Expression And Function Analysis Of TGME49₂09950 Protein Of Toxoplasma Gondii

Toxoplasma gondii is a protozoan of the apicomplexa and is an intracellular parasite that can infect nearly all nucleated cells of warm-blooded animals including humans.Toxoplasmosis is a zoonosis widely distributing in the world,which infects approximately one-third people worldwide.The symptoms of people who are infected for the first time are not obvious enough to be noticed,however,some unusual syndromes such as non-characterized influenza and AIDS may be related to the first infection with Toxoplasma gondii.Infection with Toxoplasma gondii can cause many syptoms,including miscarriage and stillbirth.Moreover,the recessive infection of Toxoplasma gondii can lead to the formation of cysts which can cause terrific harm to food safety.Toxoplasma gondii invades nucleated cells relying on the interaction between some ligands and receptors.After the invasion into the cells,Toxoplasma gondii proliferates largely,resulting in the occurrence of disease.There are many reports about toxoplasma gondii rod-shaped,membrane surface,dense granule proteins which have been chosen as candidate antigens against toxoplasmosis,but these candidate antigens are not ideal for the protection of the host.The previous research conducted by our research group showed that the Plasmodium and heparin interacting proteins play a crucial role in the development and proliferation of parasites,as Toxoplasma gondii and Plasmodium both belong to apicomplexa,the parasite heparin-binding protein PF3D7₁104400 is used as a template.The TGME49-209950 protein was screened by BLAST alignment in ToxoDB.This protein has a disulfide active site motif-CXXC-in Cys65 to Cys68.This active site is important for Trx to function in the worm body.We therefore speculate the screened protein plays an important role in the reproduction of Toxoplasma gondii.The bioinformatics analysis revealed that TGME49-209950 protein has five glycosaminoglycan binding motifs and a highly conserved Trx domain.According to the position of glycosaminoglycan binding motifs and thioredoxin functional domains,the protein was truncated into two segments,one is TGME49₂09950-N762 protein with Trx domain and glycosaminoglycan binding motif,the other is C-terminal protein containing only glycosaminoglycan named TGME49₂09950-C612.The two gene sequences were respectively expressed in prokaryotic expression protein purification.The experimental animals were immunized with a His-tagged recombinant protein to prepare a polyclonal antibody,and then the polyclonal antibody was specifically detected for the target antigen by Western-blot,and the protein was invaded by the indirect immunofluorescence（IFA）method.During the process of cell isolation and tracking in the invasive phase,partial invasion phase,and complete invasion phase,it was found that the protein was expressed in the microsomes and dense particles of the parasite at different stages.The pull down and IFA experiments verified that the target protein adhered to the host cell;in vitro antioxidant experiments demonstrated that the N segment protein with Trx domain has anti-oxidation function;in vitro protection of plasmid DNA experiments found that the protein has an attack against oxygen free radicals The function of protecting the integrity of the plasmid.