movement of Turnip crinkle virus is controlled by two small open reading frames that function in trans. Virology 244:405–416. Mackenzie, A. M., Nolan, M., Wei, K.-J., Clements, M. A., Gowanlock, D., Wallace, B. J., and Gibber, A. J. 1998. Ceratobium mosaic potyvirus: another virus from orchids. Arch. Virol. 143:903-914. Merits, A., Guo, D., and Saarma, M. 1998.VPg, coat protein and five non-structural proteins of potato A potyvirus bind RNA in a sequence-unspecific manner. J. Gen. Virol. 79: 3123-3127. Pearson, M. N., and Pone, S. P. 1988. Viruses of Vanilla in Kingdom of Tonga. Austral. Plant Pathol. 17:59-60. Peters, K. R. 1974. Charakterisierung schwer nachweisbarer phytopathogener Viren mit physikalischen und elektronenmikroskopischen Methoden. Diss. Hamburg, German, 93 pp. Qu, F., Ren, T., and Morris, T. J. 2003. The coat protein of Turnip crinkle virus suppresses posttranscriptional gene silencing at an early initiation step. J. Virol. 77:511–522. Rajamaki, M.-L., and Valkonen, J. P. T. 1999. The 6K2 Protein and the VPg of Potato Virus A Are Determinants of Systemic Infection in Nicandra physaloides. Mol. Plant-MicrobeInteract. 12: 1074-1081. Rajamaki, M.-L., and Valkonen, J. P. T. 2002. Viral Genome-Linked Protein (VPg) Controls Accumulation and Phloem-Loading of a Potyvirus in Inoculated Potato Leaves. J. Gen. Virol. 73: 1-16. Rojas, M. R., Zerbini, F. M., Allison, R. F., Gilbertson, R. L., and Lucas, W. J. 1997. Capsid protein and helper component-proteinase function as potyvirus cell-to-cell movement proteins. Virology 237: 283-295. Ruiz, V., Boskovic, J., Alfonso, C., Rivas, G., Llorca, O., Lopez, D., and Lopez, J. J. 2005. Structural analysis of Tobacco Etch Potyvirus HC-Pro oligomers involved in aphid transmission. J. Virol. 79:3758-3765. Saenz, P., Cervera, M. T., Dallot, S., Quiot, L., Quiot, J.-B., Riechmann, J. L., and Garcia, J. A. 2000. Identification of a pathogenicity determinant of Plum pox virus in the sequence encoding the C-terminal region of protein P3+6K1. J. Gen. Virol. 81: 557-566. Schaad, M. C., Jensen1, P. E., and Carrington, J. C. 1997. Formation of plant RNA virus replication complexes on membranes: role of an endoplasmic reticulum-targeted viral protein. EMBO J. 16: 4049-4059. Shukla, D.D., and Ward, C. W. 1989. Structure of potyvirus coat proteins and its application in the taxonomy of the potyvirus group. Adv. Virus Res. 36: 273-314. Silvio, U. I., Haenni, A. L., and Bernardi, F. 2001. Potyvirus protease: awealth of functions. Virus Res. 74: 157-175. Skelton, A., Daly, M., Nixon, T., Harju, V., and Mumford, R. A. 2007. First record of Bean yellow mosaic virus infecting a member of the orchid genus Dactylorhiza. Plant Pathol. 