The production of monoclonal antibody with broad spectrum reactivity against calla lily-infecting potyviruses using recombinant capsid protein
| Autor: | Wei-Fang Lin, 林為方 |
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| Rok vydání: | 2007 |
| Druh dokumentu: | 學位論文 ; thesis |
| Popis: | 95 Calla lily originated from southern Africa belongs to the genus Zantedeschia, the family Araceae. Recently calla lily has become one of economically important flowering plants and continues to increase in popularity because of its unique shape, spathe coloration and long-term lifespan. During the cultivation of calla lily, viral disease is one of the limiting factors in Taiwan as well as in other countries. Potyviruses are the major viruses infecting calla lily and often cause symptoms of stunt, mosaic, growth decline and flower deformation. The yield and quality of cut flowers are seriously affected. At present, detection methods for potyviruses in calla lily such as reverse transcription-polymerase chain reaction (RT-PCR) and enzyme-linked immunosorbent assay (ELISA) are commonly employed. Interestingly, ELISA is considered by the industry to be more suitable than RT-PCR as routine indexing technique of large amount of samples. To save the materials, labors and time in detection, therefore in this study, we tried to generate a monoclonal antibody which can detect as many potyviruses as possible. The highly conserved region of capsid protein (CP) gene of calla lily-infecting potyviruses reported in Taiwan including Calla lily latent virus (CLLV), Dasheen mosaic virus (DsMV), Turnip mosaic virus (TuMV), Zantedeschia mosaic virus (ZaMV) and Zantedeschia mild mosaic virus (ZaMMV) was selected after amino acid sequences aligned. The conserved region of 121 amino acids in length was PCR amplified by specific primers of each virus. The PCR fragments were ligated to construct two kinds of expression vectors, recombinant proteins were expressed in E. coli expression system and then purified as antigens for immunization. After cell fusion, we used the expressed proteins and potyvirus-infected plant samples as antigens to screen the hybridoma cell lines by indirect-ELISA. One stable cell line secreting potyvirus-specific antibodies named as MAb C12-C4 with best reactivity and was used for ascites production. The mouse ascites from MAb C12-C4 gave well detectable reactions to antigens at dilution up to 105 times. In the spectrum test, this MAb could detect at least ten other potyviruses by indirect-ELISA. From our experimental results, MAb C12-C4 has the potential to be used for further researches and applications. |
| Databáze: | Networked Digital Library of Theses & Dissertations |
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