| Porcine parvovirus (PPV) is the major causative agent in a syndrome of reproductive failure in sows. Porcine circovirus type 2(PCV2) causes Porcine Circovirus type 2-associated diseases (PCVD), and particularly postweaning multisystemic wasting syndrome(PMWS). These two diseases which have caused huge economic losses, are worldwide distribution; At present, vaccination is the major method to prevent them. However, porcine parvovirus inactivated vaccine which has been widely used, has the risk of not incompletely inactivated, as well as porcine parvovirus is difficultly cultivation in vitro which causes low titer proliferation. These above short comings bring many difficulties to product porcine parvovirus inactivated vaccine. Meanwhile, porcine circovirus type 2 also has low titer proliferation in vitro, which lead to hardly develop the traditional vaccines. These two kinds of infectious diseases, which have synergistic effect, often co-infect swine herds. If a vaccine to simultaneously prevent these two diseases could be developed, it will have vital significance. In this work, antigen epidemiology of porcine parvovirus and porcine circovirus type 2 in Sichuan province were investigated; DNA vaccines against porcine parvovirus and porcine circovirus type 2 as well as a recombinant pseudorabies virus that could express PPV VP2 protein and PCV2 ORF2 protein were constructed, and their immunogenicity was further demonstrated. These experiments were aiming to provide a novel vaccine to be used in prevention and control these above diseases in the future. The main research contents are the following:1. Antigen Epidemiology Survey of Porcine Parvovirus and Porcine Circovirus Type 2 in Sichuan ProvinceTwo hundred and seventy-three samples were collected from 21 large pig farms in Sichuan province, and antigen epidemiology of porcine parvovirus and porcine circovirus type 2 were investigated by PCR detecting. The antigen positive rate of porcine parvovirus was 17.22%(47/273), and positive rate of pig farms was 38.1% (8/21), which displaied a feature that infection rate of boar was much higher than that of piglets; The antigen positive rate of porcine circovirus type 2 was 52.38% (143/273), and positive rate of pig farms was 85.7% (18/21), which showed a trend that the infection rate of porcine circovirus type 2 was rising with the piglets growth. The co-infection rate of porcine parvovirus and porcine circovirus type 2 was 10.62% (29/273), and co-infection rate of pig farms was 28.7% (6/21). Co-infection was mainly take place in the sow and nursing piglets.2. Construction of DNA Vaccines of Porcine Parvovirus and Porcine Circovirus Type 2Porcine parvovirus VP2 gene and porcine circovirus type 2 ORF2 gene were amplified by PCR, then were inserted into eukaryotic expression vector pCI-neo to constructed pCI-VP2 and pCI-ORF2 respectively. Meanwhile, these two genes were linked by high hydrophilia amino acid (Gly-Gly-Gly-Ser) encoding nucleotides, and then were also inserted into pCI-neo to constructed pCI-VP2-ORF2. Plasmids pCI-VP2, pCI-ORF2 and pCI-VP2-ORF2 were transfected to Vero cells, and target gene expression analysis was detected by PCR, Western blotting and IFA assay. The results suggested that the three kinds of DNA vaccines were successfully constructed and expressed.3. Immunogenicity of DNA Vaccines in MiceVaccinate healthy Kunming mouse with DNA vaccines pCI-VP2, pCI-ORF2 and pCI-VP2-ORF2 in the dose of 100μg, and booster injection was given with the same dose at 14 days post-vaccination. Collected mouse serum by tail cutting for antibodies detection at 0,7,14,21,28,42 and 56 days post-vaccination. Specific antibodies of PPV and PCV2 could be detected on 14 days post-vaccination. After 7 days of booster immunization, antibodies converted into positive, but the titer of immunized DNA vaccines groups were lower than that of inactivated vaccine groups. Collected mouse serum for cytokine detection at 0,21 and 56 days post-vaccination, and comparative high-level of IL-2, IL-4, IFN-y were induced by these three kinds of DNA vaccines. Collected anti-coagulated blood for T-lymphocyte subsets detection at 56 days post-vaccination, and CD4+/CD8+ was declined evidently. At 56 days post-vaccination, killed a mice by cervical dislocation in a random way to prepare splenic cell suspension which were then stimulated with ConA, and examined the stimulation index. The result showed that ConA had an marked effect on promoting spleen cell proliferation. Collected the murine parenchymal organs for the potential integration detection of DNA between the exogenous gene of the 3 kinds of DNA vaccines and the mice genome at 56 days post-vaccination, and the result indicated that the 3 kinds of DNA vaccines were security for the experimental animals. All the above results demonstrated that three kinds of DNA vaccines had high security, and could induce strong cell mediated immune response as well as humoral immune response. 4. Construction of the Triple Genetic Engineering Vaccine Strain PRV SA215 (D1)Cut off the VP2-ORF2 fusion gene from pCI-VP2-ORF2, and connected into a universal transfer vector pPI-2.EGFP of pseudorabies virus, then eukaryotic expression transfer vector pPI-2.EGFP-VP2-ORF2 was successfully constructed. PRV SA215 genomic DNA and the transferred plasmid pPI-2.EGFP-VP2-ORF2 were co-transfected into Vero cells to construct recombinant virus via homologous recombination. Then the recombinant virus PRV SA215 (D1) was purified by plaque purification using green fluorescent assay and PCR identification, and confirmed by detecting the presence of VP2 gene by PCR, Western blotting analysis and IFA assay. The results suggested the recombinant PRV SA215 (D1) strain could steadily express exogenous protein, and the fusion protein had good Immunogenicity.5. Study on the Partial Biological Characteristics of PRV SA215 (D1)Vero cells were infected with the recombinant PRV SA215 (D1), then cytopathic effect(CPE) and the morphology of virus were observed, and one step growth curve was determined. The results showed that the insertion of exogenous PPV VP2 and PCV2 ORF2 gene do not affected the proliferation and packaging of recombinant virus. Vero cells inoculated PRV SA215(D1), and then subcultured 6 rounds, which found that PPV VP2 and the PCV2 ORF2 gene did not loss. These results suggested that PRV SA215 (D1) was genetic stability. Healthy Kunming mouse were immunized with 104 PFU PRV SA215 (D1), found that all immunized mouse were alive which suggesting good security. Mouse immunized with the recombinant developed comparable PRV-specific humoral immune responses and provided complete protection against a lethal dose of 106 PFU PRV challenge. Piglets immunized with 105 PFU PRV SA215 (D1) induced significant cell mediated immune response and high levels of PRV, PPV and PCV2 antibodies. CD4+ T lymphocytes in peripheral blood was significantly increased, and CD8+T lymphocytes in peripheral blood was relatively reduced. High levels of cytokines IL-2, IL-4 and IFN-γshowed that PRV SA215 (D1) can significantly enhance Thl and Th2 immune response. The PRV antibody level induced by PRVSA215 gourp was equal to that of their parents strain PRV SA215, as well as PPV and PCV antibody levels were slightly lower than that of inactivated vaccine group, respectively; All above-mentioned results suggested that PRV SA215 (D1) is expected as the candidate strain of trivalent genetic engineering live vaccine to prevent and control PRV, PPV and PCV2. |
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