Evaluation Of The Immune Efficacy Of ASFV Subunit And Peptide Vaccine

African swine Fever(ASF)is a highly infectious swine viral disease caused by African swine fever virus(ASFV),which can cause high mortality in infected pigs.ASFV is the only Arthropod-transmitted DNA virus whose virion structure is complex.So far,no commercial vaccine against ASF has been developed,and prevention entirely depends on preventing contact between the virus and susceptible hosts.At present,identification of proteins that may induce protective responses has become one of the directions of research on ASF vaccine.Both subunit vaccine and synthetic peptide vaccine are vaccines based on viral proteins,which can induce strong immune responses in the body,and both vaccines have high safety.Therefore,the aim of this study was to prepare ASFV synthetic peptide vaccine and subunit vaccine encoding P72,CD2V and EP53R proteins,and to evaluate the immune efficacy of the vaccine by immunizing mice.To lay the foundation for the development of ASF vaccine.【Method】:In this study,bioinformatics software such as ProtParam,SOPMA,ABCpred,SVMtrip and IEDB were used to screen the physical and chemical properties,structural information and epitopes of T and B cells of P72 protein,and the dominant epitopes were integrated to synthesize polypeptides for immune evaluation test.In addition,P72,CD2V and EP153R proteins,which play an important role in the process of ASFV replication,attachment,and packaging,were induced and expressed through the prokaryotic system.The codon optimization of the three proteins was carried out according to the codon bias of EScherichia coli.To improve the expression level and solubility of the proteins,pcold-tf-P72,pcold-tf-CD2V and pcold-tf-EP153R plasmids were constructed using pcold-tf as expression vectors.The constructed vectors were induced to express respectively.The induction conditions were screened,and the induced proteins were verified by Western blot,and the correct plasmid was induced and purified in large quantities.Synthesize purified protein and peptide by intraperitoneal injection of Freud’s adjuvant immune mice,divided into three times to strengthen immunity,immune intervals for 14 days,mice immune detection after the completion of the specificity of the antibodies,and the T lymphocyte subsets,spleen lymphocyte proliferation,cytokine interleukin 4(IL-4),interleukin 2(IL-2)and interferon(IFN-γ),To evaluate the immune efficacy of synthetic peptides from the perspective of humoral and cellular immunity.【Results】:Comprehensive analysis showed that P72 protein was a stable hydrophilic protein with 10.68%ofα-helical and 64.40%of irregular crimp structures.Eight dominant epitopes of protein P72 were selected by comprehensive software analysis,T cell epitopes(520-528,203-212,298-306,626-634),B cell epitopes(587-606,39-58,232-251,110-129),and two peptides(named P72-1 and P72-2,respectively)were synthesized by integrating the dominant epitopes together.The synthetic peptide vaccines P72-1 and P72-2 were prepared by dissolving in PBS solution and mixing with Fredrin’s adjuvant in equal amount.The plasmid pcold-tf-P72,pcold-tf-CD2V and pcold-tf-EP153R constructed by double enzyme digestion were consistent with the expected results,and the sequencing results were consistent with Gen Bank.The proteins P72,CD2V and EP153R were successfully induced by prokaryotic system.The optimal induction temperature was37°C and the optimal IPTG concentration was 0.5 m M.The purified proteins were obtained by nickel ion metal chelation affinity chromatography.Subunit vaccines P72,CD2V and EP153R were successfully prepared by mixing protein solution with Freund’s adjuvant.The specific antibodies of P72-1,P72-2,P72,CD2V and EP153R could be detected in mice immunized with synthetic peptide vaccine and subunit vaccine respectively at 14d after immunization,and the highest value was reached at 28 d,and the antibody titer was the highest1:51 200 for subunit vaccine P72;CD4~+/CD8~+of T lymphocyte subsets increased significantly after immunization(P<0.01).Spleen lymphocyte proliferation test showed that the number of lymphocytes in immune group was significantly increased(P<0.01).Cytokines IL-4,IL-2 and IFN-γwere also significantly increased(P<0.01).【Conclusion】:In this study,the antigenic epitopes of P72 protein were predicted by bioinformatics methods,and the two and synthetic peptide vaccines of ASFV were successfully constructed.At the same time,the proteins of P72,CD2V and EP153R were expressed and purified by prokaryotic system,and the subunit vaccines of ASFV were successfully constructed.By immunizing mice,it was found that both vaccines could produce high level of specific antibodies,stimulate the proliferation of splenic lymphocytes,and induce the production of cytokines IL-4,IL-2 and IFN-γ,which laid a technical foundation for the development of African swine fever vaccine.