臺灣博碩士論文加值系統

論文摘要

近年來，隨著芝麻產品的普及，世界各國尤其是歐美地區陸續都有芝麻過敏的報導。芝麻主要的過敏蛋白是種子儲存蛋白中含量最豐富的11S球蛋白與2S清蛋白，分別佔芝麻種子總蛋白質的60-70%與15-25%。由於種子的儲存蛋白是由基因家族所演譯的同功蛋白質所組成，藉由搜尋芝麻種子3,328的表達序列標記片段，分別發現有四種不同的11S球蛋白和三種不同的2S清蛋白的表達序列標記片段，為了提供這二個過敏蛋白家族的完整資料，本研究針對未知的芝麻11S球蛋白和2S清蛋白的同功蛋白：11S-3球蛋白、11S-4球蛋白和2S-3清蛋白進行基因之選殖及定序分析，希望能提供潛在的過敏蛋白的知識，做為未來鑑定芝麻過敏原的參考及序列的比對。

Abstract

Cases of allergy to sesame have been progressively reported in the past decade possibly due to the spread of sesame seed products into new areas of the world and consequent exposure of new populations. The allergic factors in sesame are frequently ascribed to the two abundant seed storage proteins, 11S globulin and 2S albumin, representing 60-70% and 15-25% of total sesame proteins, respectively. To facilitate the identification and detailed investigation of these two classes of sesame allergic factors, gene families encoding four 11S globulin and three 2S albumin isoforms were identified in a database search of 3,328 EST sequences from maturing sesame seeds. Full-length cDNA sequences of these two gene families were completed by PCR using a maturing sesame cDNA library as the template. The results provide the molecular information on sesame storage proteins potentially related to allergy.

目錄
頁 次
目錄………………………...……………………………………………………….I
圖目錄………………………...…………………………………………………...III
表目錄………………………...………………………………………………….. IV
縮寫表………………………………………………………….…………………...V
第一章 緒論……………………………………………………………………….1
1.1芝麻之介紹…………………………………………………………………….1
1.1.1台灣栽培品種－黑芝麻「台南一號」……………………………………2
1.1.2芝麻種子基礎研究………………………………………………………...3
1.2芝麻種子的過敏蛋白………………………………………………………….4
1.2.1芝麻11S球蛋白…………………………….………….. …………………5
1.2.2芝麻2S清蛋白…….………………...……….……..…. . ………………...6
1.3芝麻過敏的症狀、診斷與治療……………………………………………….7
1.3.1芝麻過敏症狀……………………………………………………………...7
1.3.2芝麻過敏的診斷…………………………………………………………...7
1.3.3芝麻過敏的治療原則……………………………………………………...8
1.4 實驗緣由與目的……………………………………………………………...9
第二章 材料與方法………………………………………………...……………..23
2.1實驗材料………..……………………………………………...……………..23
2.1.1 植物材料…………………………………………………..……………..23
2.1.2 質（載）體與菌株………………………………………..……………..23
2.2藥品………………………………………….…...…………………….……..23
2.3儀器與設備…………………………………………….……………………..24
2.4實驗方法……..……………………………………………. ……………….24
2.4.1 引子的設計……………………………………………. …………….….24
2.4.2 聚合酶連鎖反應…………………………………………………………26
2.4.3 DNA電泳………………………………………………………………27
2.4.4 DNA片段之回收………………………………………………………28
2.4.5 質體DNA之純化……………………………………………………….28
2.4.6 內切限制酶之剪切………………………………………………………29
2.4.7 DNA片段之接合………………………………………………………29
2.4.8 質體的轉形………………………………………………………………29
2.4.9 重組質體之篩選…………………………………………………………30
2.4.10 核酸定序………………………………………………………………..30
第三章 結果與討論……………………………………………………. ………...31
3.1 芝麻主要過敏蛋白，11S球蛋白與2S清蛋白基因家族的搜尋……….…31
3.2 芝麻11S-3球蛋白、11S-4球蛋白與2S-3清蛋白基因之選殖……….…32
3.3 芝麻11S-3球蛋白、11S-4球蛋白與2S-3清蛋白之cDNA序列與
胺基酸序列之分析………………………………………………………..32
3.4 芝麻11S球蛋白與2S清蛋白之胺基酸組成的分析…………….………34
3.5 芝麻11S-3球蛋白、11S-4球蛋白與2S-3清蛋白胺基酸序列與已知
過敏原序列的比對分析…………………………………………………..34
第四章 結論………………………………………………………………..……..52
參考文獻…………………………………………………………………………...53

圖目錄
圖1-1 線性抗原決定部位與結構抗原決定部位…………………………..…...10
圖1-2 IgE與過敏蛋白作用後，使肥大細胞釋出發炎媒介物……. ………….11
圖1-3 芝麻植株與花…………………………………………………………….12
圖1-4 芝麻種子細胞的電顯圖………………………………………………….14
圖1-5 芝麻油體的模型圖……………………………………………………….15
圖1-6 芝麻過敏蛋白的胺基酸序列…………………………………………….17
圖1-7 芝麻種子的蛋白質電泳圖……………………………………………….18
圖1-8 芝麻11S球蛋白的形成過程…………………………………………….19
圖1-9 芝麻2S清蛋白的形成過程…………………………………. ………….20
圖1-10 芝麻2S清蛋白(Ses i 1)四對雙硫鍵鍵結的二級結構圖…………….….21
圖3-1 芝麻11S-1球蛋白表達序列標記片段搜尋…………………..…………36
圖3-2 PCR產物電泳圖……………………………………………..……..….…39
圖3-3 重組質體的構築……………………………………………..……..….…40
圖3-4 重組質體以限制酶EcoR I剪切的DNA電泳圖………….……..…..….41
圖3-5 芝麻11S-3球蛋白cDNA與所演譯的胺基酸序列………….…..…..…42
圖3-6 芝麻11S-3球蛋白訊息肽切位位置………………….……..…..…….…43
圖3-7 芝麻11S-4球蛋白cDNA與所演譯的胺基酸序列……………..…….…44
圖3-8 芝麻11S-4球蛋白訊息肽切位位置………………………….…….……45
圖3-9 芝麻11S球蛋白胺基酸序列比對…………………………….…….……46
圖3-10 芝麻2S-3清蛋白cDNA與所演譯的胺基酸序列..……………..….……47
圖3-11 芝麻2S-3清蛋白訊息肽切位位置……………………………..…..……48
圖3-12 芝麻2S清蛋白胺基酸序列比對………………………………..….……49

表目錄
表1-1 芝麻（台南一號）性狀…………………………………………….……13
表1-2 芝麻的過敏蛋白…………………………………………………….……16
表1-3 芝麻過敏的症狀………………………………………………………….22
表2-1 選殖11S-3球蛋白、11S-4 球蛋白和2S-3清蛋白cDNA基因所用
之專一性引子………………………………………….………………..25
表3-1 芝麻11S球蛋白的表達序列標記片段……………………….…………37
表3-2 芝麻2S清蛋白的的表達序列標記片段…………………………………38
表3-3 芝麻11S球蛋白與2S清蛋白胺基酸組成的分析………………………50
表3-4 芝麻11S-3球蛋白、11S-4球蛋白與2S-3清蛋白胺基酸與已知過敏
原序列的比對………………………………………………….………….51

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