Toxoplasma gondii in-vitro merozoite induction model and construction method thereof

Abstract

The invention belongs to the technical field of biological medicine, and discloses a toxoplasma gondii in-vitro merozoite induction model and a construction method thereof. The invention discloses application of a glutamine antagonist to induction of differentiation of toxoplasma gondii tachyzoite to merozoite or preparation of a product for induction of differentiation of toxoplasma gondii tachyzoite to merozoite, and finds that the glutamine antagonist JHU083 can induce differentiation of toxoplasma gondii virulent strain RH and attenuated strain ME49 tachyzoite into merozoite under in-vitro conditions for the first time. A reliable in-vitro model is provided for researching the key initial stage of sexual reproduction of the toxoplasma gondii, and an irreplaceable research foundation is laid for the key link of targeted blocking of parasite transmission.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of biological medicine, and particularly relates to a Toxoplasma gondii in-vitro bradyzoite induction model and a construction method thereof. BACKGROUND

[0002] Toxoplasma gondii is a widely distributed obligate intracellular parasite that can infect almost all warm-blooded animals, including humans. Although most healthy people have no obvious symptoms after infection, in immunocompromised populations (such as AIDS patients, organ transplant recipients), Toxoplasma infection can cause serious diseases such as encephalitis, retinochoroiditis, etc., posing a serious threat to human health. The life cycle of Toxoplasma gondii includes bradyzoite, bradyzoite, schizont and oocyst, etc. Among them, bradyzoite is the main form causing acute infection, and bradyzoite is related to chronic infection. Toxoplasma schizont is an important stage in its life cycle, which occurs in the intestinal tissue of the cat family, and the formation of Toxoplasma schizont marks the beginning of sexual reproduction, so its differentiation process is crucial to understanding the life cycle and pathogenic mechanism of Toxoplasma gondii. The mechanism of Toxoplasma gondii differentiation into schizonts in the small intestine epithelial cells of the cat family is unknown.

[0003] Given that the sexual reproduction and schizont differentiation of Toxoplasma gondii in the cat family is a key link in its transmission, but in-vivo research is extremely difficult, leading to an unknown molecular mechanism of the process and a serious lack of intervention strategies that can effectively block the transmission of the parasite.

[0004] Currently, research on Toxoplasma gondii has mainly focused on its infection mechanism, immune response, and drug treatment, etc., while the in-vitro induction and differentiation mechanism of Toxoplasma gondii schizonts is relatively less studied. Due to the complexity and uncontrollability of the in-vivo environment, direct research on the differentiation process of Toxoplasma gondii schizonts in the cat family faces many challenges. Therefore, establishing an in-vitro Toxoplasma gondii schizont induction model to simulate the in-vivo environment and further study its differentiation mechanism is of great significance to revealing the life cycle and pathogenic mechanism of Toxoplasma gondii and developing new intervention strategies. SUMMARY

[0005] The present application aims to at least solve one of the technical problems existing in the prior art. A method for converting Toxoplasma gondii bradyzoites into schizonts in-vitro using glutamine antagonist JHU083 is provided, which is of great value for elucidating the mechanism of Toxoplasma gondii differentiation into schizonts in the small intestine epithelial cells of the cat family and for studying anti-Toxoplasma gondii transmission drugs.

[0006] The first aspect of the present application aims to provide the use of a glutamine antagonist in inducing Toxoplasma gondii bradyzoites to differentiate into schizonts or in preparing a product for inducing Toxoplasma gondii bradyzoites to differentiate into schizonts.

[0007] The second aspect of the present application aims to provide a method for inducing differentiation of Toxoplasma tachyzoite into Toxoplasma schizont.

[0008] The third aspect of the present application aims to provide an in vitro induced differentiation model of Toxoplasma schizont.

[0009] The fourth aspect of the present application aims to provide an application of the method of the second aspect of the present application or the in vitro induced differentiation model of Toxoplasma schizont of the third aspect of the present application.

[0010] To achieve the above-mentioned purposes, the technical solutions adopted by the present application are as follows: The first aspect of the present application provides an application of glutamine antagonist in inducing differentiation of Toxoplasma tachyzoite into Toxoplasma schizont or preparing a product for inducing differentiation of Toxoplasma tachyzoite into Toxoplasma schizont.

[0011] In some embodiments of the present application, the glutamine antagonist is JHU083, CAS No: 1998725-11-3.

[0012] In some embodiments of the present application, the effective dose of JHU083 in inducing differentiation of Toxoplasma tachyzoite into Toxoplasma schizont is 1-20 μM, such as any one of 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 or a range value formed by any two of them.

[0013] In some embodiments of the present application, the product includes a reagent, a kit or a drug.

[0014] The second aspect of the present application provides a method for inducing differentiation of Toxoplasma tachyzoite into Toxoplasma schizont, which comprises the step of treating Toxoplasma with glutamine antagonist, wherein the glutamine antagonist is JHU083.

[0015] In some embodiments of the present application, the method specifically comprises the following steps: co-culturing Toxoplasma with host cells to allow Toxoplasma to invade host cells; pre-culturing Toxoplasma that has invaded host cells; co-culturing JHU083 with pre-cultured Toxoplasma.

