Polypeptide and application thereof in preparation of toxoplasma gondii infection resistant product

Abstract

The invention provides a polypeptide and application thereof in preparation of a toxoplasma gondii infection resistant product. The amino acid sequence of the polypeptide is shown as SEQ ID NO: 1. The polypeptide provided by the invention has the obvious advantage of multi-dimensional synergistic toxoplasma gondii infection resistance, can effectively inhibit growth and reproduction of toxoplasma gondii in vitro and in vivo, reduce the polypide load of a host, relieve excessive inflammation caused by infection, promote damage repair of tissues including intestinal tracts, and contribute to improvement of intestinal flora balance. The invention provides a new technical strategy for developing a safe and comprehensive toxoplasma gondii infection prevention and treatment and prognosis improvement anti-toxoplasma gondii product in the field, and has popularization and application values.

Description

Technical Field

[0001] This invention relates to the field of polypeptide technology, and more particularly to a polypeptide and its application in the preparation of products for treating Toxoplasma gondii infection. Background Technology

[0002] Toxoplasma gondii ( Toxoplasma gondii Toxoplasmosis is a widespread obligate intracellular parasitic protozoan that is a foodborne zoonotic protozoan that can infect most warm-blooded animals, including humans. In immunocompetent individuals, infection with Toxoplasma gondii often presents as a latent or mild, self-limiting disease. However, in immunocompromised individuals (such as HIV patients and organ transplant recipients) and fetuses, Toxoplasmosis infection can lead to life-threatening complications such as toxoplasmic encephalitis, necrotizing retinochoroiditis, and congenital malformations.

[0003] The life cycle of Toxoplasma gondii is complex, with multiple morphologies including tachyzoites and bradyzoites (cysts). Current mainstream treatments (such as combined sulfadiazine and pyrimethamine) primarily target the parasite's folate metabolism pathway, proving effective only during the rapidly proliferating tachyzoite stage. However, they are largely ineffective against the long-term latent cysts within tissues, limiting treatment to acute infections and failing to eradicate latent or chronic infections, thus posing a risk of relapse. Furthermore, these drugs have significant toxic side effects (such as bone marrow suppression, hepatotoxicity, nephrotoxicity, and severe allergic reactions), require long treatment cycles, and have poor patient compliance, making them unsuitable for safe and effective treatment in special populations such as pregnant women. Current treatment strategies largely focus on killing the parasite, lacking effective interventions for key pathological aspects such as excessive host inflammatory response, tissue damage, and secondary gut microbiota dysbiosis caused by infection, impacting overall efficacy and prognosis.

[0004] Given the increasingly stringent national regulations on drug safety and efficacy, and the urgent need to overcome existing treatment bottlenecks in clinical practice, the development of anti-Toxoplasma gondii drugs with novel mechanisms of action is particularly pressing. It is necessary to explore multi-dimensional synergistic strategies that can directly and efficiently eliminate pathogens, regulate host immune pathological damage, and take into account the balance of the microecology, thereby achieving safer and more comprehensive therapeutic effects. Summary of the Invention

[0005] To address the aforementioned problems in the prior art, this invention provides a polypeptide and its application in the preparation of anti-Toxoplasma gondii products.

[0006] In a first aspect, the present invention provides a polypeptide whose amino acid sequence is shown in SEQ ID NO:1. In the present invention, the polypeptide is polypeptide YAMP1.

[0007] Secondly, the present invention provides a nucleic acid molecule that encodes the aforementioned polypeptide.

[0008] Thirdly, the present invention provides a conjugate comprising the aforementioned polypeptide and a chemical moiety conjugated thereto.

[0009] Fourthly, the present invention provides a composition comprising the aforementioned polypeptide.

[0010] The composition provided by the present invention preferably further includes other drugs against Toxoplasma gondii infection.

[0011] Fifthly, the present invention provides a biomaterial containing the aforementioned nucleic acid molecule, wherein the biomaterial is recombinant DNA, expression cassette, transposon, vector, microorganism, or cell.

[0012] In a sixth aspect, the present invention provides the use of the polypeptide, the nucleic acid molecule, the conjugate, the composition, or the biomaterial in the preparation of products that inhibit the growth and / or reproduction of Toxoplasma gondii, resist Toxoplasma gondii infection, and prevent and / or treat diseases related to Toxoplasma gondii infection.

