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Molecular analysis of astacin-like metalloproteases of Ostertagia ostertagi

Published online by Cambridge University Press:  13 December 2004

V. DE MAERE ,
I. VERCAUTEREN ,
P. GELDHOF ,
K. GEVAERT ,
J. VERCRUYSSE  and
E. CLAEREBOUT
V. DE MAERE
Affiliation:
Laboratory of Parasitology, Faculty of Veterinary Medicine, Ghent University, Salisburylaan, 133, B-9820 Merelbeke, Belgium
I. VERCAUTEREN
Affiliation:
Laboratory of Parasitology, Faculty of Veterinary Medicine, Ghent University, Salisburylaan, 133, B-9820 Merelbeke, Belgium
P. GELDHOF
Affiliation:
Laboratory of Parasitology, Faculty of Veterinary Medicine, Ghent University, Salisburylaan, 133, B-9820 Merelbeke, Belgium Current address: Moredun Research Institute, Edinburgh EH26 OPZ, Scotland, UK.
K. GEVAERT
Affiliation:
Department of Medical Protein Research, Flanders Interuniversity Institute for Biotechnology and Ghent University, Albert Baertsoenkaai, 3, B-9000 Ghent, Belgium
J. VERCRUYSSE
Affiliation:
Laboratory of Parasitology, Faculty of Veterinary Medicine, Ghent University, Salisburylaan, 133, B-9820 Merelbeke, Belgium
E. CLAEREBOUT
Affiliation:
Laboratory of Parasitology, Faculty of Veterinary Medicine, Ghent University, Salisburylaan, 133, B-9820 Merelbeke, Belgium
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Abstract

In this study, we describe the molecular analysis of zinc-metalloproteases from the abomasal nematode Ostertagia ostertagi which were exclusively recognized by local antibodies of immune cattle. Full-length or partial coding sequences of 4 different zinc-metalloprotease cDNAs of Ostertagia (met-1, -2, -3 and -4) were amplified using gene-specific primers using the 3′- and 5′-Rapid Amplification of cDNA Ends (RACE) technique. Sequence analysis identified the cDNAs as encoding zinc-metalloproteases, which showed between 62% and 70% homology to a metalloprotease 1 precursor of Ancylostoma caninum. The full-length cDNA of met-1 consists of an open reading frame (ORF) of 586 amino acids which contains 5 potential N-glycosylation sites and a predicted zinc-binding domain (HEBXHXBGFXHEXXRXDRD). The complete coding sequence of met-3 contains an ORF of 508 aa and the same conserved zinc-binding domain. These domains are signature sequences of the astacin family of the superfamily of metzincin metalloproteases. The presence of a threonine amino acid after the third histidine in MET-1 and MET-3, however, may place them in a new family or subfamily. Real-time PCR analysis of L3, exsheathed L3, L4 and adult cDNA identified transcription of the 4 metalloproteases in different life-stages. The protein MET-1 was expressed in insect cells using the baculovirus expression system but the immunization of calves with this molecule did not lead to protection against challenge infection.

Keywords

Ostertagia ostertagimetalloproteasesbaculovirus expressionvaccinationcattle
Type
Research Article
Information
Parasitology , Volume 130 , Issue 1 , January 2005 , pp. 89 - 98
Copyright
© 2004 Cambridge University Press

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