Background The trypsin-like protein Der p 3 is a major allergen of Dermatophagoides pteronyssinus. Like other vertebrate and invertebrate trypsin-like molecules, isoelectricfocusing studies with the natural Der p 3 protein have indicated that several isoforms exist.
Objective To determine the extent of the sequence variation of the Der p 3 allergen and distinguish at the molecular level, whether the sequence isoforms represent allelic variants or multiple genes of the allergen.
Methods Five cDNA clones of Der p 3 have been isolated from a λ gt 10 D. pteronyssinus library, using a radiolabelled polymerase chain reaction (PCR) Der p 3 P3WS1 probe and sequenced. Southern blot and inverse PCR analysis of Eco RI digested genomic DNA was performed.
Results Southern blot analysis of Eco RI digested genomic DNA showed that the DNA encoding Der p 3 was located on a single 3.5 kb fragment and inverse polymerase chain reaction analysis (PCR) of this DNA showed that there was only a single Der p 3 gene on this 3.5 kb fragment. The nucleotide sequence of one of the clones was identical to the original Der p 3 P3WS1 clone and two clones ditfered only in their 3′untranslated sequences. The other two contained nucleotide changes which lead to several substitutions at the amino acid level, both conservative and non conservative. Clone 3 had 98.7% identity with Der p 3 P3WS1. One clone for which the full sequence was not available (clone 4) had only 84.4% identity with the original clone and is therefore consistent with an isoallergen.
Conclusions These data along with our previous genomic sequence shows that for the most part, the Der p 3 allergen has only minor sequence variations (variants) although the isoallergen indicated by clone 4 needs further investigation. It is now evident that Der p 3 is encoded by a single gene and that most cDNA clones constructed from commercial mites show only minor sequence variation similar to that observed for the group I and group 2 house dust mite allergens.