Analysis of sequence polymorphism of a major mite allergen, Der p 2

The major house dust mite allergen Der p 2 has been regarded as an important allergen involved in the immunopathogenesis of allergic asthma and eczema.

To determine the degree of sequence polymorphism exists in Der p 2 at both the genomic DNA and cDNA levels.

Isolation of cDNA clones was performed by screening the cDNA libraries with Der p 2 cDNA probe and the genomic sequences for Der p 2 were obtained by PCR amplification from environmental dust mites with Der p 2-specific primers. DNA sequencing was carried out by the dideoxynucleotide chain termination method. The study of Der p 2 isoforms was performed by 2-D gel immunoblot analysis using mouse anti-Der p 2 serum.

In this study, we have characterized the sequences of three different gene alleles coding for the major house dust mite allergen Der p 2 at the cDNA level. The translated polypeptides from these clones differed from each other by 3-4 amino-acid residues. These polymorphic residues determined were also found in Der f 2 and they were located in regions containing T-epitopes. In addition, the genomic DNA sequences of Der p 2 which were obtained by PCR amplification using the environmental mites isolated from Taiwan and Australia have helped to confirm the authenticity of the polymorphisms detected in the cDNA clones generated from CSL cultured mites. Furthermore, 2D- immunoblot analysis indicated that there were at least 10 different isoforms (p 1 values range from greater than 7.0-5.9) of Der p 2 proteins produced by CSL cultured mites.

The results showed that there was a small but significant degree of sequence polymorphisms exists in Der p 2 gene alleles. Interestingly, the polymorphic residues were found in regions containing previously determined T-epitopes. The polymorphism data reported here will be important for the understanding of the immune responses of mite allergens as well as for the development of the peptide-based immunotherapeutic reagents for mite allergy.