Construction method and application of toxoplasma gondii polygene deletion vaccine strain

Abstract

The invention discloses a construction method and application of a toxoplasma gondii polygene deletion vaccine strain. According to the invention, a TgBFD1 gene and a TgDAHPS gene are respectively knocked out from an ME49 [delta] 6pgdh1 insect strain through a gene editing technology, and a polygene deleted insect strain [delta] 6pgdh1 [delta] bfd1 [delta] dahps is successfully constructed; the strain can be subcultured in vitro, has the characteristics of no toxicity and no formation of tissue cysts in a host body, has better immune protection after immunization and inoculation, and can resist lethal infection of highly toxic strains. The toxoplasma gondii polygene deletion strain provided by the invention has better safety than ME49 delta 6pgdh1 strain, the toxicity of the toxoplasma gondii polygene deletion strain is obviously reduced, tissue cysts are not formed, the toxoplasma gondii polygene deletion strain can effectively resist infection of wild poisonous strains, the toxoplasma gondii polygene deletion strain has good immune protection capability, the defects that ME49 delta 6pgdh1 still has toxicity and can form tissue cysts are overcome, and the toxoplasma gondii polygene deletion strain has good application prospects. A method is provided for developing a safe and efficient toxoplasma gondii vaccine.

Description

Technical Field

[0001] The present invention belongs to the technical fields of biomedicine and genetic engineering. More specifically, it relates to a construction method and application of a multi-gene deletion vaccine strain of Toxoplasma gondii. Background Art

[0002] Toxoplasma gondii is an obligate intracellular parasite that can infect almost all warm-blooded animals, and the general population is susceptible. Feline animals are the definitive hosts of Toxoplasma gondii, in which Toxoplasma gondii can complete sexual reproduction and excrete oocysts with feces. Toxoplasma gondii is highly harmful to pregnant women, which can cause miscarriage or stillbirth. Fetal infection with Toxoplasma gondii may lead to congenital toxoplasmosis, causing fetal optic nerve damage, hydrocephalus, etc.

[0003] Toxoplasma gondii is the culprit of miscarriage, bringing great harm to human health and the development of the livestock and poultry breeding industry, triggering serious social problems and causing huge economic losses. In the treatment of toxoplasmosis, the most commonly used drugs are pyrimethamine and sulfadiazine, but these drugs are only effective against tachyzoites in the acute infection stage, ineffective against bradyzoites or tissue cysts in the chronic infection stage, and there are also toxic side effects. Vaccination is of great significance for blocking congenital infection and preventing acquired infection. However, currently only the passaged and attenuated S48 strain has been prepared into a live attenuated vaccine of Toxoplasma gondii. Since the mechanism of virulence attenuation of the S48 strain is still unclear, and there is a risk of reversion to virulence and restoration of oocyst formation, its wide application is limited.

[0004] Compared with other types of vaccines, the gene deletion live vaccine constructed using the CRISPR / Cas9 gene editing technology has the advantages of clear genetic background, good safety, strong immune protection, etc., bringing new hope for the development of new anti-toxoplasmosis vaccines. The gene deletion strain ME49Δ6pg dh1 (CN114933970A) developed and prepared by the applicant's team in the early stage has reduced virulence in vivo and high immune protection for animals, and can be used to prepare candidate Toxoplasma gondii vaccines. However, considering that ME49Δ6pgdh1 still has a certain degree of acute virulence and can still form a certain number of tissue cysts in the host, there is a certain risk of pathogen transmission, which will hinder its clinical use in livestock and pets. Therefore, it is necessary to continue to optimize and transform it to reduce the virulence of the candidate vaccine strain and the risk of cyst formation, in order to overcome the problems existing in the ME49Δ6pgdh1 strain and obtain more safe and efficient candidate vaccine strains that are non-toxic and do not form cysts. Summary of the Invention

[0005] The technical problem to be solved by the present invention is to overcome the deficiencies of the existing Toxoplasma gondii ME49Δ6pgdh1 strain, which has acute virulence, can form tissue cysts, and has a risk of pathogen transmission, and to provide a Toxoplasma gondii multi-gene deletion vaccine strain that is almost non-toxic and does not form tissue cysts, as well as its construction method and application.

[0006] The first object of the present invention is to provide a Toxoplasma gondii multi-gene deletion strain.

[0007] The second object of the present invention is to provide a construction method for the Toxoplasma gondii multi-gene deletion strain.

[0008] The third object of the present invention is to provide an application of the Toxoplasma gondii multi-gene deletion strain.

[0009] The fourth object of the present invention is to provide a Toxoplasma gondii vaccine.

[0010] The fifth object of the present invention is to provide an application of a reagent lacking the Toxoplasma gondii Myb-like transcription factor TgBFD1 gene and the 3-deoxy-D-arabino-heptulosonate-7-phosphate synthase TgDAHPS gene.

[0011] The above objects of the present invention are achieved by the following technical solutions:

[0012] The present invention provides a Toxoplasma gondii multi-gene deletion strain, which is obtained by knocking out the 6-phosphogluconate dehydrogenase Tg6PGDH1 gene, the Myb-like transcription factor TgBFD1 gene, and the 3-deoxy-D-arabino-heptulosonate-7-phosphate synthase TgDAHPS gene of Toxoplasma gondii by gene editing technology; the gene ID of the Tg6PGDH1 gene is: TGME49_242600, the gene ID of the TgBFD1 gene is: TGME49_200385, and the gene ID of the TgDAHPS gene is: TGME49_221260; the nucleotide sequences of the TgBFD1 gene and the TgDAHPS gene are shown in SEQ ID NO.1 to 2 in sequence.

