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標題: 以大腸桿菌表現豬抗菌蛋白protegrin-1以其功能分析
E. coli expression and fundctional study of porcine antimicrobial protegrin-1
作者: 余佩芳
Yu, Pei-Fang
關鍵字: 豬抗菌蛋白;大腸桿菌表現
出版社: 獸醫微生物學研究所
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。

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.
Appears in Collections:微生物暨公共衛生學研究所

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