Construction And Mice Immunogenicity Analysis Of Orf Virus Polypeptide Vaccine

Orf is an infectious disease caused by the presence of pustules and papules in the nostrils and lips caused by the Orf Virus(ORFV).The B2 L and F1 L proteins encoded by ORFV 011 and ORFV 059,that can induce a protective immune response and that are the main candidate genes for the development of subunit vaccines for the orf virus.Ferritin nanoparticles are often used as antigen scaffolds to improve the immunogenicity of subunit vaccines.1.Construction and expression of recombinant vectors of B2 L and F1 L polypeptide fragments of Orf virus: Design primers according to the B2 L and F1 L gene sequences of Orf virus NZ2 strain(Gen Bank: DQ184476.1)to amplify the target genes B2 L and F1 L,respectively,and iron Protein gene fusion,PCR was connected in tandem to the prokaryotic expression vector p ET-28 a to obtain the vector p ET-Fe-F1 L and p ET-Fe-B2 L,and p ET-Fe-F1 L was digested with B2 L gene to obtain p ET-Fe-F1L-B2 L.The three recombinant plasmids sequenced correctly were transformed into BL21(DE3)cells,and IPTG was used to induce the expression of Fe-F1 L,Fe-B2 L and Fe-F1L-B2 L recombinant proteins.Western blot confirmed the high expression of recombinant proteins Fe-F1 L,Fe-B2 L and Fe-F1L-B2 L.Solubility analysis confirmed that the recombinant proteins Fe-F1 L,Fe-B2 L and Fe-F1L-B2 L mainly existed as inclusion bodies,and then used urea The purification method purifies the inclusion body protein.Western blotting results showed that the expression of recombinant proteins Fe-F1 L,Fe-B2 L and Fe-F1L-B2 L all had good reactogenicity.2.Immunogenicity analysis of orf virus B2 L and F1 L peptide vaccines: First,the purified recombinant proteins Fe-F1 L,Fe-B2 L and Fe-F1L-B2 L were prepared into peptide vaccines.Then,a total of 40 female 5-week-old BALB / c mice were randomly divided into 5 groups.Finally,mice were immunized with the peptide vaccine.In the third week after immunization,BALB / c mice of each test group were subjected to intravenous blood collection to separate serum.Then,ORFV specific antibody levels and cytokine(IL-4 and IFN-?)secretion levels were examined.The results showed that the recombinant proteins Fe-F1 L,Fe-B2 L and Fe-F1L-B2 L could induce the experimental mice to produce specific antibodies against ovine ulcer virus,and the antibody levels and their levels were significantly higher than those in the PBS group(P <0.01);The secretion levels of IL-4 and IFN-? were also significantlyhigher than those in the PBS group(P <0.05),indicating that the mutton ferritin nanoparticle vaccine constructed in this study has good immunogenicity and can effectively induce specific humoral immunity and cells immunity.In summary,three recombinant expression plasmids p ET-Fe-F1 L,p ET-Fe-B2 L and p ET-Fe-F1L-B2 L were constructed in this study,which can express the antigen protein Fe-F1 L of orf virus in E.coli on the high levels,Fe-B2 L and Fe-F1L-B2 L,the ferritin nanoparticle peptide vaccine developed on this basis,can induce and induce the test mice to produce specific antibodies against the orf virus and a high level of cellular immune response.This study laid the foundation for the further development of genetically engineered vaccines for orf virus.