An in vitro merozoite induction model of Toxoplasma gondii and its construction method

Abstract

This invention belongs to the field of biomedical technology and discloses an in vitro merozoite induction model of *Toxoplasma gondii* and its construction method. This invention discloses the application of glutamine antagonists in inducing the differentiation of *Toxoplasma gondii* tachyzoites into merozoites or in preparing products that induce this differentiation. This invention is the first to discover that the glutamine antagonist JHU083 can induce the differentiation of tachyzoites of the highly virulent *Toxoplasma gondii* strain RH and the attenuated strain ME49 into merozoites under in vitro conditions. This provides a reliable in vitro model for studying the key initial stage of sexual reproduction in *Toxoplasma gondii* and lays an irreplaceable research foundation for targeting and blocking key links in parasite transmission.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to an in vitro merozoite induction model of Toxoplasma gondii and its construction method. Background Technology

[0002] Toxoplasma gondii is a widely distributed obligate intracellular parasite that can infect almost all warm-blooded animals, including humans. While most healthy individuals are asymptomatic after infection, in immunocompromised populations (such as HIV patients and organ transplant recipients), Toxoplasma gondii infection can cause serious illnesses such as encephalitis and retinochoroiditis, posing a serious threat to human health. The life cycle of Toxoplasma gondii includes various morphologies, including tachyzoites, bradyzoites, merozoites, and oocysts. Tachyzoites are the primary form causing acute infection, while bradyzoites are associated with chronic infection. The merozoon stage is a crucial phase in its life cycle, occurring in the intestinal tissue of the definitive host, the feline. The formation of merozoites marks the beginning of sexual reproduction; therefore, its differentiation process is essential for understanding the life cycle and pathogenic mechanisms of Toxoplasma gondii. Currently, the mechanism by which Toxoplasma gondii differentiates into merozoites in the small intestinal epithelial cells of felines remains unclear.

[0003] Given that sexual reproduction and merozoon differentiation in felines are the core links in the transmission of Toxoplasma gondii, in vivo studies are extremely difficult, resulting in a lack of understanding of the molecular mechanisms of this process and a severe lack of intervention strategies that can effectively block the transmission of the parasite.

[0004] Currently, research on Toxoplasma gondii mainly focuses on its infection mechanisms, immune responses, and drug treatments, while studies on the in vitro induction and differentiation mechanisms of Toxoplasma gondii merozoites are relatively limited. Due to the complexity and uncontrollability of the in vivo environment, directly studying the differentiation process of Toxoplasma gondii merozoites in felines presents numerous challenges. Therefore, establishing an in vitro merozoite induction model of Toxoplasma gondii to simulate the in vivo environment and to deeply study its differentiation mechanisms is of great significance for revealing the life cycle and pathogenic mechanisms of Toxoplasma gondii, as well as developing new intervention strategies. Summary of the Invention

[0005] This invention aims to at least solve one of the technical problems existing in the prior art. It provides a method for converting Toxoplasma gondii tachyzoites into merozoites in vitro using the glutamine antagonist JHU083, which is of great value for elucidating the mechanism of Toxoplasma gondii differentiation into merozoites in the small intestinal epithelial cells of felines and for researching drugs against Toxoplasma gondii transmission.

[0006] The first aspect of this invention aims to provide the use of glutamine antagonists in inducing the differentiation of Toxoplasma gondii tachyzoites into merozoites or in preparing products that induce the differentiation of Toxoplasma gondii tachyzoites into merozoites.

[0007] The second objective of this invention is to provide a method for inducing the differentiation of Toxoplasma gondii tachyzoites into merozoites.

[0008] The third objective of this invention is to provide an in vitro induced differentiation model for Toxoplasma gondii merozoites.

[0009] The fourth aspect of this invention aims to provide the application of the method of the second aspect of this invention or the in vitro induced differentiation model of Toxoplasma gondii merozoites of the third aspect of this invention.

[0010] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0011] In a first aspect, the invention provides the use of a glutamine antagonist in inducing the differentiation of Toxoplasma gondii tachyzoites into merozoites or in the preparation of products that induce the differentiation of Toxoplasma gondii tachyzoites into merozoites.

[0012] In some embodiments of the present invention, the glutamine antagonist is JHU083, CAS No: 1998725-11-3.

[0013] In some embodiments of the present invention, the effective dose of JHU083 in inducing differentiation of Toxoplasma gondii tachyzoites into merozoites is 1-20 μM, such as any value or a range formed by any two of the following: 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, 10.5, 11, 11.5, 12, 12.5, 13, 13.5, 14, 14.5, 15, 15.5, 16, 16.5, 17, 17.5, 18, 18.5, 19, 19.5, and 20 μM.

[0014] In some embodiments of the present invention, the product includes reagents, kits, or drugs.

[0015] A second aspect of the present invention provides a method for inducing the differentiation of Toxoplasma gondii tachyzoites into merozoites, comprising the step of treating Toxoplasma gondii with a glutamine antagonist, wherein the glutamine antagonist is JHU083.

[0016] In some embodiments of the present invention, the method specifically includes the following steps:

[0017] Co-culturing Toxoplasma gondii with host cells allows Toxoplasma gondii to invade the host cells.

[0018] Pre-culture of Toxoplasma gondii that has already invaded host cells;

[0019] JHU083 was co-cultured with pre-cultured Toxoplasma gondii.

[0020] In some embodiments of the present invention, the final concentration of JHU083 in the co-culture system is 1-20 μM.

[0021] In some preferred embodiments of the present invention, the final concentration of JHU083 in the co-culture system is 2-15 μM, such as any value or a range formed by any two of the following: 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, 10.5, 11, 11.5, 12, 12.5, 13, 13.5, 14, 14.5, and 15 μM.

[0022] In some embodiments of the present invention, the Toxoplasma gondii is Toxoplasma glabrata, including the RH strain (a highly virulent Type I strain) and the ME49 strain (a weakly virulent Type II strain).

