Expression Of Toxoplasma Gondii Rops And Screening Of The Host Interacting Proteins

Toxoplasmosis is a parasitic zoonosis caused by Toxoplasma gondii. T. gondii belonging to theapicomplexan can be parasitic in almost all nucleated cells in addition to red blood cells. It is estimatedthat people in about 1/3 of the world’s population has infected with T. gondii. Following thedevelopment of cities and pets number continues to expand, the risk of T. gondii infection is tending toincrease. Rhoptry proteins(Rops) are the main effectors of T. gondii secreted into the host cells. Ropslocalize in different parts of the host cells and intensively interact with the proteins in host cells. Ropsaffects the biological activity of host cells through the regulation of membrane system and skeletonstructure, thereby blocking its internal defense mechanism, to complete the invasion, proliferation, and aseries of physiological process.Many Rops of T. gondii express differently between the tachyzoite and bradyzoite stages. Amongthem, Rop28 and Rop38 display more obvious changes. In this study, the full length of Rop28 andRop38 sequences were amplified by RT-PCR and cloned into the prokaryotic expression vector, andsuccessfully expressed the E. coli cells. Polyclonal antibodies were generated with purified recombinantRop38 and Rop28. In order to identify the interacting proteins, the eukyoriotic vectors expressingRop38 and Rop28 s were constructed and successfully expressed in 293 T cells.In the study, a cDNA library of the Human fibroblasts(HFF) and a T cell library(CEM) wereconstructed. The titer of the libraries is 1.2 x 108 cfu/ml and 1.38 x 108cfu/ml respectively. By using thebait plasmid to screen the HFF library, we obtained plenty of proteins intercting with Rop2, Rop5,Rop16, Rop17, Rop32 and Rop38. The bite vector expressing Rop38 was used to screen the CEMlibrary as well. The results indicated that Rops interatct with host cell proteins intensively. Furtherhybridization experiments confirmed that Rop2 B interact with 7 host cell proteins; Rop5 interact with 7proteins; Rop16 interact with14 proteins; Rop17 interact with 7 proteins; Rop32 interact with 12proteins; Rop38 interact with 11 proteins in HFF library and 20 proteins in the CEM library. Theprevious reported that upregulation of Rop38 could suppress the transcriptional expression of host cells.In this study, Rop38 interact with PRMT7 in both libraries. PRMT7 is an arginine methyltransferase, animportant member of PRMTs family. This molecule is involved in many processes including regulationof gene expression, translocation and differentiation of cells and development of individules. Our resultsmight indicate that PRMT7 could play an important role in the regulation of host cells transcription.The purpose of this project is to investigate the molecular mechanism of the pathogenicity T.gondii and interaction with host cells through screening the interacting proteins of Rops. The data ofthis study will provide the theory basis for the development of vaccines and drugs againsttoxoplasmosis.