Construction method and application of a Toxoplasma gondii multi-gene deletion vaccine strain

Abstract

This invention discloses a method for constructing and applying a Toxoplasma gondii multi-gene deletion vaccine strain. Using gene editing technology, the TgBFD1 and TgDAHPS genes were knocked out in the ME49Δ6pgdh1 strain, successfully constructing the multi-gene deletion strain Δ6pgdh1Δbfd1Δdahps. This strain can be passaged in vitro and exhibits non-toxicity and non-cyst-forming properties in the host. After immunization, it provides good immunoprotection against lethal infection by highly virulent strains. The Toxoplasma gondii multi-gene deletion strain provided by this invention has better safety than the ME49Δ6pgdh1 strain, with significantly reduced virulence and no cyst formation, effectively resisting infection by wild-type strains and providing good immunoprotection. It overcomes the shortcomings of ME49Δ6pgdh1, which still retains virulence and can form cysts, providing a method for developing safe and efficient Toxoplasma gondii vaccines.

Description

Technical Field

[0001] This invention belongs to the field of biomedicine and genetic engineering technology. More specifically, it relates to a method for constructing and applying a Toxoplasma gondii multi-gene deletion vaccine strain. Background Technology

[0002] Toxoplasma gondii is an obligate intracellular parasite that can infect almost all warm-blooded animals, and humans are generally susceptible. Felines are the definitive hosts of Toxoplasma gondii, in which the parasite can complete sexual reproduction and excrete oocysts in feces. Toxoplasmosis poses a significant risk to pregnant women, potentially causing miscarriage or stillbirth. Fetal infection with Toxoplasma gondii can lead to congenital toxoplasmosis, causing damage to the optic nerve, hydrocephalus, and other complications.

[0003] Toxoplasmosis is a major cause of miscarriage, posing a significant threat to human health and the livestock industry, leading to serious social problems and substantial economic losses. The most commonly used drugs for treating toxoplasmosis are pyrimethamine and sulfadiazine, but these drugs are only effective against tachyzoites in the acute infection phase, and ineffective against bradyzoites or tissue cysts in the chronic infection phase, while also having toxic side effects. Vaccination is crucial for blocking congenital infection and preventing acquired infection. However, currently only the attenuated S48 strain has been prepared into a live attenuated Toxoplasma gondii vaccine. Because the mechanism of virulence attenuation in the S48 strain is unclear, and there is a risk of virulence reversion and restoration of oocyst formation, its widespread application is limited.

[0004] Compared to other types of vaccines, gene-deleted live vaccines constructed using CRISPR / Cas9 gene-editing technology have advantages such as clear genetic background, good safety, and strong immunoprotective efficacy, bringing new hope for the development of novel anti-toxoplasmosis vaccines. The gene-deleted strain ME49Δ6pgdh1 (CN114933970A) previously developed and prepared by our team exhibits weakened virulence in vivo and high immunoprotective efficacy in animals, making it suitable for preparing candidate Toxoplasma gondii vaccines. However, considering that ME49Δ6pgdh1 still possesses a certain degree of acute virulence and can still form a certain number of tissue cysts in the host, posing a certain risk of pathogen transmission, it hinders its clinical use in livestock and pets. Therefore, further optimization and modification are needed to reduce the virulence and cyst formation risk of the candidate vaccine strain, aiming to overcome the problems of the ME49Δ6pgdh1 strain and obtain more non-toxic, non-cyst-forming, safe, and highly effective candidate vaccine strains. Summary of the Invention

[0005] The technical problem to be solved by this invention is to overcome the shortcomings of the existing Toxoplasma gondii ME49Δ6pgdh1 strain, which has acute virulence, forms tissue cysts, and poses a risk of pathogen transmission. This invention provides 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 objective of this invention is to provide a Toxoplasma gondii strain with multiple gene deletions.

[0007] The second objective of this invention is to provide a method for constructing Toxoplasma gondii multi-gene deletion strains.

[0008] A third objective of this invention is to provide applications for Toxoplasma gondii multi-gene deletion strains.

[0009] The fourth objective of this invention is to provide a Toxoplasma gondii vaccine.

[0010] The fifth objective of this invention is to provide the application of a reagent that deletes the Toxoplasma gondii Myb-like transcription factor TgBFD1 gene and the 3-deoxy-D-arabinohepenosaccharide-7-phosphate synthase TgDAHPS gene.

