A Toxoplasma gondii succinyl-CoA synthetase α-subunit gene-deficient vaccine strain and its application

Abstract

The present invention discloses a Toxoplasma succinyl-CoA synthetase α subunit gene-deficient vaccine strain and its application. The present invention provides a Toxoplasma gene-deficient vaccine strain (Δscsα), which is obtained by knocking out the Toxoplasma succinyl-CoA synthetase α subunit (TgSCSα) gene through gene editing technology; Toxoplasma can still be cultured in vitro after TgSCSα is knocked out and has almost non-toxic characteristics in vivo; after animals are immunized with Δscsα, they can produce good immune protection and effectively resist infection with wild-type Toxoplasma. TgSCSα has the potential to be used as a target for Toxoplasma vaccine design and can be used to prepare Toxoplasma gene-deficient vaccine strains. Δscsα is a candidate vaccine strain with good application prospects, which can provide more options and strategies for the prevention and control of toxoplasmosis.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and more specifically, relates to a Toxoplasma gondii succinyl-CoA synthetase α subunit gene-deficient vaccine strain and its application. Background Art

[0002] Toxoplasma gondii is an important zoonotic parasite that can infect all warm-blooded animals, including humans, causing adverse consequences such as miscarriage and stillbirth in pregnant women or livestock, and even leading to death in immunocompromised and immunodeficient people. Toxoplasmosis is widespread and harmful worldwide, but there are currently no ideal drugs and vaccines. The prevention and treatment of toxoplasmosis mainly use pyrimethamine and sulfadiazine, but they are only effective against Toxoplasma tachyzoites in the acute infection period, ineffective against tissue cysts in the chronic infection period, and have certain side effects. Currently, only Toxovax, a Toxoplasma vaccine, is approved for the prevention of toxoplasmosis in sheep. It is made from the attenuated S48 strain, but the vaccine strain has the risk of reversion to virulence and restoration of oocyst formation ability, which limits its widespread use. Therefore, it is urgent to develop a safe and efficient Toxoplasma vaccine.

[0003] Metabolic pathways are closely related to the growth, reproduction and virulence of the parasite and are potential targets for drug and vaccine development. The tricarboxylic acid cycle (TCA) is an important metabolic pathway and energy source in Toxoplasma gondii. Succinic acid is one of the intermediate products in the TCA cycle. Succinyl-CoA can be converted into succinic acid under the catalysis of succinyl-CoA synthetase, promoting the TCA cycle and energy production, providing energy support and metabolic intermediates for the life activities of Toxoplasma gondii. Previous studies have used a tetracycline-mediated conditional knockdown system to conditionally knock down the α subunit of Toxoplasma succinyl-CoA synthetase (SCSα). They found that conditional knockdown of SCSα had no significant effect on the differentiation of parasite bradyzoites and mitochondrial membrane potential, and only slightly slowed down the proliferation rate of the parasite in vitro (the growth rate decreased by about 30%) (Fleige T, Pfaff N, Gross U, et al. Localisation of gluconeogenesis and tricarboxylic acid (TCA)-cycle enzymes and first functional analysis of the TCA cycle in Toxoplasma gondii. [J]. International Journal for Parasitology, 2008, 38 (10): 1121-1132. DOI: 10.1016 / j.ijpara.2008.01.007.). This study did not reveal the relationship between SCSα and the reproduction and virulence of the parasite in vivo, and it is also unknown whether it can be used as a vaccine target. Currently, there is a severe lack of vaccines or effective vaccine strains that can be used to prevent toxoplasmosis. There is an urgent need to develop more vaccines and strains that are safe, non-toxic and effective in preventing toxoplasmosis, and to provide more methods and technical support for preventing and controlling Toxoplasma infection. Summary of the invention

[0004] The technical problem to be solved by the present invention is to overcome the deficiencies of existing vaccine strains or vaccines against toxoplasmosis, and to provide a Toxoplasma gondii succinyl-CoA synthetase α subunit gene-deficient vaccine strain and its application.

[0005] The purpose of the present invention is to provide a Toxoplasma gondii succinyl-CoA synthetase α-subunit gene-deficient vaccine strain.

[0006] Another object of the present invention is to provide a method for preparing a Toxoplasma gondii succinyl-CoA synthetase α subunit gene-deficient vaccine strain and its application.

