| Porcine epidemic diarrhea virus(PEDV)is a causative agent of acute,highly contagious porcine epidemic diarrhea(PED).It infects pigs of all ages and results in high mortality rates particularly in suckling piglets.In this study,structural or no-structural proteins encoded by PEDV genome were expressed and purified;based on that,sensitive and efficient serological assays were established and applied for surveillance and evaluation of humoral responses in pigs at post-vaccination or infection.Meanwhile,mechanism of PEDV invasion was investigated using the anti-PEDV monoclonal antibodies which were developed in this study.Specific primers were designed in according to PEDV G2(GenBank accession no.KU558701)and its cDNA was used as a template for cloning of various PEDV genes,including those encoding ORF3C,E,nspl,nsp2 peptides and some domains of nsp3 peptide;Recombinant eukaryotic or prokaryotic expression plasmids were constructed and proteins were correspondingly expressed in the relative systems.The proteins were then purified by affinity,tested by SDS-PAGE and Western blot,which all showed they were in correct form.Then these proteins were used as immunogens and polyclonal antibodies were generated respectively using New Zealand white rabbits.These antibodies were then identified by Western blot and IFA,which all showed good reactivity and specificity,and the ELISA titers could be more than 1:100,000.All data suggested that these antibodies could meet the requirements of later experiments.PEDV G2(ZJU/G2/2013 strain;GenBank accession KU558701)was propagated in Vero cells and purified by ultracentrifugation on a sucrose-gradient.Indirect ELISA assays based on the whole virus(WV)?S1?ORF3C and E protein were respectively developed.As the antigen concentration and dilutions of sera and HRP-conjugated antibodies were optimized by checkerboard titrations;the optimal amount of PEDV antigens used for coating were 5 ?g/mL?4.4 ng/mL?15.6 ng/mL and 7.8 ng/mL corresponding to PEDV whole virus?S1?ORF3C and E protein;the best dilutions for prnmary antibody and secondary antibody were 1:100 and 1:10,000 respectively.The coating conditions,incubation time and temperature for antibodies were all optimized,and the indirect ELISAs were finally established.The PEDV whole virus and S1 protein based ELISAs were used to detect IgG and IgA antibodies against PEDV;The ORF3C and E proteins based ELISAs were applied for detecting IgG antibodies against PEDV;and antibodies in the samples including colostrum,milk,serum,and fecal from different ages of diarrhea pigs from different pig farms in Zhejiang province were detected using the ELISAs.Results showed that antibodies against PEDV existed universally and the level in sows was relatively higher,piglets of 0-1 week age also had a high antibody level;while it declined in pigs of 3 week age,raised in pigs of 7 week age,but kept constant in pigs of 13 and 17 week age;The results coincided with current conditions of antibodies in PEDV infecting pigs of different ages.Meanwhile,neutralizing antibodies against PEDV in serums of some pigs were detected randomly.The results indicated that the samples which had high antibody levels also had high neutralizing antibody levels against PEDV,which suggested that results detected via ELISAs were consist with that of neutralization assays.Above all,the indirect ELISAs established in this study were reliable.The PEDV whole virus(WV)particles and recombinant PEDV proteins:spike(SI),ORF3,envelope(E),nonstructural protein 1(nspl),nsp2,Ac(acidic domain of nsp3),and nsp3-ADRP(ADP-ribose-1-monophosphatase domain)were tested for reactivity with sera from PEDV-infected pigs by ELISA and/or western blot analysis.According to western blots,antibody interactions with the S1 protein were relatively more sensitive and specific,although both S1 and Ac proteins were detectable by immunofluorescence(IFA)in vitro.Furthermore,a total of 951 serum samples from pigs with undetermined infection status were analyzed by ELISA,with most showing immune-reactivity towards the viral proteins described above.The earliest IgG antibody response was observed in the 1 week-old piglets,with similar antibody ontogeny and patterns of seroconversion for S1,ORF3C,E,and WV antigens.Immunoreactivity with the recombinant proteins was highly specific,as sera from 30 unaffected piglets did not react with any of them.All data indicated that the recombinant S1 protein may be useful as an antigen for development of serological assays for detection of PEDV.The PEDV S1 protein was used as immunogen to immune BABL/C mice of 6-8 week ages;and traditional hybridoma technology was used to achieve monoclonal antibodies against PEDV S1 protein.Four hybridoma cell lines which could secret monoclonal antibodies stably were developed and named as 2C2G11?2G4C5?3B10G8 and 7D7G1.The subtypes of the monoclonal antibodies were IgG1,ELISA titers of hybridoma supernatants and the ascites could reach more than 1:10 and 1:1,000 respectively.Their neutralization activities were tested using neutralization assays in vitro,and the neutralizing titers were calculated by Reed-Muench methods.The results showed that all of them have certain neutralizing activities,and the neutralization titers of 2C2G11 and 3B10G8 could be more than 1:200.Specific bands appeared in Western blot tests and specific fluorescence was observed in IF>dA;The results indicated that the monoclonal antibodies achieved in our study could recognize natural conformation of PEDV and all of them had good reactivities.Above all,the monoclonal antibodies against PEDV S1 protein could be used as important tools in the later study of PEDV cell entry.PEDV S1 truncated proteins including S10(aa 1?219)?OA(aa 1?504)?B(aa 510?640)and CD(aa 639-729)were expressed in eukaryotic system,purified by Protein A affinity,and detected by Western blot.The main antigenic epitopes of PEDV Sl protein were identified by ELISA using the neutralizing monoclonal antibodies.Meanwhile,the protein OA and CD were used to inhibit virus entry of the target cells.Results showed that the truncated proteins all expressed correctly and had good reactivity.The neutralization epitopes of PEDV S1 protein may exist in the regions of OA and CD;and they all could inhibit virus entry;the receptor binding domain may exist in the CD domain,which suggested that the neutralization epitope of PEDV S1 protein may also be its receptor binding domain. |
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