Toxoplasma gondii DAHPS gene deletion vaccine strain and application thereof

Abstract

The invention discloses a toxoplasma gondii DAHPS gene deletion vaccine strain and an application thereof. The invention provides a toxoplasma gondii gene knockout strain, the toxoplasma gondii gene knockout strain is obtained by directly knocking out a 3-deoxy-D-arabinoheptulose-7-phosphate synthase TgDAHPS gene of toxoplasma gondii through a gene editing technology, the gene ID of the TgDAHPS gene is TGGT1221260, and the nucleotide sequence of the TgDAHPS gene is shown as SEQ ID NO.1. The toxoplasma gondii gene knockout strain has the advantages that the toxoplasma gondii gene knockout strain can be used for preparing the toxoplasma The gene-deleted insect strain delta dahps provided by the invention can normally grow in vitro, does not propagate in a host and is almost non-toxic, and after the delta dahps insect strain is immunized, the host can be induced to generate good immune protection force. As a toxoplasma gondii attenuated vaccine strain, the delta dahps has the advantages of weak toxicity and almost no reproduction in vivo, can improve the resistance of a host to wild toxoplasma gondii, can be used for preventing human and animals from being infected with toxoplasma gondii, and has important significance for developing more toxoplasma gondii vaccines.

Description

Technical Field

[0001] The present invention belongs to the technical field of genetic engineering. More specifically, it relates to a Toxoplasma gondii DAHPS gene-deleted vaccine strain and its application. Background Art

[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 mainly infected by ingesting meat containing Toxoplasma gondii tissue cysts or vegetables and fruits contaminated with oocysts. Toxoplasma gondii infection can cause miscarriage, stillbirth or deformed fetuses in pregnant women or pregnant livestock, and poses a lethal risk to individuals with immune function deficiencies. There is still no safe and highly effective vaccine for the prevention and control of toxoplasmosis. The only commercially available veterinary live attenuated vaccine (Toxovax) has an unclear attenuation mechanism and there is a risk of reversion to virulence and restoration of oocyst production, which limits its widespread use. Inactivated vaccines, exosome / nanoparticle / DNA / mRNA vaccines, etc. can only partially reduce animal mortality, and the immune protection is limited. Therefore, exploring potential vaccine targets and developing candidate Toxoplasma gondii vaccines are of great significance for economic development and public health safety, etc.

[0003] The metabolic pathway is closely related to the growth and reproduction process of Toxoplasma gondii. The shikimate pathway exists in plants, fungi, bacteria and parasitic protozoa, but does not exist in mammals, making the enzymes in the shikimate pathway promising potential drug and vaccine targets. 3-Deoxy-D-arabino-heptulosonate-7-phosphate synthase (DAHPS), as the entry enzyme of the shikimate pathway, is considered to have the potential to regulate the carbon flux into the shikimate pathway. Existing studies have disclosed that the herbicide glyphosate is an effective inhibitor of the shikimate pathway enzyme EPSP synthase, and it has been proven to inhibit the in vivo growth of the apicomplexan protozoa Toxoplasma gondii, Plasmodium falciparum and Cryptosporidium parvum. However, the current research on the biological role of DAHPS in Toxoplasma gondii is still scarce, its druggability is unclear, and it is also unknown whether it can be used for the preparation of Toxoplasma gondii vaccines. Therefore, in order to develop more Toxoplasma gondii vaccines, it is necessary to discover more vaccine targets, which is of great significance for the prevention and treatment of toxoplasmosis and the development of vaccines. Summary of the Invention

[0004] The technical problem to be solved by the present invention is to overcome the deficiencies of existing vaccines for the prevention and treatment of toxoplasmosis, and provide a Toxoplasma gondii DAHPS gene-deleted vaccine strain and its application.

[0005] The first object of the present invention is to provide a Toxoplasma gondii gene knockout strain.

[0006] The second object of the present invention is to provide a method for constructing a Toxoplasma gondii gene knockout strain.

[0007] The third object of the present invention is to provide the application of the Toxoplasma gondii gene knockout strain.

[0008] The fourth object of the present invention is to provide a vaccine against Toxoplasma gondii infection.

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

[0010] The above objects of the present invention are achieved by the following technical solutions:

[0011] The present invention provides a Toxoplasma gondii gene knockout strain, wherein the strain directly knocks out the 3-deoxy-D-arabinoheptulosonate-7-phosphate synthase TgDAHPS gene of Toxoplasma gondii through gene editing technology; the gene ID of the TgDAHPS gene is TGGT1_221260, and its nucleotide sequence is as shown in SEQ ID NO.1.