[0007] Another object of the present invention is to provide a Toxoplasma gondii vaccine.

[0008] Another object of the present invention is to provide the application of the Toxoplasma gondii succinyl-CoA synthetase α subunit gene or its deletion or knockout reagent.

[0009] The above-mentioned purpose of the present invention is achieved through the following technical solutions:

[0010] The present invention provides a Toxoplasma gondii succinyl-CoA synthetase α subunit gene-deficient vaccine strain, which is obtained by directly knocking out the Toxoplasma succinyl-CoA synthetase α subunit gene TgSCSα through gene editing technology, and the nucleotide sequence of the TgSCSα gene is shown in SEQ ID NO.1.

[0011] The present invention focuses on the Toxoplasma succinyl-CoA synthetase α subunit (TgSCSα) gene, evaluates the role of TgSCSα in parasite growth and pathogenicity, and reveals the potential of TgSCSα as a drug target or vaccine design target. After knocking out the TgSCSα gene, the constructed deletion strain (Δscsα) can still be cultured in vitro, and the deletion will significantly affect the in vitro growth rate of Toxoplasma and has almost non-toxic characteristics in vivo; after animal immunization, it has good immune protection, and the immunized animals gain immunity against wild-type Toxoplasma infection and have the advantage of reduced virulence. As a target for Toxoplasma vaccine design, it can be used to prepare Toxoplasma gene knockout attenuated live vaccine strains, providing effective means and methods for preventing and treating toxoplasmosis.

[0012] Furthermore, through CRISPR / Cas9 gene editing technology, TgSCSα was knocked out of the wild-type DiCre strain to obtain the gene-deficient vaccine strain Δscsα.

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

[0014] (1) Using the pCas9-U6-sgMIC3 plasmid as a template, the gRNA primer of the TgSCSα gene was used for homologous recombination to construct the pCas9-U6-5UTR-sgSCSα plasmid and the pCas9-U6-3UTR-sgSCSα plasmid, respectively, and then used them as templates to construct the pCas9-gRNA1-gRNA2-SCSα plasmid;

[0015] (2) Using the cDNA of the DiCre strain as a template, amplify the CDS of the TgSCSα gene; using the pTub-loxp-YFP-DHFR*-loxp plasmid as a template, amplify the plasmid backbone; connect the backbone with the CDS to construct the pTub-loxp-SCSα-loxp-YFP-DHFR* plasmid; using the constructed pTub-loxp-SCSα-loxp-YFP-DHFR* plasmid as a template, use homologous fragment amplification primers to amplify the Tub::loxp-SCSα-loxp::YFP::DHFR* homologous recombination template;

[0016] (3) The pCas9-gRNA1-gRNA2-SCSα plasmid and the homologous recombination template constructed in step (2) were co-electroplated into the wild-type Toxoplasma gondii strain, and the Toxoplasma gondii TgSCSα gene-deficient strain was obtained through drug screening and PCR identification.

[0017] Preferably, the wild-type Toxoplasma gondii strain in step (3) is a wild-type DiCre strain.

[0018] More preferably, the gRNA primer sequence of the TgSCSα gene in step (1) is as shown in SEQ ID NO.2 and SEQ ID NO.3.

[0019] More preferably, the sequences of primers for amplifying the Tub::loxp-SCSα-loxp::YFP::DHFR* homologous fragment in step (2) are as shown in SEQ ID NOs. 12 to 13.

[0020] The present invention provides the use of a Toxoplasma gondii succinyl-CoA synthetase alpha subunit gene-deficient vaccine strain in the preparation of an anti-toxoplasmosis drug.

[0021] The invention provides a drug for resisting toxoplasmosis, comprising a vaccine strain of Toxoplasma gondii with a gene deletion of α-subunit of succinyl-CoA synthetase.

[0022] The invention provides a toxoplasma attenuated live vaccine, comprising a Toxoplasma succinyl-CoA synthetase alpha subunit gene-deficient vaccine strain.

[0023] The present invention provides the use of a Toxoplasma gondii succinyl-CoA synthetase alpha subunit gene as a target in screening or preparing drugs or vaccines against toxoplasmosis.

[0024] The present invention also provides the use of a reagent for deleting or knocking out the α subunit gene of Toxoplasma gondii in constructing a Toxoplasma gene-deficient strain or in preparing a Toxoplasma vaccine.