[0016] In some embodiments of the present application, the final concentration of JHU083 in the co-culturing system is 1-20 μM.

[0017] In some preferred embodiments of the application, the final concentration of JHU083 in the co-culture system is any one of, or a range formed by any two of, 2-15 μΜ, such as 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 μΜ.

[0018] In some embodiments of the application, the Toxoplasma is T. gondii, including the RH strain (Type I virulent strain) and the ME49 strain (Type II avirulent strain).

[0019] In some embodiments of the application, the co-culture of JHU083 and the pre-cultured Toxoplasma is for a period of 20-60 h.

[0020] In some preferred embodiments of the application, the co-culture of JHU083 and the pre-cultured Toxoplasma is for a period of 20-50 h, such as any one of, 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.

[0021] In some embodiments of the application, the pH in the co-culture system of JHU083 and the pre-cultured Toxoplasma is 6.5-8.5.

[0022] In some preferred embodiments of the application, the pH in the co-culture system of JHU083 and the pre-cultured Toxoplasma is 7-8.5, such as any one of, or a range formed by any two 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, 8.2, 8.4, and 8.5.

[0023] In some embodiments of the application, the pre-culturing is for a period of 8-55 h.

[0024] In some preferred embodiments of the application, the pre-culturing is for a period of 10-50 h, such as any one of, or a range formed by any two of, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, and 50 h.

[0025] According to different strains of Toxoplasma, different pre-culturing time and co-culturing time are selected. When the Toxoplasma is RH strain, the pre-culturing time is 10-15 h, and the co-culturing time of JHU083 and the pre-cultured Toxoplasma is 20-30 h. When the Toxoplasma is ME49 strain, the pre-culturing time is 45-50 h, and the co-culturing time of JHU083 and the pre-cultured Toxoplasma is 40-50 h.

[0026] In some embodiments of the present application, the MOI of the Toxoplasma to the host cell is 0.5-2.5.

[0027] In some preferred embodiments of the present application, the MOI of the Toxoplasma to the host cell is 0.5-2, such as any one 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 or a range value formed by any two of them.

[0028] In some embodiments of the present application, the host cell is a nucleated cell.

[0029] In some embodiments of the present application, the host cell comprises at least one of immune system cells (such as macrophages, dendritic cells, monocytes, neutrophils, T lymphocytes, etc.), central nervous system cells (such as neurons, astrocytes, microglia, etc.), muscle cells (such as skeletal muscle cells and cardiac muscle cells, etc.), epithelial and endothelial cells (such as intestinal epithelial cells, vascular endothelial cells, etc.), human normal 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.).

[0030] In some embodiments of the present application, the co-culturing condition of the Toxoplasma and the host cell is 30-40℃ for 0.5-2 h.

[0031] In some preferred embodiments of the present application, the co-culturing condition of the Toxoplasma and the host cell is 35-40℃ (such as any one of 35, 36, 37, 38, 39 and 40℃ or a range value formed by any two of them) for 0.5-1.5 h (such as any one 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 a range value formed by any two of them).

[0032] In some embodiments of the present application, the Toxoplasma comprises Toxoplasma with specific markers.

[0033] In order to facilitate accurate tracking and identification of the meront development process, a gene editing technique (such as CRISPR / Cas9) can be used to construct a specific marker for Toxoplasma.

[0034] In some embodiments of the present application, the specific marker comprises at least one of gra11b 、 gra80 and imc7 .

[0035] Since the sexual reproduction and schizont differentiation of Toxoplasma in felines is the core link of its transmission, but in vivo research is extremely difficult, the molecular mechanism of this process is unknown, and there is a serious lack of intervention strategies that can effectively block the transmission of the parasite. The present application first uses the glutamine antagonist JHU083 to successfully induce the in vitro model of tachyzoite differentiation into schizont, which not only provides an irreplaceable technical platform for elucidating the molecular mechanism of the initiation of sexual reproduction of Toxoplasma, but also provides a reliable model for the development of innovative drugs targeting the key link of parasite transmission-schizont formation.

[0036] In a third aspect of the present application, an in vitro induced differentiation model of Toxoplasma schizont is provided, which is constructed by the method of the second aspect of the present application.

[0037] In a fourth aspect of the present application, the method of the second aspect of the present application or the in vitro induced differentiation model of Toxoplasma schizont of the third aspect of the present application is applied in (1) or (2): (1) screening drugs for preventing or treating Toxoplasma infection; (2) studying the molecular mechanism of disease occurrence and development.

[0038] The beneficial effects of the present application are: The present application first discovers that the glutamine antagonist JHU083 can induce the tachyzoite of Toxoplasma RH strain and ME49 strain to differentiate into schizont under in vitro conditions, providing a reliable in vitro model for studying the key initiation stage of sexual reproduction of Toxoplasma, and laying an irreplaceable research foundation for targeting and blocking the key link of parasite transmission.

