Brasiliensin:: A novel intestinal thrombin inhibitor from Triatoma brasiliensis (Hemiptera: Reduviidae) with an important role in blood intake
Brasiliensin:: A novel intestinal thrombin inhibitor from Triatoma brasiliensis (Hemiptera: Reduviidae) with an important role in blood intake
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DOI：
10.1016/j.ijpara.2007.04.017
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发表时间：
2007-10-01
影响因子：
4
通讯作者：
Pereira, M. H.
中科院分区：
文献类型：
--
作者：
Araujo, R. N.;Campos, I. T. N.;Pereira, M. H.
迄今为止研究的每一种吸血无脊椎动物都产生至少一种凝血抑制剂。其中，凝血酶抑制剂最常被分离。为了研究巴西锥蝽凝血酶抑制剂的生物学意义，我们对巴西锥蝽凝血酶抑制剂基因进行了序列测定，并对其生物学功能进行了评价。霸王对巴西肠凝血酶抑制剂（brasiliensin）进行了测序，并设计了合成双链RNA（dsRNA）的引物。通过两次注射15 μ g dsRNA到四龄幼虫中诱导基因敲除（RNAi）。第二次注射后48小时，每组的虫子被允许以仓鼠为食。PCR结果显示，与对照组相比，注射dsRNA使敲除大肠杆菌中前中肠中的brasiliensin表达降低约71%。通过凝血酶抑制活性测定和柠檬酸化血浆凝固时间测定证实了基因表达的减少，这两项测定分别显示活性减少了18倍和3.5倍。敲除小鼠比对照组少摄入约39%的血液。为了证实巴西林菌素在血液摄取中的重要性，在血液喂养之前，允许四龄幼虫单独摄取喂养溶液或含有15 U凝血酶的喂养溶液。55%的血液摄入量减少了凝血酶喂养前的血液喂养。结果表明，中肠中的抗凝活性是从宿主身上取血量的重要决定因素。在血液摄入过程中抗凝剂的作用进行了讨论，根据这种新的见解。(c)2007年澳大利亚寄生虫学会由爱思唯尔有限公司出版。保留所有权利。
Every hematophagous invertebrate studied to date produces at least one inhibitor of coagulation. Among these, thrombin inhibitors have most frequently been isolated. In order to study the thrombin inhibitor from Triatoma brasiliensis and its biological significance for the bug, we sequenced the corresponding gene and evaluated its biological function. The T. brasiliensis intestinal thrombin inhibitor, termed brasiliensin, was sequenced and primers were designed to synthesize double strand RNA (dsRNA). Gene knockdown (RNAi) was induced by two injections of 15 mu g of dsRNA into fourth instar nymphs. Forty-eight hours after the second injection, bugs from each group were allowed to feed on hamsters. PCR results showed that injections of dsRNA reduced brasiliensin expression in the anterior midgut by approximately 71% in knockdown nymphs when compared with controls. The reduction in gene expression was confirmed by the thrombin inhibitory activity assay and the citrated plasma coagulation time assay which showed activity reductions of similar to 18- and similar to 3.5-fold, respectively. Knockdown nymphs ingested approximately 39% less blood than controls. In order to confirm the importance of brasiliensin in blood ingestion, fourth instar nymphs were allowed to ingest feeding solution alone or feeding solution containing 15 U of thrombin prior to blood feeding. Fifty-five percent less blood was ingested by nymphs which were fed thrombin prior to blood feeding. The results suggest that anticoagulant activity in the midgut is an important determinant of the amount of blood taken from the host. The role of anticoagulants during blood ingestion is discussed in the light of this novel insight. (c) 2007 Australian Society for Parasitology Inc. Published by Elsevier Ltd. All rights reserved.