[0011] The above-mentioned objective of this invention is achieved through the following technical solution:

[0012] This invention provides a Toxoplasma gondii strain with multiple gene deletions, obtained by knocking out the Tg6PGDH1 gene of 6-phosphoglucuronide dehydrogenase, the TgBFD1 gene of Myb-like transcription factor, and the TgDAHPS gene of 3-deoxy-D-arabinohepulose-7-phosphate synthase using gene editing technology. The gene IDs of the Tg6PGDH1 gene, TgBFD1 gene, and TgDAHPS gene are TGME49_242600, TGME49_200385, and TGME49_221260, respectively. The nucleotide sequences of the TgBFD1 and TgDAHPS genes are shown in SEQ ID NO. 1-2, respectively.

[0013] This invention focuses on the TgBFD1 gene, a regulatory factor for the differentiation and development of Toxoplasma gondii, and the TgDAHPS gene, which is present in Toxoplasma gondii but lacks in the host. Based on the existing Toxoplasma gondii gene-deleted strain ME49Δ6pgdh1, the Myb-like transcription factor (bradyzoite-formation deficient protein) TgBFD1 gene (gene ID: TGME49_200385) and the 3-deoxy-D-arabinohepenosaccharide-7-phosphate synthetase (Class-IIDAHP synthetase family protein) TgDAHPS gene (gene ID: TGME49_221260) of Toxoplasma gondii were further knocked out using CRISPR / Cas9 gene editing technology, resulting in a Toxoplasma gondii multi-gene deletion strain. Knocking out TgBFD1 from the ME49Δ6pgdh1 strain yielded the Δ6pgdh1Δbfd1 strain, which showed almost no tissue cyst formation but still exhibited some acute virulence. Further knockout of the TgDAHPS gene yielded the Δ6pgdh1Δbfd1Δdahps strain, which was found to be capable of in vitro passage and virtually non-virulent in vivo at an infectious dose of 10... 5 Under the condition of Δ6pgdh1Δbfd1Δdahps / mouse, all mice still survived; immunization with this strain effectively resisted the highly virulent Chinese I strain, with a 100% survival rate in immunized mice. Based on previous research, this invention further overcomes 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] This invention provides a method for constructing the Toxoplasma gondii multi-gene deletion vaccine strain, comprising the following steps:

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

[0016] S2. Knock out the TgDAHPS gene in the Δ6pgdh1Δbfd1 strain to obtain the Toxoplasma gondii multi-gene deletion strain Δ6pgdh1Δbfd1Δdahps.

[0017] Preferably, the construction method of the Δ6pgdh1Δbfd1 insect strain in step S1 is as follows: using pSAG1-Cas9-TgU6-sgTgUPRT plasmid as a template, a TgBFD1-specific CRISPR / Cas9 plasmid pSAG1-Cas9-TgU6-dgTgBFD1 is constructed; and a homologous template plasmid containing upstream and downstream homologous arms of the TgBFD1 gene is constructed. Then, the homologous fragment is amplified using this plasmid as a template. 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-electrotransfected into the ME49Δ6pgdh1 insect strain, and drug screening and PCR identification are performed to obtain the Δ6pgdh1Δbfd1 insect strain.

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

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

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

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

[0022] Preferably, flow cytometry is performed using green fluorescence.

[0023] This invention provides the application of reagents for deleting or knocking out the Toxoplasma gondii Myb-like transcription factor TgBFD1 gene and the 3-deoxy-D-arabinohepenosaccharide-7-phosphate synthase TgDAHPS gene in constructing Toxoplasma gondii multi-gene deletion strains.

[0024] This invention provides the application of reagents for deleting or knocking out the Toxoplasma gondii Myb-like transcription factor TgBFD1 gene and the 3-deoxy-D-arabinohepenosaccharide-7-phosphate synthase TgDAHPS gene in the preparation of Toxoplasma gondii vaccines.

[0025] This invention provides the application of Toxoplasma gondii multi-gene deletion strains in the preparation of Toxoplasma gondii vaccines.

[0026] This invention provides the application of the above method in the preparation of Toxoplasma gondii vaccines.

[0027] This invention provides a Toxoplasma gondii vaccine containing the above-mentioned Toxoplasma gondii multi-gene deletion strain.

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

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

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

[0031] The *Toxoplasma gondii* multi-gene deletion strain Δ6pgdh1Δbfd1Δdahps provided by this invention exhibits better safety than the original strains ME49Δ6pgdh1 and Δ6pgdh1Δbfd1, with significantly reduced virulence and no tissue cyst formation, overcoming the defects of the ME49Δ6pgdh1 strain. Immunization with Δ6pgdh1Δbfd1Δdahps can resist lethal infection by the highly virulent strain Chinese I, demonstrating good immunoprotective efficacy and providing methods and means for developing more safe and effective toxoplasmosis vaccines. Attached Figure Description

[0032] Figure 1 A schematic diagram for constructing the Toxoplasma gondii Δ6pgdh1Δbfd1 strain.

