應用轉殖胡蘿蔔表達禽流感病毒疫苗基因之研究

Studies on Expression of Avian Influenza Virus Vaccine Gene in Transgenic Carrots

杜韋萱(Wei-Xain Tu)

指導教授 ： 黃鵬林

共同指導教授 ： 杜宜殷(Yi-Yin Do)

國立臺灣大學/生物資源暨農學院/園藝學研究所/碩士(2010年)

https://doi.org/10.6342/NTU.2010.10170

摘要

本研究將禽流感病毒 A/ duck/ Yunlin/ 04/ (H5N2) 表面的抗原，編碼為醣蛋白之 ha 基因，轉殖入胡蘿蔔，期望生產植物型口服疫苗，以預防禽流感。以南方氏雜交法分析經農桿菌媒介法得到的胡蘿蔔轉殖株，可偵測到 ha 基因，確認已嵌入至胡蘿蔔核基因組內；另外，以免疫轉漬分析法可偵測到轉殖株中可表達 65 kDa 之 HA 外源蛋白。再利用酵素免疫連結分析 HA 蛋白含量，結果顯示在胡蘿蔔葉片中 HA 蛋白含量較根與癒傷組織高。且在胡蘿蔔根部中，HA 蛋白含量最高佔總可溶性蛋白 0.0601％。另外，為了提高外源蛋白在轉殖株的表達量，本研究構築了葉綠體基因轉殖表達質體，以基因槍法轉殖至胡蘿蔔癒傷組織中，槍擊後3周以螢光顯微鏡鏡檢，可觀察到紅色螢光蛋白，篩選 6 個月後以聚合酶連鎖反應檢測擬轉殖細胞團塊，可偵測到 ha 基因，目前繼續在含有高抗生素濃度之再生培養基進行植株誘導，以得到均質性的轉殖株。

關鍵字

家禽流行性感冒 ； 血球凝集素 ； 疫苗 ； 葉綠體基因轉殖 ； 胡蘿蔔

並列摘要

This study focuses on using transgenic carrots (Daucus carota L. cv. Sunlight Ginseng) as an antigen-delivery system for subunit vaccines against AIV. The foreign ha gene, the AIV surface glycoprotein hemagglutinin, was detected in the genome of Agrobacterium-mediated transgenic carrots by Southern blot analysis. HA protein was expressed and detected by immunoblotting analysis in transgenic carrot. Enzyme-linked immunoassays revealed that HA protein expression in carrot leaves is higher than in roots and calli. The maximal level of HA protein was 0.0601％ of the total soluble protein in roots of transgenic plants. In addition, in order to enhance foreign protein expression in transgenic carrot, chloroplast expression plasmid was constructed, and transformed to carrot calli by paticle bombardment. After 3 weeks of selection by antibiotics, the red fluorescence could be observed from bombarded carrot calli. HA gene could be synthesized using genomic DNA extracted from transgenic calli after 6 months of slection on antibiotics-containing medium as template by polymerase chain reaction. To obtain homoplasmic transplastomic lines from somatic embryos, these calli were transferred into the medium containing high concentration of antibiotics for regeneration.

並列關鍵字

avian influenza ； hemagglutinin ； vaccine ； plastid transformation ； carrot

參考文獻

陳建豪. 2006. 家禽流行性感冒病毒血球凝集素蛋白之表現及其應用. 台灣大學獸醫學研究所碩士論文.

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