The Effect Of Toxoplasma Gondii In Vivo Induced Antigen Genes C-18、Mic11、PI To The NF-κB Signal Pathway

Toxoplasma gondii is a worldwide opportunistic protozoan that can parasite in the nucleated cell of human and many vertebrates. Toxoplasma gondii can infect human, swine, cattle, horse, sheep, deer and some other animals, lead to abortion, stillbirth, monster and mentally children in the pregnant woman or dam, it is a great threaten to the life and health of human beings and the development of animal husbandy. For the reason of high infection rates of Toxoplasma gondii, and also its important role in medical and animal medicine, it is extremely necessary to learn the pathogenesis and find out safe and effective vaccine of Toxoplasma gondii, this is also a important measure to prevent and control the human and animal toxoplasmosis.As a result, we infected293T cells with RH strain Toxoplasma gondii tachyzoites, in order to verify whether the tachyzoites can activate the NF-kB signal pathway in293T cells or not; Checked out the different expression of four in vivo induced antigen genes in vivo and in vitro by realtime PCR; Constructed eukaryotic expression plasmids of the seven in vivo induced antigens, and measured their effect to the NF-kB signal pathway in293T cells; Built DNA vaccine by Toxoplasma gondii gene of in vivo induced antigens JK483877、 JK483898, and detected immunizing protection in BALB/c mouse.(1) Effect of Toxoplasma gondii tachyzoites to NF-kB signal pathway in293T cellsCollected purified RH strain Toxoplasma gondii tachyzoites and infected293T cells which were cultured in vitro. According to the analyze of double fluorescent numerical, the tachyzoites can observably activate the NF-kB signal pathway in293T cells; Collected purified RH strain Toxoplasma gondii tachyzoites and infected293T cells which were cultured in vitro, stimulated the293T cells by TNF-α, According to the analyze of double fluorescent numerical, the tachyzoites can not inhibit the activation of NF-kB induced by TNF-a, the more the tachyzoites infected, the better the NF-kB activated.(2) Verified the different expression of four in vivo induced antigen genes in vivo and in vitro by realtime PCRDesigned primers according to the sequences of in vivo induced antigen genes JK483877、 JK483880、 JK48388、 JK483898, validated the tachyzoites infected the BALB/c mouse for72hours in vivo and BHK cells for72hours in vitro by realtime PCR that the four in vivo induced antigen genes expressed higher in vivo than that in vitro (P<0.05) (3) Constructed eukaryotic expression plasmids of in vivo induced antigen genesDesigned primers according to the sequences of three functional genes and four hypothetical protein genes, amplified the target fragments through PCR, connected them on cloning vector of pMD-19-T, after identification, linked the target fragments to expression vector of PCMV-Tag2B, and finally successfully constructed the eukaryotic expression plasmids.(4) Verified the effect of the recombinant expression plasmids to NF-kB signal pathway in293T cellsTransfected the recombinant plasmid to293T cells, according to the analyze of double fluorescent numerical, the seven eukaryotic expression plasmids can not activate the NF-kB signal pathway in293T cells, and also they have no inhibition to the NF-kB signal pathway in293T cells, although some genes have tendency of inhibition, but not remarkable.(5) Immunogenicity of in vivo induced antigen JK483877、 JK483898The recombinant plasmid can be recognized by Toxoplasma gondii rabbit polyclonal serum by IFA, and it had better antigenicity. No specific TLA antibody can be found in the serum of inoculated BLAB/c mice, and also no high level of IL-2、 IL-4、 IL-12and IFN-y by cytokine ELISA. In addition, we can not see the proliferation of splenic lymphocyte in experimental groups. Mice were challenged with T. gondii RH strain tachyzoites at day14after the final immunization. All mice in the blank vector control groups died within8days post infection, while8.3%of the mice immunized with PCMV-Tag2B/JK483877and PCMV-Tag2B/JK483898were survived to day15after the parasite challenging. But there are still16.7%of the PBS control groups survived to day15after the parasite challenging. Above all, the DNA vaccine can not induce specific antibody of Toxoplasma gondii, and had no protecting force.This study verified the effect of recombinant plasmid to the NF-kB signal pathway, and identified the immunogenicity of JK483877and JK483898, so we can get more about the activation of NF-kB pathway and lay the foundation for the study of Toxoplasma gondii vaccine.