Construction Of Recombinant Pseudorabies Virus Expressing Structural Protein Of African Swine Fever Virus And Analysis Of Its Biological Characteristics

African swine fever(ASF)is an acute contagious disease of pigs caused by African swine fever virus,and characterized by high fever,bleeding,neurological symptoms,and high morbidity and mortality,which causing great losses to the pig industry.Since the first ASF outbreak reported in 1921 in Kenya,there has been no effective vaccine developed in the world.The first ASF case in China was reported in August 2018,and soon after the disease spread to most of provinces and regions across the country,severely threatening the domestic pig population.Therefore,this study intends to develop virus-vectored vaccine candidates by inserting ASFV structural protein genes into the genome of a gene-deleted pseudorabies virus and lay the foundation for further development of a safe and effective ASF vaccine.Selection of ASFV structural proteins.The p72,p54,p30,CD2 v and p EP153 R selected in this experiment are all important proteins of ASFV.The p72 protein is located on the nucleocapsid of the virus,which is produced in the late stage of viral infection and has good antigenicity,and is often used for serological diagnosis.The p54 protein is located on the inner membrane of the virus and participates in the process of virus adsorption and entry into the cell,which plays an important role in the early infection of the virus.The p30 protein is located on the inner membrane of the virus,which is produced early in the viral infection and has excellent antigenicity,and is also involved in ASFV adsorption to susceptible cells.The CD2 v protein is located on the outer membrane of the virus and is very similar to the CD2 protein of the host cell.It plays an important role in the virus' s adsorption to red blood cells and its spread in the host.p EP153 R protein is a non-essential protein of ASFV,also known as C-type leucinelike protein,which is involved in the adsorption of infected cells and red blood cells.Previous studies have shown that vaccines consist of p72,p54,p30,CD2 v,and p EP153 R proteins can induce certain protective immune responses.Cloning and expression of p30 gene and preparation of polyclonal antibody.The structural protein p30 gene(E183L)of African swine fever virus SY18 was amplified by PCR and cloned into the prokaryotic expression vector p Cold I,generating a recombinant plasmid p Cold I-p30.The recombinant plasmid was transformed into E.coli BL21(DE3)competent cells and induced expression by IPTG.SDS-PAGE analysis showed that p30 expressed in E.coli.The purified p30 protein was used as an antigen to immunize mice.The indirect immunofluorescence and Western blot results showed that the sera collected from immunized mice contain specific antibodies to p30.This provides the basis for the subsequent identification of recombinant viral protein expression.Construction of recombinant pseudorabies virus.Using the g E and g I double gene-deleted pseudorabies virus strain(JS-2012-?g E/g I)constructed previously in our laboratory as the backbone,the virus genomic DNA was extracted,and then two Cas plasmids p Cas9-TK1 and p Cas9-TK2,which could specifically recognize and splice TK gene of JS-2012-?g E/g I were constructed.At meanwhile,five transfer vectors with different viral genes were generated as p BS-p72,p BS-p54,p BS-p30,p BSCD2 v and p BS-p EP153 R.The five viral genes were inserted,respectively,into JS-2012-?g E/g I through CRISPR / Cas9 gene editing technology and homologous recombination,and the recombinant viruses named r PRV-p72,r PRV-p54,r PRV-p30,r PRV-CD2 v and r PRV-p EP153 R were successfully rescued.By drawing the growth curve,it was found that the recombinant virus has similar growth characteristics as their parental virus.Identification of foreign gene expression of recombinant viruses.After infecting Vero cells with the five kinds of recombinant viruses,p72,p54 and p30 proteins are expressed in the infected cells,confirmed by indirect immunofluorescence detection and Western blotting.In summary,this study successfully constructed five recombinant pseudorabies viruses expressing different ASFV proteins using CRISPR / Cas9 gene editing technology,which laid an important foundation for the further development of the African swine fever vaccine or vaccine combination.