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研究生:王俊傑
研究生(外文):Chun-Chieh Wang
論文名稱:海鱺rag-1與Ig基因之選殖及其在發育過程之表現
論文名稱(外文):Cloning of rag-1 and Ig gene from cobia, Rachycentron canadum, and their expression during ontogenesis
指導教授:楊惠郎
學位類別:碩士
校院名稱:國立成功大學
系所名稱:生物科技研究所碩博士班
學門:生命科學學門
學類:生物科技學類
論文種類:學術論文
論文出版年:2002
畢業學年度:90
語文別:中文
論文頁數:78
中文關鍵詞:重組蛋白脢海鱺免疫球蛋白
外文關鍵詞:cobiarag-1immunoglobulin
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  B淋巴細胞分泌的免疫球蛋白是生物體內參與體液性免疫反應最重要的蛋白質,而免疫球蛋白在B淋巴細胞發育過程會進行V、D、J基因片段重組來組成多變區,藉以產生大量不同的抗原受器來保護個體。V(D)J基因片段重組過程主要受到rag-1與rag-2兩個重組酵素的嚴格調控。由於rag-1序列在不同物種間包括從硬骨魚至哺乳動物具有高度保留的特性,且是B淋巴細胞在免疫器官發育成熟過程中相當重要的蛋白,故rag-1基因的表現是用來鑑定免疫器官相當理想的標籤。另外,為了探討海鱺免疫成熟期與幼苗發育時程上的相對關係,我們亦檢測rag-1與Ig基因在海鱺幼苗發育不同階段(從魚卵至幼魚)的表現情形。
  藉由RT-PCR方法我們已成功地選殖出海鱺片段rag-1基因與Ig基因,片段大小分別是637bp與225bp,另外,我們亦選殖海鱺b-actin基因作為基因表現的控制組。當比較海鱺與不同魚種間的rag-1與Ig基因序列發現,rag-1基因的相似度高達87﹪,而Ig基因的序列至少也有37﹪以上相似度。利用北方點墨法與RT-PCR方法分析海鱺不同器官表現rag-1基因的結果發現,rag-1基因在海鱺胸腺器官有最強之訊號表現,在脾臟與腎臟則相對表現出少量rag-1基因,此結果暗示海鱺的胸腺器官較脾臟與腎臟有最重要的免疫孕育地位。在抗體表現與幼苗發育時程相對關係上,我們可經由RT-PCR方法檢測出在孵化後第七天左右的海鱺幼苗有rag-1與Ig基因的表現,暗示此生長階段的幼魚已具備成熟之免疫能力來製造抗體。
  Immunoglobulin produced by B lymphocytes is a major protein involved in the humoral immune response. The variable region of Ig gene is assembled during B lymphocyte development by the recombination of multiple V, D, J segments. This V(D)J rearrangement process generates a vast array of antigen receptors and is strictly mediated by RAG recombinase, encoded by recombination activation gene-1 and -2. Because rag-1 gene, especially in the enzyme-activating core region, is highly conserved among species from teleost to mammals and is critical to the differentiation of pre-B cells, its expression within an associated primary lymphoid organ can serve as an ideal developmental marker to determine the location of immune organs. Besides, in order to study the maturity level of immune system with respect to time during cobia ontogeny, the expression patterns of rag-1 and Ig gene at different cobia growth stages (from embryo to fry stages) were examined.
  In addition to the use of β-actin gene as an internal control, partial rag-1 and Ig genes form cobia, Rachycentron canadum, each with an amplified size 637bp and 225bp respectively, were successfully clones. When compared with other previously reported rag-1 or Ig sequences of different fish species, the predicted rag-1 and Ig amino acid sequences of cobia displayed a minimum of 87% and 33% similarity. Tissue-specific expression of rag-1 was examined both by northern blotting and RT-PCR assay. The highest level of rag-1 expression was observed in the thymus, and a weaker expression was observed in spleen and kidney. This result indicated that thymus might be the most important immune organ compared to spleen and kidney.The expression patterns of rag-1 and Ig genes were both detected on the 7th day after hatching by RT-PCR assay. This suggested that cobia at this stage might possess mature immunity and is able to produce immunoglobulin.
目 錄
中文摘要…………………………………………………….……...I
英文摘要…………………………………………...…………..…..II
目錄………………………………………….……………………III
圖表目錄…………………………..………………………………V
縮寫表………………………….………………………..………VII