[0033] Figure 2 The results of PCR identification of the Δ6pgdh1Δbfd1 monoclonal strain.

[0034] Figure 3 A schematic diagram for constructing the Toxoplasma gondii strain Δ6pgdh1Δbfd1Δdahps.

[0035] Figure 4 PCR identification results for the Δ6pgdh1Δbfd1Δdahps monoclonal strain.

[0036] Figure 5 The results of the plaque test for the Δ6pgdh1Δbfd1 insect strain are shown.

[0037] Figure 6 The results of the plaque test for the Δ6pgdh1Δbfd1Δdahps insect strain are shown.

[0038] Figure 7 The results of the virulence gradient test for the Δ6pgdh1Δbfd1Δdahps strain are shown.

[0039] Figure 8 The results of the brain cyst formation experiment of the Δ6pgdh1Δbfd1 strain.

[0040] Figure 9 The results of the brain cyst formation experiment of the Δ6pgdh1Δbfd1Δdahps strain.

[0041] Figure 10 The experimental results show that immunizing mice with the Δ6pgdh1Δbfd1Δdahps strain effectively resists lethal infection by the Chinese I strain. Detailed Implementation

[0042] The present invention will be further described below with reference to the accompanying drawings and specific embodiments, but the embodiments do not limit the present invention in any way. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in this technical field.

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

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

[0045] The My b-like transcription factor (TgBFD1, bradyzoite-formation deficient protein) gene (gene ID: TGME49_200385) in Toxoplasma gondii strains was searched in the ToxoDB database (www.toxodb.org). Its nucleotide sequence is shown in SEQ ID NO.1.

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

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

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

[0049]

[0050] (2) Using pSAG1-Cas9-TgU6-sgTgUPRT plasmid (purchased from http: / / www.addgene.org) as a template, a specific CRISPR / Cas9 plasmid (sgRNA) for TgBFD1 was constructed. The reaction system and procedure for PCR amplification using Phanta high-fidelity enzyme (product number P505, Nanjing Novizan Biotechnology Co., Ltd.) and primers of SEQ ID NO. 5-6 in a PCR instrument (model Biometra Tone 96G, Jena Analytical Instruments AG, Germany) are shown in Tables 2 and 3.

[0051] Table 2 PCR amplification reaction system

[0052]

[0053] Table 3 PCR amplification reaction procedure

[0054]

[0055] After the reaction was completed, the linearized fragment was digested with Dpn I (catalog number 1609, Baori Biotechnology Co., Ltd.) to remove the template plasmid. The digestion reaction system and procedure are shown in Tables 4 and 5.

[0056] Table 4. PCR product digestion reaction system

[0057]

[0058] Table 5. PCR product digestion reaction procedure

[0059]

[0060] After the reaction was completed, the PCR products were recovered using a gel recovery kit (catalog number DC301-01, Nanjing Novizan Biotechnology Co., Ltd.), and the concentration of the recovered products was then determined using an ultra-micro UV spectrophotometer (model NanoDrop One, Thermo Fisher Scientific, USA).

[0061] (3) Using Exnase II enzyme (C112-02, Nanjing Novizan Biotechnology Co., Ltd.), prepare the ligation reaction system as shown in Table 6 in a sterile PCR tube. Ligate the recovered product from step (2) above with the primers SEQ ID NO.7 and SEQ ID NO.8 in Table 1 respectively. After mixing the liquid, place it in a PCR instrument at 37℃ for 30 min.

[0062] Table 6. PCR product ligation reaction system

[0063]

[0064] (4) In 10 μL of the PCR product from step (3) above was added to 5α chemicompetent cells (catalog number TSC-C01, Beijing Qingke Biotechnology Co., Ltd.) for transformation. After heat shock at 42℃ for 1 min, solid LB / c ampoules were used for the transformation. + The culture medium was incubated overnight, approximately 12 hours; single colonies were then placed in LB / Amp culture medium. + The samples were cultured in liquid culture medium and then sent for analysis. The sequencing results were analyzed, and if the sequence alignment showed that the target sequence was completely covered, it meant that the pSAG1-Cas9-TgU6-gRNA1-sgTgBFD1 and pSAG1-Cas9-TgU6-gRNA2-sgTgBFD1 plasmids were successfully constructed.

