Langner Kathrin F A, Darpel Karin E, Denison Eric, Drolet Barbara S, Leibold Wolfgang, Mellor Philip S, Mertens Peter P C, Nimtz Manfred, Greiser-Wilke Irene
USDA-ARS, Arthropod-Borne Animal Diseases Research Laboratory, 1000 E. University Ave., Laramie, WY 82071, USA.
J Med Entomol. 2007 Mar;44(2):238-48. doi: 10.1603/0022-2585(2007)44[238:caaosp]2.0.co;2.
Salivary proteins of hematophagous Culicoides spp. are thought to play an important role in pathogen transmission and skin hypersensitivity. Analysis of these proteins, however, has been problematic due to the difficulty in obtaining adequate amounts of secreted Culicoides saliva. In the current study, a collection method for midge saliva was developed. Over a 3-d period, 3- to 5-d-old male and female Culicoides nubeculosus Meigen (Diptera: Ceratopogonidae) were repeatedly placed onto the collection system and allowed to deposit saliva into a filter. Salivary products were eluted from the filters and evaluated by gel electrophoresis and mass spectrometry as well as by intradermal testing and determination of clotting time. Gel electrophoresis revealed approximately 55 protein spots displaying relative molecular masses from 5 to 67 kDa and isoelectric points ranging from 4.5 to 9.8. The majority of molecular species analyzed by mass spectrometry showed high convergence with salivary proteins recently obtained from a cDNA library of Culicoides sonorensis Wirth & Jones, including proteins involved in sugarmeal digestion, defense, and coagulation inhibition as well as members of the D7 family and unclassified salivary proteins. In addition, the proteome analysis revealed a number of peptides that were related to proteins from insect species other than Culicoides. Intradermal injection of the saliva in human skin produced edema, vasodilatation, and pruritus. The anticoagulant activity of the saliva was demonstrated by significantly prolonged clotting times for human platelets. The potential role of the identified salivary proteins in the transmission of pathogens and the induction of allergies is discussed.
吸血蠓类的唾液蛋白被认为在病原体传播和皮肤超敏反应中起重要作用。然而，由于难以获得足够量的分泌型蠓唾液，对这些蛋白质的分析一直存在问题。在本研究中，开发了一种蠓唾液的收集方法。在3天的时间里，将3至5日龄的雄性和雌性淡黄库蠓（双翅目：蠓科）反复放置在收集系统上，使其将唾液沉积到过滤器中。从过滤器中洗脱唾液产物，并通过凝胶电泳、质谱分析以及皮内试验和凝血时间测定进行评估。凝胶电泳显示约55个蛋白点，其相对分子质量为5至67 kDa，等电点范围为4.5至9.8。通过质谱分析的大多数分子种类与最近从索诺拉库蠓的cDNA文库中获得的唾液蛋白高度一致，包括参与糖餐消化、防御和凝血抑制的蛋白质以及D7家族成员和未分类的唾液蛋白。此外，蛋白质组分析还揭示了一些与蠓以外昆虫物种的蛋白质相关的肽段。将唾液皮内注射到人体皮肤中会产生水肿、血管扩张和瘙痒。唾液对人血小板的凝血时间显著延长，证明了其抗凝活性。文中讨论了已鉴定的唾液蛋白在病原体传播和过敏诱导中的潜在作用。