[0013] The use of the polypeptide, nucleic acid molecule, conjugate, composition, or biomaterial provided by the present invention in the preparation of products for inhibiting the growth and / or reproduction of Toxoplasma gondii, resisting Toxoplasma gondii infection, and preventing and / or treating Toxoplasma gondii-related diseases, preferably, the products are used to achieve any one or more of the following objectives: (1) It disrupts the physiological structure of Toxoplasma gondii; (2) Reduces the inflammatory response caused by Toxoplasma gondii infection; (3) Repair tissue damage caused by Toxoplasma gondii infection; (4) Reduce the load level of Toxoplasma gondii in the host; (5) Improves intestinal flora imbalance caused by Toxoplasma gondii infection.

[0014] The use of the polypeptide, nucleic acid molecule, conjugate, composition or biomaterial provided by the present invention in the preparation of products that inhibit the growth and / or reproduction of Toxoplasma gondii, resist Toxoplasma gondii infection, and prevent and / or treat Toxoplasma gondii infection-related diseases, preferably, the polypeptide is used to inhibit the growth, reproduction and / or infection of Toxoplasma gondii in vitro and / or in vivo.

[0015] The use of the polypeptide, nucleic acid molecule, conjugate, composition or biomaterial provided by the present invention in the preparation of products that inhibit the growth and / or reproduction of Toxoplasma gondii, resist Toxoplasma gondii infection, and prevent and / or treat Toxoplasma gondii infection-related diseases, preferably, the products include pharmaceuticals or disinfectants.

[0016] More preferably, the drug further includes a pharmaceutically acceptable carrier.

[0017] More preferably, the dosage form of the drug is a powder, tablet, granule, capsule, solution, emulsion or suspension.

[0018] The application of the polypeptide, nucleic acid molecule, conjugate, composition or biomaterial provided by the present invention in the preparation of products that inhibit the growth and / or reproduction of Toxoplasma gondii, resist Toxoplasma gondii infection, and prevent and / or treat Toxoplasma gondii infection-related diseases, preferably, the effective concentration of the polypeptide is 2.5 μg / ml to 80 μg / ml, or 5 mg / kg to 20 mg / kg.

[0019] More preferably, the effective concentration of the polypeptide is 40 μg / ml or 10 mg / kg.

[0020] More preferably, the effective in vitro concentration of the polypeptide is 2.5 μg / ml to 80 μg / ml.

[0021] More preferably, the effective in vitro concentration of the polypeptide is 40 μg / ml.

[0022] More preferably, the effective in vivo concentration of the polypeptide is 5 mg / kg to 20 mg / kg.

[0023] More preferably, the effective in vivo concentration of the polypeptide is 10 mg / kg. In a seventh aspect, the present invention provides a method for preparing the polypeptide, which is prepared by solid-phase synthesis according to the amino acid sequence shown in SEQ ID NO:1.

[0024] In this invention, unless otherwise stated, the term Toxoplasma gondii refers to Toxoplasma gondii.

[0025] The present invention has the following beneficial effects: The polypeptide YAMP1 provided by this invention has significant advantages in multi-dimensional synergistic anti-Toxoplasma gondii infection. It effectively inhibits the growth and reproduction of Toxoplasma gondii both in vitro and in vivo, reduces the host's parasite burden, alleviates excessive inflammation caused by infection, promotes tissue repair including in the intestines, and helps improve the balance of intestinal flora. This provides a new technical strategy for developing safer and more comprehensive anti-Toxoplasma gondii products for the prevention and treatment of Toxoplasma gondii infection and for improving prognosis, and has significant application value. Attached Figure Description

[0026] Figure 1 This is the mass spectrometry identification result of the polypeptide YAMP1 provided in Example 1 of the present invention.

[0027] Figure 2 This is a schematic diagram of the spatial conformation of the polypeptide YAMP1 provided in Example 1 of the present invention.

[0028] Figure 3The results of circular dichroism spectroscopy measurements of the polypeptide YAMP1 provided in Example 1 of this invention in hydrophilic (PBS) and hydrophobic (50% TFE) environments are shown.

[0029] Figure 4 A is a schematic diagram of the molecular docking of the polypeptide YAMP1 provided in Example 1 of the present invention with the Toxoplasma gondii SAG1 protein; A is a schematic diagram of the hydrogen bond interaction between the two; B is a schematic diagram of the hydrophobic interaction between the two.

[0030] Figure 5 The results are molecular dynamics simulation analysis results of the polypeptide YAMP1 and Toxoplasma gondii SAG1 protein provided in Example 1 of this invention; A is the RMSD result; B is the number of hydrogen bonds; C is the area of ​​the protein surface accessible to the solvent; D is the binding free energy.

[0031] Figure 6 These are the statistical results of the survival rate of Toxoplasma gondii tachyzoites under different treatments provided in Example 2 of this invention; different lowercase letters indicate significant differences. p <0.05.