[0065] (5) Using pSAG1-Cas9-TgU6-gRNA1-sgTgBFD1 plasmid as a template, PCR amplification was performed using primers with SEQ ID NO. 9-10 in Table 1, following the reaction system and procedure in Tables 2-5. Using pSAG1-Cas9-TgU6-gRNA2-sgTgBFD1 plasmid as a template, PCR amplification was performed using primers with SEQ ID NO. 11-12 in Table 1, following the reaction system and procedure in Tables 2-3. After the reaction, the two PCR products were recovered using a gel extraction kit, and the concentration of the recovered products was determined using an ultra-micro UV spectrophotometer.

[0066] (6) Refer to Table 6 and use the two recovered products from step (5) above to prepare the ligation reaction system. The optimal amount of linear cloning vector = [number of fragment base pairs × 0.02] ng, and the optimal amount of insert fragment amplification product = [number of fragment base pairs × 0.04] ng. After mixing the liquid, place it in a PCR instrument at 37℃ for 30 min to perform ligation.

[0067] (7) Following the transformation method in step (4) above, transform the 10 μL PCR product from step (6) above; take a single colony and place it in an LB / c container. + The plasmid was cultured in liquid culture medium and then analyzed based on the sequencing results. If the comparison showed that the gRNA2 fragment was inserted into the pSAG1-Cas9-TgU6-gRNA1-sg TgBFD1 plasmid, it indicated that the pSAG1-Cas9-TgU6-dgTgBFD1 plasmid was successfully constructed. The plasmid concentration was then determined using an ultra-micro UV spectrophotometer.

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

[0069] (1) Using Phanta high-fidelity enzyme and primers in Table 7, and referring to the reaction system and procedure in Tables 2-3, the 5' homologous arm fragment (TgBFD1-5UTR, using primers of SEQ ID NO.13-14) and the 3' homologous arm fragment (TgBFD1-3UTR, using primers of SEQ ID NO.15-16) were amplified from the CAT-containing plasmid (awarded by Huazhong Agricultural University) (using primers of SEQ ID NO.17-18); the pUC19 vector (purchased from http: / / www.addgene.org) (PUC19, using primers of SEQ ID NO.19-20) was linearized using the designed specific primers, and the above four PCR products were recovered using a gel recovery kit, and the concentration of the recovered products was determined using an ultra-micro UV spectrophotometer.

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

[0071]

[0072] (2) Use Exnase MultiS enzyme (Catalog No. C113-02, Nanjing Novizan Biotechnology Co., Ltd.) to prepare the ligation reaction system in Table 8 in a sterile PCR tube to ligate the four recovered products in step (1) above. After mixing the liquid, place it in a PCR instrument 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 section 1 above, the PCR product from step (2) above was added to Trans1-T1 Phage Resistant chemocompetent cells (catalog number CD501-03, Beijing TransGen Biotech Co., Ltd.) for transformation. Single colonies were then placed in LB / Amp + The samples were cultured in liquid culture medium and sent for analysis. The sequencing results were analyzed. If the TgBFD1-5UTR and TgBFD1-3UTR fragment sequences were completely matched after comparison, it indicates that the pTgBFD1-5UTR::CAT::TgBFD1-3UTR-PUC19 homologous template plasmid was successfully constructed.

[0076] (4) Amplify homologous fragments of TgBFD1-5UTR::CAT::TgBFD1-3UTR.

[0077] Using the pTgBFD1-5UTR::CAT::TgBFD1-3UTR-PUC19 homologous template plasmid as a template, PCR amplification was performed using the primers shown in Table 9 and referring to the reaction system and procedure in Tables 2-3. After amplification, the TgBFD1-5UTR::CAT::TgBFD1-3UTR homologous fragment (sequence shown as SEQ ID NO.3) with the correct band size was recovered using a gel recovery kit, and the concentration of the recovered product was determined using an ultra-micro UV spectrophotometer.

[0078] Table 9 Primers for amplifying homologous fragments of TgBFD1-5UTR::CAT::TgBFD1-3UTR.

[0079]

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

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

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

[0083] (2) Add 50 μL Cytomix, 1500 ng of TgBFD1-5UTR::CAT::TgBFD1-3UTR homologous fragment, 7500 ng of pSAG1-Cas9-TgU6-dgTgBFD1 plasmid and the resuspended worms from step (1) above 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 the 4mm electroporation cuvette, being careful not to generate air bubbles; use a BTX electroporator (model Gemini X2, BTX Corporation, USA) for electroporation, aligning the metal surfaces of the two sides of the electroporation cuvette with the metal electrode in the instrument: 1600V, 25μF, 50Ω, 4mm, once, gently shake the electroporation cuvette to mix the liquid; 1500V, 25μF, 50Ω, 4mm, once, gently shake the electroporation cuvette to mix the liquid; 1500V, 25μF, 50Ω, 4mm, once, let stand for 2 minutes, and then add the liquid in the electroporation cuvette to HFF cells (purchased from ATCC Cell Bank) containing 2% FBSDMEM medium for culture.

