title: Constitutive expression and characterization of a surface SRS (NcSRS67) protein of Neospora caninum with no orthologue in Toxoplasma gondii.
abstract: Neospora caninum is a parasite of the Apicomplexa phylum responsible for abortion and losses of fertility in cattle. As part of its intracellular cycle, the first interaction of the parasite with the target cell is performed with the surface proteins known as the SRS superfamily (Surface Antigen Glycoprotein - Related Sequences). SAG related or SRS proteins have been a target of intense research due to its immunodominant pattern, exhibiting potential as diagnostic and/or vaccine candidates. The aim of this study was the cloning, expression and characterization of the gene NcSRS67 of N. caninum using a novel designed plasmid. The coding sequence of NcSRS67 (without the signal peptide and the GPI anchor) was cloned and expressed constitutively instead of the ccdB system of pCR-Blunt II-TOPO. The protein was purified in a nickel sepharose column and identified by mass spectrometry (MS/MS). The constitutive expression did not affect the final bacterial growth, with a similar OD 600nm compared to the non-transformed strains. The recombinant NcSRS67 was over expressed and the native form was detected by the anti-rNcSRS67 serum on 1D western blot as a single band of approximately 38kDa as predicted. On an in vitro assay, the inhibitory effect of the polyclonal antiserum anti-rNcSRS67 was nearly 20% on adhesion/invasion of host cells. The NcSRS67 native protein was localised on part of the surface of N. caninum tachyzoite when compared to the nucleus by confocal immunofluorescence.
authors.affiliation: Faculdade de Ciências Farmacêuticas de Ribeirão Preto, Universidade de São Paulo, Av do café, s/n, 14040-903 Ribeirão Preto, SP, Brazil.
authors.forename: Marcos Alexandre
authors.initials: MA
authors.lastname: Bezerra
authors.affiliation: Faculdade de Ciências Farmacêuticas de Ribeirão Preto, Universidade de São Paulo, Av do café, s/n, 14040-903 Ribeirão Preto, SP, Brazil.
authors.forename: Luiz Miguel
authors.initials: LM
authors.lastname: Pereira
authors.affiliation: Faculdade de Ciências Farmacêuticas de Ribeirão Preto, Universidade de São Paulo, Av do café, s/n, 14040-903 Ribeirão Preto, SP, Brazil.
authors.forename: Aline
authors.initials: A
authors.lastname: Bononi
authors.affiliation: Faculdade de Ciências Farmacêuticas de Ribeirão Preto, Universidade de São Paulo, Av do café, s/n, 14040-903 Ribeirão Preto, SP, Brazil.
authors.forename: Carla Agostino
authors.initials: CA
authors.lastname: Biella
authors.affiliation: Faculdade de Ciências Farmacêuticas de Ribeirão Preto, Universidade de São Paulo, Av do café, s/n, 14040-903 Ribeirão Preto, SP, Brazil.
authors.forename: Luciana
authors.initials: L
authors.lastname: Baroni
authors.affiliation: Núcleo de Apoio à Pesquisa em Produtos Naturais e Sintéticos, Universidade de São Paulo, Ribeirão Preto, SP, Brazil.
authors.forename: Leticia
authors.initials: L
authors.lastname: Pollo-Oliveira
authors.affiliation: Faculdade de Ciências Farmacêuticas de Ribeirão Preto, Universidade de São Paulo, Av do café, s/n, 14040-903 Ribeirão Preto, SP, Brazil; Núcleo de Apoio à Pesquisa em Produtos Naturais e Sintéticos, Universidade de São Paulo, Ribeirão Preto, SP, Brazil. Electronic address: ayatsuda@fcfrp.usp.br.
authors.forename: Ana Patrícia
authors.initials: AP
authors.lastname: Yatsuda
mesh_terms: D000818:Animals; D000913:Antibodies, Protozoan / Q000276:immunology; D000953:Antigens, Protozoan / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification / Q000378:metabolism; D000954:Antigens, Surface / Q000737:chemistry / Q000235:genetics / Q000276:immunology; D002417:Cattle; D002522:Chlorocebus aethiops; D003001:Cloning, Molecular; D015870:Gene Expression; D017125:Genes, Protozoan; D008565:Membrane Proteins / Q000235:genetics / Q000302:isolation & purification*; D051379:Mice; D018688:Neospora / Q000737:chemistry / Q000235:genetics* / Q000276:immunology; D016366:Open Reading Frames / Q000235:genetics; D010957:Plasmids / Q000235:genetics; D021382:Protein Sorting Signals / Q000235:genetics; D015800:Protozoan Proteins / Q000235:genetics* / Q000276:immunology / Q000302:isolation & purification* / Q000378:metabolism; D053719:Tandem Mass Spectrometry; D014122:Toxoplasma / Q000737:chemistry / Q000235:genetics*; D014709:Vero Cells
chemical_list: D000913:Antibodies, Protozoan; D000953:Antigens, Protozoan; D000954:Antigens, Surface; D008565:Membrane Proteins; D021382:Protein Sorting Signals; D015800:Protozoan Proteins
keywords: Apicomplexa; NcSRS67; Neospora caninum; Plasmid pCR-Blunt II-TOPO; SRS proteins; Surface protein