Toxoplasma gondii DAHPS gene deletion vaccine strain and application thereof

Abstract

The application discloses a Toxoplasma gondii DAHPS gene deletion vaccine strain and application thereof. The application provides a Toxoplasma gondii gene knockout strain, which is obtained by directly knocking out a 3-deoxy-D-arabino-heptulosonate-7-phosphate synthase TgDAHPS gene of the Toxoplasma gondii through a gene editing technology, the gene ID of the TgDAHPS gene is TGGT1_221260, and the nucleotide sequence is shown as SEQ ID NO. 1. The gene deletion strain Δdahps provided by the application can grow normally in vitro, does not reproduce in a host in vivo, and is almost non-toxic. After immunization of the Δdahps strain, the host can be induced to have good immune protection. The Δdahps has the advantages of weak virulence and almost no reproduction in vivo as the attenuated vaccine strain of the Toxoplasma gondii, can improve the resistance of the host to wild Toxoplasma gondii, and can be used for preventing the infection of the Toxoplasma gondii in humans and animals, and has important significance for developing more Toxoplasma gondii vaccines.

Description

Technical Field

[0001] This invention belongs to the field of genetic engineering technology. More specifically, it relates to a Toxoplasma gondii DAHPS gene deletion vaccine strain and its application. Background Technology

[0002] Toxoplasma gondii is an important zoonotic parasitic protozoan that can infect almost all warm-blooded animals, including humans, causing toxoplasmosis. Toxoplasma gondii has multiple transmission routes; humans and animals are primarily infected through ingesting meat containing Toxoplasma gondii tissue cysts or vegetables and fruits contaminated with oocysts. Toxoplasmosis infection can cause miscarriage, stillbirth, or birth defects in pregnant women and animals, and poses a fatal risk to immunocompromised individuals. Currently, there is no safe and effective vaccine for toxoplasmosis. The only commercially available live attenuated veterinary vaccine (Toxovax) has an unclear attenuation mechanism and carries the risk of virulence reversion and restoration of oocyst production, limiting its widespread use. Inactivated vaccines, exosome / nanoparticle / DNA / mRNA vaccines, etc., can only partially reduce animal mortality and offer limited immunoprotective efficacy. Therefore, identifying potential vaccine targets and developing candidate vaccines for Toxoplasma gondii are of great significance for economic development and public health security.

[0003] Metabolic pathways are closely linked to the growth and reproduction of Toxoplasma gondii. The shikimic acid pathway exists in plants, fungi, bacteria, and parasitic protozoa, but not in mammals, making enzymes in this pathway potential drug and vaccine targets. 3-Deoxy-D-arabinohepulose-7-phosphate synthase (DAHPS), as the entry enzyme in the shikimic acid pathway, is considered to have the potential to regulate carbon flux into the pathway. Existing research has shown that the herbicide glyphosate is an effective inhibitor of the shikimic acid pathway enzyme EPSP synthase, demonstrating its ability to inhibit the in vivo growth of Toxoplasma gondii, Plasmodium falciparum, and Cryptosporidium microsporum. However, current research on the biological role of DAHPS in Toxoplasma gondii and its targeting properties is limited; its potential for use in Toxoplasma gondii vaccine development remains unclear. Therefore, to develop more Toxoplasma gondii vaccines, it is necessary to identify more vaccine targets, which is of great significance for the prevention and control of toxoplasmosis and vaccine development. Summary of the Invention

[0004] The technical problem to be solved by this invention is to overcome the shortcomings of existing toxoplasmosis prevention and control vaccines, and to provide a Toxoplasma gondii DAHPS gene deletion vaccine strain and its application.

[0005] The first objective of this invention is to provide a Toxoplasma gondii gene knockout strain.

[0006] The second objective of this invention is to provide a method for constructing Toxoplasma gondii gene knockout strains.

[0007] A third objective of this invention is to provide applications for Toxoplasma gondii gene knockout strains.

[0008] A fourth objective of this invention is to provide a vaccine against Toxoplasma gondii infection.

[0009] The fifth object of this invention is to provide the application of a reagent for knocking out the Toxoplasma gondii 3-deoxy-D-arabinohepenosaccharide-7-phosphate synthase TgDAHPS gene.

