Studies On Gene-sequencing And Immunoprotective Efficacy Of TgGRA 24 And 25

As an obligate intracellular protozoan,Toxoplasma gondii can infect almost warm-blooded animals including humans.About one third of the population in the world has been infected with T.gondii,while it is 8% in China.It has become a serious problem to human life and health.In pregnant women,it can cause miscarriage,fetal malformations,low immune function,even death.It also causes larger economic losses to husbandary production for fetal malformations and death every year.However,there is still no chemical compounds to cure it completely,and no safe and effective vaccines have been developed to control and protect it,hencce,it is vey important to screen antigen molecule from Toxoplasma gondii as vaccine candidate.All dense granule proteins(GRAs)are excreted and secreted antigen(ESA).ESA are highly immunogenic,can induce protective immunity through antibody-dependent or cell-mediated immune response,and participat in the growth and translation of the parasite in host cell.Once GRA24 invade into host cells,it can continously cause host cells autophosphorylation and nuclear translation through p38?MAP kinase.Not only can GRA24 control T.gondii invade intointestinal cell,but also it can increase the secretion of host chemokine and regulate the copy of its tachyzoitesat early stages.In addition,GRA24 also can stimulate the activation and proliferation of T cells,and induce Th1-type immune response.GRA25 is an essential virulence factor of T.gondii,which can take effect on production of CCL2 and CXCL1 by macrophages after infected with T.gondii.In this study,sequence variations in GRA24 gene and GRA25 gene of T.gondii from different hosts and geographic isolates were evaluated.The results showed that the size of948 bp and 6264/6261 bp could be amplified by PCR technique according to the expected GRA24 and GRA25 sequence,consistent with the expected results.Nucleic acid gene mutation rate of GRA24 was 0.05 to 0.2%,and the constructed phylogenetic tree of GRA24 by BI?MP?ML was not useful in grouping classical Type I,Type II and Type III of T.gondii.Nucleic acid gene mutation rate of GRA25 was 0-4.4%.Phylogenetic analyses showed that strains belonging to the classical Type I?Type II and Type III were not grouped into their own branches based on the GRA25 sequences.Therefore,both GRA24 and GRA25 genes are not suitable as effective genetic marker.In this study,the protection induced by the constructed DNA vaccine of pVAX-GRA24 and pVAX-GRA25 were evaluted by detecting the humoral and cellular immune-related indicators.The results indicated that the IgG antibodies and antibody subclasses of IgG1,IgG2 a significantly increased,and cytokines IFN-?? IL-2 ? IL-12 and IL-23 significantly increased after mice were immunized,meanwhile CD3+CD4+CD8-helper T cells and CD3+CD8+CD4-T cells were higher than that of the control,the difference was significant.The conclusion could be made that pVAX-GRA24 and pVAX-GRA25 could induce high levels of Th1 type cellular and humoral immune responses in KM mice.In addition,after challenge infection with T.gondii tachyzoites(RH),the survival time of KM mice in single gene and multi-gene immunized group got prolonged,and after infected with T.gondii cysts(PRU),the cysts in mice brain were significantly reduced in single gene and multi-gene immunized group,the difference was siginificantly compared with that of the control.The above results suggest that,Toxoplasma gondii dense granule protein GRA24 and GRA25 are good preventive vaccine candidate antigens of Toxoplasma gondii infection.