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標題: 利用矮南瓜黃化嵌紋病毒載體在矮南瓜上表現塵蟎第二群及第五群過敏原蛋白
Expression of house dust mite group 2 and group 5 allergens by Zucchini yellow mosaic virus vector in squash
作者: 鄭浩文
Jheng, Hao-Wen
關鍵字: monoclonal antibody;單株抗體;viral vector;house dust mite;免疫療法;過敏;塵蟎;病毒載體
出版社: 植物病理學系
引起氣喘的過敏原的種類甚多,常見有包括花粉、動物毛屑、及塵蟎等。隨著生活環境的逐漸惡化,全球罹患過敏性氣喘的人口也日漸增加,在台灣又以塵蟎為最主要的過敏原。醫學上對於塵蟎過敏的治療常使用減敏療法 (allergen immunotherapy),以長時間皮下注射或口服過敏原的方式,來鈍化人體內與過敏相關的免疫反應產生,對過敏原產生耐受性 (tolerance),藉以降低過敏發生的頻率及嚴重度。本實驗室業已完成利用矮南瓜黃化嵌紋病毒 (Zucchini yellow mosaic virus, ZYMV)載體於受感染的矮南瓜植株內表現塵蟎第五群過敏原 (Der p 5),並在老鼠模式的動物試驗中以餵食粗萃材料的方式,獲得大幅降低過敏反應指標的結果。為了提高療效,本研究以ZYMV同時表現塵蟎第二及第五群過敏原的重組蛋白 (Der p 2&5),作為日後提供動物實驗之用。此外,亦利用細菌蛋白表現系統所生產大量的Der p 5蛋白,經純化後免疫小鼠,以感染ZYMV-Der p 5的植物組織作為篩選融合瘤細胞株(hybridoma cells)之材料,以製備高專一性之單株抗體。經過篩選後共得到十個對Der p 5具有正反應的細胞株,其中有四個用以產生腹水,腹水的力價可達2 × 10 6 ,並且具有高度的專一性。利用所製備的單株抗體以西方轉漬法 (western blot)定量感染重組病毒的矮南瓜上的Der p 2&5含量,可知每一百克感染植物組織中含有1.83 mg 的Der p 2&5。利用植物病毒載體在植物上表現外源蛋白已是經常被使用的策略,以本研究的矮南瓜黃化嵌紋病毒載體可成功的在矮南瓜上表現重組的塵蟎過敏原 Der p 2&5,並且已製備針對過敏原Der p 5的單株抗體,可作為一專一性的定量工具。

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.
Appears in Collections:植物病理學系

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