Janwan Penchom, Intapan Pewpan M, Laummaunwai Porntip, Rodpai Rutchanee, Wongkham Chaisiri, Insawang Tonkla, Thanchomnang Tongjit, Sanpool Oranuch, Maleewong Wanchai
Department of Medical Technology, School of Allied Health Sciences and Public Health, Walailak University, Nakhon Si Thammarat 80161, Thailand; Research and Diagnostic Center for Emerging Infectious Diseases, Faculty of Medicine, Khon Kaen University, Khon Kaen 40002, Thailand.
Research and Diagnostic Center for Emerging Infectious Diseases, Faculty of Medicine, Khon Kaen University, Khon Kaen 40002, Thailand; Department of Parasitology, Faculty of Medicine, Khon Kaen University, Khon Kaen 40002, Thailand.
Exp Parasitol. 2015 Dec;159:53-8. doi: 10.1016/j.exppara.2015.08.010. Epub 2015 Aug 25.
Gnathostoma spinigerum is the causative agent of human gnathostomiasis. The advanced third stage larva (AL3) of this nematode can migrate into the subcutaneous tissues, including vital organs, often producing severe pathological effects. This study performed immuno-proteomic analysis of antigenic spots, derived from G. spinigerum advanced third stage larva (GSAL3) and recognized by human gnathostomiasis sera, using two-dimensional (2-DE) gel electrophoresis based-liquid chromatography/tandem mass spectrometry (LC/MS-MS), and followed by the aid of a database search. The crude GSAL3 extract was fractionated using IPG strips (pH 3-11NL) and followed by SDS-PAGE in the second dimension. Each gel was stained with colloidal Coomassie blue or was electro-transferred onto a nitrocellulose membrane and probed with gnathostomiasis human sera by immunoblotting. Individual Coomassie-stained protein spots corresponding to the antigenic spots recognized by immunoblotting were excised and processed using LC/MS-MS. Of the 93 antigenic spots excised, 87 were identified by LC/MS-MS. Twenty-seven protein types were found, the most abundant being Ascaris suum37. Six spots showed good quality spectra, but could not be identified. This appears to be the first attempt to characterize antigenic proteins from GSAL3 using a proteomic approach. Immuno-proteomics shows promise to assist the search for candidate proteins for diagnosis and vaccine/drug design and may provide better understand of the host-parasite relationship in human gnathostomiasis.
棘颚口线虫是人类颚口线虫病的病原体。这种线虫的晚期第三期幼虫（AL3）可迁移至皮下组织，包括重要器官，常产生严重的病理影响。本研究采用基于二维（2-DE）凝胶电泳的液相色谱/串联质谱（LC/MS-MS），并借助数据库搜索，对源自棘颚口线虫晚期第三期幼虫（GSAL3）且被人类颚口线虫病血清识别的抗原斑点进行免疫蛋白质组学分析。GSAL3粗提物使用IPG条（pH 3-11NL）进行分级分离，然后在第二维进行SDS-PAGE。每块凝胶用考马斯亮蓝胶体染色，或电转移到硝酸纤维素膜上，通过免疫印迹法用颚口线虫病人血清进行检测。切除与免疫印迹法识别的抗原斑点相对应的考马斯亮蓝染色的单个蛋白质斑点，并使用LC/MS-MS进行处理。在切除的93个抗原斑点中，有87个通过LC/MS-MS鉴定出来。共发现27种蛋白质类型，其中最丰富的是猪蛔虫37。有6个斑点显示出高质量的光谱，但无法鉴定。这似乎是首次尝试使用蛋白质组学方法对GSAL3的抗原蛋白进行表征。免疫蛋白质组学有望协助寻找诊断和疫苗/药物设计的候选蛋白质，并可能更好地理解人类颚口线虫病中的宿主-寄生虫关系。
