摘要
對抗多重抗藥性細菌的新型藥物中，抗菌胜肽的研發在其中扮演著相當重要的角色。抗菌胜肽是由12〜100個氨基酸組成的分子，具有雙極性且帶有正電荷，藉由氨基酸序列與分子結構的不同而能廣泛抑，包含細菌，原蟲，真菌，病毒，甚至腫瘤細胞的生長。大部分抗菌胜肽的作用機轉是經由非專一性機制與細胞的細胞膜表面產生交互作用改變膜的結構，使膜的結構不穩定進而瓦解，最終導致細菌溶解死亡，但在某些情況下也有可能激發先天免疫反應。許多抗菌胜肽已經進入臨床前和臨床試驗，包括治療導管部位感染，囊腫性纖維化，痤瘡，傷口癒合和正在接受幹細胞移植的患者。Blo t19 是一種從熱帶無爪蟎 (Blomia tropicalis) 分離出來的過敏原，同時與豬蛔蟲類抗菌胜肽 (Ascaris suum antibacterial factor，ASABF) 具少許的同源性。本研究的目的是探討Blo t19是否也具有抗菌活性。實驗首先利用大腸桿菌 (Escherichia coli) 系統大量表現重組胜肽Blo t19，本研究分別選用大腸桿菌 BL21和Origami 表達大量的Blo t19再進行純化。實驗結果顯示，雖然重組 Blo t19在大腸桿菌BL21的表現量高於在大腸桿菌Origami的表現量，但Origami表現的 Blo t19卻較BL21所表現的Blo t19具有較高的活性與穩定性，因此後續所有研究將會使用由Origami菌株所表現的 Blo t19。0.4 µg/ml的 Blo t19能夠抑制50% 的綠膿桿菌 (P. aeruginosa) 生長，50 µg/ml 處理2小時則能夠殺死99% 的P. aeruginosa 。其 Blo t19 濃度與抑菌效果間具有正相關的線性關係，但其殺菌活性卻會被氯化鈣抑制。值得注意的是Blo t19 並不會對哺乳動物細胞產生細胞毒性。根據上述的實驗結果，Blo t19抗菌肽有可能發展成為一種不具細 胞毒性的、並致力於治療P. aeruginosa 感染的藥物發展。

Abstract
The discovery of novel antimicrobial peptides plays an important role in the development of new antimicrobials for treatment of multidrug-resistant bacteria. Antimicrobial peptides are relatively small (12 to 100 amino acids), amphipathic, and positively charged molecules of variable length, sequence and structure with activity against a wide range of microorganisms including bacteria, protozoa, fungi, viruses and even tumor cells. They usually act through relatively non-specific mechanisms resulting in membranolysis but can also stimulate innate immune responses. Several antimicrobial peptides have already entered pre-clinical and clinical trials for the treatment of catheter site infections, cystic fibrosis, acne, wound healing and infections in patients undergoing stem cell transplantation. Previously it has been shown that Blo t19 - one of the allergens isolated from Blomia tropicalis exhibits weak homology with a characterized antimicrobial peptide ASABF. The aim of this study is to investigate whether Blo t19 also possesses an antimicrobial activity. In the present study, Blo t19 was overexpressed in E. coli BL21 and Origami strains and purified to homogeneity. Although the expression level of recombinant Blo t19 in E. coli BL21 was higher than in the Origami strain, the Origami-synthesized Blo t19 seemed to be more active and stable than that produced in BL21 and was therefore used for all subsequent studies. Recombinant Blo t19 is able to inhibit 50% of Pseudomonas aeruginosa growth at 0.4 µg/ml and kill 99% of P. aeruginosa at 50 µg/ml in 2 h incubation. The antibacterial activity was dose-dependent and could be inhibited by CaCl2. Moreover, this peptide did not exhibit detectable cytotoxicity to cultured mammalian cells. In conclusion, we demonstrated in this study that Blo t19 is a novel antimicrobial peptide and has the potential to be developed into a new drug for the treatment of P. aeruginosa infections.

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