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研究生:譚德昌
論文名稱:利用大腸桿菌表現Indolicidin類似物及功能分析
論文名稱(外文):Expression and functional study of an Indolicidin analog in E. coli
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:獸醫學系
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:65
中文關鍵詞:大腸桿菌表現抗菌蛋白
外文關鍵詞:Escherichia coliindolicidinexpression
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抗菌蛋白為哺乳動物免疫系統的重要成員。Indolicidin屬於cathelicidin家族,為分離自牛嗜中性球的陽性抗菌蛋白。Indolicidin具快速之殺菌作用且具廣泛抗微生物效力。而I-13蛋白為基於indolicidin胺基酸序列所設計的抗菌蛋白,其保留了indolicidin對葡萄球菌屬的抗菌效力,但對格蘭氏陰性菌的抗菌效力較indolicidin強且細胞毒性較低。由於I-13具有抗菌效力強及抗菌譜廣泛等優點,故可能可應用於人類及動物病原微生物感染之防治。因此,實驗目的即為表現與純化I-13蛋白,並進行功能分析。實驗將含I-13基因之DNA片段構築於pET-32a(+)表現質體,並將所構築之表現質體轉形至E. coli BL21(DE3)表現宿主。經乳糖誘導I-13融合蛋白表現後,以鎳離子親和性管柱純化融合蛋白;並於I-13融合蛋白經腸激酵素處理後測定其抗菌效力。根據最小抑菌濃度試驗的結果,合成的I-13蛋白對標準菌株E. coli (ATCC 25922)及S. aureus (ATCC 25923)之最小抗菌濃度皆為8 μM。而表現之I-13融合蛋白經腸激酵素處理後於濃度128 μM對E. coli (ATCC 25922)具生長抑制效果,而對S. aureus (ATCC 25923)之最小抑菌濃度則為64 μM。根據上述結果,I-13抗菌蛋白可利用基因重組技術於原核表現系統表現。

Antimicrobial peptides are important components of the innate defenses of mammalians. Indolicidin, a cationic antimicrobial peptide isolated from bovine neutrophils, is a member of cathelicidin family. The action of indolicidin is fast and lethal to a broad spectrum of pathogens. A novel cationic peptide, I-13, based on the primary structure of the indolicidin, has improved activity against Gram-negative bacteria, while it maintains the activity of indolicidin against staphylococci and demonstrates a reduced cytotoxicity. Since I-13 shows impressive in vitro activity against microorganisms, it may be a good candidate for clinical use in humans and animals. In this study, the expression, purification and functional study of I-13 were investigated. DNA fragment encoding I-13 gene was synthesized and inserted into pET32a(+) expression vector and transformed into E. coli BL21(DE3). The I-13 fusion proteins were expressed by induction with 1% lactose and purified by Ni2+-chelating affinity chromatography. The purified I-13 fusion proteins were cleaved by enterokinase to remove fusion partner and the antimicrobial activity of the recombinant I-13 was determined. It was found that the MICs of synthetic I-13 against E. coli (ATCC 25922) and S. aureus (ATCC 25923) were both 8 μM. The MIC of recombinant I-13 against S. aureus (ATCC 25923) was 64 μM and recombinant I-13 could inhibit the growth of E. coli (ATCC 25922) at 128 μM. These results demonstrated that antimicrobial peptide I-13 could be produced by fusion protein technology in prokaryotic expression system.