[0013] The present invention focuses on the regulatory factor TgBFD1 gene involved in the differentiation and development of Toxoplasma gondii and the TgDAHPS gene that exists in Toxoplasma gondii but is absent in the host. Based on the existing Toxoplasma gondii gene knockout strain ME49Δ6pgdh1, through the CRISPR / Cas9 gene editing technology, the Myb-like transcription factor (bradyzoite-formation deficient protein) TgBFD1 gene (gene ID: TGME49_200385) and the 3-deoxy-D-arabino-heptulosonate-7-phosphate synthase (Class-IIDAHP synthetase family protein) TgDAHPS gene (gene ID: TGME49_221260) of Toxoplasma gondii were further knocked out to obtain a Toxoplasma gondii multi-gene knockout strain. After knocking out TgBFD1 based on the ME49Δ6pgdh1 strain, the Δ6pgdh1Δbfd1 strain was obtained, and it was found that this strain hardly formed tissue cysts but still had a certain acute virulence; after further knocking out the TgDAHPS gene, the Δ6pgdh1Δbfd1Δdahps strain was obtained, and it was found that this strain could be passaged in vitro, was almost non-toxic in vivo, and when the infection dose was 10 5 Δ6pgdh1Δbfd1Δdahps / mouse, all the mice still survived; immunization with this strain could effectively resist the virulent strain Chinese I, and the survival rate of the immunized mice was 100%. Based on the previous research, the present invention further overcame the problems of virulence and tissue cyst formation of the ME49Δ6pgdh1 strain, providing methods and means for developing more safe, efficient and non-toxic Toxoplasma gondii vaccines.

[0014] The present invention provides a method for constructing the above-mentioned Toxoplasma gondii multi-gene knockout vaccine strain, which comprises the following steps:

[0015] S1. Using the ME49Δ6pgdh1 strain as the starting strain, knocking out the TgBFD1 gene to construct the Δ6pgdh1Δbfd1 strain;

[0016] S2. Knocking out the TgDAHPS gene on the Δ6pgdh1Δbfd1 strain to obtain the Toxoplasma gondii multi-gene knockout strain Δ6pgdh1Δbfd1Δdahps.

[0017] Preferably, the method for constructing the Δ6pgdh1Δbfd1 strain in step S1 is as follows: Using the pSAG1-Cas9-TgU6-sgTgUPRT plasmid as a template, construct the TgBFD1-specific CRISPR / Cas9 plasmid pSAG1-Cas9-TgU6-dgTgBFD1; construct a homologous template plasmid containing the upstream and downstream homologous arms of the TgBFD1 gene, and then amplify the homologous fragment using this plasmid as a template. The nucleotide sequence of the homologous fragment is as shown in SEQ ID NO.3; finally, co-electroporate the TgBFD1-specific CRISPR / Cas9 plasmid and the homologous fragment into the ME49Δ6pgdh1 strain, perform drug screening and PCR identification to obtain the Δ6pgdh1Δbfd1 strain.

[0018] More preferably, the primer sequences for constructing the TgBFD1-specific CRISPR / Cas9 plasmid are as shown in SEQ ID NO.7-8; the primer sequences for amplifying the TgBFD1-5UTR::CAT::TgBFD1-3UTR homologous fragment are as shown in SEQ ID NO.21-22.

[0019] Preferably, chloramphenicol is used for drug screening.

[0020] The method for constructing the Δ6pgdh1Δbfd1Δdahps strain in step S2 is as follows: Using the pSAG1-Cas9-TgU6-sgTgUPRT plasmid as a template, construct the TgDAHPS-specific CRISPR / Cas9 plasmid pSAG1-Cas9-TgU6-dgTgDAHPS; construct a homologous template plasmid containing the upstream and downstream homologous arms of the TgDAHPS gene, and amplify the homologous fragment using this plasmid as a template. The nucleotide sequence of the homologous fragment is as shown in SEQ ID NO.4; then co-electroporate the TgDAHPS-specific CRISPR / Cas9 plasmid and the homologous fragment into the Δ6pgdh1Δbfd1 strain, perform flow sorting and PCR identification to obtain the Δ6pgdh1Δbfd1Δdahps strain.

[0021] More preferably, the primer sequences for constructing the TgDAHPS-specific CRISPR / Cas9 plasmid are as shown in SEQ ID NO.29-30; amplify TgDAHPS-5UTR::YFP-DHFR * ::TgDAHPS-3UTR homologous fragment primer sequences are as shown in SEQ ID NO.37-38.

[0022] Preferably, green fluorescence is used for flow sorting.

[0023] The present invention provides the application of a reagent for deleting or knocking out the Myb-like transcription factor TgBFD1 gene and the 3-deoxy-D-arabino-heptulosonate-7-phosphate synthase TgDAHPS gene of Toxoplasma gondii in constructing a multi-gene deletion strain of Toxoplasma gondii.

[0024] The present invention provides the application of a reagent for deleting or knocking out the Myb-like transcription factor TgBFD1 gene and the 3-deoxy-D-arabino-heptulosonate-7-phosphate synthase TgDAHPS gene of Toxoplasma gondii in the preparation of a Toxoplasma gondii vaccine.

[0025] The present invention provides the application of a multi-gene deletion strain of Toxoplasma gondii in the preparation of a Toxoplasma gondii vaccine.

[0026] The present invention provides the application of the above method in the preparation of a Toxoplasma gondii vaccine.

