Expression and characterisation of a Psoroptes ovis glutathione S-transferase.
The astigmatid mite Psoroptes ovis is the causative agent of sheep scab, a highly contagious parasitic disease of sheep. Infection causes severe allergic dermatitis, resulting in damage to the fleece and hide, loss of condition and occasional mortality. Interest in the P. ovis allergens led us to characterise a glutathione S-transferase (GST) which displays homology to GST allergens isolated from the house dust mite, Dermatophagoides pteronyssinus and the cockroach, Blatella germanica. A cDNA encoding a mu-class GST from P. ovis was expressed in Escherichia coli and the recombinant protein purified for biochemical analysis. SDS-PAGE analysis indicated that the purified product was homogeneous and had an apparent molecular weight of 30 kDa. The recombinant GST (rGST) is active towards the substrate 1-chloro-2,4-dinitrobenzene (CDNB), whereas 1,2-dichloro-4-nitrobenzene (DCNB) is a poor substrate. The recombinant protein was also tested for recognition by IgE and IgG antibodies in serum from P. ovis naïve and P. ovis infested sheep. Neither IgE nor IgG antibodies were detected to the rGST. Prausnitz--Küstner testing with rGST did not provoke a characteristic weal and flare response. Biopsies collected at the PK test sites were stained for eosinophils, neutrophils, mast cells and basophils. Neutrophil, mast cell and basophil counts were not significantly different to the controls. Eosinophil numbers were significantly higher than controls, but were not due to an IgE response.
Lee
Alison J
AJ
School of Biology, The University of Leeds, LS2 9JT, Leeds, UK. a.j.lee@leeds.ac.uk
Huntley
John
J
Van den Broek
Adri
A
Coates
David
D
Isaac
R Elwyn
RE
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D015153:Blotting, Western / Q000662:veterinary; D018076:DNA, Complementary / Q000737:chemistry; D004591:Electrophoresis, Polyacrylamide Gel / Q000662:veterinary; D015971:Gene Expression Regulation, Enzymologic; D015723:Gene Library; D005982:Glutathione Transferase / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics / Q000276:immunology; D008924:Mite Infestations / Q000201:enzymology / Q000276:immunology / Q000662:veterinary*; D008925:Mites / Q000201:enzymology* / Q000235:genetics / Q000276:immunology; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010802:Phylogeny; D016133:Polymerase Chain Reaction; D012756:Sheep; D012757:Sheep Diseases / Q000201:enzymology / Q000276:immunology / Q000469:parasitology*
D018076:DNA, Complementary; D005982:Glutathione Transferase