| Procine parvovirus (PPV) is one of the major etiological agent of reproductive failure in pigs.It belongs to the family Parvoviridae.Pigs of all ages are susceptible to the viruS,The clinical signs of PPV are characterized by abortion,fetal abnormality,mummification and aciesis. PCV2 is the main cause for Pigs multiple system failure syndromes after weaned (PMWS), which clinical manifestations are weight loss, pale skin, or jaundice. At present, these three kinds of infectious diseases often co-infect or secondarily infect swine herds and cause significant economic losses. Therefore, safe and effective vaccine is a key point in preventing and controlling clinical diseases. Interferon-gamma (IFN-y) plays a major role in immunoregulation. Its immune modulation includes activating macrophages, up-regulating the expression of MHC classâ… and classâ…¡molecules, prompting the presentation of antigens, and inducing Th1 cell growth and differentiation.It is widely used in the study of DNA vaccine adjutants.In this experiment, IN order to construct the eukaryotic expression plasmid pCI-OV, the B lymphocyte epitope gene of PCV2 0RF2 was recombined with the N end of PPV VP2 gene. And to constrcted the expression plasmid pCI-IOV, the porcine IFN-y gene was cloned into the eukaryotic expression vector pCI-OV through gene linker. Then the eukaryotic expression recombinant plasmids were transfected into Vero cells by Lipofectamine. RT-PCR and the immuno-fluorescent antibody technique were used to detect and identify the expressed products in mammalian cells;the bioactivity of the products were detected by MTT assay. The results indicated that the interest genes were amplified and the specific proteins were detected and possessed obvious bioactivity. Expression of the specific protein was detected too.The recombinant plasmid pCI-OV and pCI-IOV,the control plasmid PCI-neo,PBS, the PPV inactivated vaccine and PCV2 subunit vaccine were injected into mice by intramuscular method. The lymphocyte transformation function of immuned mice was detected; In the different periods, the dynamic variation of blood T lymphocytes were assayed; PPV and PCV2 antibodies were measured too. The results indicated that the reactive of the splenic T lymphocytes to the ConA stimulation,immune function of inducing the CD4+, CD8+T lymphocyte and the PPV/PCV2 antibody levels of the pCI-IOV group were significantly higher than the PPV vaccine group,PCV2 vaccine group and pCI-OV group after three weeks immuned. The result indicated that the IFN-y can enhance the immunity of ORF2/VP2 DNA vaccine as cytokine adjuvant. The results provide reference data for the new generation vaccine research of PPV and PCV2.The heart, liver, spleen, lung, kidney, muscle of the injected site were sampled at 24h,7d,14d,21d,28d,35d,49d and 105d after the first immunization, for biodistribution and safety study by PCR method. Genome DNA were extracted for PCR assay. recombinant plasmids can distribute in different tissues from 24 h to 49 d after the first immunization. but the plasmid couldn't exist at 105 d. The safety experiment indicated that each gel-purified genomic DNA was negative for PCR assay. No genomic integration of DNA vaccine was detected in the immunized mice. |
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