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 (146-156aa), four recognized GP5EP5 (164-180aa) and nine recognized GP5EP7 (192-200aa). After precise analysis by sequential deletion of the terminal amino acid residues, the three minimal epitopes (R(152)LYRWR(156), 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.
Zhou
Yan-Jun
YJ
Division of Swine Infectious Diseases, Shanghai Veterinary Research Institute, Chinese Academy of Agricultural Sciences, No. 518, Ziyue Road, Minhang District, Shanghai 200241, China.
Yu
Hai
H
Tian
Zhi-Jun
ZJ
Liu
Jin-Xia
JX
An
Tong-Qing
TQ
Peng
Jin-Mei
JM
Li
Guo-Xin
GX
Jiang
Yi-Feng
YF
Cai
Xue-Hui
XH
Xue
Qiang
Q
Wang
Mei
M
Wang
Yun-Feng
YF
Tong
Guang-Zhi
GZ
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000818:Animals; D000911:Antibodies, Monoclonal / Q000235:genetics* / Q000276:immunology; D000918:Antibody Specificity; D000956:Antigens, Viral / Q000235:genetics; D017124:Conserved Sequence; D017931:DNA Primers; D000939:Epitopes / Q000235:genetics* / Q000276:immunology; D004926:Escherichia coli / Q000235:genetics; D005822:Genetic Vectors; D016366:Open Reading Frames / Q000235:genetics; D010446:Peptide Fragments / Q000737:chemistry; D010957:Plasmids / Q000235:genetics; D019318:Porcine Reproductive and Respiratory Syndrome / Q000276:immunology*; D019316:Porcine respiratory and reproductive syndrome virus / Q000235:genetics* / Q000276:immunology*; D015183:Restriction Mapping; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013552:Swine; D014759:Viral Envelope Proteins / Q000235:genetics*
D000911:Antibodies, Monoclonal; D000956:Antigens, Viral; D017931:DNA Primers; D000939:Epitopes; D010446:Peptide Fragments; D014759:Viral Envelope Proteins; C114789:glycoprotein 5, PRRSV