Fusion Expression And Immune Responses Of PCV2 Cap Protein And Vibrio Vulnificus Flagellin

Porcine circovirus type 2(PCV2)is the major pathogen of porcine circovirus disease(PCVD),leading to immunosuppression,secondary infections and heavy economic losses to the pig industry.PCV2 Cap protein is the the main structural and immunogenic protein with an important role in immune protection.Bacterial flagellin such as flaB of V.vulnificus is the stimulant of TLR5 and can act as a molecular adjuvant.In this study,the sequences of FlaB and Cap genes were optimized to E.coli codon usage,and the synthetic sequences were cloned into self-aggregating peptide ELK 16 fusion expression vector pET-ELK 16.The recombinant vectors pELK16-FlaB-Cap,pELK16-FlaB and pELK 16-Cap were transformed into BL21(DE3)E.coli,and the fusion protein expression was induced with IPTG.SDS-PAGE analysis showed that the three fusion proteins were expressed as active inclusion bodies.After centrifugation and washing with 0.5%Triton X-100,the purities of ELK16-FlaB-Cap,ELK16-FlaB and ELK16-Cap fusion proteins were up to 90%,96%and 93%.The mice were immunized with ELK16-Cap + IFA(incomplete Freund’s adjuvant),ELK16-Cap + ELK16-FlaB,ELK16-FlaB-Cap or PBS as the control.At days 7,14,21,and 28 after primary immunization,the serum samples were tested for Cap-specific antibodies by indirect ELISA.The results showed that,from day 21 after primary immunization,all of the immunized groups were positive for Cap-specific antibodies with the highest antibody level in ELK 16-Cap+ELK16-FlaB immunization group,followed by IFA +ELK16-Cap immunization group and ELK16-FlaB-Cap immunization group.On day 14 after boosting immunization,the splenic lymphocytes were collected from the sacrified mice and stimulated with ConA(5 μg/mL)or Cap fusion protein(10 μg/mL)before cytokine detection.The results showed that,compared with the control group,the levels of TNF-a,IL-12,and IFN-y in the immunized groups increased significantly.TNF-a concentrations were much higher after ELP-Cap stimulation than that after Con A stimulation,while both IL-12 and IFN-y were comparable between the two groups.These data indicate that mixed FlaB has a significantly greater stimulatory effect on PCV2 Cap ELISA antibody production than IFA,but the stimulatory effect of fused flaB is relatively weak.The fused FlaB has stronger stimulatory effect on IL-12 production,whereas mixed FlaB has a stronger stimulatory effect on TNF-a and IFN-y production.To improve the antigenicity of PCV2 recombinant antigen and to compare the effects of purification tags on their immunostimulatory effects,the coding sequences for neutralizing epitopes of PCV-2a,PCV-2d and PCV-2e were fused with PCV-2b Cap gene(4Cap)and expressed as a His-tagged protein.In addition,both FlaB and His-4Cap were expressed as His-tagged proteins,and FlaB was expressed as an ELP-tagged protein.The fusion proteins were purified by affinity chromatography or phase transition cycling.Thirty-six mice were divided into 6 groups,including PBS control group,His-4Cap,His-4Cap+ELP-FlaB,His-4Cap+His-FlaB,His-4Cap+ELK16-FlaB and His-4Cap+IFA immunization groups.Serum samples were collected at 7,14,21,and 28 days after primary immunization for Cap-specific antibody detection by indirect ELISA.On day 14 after boosting immunization,splenic lymphocytes were taken from sacrificed mice and stimulated with ConA(5 μg/ml)or 4Cap(10 μg/mL)for cytokine detection.On day 14 after boosting immunization,three mice from each group were challenged with 103 TCID50 PCV2b,and the viremia was measured by qPCR at day 3 or 7 after challenge.The results showed that,on day 7 after primary immunization,only His-4Cap + ELP-FlaB immunization group was positive for Cap-specific antibody.From day 14,antibody levels of all immunization groups were increased gradually.At day 21 after primary immunization,His-4Cap+ His-FlarB immunization group had the highest antibody level.Compared to the control group,the expression levels of TNF-α,IL-12 and IFN-y in the five immunization groups were significantly elevated.The TNF-a levels of ELP-Cap stimulation were higher than that of Con A stimulation,while both IL-12 and IFN-y were comparable between the two stimulation groups.These results indicate that His,ELP and ELK 16 tags have no significant effect on flaB stimulation of ELISA antibody response against PCV2 Cap protein,and all of the tagged flaB can stimulate TNF-a,IL-12 and IFN-y production in mice.The qPCR results showed that virrmia of all immunized mice was reduced,with the lowest level in His-4Cap + ELP-FlaB immunization group.