[0023] In some embodiments of the present invention, the JHU083 and the pre-cultured Toxoplasma gondii are co-cultured for 20-60 hours.

[0024] In some preferred embodiments of the present invention, the co-culture time of JHU083 and the pre-cultured Toxoplasma gondii is 20-50 h, such as any value or a range formed by any two of 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48 and 50 h.

[0025] In some embodiments of the present invention, the pH value in the co-culture system of JHU083 and pre-cultured Toxoplasma gondii is 6.5-8.5.

[0026] In some preferred embodiments of the present invention, in the co-culture system of JHU083 and pre-cultured Toxoplasma gondii, the pH value is 7-8.5, such as any one of 7, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8, 8.1, 8.2, 8.4, and 8.5, or a range formed by any two of them.

[0027] In some embodiments of the present invention, the pre-culture time is 8-55 hours.

[0028] In some preferred embodiments of the present invention, the pre-culture time is 10-50 h, such as any value or a range formed by any two of the following: 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48 and 50 h.

[0029] Depending on the strain of *Toxoplasma gondii*, different pre-culture and co-culture times were selected. When *Toxoplasma gondii* was an RH strain, the pre-culture time was 10-15 hours, and the co-culture time between JHU083 and the pre-cultured *Toxoplasma gondii* was 20-30 hours. When *Toxoplasma gondii* was an ME49 strain, the pre-culture time was 45-50 hours, and the co-culture time between JHU083 and the pre-cultured *Toxoplasma gondii* was 40-50 hours.

[0030] In some embodiments of the present invention, the MOI between the Toxoplasma gondii and the host cell is 0.5-2.5.

[0031] In some preferred embodiments of the present invention, the MOI of the Toxoplasma gondii with the host cell is 0.5-2, such as any value or a range formed by any two of 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9 and 2.

[0032] In some embodiments of the present invention, the host cell is a nucleated cell.

[0033] In some embodiments of the present invention, the host cell includes at least one of the following: immune system cells (such as macrophages, dendritic monocytes, neutrophils, T lymphocytes, etc.), central nervous system cells (such as neurons, astrocytes, microglia, etc.), muscle cells (such as skeletal muscle cells and cardiomyocytes, etc.), epithelial cells and endothelial cells (such as intestinal epithelial cells, vascular endothelial cells, etc.), normal human skin cell lines (such as human skin fibroblasts, foreskin fibroblasts, etc.), and other parenchymal cells (such as stem cells, lung cells, retinal cells, and placental trophoblast cells, etc.).

[0034] In some embodiments of the present invention, the conditions for co-culturing the Toxoplasma gondii with the host cells are 30-40°C for 0.5-2 hours.

[0035] In some preferred embodiments of the present invention, the conditions for co-culturing Toxoplasma gondii with the host cells are 35-40°C (such as any value of 35, 36, 37, 38, 39 and 40°C or any range formed by both) for 0.5-1.5 h (such as any value of 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4 and 1.5 h or any range formed by both).

[0036] In some embodiments of the present invention, the Toxoplasma gondii includes Toxoplasma gondii with a specific marker.

[0037] To facilitate precise tracking and identification of the schizont development process, gene editing technologies (such as CRISPR / Cas9) can be used to construct Toxoplasma gondii with specific markers.

[0038] In some embodiments of the present invention, the specific marker includes gra11b , gra80 and imc7 At least one of them.

[0039] Given that sexual reproduction and merozoite differentiation in felines are the core processes of Toxoplasma gondii's transmission, in vivo studies are extremely difficult, leading to a lack of clarity regarding the molecular mechanisms of this process and a severe shortage of effective intervention strategies to block parasite transmission. This invention, for the first time, successfully induces an in vitro model of tachyzoite differentiation into merozoites using glutamine antagonism against JHU083. This model not only provides an irreplaceable technical platform for elucidating the molecular mechanisms of the initiation of sexual reproduction in Toxoplasma gondii, but also offers a reliable model for developing innovative drugs targeting the key stage of parasite transmission—merozoite formation.

[0040] A third aspect of the present invention provides an in vitro induced differentiation model of Toxoplasma gondii merozoites, which is constructed by the method of the second aspect of the present invention.

[0041] A fourth aspect of the present invention provides the application of the method of the second aspect of the present invention or the Toxoplasma gondii merozoite in vitro induced differentiation model of the third aspect of the present invention in (1) or (2):

[0042] (1) Screening for drugs to prevent or treat toxoplasmosis infection;

[0043] (2) Study the molecular mechanisms of disease occurrence and development.

[0044] The beneficial effects of this invention are:

[0045] This invention is the first to discover that the glutamine antagonist JHU083 can induce the differentiation of tachyzoites of Toxoplasma gondii RH strain and ME49 strain into merozoites under in vitro conditions. This provides a reliable in vitro model for studying the key initial stage of sexual reproduction of Toxoplasma gondii and lays an irreplaceable research foundation for targeting and blocking key links in the transmission of the parasite.

[0046] This invention is the first to utilize the glutamine antagonist JHU083 to establish an in vitro induced differentiation model of Toxoplasma gondii merozoites that does not depend on a feline host. It is the first to achieve efficient in vitro induction of Toxoplasma gondii merozoites, filling a long-standing technical gap in this research field and providing an irreplaceable platform for elucidating the formation of the key initial stage of sexual reproduction in Toxoplasma gondii.

[0047] By combining indirect immunofluorescence and transmission electron microscopy, the successful induction of merozoites was verified at both the protein localization and ultrastructural levels. The in vitro merozoite differentiation model provided by this invention can not only be used to study the molecular mechanisms of merozoite differentiation, but also provides an effective tool for screening antiparasitic drugs targeting the key initiation stages of Toxoplasma gondii sexual reproduction. Attached Figure Description

[0048] The present invention will be further described below with reference to the accompanying drawings and embodiments, wherein:

[0049] Figure 1 Illustration of tag insertion and identification strategies.

