An aspartyl protease inhibitor of Ostertagia ostertagi:: Molecular cloning, analysis of stage and tissue specific expression and vaccine trial

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 bacteria] 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 L-3, L-4 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 L-3, the intestinal cells of L-4, 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. (c) 2005 Elsevier B.V. All rights reserved.