Development And Preliminary Application Of Toxoplasma Gondii PSMART2b-SAG3 DNA Vaccine

Toxoplasmosis is a kind of serious zoonosis,which can induce the abortion in pregnant woman and animal.The disease is caused by toxoplasma gondii,a intracellular parasite,and it is the second type of animal disease in China.Currently,sulfa dr?gs and antibacterial Synerists still play important role in the prevention and treatment of toxoplasmosis.But long term use of dr?gs has a great impact on public health security.What's more,there are almost no vaccine for the prevention and control of toxoplasmosis.To solve this situation,in this study,recombinant plasmid pSMART2b-SAG3 was constructed,and the fusion protein could interact with rabbit anti-SAG3 protein serum,which confirm the immunogenicity of the protein.Then the recombinant pSMART2b-SAG3 plasmid was injected into the hindlimb of mices.The cellular immunity and humorol immunity were tested and the protective effect of the plasmid for the infection of Toxoplasma gondii RH strain tachyzoite was analyzed,To explore the potential of the recombinant plasmid as a DNA vaccine preliminarily.The construction,expression and identification of recombinant p SMAET2b-SAG3 plasmid Toxoplasma gondii RH strain tachyzoites were purified,the total RNA was extracted with Trizol method and reverse transcripted into c DNA.Take the c DNA as the template and amplify the CDS of SAG3 gene.The SAG3 gene without signal peptide gene was successfully amplified and the length was 1068 bp.Then it was connected to p MD-18 T clone vector and alphavirus replicon-vectored plasmid pSMART2 b.At last,the recombinant plasmid pSMART2b-SAG3 and pSMART2 b plasmid were respectively transfected into 293 T cells and the immunogenicity of was confirmed by the test of the rabbit anti-SAG3 protein serum.The study of the protective effect of plasmid pSMART2b-SAG3 on mices The reconbinant plasmid pSMART2b-SAG3 and pSMART2 b were injected respectively into the hindlimb,the immune doses were 100?g per mice and 50?g per mice.The mices were immunized for 3 times,then the cellular immunity level and humorol immunity level were evaluated.The proliferation of spleen lymphocytes in mice was tested with MTT method and the levels of CD4+ and CD8+ were tested by flow cytometry,summarize and analyze the weight change and survival rate of every group.The number of Toxoplasma gondii was tested with the q PCR about the total genome of brain tissue.In the end,the result showed that after the inoculation of recombiant plasmid,with the increase of the immunization times,the antibody titer,IL-2 ? IFN-? ? SI and the levels of CD4+ and CD8+ all increased gradually,compared with the control group,the difference was significant.After the immunization,challenge every mices with 1×103 Toxoplasma gondii RH strain(except negative control groups).Two weeks after challenge,the survival rate of the pSMART2b-SAG3(50?g)group was highest(18.75%),the survival rate of the pSMART2b-SAG3(100?g)group was 6.25%,none survive in the other groups;with the result statistic analysis,we found that the average weight of mice in every groups increase slowly.The protective effect on the mices which were inoculated 50?g recombinant plasmids pre mice was better than the mices which were inoculated with 100?g recombinant plasmids per mice.Then the number of Toxoplasma gondii tachyzoites in the lungs of mices in every groups was tested by q PCR.As a result,the number of pSMART2b-SAG3(50?g)group was the lowest at 105.02,lower than the positive control 25.95%.In conclusion,the recombinant plasmid pSMART2b-SAG3 can to some extent protect mices against the infection of Toxoplasma gondii,which indicate that SAG3 can be take as a candidate antigen for the vaccine.