[0050] Figure 2 The images show merozoites observed by indirect immunofluorescence after 48 hours of induction with 10 μM JHU083 in strain M4; A represents endobronchial budding of strain ME49 after 48 hours of induction with 10 μM JHU083, with a scale bar of 10 μm. B represents the expression of GRA80 in strain ME49 as detected by indirect immunofluorescence, with a scale bar of 10 μm.

[0051] Figure 3 Mature schizonts observed by indirect immunofluorescence 48 h after JHU083 induction of RH strain and ME49 strain. Scale bar is 10 μm.

[0052] Figure 4 Polyploid schizonts observed by indirect immunofluorescence after RH strains were induced with JHU083 for 48 h; where A represents polyploid schizonts observed after treatment with 10 μM JHU083, with a scale bar of 10 μm. B represents polyploid schizonts co-stained with merozoite markers, with a scale bar of 10 μm.

[0053] Figure 5 Brachyzoites and merozoites observed by indirect immunofluorescence 48 h after induction of ME49 strain by JHU083. Scale bar is 10 μm.

[0054] Figure 6 To detect the expression of GRA11B in ME49 under different conditions, the scale bar was 10 μm.

[0055] Figure 7 The transformation rate of merozoites induced by ME49 strain under different induction conditions is shown.

[0056] Figure 8 To detect the expression of GRA11B and IMC1 in ME49 cells by immunofluorescence under pH 7.4 culture conditions, the scale bar is 10 μm.

[0057] Figure 9The expression of GRA11B and IMC1 in ME49 was detected by immunofluorescence under pH 8.2 culture conditions, with a scale bar of 10 μm.

[0058] Figure 10 To detect the expression of GRA11B and IMC1 in ME49 cells during continuous culture at pH 8.2 using immunofluorescence, the scale bar was 10 μm.

[0059] Figure 11 To observe the morphology of ME49 and RH strains after treatment with 10 μM JHU083 using transmission electron microscopy; where A represents endogenous budding in the vesicles of ME49 strain (scale bar: 1 μm), B represents multinucleated mother cells of ME49 strain, C represents endogenous budding in the vesicles of RH strain, and DE represents multinucleated mother cells of RH strain (scale bar: 500 nm). Detailed Implementation

[0060] The following will describe the concept and technical effects of the present invention clearly and completely with reference to embodiments, so as to fully understand the purpose, features and effects of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. Other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are all within the scope of protection of the present invention.

[0061] Unless otherwise specified in the examples, the procedures should be performed under standard conditions or conditions recommended by the manufacturer. Reagents or instruments whose manufacturers are not specified are all commercially available products.

[0062] First, using CRISPR / Cas9 gene editing technology, a series of gene marker strains for tracking schizont development were successfully constructed, including RH / ME49- gra11b -HA、RH / ME49- gra80 -HA and RH / ME49- imc7 -HA; its construction process includes designing and constructing sgRNA plasmids (pSAG1::CAS9-U6::) targeting the gene. sggra11b / gra80 / imc7 (etc.) and homologous recombination templates, and import the editing system into RHΔ via electroporation. ku80 / ME49Δ ku80The parasite strains were selected and screened for drugs and sorted into single-clone strains. The correct recombinant tagged strains were verified by specific PCR. Subsequently, an in vitro induction model was established based on this: the tagged strains were infected into host cells at a specific multiple of infection (MOI=1), and after different culture times (12h for RH strain, 48h for ME49 strain), the cells were induced with complete medium containing a series of concentrations of JHU083 (0, 2.5, 5, 10 μM) and cultured for a further specific time (24h for RH strain, 48h for ME49 strain). Indirect immunofluorescence was used to label the parasites with anti-HA and anti-IMC1, GAP45 antibodies, and the specific proteins of merozoites in the vacuoles were statistically analyzed. gra11b The proportion of positive signals is used to calculate the merozoite transformation rate; by comparing the transformation rates under different conditions, the optimal induction conditions are determined, thereby establishing the most efficient in vitro induced differentiation model. Furthermore, this method includes morphological verification of the induced merozoites using transmission electron microscopy. This invention provides a key technical platform and experimental tools for studying the developmental biology of the sexual reproduction initiation stage of Toxoplasma gondii.

[0063] The features and performance of the present invention will be further described in detail below with reference to embodiments.

[0064] Example 1: Construction of a gene-edited Toxoplasma gondii strain using CRISPR / Cas9 technology

[0065] The only recognized biomarker for Toxoplasma gondii merozoites to date. gra11b It was used to track the schizogony and development of Toxoplasma gondii in the intestines of infected cats. gra80 It was also used to identify mature schizonts. To this end, the inventors first constructed RH- gra11b -HA、ME49- gra11b -HA、RH- gra80 -HA and ME49- gra80 -HA-tagged strains. Toxoplasma gondii endometrial complexes. IMC7 It is commonly used as a marker for daughter cell formation during Toxoplasma gondii division, and therefore RH- imc7 -HA and ME49- imc7 -HA monoclonal tagged strains were used to observe the effect of the glutamine antagonist JHU083 (CAS No: 1998725-11-3) on the treatment of Toxoplasma gondii (Gnaphalium affine), Toxoplasma gondii ) The schizotypal proliferation of Toxoplasma gondii.

