Construction And Immunogenicity Of Recombinant Pseudorabies Virus Expressing PCV2-cap Protein And PPV1-VP2 Protein

Porcine circovirus type 2?PCV2?,porcine parvovirus type 1?PPV1?and pseudorabies virus?PRV?are the three most important pathogens of swine.They often induce mixed infection and cause huge economic losses to the pig industry.Moreover,the emergent PRV variant strains pose a new challenge to the clinical prevention and control of pseudorabies in recent years.Therefore,it is of great scientific value to develop a safe and effective vaccine for the prevention of these three diseases.Cap protein and VP2 protein are the main protective antigens of PCV2 and PPV1,respectively,and are ideal targets for the development of genetic engineering vaccine.The expression of exogenous protective antigens by PRV as a living vector has become a hotspot in genetic engineering vaccine.The purpose of this study was to construct recombinant PRV expressing PCV2-Cap protein and PPV1-VP2 protein at high level and to provide genetic engineering vaccine candidate for the prevention and control of these three viral diseases.To construct the recombinant virus expressing PCV2-Cap protein and PPV1-VP2 protein,the nonessential gene TK was selected as the insertion site.Firstly,the EGFP gene was replaced by the codon optimized PPV1-VP2 gene,and the recombinant plasmid pMD18T-LR?TK?-VP2_opti was then constructed using the recombinant plasmid pMD18T-LR?TK?-EGFP as the skeleton.The PRV-gG signal peptide sequence was coupled with the codon optimized PPV1-VP2 gene,and another recombinant plasmid pMD18T-LR?TK?-VP2_opti?SP?was also constructed as above.Classical homologous recombination technique was used for construction of recombinant virus.Recombinant virus rPRV-TK^-/gE^-/gG^-/3Cap⁺genome and pMD18T-LR?TK?-EGFP were co-transfected into PK-15 cells with calcium phosphate transfection kit.Recombinant virus rPRV-TK^-/EGFP⁺/gE^-/gG^-/2Cap⁺was purified after 16 circles of plaque purification marked with EGFP and identification by PCR.Its genome and pMD18T-LR?TK?-VP2_opti or pMD18T-LR?TK?-VP2_opti?SP?were co-transfected into PK-15 cells.Rrecombinant virus rPRV-TK^-/VP2⁺/gE^-/gG^-/2Cap⁺and rPRV-TK^-/VP2⁺?SP?/gE^-/gG^-/2Cap⁺were purified after 6 circles of plaque purification without EGFP and identification by PCR,respectively.Two constructed recombinant viruses were identified by IPMA and IFA,respectively.The results showed that the two recombinant viruses can express PCV2-Cap protein and PPV1-VP2 protein by IPMA.PPV1-VP2 protein expressed by the recombinant virus rPRV-TK^-/VP2⁺/gE^-/gG^-/2Cap⁺was found to locate in the nucleus and cytoplasm,while PPV1-VP2 protein expressed by the recombinant virus rPRV-TK^-/VP2⁺?SP?/gE^-/gG^-/2Cap⁺was found to locate in the cytoplasm by IFA.The results of growth kinetics test showed that two recombinant viruses had high proliferation titers in PK-15 cells,but that of them were lower than parent PRV-JF strain.To determine the immunogenicity of two recombinant viruses rPRV-TK^-/VP2⁺/gE^-/gG^-/2Cap⁺and rPRV-TK^-/VP2⁺?SP?/gE^-/gG^-/2Cap⁺in vivo,6⁸-weeks-old healthy female Balb/c mice were used to carry out the vaccination test.Immunogenicity of two constructed recombinant viruses in mice was evaluated based on the observation of clinical symptoms and the detection of anti-PRV,PCV2 or PPV1antibodies level.Mice immunized with two live recombinant viruses did not show obvious clinical symptoms,indicating that two constructed recombinant viruses were safe.Both live and inactivated recombinant viruses could induce PRV neutralizing antibodies in mice by microneutralization test,and PCV2 and PPV1 antibodies were detected by IPMA.The results showed that low levels of PCV2 and PPV1 antibodies could be detected in mice immunized with inactivated recombinant viruses,while corresponding antibodies did not detected in mice immunized with live recombinant viruses.In this study,two recombinant viruses expressing PCV2-Cap protein and PPV1-VP2 protein were successfully constructed.The antigenic reaction activity of PCV2-Cap protein and PPV1-VP2 protein expressed by recombinant virus was confirmed in vitro.Both live and inactivated recombinant viruses could induce PRV antibodies in mice,while only inactivated recombinant virus could induce low level PCV2 and PPV1 antibodies in mice.The causes of the poor immunogenicity of PCV2-Cap protein and PPV1-VP2 protein expressed by recombinant PRV need to be further explored.