| Porcine reproductive and respiratory syndrome (PRRS) is an infectious disease found in swine farms worldwide and it is characterized by reproductive failure such as late-term abortions in sows and respiratory illness and mortality in young pigs. In this study, IFN-r gene and IL-2 gene of swine were amplified and cloned into the mammalian expression vector pcDNA3.1(+), respectively. In addition, porcine ubiquitin gene and PRRSV GP5 gene were amplified. The DNA recombinant plasmids expressing PRRSV GP5 (pCI-GP5) and expressing a fusion protein of ubiquitin and GP5 (pcDNA-U-GP5) were constructed, respectively. Furthermore, Balb/c mice were immunized with these DNA recombinant plasmids and the immune responses were exa mined. SPF piglets were vaccinated with pCI-GP5 by codelivery of pcDNA-IFN-y, pcDNA-IL-2 and pcDNA-IL-4. The protective efficacy was also evaluated.IFN-y gene and IL-2 gene of Chang-Bai swine were amplified by RT-PCR, and recombinant eukaryotic expression plasmids pcDNA-IFN-y and pcDNA-IL-2 were produced by inserting them into pcDNA3.1(+), respectively. The recombinant plasmids were transfected into COS7 cells and the bioactivities of cultures were assayed. The supernatant of pcDNA-IFN-y-transfected cells could inhibit the replication of PRRSV. After COS7 cells were transfected with pcDNA-IL-2, the amount of IL-2 in the supernatant was 24 units per mililiter.GP5 gene of PRRSV BJ-4 was inserted into mammalian expression plasmid pCI-neo and resulted in the production of pCI-GP5 construct A fused gene of porcine ubiquitin gene & PRRSV GP5 gene was inserted into pcDNA4.0 HIS MAX A vector to construct a recombinant plasmid pcDNA-U-GP5. The recombinant constructs were transfected into COS7 cells. In vitro expression of GP5 in pCI-GP5 was deter mined by indirect immune fluorescent assay, while expression of GP5 in pcDNA-U-GP5 was unable to be detected due to the rapid degradation of ubiquitin-GP5.Balb/c mice were vaccinated with pCI-GP5 or pcDNA-U-GP5 by intramuscular injection, then the humoral and cellular immune responses were analyzed. Five weeks after the third immunization, pigs in pCI-GP5 group exhibited much higher antibody levels and percentage of NK lysis than those in pcDNA-U-GP5 group. A significantly enhanced stimulation index and a significantly increased percentage of CD4~CD8+T cells were observed in pcDNA-U-GP5 group compared with pCI-GP5 group. The Results demonstrated that DNA immunization against PRRSV induced the production of both humoral and cell mediated immune responses in mice. The pcDNA-U-GP5 could elicit much stronger specific cellular immune response.SPF piglets were immunized with pCI-GP5 by co-administration of pcDNA-IFN-y, pcDNA-IL-2 and pcDNA-IL-4 every 2 weeks for three inoculations. The vaccinated pigs were challenged intranasally with PRRSV BJ-4. DNA immunization with pCI-GP5 resulted in the production of both neutralizing antibodies (1:19.2) and cellular immune responses two weeks after the third immunization. The pCI-GP5 showed significantly better protection from PRRSV challenge compared with the controlplasmid. The protection was characterized with a decrease of 39% PRRSV-positive tissues. In addition, the co-administration of pcDNA-IL-4 increased the immune response induced by pCI-GP5. This immune response was characterized by a significant enhancement of lymphoproliferative response, a higher percentage of CD4-CD8+T cell, showing a higher viral neutralization titer of 1:35.5, a lower percentage of PRRSV-positive tissues and a significantly decreased frequency of viraemia (a drop of 64% and a drop of 17%, respectively, compared with the control group). pcDNA-IL-2 codelivery with pCI-GP5 resulted in enhanced cellular immune responses, remarkblely reduced percentage of PRRSV-positive tissues and frequency of viraemia (a drop of 59% and a drop of 50%, respectively, compared with the control group). In contrast, the co-administration of pcDNA-IFN-y had no adjuvant effects even if an increased percentage of CD4-CD8+ lymphocyte subset appeared. |
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