[0027] The present invention provides a Toxoplasma gondii vaccine containing the above multi-gene deletion strain of Toxoplasma gondii.

[0028] Preferably, the vaccine is a live attenuated vaccine.

[0029] More preferably, the vaccine further contains a medically acceptable vaccine adjuvant.

[0030] The present invention has the following beneficial effects:

[0031] The multi-gene deletion strain of Toxoplasma gondii Δ6pgdh1Δbfd1Δdahps provided by the present invention has better safety than the parental strains ME49Δ6pgdh1 and Δ6pgdh1Δbfd1, its virulence is significantly decreased, and it does not form tissue cysts, overcoming the defects existing in the ME49Δ6pgdh1 strain. Immunization with Δ6pgdh1Δbfd1Δdahps can resist the lethal infection of the virulent strain Chinese I and has good immune protection, providing methods and means for developing more safe and efficient Toxoplasma gondii vaccines. Description of the Drawings

[0032] Figure 1 It is a schematic diagram for constructing the Toxoplasma gondii Δ6pgdh1Δbfd1 strain.

[0033] Figure 2 It is the PCR identification result of the Δ6pgdh1Δbfd1 monoclonal strain.

[0034] Figure 3 It is a schematic diagram for constructing the Toxoplasma gondii Δ6pgdh1Δbfd1Δdahps strain.

[0035] Figure 4 It is the PCR identification result of the Δ6pgdh1Δbfd1Δdahps monoclonal strain.

[0036] Figure 5 Plaque assay results of the Δ6pgdh1Δbfd1 strain.

[0037] Figure 6 Plaque assay results of the Δ6pgdh1Δbfd1Δdahps strain.

[0038] Figure 7 Virulence gradient test results of the Δ6pgdh1Δbfd1Δdahps strain.

[0039] Figure 8 Results of the cerebral cyst formation assay of the Δ6pgdh1Δbfd1 strain.

[0040] Figure 9 Results of the cerebral cyst formation assay of the Δ6pgdh1Δbfd1Δdahps strain.

[0041] Figure 10 Results of the experiment that mice immunized with the Δ6pgdh1Δbfd1Δdahps strain can effectively resist lethal infection of the Chinese epidemic strain Chinese I. Detailed implementation manners

[0042] The present invention will be further described below in conjunction with the accompanying drawings of the specification and specific embodiments, but the embodiments 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 technical field.

[0043] Unless otherwise specified, the reagents and materials used in the following examples are all commercially available.

[0044] Example 1 Construction of a Toxoplasma gondii double gene deletion strain

[0045] Search for the Myb-like transcription factor (TgBFD1, bradyzoite-formation deficient protein) gene (gene ID: TGME49_200385) in the Toxoplasma gondii database ToxoDB (www.toxodb.org) of the Toxoplasma gondii type II strain, and its nucleotide sequence is shown in SEQ ID NO.1.

[0046] 1. Construction of the pSAG1-Cas9-TgU6-dgTgBFD1 plasmid

[0047] (1) Using the TgBFD1 genomic sequence, search for the sequences of the target site gRNAs through the E-CRISP Design website (http: / / www.e-crisp.org / E-CRISP / designcrispr.html), and design primers based on the gRNA sequences. The designed target primer sequences are shown as SEQ ID NO.7 - 8 in Table 1.

[0048] Table 1 Primers for constructing the CRISPR / Cas9 plasmid of TgBFD1

[0049]

[0050] (2) Using the pSAG1-Cas9-TgU6-sgTgUPRT plasmid (purchased from http: / / www.addgene.org) as a template, construct the specific CRISPR / Cas9 plasmid (sgRNA) of TgBFD1. The reaction system and procedure for PCR amplification using Phanta high-fidelity enzyme (product number P505, Nanjing Novoprotein Scientific Co., Ltd.) and the primers of SEQ ID NO.5 - 6 in a PCR instrument (model Biometra Tone96G, Analytik Jena AG, Germany) are shown in Table 2 and Table 3 respectively.

[0051] Table 2 Reaction system for PCR amplification

[0052]

[0053] Table 3 Reaction procedure for PCR amplification

[0054]

[0055] After the reaction, use Dpn I (product number 1609, Takara Biotechnology Co., Ltd.) to digest the linearized fragment to remove the template plasmid. The digestion reaction system and procedure are shown in Table 4 and Table 5 respectively.

[0056] Table 4 Reaction system for digesting PCR products

[0057]

[0058] Table 5 Reaction procedure for digesting PCR products

[0059]

[0060] After the reaction was completed, a gel extraction kit (product number DC301-01, Nanjing Novoprotein Scientific Inc.) was used to extract the PCR products from the gel, and then a NanoDrop One ultra-micro ultraviolet spectrophotometer (Thermo Fisher Scientific, USA) was used to measure the concentration of the recovered products.

[0061] (3) Use ExnaseⅡ enzyme (product number C112-02, Nanjing Novoprotein Scientific Inc.) to prepare the ligation reaction system in Table 6 in a sterilized PCR tube. Ligate the recovered products from step (2) above with the primers of SEQ ID NO.7 and SEQ ID NO.8 in Table 1 respectively. After mixing the liquid evenly, place it in a PCR instrument and react at 37°C for 30 min.

