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研究生:賴欣怡
研究生(外文):Hsin-Yi Lai
論文名稱:點帶石斑及龍膽石斑免疫球蛋白基因選殖及特性研究
論文名稱(外文):Molecular cloning and characterization of grouper (epinephelus coioides and epinephelus lanceolatus) immunoglobulin gene
指導教授:林正輝林正輝引用關係
學位類別:碩士
校院名稱:國立臺灣海洋大學
系所名稱:水產養殖學系
學門:農業科學學門
學類:漁業學類
論文種類:學術論文
論文出版年:2005
畢業學年度:93
語文別:中文
論文頁數:88
中文關鍵詞:點帶石斑龍膽石斑免疫球蛋白
外文關鍵詞:Epinephelus coioidesEpinephelus lanceolatusimmunoglobulin
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免疫球蛋白(immunoglobulin),是脊椎動物體內對外來異原分子所產生的一種具防禦功能的蛋白質,基本結構為Y字形。在哺乳類免疫球蛋白早已研究許久,然而在魚類則是近數十年才被發現類似哺乳動物免疫球蛋白IgM的存在,有單體(monomer)及四體(tetramer)兩種型態。本實驗在基因選殖方面,利用PCR或RT-PCR從點帶石斑頭腎library及龍膽石斑頭腎選殖出免疫球蛋白cDNA重鏈及輕鏈基因序列,並進行分析。在點帶石斑方面,重鏈基因選殖出一全長序列,共1940 bp,預測演譯出590個胺基酸,扣除signal peptide後,分子量大小約為64 kDa。輕鏈基因則選殖出一全長序列,共1066 bp,預測演譯出243個胺基酸,扣除signal peptide後,分子量大小約為24 kDa。以此估算點帶石斑免疫球蛋白四體大小約為704 kDa。而龍膽石斑方面,重鏈基因選殖出兩個3’RACE clones(ELH3、ELH4),長為847 bp以及850 bp,分別預測演譯(deduced)出243、243個胺基酸。輕鏈基因則選殖出三個3’RACE clones(ELL3、ELL6、ELL8),長為512 bp、501 bp、493 bp,分別預測演譯出107、96、101個胺基酸。在蛋白質純化方面,由點帶石斑及龍膽石斑血清中,利用非還原態之SDS-PAGE純化出免疫球蛋白IgM,再利用SDS-PAGE分別評估點帶石斑重鏈(heavy chain)分子量大小約為80 kDa,龍膽石斑重鏈分子量大小約為78 kDa,點帶石斑輕鏈(light chain)的分子量大小約為30 kDa,龍膽石斑輕鏈的分子量大小約為30 kDa。
謝辭………………………………………………………………………...i
摘要………………………………………………………………………...ii
Abstract……………………………………………………………………iii目錄………………………………………………………………………..iv
圖表頁次………………………………………………………………….vii
壹、前言……………………………………………………………………1
一、石斑魚的生物學及養殖.........................................................................1
二、免疫系統概述.........................................................................................3
三、魚類免疫系統與免疫球蛋白………………………………………….6
四、研究目的……………………………………………………………….8
貳、材料方法………………………………………………………………9
一、點帶石斑免疫球蛋白基因選殖………………………………………9
1-1 分離點帶石斑免疫球蛋白基因之部分cDNA序列…….………9
1-1-1 點帶石斑total RNA萃取……………………………….………9
1-1-2 RNA膠體電泳之分析…………………………………………..9
1-1-3 點帶石斑免疫球蛋白重鏈基因引子設計…………………...10
1-1-4 反轉錄酶反應………………………………………………...10
1-1-5 聚合酶鏈鎖反應……………………………………………...10
1-1-6 瓊脂膠體萃取………………………………………………...11
1-1-7 定序載體的構築與製備……………………………………...11
1-1-8質體轉形作用…………………………………………………11
1-1-9小量質體DNA之製備………………………………………..12
1-1-10 限制內切酵素之質體DNA確認…………………………...13
1-1-11 PCR產物之定序……………………………………………...13
1-2點帶石斑免疫球蛋白重鏈及輕鏈基因之選殖………………...13
1-2-1 點帶石斑免疫球蛋白重鏈及輕鏈基因選殖引子設計……...13
1-2-2 點帶石斑頭腎cDNA library擴增…………………………...14
二、龍膽石斑免疫球蛋白基因選殖…………………………………….15
2-1 分離龍膽石斑免疫球蛋白基因之部分cDNA序列……..……..15
2-1-1 龍膽石斑total RNA萃取………………………………..……15
2-1-2龍膽石斑免疫球蛋白重鏈及輕鏈基因引子設計……….……15
2-1-3 龍膽石斑免疫球蛋白重鏈及輕鏈基因片段之選殖…….…..16
2-2龍膽石斑免疫球蛋白重鏈及輕鏈基因之3’ RACE……….……16
2-2-1反轉錄酶反應…………………………………………….……16
三、點帶石斑及龍膽石斑免疫球蛋白質純化…………………………..16
3-1石斑血清分離……………………………………………….…...16
3-2免疫球蛋白之純化……………………………………………....17
3-2-1血清蛋白之粗純化…………………………………………….17
3-2-2膠體萃取……………………………………………………….17
3-3還原態之SDS-聚丙烯胺膠體電泳……………………………...18
3-4非還原態之SDS-聚丙烯胺膠體電泳…………………………...18
3-5銀染………………………………………………………………19
3-6蛋白質濃度測定…………………………………………………20
3-7西方點墨法之分析………………………………………………20
參、實驗結果………………………………………………………..…….22
一、點帶石斑免疫球蛋白重鏈基因序列…………………………..……22
二、點帶石斑免疫球蛋白重鏈基因比較結果…………………………..23
三、點帶石斑免疫球蛋白輕鏈基因序列………………………………..24
四、點帶石斑免疫球蛋白輕鏈基因比較結果…………………………..24
五、龍膽石斑免疫球蛋白重鏈部分基因序列與比較結果……………..25
六、龍膽石斑免疫球蛋白輕鏈部分基因序列與比較結果……………...26
七、點帶石斑免疫球蛋白及龍膽石斑免疫球蛋白純化結果…………...26
肆、討論…………………………………………………………………..28
一、點帶石斑免疫球蛋白重鏈與輕鏈基因序列與比對結果…………..28
二、龍膽石斑免疫球蛋白重鏈與輕鏈部分基因序列與比對結果……..29
三、點帶石斑與龍膽石斑免疫球蛋白純化結果………………………..30
伍、參考文獻……………………………………………………………..32
陸、圖表…………………………………………………………………..36
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