中文摘要……………………………………………………………I
英文摘要……………………………………………………………II
目次…………………………………………………………………III
圖次…………………………………………………………………VII
表次…………………………………………………………………IX
壹、緒言……………………………………………………………01
貳、文獻探討………………………………………………………03
一、抗菌蛋白………………………………………………….. 03
二、Cathelicidins家族………………..………………………04
三、Indolicidin之發現與表現………………………………… 07
四、Indolicidin之蛋白結構特性……………………………… 06
五、Indolicidin之作用機制…………………………………… 09
六、Indolicidin之抗微生物功能……………………………… 11
1. 抗菌活性分析………………………………………… 11
2. 抗黴菌與原蟲活性分析……………………………… 12
3. 抗病毒活性分析……………………………………… 13
七、Indolicidin的其他生物活性……………………………… 13
1. 溶血作用 (hemolytic activity) ………………………. 13
2. 細胞毒性作用 (cytotoxicity) ………………………... 14
八、抗菌蛋白生產方式………………………………………. 14
參、材料與方法……………………………………………………16
一、藥品與試劑………………………………………………. 11
二、培養基、試劑與緩衝溶液………………………………. 17
三、基因片段的設計與製備…………………………………. 17
四、pET32a/ I-13表現質體的構築與確認…………………… 18
(一)勝任細胞 (competent cell)之製備………………. 18
(二)pET32a(+)質體之轉形 (transformation)………… 19
(三)pET32a(+)質體之製備…………………………… 19
(四)pET32a/ I-13表現質體之構築…………………… 20
(五)構築完成之pET32a/ I-13表現質體之轉形…….. 22
(六)構築完成之pET32a/ I-13表現質體之確認…….. 22
五、pET32a/ I-13表現質體的轉形與表現…………………… 22
(一)轉形用pET32a/ I-13表現質體的製備………….. 22
(二) pET32a/ I-13表現質體的轉形…………………. 23
(三) pET32a/ I-13表現質體於BL21(DE3)表現系統的表現與分析………………………………….. 23
(四) I-13融合蛋白之純化、確認與定量…………… 24
(五) I-13融合蛋白之再摺疊………………………… 25
五、抗菌蛋白活性試驗………………………………………. 26
(一)影響宿主細胞生長之試驗………………………. 26
(二)抗生素敏感性試驗………………………………. 26
(三)抗菌試驗…………………………………………. 27
肆、結果……………………………………………………………28
一、pET32a/ I-13基因之表現質體構築與確認……………… 28
二、pET32a/ I-13基因表現質體的轉形與表現……………… 32
(一) pET32a/ I-13基因表現質體於大腸桿菌BL21(DE3)株的表現與分析……………………32
(二) I-13融合蛋白之純化與定量…………………….. 33
五、抗菌蛋白活性試驗……………………………………….. 39
(一)影響宿主細胞生長之試驗………………………. 39
(二)抗生素敏感性試驗……………………………….. 39
(三)抗菌試驗………………………………………….. 40
伍、討論與結論……………………………………………………44
柒、參考文獻………………………………………………………53
附錄…………………………………………………………………60
附錄一、以Gene-Spin™ Miniprep Purification kit純化質體DN60
附錄二、組胺酸親和性管柱純化步驟……………………………61
附錄三、BCA蛋白質分析方法……………………………………62
附錄四、實驗使用之培養基、試劑與緩衝液配方………………63
圖次
圖一、Cathelicidins家族的pre-pro-peptide基因結構圖……06
圖二、pre-pro-indolicidin之胺基酸序列……….…………………08
圖三、合成的I-13基因片段及其相對應之胺基酸序列與限制酵素切割位……………………………………………………. 18
圖四、pET32a(+)表現質體與pET32a/ I-13表現質體構造圖…21
圖五、pET32a/ I-13表現質體基因構築簡圖……………………21
圖六、pET32a(+)表現質體與pET32a/ I-13表現質體之Bgl I限制酵素切割圖譜………………………………………………. 29
圖七、以Bgl I限制酵素截切pET32a(+)與pET32a/ I-13表現質體之瓊脂凝膠電泳圖………………………………………. 30
圖八、pET32a/ I-13定序結果……………………………………31
圖九、以乳糖誘導pET32a/ I-13表現質體於BL21(DE3)之表現結果……………………………………………………..…..34
圖十、以乳糖誘導pET32a(+)質體於BL21(DE3)之表現結果…35
圖十一、pET32a(+)與pET32a/ I-13表現質體於BL21(DE3)之表現結果……………………………………………………36
圖十二、以IPTG或乳糖誘導pET32a/ I-13表現質體於BL21(DE3)之表現果……………………………………. 37
圖十三、以乳糖誘導I-13融合蛋白表現與純化結果……………38
圖十四、含pET32a(+)質體與pET32a/ I-13表現質體之BL21(DE3)菌株,於各種不同誘導條件之生長曲線圖..41

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