[0039] The present application first uses the glutamine antagonist JHU083 to establish an in vitro induced differentiation model of Toxoplasma schizont that does not depend on feline hosts, and first realizes the efficient in vitro induction of Toxoplasma schizont, which fills the long-standing technical gap in this field and provides an irreplaceable platform for analyzing the formation of the key initiation stage of sexual reproduction of Toxoplasma.

[0040] The successful induction of the meronts is verified from two aspects of protein localization and ultrastructure by combining indirect immunofluorescence with transmission electron microscopy. The in vitro induction and differentiation model of the Toxoplasma gondii meronts provided by the application can be used for researching the molecular mechanism of the meront differentiation, and also provides an effective tool for screening anti-parasite drugs targeting the key initial stage of the sexual reproduction of the Toxoplasma gondii. BRIEF DESCRIPTION OF DRAWINGS

[0041] The application will be further described below in combination with the drawings and examples, in which: Figure 1 A schematic diagram of the tag insertion and identification strategy.

[0042] Figure 2 Meronts observed by indirect immunofluorescence after 48 h of induction of the M4 strain by 10 μM JHU083; wherein, A is the internal binary budding of the ME49 strain after 48 h of induction by 10 μM JHU083, and the scale is 10 μm. B is the expression of GRA80 of the ME49 strain detected by indirect immunofluorescence, and the scale is 10 μm.

[0043] Figure 3 Mature meronts observed by indirect immunofluorescence after 48 h of induction of the RH strain and the ME49 strain by JHU083, and the scale is 10 μm.

[0044] Figure 4 Polyploid meronts observed by indirect immunofluorescence after 48 h of induction of the RH strain by JHU083; wherein, A is the polyploid meronts observed after treatment by 10 μM JHU083, and the scale is 10 μm. B is the polyploid meronts co-stained with a meront marker, and the scale is 10 μm.

[0045] Figure 5 Bradyzoites and meronts observed by indirect immunofluorescence after 48 h of induction of the ME49 strain by JHU083, and the scale is 10 μm.

[0046] Figure 6 Expression of GRA11B in the ME49 under different conditions detected by immunofluorescence, and the scale is 10 μm.

[0047] Figure 7 Meront induction conversion rates of the ME49 strain under different induction conditions.

[0048] Figure 8 Expression of GRA11B and IMC1 in the ME49 detected by immunofluorescence under the culture condition of pH 7.4, and the scale is 10 μm.

[0049] Figure 9Expression of GRA11B and IMC1 in ME49 was detected by immunofluorescence under pH 8.2 culture condition, scale bar, 10 μm.

[0050] Figure 10 Expression of GRA11B and IMC1 in ME49 was detected by immunofluorescence under pH 8.2 culture condition, scale bar, 10 μm.

[0051] Figure 11 Transmission electron microscopy observation of ME49 and RH strains treated with 10 μM JHU083, A, endopolygeny in the trophozoite of ME49 strain, scale bar, 1 μm, B, multinucleated amastigote of ME49 strain, C, endopolygeny in the trophozoite of RH strain, D-E, multinucleated amastigote of RH strain, scale bar, 500 nm. DETAILED DESCRIPTION

[0052] The concept and the technical effects of the present application will be described below in conjunction with the embodiments so as to fully understand the purpose, features and effects of the present application. Obviously, the described embodiments are only some of the embodiments of the present application, but not all the embodiments. Based on the embodiments of the present application, other embodiments obtained by those skilled in the art without creative labor are within the protection scope of the present application.

[0053] The specific conditions not mentioned in the embodiments are carried out according to the conventional conditions or the conditions recommended by the manufacturer. The reagents or instruments not mentioned by the manufacturer are all conventional products that can be purchased on the market.

[0054] Firstly, a series of gene marker strains for tracking schizogony development were successfully constructed by using CRISPR / Cas9 gene editing technology, including RH / ME49- gra11b -HA, RH / ME49- gra80 -HA and RH / ME49- imc7 -HA; the construction process includes designing and constructing sgRNA plasmid (pSAG1::CAS9-U6: sggra11b / gra80 / imc7 and homologous recombination template, the editing system was introduced into RHΔ ku80 / ME49Δ ku80The correct recombinant tagged strain was obtained by specific PCR verification after drug screening and sorting of the monoclonal strain. Subsequently, an in vitro induction model was established based thereon: the tagged strain was used to infect host cells at a specific multiplicity of infection (MOI = 1), and after being cultured for different time periods (12 h for the RH strain and 48 h for the ME49 strain), the complete culture medium containing a series of concentrations of JHU083 (0, 2.5, 5, and 10 μM) was used for induction, and the specific time periods (24 h for the RH strain and 48 h for the ME49 strain) were continued. By means of indirect immunofluorescence technology, the anti-HA and anti-IMC1 and GAP45 antibodies were used for labeling, and the conversion rate of the schizonts was calculated by means of the proportion of the positive signals; by comparing the conversion rates under different conditions, the optimal induction condition was determined, so as to establish the most efficient in vitro induction and differentiation model. In addition, the method also includes morphological verification of the schizonts induced by using a transmission electron microscope. The present application provides a key technical platform and experimental tool for the developmental biology of the initiation stage of the sexual reproduction of Toxoplasma. gra11b

[0055] The features and performances of the present application are further described in detail below in combination with embodiments.

