| Foot and mouth disease (FMD) is one of the most acute, febricity, high contagious disease of cloven-hoofed livestock which is caused by FMDV. Once the disease breakout, countries will have enormous economic and politic disaster. To date, the most widely used FMD vaccine is a chemically inactivated whole viral preparation that is formulated with oil adjuvant. Although it has an influential role, the use of the killed FMDV as vaccine is not completely safe due to the case of incomplete inactivation and linkage during its manufacturing, and it is necessary to develop a security, high performed new-type FMD vaccine. At present, the most frequently used new-type FMD vaccine is polypeptide vaccine. Polypeptide has many advantage, for example, security, stabilize, easy dilivery and high direction, but its immunogenicity usually poor, therefore, polypeptide often used with some adjuvant. In our research, to study the effect of bovine IL-2 and IFN-γon the immune response of FMDV G-H peptide (137-158aa from the VP1 protein of FMDV) in mice, BoIL-2, BoIFN-γeukaryotic expression plasmid and bovine IFN-γmature protein (pr-mBoIFN-γ) were respectively combined with FMDV G-H peptide fusion protein (G-H) and immunized to the Kunming mice.In the study, VP1 protein, G-H and pr-mBoIFN-γwere successfully prokaryotic expressed and purified, and eukaryotic expression plasmid encoding for BoIL-2 and BoIFN-γwere constructed. In order to confirm that the two eukaryotic expression plasmids would express correctly in eukaryotic systems, they were transfected into BHK21 cells, and the plasmid pcDNA3.1 was used as negative control. The results show that BHK21 cells infected with pcDNA3.1-pBoIL-2 and pcDNA3.1-pBoIFN-γcould respectively be stained with mouse anti-pr-mBoIFN-γand anti-pr-mBoIL-2 polyclonal antibody and goat anti-mouse IgG conjugated with ?uorescence, whereas no ?uorescence was observed in pcDNA3.1 infected BHK21cells. The two eukaryotic expression plasmids, pr-mBoIFN-γand free vector pcDNA3.1 were combined with G-Hpeptide respectively, and negative and positive control were set up. The humoral immunity was monitored by indirect ELISA and Virus Neutralization Test (VNT). The results indicated that after the primary immunization, serum IgG levels were not apparently increased in each group compared to that of controls. After the second and third immunization, the levels of the groups inoculated with G-H+pcDNA3.1-pBoIFN-γ, G-H+pr-mBoIFN-γand G-H+pcDNA3.1-pBoIL-2 is significantly higher compared to that of controls (P<0.05) but significantly lower than that of killed vaccine groups (P<0.05). Furthermore, the neutralizing antibody titer (NAT) of G-H+pcDNA3.1-pBoIL-2 and G-H+ pcDNA3.1-pBoIFN-γare significantly higher than that of the group G-H+FA (P < 0.05), which indicated that rBoIFN-γ, rBoIL-2 as a molecular adjuvant, may perform better application than the classic immunoadjuvant. Lymphocyte proliferation assay show that 35 days after the first immunization, the group inoculated with G-H+pcDNA3.1-pBoIFN-γshows the highest level, followed by G-H+pcDNA3.1-pBoIL-2 and G-H+Pr-mBoIFN-γgroups, and they significantly higher than the controls. In all the groups, the killed vaccine group induced the highest level of IL-4, and the group with G-H+Pr-mBoIFN-γinduced the highest level of IFN-γ. These results are conformed to the results of IELISA assay and Lymphocyte proliferation assay. Compared to the group with G-H+pcDNA3.1, not only the IL-4 but also the IFN-γrelative expression levels of the group co-inoculated with G-H+pcDNA3.1-pBoIL-2, pcDNA3.1-pBoIFN-γor G-H+Pr-mBoIFN-γwas elevated, which further indicated that rBoIFN-γand rBoIL-2 have enhancement property to the immune response of FMDV G-H loop peptide in mice.In conclussion, the study offered the theory information and supported material for the research and application of G-H loop peptide of FMDV Type Asia1 in the immunization of FMD. The results implied that BoIL-2 and BoIFN-γmay represent an effective adjuvant in the future. |
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