| Vaccine is one of the most effective methods for control of toxoplasmosis. By now, the whole development history of Toxoplasma gondii vaccine contains four phases: live and killed Toxoplasma gondii vaccines, excreted/secreted antigens as vaccine components, recombinant antigens vaccines and DNA vaccines. Although the DNA vaccines of Toxoplasma gondii have much more advantages, compared with other traditional vaccines, the research of these vaccines is still in its infancy. Monovalent DNA vaccines can induce the cell mediated immunity and the humoral-mediated immunity against the infection of Toxoplasma gondii in organism.These effect are not ideal. Therefore, it is a popular trend for Toxoplasma gondii vaccines to develop the combined and multivalent DNA vaccines. In addition, the security and the mechanism of its immunoprotection are not very clear. To investigate the vaccine immunologic mechanism and The effects on the gene expression profile of eukaryotic cell will be helpful to develop the efficient, safe and practical DNA vaccines of Toxoplasma gondii.1. Construction of DNA vaccines pVAX1-p30,pVAX1-p24 against Toxoplasma gondii with p30,p24 gene and the study on immune-protection In this section, Toxoplasma gondii p24/p30 gene fragments were amplified by PCR, which were inserted into the eukaryotic expression vector pVAX1. Recombinant plasmid pVAX1-p24/pVAX1-p30 were Identified by PCR amplification, restriction endonuclease digestion and sequence analysis. The recombinant plasmid pVAX1-p24/pVAX1-p30 and the vector pVAX1 were transfected into mouse macrophage(RAW264.7)to establish the vitro eukaryotic cell temporary expression system, and detecting the expression of p24/p30 antigen protein by immunocytochemistry. BALB/c mice were immunized intra-muscularly with the recombinant plasmid pVAX1-p24/pVAX1-p30 to establish the model of Toxoplasma gondii infection. Serum IgG antibodies and cytokines IFN-γ,IL-2,IL-4 were demonstrated by ELISA and the T lymphocyte subsets assayed by flow cytometry. The survival times of the infected mice were observed and the killing activity of NK cells tested by MTT assay.The results showed that the recombinant plasmids pVAX1-p24/pVAX1-p30 were constructed successfully, and the p24/p30 antigen protein can be expressed in the mouse macrophage. The productions of IgG antibodies,IFN-γand IL-2 were more obvious in mice immunized with the compound pVAX1-p24 and pVAX1-p30 DNA vaccines than other experimental groups. There were decrease of CD4+ cells and increase of CD8+ cells resulting in a descent of CD4+/ CD8+ ratio. The survival times was considerably prolonged, and the killing activity of NK cells was increased of mice immunized with the compound pVAX1-p24 and pVAX1-p30 DNA vaccines in comparison with that of mice immunized with monovalent DNA vaccine.Tt is evident from this study that the compound pVAX1-p24 and pVAX1-p30 DNA vaccines can induce the cell mediated immunity and the humoral-mediated immunity against Toxoplasma gondii infection, and elicit a better immuno-protection in mice than the monovalent DNA vaccine.2. Construction of the compound DNA vaccines pBudCE-p24-p30 against Toxoplasma gondii with p30,p24 gene and the effect of pBudCE-p24-p30 on gene expression profile of mouse macrophageIn this section, Toxoplasma gondii p24/p30 gene fragments were amplified by PCR, which were inserted into the eukaryotic expression vector pBudCE4.1. Recombinant plasmid pBudCE-p24, pBudCE-p30 and pBudCE-p24-p30 were Identified by PCR amplification, restriction endonuclease digestion and sequence analysis. The recombinant plasmid pBudCE-p24,pBudCE-p30,pBudCE-p24-p30 and the vector pBudCE4.1 were transfected into mouse macrophage(RAW264.7)to establish the vitro eukaryotic cell temporary expression system, and detecting the expression of p24/p30 antigen protein by immunocytochemistry. Gene expression profile of differential expression of mouse macrophage transfected with pBudCE-p24-p30 were analyzed by using Affymetrix genechip. RT-PCR was used to confirm some up and down regulated expressions of related genes.The results showed that the recombinant plasmids pBudCE-p24,pBudCE-p30 and pBudCE-p24-p30 were constructed successfully, and the p24/p30 antigen protein can be expressed in the mouse macrophage. Among the total 22 000 tested genes, the expression of 16 genes was either up-regulated or down-regulated more than 3 fold in mouse macrophage transfected with pBudCE-p24-p30.The detected 16 genes fell into 3 categories: gene transcription, signal transduction, unknown function. 9 genes were up-regulated, including Pdlim4, Pcdh7, CAMK4, Nudcd2 etc, while 7 genes were down-regulated, including E2F5, Rpn1, Sh3bgrl etc. The results of RT-PCR were coincident with that of gene expression profile. This indicated that the effect of compound DNA vaccines pBudCE-p24-p30 on gene expression profile of mouse macrophage was not obviously.
|
PDF Full Text Request