title: Gene cloning and characterization of the protein encoded by the Neospora caninum bradyzoite-specific antigen gene BAG1.
abstract: Neospora caninum is an Apicomplexan parasite that causes repeated abortion and stillbirth in cattle. The aim of this study was to clone the gene encoding the N. caninum orthologue (NcBAG1) of the Toxoplasma gondii bradyzoite-specific protein TgBAG1 and characterize its expression pattern in the parasite. Isolation of the full-length 684-bp gene revealed that it shared 78.3% sequence similarity with TgBAG1. NcBAG1 encodes a predicted protein of 227 amino acids with 80.3% similarity to TgBAG1. A putative signal peptide sequence and an invariant GVL motif characteristic of small heat-shock proteins were identified in the predicted N. caninum amino acid sequence. We expressed the NcBAG1 gene as a recombinant glutathione S-transferase fusion protein (rNcBAG1) in Escherichia coli and used the purified 60 kDa protein to obtain a monoclonal antibody (Mab). rNcBAG1 reacted to Mabs specific for NcBAG1 and TgBAG1. No reaction between the NcBAG1 Mab and N. caninum tachyzoites was observed. Although the predicted molecular mass of NcBAG1 is 25 kDa, Western blot analysis of parasite lysates using the NcBAG1 Mab revealed a cross-reactive protein of approximately 30 kDa. Additionally, immunofluorescence assays using the tachyzoite-specific Mab for NcSAG1 and the bradyzoite-specific Mab for TgBAG1 or NcSAG4 revealed NcBAG1-specific expression in bradyzoites in cultures exposed to sodium nitroprusside, a reagent that increases the frequency of bradyzoites. Interestingly, the NcBAG1 protein was identified in the cytoplasm of the bradyzoite-stage parasites. This preliminary analysis of the NcBAG1 gene will assist investigations into the role of this protein in N. caninum .
authors.affiliation: Laboratory of Veterinary Parasitology, Department of Veterinary Medicine, Faculty of Veterinary Science, Nippon Veterinary and Life Science University, 1-7-1 Kyonan, Musashino, Tokyo 180-8602, Japan.
authors.forename: T
authors.initials: T
authors.lastname: Kobayashi
authors.forename: S
authors.initials: S
authors.lastname: Narabu
authors.forename: Y
authors.initials: Y
authors.lastname: Yanai
authors.forename: Y
authors.initials: Y
authors.lastname: Hatano
authors.forename: A
authors.initials: A
authors.lastname: Ito
authors.forename: S
authors.initials: S
authors.lastname: Imai
authors.forename: K
authors.initials: K
authors.lastname: Ike
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology; D000953:Antigens, Protozoan / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D002522:Chlorocebus aethiops; D003001:Cloning, Molecular; D016054:DNA, Protozoan / Q000737:chemistry; D005260:Female; D005786:Gene Expression Regulation / Q000187:drug effects*; D006360:Heat-Shock Proteins / Q000235:genetics; D051379:Mice; D008807:Mice, Inbred BALB C; D018688:Neospora / Q000235:genetics* / Q000276:immunology / Q000378:metabolism; D020030:Nitric Oxide Donors / Q000494:pharmacology; D009599:Nitroprusside / Q000494:pharmacology; D016133:Polymerase Chain Reaction; D015800:Protozoan Proteins / Q000737:chemistry / Q000235:genetics* / Q000276:immunology / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000276:immunology / Q000378:metabolism; D013045:Species Specificity; D014709:Vero Cells
chemical_list: D000911:Antibodies, Monoclonal; D000953:Antigens, Protozoan; C106179:BAG1 protein, Toxoplasma; D016054:DNA, Protozoan; D006360:Heat-Shock Proteins; D020030:Nitric Oxide Donors; D015800:Protozoan Proteins; D011993:Recombinant Fusion Proteins; C115390:SAG1 protein, Neospora caninum; D009599:Nitroprusside