Study On Construction And Immunological Responses To Eukaryotic Recombinant Expression Vector Containing PRRSV ORF5 And CpG Motifs

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.Bacterial DNA has immunostimulatory effects and adaptive immune responses in numerous species.It has been proved that synthetic CpG-ODN motifs could mimic bacterial DNA to induce a potent Th1 immune response. In the study, the CpG-ODN of the strongest immunostimulatory activity on the swine was screened out.PRRSV ORF5 gene of swine were amplified and cloned into the mammalian eukaryotic expression vector pVAX1 and constructed the eukaryotic recombinant expression vector plasmid pVAX1-ORF5. The DNA recombinant plasmids CpG-pVAX1-ORF5 containing ORF5 gene coding region and CpG motifs were constructed, respectively. Furthermore, In order to detect the immunogenicity of CpG-pVAX1-ORF5. SPF mice were immunized with these DNA recombinant plasmids and the immune responses were examined. Piglets were vaccinated with CpG-pVAX1-ORF5. The protective efficacy was also evaluated.1,The Preliminary Study of Different CpG-ODN Sequences with Immunostimulatory in Peripheral Blood Lymph cells and Spleen Cells of Swine.In the study, nine different artificial unmethylated oligodexynucleotides (CpG-ODN) sequences based on the swine peripheral blood lymph cells and spleen cells respectively. The effect of the invitro lymphocyte transformation was detected by MTT assay. According to the tested OD ,calculated the lymph cells SI to study the immune effect of CpG-ODN. It is showed that the nine CpG-ODNs had different extent of immunostimulatory effects on the swine peripheral blood lymph cells. The stimulation index of CpG-ODN was screened out . After elaborate analysis ,the conclusion was drawn that the CpG-ODN7 had the strongest immunostimulatory activity on the swine peripheral blood lymph cells(SI=3.5). and CpG-ODN6 had the biggest immunostimulatory index on the swine spleen immue cells (SI=2.7) .This would be useful for study of effective CpG-ODN immune adjunct for gene vaccination in future.2,Construction and express of eukaryotic expression vector containing PRRSV SD1 ORF5 Gene and CpG motifs. PRRSV ORF5 gene of swine were amplified by the recombinant expression vector plasmid PIRORF5S-ORF5 and were chosen two new enzyme sites; Then ORF5 gene was cloned into the mammalian eukaryotic expression vector pVAX1 and constructed the eukaryotic recombinant expression vector plasmid pVAX1-ORF5. CpG-ODN of the strongest immunostimulatory activity on the swine was cloned into recombinant expression vector plasmid pVAX1-ORF5; The DNA recombinant plasmids CpG-pVAX1-ORF5 containing ORF5 gene coding region and CpG motifs were constructed, respectively. The DNA recombinant plasmids CpG-pVAX1-ORF5 was identified by enzyme analysis and nucleotide sequencing test .To improve the effect of the gene immunization against porcine reproductive and respiratory syndrome virus(PRRSV)SD1 the recombinant expression vector plasmid CpG-pVAX1-ORF5 was transfected into COS-7 cells by using lipofectamine methods.The ORF5 mRNA of transfected cells were detected by RT-PCR. The transient expression of PRRSV SD1 ORF5 nucleocapsid protein was detected by indirect immunofluorescence assay(IFA). In some transfected COS-7 cells, the green fluorescence was showed, thus we can conclude that the eukaryotic expression vector CpG-pVAX1-ORF5 was successfully constructed and expressed in vitro. This would place the base for study of CpG-ODN immune adjunct effective and intensity of immue protective reaction on piglets for gene vaccination in future.3,Studies of immunogenicity responses of mice immuned with eukaryon recombinant expression plasmid CpG-pVAX1-ORF5.In order to detect its immunogenicity level ,the recombinant expression plasmid was inoculated into SPF female mice(22-26).The PRRSV antibody protein in serum were confirmed by ELISA. The number of CD4+,CD8 + and the lymphocyte proliferation test(MTT) in peripheral blood of mice vaccinated were used to detect. The results showed that the recombinant plasmid CpG-pVAX1-ORF5 could induce higher response of cellular immune response and specific humoral immune responses in mice . CpG-ODN was effective adjuvant which could significantly enhance the immunogenicity of PRRSV SD1 gene vaccine .It will lay a foundation for further studying on pig immunoreaction intensity and the protective reaction induced by PRRSV SD1 ORF5 gene vaccine .4,Study on immunological responses to eukaryotic recombinant expression vector plasmid CpG-pVAX1-ORF5 on pigs. piglets were immunized with DNA recombinant plasmids which expresses PRRSV GP5 for three immunity.The PRRSV antibody in serum, the concentration of IL-2 and the lymphocyte proliferation test(MTT) in peripheral blood of piglets vaccinated were used to detect.The vaccinated pigs were challenged intranasally with PRRSV SD1. The results showed that GP5 DNA immunization with CpG resulted in the production of both PRRSV antibodies and cellular immune(a significant enhancement of a lymphoproliferative response ) . The CpG-pVAX1-ORF5 showed significantly better protection from the PRRSV challenge compared with the control plasmid. This immune response was characterized by a significantly decreased frequency of viraemia with a decrease of 80% compared with the control group.There was little clinic symptom and protection in part to lungs. The results indicated that CpG-ODN could be used as immune adjuvant of PRRSV DNA vaccine.