[0012] The present invention directly knocks out the 3-deoxy-D-arabinoheptulosonate-7-phosphate synthase (TgDAHPS) gene in Toxoplasma gondii through the CRISPR / Cas9 technology, and finally obtains the Toxoplasma gondii TgDAHPS gene knockout vaccine strain Δdahps, and verifies the potential of the Δdahps strain in the preparation of Toxoplasma gondii vaccine through in vitro and in vivo experiments. The research shows that there is almost no difference in the plaque size between the DiCre and Δdahps strains, indicating that the deletion of TgDAHPS does not affect the growth of Toxoplasma gondii under normal culture conditions, and the Δdahps strain can grow normally in vitro; the parasite load in the ascites of mice inoculated with the Δdahps strain is significantly lower than that of the control group mice and is lower than the detection threshold, indicating that Toxoplasma gondii lacking TgDAHPS does not reproduce in the host; all the mice inoculated with different doses of the Δdahps strain survived within 30 days, and the survival rate was 100%, while all the mice infected with the parental DiCre strain died within 11 days, and the survival rate was 0%, indicating that Toxoplasma gondii lacking TgDAHPS has almost no virulence. Finally, the immune protection test of the Δdahps strain on mice was carried out, and the results showed that immunization with the Δdahps strain had good protection against the infection of wild-type Chinese I strain in mice. Therefore, the TgDAHPS gene knockout strain Δdahps can be well used as a Toxoplasma gondii vaccine to prevent Toxoplasma gondii infection.

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

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

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

[0016] (3) Using the pTub-YFP-DHFR * plasmid as a template, whose nucleotide sequence 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 co-electroporated into the parental strain, and the TgDAHPS gene knockout strain Δdahps was obtained through pyrimethamine screening 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 - 6; the primer sequences for constructing the pSAG1-Cas9-TgU6-dgTgDAHPS plasmid are shown in SEQ ID NO.7 - 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 label.

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

[0023] The present invention provides the application of a Toxoplasma gondii gene knockout strain in the preparation of a Toxoplasma gondii vaccine.

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

[0025] The present invention provides the application of the Toxoplasma gondii 3-deoxy-D-arabino-heptulosonate-7-phosphate synthase TgDAHPS gene in the construction of a gene knockout strain or a Toxoplasma gondii vaccine.

[0026] The present invention also provides the application of a reagent for knocking out the Toxoplasma gondii 3-deoxy-D-arabino-heptulosonate-7-phosphate synthase TgDAHPS gene in the construction of a gene knockout strain or a Toxoplasma gondii vaccine.

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

[0028] The Toxoplasma gondii DAHPS gene deletion vaccine strain provided by the present invention has a simple and convenient preparation method. The DAHPS of Toxoplasma gondii is directly knocked out by using gene editing technology. The constructed Toxoplasma gondii gene knockout strain Δdahps can grow normally in vitro, does not reproduce in the host body and has almost no virulence. After immunization with the Δdahps strain, it can induce good immune protection in the host. As a Toxoplasma gondii attenuated vaccine strain, Δdahps has the advantages of weak virulence and almost no reproduction in the body, and can also improve the resistance of the host to wild Toxoplasma gondii. It can be used to prevent Toxoplasma gondii infection in humans and animals, and can be used to prepare a Toxoplasma gondii vaccine, bringing new hope for the development of a Toxoplasma gondii vaccine. BRIEF DESCRIPTION OF THE DRAWINGS

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

[0030] Figure 2 It is the PCRs result of the Δdahps monoclonal strain.

[0031] Figure 3 It is the result of the plaque assay of the Δdahps strain.

[0032] Figure 4 It is the result of the virulence gradient test of the Δdahps strain.

[0033] Figure 5 It is the result of the parasite load test of the Δdahps strain.

[0034] Figure 6 It is the result of the immune protection test after immunization with the Δdahps strain and intraperitoneal inoculation with the Chinese epidemic strain Chinese I. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0035] The present invention will be further described below in conjunction with the accompanying drawings of the specification and specific embodiments. However, the embodiments do not impose any form of limitation on the present invention. Unless otherwise specified, the reagents, methods, and equipment used in the present invention are conventional reagents, methods, and equipment in the technical field.

