Epitope analysis of glycoprotein I of pseudorabies virus.
A panel of 11 monoclonal antibodies (MAbs) raised against pseudorabies virus (PRV) was used to map epitopes on the virus glycoprotein I (gI). We employed three approaches to map epitopes on gI. By a competition binding assay, six groups of MAbs were defined as reacting with distinct antigenic domains on gI. To identify regions along the gI polypeptide chain encompassing the domains recognized by these MAbs, DNA fragments derived from the gI-coding region were cloned into pEX expression plasmids. The antigenic reactivity of the fusion proteins expressed in Escherichia coli was analysed by immunoblotting. Five antigenic domains were mapped within the first 238 amino acids of gI: domains A, B and D were mapped between amino acids 52 and 123 and domains C and E between amino acids 78 and 238. One MAb, representing domain F, did not react with the expressed fusion proteins. To assess the precise location and amino acid sequences of the epitopes, overlapping nonapeptides covering the amino acid sequence 52 to 238 were synthesized. The antibody-binding activity of these peptides was tested by an ELISA (Pepscan-method). Three antigenic domains were mapped: domain A was localized to amino acids 64 to 73 and 75 to 84, domain B to amino acids 52 to 67 and domain D to amino acids 68 to 82. Four MAbs representing antigenic domains C, E and F did not react in the Pepscan. Finally, sera from pigs infected experimentally with PRV reacted with the fusion protein of plasmid ps 1 (amino acids 52 to 238).
Jacobs
L
L
Central Veterinary Institute, Department of Virology, Lelystad, The Netherlands.
Meloen
R H
RH
Gielkens
A L
AL
Van Oirschot
J T
JT
D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology; D000918:Antibody Specificity; D000956:Antigens, Viral / Q000032:analysis* / Q000235:genetics; D001667:Binding, Competitive; D015153:Blotting, Western; D003001:Cloning, Molecular; D004279:DNA, Viral / Q000032:analysis; D004797:Enzyme-Linked Immunosorbent Assay; D000939:Epitopes / Q000032:analysis / Q000235:genetics; D011558:Herpesvirus 1, Suid / Q000235:genetics / Q000276:immunology*; D007106:Immune Sera / Q000276:immunology; D008969:Molecular Sequence Data; D010449:Peptide Mapping; D015183:Restriction Mapping; D013552:Swine; D014759:Viral Envelope Proteins / Q000235:genetics / Q000276:immunology*
D000911:Antibodies, Monoclonal; D000956:Antigens, Viral; D004279:DNA, Viral; D000939:Epitopes; D007106:Immune Sera; D014759:Viral Envelope Proteins; C042966:pseudorabies virus glycoproteins