Cloning and characterisation of a group II allergen from the dust mite Tyrophagus putrescentiae

The complete cDNA encoding a major allergen from the dust mite Tyrophagus putrescentiae, Tyr p 2, has been sequenced and expressed. A degenerate primer was designed to the N-terminal amino acid sequence of the 16-kDa protein. The complete cDNA sequence was achieved by using reverse transcriptase PCR, PCR+1, standard cloning and sequencing techniques. The cDNA of Tyr p 2 is 552 nucleotides in length from the start codon including 126 nucleotides after the stop codon up to the beginning of the poly(A) tail. The leader sequence consists of 15 amino acids. Regarding the predicted amino acid sequence, there are no potential N glycosylation sites (N-X-S/T). The sequence showed similarity to group II allergens from other mite species, and some regions are completely conserved. To show that the cloned cDNA sequence was coding for an allergen, Tyr p 2 was expressed in Escherichia coli and shown to react with a T. putrescentiae-positive serum pool.