Establishment of a two‐dimensional electrophoresis map forNeospora caninumtachyzoites by proteomics

Abstract

Expressed proteins and antigens from Neospora caninum tachyzoites were studied by two-dimensional gel electrophoresis and immunoblot analysis combined with matrix-assisted laser desorption/ionization-time of flight mass spectrometry. Thirty-one spots corresponding to 20 different proteins were identified from N. caninum tachyzoites by peptide mass fingerprinting. Six proteins were identified from a N. caninum database (NTPase, 14-3-3 protein homologue, NcMIC1, NCDG1, NcGRA1 and NcGRA2), and 11 proteins were identified in closely related species using the T. gondii database (HSP70, HSP60, pyruvate kinase, tubulin alpha- and beta-chain, putative protein disulfide isomerase, enolase, actin, fructose-1,6-bisphosphatase, lactate dehydrogenase and glyceradehyde-3-phosphate dehydrogenase). One hundred and two antigen spots were observed using pH 4-7 IPG strips on immunoblot profiles. Among them, 17 spots corresponding to 11 antigenic proteins were identified from a N. caninum protein map. This study involved the construction of in-depth protein maps for N. caninum tachyzoites, which will be of value for studies of its pathogenesis, drug and vaccine development, and phylogenetic studies.

Efficacy of protein A-HRP in an immunological study of black rockfish (Sebastes schlegeli Higendorf) humoral immune responses

“…The protein content was determined according to the method of Bradford [27] and adjusted to 60 mg ml ÿ1 with rehydration buffer (9 M urea, 2% CHAPS, 0.4% DTT, 0.5% IPG buffer, and 0.002% bromophenol blue) for isoelectric focusing (IEF). IEF was performed using the IPGphorÔ system (Amersham Bioscience) according to the method of Lee et al [29], with IPG strips (Immobiline DryStripÔ, pH 4e7, 13 cm; Amersham Bioscience). The IEF was carried out at 20 C using an automated programmer; the steps included rehydration for 14 h (7 h at 30 V and 7 h at 60 V) followed by focusing for 86.1 kV h as follows: 2 h at 200 V, 1 h at 500 V and 1000 V, 1 h at 2000 V, 2 h at 4000 V and 10 h at 8000 V. After IEF, the IPG strips were equilibrated with 10 mg ml ÿ1 DTT in equilibration buffer (6 M urea, 2% SDS, 30% glycerol, 0.002% bromophenol blue and 50 mM TriseHCl, pH 8.8) for 15 min followed by 15 min incubation at RT with 40 mg ml ÿ1 iodoacetamide equilibration buffer.…”

Identification of the cross-reactive and species-specific antigens between Neospora caninum and Toxoplasma gondii tachyzoites by a proteomics approach

“…Spectra were obtained by the sum of 1,000 and 4,000 consecutive laser shots in the MS and MS/MS modes, respectively. For the MALDI-TOF MS analysis, protein identification by MS was performed as described by Lee et al (2003). The monoisotopic peptide masses were selected in a range between 300 and 3,800 Da.…”

“…The proteomics approach has been found to be useful for analyzing the proteomes of some parasitic organisms, e.g., Plasmodium (Choumet et al 2007), Toxoplasma (Kawase et al 2007), Trichomonas (De Jesus et al 2007), Leishmania (Brobey et al 2006), and Trypanosoma (Foucher et al 2006) species, including their development, evolution, and pathogenicity. The first proteomics analysis in N. caninum was published by Lee et al (2003). Reports on the identification of new antigens to improve serodiagnosis and the definition of the molecular difference between N. caninum and T. gondii via the application of proteomics, as well as on the identification of a number of homologue proteins between N. caninum and T. gondii tachyzoites, such as HSP70, tubulin (α-and β-chains), PDI, and actin, as well as enolase, which were believed to be common antigens in both parasites, were also published by Lee et al (2004Lee et al ( , 2005.…”

Development of competitive ELISA for neosporosis by employing immunoproteomics

“…Silver staining of gels, image analysis, in-gel digestion of protein spots on gels, matrixassisted laser desorption/ionization (MALDI)-time of flight (TOF) mass spectrometry (MS) and database searches were performed as Lee et al (13). For more precise discussing, gel staining, image analysis, and protein identification were performed a minimum of three times and several minor stained bands and spots were not analyzed.…”

“…Determinations of antigenic proteins that are competing against each other in cross-reactivity and their use in ELISA have been reported for neosporosis (10,11). Recently, immunoproteomics (twodimensional gel electrophoresis (2-DE) and its immunoblot studies) was applied in an attempt to reveal antigenic proteins of N. caninum by using rabbit anti-N. caninum serum, and the number of spots of N. caninum antigenic proteins was crossly reacted with rabbit anti-T. gondii serum (12,13).…”