[0025] The present invention has the following beneficial effects:

[0026] The present invention provides a toxoplasma gene knockout attenuated live vaccine strain, which is obtained by directly knocking out the toxoplasma succinyl-CoA synthetase α subunit (TgSCSα) gene through gene editing technology; after the TgSCSα gene is knocked out, the toxoplasma can still be cultured in vitro and has almost non-toxic characteristics in vivo; after the animal is immunized with Δscsα, good immune protection is generated, and the ability to resist wild-type toxoplasma infection is obtained. TgSCSα can be used as a target for toxoplasma vaccine design, can be used to prepare toxoplasma gene-deficient vaccine strains, and provide effective means and methods for preventing and treating toxoplasmosis. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 This is a schematic diagram for knocking out Toxoplasma Δscsα.

[0028] Figure 2 These are the PCR results of the Δscsα monoclonal strain.

[0029] Figure 3 These are the results of the plaque assay of the Δscsα strain.

[0030] Figure 4 These are the results of the insect load experiment on the Δscsα strain.

[0031] Figure 5 These are the results of the virulence gradient experiment of the Δscsα strain.

[0032] Figure 6 These are the results of the immune protection experiment when mice immunized with the Δscsα strain were infected with the wild-type ME49 strain. DETAILED DESCRIPTION

[0033] The present invention is further described below in conjunction with the accompanying drawings and specific examples, but the examples do not limit the present invention in any form. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in the art.

[0034] Unless otherwise specified, the reagents and materials used in the following examples are commercially available.

[0035] Example 1 Construction of Toxoplasma gondii gene knockout Δscsα strain

[0036] In this example, gene knockout was performed on the basis of the parent strain of Toxoplasma gondii DiCre (kindly donated by Huazhong Agricultural University). The succinyl-CoA-synthetase alpha subunit gene TgSCSα (succinyl-CoA-synthetase alpha, gene ID: TGGT1_290600) in the ToxoDB (www.toxodb.org) website was searched for in the Toxoplasma gondii type I strain. The nucleotide sequence of the TgSCSα gene is shown in SEQ ID NO.1.

[0037] 1. Construction of pCas9-gRNA1-gRNA2-SCSα plasmid

[0038] (1) Design TgSCSα gene-specific gRNA primers through the E-CRISP Design website (http: / / www.e-crisp.org / E-CRISP / designcrispr.html), as shown in Table 1, and design specific plasmids according to the gRNA sequence.

[0039] Table 1 Primers for constructing pCas9-U6-5UTR-sgSCSα plasmid and pCas9-U6-3UTR-sgSCSα plasmid

[0040]

[0041] (2) Using pCas9-U6-sgMIC3 plasmid (kindly provided by Huazhong Agricultural University) as a template, pCas9-U6-5UTR-sgSCSα plasmid and pCas9-U6-3UTR-sgSCSα plasmid backbone fragments were amplified to construct TgSCSα-specific CRISPR / Cas9 plasmids. The amplification primers are shown in Table 2. The backbone fragments were ligated with TgSCSα-specific primers in Table 1, and the ligation system was shown in Table 3. The ligation was carried out at 37°C for 30 min to construct pCas9-U6-5UTR-sgSCSα plasmid and pCas9-U6-3UTR-sgSCSα plasmid.

[0042] Table 2 Primers for amplifying backbone fragments of pCas9-U6-5UTR-sgSCSα plasmid and pCas9-U6-3UTR-sgSCSα plasmid

[0043]

[0044] Table 3 Reaction system

[0045]

[0046] (3) The product of the previous step was added to Escherichia coli DH5α competent cells, first ice-bathed and then heat-shocked at 42°C, and then ice-bathed again and spread on an ampicillin-resistant solid culture plate, cultured overnight, and a single colony was picked and placed in a liquid culture medium containing ampicillin for expansion. If the sequencing result of the bacterial solution was correct, it means that the pCas9-U6-5UTR-sgSCSα plasmid and the pCas9-U6-3UTR-sgSCSα plasmid were successfully constructed.

[0047] (4) Use the primers in Table 4 to amplify the backbone and insert fragments required for constructing the TgSCSαCRISPR / Cas9 plasmid, and connect the linearized backbone and insert fragments according to the method in step (2).

