Preparation And Immune Effect Of Structural Protein Of African Swine Fever Virus With Intramolecular Adjuvants

African swine fever(ASF)is a severe infectious disease caused by African swine fever virus(ASFV)and endangers the healthy development of the world’s swine industry,with a 100%mortality.At present,there is no vaccine against ASF in the world.ASFV inactivated vaccine has been proved not to be fully protective.Live attenuated vaccine with gene deletion has protection in challenge,but its safety needs to be evaluated.The research and development of new vaccines has become an urgent scientific and technological problem in the industry.Therefore,the route of subunit vaccines is adopted for research.The subunit vaccine is safe,efficient and can be mass production.The disadvantage is that it causes weak cellular immunity.Among the known ASFV structural proteins,genetic engineering technology is used to engineer or modify the effective region and the dominant antigenic epitope.Molecular adjuvants are added to enhance the continuous stimulation of antigens in vivo and activate humoral and cellular immune responses.In this study,the truncated main capsid protein p72(185-405aa)and full-length pentontal protein H240R of ASFV were named as P protein and H protein respectively,with molecular weights of about 27 k Da and 31 k Da.At the C-terminal of truncated p72 protein,the flexible linker was used to connect intramolecular adjuvant tuftsin to tetanus toxin T cell epitope P2,which was P-T protein with a molecular weight of about 35 k Da.The target proteins were expressed by prokaryotic expression and purified by affinity chromatography.The results showed that P-T protein,P protein and H protein existed in inclusion body protein form at different induction temperatures,all at 37℃,IPTG concentration of 0.5 mmol/L,and speed of 210 rpm.After purification and renaturation,the purity of the three proteins could reach more than 90%.Western blot and Dot-ELISA showed that the hyperimmune serum could combine with the three proteins to develop color,indicating that the target protein was expressed correctly,and the renatured protein had activity and immunogenicity.After the endotoxin was removed by tritonx-114liquid phase extraction method,the endotoxin content detected by limulus lysate was less than 50 EU/m L,which met the standard requirements of"Chinese veterinary pharmacopoeia".The three proteins were emulsified with W/O/W ISA 201 VG adjuvant for 15min at 30℃,and the immunological preparation after emulsification had good stability.The optimal injection dose at the time of immunization was 50μg per mouse,and evaluation of the immune effects of these proteins in mice all stimulated the body to mount an immune response.The H protein immunization group reached the plateau on the 28th day of immunization,with a titer of 1:51200,while the other groups reached the plateau on the 35th day of immunization.The titer of the P-T immunization group was 1:96000,and the P-T+H mixed immunization group was1:128000,the titer of P protein was only 1:32000.The P-T and P-T+H groups induced specific cellular immune responses and induced the production of cytokines(IL-2,IFN-γ,TNF-α)and lymphocyte proliferation levels higher than those in P and H groups alone(P<0.01).It showed that molecular adjuvant could improve humoral immunity and Th1 cellular immune response of protein in the body.The differentiation of splenic T lymphocytes was assessed by flow cytometry.The results showed that the proportion of CD3~+and CD4~+T lymphocytes in P-T and P-T+H groups was significantly higher than that in other immune groups(P<0.01).But there was no significant difference between the two groups.It was proved that H protein could assist P-T protein to enhance the body’s humoralimmune response.It was further verified that the molecular adjuvant could promote the immune function of protein in the body.In conclusion,the three recombinant proteins expressed in prokaryotic cells in this study produced a good immune response in mice.Molecular adjuvants can enhance the humoral and cellular immune effects of the protein.There is an interaction effect between H240R and p72 protein,which can promote the systemic immune ability of p72 protein in the body,which provides ideas for the further research and development of vaccine.