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Cloning an antimicrobial peptide gene from the oriental fly, Bactrocera dorsalis (Hendel) and expressing in E. coli
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被東方果實蠅 (Bactrocera dorsalis Hendel) 產卵的果肉不易受微生物感染，引發本研究探討蟲卵表面抗生物質的本質與特性的動機。首先利用洋菜膠抑菌環試驗證實蟲卵洗滌液具有抗生物質。藉由聚丙烯醯胺凝膠電泳 (SDS-PAGE) 分析，認為此抗生物質可能是一抗菌胜肽分子，大小約3 kDa。經質譜定序的結果顯示，此抗菌胜肽含28個胺基酸，可能源自於sarcocystatin-like protein的降解。接著，參考sarcocystatin-like protein的基因序列設計引子，經RT-PCR及RACE方法進行完整基因的選殖，最後選殖出一全長489 bp的cDNA序列，命名為Bdscys-A (accession number MG231276)。此基因可轉譯出124個胺基酸，Met1-Ala26為訊息胜肽序列，Leu45至Ser115為cystatin保守區域。Bdscys-A序列具有數個疑似可被胰蛋白酶 (trypsin) 切割的位點，推論可經胰蛋白酶切割切割產生28個胺基酸之α螺旋結構抗菌胜肽。檢測Bdscys-A基因在蟲體的表現區域，結果顯示不論在雌、雄成蟲的頭、胸及腹部皆有表現，而以腹部的表現量較頭、胸部為高；在雌蟲的表現量也明顯的較雄蟲高。在雄蟲，馬氏管 (Malpighian tubule) 有較高的表現，然其表現量並不多；在雌蟲則以生殖副腺 (reproductive accessory gland) 表現量最高。利用大腸桿菌 (Escherichia coli, E. coli) 蛋白質表現系統來大量表現Bdscys-A時，顯示訊息胜肽序列會完全阻礙Bdscys-A在E. coli的表現。刪除訊息胜肽序列的載體pET29a-NSPSA，在IPTG的誘導下，會大量表現Bdscys-A。最後，Bdscys-A蛋白於抗菌試驗顯示抗菌活性並不強，推測可能需經酵素切割後所產生之抗菌胜肽可能具抗菌效果，期望將來可透過胺基酸的取代使抗菌胜肽更穩定，能開發成應用廣泛的抗菌劑。
Fruit would be reduced from microbial infection when oriental fruit fly (Bactrocera dorsalis Hendel) laid eggs inside the fruit, triggering the investigation on the anti-microbial material associated with eggs. Firstly, it was confirmed that the egg washing solution contains antibacterial material according to the positive result of antibacterial ring test; subsequently, the SDS-PAGE analysis showed that it might be an antibacterial peptide (AMP) with a size of about 3 kDa. The mass spectrometry analysis further revealed that the AMP consisted of 28 amino acids, which is possibly derived from digestion of sarcocystatin-like protein. After that, based on the sequence of sarcocystatin-like protein gene, and followed a series of RT-PCR and RACE reactions; and finally, a full-length 489-bp cDNA sequence was completed and named Bdscys-A with an accession number MG231276 in GenBank. The Bdscys-A can transduce a protein consists of 124 amino acids, containing a signal peptide from Met1 to Ala26 and conserved domain of cystatin from Leu45 to Ser115. Furthermore, there are two suspected trypsin digestion sites located in Bdscys-A; therefore, it is speculated that the 28 amino acid comprised an antibacterial peptide with α helix structure was derived from Bdscys-A by trypsin digestion. The gene expression in adult fly showed that Bdscys-A is expressed in all of head, thorax and abdomen regions of both males and females, while the expression of the abdomen was higher than that of the head and thorax. Moreover, females expressed higher than males. In males, the Malpighian tubules showed the highest Bdscys-A, though its expression was very low; in females, the reproductive accessory glands were the highest in Bdscys-A expression compared to other organs. When Bdscys-A was expressed using E. coli protein expression system, the cDNA with the complete open reading frame (pET29a-NSPSA) could not be induced by IPTG to express recombinant protein; conversely, the signal peptide-deleted cDNA (pET29a-NSPSA) could successfully express a considerable amount of Bdscys-A protein. These results show that the signal peptide sequence of Bdscys-A completely blocked the protein synthesis in E. coli. Finally, the antimicrobial test results showed that the antimicrobial activity of Bdscys-A was not significant, and it is speculated that the antimicrobial effect of Bdscys-A could be enhanced after enzymatic digestion. It is expected, in the future, that the antimicrobial peptide would be stabilized through the substitution of amino acids, and could be developed into a widely used antimicrobial agent.
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