[0062] Table 6 PCR product ligation reaction system

[0063]

[0064] (4) Add 10 μL of the PCR product from step (3) above to 5α chemically competent cells (product number TSC-C01, Tsingke Biotechnology Co., Ltd., Beijing) for transformation. After heat shock at 42°C for 1 min, use solid LB / Amp + medium for overnight culture for about 12 h; pick monoclonal colonies and place them in LB / Amp + liquid medium for culture and then send for sequencing. Analyze according to the sequencing results. If sequence alignment shows that the target sequence has been completely covered, it indicates that the plasmids pSAG1-Cas9-TgU6-gRNA1-sgTgBFD1 and pSAG1-Cas9-TgU6-gRNA2-sgTgBFD1 have been successfully constructed.

[0065] (5) Using the pSAG1-Cas9-TgU6-gRNA1-sgTgBFD1 plasmid as a template, use the primers with primer sequences SEQ ID NO.9-10 in Table 1, and refer to the reaction systems and procedures in Tables 2-5 for PCR amplification; using the pSAG1-Cas9-TgU6-gRNA2-sgTgBFD1 plasmid as a template, use the primers of SEQ ID NO.11-12 in Table 1, and refer to the reaction systems and procedures in Tables 2-3 for PCR amplification. After the reaction was completed, the above 2 PCR products were subjected to gel extraction using a gel extraction kit, and a NanoDrop One ultra-micro ultraviolet spectrophotometer was used to measure the concentration of the recovered products.

[0066] (6) Prepare a ligation reaction system using the 2 recycling products in the above step (5) with reference to Table 6. The optimal dosage of the linear cloning vector = [number of base pairs of the fragment × 0.02] ng, and the optimal dosage of the amplified product of the inserted fragment = [number of base pairs of the fragment × 0.04] ng. After mixing the liquid, place it in a PCR instrument and react at 37 °C for 30 min for ligation.

[0067] (7) Refer to the transformation method in the above step (4) to transform the 10 μL PCR product in the above step (6); pick a monoclonal colony and place it in LB / Amp + Liquid medium for culture and send for testing. Analyze according to the sequencing results. If it shows that the fragment of gRNA2 is inserted into the pSAG1-Cas9-TgU6-gRNA1-sgTgBFD1 plasmid after comparison, it means that the pSAG1-Cas9-TgU6-dgTgBFD1 plasmid is successfully constructed, and use a ultra-micro ultraviolet spectrophotometer to measure the plasmid concentration.

[0068] 2. Amplification of TgBFD1-5UTR::CAT::TgBFD1-3UTR homologous fragment

[0069] (1) Use Phanta high-fidelity enzyme and the primers in Table 7, refer to the reaction systems and procedures in Tables 2 - 3, and amplify the 5' homologous arm fragment (TgBFD1-5UTR, using the primers of SEQ ID NO.13 - 14) and the 3' homologous arm fragment (TgBFD1-3UTR, using the primers of SEQ ID NO.15 - 16) from Toxoplasma gondii ME49 genomic DNA respectively; amplify the CAT fragment (amplified using the primers of SEQ ID NO.17 - 18) from the plasmid with CAT (kindly provided by Huazhong Agricultural University); use the designed specific primers to linearize the pUC19 vector (purchased from http: / / www.addgene.org) (PUC19, using the primers of SEQ ID NO.19 - 20). Gel-recover the above 4 PCR products using a gel recovery kit respectively, and use a ultra-micro ultraviolet spectrophotometer to measure the concentration of the recycling products.

[0070] Table 7 Primers for constructing pTgBFD1-5UTR::CAT::TgBFD1-3UTR-PUC19 homologous plasmid

[0071]

[0072] (2) Prepare the ligation reaction system in Table 8 in a sterilized PCR tube using Exnase MultiS enzyme (product number C113-02, Nanjing Novoprotein Scientific Co., Ltd.), ligate the 4 recycling products in the above step (1), after mixing the liquid, place it in a PCR instrument and react at 37 °C for 30 min.

[0073] Table 8 PCR product ligation reaction system

[0074]

[0075] (3) Referring to the transformation method in step (4) of the above (1), add the PCR product in the above step (2) to Trans1-T1 Phage Resistant chemically competent cells (product number CD501-03, Beijing TransGen Biotech Co., Ltd.) for transformation, pick monoclonal colonies and place them in LB / Amp + Liquid medium for culture and send for testing. Analyze according to the sequencing results. If the TgBFD1-5UTR and TgBFD1-3UTR fragment sequences are completely matched after comparison, it indicates that the pTgBFD1-5UTR::CAT::TgBFD1-3UTR-PUC19 homologous template plasmid is successfully constructed.

[0076] (4) Amplify the TgBFD1-5UTR::CAT::TgBFD1-3UTR homologous fragment

[0077] Using the pTgBFD1-5UTR::CAT::TgBFD1-3UTR-PUC19 homologous template plasmid as a template, use the primers shown in Table 9, and refer to the reaction systems and procedures in Tables 2-3 for PCR amplification. After amplification, use a gel extraction kit to extract the TgBFD1-5UTR::CAT::TgBFD1-3UTR homologous fragment with the correct band size (the sequence is shown in SEQ ID NO.3), and use a ultra-micro ultraviolet spectrophotometer to measure the concentration of the recovered product.

[0078] Table 9 Primers for amplifying the TgBFD1-5UTR::CAT::TgBFD1-3UTR homologous fragment

[0079]

[0080] 3. Construction of the Toxoplasma gondii double gene deletion strain Δ6pgdh1Δbfd1

[0081] Using the publicly available ME49Δ6pgdh1 in the prior art (constructed according to the method disclosed in the prior art: CN114933970A, gene ID of the Tg6PGDH1 gene: TGME49_242600) as the starting strain, the construction schematic diagram of the Toxoplasma gondii Δ6pgdh1Δbfd1 strain is as Figure 1 shown.

