Preliminary Study Of Recombinant Adenoviral Vector Vaccine Expressing ASFV Protective Antigens

African swine fever(ASF)is a disease caused by African swine fever virus(ASFV),which caused the high mortality of domestic pigs and wild boars.ASFV is a large double stranded DNA virus with approximately 180 kb in length and encodes up to 180 proteins.ASFV is mainly transmitted through contact,and can also be transmitted through the air within a short distance.The infection of swine alveolar macrophages causes acute hyperthermia in domestic pigs and wild boars.At present,ASFV has spread to most provinces and cities in China,causing huge economic losses.To date,there is no effective vaccine against ASFV,the recombinant Adenoviral vector vaccines are capable of inducing strong cellular and humoral immune responses with greater safety.Therefore,this study aims to construct recombinant Adenoviral vectors expressing a variety of ASFV protective antigens,and provide foundation for the study of ASF recombinant Adenoviral vector vaccine.EP153R(EP153R),CD2v(EP402R),p54(E183L)and p72(B646L)proteins play important roles in ASFV replication and packaging,which are important protective antigens for ASFV.pShuttle-EGFP-EP 153R-EP402R,pShuttle-EGFP-EP153R-E183L and pShuttle-EGFP-B646L were constructed using EGFP as a tag protein.To enhance the immune effect,pShuttle-CTLA4-EP153R-EP402R,pShuttle-CTLA4-EP153R-E183L and pShuttle-CTLA4-B646L were constructed with the partial sequence of CTLA4 as the leader sequence,then the constructed vectors were transfected into 293T cells respectively,and the expression of fusion protein was verified by western blot(the expression of CTLA4-B646L was not observed).To obtain pAd-EGFP/CTLA4-EP153R-EP402R,pAd-EGFP/CTLA4-EP153R-E183L and pAd-EGFP-B646L,the corresponded plasmid was transformed into BJ5183(the recombinant strains of E.coli)and these plasmids were transfected into 293A to obtain recombinant Adenoviruses.Recombinant Adenovirus was injected subcutaneously into mice for prime immunization,and boost immunization after 7 days.The sera were collected at 14 days,30 days,and 46 days after prime immunization.The EP153R and EGFP were expressed in E.coli and used to detect antibody titer in sera.The spleen and lymph nodes in armpit and groin were collected at 50 days post prime immunization.Lymphocytes were stimulated with EP153R and EGFP to detect the proliferation of spleen and lymph node cells,respectively.IFN-?+/CD3 was detected by flow cytometry to analyze the recognition and response of alloantigens post immunization.The results showed that the five recombinant Adenoviruses were constructed,which expressing predicted ASFV protective antigens,could induce specific antibodies in mice,and the recombinant Adenovirus immunization groups with CTLA4 could induce better humoral immune responses.In Addition,the EP153-CD2v incduced immune response was better than recombinant EP153R-p54,and the humoral immune response caused by EGFP-p72 protein was weak.The lymphocyte proliferation experiment revealed that the Ad-EGFP-p72 could stimulate lymphocytes proliferation,while no proliferation was observed by EP153R immunization.The flow cytometry results were consistent with lymphocyte proliferation experiments.The Ad-EGFP-p72 immunization group secreted the specific IFN-y upon EGFP stimulation,and the other groups could not induce IFN-y secretion after EP153R stimulation.