[0056] Example 1: Construction of a gene edited strain of Toxoplasma gondii by CRISPR / Cas9 technology To date, the only recognized marker for Toxoplasma schizonts gra11b has been used to follow the process of schizogony of Toxoplasma in the intestinal tract of infected cats. gra80 It has also been 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 inner membrane complex IMC7 is usually used as a marker for daughter cell formation during the division of Toxoplasma, and therefore, RH- imc7 -HA and ME49- imc7 -HA monoclonal tagged strains were also constructed for observing the schizogony of Toxoplasma (Toxoplasma gondii, Toxoplasma gondii ) after treatment with the glutamine antagonist JHU083 (CAS No: 1998725-11-3).

[0057] The specific experiments are as follows: 1. Construction of the tagged strain (1) Construction of the CRISPR / CAS9 system plasmid pSAG1::CAS9-U6: sggra11b / gra80 / imc7 (2) Construction of the plasmid pSAG1::HA ​The gRNA of the UPRT gene was replaced with the gRNA of the gene using the Multi-Segment Seamless Cloning Kit (Seamless Cloning and Assembly Kit) of TransGen Biotech Co., Ltd. (Catalog No: CU201-02) with the pSAG1::CAS9-U6::sgUPRT plasmid (https: / / www.addgene.org / 54467 / ) as the template, and the gRNA of the gene was replaced with the gRNA of the gene gra11b The gRNA of the gene was replaced with the gRNA of the gene, and the specific operation steps are as follows: 1) Fragment amplification The CRISPR / CAS9 backbone was amplified in three fragments, and the primer sequences were as follows: Backbone 1 fragment: Upstream primer: Backbone-1F: 5'-GTTTTAGAGCTAGAAATAGCAAG-3' (SEQ ID NO: 1); Downstream primer: Backbone-1R: 5'-CCTTCTAGACGCATGGTAAA-3' (SEQ ID NO: 2); Backbone 2 fragment: Upstream primer: Backbone-2F: 5'-TTTACCATGCGTCTAGAAGGT-3' (SEQ ID NO: 3); Downstream primer: Backbone-2R: 5'-CTGTCTTGTGTATTGACCCATGT-3' (SEQ ID NO: 4); sgRNA backbone 3 fragment: Upstream primer: gra11b-sgRNA-F: 5'-CCACATGGGTCAATACACAAGACAGCTA-3' (SEQ ID NO: 5); Downstream primer: gra11b-sgRNA-R: 5'-TTCTAGCTCTAAAACGAAGCCTGCCTGAATTGATCAACTTGACATCCC-3' (SEQ ID NO: 6); gra80-sgRNA-R: 5'-TTCTAGCTCTAAAACTCGCCTCTCGCTGCGCGAACAACTTGACATCCC-3' (SEQ ID NO: 7); imc7-sgRNA-R: 5'-TTCTAGCTCTAAAACagaggatggtgacggtgatgAACTTGACATCCC-3' (SEQ ID NO: 8); All the above DNA fragments were amplified with Phanta High-fidelity DNA Polymerase (2 × Phanta Flash Master Mix (Dye Plus)) of Vazyme Company, gra11b / gra80 / imc7 The PCR (Polymerase Chain Reaction) reaction system of sgRNA is shown in Table 1, and the PCR reaction parameters are shown in Table 2. After the PCR reaction, the target fragments were recovered by running the gel, and the gel purification was performed using the Quick Gel Extraction Kit (Catalog No: EG101-01) of TransGen Biotech Company to purify and recover each DNA fragment.

[0058] Table 1 gra11b / gra80 / imc7 PCR reaction system of sgRNA

[0059] Table 2 PCR reaction parameters

[0060] 2) Fragment ligation The purified DNA product was ligated using the Seamless Cloning and Assembly Kit of TransGen Biotech Company. The reaction system is shown in Table 3.

[0061] Table 3 Reaction system of multi-fragment seamless ligation

[0062] The reaction system was gently mixed, and the reaction was carried out at 50°C for 15 min, and then cooled on ice. The product was transferred to 50 μL of Trans1-T1 competent cells (TransGen Biotech, Trans1-T1 Phage Resistant Chemically Competent Cell), gently mixed, and placed on ice for 30 min. Then, it was transferred to a 42°C water bath for heat shock for 45 s, and then immediately transferred to ice for 2 min. 500 μL of LB medium was added, and the mixture was incubated at 37°C on a shaker at 250 rpm for 1 h. 200 μL of the mixture was evenly coated on an ampicillin-resistant plate, and after 14-16 h, four single colony strains were picked from the solid culture plate and cultured in ampicillin liquid medium. 300 μL of the bacterial solution was mixed with 500 μL of 80% glycerol at a ratio of 1:1, and stored at -25°C for standby. The remaining bacterial solution was extracted to obtain plasmids, which were sent to GenScript Biotech Corporation for sequencing. The sequencing primers were M13F and M13R, each for one reaction. The sequencing results showed that the target sequence was gra11b / gra80 / imc7The sgRNA sequence was correct and there were no errors at the plasmid ligation site; therefore, plasmid construction was successful, meaning pSAG1::CAS9-U6:: was successfully obtained. sggra11b / gra80 / imc7 Plasmid.

