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研究生:余佩芳
研究生(外文):Yu, Pei-Fang
論文名稱:以大腸桿菌表現豬抗菌蛋白protegrin-1以其功能分析
論文名稱(外文):E. coli expression and fundctional study of porcine antimicrobial protegrin-1
指導教授:毛嘉洪
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:66
中文關鍵詞:豬抗菌蛋白大腸桿菌表現
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中文摘要
哺乳動物受到外來病源菌感染時,吞噬細胞和上皮細胞會產生及分泌抗菌蛋白。而protegrin-1(PG-1)是從豬的白血球分離,是個具有18個胺基酸且富含半胱胺酸,呈現β-sheet結構的抗菌蛋白。PG-1是以沒有活性的preform形式存在於嗜中性球中,經由嗜中性球的彈力蛋白酵素作用才呈現出有活性的PG-1,而PG-1的抗菌範圍非常廣泛。而本研究目的是希望能利用大腸桿菌表現系統表現具有活性PG-1,能進一步應用於豬隻的疾病。本研究使用反轉錄聚合酵素連鎖反應(RT-PCR),從仔豬的嗜中性球選殖出preproPG-1(ppPG-1),嵌入pET32a(+)載體中,轉形至大腸表現菌株AD494(DE3)pLysS中進行重組蛋白質表現。經1 mM IPTG誘導ppPG-1融合蛋白產生,利用組胺酸親和性管柱純化,所得的蛋白經由彈力蛋白酵素切割後進行抗菌實驗。結果顯示,40μM的彈力蛋白酵素切割的ppPG-1融合蛋白能延遲標準菌株E. coli (ATCC 25922)及S. aureus (ATCC 25923)的生長。因此,本研究證實可以利用大腸桿菌表現系統表現具活性PG-1。

Abstract
The mammalian host response to infection includes the production and secretion of antimicrobial peptides from phagocytes and epithelial cell. Protegrin-1 (PG-1) is a cysteine-rich, 18-residue β-sheet peptide isolated from porcine leukocytes, with antimicrobial activity against a broad range of microorganism. It were found to be stored as inactive performs in porcine neutrophil granules but could be activated extracellularly by neutrophil elastase. In this study, PG-1 were expressed and purified to improve the diarrhea symptom of piglets. The cDNA for porcine pre-pro-PG-1 (ppPG-1) were cloned and inserted into pET32a(+) expression vector and transformed into E. coli AD494 (ED3) pLysS. The ppPG-1 fusion proteins were expressed by induction with 1 mM IPTG and purified by Ni2+-chelating affinity chromatography. The purified ppPG-1 fusion proteins were cleaved by enterokinase to remove fusion partner.ppPG-1 retarded the growth of E. coli (ATCC 25922) and S. aureus (ATCC 25923) at 40 μM. These results demonstrated that antimicrobial peptide PG-1 could be produced by fusion protein technology in prokaryotic expression system.

