Research On GRA1/MIC6Bivalent DNA Vaccine And Subunit Vaccine Against Toxoplasma Gondii

Toxoplasmosis caused by the obligate intracellular protozoan parasiteToxoplasma gondii with an exceptionally broad host range is a life-threateningdisease,infecting alost every kind of warm-blooded species including man.Thus,thedevelopment of an effective vaccine against T.gondii would be of great value for theeffective control of T.gondii infection in human and animals.Research institutions ona variety of research value of Toxoplasma gondii vaccine genes.However,the actualprotective effect is not very well.In this study,selected of Toxoplasma GRA1gene andMIC6gene preparation of bivalent nucleic acid vaccines and subunit vaccines.Toxoplasma invasion of host cells after the dense granules of Toxoplasma gondiiGRA1gene encoding the protein in humans and animals experimentally infected witha strong immunogenicity.It was used as a vaccine important candidate factor.MIC6istransmembrane protein of typeⅠ which plays an importantrole in adhesion andinvasion.It adhesion receptors on the identification of the host cell membrane.MIC6was also used as a vaccine important candidate factor.Use the GRA1and MIC6genessingle and tandem with eukaryotic expression vector and prokaryotic expressionvector connected to the building of nucleic acid vaccines and subunit vaccines.Thenused the vaccine immunized animals.All the groups have a certain degree ofprotective immunity.Five DNA fragments were amplified by PCR.Then the DNA fragments wereinserted into pMD18-T vector and constructed the recombinant plasmids:pMD18-T-GRA1,pMD18-T-MIC6,pMD18-T-L-GRA1,pMD18-T-L-MIC6and pMD18-T-C-MIC6.The sequencing results by Blast comparison with the original sequence,the homology was100%.The recombinant plasmids pVAX1-GRA1,pVAX1-MIC6,pVAX1-L-MIC6-L-GRA1and pVAX1-C-MIC6-L-GRA1were constructed and expressed in Hela cells.By Western blot verified protein expression.The recombinant plasmids pET-28a-GRA1,pET-28a-MIC6,pET-28a-L-MIC6-L-GRA1and pET-28a-C-MIC6-L-GRA1were constructed and expressed in DE3cells.The protein was reactogenicity confirmed from Western blot.The BALB/c mice were immunized with recombinant plasmids. After the thirdtime immunity,the IgG tite of all experimental groups were significantly increasedcompared to the control group (p<0.01). The detection of cellular immunity stated thatthe value of CD4+and CD8+T lymphocyte significantly increased compared with thepVAX1control group and PBS control group (p<0.01).Also have a significant levelsof IFN-γ and IL-2production,compared with the groups immunized with pVAX1control group or PBS control group (p<0.01).Among those four experimentalgroups,the productions of IgG antibodies and the percentage of CD4+cells in micevaccinated with pVAX1-C-MIC6-GRA1were higher than with pVAX1-GRA1andpVAX1-MIC6(p<0.05).The levels of IFN-γ and IL-2in the pVAX1-C-MIC6-GRA1group was also higher than other experimental groups.After lethal challenge, the miceimmunized with pVAX1-C-MIC6-GRA1showed an increased survival timecomparedwith control groups.The BALB/c mice were immunized with recombinant protein.After the thirdtime immunity,the IgG tite of all experimental groups were significantly increasedcompared to the control group (p<0.01).The detection of cellular immunity stated thatthe value of CD4+and CD8+T lymphocyte significantly increased compared with theadjuvant control group and PBS control group (p<0.01).Also have a significant levelsof IFN-γ and IL-2production, compared with the groups immunized with adjuvantcontrol group and PBS control group (p<0.01).Among those four experimentalgroups,The productions of IgG antibodies and the percentage of CD4+cells in micevaccinated with C-MIC6-GRA1were higher than with GRA1and MIC6(p<0.05).The levels of IFN-γ and IL-2in the C-MIC6-GRA1group was also higher thanother experimental groups.After lethal challenge, the mice immunized with C-MIC6-GRA1showed an increased survival timecompared with control groups.