A cDNA clone coding for a novel allergen, Cla h III, of Cladosporium herbarum identified as a ribosomal P2 protein.
Mold allergens represent a major cause of atopic disorders. Progress in the molecular characterization of allergens has been hampered by batch-to-batch variation and poor yields in mold extracts. In the present study, we established a cDNA library in lambda ZAP II by using mRNA isolated from a major allergen-producing mold, Cladosporium herbarum. From this library, a novel allergen has been cloned and sequenced. The clone encodes a full length protein of 111 amino acids with a molecular mass of 11.1 kDa and pI of 3.94. By using sequence homology analysis, the allergen was found to belong to the ribosomal P2 protein family. Approximately 60% peptide sequence homology was found between the cloned protein and other known fungal ribosomal P2 proteins. In addition to conserved C-terminal sequences and serine blocks for phosphorylation in all eukaryotic ribosomal P2 proteins, this protein also contains a potential N-glycosylation site at position 15-17. Northern blots demonstrated that mRNA molecules of this gene are present even at late stages of culture. The gene was expressed in Escherichia coli by using the pMAL-2c system, and murine antisera against the recombinant allergen (rCh2.1) were generated. The antisera revealed that the native allergen corresponding to rCh2.1 was present in extracts prepared by grinding mycelia under liquid nitrogen in the presence of protease inhibitors, but absent in extracts prepared by conventional methods. This result indicates the usefulness of recombinant allergens in characterization and standardization of mold allergenic extracts.
Zhang
L
L
Life Sciences Division, Health Canada, Ottawa, Ontario.
Muradia
G
G
Curran
I H
IH
Rode
H
H
Vijay
H M
HM
D000485:Allergens / Q000235:genetics*; D000595:Amino Acid Sequence; D000818:Animals; D000946:Antigens, Fungal / Q000235:genetics*; D001483:Base Sequence; D015152:Blotting, Northern; D015153:Blotting, Western; D002963:Cladosporium / Q000235:genetics* / Q000276:immunology*; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000032:analysis; D015723:Gene Library; D006801:Humans; D007073:Immunoglobulin E / Q000276:immunology; D051379:Mice; D008969:Molecular Sequence Data; D012269:Ribosomal Proteins / Q000235:genetics*; D017386:Sequence Homology, Amino Acid
D000485:Allergens; D000946:Antigens, Fungal; D018076:DNA, Complementary; D012269:Ribosomal Proteins; C091443:allergen Cla h III; D007073:Immunoglobulin E