Role Of Surface Antigen SAG3 On Invasion And Reproduction Of Toxoplasma Gondii

Toxoplasma gondii is a widespread obligate intracellular , opportunistic protozoan parasite and the etiological agent of toxoplasmosis.The healthy people are asymptomatic after infect parasite, but in immunocompromised patient, such as patients with tumor, AIDS patients and patient with organ transplant ,it could cause a series of serious diseases even death .T.gondii could be transmitted vertically to foetus through placenta and cause premature birth, abortion, fetal death and teras.The infection of T.gondii has become a serious public heathy problem with the epidemic of AIDS and tumor and the growing number of raising pet in the recent years.Up to date,there is no effective and safe drug for toxoplasmosis, and further understanding of its pathogenic mechanism is with great significance for the prevention and treatment.Invasion of host cells and rapid reproduction in host cells is the premise in the pathogenesis of T.gondii.Up to date,we still haven't clarify completely the mechanism of invasion and replication. Previous studies indicate SRS(SAG1-related sequence) protein superfamily play a critical role in the process.SAG3 is an important membrane of SRS protein superfamily, which is expressed by all invasive stages with low immunogenicity. Previous studies imply SAG3 is invovled in invasion and reproduction of T.gondii,but research about SAG3 is so few that the role of SAG3 in pathogensis of T. gondii is unclear. Therefore, the further research of SAG3 function will provide a cue to illuminate pathogenic mechanism of T.gondii.ObjectiveTo determinate the influence of inhibited expression of SAG3 to Toxoplasma gondii RH strain invasion and reproduction with RNAi technology for further understanding SAG3 function and pathogenic mechanism of T.gondii.ContextPart I Role of SAG3 in invasion of Toxoplasma gondii:dsRNA-UTR of SAG3 were transfected Toxoplasma gondii RH strain by electroporation, Total RNA of transformed parasites and hela cells were extracted in 5min,30min,1h and 2h after treated parasites infect cells in vitro.Expression level of MIC2, ROP2 and GRA2, three invasion-associated gene, were detected by semi-quantitative RT-PCR, the green fluorescence protein(GFP) were used as controls in the transformation experiments as well as blank controls.PartII Role of SAG3 in reproduction of Toxoplasma gondii:dsRNA-UTR and dsRNA-CDS of SAG3 gene were transfected Toxoplasma gondii RH strain by electroporation, Total RNA of transformed parasites and hela cells were extracted in 12h,24h and 48h after treated parasites infect cells in vitro.Expression level of AK and HXGPRT of purine salvage enzymes were detected by semi-quantitative RT-PCR, the green fluorescence protein(GFP) were used as controls in the transformation experiments as well as negative control and blank control.Results1) Parasites transfected with dsRNA of SAG3 gene were added to infect hela cell in vitro,and three genes of MIC2, ROP2 and GRA2 which expression level were lower than control groups.2) Parasites transfected with dsRNA of SAG3 gene were added to infect hela cell in vitro,and AK and HXGPRT which expression level were lower than control groups.Conclusion: Invasion and replication are partly inhibited while Toxoplasma gondii SAG3 gene's expression is knock down with RNAi, laying a foundation for further research of SAG3 function and clarification of pathogenic mechanism of T.gondii invading host cell...