[0032] Figure 7 These are scanning electron microscope images of Toxoplasma gondii tachyzoites under different treatments provided in Example 2 of this invention; A and B are untreated tachyzoites under different fields of view; C to F are tachyzoites treated with 40 μg / mL polypeptide YAMP1 under different fields of view.

[0033] Figure 8 These are transmission electron microscope images of Toxoplasma gondii tachyzoites under different treatments provided in Example 2 of this invention; A and B are untreated tachyzoites under different fields of view; C to F are tachyzoites treated with 40 μg / mL polypeptide YAMP1 under different fields of view.

[0034] Figure 9 These are the Western blot (WB) results of Toxoplasma gondii tachyzoites under different treatments provided in Example 2 of this invention; A is a protein banding diagram; B is the intensity analysis result of the bands in A; **** p <0.0001.

[0035] Figure 10 This invention provides the effects of NC, IC, and YAMP1 mice at 10 dpi on tissue inflammation and parasitic load in Example 3 of this invention; A shows the H&E staining of small intestinal tissue in each group of mice; B shows the Toxoplasma gondii load levels in the brain, heart, lungs, liver, kidneys, and spleen of each group of mice; C shows the H&E staining of brain tissue specimens from each group of mice (circles indicate cysts, arrows indicate inflammation); D shows the brain cyst count results for each group of mice; data in the figure are expressed as mean ± SD, and ns represent... p >0.05, *** p <0.001, ****p <0.0001.

[0036] Figure 11 This is the statistical result of serum inflammatory cytokine levels in mice of the NC group, IC group, and YAMP1 group at 10 dpi and 30 dpi provided in Example 3 of the present invention; A is IFN-γ; B is IL-12p70; C is IL-10; D is TNF-α; E is IL-4; F is IL-6; data in the figure are expressed as mean ± SD; ns represent p >0.05, * p <0.05,** p <0.01, *** p <0.001, *** p <0.0001.

[0037] Figure 12 The results of gut microbiota analysis of mice in the NC, IC, and YAMP1 groups provided in Example 3 of this invention are as follows: A represents the relative abundance of phylum-level microbiota in each group of mice at 10 dpi; B represents the relative abundance of genus-level microbiota in each group of mice at 10 dpi; C represents the relative abundance of phylum-level microbiota in each group of mice at 30 dpi; D represents the relative abundance of genus-level microbiota in each group of mice at 30 dpi; E and F represent the number of shared and independent core otues in each group of mice at 10 dpi and 30 dpi, respectively.

[0038] Figure 13 The results are the metabolic level analysis results of mice in the NC group, IC group and YAMP1 group provided in Example 3 of the present invention; A and B are the positive ion multivariate statistical partial least squares discriminant analysis (PLS-DA) results of mice in each group at 10 dpi and 30 dpi, respectively; C and D are the negative ion multivariate statistical partial least squares discriminant analysis (PLS-DA) results of mice in each group at 10 dpi and 30 dpi, respectively; E and F are the inter-group overlapping differential metabolites of mice in each group at 10 dpi and 30 dpi, respectively.

[0039] Figure 14 The results are the biosafety evaluation results of peptide YAMP1 provided in Example 5 of this invention; A is the statistical result of the hemolysis rate of peptide YAMP1, and Control indicates PBS containing 1% Triton X-100 as a positive control; B is the statistical result of the inhibition rate of cytotoxicity of peptide YAMP1 detected by CCK-8 assay, and Control indicates PBS containing 1% Triton X-100 as a positive control; 2.5, 5, 10, 20 and 40 represent the working concentration of peptide YAMP1, in μg / mL. Detailed Implementation

[0040] To make the objectives, technical solutions, and advantages of this invention clearer, the technical solutions of this invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some, not all, of the embodiments of this invention. All other embodiments obtained by those skilled in the art based on the embodiments of this invention without creative effort are within the scope of protection of this invention.

[0041] Unless otherwise specified, the experimental methods involved in the following embodiments are conventional methods in the art. For example, you can refer to the experimental manual in the art or follow the conditions recommended in the manufacturer's instructions.

[0042] Unless otherwise specified, all experimental materials and reagents used in the following examples are commercially available.

[0043] The experimental animals used in the following examples are as follows: Toxoplasma gondii: tachyzoites and cysts, kindly provided by the Animal Protozoan Laboratory of China Agricultural University, passaged and preserved by the inventor's laboratory, and the biological material is available to the public from the applicant; Mice: breed: C57BL / 6, 6-week-old SPF-grade females, weighing 18-22 g, purchased from Beijing Spaford Technology Co., Ltd.