56:344. Styaa, M., Wahla, R., and Beierwaltes, W. H. 1984. Dot-based ELISA and RIA: Two rapid assays that screen hybridoma supernatants against whole live cells. J. Immunol. Meth. 73: 75-81. Stocker, J. W., and Heusser, C. H. 1979. Methods for binding cells to plastic: Application to a solid-phase radioimmunoassay for cell-surface antigens. J. Immunol. Meth. 26: 87-95. Tanaka, S., Nishii, H., Ito, S., and Kameya, M. 1997. Detection of Cymbidium mosaic potexvirus and Odontoglossum ringspot tobamovirus from Thai orchids by rapid immunofilter Paper Assay. Plant Dis. 81:167-170. Thomas, C. L., Leh, V., Lederer, C., and Maule, A. J. 2003. Turnip crinkle virus coat protein mediates suppression of RNA silencing in Nicotiana benthamiana. Virology 306:33–41. Tsuda, S., Kameya, M., Hanada, K., Kouda, Y., Nikada, M., and Tomaru, K. 1992. A novel detection and identification technique for plant viruses: Rapid immunofilter paper assay (RIPA). Plant Dis. 76:466-469. Uchanska-Ziegler B., Werneta, P., and Ziegler, A. 1980. Rapid preparation of multiple cell samples for immunofluorescence analysis using microtiter plates. J. Immunol. Meth. 39: 85-93. Verchot, J., Koonin, E.V., and Carrigyon, J. C. 1991. The 35-kDa protein from the N-terminus of the potyirus polyprotein functions as a third virus encoded proteinase. Virology 185: 527-535. Waltermann, A., and Maiss, E. 2006. Detection of 6K1 as a mature protein of 6 kDa in plum pox virus-infected Nicotiana benthamiana. J.Virol.87: 2381-2386. Wang, Y.-Y., Beck, D., Gardner, R. C., and Pearson, M. N. 1993. Nucleotide sequence, serology and symptomatology suggest that Vanilla necrosis potyvirus is a strain of Watermelon mosaic virus II. Arch. Virol. 129: 93-103. Webster, C. G., Wylie, S. J., and Jones, M. G. K. 2004. Diagnosis of plant viral pathogens. Curr. Sci. 86:1604-1607. Wey, G.-C. 1988. Occurrence and investigation of important disease of Phalaenopsis in Taiwan. Rep. Taiwan Sugar Res. Inst. 122:31-41. White, K. A., J. M. Skuzeski, W.-Z. Li, and T. J. Morris. 1995. Immunodetection, expression strategy and complementation of tTurnip crinkle virus p28 and p88 replication components. Virology 211:525–534. Wisler, G. C., Zettler, F. W., and Mu, L. 1987. Virus infections of Vanilla and other orchids in French Polynesia. Plant Dis. 71:1125-1129. Yamamoto, T., and Ishii, M. 1981. Mosaic disease of Calanthe discolor Lind. Proc. Assoc. Plant Port. Shikoku 16:75-79. Yeh, S.-D., and Gonsalves, D. 1984a. Purification and immunological analysis of cylindrical-inclusion protein induced by Ppaya ringspot virus and Wtermelon mosaic virus 1. Phytopathology 74:1273-1278. Yeh, S. D., Gonsalves, D., and Provvidenti, R. 1984b. Comparative studies on host range and serology of Papaya ringspot virus and Watermelon mosaic virus 1. Phytopathology 74: 1081. Yora, K., Saito, Y., Doi, Y., Inouye, T., and Tomaru, K. 1983. Handbook of Plant Viruses. Asakura Shoten, Tokyo, Japan. 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. 74:621-626. Zheng, Y.-X., Chen, C.-C., Chen, Y.-K., and Jan, F.-J. 2008a. Identification and characterization of a potyvirus causing chlorotic spots on Phalaenopsis orchids. Eur. J. Plant Pathol.121:87-95. Zheng, Y.-X., Chen, C.-C., Yang, C.-J., Yeh, S.-D., and Jan, F.-J. 2008b. Identification and characterization of a tospovirus causing chlorotic ringspots on Phalaenopsis orchids. Eur. J. Plant Pathol.120:199-209.
