The Location Of Invadsion-related Protein MIC3 Of Toxoplasma Gondii And Protective Effect Of Its Vaccine In Mice

Objective:To observe the distribution of the Microneme protein in T.gondii tachyzoites and the immunity protection effect in mice.Methods:(A)The gene encoding MIC3 was amplified from Microneme of T.gondii RH strain by using RT-PCR,subcloned into eukaryotic expression vector,thus two eukaryotic expression plasmids pcDNA3-MIC3 and pEGFP-C2-MIC3 were constructed.(B)Two plasmids pcDNA3-MIC3 and pEGFP-C2-MIC3 were transfected into COS-7 cells by the use of liposomes in order to acquire its recombinant protein expression and indentify it through SDS-PAGE and Western-blot;and to survey the location of MIC3 in eukaryotic cells by using the fluorescent protein EGFP as label.(C)The plasmid pEGFP-C2-MIC3 was tranfected into T.gondii through using electroporation in order to observe the specific location in insects.(D)After preparation and purification of recombinant plasmids pcDNA3-MIC3,mice were inoculated intramuscularly third.To clarify the effect of the immune system,the survival time of challenged mice was observed.Lymphocyte transformation rate, CD₄⁺ and CD₈⁺ lymphocyte ratio of the spleen cells and specific antibody in serum of the immunized animals were examined.Result:(A)Two recombinant plasmids pcDNA3-MIC3 and pEGFP-C2-MIC3 were successfully constructed.DNA sequencing results showed that the size of the MIC3 for 1080 bp fragments were cloned into the Hindâ…¢/EcoRâ…¤sites in pcDNA3 and the Bglâ…¡/Hindâ…¢sites in pEGFP-C2 respectively.(B)The expression product of pcDNA3-MIC3 into COS-7 cells showed the 42KDa zone by SDS-PAGE.Western-blot and ELISA further confirmed MIC3 protein has a good immunological activity.(C)The fusion protein MIC3-EGFP were evenly distributed COS-7 cells in the cytoplasm and the green fluorescence was gathered like an english letter "C" around the nuclear membrane by transfection,but these phenomenons didn't appear in the nucleus and membrane. MIC3-EGFP tranfected into T.gondii by electroporation could be focused on the top of the insect,which illustrated MIC3 could accurately targeted to the top organelle.(D)The DNA vaccines of the gene MIC3 was successfully constructed. Protective experiments of DNA vaccine pcDNA3-MIC3 in animals showed that the vaccine could prolong the survival time of the mice challenged by virulent RH strains of T.gondii.The study on the impact to the immune system indicated that,pcDNA3-MIC3 vaccine could stimulate the animal to produce high level specific antibody and could induce the decrease of the CD₄⁺/CD₈⁺ ratio in the experimental mice.These results indicated that the DNA vaccine could stimulate the effect of the host against Toxoplasma mainly by the way of CD₈⁺ CTL cells.Conclusion:(1)The eukaryotic expression vectors of pcDNA3-MIC3 and pEGFP-C2-MIC3 were successfully constructed.(2)pcDNA3-MIC3 was successfully transfected into COS-7 cells and the MIC3 protein was acquired.ELISA and Western-blot analysis showed that recombinant protein had the specific immunogenicity.(3)pEGFP-C2-MIC3 was successfully transfected into COS-7 cells and T.gondii respectively.The former showed that MIC3-EGFP located in the cytoplasm of eukaryotic cells.The latter showed that MIC3-EGFP was correctly targeted to microneme.(4)The DNA vaccine of MIC3 were constructed successfully. Protective experiments in animals showed that the vaccine could significantly prolong the survival time of the mice challenged with virulent RH strains of Toxoplasma.The immue mechanism is mainly by the way of CD₈⁺ CTL cell.