[0048] Table 4 Primers for constructing TgSCSαCRISPR / Cas9 plasmid

[0049]

[0050]

[0051] (5) Referring to step (3), the pCas9-gRNA1-gRNA2-SCSα plasmid was successfully constructed.

[0052] 2. Amplify Tub::loxp-SCSα-loxp::YFP::DHFR* homologous fragment

[0053] Using the cDNA of the DiCre strain as a template, the primers in Table 5 were used to amplify the CDS of the TgSCSα gene; using the pTub-loxp-YFP-DHFR*-loxp plasmid (kindly provided by Huazhong Agricultural University) as a template, the primers shown in SEQ ID NO.8-9 were used to amplify the plasmid backbone; referring to the methods mentioned in (2) and (3) in step 1 above, the backbone and CDS were connected to construct the pTub-loxp-SCSα-loxp-YFP-DHFR* plasmid;

[0054] The pTub-loxp-SCSα-loxp-YFP-DHFR* plasmid was used as a template for homologous fragment amplification, the primers were as shown in Table 6, and the PCR amplification product Tub::loxp-SCSα-loxp::YFP::DHFR* homologous fragment was recovered by gel recovery.

[0055] Table 5 Primers for constructing pTub-loxp-SCSα-loxp-YFP-DHFR* plasmid

[0056]

[0057] Table 6 Primers for amplifying Tub::loxp-SCSα-loxp::YFP::DHFR* homologous fragments

[0058]

[0059] 3. Construction of the Toxoplasma gondii knockout strain Δscsα

[0060] (1) DiCre tachyzoites were collected by centrifugation at 3000 rpm for 7 min, and the worms were resuspended in 5 mL Cytomix (120 mM KCl, 0.15 mM CaCl2, 10 mM K2HPO4 / KH2PO4, 25 mM HEPES, 2 mM EGTA, 5 mM MgCl2, pH = 7.6). The worms were centrifuged again at 3000 rpm for 8 min and resuspended in 300 μL cytomix.

[0061] (2) Add 1500 ng of Tub::loxp-SCSα-loxp::YFP::DHFR* homologous fragment and 7500 ng of pCas9-gRNA1-gRNA2-SCSα plasmid, as well as the resuspended insect body in step (1) into a 1.5 ml EP tube, pipette and mix well, and transfer all to a 4 mm electric rotating cup.

[0062] (3) Electrotransfection was performed using a Bio-Rad electroporator using the following conditions: 1600 V, 25 μF, 50 Ω, 4 mm once, and gently shake the liquid to mix; 1500 V, 25 μF, 50 Ω, 4 mm once, and gently shake the liquid to mix; 1500 V, 25 μF, 50 Ω, 4 mm once, and then let it stand. Finally, all the liquid in the electroporation cup was added to human fibroblast HFF cells (purchased from ATCC cell bank) for culture.

[0063] (4) 1 μM pyrimethamine was used for drug screening. After 2-3 rounds of pyrimethamine screening culture, the wild-type Toxoplasma gradually decreased. At this time, the electroporated strain library was identified by PCR using the primers in Table 6. The results showed that both PCR1 and PCR2 of the electroporated strain library had bands at the target size, while the wild-type DiCre had no bands, indicating that the electroporated strain library contained strains with the TgSCSα gene successfully knocked out. The construction model of the Toxoplasma Δscsα gene knockout strain is shown in the figure below. Figure 1 shown.

[0064] Table 7 PCR primers for identification of monoclonal strains of Δscsα

[0065]

[0066] The electroporated strain library was treated with rapamycin for 3 days to remove the CDS of the TgSCSα gene in the strain. The tachyzoites of the electroporated strain library were collected and limited dilution was performed. One Toxoplasma tachyzoite was added to each well of a 96-well plate filled with cells. After the strains grew for about a week, the single-spotted strains were observed and selected under a microscope. Half of the tachyzoites in the wells were collected to extract DNA for strain identification, and the other half were subcultured and expanded.

[0067] (7) PCR identification was performed on the monoclonal strain selected in the previous step using the primers in Table 6. The results are as follows: Figure 2 As shown, PCR1 and PCR2 of the monoclonal strain have bands at the target size, but PCR3 has no band; while PCR1 and PCR2 of the wild-type DiCre have no bands at the target size, and PCR3 amplifies a band, indicating that the TgSCSα gene has been knocked out in the strain, and the Δscsα monoclonal strain has been successfully obtained.

