| Summary: | 碩士 === 國立高雄師範大學 === 生物科學研究所 === 90 === Cymbidium mosaic virus（CyMV） is a major limiting factor for orchid plants. Infected plants are unhealthy and produce lower quality flowers. Papaya ringspot virus（PRSV）is a significant pathogen in papaya. The cDNAs of VH and VL of CyMV and PRSV antibody genes were obtained by using polymerase chain reaction. ScFv（single-chain variable fragment）of CyMV and PRSV were constructed by gene splicing by overlap extension (gene SOEing). Results of nucleotide sequences analysis revealed 723 and 726 nucleotides encoding for 241 and 242 amino acids, respectively, for scFv-CyMV and scFv-PRSV. In order to express scFv-CyMV and scFv-PRSV antibody, pCANTAB-CyMV and pCANTAB-PRSV were constructed for transformation into E. cloi TG1 and 570 and 1242 of colonies, respectively, from CyMV and PRSV phage display system were obtained. Twelve scFv-CyMV and five scFv-PRSV colonies were selected with positive reactions in indirect ELISA. 744 and 747 nucleotides fragments of scFv-CyMV and scFv-PRSV, respectively, were cloned from those positively selected colonies by PCR with specific primers. However, two of 1500 nucleotide fragments were obtained for scFv-CyMV and scFv-PRSV, respectively, by using SfiΙand NotΙ enzyme digestion. C6, C10 and C11 of scFv-CyMV were selected for transformation into E. coli HB2151 for monitoring the locations of scFv-CyMV antibody in E. coli cell including supernatant, periplasm and cytosol. The result showed that scFv-CyMV antibody was most aboundant in supernatant. ScFv-CyMV and scFv-PRSV nucleotide fragments were also cloned by PCR with specific primers for inserting into pBin-19 use for transformation of plants and pIVEX-2.4a use for large scale protein expression.
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