| Despite great efforts have been made to control and eradicate porcine circovirus 2(PCV2)and pseudorabies virus(PRV),PCV2 and PRV are still prevalent in pig farms in China.PCV2 infection can lead to the occurrence of porcine circovirusassociated disease(PCVAD),while PRV infection can cause high mortality of piglets and reproductive failure of breeding pigs,PCVAD and PR seriously affect the development of pig industry.The co-infection of PCV2 and PRV has been reported many times,and it has been confirmed that can cause more serious lung and brain tissue damage.In addition,in recent years,the PCV2 predominant genotype has been gradually changed from PCV2b to PCV2d in China,which brings new challenges to the prevention and control of PCV2.The prevention and control of PCV2 and PRV is mainly through vaccination.However,the vaccines currently on the market have problems such as high cost,cumbersome operation and potential safety hazards.Therefore,the development of safe and effective bivalent vaccine is of great significance for preventing PCV2 and PRV infection,simplifying immunization procedure and reducing production cost.The recombinant virus rPRV-2d expressing PCV2d Cap protein and recombinant virus rPRV-2d-18 co-expressing PCV2d Cap protein and porcine IL-18 protein were constructed in this study.The biological characteristics and immunogenicity of the recombinant virus were studied to screen candidate strains of recombinant virus live vector vaccine with safe,stable and good immunogenicity,which can help to prevent and purify PCV2 and PRV in China.In this study,PCV2d ORF2 gene was inserted into PRV general transfer plasmid PG to construct the recombinant plasmid pG-2d-EGFP.Then,the gE-/gI-/TK-PRV NY parent strain and the recombinant plasmid pG-2d-EGFP were transfected into ST cells.PCV2d ORF2 Gene and EGFP expression cassette were inserted into the gG glycoprotein of gE-/gI-/TK-PRV NY by homologous recombination.The lesions with green fluorescence were selected and purified to obtain recombinant virus rPRV-2dEGFP.The EGFP gene double knockout plasmid PX459-gRNAl-EZ-gRNA2 and the recombinant virus rPRV-2d-EGFP were transfected into ST cells.The EGFP gene was knocked out about 1.2 kb using CRISPR/Cas9 technology,and the lesions without green fluorescence were selected and purified to obtain recombinant virus rPRV-2d.RT-PCR,Western blot and indirect immunofluorescence assay showed that the Cap protein of PCV2d was successfully expressed in ST cells.We inserted porcine IL-18 gene into eukaryotic expression vector pBApoEF1?_Pur_DNA to construct eukaryotic expression plasmid pBA-18.Then the whole expression box containing EF1? promoter,porcine IL-18 gene and HSV TK polyA was integrated into the recombinant plasmid pG-2d-EGFP to obtain recombinant plasmid pG-2d-18-EGFP through seamless cloning technology.The recombinant virus rPRV2d-18-EGFP with green fluorescence was obtained by transfection and plaque purification.The EGFP gene was knocked out by CRISPR/Cas9 technology,and the recombinant virus rPRV-2d-18 without green fluorescence was obtained by plaque purification.The results of transcription and expression analysis showed that porcine IL-18 protein was successfully expressed in ST cells and cleaved into mature IL-18 protein with biological activity.The culture and proliferation characteristics of the recombinant viruses rPRV-2d and rPRV-2d-18 were studied by ST,PK-15,IPEC-J2 and Vero cells.The physicochemical properties,genetic stability,safety and immunogenicity of the recombinant viruses were also studied.The results showed that the recombinant virus rPRV-2d and rPRV-2d-18 had similar proliferation characteristics with gE-/gI-/TK-PRV NY parental strain,and the highest titer was found in ST cells(106.25TCID50/0.1mL and 106.00TCID50/0.1 mL,respectively).The recombinant virus has the general physical and chemical characteristics of PRV,and has good genetic stability and safety.It can stimulate mice to produce the same immune response as the PCV2 commercial vaccine or gE-gI-/TK-PRV NY parent strain,and protect mice against the attack of PCV2 DF virulent strain and PRV NY virulent strain.However,the recombinant virus rPRV-2d18 did not show stronger immune effect than the recombinant virus rPRV-2d,so further experiments are needed to optimize it.In conclusion,the recombinant viruses rPRV-2d and rPRV-2d-18 constructed in this study can correctly express PCV2d Cap protein and porcine IL-18 protein.The insertion of foreign gene had no significant effect on the biological characteristics of the recombinant virus.The recombinant virus can successfully induce PCV2 and PRV specific immune responses in mice.The recombinant virus can also provide complete protection against lethal attack of PRV and effectively inhibit the proliferation of PCV2 in various organs.The recombinant viruses constructed in this study is expected to be a candidate vaccine strain for prevention and control of PCV2 and PRV,which provides a powerful tool for prevention,control and purification of PCV2 and PRV,and provides some theoretical basis for the development of recombinant virus live vector vaccine. |
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