[0066] The specific experiment is as follows:

[0067] 1. Construction of tagged insect strains

[0068] (1) CRISPR / CAS9 system plasmid pSAG1::CAS9-U6:: sggra11b / gra80 / imc7 Construction

[0069] Using the pSAG1::CAS9-U6::sgUPRT plasmid (https: / / www.addgene.org / 54467 / ) as a template, the gRNA of the UPRT gene was replaced with [missing information - likely a specific gene or component] using the TransGen Biotech Seamless Cloning and Assembly Kit (catalog number: CU201-02). gra11b The specific steps for manipulating gRNA in genes are as follows:

[0070] 1) Fragment amplification

[0071] The CRISPR / CAS9 backbone is amplified in three segments, and its primer sequences are as follows:

[0072] Skeleton 1 fragment:

[0073] Upstream primer: Backbone-1F: 5'-GTTTTAGAGCTAGAAATAGCAAG-3' (SEQ ID NO:1);

[0074] Downstream primer: Backbone-1R: 5'-CCTTCTAGACGCATGGTAAA-3' (SEQ ID NO:2);

[0075] Skeleton 2 fragment:

[0076] Upstream primer: Backbone-2F: 5'-TTTACCATGCGTCTAGAAGGT-3' (SEQ ID NO:3);

[0077] Downstream primer: Backbone-2R: 5'-CTGTCTTGTGTATTGACCCATGT-3' (SEQ ID NO:4);

[0078] sgRNA backbone fragment 3:

[0079] Upstream primer: gra11b-sgRNA-F: 5'-CCACATGGGTCAATACACAAGACAGCTA-3' (SEQ ID NO:5);

[0080] Downstream primer:

[0081] gra11b-sgRNA-R: 5'-TTCTAGCTCTAAAACGAAGCCTGCCTGAATTGATCAACTTGACATCC C-3' (SEQ ID NO: 6);

[0082] gra80-sgRNA-R: 5'-TTCTAGCTCTAAAACTCGCCTCTCGCTGCGCGAACAACTT GACATCCC-3' (SEQ ID NO: 7);

[0083] imc7-sgRNA-R: 5'-TTCTAGCTCTAAAACagaggatggtgacggtgatgAACTTGACA TCCC-3' (SEQ ID NO: 8);

[0084] All the above DNA fragments were amplified using Phanta high-fidelity DNA polymerase (2 × Phanta Flash Master Mix (Dye Plus)) from Vazyme. gra11b / gra80 / imc7 The PCR (Polymerase Chain Reaction) system for -sgRNA is shown in Table 1, and the PCR reaction parameters are shown in Table 2. After the PCR reaction, the target fragment was recovered by gel extraction. The DNA fragments were purified and recovered using the Quick Gel Extraction Kit (catalog number: EG101-01) from TransGen Biotech.

[0085] Table 1 gra11b / gra80 / imc7 PCR reaction system for -sgRNA

[0086]

[0087] Table 2 PCR reaction parameters

[0088]

[0089] 2) Fragment connection

[0090] The purified DNA product was ligated using the TransGen Biotech Seamless Cloning and Assembly Kit. The reaction system is shown in Table 3.

[0091] Table 3. Reaction systems with seamless multi-segment connections

[0092]

[0093] Gently mix the reaction mixture and react at 50°C for 15 min. After the reaction, cool on ice. Transfer the product to 50 μL of Trans1-T1 competent cells (TransGen Biotech, Trans1-T1 Phage-Resistant Chemically Competent Cell), gently mix, incubate on ice for 30 min, heat shock in a 42°C water bath for 45 s, and then immediately transfer to ice for 2 min. Add 500 μL of LB medium and incubate at 37°C on a shaker at 250 rpm for 1 h. Spread 200 μL evenly on an ampicillin-resistant plate. After 14-16 h, pick 4 single colonies from the solid culture plate and expand them in ampicillin liquid medium. Mix 300 μL of the bacterial culture with 500 μL of 80% glycerol at a 1:1 ratio and store at -25°C for later use. Extract plasmids from the remaining bacterial culture and send it to Qingke Biotechnology Co., Ltd. for sequencing. Sequencing primers were used, with one reaction each from universal primers M13F and M13R. Sequencing results showed the target sequence was... gra11b / gra80 / imc7 The sgRNA sequence was correct and there were no errors at the plasmid ligation site, indicating successful plasmid construction, i.e., pSAG1::CAS9-U6:: sggra11b / gra80 / imc7 Plasmid.

[0094] 3) pSAG1::CAS9-U6:: sggra11b / gra80 / imc7 plasmid extraction

[0095] Using the Kangwei Bio Endotoxin Removal and Plasmid Extraction Kit (Catalog No.: CW2105S), the specific steps are as follows: ① Collect the precipitated bacteria, add 500 μL of P1 Buffer (with added RNase A) to fully resuspend, and transfer to a 2 mL centrifuge tube; ② Add 500 μL of P2 Buffer, gently invert to mix, and incubate at room temperature for 3-5 min; ③ Add 500 μL of P3 Buffer, invert to mix, and centrifuge at 13000g for 5 min until a white flocculent precipitate is visible; ④ Transfer the supernatant to a filter column and centrifuge at 13000g for 1 min; ⑤ Transfer the filtrate to a new centrifuge tube, add 450 μL of isopropanol, and vortex to mix; ⑥ Add 200 μL of PS Buffer to the binding column, centrifuge at 13000g for 1 min, and discard the filtrate; ⑦ Add the mixture from step ⑤ to the binding column, with a maximum volume of 750 μL each time, and discard the filtrate; repeat step ⑦ until all the mixture has been transferred; ⑧ Add 600 μL of PW... Buffer, centrifuge at 13000g for 1 min, discard the filtrate; ⑨ Repeat step ⑧; ⑩ Centrifuge in an empty tube at 13000g for 2 min; Transfer the binding column to a 1.5 mL EP tube, open the cap to evaporate the alcohol for 1 min, add 80-100 μL of preheated ddH2O to the center of the column, incubate for 2 min, and then centrifuge at 13000g for 1 min. Measure the concentration and store at -20℃ for long-term storage.

[0096] (2) 5'UTR- gra11b / gra80 / imc7 Preparation of HA-DHFR-3'UTR homologous template

[0097] Using the HA-DHFR (nucleotide sequence as shown in SEQ ID NO:28) preserved in the laboratory as a template, the corresponding DNA fragments were amplified using the following primer sequences.