[0010] The above-mentioned objective of this invention is achieved through the following technical solution:

[0011] This invention provides a Toxoplasma gondii gene knockout strain, wherein the 3-deoxy-D-arabinohepnisyl-7-phosphate synthase TgDAHPS gene of Toxoplasma gondii was directly knocked out using gene editing technology; the gene ID of the TgDAHPS gene is TGGT1_221260, and its nucleotide sequence is shown in SEQ ID NO.1.

[0012] This invention utilizes CRISPR / Cas9 technology to directly knock out the 3-deoxy-D-arabinohepenosaccharide-7-phosphate synthase (TgDAHPS) gene in Toxoplasma gondii, ultimately obtaining the TgDAHPS gene knockout vaccine strain Δdahps. In vitro and in vivo experiments have verified the potential of the Δdahps strain in the preparation of Toxoplasma gondii vaccines. Studies have shown that the plaque size of the DiCre and Δdahps strains was almost identical, indicating that the absence of TgDAHPS does not affect the growth of Toxoplasma gondii under normal culture conditions, and the Δdahps strain can grow normally in vitro. Mice inoculated with the Δdahps strain had significantly lower levels of cephalopods in ascites than the control group, and these levels were below the detection threshold, indicating that Toxoplasma gondii without TgDAHPS does not reproduce in the host. Mice inoculated with different doses of the Δdahps strain all survived within 30 days (100% survival rate), while mice infected with the parental DiCre strain all died within 11 days (0% survival rate), indicating that Toxoplasma gondii without TgDAHPS has almost no virulence. Finally, an immunoprotective efficacy test of the Δdahps strain in mice was conducted, showing that immunization with the Δdahps strain provided good protection against wild-type Chinese I strain infection in mice. Therefore, the TgDAHPS knockout strain Δdahps can be effectively used as a Toxoplasma gondii vaccine for the prevention of Toxoplasma gondii infection.

[0013] This invention provides a method for constructing a Toxoplasma gondii gene knockout strain, comprising the following steps:

[0014] (1) Using pSAG1-Cas9-TgU6-sgTgUPRT plasmid as a template, gRNA sequences targeting the 5' and 3' uncoding regions of TgDAHPS were designed to construct pSAG1-Cas9-TgU6-5UTR-sgTgDAHPS and pSAG1-Cas9-TgU6-3UTR-sgTgDAHPS plasmids;

[0015] (2) By using the homologous and homogroup method, the pSAG1-Cas9-TgU6-5UTR-sgTgDAHPS and pSAG1-Cas9-TgU6-3UTR-sgTgDAHPS plasmids were merged to construct the CRISPR / Cas9 plasmid pSAG1-Cas9-TgU6-dgTgDAHPS that simultaneously targets the 5' and 3' uncoding regions of TgDAHPS;

[0016] (3) pTub-YFP-DHFR * Using a plasmid as a template, the nucleotide sequence of which is shown in SEQ ID NO.2, the homologous recombination template Ko-DAHPS-YFP-DHFR was amplified. * ;

[0017] (4) The CRISPR / Cas9 plasmid constructed in step (2) and the homologous recombination template amplified in step (3) were electrotransfected into the starting strain. The TgDAHPS gene knockout strain Δdahps was obtained by screening with pyrimethamine and PCR identification.

[0018] Preferably, the gRNA sequences used in step (1) are shown in SEQ ID NO.3 and SEQ ID NO.4.

[0019] More preferably, the primer sequences for constructing the pSAG1-Cas9-TgU6-5UTR-sgTgDAHPS and pSAG1-Cas9-TgU6-3UTR-sgTgDAHPS plasmids are shown in SEQ ID NO. 3 to 6; and the primer sequences for constructing the pSAG1-Cas9-TgU6-dgTgDAHPS plasmid are shown in SEQ ID NO. 7 to 10.

[0020] Preferably, the primer sequences used for the homologous recombination template in step (3) are shown in SEQ ID NO.11 and SEQ ID NO.12.

