Construction And Evaluation Of A Self-assembling Nanoparticle Vaccine Against Classical Swiae Fever

Classical swine fever(CSF)is a severe infectious disease that seriously damages the pig industry.CSF is a World Organisation for Animal Health(OIE)-listed.CSF is caused by classical swine fever virus(CSFV),whose genome length is 12.3kb.E2 protein is the most important protective antigen protein of CSFV,which can induce neutralizing antibodies upon CSFV challenge,and therefore E2 protein is the main target for CSF vaccine development.Ferritin(FR)is a nanoparticle that can spontaneously assemble into nanoparticles.Ferritin(12.2 nm in diameter)forms a nearly spherical particle composed of 24 subunits arranged with octahedral symmetry around a hollow interior.The ferritin nanoparticle has been used to present the antigens designed.FR nanoparticles as antigen display platform can greatly improve the density of the antigen to stimulate animal to produce a higher level of immune response.Therefore,it can be used as a vaccine design to develop safe and efficient vaccines.In order to develop an excellent CSFV marker vaccine,this study used FR as a display platform,and connects the CSFV E2 gene with the Ferritin gene,then cloned into the expression plasmid.After transient transfection,it proved that FR-E2 nanoparticles can be successfully express and secreted into the supernatant using the CSFV E2 antigen capture ELISA kit.Subsequently,the expression of the Ferritin(FR-SP1 and ER-GT6)and FR-E2 nanoparticles(FR-E2-SP1 and FR-E2-GT6)was successfully detected using the anti-His monoclonal antibody by Western blotting.Further,this study proved that the FR-E2 nanoparticles(FR-E2-SP1 and FR-E2-GT6)successfully expressed,and which were recognized by CSFV specific monoclonal antibody WH303.And after being negatively stained,FR-E2 nanoparticles wre successfully observed with a diameter of 10-15 nm using transmission electron microscope.Subsequently,two ExpiCHO suspension cell lines stably expressing FR-E2 nanoparticles(FR-E2-SP1 and FR-E2-GT6)were constructed and increased the positive rate of these cell lines by flow cytometry.Next,the antigenicity were compared of FR-E2 nanoparticles with CSFV HCLV(C)strain E2 and Shimen strain E2 protein by an indirect ELISA(iELISA).The results showed that FR-E2 nanoparticles have similar antigenicity to the E2 protein of the C strain and Shimen strain.In order to develop an excellent CSFV marker vaccine,mouse were vaccinated FR-E2 nanoparticles mixed sigma adjuvant system(SAS)at a ratio of 1:1(v/v),and detected antibody levels.The results showed that FR-E2 nanoparticles had better immunogenicity than E2 protein,and induce higher levels of neutralizing antibodies.In conclusion,the FR-E2 self-assembling nanoparticles constructed in this study are promising vaccine candidates and have the potential to be developed as diagnostic antigens for CSF.