[0044] Example 1: Preparation and structural analysis of polypeptide YAMP1 1. Preparation of peptide YAMP1 Beijing Solarbio Science & Technology Co., Ltd. synthesized the polypeptide YAMP1 with the amino acid sequence QGHLNSPTTVNRSLNALR (SEQ ID NO:1) using a standard solid-phase synthesis method with L-type amino acids as raw materials. The polypeptide was purified by high-performance liquid chromatography and then identified by mass spectrometry. Figure 1 As shown, the actual molecular weight of the purified polypeptide YAMP1 is consistent with the theoretical value, and the purity is greater than 90%.

[0045] 2. Structural analysis of peptide YAMP1 (1) Spatial conformation prediction of peptide YAMP1 This invention uses the alphafold3 platform to predict the structure of the peptide YAMP1 based on its amino acid sequence.

[0046] like Figure 2 As shown, the polypeptide YAMP1 has an α-helix as its main conformation.

[0047] (2) Circular dichroism spectroscopy test The synthesized peptide YAMP1 was dissolved in PBS and 50% trifluoroethanol (TFE) at a concentration of 0.5 mg / mL to simulate hydrophilic and hydrophobic environments, respectively, and used for circular dichroism spectroscopy experiments. Wavelength scans were performed in the range of 190–240 nm. The same sample was scanned four times, and the mean values ​​were calculated and the data were fitted.

[0048] like Figure 3 As shown, the polypeptide YAMP1 exhibits a random coil structure in a hydrophilic environment and an α-helix structure in a hydrophobic environment. This indicates that polypeptide YAMP1 is a dipolar molecule that can form an amphiphilic α-helix structure under appropriate conditions.

[0049] 3. Interaction analysis between peptide YAMP1 and Toxoplasma gondii surface antigen 1 protein (SAG1) The SAG1 protein is a key protein for Toxoplasma gondii infection of the host. This invention analyzes the interaction between the peptide YAMP1 and the SAG1 protein through molecular docking and molecular dynamics simulation.

[0050] (1) Molecular docking The sequence and 3D structure of Toxoplasma gondii SAG1 protein were obtained using the NCBI database and OpenBabel 2.4.1 software. Molecular docking and visualization of the peptide YAMP1 and SAG1 protein were performed using AutoDockTools 1.5.6 and PyMOL software.

[0051] like Figure 4 As shown in A and B, LYS2223, THR2218, ASP2214, TRP2080, and SER2079 in protein SAG1 form hydrogen bonds with GLN1, THR8, ASN15, ARG18, and LEU17 in the peptide, while PRO2160, MET2163, VAL2139, ILE2099, PRO2098, VAL2097, and TRP2080 form hydrophobic interactions with LEU4, PRO7, LEU14, and LEU17 in the peptide.

[0052] (2) Molecular dynamics simulation Molecular dynamics simulations were performed using Amber24 software, with the ff19SB molecular force field selected and the OPC water model employed.

[0053] A simulated nonvalent complex (i.e., a complex) formed by the binding of peptides YAMP1 and SAG1 proteins through intermolecular interactions (such as hydrogen bonding, hydrophobic interactions, and electrostatic interactions) was placed in a cubic water tank. The cutoff distances for both electrostatic and van der Waals interactions were set to 1.0 nm, and the time step was set to 2 fs. Long-range corrections for electrostatic interactions were performed using the PME method. The system temperature was set to 300 K, and the pressure to 1 bar. One complex system was constructed.

[0054] After establishing the composite system, energy minimization was first performed, followed by NVE equilibrium kinetics at 200 ps and NPT equilibrium kinetics at 100 ps. The temperature of the thermocoupled system was controlled using the V-rescale method, and the pressure was controlled using the Parrinello-Rahman method. Finally, molecular dynamics sampling was performed for 100 ns. RMSD, RMSF, gyroscope radius, and number of hydrogen bonds were calculated using Amber's built-in modules, Cpptraj, and Python scripts.

[0055] RMSD results as follows Figure 5 As shown in A, the complex exhibits significant conformational fluctuations between 0 and 80 ns, and tends to reach equilibrium between 80 and 100 ns, indicating that the overall conformation of the complex system of peptides YAMP1 and SAG1 is stable in the later stages.

[0056] like Figure 5 As shown in B, the complex system of peptide YAMP1 and SAG1 protein can form hydrogen bond interactions, with the number of hydrogen bonds ranging from 0 to 7, which contributes to the binding.

[0057] like Figure 5 As shown in C, the solvent-accessible surface area of ​​the entire system is relatively stable.

[0058] like Figure 5 As shown in D, the binding free energy of the complex changes with time. The lower the binding free energy, the stronger the binding force between the two.