[0021] Preferably, the homologous recombination template in step (3) contains a pyrimethamine drug screening tag.

[0022] Preferably, the starting strain used in step (4) is the DiCre strain.

[0023] This invention provides the application of Toxoplasma gondii gene knockout strains in the preparation of Toxoplasma gondii vaccines.

[0024] This invention provides a vaccine against Toxoplasma gondii infection, containing the above-mentioned Toxoplasma gondii gene knockout strain.

[0025] This invention provides the application of the Toxoplasma gondii 3-deoxy-D-arabinohepenosaccharide-7-phosphate synthase TgDAHPS gene in the construction of gene knockout strains or Toxoplasma gondii vaccines.

[0026] This invention also provides the application of reagents for knocking out the Toxoplasma gondii 3-deoxy-D-arabinohepenosaccharide-7-phosphate synthase TgDAHPS gene in the construction of gene knockout strains or Toxoplasma gondii vaccines.

[0027] The present invention has the following beneficial effects:

[0028] The Toxoplasma gondii DAHPS gene-deleted vaccine strain provided by this invention has a simple and convenient preparation method. It utilizes gene editing technology to directly knock out the DAHPS gene of Toxoplasma gondii. The constructed Toxoplasma gondii gene knockout strain Δdahps can grow normally in vitro, does not reproduce in the host, and is almost non-virulent. Immunization with the Δdahps strain can induce good immune protection in the host. As an attenuated Toxoplasma gondii vaccine strain, Δdahps has the advantages of low virulence and almost no reproduction in the body. It can also enhance the host's resistance to wild-type Toxoplasma gondii and can be used to prevent Toxoplasma gondii infection in humans and animals. It can be used to prepare a Toxoplasma gondii vaccine, bringing new hope to the development of Toxoplasma gondii vaccines. Attached Figure Description

[0029] Figure 1 This is a schematic diagram of the construction of the Toxoplasma gondii Δdahps strain.

[0030] Figure 2 The results of PCRs for the Δdahps monoclonal strain are shown.

[0031] Figure 3 The results are from the plaque phagocytosis test of the Δdahps insect strain.

[0032] Figure 4 The results are from the virulence gradient test of the Δdahps strain.

[0033] Figure 5 The results are from the Δdahps insect load test.

[0034] Figure 6 The results of an immunoprotective test of intraperitoneal inoculation with the Chinese prevalent strain Chinese I after immunization with the Δdahps strain. Detailed Implementation

[0035] The present invention will be further described below with reference to the accompanying drawings and specific embodiments, but the embodiments do not limit the present invention in any way. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in this technical field.

[0036] Unless otherwise specified, all reagents and materials used in the following examples are commercially available.

[0037] Example 1: Construction of Toxoplasma gondii gene knockout plasmid

[0038] Based on the parent strain DiCre (a gift from Huazhong Agricultural University), the DAHPS gene of Toxoplasma gondii was directly knocked out. DiCre is a type I strain of Toxoplasma gondii in the family Toxoplasmidae of the order Coccidia. The full name of the DAHPS gene is 3-deoxy-D-arabinohepulose-7-phosphate synthetase (Class-IIDAHP synthetase family protein, TgDAHPS, gene ID: TGGT1_221260), and its nucleotide sequence is shown in SEQ ID NO.1.

[0039] 1. Construction of pSAG1-Cas9-TgU6-5UTR-sgTgDAHPS and pSAG1-Cas9-TgU6-3UTR-sgTgDAHPS plasmids

[0040] (1) Design of specific sgRNA

[0041] Specific sgRNA primers for the 5' and 3' untranslated regions of the TgDAHPS gene were designed using the sgRNA website (http: / / www.e-crisp.org / E-CRISP / designcrispr.html), as shown in Table 1.