[0082] (1) Collect Toxoplasma gondii ME49Δ6pgdh1 tachyzoites and centrifuge them at 3000 rpm for 8 min at room temperature; then resuspend the parasites in 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), and after resuspension, centrifuge at 3000 rpm for 8 min at room temperature; resuspend the parasites in 250 μL of Cytomix.

[0083] (2) Add 50 μL of Cytomix, 1500 ng of the TgBFD1-5UTR::CAT::TgBFD1-3UTR homologous fragment, 7500 ng of the pSAG1-Cas9-TgU6-dgTgBFD1 plasmid, and the resuspended parasites from the above step (1) into a 1.5 mL EP tube.

[0084] (3) Use a pipette to thoroughly mix the liquid in the EP tube and add it all to a 4 mm electroporation cuvette, taking care not to generate bubbles; perform electroporation using a BTX electroporator (model Gemini X2, BTX, USA), align the metal faces on both sides of the electroporation cuvette with the metal sheet electrodes in the instrument: give one shock at 1600 V, 25 μF, 50 Ω, 4 mm, gently stir the liquid in the electroporation cuvette to mix; give one shock at 1500 V, 25 μF, 50 Ω, 4 mm, gently stir the liquid in the electroporation cuvette to mix; give one shock at 1500 V, 25 μF, 50 Ω, 4 mm, after standing for 2 min, add the liquid in the electroporation cuvette to HFF cells (purchased from the ATCC cell bank) cultured in 2% FBS DMEM medium for culture.

[0085] (4) When large parasitophorous vacuoles are visible in the cells, dilute chloramphenicol to a working concentration of 30 μM with 2% FBS DMEM medium to prepare a drug screening medium, and use this medium to culture the parasites for drug screening.

[0086] (5) Thereafter, for each subsequent subculture, use HFF cells in 2% FBS DMEM medium containing 30 μM chloramphenicol. After 5 - 6 rounds of chloramphenicol screening culture, use Rapid Taq enzyme (P222, Nanjing Novoprotein Scientific Co., Ltd.) and the primers shown in Table 10 to perform PCR identification on the electroporated parasite strain library. The PCR identification reaction system is shown in Table 11, and the reaction program refers to Table 3.

[0087] Table 10 PCR identification primers for Δ6pgdh1Δbfd1 parasite strains

[0088]

[0089] Table 11 PCR identification reaction system

[0090]

[0091] (6) Results showed that there was a band at the target size in PCR3 of the parental strain ME49Δ6pgdh1, while there were bands at the target size in PCR1, PCR2, and PCR3 of the electroporated parasite library, indicating that the electroporated parasite library contained parasites with successful knockout of the TgBFD1 gene.

[0092] (7) The electroporated parasite library was collected for dilution counting and added to a 96-well plate containing HFF cells, with 1 tachyzoite of Toxoplasma gondii added to each well. After culturing for 7 - 10 days, whether there were monoclonal parasite strains in the 96-well plate was observed. The HFF cells containing the monoclonal parasite strains were scraped off with a pipette tip and all added to a 24-well plate containing HFF cells for continued culturing. When 50% of the tachyzoites in the parasitophorous vacuoles in the 24-well plate escaped, the cells in the wells were scraped off. Half of them was used to extract gDNA (product number DP348, Tiangen Biochemical Technology Co., Ltd., Beijing) for identification of the monoclonal parasite strains, and the remaining part was passaged to a new 24-well plate for continued culturing.

[0093] (8) The monoclonal parasite strains were identified by PCR using the primers shown in Table 10, and the results were as Figure 2 shown. The presence of a PCR1 band indicated that the 5' homologous arm of TgBFD1 was integrated into the genome of Toxoplasma gondii, and the presence of a PCR2 band indicated that the 3' homologous arm of TgBFD1 was integrated into the genome of Toxoplasma gondii; PCR3 was used to detect whether TgBFD1 was still in the genome. If there was a PCR3 band in the control group ME49Δ6pgdh1 but no PCR3 band in the transfected experimental group, it indicated that TgBFD1 was successfully knocked out, and the Δ6pgdh1Δbfd1 parasite strain was constructed.

[0094] Example 2 Construction of a Toxoplasma gondii multi-gene deletion parasite strain

[0095] First, a plaque and bradyzoite cyst formation experiment was performed on the Δ6pgdh1Δbfd1 parasite strain constructed in Example 1 (for the specific experimental method steps, see Examples 3 and 5 in detail). The results showed that knocking out TgBFD1 had no obvious effect on the in vitro growth of Toxoplasma gondii, and almost no bradyzoite cysts were detected in the in vivo inoculation. Based on this, in this example, the Toxoplasma gondii Δ6pgdh1Δbfd1 constructed in Example 1 was used as the starting parasite strain, and the 3-deoxy-D-arabino-heptulosonate-7-phosphate synthase (Class-IIDAHP synthetase family protein) TgDAHPS gene (gene ID: TGME49_221260) was searched from the Toxoplasma gondii database ToxoDB. Its nucleotide sequence was as shown in SEQ ID NO.2, and a multi-gene deletion parasite strain Δ6pgdh1Δbfd1Δdahps was further constructed. The construction schematic diagram was as Figure 3 shown.

[0096] 1. Construction of pSAG1-Cas9-TgU6-dgTgDAHPS plasmid

[0097] (1) Using the TgDAHPS genomic sequence, search for the sequence of the targeting site gRNA through the E-CRISP Design website, and design primers according to the gRNA sequence. The specific primer sequences of the designed target sites are shown in Table 12.

