AN ALLERGENIC POLYPEPTIDE REPRESENTING A VARIABLE REGION OF HSP-70 CLONED FROM A CDNA LIBRARY OF CLADOSPORIUM-HERBARU

Background Extracts of Cladosporium herbarum, a major source of fungal aeroallergens, exhibit a complex profile of IgE-binding proteins. Yie lds of conventionally purified allergens from this mold have been insu fficient to permit further molecular analyses. Objective To enhance an d simplify the purification of allergens from C. herbarum, we have sou ght to use recombinant DNA techniques to clone, identify and bacterial ly express immunoselected C. herbal um allergens. Methods We construct ed a cDNA library in lambda ZAP II using mRNA isolated from C. herbaru m. From this library, phage clones encoding a new allergen were immuno selected using pooled human atopic IgE. The cloned cDNA was excised fr om the phage vector as a recombinant pBluescript II SK-phagemid and se quenced. Expression of the recombinant allergen was carried out in E. coli XL1-blue transformants of the phagemid. Bacterial lysates from ce lls induced to express the cloned allergen were immunoblotted and prob ed with individual human atopic IgEs. Results The cDNA clone encodes a 278 amino acid polypeptide homologous to the C-terminal portion of 70 kDa heat shock protein (hsp 70). The polypeptide possesses features c ommon to other hsps 70, i.e. a similar hydropathic profile and a varia ble C-terminal region with conserved sequence at the very C-terminus. Binding of the recombinant peptide to IgE from 38% of atopic sera or p lasma from individuals allergic to C. herbarum was demonstrated. Concl usion These results indicate that amino acid substitutions are relativ ely conserved even in the variable C-terminal regions of hsp 70 specie s. Thus, this study should draw attention to the possibility of induct ion of anaphylactic responses in a sensitized individual when hsp 70 f rom any pathogenic species is administered for vaccination.