[0059] The above results indicate that the peptides YAMP1 and SAG1 bind relatively stably to each other, with multiple amino acids involved in the interaction, primarily through hydrogen bonding and hydrophobic interactions.

[0060] Example 2: In vitro killing effect of polypeptide YAMP1 on Toxoplasma gondii 1. Flow cytometry analysis (1) Treatment of Toxoplasma gondii tachyzoites Treat 1×10 with 1 mL PBS 6 One tachyzoite was designated as the Control group, serving as a negative control; 1 × 10⁶ cells were treated with PBS containing 400 μM sulfadiazine (SFZ).6 One tachyzoite was designated as the SFZ group, serving as a positive control; each 1×10⁶ molecule was treated with PBS containing different working concentrations (2.5 μg / ml, 5 μg / ml, 10 μg / ml, 20 μg / ml, 40 μg / ml, and 80 μg / ml) of peptide YAMP1. 6 These tachyzoites are denoted as YAMP1 group.

[0061] The Control, SFZ, and YAMP1 groups were treated at 37°C for 30 min. Then, they were centrifuged (2000 r / min, 10 min) to remove the supernatant and collect the tachyzoite precipitate.

[0062] (2) Detection Add 1 mL of propidium iodide working solution (3 μM / mL) to stain tachyzoites, incubate at room temperature for 15 min, and then perform flow cytometry analysis.

[0063] like Figure 6 As shown, different concentrations of peptide YAMP1 have a lethal effect on Toxoplasma gondii tachyzoites. The number of tachyzoites decreases with increasing concentration of peptide YAMP1. The optimal insecticidal effect is achieved when the working concentration of peptide YAMP1 is 40 μg / mL.

[0064] 2. Electron microscopy observation (1) Treatment of Toxoplasma gondii tachyzoites 5 × 10⁵ cells were treated with YAMP1 at a working concentration of 40 μg / mL. 7 One tachyzoite of *Toxoplasma gondii* was treated at 37°C for 30 min. Untreated tachyzoites of *Toxoplasma gondii* served as a control.

[0065] Centrifuge (2000 r / min, 10 min) to remove the supernatant and collect the tachyzoite precipitate.

[0066] (2) Observation by scanning electron microscopy (SEM) The tachyzoite precipitate was washed twice with PBS and then fixed overnight with 4% glutaraldehyde at 4°C. The tachyzoites were dehydrated by treatment with ethanol of different concentrations (30%, 50%, 70%, 90%, 95%, and 100%, respectively) three times for 10 minutes each time, and then covered with metal foil. Morphological differences in tachyzoites under different treatments were analyzed using a Hitachi SU8100 (Japan) scanning electron microscope.

[0067] like Figure 7 As shown in A and B, untreated tachyzoites are meniscus-shaped with a smooth, flat surface. Figure 7 As shown in D and F, after treatment with the peptide YAMP1, the morphology of tachyzoites was disrupted, and the complete crescent shape was no longer observed. Figure 7 As shown in C and E, the surface of the tachyzoite folds and deforms after treatment with the peptide YAMP1.

[0068] (3) Observation by transmission electron microscopy (TEM) The tachyzoite precipitate was washed twice with PBS, centrifuged at 3000×g for 10 minutes, and fixed overnight at 4°C with 4% glutaraldehyde. Subsequently, the tachyzoites were washed twice with 100 mM calcium acetate buffer and fixed at 4°C for 2 hours in 100 mM sodium dimethylarsinate buffer containing 1% osmium tetroxide. The tachyzoites were then washed three times with distilled water and treated three times each with different concentrations (30%, 50%, 70%, 90%, 95%, and 100%) of ethanol for 10 minutes each time to dehydrate them. The tachyzoite samples were then embedded in epoxy resin and polymerized at 60°C for 48 hours. Ultrathin sections were cut using a Leica EM UC7 microtome and a diamond blade. The sections were then double-stained with uranium acetate and lead citrate, and the internal and cross-sectional structures of the tachyzoites were analyzed under a Hitachi HT7800 (Japan) transmission electron microscope.

[0069] like Figure 8 As shown in A and B, the untreated tachyzoites have a normal internal structure and intact organelles. Figure 8 As shown in C and D, after treatment with the peptide YAMP1, tachyzoites undergo abnormal deformation, with altered internal organelle structures and cytoplasmic vacuolation. Furthermore, as... Figure 8 As shown in E and F, the biological characteristics of most tachyzoites are destroyed after treatment with the peptide YAMP1.

[0070] 3. Western blot (WB) assay (1) Treatment of Toxoplasma gondii tachyzoites Treatment with YAMP1 at a working concentration of 40 μg / mL for 5 × 10⁵ days 7 One tachyzoite was used as the treatment group (YAMP1) and incubated at 37°C for 30 min. Tachyzoites treated with an equal volume of PBS served as the control group (NC).

