title: Molecular cloning and characterization of gut-derived cysteine proteinases associated with a host protective extract from Haemonchus contortus.
abstract: Cysteine proteinases have been implicated in the protection conferred by vaccination with detergent-soluble extracts of Haemonchus contortus. In the present study, antisera from sheep refractory to Haemonchus challenge following vaccination with a 'proteinase-enriched' Haemonchus gut membrane extract, were employed to screen a cDNA expression library of the adult parasite. This resulted in the isolation of 3 cDNAs (designated hmcp1, 4 and 6) encoding cathepsin B-like cysteine proteinases. Immunocytochemical studies specifically localized the products of these genes to the microvillar surface of the parasite's gut and RT-PCR experiments revealed that these were developmentally regulated, being expressed exclusively during the blood-feeding parasitic stages. In addition, a generic PCR approach was adopted in order to identify the predominant cysteine proteinases in a UK strain of Haemonchus. A panel of 5 cDNAs, including hmcp1 and 4, was amplified in this way. Genomic Southern blot analysis indicated that some of these enzymes were encoded by single-copy genes, whereas others were encoded by multi-copy genes. Subsequent sequence analysis revealed that the proteases identified in this study were distinct from those previously reported in USA strains of the parasite.
authors.affiliation: Moredun Research Institute, International Research Centre, Penicuik, Midlothian.
authors.forename: P J
authors.initials: PJ
authors.lastname: Skuce
authors.forename: D L
authors.initials: DL
authors.lastname: Redmond
authors.forename: S
authors.initials: S
authors.lastname: Liddell
authors.forename: E M
authors.initials: EM
authors.lastname: Stewart
authors.forename: G F
authors.initials: GF
authors.lastname: Newlands
authors.forename: W D
authors.initials: WD
authors.lastname: Smith
authors.forename: D P
authors.initials: DP
authors.lastname: Knox
mesh_terms: D000818:Animals; D003546:Cysteine Endopeptidases / Q000235:genetics* / Q000302:isolation & purification; D004064:Digestive System / Q000201:enzymology*; D015723:Gene Library; D006188:Haemonchiasis / Q000517:prevention & control / Q000662:veterinary*; D006189:Haemonchus / Q000201:enzymology*; D015801:Helminth Proteins / Q000235:genetics* / Q000302:isolation & purification; D007150:Immunohistochemistry; D008969:Molecular Sequence Data; D012756:Sheep; D012757:Sheep Diseases / Q000276:immunology* / Q000517:prevention & control; D014018:Tissue Distribution; D014611:Vaccination; D014612:Vaccines / Q000276:immunology
chemical_list: D015801:Helminth Proteins; D014612:Vaccines; D003546:Cysteine Endopeptidases