Research On Construction And Biological Function Of PLP1 Gene Deletion Strain From Toxoplasma Gondii

Toxoplasma gondii is an obligate intracellular apicomplexan parasite,which can infect the nucleated cells of almost all warm blooded animals,including human.About a third of the world’s population is infected with T.gondii.There are many studies on the life cycle of T.gondii,but little is known about the mechanism of its escaping from host cells.A microneme protein called PLP1 plays an important role in the escape of T.gondii from host cells,which has the function of perforation and provides conditions for the rapid escape from host cells.T.gondii CDPK1 regulates the secretion of microneme proteins and plays an important role in the stages of motion,invasion and escape.In view of the lack of effective vaccine for toxoplasmosis at present,this study aimed to explore the biological function of PLP1 gene of T.gondii,establishing the foundation for its in-depth study,and provide the basis for the selection of candidate antigens of T.gondii vaccine.The following experiments were carried out.First,expression and preparation of polyclonal antibodies of PLP1 and CDPK1 proteins were carried out.Truncated PLP1 gene fragments and truncated CDPK1 gene fragments were amplified by PCR,then were connected to prokaryotic expression vector,respectively.Recombinant vectors pET-28a(+)-PLP1 and pGEX-6p-1-CDPK1 were constructed and induced to expression.pET-28a(+)-PLP1 was expressed in the form of inclusion body,and the recombinant protein was purified by KCl staining and gel cutting method,and the mouse polyclonal antibody was prepared by immunizing 6~8 weeks Kunming mouse.pGEX-6p-1-CDPK1 was expressed in soluble form,and the recombinant protein was purified by GST tag affinity chromatography,and the rabbit polyclonal antibody was prepared by immunizing 8-week New Zealand rabbit.In this study,the recombinant protein and polyclonal antibody of PLP1 gene and CDPK1 gene were successfully obtained,which laid a foundation for the study of the function of PLP1 gene.To further study the function of PLP1 gene,we successfully obtained PLP1 gene deletion strain(PLP1-KO)and PLP1 complemaentary strain(PLP1-CO)strains by CRISPR/Cas9 gene editing technology.Compared with RH strain and PLP1-CO strain,PLP1-KO strain had no significant changes invasion efficiency,and lightly affected proliferation in vitro.However,the size and number of plaque formation were significantly reduced,and the ability of escaping host cells was significantly reduced.Lethal assay in vivo showed that 50% of the mice inoculated with 1000 PLP1-KO strain tachyzoites survived.The results showed that PLP1 gene deletion can not only significantly reduce the ability of T.gondii to escape from the host cells,but also significantly reduce the virulence to mice.Finally,the causes of survival of mice infected with PLP1-KO strain was studied by detecting the serum antigen level and antibody level.In mice infected with 1000 and 100 PLP1-KO strain,serum circulating antigen of the mice reached a peak at 7th and 10 th day after infection,and the level of tachyzoite antigen of 100 PLP1-KO strain was significantly lower than that of those infected with 1000 PLP1-KO strain.Further detection of antibody levels found that the antibody levels of mice at both infection doses showed a consistent upward trend,and continued to increase within 20 days after infection.The results showed that after infection with 1000 tachyzoites,due to the large number of infected parasites,the number of parasites had reached the peak 7 days after infection.At this time,some mice died because they did not produce enough antibodies to resist T.gondii.However,in mice infected with 100 tachyzoites,due to the small infection base,the immune system of mice can inhibit the proliferation of T.gondii in vivo,and gradually eliminate T.gondii with the increase of antibody level,so its survival rate(80%)is higher than that of mice infected with 1000 PLP1-KO strain.The results showed that the lethality of PLP1 gene deficient strain to mice was closely related to the initial infection dose and the immunity of the mice.