[0071] (2) Detection Total protein was extracted from the cell pellet using radioimmunoprecipitation analysis of the lysis buffer. Protein concentration in each sample was measured at 562 nm using a colorimetric biuret assay.

[0072] Protein samples were separated by electrophoresis on a sodium dodecyl sulfate polyacrylamide gel (SDS-PAGE) and transferred to a polyvinylidene fluoride membrane. After blocking the membrane with 5% skim milk powder for 1 hour, it was incubated overnight at 4°C with primary antibodies against SAG1 (1:2000, Thermo Fisher) and fructose-1,6-bisphosphate aldolase (ALD) (1:2000, Thermo Fisher). After washing the membrane with 1×PBS containing 0.05% Tween-20, horseradish peroxidase-conjugated secondary antibody was added, and the membrane was incubated at room temperature for 1 hour. After repeating the washing steps, the membrane was developed using an enhanced chemiluminescence immunoblotting assay kit.

[0073] ALD is a core housekeeping protein in the glycolysis pathway. Toxoplasma gondii tachyzoites rely on glycolysis for energy, and the transcription and translation levels of its encoding gene fluctuate very little during the tachyzoite's infection, proliferation, and other physiological processes. In this embodiment, ALD was used as an internal control to ensure the accuracy of the calibration of sample loading amount and protein extraction efficiency. ImageJ software was used for quantitative analysis of the band intensity.

[0074] like Figure 9 As shown in A and B, the SAG1 protein content was significantly reduced after YAMP1 treatment compared to the control group.

[0075] The above results indicate that the polypeptide YAMP1 can disrupt the morphology and internal structure of tachyzoites, causing cytoplasmic vacuolation and leakage of intracellular solutes, and can directly kill Toxoplasma gondii in vitro.

[0076] Example 3: In vivo inhibition of Toxoplasma gondii infection by polypeptide YAMP1 1. Animal experimental methods Six-week-old SPF-grade female C57BL / 6 mice were selected and randomly divided into three groups of 12 mice each: blank control group (NC), infection control group (IC), and peptide YAMP1 treatment group (YAMP1).

[0077] Each mouse in the IC and YAMP1 groups was infected by oral gavage with 5 Toxoplasma gondii cysts, while each mouse in the NC group was infected by gavage with 200 μL. 24 hours later, each mouse in the YAMP1 group was injected by gavage with 200 μL of YAMP1 solution (i.e., the dose of YAMP1 was 10 mg / kg), and each mouse in the NC and IC groups was injected by gavage with 200 μL of PBS.

[0078] Observe each group of mice daily and record their weight and condition.

[0079] Ten days post-infection (10 dpi), six mice from each group were randomly selected and euthanized. Fecal, blood, and organ samples (brain, small intestine, colon, heart, liver, spleen, lung, and kidney) were aseptically collected. At 30 days post-infection (30 dpi), all remaining mice were euthanized, and fecal, blood, and organ samples were aseptically collected from each group.

[0080] 2. H&E staining Small intestinal tissues from mice in each group at 10 dpi and brain tissues from mice at 30 dpi were fixed overnight in 4% paraformaldehyde, followed by H&E staining, and pathological changes were observed under an optical microscope.

[0081] like Figure 10 As shown in Figure A, compared to the NC group, the IC group mice exhibited severe inflammatory infiltration in their intestines, with shortened villi, shallower crypts, and destroyed goblet cells. In contrast, the YAMP1 group mice showed more intact villi and intestinal epithelial structure, exhibiting milder inflammatory infiltration and a milder inflammatory response. Figure 10 As shown in Figure C, H&E staining of brain tissue revealed that, compared with the NC group, the IC group showed inflammatory infiltration and vascularization in the brain, while the YAMP1 group of mice had milder inflammatory response and fewer cysts in their brain tissue.

[0082] 3. qRT-PCR detection of Toxoplasma gondii load level Tissue DNA was extracted from each group of mice (brain, heart, liver, spleen, lung, and kidney) at 10 dpi using a tissue DNA extraction kit (Solarbio, Beijing, China). Using 1 μL of tissue DNA as a template, qRT-PCR was performed with primers B1-F: 5'-GCATAGGTTGCAGTCACTGACGAG-3' (SEQ ID NO:2) and B1-R: 5'-AGGCGACCAATCTGCGAATACAC-3' (SEQ ID NO:3) to detect the copy number of the B1 gene of *Toxoplasma gondii* and quantify the *Toxoplasma gondii* load in the tissues of each group of mice.

