Toxoplasma gondii gene deletion strain with immune protection effect and application of Toxoplasma gondii gene deletion strain

Abstract

The invention provides a toxoplasma gondii gene deletion strain with an immune protection effect and application of the toxoplasma gondii gene deletion strain, and belongs to the technical field of veterinary parasitology, veterinary immunology and parasite molecular biology. According to the toxoplasma gondii gene deletion strain, a gamma-glutamyl hydrolase gene is deleted. The invention also provides a method for constructing the insect strain. The method comprises the following steps: constructing a circular plasmid pSAG1-Cas9-U6-sgGGH; constructing a repair template containing upstream and downstream homologous arms of the gamma-glutamyl hydrolase gene and a DHFR resistance gene; co-transfecting the annular plasmid and the repair template to a toxoplasma gondii ME49 strain; performing drug screening on pyrimethamine to obtain positive monoclone; and confirming that the GGH gene is completely knocked out through PCR (Polymerase Chain Reaction) detection, sequencing analysis and immunofluorescence detection. The toxoplasma gondii gene deletion strain can provide a remarkable protection effect on toxoplasma gondii infection and is high in safety.

Description

Technical Field

[0001] This invention belongs to the fields of veterinary parasitology, veterinary immunology and parasitic molecular biology, and specifically relates to a Toxoplasma gondii gene-deleted strain with immune protection and its application. Background Technology

[0002] Toxoplasmosis is caused by Toxoplasma gondii (… Toxoplasma gondii Protozoan diseases caused by parasites in humans and various animals seriously endanger livestock production and public health safety. T. gondii Toxoplasmosis is an obligate intracellular parasitic protozoan belonging to the phylum Apicocomplexa. It has a wide host range, infecting almost all warm-blooded animals and exhibiting a worldwide distribution. It can cause miscarriage in pregnant women, neurological damage in fetuses, and even death. Primary toxoplasmosis infection is often asymptomatic, but poses a significant threat to immunocompromised individuals (such as HIV / AIDS patients), leading to high mortality rates. In livestock production, toxoplasmosis infection can cause miscarriage or stillbirth in female animals, immunosuppression, and even acute death, resulting in substantial economic losses. Currently, there are very limited drugs for treating toxoplasmosis. A common treatment is a combination of pyrimethamine and sulfadiazine, but this only kills tachyzoites and is ineffective against bradyzoites, and can cause significant side effects. Vaccination is considered the most promising and effective immunization measure against toxoplasmosis infection, particularly suitable for specific populations such as pregnant women and HIV-infected individuals; however, human vaccines are still under development. Currently, the only commercially available toxoplasmosis vaccine for livestock and poultry is Toxovax®, a live attenuated strain of Toxoplasma gondii S48, which is approved for use in Europe and New Zealand for the prevention and treatment of congenital toxoplasmosis in sheep. However, the protective mechanism of this vaccine is not fully understood, and the S48 strain carries a high risk of retrovirus infection. Therefore, developing an ideal toxoplasmosis vaccine, especially one suitable for livestock and poultry production, remains a significant challenge.

[0003] Currently, the development of Toxoplasma gondii vaccines includes inactivated vaccines, subunit vaccines, live attenuated vaccines, DNA vaccines, epitope vaccines, and mRNA vaccines. Among these, live attenuated vaccines are considered the most effective strategy for Toxoplasma gondii vaccination. Oral administration of live attenuated vaccines can mimic the natural infection state of Toxoplasma gondii, inducing cellular and humoral immunity against Toxoplasma gondii in the host without causing disease. However, safe and highly effective live attenuated vaccines are currently lacking. Summary of the Invention

[0004] The purpose of this invention is to provide a Toxoplasma gondii gene-deleted strain with immune protection, which has high safety and good immune effect.

[0005] The objective of this invention is achieved through the following technical solution: A Toxoplasma gondii gene-deleted strain with immune protection, wherein the strain lacks the γ-glutamyl hydrolase gene.

[0006] In this invention, the coding sequence of the γ-glutamyl hydrolase gene is shown in SEQ ID NO.3, and the amino acid sequence is shown in SEQ ID NO.4.

[0007] The present invention also provides a method for the aforementioned insect strain, comprising: (1) Construct the circular plasmid pSAG1-Cas9-U6-sgGGH; (2) Construct a repair template containing upstream and downstream homologous arms of the γ-glutamyl hydrolase gene and the DHFR resistance gene; (3) The circular plasmid pSAG1-Cas9-U6-sgGGH and the repair template were co-transfected into the ME49 strain of Toxoplasma gondii by electroporation. (4) Positive monoclonal antibodies were obtained through screening with pyrimethamine; (5) Complete knockout of the GGH gene was confirmed by PCR detection, sequencing analysis and immunofluorescence detection.

