ABSTRACT
House dust mite antigens have been used for decades to diagnose allergic diseases in humans and animals. The objective of this study was to identify allergens in commercial Dermatophagoides farinae and Blomia tropicalis extracts by immunoblotting using sera from allergic dogs and anti-dog IgE conjugate. The analysis of antigens present in the D. farinae extract (FDA Allergenic) using sera from 10 dogs allergic to D. farinae showed that eight sera recognized a band of approximately 102 kDa, eight recognized two bands of 52 to 76 kDa, five recognized one band of approximately 76 kDa, four recognized one band of 31 to 38 kDa, and two recognized one band of 12 to 17 kDa. Immunoblot assays of the B. tropicalis extract (FDA Allergenic) using sera from 10 animals allergic to B. tropicalis showed that five sera recognized two bands of 52 to 76 kDa. These results demonstrate the importance of the two house dust mite species for the pathogenesis of canine atopic dermatitis in Brazil. In addition, the results indicate which allergens should be present in allergenic extracts used for diagnosis and allergen-specific immunotherapy.
Antígenos de ácaros da poeira domiciliar são utilizados por décadas para diagnóstico de doenças alérgicas em seres humanos e animais. O objetivo do presente trabalho foi identificar proteínas alergênicas presentes em extratos de Dermatophagoides farinae e Blomia tropicalis através de "immunoblotting" utilizando-se soros de animais alérgicos e conjugado anti-IgE canina. A análise por "immunoblotting" dos antígenos presentes no extrato de D. farinae (FDA Allergenic), utilizando soros de dez animais alérgicos, mostrou que oito soros reconhecem uma banda com peso molecular de aproximadamente 102 kDa; oito soros duas bandas entre 52 e 76 kDa; cinco soros uma banda com aproximadamente 76 kDa; quatro soros uma banda entre 31 e 38 kDa; e dois soros uma banda entre 12 e 17 kDa. A análise por "immunoblotting" dos antígenos do extrato de B. tropicalis (FDA Allergenic) mostrou que cinco soros reconhecem duas bandas com pesos moleculares entre 52 e 76 kDa. Esses resultados demonstram a importância dessas duas espécies de ácaros da poeira domiciliar na patogênese da dermatite atópica canina no Brasil, assim como indicam alérgenos que devem estar presentes nos extratos alergênicos utilizados para diagnóstico e imunoterapia alérgeno-específica.
Subject(s)
Animals , Dogs , Antigens, Dermatophagoides/isolation & purification , Dogs/immunology , Desensitization, Immunologic , Dermatitis, Atopic/veterinary , Immunoglobulin E/isolation & purification , Immunotherapy/veterinary , Allergens , Antigens , Mites/immunology , Dust/immunologyABSTRACT
Our objective was to clone, express and characterize adult Dermatophagoides farinae group 1 (Der f 1) allergens to further produce recombinant allergens for future clinical applications in order to eliminate side reactions from crude extracts of mites. Based on GenBank data, we designed primers and amplified the cDNA fragment coding for Der f 1 by nested-PCR. After purification and recovery, the cDNA fragment was cloned into the pMD19-T vector. The fragment was then sequenced, subcloned into the plasmid pET28a(+), expressed in Escherichia coli BL21 and identified by Western blotting. The cDNA coding for Der f 1 was cloned, sequenced and expressed successfully. Sequence analysis showed the presence of an open reading frame containing 966 bp that encodes a protein of 321 amino acids. Interestingly, homology analysis showed that the Der p 1 shared more than 87 percent identity in amino acid sequence with Eur m 1 but only 80 percent with Der f 1. Furthermore, phylogenetic analyses suggested that D. pteronyssinus was evolutionarily closer to Euroglyphus maynei than to D. farinae, even though D. pteronyssinus and D. farinae belong to the same Dermatophagoides genus. A total of three cysteine peptidase active sites were found in the predicted amino acid sequence, including 127-138 (QGGCGSCWAFSG), 267-277 (NYHAVNIVGYG) and 284-303 (YWIVRNSWDTTWGDSGYGYF). Moreover, secondary structure analysis revealed that Der f 1 contained an a helix (33.96 percent), an extended strand (17.13 percent), a ß turn (5.61 percent), and a random coil (43.30 percent). A simple three-dimensional model of this protein was constructed using a Swiss-model server. The cDNA coding for Der f 1 was cloned, sequenced and expressed successfully. Alignment and phylogenetic analysis suggests that D. pteronyssinus is evolutionarily more similar to E. maynei than to D. farinae.