[0085] (4) When large vesicles are visible in the cells, dilute chloramphenicol to a working concentration of 30 μM in 2% FBSDMEM medium to prepare a drug screening medium. Use this medium to culture the worms for drug screening.

[0086] (5) HFF cells in 2% FBSDMEM medium containing 30 μM chloramphenicol were used for each subsequent passage culture. After 5 to 6 chloramphenicol screening cultures, the electrically converted insect strain library was identified by PCR using Rapid Taq enzyme (P222, Nanjing Novizan Biotechnology Co., Ltd.) and the primers shown in Table 10. The PCR identification reaction system is shown in Table 11, and the reaction procedure is shown in Table 3.

[0087] Table 10 Primers for PCR identification of the Δ6pgdh1Δbfd1 strain

[0088]

[0089] Table 11 PCR Identification Reaction System

[0090]

[0091] (6) The results showed that PCR3 of the parental strain ME49Δ6pgdh1 had a band at the target size, while PCR1, PCR2 and PCR3 of the electrically transferred strain library all had bands at the target size, indicating that the electrically transferred strain library contained strains with the TgBFD1 gene successfully knocked out.

[0092] (7) Collect the electroporated parasite library, dilute and count it, and add it to a 96-well plate containing HFF cells. Add one Toxoplasma gondii tachyzoite to each well. After culturing for 7-10 days, observe whether there are monoclonal parasites in the 96-well plate. Use a pipette tip to scrape off the HFF cells containing monoclonal parasites and add them all to a 24-well plate containing HFF cells. Continue culturing. When 50% of the tachyzoites in the vesicles of the parasites in the 24-well plate have escaped, scrape off the cells in the well. Use half of the cells to extract gDNA (product number DP348, Beijing Tiangen Biotech Co., Ltd.) for monoclonal parasite identification. Passage the remaining part to a new 24-well plate for continued culturing.

[0093] (8) Monoclonal strains were identified by PCR using the primers shown in Table 10. The results are as follows: Figure 2 As shown, the presence of a PCR1 band indicates that the 5' homologous arm of TgBFD1 has integrated into the Toxoplasma gondii genome, and the presence of a PCR2 band indicates that the 3' homologous arm of TgBFD1 has integrated into the Toxoplasma gondii genome. PCR3 detects whether TgBFD1 is still present in the genome. If the control group ME49Δ6pgdh1 shows a PCR3 band, while the transfected experimental group does not, it indicates that TgBFD1 has been successfully knocked out, and the Δ6pgdh1Δbfd1 strain has been constructed.

[0094] Example 2: Construction of Toxoplasma gondii multi-gene deletion strains

[0095] First, plaque and brain cyst formation experiments were conducted on the Δ6pgdh1Δbfd1 strain constructed in Example 1 (details of the experimental methods are provided in Examples 3 and 5). The results showed that knocking out TgBFD1 had no significant effect on the in vitro growth of Toxoplasma gondii, and brain cysts were almost undetectable after in vivo inoculation. Based on this, this example uses the Toxoplasma gondii Δ6pgdh1Δbfd1 strain constructed in Example 1 as the starting strain. The 3-deoxy-D-arabinohepenosaccharide-7-phosphate synthetase (Class-IIDAHP synthetase family protein) TgDAHPS gene (gene ID: TGME49_221260) was searched in the Toxoplasma gondii database ToxoDB. Its nucleotide sequence is shown in SEQ ID NO.2. The multi-gene deletion strain Δ6pgdh1Δbfd1Δdahps was further constructed, and the construction diagram is shown in the figure. Figure 3 As shown.

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

[0097] (1) Using the TgDAHPS genome sequence, the sequence of the target site gRNA was found through the E-CRISP Design website, and primers were designed based on the gRNA sequence. The target-specific primer sequences are shown in Table 12.

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

[0099]

[0100] (2) CRISPR / Cas9 plasmids were constructed and sequenced according to the construction method in Example 1. If the gRNA2 fragment is inserted into the pSAG1-Cas9-TgU6-gRNA1-sgTgDAHPS plasmid after comparison, it indicates that the pSAG1-Cas9-TgU6-dgTgDAHPS plasmid was successfully constructed.