[0098] ME49 / RH -gra11b -HA-DHFR-F: 5'-GATCCCGAAACAAACTTGTTAGAGGAAGAGGACGAGTTGAAGCCAtacccttacgatgtaccgg-3' (SEQ ID NO: 9);

[0099] MR49 / RH-DHFR- gra11b -3'UTR-R: 5'-CCGACCAGGCTGAGAGCGCACCTGCTC CTGCGGCTGTGTCTCGtCctgcaaGtgcatagaagg-3' (SEQ ID NO: 10);

[0100] ME49- gra80 -HA-DHFR-F: 5'-CCTCTCGGCCCCCGTGGGCTCCGGGAGCAGGGCCCGAGAACtacccttacgatgta-3' (SEQ ID NO: 11);

[0101] ME49- gra80 -HA-DHFR-R: 5'-GAAGTCAAGCAACGGGCGCGACTCGTCATTTCTTCTGAACCGtCctgcaaGtgcat-3' (SEQ ID NO: 12);

[0102] RH- gra80- HA-DHFR-F: 5'-CCTCTCTGCCCCCGTGGGCTCCGGGAGCAGGGCCCGAGAACtacccttacgatgta-3' (SEQ ID NO: 13);

[0103] RH- gra80-HA-DHFR-R: 5'-GAAGTCAAGCAACGGGCGCGACTCGTCATTTCTTCTGAACCGtCctgcaaGtgcat-3' (SEQ ID NO: 12);

[0104] MR49 / RH- imc7 -HA-DHFR-F: 5'-GCTCAACGCGGAGGAACAGACCACCGCTGTCCCAATCGCTGCGtacccttacgatgtaccgg-3' (SEQ ID NO: 14);

[0105] MR49 / RH-DHFR- imc7 -3'UTR-R: 5'-ctcttattgggagtccccactgtccccacacttctctgtctccgtCctg caaGtgcatagaagg-3' (SEQ ID NO: 15);

[0106] All the above DNA fragments were amplified using Phanta high-fidelity DNA polymerase (2 × Phanta Flash Master Mix (Dye Plus)) from Vazyme, 5'UTR- gra11b / gra80 / imc7 The PCR reaction system for the HA-DHFR-3'UTR homologous template is shown in Table 4, and the PCR reaction parameters are shown in Table 5. After the PCR reaction, the target fragment was recovered by gel extraction. The DNA fragments were purified and recovered using the Quick Gel Extraction Kit from TransGen Biotech.

[0107] Table 4 PCR reaction system

[0108]

[0109] Table 5 PCR reaction parameters

[0110]

[0111] (3) Electroporation

[0112] ① From RHΔ in good growth condition ku80 / ME49Δ ku80 Cells were scraped from a T25 culture flask (preserved from the Department of Pathogenic Biology, Southern Medical University) using a cell scraper. The collected suspension was repeatedly aspirated 6–7 times with a 5 mL syringe to achieve sufficient mechanical lysis and release of the parasites. Approximately 107 Toxoplasma gondii were collected and transferred to a 15 mL centrifuge tube. The tube was centrifuged at 3000 rpm for 10 min.

[0113] ② After centrifugation, discard the supernatant and add 600 μL of cytomix buffer (containing 0.174 g K₂HPO₄, 0.136 g KH₂PO₄, 0.895 g KCl, 0.001665 g CaCl₂, 0.0476 g MgCl₂, 0.59575 g HEPES, 0.0074448 g EDTA·2Na·2H₂O, 0.12195 g ATP, and 0.14634 g glutathione; bring the volume to 100 mL with deionized water; adjust the pH to 7.4 with NaOH after preparation; filter through a 0.22 μm filter and aliquot for storage at -20℃) to resuspend the precipitate.

[0114] ③ Add pSAG1::CAS9-U6 ::sggra11b / gra80 / imc7 (50μg) and 5'UTR- gra11b / gra80 / imc7 The template for homologous recombination of HA-DHFR-3'UTR (12 μg) was mixed evenly and added to an electroporation cup with a diameter of 4 mm.

[0115] ④ Set the electroporation apparatus program: voltage 1700V, capacitor 25F, resistance ∞. After transfection with Toxoplasma gondii, the cells were inoculated into HFF cells and cultured. After 48 hours, the culture medium was replaced with medium containing a final concentration of 3 μM pyrimethamine.

[0116] ⑤ Screening for Monoclonal Cells: After three generations of drug screening, monoclonal cell screening is performed. A flask filled with HFF cells (T25) is digested and added to a 96-well plate (200 μL per well). The worms are collected, counted using a cell counting chamber, and diluted to 1000 tachyzoites / mL using limiting dilution. 200 μL (approximately 2 Toxoplasma gondii per well) is transferred to a pipette, and the volume is brought to 20 mL. The original culture medium in the 96-well plate is discarded, and the diluted worm solution is inoculated into each well at a rate of 200 μL.

[0117] ⑥ Culture in a cell culture incubator (37℃, 5% CO2) for 7 days. After the culture is completed, select a well with only one plaque, scrape off the cell surface thoroughly with a pipette tip, transfer the suspension to a 1.5mL EP tube, add 800μL of 3μM pyrimethamine medium, mix well by pipetting, seed 500μL into a 24-well plate for further culture, and use the remaining 500μL for genomic extraction and PCR verification.

