臺灣博碩士論文加值系統

家禽流行性感冒 (禽流感) 病毒為傳染性的呼吸疾病，威脅人類的健康並影響全球的經濟發展。利用基因轉殖植物已可大量生產有經濟價值和安全性的醫藥蛋白質，例如抗體、抗原和荷爾蒙等。本論文將禽流感病毒表面的抗原決定位，編碼為醣蛋白之ha基因，利用農桿菌媒介法轉殖入胡蘿蔔，期望製成植物型的口服疫苗，以預防禽流感。為了提高基因的表現，利用2倍的CaMV 35S啟動子和促進轉譯作用的因子調控；並在基因末端接上內質網保留訊號-HDEL和KDEL，期望引導蛋白滯留在內質網中穩定累積。轉殖處理之胡蘿蔔下胚軸，在含有抗生素的篩選培養基中誘導產生癒合組織，經由體胚發生後產生抗性植株。進一步利用聚合酶連鎖反應偵測到ha基因，確認已嵌入至轉殖胡蘿蔔的基因組中；萃取轉殖胡蘿蔔葉片的總蛋白，以免疫轉漬分析可偵測到65 kDa 之HA外源蛋白。再利用酵素免疫連結分析HA蛋白含量，結果顯示含KDEL蛋白在轉殖胡蘿蔔的含量較含HDEL蛋白高，且最高占總可溶性蛋白之0.074%。顯示首度利用植物表現系統可表達HA蛋白。

Avian influenza virus（AIV）is an infectious respiratory pathogen that threats human health and affects global economies. Transgenic plants have been employed successfully as a low-cost and large-scale production system of safe and biological therapeutically proteins, including antibodies, antigens and hormones. This study focuses on using transgenic carrots (Daucus Carota L. cv. ‘sunlight ginseng’ ) as an antigen-delivery system for subunit vaccines against AIV. The gene encoding the AIV surface glycoprotein (hemagglutinin, HA), a major antigen of AIV agent, was introduced into carrots by Agrobacterium tumefaciens-mediated transformation methods. In order to optimize the expression of this gene in transgenic plants, ha gene was under control of the duplicated cauliflower mosaic virus (CaMV) 35S promoter and tobacco etch virus (TEV) enhancer. Coding sequence of endoplasmic reticulum (ER) retention signals, HDEL and KDEL, were added to the 3’ end of ha gene. The regenerated plants with kanamycin resistance were obtained through somatic embryogenesis from the embryogenic callus formed on the selection medium. The foreign ha gene was synthesized by polymerase chain reaction from transgenic plants. According to the results of immunoblotting analysis, HA protein was expressed and detected by a specific monoclonal antibody in transgenic plants. Enzyme-linked immunoassays revealed that KDEL confers a higher level expression of HA protein than HDEL does. Therefore, the maximal level of HA protein was 0.074% of the soluble protein in leaves of transgenic plant. The results demonstrate the feasibility of producing HA in transgenic plants.

摘要………………………………………………………………………i
Abstract…………………………………………………………………ii
壹、 前言………………………………………………………………………1
貳、 前人研究…………………………………………………………2
一、 植物表現系統……………………………………………………2
二、 以植物表現疫苗之機制與策略…………………………………3
三、 葉綠體基因轉殖………………………………………………4
四、 植物生產疫苗之發展現況 ……………………………………6
五、 家禽流行性感冒病毒…………………………………………7
(一) 家禽流行性感冒病毒之性質……………………………7
(二) 治療家禽流行性感冒病毒之疫苗………………………8
參、 材料與方法……………………………………………………10
一、 植物材料………………………………………………………10
二、 試驗方法………………………………………………………10
(一) 質體DNA之小量製備……………………………………10
(二) DNA片段之回收…………………………………………11
(三) 接合反應與質體DNA之轉型……………………………11
(四) 大腸桿菌待轉型細胞 (competent cell) 之製備……11
(五) 農桿菌電穿孔轉型法……………………………………12
(六) 農桿菌質體DNA之小量製備……………………………12
三、 表現載體之構築………………………………………………13
(一) 質體材料…………………………………………………13
(二) 聚合酶連鎖反應（polymerase chain reaction, PCR) ………………………………………………13
(三) HA基因之構築…………………………………………15
(四) 葉綠體表現載體之構築………………………………15
四、 農桿菌轉殖及植株再生……………………………………16
五、 擬轉殖植株之鑑定…………………………………………17
(一) GUS活性組織化學染色分析…………………………17
(二) 植物基因組DNA之萃取………………………………17
(三) 雜交探針製備…………………………………………17
(四) 南方氏雜交分析（Southern blot analysis）……18
(五) 植物可溶性蛋白之萃取………………………………19
(六) 聚丙烯醯胺膠體電泳（SDS-Polyacrylamide Gel Eletrophoresis, SDS-PAGE)……………………………………19
(七) 免疫轉漬分析（Immunoblotting analysis）………19
(八) 酵素免疫分析法（Enzyme-linked immunoassay, ELISA)……………………………………20
肆、 結果 ……………………………………………………………28
一、 轉殖植株的篩選和再生………………………………………28
二、 轉殖植株分析…………………………………………………28
伍、 討論 ……………………………………………………………44
一、 禽流感的防治策略……………………………………………44
二、 胡蘿蔔的基因轉殖與再生 ……………………………………45
三、 植物生產疫苗之可行性………………………………………46
四、 轉殖基因表現量之影響因子…………………………………47
五、 葉綠體基因轉殖………………………………………………50
參考文獻……………………………………………………………51

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