Smith W A, Thomas W R
TVW Telethon Institute for Child Health Research, Perth, Australia.
Clin Exp Allergy. 1996 May;26(5):571-9.
The trypsin-like protein Der p 3 is a major allergen of Dermatophagoides pteronyssinus. Like other vertebrate and invertebrate trypsin-like molecules, isoelectric-focusing studies with the natural Der p 3 protein have indicated that several isoforms exist.
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.
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 PCR analysis of Eco R1 digested genomic DNA was performed.
Southern blot analysis of Eco R1 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 differed 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.
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 1 and group 2 house dust mite allergens.
类胰蛋白酶蛋白Der p 3是粉尘螨的主要变应原。与其他脊椎动物和无脊椎动物的类胰蛋白酶分子一样，对天然Der p 3蛋白进行的等电聚焦研究表明存在几种同工型。
确定Der p 3变应原的序列变异程度，并在分子水平上区分序列同工型是代表变应原的等位基因变体还是多个基因。
使用放射性标记的聚合酶链反应（PCR）Der p 3 P3WS1探针，从λgt10粉尘螨文库中分离出5个Der p 3的cDNA克隆并进行测序。对经Eco R1消化的基因组DNA进行Southern印迹和反向PCR分析。
对经Eco R1消化的基因组DNA进行的Southern印迹分析表明，编码Der p 3的DNA位于一个3.5 kb的单一片段上，对该DNA进行的反向聚合酶链反应分析（PCR）表明，在这个3.5 kb的片段上只有一个Der p 3基因。其中一个克隆的核苷酸序列与原始的Der p 3 P3WS1克隆相同，另外两个克隆仅在其3'非翻译序列上有所不同。另外两个克隆含有核苷酸变化，导致在氨基酸水平上有几个保守和非保守的替换。克隆3与Der p 3 P3WS1的同一性为98.7%。一个无法获得完整序列的克隆（克隆4）与原始克隆的同一性仅为84.4%，因此与一种异源变应原一致。
这些数据以及我们之前的基因组序列表明，在大多数情况下，Der p 3变应原只有微小的序列变异（变体），尽管克隆4所示的异源变应原需要进一步研究。现在很明显，Der p 3由一个单一基因编码，并且从商业螨虫构建的大多数cDNA克隆仅显示出与1组和2组屋尘螨变应原类似的微小序列变异。
