Importance of M-protein C terminus as substrate antigen for serodetection of equine arteritis virus infection.
Equine arteritis virus (EAV), an enveloped positive-stranded RNA virus, is the prototype of the arterivirus group. In a previous paper (A. Kheyar, S. Martin, G. St.-Laurent, P. J. Timoney, W. H. McCollum, and D. Archambault, Clin. Diagn. Lab. Immunol. 4:648-652, 1997), we have shown that the unglycosylated membrane (M) protein, which is composed of 162 amino acids (aa), is a major target of equine antibody to EAV. In order to determine the antigenic regions of the M protein, the cDNA encoding the M protein of EAV was inserted into the procaryotic expression vector pGEX-4T-1 to produce recombinant glutathione S-transferase-M fusion protein. Various deletion mutant clones, which covered the entire sequence of the M protein, were then generated by inverse PCR and expressed in Escherichia coli to examine, by a Western blot assay, the antigenic reactivity of the clone-derived truncated M proteins with sera from horses either experimentally or naturally infected with EAV. Deletion of the hydrophobic N-terminal 87 aa did not abolish immune reactivity of the protein with serum antibodies to EAV, thereby demonstrating the antigenicity of the C-terminal region (aa 88 to 162) of the M protein. Further truncations of the M-protein C-terminal domain defined particular linear epitope-containing amino acid sequence regions. However, only the M-protein C-terminal region was readily recognized by all EAV-specific horse antisera tested in this study. Based on these findings, only the M-protein C-terminal polypeptide composed of aa 88 to 162 is necessary to identify horse serum antibodies specific to the EAV M protein. Thus, this polypeptide might be useful for serodetection of EAV-infected animals.
Jeronimo
Célia
C
Laboratory of Molecular Virology and Immunology, Department of Biological Sciences, University of Québec at Montréal, Succursale Centre-Ville, Montréal, Québec, Canada H3C 3P8.
Archambault
Denis
D
D000818:Animals; D000914:Antibodies, Viral / Q000097:blood* / Q000276:immunology; D000956:Antigens, Viral / Q000235:genetics / Q000276:immunology*; D018174:Arterivirus Infections / Q000097:blood / Q000276:immunology / Q000662:veterinary* / Q000821:virology; D015153:Blotting, Western / Q000379:methods; D018985:Epitopes, B-Lymphocyte / Q000235:genetics / Q000276:immunology*; D007240:Equartevirus / Q000276:immunology*; D004926:Escherichia coli; D015870:Gene Expression; D005982:Glutathione Transferase / Q000235:genetics; D006734:Horse Diseases / Q000097:blood / Q000175:diagnosis* / Q000276:immunology / Q000821:virology; D006736:Horses; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000276:immunology; D012698:Serologic Tests; D014759:Viral Envelope Proteins / Q000235:genetics / Q000276:immunology*
D000914:Antibodies, Viral; D000956:Antigens, Viral; D018985:Epitopes, B-Lymphocyte; D011993:Recombinant Fusion Proteins; D014759:Viral Envelope Proteins; D005982:Glutathione Transferase