[0063] 3) pSAG1::CAS9-U6:: sggra11b / gra80 / imc7 plasmid extraction 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.

[0064] (2) 5'UTR- gra11b / gra80 / imc7 Preparation of HA-DHFR-3'UTR homologous template 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.

[0065] ME49 / RH -gra11b -HA-DHFR-F: 5'-GATCCCGAAACAAACTTGTTAGAGGAAGAGGACGAGTTGAAGCCAtacccttacgatgtaccgg-3' (SEQ ID NO: 9); MR49 / RH-DHFR- gra11b-3'UTR-R:5'-CCGACCAGGCTGAGAGCGCACCTGCTC CTGCGGCTGTGTCTCGtCctgcaaGtgcatagaagg-3'(SEQ ID NO:10) ME49- gra80 -HA-DHFR-F:5'-CCTCTCGGCCCCCGTGGGCTCCGGGAGCAGGGCCCGAGAACtacccttacgatgta-3'(SEQ ID NO:11) ME49- gra80 -HA-DHFR-R:5'-GAAGTCAAGCAACGGGCGCGACTCGTCATTTCTTCTGAACCGtCctgcaaGtgcat-3' (SEQ ID NO:12) RH- gra80- HA-DHFR-F: 5'-CCTCTCTGCCCCCGTGGGCTCCGGGAGCAGGGCCCGAGAACtacccttacgatgta-3' (SEQ ID NO:13) RH- gra80 -HA-DHFR-R:5'-GAAGTCAAGCAACGGGCGCGACTCGTCATTTCTTCTGAACCGtCctgcaaGtgcat-3' (SEQ ID NO:12) MR49 / RH- imc7 -HA-DHFR-F:5'-GCTCAACGCGGAGGAACAGACCACCGCTGTCCCAATCGCTGCGtacccttacgatgtaccgg-3' (SEQ ID NO:14) MR49 / RH-DHFR- imc7 -3'UTR-R:5'-ctcttattgggagtccccactgtccccacacttctctgtctccgtCctg caaGtgcatagaagg-3'(SEQ ID NO:15) Available DNA Detection Protein(Vazyme) and Phanta Radical Dye Diluent(2×Phanta Flash Master Mix(Dye Plus)) with 5'UTR-1. gra11b / gra80 / imc7The PCR reaction system of 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 is recovered by running the gel, and the gel is purified by using the Quick Gel Extraction Kit of TransGen Biotech Co., Ltd. to recover each DNA fragment.

[0066] Table 4 PCR reaction system

[0067] Table 5 PCR reaction parameters

[0068] (3) Electroporation ① From the T25 culture flask of RHΔ ku80 / ME49Δ ku80 (saving from the Department of Pathogenic Biology, Southern Medical University), the cell scraper was used to scrape the cells, and the collected suspension was repeatedly aspirated 6-7 times with a 5 mL syringe to achieve sufficient mechanical lysis to release the worms. About 107 Toxoplasma were collected and transferred to a 15 mL centrifuge tube, and centrifuged at 3000 rpm for 10 min.

[0069] ② After centrifugation, the supernatant was discarded, and 600 μL of cytomix buffer (K2HPO40.174 g, KH2PO40.136 g, KCl 0.895 g, CaCl20.001665 g, MgCl20.0476 g, HEPES 0.59575 g, EDTA·2Na·2H2O 0.0074448 g, ATP 0.12195 g, and glutathione 0.14634 g, deionized water to 100 mL, after preparation, adjust the pH to 7.4 with NaOH, 0.22 μm filter, and store at -20°C.) was added to resuspend the precipitate.

[0070] ③ Add pSAG1::CAS9-U6 ::sggra11b / gra80 / imc7 (50 μg) and 5'UTR- gra11b / gra80 / imc7 -HA-DHFR-3'UTR homologous recombination template (12 μg), mix well, and add a 4 mm diameter electroporation cup.

[0071] ④ Set the electroporator program: voltage 1700 V, capacitance 25 F, resistance ∞. After transfection of Toxoplasma, inoculate into HFF cells for culture, and replace with medium containing a final concentration of 3 μM ethylamine 48 h later.

[0072] V. Screening of monoclonal: After three generations of drug screening, monoclonal screening was performed. The digested HFF cells (T25) were added to the 96-well plate (200 μL per well). The worms were collected and counted using a cell counting plate, diluted to 1000 tachyzoites / mL by limiting dilution method, 200 μL (about 2 Toxoplasma per well) was transferred to the pipette slot, the volume was supplemented to 20 mL, the original culture medium in the 96-well plate was sucked off, and the diluted worm liquid was inoculated into the 96-well plate at 200 μL per well.

