| Toxoplasma gondii,an obligate intracellular protozoan,infects almost all warm blooded animals,including humans,and causes toxoplasmosis.At present,the double positive of anti-T.gondii Ig M and Ig G in serum is the main way to diagnose acute toxoplasmosis.However,the high false positive of Ig M makes the detection have certain limitations.Circulating antigens(CAgs)has been proved to be a reliable diagnostic indicators of acute infection and contains a large number of reliable vaccine development targets.However,the literature has limited studies on CAgs.Exploring the diagnostic components of CAgs is of great significance to improve the accuracy of acute toxoplasmosis detection,especially the current therapeutic drugs are only effective for acute toxoplasmosis.In this study,LC-MS/MS was used to identify CAgs in the serum of mice and piglets with acute T.gondii infection,screened proteins with diagnostic value,and identified possible virulence factors through CRISPR/CAS9 gene editing technology,so as to provide a theoretical basis for improving the accuracy of acute toxoplasmosis detection and the target of vaccine development.Identification of CAgs in serum of infection models:ESA(excretory secretory antigen),the main component of CAgs,was collected and immunized to mice and rabbits to obtain mouse and rabbit ESA multi-antibodies.Then,a double antibody sandwich ELISA was established to detect CAgs;Mice(1×10~7/piece)and piglets(6×10~8/head)acute infection models were established by intraperitoneal inoculation of tachyzoites.The results of sandwich ELISA and Nest-PCR showed that CAgs in serum was consistent with the emergence time of the parasites in blood,and disappeared with the disappearance of parasites.Then,mouse ESA polyclonal antibody was mixed with infected serums for immunoprecipitation,and a total of 31 CAgs or 28 CAgs with high reliability were identified in the serum of infected mice or infected piglets;In addition,ESA was subjected to WB by two-dimensional electrophoresis(2DE)with the serum of infected piglets for 18 days as the primary antibody.A total of 8matching gel spots were obtained,of which 5 proteins were identified in both methods.Subcellular localization and immunoprotected effect of some new CAgs:Six novel CAgs were selected for prokaryotic expression,of which four were inclusion body expression(r TgRuv BL1,r TgRnase H1,r TgRPS2 and r TgDPM),and two were soluble expression(r TgZFLP3 and r TgRPL7),and immunized mice to obtain polyclonal antibodies,respectively;ELISA and IFA showed that the six multi-antibodies had excellent reactivity with ESA and tachyzoites.Moreover,these six CAgs were localized in different subcellular compartments within T.gondii tachyzoites,including the nucleus(TgRuv BL1),the apical end(TgRNase H1and TgRPS2),the membrane surface(TgDPM and TgRPL7),and the apical(TgZFLP3);WB results further confirmed that TgRuv BL1,TgRNase H1 and TgRPS2 existed in the serum of infected mice,and TgDPM,TgRPL7 and TgZFLP3 existed in the serum of infected piglets;Indirect ELISA showed that the levels of TgZFLP3 in the sera of the infected piglets increased remarkably at days 2–12 after infection(P<0.05 or P<0.01),indicating that that TgZFLP3 has the potential to be used as a target for the detection of acute toxoplasmosis in piglets;In addition,the immune protection experiment showed that these six proteins may not be useful as candidate vaccine against toxoplasmosis.Diagnostic value of TgSAG1 and TgHSP70 in toxoplasmosis:By comparing and analyzing the serum CAgs of infected mice,piglets and dogs.A total of 18 CAgs appeared in the three infected animals,and 8 had high reliability;TgHSP70 and TgSAG1 were selected for prokaryotic expression(both soluble expression)to prepare mouse polyclonal antibody;WB results showed that the sera of infected piglets contained specific anti-r TgHSP70 and anti-r TgSAG1 Ig G;Indirect ELISA showed that both of them could describe the changes of antibodies in the sera of infected piglets;However,the subsequent yin-yang coincidence rate experiment showed that the yin-yang coincidence rate of r TgHSP70 was low,indicating that TgHSP70 was not suitable for the detection of chronic toxoplasmosis;Further experiments showed that TgHSP70 and TgSAG1 existed in the sera of infected dogs,mice and piglets;ELISA results showed that the changes of TgHSP70 and TgSAG1 in the sera of infected piglets were consistent with the results of Nest-PCR,proved that TgHSP70 and TgSAG1 have the potential to be used as serum diagnostic markers of acute toxoplasmosis in different animals.Construction and phenotypic experiment of RHΔSRS55M,RHΔOR,RHΔZFLP3 and RHΔTKL1strains:CRISPR/CAS9 gene editing technology was used to construct the single gene deletion strains of the selected possible virulence factors TgSRS55m,TgOR,TgZFLP3 and TgTKL1;Simultaneously,the prokaryotic expression of TgSRS55m,TgOR and TgTKL1 were carried out to prepare mouse polyclonal antibodies to verify the deletion strains;PCR,IFA or WB results showed that RHΔSRS55M,RHΔOR,RHΔZFLP3 and RHΔTKL1 strains were successfully constructed;Phenotypic and virulence experiment showed that RHΔSRS55M strain had no phenotypic and virulence defects;RHΔZFLP3strain had no phenotypic defects,but the virulence decreased;The invasion and proliferation ability of RHΔOR strain were decreased,and the virulence decreased significantly;The invasion ability of RHΔTKL1 strain was significantly deficient,and its virulence to mice was completely lost;In addition,the sensitivity of RHΔOR strain to hydrogen peroxide increased,the level of ROS and MDA in RHΔOR strain were increased significantly(P<0.01),and the total antioxidant capacity decreased,suggesting that TgOR may be involved in T.gondii resistance to host oxidative damage.Immune protection of RHΔTKL1 strain and exploration of TgTKL1 interaction protein:WB and ELISA showed that the serum of mice inoculated with RHΔTKL1 strain(10~3)contained a large amount of specific anti TLA Ig G;Further detection of Ig G subtypes showed that the serum of mice contained a large number of specific anti-TLA Ig G1 and Ig G2a,which involved predominant Ig G2a production,suggesting a slight bias toward a Th1-type response;Flow cytometry showed that CD3~+,CD4~+and CD8~+T cells in spleen of mice inoculated with RHΔTKL1 strain increased significantly(P<0.05),and the proportion of CD8~+T cells also increased significantly(P<0.05);The results of ELISA showed that splenocytes stimulated by TLA produced a large number of IL-2,IL-12 and IFN-γ(P<0.01);Moreover,all the surviving mice survived after re-inoculation with RH strain,indicating that RHΔTKL1strain has the potential to be used as a attenuated vaccine;Using Turbo ID proximity labeling technology to construct the TKL1-TUR stain to identify TgTKL1 interaction proteins,and a total of 51 potential interaction proteins were identified,and most of them were involved in important biological processes,such as protein deubiquitylation;Further,HA tag was labeled on the tail of TgUCTH gene(potential interaction protein)base on TKL1-TUR strain to construct TKL1-UCTH stain;IFA showed that TgTKL1 and TgUCTH were co-located in the nucleus,and CO-IP test showed that there was an interaction relationship between them.To sum up,this study confirmed that larger difference of CAgs appeared in the serum of different animals after acute infection with T.gondii,and TgSAG1 and TgHSP70 have potential as serum diagnostic markers of acute toxoplasmosis in different animals,and RHΔTKL1 strain has the potential to be used as T.gondii attenuated strain vaccine,which provides a strong theoretical basis for the development of acute T.gondii diagnostic kit and T.gondii attenuated strain vaccine. |
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