[0042] Table 1 Primers for constructing TgDAHPS-specific CRISPR / Cas9 plasmids

[0043]

[0044] (2) Template plasmid linearization

[0045] Using the pSAG1-Cas9-TgU6-sgTgUPRT plasmid (purchased from http: / / www.addgene.org) as a template, the CRISPR / Cas9 backbone was amplified using primers as shown in Table 2 and Phanta high-fidelity enzyme (catalog number P505, Nanjing Novizan Biotechnology Co., Ltd.). The PCR amplification reaction system and procedure are shown in Tables 3 and 4. The linearized fragment was digested with DpnI (catalog number 1609, Baori Biotechnology Co., Ltd.) to remove the template plasmid. After the reaction, the above products were detected by agarose gel electrophoresis. The target fragment was extracted and recovered using a gel extraction kit (catalog number DC301-01, Nanjing Novizan Biotechnology Co., Ltd.). The concentration of the recovered product was determined using a NanoDrop One ultraviolet spectrophotometer (model NanoDrop One, Thermo Fisher Scientific, USA). The product was stored at -20℃ for later use.

[0046] Table 2 Primers for amplifying the CRISPR / Cas9 backbone

[0047]

[0048] Table 3 PCR amplification reaction system

[0049]

[0050] Table 4 PCR amplification reaction procedure

[0051]

[0052] (4) Construction of plasmids

[0053] The gRNA primers designed above were ligated to the linearized backbone using Exnase II enzyme (catalog number C112-01, Nanjing Novizan Biotechnology Co., Ltd.). The reaction system was prepared as shown in Table 5.

[0054] Table 5 Single-fragment linkage reaction system

[0055]

[0056] The optimal amount of linear cloning vector = [number of fragment base pairs × 0.02] ng; the optimal amount of insert fragment amplification product = [number of fragment base pairs × 0.04] ng.

[0057] After gently mixing the reaction mixture, react at 37°C for 30 min. Once the reaction is complete, add all the product to a 50 μL container. In 5α chemocompetent cells (catalog number TSC-C01, Beijing Qingke Biotechnology Co., Ltd.), the cells were incubated on ice for 5 min, followed by heat shock at 42℃ for 1 min. After the ice incubation, the bacterial culture was evenly spread on LB / Am p+ Incubate overnight at 37°C inverted on a plate; randomly select 5 single colonies for expansion culture and sequence 2 of them. If the sequencing shows that the designed target sgRNA successfully replaces the UPR T-sgRNA of the original template plasmid, it indicates that the pSAG1-Cas9-TgU6-5UTR-sgTgDAHPS and pSAG1-Cas9-TgU6-3UTR-sgTgDAHPS plasmids have been successfully constructed.

[0058] 2. Construction of pSAG1-Cas9-TgU6-dgTgDAHPS CRISPR / Cas9 plasmid

[0059] (1) Using the primers in Table 6, the temp-gRNA-3UTR-TgDAHPS fragment was amplified from the pSAG1-Cas9-TgU6-3UTR-sgTgDAHPS plasmid, and the Vec-gRNA-5UTR-TgDAHPS fragment was amplified from the pSAG1-Cas9-TgU6-5UTR-sgTgDAHPS plasmid. The fragments were then gel-cleaved and their concentrations were determined. The same procedure was followed. The two amplified fragments were then ligated, transformed, picked, and sequenced.

[0060] Table 6 Primers for amplifying temp-gRNA-3UTR-TgDAHPS and Vec-gRNA-5UTR-TgDAHPS fragments

[0061]

[0062]

[0063] (2) If the sequencing results show that the temp-gRNA-3UTR-TgDAHPS fragment is inserted into the Vec-gRNA-5UTR-TgDAHPS plasmid, it indicates that the pSAG1-Cas9-TgU6-dgTgDAHPS CRISPR / Cas9 plasmid has been successfully constructed.

[0064] 3. Amplification of Ko-DAHPS-YFP-DHFR * Homologous fragments

[0065] With Tub-YFP-DHFR * (This team constructed the plasmid, article doi:10.1038 / s41467-024-47097-8, its sequence is shown in SEQ ID NO.2) Using the plasmid as a template, PCR amplification was performed using Phanta high-fidelity enzyme according to the primers in Table 7, and the amplification product was purified.