[0083] like Figure 10 As shown in B, the amount of Toxoplasma gondii B1 gene in the brain, heart, and lungs of IC group mice was higher, while the copy number of Toxoplasma gondii B1 gene in the brain, heart, liver, kidney, and spleen of YAMP1 group mice was significantly lower than that in IC group (P<0.05).

[0084] 4. Brain tissue capsule count After euthanasia, complete brain tissue was taken from the mice, cut along the midline of the sagittal plane, and half of the brain tissue was homogenized in 4 mL of PBS solution. 20 μL of the homogenate was placed on a glass slide and placed under an optical microscope to count the number of capsules. Each sample was counted 4 times, and the average value was taken. Finally, the number of capsules in the complete brain tissue of the mice was evaluated based on the obtained data.

[0085] like Figure 10 As shown in D, the brain tissue cyst count results showed that the number of cysts in the brain tissue of the YAMP1 group mice was reduced by about 60% compared with the IC group, indicating that the infection degree of the YAMP1 group mice was significantly reduced.

[0086] 5. Serum inflammatory cytokine levels The levels of inflammatory cytokines (IFN-γ, IL-12p70, IL-10, TNF-α, IL-4 and IL-6) in the serum of mice in each group were detected at 10 dpi and 30 dpi.

[0087] like Figure 11 As shown in Figures A through F, on day 10 post-infection, the serum levels of IL-12p70, IFN-γ, IL-10, and TNF-α in the YAMP1 group were significantly higher than those in the IC group; on day 30 post-infection, the serum levels of IL-12p70, IFN-γ, IL-6, IL-10, and TNF-α in the YAMP1 group were significantly higher than those in the IC group. This indicates that YAMP1 supplementation promotes the host immune response and facilitates inflammation resolution.

[0088] 6. Fecal intestinal microbial analysis 16S rRNA sequencing was performed on fecal samples from mice in each group 10 and 30 days after infection to analyze the composition of fecal microorganisms.

[0089] like Figure 12 As shown in A, regarding species composition, at the phylum level on day 10 post-infection, supplementation with peptide YAMP1 significantly modulated the Bacteroidetes phylum ( Bacteroidota ) and Proteobacteria ( Proteobacteria The restoration of ( ). For example Figure 12 As shown in B, at the genus level, supplementation with the peptide YAMP1 significantly reduced Escherichia coli (Escherichia coli spp.) Escherichia The level of ) and increased Akkermania ( Akkemaansia The abundance of ). For example, Figure 12 As shown in C and D, supplementation with peptide YAMP1 on day 30 post-infection promoted the recovery of bacterial abundance at all phyla and significantly increased the abundance of Lactobacillus.

[0090] Alpha diversity analysis showed that Toxoplasma gondii infection and YAMP1 supplementation did not show significant differences in species richness (Chao 1 index) and diversity (Simpson index).

[0091] Principal coordinate analysis (PCoA) based on Bray-Curtis distances showed that with the addition of peptide YAMP1, the IC group and the YAMP1 group exhibited significant separation at the genus level.

[0092] like Figure 12 As shown in E and F in the diagram, Venn diagrams indicate that the composition of the microbial community exhibits significant heterogeneity among different groups, and that supplementation with the peptide YAMP1 can significantly alter the community structure.

[0093] LEfSe analysis explored the differences in microbial communities among different groups. At 10 dpi, the IC group had p__ Proteobacteria g__ Helicobacter The YAMP1 group exhibits f__ characteristics. Enterobacteriaceae _A、g__ Escherichia Unique groups; at 30 dpi, p__ in the IC group Campylobacterota b__p__ Desulfobacterota The _G group is representative, and the YAMP1 group presents a pattern like g__ Romboutsia The unique groups such as _B and g__CAG_793 reflect the specificity of the microbial community structure in different groups.

[0094] 7. Fecal metabolomics analysis Fecal samples from mice in each group were collected 10 and 30 days post-infection, and metabolites were detected using positive ion mode and negative ion mode to analyze the composition of fecal metabolites.

[0095] Table 1. Summary of differentially expressed metabolites in feces of mice in each group on day 10 post-infection.

[0096] Table 2. Summary of differentially expressed metabolites in feces of mice in each group on day 30 post-infection.

[0097] As shown in Tables 1 and 2, and Figure 13As shown in A-F, on days 10 and 30 post-infection, nearly a quarter of the metabolites in the feces of mice in different groups were significantly regulated, with 64 (10 dpi) and 62 (30 dpi) overlapping metabolites being regulated, respectively.

