Expression Of Spike Protein Of Porcine Epidemic Diarrhea Virus And Development Of Monoclonal Antibody Against Spike Protein

Porcine epidemic diarrhea(PED),caused by the porcine epidemic diarrhea virus(PEDV),is an acute and highly infectious disease.Infected piglets show clinical symptoms such as enteritis,vomiting,and watery diarrhea.The mortality in piglets is about 100%when they are infected at the age of 1 to 3 days,and then decreases to almost 10%by the age of elder,the average mortality is 50%.PED was first reported in China in 1976,and outbroke in 2010,which has caused huge economic losses to the swine industry.PEDV can be divided into G1 subtype and G2 subtype according to the S protein.The vaccine strain CV777 belongs to the G1 subtype,while the strains of PEDV appear in China since 2010 usually belong to the G2 subtype.Therefore,the traditional vaccine cannot protect pigs well.The purpose of this study is to develop a monoclonal antibody against PEDV S protein.After that,we will build an ELISA method for detecting PEDV in the future.1.The expression of PEDV S protein in vitro.In this study,compared the S protein of G1 and G2 subtype strains,we found that the S protein of G2 subtype strains has deletions,insertions and mutations,which are mainly located at the 27-266AA.In order to obtain prokaryotic and eukaryotic plasmids that expressed S protein of PEDV,the PEDV S gene was amplified by PCR with two pairs of conservative primers.And then it was cloned into the prokaryotic expression vector pGEX-6P-1 and eukaryotic expression vector pcDNA3.1,named pGEX-S and pcDNA3.1.pGEX-S was transformed into BL21(DE3)competent cells,and induced with 0.25 mmol/L IPTG at 37? and 225 rpm for 5h.SDS-PAGE and Western-blot results showed that the fusion protein was expressed and its molecular weight was about 59kDa.After optimizing the induction conditions,the recombinant protein could be expressed in the supernatant.Soluble protein was purified by GSTrap-FF column,and verified by SDS-PAGE and Western-blot.The eukaryotic expression recombinant plasmid pcDNA3.1-S was transfected into 293T cells and verified by IFA and Western-blot.The results showed that S protein could be expressed in 293T cells.These results provided materials for development of monoclonal antibodies against PEDV S protein.2.Development of monoclonal antibody against PEDV S proteinIn this study,8-week-old BALB/c mice were immunized with the fusion protein every 14 days.The cell fusion was made after the 4th imUunization.The 293T cells transfected with pcDNA3.1-S were used for screening the specific monoclonal antibody.The analysis of IFA and Western-blot both showed that three hybridoma cells could secrete monoclonal antibody against PEDV S protein,named PEDV-2B10?PEDV-4C3 and PEDV-5B7.PEDV-2B10 and PEDV-4C3 were IgG1 and PEDV-5B7 was IgM.Specific identification of monoclonal antibody showed that both PEDV-2B10 and PEDV-4C3 were against G2 subtype of PEDV.The ascites titer of PEDV-2B10 was 3200 and PEDV-4C3 was 6400.Four eukaryotic expression plasmids were constructed and transfected into 293T cell for analyzing the epitopes of antibodies.The results showed that the epitope of PEDV-2B10 was located at 18?88AA,and PEDV-4C3 was at 248-312AA.Our results laid the foundation for further research of PEDV S protein and establishment of an ELISA method for detecting PEDV S protein.