Construction Of Alphavirus Replicate DNA Vaccine And Recombinant Adenovirus Vector Vaccine For PRRS And Evaluation Of Combined Immunization Effect

Porcine reproductive and respiratory syndrome,commonly known as PRRS,is one of the most economically devastating viral diseases in the global pig industry caused by the Porcine Reproductive and Respiratory Syndrome virus.PRRS is a highly contagious disease with complex infectious characteristics and highly heterogeneous genetic and recombinant features.The mayor clinical symptoms are reproductive disorders in sows and respiratory diseases in piglets.Porcine is the only known host for the disease,and porcine monocytes are target cells of PRRS virus infection,such as porcine alveolar macrophages and dendritic cells.The genome of PRRS V is a single-stranded positive-stranded RNA with a gene structure of 5’UTR-ORF1a/ORF1b-ORF2a/ORF2b-ORF3-ORF4-ORF5/ORF5a-ORF6-ORF7-3’UTR,with overlapping regions between adjacent reading frames.GP2a,GP3,and GP4 belong to N glycosylated proteins,which are encoded by ORF2a,ORF3 and ORF4,respectively,and are present on the viral envelope.GP2a,GP3 and GP4 can form heterotrimeric structures,and heterotetramer complexes can be formed with non-glycosylated E proteins in infected cells.In this study,ORF2a,ORF3 and ORF4 genes were cloned in series on alphavirus replicate vectors and adenovirus vectors,respectively,to obtain alphaviral replicate vector DN A vaccine and recombinant adenovirus vector vaccine.Adenovirus vaccine can stimulate rapid and sustained induction of humoral and cellular immunity in pigs;The alphavirus replicate vector system has high expression of foreign genes and has a high safety profile.In this study,the heterologous prime-boost strategy of recombinant adenovirus vaccine and alphavirus replicate vector vaccine was used.The heterologous prime-boost strategy reduces the immune response of the vaccine vector,while the combination between vaccines of different mechanisms increases the strength,breadth and durability of the immune response.A summary of the study is as follows:1.Construction and preparation of alphavirus replicate vector vaccineThe SFV replicon of the pSFV vector was inserted into the pShuttle-CMV vector in two segments,and the 432bp fragment from the 5’ end to the BsiWI site was first amplified by PCR,and connected to the pShuttle-CMV vector to obtain pShuttle-SFV1.The remaining fragments in pSFV3 were then cut by BsiWI and SpeI.and cloned to pShuttle-SFV1 and named pShuttle-SFV.In order to obtain the highly expressed target gene GP234 alphavirus replicate vector vaccine,the glycosylation sites of GP2,GP3 and GP4 gene sequences were mutated and pig-derived codon preferences optimized,and P2A sequences were inserted between genes and cloned to the pShuttle-SFV vector containing alphavirus replicators named pSFV-GP234.The recombinant plasmid was transfected with 293A cells,and the cells were transfected by Western blot detection by anti-tag antibody,and the results showed that there were additional specific bands of 29.3KDa,28.6KDa,and 19.3KDa proteins in the swimlane,corresponding to GP2,GP3 and GP4,respectively,indicating that the target gene could express and cut completely.2.Construction and preparation of recombinant adenovirus vaccineThe gene of interest GP234 was inserted into the laboratory to construct a pShuttle-Hsp70 vector containing Hsp70,named pShuttle-GP234-Hsp70.The recombinant plasmid pShuttleGP234-Hsp70 was digested with PmeI.and transformed into BJ5183-pAdEasy-1 recombinant bacteria to obtain the recombinant plasmid pAd-GP234-Hsp70,and pAd-GP234-Hsp70 was transfected into HEK293A cells to obtain recombinant adenovirus rAd-GP234-Hsp70.HEK293A cells infected with recombinant adenovirus were identified by indirect immunofluorescence and showed that the tags embedded with all three proteins bound to the corresponding antibodies.HEK293A cells infected with recombinant adenovirus were identified by Western-blot and showed additional 29.3kDa,28.6kDa,and 19.3kDa proteins in the lane.The obtained recombinant adenovirus is amplified on HEK293A cells to obtain hightiter recombinant adenovirus in preparation for animal experiments.3.Recombinant adenovirus and alphavirus vaccine combined immunization infected mice and their effect detectionThe mice were divided into four groups,six in each group,each mouse was immunized twice,the first group was vaccinated with recombinant adenovirus vaccine rAd-GP234-Hsp70,the second group was vaccinated with alphavirus replicate vaccine pSFV-GP234,the third group was first vaccinated with pSFV-GP234 booster injection rAd-GP234-Hsp70,and the fourth group was injected with PBS.Serum is collected through the orbit every 7 days and serum antibody levels are measured.On the 28th day,mouse splenocytes were collected,and splenocytes were stimulated with prokaryotic expression proteins ELP-GP2,ELP-GP3,ELPGP4,ConA,PBS and ELP proteins,and tumor necrosis factor α,interleukin 4 and interferonγin the supernatant were detected,and the results showed that the immune group with pSFVGP234 booster immunity rAd-GP234-Hsp70 had a good effect.In summary,the alphavirus replicate DNA vaccine and recombinant adenovirus vaccine constructed in this study adopt the heterologous-primary immunity strengthening strategy,which provides a new option for the prevention and control of PRRSV,which is conducive to promoting the healthy development of the aquaculture industry.