Medical and Forensic Entomology Research Group, School of Medicine and Health Sciences, Universidad del Rosario, Bogotá, Colombia.
Acta Trop. 2013 Dec;128(3):686-91. doi: 10.1016/j.actatropica.2013.09.020. Epub 2013 Sep 25.
Sarconesiopsis magellanica (Diptera: Calliphoridae) is a medically important necrophagous fly which is used for establishing the post-mortem interval. Diptera maggots release proteolytic enzymes contained in larval excretion and secretion (ES) products playing a key role in digestion. Special interest in proteolytic enzymes has also been aroused regarding understanding their role in wound healing since they degrade necrotic tissue during larval therapy. This study was thus aimed at identifying and characterising S. magellanica proteolytic enzyme ES products for the first time. These products were obtained from first-, second- and third-instar larvae taken from a previously-established colony. ES proteins were separated by SDS-PAGE and their proteolytic activity was characterised by zymograms and inhibition assays involving BAPNA (Nα-benzoyl-dl-Arg-p-nitroanilide) and SAPNA substrates, using synthetic inhibitors. The protein profile ranged from ∼69kDa to ∼23kDa; several of them coincided with the Lucilia sericata ES protein profile. Serine-protease hydrolysis activity (measured by zymogram) was confirmed when a ∼25kDa band disappeared upon ES incubation with PMSF inhibitor at pH 7.8. Analysis of larval ES proteolytic activity on BAPNA and SAPNA substrates (determined by using TLCK and TPCK specific inhibitors) suggested a greater amount of trypsin-like protease. These results support the need for further experiments aimed at validating S. magellanica use in larval therapy.
麦加利蝇（双翅目：丽蝇科）是一种重要的医学性嗜尸性蝇类，可用于推断死亡时间。双翅目蝇蛆释放的蛋白酶包含在幼虫排泄和分泌物（ES）产物中，在消化过程中起着关键作用。由于蛋白酶在幼虫治疗过程中降解坏死组织，因此人们对其在伤口愈合中的作用也产生了特别的兴趣。因此，本研究首次旨在鉴定和表征麦加利蝇的蛋白酶 ES 产物。这些产物是从小麦加利蝇先前建立的品系的一龄、二龄和三龄幼虫中获得的。ES 蛋白通过 SDS-PAGE 分离，其蛋白酶活性通过使用合成抑制剂的 BAPNA（Nα-苯甲酰基-dl-精氨酸-对硝基苯胺）和 SAPNA 底物的酶谱和抑制试验进行表征。蛋白图谱范围从约 69kDa 到约 23kDa；其中一些与丝光绿蝇 ES 蛋白图谱重合。当 ES 在 pH 7.8 下与 PMSF 抑制剂孵育时，约 25kDa 带消失，证实了丝氨酸蛋白酶水解活性（通过酶谱测量）。对 BAPNA 和 SAPNA 底物的幼虫 ES 蛋白酶活性分析（使用 TLCK 和 TPCK 特异性抑制剂确定）表明存在更多的胰蛋白酶样蛋白酶。这些结果支持需要进一步实验来验证麦加利蝇在幼虫治疗中的应用。
