Construction And Immunogenicity Of Genetic Engineering Vaccines Against European Type PRRSV

Porcine reproductive and respiratory syndrome (PRRSV) causes severe disease that ischaracterized by respiratory problems in all-ages pigs as well as reproductive failure in pregnantsows. PRRSV shows a high degree of genetic variation. Since the emergence of highlypathogenic NA genotype PRRSV in2006in Asian countries, the genetic divergences of EUgenotype PRRSV have increased in various European countries. With the spread of PRRSVbreaking the existing geographical restrictions, both of North American type and European typehave coexisted. This epidemic situation make the prevention and control of disease moredifficulty and complexity than before. In order to control European type PRRSV, this study wastargeted at ORF3, ORF5gene of Lelystad virus，represented European type prototypes. On thebasis, prokaryotic expression plasmids that expressed ORF3,ORF5gene were constructed ofpET-28a-ORF3and pGEX-4T-ORF5.Three recombinant eukaryotic expression plasmids wereconstructed pVAX-ORF3,pAX-ORF5and pVAX-ORF3-ORF5. The immunogenicity efficiencyof recombinant plasmids were further evaluated in mice and pigs.The ORF3, ORF5genes were respectively inserted into the prokaryotic expression vectorspET-28a (+), pGEX-4T-1.Then they were induced by IPTG. SDS-PAGE and Westem-Blotshowed that the fusion protein were expressed mainly in the inclusion bodies forms and reactedwith the porcine polyclonal antibody against PRRSV. Pigs immunological results showed thatORF3and ORF5protein can stimulate pigs to produce specific antibodies, immune cells resultsshowed that piglets can stimulate CD4+、CD8+T lymphocyte proliferation and differentiation.Cytokine levels results showed that subunit vaccine can induce the class Thl and Th2cells in theimmune response. Viral load results showed that ORF3and ORF5subunit vaccine againstPRRSV of protection was limited in pigs.LV ORF3, ORF5gene were cloned into the eukaryotic expression vector pVAX1.Threerecombinant eukaryotic expression piasmids were constructed to express various proteins ofPRRSV, named as pVAX-ORF3, pVAX-ORF5and pVAX-ORF3-ORF5.The ability of thepiasmids to express the correspondent virus genes in eukaryotic, which were verified by IFA andRT-PCR.Mice immunological results showed that from the beginning of immunization, the micebegan to appear PRRSV-specific antibodies in the sera and the antibody levels continued to riseafter immunization, reaching a peak at35days.The immune cells results showed that DNAvaccine stimulated T lymphocyte subsets proliferation and differentiation pVAX and PBSsignificantly higher than the control group. Cytokine results showed cytokine IFN-γ, IL-2 increased significantly, inducing cell-mediated and humoral immunity on14and35days.The results proved that pigs immunized recombinant nucleic acid vaccines can inducePRRSV-specific antibodies and neutralizing antibodies.Levels of cytokines significantlyincreased, indicating that DNA vaccines can mediate better class Thl and Th2type immuneresponses. Challenge results indicated that the recombinant DNA vaccine have a protectiveefficacy in pigs. Among the recombinant DNA vaccines，pVAX-ORF3-ORF5is andidategenetically vaccine for further research.In this study, DNA vaccine and subunit vaccines have laid a good foundation for the furtherdevelopment of genetic engineering vaccine research.