The NcGRA7gene encodes the immunodominant 17 kDa antigen ofNeospora caninum

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.

First epidemiological study on exposure to Neospora caninum in different canine populations in the Algiers District (Algeria)

“…The 29-33 and 40-42 kDa proteins corresponded most probably to the two major surface antigens NcSAg1 and NcSRS2, respectively [45]. The 14-22 kDa band had a molecular weight compatible with Gra7 antigen [46]. The heaviest bands (120-138 and 180 kDa) were maybe due to an incomplete protein denaturation.…”

Cloning and expression of Neospora caninum dense-granule 7 in E. coli

“…Previous studies revealed that NcGRA7 might be involved in the initial host cell invasion process of N. caninum (Augustine et al 1999;Cho et al 2005). NcGRA7 is an immunodominant antigen in both of tachyzoite and bradyzoite stages (Vonlaufen et al 2004;Alvarez-Garcia et al 2007), unlike NcSAG1, which is only expressed in tachyzoites (Fuchs et al 1998) and it seems to be a good marker in detecting antibodies against both stages (Huang et al 2007). The main objective of our study was to clone and express the NcGRA7 gene using an efficient prokaryotic expression vector, in order to produce a sufficient amount of the protein for developing diagnostic kits.…”

A vaccine formulation combining rhoptry proteins NcROP40 and NcROP2 improves pup survival in a pregnant mouse model of neosporosis

“…NcROP2 sequence was amplified using as DNA template the pQE30-NcROP2 plasmid (Debache et al, 2008). NcNTPase was amplified from NcLiv cDNA, and NcGRA7 was cloned as previously described (Álvarez-García et al, 2007;Jiménez-Ruiz et al, 2012).…”

“…coli BL21(DE3)pLysS competent cells (Agilent Technologies) were transformed with the resulting expression vectors and foreign expression of rNcROP40, rNcROP2 and rNcNTPase as a (His) 6 -tagged fusion proteins was carried out following standard procedures (Álvarez-García et al, 2007). Transformed bacteria were cultured in selective medium (Luria Bertoni supplemented with 10 g/l glucose, 100 mg/ml ampicillin and 34 mg/ml chloramphenicol) until the OD 600 reached 0.6.…”