Expression,secretion And Immunogenicity Study Of PEDV S And M Protein In Sf9 Cells

Porcine epidemic diarrhea(PED)is an enteric epidemic swine disease caused by porcine epidemic diarrhea virus(PEDV),which manifested clinically by watery diarrhea,vomiting,anemia,dehydration and weight loss.Pigs of all ages can be infected with PEDV,especially the piglets are most susceptible,and the mortality of piglets can be up close to 100%.PEDV is an enveloped virus belonging to α coronaviruses of coronaviruses family.Its main structural protein Spike protein is embedded on the surface of viral particles,which is responsible for binding of virus to specific cellular receptors and mediating virus-cell membrane fusion and cell entry.It is also the key structural protein to induce neutralizing antibodies.M protein plays an indisppensable role in PEDV virus assembly,budding and host immune regulation.Since 2010,PEDV mutant strains have appeared in many provinces and cities in China,posing challenging obstacles to prevention and eradication of PED.Therefore,further studies on the expression methods of the main structural proteins S and M of PEDV is needed.In order to invistigate the expressing,secretory and immunogenic properties of PEDV structural proteins S,M and E expressed in Sf9 cells,these proteins were expressed in baculovirus insect cell expression system,and their expressing and secretory properties explored.Medium supernatant were concentrated by sucrose density gradient centrifugation,and the morphology of S-M protein was observed under transmission electron microscopy(TEM).Reactivity of S,M and S-M proteins against PEDV positive pig sera were tested by Enzyme-linked immunosorbent assay(ELISA),and the immunogenicity of proteins or virus-like particles(VLPs)were evaluated by mice immunoassay.1.Expression and secretion of PEDV structural proteins S,M and E in insect cellsSpecific primers were designed to introduce different restriction enzyme sites into M and E gene fragments,and M and E genes were inserted into the expression vectors containing S gene,respectively.Then the ligation products were transformed into DH5αcompetent cells,and the recombinant plasmids were extracted and verified by digestion and sequencing analysis.Seven recombinant plasmids(p Fast Bac-S、p Fast Bac-M、p Fast Bac-E、p Fast Bac-S-M、p Fast Bac-S-E、p Fast Bac-M-E and p Fast Bac-S-M-E)were successfully obtained.The positive plasmids were transformed into DH10 Bac competent cells.Pure white recombinant bacmids were picked through blue-and-white screening and cultured overnight to extract recombinant bacmids.Results of PCR and electrophoresis identification showed that recombinant bacmids were successfully constructed.The recombinant bacmids were transfected into Sf9 cells,and cell pathological effect were observed(swelling,enlargement,rounder edges,etc.),which showed that the recombinant baculovirus was successfully revived.The recombinant baculoviruses(r BV-S、r BV-M、r BV-E、r BV-S-M、r BV-S-E、r BV-M-E and r BV-S-M-E)were successfully obtained.The expressing properties of recombinant proteins S,M and E in Sf9 cells were detected by Indirect immunofluorescence Assay(IFA).Green and red fluorescence signals were detected in infected Sf9 cells,while no fluorescence signal was detected in healthy Sf9 cells(negative control group),which showed that S,M and E proteins were all expressed in Sf9 cells.Western blotting results showed that S,M and E proteins could be detected in the cell pellets,further indicating that these proteins could be expressed alone or together in Sf9 cells.Moreover,S,M and S-M protein could be detected in the concentrated medium supernatant,and S or M proteins could be detected in the co-expressed S-E and M-E proteins,while E protein was not detected.These results showed that S,M and S-M proteins could be secreted into the medium.2.Identification of S-M virus-like particles and preliminary evaluation of their immunogenicityBy optimizing the MOI and TOH parameters when S and M proteins were expressed alone or co-expressed in Sf9 cells,the secretory expression system of S and M proteins in Sf9 cells was initially established.The medium supernatant was concentrated by sucrose gradient centrifugation,and secretory S-M VLPs was obtained.The expressing and secretory properties of S and M proteins were analyzed by western-blotting,the morphology of S-M VLPs were observed by transmission electron microscopy,and the reactivity with PEDV positive pig sera was tested by indirect ELISA,and the immunogenicity was tested after immunizing mice.The IFA results showed that the recombinant proteins were expressed in Sf9 and located near the cell membrane.Western blotting and TEM results further demonstrated that S-M protein could be secreted and assembled into VLPs formed by S-M protein with a diameter about 90-190 nm.Indirect ELISA results showed that S,M and S-M proteins could react specifically with PEDV positive pig sera,and high level of specific Ig G and neutralizing antibodies could be induced in mice after immunization for 3 times.This study explored the expression methods and purification techniques of PEDV structural proteins S,M and E in Sf9 cells.These results indicated that secretory S-M VLPs with good immunogenicity can be produced by co-expression of S and M proteins in Sf9 insect cells.These results will lay an experimental foundation for invistigating the assembly mechanism of PEDV VLPs and developing the subunit vaccines.