[0008] In this invention, the construction method in step (1) is as follows: using pSAG1-Cas9-U6-sgUPRT plasmid as a template, PCR amplification is performed using primers shown in SEQ ID NO.1 and SEQ ID NO.2, and the circular plasmid pSAG1-Cas9-U6-sgGGH is constructed through self-homological recombination.

[0009] In this invention, the DHFR resistance gene in the repair template described in step (2) is located between the upstream and downstream homologous arms of the γ-glutamyl hydrolase gene; the sequences of the upstream and downstream homologous arms of the γ-glutamyl hydrolase gene are shown in SEQ ID NO.21 and SEQ ID NO.22 respectively, and the sequence of the DHFR resistance gene is shown in SEQ ID NO.23.

[0010] The present invention also provides the application of the aforementioned insect strain in the preparation of attenuated live vaccines.

[0011] The present invention also provides a live attenuated Toxoplasma gondii vaccine containing the aforementioned strain.

[0012] This invention utilizes CRISPR / Cas9 gene editing technology to delete the γ-glutamyl hydrolase (GGH) gene, thereby obtaining the Toxoplasma gondii gene-deleted strain (ME49). ΔGGH (The strain with the Toxoplasma gondii gene deletion) This Toxoplasma gondii gene deletion strain exhibits significantly reduced virulence and replication capacity, good genetic stability, and high safety. This deletion strain provides significant protection against infection by both RH and ME49 strains, thus demonstrating good immunization efficacy. ME49 ΔGGHThe strain can be used to prepare attenuated live vaccines and can also serve as an important tool for studying the replication mechanism of Toxoplasma gondii and the metabolic regulation mechanism of the parasite, thus having multiple scientific research applications. Attached Figure Description

[0013] Figure 1 This is a schematic diagram of the construction of a GGH gene deletion insect strain provided in an embodiment of the present invention, wherein PCR1, PCR2, and PCR3 represent PCR1 fragment, PCR2 fragment, and PCR3 fragment, respectively, for identifying the deletion strain.

[0014] Figure 2 The gel electrophoresis results of the pSAG1-Cas9-U6-sgGGH linearized vector are provided for embodiments of the present invention. M: DNA molecular weight standard DL 5000; 1: pSAG1-Cas9-U6-sgGGH.

[0015] Figure 3 The gel electrophoresis results of the repair template are provided for the embodiments of the present invention. Lane 1 is the 5'UTR-GGH amplification product; lane 2 is the 3'UTR-GGH amplification product; lane 3 is the DHFR amplification product; lane 4 is the pUC-19 linearized vector amplification product; and lane 5 is the repair template (5'UTR-DHFR-3'UTR amplification product).

[0016] Figure 4 This is a PCR identification diagram showing the successful confirmation of GGH gene deletion by PCR detection in an embodiment of the present invention. M: DNA Marker, DL 5000; Lane 1 is ME49. ΔGGH Lane 1 shows the PCR1 fragment amplification product of the ME49 strain; Lane 2 shows the PCR1 fragment amplification product of the ME49 strain; Lane 3 shows the PCR1 fragment amplification product of the ME49 strain. ΔGGH Lane 4 contains the PCR2 fragment amplification product of the ME49 strain; Lane 5 contains the PCR2 fragment amplification product of the ME49 strain. ΔGGH Lane 6 contains the PCR3 fragment amplification product of the ME49 strain.

[0017] Figure 5 The image provided in this embodiment of the invention shows the expression of GGH protein detected by IFA, in which Hoechst is used to label the cell nucleus, GAP45 is used to label the Toxoplasma gondii cell membrane, and GGH is used to label the Toxoplasma gondii GGH protein.

[0018] Figure 6 This is a graph showing the phenotypic changes in virulence of the GGH gene-deleted strain compared to the ME49 parent strain, as provided in this embodiment of the invention. The vertical axis represents the number of plaques after Toxoplasma gondii infection. ** indicates... p < 0.01.

[0019] Figure 7This is a graph showing the phenotypic changes in the replication ability of the GGH gene-deleted strain compared to the ME49 parent strain provided in this embodiment of the invention. The vertical axis represents the percentage of the total number of vacuoles containing different tachyzoites; ** indicates p <0.01, ## indicates p < 0.01.

[0020] Figure 8 This is a comparison graph showing the effect of the GGH gene-deleted parasite strain and the ME49 parental strain on mouse survival rate according to embodiments of the present invention. The numbers in parentheses represent the inoculation dose. * indicates... p < 0.05.

[0021] Figure 9 This graph shows the changes in mouse survival rates after immunization with the GGH gene-deleted parasite strain provided in this embodiment of the invention, followed by challenge with different wild-type parasite strains. * indicates... p < 0.05. Detailed Implementation

[0022] Unless otherwise stated, all technical and scientific terms used in this invention have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. While only preferred methods and materials have been described in this invention, any methods and materials similar to or equivalent to those described herein may be used in the implementation or testing of this invention.

