Mapping of the antigenic and allergenic epitopes of Lol p VB using gene fragmentation.
The recombinant proteins of Lol p VA and Lol p VB expressed in E. coli reacted with IgE antibodies from sera of allergic patients and mAbs FMC A7 and PpV1. Cross-absorption analyses using these recombinant proteins showed that Lol p VA and Lol p VB possess both similar and unique IgE binding determinants. Gene fragmentation was utilized to localize the antigenic and allergenic determinants of Lol p VB. When full-length cDNA of Lol p VB was digested into three fragments and expressed as the fusions from the glutathione transferase of pGEX vectors, fragments Met1-Val196 and Asp197-Val339 bound IgE while fragment Met1-Pro96 did not. The data suggest that there are at least two IgE binding determinants in Lol p VB. In addition, only fragment Met1-Val196 reacted with mAb PpV1. The localization of these determinants was further resolved using random fragment expression libraries. The mAb PpV1 determinant was near the N-terminal region of Lol p VB molecule. The IgE binding determinants were distributed in the central region: region I (amino acids 111-195) and II (199-254). These IgE binding determinants are conserved in Lol p VA.
Ong
E K
EK
School of Botany, University of Melbourne Victoria, Australia.
Knox
R B
RB
Singh
M B
MB
D000485:Allergens / Q000235:genetics / Q000276:immunology*; D052179:Antigens, Plant; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000235:genetics; D018604:Epitope Mapping; D015723:Gene Library; D006801:Humans; D006967:Hypersensitivity / Q000276:immunology*; D008129:Lolium / Q000276:immunology; D010446:Peptide Fragments / Q000737:chemistry / Q000276:immunology; D010940:Plant Proteins / Q000235:genetics / Q000276:immunology*; D011058:Pollen / Q000276:immunology; D013329:Structure-Activity Relationship
D000485:Allergens; D052179:Antigens, Plant; D018076:DNA, Complementary; C084941:Lol p V protein, Lolium perenne; D010446:Peptide Fragments; D010940:Plant Proteins