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

[0102] (1) Using Phanta high-fidelity enzyme and primers in Table 13, and referring to the reaction systems and procedures in Tables 2-3, the 5' homologous arm (TgDAHPS-5UTR, using primers SEQ ID NO. 31-32) and 3' homologous arm fragment (TgDAHPS-3UTR, using primers SEQ ID NO. 33-34) of TgDAHPS were amplified from the ME49 genomic DNA of Toxoplasma gondii. * Plasmid (constructed using conventional methods in the field, with Tub as the promoter, YFP as the fluorescent reporter gene, and DHFR as the promoter) * YFP-DHFR was obtained by amplification using a gene (used for screening pyrimethamine drugs) as a template. * The fragments (amplified using primers SEQ ID NO. 35-36) were extracted using a gel extraction kit, and the concentration of the extracted products was determined using an ultra-micro UV spectrophotometer.

[0103] Table 13 Constructing pTgDAHPS-5UTR::YFP-DHFR * ::TgDAHPS-3UTR-PUC19 homologous template plasmid primers

[0104]

[0105] (2) Referring to the method in step 2 of Example 1, and using the PUC19 fragment amplified in that step, pTgDAHPS-5UTR::YFP-DHFR was successfully constructed. * Using the homologous template plasmid ::TgDAHPS-3UTR-PUC19 as a template, PCR amplification was performed using Phanta enzyme and primers shown in Table 14, following the reaction system and procedure in Tables 2-3. After amplification, the correct band size of TgDAHPS-5UTR::YFP-DHFR was extracted using a gel extraction kit. * The homologous fragment of ::TgDAHPS-3UTR (sequence shown in SEQ ID NO.4) was recovered by gel extraction, and the concentration of the recovered product was determined using an ultra-micro UV spectrophotometer.

[0106] Table 14 Amplification of TgDAHPS-5UTR::YFP-DHFR * ::TgDAHPS-3UTR homologous fragment primers

[0107]

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

[0109] (1) Following the same method as in step 3 of Example 1, after resuspending Toxoplasma gondii Δ6pgdh1Δbfd1 tachyzoites in 250 μL of Cytomix, add 50 μL of Cytomix, 7500 ng of pSAG1-Cas9-TgU6-dgTgDAHPS plasmid, and 1500 ng of TgDAHPS-5UTR::YFP-DHFR * The ::TgDAHPS-3UTR homologous fragment was thoroughly mixed and electroporated. After electroporation, it was added to a T25 culture flask containing HFF cells for culture.

[0110] (2) When many spontaneous green fluorescent vacuoles are visible in the cells, the electroporated insect library is identified by PCR using Rapid Taq enzyme and primers shown in Table 15. The PCR identification reaction system and procedure are shown in Table 11 and Table 3. The results showed that PCR3 of the parent insect strains Δ6pgdh1Δbfd1 had a band at the target size, while PCR1, PCR2 and PCR3 of the electroporated insect library all had bands at the target size, indicating that the electroporated insect library contained an insect strain with the TgDAHPS gene successfully knocked out.

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

[0112]

[0113] (3) Collect the electroporation library of worm strains and sort them using a flow cytometer (model BD FACSAria, BD Corporation, USA). Add the strains to 96-well plates containing adherent HFF cells, and add one Toxoplasma gondii tachyzoite to each well. After culturing for 7-10 days, observe whether there are autoluminescent monoclonal worm strains in the 96-well plates. Use a pipette tip to scrape off the HFF cells from the wells containing autoluminescent monoclonal worm strains and add them all to 24-well plates containing HFF cells for expansion culture. When 50% of the tachyzoites in the vesicles of the worms in the 24-well plates have escaped, scrape off the cells from the wells. Use half of the cells to extract gDNA for monoclonal worm strain identification, and passage the remaining part to new 24-well plates for continued culture.

[0114] (4) Monoclonal strains were identified by PCR using the primers shown in Table 15. The results are as follows: Figure 4 As shown, the presence of PCR1 band indicates that the 5' homologous arm of TgDAHPS is integrated into the Toxoplasma gondii genome, and the presence of PCR2 band indicates that the 3' homologous arm of TgDAHPS is integrated into the Toxoplasma gondii genome. PCR3 detects whether TgDAHPS is still in the genome. If the control group Δ6pgdh1Δbfd1 has a PCR3 band, while the transfection experimental group does not have a PCR3 band, it indicates that TgDAHPS has been successfully knocked out, and the Δ6pgdh1Δbfd1Δdahps strain has been constructed.