[0023] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be apparent to those skilled in the art. This specification and embodiments are merely exemplary.

[0024] The experimental materials and tools used in this embodiment of the invention are sourced from: Experimental animals: ICR mice, 7 weeks old, purchased from Henan Skobes Company; Tools and reagents: Phanta Max high-fidelity enzyme, Tag enzyme, Trizol, DNA Marker, and homologous recombination kit were purchased from Nanjing Novizan Biotechnology Co., Ltd.; gel extraction kit, plasmid extraction kit, endotoxin-free plasmid extraction kit, and DNA extraction kit were purchased from Omega Biotechnology Co., Ltd.; T25 cell culture flasks, 12-well cell culture plates, and 6-well cell culture plates were purchased from Nest Biotechnology Co., Ltd.; fetal bovine serum was purchased from Cytiva Biotechnology Co., Ltd.; 0.4 cm electroporation cuvettes were purchased from Bio-Rad Biotechnology Co., Ltd.

[0025] Main instruments and equipment: PCR amplification instrument (TaKaRa), benchtop refrigerated centrifuge (Eppendorf), cell culture incubator (Thermo Fisher Scientific); gel imaging system, electroporation instrument (Bio-Rad).

[0026] The Cytomix buffer (pH 7.6) was prepared as follows: Weigh 1.02 g magnesium chloride (MgCl2), 22.1 mg calcium chloride (CaCl2), 8.95 g potassium chloride (KCl), 5.96 g HEPES (4-hydroxyethylpiperazine ethanesulfonic acid), 744 mg ethylenediaminetetraacetic acid (EDTA), and 100 mL of K2HPO4 / KH2PO4 solution. Dilute to 1000 mL with double-distilled water and adjust the pH to 7.6. Filter through a 0.22 µm filter and store at 4°C. The K2HPO4 / KH2PO4 solution was prepared as follows: Mix 10 mL of 1 M K2HPO4 aqueous solution and 10 mL of 1 M KH2PO4 aqueous solution, and dilute to 100 mL with water.

[0027] Example 1 ME49 ΔGGH Construction of Toxoplasma gondii 1. Materials and Methods Using Toxoplasma gondii strain ME49 (published in Saeij JP, Boyle JP, Boothroyd JC). Differences among the three major strains of Toxoplasma gondii and their specific interactions with the infected host. Trends Parasitol. 2005;21(10):476-81.) as parent strain.

[0028] The pSAG1-Cas9-U6-sgUPRT plasmid and pUPRT-DHFR-D plasmid are disclosed in Zhang Weichao. Application of TALEN and CRISPR technology in the study of Toxoplasma gondii pathogenesis mechanism [D]. Huazhong Agricultural University, 2016.

[0029] pUC-19 plasmid was purchased from Shanghai Sangon Biotech Co., Ltd.

[0030] The amplification primers used in this invention are shown in Table 1.

[0031] Table 1. Amplification primers used in this invention

[0032] The nucleotide sequence of 5'UTR-GGH (upstream homologous arm of the GGH gene) is shown in SEQ ID NO.21; the nucleotide sequence of 3'UTR-GGH (downstream homologous arm of the GGH gene) is shown in SEQ ID NO.22; the nucleotide sequence of the DHFR resistance gene is shown in SEQ ID NO.23; the sequence of PCR1 fragment for identifying the deletion strain is shown in SEQ ID NO.24; the sequence of PCR2 fragment for identifying the deletion strain is shown in SEQ ID NO.25; and the sequence of PCR3 fragment for identifying the deletion strain is shown in SEQ ID NO.26.

[0033] The gene encoding γ-glutamyl hydrolase (GGH) of Toxoplasma gondii ME49 is shown in SEQ ID NO.3, and the amino acid sequence of the protein encoded by the GGH gene is shown in SEQ ID NO.4.

[0034] 2. Extraction of total DNA from Toxoplasma gondii tachyzoites (1) Toxoplasma gondii ME49 tachyzoites were inoculated into HFF-1 cells (Chinese Academy of Sciences Stem Cell Bank) using conventional methods. The tachyzoites proliferated rapidly through internal budding. When about 50% of the parasites escaped from the cells, the remaining adherent cells were scraped off with a cell scraper. The suspension in the bottle (including the scraped-off adherent cells) was transferred to a 50 mL centrifuge tube with a pipette. Then, the suspension was repeatedly blown 8-10 times with a 5 mL syringe to completely break down the cells and release the intracellular parasites.

[0035] (2) The worm body was purified by filtration with a sterile 5 μm filter, retaining host cell fragments and unbroken cells. The filtrate containing the worm body was collected into a new 50 mL centrifuge tube, centrifuged at 1000 g at room temperature for 10 min, the supernatant was discarded, the worm body was resuspended and washed with 10 mL PBS buffer, and centrifuged at 1000 g for 10 min.