[0068] Example 2 In vitro plaque assay of Δscsα strain

[0069] (1) Washing the extracellular tachyzoites with PBS, scraping and blowing HFF cells to make the parasites escape, and collecting the intracellular tachyzoites of the DiCre strain and the Δscsα strain constructed in Example 1 by differential centrifugation;

[0070] (2) Add 100 tachyzoites collected in the previous step to each well of a 6-well plate filled with HFF cells and culture in a cell culture incubator for one week.

[0071] (3) After washing with PBS, fix with 4% PFA at 37°C for half an hour;

[0072] (4) Discard the fixative and stain with 0.1% crystal violet at 37°C for half an hour;

[0073] (5) After washing with PBS and drying, the plaque images were scanned, the plaques were counted and the area was measured, and the statistical results were analyzed using Student's t test.

[0074] The results are as follows Figure 3 As shown, compared with the wild-type DiCre strain, knocking out the TgSCSα gene significantly affected the in vitro growth rate of Toxoplasma gondii, but the Δscsα strain could still grow and form visualized plaques.

[0075] Example 3 In vivo proliferation experiment of Δscsα strain

[0076] (1) Use DMEM medium without FBS to collect intracellular tachyzoites of the Δscsα strain and the wild-type DiCre strain;

[0077] (2) Count and dilute the tachyzoites. Δscsα and DiCre strains were diluted to 1×10 4 Five experimental mice (Guangzhou Ruige Biotechnology Co., Ltd.) were inoculated with each dose. Five days after infection, normal saline was injected into the peritoneal cavity of the mice to collect the parasites in the peritoneal cavity. The mixture of normal saline and ascites in the peritoneal cavity of the mice was centrifuged at 3000rpm for 8min, and a small amount of supernatant was discarded until 2mL of liquid remained in the tube. The tube was resuspended and 200μL of liquid was drawn for gDNA extraction. The blank mice were injected with normal saline intraperitoneally, and the negative control liquid was extracted. 0, 10, 10 2 , 10 3 , 10 4 , 10 5 , 10 6 Each DiCre tachyzoite was added to 2 mL of negative control solution, and 200 μL of liquid was aspirated for gDNA extraction to prepare a standard curve; the worm load in mice was detected by real-time fluorescence quantitative PCR (qPCR), the detection primers are shown in Table 7, the qPCR reaction system is shown in Table 8, and the reaction procedure is shown in Table 9.

[0078] Table 7 Primers used for qPCR

[0079]

[0080] Table 8 qPCR reaction system

[0081]

[0082] Table 9 qPCR reaction procedure

[0083]

[0084] The worm-loaded samples were tested using LC480, and the Ct values ​​of the worm-loaded samples were substituted into the standard curve to calculate the specific worm-loaded number of mice, and a bar graph was drawn to statistically show the results.

[0085] Test results such as Figure 4 As shown, the wild-type DiCre strain reproduced in large numbers in mice, and the number of Δscsα strains in mice was significantly reduced compared with the wild-type strain, indicating that the loss of TgSCSα significantly affects the growth ability of the worm in vivo.

[0086] Example 4 In vivo toxicity experiment of Δscsα strain

[0087] DiCre and Δscsα tachyzoites were collected and diluted in DMEM medium without FBS. 100 DiCre tachyzoites / mouse were inoculated into 8 experimental mice. 1000 and 10000 tachyzoites of Δscsα strain were inoculated into 8 experimental mice respectively. 1×10 5 , 1×10 6 Five experimental mice were inoculated with each dose. The mental state and survival status of the mice were recorded. After two weeks, blood was collected from the retro-orbital vein of the mice, and the serum of the mice was tested by indirect fluorescent immunoassay, and the serum-negative mice were eliminated. The mortality data of the mice were analyzed using Prism software.

[0088] The results are as follows Figure 5 As shown in the figure, mice inoculated with wild-type DiCre strains had some dead individuals on the 8th day after infection, and all mice died on the 15th day after infection; while the inoculation dose was 1×10 3 , 1×10 4 , 1×10 5 , 1×10 6 All mice carrying Δscsα parasites survived, indicating that the Δscsα strain is almost non-toxic in mice.

