title: An aspartyl protease inhibitor of Ostertagia ostertagi: molecular cloning, analysis of stage and tissue specific expression and vaccine trial.
abstract: Protease inhibitors are thought to protect intestinal parasitic nematodes from their hostile proteolytic environment. In a previous study, screening of Ostertagia ostertagi cDNA libraries with local antibody probes of the abomasal lymph nodes and mucus revealed a (28 kDa) aspartyl protease inhibitor (API), which was exclusively recognised by antibodies from immune calves. Here we report the molecular characterization of Oo-API (sequence analysis, developmental expression and localization) and a vaccine trial in cattle with the native and recombinant baculo-expressed antigen. The full-length open reading frame of api encodes a protein of 28 kDa. The sequence showed 82% significant homology to an Aspin homologue from Trichostrongylus colubriformis (AA034715). The cDNA encoding the full-length sequence was cloned in a bacterial pET expression vector and the pVec 35 baculovirus vector. Polyclonal rabbit serum against the Escherichia coli-expressed protein was used to develop Western Blots of extracts and ES and to localize the antigen on L3, L4 and adult worm sections. The protein was expressed in all life stages, which was confirmed by real-time polymerase chain reaction (RT-PCR), and was mainly localized in the cuticle of L3, the intestinal cells of L4, and the gut and sphincter of adult worms. Polyclonal serum was also used to affinity purify the native protein. Vaccination of calves with native Oo-API and baculovirus-expressed Oo-rbAPI in combination with QuilA resulted in no protection against Ostertagia challenge infections.
authors.affiliation: Laboratory of Parasitology, Faculty of Veterinary Medicine, Ghent University, Salisbwylaan 133, B-9820 Merelbeke, Belgium. veerle.demaere@UGent.be
authors.forename: Veerle
authors.initials: V
authors.lastname: De Maere
authors.forename: Isabel
authors.initials: I
authors.lastname: Vercauteren
authors.forename: Kris
authors.initials: K
authors.lastname: Gevaert
authors.forename: Jozef
authors.initials: J
authors.lastname: Vercruysse
authors.forename: Edwin
authors.initials: E
authors.lastname: Claerebout
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D000909:Antibodies, Helminth / Q000096:biosynthesis; D000947:Antigens, Helminth / Q000096:biosynthesis* / Q000235:genetics; D016282:Aspartic Acid Endopeptidases / Q000037:antagonists & inhibitors* / Q000235:genetics; D002417:Cattle; D003001:Cloning, Molecular; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004926:Escherichia coli / Q000378:metabolism; D005260:Female; D005822:Genetic Vectors; D008297:Male; D008969:Molecular Sequence Data; D010028:Ostertagia / Q000187:drug effects* / Q000502:physiology; D010029:Ostertagiasis / Q000276:immunology / Q000517:prevention & control; D011994:Recombinant Proteins / Q000494:pharmacology; D016415:Sequence Alignment; D014611:Vaccination
chemical_list: D000909:Antibodies, Helminth; D000947:Antigens, Helminth; D004791:Enzyme Inhibitors; D011994:Recombinant Proteins; D016282:Aspartic Acid Endopeptidases