Exploration Of The Immunogenicity Of Structural Proteins Of African Swine Fever Virus

African swine fever(ASF)is a widespread and highly contagious swine infectious disease caused by African swine fever virus(ASFV).Pigs of all breeds and different ages can be infected,domestic pigs are highly susceptible,with an incidence rate and mortality of up to 100%,threatening the global pig industry.There is no effective vaccine against ASF till now.Knowing the antigenicity of viral proteins is great helpful for developing effective and safe vaccine.In order to study which structural proteins of ASFV can be recognized by immune response,ASFV structural protein p12,p14.5,p17,p22,p30,p54 and p E199 L genes were amplified from the genome of ASFV Pig/HLJ/2018.It was cloned into p ET-32a(+)prokaryotic expression vector and p CAGGS eukaryotic expression vector for expression.After the expressed protein was identified by SDS-PAGE and Western-blot,the purified recombinant prokaryotic proteins were used as the coating antigen,we used matrix method to optimize the reaction conditions and establish indirect ELISA method successfully.Meanwhile,the purified eukaryotic recombinant protein was used as immunogen to carry out animal experiments.The established indirect ELISA method was used to detect the level of specific antibodies in the serum of mice,the ELISPOT method was used to detect the secretion of IFN-γ in the spleen cells of the mice,the immunogenicity of p12,p14.5,p17,p22,p30,p54 and p E199 L were analyzed by antibody level and cellular immune response.The results showed that seven prokaryotic expression plasmids and seven eukaryotic expression plasmids were successfully constructed.SDS-PAGE results showed that seven recombinant proteins were successfully expressed and purified.The Western-blot results showed that the recombinant protein had good reactogenicity.Using the purified product of recombinant prokaryotic protein as the coating antigen,optimized reaction conditions and established indirect ELISA method;the purified recombinant eukaryotic proteins and different protein combinations were used as immunogens to immunize mice.The sera of immunized mice were collected and analyzed for their immune response.The ELISA data showed that p12,p14.5,p17,p22,p30,p54 and p E199 L proteins can induce mice to produce specific antibodies against the immunogen,which have good immunogenicity.For the groups of combination of these proteins,the antibody level of certain immunogen was various with that immunized with a single immunogen.The level of antibodies against p17 and p22 in the group that immunized with multi protein was significantly lower than that immunized with single protein immunogen.In the combined protein immunized group,p12 and p14.5 protein significantly affected the antibody level against single protein p12,p14.5,p17 and p22,and p12 affected the antibody level against p54 and p E199 L protein.The results of ELISPOT showed that these proteins did not induced a significant IFN-γ dot response in the mice model.Using optimized indirect-ELISA system,we also measured swine sera that collected from experimental pigs that inoculated with ASFV attenuated vaccine which developed by researchers of Harbin Veterinary Research Institute.The results showed that all antibodies against these seven protein immunogens can be readily detected.In conclusion,ASFV structural proteins p12,p14.5,p17,p22,p30,p54 and p E199 L all have good immunogenicity.Outer membrane protein p12 and/or capsid protein p14.5 can affect the antibody level of single protein p12,p14.5,p17,p22,p54 and p E199 L after multi protein combination.This study provides important reference data for exploring the immunogenicity of ASFV important functional proteins.