Construction And Immunogenicity Of Recombinant Plasmid Of Porcine Parvovirus VP2 Gene Carrying An Exogenous T Cell Epitope

Porcine parvovirus ( Porcine parvovirus , PPV) is the main cause of reproductive failure in gilts . PPV In addition to the infected pigs alone , the virus and porcine circovirus (Porcine circovirus Type2, PCV-2) , mixed infections are much more common. The two kinds of infectious diseases in the pig concurrent or secondary infection is more prevalent, causing huge economic losses to the pig industry . VP2 protein of PPV is major protective antigen , and is highly conserved . PCV-2 genome is located within the 201-220 amino acids of the ORF1 polypeptide P21, an immunodominant T cell epitopes , after immune animals , the lymphocytes of the animals increased significantly,and PCV-2 -specific antibody levels in the serum of immunized animals was significantly increased,the advantage of the development of epitope vaccines target epitopes . In this study, porcine parvovirus VP2 gene and the porcine circovirus type 2 peptide P21 were selected as the target gene. In this study, porcine parvovirus VP2 gene and the porcine circovirus type 2 peptide P21 were selected as the target gene.The recombinant plasmid pcDNA- P21- VP2 was constructed to prevent PPV and PCV-2 virus infection , the test was constructed co-expression of the reorganization plasmid pcDNA-P21-VP2 and the recombinant plasmid were immune tests , analysis of the recombinant plasmid was induced in mice of both humoral and cellular immune response .In this study, PPV were treated and inoculated into PK-15 cell. using the Primer 5.0 software , a pair of primers were designed and synthesized the PPV virus DNA as a template , VP2 gene fragment was amplified by PCR , the gene was cloned into pMD-18T vector and enzyme digestion, PCR identification and sequencing . VP2 gene was cloned into the eukaryotic expression vector pcDNA3.1 (+) , porcine circovirus type 2 T cell epitope gene of P21 was Inserted into the vector, constructed the recombinant plasmid pcDNA -P21 -VP2 . The results showed that the VP2 gene and P21 gene were correctly inserted into the vector by restriction endonuclease .The recombinant plasmid of pcDNA -P21 -VP2 , by the calcium phosphate -mediated transfection of HEK293T cells for expression in eukaryotic cells , analysis of the expression of recombinant proteins by SDS - PAGE and Western - blot analysis . The test results show that the size of the recombinant protein of approximately 67kDa , Consistent with the predicted protein size , and the expressed protein can be identified by mouse source PPV antiserum and murine PCV-2 antiserum. Intramuscular injection of pcDNA -P21 -VP2 positive plasmids on Balb / c mice , antibody testing by ELISA , MTT assay to detect immune spleen lymphocyte proliferation activity . pcDNA -P21 -VP2 plasmid can effectively induce a significant immune response in mice,and significant role in promoting the proliferative response of lymphocytes . Provide an experimental basis for the development of against PPV /PCV-2.