請用此 Handle URI 來引用此文件: http://hdl.handle.net/11455/12674
標題: 利用大腸桿菌表現Indolicidin類似物及功能分析
Expression and functional study of an Indolicidin analog in E. coli
作者: 譚德昌
關鍵字: Escherichia coli
大腸桿菌
indolicidin
expression
表現
抗菌蛋白
出版社: 獸醫學系
摘要: 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.
URI: http://hdl.handle.net/11455/12674
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