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Archives of Virology

, Volume 163, Issue 7, pp 1877–1887 | Cite as

Utilization of phage display to identify antigenic regions in the PCV2 capsid protein for the evaluation of serological responses in mice and pigs

  • Marcus Rebouças Santos
  • Viviane Sisdelli Assao
  • Fabiana de Almeida Araújo Santos
  • Rafael Locatelli Salgado
  • Ana Paula Carneiro
  • Juliana Lopes Rangel Fietto
  • Gustavo Costa Bressan
  • Márcia Rogéria de Almeida
  • Zelia Inês Portela Lobato
  • Carlos Ueira-Veira
  • Luíz Ricardo Goulart
  • Abelardo Silva-Júnior
Original Article

Abstract

Porcine circovirus 2 (PCV2) is associated with a series of swine diseases. There is a great interest in improving our understanding of the immunology of PCV2, especially the properties of the viral capsid protein Cap-PCV2 and how they relate to the immunogenicity of the virus and the subsequent development of vaccines. Phage display screening has been widely used to study binding affinities for target proteins. The aim of this study was to use phage display screening to identify antigenic peptides in the PCV2 capsid protein. After the selection of peptides, five of them presented similarity to sequences found in cap-PCV2, and four peptides were synthesized and used for immunization in mice: 51–CTFGYTIKRTVT-62 (PS14), 127-CDNFVTKATALTY-138 (PS34), 164-CKPVLDSTIDY-173 (PC12), and 79-CFLPPGGGSNT-88 (PF1). Inoculation with the PC12 peptide led to the highest production of antibodies. Furthermore, we used the PC12 peptide as an antigen to examine the humoral response of swine serum by ELISA. The sensitivity and specificity of this assay was 88.9% and 92.85%, respectively. Altogether, characterization of immunogenic epitopes in the capsid protein of PCV2 may contribute to the improvement of vaccines and diagnostics.

Notes

Compliance with ethical standards

Funding

We thank the Brazilian Government Agencies. This research was funded by the Coordination for the Improvement of Higher Education Personnel - CAPES (grant number 23038.004678/2015-24), National Council for Scientific and Technological Development - CNPq (grant number 304727/2016-4), Foundation for Research Support of the State of Minas Gerais - FAPEMIG (grant numbers PPM-00796-15, CVZ-APQ-01327-14, CBB-RED-00005/14).

Conflict of interest

The authors declare no conflicts of interest.

Ethical approval

This project complied with the principles of the Commission for Ethics in Animal Experimentation of the Federal University of Viçosa (UFV) under protocol n°39/2012. All authors contributed to this work and agreed to its publication.

Supplementary material

705_2018_3816_MOESM1_ESM.jpg (1018 kb)
Supplementary Fig 1S. Expression of scFv clones. 96-well plates were sensitized with the supernatant from the clones, incubated with anti-HA and developed with OPD solution.
705_2018_3816_MOESM2_ESM.jpg (360 kb)
Supplementary Fig 2S. Specificity of scFv fragments against Cap-PCV2 and complementarity determining regions (CDR). A) Binding capacity of scFv fragments to Cap-PCV2 and BSA determined by ELISA. B) Binding capacity of F1 and F5 scFv fragments to Cap-PCV2 assessed by Western blotting. Arrows indicate the stained Cap-PCV2 band (about 30 kDa). C+: Pig anti-PCV2 polyclonal antibodies; C-: negative control for primary antibody (PBS); PM: molecular weight standard. C) CDR sequences of the selected scFv fragments. *It was not possible to identify CDR3 sequences according to IgBlast database.
705_2018_3816_MOESM3_ESM.doc (76 kb)
Supplementary Table 1. Selection of peptide sequences according to the PCV2 antibody/fragment used.

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Copyright information

© Springer-Verlag GmbH Austria, part of Springer Nature 2018

Authors and Affiliations

  • Marcus Rebouças Santos
    • 1
  • Viviane Sisdelli Assao
    • 1
  • Fabiana de Almeida Araújo Santos
    • 2
  • Rafael Locatelli Salgado
    • 3
  • Ana Paula Carneiro
    • 2
  • Juliana Lopes Rangel Fietto
    • 3
  • Gustavo Costa Bressan
    • 3
  • Márcia Rogéria de Almeida
    • 3
  • Zelia Inês Portela Lobato
    • 4
  • Carlos Ueira-Veira
    • 2
  • Luíz Ricardo Goulart
    • 1
  • Abelardo Silva-Júnior
    • 1
  1. 1.Laboratory of Animal Virology, Department of VeterinaryFederal University of ViçosaViçosaBrazil
  2. 2.Institute of Genetics and BiochemistryFederal University of UberlândiaUberlândiaBrazil
  3. 3.Laboratory of Animal InfectologyBIOAGRO, Federal University of ViçosaViçosaBrazil
  4. 4.Department of Preventive Veterinary MedicineFederal University of Minas GeraisBelo HorizonteBrazil

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