Expression Of Porcine Epidemic Diarrhea Virus Spike Protein In The Baculovirus Expression System And Development Of Monoclonal Antibodies Against The Spike Protein

Porcine epidemic diarrhea virus（PEDV）can cause acute watery diarrhea,vomiting and dehydration with high morbidity and mortality in suckling piglets.In 2010,the variant PEDV infection-induced porcine epidemic diarrhea（PED）outbreaks took place and later widely spread to most regions of China,resulting in huge economic losses to the pig industry.In order to investigate the epidemiological and etiological characterization of the variant PEDV,77 diarrhea samples were collected from different pig farms in Beijing,Shanghai,Zhejiang,Henan and Shandong provinces,and diagnosed by our established RT-PCR method to detect PEDV’s genetic material.The results showed74.0%of them were PEDV-positive.Then the different S genes were cloned and sequenced from some of the positive samples,and used for sequence alignment and genetic variation analysis by comparing with the counterparts of the representative strains isolated in China and other counties.The results showed the nucleotide sequence homology between the S genes of our isolates and the emerging variant strains ranged from 97.1%to 99.4%,while the homology between the S genes of our isolates and the classical strains was as low as 93.9%⁹4.3%.It also showed that there were stable mutations in the S protein–five amino acid insertions(⁵⁹QGVN⁶² and N¹⁴⁰)and two deletions(¹⁶³NI¹⁶⁴/¹⁶³DI¹⁶⁴),compared with the classical strains,while two stable amino acid substitutions,i.e.,site 182（Y→H）and 345（F→L）occurred in the S protein compared with the previously reported variant strains.The phylogenetic analysis based on the S genes showed the PEDV strains obtained in this study were closely related to the other variant PEDV strains and they all clustered on the same branch.All these suggested that the PEDV strains we obtained in this study were PEDV variant strains having obvious amino acid mutations on the S protein.In addition,some novel mutations were observed on the S protein of PEDV variant strains circulating during 2016-2017 in comparison with those that emerged in 2010.In order to investigate the antigenicity of S protein,in this study,we first designed the primers based on the cleavage site of S1/S2.Then,the S1 gene of PEDV was amplified by RT-PCR and cloned into pFastBacHTA vector and the recombinant plasmid pFastBacHTA-S1 was acquired.It was then transformed into DH10Bac competent cells and selected by the blue-white screening and PCR.The positive recombinant shuttle plasmid which was designated rBacmid-S1 was transfected into the Sf9cells that later developed visible cytopathic effect（CPE）at 48 hours post transfection（hpt）.The first passage（rBac-S1-P1）was obtained at 72 hpt and was serially passaged to P3 in the Sf9 cells.After that,the P3 was inoculated into the Sf9 cells for PEDV identification.Indirect immunofluorescence（IFA）result confirmed that the cells infected by rBac-S1-P3 recombinant virus showed bright green fluorescence,suggesting the candidate recombinant protein could be specifically identified by the PEDV pig antisera and the monoclonal antibody（MAb）against the PEDV S1 protein.The SDS-PAGE analysis showed a specific band at around 120kDa corresponding to the molecular weight of candidate recombinant protein,which could also recognized by MAb against S1 protein using western blotting.On the other hand,in order to produce MAbs against the PEDV S protein,we immunized BALB/c micewith highly purified PEDV particles and obtained 12 MAbs that could specially recognize the PEDV-infected cells by hybridoma technology.Further,based on the rBac-S1 infected Sf9 cells,we acquired 5 MAbs that could recognize the PEDV S1 protein using IFA.This study provides the basis for further investigation on the PEDV antigenicity.