[0066] Table 7 Amplification of Ko-DAHPS-YFP-DHFR* Primers for the fragment

[0067]

[0068] Example 2: Construction of the Toxoplasma gondii gene knockout strain Δdahps

[0069] (1) Collect the parental DiCre strains that are about to escape, filter them with a sterile filter membrane with a pore size of 3 μm to remove host cell debris, and centrifuge at 3000 rpm for 8 min; discard the supernatant, add 8 mL of Cytomix (120 mM KCl, 0.15 mM CaCl2, 10 mM K2HPO4 / KH2PO4, 25 mM HEPES, 2 mM EDTA, 5 mM MgCl2, pH=7.6) to resuspend the strains, centrifuge again at 3000 rpm for 8 min, and discard the supernatant.

[0070] (2) Resuspend the worms in 250 μL of Cytomix and add 1500 ng of Ko-DAHPS-YFP-DHFR * Homologous fragments and 7500 ng of pSAG1-Cas9-TgU6-dgTgDAHPS CRISPR / Cas9 plasmid were added to a sterile 1.5 mL EP tube and mixed thoroughly. The mixture was then transferred to a 4 mm electroporation cuvette and allowed to stand for 2 min. Subsequently, the cuvette was placed in a BTX electroporator (Gemini X2, BTX Corporation, USA) and the following program was set: 1600 V, 25 μF, 50 Ω, 4 mm for one electroporation; 1500 V, 25 μF, 50 Ω, 4 mm for two electroporations. After electroporation, the entire suspension of the parasites was added to human foreskin fibroblasts (HFF cells, purchased from ATCC Cell Bank) for culture and observation.

[0071] (3) Because the strain carries the YFP tag, when large green fluorescent vesicles are observed under a fluorescence microscope, the culture medium can be replaced with a drug screening medium containing pyrimethamine. When the strain containing the YFP tag is relatively stable, single clones can be selected in a 96-well plate.

[0072] (4) Collect the intracellular tachyzoites from step (3), dilute and count the worm suspension, add 1 Tg / 150 μL / well to a 96-well plate, and incubate at 37°C in a constant temperature incubator containing 5% CO2. After 7 days, observe whether there are monoclonal cells under an inverted fluorescence microscope (model ECLIPSE Ts2-FL, Nikon Corporation, Japan) and an inverted optical microscope (model ECLIPSE Ts2, Nikon Corporation, Japan). Use a sterile pipette tip to scrape the host cells corresponding to the monoclonal cells from the wells and add them to a 24-well plate containing HFF cells for propagation. When most of the tachyzoites in the vesicles of the 24-well plate have escaped, scrape off the cells from the wells. Half of them are used to extract gDNA for monoclonal worm identification, and the remaining part is passaged into a new 24-well plate for further culture.

[0073] (5) Use the primers in Table 8 to identify the monoclonal strain by PCR. The PCR reaction system and procedure are the same as those in Tables 3 and 4.

[0074] Table 8 Primers for PCR identification of Δdahps monoclonal strains

[0075]

[0076] The schematic diagram of the Toxoplasma gondii Δdahps strain is shown below. Figure 1 As shown, the PCR identification results are as follows: Figure 2 As shown, PCR1 and PCR2 of the monoclonal strain contain bands of the target size, while PCR3 does not contain a target band, indicating that the TgDAHPS gene has been successfully knocked out in the DiCre strain and the Δdahps strain has been obtained.

[0077] Example 3: Effect of the Δdahps vaccine strain

[0078] To verify whether the Toxoplasma gondii vaccine strain Δdahps, which lacks the TgDAHPS gene and was constructed in Example 2 above, can be used for vaccine design, in vitro and in vivo experiments were conducted respectively:

[0079] 1. In vitro plaque assay of Δdahps strain

[0080] (1) When the DiCre and Δdahps strains are about to escape from the in vitro culture, discard the original culture medium, wash twice with PBS, add fresh medium containing 2% FBSDMEM, scrape off the cells along with the insects with a cell scraper, and then repeatedly blow with a 5mL syringe to rupture the vacuoles and release the insects. The insect suspension is filtered and purified using a 3μm pore size filter membrane, and the insects are diluted 10 times and counted.

[0081] (2) 100 Tg / 3 mL / well was seeded into a 6-well plate with HFF cells and cultured in a 37℃, 5% CO2 incubator for 7 days.