[0073] VI. Incubation in the cell culture box (37°C, 5% CO2) for 7 days. After incubation, only one plaque was selected, the cell surface was scraped with a gun head, and the suspension was transferred to a 1.5 mL EP tube. 800 μL of 3 μM allopurinol culture medium was added, mixed well by blowing, and 500 μL of it was inoculated into a 24-well plate for further culture. The remaining 500 μL was used for genomic extraction for PCR verification.

[0074] VII. The genome of the worm strain to be verified was extracted according to the method mentioned above, RHΔ ku80 As a negative control, ddH2O was used as a blank control. The primer sequences for verification are as follows: gra11b-P1-F: 5'-CTCGATGAGGCTCTCGAGAC-3' (SEQ ID NO: 16); gra11b-P1-R: 5'-gacgactcacgggatttacagc-3' (SEQ ID NO: 17); gra11b-P2-F: 5'-GGCGCGGCATACAAAGAC-3' (SEQ ID NO: 18); gra11b-P2-R: 5'-GTAGACCTAAACCCTGTCGACG-3' (SEQ ID NO: 19); gra11b-P3-F: 5'-CTCGATGAGGCTCTCGAGAC-3' (SEQ ID NO: 16); gra11b-P3-R: 5'-GTAGACCTAAACCCTGTCGACG-3' (SEQ ID NO: 19); gra80-P1-F: 5'-CTGCCCTTTACCAGGTGATC-3' (SEQ ID NO: 20); gra80-P1-R: 5'-cagacagtacgcgtctactaag-3' (SEQ ID NO: 21); gra80-P2-F: 5'-GTTGCACACGTCTGCAAC-3' (SEQ ID NO: 22); gra80-P2-R: 5'-GTCAAGAAGCGTCGTGGT-3' (SEQ ID NO: 23); gra80-P3-F: 5'-CTGCCCTTTACCAGGTGATC-3' (SEQ ID NO: 20); gra80-P3-R: 5'-GTCAAGAAGCGTCGTGGT-3' (SEQ ID NO: 23); imc7-P1-F: 5'-GGCAAGGAGCTTCGGAGC-3' (SEQ ID NO: 24); imc7-P1-R: 5'-ctcagtcaggcagtctccc-3' (SEQ ID NO: 25); imc7-P2-F: 5'-cacacagtctcacctcgc-3' (SEQ ID NO: 26); imc7-P2-R: 5'-ggagacaagcagtccacctc-3' (SEQ ID NO: 27); imc7-P3-F: GGCAAGGAGCTTCGGAGC-3' (SEQ ID NO: 24); imc7-P3-R: 5'-ggagacaagcagtccacctc-3' (SEQ ID NO: 27); Tag insertion and identification strategy (similar to the following gene edited worm strain strategy) Figure 1 (GOI represents gene of interest, in the present application refers to gra11b, gra80 and imc7 (Gene of Interest), PCR1, PCR2 and PCR3 represent fragments that need to be verified; DHFR is an ethylenimine drug screening tag; strong promoter is used to start the transcription and translation of the drug screening tag) as shown, where the knockout worm strain PCR1 / PCR2 has a purpose band, PCR3 has no purpose band, the wild type worm strain PCR1 / PCR2 has no purpose band, PCR3 has a purpose band, and the blank control group has no any band. The correct worm strain is named RH / ME49- gra11b / gra80 / imc7 -HA worm strain.

[0075] Example 2 Efficient in vitro induced differentiation model of Toxoplasma tachyzoites based on indirect immunofluorescence experiment This embodiment is based on the indirect immunofluorescence experiment to efficiently construct the Toxoplasma tachyzoite in vitro induction differentiation model, and the specific process is as follows: (1) Cell preparation Put several 14mm cell climbing sheets in 24 empty plates, and divide them into different concentration groups of 2.5μM glutamine antagonist JHU083, 5μM glutamine antagonist JHU083, 10μM glutamine antagonist JHU083, anhydrous ethanol and 5μM ethyl pyrimidine (the solvent is anhydrous ethanol), and set three duplicate wells for each group.

[0076] According to the needs, a certain number of HFF cells (ATCC number SCRC-1041) in good growth state (T25 culture bottle) are selected, the original culture medium is discarded, and then 1mL PBS buffer is used for washing once, and then the PBS is aspirated. 1mL 0.25% Trypsin-EDTA digestion solution is added to each bottle, and the digestion is carried out at 37°C for 2min. After digestion, 1mL of Gibco DMEM medium containing 15% fetal bovine serum (FBS) (hereinafter referred to as D15 medium) is added to terminate the digestion reaction. The cell suspensions of two bottles are collected into a 15mL centrifuge tube and centrifuged at 800rpm for 5min. After centrifugation, the supernatant is removed, and 2mL of D15 medium is added to resuspend the cell precipitate. Then, the resuspension is completely transferred to a pipette tank which has been pre-added with a certain volume (determined according to the number of set wells, 1mL per well) of D15 medium, and a 1000μL pipette is used to fully blow and mix. Finally, 1mL of cell suspension is inoculated into the 24-well plate where the cell climbing sheet has been placed, and the culture is continued until the cell density reaches 100%, which can be used for subsequent experiments.

