Construction Of Recombinant DNA Vaccine Of Orf Virus B2L And Sheep Pox Virus P32 Genes

Orf virus（ORFV）is an important pathogen that causes "aphthous ulcers" in animals such as sheep and goats.Clinically,it is mainly characterized by papules,erythema,blisters,pustules and crusts on the skin/mucous membrane of the mouth and lips of infected animals.The disease is highly contagious and can be infected through direct and indirect contact.Lambs are the most susceptible and seriously affect the growth performance of lambs.Sheep pox virus（SPPV）is a member of the genus Capripoxvirus of the Poxviridae family.Sheep pox（SP）caused by SPPV is an acute,febrile,and highly contagious infectious disease,which is mainly characterized by the formation of typical nodular lesions on the skin/mucous membranes of the whole body,is highly infectious,and has a high fatality rate.At present,Orf and SP have become common viral diseases in sheep,which seriously restrict the development of sheep farming.In view of the prominent harm of Orf and SP to the sheep industry,and the lack of safe and effective vaccines for the prevention and treatment of the above two diseases,this study carried out the research on the DNA vaccine with the main immunogen gene tandem of ORFV and SPPV.First,the ORFV B2 L and SPPV P32 genes with good immunogenicity were used as candidate antigen genes,and the DNA fragment of the self-cleaving peptide P2 A was used to concatenate the B2 L gene and the P32 gene by overlapping extension PCR technology,and then cloned by seamless cloning technology to eukaryotic expression vectors.Then,the recombinant plasmid expressing B2L-P2A-P32 was transfected into eukaryotic cells,and Western Blot analysis was performed 48 hours after transfection.The results showed that the self-cleavage of the translated polypeptide at P2 A enabled the independent expression of B2 L and P32 proteins in eukaryotic cells.In order to clarify the immune protection effect of pc DNA3.1-B2L-P2A-P32 recombinant vaccine plasmid and its application prospect as a vaccine candidate strain,in this study,the prepared recombinant DNA vaccine plasmid was mixed with adjuvant to immunize mice.The immunization program was the first immunization on the 0th day,the booster immunization on the 21 st day,and the challenge protection test was performed 14 days after the second immunization.Detection of serum specific antibody,neutralizing antibody,immunoglobulin Ig G1 and Ig G2 a levels showed that,pc DNA3.1-B2L-P2A-P32 recombinant vaccine plasmid combined adjuvant immunization can induce specific antibodies against ORFV and SPPV,and it can induce the production of anti-SPPV neutralizing antibodies.The cytokines in serum were detected by ELISA.The results showed that the pc DNA3.1-B2L-P2A-P32 recombinant vaccine plasmid combined adjuvant immunization group could induce higher levels of IFN-γ and IL-4,and the difference was statistically significant.The above results show that the mixed immunization of pc DNA3.1-B2L-P2A-P32 recombinant vaccine plasmid and adjuvant can effectively induce the body’s immune response.The results of animal challenge protection test showed that the pc DNA3.1-B2L-P2A-P32 recombinant vaccine plasmid combined adjuvant immunization group could effectively reduce the viral load in the lungs of mice,which indicated that the recombinant vaccine plasmid could provide good immune protection.In conclusion,the recombinant vaccine plasmid expressing ORFV B2 L and SPPV P32 genes in tandem is expected to be used in the preparation of DNA vaccines,providing an important technical means for the prevention and control of Orf and SP in clinical practice.