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

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

[0038] Based on the parental strain DiCre (kindly provided by Huazhong Agricultural University), the Toxoplasma gondii DAHPS gene was directly knocked out. DiCre is a type I strain of the genus Toxoplasma in the family Toxoplasmatidae of the order Coccidia. The full name of the DAHPS gene is 3-deoxy-D-arabinoheptulosonate-7-phosphate synthase (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-sgTgD AHPS plasmids

[0040] (1) Design of specific sgRNA

[0041] Specific sgRNA primers were designed for the 5' and 3' non-coding regions of the TgDAHPS gene through 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) Linearization of the template plasmid

[0045] Using the pSAG1-Cas9-TgU6-sgTgUPRT plasmid (purchased from http: / / www.addgene.org) as a template, the CRISPR / Cas9 backbone was amplified using the primers shown in Table 2 and Phanta High-Fidelity Enzyme (product number P505, Nanjing Novoprotein Scientific Co., Ltd.). The PCR amplification reaction system and procedure are shown in Tables 3 and 4. DpnⅠ (product number 1609, Takara Biotechnology Co., Ltd.) was used to digest the linearized fragment to remove the template plasmid. After the reaction, the above products were detected by agarose gel electrophoresis. The target fragment was excised and recovered using a Gel Extraction Kit (product number DC301-01, Nanjing Novoprotein Scientific Co., Ltd.), and the concentration of the recovered product was measured using a NanoDrop One ultra-micro ultraviolet spectrophotometer (Thermo Fisher Scientific, USA), and stored at -20°C 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 Plasmid

[0053] The designed gRNA primers were respectively ligated with the linearized backbone using ExnaseⅡ enzyme (product number C112-01, Nanjing Novoprotein Scientific Co., Ltd.). The reaction system was prepared as shown in Table 5.

[0054] Table 5 Single-Fragment Ligation Reaction System

[0055]

[0056] Optimal dosage of linear cloning vector = [number of fragment base pairs × 0.02] ng, optimal dosage of insert fragment amplification product = [number of fragment base pairs × 0.04] ng

[0057] After gently mixing the reaction system, react at 37°C for 30 min. After the reaction, add all the products to 50 μL of 5α chemically competent cells (product number TSC-C01, Beijing Tsingke Biotechnology Co., Ltd.). After ice-bathing for 5 min, heat-shock at 42°C for 1 min. After ice-bathing, spread the bacterial strain evenly on LB / Amp+ On the plate, incubate overnight at 37 °C in an inverted position; randomly pick 5 single colonies for expanded culture and sequence 2 of the bacterial solutions. If the sequencing shows that the designed target sgRNA successfully replaces the UPR T-sgRNA of the original template plasmid, it indicates that the plasmids pSAG1-Cas9-TgU6-5UTR-sgTgDAHPS and pSAG1-Cas9-TgU6-3UTR-sgTgDAHPS are successfully constructed.

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

[0059] (1) Use the primers in Table 6 to amplify the temp-gRNA-3UTR-TgDAHPS fragment from the pSAG1-Cas9-TgU6-3UTR-sgTgDAHPS plasmid, and amplify the Vec-gRNA-5UTR-TgDAHPS fragment from the pSAG1-Cas9-TgU6-5UTR-sgTgDAHP S plasmid. Then, perform gel cutting recovery and concentration measurement on the above fragments. The method steps are the same as above. Ligate, transform, pick colonies, and sequence the two amplified fragments.

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

[0061]

[0062]

[0063] (2) If the sequencing result shows 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. Amplify Ko-DAHPS-YFP-DHFR * Homologous fragment

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

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

[0067]

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

[0069] (1) Collect the parental DiCre strain about to escape, filter out host cell debris with a sterile filter membrane with a pore size of 3 μm, 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 parasites, centrifuge again at 3000 rpm for 8 min, and discard the supernatant.

[0070] (2) Resuspend the parasites with 250 μL of Cytomix, add 1500 ng of the Ko-DAHPS-YFP-DHFR * homologous fragment and 7500 ng of the pSAG1-Cas9-TgU6-dgTgDAHPS CRISPR / Cas9 plasmid into a sterile 1.5 mL EP tube and mix well. Transfer all to a 4 mm electroporation cuvette and let stand for 2 min. Then place it in a BTX electroporator (model Gemini X2, BTX, USA), and set the program: 1600 V, 25 μF, 50 Ω, 4 mm shock once; 1500 V, 25 μF, 50 Ω, 4 mm shock twice. After the electroporation is completed, add all the parasite suspension to human foreskin fibroblast HFF cells (purchased from the ATCC cell bank) for culture and observation.

