Objective:To characterize the major allergens of Macrobrachium rosenbergii.(giant freshwater prawn).Methods:Raw and cooked extracts of the giant freshwater prawn were prepared.The IgE reactivity pattern was identified...Objective:To characterize the major allergens of Macrobrachium rosenbergii.(giant freshwater prawn).Methods:Raw and cooked extracts of the giant freshwater prawn were prepared.The IgE reactivity pattern was identified by sodium dodecyl sulfate polyaerylamide gel electrophoresis(SDS-PAGE)and immunoblotting technique with the sera of 20 skin prick test(SPT)positive patients.The major allergen identified was then characterized using the proteomics approach involving a combination of two-dimensional(2-DE)electrophoresis,mass spectrometry and bioinformatics tools.Results:SDS-PAGE of the raw extract showed 23 protein bands(15-250 kDa)but those ranging from 40 to 100 kDa were not found in the cooked extract.From immunoblotting experiments,raw and cooked extracts demonstrated 11 and 5 IgE-binding proteins,respectively,with a molecular mass ranging from 15 to 155 kDa.A heat-resistant 36 kDa protein was identified as the major allergen of both extracts.In addition,a 42 kDa heat-sensitive protein was shown to be a major allergen of the raw extract.The 2-DE gel fractionated the prawn proteins to more than 50 different protein spots.Of these,10 spots showed specific:IgE reactivity with patients'sera.Matrix assisted laser desorption/ionization-lime of flight(MALDI-TOF)analysis led to identification of 2 important allergens,tropomyosin and arginine kinase.Conclusions:It can be concluded that the availability of such allergens would help in component-based diagnosis and therapy of prawn allergies.展开更多
黑腹果蝇(Drosophila melanogaster)是研究宿主与病毒互作机制的模式生物,RNA干扰(RNA interference,RNAi)是果蝇最主要的抗病毒机制。果蝇C病毒(Drosophila C virus,DCV)是天然感染果蝇的正链RNA病毒,通过编码RNA沉默抑制子(viral supp...黑腹果蝇(Drosophila melanogaster)是研究宿主与病毒互作机制的模式生物,RNA干扰(RNA interference,RNAi)是果蝇最主要的抗病毒机制。果蝇C病毒(Drosophila C virus,DCV)是天然感染果蝇的正链RNA病毒,通过编码RNA沉默抑制子(viral suppressors of RNAi,VSR)1A蛋白(DCV1A)来抑制果蝇RNAi通路。本研究以DCV1A为诱饵蛋白,通过免疫共沉淀(co-immunoprecipitation,Co-IP)和质谱分析,获得了与DCV1A可能存在互作的108个靶蛋白。通过免疫共沉淀和Western blot分析,在果蝇S2细胞中验证了DCV1A与原肌球蛋白2(tropomyosin 2,TM2)存在互作。当细胞感染DCV后,细胞内Tm2的表达水平被显著诱导上调;通过RNAi干扰细胞中Tm2的表达后DCV复制水平显著增加,过表达Tm2基因后DCV复制水平有所下降。在S2细胞中共表达DCV1A和Tm2基因时,DCV1A蛋白水平显著下调,DCV复制也受到抑制。在Tm2突变型果蝇和野生型果蝇的胸腔内注射DCV,监测存活率发现,与野生型果蝇相比,Tm2基因突变体果蝇对DCV更为敏感。研究结果表明,在果蝇细胞水平和成虫水平上Tm2均发挥抑制DCV复制的作用。展开更多
基金Supported by a research grant from UPSI(grant No.UPSI2011-0018-102-01)
文摘Objective:To characterize the major allergens of Macrobrachium rosenbergii.(giant freshwater prawn).Methods:Raw and cooked extracts of the giant freshwater prawn were prepared.The IgE reactivity pattern was identified by sodium dodecyl sulfate polyaerylamide gel electrophoresis(SDS-PAGE)and immunoblotting technique with the sera of 20 skin prick test(SPT)positive patients.The major allergen identified was then characterized using the proteomics approach involving a combination of two-dimensional(2-DE)electrophoresis,mass spectrometry and bioinformatics tools.Results:SDS-PAGE of the raw extract showed 23 protein bands(15-250 kDa)but those ranging from 40 to 100 kDa were not found in the cooked extract.From immunoblotting experiments,raw and cooked extracts demonstrated 11 and 5 IgE-binding proteins,respectively,with a molecular mass ranging from 15 to 155 kDa.A heat-resistant 36 kDa protein was identified as the major allergen of both extracts.In addition,a 42 kDa heat-sensitive protein was shown to be a major allergen of the raw extract.The 2-DE gel fractionated the prawn proteins to more than 50 different protein spots.Of these,10 spots showed specific:IgE reactivity with patients'sera.Matrix assisted laser desorption/ionization-lime of flight(MALDI-TOF)analysis led to identification of 2 important allergens,tropomyosin and arginine kinase.Conclusions:It can be concluded that the availability of such allergens would help in component-based diagnosis and therapy of prawn allergies.
文摘黑腹果蝇(Drosophila melanogaster)是研究宿主与病毒互作机制的模式生物,RNA干扰(RNA interference,RNAi)是果蝇最主要的抗病毒机制。果蝇C病毒(Drosophila C virus,DCV)是天然感染果蝇的正链RNA病毒,通过编码RNA沉默抑制子(viral suppressors of RNAi,VSR)1A蛋白(DCV1A)来抑制果蝇RNAi通路。本研究以DCV1A为诱饵蛋白,通过免疫共沉淀(co-immunoprecipitation,Co-IP)和质谱分析,获得了与DCV1A可能存在互作的108个靶蛋白。通过免疫共沉淀和Western blot分析,在果蝇S2细胞中验证了DCV1A与原肌球蛋白2(tropomyosin 2,TM2)存在互作。当细胞感染DCV后,细胞内Tm2的表达水平被显著诱导上调;通过RNAi干扰细胞中Tm2的表达后DCV复制水平显著增加,过表达Tm2基因后DCV复制水平有所下降。在S2细胞中共表达DCV1A和Tm2基因时,DCV1A蛋白水平显著下调,DCV复制也受到抑制。在Tm2突变型果蝇和野生型果蝇的胸腔内注射DCV,监测存活率发现,与野生型果蝇相比,Tm2基因突变体果蝇对DCV更为敏感。研究结果表明,在果蝇细胞水平和成虫水平上Tm2均发挥抑制DCV复制的作用。
