Construction And Biological Function Research Of Toxoplasma Gondii TR And ROP18 Gene Deletion Strains

Toxoplasma gondii can parasitize in eukaryotic cell and cause toxoplasmosis.At present,there is no effective treatment for toxoplasmosis,so it is necessary to develop a specific vaccine against toxoplasmosis.For the development of new vaccines,the study of the immune evasion mechanism of T.gondii is a crucial step.The infection of T.gondii can trigger the host's natural immune response: on the one hand,excessive reactive oxygen species(ROS)in macrophages and other cells can be produced while signal transduction and transcription activator 1(STAT1)was activated;on the other hand,IRGs can be recruited to the parasitophorous vacuole membrane(PVM)by activating IRGs signaling pathway,the parasitophorous vacuole(PV)are lysed and then removed by autophagy of cells.However,T.gondii can secrete many factors to avoid immune attack: On the one hand,T.gondii can produce a variety of peroxidases to resist the damage of ROS.Previous studies in our laboratory have shown that there are a large number of thioredoxin reductase(TR)in the excretory antigen(ESA)secreted by T.gondii,which plays an important role in the escape of active oxygen damage,and this is a key that makes T.gondii survive and reproduce in the host.On the other hand,some studies have shown that ROP18 can specifically bind IRGs in host cells to prevent their accumulation on PVM,thus avoiding the exposure of T.gondii,the attack of ROS and the elimination of autophagy.Therefore,we speculate that ROP18 and TR may play a synergistic role in immune clearance of T.gondii.Based on this hypothesis,we conducted the following experiments.Firstly,the expression of ROP18 protein and the generation of polyclonal antibody were carried out.The truncated gene of ROP18 was amplified by PCR,and the recombinant plasmid pET-30a-ROP18 was constructed for protein expression.ROP18 protein was expressed as insoluble construct,which was purified by KCl staining and gel cutting method,and polyclonal antibody was prepared by immunizing female Kunming mice.Indirect immunofluorescence assay(IFA)showed that ROP18 was mainly distributed in the cytoplasm of T.gondii,and the location of the strongest fluorescence intensity is exactly where the rhoptry exist.In this study,the polyclonal antibodies and the recombinant protein ROP18 were successfully obtained,which laid a foundation for the study of the characteristics and functions of ROP18.Secondly,the ROP18 gene deletion and complementary strains were constructed and their biological functions were studied.ROP18 deletion and complementary strains were successfully obtained by using CRISPR/Cas9 gene edit technique.Compared to the RH strain,RH-ROP18-KO strain had lower invasive and proliferative ability in vitro,the survival time of mice which was injected with the RH-ROP18-KO strain was prolonged.Meanwhile,Irga6 gene of macrophages was amplified by RT-PCR,and pGEX-6p-1-Irga6 recombinant plasmid was constructed for protein expression.Polyclonal antibodies and recombinant proteins Irga6 were successfully obtained in this study,IFA assay showed that the fluorescence intensity of Irga6 gathered on the PV surface of RH-ROP18-KO strain was higher than that of RH strain,which confirmed that ROP18 could prevent the aggregation of Irga6 on PVM through phosphorylation,preventing PV from being destroyed.Finally,in order to explore whether ROP18 and TR play a synergistic role in the immune clearance process of T.gondii resistance to host,the TR gene deletion strain?the ROP18 and TR double gene deletion strain were constructed.TR gene was deleted on the basis of RH strain,and RH-TR-KO strain was screened by pyrimidine.Meanwhile,we constructed a chloromycetin-resistant ROP18 gene deletion strain by using CRISPR/Cas9 gene edit technology,then TR gene was deleted on the basis of this strain,and RH-ROP18-TR-KO strain was obtained by screening with pyrimidine.The invasion ability and proliferation rate of RH-ROP18-TR-KO strain in vitro were lower than those of RH strain and single gene deletion strain.Toxicity experiments in mice showed that mice which was injected with tachyzoite RH-ROP18-TR-KO survived longer than mice which was injected with tachyzoite that knocked out one of them alone.These studies showed that ROP18 and TR could evade host immune attack through different mechanisms with synergistic effects.