Lucilia porphyrina幼蟲體內之抑菌性胜肽的純化及鑑定

Purification and identification of antimicrobial peptides in larval stage of Lucilia porphyrina

周季賢(Chi-Hsien Chou)

指導教授 ： 邱顯肇

高雄醫學大學/醫學院/生物化學研究所/碩士(2005年)

摘要

當昆蟲受到外來病原菌的感染後，會產生大量的抑菌性胜肽(antimicrobial peptides, AMPs)。AMPs在缺乏後天性免疫防禦機制的昆蟲體內扮演著重要的角色，以避免受到病原菌的嚴重侵襲。根據研究指出，AMPs對於大部分的病原菌，如細菌、黴菌、病毒等，甚至抗藥性菌株均有抑制作用。迄今為止，已超過800種的AMPs分別從不同的物種中被分離出來。
在本研究中，欲從金蠅屬(Chrysomya)及綠蠅屬(Lucilia)的幼蟲體內，分離出具有抑菌活性的胜肽。分離純化的流程採用一連串的層析方法，包括Sep-Pak prepurification、逆相高壓液相層析法、陽離子交換樹脂層析法。抑菌活性試驗則採用Plate-growth inhibition zone assay、Liquid-growth inhibition assay、Native gel overlay assay等方法。初步的結果證實，Chrysomya megacephala與Lucilia porphyrina 的粗萃物對於大腸桿菌、金黃色葡萄球菌、枯草桿菌均有抑制效果，顯示粗萃物具有廣效性的抑菌活性。在C. megacephala的幼蟲體內，抑菌物質的產生是受到感染後而大量表現；在L. porphyrina的幼蟲體內，某些抑菌物質是持續表現的，但當受到病原菌感染後原先之抑菌物質可能會大量產生，並可能誘導其他種抑菌物質的表現。另外，L. porphyrina的粗萃物對於幾種抗藥性菌株也有抑制活性。在一連串的分離純化過程中，我們證實了L. porphyrina體內的抑菌物質確實為AMPs；之後發現一些AMPs明顯地對革蘭氏陰性菌有抑制作用；另一些AMPs則選擇性地抑制革蘭氏陽性菌。因此，在L. porphyrina的幼蟲體內可以產生多種的AMPs，且彼此之間具有加成協同作用，以達到最佳的抑菌效果。最後，我們從L. porphyrina幼蟲體內分離純化出一種對金黃色葡萄球菌有抑菌作用的AMP，經由質譜儀分析其分子量為4,061.06 Da，並利用Edman胺基酸定序儀測得三十八個胺基酸序列，其序列與Phormia terranovae體內所產生的defensin A完全相同。

關鍵字

純化鑑定 ； 抑菌性胜?

並列摘要

Insects produce a variety of antimicrobial peptides when they are infected. Antimicrobial peptides play a pivotal role in the defenses of insects that lack an acquired immune system and protect them from invading microorganisms. They are known to be effective against a wide range of pathogens including bacteria, fungi, virus, and even antibiotic-resistant bacterial species. More than 800 different antimicrobial peptides have been isolated from species of all kingdoms.
In this study, we want to find out a family of antimicrobial peptides (AMPs) from Chrysomya and Lucilia larvae. The purification procedures include a sequence of chromatographic methods: Sep-Pak prepurification, reverse-phase HPLC chromatography, cation-exchange chromatography. The antibacterial activity is examed by plate-growth inhibition zone assay, liquid-growth inhibition assay, and native gel overlay assay. The preliminary results revealed that the crude extract of Chrysomya megacephala and Lucilia porphyrina represent a broad range of antibacterial spectrum. Production of AMPs in C. megacephala is inducible by infection of E.coli. Production of some AMPs from L. porphyrina is constitutive, whereas the others are inducible triggered by E. coli infection. The crude extract of L. porphyrina is active against several antibiotic-resistant bacterial strains as well. In the purification procedures, we find that some AMPs are predominantly active against Gram-negative strains, and some AMPs are selectively active against Gram-positive strains. Hence, there are more than one AMPs in L. porphyrina, and they may have synergistic effects to kill invading microorganisms. Finally, a pure AMP effective against S. aureus was isolated in the present study. The calculated molecular mass measured by nano-ESI-Q TOF is 4,061.06 Da, and the N-terminal sequence is obtained by Edman degradation. The molecular mass and partial amino acid sequence are completely identical with defensin A found in Phormia terranovae.

並列關鍵字

antimicrobial peptides ； Lucilia porphyrina

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