[0118] ⑦ Extract the genome of the strain to be verified using the method mentioned above, RHΔ ku80 As a negative control, ddH2O was used as a blank control. The verification primer sequences are as follows:

[0119] gra11b-P1-F:5’-CTCGATGAGGCTCTCGAGAC-3’(SEQ ID NO:16);

[0120] gra11b-P1-R:5’-gacgactcacgggatttacagc-3’(SEQ ID NO:17);

[0121] gra11b-P2-F:5’-GGCGCGGCATACAAAGAC-3’(SEQ ID NO:18);

[0122] gra11b-P2-R:5’-GTAGACCTAAACCCTGTCGACG-3’(SEQ ID NO:19);

[0123] gra11b-P3-F:5’-CTCGATGAGGCTCTCGAGAC-3’(SEQ ID NO:16);

[0124] gra11b-P3-R:5’-GTAGACCTAAACCCTGTCGACG-3’(SEQ ID NO:19);

[0125] gra80-P1-F:5’-CTGCCCTTTACCAGGTGATC-3’(SEQ ID NO:20);

[0126] gra80-P1-R:5’-cagacagtacgcgtctactaag-3’(SEQ ID NO:21);

[0127] gra80-P2-F:5’-GTTGCACACGTCTGCAAC-3’(SEQ ID NO:22);

[0128] gra80-P2-R:5’-GTCAAGAAGCGTCGTGGT-3’(SEQ ID NO:23);

[0129] gra80-P3-F:5’-CTGCCCTTTACCAGGTGATC-3’(SEQ ID NO:20);

[0130] gra80-P3-R:5’-GTCAAGAAGCGTCGTGGT-3’(SEQ ID NO:23);

[0131] imc7-P1-F:5’-GGCAAGGAGCTTCGGAGC-3’(SEQ ID NO:24);

[0132] imc7-P1-R: 5'-ctcagtcaggcagtctccc-3' (SEQ ID NO: 25);

[0133] imc7-P2-F: 5'-cacacagtctcacctcgc-3' (SEQ ID NO: 26);

[0134] imc7-P2-R: 5'-ggagacaagcagtccacctc-3' (SEQ ID NO: 27);

[0135] imc7-P3-F: GGCAAGGAGCTTCGGAGC-3' (SEQ ID NO: 24);

[0136] imc7-P3-R: 5'-ggagacaagcagtccacctc-3' (SEQ ID NO: 27);

[0137] Tag insertion and identification strategies (the gene-edited insect strain strategies described below are similar) are as follows: Figure 1 (GOI represents the gene of interest, and in this invention refers to...) gra11b, gra80 and imc7 (Gene of Interest), PCR1, PCR2, and PCR3 represent the fragments to be validated; DHFR is the pyrimethamine drug screening tag; a strong promoter is used to initiate the transcription and translation of the drug screening tag. As shown, the knockout strain PCR1 / PCR2 showed the target band, while PCR3 showed no target band. The wild-type strain PCR1 / PCR2 showed no target band, while PCR3 showed the target band. The blank control group showed no bands. The correctly identified strain was named RH / ME49- gra11b / gra80 / imc7 -HA bug strain.

[0138] Example 2: Establishment of an efficient in vitro differentiation model of Toxoplasma gondii merozoites based on indirect immunofluorescence experiments

[0139] This embodiment demonstrates an efficient in vitro differentiation model of Toxoplasma gondii merozoites based on indirect immunofluorescence experiments, as detailed below:

[0140] (1) Cell preparation

[0141] Several 14mm cell smears were placed in 24-cell plates and divided into different concentration groups: 2.5μM glutamine antagonist JHU083, 5μM glutamine antagonist JHU083, 10μM glutamine antagonist JHU083, anhydrous ethanol, and 5μM pyrimethamine (all in anhydrous ethanol). Each group had three replicates.

[0142] Select a suitable number of HFF cells (ATCC catalog number SCRC-1041) in good growth condition (T25 culture flasks) as needed. After discarding the original culture medium, rinse each flask once with 1 mL of PBS buffer, then discard the PBS. Add 1 mL of 0.25% Trypsin-EDTA digestion solution to each flask and digest at 37°C for 2 min. After digestion, add 1 mL of Gibco DMEM medium containing 15% fetal bovine serum (FBS) (hereinafter referred to as D15 medium) to terminate the digestion reaction. Collect the cell suspension from both flasks into 15 mL centrifuge tubes and centrifuge at 800 rpm for 5 min. After centrifugation, remove the supernatant and resuspend the cell pellet in 2 mL of D15 medium. Then, transfer the entire resuspended cell pellet to a pipette pre-filled with a certain volume (1 mL per well, depending on the number of wells) of D15 medium and mix thoroughly using a 1000 μL pipette. Finally, the cell suspension was seeded into each well of a 24-well plate containing cell spreaders at a rate of 1 mL. The cells were cultured until the cell density reached 100%, at which point they were ready for subsequent experiments.

[0143] (2) Drug preparation

[0144] The 1 mM JHU083 stock solution was diluted to the corresponding concentration using anhydrous ethanol as the solvent and Gibco DMEM medium containing 1% FBS (hereinafter referred to as D1 medium). The drug-containing medium was prepared with the lights off throughout the experiment to avoid light exposure. The UT group used D1 medium diluted 1:100 with anhydrous ethanol.

[0145] (3) Toxoplasma gondii infection of cells

[0146] From RH- in good growth condition gra11b -HA strain (RH- gra80 -HA、RH- imc7 -HA strains are the same as RH- gra11b In a T25 culture flask containing the HA strain, cells were scraped off using a cell scraper. The collected suspension was repeatedly aspirated 6-7 times with a 5mL syringe to achieve thorough mechanical lysis and release the parasites. The lysate was then transferred to a 15mL centrifuge tube and centrifuged at 3000rpm for 10min. During centrifugation, approximately 100μL of the residual liquid in the original culture flask was transferred to a new HFF cell culture flask for strain preservation. A cell counting chamber was prepared and coverslips were placed. After centrifugation, the supernatant was discarded, and the pellet was resuspended in 3mL of Gibco DMEM medium containing 1% FBS (hereinafter referred to as D1 medium). 10μL of the resuspended solution was added to the edge of the coverslip on the counting chamber, and Toxoplasma gondii were counted under a microscope. Based on the counting results, 1×10⁶ cells were infected per well. 5Toxoplasma gondii (MOI = 1) concentration: Aspirate an appropriate volume of parasite suspension into a new 15 mL centrifuge tube for limiting dilution. Then, bring the total volume to a desired level using D1 medium (1 mL per well, depending on the number of wells). Remove the pre-coated 24-well plate and aspirate the remaining medium from each well using a pipette. Inoculate each well with 500 μL of the diluted parasite suspension, gently agitating the plate horizontally to ensure even distribution. Incubate at 37°C for 1 hour to allow for complete Toxoplasma gondii invasion.

