| Summary: | 碩士 === 國立中興大學 === 植物病理學系 === 93 === There are many allergens in our surroundings such as pollens, hairs of pets, and house dust mites etc. House dust mite (HDM) allergens are the major allergens and cause serious allergenic asthma worldwide. Allergen immunotherapy is a general way to treat the HDM allergenic patients. The purified allergen is administrated by subcutaneous injection or oral delivery weekly to induce tolerance to specific allergen and has to be persisted for two to three years for the reducing of symptoms and frequency of allergenic asthma. In animal test we have demonstrated that the allergic reactions of sensitized mice was reduced by oral feeding with the extract from squash plants infected with Zucchini yellow mosaic virus (ZYMV) vector carrying Der p 5 allergen. In this study, the house dust mite Dermatophagoides peteronyssinus group 2 and group 5 allergens, constructed together as a fusion protein Der p 2&5, were introduced into ZYMV vector, and expressed on zucchini squash infected by the recombinant ZYMV-Der p 2&5 recombinant virus. In order to quantitatively determine the expression of Der p 2&5 protein by ZYMV vector in squash plants, the monoclonal antibodies (MAbs) against the HDM allergen are produced. Using bacterial-expression Der p 5 protein immunized BALB/c mice, the hybridoma cell lines were screened by the extracts from plant infected with ZYMV-Der p 5. Ten hybridoma cell lines of MAbs secreting against Der p 5 protein were obtained, and the ascitic fluids of four cell lines were collected. The titer of the ascitic fluids was determined as to 2 × 106 folds by indirect ELISA. The yields of Der p2&5 expressed by ZYMV vector was estimated as 1.83 mg of 100 gram plant tissues by the MAb 8E12A11. In addition, the MAbs produced against Der p 5 can be used as a quantitative tool for detecting the amount of Der p 2&5 in squash plants for the animal test.
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