Establishment Of Indirect ELISA For Antibody Against Classical Swine Fever Virus And Study On The Immunological Characteristics Of Bacterial Outer Membrane Vesicles Presenting Its E2 Protein

Classical swine fever,symbolized high morbidity and mortality,is a highly contagious disease caused by classical swine fever virus(CSFV).It causes huge economic loss in swine industry every year.The E2 protein of CSFV is the most important protective antigen.Therefore,The E2 protein of CSFV is the most important target protein for establishing CSFV detection methods and constructing novel vaccines.The establishment of a rapid and effective CSFV antibody by indirect ELISA method can help to monitor the CSFV antibody level in pigs so that we can grasp the immune status timely and adjust the immunization program.The E2 protein vaccine of CSFV has attracted more attention because of its strong antigenicity,stable content,and advantages of distinguishing wild virus infection and vaccination.The study was described as following three parts:1.To reanize ?E2(the non-transmembrane region of E2 protein protein of CSFV)expression,a recombant p ET32a-?E2 expression plasmid was constructed and transfected into E.Coli BL21(DE3).Induced by IPTG,the ?E2 protein expressed in the form of inclusion bodies mainly.The inclusion body was denatured with 8mol/L urea and the denatured protein was renatured using a stepwise dialysis method.The renatured ?E2 protein can be recognized by CSFV positive sera and has good reactogenicity.2.The ?E2 protein obtained in the previous experiment was used as an antigen to coat microtiter plate.After conditions were optimized,an indirect ELISA method for detecting serum CSFV in swine was established.The sensitivity of the method was 81.58%,the specificity was 91.67%,and the agreement rate with the IDEXX CSFV antibody ELISA test kit was 84.24%.This study provides a reference method for the detection of serum CSFV antibodies in clinical pigs.3.This study firstly linked the N-terminus of ?E2 gene to the C-terminus of the Cly A gene and cloned into the expression vector p BAD18-Cm to establish the recombinant expression plasmid p BAD18-Cm-Cly A-?E2.And transformed E.coli DH5?.The recombinant bacteria were induced with 0.2% arabinose,and outer membrane vesicles(OMVs)presenting the CSFV ?E2 protein were extracted by ultracentrifugation.Transmission electron microscopy,mass spectrometry,SDS-PAGE and Western-blot analysis revealed that the recombinant strain secreted recombinant OMVs,and the ?E2 protein was presented on the outer surface of recombinant OMVs.The results show that the CSFV ?E2 protein can be presented on the surface of OMVs using the Cly A gene as a guide sequence.Recombinant ?E2 protein,recombinant OMVs and a rabbit attenuated strain of swine fever virus were used to immunize rabbits.The immune characteristics of recombinant OMVs presenting CSFV ?E2 protein were mainly explored.The results showed that compared with the other two vaccines,recombinant OMVs presenting CSFV ?E2 protein could rapidly stimulate rabbits to produce antibodies(P<0.05),and could better stimulate cellular immunity(P<0.01).The CSFV ?E2 protein presented by OMVs can be used as a candidate vaccine for CSFV.