[0036] (3) Discard the supernatant, resuspend the worms in 250 μL PBS buffer, and transfer them to a centrifuge tube.

[0037] (4) Use a DNA extraction kit to extract total DNA from the worm body to obtain the total DNA of ME49 Toxoplasma gondii tachyzoites.

[0038] 3. Construction of repair template and pSAG1-Cas9-U6-sgGGH plasmid specifically targeting the GGH gene transcription region (1) Amplification of repair template Using the total DNA of ME49 Toxoplasma gondii tachyzoites as a template, and primers F1 (SEQ ID NO. 5) and R1 (SEQ ID NO. 6) as primers, 5'UTR-GGH (SEQ ID NO. 21) was amplified. Using the total DNA of ME49 Toxoplasma gondii tachyzoites as a template, and primers F2 (SEQ ID NO. 9) and R2 (SEQ ID NO. 10) as primers, 3'UTR-GGH (SEQ ID NO. 22) was amplified.

[0039] Using pUPRT-DHFR-D plasmid as a template and F3 (SEQ ID NO.7) and R3 (SEQ ID NO.8) as primers, the DHFR resistance gene (SEQ ID NO.23) was amplified; using pUC-19 plasmid as a template and F4 (SEQ ID NO.11) and R4 (SEQ ID NO.12) as primers, PCR amplification was performed to obtain the pUC-19 linearized vector.

[0040] The total PCR amplification system was as follows: 1.0 μL of ME49 Toxoplasma gondii tachyzoite total DNA / plasmid template, 25 μL of 2 × PhantaMax (Novizan), 2 μL of upstream primer F (10 pM), 2 μL of downstream primer R (10 pM), and 20 μL of sterile ultrapure water. The mixture was thoroughly mixed. The PCR program was as follows: pre-denaturation at 95℃ for 3 min; denaturation at 95℃ for 15 s, annealing at 58℃ for 15 s, extension at 72℃ for 2 min, for 35 cycles; final extension at 72℃ for 5 min.

[0041] After each PCR amplification reaction, 50 μL of the PCR product was taken and electrophoresed on a 1.5% agarose gel. The results are as follows. Figure 3 The 5'UTR-GGH amplification product was 669 bp, the 3'UTR-GGH amplification product was 1073 bp, the DHFR amplification product was 3163 bp, and the pUC-19 amplification product was 2662 bp, all of which met expectations.

[0042] The target fragment was recovered and purified using an OMEGA gel recovery kit.

[0043] Since the 5'UTR-GGH, 3'UTR-GGH, DHFR resistance genes, and pUC-19 linearized vector obtained from the above amplifications all possess homologous arms, the four PCR products were constructed into a circular plasmid containing the repair template using a homologous recombination kit (ClonExpress Ultra One Step Cloning Kit V3, Novizan). After transforming this circular plasmid into *E. coli* (DH5α), positive clones were selected, and sequencing confirmed their correct sequence. Using the circular plasmid containing the repair template as a template, PCR amplification was performed using F6 (SEQ ID NO. 13) and R6 (SEQ ID NO. 14) primers. Electrophoresis on a 1.5% agarose gel yielded the following results: Figure 3 In lane 5 of the middle swimming pool, a target band of 4905 bp was visible. After gel recovery, the repair template was obtained. In the repair template, the DHFR resistance gene was located between the upstream and downstream homologous arms of the γ-glutamyl hydrolase gene.

[0044] (2) Constructing a pSAG1-Cas9-U6-sgGGH plasmid that specifically targets the transcriptional region of the GGH gene. PCR amplification was performed using pSAG1-Cas9-U6-sgUPRT as a template and F5 (SEQ ID NO.1) and R5 (SEQ ID NO.2) as primers. The total PCR amplification system and PCR program were the same as those used in the template amplification and repair section. After PCR amplification, 50 μL of the PCR product was electrophoresed on a 1.5% agarose gel. The results are shown in the figure below. Figure 2 The amplification product was 9674 bp. Both SEQ ID NO.1 and SEQ ID NO.2 had a homologous arm sequence targeting the GGH gene transcription region introduced at their 5' ends, and the two homologous arm sequences were inversely complementary. After the PCR amplification product was digested with Dpn I enzyme (Novizan) to eliminate original plasmid contamination and purified, homologous recombinase (ClonExpress Ultra One Step Cloning Kit V3, Novizan) was added, and the mixture was incubated at 37°C for 30 min in vitro. Self-homological recombination was then performed using the complementary homologous arms at both ends of the amplification product to form a circular pSAG1-Cas9-U6-sgGGH plasmid specifically targeting the GGH gene transcription region.

[0045] After transforming the circular pSAG1-Cas9-U6-sgGGH plasmid into Escherichia coli (DH5α), positive clones were picked and sequenced correctly.

