Cloning and expression of Blo t 1, a novel allergen from the dust mite Blomia tropicalis, homologous to cysteine proteases.
BACKGROUND
House dust mite allergens have been shown to be a very important stimulus in the causation of asthma and triggers for the exacerbation of symptoms. Therefore, characterization of mite-derived allergens at the molecular level is an important step for the development of effective diagnostic and therapeutic approaches, as well as for epidemiological studies.


OBJECTIVE
To clone, express and characterize at the molecular level the cysteine protease from Blomia tropicalis (Bt).


METHODS
A full-length cDNA encoding Blo t 1 was cloned from a Bt cDNA library using a PCR and RACE-based strategy. The cDNA was PCR-amplified, sequenced and subcloned into a prokaryotic expression vector. The allergenicity of the recombinant Blo t 1 was evaluated for IgE reactivity by Western blot.


RESULTS
Blo t 1 cDNA encodes a 221 amino acids polypeptide with an estimated molecular weight of 25 kDa. The recombinant protein is 35% identical to other mite cysteine proteases. Recombinant Blo t 1 (rBlo t 1) bound IgE from 62% of Bt skin test-positive serum. Dermatophagoides pteronyssinus (Dp) skin test-positive sera did not react with rBlo t 1, indicating the possible presence of unique IgE epitopes on the rBlo t 1 molecule. A three-dimensional image of Blo t 1, constructed based on predicted analysis, showed conserved secondary and tertiary structure with other cysteine proteases.


CONCLUSIONS
We report the cloning, expression and IgE reactivity of Blo t 1, a novel allergen from the domestic mite Blomia tropicalis (Bt), highly homologous to cysteine proteases. This putative cysteine protease, designated Blo t 1, may play a major role as an immunodominant allergen involved in dust mite-specific IgE-mediated hypersensitivity.
Mora
C
C
Department of Microbiology, University of Puerto Rico, Medical Sciences Campus, School of Medicine, San Juan, Puerto Rico.
Flores
I
I
Montealegre
F
F
Díaz
A
A
D000485:Allergens / Q000235:genetics*; D000595:Amino Acid Sequence; D000818:Animals; D000937:Antigen-Antibody Reactions; D052179:Antigens, Plant; D001483:Base Sequence; D015153:Blotting, Western; D003001:Cloning, Molecular; D003546:Cysteine Endopeptidases / Q000235:genetics*; D004391:Dust; D004926:Escherichia coli / Q000276:immunology; D007073:Immunoglobulin E / Q000276:immunology; D008925:Mites; D008969:Molecular Sequence Data; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid
D000485:Allergens; D052179:Antigens, Plant; C473655:Blo t 1allergen, Blomia tropicalis; D004391:Dust; D007073:Immunoglobulin E; D003546:Cysteine Endopeptidases