[0098] KEGG enrichment analysis showed that on day 10 post-infection, the primary bile acid biosynthesis pathway was significantly enriched, and lipid metabolism and amino acid metabolism-related pathways were significantly affected; on day 30 post-infection, the amino acid metabolism pathway was significantly enriched, and glucose metabolism and energy production, as well as amino acid metabolism, were significantly affected.

[0099] Example 4: Biosafety evaluation of peptide YAMP1 1. Hemolysis test The hemolytic activity of peptide YAMP1 was evaluated using 4% rabbit erythrocytes (RBEs, S9452, Solarbio, China).

[0100] The working concentrations of peptide YAMP1 were adjusted to 2.5 μg / ml, 5 μg / ml, 10 μg / ml, 20 μg / ml, and 40 μg / mL using PBS. Then, 100 μL of each concentration was added to each well of a 96-well plate containing 100 μL of RBEs. The plates were incubated at 37°C for 30 min. Sterile PBS was used as a blank background to prevent hemolysis, and RBEs were treated with PBS containing 1% Triton X-100 as a positive control.

[0101] Collect RBEs from different treatments and centrifuge at 1000 rpm for 5 min.

[0102] 100 μL of the supernatant after centrifugation was transferred to a 96-well plate, and the absorbance was measured at 540 nm. The hemolysis of RBEs was evaluated based on the amount of hemoglobin released, and the hemolysis rate (%) was calculated.

[0103] like Figure 14 As shown in A, different concentrations of the peptide YAMP1 had virtually no hemolytic effect on RBEs, with hemolysis rates all below 0.004%.

[0104] 2. CCK8 assay for cytotoxicity The CCK8 assay was used to detect the cytotoxicity of YAMP1 on Ana-1 cells.

[0105] Ana-1 cells were cultured in RPMI complete medium at a concentration of 1×10⁻⁶. 5Ana-1 cells were seeded in 96-well plates and cultured for 3 h to allow them to adhere. Ana-1 cells were then treated with different working concentrations of YAMP1 (2.5 μg / mL, 5 μg / mL, 10 μg / mL, 20 μg / mL, and 40 μg / mL) at 37°C for 24 h. Following this, 10 μL of CCK8 solution was added to each well, and the cells were incubated at 37°C for another 2 h. RPMI complete medium was used as a blank background, and Ana-1 cells treated with PBS containing 1% Triton X-100 served as a positive control.

[0106] After incubation, cell supernatant was collected, and absorbance at 450 nm was measured using an ELISA reader. Cell inhibition rate (%) was then calculated.

[0107] like Figure 14 As shown in B, different concentrations of the peptide YAMP1 had virtually no inhibitory effect on the activity of Ana-1 cells, with inhibition rates all below 0.06%, indicating that YAMP1 has low cytotoxicity.

[0108] The above results indicate that the peptide YAMP1 exhibits excellent biocompatibility within the working concentration range of 2.5 μg / ml to 40 μg / mL.

[0109] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A polypeptide, characterized in that, Its amino acid sequence is shown in SEQ ID NO:

1.

2. A nucleic acid molecule, characterized in that, It encodes the polypeptide described in claim 1.

3. A conjugate, characterized in that, It comprises the polypeptide of claim 1, and the chemical moiety conjugated thereto.

4. A composition, characterized in that, It comprises the polypeptide of claim 1; preferably, it also comprises other drugs against Toxoplasma gondii infection.

5. A biomaterial, characterized in that, The biological material contains the nucleic acid molecule of claim 2, wherein the biological material is recombinant DNA, expression cassette, transposon, vector, microorganism or cell.

6. The use of the polypeptide of claim 1, the nucleic acid molecule of claim 2, the conjugate of claim 3, the composition of claim 4, or the biomaterial of claim 5 in the preparation of products that inhibit the growth and / or reproduction of Toxoplasma gondii, resist Toxoplasma gondii infection, and prevent and / or treat diseases related to Toxoplasma gondii infection.

7. The application according to claim 6, characterized in that, The product is used to achieve one or more of the following purposes: (1) It disrupts the physiological structure of Toxoplasma gondii; (2) Reduces the inflammatory response caused by Toxoplasma gondii infection; (3) Repair tissue damage caused by Toxoplasma gondii infection; (4) Reduce the load level of Toxoplasma gondii in the host; (5) Improves intestinal flora imbalance caused by Toxoplasma gondii infection.

8. The application according to claim 6 or 7, characterized in that, The products include medicines or disinfectants.

9. The application according to claim 8, characterized in that, The drug also includes a pharmaceutically acceptable carrier.

10. The method for preparing the polypeptide according to claim 1, characterized in that, Prepared using solid-phase synthesis based on the amino acid sequence shown in SEQ ID NO:1.

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