[0098] Table 12 Primers for constructing TgDAHPS-specific CRISPR / Cas9 plasmid

[0099]

[0100] (2) Refer to the construction method of Example 1 to construct the CRISPR / Cas9 plasmid and perform sequencing. If the alignment shows that the fragment of gRNA2 is inserted into the pSAG1-Cas9-TgU6-gRNA1-sgTgDAHPS plasmid, it indicates the successful construction of the pSAG1-Cas9-TgU6-dgTgDAHPS plasmid.

[0101] 2. Amplification of TgDAHPS-5UTR::YFP-DHFR * ::TgDAHPS-3UTR homologous fragment

[0102] (1) Using Phanta high-fidelity enzyme and the primers in Table 13, respectively amplify the 5' homologous arm of TgDAHPS (TgDAHPS-5UTR, using the primers of SEQ ID NO.31 - 32) and the 3' homologous arm fragment (TgDAHPS-3UTR, using the primers of SEQ ID NO.33 - 34) from Toxoplasma gondii ME49 genomic DNA with reference to the reaction systems and procedures in Tables 2 - 3; amplify the YFP-DHFR * fragment (amplified using the primers of SEQ ID NO.35 - 36) with the pTub-YFP-DHFR * plasmid (constructed by conventional methods in the art, Tub is the promoter, YFP is the fluorescent reporter gene, and DHFR * is the pyrimethamine drug screening gene) as the template. Gel recovery kits were used to recover the above 3 PCR products respectively, and a ultra-micro ultraviolet spectrophotometer was used to measure the concentration of the recovered products.

[0103] Table 13 Primers for constructing pTgDAHPS-5UTR::YFP-DHFR * ::TgDAHPS-3UTR-PUC19 homologous template plasmid

[0104]

[0105] (2) Refer to the method in step 2 of Example 1 and use the PUC19 fragment amplified in this step to successfully construct the plasmid pTgDAHPS-5UTR::YFP-DHFR * ::TgDAHPS-3UTR-PUC19 homologous template plasmid. Using this plasmid as a template, perform PCR amplification with Phanta enzyme and the primers shown in Table 14, referring to the reaction systems and procedures in Tables 2 - 3. After amplification, use a gel extraction kit to extract the TgDAHPS-5UTR::YFP-DHFR * ::TgDAHPS-3UTR homologous fragment (the sequence is shown in SEQ ID NO.4) from the gel, and use a ultra-micro ultraviolet spectrophotometer to measure the concentration of the recovered product.

[0106] Table 14 Primers for amplifying the TgDAHPS-5UTR::YFP-DHFR * ::TgDAHPS-3UTR homologous fragment

[0107]

[0108] 3. Construction of the Toxoplasma gondii multi-gene deletion strain Δ6pgdh1Δbfd1Δdahps

[0109] (1) Using the method in step 3 of Example 1, after resuspending the Toxoplasma gondii Δ6pgdh1Δbfd1 tachyzoites with 250 μL of Cytomix, mix them thoroughly with 50 μL of Cytomix, 7500 ng of the plasmid pSAG1-Cas9-TgU6-dgTgDAHPS, and 1500 ng of the TgDAHPS-5UTR::YFP-DHFR * ::TgDAHPS-3UTR homologous fragment, and perform electroporation. After electroporation, add the mixture to a T25 culture flask containing HFF cells for culture;

[0110] (2) When there are many spontaneously green fluorescent parasitophorous vacuoles visible in the cells, use Rapid Taq enzyme and the primers shown in Table 15 to perform PCR identification on the electroporated parasite strain library. The reaction systems and procedures for PCR identification refer to Tables 11 and 3; the results show that PCR3 of the parental strain Δ6pgdh1Δbfd1 has a band at the target size, while PCR1, PCR2, and PCR3 of the electroporated parasite strain library all have bands at the target size, indicating that the electroporated parasite strain library contains parasite strains with the TgDAHPS gene successfully knocked out.

[0111] Table 15 Primers for PCR identification of the Δ6pgdh1Δbfd1Δdahps strain

[0112]

[0113] (3) Collect the electrotransformed parasite stock, and perform flow sorting using a flow cytometer (model BD FACSAria, BD Biosciences, USA). Add it to a 96-well plate containing adherent HFF cells, and add 1 Toxoplasma gondii tachyzoite to each well. After culturing for 7 - 10 days, observe whether there are monoclonal parasite strains with spontaneous green fluorescence in the 96-well plate. Use a pipette tip to scrape off the HFF cells in the well containing the monoclonal parasite strain with spontaneous green fluorescence, and add all of them to a 24-well plate containing HFF cells for expansion culture. When 50% of the tachyzoites in the parasitophorous vacuoles in the 24-well plate have escaped, scrape off the cells in the well. Half of them are used to extract gDNA for identification of the monoclonal parasite strain, and the remaining part is passaged to a new 24-well plate for continued culture.

[0114] (4) The monoclonal parasite strains are identified by PCR using the primers shown in Table 15. The results are as Figure 4 shown. One PCR band indicates that the 5' homologous arm of TgDAHPS is integrated into the Toxoplasma gondii genome, and two PCR bands indicate that the 3' homologous arm of TgDAHPS is integrated into the Toxoplasma gondii genome. PCR3 is used to detect whether TgDAHPS is still in the genome. If the control group Δ6pgdh1Δbfd1 has a PCR3 band while the transfected experimental group does not, it indicates that TgDAHPS has been successfully knocked out, and the Δ6pgdh1Δbfd1Δdahps parasite strain is constructed.

