Effect of the glycine-rich region deleted PPV VP2 to the VLPs / 生物工程学报

The N-terminal of porcine parvovirus (PPV) viral protein 2 (VP2) links a glycine-rich domain which is a cleavage site of PPV VP3.In order to confirm that the glycine-rich domain was essential for the self-assembling of virus-like particles (VLPs).The VP2 gene with glycine-rich domain deleted and the complete VP2 gene were inserted to eukaryotic expression vector pCI-neo and were named pCI-AVP2 and pCI-VP2. Then, pCI-delta VP2, pCI-VP2 and pCI-neo were transferred into Vero Cells by liposome and the VLPs was detected by SDS-PAGE, Western blotting, indirect immunofluorescence and immunoelectron microscopy. Furthermore, 56 female Kunming mice were divided into 5 groups and injected intramuscularly with pCI-delta VP2, pCI-VP2 and pCI-neo as DNA vaccine, PPV inactivated vaccine and normal saline separately. The peripheral blood of the mice was collected to analyze the subgroups of the peripheral blood mononuclear cell by flow cytometry, to detect the antibody and lymphocyte proliferation by indirect-ELISA and MTT assay separately. The results show that the VLPs were observed both in the pCI-delta VP2 and pCI-VP2 transferred Vero Cells. The two VLPs could agglutinate guinea pig erythrocytes. The results also show that both the pCI-delta VP2 and pCI-VP2 vaccine induced special cellular and humoral immunity effectively. Those results revealed that the glycine-rich domain is not essential for the VPL's self-assembling. This study provides a new theoretical evidence for the relationship between the gene structure and protein function of VP2.

Construction of a dual-promoter expression plasmid delivered by Salmonella choleraesuis C500 / 生物工程学报

Salmonella choleraesuis C500 strain is an attenuated vaccine preventing piglet from paratyphoid and can also be used as a live vector of other DNA vaccines. Through mucosal immunization, immune response to specific antigens carried by it can be induced. To enhance the immune efficiency of DNA vaccine it carried, promoter Ptrc was inserted into the down stream of the human cytomegalovirus (CMV) immediate early promoter of eukaryotic expression plasmid pEGFP-C1. Then transcription terminator rrnbT1T2 was inserted into down stream of the multiple clone sites of pEGFP-C1, and the dual-promoter expression vector pEGFPPtrcR was constructed. Using 1xTSS method, we transformed the recombinant plasmid into C500, and obtained C500/pEGFPPtrcR. We used SDS-PAGE and Western blotting to detect the expression of report gene EGFP. Strong green fluorescence was observed under fluorescent microscope. The stable passages of this recombinant bacterium were at least 20 generations in vitro. Using liposome we transfected plasmid pEGFPPtrcR into Vero cell. After 24 h, green fluorescent was observed, showing the expression of EGFP in nuclei and endochylema. The construction of dual-promoter expression vector pEGFPPtrcR was successful. The foreign gene was expressed in Salmonella strain C500 and somatocytes, resulting in increased antigen expression. This research provides a foundation for the research of new DNA vaccines which use Salmonella C500 as carrier.

Obtaining of union expression virus-like particle of PPV VP2 and PCV2 ORF2 and its immunogenicity / 中国兽医学报

The amplified VP2 gene (PPV strain SC-1) and PCV20RF2 gene were inserted into the eukaryotic expression vector pPI-2.EGFP.Then the recombinant plasmid named pPI-2.EGFP.VP2.ORF2 was obtained.Mediated by liposome,the recombinant plasmid was transfected into Veto cells and expressed.Using immunofluorescence assay,the fluorescence of expression products were first detected at 20 h after transfection and peaked 36 h.Under electronmicroscope,virus-like particles (VLPs) can be observed in the transfected cells.To confirm the obtained VLPs to be recombinant particles,piglets were immunized using purified VLPs.The dynamic variation of blood T lymphocytes and serum antibody level of PPV and PCV2 were measured.The results showed that the ratio of CD3+,CD4+ T lymphocyte in peripheral blood lymphocyte of immunized piglets raised in a certain degree,the number of CD8+ T lymphocytes fell at 7-14 d after immunization,and then raised.Relatively high level of PP-V,PCV2 specific antibody could be detected.This indicated that the expression of recombinant plasmid pPI-2.EGFP.VP2.ORF2 was successful,the virus-like particles were formed and showed favourable immunogenicity.

Evaluation of the vaccinations with three strains of gene-deleted mutants from pseudorabies virus, PRV TK-, PRV gE-/gI and PRV TK-/gE-/gI- after exposure to the wild Fa strain / 中国人兽共患病学报

To evaluate the value of the vaccinations with 3 strains of gene-deleted mutants from pseudorabies virus(PRV), PRV TK-, PRV gE-/gI- and PRV TK-/gE-/gI- after to exposure to the wild Fa strain, these mutant strains from the PVR reference isolate Fa were used to vaccinate 4 weeks old PRV-free pigs with a dosage of 105 PFU each ,and followed by nasally challenged by the parental Fa strain with a dosage of 107 PFU at 14 days post vaccination. The pathological changes, virus discharge and distribution were evaluated after vaccination and challenge. It was found that the histopathological observations in the 10 collected samples including cerebrum, cerebellum, heart, liver, lungs, spleen, kidneys, tonsils, lymph nodes and trigeminal ganglion from these 3 mutant strains showed that the rates of occurrence of pathological changes in various organs were 4/10, 3/10 and 4/10 respectively, whereas that of the positive controls were 9/10. The damage in lungs was more serious in pigs vaccinated with PRV TK-mutant and positive control in comparison with other groups of pigs inoculated, and the damages in cerebrum, cerebellum and trigeminal ganglion in positive controls were more serious than those of pigs vaccinated with the 3 gene-deleted mutants. However, the tonsils, the main organ for latent infection were damaged mildly in the pigs inoculated with these 3 gene-deleted mutants in comparison with that of the positive controls. As demonstrated by Southern blot analysis, all the vaccinated pigs could discharge viruses by secretion through nasal cavity, but the soldier pigs were not infected successively by the gene-deleted mutants and the gene-deleted mutants were also unable to establish infection in cerebrum and cerebellum. Nevertheless, they could not effectively block discharge of PRV Fa after exposure to Fa virus, but could block effectively the virulent Fa virus invading into cerebrum and cerebellum. From these observation, it is evident that the deleted mutants of the TK, gE/gI , TK/gE/gI genes can block the invasion of virulent Fa virus into cerebrum and cerebellum and lessen the damages on multiple organs or tissues ,indicating that the deleted mutant of TK/gE/gI gene may be the most promising candidate of vaccine strain for development of the commercial vaccine.