Identification of an abundant allergen from the sheep louse, Bovicola ovis.
Infestation of sheep with the louse Bovicola ovis is common worldwide and leads to an allergic dermatitis referred to as 'scatter cockle'. IgE from an infested lamb was used in immunoaffinity chromatography to purify allergens from crude preparations of whole B. ovis and its faeces. SDS-PAGE of the affinity-purified eluates from both preparations showed a dominant band with M(r) of 28.5 kDa. Spleen cells from a mouse immunised with B. ovis faecal antigens were used to produce hybridomas which were screened by ELISA to identify those producing monoclonal antibodies (mAb) to the allergens purified by IgE immunoaffinity chromatography. Western blotting demonstrated that all of the mAbs examined recognised the 28.5 kDa allergen. The allergen, purified using immunoaffinity columns constructed with one of the specific mAbs, was shown to cause immediate and late-phase responses on intradermal skin testing in B. ovis-infested but not in naïve lambs. Levels of serum IgE specific for the purified allergen were significantly higher in infested than in naïve lambs (P < or = 0.0025). N-terminal and internal amino acid (aa) sequences obtained from the purified 28.5 kDa allergen were used to design primers to amplify a partial cDNA probe from B. ovis cDNA by PCR. The amplified probe was radiolabeled and used to screen a B. ovis cDNA library. The complete nucleotide sequence of the allergen was determined from the sequences of the positive clones from the library. The full-length cDNA encodes a 255 aa protein including a secretory leader sequence of 26 aas and a mature protein of 229 aas. The encoded protein showed strong homology to several hypothetical proteins of unknown function from diverse species and weak homology with lipid-binding proteins of Xenopus tropicalis and Galleria mellonella. This is the first allergen to be identified from a louse and it has been designated Bov o 1 in accordance with the criteria of the World Health Organization/International Union of Immunological Societies Allergen Nomenclature Subcommittee.
Pfeffer
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Hopkirk Research Institute, AgResearch Ltd., Private Bag 11008, Palmerston North 4442, New Zealand. alex.pfeffer@agresearch.co.nz
Shoemaker
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CB
Shaw
R J
RJ
Green
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RS
Shu
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D000485:Allergens / Q000737:chemistry / Q000276:immunology* / Q000302:isolation & purification; D000818:Animals; D002846:Chromatography, Affinity; D017931:DNA Primers / Q000235:genetics; D018076:DNA, Complementary / Q000235:genetics / Q000302:isolation & purification; D004591:Electrophoresis, Polyacrylamide Gel; D007073:Immunoglobulin E / Q000097:blood; D019476:Insect Proteins / Q000737:chemistry / Q000276:immunology* / Q000302:isolation & purification; D051379:Mice; D008807:Mice, Inbred BALB C; D008969:Molecular Sequence Data; D008970:Molecular Weight; D008003:Phthiraptera / Q000276:immunology*; D017422:Sequence Analysis, DNA; D020539:Sequence Analysis, Protein; D012756:Sheep; D012757:Sheep Diseases / Q000175:diagnosis / Q000276:immunology / Q000469:parasitology; D012882:Skin Tests
D000485:Allergens; D017931:DNA Primers; D018076:DNA, Complementary; D019476:Insect Proteins; D007073:Immunoglobulin E