| Toxoplasma gondii(T.gondii)is a specialized intracellular parasitic protozoan.Infection of T.gondii can lead to a zoonotic toxoplasmosis and will not only cause huge economic losses to animal husbandry,but also bring potential health risks to human beings.Because T.gondii has a complex life and it can develop chronic infections in most hosts,prevention or treatment is difficult.So far,the main drugs used to treat toxoplasmosis is a combination of pyrimidine and sulfadiazine.However,these drug treatments are only effective in the acute phase of infection and can ca-use serious side effects.Therefore,the development of a vaccine for T.gondii is of great importance.Pyrimidines play an important role in the proliferation of T.gondii and uridine monophosphate(UMP),as the precursor of all pyrimidines,is one of the most important nucleotides in RNA for T.gondii.Through de novo biosynthesis or salvage pathways,T.gondii can obtain sufficient UMP for reproduction.The orotate nucleoside-5-phosphate decarboxylase(OMPDC)and uracil phosphoribosyltransferase(UPRT)of T.gondii are key enzymes in the UMP de novo synthesis and salvage pathways.Knockdown OMPDC and UPRT genes can completely block the synthesis pathway of uridine monophosphate,which can be used as a gene replication-deficient vaccine candidate.In this study,we constructed RHΔompdcΔuprt strain with dual deletion of OMPDC and UPRT genes and investigated the biological characteristics and immune protection of this strain.The main points are as follows:1.Construction of T.gondii OMPDC and UPRT double knockout strainThe knockout plasmids p SAG1::CAS9-U6::sg HXGPRT and p SAG1::CAS9-U6::sg OMPDC were constructed using CRISPR technology.p SL10-6x HA-Lox P-HXLox P-T7 plasmid was used as a template to amplify the HXGPRT donor fragment with uprt and ompdc homologous arms.The knockdown of uprt and ompdc genes was performed in T.gondii RHΔku80 strain,and the gene deletion strain RHΔompdcΔuprt was successfully obtained after PCR,q RT-PCR and Western blot validation.2.Biological characterization of T.gondii OMPDC and UPRT double knockout strainsAfter obtaining the RHΔompdcΔuprt strain,the wild strain and the knockout strain were subjected to plaque assay,invasion assay,intracellular proliferation assay and virulence assay,respectively.In vitro experiments showed that without uracil and UMP,compared with wild strains,the intracellular proliferation rate of knocked out strains was significantly slowed down and no plaque was formed.However,there was no difference between its invasive ability and that of wild strains.With the addition of uracil and UMP,the RHΔompdcΔuprt strain could proliferate and form plaque normally.In vivo experiments showed that the mutant strain did not proliferate in mice and did not cause mice death.BALB/c and BALB/C-nu mice were intraperitoneally injected with different doses of mutant strain and wild strain.The mice inoculated with the wild strain died while the mice inoculated with the mutant strain survived.In addition,the same doses of mutant and wild strains were intraperitoneally injected the mice,and it was observed that the intraperitoneal parasite load of the mice inoculated with the wild strain increased day by day,and the liver and lung tissues contained a large number of parasites.While the parasite load of the mice inoculated with the mutant strain decreased day by day and was reduced to a very low amount by day 5,and the liver and lung tissues were almost free of parasite.HE staining of liver,spleen and lung tissues showed that the tissues of mice inoculated with the wild strain were severely diseased while those of mice inoculated with the mutant strain were not different from those of normal mice.After intramuscular injection of the mutant strain into cats,the absence of oocysts in the feces of cats for 10 days.In conclusion,the RHΔompdcΔuprt strain was safe in mice and cats,which laid the foundation for the subsequent use of the RHΔompdcΔuprt strain as a live attenuated vaccine.3.Immunoprotection analysis of T.gondii RHΔompdcΔuprt live attenuated vaccine in miceAfter three immunizations of BALB/c mice with the RHΔompdcΔuprt strain,the antibody potency in the sera of mice was measured by indirect ELISA.The results showed that the potency of antibodies(Ig G,Ig G1,Ig G2a)in the immunized group was significantly higher than that the control group and the Ig G2a/Ig G1 ratio was increased,suggesting that the induced immunity was a mixed immunity dominated by Th1 type.The T-cell subsets of splenic lymphocytes and the cytokine produced after STAg stimulation were subsequently measured using a flow cytometer.The results showed that the number of CD4+,CD8+ T cells and the levels of IL-2,IL-4,IL-10,IL-12,and IFN-γ were significantly higher in the immunized group than the control group.Mice were challenged with RH,ME49 and WH6 strains,all mice in the immunized group survived,while all mice in the control group died.HE staining of the tissues and organs of the immunized mice showed no lesions.In summary,the live attenuated vaccine could provide 100% protection to mice.After adoptive immunization with immunized serum and spleen cells,it was observed that the mice with adoptive immunization could prolong the survival time to a certain extent,indicating that the adoptive immunization could not provide complete protection.4.Analysis of the immune protection of T.gondii RHΔompdcΔuprt live attenuated vaccine in catsAfter two immunizations of cats with the RHΔompdcΔuprt strain,the potency of Ig G antibodies in the sera of cats were measured by indirect ELISA.The results showed that the antibody potency of the immunized group was significantly higher than that of the control group.ELISA kit was used to detect the cytokines in cat serum.The results showed that the levels of IL-2 and IFN-γ were significantly higher than those in the control group.After the cats were challenged with ME49 cysts,it was found that the control group cats started to shedding oocysts on day 3 and lasted for 11 days;the immunized group cats started to discharge oocysts on day 3 and lasted for 6 days;and the total number of oocysts shedding in the immunized group was significantly lower than that in the control group,and the rate of oocyst reduction was 95.3%.It showed that RHΔompdcΔuprt live attenuated vaccine had some protective effect on cats.In conclusion,we successfully constructed a strain of RHΔompdcΔuprt and deeply researched its biological characteristics,immune protection and immune protection mechanism in mice.We also preliminarily proved the feasibility of this strain as a vaccine for T.gondii in cats.We found that this strain has some protection against T.gondii infection in cats,laying a foundation for the development of T.gondii vaccine. |
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