[0089] Example 5 Experiment on the immune protection of Δscsα strain on mice

[0090] (1) The extracellular tachyzoites were washed off with PBS, and the intracellular tachyzoites of Toxoplasma gondii ME49 strain (kindly donated by Huazhong Agricultural University) were collected by adding DMEM medium without FBS. The collected liquid was diluted 10 times and counted under a microscope using a cell counting plate. After counting, the tachyzoites were diluted;

[0091] (2) In the virulence gradient experiment of Example 3, all 8 mice inoculated with 10,000 Δscsα strains survived; one month after inoculation, the surviving mice and 5 blank mice were inoculated with 10 4 The ME49 experiment was performed by intraperitoneal inoculation at different doses of tachyzoites / mouse, and the mouse survival rate curve was drawn using prism5.

[0092] The results are as follows Figure 6 As shown, unimmunized control mice were infected for 10 4 After the ME49 strain was introduced, the survival rate was only 20% within one month; 4 The survival rate of mice immunized with Δscsα tachyzoites was 100% within one month, indicating that vaccination with the Δscsα strain has good immune protection for mice against infection with wild-type Toxoplasma gondii strains and can be used to prepare Toxoplasma vaccines.

[0093] In summary, the present invention provides a Toxoplasma gene-deficient vaccine strain, which is obtained by directly knocking out the Toxoplasma succinyl-CoA synthetase α subunit (Δscsα) gene through gene editing technology; Toxoplasma can still be cultured in vitro after the TgSCSα gene is knocked out, and has almost non-toxic characteristics in vivo; after animals are immunized with Δscsα, they can produce good immune protection to resist infection with wild-type Toxoplasma. TgSCSα can be used as a target for Toxoplasma vaccine design and can be used to prepare Toxoplasma gene-deficient vaccine strains. The Toxoplasma gene-deficient vaccine strain (Δscsα) provided by the present invention provides a safe and effective vaccine candidate for the prevention and treatment of toxoplasmosis, and provides more methods for the research and development of anti-toxoplasmosis vaccines.

[0094] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be equivalent replacement methods and are included in the protection scope of the present invention.

Claims

1. Use of a Toxoplasma gondii succinyl-CoA synthetase α subunit gene-deficient vaccine strain in the preparation of a Toxoplasma gondii vaccine, characterized in that: Through CRISPR / Cas9 gene editing technology, DiCre Knockout TgSCSα Gene, to obtain gene-deficient vaccine strains Δscsα ; TgSCSα The nucleotide sequence of the gene is shown in SEQ ID NO.

1.

2. The use according to claim 1, characterized in that: The method for preparing the Toxoplasma gondii succinyl-CoA synthetase α subunit gene-deficient vaccine strain comprises the following steps: (1) Using pCas9-U6-sgMIC3 plasmid as template, TgSCSα The gRNA primers of the gene were used for homologous recombination to construct pCas9-U6-5UTR-sg SCSα Plasmids and pCas9-U6-3UTR-sg SCSα plasmid, and then used it as a template to construct pCas9-gRNA1-gRNA2- SCSα Plasmids; (2) DiCre The cDNA of the insect strain was used as a template to amplify TgSCSα CDS of gene; pTub-loxp-YFP- DHFR* -loxp plasmid was used as a template to amplify the plasmid backbone; the above backbone was connected to CDS to construct pTub-loxp- SCSα -loxP-YFP- DHFR* Plasmid; constructed pTub-loxp- SCSα -loxP-YFP- DHFR* The plasmid was used as a template and homologous fragment amplification primers were used to amplify Tub::loxp- SCSα -loxp::YFP:: DHFR* Homologous recombination template; (3) Place pCas9-gRNA1-gRNA2- SCSα The plasmid and the homologous recombination template constructed in step (2) were co-electroplated into Toxoplasma gondii DiCre Among the strains, Toxoplasma gondii was obtained through drug screening and PCR identification. TgSCSα Gene deletion strains.

3. A live attenuated Toxoplasma vaccine, characterized in that: Contains the gene-deficient vaccine strain as claimed in claim 1.

4. Use of a reagent for deleting or knocking out the α subunit gene of Toxoplasma gondii succinyl-CoA synthetase in the preparation of a Toxoplasma gondii vaccine, characterized in that: The nucleotide sequence of the gene is shown in SEQ ID NO.1.

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