[0147] One hour after Toxoplasma gondii invasion, the inoculum was removed and replaced with D1 medium for further culturing. RH- gra11b -HA、RH- gra80 -HA and RH- imc7 -HA strain cultured for 12 hours; while ME49- gra11b -HA、ME49- gra80 -HA and RH- imc7 - HA strains were cultured for 48 hours. After the culture was completed, the medium was replaced with D1 medium containing different concentrations of JHU083 (0, 2.5, 5, 10 μM) and cultured for another 24 hours (RH strain) or 48 hours (ME49 strain).

[0148] (4) Detection of merozoite markers by indirect immunofluorescence (IF) assay

[0149] After culture, the culture medium was discarded, and the cells were rinsed with pre-cooled PBS. Then, 500 μL of 4% paraformaldehyde was added, and the cells were fixed at room temperature for 15 min. After fixation, the cells were rinsed with PBS, and then permeabilized with 500 μL of 0.5% Triton X-100 (diluted with PBS) for 10 min. After permeabilization, the cells were washed three times with PBS for 3 min each time. Next, 500 μL of 10% BSA blocking buffer was added to each well, and the cells were blocked at 37°C for 1 h. After blocking, rabbit / mouse HA monoclonal antibody and mouse IMC1 polyclonal antibody / rabbit GAP45 polyclonal antibody were diluted 1:1000 using the same blocking buffer. 200 μL of the diluted primary antibody was added to each well, and the cells were incubated overnight (>8 h) at 4°C. After incubation, the primary antibody was recovered, and the cells were washed three times with PBS for 3 min each time. Subsequently, secondary antibodies containing Alexa Fluor 594-labeled goat anti-mouse IgG and Alexa Fluor 488-labeled goat anti-rabbit IgG were diluted 1:1000 using blocking buffer, with 200 μL added to each well. The mixture was incubated at 37°C in the dark for 50 min. After incubation, the secondary antibodies were discarded, and the slides were washed three times with PBS. Finally, the slides were mounted using mounting medium containing DAPI and observed under a fluorescence microscope. gra11b or gra80The presence or absence of a positive result in a vacuolar vesicle determines whether the vesicle is a schizont. This is determined by calculating the number of vacuolar vesicles (PVs) out of 100 PVs. gra11b The percentage of positive Toxoplasma gondii vesicles represents the merozoite transformation rate under the given time and drug concentration conditions. Experiments and calculations were conducted on merozoite transformation rates under different conditions (culture medium pH=7.4 and pH=8.2). The condition with the highest transformation rate in D1 culture medium at pH 7.4 containing 10 μM JHU083 is the most efficient in vitro differentiation model based on JHU083 Toxoplasma gondii merozoites under this invention.

[0150] Experimental results are as follows Figures 2-5 As shown.

[0151] Figure 2 Figure A shows the endogenous budding of ME49 strain after induction with 10 μM JHU083 for 48 h. In the figure, the green fluorescent marker is the mother cell marker IMC7, and the red fluorescent marker is the daughter cell marker IMC1. Colocalization of red and green fluorescence indicates that the vesicle undergoes endogenous budding, while incomplete colocalization of red and green fluorescence indicates that the vesicle undergoes endogenous multiple budding. Figure 2 Figure B shows the expression of GRA80 in strain ME49. In the figure, GRA80 is a schizogony-specific marker with green fluorescence, and IMC1 is a marker with red fluorescence. Among them, the green fluorescent positive vesicles are mature schizogonies, while the green fluorescent negative vesicles are not mature schizogonies.

[0152] Figure 3 Mature schizonts observed by indirect immunofluorescence 48 h after induction of RH strain and ME49 strain by JHU083 are shown. Green fluorescence indicates the mature schizont marker GRA11B, while red fluorescence indicates the maternal cell marker GAP45. Green fluorescence-positive vesicles are mature schizonts, while green fluorescence-negative vesicles are not.

[0153] Figure 4 Polyploid schizonts observed by indirect immunofluorescence in RH strains induced by JHU083 for 48 h are shown. Figure 4 In A, the green fluorescent marker is the mother cell marker. imc7 The red fluorescent marker is the daughter cell marker IMC1. UT group imc7 The complete colocalization with IMC1 indicates that the proliferation within the vesicle belongs to synchronous internal budding. Figure 4 In B, the green fluorescent marker is a merozoite-specific marker. gra80 The red fluorescent marker is the daughter cell marker IMC1.

[0154] Figure 5The image shows bradygonians and merozoites observed by indirect immunofluorescence 48 hours after JHU083 induction in ME49 strain. The red markers indicate bradygonian stage-specific markers MAG2 or BAG1, while the green markers indicate merozoite stage-specific markers GRA11B or... gra80 .

[0155] The results showed that after induction with JHU083, the endogenous budding of ME49 strain was disrupted after 48 h of induction with 10 μM JHU083, and asynchronous division occurred within the same nacelle vesicle. Figure 2 Indirect immunofluorescence assays can detect the expression of GRA80 in the ME49 strain (A). Figure 2 Indirect immunofluorescence assays can detect the expression of GRA11B in the ME49 strain (B); Figure 3 In the untreated group, no positive signals of GRA11B and GRA80 were detected. In the RH strain, after induction with 10 μM JHU083 for 48 h, neither GRA11B nor GRA80 showed positive signals. Figure 3 ) and GRA80 ( Figure 4 All showed positive signals, while no positive signals for GRA11B and GRA80 were detected in the untreated group. Furthermore, ME49 ( ) after 48 h of JHU083 treatment were detected using the bradygenesis-specific markers MAG2 and BAG1, and the merozoogenesis-specific markers GRA11B and GRA80. Figure 5 The inventors discovered that bradyzoite-specific markers and merozoite-specific markers do not appear in the same vesicle. These results indicate that JHU083 can successfully induce the transformation of RH strain and ME49 strain into merozoites under in vitro conditions outside the feline gut.

