title: Isolation and immunolocalization of a putative protective antigen (p26/23) from adult Haemonchus contortus.
abstract: The putative protective antigen, p26/23, from adult Haemonchus contortus was isolated. A soluble extract from adult helminths obtained from the abomasa of hyperinfected (12,000 infective larvae) female Manchego lambs and treated with a mixture of protease inhibitors was subjected to affinity chromatography (hexylgluthatione) to eliminate the enzyme gluthatione S-transferase. The eluate was analyzed by electrophoresis under denaturing and reducing conditions (sodium dodecyl sulfate polyacrylamide gel electrophoresis), electrotransferred to nylon membranes, and assayed by Western blot with sera from immunized lambs. The bands recognized by the lambs' sera corresponding to proteins with a molecular weight of 23-26 kDa (p26/23) were excised, eluted, and separated by reverse-phase chromatography. This allowed the isolation of a single protein, which was expressed in both infective larvae (L3) and the adult stage of the parasite. The first 20 amino acids of the N-terminal end of the purified protein were determined. The partial amino acid sequence revealed a 100% identity with the N terminus of one of the peptides present in the p26/23 immunoprotective fraction previously tested by us against sheep haemonchosis. No significant homology with any reported sequence was found except for the deduced sequence of a hypothetic H.contortus protein (HCC00515). Immunolocalization studies showed that the protein was expressed in the hypodermic chords of the nematode.
authors.affiliation: Department of Animal Health, Faculty of Veterinary Science, University Complutense of Madrid, Avda. Puerta de Hierro s/n, 28040 Madrid, Spain.
authors.forename: L
authors.initials: L
authors.lastname: García-Coiradas
authors.forename: F
authors.initials: F
authors.lastname: Angulo-Cubillán
authors.forename: S
authors.initials: S
authors.lastname: Méndez
authors.forename: V
authors.initials: V
authors.lastname: Larraga
authors.forename: C
authors.initials: C
authors.lastname: de la Fuente
authors.forename: M
authors.initials: M
authors.lastname: Cuquerella
authors.forename: J M
authors.initials: JM
authors.lastname: Alunda
mesh_terms: D000818:Animals; D000909:Antibodies, Helminth / Q000276:immunology; D000947:Antigens, Helminth / Q000032:analysis / Q000737:chemistry / Q000276:immunology* / Q000302:isolation & purification*; D015153:Blotting, Western; D004591:Electrophoresis, Polyacrylamide Gel; D006189:Haemonchus / Q000737:chemistry / Q000276:immunology*; D008969:Molecular Sequence Data; D008970:Molecular Weight; D011817:Rabbits; D020539:Sequence Analysis, Protein
chemical_list: D000909:Antibodies, Helminth; D000947:Antigens, Helminth