Screening And Characterization Of RS1Protein Affinity Peptides Of Porcine Epidemic Diarrhea Virus

Porcine epidemic diarrhea (PED) is a highly contagious, enteric disease of swine caused byporcine epidemic diarrhea virus (PEDV). The virus is ubiquitous among swine throughout theworld and is enzootic in most herds that have been tested. Thus，there is one of the hot spots of thePEDV study. Its prevailence has resulted in heavy economic losses in swine industry, especially inpiglets.The S protein of PEDV is a glycoprotein localized on the virion surface, which plays a keyrole, because of possessing the neutralizing epitopes and receptor binding domain. Given this,screening and identification of the receptor binding domain and antigenic epitopes were carried outin this study. It is necessary to provide some valuable information for development of diagnosistechnology and novel vaccine, and for designing antiviral immunization strategy of PEDV.The test successfully amplified PEDV rS1gene was cloned into prokaryotic expression vectorpGEX-6P-1, resulting in a recombinant plasmid pGEX-6P-1-rS1followed by expression in E.coliBL21. The recombinant protein was expressed in the form of inclusion bodies, which was purifiedand used as immunogen to generate rabbit anti-rS1polyclonal antibody. Indirect ELISA analysisshowed that the titer of the polyclonal antibody was approximately1:65536. Western-blot showedthat the polyclonal antibody could specifically bind to PEDV.Random12-mer Phage Display Peptide Library was used to panning the rS1protein for fiverounds. In the four rounds, tagged protein of pGEX-6P-1was used for a subtraction panning.Finally, ten monoclonal phages were selected randomly and subjected to sequence comparison Theresults show that ten positive phage clones were achieved. Sequencing results showed that sixphage clones had consensus sequences–SSTPSGHWHVSW-, five different peptides werescreened by panning on the rS1recombinat protein.The inhibition of the phage bearing peptide S to PEDV was evaluated in Vero-E6cell byImmunofluorescence and real time quantitative PCR. The results indicated PEDV and peptideincubation led to a decreased infectivity of PEDV. The current study may be helpful for designpreventive strategies to PEDV infection based on phage display techniques.