Clinical & Experimental Allergy

An allergenic polypeptide representing a variable region of hsp 70 cloned from a cDNA library of Cladosporium herbarum

Authors

  • L. ZHANG,

    1. Life Sciences Division, Bureau of Drug Research, Health Protection Branch. Health Canada, Ottawa, Canada
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    • Current address: Immunochemislry Division. National Research Council ol Canada. 100 Sussex Drive, Ottawa. ON KIA 0R6. Canada.

  • G. MURADIA,

    1. Life Sciences Division, Bureau of Drug Research, Health Protection Branch. Health Canada, Ottawa, Canada
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  • M. W. DE VOUGE,

    1. Life Sciences Division, Bureau of Drug Research, Health Protection Branch. Health Canada, Ottawa, Canada
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  • H. RODE,

    1. Bureau of Biologics, Drugs Directorate, Health Protection Branch. Health Canada, Ottawa, Canada
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  • H. M. VIJAY

    Corresponding author
    1. Life Sciences Division, Bureau of Drug Research, Health Protection Branch. Health Canada, Ottawa, Canada
      Dr H. M. Vijay, Life Sciences Division. Bureau of Drug Research. Health Canada. Postal Locator 2201C. Ottawa. ON. Canada KIA 0L2.
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Dr H. M. Vijay, Life Sciences Division. Bureau of Drug Research. Health Canada. Postal Locator 2201C. Ottawa. ON. Canada KIA 0L2.

Summary

Background Extracts of Clado.sporium herharum, a major source of fungal aeroaller-gens. exhibit a complex profile of IgE-binding proteins. Yields of conventionally purified allergens from this mold have been insufficient to permit further molecular analyses.

Objective To enhance and simplify the purification of allergens from C. herbarum, we have sought to use recombinant DNA techniques to clone, identify and bacterialiy express immunoselected C. herbarum allergens.

Methods We constructed a cDNA library in AZAP II using mRNA isolated from C. herbarum. From this library, phage clones encoding a new allergen were immunoselected using pooled human atopic IgE. The cloned cDNA was excised from the phage vector asa recombinant pBluescript II SK-phagemid und sequenced. Expression of the recombinant allergen was carried out in E. coli XLl-blue transformants of the phagemid. Bacterial lysates from cells induced to express the cloned allergen were immunoblotted and probed 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 common to other hsps 70. i.e. a similar hydropathic profile and a variable C-terminal region with conserved sequence at the very C-terminus. Binding of the recombinant peptide to IgE from 38% of atopic sera or plasma from individuals allergic to C. herbarum was demonstrated.

Conclusion These results indicate that amino acid substitutions are relatively conserved even in the variable C-tcrminal regions ofhsp 70 species. Thus, this study should draw attention to the possibility of induction of anaphylactic responses in a sensitized individual when hsp 70 from any pathogenic species is administered for vaccination.

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