[0071] (3) Since this strain carries a YFP tag, when large parasitophorous vacuoles with green fluorescence are seen under a fluorescence microscope, the culture medium can be changed to a drug screening medium containing pyrimethamine. When the strain carrying the YFP tag is relatively stable, monoclonal selection can be carried out in a 96-well plate.

[0072] (4)Collect the intracellular tachyzoites from step (3), dilute and count the parasite suspension, add it to a 96-well plate at 1 Tg / 150 μL / well, and culture it in a constant temperature incubator at 37 °C with 5% CO2. After 7 days, observe whether there are monoclonal colonies under an inverted fluorescence microscope (model ECLIPSE Ts2-FL, Nikon, Japan) and an inverted optical microscope (model ECLIPSE Ts2, Nikon, Japan). Use a sterile pipette tip to scrape the host cells in the well corresponding to the monoclonal colony and add them to a 24-well plate containing HFF cells for expansion; when most of the tachyzoites in the parasitophorous vacuoles in the 24-well plate escape, scrape the cells in the well. Half of them are used to extract gDNA for monoclonal parasite strain identification, and the remaining part is passaged to a new 24-well plate for continued culture.

[0073] (5)Use the primers in Table 8 to perform PCR identification on the monoclonal parasite strains. 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 parasite strains

[0075]

[0076] The construction pattern diagram of the Toxoplasma gondii Δdahps strain is as Figure 1 shown, and the PCR identification results are as Figure 2 shown, showing that there are bands of the target size in PCR1 and PCR2 of the monoclonal parasite strain, while there is no band of the target size in PCR3, 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 lacking the TgDAHPS gene constructed in Example 2 above can be used for vaccine design, in vitro and in vivo tests were carried out respectively:

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

[0080] (1)When the DiCre and Δdahps strains are about to escape during in vitro culture, discard the original medium, wash twice with PBS, add fresh DMEM medium containing 2% FBS, use a cell scraper to scrape the cells and the parasites together, and then repeatedly pipette with a 5 mL syringe to break the parasitophorous vacuoles and let the parasites escape. The parasite suspension is filtered and purified through a filter membrane with a pore size of 3 μm, and the parasites are diluted 10-fold and counted.

[0081] (2)Inoculate 100 Tg / 3 mL / well on a 6-well plate filled with HFF cells and culture it in a 37 °C, 5% CO2 incubator for 7 days.

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

[0083] The results are as Figure 3 shown. Under normal culture conditions, there was basically no difference in the size of the visible plaques formed by the parental strain DiCre and the knockout strain Δdahps, indicating that the deletion 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] Similarly, collect and count the intracellular DiCre and Δdahps strains using DMEM medium without FBS. The DiCre strain was inoculated into 7-week-old female ICR mice (purchased from Guangzhou Regene Biotechnology Co., Ltd.) by intraperitoneal injection at a dose of 1×10 3 Tg / 200 μL / mouse, while the Δdahps strain was inoculated and infected at doses of 1×10 3 、1×10 4 、1×10 5 、1×10 6 tachyzoites / mouse. Six mice were inoculated in each group, and the survival of the mice was recorded daily. After 30 d, the results were statistically analyzed.

[0086] The results are as Figure 4 shown. It can be seen that all the mice inoculated with the parental DiCre strain died within 11 d, while the mice inoculated with different infection doses of the Δdahps strain did not die within 30 d, and the survival rate was 100%, indicating that Toxoplasma gondii lacking TgDAHPS has almost no virulence.

[0087] 3. In vivo parasite load test of Toxoplasma gondii Δdahps

[0088] Similarly, collect and count the intracellular DiCre and Δdahps strains. Both the DiCre and Δdahps strains were infected into 7-week-old female ICR mice by intraperitoneal injection at a dose of 1×10 4 Tg / 200 μL / mouse. Five mice were inoculated in each group. After 5 d, the mice were euthanized, 5 mL of normal saline was injected intraperitoneally, mixed well, the ascites of the mice was collected, centrifuged at 3500 r / min at room temperature for 6 min, the supernatant was discarded and the volume was fixed to 2 mL. 1 / 10 of the resuspended solution was taken to extract gDNA (product number DP348, Tiangen Biochemical Technology (Beijing) Co., Ltd.).

