Biological Characteristics And Functions Of Toxoplasma Gondii Thioredoxin

Toxoplasma gondii?T.gondii?,a apicomplexan parasite,is a food-borne intracellular parasitic protozoa with many host types and can infect nearly 300 species of organisms,including humans.The disease caused by T.gondii infection is called toxoplasmosis,a serious zoonotic parasitic disease,which is a worldwide epidemic and poses a huge threat to human health,the development of animal husbandry and food safety.It is necessary to continuously find effective vaccines or methods to control T.gondii infection.The invasion of the host by apicomplexan parasites is a fast and continuous process,and the primary condition for invasion is that the ligands of the parasites and the host's related receptors are bound to each other.Previous research in our laboratory found that heparin glycosaminoglycan is one of the main receptor molecules during the invasion of the host by Plasmodium,but the mechanism is not clear.The related molecular mechanism of T.gondii,which is an intracellular parasite,needs to be studied,and there are few reports about Toxoplasma heparin binding protein.Therefore,this research will systematic research on the biological characteristics and functions of Toxoplasma heparin-binding protein,Toxoplasma thioredoxin?Thioredoxin,Trx?.The basic biological information and conserved domain of T.gondii Trx were analyzed and predicted firstly,and the results showed that T.gondii Trx has a sigal peptide without transmembrane region,which is a kind of secreted protein.The protein also contains a typical redox motif“-CXXC-”,and it is speculated that the protein may play a role in redox-related processes in T.gondii.In order to better analyze the function of T.gondii Trx,different prokaryotic expression vectors were constructed by segmenting this protein,and His-tag and GST-tag recombinant proteins were expressed and purified in this study.Meanwhile,specific polyclonal antibodies were obtained by immunizing experimental animals.The subcellular localization using indirect immunofluorescence experiments showed that T.gondii Trx was widely distributed in the cytoplasm of the T.gondii,and the fluorescence intensity of the parasite in cells was higher than that of free parasites,which may be involved in the process of T.gondii proliferation and interaction with the hosts.Transcription and expression levels of Trx of T.gondii were analyzed by real-time quantitative PCR and Western blot experiments respectively,and the results showed that the transcription and expression levels in the ME49strain were significantly higher than those in the RH strain?P?0.01?.The in vitro heparin-binding test and cell adhesion test showed that T.gondii Trx can specifically bind to heparin molecules and adhere obviously to a variety of cells.And the results of in vitro antioxidation test showed that T.gondii Trx has a certain protective ability to the oxidatively damaged plasmid,and the antioxidant ability disappeared after mutation of specific amino acid sites of T.gondii Trx,meanwhile T.gondii Trx can resist the oxidation of H₂O₂to a certain extent?P?0.01?.In order to further research the function of T.gondii Trx,CRISPR/Cas9 gene editing technology was used to construct a T.gondii Trx overexpression strain.By comparing its invasion rate,proliferation rate,transcription level and expression level,and redox index with wild types,the results showed that overexpression strains have significantly higher invasion and proliferation rates than wild types,and transcription and expression level was higher than wild types also?P?0.01?.Overexpression strains T-AOC level increased significantly,and ROS and MDA levels decreased significantly?P?0.01?.In summary,in this research it was found that T.gondii heparin-binding protein,T.gondii Trx,has heparin binding ability and cell adhesion ability,with a certain ability to resist oxidative damage.The invasion rate,proliferation rate and some redox indexes changes of overexpression strains increased significantly.The research results will lay the foundation for revealing the interaction mechanism of T.gondii with host cells,and also provides a certain reference value and theoretical basis for the development of new toxoplasma vaccines and the screening of drug targets.