| Summary: | 碩士 === 國立中興大學 === 植物病理學系 === 90 === Abstract
The Taiwan isolate of Papaya leaf-distortion mosaic virus (PLDMV), a member of the genus Potyvirus of the family Potyviridae, only infects plants of Carica papaya, which was not the proper propagation host for virus purification. For antiserum production, the complete reading frame of the coat protein (CP) gene of Papaya leaf-distortion mosaic virus (PLDMV) was amplified from the total RNA extracted from virus-infected leaves of papaya plants by reverse transcription-polymerase chain reaction (RT-PCR) with the CP-gene specific primers. The amplified DNA fragment was cloned, sequenced, and transferred to the bacteial expression vector pET-32a (+) vector (Novagen) in this investigation. The PLDMV CP was expressed as a 51.8-kDa fusion protein containing the 109 aa thioredoxin protein and a N-terminal histidine tag in the E. coli cells. This fusion protein reacted with PLDMV-specific antiserum As48, previously produced against the bacterial expressed PLDMV CP protein that was fused with a cellulose binding domain, and the histidine tag-specific monoclonal antibody in western blotting. The target protein of PLDMV CP was collected and purified under denaturing conditions, in buffers containing 6 M urea, by Ni2+-NTA affinity chromatography. The denatured target protein was used for antiserum production after dialysis. Most of the fusion protein was not refolded during the process of dialysis. The antiserum As59 produced by immunizing a New Zealand white rabbit with the purified fusion protein reacted with the boiled extracts of PLDMV-infected papaya plants in ELISA test with high sensitivity. It also was able used to detect the CP of PLDMV in the infected leaves by Western blotting. In addition, the reading frame of PLDMV CP was in frame inserted into the Zucchini yellow mosaic virus (ZYMV) vector at the N-terminal region of the HC-Pro protein, with additional six histidine residues to facilitate the purification and a NIa protease cleavage site to process the free form proteins. The recombinant virus ZYMVPCP4-6 carrying the CP of PLDMV induced milder symptoms of yellow mosaic with vein-banding on zucchini squash plants than those induced by the wild type of ZYMV. The recombinant ZYMVPCP4-6 also induced mottling and mild vein-banding symptoms on plants of horn melon and local lesions on plants of Chenopodium quinoa. The expression efficiency of PLDMV CP in the ZYMVPCP4-6 infected squash plants is discussed.
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