Screening And Characterization Of GP5 Protein Affinity Peptides Of Porcine Reproductive And Respiratory Syndrome Virus

According to the GP5 gene sequence of PRRSV strain VR-2332 published in GenBank, a pair of primers was designed. The GP5 gene of a PRRSV field isolate designated as HH08 was amplified by RT-PCR. There are 603 base pairs of the gene in length. The homology identity between the GP5 gene and that of VR-2332 and CH-1αis 91.5% and 99.2%, respectively.Using two pairs of primers, the N terminal and C terminal parts of GP5 from PRRSV isolate were amplified. By such amplification, the signal peptide sequence and transmembrane regions in the GP5 were deleted. Both gene fragments were linked using three repeated peptide sequences and cloned into prokaryotic expression vector, resulting in the PET-GP5 prokaryotic expression plasimid. The protein of interest was expressed in E.Coli Rosetta system in LB medium conventionally. SDS-PAGE indicates that the GP5 protein is approximately 18 Ku as expected. Western blot indicates that the protein can be recognized by swine anti-PRRSV serum. Meanwhile, the inhibitory ability of the GP5 protein to Marc-145 cell infection by PRRSV was confirmed. The GP5 was then used as an immunogen to inoculate a rabbit to generate polyclonal serum. Western-blot, ELISA and immunofluorescence indicate that the achieved serum can be used to detect PRRSV GP5 protein. Moreover, the inhibitory activity of the serum to PRRSV infection was confirmed.Random 12-mer Phage Display Peptide Library was used to panning the GP5 protein for four rounds. Twenty monoclonal phages were selected randomly and subjected to sequence comparison. The results show that eight different peptides were screened by panning on the GP5 recombinat protein. Two peptides that have the highest binding activity to the GP5 were synthesized. their sequences are KHMHWHPPALNT named K and HWGNHSKSHPQR named H, respectively. ELISA shows that both peptides can bind to the GP5 protein and inhibit cell infection by PRRSV.The immune responses of the two phages bearing either K or H peptide were evaluated in mice. The BALB/c mice were divided into five groups (twelve/group), namely, the V+K group inoculated with the PRRSV and phages bearing peptide K, the V+H group inoculated with the PRRSV and phages bearing peptide H, the V+S group inoculated with the PRRSV andl phages bearing a control peptide (S), the V+P group inoculated with the PRRSV and PBS and the C group (blank control). Serum, spleens and blood of mice were collected before immunization and 7,14,21,28 days after immunization respectively. ELISA was used to detect the immune response of different groups. Finally the neutralizing antibody of immunized mice was analyzed in PRRSV GP5-based ELISA. The results show that IgG titers of serum in mice given V+K group or V+H group were lowered than V+S group,V+P group. The current study may be helpful for preventing PRRSV infection based on phage display techniques.