[0046] 4. ME49 ΔGGH Monoclonal screening ME49 ΔGGH See the schematic diagram of gene-deleted insect strain construction. Figure 1Using CRISPR / Cas9 gene editing technology, the γ-glutamyl hydrolase (GGH) gene in the Toxoplasma gondii ME49 strain genome was knocked out and replaced with a drug resistance selection marker (DHFR gene) through homologous recombination repair, thereby obtaining a stably inherited gene-deleted attenuated strain. The specific method is as follows: (1) Culture ME49 tachyzoites of Toxoplasma gondii in HFF-1 cells. When about 50% of the parasites have escaped from the cells, scrape off the remaining adherent cells with a cell scraper. Transfer the suspension (including the scraped-off adherent cells) in the bottle to a 50 mL centrifuge tube with a pipette. Then, repeatedly blow the suspension 8-10 times with a 5 mL syringe to completely break down the cells and release the intracellular parasites.

[0047] (2) The worms were purified by filtration through a sterile 5 μm filter, retaining host cell fragments and unbroken cells. The filtrate containing the worms was collected into a new 50 mL centrifuge tube and centrifuged at 1000 g / min at room temperature for 10 min. The supernatant was discarded, and the worms were resuspended and washed with 10 mL Cytomix buffer (pH 7.6). The worms were centrifuged at 1000 g for 10 min.

[0048] (3) Discard the supernatant, resuspend the worms in 250 μL Cytomix buffer, and transfer them to a centrifuge tube.

[0049] (4) Add to the suspension containing worms (containing 1×10 7 Add 1500 ng of the prepared repair template and 7500 ng of the circular pSAG1-Cas9-U6-sgGGH plasmid (sterilized in a 70℃ metal bath for 10 min beforehand) to a centrifuge tube containing one worm body. Mix thoroughly and transfer the entire liquid to a 4 mm electroporation cup.

[0050] (5) Place it in an electro-rotator for electro-rotation under the following conditions: 1600 V, 25 μF, 50 Ω, and electro-rotate 2 to 3 times.

[0051] (6) The electroporated insects were directly added to the prepared HFF-1 cells for normal culture.

[0052] (7) After culturing for 24 h, add 3 μM pyrimethamine to the culture medium for drug screening.

[0053] (8) After 50% of the transfected parasites have escaped from HFF-1 cells, a small number of parasites can be passaged into new HFF-1 cells (the culture medium contains pyrimethamine at a final concentration of 3 μM) for 2-3 generations of drug screening. When all parasites no longer die under the action of the drug, the parasites can be purified, and DNA can be extracted from a portion of the parasites. Preliminary PCR identification can be performed using primers for identifying the PCR1 and PCR2 fragments of the deletion strain. The remaining parasites can be passaged and single-clone strains can be screened. Normal parental parasite DNA as a template produces no amplification product, while GGH gene deletion positive parasite DNA as a template can amplify bands of 995 bp and 1125 bp, respectively.

[0054] (9) Single-clone screening: Most of the parasites initially screened as GGH gene deletion positive were passaged, and a small number of parasites were counted. After serial dilution, the parasites were placed in 2-3 96-well plates pre-conjugated with HFF-1 cells for single-clone screening. The peripheral wells were not screened. The remaining wells were filled with 200 μL of complete medium containing 3 μM pyrimethamine (DMEM high glucose medium containing 10% fetal bovine serum and 1% penicillin-streptomycin solution (100 U / mL penicillin, 100 μg / mL streptomycin, all purchased from Gibco). 0.5 / 1 / 2 / 3 parasites were serially seeded per well and placed in a 37℃, 5% CO2 cell culture incubator for 10-14 days.

[0055] (10) After culturing in a 96-well plate for about 10 to 14 days, observe the number of Toxoplasma gondii plaques in different wells and carefully select HFF-1 cell wells with a single plaque. If the plaque is small, the culture time can be extended appropriately to avoid counting errors due to some plaques being too small to be overlooked.

[0056] (11) Using a pipette tip, scrape off the adherent cells at the bottom of the cells containing the monoclonal insect strain and blow them off. Transfer the cells and the insect together to a 24-well plate filled with HFF-1 cells (complete medium containing 3 μM pyrimethamine) and continue culturing.

[0057] (12) When 80% of the HFF-1 cells in the 24-well plate are destroyed and lysed by the parasites, a small amount of parasites are taken into a new 24-well plate filled with HFF-1 cells (containing 3 μM pyrimethamine) for culture and labeled with serial numbers. The remaining parasites are used for DNA extraction.

[0058] (13) The deletion of the GGH gene was verified by PCR and sequencing, and the positive strain identified as a monoclonal strain was named ME49. ΔGGH The insect strains were passaged, cultured, and cryopreserved.