[0082] (3) Discard the culture medium in the 6-well plate and wash twice with PBS; after fixing with 4% paraformaldehyde, stain the cells with 0.1% crystal violet, wash with PBS 1-2 times after 20 min, air dry, and then scan the 6-well plate with a scanner (ScanMaker i600, Shanghai Zhongjing Technology Co., Ltd.).

[0083] The results are as follows Figure 3 As shown, under normal culture conditions, the size of the visual patches formed by the parent strain DiCre and the knockout strain Δdahps is basically the same, indicating that the absence of TgDAHPS does not affect the growth of Toxoplasma gondii under normal culture conditions.

[0084] 2. In vivo virulence gradient test of Toxoplasma gondii Δdahps in mice

[0085] As above, intracellular DiCre and Δdahps strains were collected and counted using FBS-free DMEM medium. DiCre strains were counted at 1×10⁻⁶. 3 Seven-week-old female ICR mice (purchased from Guangzhou Ruige Biotechnology Co., Ltd.) were injected intraperitoneally with a dose of Tg / 200 μL / mouse, while the Δdahps strain was administered at a dose of 1×10⁻⁶. 3 1×10 4 1×10 5 1×10 6 Six mice were inoculated with the parasite / mouse. The survival rate of the mice was recorded daily, and the results were statistically analyzed after 30 days.

[0086] The results are as follows Figure 4 As shown, all mice inoculated with the parent DiCre strain died within 11 days, while mice inoculated with different infection doses of the Δdahps strain did not die within 30 days, with a survival rate of 100%, indicating that Toxoplasma gondii lacking TgDAHPS is almost non-virulent.

[0087] 3. Toxoplasma gondii Δdahps in vivo load test

[0088] As above, intracellular DiCre and Δdahps strains were collected and counted. Both DiCre and Δdahps strains were counted at a rate of 1 × 10⁻⁶. 4 Seven-week-old female ICR mice were infected via intraperitoneal injection at a dose of Tg / 200 μL / mouse. Five mice were inoculated in each group. Five days later, the mice were euthanized, and 5 mL of physiological saline was injected intraperitoneally. The mixture was then collected, and the ascites fluid was centrifuged at 3500 r / min for 6 min at room temperature. The supernatant was discarded, and the volume was adjusted to 2 mL. gDNA (catalog number DP348, Beijing Tiangen Biotech Co., Ltd.) was extracted from 1 / 10 of the resuspended fluid.

[0089] In addition, 5 mL of physiological saline was injected intraperitoneally into blank ICR mice, and the mixed solution was used as a negative control; 0, 10, and 10 mL of saline were injected intraperitoneally into the mice. 2 10 3 10 4 10 5 10 6 One parental DiCre tachyzoite was added to 2 mL of negative control solution, mixed well, and 1 / 10 of the mixture was taken to extract gDNA for the preparation of the standard curve. The amount of *Ceratophyllum demersum* in mouse ascites was detected using a real-time quantitative PCR (qPCR) instrument (catalog number 05015278001, Roche LightCycler 480 II). The detection primers are shown in Table 9. The qPCR reaction system was prepared in a 96-well plate as shown in Table 10, and the reaction procedure is shown in Table 11.

[0090] Table 9 Primers used for qPCR

[0091]

[0092] Table 10 qPCR reaction system

[0093]

[0094] Table 11 qPCR reaction procedure

[0095]

[0096] qPCR results as follows Figure 5 As shown, the amount of *Toxoplasma gondii* in mice inoculated with the Δdahps knockout strain was significantly lower than that in mice in the parental DiCre control group, and was far below the detection threshold, indicating that *Toxoplasma gondii* without TgDAHPS does not reproduce in vivo.

[0097] 4. Immunoprotection test

[0098] (1) With 1×10 2 Five 7-week-old mice were intraperitoneally inoculated with Δdahps parasites; 72 days later, immunized and unimmunized mice were inoculated with 1×10⁻⁶ parasites. 4 A Chinese I tachyzoite strain (a gift from Huazhong Agricultural University, representing a dominant and highly virulent strain in my country) was used to observe and record mouse mortality. The survival rate of mice was calculated after 30 days.

