Construction Of Eucaryon Plamids And Study On Immunogenicity Of The Porcine Interleukin-15 And Porcine Reproductive And Respiratory Syndrome Virus ORF5 Gene

In this study, PRRSV ORF5 gene and porcine IL-15 gene were inserted into the eukaryotic expression vector, pVAX1, resulting in recombinant plasmids, pVAX1-PRRSV ORF5 and pVAX1-pIL-15. The plasmids were transfected into Vero cells and the results showed that the transient expression of both genes was confirmed by respective antibodies.Experimental mice were divided into 7 groups. They were pVAX1-PRRSV ORF5 group, pVAX1-pIL-15 group, pVAX1-PRRSV ORF5 (100μg) and pVAX1-pIL-15 (100μg) group, pVAX1-PRRSV ORF5 (100μg) and pVAX1-pIL-15 (200μg) group, pVAX1-PRRSV ORF5 (100μg) and pVAX1-pIL-15 (50μg) group, vector pVAX1 control group and PBS control group. Lymphocyte proliferative assay, enzyme linkered immunosorbent assay and flow cytometric technique were used to analyze the immune response of different groups.The results showed that the general immune responses of five immunized groups are higher than the empty pVAX1 vector and PBS control groups. Especially, the mice inoculated with the pVAX1-PRRSV ORF5 and pVAX1-pIL-15 had higher immune response than other groups in terms of the T lymphocyte proliferative function and the number of CD4+ and CD8+ in peripheral blood and spleen as well as the levels of IFN-γin peripheral blood. In addition, the pVAX1-pIL-15 (100μg) group had the highest cellular immune responses. The pVAX1-pIL-15 (50μg) group is better than the pVAX1-pIL-15 (200μg) group in terms of the stimulation of cellular immune responses. However, the highest level of specific anti-PRRSV ORF5 antibody in peripheral blood of immunized mice was stimulated by the combinaiton of the pVAX1-PRRSV ORF5 (100μg) and pVAX1-pIL-15 (200μg). These results are helpful for analyzing the adjuvant function of pIL-15.After inoculating different doses plasmids of pVAX1-PRRSV ORF5 and pVAX1-pIL-15 into the mice, tissue samples from different time points were collected and subjected to PCR amplifying PRRSV ORF5 gene. The results showed that the ORF5 gene is detectable shortly in mice after intramuscular injection, and it mainly can be detected in brain, heart, liver, lung and kidney from 3 h to 42 d post-immunization.At the same time, we designed two paris of primers to amplify two truncated pIL-15 genes with deletion of either N-terminal 29 aa signal peptide or N-terminal 48 aa signal peptide. The maximum expression of the pIL-15 was determined after cloning and expression of both genes in E.coli. The pIL-15 protein was used as an immunogen to inoculate a rabbit for generation of specific polyclonal antiserum. Our results showed that both the pIL-15 and its antiserum are biologically active.To sum up, our results indicate that pIL-15 can enhance the immune responses of DNA plasmid encoding the PRRSV ORF5 in mice. The optimal dose of the molecular adjuvant, pIL-15, is important for improvement of the DNA vaccination of PRRSV ORF5. The current study provide useful data for rational immunization of PRRSV ORF5 DNA.