RESUMO
Recombinant production in bacteria of soluble and monomeric Phl p 1, a major allergen of Timothy grass pollen, has proved to be very problematic. In order to facilitate expression and purification of this allergen, a recombinant variant was designed with a single amino acid substitution. Several comparative analyses with natural counterparts using electrophoretic and HPLC separations, together with immunological assays, demonstrated high equivalence. This is the first description of an approach aiming at an improvement of a natural like recombinant allergen.
Assuntos
Alérgenos/metabolismo , Proteínas de Plantas/metabolismo , Alérgenos/química , Alérgenos/genética , Sequência de Aminoácidos , Cromatografia Líquida de Alta Pressão , Dicroísmo Circular , Eletroforese em Gel de Poliacrilamida , Imuno-Histoquímica , Dados de Sequência Molecular , Peso Molecular , Mapeamento de Peptídeos , Proteínas de Plantas/química , Proteínas de Plantas/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Homologia de Sequência de AminoácidosRESUMO
Allergic airway diseases are often caused by house dust mites (HDM) and storage mites (SM), respectively, and we were recently able to demonstrate that symptomatic German farmers are frequently sensitized to different mite species. The present study aimed to obtain information on the protein and immunobiochemical characteristics of the extracts of the following mites: Dermatophagoides pteronyssinus, Dermatophagoides farinae, Acarus siro, Acarus farris, Lepidoglyphus destructor, Tyrophagus putrescentiae, Blomia tjibodas, Blomia topicalis, Blomia kulagini, Glycyphagus domesticus, Thyreophagus entomophagus, and Cheyletus eruditus. Specific IgE determinations were performed with EAST. The protein patterns of the mite extracts were studied by SDS-PAGE estimating the weighted-average molecular weights of the proteins. Using the Western blot technique, we determined the allergen pattern in several mite extracts. The allergens in each extract were classified in terms of the frequency of sensitization as major, intermediate, and minor allergens using allergograms. As already reported, a positive EAST to at least one mite was measured in 31/86 patients, and most sera were positive with several mite species. The majority of the mite extracts exhibited a very complex protein pattern according to SDS-PAGE. An allergen was found in Western blots of nearly all species equivalent to a molecular weight of 14 to 15 kD. This was, in our opinion, the first time that this particular allergen was detected in Blomia tjibodas and Glycyphagus domesticus. In conclusion, using EAST we were able to show that 36% of the German farmers tested were sensitized to SM. All mite extracts showed a complex protein pattern in the molecular weight range -95 kD to -10 kD. A common allergen band in the region of -14 to -15 kD was found in the majority of the mites studied, and it can be assumed that this corresponds with the group 2 allergen.
