Selective cloning of peanut allergens, including profilin and 2S albumins, by phage display technology.
BACKGROUND
Peanut kernels contain many allergens able to elicit IgE-mediated type 1 allergic reactions in sensitized individuals. Sera from sensitized patients recognize variable patterns of IgE-binding proteins. The identification of the IgE-binding proteins of peanut extract would faciliate improvement of diagnostic and immunotherapeutic approaches as well as development of sensitive test systems for the detection of hidden peanut allergens present as additives in various industrial food products and the investigation of their stability during processing of food products.


METHODS
We applied the pJuFo cloning system based on the phage surface display of functional cDNA expression products to clone cDNAs encoding peanut allergens. Sera (n = 40) of peanut-allergic individuals were selected according to case history, radioallergosorbent test and immunoblot analysis to demonstrate IgE binding towards the newly identified recombinant allergens.


RESULTS
In addition to the known allergens Ara h 1 and Ara h 2 we were able to identify four allergens with estimated molecular weights of 36, 16, 14.5 and 14 kDa. Three of them formally termed Ara h 4, Ara h6 and Ara h 7 show significant sequence similarities to the family of seed storage proteins and the fourth (Ara h 5) corresponds to the well-known plant allergen profilin. Immunoblotting of the six expressed recombinant allergens with 40 patients sera shows 14 individual recognition patterns and the following frequency of specific IgE binding: Ara h 1 was recognized by 65%, Ara h 2 by 85%, Ara h 4 by 53%, Ara h 5 by 13%, Ara h 6 by 38% and Ara h 7 by 43% of the selected sera.


CONCLUSIONS
All of the selected peanut-positive sera can detect at least one of the six identified recombinant allergens which can be used to establish individual patients' reactivity profiles. A comparison of these profiles with the clinical data will possibly allow a further insight into the relationship between clinical severity of the symptoms and specific IgE levels towards the six peanut allergens.
Kleber-Janke
T
T
Research Center Borstel, Germany. tkleber@fz-borstel.de
Crameri
R
R
Appenzeller
U
U
Schlaak
M
M
Becker
W M
WM
D055516:2S Albumins, Plant; D000485:Allergens / Q000235:genetics* / Q000276:immunology; D000595:Amino Acid Sequence; D052179:Antigens, Plant; D010367:Arachis / Q000737:chemistry* / Q000276:immunology; D010582:Bacteriophage lambda / Q000235:genetics*; D015152:Blotting, Northern; D015153:Blotting, Western; D003001:Cloning, Molecular / Q000379:methods; D003285:Contractile Proteins; D018076:DNA, Complementary / Q000032:analysis; D015723:Gene Library; D006023:Glycoproteins / Q000235:genetics / Q000276:immunology; D006801:Humans; D007073:Immunoglobulin E / Q000378:metabolism; D008565:Membrane Proteins; D008840:Microfilament Proteins / Q000235:genetics* / Q000276:immunology; D008969:Molecular Sequence Data; D010940:Plant Proteins / Q000235:genetics* / Q000276:immunology; D051304:Profilins; D011498:Protein Precursors / Q000235:genetics* / Q000276:immunology; D011994:Recombinant Proteins / Q000378:metabolism; D055314:Seed Storage Proteins
D055516:2S Albumins, Plant; D000485:Allergens; D052179:Antigens, Plant; C500889:Ara h 1 protein, Arachis hypogaea; C500890:Ara h 2 allergen, Arachis hypogaea; C078642:Ara h II protein, Arachis hypogaea; D003285:Contractile Proteins; D018076:DNA, Complementary; D006023:Glycoproteins; D008565:Membrane Proteins; D008840:Microfilament Proteins; D010940:Plant Proteins; D051304:Profilins; D011498:Protein Precursors; D011994:Recombinant Proteins; D055314:Seed Storage Proteins; C118207:allergen Ara h3; D007073:Immunoglobulin E