SEQUENCE POLYMORPHISMS OF THE DER-P-3 HOUSE-DUST MITE ALLERGEN

Background The trypsin-like protein Der p 3 is a major allergen of Der matophagoides pteronyssinus. Like other vertebrate and invertebrate tr ypsin-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 lambda gt10 D. pteronyssinus library, using a radiolabelled polymerase chain reaction (PCR) Der p 3 P3WS1 probe and sequenced. Southern blot and inverse PC R analysis of Eco R1 digested genomic DNA was performed. Results South ern blot analysis of Eco R1 digested genomic DNA showed that the DNA e ncoding Der p 3 was located on a single 3.5 kb fragment and inverse po lymerase chain reaction analysis (PCR) of this DNA showed that there w as 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 P3 WS1 clone and two clones differed only in their 3' untranslated sequen ces. The other two contained nucleotide changes which lead to several substitutions at the amino acid level, both conservative and non conse rvative. 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% ide ntity with the original clone and is therefore consistent with an isoa llergen. Conclusions These data along with our previous genomic sequen ce shows that for the most part, the Der p 3 allergen has only minor s equence variations (variants) although the isoallergen indicated by cl one 4 needs further investigation. It is now evident that Der p 3 is e ncoded by a single gene and that most cDNA clones constructed from com mercial mites show only minor sequence variation similar to that obser ved for the group 1 and group 2 house dust mite allergens.