Study On The Functions Of Toxoplasma Gondii Protein Phosphatases PPM2A And PPM2B

Toxoplasma gondii is an obligate intracellular parasitic opportunistic pathogen that can infect all nucleated cells and can cause miscarriages and miscarriage in pregnant women or animals.It has a complicated life history,multiple infection routes,a wide range of infection hosts,and a high positive rate of infection.It can cause more serious damage to the development of animal husbandry,and it has also caused problems for human health.However,there is currently no effective vaccine against Toxoplasma gondii infection.Molecules involved in protein modification and removing protein modification have always been important targets for drug development,As the phosphatase PPMs protein with protein dephosphorylation modification function plays an important role in the stress resistance of animals and plants,Toxoplasma gondii is transformed from tachyzoites to the bradyzoite process is the biological basis of the disease of Toxoplasma gondii,which is also a form of stress resistance of Toxoplasma gondii.Therefore,understanding whether the toxoplasma protein phosphatase PPMs participate in this conversion process is of great significance for the development of toxoplasma drug targets.However,it is not entirely clear whether phosphatase PPMs play an important role in the growth and development of Toxoplasma gondii.In this study,Toxoplasma gondii protein phosphatases PPM2 A and PPM2 B were used as the research object,using CRISPR / Cas9 technology to perform single gene knockout and double gene knockout on PPM2 A and PPM2 B in RH ? HX strains respectively,and compare each gene knockout strain growth and development differences and virulence changes;At the same time,IFA technology was used to detect the position of the protein in Toxoplasma gondii,yeast two-hybrid technology was used to investigate whether PPM2 A interacts with Ca M,and PPM2 A was detected protein phosphatase activity in vitro,with a view to elaborating the function of Toxoplasma gondii protein phosphatases PPM2 A and PPM2 B in the growth and development of Toxoplasma gondii,providing a certain screening reference for drug research or vaccine research to Toxoplasma gondii protein phosphatase targets.The specific experimental contents are as follows :(1)Location experiment of PPM2 A and PPM2BUsing CRISPR / Cas9 technology,gene targeting was performed at the proximal end of the target gene downstream of the RH ? KU80 strain,and then the localization tag containing HA was homologously recombined to the target gene downstream.Through indirect immunofluorescence experiments,the target protein was detected in Toxoplasma gondii.The staining results showed that PPM2 A was dispersed throughout the body of Toxoplasma gondii,and PPM2 B was dispersed in the cytoplasm of Toxoplasma gondii.(2)Construction of PPM2 A and PPM2 B gene single-deletion and double-deletion strainsUsing CRISPR / Cas9 technology,targeting to the target gene of the RH ? HX strain,and then homologously recombine the drug screening tag containing DHFR to the position of the target gene,through pyrimethamine drug screening,monoclonal PCR identification,Finally,the RH ? HX ? PPM2 A and RH ? HX ? PPM2 B monoclonal strains were successfully obtained;Corresponding phenotypic experiments of obtained RH ? HX ? PPM2 A and RH ? HX ? PPM2 B monoclonal strains showed no significant difference in the growth and virulence,so the two proteins were subjected to a double gene deletion experiment by us.Targeting was performed in the middle of the PPM2 B gene sequence of the RH ? HX ? PPM2 A strain,and then the drug screening tag containing CAT was homologously recombined into the PPM2 B gene,through chloramphenicol drug screening,monoclonal PCR identification,finally successfully obtained RH ? HX ? PPM2 A ? PM2 B monoclonal strain.(3)Phenotype experiments of PPM2 A and PPM2 B knockout strainsAfter obtaining the corresponding monoclonal strains,the monoclonal strains were subjected to replication experiments,plaque experiments and virulence experiments,respectively.In the replication experiment,the parasites were fixed and stained on the cell slide for 24h;in the plaque experiment,the parasites were fixed and stained on a six-well plate for 7 days;the virulence test was performed by intraperitoneal injection,8-9 weeks old female ICR mice were injected with 200 parasites per mouse,and the survival status of the mice was recorded within 30 days.Through replication experiments and plaque experiments,we observed that the replication ability of the RH ? HX ? PPM2 B and RH ? HX ? PPM2 A ? PPM2 B monoclonal strains did not significantly decrease,and the replication ability of the RH ? HX ? PPM2 A monoclonal strains slightly decreased;through virulence experiments,we found that the virulence of RH ? HX ? PPM2 A,RH ? HX ? PPM2 B,RH ? HX ? PPM2 A ? PPM2 B monoclonal knockout strains were not significantly weakened compared with RH ? HX.(4)PPM2A enzyme activity experiment in vitroConstruct the p E-SUMO-PPM2 A plasmid,transfer the plasmid into E.coli BL21 for large-scale expression,and then purify the PPM2 A protein in vitro,and finally use the Biyuntian alkaline phosphatase detection kit(P0321)to detect the PPM2 A protein phosphatase activity,When the standard or the well with alkaline phosphatase activity shows different shades of yellow,the phosphatase activity of PPM2 A was calculated by measuring the absorbance at 405 nm,but the phosphatase activity of PPM2 A protein was not detected in this study.(5)PPM2A and Ca M yeast two-hybrid experimentConstruct pGBKT7-PPM2 A plasmid and pGADT7-Ca M plasmid,transfer pGBKT7-PPM2 A plasmid into yeast Y2 HGold competent cells and spread on SD /-Trp plate,transform pGADT7-Ca M into yeast Y187 competent cells and coat SD /-Leu plate,pick single colonies on two plates and place them in the same centrifuge tube for conjugation culture,then take the conjugated culture and apply to DDO / X / A plate for 3-5 days,we found the blue colonies grew on the DDO / X / A plate,and it was verified that the pGBKT7-PPM2 A plasmid had no self-activation phenomenon,indicating that there was an interaction between PPM2A and CaM.