[0059] The PCR verification method is as follows: Using purified insect DNA as a template, PCR amplification was performed using primers (F7 and R7) for identifying the PCR1 fragment (SEQ ID NO. 24), primers (F8 and R8) for identifying the PCR2 fragment (SEQ ID NO. 25), and primers (F9 and R9) for identifying the PCR3 fragment (SEQ ID NO. 26). Additionally, DNA from the ME49 strain was used for the same PCR amplification as a control. The PCR products were then detected by 1.5% agarose gel electrophoresis. The results are shown below. Figure 4 As shown, ME49 ΔGGH The DNA of the parent strain could amplify PCR1 (995 bp) and PCR2 (1125 bp) fragments, but PCR3 (1300 bp) was missing. However, the DNA of the ME49 strain did not amplify PCR1 and PCR2 bands, but the PCR3 band was detectable and its size was as expected. The three amplification products were sent to the company for sequencing, and the results showed that, unlike the parent strain, ME49… ΔGGH In monoclonal strains GGH The gene was replaced with the DHFR resistance gene.

[0060] (14) ME49 was detected by indirect immunofluorescence using rat anti-GGH polyclonal antibody. ΔGGH Insect-infested strains, as shown in the following results Figure 5 As shown, compared to the parent strain, ME49 ΔGGH No green fluorescence was detected in the insect strain, further confirming that GGH protein was not expressed at all, thus successfully obtaining a stable GGH gene knockout strain ME49. ΔGGH .

[0061] The rat anti-GGH polyclonal antibody was prepared as follows: the coding sequence of the γ-glutamyl hydrolase (GGH) gene (SEQ ID NO.3) was inserted into the pET-28a plasmid (Novagen®), and then transformed into E. coli for prokaryotic induction expression. The recombinant GGH protein was purified by nickel column chromatography, and the purified recombinant GGH protein was fully immunized with adjuvant before immunizing rats. After three immunizations, the rat serum was collected, which is the rat anti-GGH polyclonal antibody.

[0062] The indirect immunofluorescence detection method is as follows: (1) Seed an appropriate amount of HFF-1 cells into a 12-well plate containing a 20 mm cell spreader and culture until the cell density reaches about 80%~90%.

[0063] (2) Inoculate with an appropriate amount of insects (1×10⁻⁶) 5 ~1×10 6(Either can be placed on a cell slide that is nearly confluent with HFF-1 cells and incubated at 37°C in a 5% CO2 cell culture incubator for 24-48 hours.)

[0064] (3) Discard the cell culture medium, add 1 mL PBS to the well containing the cell slab, gently shake the cell plate for 30 seconds, and repeat the washing 2-3 times.

[0065] (4) Add 400 μL of 4% paraformaldehyde to each well and fix at room temperature for 15 min.

[0066] (5) Discard the fixative and wash 3 times with PBS.

[0067] (6) Add 400 μL of 0.1% TritonX-100 and incubate at room temperature for 15 min to permeate.

[0068] (7) Discard the permeation solution and wash 3 times with PBS.

[0069] (8) Incubate at room temperature for 30 min using 5% BSA (diluted with PBS).

[0070] (9) Rat anti-GGH polyclonal antibody and rabbit anti-TgGAP45 polyclonal antibody were prepared respectively (see the literature Zhou P, Yu Y, Qi W, Wang X, Yu Y, Wang J, Zhang L, Yu Z, Liu T. PLGA nanoparticles as an efficient carrier in Toxoplasma GAP45: a more effective vaccine against acute toxoplasmosis than traditional ones. Front Immunol. 2025;16:1600399.) was added to diluent (5% BSA) (1:1000 dilution) to prepare a polyclonal antibody mixture. 500 μL of the polyclonal antibody mixture was added to wells containing cell smears and incubated at room temperature for 1 h or overnight.

[0071] (10) Discard the primary antibody solution and wash with PBS 3-5 times.

[0072] (11) FITC-labeled Goat Anti Rabbit IgG (H+L) (Biolegend, 405404) and Cy3-labeled Goat Anti Rabbit IgG (H+L) (Beyotime, A0507) were diluted 1:5000 using 5% BSA. 500 μL of the two labeled antibody mixture was added to each well containing cell smears and incubated at room temperature in the dark for 1 h.

[0073] (12) Discard the secondary antibody solution and wash with PBS 3-5 times (avoid light).

[0074] (13) Take 10 μL of Hochest-containing antiquenching solution on a glass slide in advance.

[0075] (14) Gently place the cell-attached slide onto the area of ​​the glass slide where the antiquenching solution has been added (the side of the cell-attached slide is in contact with the glass slide).

[0076] (15) After absorbing excess liquid with absorbent paper, seal the slide with sealing film.

[0077] (16) Use laser confocal microscopy to observe and photograph cell slides.

