Monoclonal antibodies and conserved antigenic epitopes in the C terminus of GP5 protein of the North American type porcine reproductive and respiratory syndrome virus

Glycoprotein 5 (GP5) is the major glycoprotein of porcine reproductive and respiratory syndrome virus (PRRSV). In this study, the gene encoding rtGP5, lacking signal peptide sequence, was expressed as GST-fusion protein in E. coli. Fifteen monoclonal antibodies (MAbs) against rtGP5 were developed and used to probe a series of GP5 peptides by ELISA, in which two MAbs specifically recognized the epitope GP5EP3 (146-156 aa), four recognized GP5EP5 (164-180 aa) and nine recognized GP5EP7 (192-200 aa). After precise analysis by sequential deletion of the terminal amino acid residues, the three minimal epitopes ((RLYRWR156)-L-152, E(169)GHLIDLKRV(178) and Q(196)WGRL(200)) were determined, which were highly conserved among the North American type isolates, with the exception of one amino acid mutation (L-200 to P-200). Mutational analysis showed that the mutant (Q(196)WGRP(200)) could be recognized by four of nine anti-GP5EP7 MAbs, indicating Q(196)WGRP(200) was also one minimal epitope. Western blot analysis showed that GP5EP5 and GP5EP7 (L-200 or P-200) could be recognized by PRRSV-positive sera of CH-1a and/or BJ-4, suggesting GP5EP5 and GP5EP7 (L-200 or P-200) were antigenic epitopes in the PRRSV-infected pigs. MAbs against GP5EP3, GP5EP5, and GP5EP7 could react with MARC-145 cells infected with the North American type isolates from China in IFA. However, very interestingly, when the highly pathogenic PRRSV, represented by HUN4, was passaged in MARC-145 cells, MAbs against GP5EP7 did not react with HUN4-F20-HUN4-F112 (20-112th passage virus), where Q(196)WGRL(200) had mutated to R(196)WGRL(200). Due to no mutations observed in GP5EP3 and GP5EP5, MAbs against GP5EP3 and GP5EP5 could recognize HUN4-F20-HUN4-F112. All the results herein might deepen the understanding of the antigen structure of in the C terminus of GP5 and facilitate the development of diagnostic antigens of the North American type PRRSV in China. (C) 2009 Published by Elsevier B.V.