[0115] Example 3: In vitro plaque experiment of gene-deleted insect strains

[0116] (1) Wild-type ME49 insect strain, Δ6pgdh1Δbfd1 insect strain constructed in Example 1, and Δ6pgdh1Δbfd1Δdahps insect strain constructed in Example 2 were cultured separately, and intracellular tachyzoites were collected.

[0117] (2) Add the collected tachyzoites to each well of a 6-well plate containing HFF cells, with 100 tachyzoites added to each well, and incubate at 37°C for 7–14 days.

[0118] (3) Wash twice with PBS, 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 empty spots using a scanner (ScanMaker i600, Shanghai Zhongjing Technology Co., Ltd.) and save the image.

[0121] The results of the empty spots are as follows Figure 5 and Figure 6As shown, the plaque size of ME49 and Δ6pgdh1Δbfd1 strains is almost the same, as is the plaque size of Δ6pgdh1Δbfd1 and Δ6pgdh1Δbfd1Δdahps strains. This indicates that knocking out TgBFD1 and TgDAHPS on the basis of ME49Δ6pgdh1 strain will not affect the in vitro growth of Toxoplasma gondii.

[0122] Example 4: In vivo toxicity experiment of gene-deleted insect strains

[0123] The Δ6pgdh1Δbfd1 and Δ6pgdh1Δbfd1Δdahps insect strains constructed in Examples 1 and 2 were used to dilute tachyzoites in FBS-free DMEM medium. Both strains were diluted with 1×10⁻⁶ mol / L. 2 Eight mice (ICR strain, Guangzhou Ruige Biotechnology Co., Ltd.) were inoculated with a dose of 1 parasite / mouse. Simultaneously, the Δ6pgdh1Δbfd1Δdahps strain was administered at a dose of 1×10⁻⁶. 4 1×10 5 Five mice were inoculated with a dose of 1 parasite / mouse. The mice's mental status and survival rate were recorded. Blood samples were collected from the mice after 21 days, and indirect immunofluorescence assay (IFA) was used to determine successful infection. Uninfected mice were culled. Mouse survival curves were analyzed using GraphPad Prism 9.5 software, and Kaplan-Meier survival plots were generated.

[0124] The results are as follows Figure 7 As shown, within 30 days, at 1×10 2 Mice inoculated with the Δ6pgdh1Δbfd1 strain at a dose of 1 × 10⁻⁶ parasites / mouse had a survival rate of 62.5%, while mice inoculated with the Δ6pgdh1Δbfd1Δdahps strain had a survival rate of 62.5% at a dose of 1 × 10⁻⁶ parasites / mouse. 2 1×10 4 1×10 5 All of them survived and did not exhibit clinical symptoms of toxoplasmosis such as rough hair or lethargy, indicating that the Δ6pgdh1Δbfd1Δdahps strain has almost no virulence in vivo.

[0125] Example 5: Brain cyst formation experiment of gene-deleted parasite strains

[0126] (1) Using the parental insect strain ME49Δ6pgdh1 and the Δ6pgdh1Δbfd1 insect strain constructed in Example 1, the tachyzoites were diluted with FBS-free DMEM medium, with each diluted to 1×10⁻⁶. 2Eight mice were inoculated with a dose of 1 parasite / mouse. Blood samples were collected from the mice 21 days later, and indirect immunofluorescence assay (IFA) was used to determine whether infection was successful. Uninfected mice were culled. Brain tissue from mice still alive after 30 days was stained with DBA, the number of brain cysts was counted, and the results were analyzed using Student's t-test.

[0127] (2) Using Example 4 with 1×10 2 Mice that survived 30 days after being inoculated with Δ6pgdh1Δbfd1 and Δ6pgdh1Δbfd1Δdahps strains were subjected to DBA staining of their brain tissue, and the number of brain cysts was counted. The results were analyzed using Student's t-test.

[0128] The results are as follows Figure 8 and Figure 9 As shown, the Δ6pgdh1Δbfd1 strain exhibited a significantly reduced number of brain cysts compared to the control parental strain, with almost no brain cysts forming. Furthermore, no brain cysts were detected in mice inoculated with the Δ6pgdh1Δbfd1Δdahps strain, indicating that the Δ6pgdh1Δbfd1Δdahps strain almost completely fails to form cysts in mice.