[0089] In addition, 5 mL of normal saline was intraperitoneally injected into blank ICR mice, and the mixed solution was aspirated as the negative control; 0, 10, 10 2 , 10 3 , 10 4 , 10 5 , 10 6 parental DiCre tachyzoites were added to 2 mL of the negative control solution, mixed well, and 1 / 10 of the mixed solution was taken separately to extract gDNA for the preparation of the standard curve. The parasite load in the ascites of mice was detected using a real-time quantitative PCR (qPCR) instrument (product number 05015278001, Roche LightCycler 480 Instrument II, USA). The detection primers are shown in Table 9. The qPCR reaction system shown in Table 10 was prepared in a 96-well plate, and the reaction program 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 program

[0095]

[0096] The qPCR results are as Figure 5 shown, indicating that the parasite load in the ascites of mice inoculated with the Δdahps knockout strain was significantly lower than that of the parental DiCre control group, and far lower than the detection threshold, suggesting that Toxoplasma gondii lacking TgDAHPS does not reproduce in vivo.

[0097] 4. Immunoprotection test

[0098] (1) Five 7-week-old mice were intraperitoneally inoculated with 1×10 2 Δdahps tachyzoites / mouse; 72 days later, the immunized mice and non-immunized mice were inoculated with 1×10 4 Chinese I tachyzoites of the Chinese epidemic strain (kindly provided by Huazhong Agricultural University, which is a dominant virulent strain in China), and the death of the mice was observed and recorded. The survival rate of the mice was statistically analyzed 30 days later.

[0099] The results are as Figure 6 shown. All the control mice inoculated with Chinese I died within 10 days, and 100% of the mice immunized with Δdahps survived after reinfection with a high dose of Chinese I tachyzoites within 30 days, indicating that inoculation with the Δdahps strain can generate good immunoprotection.

[0100] In summary, the present invention provides a Toxoplasma gene knockout strain. By means of gene editing technology, the 3-deoxy-D-arabinoheptulosonate-7-phosphate synthase gene (TgDAHPS) of Toxoplasma was directly knocked out on the parental strain DiCre, thereby obtaining the Δdahps strain. This strain can grow normally in vitro, cannot reproduce in the host body and has almost no virulence; the Δdahps strain can induce immune protection in mice after infecting them, and can be used to prepare a genetic engineering vaccine against toxoplasmosis, 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 other changes, modifications, substitutions, combinations, and simplifications made without departing from the spirit and principle of the present invention shall be equivalent replacement methods and are all included in the protection scope of the present invention.

Claims

1. A Toxoplasma gondii gene knockout strain, characterized in that: The strain is obtained by directly knocking out the 3-deoxy-D-arabinoheptulose-7-phosphate synthase TgDAHPS gene of Toxoplasma gondii through gene editing technology; the gene ID of the TgDAHPS 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: The following steps are involved: (1) Using the pSAG1-Cas9-TgU6-sgTgUPRT plasmid as a template, gRNA sequences targeting the 5' and 3' non-coding regions of TgDAHPS were designed to construct pSAG1-Cas9-TgU6-5UTR-sgTgDAHPS and pSAG1-Cas9-TgU6-3UTR-sgTgDAHPS plasmids; (2) Using the homology and homology 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' UTRs of TgDAHPS; (3) pTub-YFP-DHFR * The plasmid was used as a template, and its nucleotide sequence is shown in SEQ ID NO.2 to amplify the homologous recombination template Ko-DAHPS-YFP-DHFR * ; (4) The CRISPR / Cas9 plasmid constructed in step (2) and the homologous recombination template amplified in step (3) were co-transfected into the starting insect strain, and the TgDAHPS gene knockout strain Δdahps was obtained through pyrimethamine screening and PCR identification.

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: The primer sequences used to construct the CRISPR / Cas9 plasmid pSAG1-Cas9-TgU6-dgTgDAHPS in step (2) are shown in SEQ ID NOs. 5 to 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 the DiCre strain.

7. 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. Use of the Toxoplasma 3-deoxy-D-arabinoheptulose-7-phosphate synthase TgDAHPS gene in constructing a gene knockout strain or a Toxoplasma vaccine, characterized in that: The gene ID of the TgDAHPS gene is TGGT1_221260, and its nucleotide sequence is shown in SEQ ID NO.

1.

10. Use of a reagent for knocking out the Toxoplasma 3-deoxy-D-arabinoheptulose-7-phosphate synthase TgDAHPS gene in constructing a gene knockout strain or a Toxoplasma vaccine, characterized in that: The gene ID of the TgDAHPS gene is TGGT1_221260, and its nucleotide sequence is shown in SEQ ID NO.1.

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