Cloning of three new allergens from the dust mite Lepidoglyphus destructorusing phage surface display technology

The dust mite Lepidoglyphus destructor is a common species in Europe and a major cause of dust mite allergy in rural surroundings, but it also contrib utes to dust mite allergy in urban areas. One major allergen, Lep d 2, has been expressed as a recombinant protein and evaluated both in vivo and in v itro and shown to detect 60% or more of L. destructor-sensitized subjects. Additional recombinant allergens are needed to obtain a reliable diagnostic tool for L. destructor allergy. The aim of this study was to clone and exp ress new allergens from L. destructor and determine their recognition frequ ency among sensitized individuals. A phage display cDNA expression library was constructed and screened with sera from L. destructor-sensitized indivi duals. The cDNAs encoding the allergens were cloned into the pET17b vector and subsequently expressed in Escherichia coli as C-terminal His(6)-tagged proteins. Immunoblotting of the recombinant proteins was performed using se ra from 45 subjects allergic to L. destructor. Three new allergens from L. destructor, Ld 5 (originating from a partial Lep d 5 clone), Lep d 7 and Le p d 13, were identified and recognized by 4/45 (9%), 28/45 (62%) and 6/45 ( 13%) sera from L. destructor-sensitized subjects, respectively.