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The NcGRA7 gene encodes the immunodominant 17 kDa antigen of Neospora caninum

Published online by Cambridge University Press:  11 October 2006

G. ÁLVAREZ-GARCÍA ,
A. PITARCH ,
A. ZABALLOS ,
A. FERNÁNDEZ-GARCÍA ,
C. GIL ,
M. GÓMEZ-BAUTISTA ,
A. AGUADO-MARTÍNEZ  and
L. M. ORTEGA-MORA
G. ÁLVAREZ-GARCÍA
Affiliation:
Grupo SALUVET, Departamento de Sanidad Animal, Facultad de Veterinaria, Universidad Complutense de Madrid, 28040-Madrid, Spain
A. PITARCH
Affiliation:
Departamento de Microbiología II, Facultad de Farmacia, Universidad Complutense de Madrid, Plaza Ramón y Cajal s/n, 28040-Madrid, Spain
A. ZABALLOS
Affiliation:
Departamento de Inmunología y Oncología. Centro Nacional de Biotecnología. Universidad Autónoma de Madrid, Campus de Cantoblanco, 28049-Madrid, Spain
A. FERNÁNDEZ-GARCÍA
Affiliation:
Grupo SALUVET, Departamento de Sanidad Animal, Facultad de Veterinaria, Universidad Complutense de Madrid, 28040-Madrid, Spain
C. GIL
Affiliation:
Departamento de Microbiología II, Facultad de Farmacia, Universidad Complutense de Madrid, Plaza Ramón y Cajal s/n, 28040-Madrid, Spain
M. GÓMEZ-BAUTISTA
Affiliation:
Grupo SALUVET, Departamento de Sanidad Animal, Facultad de Veterinaria, Universidad Complutense de Madrid, 28040-Madrid, Spain
A. AGUADO-MARTÍNEZ
Affiliation:
Grupo SALUVET, Departamento de Sanidad Animal, Facultad de Veterinaria, Universidad Complutense de Madrid, 28040-Madrid, Spain
L. M. ORTEGA-MORA
Affiliation:
Grupo SALUVET, Departamento de Sanidad Animal, Facultad de Veterinaria, Universidad Complutense de Madrid, 28040-Madrid, Spain
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Abstract

A Neospora caninum 17–19 kDa antigenic protein fraction (p17) in one-dimensional polyacrylamide gel electrophoresis (SDS-PAGE) is the immunodominant antigen recognized by sera from bovines naturally infected by N. caninum. To identify the proteins making up the p17 fraction, we screened a new N. caninum tachyzoite cDNA library with an affinity-purified antibody against p17 (APA17). We isolated several cDNA clones with 100% sequence identity to the NcGRA7 gene. This previously described gene encodes a dense granule protein with an apparent molecular mass of 33 kDa. A second line of evidence emerged through a combined proteomic approach associating two-dimensional PAGE (2D-PAGE) to Western blotting and to mass spectrometry to characterize the p17 fraction. Two acidic immunodominant but minority protein spots were recognized by APA17 and by bovine sera. These antigens of 17 and 33 kDa are respectively composed of 4 and 2 isoforms. Furthermore, p17 isolation by 2D-PAGE and peptide sequencing by tandem mass spectrometry yielded a partial sequence of 17 amino acids, which allowed the putative amino terminal region of the NcGRA7 protein to be identified unambiguously.The NcGRA7 protein, without the putative signal peptide at the NH2-terminus, was cloned and expressed in Escherichia coli and when the purified recombinant protein (rNcGRA7) was analysed by SDS-PAGE and mass spectrometry, 2 bands of 24 and 33 kDa were resolved and identified as NcGRA7. These results demonstrate that the immunodominant 17 kDa antigen of N. caninum is encoded by the NcGRA7 gene.

Keywords

Neospora caninumimmunodominant antigenp17NcGRA7
Type
Research Article
Information
Parasitology , Volume 134 , Issue 1 , January 2007 , pp. 41 - 50
Copyright
© 2006 Cambridge University Press

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