[0156] By comparing the merozoite conversion rate at different pH values, the results are as follows: Figures 6-10 As shown, the optimal transformation rate of Toxoplasma gondii merozoites was achieved at pH 7.4 after treatment with 10 μM JHU083 for 48 h.

[0157] Figure 6 The green fluorescent marker is GRA11B, a merozoogenesis-specific protein, while the red fluorescent marker is IMC1.

[0158] Example 3: Transmission electron microscopy observation of in vitro merozoites

[0159] Toxoplasma gondii strain ME49 or RH strain was inoculated into HFF cells in T25 culture flasks at an MOI of 1. After 12 h of growth for RH strain or 48 h for ME49 strain, the original culture medium was discarded, and the cells were cultured under the experimental conditions of the most efficient in vitro differentiation model of merozoites obtained in Example 2. After culture, the culture medium was discarded, 1 mL of pre-cooled PBS was added, and cells were scraped off with a cell scraper and transferred to 1.5 mL EP tubes. The cells were centrifuged at 1000 rpm for 5 min to form cell clumps of about 0.5-1 mm in height. The PBS was discarded, and 1 mL of 2.5% glutaraldehyde electron microscopy fixative at room temperature was slowly added. After fixation at room temperature for 1 h, the cells were transferred to 4°C and fixed overnight. The fixative was discarded, 1 mL of PBS was added, and the cells were incubated at 4°C. The cells were then sent to the Central Laboratory of Southern Medical University for slide preparation and observation.

[0160] The results are as follows Figure 11 As shown in the figure, PV represents the Toxoplasma gondii vesicle, HC represents the host cell, and n1-n6 represent the number of multinuclei within a single cell. Figure 11 In the DE section, the red arrow marks the dividing cell nucleus.

[0161] After treating RH and ME49 strains with 10 μM JHU083 for 48 h, distinct multinucleated schizonts were observed, with more than two nuclei contained within a single organism. This also confirmed that drug treatment disrupted internal budding, leading to internal polybudding. Transmission electron microscopy results were completely consistent with indirect immunofluorescence results, indicating that JHU083 treatment of ME49 and RH strains resulted in schizogony.

[0162] In summary, this invention achieves, for the first time, highly efficient in vitro induction of Toxoplasma gondii merozoites: Utilizing the glutamine antagonist JHU083, a novel in vitro differentiation model for Toxoplasma gondii merozoites, independent of feline hosts, was established, filling a long-standing technological gap in this research field and providing an irreplaceable platform for elucidating the formation of key initial stages of sexual reproduction in Toxoplasma gondii. A comprehensive merozoite marker and identification system was also constructed: Through CRISPR / Cas9 gene editing technology, a complete merozoite marker and identification system was successfully constructed, including... gra11b -HA、 gra80 -HA and imc7A series of specific marker strains, including HA, enabled precise tracking and identification of the schizont development process. A standardized and quantifiable in vitro induction and evaluation system was established: the optimal induction concentration of JHU083 (2.5-10 µM) and the culture time windows for different strains (RH / ME49) were determined, and a statistical method for merozoite transformation rate based on indirect immunofluorescence was established, giving the model high reproducibility and quantitative assessment capabilities. The model has the potential for multi-dimensional validation and wide application: combining indirect immunofluorescence and transmission electron microscopy, the successful induction of merozoites was verified at both the protein localization and ultrastructural levels. This model can not only be used to study the molecular mechanisms of merozoite differentiation, but also provides an effective tool for screening antiparasitic drugs targeting the key initiation stages of Toxoplasma gondii sexual reproduction.

[0163] The embodiments of the present invention have been described in detail above with reference to the accompanying drawings. However, the present invention is not limited to the above embodiments, and various changes can be made within the scope of knowledge possessed by those skilled in the art without departing from the spirit of the present invention. Furthermore, the embodiments of the present invention and the features thereof can be combined with each other unless otherwise specified.

Claims

1. Use of a glutamine antagonist, JHU083, in inducing differentiation of Toxoplasma gondii tachyzoites into bradyzoites or in preparing a product for inducing differentiation of Toxoplasma gondii tachyzoites into bradyzoites.

2. A method for inducing differentiation of Toxoplasma gondii tachyzoites into bradyzoites, comprising the step of treating Toxoplasma gondii with a glutamine antagonist, JHU083.

3. The method of claim 2, wherein, The method specifically comprises the following steps: co-culturing Toxoplasma gondii with host cells, so that the Toxoplasma gondii invades the host cells; pre-culturing the Toxoplasma gondii that has invaded the host cells; co-culturing JHU083 and the pre-cultured Toxoplasma gondii.

4. The method of claim 3, wherein, In the co-culturing system of JHU083 and the pre-cultured Toxoplasma gondii, the final concentration of JHU083 is 1-20 μM; and / or, the co-culturing time of JHU083 and the pre-cultured Toxoplasma gondii is 20-60 h; and / or, in the co-culturing system of JHU083 and the pre-cultured Toxoplasma gondii, the pH value is 6.5-8.5; and / or, the pre-culturing time is 8-55 h.

5. The method of claim 4, wherein, The MOI of Toxoplasma gondii and host cells is 0.5-2.5; and / or, the host cells are nucleated cells.

6. The method according to any one of claims 2-5, characterized in that, The Toxoplasma gondii comprises Toxoplasma gondii with specific markers.

7. The method of claim 6, wherein, The specific markers include gra11b , gra80 , imc7 at least one of.

8. An in vitro induced differentiation model of Toxoplasma gondii bradyzoites, which is constructed by the method of any one of claims 2-7.

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