The Preliminary Study On The Immune Response Of DC Against Toxoplasma Gondii ROP38 Mediated By TLR4

Toxoplaysma gondii belongs to the Apicomplexanparasites.Toxoplasmosis is a n immunosuppressive disease,The invasion of Toxoplasma gondii has a close relationship with the host's immune status,and the toxoplasmosis can cause se rious damage when the host's immune function is low.It is worth mentioning that toxoplasmosis can be transmitted to the fetus through the placenta,and m ost of the infected fetuses show negative infections before they show symptom s.In animal husbandry production,the risk of toxoplasmosis is also serious,es pecially for the swine industry,which often causes huge economic losses.Howe ver,until now,there are no drugs that can cure toxoplasmosis.The sulfa drug s used in clinical practice can only affect the tachyzoite morphology,but hav e no ideal therapeutic effect on chronic infection caused by Toxoplasma gondii.The study of Toxoplasma vaccine has become a hot issue in Toxoplasma rese arch,and the understanding of the innate immune mechanism of Toxoplasma g ondii is particularly important.MAPKs is a group of serine-threonine protein kinases that are activated by different extracellular stimuli and is an important component of the signal tran sduction of organisms.ROP38 can inhibit the transcription of host cells by do wn-regulating MAPK signal transduction pathway.The expression levels of RO P38 are different.The expression of ROPs is down-regulated during the conv ersion of Toxoplasma gondii to tachyzoites,but the expression of ROP38 is i ncreasing.At present,the basic biological research on Toxoplasma gondii has b een very mature,but the conversion mechanism between Toxoplasma gondii ta chyzoites and tachyzoites is still unclear.The response function of DCs to Toxo plasma gondii has not been reported in depth.The role of TLR4,which plays an important role in the innate immune system,in the immune response caus ed by Toxoplasma gondii invasion remains to be further studied.Firstly,The total RNA from T.gondii RH strain was extracted by guanidine thiocyanate method,then cDNA was synthesized with reverse transcription rea ction.After ROP38 gene was amplified with PCR technique,the recombinant pGEX-4T-ROP38 was constructed and expressed with IPTG induction.The rec ombinant ROP3 8 protein was detected by SDS-PAGE and Western blot analysi s.The recombinant plasmid pEGFP-Nl-ROP38 was constructed and successfully transfected and expressed in HEK293.To lay the foundation for the study of Toxoplasma nucleic acid vaccine.Then,TLR4-mediated DCs infection of ROP38 antigen was to carry out.The experiment was divided into 3 groups of 3 replicates.The DCs cultured to t he 7th day were collected,and 0.5 mL TLR4 antibody was added to the TLR 4 antibody seal group,And 1 mL ROP38 recombinant antigen;TLR4 antibody unseal group added 1 mL ROP38 and 0.5mL RPMI 1640 medium;blank con trol group added 1.5mL RPMI 1640 medium.Flow cytometry detected CDllc+expression levels and Flowjo software analyzes the data.the expression level of CD11c+was 69.0±2.7,59.6±1.2 and 22.1±0.6 among the TLR4 antibody uns eal group,TLR4 antibody seal group and the control group respectively,and t he difference analysis revealed that the expression level of CDllc+ in TLR4 a ntibody unseal group was significantly higher than that of the others(P<0.05).Finally,in vivo,Toxoplasma gondii infected mice by TLR4-mediated.4 grou ps were set up,namely the group of blank control(BC),the group of TLR4 antibody seal(TAS),the group of TLR4 antibody unseal(TAU)and The group of infected by toxoplasma gondii(IYG).The BC was not treated.The mice in the other groups were intraperitoneally injected with 1×104 tachyzoites of Toxo plasma gondii RH strain.the proliferation of T lymphocytes from spleen after 3d,and the expression of IL-6,IL-12,IL-18 and IFN-? from peripheral blood was determined to explore the relation of TLR4 to Toxoplasma gondii infectio n.The results showed that CD4+CD8-/CD8+CD4-expression between BC,TAS and ITG separately was 12.3±0.21/4.4±0.01,21.1±4.03/8.21±0.18 and 30.2±0.07/15.6±0.14,the difference between ITG and BC were obvious(P<0.05),the difference in CD8+CD4-was obvious between ITG and TAS(P<0.05),but t he difference of CD4+CD8-in the TAS was not significant compared with the BC(P>0.05).It is suggested that mice infected with Toxoplasma gondii can stimulate the body to produce high levels of CD8+CD4-T lymphocytes throug h TLR4.Real-time PCR showed that the expression levels of IL-12,IL-18 and IFN-y in the ITG were up-regulated by 2.3,5.5 and 3.2 times,respectively,c ompared with the TAS,suggesting that Toxoplasma gondii induce Thl immune response effectively through TLR4.TLR4 distribution and expression in diffe rent organs was detected by immunohistochemistry,the results indicated TLR4 expression level in ITG was the highest one,following as BC,TAS by immu nohistochemistry.TLR4 could induce DC maturation with ROP38 antigen stimulation,Thl im munoreaction by TLR4 induction to Toxoplasma gondii was the main path.It could provide theoretical basis to understand the mechanism on innate immunit y of Toxoplasma gondii.