[0077] (2) Drug preparation 1mM JHU083 stock solution is diluted to the corresponding concentration with 1% FBS-containing Gibco DMEM medium (hereinafter referred to as D1 medium) using anhydrous ethanol as the solvent. The preparation of the drug-containing medium is carried out in the dark on the test bench, and attention should be paid to avoid light. The UT group is 1:100 D1 added with anhydrous ethanol.

[0078] (3) Toxoplasma infection of cells The RH- gra11b -HA strain (RH- gra80 -HA, RH- imc7 -HA strain and RH- gra11b- HA strain) T25 flask, the cells were scraped with a cell scraper and the collected suspension was pipetted 6-7 times with a 5 mL syringe to achieve a full mechanical lysis, releasing the worms. Subsequently, the lysate was transferred to a 15 mL centrifuge tube and centrifuged at 3000 rpm for 10 min. During centrifugation, about 100 μL of the remaining liquid in the original flask was taken and transferred to a new HFF cell culture flask for strain preservation, and a cell counting plate and cover glass were prepared. After centrifugation, the supernatant was discarded and the pellet was resuspended with 3 mL of Gibco DMEM medium containing 1% FBS (hereinafter referred to as D1 medium). 10 μL of the resuspended solution was taken and added to the edge of the cover glass of the counting plate, and the Toxoplasma count was performed under a microscope. According to the counting results, the corresponding volume of the worm suspension was taken into a new 15 mL centrifuge tube for limiting dilution, with an infection ratio of 1 x 10 5 The ratio of Toxoplasma (multiplicity of infection MOI = 1) was taken into a new 15 mL centrifuge tube for limiting dilution, with an infection ratio of 1 x 10

[0079] After 1 h of Toxoplasma invasion, the inoculum was removed and replaced with D1 medium for continued culture. Among them, the RH- gra11b -HA, RH- gra80 -HA and RH- imc7 -HA strain were cultured for 12 h; while the ME49- gra11b -HA, ME49- gra80 -HA and RH- imc7 -HA strain were cultured for 48 h. After culture, D1 medium containing different concentrations of JHU083 (0, 2.5, 5, 10 μM) was replaced and continued to be cultured for 24 h (RH strain) or 48 h (ME49 strain).

[0080] (4) Indirect immunofluorescence (Immunofluorescence, IF) experiment to detect the schizont marker After the end of culture, the culture medium was discarded, the cells were rinsed with pre-cooled PBS, then 500 μL of 4% paraformaldehyde was added, and fixed at room temperature for 15 min. After fixation, PBS was used for rinsing, and then 500 μL of 0.5% Triton X-100 (diluted with PBS) was added for permeabilization treatment for 10 min. After permeabilization, PBS was used for washing 3 times, each time for 3 min. Then, 500 μL of 10% BSA blocking solution was added to each well, and blocked at 37°C for 1 h. After blocking, the same blocking solution was used to dilute rabbit / mouse HA monoclonal antibody and mouse IMC1 polyclonal antibody / rabbit GAP45 polyclonal antibody at a ratio of 1:1000, 200 μL of the diluted primary antibody was added to each well, and incubated at 4°C overnight (>8 h). After the end of incubation, the primary antibody was recovered, and PBS was used for washing 3 times, each time for 3 min. Then, the blocking solution was used to dilute Alexa Fluor 594-labeled goat anti-mouse IgG and Alexa Fluor 488-labeled goat anti-rabbit IgG at a ratio of 1:1000, 200 μL of the diluted secondary antibody was added to each well, and incubated at 37°C for 50 min in the dark. After incubation, the secondary antibody was discarded, PBS was used for washing 3 times, and finally, a mounting agent containing DAPI was used for mounting, and observed under a fluorescence microscope, and the number of positive and negative PVs was counted. gra11b or gra80 Whether the positive or negative in the nanobubble was judged as a schizont. By calculating the ratio of the positive nanobubbles (PV) in 100 nanobubbles (PV) under the condition of the time and the drug concentration, the ratio was the transformation rate of the schizont under the condition of the time and the drug concentration. The transformation rates of the schizont under different conditions (culture medium pH=7.4 and pH=8.2) were experimented and calculated, and the highest transformation rate of the D1 culture medium containing 10 μM JHU083 at pH 7.4 was the highest efficiency of the in vitro induction and differentiation model of the schizont of Toxoplasma based on JHU083. gra11b

[0081] The experimental results are shown in Figure 2 - Figure 5

[0082] Figure 2 Figure A in the middle shows the endodouble budding of the ME49 strain after being induced by 10 μM JHU083 for 48 h. In the figure, the green fluorescence is labeled with the mother cell marker IMC7, and the red fluorescence is labeled with the daughter cell marker IMC1. The co-localization of the red fluorescence and the green fluorescence indicates that the nanobubble undergoes endodouble budding, and the incomplete co-localization of the red fluorescence and the green fluorescence indicates that the nanobubble undergoes endomultibudding. Figure 2 Figure B in the middle shows the expression of GRA80 of the ME49 strain. In the figure, the green fluorescence is labeled with the schizont-specific marker GRA80, and the red fluorescence is labeled with IMC1. The nanobubble with positive green fluorescence is a mature schizont, and the nanobubble with negative green fluorescence is not a mature schizont.

