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研究生:康嘉真
研究生(外文):Chia-Chen Kang
論文名稱:人類A型流感病毒表面抗原基因轉殖至萵苣之研究
論文名稱(外文):Studies on the surface antigenic gene of Human influenza A virus transferred into lettuce(Lactuca sativa L.)
指導教授:尤進欽
指導教授(外文):Jinn-Chin Yiu
口試委員:方怡丹劉程煒郭純德尤進欽
口試委員(外文): Jinn-Chin Yiu
口試日期:2011-06-17
學位類別:碩士
校院名稱:國立宜蘭大學
系所名稱:園藝學系碩士班
學門:農業科學學門
學類:園藝學類
論文種類:學術論文
論文出版年:2011
畢業學年度:99
語文別:中文
論文頁數:93
中文關鍵詞:流感病毒
外文關鍵詞:influenza virus
相關次數:
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A型流行性感冒病毒寄主廣泛,為一種高度傳染性的疾病。目前流感的預防方式為接種疫苗,市面疫苗皆採用紅血球凝集素(Hemagglutinin;HA)作為抗原製作疫苗。由於A型流感病毒經常變異,且HA具株特異性,疫苗成分年年更迭,產量經常供不應求。本研究目的係利用A型H1N1型流感病毒(Influenza virus)表面抗原蛋白神經胺酸酶(Neuraminidase;NA),於不同亞型之活性部位皆相同的特性。將基因構築於pBI121與pCAMBIA 2301兩種植物轉殖載體,再以農桿菌媒介法轉殖至大球品種之皺葉萵苣(Grand Rapids TBR)中,並探討轉殖萵苣的轉殖效率及目標蛋白質的表現情形。
本實驗以皺葉萵苣的子葉做為培植體。誘導的芽鞘以kanamycin進行篩選,植株再生率約為7.4%~9.1%。經PCR分析,證明再生植株的基因組具有NA基因。RT-PCR分析顯示NA基因可正確轉錄出mRNA。利用西方墨點分析,確認轉殖皺葉萵苣可正確地合成目標蛋白。T0代經篩選分析後發現不同載體之再生植株的轉殖成功率介於2.9%~3.5%之間。檢測2品系的T2植株,約有80.3±0.5%的T2植株呈正反應,顯示目標基因高度穩定遺傳至後代。T2植株之葉片蛋白質進行ELISA分析,NA蛋白之表現量約佔總可溶性蛋白的0.39%~0.43%。本試驗結果建議萵苣作為一個候選者生產NA蛋白以抵抗流感病毒是可行的,未來將進行動物試驗來證實其保護效力。

The host range of influenza A viruses is very widely, that is a highly contagious diseases. At present the way to prevent influenza is by getting a flu vaccination that is using hemagglutinin (Hemagglutinin; HA) as a vaccine antigen production. Because of influenza A viruses have high variability and the surface glycoprotein HA is strain-specific, the components of vaccine changed every year, and vaccine supplies would be in adequate. The objective in the study is using influenza A H1N1 virus (Influenza virus) surface antigen neuraminidase (Neuraminidase, NA) in the characteristic that the active site of different subtypes is the same. The target gene was integrated into the plant expression vector pBI121 and pCAMBIA2301 respectively, and then transferred into lettuce via Agrobacterium tumefaciens-mediated, and to explore transformation efficiency and target protein expression level in transgenic lettuce.
In this experiment, the cotyledon of wrinkled leaf lettuce was used as explants. The induced shoots was selected by kanamycin, the plant regeneration rate was about 7.4%-9.1%.Through PCR analysis, proving that there is NA gene in the regenerated plants genomic DNA.RT-PCR analysis showed that NA gene can be transcribed into mRNA correctly. Using western blot analysis confirmed that target protein can be synthesized in transgenic lettuce properly. The transformation ratio of T0generation lettuce harboring different expression vector ranged from 2.9% to 3.5%usingmolecular analysis. Two transgenic lines of T2generation plants were selected to detect target protein. The result was revealed that about 80.3 ± 0.5% of T2transgenic plants possess positive result, indicating the transgenes were inherited in the offspring stably. Based on the ELISA results, NA protein expression level made up about0.39% to 0.43% of total soluble protein in the leavesofT2generation transgenic plants. This suggests that the feasibility that lettuce as a candidate produced NA protein to against Influenza virus, and will be tested in future animal experiments.

目錄
中文摘要
Abstract
前言
前人研究
一、流行性感冒之緣起與演變
二、流感病毒之種類
三、傳染致病的機制與臨床特徵
四、流感預防方式與治療藥物
五、疫苗種類與供需情形
六、植物生產人類與動物疫苗
七、神經胺酸酶(NA)作為流感疫苗之潛力
材料與方法
一、試驗材料
二、試驗方法
(一) 植物之轉型與再生
(二) 再生植株分子層次分析
結果
一、農桿菌媒介轉殖系統載體之確認
二、轉基因植物再生情形
(1) 萵苣再生流程
(2) 萵苣再生情形綜合分析
(3) 轉殖萵苣植株外觀比較
三、轉殖再生萵苣之篩選分析
(1) PCR分析
(2) RT-PCR分析
(3) 再生植株採種情形
四、轉植株後裔之種植與鑑定
(1) T1 代
(2) T2 代
(3) 轉殖株後裔之種子對Kanamycin的抗性測試
(4) 轉殖植株後裔之採種情形
(5) NA蛋白質含量測定
討論
一、藉由農桿菌媒介法將基因轉殖至植株之再生及採種情形
二、基因轉殖萵苣之分子分析
三、轉殖基因植株子代之種植與鑑定
四、轉殖株後裔之種子對kanamycin的抗性測試
參考文獻
附件一、pBI121-NA與pCAMBIA 2301-NA質體圖
附件二、H1N1表面抗原NA核酸序列
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