Construction And Immunogenicity Of Recombinant Plasmid Co-expressing Capsid Protein Of Porcine Circovirus Type 2 And VP2 Protein Of Porcine Parvovirus

Porcine parvovirus (PPV) and porcine circovirus type 2 (PCV2) are the two important pathogeny of reproductive failure in primiparous sows. Primiparous sows infected with PPV may product fetal death, mummified piglets, reproductive failure, and pregnant sows infected with PCV2 may have abortions, stillbirths or bear weak litters. Co-infection of PCV2 and PPV also lead to postweaning multisystemic wasting syndrome. The recombinant plasmid PIRES-VP2-ORF2 was constructed to lay the foundation for DNA vaccine against both PPV and PCV2, and tests were carried out to analyse the humoral and cellullar immunologic responses by recombinant plasmid in mice.Viral genomic DNA of PPV and PCV2 were extracted respectively. VP2 and ORF2 genes were amplified by PCR, and then inserted into the PIRES plasmid to construct three kinds of recombinant plasmids: PIRES-VP2, PIRES-ORF2 and PIRES-VP2-ORF2. The three plasmids were transfected into Chinese hamster ovary (CHO) cells, and the proteins translated were correctlly detected by western-blot.The results proved that the recombinant plasmids PIRES-VP2 and PIRES-ORF2 can express VP2 protein and Cap protein respectively in CHO, and the recombinant plasmid PIRES-VP2-ORF2 can express two proteins simultaneously.4-6 weeks aged Balb/c mice were immunized with PIRES-VP2, PIRES-ORF2, PIRES-VP2-ORF2, blank plasmid PIRES and 100 ul PBS respectively, 100ug per mouse. Each group mice were immunized three times with two-week interval. The blood was collectecd every week to examine and analyze the level of humoral immune response after first immunization. Spleen lymphocyte cells were collcted to examine the proliferation by WST-8 method and the numbers of CD4+ and CD8+ by flow cytometry test to assess the cellular immunologic responses during two weeks after the last booster immunization. Pathological detections and PCR were used to prove the safety.The ELISA results showed that the mice immunized with PIRES-VP2-ORF2 could produce antibodies against PPV and PCV2 simultaneously, and that immunized with PIRES-VP2 could produce antibodies against PPV, while that immunized with PIRES-ORF2 could produce antibodies PCV2, and blank plasmid and PBS groups produce little antibodies against PPV or PCV2. The antibodies titers of mice vaccined with PIRES-VP2, PIRES-ORF2, PIRES-VP2-ORF2 increased significantly after the second immunization. The differences in antibodies titers of them were significant between (p<0.05). While the titers of the single gene expression plasmids groups and co-expression plasmisds group had no significant difference(p>0.05).The lymphocyte proliferation test results showed that the level of spleen lymphocyte proliferation can increase significantly in recombinant plasmid group while two control groups can not proliferate obviously after viral specific stimulation. The result of flow cytometry test show that the level of CD4+ and CD8+ are improved by 10.7% and 5.3% respectively after PPV stimulus in PIRES-VP2 group, 10.1% and 4.8% respectively after PCV2 stimulus in PIRES-ORF2 group, 12.7% and3.9% respectively after PPV stimulu and 12.5% and 4.8% respectively after PCV2 stimulus in PIRES-VP2-ORF2 group. There was no obviousl change after PPV or PCV2 stimulus in PIRES group and PBS group. The immunity test results showed that the constructed recombinant plasmid can cause humoral and cellullar immunologic response in mice.The safety test showed that the various organs of mice have no lesions in recombinant plasmids groups and the recombinant plasmid PIRES-VP2-ORF2 didn't integrate into the chromosome of mice, which proved that the recombinant plasmids have good safety.In conclusion, the plasmid PIRES-VP2-ORF2 was constructed and its immunogenicity was analyzed, which proved that the plasmid PIRES-VP2-ORF2 could coexpress two types of protein simultaneously, and cause mice to induce immune response against both PPV and PCV2.