[0078] Example 2 ME49 ΔGGH In vitro toxicity detection ME49 was detected using the following method. ΔGGH In vitro toxicity: (1) An appropriate amount of HFF-1 cells were passaged into 6-well plates in DMEM high glucose medium (both purchased from Gibco) containing 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin double antibiotic solution (100 U / mL penicillin, 100 μg / mL streptomycin) and grown until the cell density reached 90% (forming a monolayer of cells). (2) Replace the cell culture medium with DMEM high glucose medium containing 2% FBS and 1% penicillin-streptomycin aqueous solution (100 U / mL penicillin, 100 μg / mL streptomycin), and then add the purified ME49 cells to the medium. ΔGGH Tachyzoites of the strain and wild-type ME49 strain were inoculated with 200 tachyzoites per well, with at least three replicate wells for each strain. Blank control wells containing only HFF-1 cells and no insect inoculation were also set up. (3) Place the inoculated 6-well plate in a 37°C, 5% CO2 cell culture incubator for 14 days.

[0079] (4) Observe the plaque formation of the worms in the 6-well plate under an optical microscope; (5) Add 1.5 mL of 4% paraformaldehyde to each well and fix at room temperature for 15 min.

[0080] (6) Discard the fixative and wash three times with PBS; (7) Add 2 mL of crystal violet staining solution to each well and incubate at room temperature for 1 h; (8) Discard the crystal violet staining solution and wash with PBS 4 to 6 times until obvious empty spots are visible; (9) After drying the 6-well plate in an oven at 37°C, the bottom of the 6-well plate was scanned with a scanner, and then the number of empty spots and the relative area of ​​each well were statistically analyzed.

[0081] The results are as follows Figure 6 As shown, ME49 ∆GGH The number of vacuoles in the insect-infested strain (31.2 ± 0.39) was significantly lower than that in the ME49 strain (44.3 ± 0.28). p < 0.01), a decrease of 30.0%.

[0082] Example 3: ME49 ΔGGH In vitro replication ability detection ME49 was detected using the following method. ΔGGH In vitro replication ability: (1) Seed an appropriate amount of HFF-1 cells into a 24-well plate containing a 12 mm cell spreader and cultured them in DMEM high glucose medium containing 10% fetal bovine serum and 1% penicillin-streptomycin solution (100 U / mL penicillin, 100 μg / mL streptomycin) until the cell density reaches 80%-90% (forming a complete monolayer of cells).

[0083] (2) The purified ME49 ΔGGH Freshly expelled tachyzoites of the ME49 strain and wild-type ME49 strain were processed at a ratio of 1×10⁻⁶. 5 ~1×10 6 The cells were seeded per well onto a slide containing HFF-1 cells and incubated at 37°C and 5% CO2 for 24 h. A blank control well containing only HFF-1 cells and no parasites was also included (to exclude interference from non-specific staining).

[0084] (3) Discard the cell culture medium, add 1 mL PBS to the well containing the cell slab, gently shake the cell plate for 15 seconds, and repeat the washing 2-3 times.

[0085] (4) Add 400 μL of 4% paraformaldehyde to each well and fix at room temperature for 15 min.

[0086] (5) Discard the fixative and wash 3 times with PBS.

[0087] (6) Add 400 μL of 0.1% Triton X-100 solution and permeate at room temperature for 15 min.

[0088] (7) Discard the permeation solution and wash 3 times with PBS.

[0089] (8) Incubate at room temperature for 30 min using 5% BSA (diluted with PBS).

[0090] (9) Add 500 μL of primary antibody dilution to each well containing a cell smear and incubate overnight at 4°C. Rabbit anti-TgGAP45 (5% BSA diluted 1:1000)

[0091] (10) Discard the primary antibody solution and wash with PBS 3-5 times.

[0092] (11) The secondary antibody Cy3-labeled Goat Anti Rabbit IgG (H+L) (Beyotime, A0507) was diluted with 5% BSA at a ratio of 1:5000. 500 μL of the secondary antibody dilution solution was added to each well containing cell smears and incubated at room temperature in the dark for 1 h.

[0093] (12) Take 10 μL of Hochest-containing anti-fluorescence quenching solution on a glass slide in advance.

[0094] (13) Gently place the cell-attached slide onto the area of ​​the glass slide where the anti-fluorescence quenching solution has been added (the cell-attached side is in contact with the glass slide).

[0095] (14) After absorbing excess liquid with absorbent paper, seal the slide with sealing film.

[0096] (15) Observe and count the number of vesicles under a fluorescence microscope. Record the number of vesicles with 1, 2, 4, 8 and 16 worms respectively. Count 150 vesicles for each crawling slide. Calculate the proportion of vesicles at different proliferation stages in each group to evaluate the in vitro proliferation capacity of the worms.

