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研究生:黃瓊賢
研究生(外文):Huang Chiung Hsien
論文名稱:雞介白素-8及雞介白素-18cDNA之選殖與重組蛋白表現
論文名稱(外文):cDNA Cloning and Recombinant Protein Expression of Chicken Interleukin-8 and Interleukin-18
指導教授:邱繡河
指導教授(外文):Shiow-Her Chiou
學位類別:碩士
校院名稱:國立中興大學
系所名稱:獸醫微生物學研究所
學門:獸醫學門
學類:獸醫學類
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:70
中文關鍵詞:雞介白素-8雞介白素-18
外文關鍵詞:chIL-8chIL-18
相關次數:
  • 被引用被引用:3
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摘要
本研究的目的是選殖(molecular cloning) 雞IL-8及雞IL-18 (chicken IL-18,chIL-18) 之cDNA,並表現此兩種細胞素以供將來進一步研究應用。先前,本實驗室以大豆素A (concanavalin A,Con A)刺激過的白血球製備雞的cDNA 基因庫(cDNA library),在本研究中,我們利用聚合酶連鎖反應(polymerase chain reaction,PCR)自cDNA library增幅得到chIL-8之cDNA。繼而,自脂多醣體(lipopolysaccharide,LPS)刺激過的白血球萃取RNA,利用反轉錄聚合酶連鎖反應(reverse transcriptase-polymerase chain reaction,RT-PCR)增幅取得chIL-18 cDNA。此外,利用RT-PCR方法,可以在五週齡雞的胸腺、肝臟、腎臟、華氏囊、脾臟、胰臟、十二指腸、盲腸扁桃等多種臟器,偵測到chIL-18的mRNA。將所選殖的chIL-8及chIL-18 cDNA進一步以pET32載體構築chIL-8及chIL-18 之原核表現質體,利用大腸桿菌(Escherichia coli)大量表現chIL-8及chIL-18的融合蛋白(fusion proteins),經過親和性液相層析法純化後,以十二烷基硫酸鈉聚丙醯胺凝膠電泳(sodium dodecyl sulfate polyacrylamide gel electrophoresis,SDS-PAGE)及西方墨點法(Western blot analysis)分析。結果顯示,所表現的chIL-8及chIL-18融合蛋白其分子量如預期,分別約為31 kDa及36 kDa。
Abstract
The purposes of this study are to clone chIL-8 and chicken IL-18 (chIL-18) cDNA, and to express both cytokines for future studies and applications. Previously, our laboratory constructed a cDNA library based on concanavalin A (Con A)-stimulated leukocytes. In this study, we isolated chIL-8 cDNA from this premade cDNA library by employing polymerase chain reaction (PCR). Subsequently, using reverse transcriptase-PCR (RT-PCR), we obtained chIL-18 cDNA from leukocytes stimulated with bacterial lipopolysaccharide (LPS). Further more, chIL-18 mRNA was detected by RT-PCR among various tissues of 5-week-old chickens, including thymus, liver, kidney, bursa, spleen, cecal tonsil, pancreas and duodenum. ChIL-8 and chIL-18 cDNA were cloned into pET32 vector to construct prokaryotic expression vectors of chIL-8 and chIL-18. Both expression vectors were introduced into Escherichia coli, and fusion proteins of chIL-8 and chIL-18 were purified by affinity chromatography. After SDS-PAGE and Western blot analyses, we confirmed that molecular weights of the expressed chIL-8 and chIL-18 fusion proteins were as expected, which were 31 kDa, and 36 kDa, respectively.
