Construction And The Immune Efficacy Evaluation Of Recombinant Adenovirus Expressing Protective Antigen Of ASFV

African Swine Fever(ASF)is an acute,virulent,highly contagious disease caused by infection with the African Swine Fever Virus(ASFV).With the speedy spread of African Swine Fever(ASF)in the world,it raises a rigorous challenge to the steady development of the global pig industry.Until now,the great challenge of prevention and control the African swine fever still not be solved because the safe vaccines and effective treatment drugs have not been produced.Therefore,the purpose of this study is to provide exploration for the development of African Swine Fever vaccine through constructing recombinant protective antigen of ASFV by adenovirus expression,and evaluate its immune effect in mice model.According to the sequence of ASFV Georgia 2007/1 gene,the E183 L,EP402R,K205 R,O61R,B646 L,A104R and B438 L genes of ASFV were synthesized.The target gene was amplified by specific primers,after that,transitional plasmids were constructed by TOPO cloning reaction and conformed by digested and sequenced.The seven successfully constructed transitional plasmids were transfected into HEK293 T cells,and the expression of protein was detected by western blot(WB)experiment.At the same time,the recombinant adenovirus plasmid were built by recombined 7 transition plasmids and p AD-CMV-3×FLAG through LR reaction.AD293 cells were transfected with recombinant adenovirus plasmid that digested with Pac?enzyme.By using immunofluorescence analysis and cytopathic experiment,we conformed that the recombinant adenovirus Ad5-ASFV-E183L?Ad5-ASFV-EP402R?Ad5-ASFV-K205R?Ad5-ASFV-O61R?Ad5-ASFV-B646L?Ad5-ASFV-A104R?Ad5-ASFV-B438 L were successfully packaged.The recombinant adenovirus were harvested and further inoculated into AD293 cells and passed to the 20 th generation.The genome of the virus was extracted and amplified by PCR to detect its genetic stability.The protein expression levels were detected by WB.The recombinant adenovirus were amplified in large quantities and purified by Cs Cl density gradient centrifugation-dialysis,and the titer was determined by measuring adenovirus hexon proteins.We selected the recombinant adenovirus Ad5-ASFV-E183L?Ad5-ASFV-EP402R?Ad5-ASFVK205 R ? Ad5-ASFV-O61 R and Ad5-ASFV-B646 L because of their titers met the immunization requirements,and evaluated the cellular immunity and humoral immunity in female BALB/c mice aged6-8 weeks.The mice were immunized twice by intramuscular injection in the thigh.The second immunization was performed at 21 d after the first time.The vena ophthalmic blood was collected at 0,7,14,21,28,and 35 d after the first immunization.The serum levels of IFN-? and IL-4 were determined by ELISA at day 14 and 35.At day 35,3 mice from each group were sacrificed and spleen lymphocytes were isolated.p72?CD2v?K205R and p54 were used as specific stimulators,and Con A as non-specific stimulators,respectively,to detect the proliferation of lymphocytes by using CCK8.By using immunofluorescence assay and cytopathic experiment,we confirmed the recombinant adenovirus Ad5-ASFV-E183L?Ad5-ASFV-EP402R?Ad5-ASFV-K205R?Ad5-ASFV-O61R?Ad5-ASFV-B646L?Ad5-ASFV-A104R?Ad5-ASFV-B438 L were all successfully packaged.Furthermore,the WB experiment results showed that the recombinant adenovirus Ad5-ASFV-E183L?Ad5-ASFV-EP402R?Ad5-ASFV-K205R?Ad5-ASFV-O61R?Ad5-ASFV-B646L?Ad5-ASFV-A104R?Ad5-ASFV-B438 L were successfully packaged.Ad5-ASFV-A104 R and AD5-ASFV-B438 L did not carry out subsequent immune evaluation experiments due to low titer.The ELISA experiment result indicated that the mice immunized with AD5-ASFV-E183L?AD5-ASFV-EP402R?AD5-ASFV-K205 R and AD5-ASFV-B646 L could produce specific antibodies and induce lymphocyte proliferation under the stimulation of specific stimulants.Recombinant adenoviruses Ad5-ASFV-EP402R?Ad5-ASFV-K205R?Ad5-ASFV-E183 L and Ad5-ASFV-O61 R could stimulate IFN-? secretion.However,it cannot stimulate the secretion of IL-4,which can cause different degrees of humoral immune response and cellular immune response in mice.Recombinant adenovirus AD5-ASFV-B646 L could not stimulate the secretion of IFN-? and IL-4,which indicated that the humoral and cellular immune responses were not stimulated.In summary,five candidate strains of recombinant adenovirus vaccine expressing different ASFV proteins were successfully constructed in this research,and humoral and cellular immune responses were slighted induced in mice model,which laid a foundation for further exploration of African classical swine fever vaccine.