Prokaryotic Expression For E2 Protein Of Classical Swine Fever Virus In E.coli And Development Of Bacterium-Like Partical Vaccine

Classical swine fever is an acute,pyretic and highly contagious disease caused by classical swine fever virus.In recent years,the epidemic characteristic of swine fever has undergone significant changes,with a co-existence of typical swine fever and atypical swine fever,and with the demonstration of recessive infection,persistent infection and mixed infection,which brings great difficulties to the prevention and control of CSFV.However,practice has proved that the vaccination of hog cholera lapinized vaccine(HCLV)is still the most effecti-ve measure to control swine fever,in spite of which can't distinguish viral infection to vaccine immunization with serum detection.Subunit vaccine which can serve as serum marker has been developed by eukaryotic expression system in foreign country,but the drawbacks of this subunit vaccine which is poor production,high costing and complicate manufacturing technique affected its further application and spread.Therefore,it is extremely urgent to develop indeed feasible new generation vaccine with the function of serum marker.GEM particle surface display system composed of lactic acid peptidoglycan skeleton Gram and protein anchor is a new antigen presentation technology developed in recent years.When co-expressing the target protein and PA in other host strain,the fusion protein can be displayed on the surface of GEM through simple incubation,and then develop the bacterium-like particle vaccine.The antigen acquired using this method is safe and efficient,and more convenient and cheap,and it has been continually reported aboard.In this study,E2 protein of CSFV fused with the protein anchor was successfully expressed in E.coli and then displayed on the GEM surface.The BLP vaccine based on E2 protein was prepared and evaluated its immunogenicity with mice.In addition,the fluorescent antibody fused by the green fluorescent protein and E2 nanobody was expressed in E.coli,and the direct immunofluorescence method for the detection of CSFV was successfully established using this fusion protein.The main contents of this study are as follows:Test ? Expression and Purification of E2 protein fused with the protein anchorIn this chapter,the front 176aa gene of CSFV E2 was fused with PA3 and expressed by CVC1103 strain.E2-PA3 was partially soluble expressed in E.coli with SDS-PAGE,and can be detected with E2 monoclonal antibody in Western blot.The fused protein was successfully displayed on the GEM surface following incubated with GEM particles.However,some E.coli protein were also be concentrated by GEM particles.In order to eliminate the interference of impure protein,pET-E2-PA3 was transferred into CVC1105 for optimization of host bacteria.The results showed that the ratio of E2-PA3 in the total protein was significantly higher when incubated with GEM,and the nonspecific is almost negligible.So the CVC1105 expression platform was chosen as expressing host for E2-PA3.Test ? Preparation and immunogenicity evaluation of BLP vaccineMice were immunized with GEM-E2-PA3 which was composed with equal proportion of BLP antigen and adjuvant 206.Meanwhile,GEM particles and PBS immunized groups were served as control.It was revealed that the blocking rate of antibody in BLP vaccine immunized group reached to 60%at 3 weeks after first immunization,and rose up to 90%at 3 weeks after second immunization;later,the antibody remained at high levels for several weeks following vaccination,while the blocking rate of control groups in GEM and PBS always remained at 20-30%,which were visibly below BLP vaccine group(P<0.05).Result showed that lymphocyte stimulating index of mice of BLP group was obviously higher than groups of GEM and PBS in of Lymphocyte proliferation test.The Real time PCR revealed that the level of IFN-? and IL-4 mRNA from mice immunized with BLP was obviously higher than other groups.In conclusion,the BLP vaccine developed in this study can not only induce high-level humoral immunity;meanwhile,it can be mediated stronger cellular immunity.Test ? Expression of fluorescent antibody SE and establishment of CSFV direct immunofluorescence methodThe fused EGFP and VHH nanobody protein against CSFV was expressed in E.coli.After purification of recombinant protein done by nickel column,the specificity of fluorescent antibody was identified by incubation with CSFV,PRV,PCV-2 and cell PK15 infected by PPV.And we also compared the efficacy of traditional CSFV immunofluorescence assay.It was revealed that fluorescent antibody can be partial soluble expressed in E.coli,and only the PK15 cell infected with CSFV can be observed obvious green fluorescence with direct immunofluorescence assay,while only few nonspecific fluorescent spot in other groups.Compared with traditional immunofluorescence assay developed by commercialized monoclonal antibody,the direct immunofluorescence assay established by SE possess stronger artculation,and SE have a great many of advantages,such as easily operation,short preparation periods and low costing.Therefore,the SE acquired in this experiment has the potential value of applying CSFV to direct immunofluorescence assay.