Preliminary Study On Recombinant Pseudorabies Vaccine Expressing CD2v And P12 Proteins Of African Swine Fever Virus

African Swine fever（ASF）and Pseudorabies（PR）are caused by African Swine fever virus（ASFV）and Pseudorabies virus（PRV）.ASFV and PRV can cause highly death rate and infectious diseases in pigs.Currently,there is no commercial African swine fever vaccine,and both diseases have caused huge economic losses to the pig industry.In this study,the classic PRV-Fa strain was used as a vector to insert ASFV（Pig/HLJ/18）CD2v（EP402R）and P12（O61R）genes through CRISPR/Cas9（Clustered Regularly Interspaced Short Palindromic Repeats/Cas9）gene editing technology.We successfully constructed a recombinant attenuated strain PRV-（35）g I-（P12）-（35）TK-（CD2v）with deletion of TK and g I gene,and respectively expression of ASFV CD2 v and P12 in the TK（UL23）and g I（US7）gene loci of Fa strain.After the virus was passed for 30 generations,it was verified by PCR amplification,Western blot and immunofluorescence experiments that CD2 v and P12 were stably expressed in African green monkey kidney epithelial cells（Vero）,indicating that the genetic traits of the recombinant strain were stable.The one-step growth curve and plaque growth assay showed that the replication ability of the recombinant strain was weaker than wild-type strain PRV-Fa and it was not lethal to mice after challenge,indicating that the virulence of the recombinant strain was significantly weakened.After pathological evaluation of mice,determination of virus copy number in mouse tissues,detection of lymphokines in mice and analysis of T cells by flow cytometry（FCM）,it was found that the recombinant strain did not cause systemic inflammation and itching in mice,and it could induce T cells Proliferate and activate T cells to facilitate cellular immune responses.In order to study whether the recombinant virus can form a protective immune function,PRV-（35）g I-（35）TK and PRV-（35）g I-（P12）-（35）TK-（CD2v）were used to immunize mice,and it was found that both were 100% Immune to the invasion of PRV-Fa.The recombinant virus can be tested in vitro to produce specific antibodies after immunization,and purified CD2 v and P12 can be used to stimulate the expression of interferon gamma（IFN-γ）in peripheral blood mononuclear lymphocytes（PBMC）of mice after immunization,indicating that PRV-（35）g I-（P12）-（35）TK-（CD2v）has high immunogenicity and can induce specific cellular and humoral immune responses in mice.The recombinant strain provides a new option for the research of new vaccines to prevent PR and ASF.