[0115] Example 3 In vitro plaque assay of gene deletion parasite strains

[0116] (1) Culture the wild-type ME49 parasite strain, the Δ6pgdh1Δbfd1 parasite strain constructed in Example 1, and the Δ6pgdh1Δbfd1Δdahps parasite strain constructed in Example 2 respectively, and collect the intracellular tachyzoites.

[0117] (2) Add the above-collected tachyzoites to a 6-well plate containing HFF cells, add 100 tachyzoites to each well, and culture at 37°C in a cell culture incubator for 7 - 14 days.

[0118] (3) Wash twice with PBS, and add 1 mL of 4% paraformaldehyde to each well, and fix at 37°C for 20 min.

[0119] (4) After washing with PBS, add 1 mL of 0.1% crystal violet to each well, and stain at 37°C for 20 min.

[0120] (5) After washing once with PBS and air-drying, scan the plaques using a scanner (model ScanMaker i600, Microtek Labscan Co., Ltd., Shanghai), and save the pictures.

[0121] The plaque results are as Figure 5 and Figure 6As shown, it can be seen that there is almost no difference in the plaque size between the ME49 and Δ6pgdh1Δbfd1 strains, and there is also almost no difference in the plaque size between the Δ6pgdh1Δbfd1 and Δ6pgdh1Δbfd1Δdahps strains, indicating that knocking out TgBFD1 and TgDAHPS on the basis of the ME49Δ6pgdh1 strain does not affect the in vitro growth of Toxoplasma gondii.

[0122] Example 4 In Vivo Virulence Experiment of Gene-Deleted Strains

[0123] Using the Δ6pgdh1Δbfd1 and Δ6pgdh1Δbfd1Δdahps strains constructed in Example 1 and Example 2, tachyzoites were diluted with DMEM medium without FBS. Both strains were inoculated into 8 mice (breed ICR, Guangzhou Regene Biotechnology Co., Ltd.) at a dose of 1×10 2 tachyzoites / mouse. At the same time, the Δ6pgdh1Δbfd1Δdahps strain was inoculated into 5 mice at doses of 1×10 4 and 1×10 5 tachyzoites / mouse respectively. The mental state and survival number of the mice were recorded. After 21 days, the mice were bled, and indirect immunofluorescence assay (IFA) was used to identify whether they were successfully infected, and the uninfected mice were eliminated. The survival curve of the mice was analyzed using GraphPad prism 9.5 software, and a Kaplan-Meier survival plot was made.

[0124] The results are as Figure 7 shown. Within 30 days, the survival rate of mice inoculated with the Δ6pgdh1Δbfd1 strain at a dose of 1×10 2 tachyzoites / mouse was 62.5%, while all the mice inoculated with the Δ6pgdh1Δbfd1Δdahps strain survived at the inoculation doses of 1×10 2 , 1×10 4 , and 1×10 5 tachyzoites / mouse, and no clinical symptoms of toxoplasmosis such as rough hair and listlessness appeared, indicating that the Δ6pgdh1Δbfd1Δdahps strain has almost no in vivo virulence.

[0125] Example 5 Brain Cyst Formation Experiment of Gene-Deleted Strains

[0126] (1) Using the parental strain ME49Δ6pgdh1 and the Δ6pgdh1Δbfd1 strain constructed in Example 1, tachyzoites were diluted with DMEM medium without FBS. Both were at a dose of 1×10 2Eight mice were inoculated with each dose of the number of parasites / mouse. After 21 days, the mice were bled, and indirect immunofluorescence assay (IFA) was used to identify whether they were successfully infected, and the uninfected mice were excluded. The brains of the surviving mice after 30 days were stained with DBA, the number of cerebral cysts was counted, and the statistical results were analyzed using Student's t test.

[0127] (2) Mice surviving 30 days after inoculation with the Δ6pgdh1Δbfd1 and Δ6pgdh1Δbfd1Δdahps strains at a dose of 1×10 2 parasites / mouse were used as in Example 4. The brains of the mice were stained with DBA, the number of cerebral cysts was counted, and the statistical results were analyzed using Student's t test.

[0128] The results are as Figure 8 and Figure 9 shown. Compared with the parental strain of the control, the number of cerebral cysts of the Δ6pgdh1Δbfd1 strain was significantly reduced, and almost no cerebral cysts were formed. Moreover, no cerebral cysts were detected in the mice inoculated with the Δ6pgdh1Δbfd1Δdahps strain, indicating that the Δ6pgdh1Δbfd1Δdahps strain hardly formed cysts in mice.

[0129] Example 6 Immunoprotective ability experiment of gene-deleted strains in mice

[0130] (1) When the Chinese I strain (kindly provided by Huazhong Agricultural University, which is a dominant virulent strain in China) cultured in HFF cells had large parasitophorous vacuoles, the extracellular tachyzoites were washed away with PBS, DMEM medium without FBS was added, and the intracellular tachyzoites were collected. 100 μL of the liquid containing tachyzoites was diluted 10 times and counted under a microscope. After counting, the tachyzoites were diluted with DMEM medium without FBS.

[0131] (2) Mice immunized with 1×10 3 Δ6pgdh1Δbfd1Δdahps parasites 42 days later and non-immunized blank mice were reinfected with the Chinese I strain (1×10 4 parasites / mouse). The mental state and survival rate of the mice were observed and recorded, and the survival curve was statistically analyzed and plotted in GraphPad prism 9.5 software.

