| Toxoplasma gondii is an obligate intracellular protoza, which can infect almost all mammals including humans. Toxoplasma infection have brought serious harm to animal and human health in the world.Currently, there is still no better vaccines and drugs in the actural applications,so the scientific issues of an effective vaccine research and finding novel drug targets should be resolved.Motigen-activated protein kinases(MAPKs) are a class of intracellular serine/threonine protein kinases. It has been confirmed, MAPKs signal transduction system can stimulate extracellular signal transduction into the cell and the nucleus, regulate important physiological process such as cell prolification, differentiation, apoptosis, transformation and stress reactions. Toxoplasma can encode Tg MAPK1 and Tg MAPK2. Tg MAPK1, a p38α MAPK homologue, expresses when tachyzoites change into bradyzoites,or when the body received external stimuli, and parasite growth and gene functions are not fully understood.To explore these scientific questions, we cloned and expressed Tg MAPK1 in this work: We induced expression and got a kind of recombinant protein called r Tg MAPK1 by constructing a prokaryotic expression vector. The use of mice which were immunized with r Tg MAPK1 was for the preparation of anti-mouse polyclonal antibodies. Using IFA to detect the expression site of Tg MAPK1 in T.gondii. We could use homologous recombination, at the same time, chose CAT for the selectable marker to construct Tg MAPK1 gene targeting vector, and chose DHFR to construct Tg MAPK1 gene overexpression vector; getting Tg MAPK1 knock out strains and complement strains of T.gondii by resistence screening; using immunofluorescence, fluorescence quantitative PCR, flow cytometry, vacuole assay to study for the phenotype changes after deleting Tg MAPK1 gene in vivo and vitro, laid a foundation for the identification of potential medical targets of toxoplasmosis and the research of diagnostic antigens as candidates for vaccine.1.Tg MAPK1 cloning, expression and bioinformatics analysis of Toxoplasma gondiiUsing RT-PCR to amplify Tg MAPK1 coding sequence, fragment size is 1599 bp. After sequence comparison and phylogenetic analysis, it showed that the MAPK1 amino acid of T. gondii are highly homologous with Plasmodium falciparum, Eimeria protoza and other protoza, but the homology between mammals like humans was low, which indicated they were distantly related. Then we cloned the Tg MAPK1 and made it express. Building a Tg MAPK1 prokaryotic expression vector p ET-28a(+)- Tg MAPK1, using IPTG to induce the expression of recombinant proteins. Using SDS-PAGE and Western blotting analysis to confirm the size of r Tg MAPK1 is 58 ku. After Ni-NTA purification, preparing r Tg MAPK1 immunized BALB/c mice to obtain polyclonal antibodies which could anti r Tg MAPK1, using IFA to detect the subcellular localization of Tg MAPK1 in the tachyzoites of Toxoplasma gondii.2.The expression change of Tg MAPK1 when inducing bradyzoities transformation in vitro.Constructing a model in which tachyzoites induced bradyzoites transformation, happened in T. gondii in vitro. Using relative fluorescence quantitative QRT-PCR to detect expression changes of BAG1, ENO1, SAG2 D, LDH2 of specific proteins of bradyzoites under conditions( temperature 41℃, alkaline p H 8.1) for judging the effect of bradyzoites transformation in vitro; Thereby detecting the Tg MAPK1 expression under conditions for a clear influence of Tg MAPK1 for the inducing differentiation.3.Building Tg MAPK1 knock out strains and complement strains of Toxoplasma gondiiCloning noncoding regions on both sides of Tg MAPK1 in T. gondii by PCR, and the wing to CAT was for screening marker, in order to construct a gene targeting vector called pmin CAT-Tg MAPK1-UTR; We could make DHFR the selectable marker to construct over-expression p DHFR – Tg MAPK1; After linearized targeting vector, transfected Tg GT1 strains by electroporation, and then monoclonal