[0129] Example 6: Immunoprotective effect of gene-deleted parasite strain on mice

[0130] (1) When the Chinese I strain (a gift from Huazhong Agricultural University, which is a superior and highly virulent strain in my country) cultured in HFF cells has large vesicles, wash away the extracellular tachyzoites with PBS, add DMEM medium without FBS and collect the intracellular tachyzoites. Take 100 μL of the liquid containing tachyzoites, dilute it 10 times and count it under a microscope. After counting, dilute the tachyzoites with DMEM medium without FBS.

[0131] (2) Immunization dose of 1×10 3 Mice inoculated with Δ6pgdh1Δbfd1Δdahps parasites for 42 days and unimmunized control mice were re-infected with the Chinese I strain (1×10⁻⁶ pgdh1Δbfd1Δdahps). 4 (Individual insects / mouse). The mental state and survival rate of the mice were observed and recorded, and the results were statistically analyzed and survival curves were plotted using GraphPad Prism 9.5 software.

[0132] The results are as follows Figure 10 As shown, unimmunized control mice were infected with 1×10 4 After one Chinese I worm was introduced, all of them died within 11 days, while after 1×10 3Mice immunized with the Δ6pgdh1Δbfd1Δdahps tachyzoite had a 100% survival rate within 30 days. This indicates that inoculation with the Δ6pgdh1Δbfd1Δdahps strain can produce good immunoprotection and can be used to prepare Toxoplasma gondii vaccines.

[0133] In summary, this invention, through knockout of the Myb-like transcription factor TgBFD1 gene and the 3-deoxy-D-arabinohepnigulipose-7-phosphate synthase TgDAHPS gene of *Toxoplasma gondii*, found that knocking out TgBFD1 in a Tg6PGDH1 knockout strain did not significantly affect the in vitro growth rate of *Toxoplasma gondii*, but it further reduced cyst formation. Furthermore, simultaneously knocking out both TgBFD1 and TgDAHPS in a Tg6PGDH1 knockout strain did not affect the in vitro growth of *Toxoplasma gondii*, but the parasite was almost non-toxic and did not form cysts. Immunization with the above-mentioned multi-gene deletion strains effectively resisted infection by highly virulent strains, demonstrating good immunoprotective effects. The multi-gene deletion strains Δ6pgdh1Δbfd1Δdahps provided by this invention offer 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 to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. A Toxoplasma gondii multi-gene deletion vaccine strain, characterized in that, The insect strains were genetically edited to knock out glucosyl 6-phosphate dehydrogenase in Toxoplasma gondii. Tg 6PGDH1 gene, Myb-like transcription factor Tg BFD1 gene and 3-deoxy-D-arabinohepenoyl-7-phosphate synthase Tg The DAHPS gene was obtained afterward; Tg The gene ID of the 6PGDH1 gene is: TGME49_242600. Tg The gene ID for BFD1 is: TGME49_200385. Tg The gene ID of the DAHPS gene is: TGME49_221260; the strain is ME49.

2. The method for constructing the Toxoplasma gondii multi-gene deletion vaccine strain according to claim 1, characterized in that, Includes the following steps: S1. ME49 Δ6pgdh1 The insect strain was the originating strain; it was knocked out. Tg BFD1 gene construction Δ6pgdh1Δbfd1 Insect strains; S2. In Δ6pgdh1Δbfd1 Knockout from insect strains Tg DAHPS gene was used to obtain Toxoplasma gondii multi-gene deletion strains. Δ6pgdh1 Δbfd1Δdahps .

3. Deletion or knockout of Toxoplasma gondii Myb-like transcription factor Tg BFD1 gene and 3-deoxy-D-arabinohepenoyl-7-phosphate synthase Tg The application of the reagent for the DAHPS gene in constructing the Toxoplasma gondii multi-gene deletion vaccine strain as described in claim 1.

4. Deletion or knockout of Toxoplasma gondii Myb-like transcription factor Tg BFD1 gene and 3-deoxy-D-arabinohepenoyl-7-phosphate synthase Tg The application of the DAHPS gene reagent in the preparation of Toxoplasma gondii vaccines containing the Toxoplasma gondii multi-gene deletion vaccine strain as described in claim 1.

5. The use of the Toxoplasma gondii multi-gene deletion strain according to claim 1 in the preparation of Toxoplasma gondii vaccine.

6. The application of the method of claim 2 in the preparation of Toxoplasma gondii vaccine.

7. A Toxoplasma gondii vaccine, characterized in that, The strain containing the Toxoplasma gondii multigene deletion as described in claim 1.

8. The vaccine according to claim 7, characterized in that, The vaccine in question is a live attenuated vaccine.

9. The vaccine according to claim 7, characterized in that, The vaccine also contains medically acceptable vaccine adjuvants.

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