第一章、前言…………………………..………………………………….1
第二章、文獻探討
一、海鱺之簡介…………………………………………………………..3
二、免疫系統之概論……………….……………………..…………….. 4
三、魚類免疫器官之簡介…………………………..……………………5
四、免疫球蛋白之簡介…………………………..………………………7
五、魚類免疫球蛋白(Ig)之研究……………..………………………. 8
六、RAG蛋白之簡介
RAG(recombination-activating gene)之發現……………………10
RAG蛋白之高保留特性………………………………………….11
RAG蛋白與V(D)J片段隨機重組機制之關係………..…………12
RAG蛋白的表現時期…………………………….…………..…..13
第三章、研究目的…………………………………………….…..…..…..15
第四章、材料與方法
一、實驗材料…..…………………………………………….……….…16
二、實驗方法……………………………………………………………21
第五章、結果與討論
一、海鱺rag-1基因之選殖…………………...……..………………..30
二、海鱺rag-1基因比對結果……………………….….………………31
三、海鱺Ig基因之選殖………………….…………………...…………32
四、海鱺Ig基因比對結果…………...……..…………….……………..34
五、海鱺b-actin基因之選殖及比對結果..……………………………..35
六、海鱺DIG-labeled rag-1、Ig與β-actin探針之合成……….………35
七、雜交溫度(Thyb)之計算………...……..………………………..…..37
八、Rag-1基因在海鱺不同器官表現之情形………….………….……38
九、海鱺rag-1與Ig基因在幼苗發育階段表現之情形….……….……40
第六章、結論………………………………………………...…….43
第七章、參考文獻……………………………………...………….45


圖 表 目 錄
表一、聚合脢連鎖反應(PCR)使用之引子對(primers)與反應條件…….54
圖一、經RT-PCR方法放大海鱺β-actin片段基因之電泳圖……………..55
圖二、經PCR方法放大海鱺rag-1片段基因之電泳圖………………...….55
圖三、經RT-PCR方法放大海鱺免疫球蛋白(Ig)片段基因之電泳圖…...56
圖四、以PCR方法合成探針(DIG-labeled probe)之電泳圖…………..…57
圖五、海鱺β-actin片段核酸(nucleic sequence)序列…………………......58
圖六、海鱺β-actin片段核酸序列經NCBI網站BLAST進行比對之結果……………………………………………………………….……59
圖七、海鱺rag-1片段核酸(nucleic sequence)序列…………….……60
圖八、海鱺rag-1片段核酸序列經NCBI網站BLAST進行比對之結果…….. …………………………………………………………..…61
圖九、海鱺與不同物種之rag-1片段胺基酸序列比對結果……………...62
圖十、海鱺與不同物種之rag-1核酸、胺基酸序列相似度百分比………64
圖十一、海鱺Ig片段核酸(nucleic sequence)序列………………..………65
圖十二、海鱺Ig片段核酸序列經NCBI網站BLAST進行比對之結果.…66
圖十三、海鱺與不同物種之Ig片段胺基酸序列比對結果………………..67
圖十四、海鱺與不同物種之Ig核酸、胺基酸序列相似度百分比…………68
圖十五、以RT-PCR方法檢測海鱺不同器官之rag-1基因表現情形….69
圖十六、以northern blotting方法檢測rag-1基因在海鱺不同器官之表現情形………………………………………………………………….70
圖十七、以RT-PCR方法檢測海鱺不同發育階段rag-1基因之表現情形…………………………………………………………………….71
圖十八、以RT-PCR方法檢測海鱺不同發育階段Ig基因之表現情形…..72
圖十九、以northern blotting方法檢測rag-1與Ig基因在海鱺不同生長階段之表現情形……………………………..……………………73

附錄一、pGEM®-T Easy Vector圖譜……………………..……………….74
附錄二、RAG蛋白表現週期與淋巴球成熟過程之相關圖…………….….75
附錄三、RAG-1與RAG-2蛋白進行切割、接合反應之過程…………...…76
附錄四、不同物種間RAG-1、RAG-2蛋白的相似度百分比…………...….77
附錄五、B細胞發育過程總覽圖…………………………………..……….78
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