chloramphenicol- resistant strains were screened, which were identified using methods like PCR, RT-PCR, Southern blotting and Western blotting. Then the overexpression vector p DHFR–Tg MAPK1 were transfected by electroporation into correct Tg MAPK1 deletion strains, screened by pyrimethamine-resistant to get resistant monoclonal strains. Then using RT-PCR, Southern blotting and Western blotting for identification.4.Functions of Tg MAPK1 of Toxoplasma gondiiStudies in vitro: At the status of cell culture in vitro, applicating IFA, QRT-PCR, FCS and vacuole test methods to compare differences of Tg MAPK1 deficient strains, the complement strains and wild type strains(Tg GT1) of T. gondii in adhesion, invasion, proliferation, differentiation, antigen gene and cytokine expression in macrophages tests to identify differences in the impact on the change of Tg MAPK1 deletion phenotype.Studies in vivo: Using different dose of tachyzoites of MAPK1 deletion strains, complement strains and the wild type to vaccinate mice. Making the statistical analysis of the average survival time of mice and survival parasites in liver, brain organization to determine the effects of Tg MAPK1 on pathogenicity and proliferation of mice in vivo. Cytokines expression of ΔTg MAPK1 were compared with the wild-type of T.gondii in the vaccinated mice on the peritoneal fluid and serum by BDTMCBA through flow cytometry in day3 and day7, suggesting that the impact of MAPK1 to the variation of vivo cytokine gene.In summary, the following conclusion of the present study :(1) Sequence alignment and phylogenetic analysis showed Tg MAPK1 has distant kinship with MAPK1, including mammals like humans, suggesting that Tg MAPK1 can act as candidates of gene therapy and drug targets.(2) Cloned and expressed Tg MAPK1, obtained recombinant protein r Tg MAPK1 and its polyclonal antibodies, and proved Tg MAPK1 expressed on the cell mambrance of T. gondii.(3)Tg MAPK1 protein expression levels increased with bradyzoites transformation in vitrand in the early induction and alkaline conditions, it showed significant differences, suggestinthat differentiation of T. gondii under arkaline conditions is more dependent on MAPKsignaling pathways.(4) Constructed gene targeting vector and over-expression vector of TgMAPK1, using electroporation, resistance screening and PCR, RT-PCR, Southern blotting,Western blotting and other identification, successfully acquired Tg MAPK1 knock out strains and complement strains of T. gondii.(5) Studies in vitro showed that:Adhesion invasive assay showed Tg MAPK1 deletion tachyzoites can reduce the ability of adhesion to the host cell but had no effect on invasion.Proliferation assay showed MAPK1 deletion can slow the proliferation of tachyzoites.Induced differentiation assay showed Tg MAPK1 knock out may reduce the effectiveness of tachyzoites converted to bradyzoites.Genes differentially expressed assay showed MAPK1 showed a negative regulation of gene expression for gene SAG3, GRA1 and MICs, but a positive regulation to the expression of ROPs.The differences of macrophage cytokine gene expression tests showed MAPK1 possibly promoted macrophage activation by regulating TNF, IL-6 and IL-10 expression.(6) Studies in vivo showed that:Pathogenicity assay showed that Tg MAPK1 deletion reduced virulence in mice, reducing its growth rate in mice,suggesting Tg MAPK1 as a new virulence gene of Toxoplasma gondii.The differences of peritoneal fluid cytokine gene expression assay showed that Tg MAPK1 probably promoted the intra-abdominal macrophage activation by regulating the expression of IL-12p70、TNF-α、IFN-γ and IL-10 production, and then kill Toxoplasma gondii.The differences of serum cytokine gene expression assay showed that TgMAPK1 probably promoted the changes of Th2-type immune response to Th1-type by regulating the expression of IL-12p70、TNF-α、IFN-γ、IL-6 and IL-10, in order to establish a latent infection or chronic infection. |
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