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Title: | 轉殖甘藷表現口蹄疫病毒結構蛋白VP4-1 Expression of Foot and Mouth Disease Virus Structural Protein VP4-1 in Transgenic Sweet Potatoes |
Authors: | Hsia-Hwa Yu 余夏華 |
Publication Year : | 2002 |
Degree: | 碩士 |
Abstract: | 本實驗主要目的在以農桿菌建立甘藷之轉殖系統,並在此甘藷中表現口蹄疫病毒結構蛋白VP4-1。利用帶有口蹄疫病毒(O Tai/97株)之VP4-1基因(即P1 fragment)的轉型農桿菌感染台農67號甘藷之葉片後,予以黑暗處理,致受感染的葉片生出毛狀根。待毛狀根生長至適當長度,轉移至LS培養基並在光照下培養,數週後自毛狀根誘導出再生植株。經由Hygromycin初步篩選出具有抗性的植株後,藉由南方轉漬分析確認p1基因及hpt II基因成功地轉殖入再生植株之基因體中,並透過北方轉漬分析偵測到p1及hpt II mRNA的表現。這些結果都顯示本研究中的甘藷轉殖系統是可行的。在西方轉漬分析方面,可能是由於P1與VP1蛋白的表現量非常低,或是在植物細胞中被很快的分解了,以致偵測不易。 為了評估此轉殖甘藷是否具有潛力以發展成為預防口蹄疫之口服疫苗,便進行免疫原性(Immunogenicity)實驗。小鼠每週餵食三次,每次餵食0.5克磨碎的轉殖蕃薯葉子加口服佐劑(cholera toxin)持續兩個月。初步的分析結果顯示經過餵食小鼠的血清中VP1抗體的量並無較未經餵食小鼠高,且樣本數過少,因此需要再進一步的研究才能釐清。 The objectives of this thesis were first to establish an Agrobacterium-mediated transformation system for sweet potato and then express foot and mouth disease virus structural protein VP4-1 (P1 protein) in the transgenic plants. I. batatas (L.) Lam. (sweet potato) cultivar TNG67 was transformed with the agropine strains of A. rhizogenes, 15834 (pCAMBIA 1390-35S-P1) and R1601 (pCAMBIA 1390-35S-P1). Hairy root formation was observed about 2 weeks later. For plant regeneration, the hairy roots (5?8 cm in length) were transferred onto LS medium and grown for (at least) 50 days, after which shoots were generated from the hairy roots. Southern blot analysis confirmed that the introduced DNA had been integrated; Northern blot analysis indicated that the four transgenic plants expressed the p1 and hpt II mRNA. These data indicated that the transformation system was genetically stable. The result of Western blot might suggested the low expression of the P1 protein in the transgenic sweet potatoes, and the protein may be degraded immediately in the plant cell. Immunogenicity test was conducted to estimate the potential of the transgenic sweet potato lines as an oral vaccine against foot and mouth disease. Mice were fed three times a week, for 2 months with approximately 0.5 g of freshly harvested leaves plus 10 μg oral adjuvant Cholera toxin. The rough data in this thesis indicated that mice fed with transgenic sweet potato leaves faired to elicit an antibody response to VP1 protein. Hence the immunogenicity of transgenic sweet potato should be further investigated. |
URI: | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/75310 |
Fulltext Rights: | 未授權 |
Appears in Collections: | 植物科學研究所 |
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