目次
中文摘要……………………………………………………….. I
英文摘要……………………………………………………….. II
目次…………………………………………………………….. III
圖次…………………………………………………………….. VII
表次…………………………………………………………….. VIII
壹、緒言………………………………………………………… 01
貳、文獻探討…………………………………………………… 02
一、抗菌蛋白……………………………………………………... 02
二、哺乳類抗菌胜太的分布及特色……………………………... 02
三、抗菌胜太的結構……………………………………………... 03
四、Cathelicidin前驅物的結構…………………………………... 03
五、Protegrins的簡介…………………………………………….. 04
六、Protegrins一般特性………………………………………….. 05
七、Protegrins的抗菌機制……………………………………….. 06
八、Protegrins在試管中或活體中修飾的過程………………….. 07
九、Peotegrins結構變化對抗菌功能的影響…………………….. 09
十、Protegrins的應用…………………………………………….. 10
參、材料與方法………………………………………………… 11
一、構築與確認TA/preproprotegrin-1(ppPG-1)重組質體……... 11
1.萃取白血球總核醣核酸…………………………………….. 11
2.製備ppPG-1的基因片段……………………………………. 11
3.構築TA/ppPG-1重組質體…………………………………... 14
4.製備勝任細胞(competent cell)…………………………… 16
5.TA/ppPG-1重組質體之轉形(transformation)與確認…… 16
6.萃取與純化TA/ppPG-1重組質體…………………………... 18
二、構築重組pET32a/ppPG-1基因表現載體與確認……… 18
1.製備ppPG-1基因片段與確認………………………………. 18
2.製備pET32a(+)載體DNA片段與確認…………………. 20
3.構築pET32a/ppPG-1基因表現載體………………………... 21
4.勝任細胞中pET32a/ppPG-1基因表現載體的轉形………... 23
5.篩選含有pET32a/ppPG-1基因表現載體的菌株…………... 23
三、表現已轉形的pET32a/ppPG-1基因表現載體………… 25
1.製備轉形用pET32a/ppPG-1基因表現載體………………... 25
2.pET32a/ppPG-1基因表現載體的轉形………………… 25
3.pET32a/ppPG-1基因於表現系統的表現與分析…….... 25
4.純化、確認與定量ppPG-1表現蛋白………………………. 26
四、抗菌蛋白活性試驗………………………………………….. 28
1.影響宿主細胞生長的試驗………………………………….. 28
2.抗菌試驗…………………………………………………….. 28
肆、結果………………………………………………………… 30
一、構築與確認重組TA /ppPG-1載體………………………….. 30
1.獲得ppPG-1的完整基因……………………………………. 30
2.確認重組TA /ppPG-1載體………………………………….. 30
二、構築重組pET32a/ppPG-1表現載體與確認………………… 34
1.製備ppPG-1基因片段和pET32a/ ppPG-1表現載體……… 34
2.確認pET32a/ ppPG-1表現載體…………………………….. 34
三、表現ppPG-1的蛋白質………………………………………. 37
四、抗菌蛋白活性測試…………………………………………... 42
1.影響宿主細胞生長之試驗………………………………….. 42
2.抗菌試驗…………………………………………………….. 44
伍、討論………………………………………………………… 45
一、構築TA/ppPG-1重組質體………………………………….. 45
二、重組pET32a/ ppPG-1表現載體之構築…………………….. 45
三、表現ppPG-1蛋白質…………………………………………. 46
1.表現宿主的選擇…………………………………………….. 46
2.融合蛋白的表現…………………………………………….. 48
3.蛋白質純化………………………………………………….. 48
四、抗菌蛋白活性測試……………………………………… 49
1.影響宿主細胞生長之試驗……………………………... 49
2.抗菌試驗…………………………………………………….. 49
陸、結論……………………………………………………….... 51
柒、參考文獻…………………………………………………… 52
附錄…………………………………………………………….. 58
附錄一:萃取白血球總核醣核酸實驗步驟………………………... 58
附錄二:RT-PCR製備ppPG-1基因使用之4組引子…………….. 59
附錄三:TOPO TA Cloning®中M13(+)和M13(-)引子序列... 60
附錄四:Gene-SpinTM Miniprep Purification Kit實驗步驟………... 61
附錄五:選殖具有Nco I和Xho I ppPG-1基因序列之核酸引子…. 62
附錄六:Gene-SpinTMGel Extraction Kit實驗步驟………………… 63
附錄七:pET32a載體上S(+)和T7(-)引子之序列………………… 64
附錄八:pET expression system之inclusion body純化步驟……… 65
附錄九:BCA蛋白質分析方法之實驗步驟………………………. 66
圖次
圖一:TA/ ppPG-1重組質體之構築流程示意圖…………………... 15
圖二:pET32a(+)載體構造圖和pET32a/ppPG-1基因表現載體簡圖…………………………………………………………. 22
圖三:pET32a/ppPG-1基因表現載體構築簡圖…………………… 22
圖四:以RT-PCR製備ppPG-1基因之瓊脂凝膠電泳分析………. 31
圖五:對於TA/ppPG-1重組質體應用M13(+)和M13(-)引子進行PCR確認之電泳分析……………………………… 32
圖六:本實驗所選殖到的ppPG-1基因序列(上),和已發表的序列之比較…………………………………………………. 33
圖七:以核酸限制酵素Bgl I截切pET32a(+)和pET32a/ppPG-1質體DNA之電泳分析……………………………………... 35
圖八:pET32a/ppPG-1定序和轉譯的結果………………………… 36
圖九:pET32a(+)載體在AD494(DE3)pLysS之表現分析…. 38
圖十:pET32a/ppPG-1表現載體在AD494(DE3)pLys之表現分析…………………………………………………………. 39
圖十一:比較pET32a(+)和pET32a/ppPG-1表現載體在AD494(DE3)pLysS之表現……………………………………. 40
圖十二:pET32a/ppPG-1表現載體在AD494(DE3)pLysS之表現、純化分析…………………………………………….. 41
圖十三:pET32a(+)和pET32a/ppPG-1在AD494(DE3)pLysS之生長曲線………………………………………………. 43
圖十四:pET32(+)和pET32a/ppPG-1在AD494(DE3)pLysS中,經1 mM IPTG誘導表現之生長曲線……………… 43
表次
表一︰Protegrins的胺基酸序列…………………………………… 5
表二:用於製備ppPG-1基因之4組寡核酸引子組………………. 12
表三:用於選殖ppPG-1基因序列之核酸引子……………………. 19
表四:表現pET32a(+)和pET32a/ppPG-1蛋白質之部分胺基酸序列且預估其分子量大小………………………………. 37
表五:pET32a融合蛋白及ppPG-1融合蛋白經由不同蛋白酵素處理之抗菌結果……………………………………………. 44

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