Construction Of Toxoplasma Gondii Carbamoyl Phosphate Synthetase Ⅱ Deletion Strain And Research On Its Immunoprotection Efficacy

Toxoplasma gondii belongs to Apicomplexa,class Sporozoa,lives parasitically in host cells.Its reproductive mode includes asexual reproduction and sexual reproduction,with a complex life history.Both humans and animals are susceptible to T.gondii,which can cause severe toxoplasmosis when the host immunity is low.T.gondii is a natural strain with purine,ornithine and arginine deficiency.It must obtain raw materials from cells to maintain its growth and development.T.gondii can synthesize uridine independently.As a precursor of other pyrimidine nucleotide synthesis in T.gondii,uridine can be used for DNA and RNA synthesis,which plays an important role in the replication and proliferation of the parasite.The limited synthesis of uridine in T.gondii may affect the proliferation and virulence of the parasite.T.gondii uridine synthesis includes de novo synthesis pathway and remedial synthesis pathway.Carbamyl phosphate synthase Ⅱ(CPS Ⅱ)is the first key enzyme in the de novo synthesis pathway of uridine acid.It contains glutamine aminotransferase domain,ATP binding domain and other subunits,which can transfer the amino group of glutamine and ATP phosphoric acid to combine with CO2 to form carbamyl phosphate.In order to explore the effect of CPSⅡ deletion on the biological characteristics of T.gondii,the CPSⅡ gene deletion strain was constructed in this study to explore its replication and proliferation ability in vitro and in vivo and the immune effect induced in mice.According to the principle of CRISPR technology,the knockout plasmid p SAG1::Cas9-U6:: SGCPS Ⅱ was constructed.Taking ps AG1 ::Cas9-U6:: sg UPRT as the template,the sg UPRT sequence on the plasmid was replaced with the sg RNA sequence targeting CPSⅡ by point mutation.The DHFR sequence with 40 bp homologous arm was amplified by PCR using the p UPRT::DHFR-D plasmid as the template.The p SAG1::Cas9-U6:: SGCPS Ⅱ plasmid and DHFR sequence were transfected into the tachyzoites of T.gondii,and the gene knockout strains were screened by the resistant drug pyrimidine.After obtaining the ΔCPSⅡ strains,Vero cells and BALB/c mice were infected with wild-strains and ΔCPSⅡ strains,respectively,to investigate the effect of CPSⅡgene deletion on the parasite.The results in vitro and in vivo showed that the proliferation rate of ΔCPSⅡ strain was significantly lower than that of the wild type strain.The ΔCPSⅡ strain could proliferate and escape normally when the culture medium was added with uracil,and the replication rate was close to that of the wild type.Without adding uracil,the multiplication rate of knockout strains was significantly reduced.BALB/c mice were inoculated with wild-type strain andΔCPSⅡ strain.The median survival time of mice inoculated with wild-strain was 4days,and the median survival time of mice inoculated with ΔCPSⅡ strain was 10 days.The CPSⅡ knockout strain could significantly improve the immune ability and prolong the survival time of mice.At 4 and 10 days after infection,the ascites and liver tissue of mice were collected,and it was found that the proliferation of ΔCPSⅡ strain was not observed in the ascites of mice at 4 days after infection,but a small number of parasite proliferate in liver.However,the proliferation of ΔCPSⅡ strain at 10 days after infection was similar to that of the wild-strain at 4 days after infection.Therefore,the proliferation ability of ΔCPSⅡ strain was limited at the early stage of infection.The ability to proliferate is restored in the late stage of infection.The results of cytokine detection,immuno-related transcription factors expression lever detection and white blood cell count in the peripheral blood of mice 4 and 10 days after infection showed that theΔCPSⅡ mutant strain could induce the expression of IFN-γ and IL-12 in mice,which indirectly indicated that the ΔCPSⅡ strain had the ability to induce the innate and adaptive immune responses of the host,and the expression difference of transcription factors was consistent with that of cytokines.The wild-type strain and ΔCPSⅡ strain significantly increased the number of neutrophils and decreased the number of lymphocytes at 4 and 10 days after infection,respectively.At 10 days after infection,the absolute value and percentage of neutrophils and lymphocytes produced by the knockout strain were significantly different from that of the PBS control group.T.gondii infection usually elicits an effective Th1 cell response,but the results of this study showed a significant decrease in the number of host lymphocytes.In view of these results,we speculated that ΔCPSⅡ strain could induce immune response in mice at the early stage of infection,but the effect was not as strong as that of RH strain.In the late stage of infection,the ΔCPSⅡ strain recovered its ability to proliferate and proliferated in large numbers,which inhibited the host immune response,so the number of lymphocytes decreased significantly and the mice died.Uridylic acid is an important nutrient for the survival and reproduction of T.gondii.The deficiency of uridic acid seriously affects the replication,proliferation and virulence of T.gondii.In this study,it was found that the deletion of CPSⅡ gene could slow down the replication and proliferation rate of the insect strain,but the parasite did not completely lose its proliferation ability and virulence,it is possible that the uridine salvage pathway plays a role.T.gondii uridine acid synthesis mechanism has not been fully elucidated,as a result,the CPSⅡ gene deletion strains of insect biology still need further study.However,the deletion of CPSⅡ gene resulted in T.gondii uridine deficiency,which resulted in reduced proliferation and virulence of the parasite,and still had the characteristics of inducing innate immunity and cellular immunity.It is expected to be an ideal model for the development of live attenuated vaccine against T.gondii,and provide theoretical and experimental basis for the long-term and effective control of T.gondii infection.