CHARACTERIZATION OF NEOSPORA CANINUM PROTEASE, NcSUB1 (NC-p65), WITH RABBIT ANTI-N54

• Published in: The Journal of parasitology Vol. 88; no. 6; pp. 1113 - 1119
• Main Authors: Louie, Kitland, Nordhausen, Robert, Robinson, Thomas W, Barr, Bradd C, Conrad, Patricia A
• Format: Journal Article
• Language: English
• Published: Lawrence, KS American Society of Parasitologists 01-12-2002; Allen Press Inc
• Subjects: Amino acids; Animals; Antibodies; Antigens, Protozoan - immunology; Antiserum; Biological and medical sciences; Cercopithecus aethiops; Electron microscopy; Endopeptidases - chemistry; Endopeptidases - immunology; Ethanol; Fetuses; Fluorescent Antibody Technique; Fundamental and applied biological sciences. Psychology; Gels; Hydrogen-Ion Concentration; Immune Sera - immunology; Immunoblotting; Immunofluorescence; Immunohistochemistry; IMMUNOLOGY; Laboratories; Life cycle. Host-agent relationship. Pathogenesis; Microscopy; Microscopy, Immunoelectron; Molecular Weight; Neospora - enzymology; Neospora - immunology; Organelles; Parasite hosts; Parasites; Parasitology; Proteins; Proteolysis; Protozoa; Rabbits; Resins; Tachyzoites; Vero Cells; Weight reduction; Protozoa; Host parasite relation; Apicomplexa; Life history; Protozoal disease; Enzyme; Antibody; Rabbit; Parasite; Parasitosis; Lagomorpha; Infection; Peptidases; Vertebrata; Mammalia; Enzymatic activity; Neospora caninum; Hydrolases; Immunoelectron microscopy; Developmental stage; Immunofluorescence; Localization
• ISSN: 0022-3395; 1937-2345
• DOI: 10.1645/0022-3395(2002)088[1113:CONCPN]2.0.CO;2

NcSUB1 (formerly known as NC-p65) is the first molecularly described proteolytic enzyme of the intracellular protozoan parasite Neospora caninum. This report describes the characterization of a rabbit anti-N54, which is an antiserum generated against an internal fragment of NcSUB1 (amino acids 649–783). In immunofluorescence studies rabbit anti-N54 labeled the apical end of the fixed parasite. By immuno-gold electron microscopy, the antibody bound primarily to the microneme organelles of the parasite. Analysis of secreted parasitic proteins indicated that a protein of molecular weight 65 kDa (reduced) or 55 kDa (nonreduced) was recognized by the antibody. The same secreted proteins were affinity purified with rabbit anti-N54–coupled resins and were shown to contain major proteolytic activity by zymography. Thus, rabbit anti-N54 is the first antibody developed for N. caninum that binds to the microneme proteins and recognizes a major secreted enzyme.