Identification Of Inducing Neutralizing Antibody Regions In PDCoV S Protein And Screening Of Interacting Proteins

Porcine deltacoronavirus(PDCo V),a member of the δ coronavirus genus,is a newly emerged coronavirus that can cause intestinal diseases in pigs.In 2014,herds first broke out in the United States and quickly spread to other regions.It mainly infects piglets,and the main clinical symptoms are vomiting,watery diarrhea and dehydration,which bring great threats and cause economic losses in swine industry.The spike(S)protein of coronavirus,one of structural proteins,is kind of glycoprotein located on the surface of virus particle envelope,which can induce neutralizing antibody in infected host.These neutralizing antibodies always play an important role in preventing PDCo V from reinfection.Therefore,it is of great significance to analyze the regions which induce neutralizing antibody in S protein and screen out the host protein interacting with these regions for the research of genetic engineering vaccine against virus invasion.In order to identify the regions that produce neutralizing antibodies,this study first constructed a full-length S eukaryotic expression plasmid and purified the full-length S protein.At the same time,sucrose gradient centrifugation was used to purify PDCo V virus particles.The results of electron microscope showed that the purification of virus was successful.The purified S protein and purified virus particles were used as immunogens to immunize mice,and three monoclonal antibodies against S protein were screened,which were 17C7,23F3 and 9E3 respectively.The virus neutralization test has verified that none of the three monoclonal antibodies have neutralizing activity,but all three monoclonal antibodies have good reactivity with the S protein in the virus,which provid a tool for S protein research in future.Secondly,there is a CO-26k(COE)region in the S protein of Transmissible gastroenteritis virus and Porcine epidemic diarrhea virus,which can mediate the production of neutralizing antibodies,and the C-terminal domain(CTD)of S1 also seems to contain neutralizing epitopes.In this study,we predicted the COE region of PDCo V S protein,constructed COE and S1-CTD expression plasmids,purified COE protein and S1-CTD protein,immunized mice,and obtained polyclonal antibodies.The virus neutralization test showed that the polyclonal antibodies produced in COE region did not have neutralizing activity,while the polyclonal antibodies produced in S1-CTD region had neutralizing activity,and the neutralizing titer was 1:64.Then we identified that S1-CTD region was the region that induced neutralizing antibodies.Finally,in order to screen out which host protein interacting with S1-CTD region,this study selected IPI-2I cells as target cells,extracted their cellular proteins,and screened out interacting host proteins by Flag-pull down experiment.According to the results of mass spectrometry,five genes with higher scores are selected for verification,and five expression plasmids are successfully constructed.After western-blot verification,Co-IP test verified that KIF1-binding protein(KIFBP)interacted with S1-CTD,and confocal microscopy test verified that they were co-located.Through overexpression of KIFBP,it down-regulated virus proliferation,and the mechanism of its down-regulation on virus proliferation needed further study.In conclusion,this study prepared the PDCo V S protein monoclonal antibodies,which have a good specific reaction with the S protein,laying the foundation for the study of the S protein.Secondly,this study identified that S1-CTD region of S protein can mediate the production of neutralizing antibody,and then screened out the interacting host protein,which provided basis for the research of pathogenic mechanism and subunit vaccine.