| Toxoplasma gondii is the aetiological agent of toxoplasmosis and is the most frequent and best known of the parasitic diseases. Recently, the development of vaccines against toxoplasmosis has progressed considerably due to the application of the technology of molecular biology.Research showed that the inactivated vaccine was insufficient to induce proper immune responses. A vaccine based on the live attenuated S48 strain was developed for veterinary uses[1]. However, it is not suitable for human use for the possibility of reverting to a pathogenic strain. Subunit vaccine using nature or recombinant parasite antigens is expensive, difficult to purified and has a short shelf life therefore it is not appropriate for wide-ranging application. Under the present scenario, development of DNA vaccine against toxoplasmosis is an attractive alternative.P30, or SAG1, is the main surface antigen of the Toxoplasma tachyzoite covered 3 to 5 percent of the gross proteins of T. gondii. Being highly immunogenic and immuno-protective, it was considered as an important candidate for effective diagnostic reagents and vaccine antigen[4,5]. More recent reports showed that ROP2, a p54 kDa protein component of the rhoptries expressed in three stages of T. gondii life cycle ,participated in the parasite invasion into host cells by mediating host organelle association with the parasitophorous vacuole membrane (PVM), and had also been proposed as a vaccine candidate against toxoplasmosis.Meanwhile, accumulating evidence indicates that vaccination with stage-specific antigens leads to stage-limited protection12'31. Based on this background, we studied the efficacy of immunization with P30-R0P2-encoding compound DNA vaccine and induced resistance against challenge with the virulent RH T. gondii strain, at the same time compared with those of a single gene vaccine encoding P30.In this study, gene engineering technology was exploited to construct the monogene eukaryotic expression plasmid pcDNA3. 1-P30. Then it was transiently introduced into Hela cells by liposomes with the recombinant eukaryotic expression plasmid pcDNA3. 1-P3O-ROP2 respectively. The total RNA of the cell culture was extracted by use of Trizol 48 hs after transfection. The eukaryotic expression vector pcDNA3. 1 was verified to have the capability to transcript and translate the P30 and the P30-R0P2 genes in mammal cells through RT-PCR methods. House-keeping gene P — actin was used as an inner control. After that abundant plasmids pcDNA3. 1-P30 and pcDNA3. 1-P3O-ROP2 were extracted by the means of alkaline lysis. BALB/c mice were intramuscularly immunized with the compound DNA vaccine encoding P30 and ROP2 genes (100 y g per animal) or equal dose of monovalent DNA vaccine encoding P30 single gene. Two booster injections were carried out at 2-week intervals. As negative controls, the empty vector pcDNA3.1 and PBS buffer were injected in the other two groups respectively. Mice were bled at day 13 (a day before the second dose), day 27(a day before the last dose), day 42(14 days after the last dose) and day 56(28 days after the last dose). All sera was analysed individually. Challenge or removal of spleens took place at day 56 unless otherwise stated. Each mice inoculated with DNA constructions or PBSbuffer were challenged intraperitoneally with 105 tachyzoite forms of T. gondii RH strain. The proliferation activity of T cells and the activity of NK cells were determined by means of MTT assay. The total IgG and cytokines of IFN-y^ IL-4 were determined by ELISA. Flow cytometry was applied to detect the subclones of T cells. Results: Both immunized groups (group injected with pcDNA3.1-P30 or pcDNA3. 1-P30-ROP2) gained the highest IgG at day 56, while a more transparent production of antigen-specific IgG was raised in the compound vaccination group compared to the single vaccination one (P<0. 01) . The pcDNA3.1-P30-ROP2 group raised higher IFN-y (P<0. 01) , CD8+ T cell proportion (P<0.01) . The proliferation activity of T cells of group pcDNA3.1-P30-ROP2 increased evidently (P<0. 01) .while neither group showed significant NKC killing activity. No IL-4 was detected in all the groups. Although no mice survived a lethal challenge with highly virulent Toxoplasma gondii RH strain, compound DNA vaccination induced a long-lasting protection.Our results showed that DNA vaccination could induced specific immune response against T. gondii, including the specific antibody and a protective Thl response, and activated the non-specific immune system such as the NK cells as well. Compound DNA vaccine of genes from different livestages of T. gondii elicit better immunoprotectivity in mice than vaccine of a single gene.
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