[0083] 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.

[0084] 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.

[0085] Figure 5 The 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 .

[0086] 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 found that the bradyzoite-specific marker and the tachyzoite-specific marker did not appear in the same nodule. These results indicated that JHU083 was able to induce the RH strain and the ME49 strain to transform into tachyzoites under in vitro conditions outside the feline intestinal tract.

[0087] By comparing the tachyzoite conversion rate at different pH values, the results are shown in Figure 6 - Figure 10 As shown in

[0088] Figure 6 The green fluorescence indicates the tachyzoite-specific protein GRA11B, and the red fluorescence indicates IMC1.

[0089] Example 3 Transmission electron microscopy observation of in vitro tachyzoites The ME49 strain or the RH strain of Toxoplasma gondii was inoculated into HFF cells in a T25 culture flask at a MOI of 1. After the RH strain grew for 12 h or the ME49 strain grew for 48 h, the original culture medium was discarded, and the cells were cultured under the experimental conditions of the highest-efficiency in vitro induction and differentiation model of tachyzoites obtained in Example 2. After the culture was completed, the culture medium was discarded, 1 mL of pre-cooled PBS was added, the cells were scraped with a cell scraper, and were transferred to a 1.5 mL EP tube. Centrifugation was performed at 1000 rpm for 5 min to form cell clumps about 0.5-1 mm high. The PBS was discarded, and 1 mL of room-temperature 2.5% glutaraldehyde electron microscopy fixing solution was slowly added. After standing for 1 h at room temperature, the cells were transferred to 4°C for overnight fixation. The fixing solution was discarded, 1 mL of PBS was added, and the cells were stored in a refrigerator at 4°C. The cells were sent to the Central Laboratory of Southern Medical University for sectioning and observation.

[0090] The results are shown in Figure 11 In the figure, PV is the Toxoplasma gondii nodule, HC is the host cell, and n1-n6 are the numbers of nuclei in a single cell. Figure 11 The red arrows in D-E indicate the dividing nuclei.

[0091] After the RH strain and the ME49 strain were treated with 10 μM JHU083 for 48 h, obvious multinucleated tachyzoites were observed, and each tachyzoite contained more than two nuclei. In addition, it was also confirmed that the endodyney was broken after drug treatment, leading to the appearance of endopolygeny. The transmission electron microscopy results were completely consistent with the indirect immunofluorescence results. The treatment of the ME49 strain and the RH strain with JHU083 induced the tachyzoites to divide in a schizogony mode.

[0092] In summary, the present application first realizes the efficient in vitro induction of Toxoplasma tachyzoites: using glutamine antagonist JHU083, a new in vitro induction and differentiation model of Toxoplasma tachyzoites independent of feline hosts is established, which fills the long-standing technical gap in this research field and provides an irreplaceable platform for analyzing the formation of the key initiation stage of Toxoplasma sexual reproduction. A perfect tachyzoite labeling and identification system is constructed: through CRISPR / Cas9 gene editing technology, a series of specific marker strains including gra11b -HA, gra80 -HA and imc7 -HA are successfully constructed, which realizes the precise tracking and identification of tachyzoite development process. A standardized and quantifiable in vitro induction and evaluation system is established: the optimal induction concentration (2.5-10 µM) of JHU083 is determined, the culture time window of different strains (RH / ME49) is established, and the tachyzoite conversion rate statistical method based on indirect immunofluorescence is established, which makes the model have high repeatability and quantitative evaluation ability. The model has the potential of multi-dimensional verification and wide application: combined with indirect immunofluorescence and transmission electron microscopy, the successful induction of tachyzoites is verified from two aspects of protein localization and ultrastructure. This model can not only be used for studying the molecular mechanism of tachyzoite differentiation, but also provides an effective tool for screening anti-parasitic drugs targeting the key initiation stage of Toxoplasma sexual reproduction.

[0093] The embodiments of the present application are described in detail above in combination with the drawings, but the present application is not limited to the above embodiments, and various changes can be made within the knowledge range of ordinary skilled persons in the art without departing from the purpose of the present application. In addition, the embodiments of the present application and the features in the embodiments can be combined with each other without conflict.

Claims

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

2. Use according to claim 1, characterized in that, The glutamine antagonist is JHU083.

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

4. The method of claim 3, 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.

5. The method of claim 4, 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.

6. 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.

7. The method according to any one of claims 3-6, characterized in that, The Toxoplasma gondii comprises Toxoplasma gondii with specific markers.

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

9. An in vitro induced differentiation model of Toxoplasma gondii bradyzoites, which is constructed by the method of any one of claims 3-8.

10. Use of the method of any one of claims 3-8 or the in vitro induced differentiation model of Toxoplasma gondii bradyzoites of claim 9 in (1) or (2): (1) screening drugs for preventing or treating Toxoplasma gondii infection; (2) studying molecular mechanisms of disease development.

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