[0132] The results are as Figure 10 shown. All the control group mice without immunization died within 11 days after infection with 1×10 4 Chinese I parasites, while after being immunized with 1×10 3All the mice immunized with Δ6pgdh1Δbfd1Δdahps tachyzoites survived 100% within 30 days. This indicates that inoculation with the Δ6pgdh1Δbfd1Δdahps strain can generate good immune protection and can be used to prepare a Toxoplasma gondii vaccine.

[0133] In summary, through the knockout of the Myb-like transcription factor TgBFD1 gene and the 3-deoxy-D-arabino-heptulosonate-7-phosphate synthase TgDAHPS gene of Toxoplasma gondii in the present invention, it was found that knocking out TgBFD1 on the basis of the Tg6PGDH1 knockout strain had no significant effect on the in vitro growth rate of Toxoplasma gondii, but could further reduce cyst formation. Meanwhile, knocking out both TgBFD1 and TgDAHPS on the basis of the Tg6PGDH1 knockout strain also did not affect the in vitro growth of Toxoplasma gondii, but the parasites were almost non-toxic and did not form cysts. Immunization with the above multi-gene deletion strains can effectively resist the infection of highly virulent strains, showing good immune protection effects. The multi-gene deletion strain Δ6pgdh1Δbfd1Δdahps provided by the present invention provides methods and means for developing more effective and safe Toxoplasma gondii vaccines.

[0134] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited by 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 multi-gene deleted vaccine strain, characterized in that: The parasite strain is obtained by knocking out the 6-phosphogluconate dehydrogenase Tg6PGDH1 gene, the Myb-like transcription factor TgBFD1 gene and the 3-deoxy-D-arabinoheptulose-7-phosphate synthase TgDAHPS gene of Toxoplasma by gene editing technology; the gene ID of the Tg6PGDH1 gene is: TGME49_242600, the gene ID of the TgBFD1 gene is: TGME49_200385, and the gene ID of the TgDAHPS gene is: TGME49_221260.

2. The method for constructing the Toxoplasma gondii multi-gene deleted vaccine strain according to claim 1, characterized in that: The following steps are involved: S1. Using the ME49Δ6pgdh1 strain as the starting strain, the TgBFD1 gene was knocked out to construct the Δ6pgdh1Δbfd1 strain; S2. The TgDAHPS gene was knocked out in the Δ6pgdh1Δbfd1 strain to obtain the Toxoplasma gondii multi-gene deletion strain Δ6pgdh1Δbfd1Δdahps.

3. The method according to claim 2, characterized in that: The method for constructing the Δ6pgdh1Δbfd1 strain in step S1 is as follows: using the pSAG1-Cas9-TgU6-sgTgUPRT plasmid as a template, constructing a TgBFD1-specific CRISPR / Cas9 plasmid pSAG1-Cas9-TgU6-dgTgBFD1; simultaneously constructing a homologous template plasmid containing upstream and downstream homologous arms of the TgBFD1 gene, and then amplifying a homologous fragment using the plasmid as a template, wherein the nucleotide sequence of the homologous fragment is shown in SEQ ID NO.3; finally, the TgBFD1-specific CRISPR / Cas9 plasmid and the homologous fragment are co-electroporated into the ME49Δ6pgdh1 strain, and drug screening and PCR identification are performed to obtain the Δ6pgdh1Δbfd1 strain; The construction method of the Δ6pgdh1Δbfd1Δdahps strain in step S2 is as follows: using the pSAG1-Cas9-TgU6-sgTgUPRT plasmid as a template, constructing a TgDAHPS-specific CRISPR / Cas9 plasmid pSAG1-Cas9-TgU6-dgTgDAHPS; simultaneously constructing a homologous template plasmid containing upstream and downstream homologous arms of the TgDAHPS gene, and then amplifying a homologous fragment using the plasmid as a template, wherein the nucleotide sequence of the homologous fragment is shown in SEQ ID NO.4; finally, the TgDAHPS-specific CRISPR / Cas9 plasmid and the homologous fragment are co-electroporated into the Δ6pgdh1Δbfd1 strain, and flow sorting and PCR identification are performed to obtain the Δ6pgdh1Δbfd1Δdahps strain.

4. Use of a reagent for deleting or knocking out the Toxoplasma Myb-like transcription factor TgBFD1 gene and the 3-deoxy-D-arabinoheptulose-7-phosphate synthase TgDAHPS gene in constructing a Toxoplasma multi-gene deletion strain, characterized in that: The gene ID of the TgBFD1 gene is TGME49_200385, and the gene ID of the TgDAHPS gene is TGME49_221260.

5. Use of a reagent for deleting or knocking out the Toxoplasma Myb-like transcription factor TgBFD1 gene and the 3-deoxy-D-arabinoheptulose-7-phosphate synthase TgDAHPS gene in the preparation of a Toxoplasma vaccine, characterized in that: The gene ID of the TgBFD1 gene is TGME49_200385, and the gene ID of the TgDAHPS gene is TGME49_221260.

6. Use of the Toxoplasma gondii multi-gene deleted strain according to claim 1 in the preparation of Toxoplasma gondii vaccine.

7. Use of the method according to claim 2 or 3 in the preparation of a Toxoplasma gondii vaccine.

8. A Toxoplasma gondii vaccine, characterized in that: Containing the Toxoplasma gondii multi-gene deletion strain as described in claim 1.

9. The vaccine according to claim 8, characterized in that The vaccine is a live attenuated vaccine.

10. The vaccine according to claim 8, characterized in that The vaccine also contains a medically acceptable vaccine adjuvant.

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