| African swine fever（ASF）is an acute,febrile,highly lethal and virulent infectious disease caused by African swine fever virus（ASFV）infecting domestic pigs or wild boars,which has brought huge economic losses to China’s pig farming industry since it was introduced to China in 2018.ASFV is a double-stranded DNA virus with a capsid,and the genome length of different isolates varies from 170-193 kb,encoding up to150-200 proteins.Due to the complex structure and large genome of ASFV,the key genes for immune protection have not been clarified,the ability to replicate in monocytes and macrophages,and host immune evasion,a safe and effective vaccine for prevention and control has not been developed.Live virus vectors,especially replication-deficient adenovirus vectors,are one of the good vectors for the development of ASF vaccines because of their high safety and efficacy.The p72（B646L）,p49（B438L）,p54（E183L）,p30（CP204L）,p A104R（A104R）and p10（K78R）proteins are structural proteins of ASFV,which are mainly involved in the process of ASFV infection,replication and packaging.In order to develop a safe and effective live vector vaccine against ASF,this study selected a replication-defictive human adenovirus type 5 expression system to construct recombinant adenoviruses expressing the above six ASFV structural proteins,and preliminary method for the identification and content detection of each of the six ASFV antigen-recombinant adenovirus mixed viruses was established,and completed laboratory studies on genetic stability,safety to target animals and non-target animals for transgenic biosafety evaluation.1.The ASFV SY18 genome was used as the template to amplify the above six target genes with 6×His tags at the 3′end and cloned into the adenovirus expression system shuttle plasmid to obtain six recombinant shuttle plasmids.The recombinant shuttle plasmids were linearized by PacⅠendonuclease and cotransfected with the adenovirus backbone plasmids,which were also linearized by PacⅠendonuclease,respectively,and the linearized recombinant shuttle plasmids were homologously recombined with the backbone plasmids to finally package the recombinant adenovirus carrying the target genes,named as r Ad5-B646L,r Ad5-B438L,r Ad5-E183L,r Ad5-CP204L,r Ad5-A104R,r Ad5-K78R.The titers of the recombinant adenovirus were above 109.0 TCID50/m L after stable proliferation;the recombinant adenovirus was correctly identified by PCR amplification and sequence determination using the identified primer Ad5CMV-F/R,which proved that the target gene was correctly recombined into the adenovirus vector;indirect immunofluorescence and Western Blot results showed that the recombinant adenovirus could effectively mediate the expression of the target protein in eukaryotic cells.2.The six recombinant adenoviruses expressing ASFV antigens were mixed at equal titers and volumes,and six recombinant adenoviruses mixed viruses（called“six antigens-recombinant adenovirus mixed viruses”）were used as models for the identification and content testing of single strains by direct multiple dilution-PCR assay,direct multiple dilution-q PCR assay and multiple dilution culture-q PCR assay.Both direct multiple dilution-PCR assay and direct multiple dilution-q PCR assay were performed by performing 10-fold multiple dilution of the mixed virus,and each dilution was detected by PCR or q PCR with each adenovirus specific primer separately;the multiple dilution culture-q PCR assay was performed by inoculating each gradient dilution of the mixed viruses into long to monolayer HEK293AD cells after 10-fold multiple dilution,cultured for 7 days and then freeze-thawed,q PCR assays were performed on the freeze-thawed solution using each adenovirus-specific primer.In contrast,the direct multiple dilution-PCR assay could only detect a 10-1 dilution gradient in the multiple dilution of the six antigens-recombinant adenovirus mix,the direct multiple dilution-q PCR assay could detect a 10-4 dilution gradient in the multiple dilution of the six antigens-recombinant adenovirus mix,while the multiple dilution culture-q PCR assay could detect a 10-7 dilution gradient,which makes the assay results more intuitive to show the content of individual viruses,and the mixed viruses after gradient dilution is re-inoculated into the cells,so that the virus proliferates in the cells,thus it can be determined that each virus is a live virus.3.The six recombinant adenoviruses were transmitted on cells for 20 generations,and the genomes of P4,P12 and P20 generations of recombinant viruses were sequenced,the titers of P4,P8,P12,P16 and P20 generations were determined,and the hemagglutination potency of P4,P12 and P20 generations was determined.The titers of different generations were stable above 109.0TCID50/ml,and the hemagglutination potency of different generations remained stable at about 1:28,which proved that the six recombinant adenoviruses had good genetic stability in vitro.4.The six heavy adenoviruses were inoculated with target animals,pigs,at overdose,and no abnormal clinical manifestations were observed within 14 days,and the average daily weight gain was not significantly different from that of the control group;the six heavy adenoviruses were inoculated with non-target animals,mice and rabbits,at overdose,and no abnormal clinical manifestations were observed within 14days,and the virus was not detected by intraperitoneal flush at 7 and 14 days.The six recombinant adenoviruses were shown to be safe for both target and non-target animals.In summary,this study（1）successfully constructed six replication-deficient human type 5 adenovirus vaccine candidates expressing different ASFV structural proteins;（2）A preliminary method was developed for the identification and content testing of single strain viruses in the combination of six antigens-recombinant adenovirus vector ASF vaccine candidate stains;（3）In vitro studies of six recombinant adenoviruses confirmed good genetic stability and safety in target and non-target animals.The above studies have laid an important foundation for further search of safe and effective live vector vaccines against ASF. |
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