Antigenic structure of the nucleocapsid protein of porcine reproductive and respiratory syndrome virus.
A collection of 12 monoclonal antibodies (MAbs) raised against porcine reproductive and respiratory syndrome (PRRS) virus was used to study the antigenic structure of the virus nucleocapsid protein (N). The full-length N gene, encoded by open reading frame 7, was cloned from the Canadian PRRS virus, PA-8. Deletions were introduced into the N gene to produce a series of nine overlapping protein fragments ranging in length from 25 to 112 amino acids. The individual truncated genes were cloned as glutathione S-transferase fusions into a eukaryotic expression vector downstream of the T7 RNA polymerase promoter. HeLa cells infected with recombinant vaccinia virus expressing T7 RNA polymerase were transfected with plasmid DNA encoding the N protein fragments, and the antigenicity of the synthesized proteins was analyzed by immunoprecipitation. Based on the immunoreactivities of the N protein deletion mutants with the panel of N-specific MAbs, five domains of antigenic importance were identified. MAbs SDOW17, SR30, and 5H2.3B12.1C9 each identified independent domains defined by amino acids 30 to 52, 69 to 123, and 37 to 52, respectively. Seven of the MAbs tested specifically recognized the local protein conformation formed in part by the amino acid residues 52 to 69. Furthermore, deletion of 11 amino acids from the carboxy terminus of the nucleocapsid protein disrupted the epitope configuration recognized by all of the conformation-dependent MAbs, suggesting that the carboxy-terminal region plays an important role in maintaining local protein conformation.
Wootton
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Department of Pathobiology, Ontario Veterinary College, University of Guelph, Guelph, Ontario N1G 2W1, Canada.
Nelson
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EA
Yoo
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D000911:Antibodies, Monoclonal / Q000276:immunology; D000918:Antibody Specificity; D000956:Antigens, Viral / Q000276:immunology*; D003001:Cloning, Molecular; D018604:Epitope Mapping; D000939:Epitopes / Q000276:immunology; D005814:Genes, Viral; D006367:HeLa Cells; D006801:Humans; D008969:Molecular Sequence Data; D019590:Nucleocapsid Proteins / Q000737:chemistry* / Q000235:genetics / Q000276:immunology* / Q000378:metabolism; D010957:Plasmids / Q000235:genetics; D019316:Porcine respiratory and reproductive syndrome virus / Q000254:growth & development / Q000276:immunology*; D011233:Precipitin Tests; D011487:Protein Conformation; D011993:Recombinant Fusion Proteins / Q000276:immunology
D000911:Antibodies, Monoclonal; D000956:Antigens, Viral; D000939:Epitopes; D019590:Nucleocapsid Proteins; D011993:Recombinant Fusion Proteins