目次
中文摘要………………………………......…………………………………... I
英文摘要……………………..………………………………………………... II
中英文縮寫對照表……………………………………………………………. III
目次…..……………………..…………………………………......................... Ⅴ
表次……..…………………..…………………………………......................... Ⅶ
圖次………..………………..…………………………………......................... Ⅶ
第一章 緒言……………………..…………………………………………. 1
第二章 文獻探討………………………………..……………..................... 3
第一節 趨化素(chemokines)的簡介……………..……………………… 3
第二節 IL-8的簡介……………..……………………………………….. 4
2-1 IL-8的發現…………………...…………………………………. 4
第三節 雞IL-8(chicken IL-8,chIL-8)的簡介………………………….. 5
3-1 chIL-8的發現……………...…………………………………….. 5
3-2 chIL-8之生物來源及蛋白質合成…...………………………….. 7
3-3 chIL-8在組織分佈情形…………………………………………. 7
3-4 分子結構……………...…………………………………………. 8
3-5 chIL-8基因的表現…...………………………………………….. 8
3-6 目前雞IL-8的應用……………………………………………… 9
第四節 IL-18的簡介………..…………………………………………… 10
4-1 IL-18的發現……………...……………………………………... 10
4-2 IL-18之生物來源、特性及功能…...…………………………… 10
4-3 IL-18在細胞及組織分佈情形………………………………….. 12
4-4 IL-18基因結構及訊息傳導途徑……………………………….. 13
4-5 IL-18蛋白質的表現與成熟..…………………………………… 14
4-6 雞IL-18的發現………………………………………………….. 15
4-7 IL-18在醫學及疫苗之相關研究………………………….......... 15
第三章 材料與方法……………………………………………………… 17
第一節 chIL-8 及chIL-8的選殖………………………………………... 17
1-1 雞隻之白血球 cDNA libary的來源……………………………. 17
1-2 雞白血球的分離、培養及刺激…………………………………. 17
1-3 萃取白血球之RNA……………………………………………... 18
1-4 雞隻臟器的採集及RNA萃取…………………………….......... 18
1-5 聚合酶連鎖反應(PCR)增幅chIL-8之cDNA………………. 18
1-6 RT-PCR增幅chIL-18之cDNA及偵測雞隻基因表現………… 19
1-6.1 引子序列………………………………………………………. 19
1-6.2 Two tube RT-PCR……………………………………………… 20
1-6.3 One tube RT-PCR……………………………............................ 20
1-7 PCR產物的純化……………………………………………… 21
1-8 勝任細胞之製備………………………………………………. 22
1-9 DNA接合作用(ligation)及轉形作用(transformation)……….. 22
1-9.1 DNA ligation……………………………………………............ 22
1-9.2 Transformation………………………………............................. 22
1-10 質體(plasmid) DNA 萃取………………………………........... 23
1-11 定序反應………………………………………………………. 23
第二節 chIL-8 及chIL-8融合蛋白的表現……………………………. 24
2-1 PCR增幅含限制酵素切位之chIL-18及chIL-18 cDNA……. 24
2-2 原核表現載體pETchIL-8及pETchIL-18的構築……………. 25
2-2.1 限制酵素切割增幅後之chIL-8及chIL-18 cDNA片段……… 25
2-2.2 限制酵素切割pET-32a表現載體…………………………….. 25
2-2.3 接合及轉型作用………………………………………………. 26
2-3 重組chIL-8及chIL-18之融合蛋白表現…………………….. 26
2-3.1 pETchIL-8及pETchIL-18送入大腸桿菌蛋白表現菌株…….. 26
2-3.2 SDS-PAGE測試融合蛋白表現情形………………………….. 26
2-3.3 西方墨點法分析………………………………………………. 27
2-4 融合蛋白之純化………………………………………………. 27
2-4.1 chIL-8融合蛋白之析出……………………………………….. 28
2-4.2 chIL-18融合蛋白之析出……………………………………… 28
第四章 結果…………………………………………………………........... 29
第一節 chIL-8 之cDNA選殖與蛋白表現…………………….......... 29
1-1 chIL-8 cDNA的選殖………………………………………......... 29
1-2 chIL-8 原核表現載體之構築………………………………....... 30
1-3 chIL-8融合蛋白之表現……………………………………......... 30
1-4 chIL-8融合蛋白之純化……………………………………......... 31
第二節 chIL-18 之cDNA選殖與蛋白表現…………………......... 31
2-1 chIL-18 cDNA的選殖……………………………………........... 31
2-2 RT-PCR偵測雞隻IL-18基因表現……………………............... 32
2-3 chIL-18原核表現載體之構築………………………………....... 32
2-4 chIL-18融合蛋白之表現…………………………………........... 33
2-5 chIL-18融合蛋白之純化…………………………………........... 33
第五章 討論與未來研究方向………………………………………........... 53
參考文獻.……………………………………………………………………… 57
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