摘要: 摘要 2003年於彰化蘭花栽培園區中於蝴蝶蘭上從葉部出現黃化斑點(chlorotic spot) 之病徵的植株發以及在葉片上產生黃化輪紋 (chlorotic ring) 病徵的植株分別發現新的病毒,分別屬於 Potyvirus 屬的蝴蝶蘭黃化斑點病毒 (Phalaenopsis chlorotic spot virus, PhCSV) 以及屬於 Carmovirus 屬的康乃馨斑駁病蝴蝶蘭分離株 (Carnation mottle virus, CarMV)。雖然近幾年對於蘭花病毒的相關研究不少,但真正進行田間檢測的病毒卻相當的少,主要是因為缺乏具專一性及靈敏度檢測的工具來進行病毒的檢測。因此,為了落實無病毒苗的觀念,因此製備對此兩種病毒具專一性的血清,希望本實驗所生產的血清能夠在業者及檢測單位進行病毒的檢測時貢獻一份力量。利用PhCSV 的病毒顆粒為抗原,對實驗小鼠進行腹腔免疫注射,以病毒顆粒刺激實驗小鼠之脾臟進而活化對此病毒具有專一性之免疫細胞,取實驗小鼠之脾臟細胞 (spleen cell) 和腫瘤細胞 (myeloma cell) 以 PEG1450 進行細胞融合產生融合瘤細胞 (hybridoma cell),利用感染 PhCSV 的圓葉煙草 (Nicotiana benthaniana) 葉片粗汁液進行單元抗體之篩選,共獲得3個細胞株分別為 PhCSV-28F5-0-D7、PhCSV-28F5-0-E8 以及 PhCSV -28F5-1-E4,此 3 個細胞株皆可在進行間接酵素連結免疫吸附法測試時能夠明顯區分感染病毒之葉片以及健康之葉片。將對病毒具專一性之細胞進行實驗小鼠之腹腔注射,使小鼠產生腹水,經過測試得知所獲得的血清力價為 106 倍,而血清之專一性在利用西方轉漬法分析後能夠在預期大小觀察到專一性的條帶;而 CarMV-Ph 的血清則是利用純化的病毒顆粒進行紐西蘭白兔的肌肉注射,經過四次注射後進行採血獲得血清,在力價測試中血清稀釋至 105 倍都能夠進行結果的判讀,利用稀釋 104 倍的血清進行靈敏度的測試,在感染病毒的葉片稀釋 104 都能夠和健康的植株葉片有兩倍以上的差距。在經過血清學測試後上述的兩個血清擁有應用於病毒檢測的能力,希望此血清的出現能夠增加台灣的蝴蝶蘭產業在國際市場上的競爭力,並降低受到 PhCSV 和 CarMV-Ph 感染的蘭苗在栽培園間出現的機會。
Abstract In 2003, an isolate of the potyvirus Phalaenopsis chlorotic spot virus (PhCSV) and a phalaenopsis isolate of the carmovirus Carnation mottle virus (CarMV-Ph) were isolated from Phalaenopsis orchid plants. On the infected plants, these virus isolates caused respectively leaf chlorotic spot and leaf chlorotic ring symptoms. Despite reported presence of these viruses in Phalaenopsis orchid for several years, these accurate detection from the infected plants was ineffective, because of lack of specific and sensitive detection tools. Hence, to develop an officient a detection tool, antibodies against PhCSV and CarMV-Ph particles were generated. Purified PhCSV was injected into BALB/c mice for the production of hybridomas from mice spleen cells with FOX-NY myeloma cells by PEG-1450, and the resultant positive hybridoma clones were screened by indirect ELISA. Three cell lines were selected and determined to be IGg1/κ isotype. The titer of the monoclonal antibody (106) and poltclonal antibody (105) was determined by indirect ELISA, and the specificity of the antibody to the antigen was determined by western blotting analysis. Polyclonal antibody against the CarMV-Ph was produced in a New Zealand white rabbit. After 4 rounds of immunization by purified CarMV-Ph virus particles, the rabbit was bled weekly and antiserum was collected. The generated PhCSV monoclonal antibody and CarMV-Ph polyclonal antibody have effective application potentials in the immunological detection and can be employed to detect these economically important viruses in Phalaenopsis orchid nursery to produce virus-free Phlalenopsis orchid seedlings.
URI: http://hdl.handle.net/11455/31194
其他識別: U0005-1102200914333500
文章連結: http://www.airitilibrary.com/Publication/alDetailedMesh1?DocID=U0005-1102200914333500
Appears in Collections:植物病理學系

文件中的檔案:

取得全文請前往華藝線上圖書館



Items in DSpace are protected by copyright, with all rights reserved, unless otherwise indicated.