[0099] The results are as follows Figure 6 As shown, all control mice inoculated with Chinese I died within 10 days, while mice immunized with Δdahps and then reinfected with a high dose of Chinese I parasites had 100% survival within 30 days, indicating that inoculation with the Δdahps parasite strain can produce good immune protection.

[0100] In summary, this invention provides a Toxoplasma gondii gene knockout strain. Through gene editing technology, the 3-deoxy-D-arabinohepnigulipose-7-phosphate synthase gene (TgDAHPS) of Toxoplasma gondii was directly knocked out in the parent strain DiCre, thereby obtaining the Δdahps strain. This strain can grow normally in vitro, cannot reproduce in the host, and is almost non-virulent. Infection of mice with the Δdahps strain can induce immune protection, and it can be used to prepare an anti-toxoplasmosis genetically engineered vaccine, providing more options for the prevention and treatment of toxoplasmosis.

[0101] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. A Toxoplasma gondii gene knockout strain, characterized in that, The strain was modified by gene editing technology to directly knock out the 3-deoxy-D-arabinoheptulose-7-phosphate synthase in Toxoplasma gondii. Tg The DAHPS gene was obtained; Tg The gene ID of the DAHPS gene is TGGT1_221260, and its nucleotide sequence is shown in SEQ ID NO.

1.

2. The method for constructing the Toxoplasma gondii gene knockout strain according to claim 1, characterized in that, Includes the following steps: (1) pSAG1-Cas9- Tg U6-sg Tg Using UPRT plasmids as templates, targeted therapies were designed. TgDAHPS The gRNA sequences of the 5' and 3' uncoding regions were used to construct pSAG1-Cas9- Tg U6-5UTR-sg Tg DAHPS and pSAG1-Cas9- Tg U6-3UTR-sg Tg DAHPS plasmid; (2) By using the homologous and homogroup method, pSAG1-Cas9- Tg U6-5UTR-sg Tg DAHPS and pSAG1-Cas9- Tg U6-3UTR-sg Tg DAHPS plasmids were merged to construct a simultaneous targeting system. TgDAHPS The CRISPR / Cas9 plasmid pSAG1-Cas9- contains the 5' and 3' uncoding regions. Tg U6-dg Tg DAHPS; (3) Using pTub-YFP- DHFR * Using a plasmid as a template, the nucleotide sequence of which is shown in SEQ ID NO.2, the homologous recombination template Ko- was amplified. DAHPS -YFP- DHFR * ; (4) The CRISPR / Cas9 plasmid constructed in step (2) and the homologous recombination template amplified in step (3) were co-electrotransfected into the starting strain, and the resulting strains were screened with pyrimethamine and identified by PCR. Tg DAHPS gene knockout strain Δdahps.

3. The construction method according to claim 2, characterized in that, The gRNA-specific primer sequences used in step (1) are shown in SEQ ID NO.3 and SEQ ID NO.

4.

4. The construction method according to claim 2, characterized in that, In step (2), the CRISPR / Cas9 plasmid pSAG1-Cas9- is constructed. Tg U6-dg Tg The primer sequences used in DAHPS are shown in SEQ ID NO.5~10.

5. The construction method according to claim 2, characterized in that, In step (3), the homologous recombination template contains a pyrimethamine drug screening tag.

6. The construction method according to claim 2, characterized in that, The starting strain used in step (4) is DiCre Insect strains.

7. The use of the Toxoplasma gondii gene knockout strain according to claim 1 in the preparation of Toxoplasma gondii vaccine.

8. A vaccine against Toxoplasma gondii infection, characterized in that, Contains the Toxoplasma gondii gene knockout strain as described in claim 1.

9. Knockout of Toxoplasma gondii 3-deoxy-D-arabinohepeptulose-7-phosphate synthase Tg The application of reagents for the DAHPS gene in constructing gene knockout strains or in preparing Toxoplasma gondii vaccines is characterized by, The Tg The gene ID of the DAHPS gene is TGGT1_221260, and its nucleotide sequence is shown in SEQ ID NO.1.

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