[0097] The results are as follows Figure 7 As shown, ME49 ΔGGH The proportion of non-replicating tachyzoites in the insect strain was 65%, which was significantly higher than the 41% proportion in the ME49 wild strain. p < 0.01), while ME49 ΔGGH The proportion of vesicles containing four tachyzoites in the insect strain was 10.4%, which was significantly lower than the 21.7% proportion in the ME49 wild strain. p < 0.01). These results suggest that ME49, during in vitro growth, ΔGGH The replication ability of the insect strain was significantly weakened compared to the parent strain.

[0098] Example 4: ME49 ΔGGH cytotoxicity testing Seven-week-old ICR mice were randomly divided into two groups of 45 mice each, labeled ME49 and ME49 respectively. ΔGGH Groups. Mice in each group were intraperitoneally inoculated with 100, 1000, or 10000 corresponding strains of the parasite (ME49 or ME49). ΔGGHThe mice were given tachyzoites (suspended in 100 μL PBS), with 15 mice per dose. They were observed for 30 days post-inoculation, and their clinical symptoms and survival were recorded. Results are as follows: Figure 8 As shown, during the 30-day observation period, the same dose (100, 1000, or 10000 doses) of ME49 was administered. ΔGGH The survival rate of the mice was significantly higher than that of the mice inoculated with the corresponding dose of ME49. p < 0.05). Among them, 100 ME49 cells were vaccinated. ΔGGH The survival rate of the tachyzoite dose group was the highest, reaching 73.3%; while the survival rate of the group inoculated with the same dose (100) of ME49 tachyzoites was only 20.0%.

[0099] Example 5: ME49 ΔGGH Detection of immune protection effect Seven-week-old ICR mice were randomly divided into an immunized group (n=40) and a non-immunized group (n=20). Each mouse in the immunized group was injected intraperitoneally with 100 ME49 molecules. ΔGGH Tachyzoites (suspended in 100 μL PBS). Thirty days after immunization, surviving immunized mice and unimmunized control mice were randomly divided into two groups of 10 mice each, for a total of four groups: ME49. ΔGGH After immunization, the RH strain group and ME49 were challenged. ΔGGH The ME49 strain group was challenged after immunization, and the RH strain group was challenged without immunization (control, see Saeij JP, Boyle JP, Boothroyd JC. Differences among the three major strains of Toxoplasma gondii and their specific interactions with the infected host. Trends Parasitol. 2005;21(10):476-81.) and the non-immunized ME49 strain group (control). Subsequently, mice in each challenge group were intraperitoneally injected with 10,000 tachyzoites of the corresponding strain (RH or ME49) (suspended in 100 μL PBS). The survival rate and clinical manifestations of the mice after challenge were recorded, and survival curves were plotted. The results are as follows Figure 9 As shown, after the above immunization procedure, the immunized mice exhibited good resistance to infection with both the RH strain and the ME49 strain, with survival rates reaching 60.0% and 80.0%, respectively, which were significantly improved compared with the corresponding unimmunized control groups. p < 0.05).

Claims

1. A Toxoplasma gondii gene-deleted strain with immunoprotective effects, characterized in that: The strain lacked the γ-glutamyl hydrolase gene.

2. The insect strain according to claim 1, characterized in that: The coding sequence of the γ-glutamyl hydrolase gene is shown in SEQ ID NO.3, and the amino acid sequence is shown in SEQ ID NO.

4.

3. A method for constructing the insect strain of claim 1, characterized in that, Includes the following steps: (1) Construct the circular plasmid pSAG1-Cas9-U6-sgGGH; (2) Construct a repair template containing upstream and downstream homologous arms of the γ-glutamyl hydrolase gene and the DHFR resistance gene; (3) The circular plasmid pSAG1-Cas9-U6-sgGGH and the repair template were co-transfected into the ME49 strain of Toxoplasma gondii by electroporation. (4) Positive monoclonal antibodies were obtained through screening with pyrimethamine; (5) Complete knockout of the GGH gene was confirmed by PCR detection, sequencing analysis and immunofluorescence detection.

4. The method according to claim 3, characterized in that... The construction method in step (1) is as follows: using pSAG1-Cas9-U6-sgUPRT plasmid as a template, PCR amplification is performed using primers shown in SEQ ID NO.1 and SEQ ID NO.2, and the circular plasmid pSAG1-Cas9-U6-sgGGH is constructed through self-homological recombination.

5. The method according to claim 4, characterized in that... In step (2), the DHFR resistance gene in the repair template is located between the upstream and downstream homologous arms of the γ-glutamyl hydrolase gene; the sequences of the upstream and downstream homologous arms of the γ-glutamyl hydrolase gene are shown in SEQ ID NO.21 and SEQ ID NO.22 respectively, and the sequence of the DHFR resistance gene is shown in SEQ ID NO.

23.

6. The use of the insect strain described in claim 1 in the preparation of a live attenuated vaccine.

7. A live attenuated Toxoplasma gondii vaccine containing the strain described in claim 1.

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