A Toxoplasma gondii triple gene knockout vaccine strain and its application

Abstract

The invention discloses a Toxoplasma triple gene knockout vaccine strain and its application. The invention knocks out three genes of Toxoplasma, namely, sedoheptulose-1,7-bisphosphatase TgSBPase, transaldolase TgTAL and ribulose-5-phosphate epimerase TgRuPE, by gene editing technology, and successfully constructs a TgSBPase, TgTAL and TgRuPE triple gene knockout strain; after knocking out these three genes, Toxoplasma can be subcultured in vitro, and has almost non-toxic characteristics in vivo, and mice can effectively resist infection of wild toxic strains after being immunized with the triple gene knockout strain, and have good immune protection, providing methods and means for developing efficient, non-toxic and safe anti-toxoplasmosis vaccines.

Description

Technical Field

[0001] The present invention belongs to the field of genetic engineering technology, and more specifically, relates to a Toxoplasma gondii triple gene knockout vaccine strain and its application. Background Art

[0002] Toxoplasmosis is a zoonotic parasitic disease caused by the apicomplexan protozoan Toxoplasma gondii, which seriously endangers human health and the sustainable development of animal husbandry. Healthy people or animals infected with Toxoplasma gondii show no symptoms or only mild clinical symptoms, but people and animals with low or defective immunity will suffer serious consequences and even endanger their lives. Toxoplasma gondii can invade the eyes, brain, etc. of the body, causing toxoplasmosis eye disease and encephalopathy, and can also cause adverse consequences such as miscarriage and stillbirth in pregnant women or pregnant livestock. The transmission routes of Toxoplasma gondii are complex and diverse, the most common of which is oral transmission. People or animals are generally infected by drinking water contaminated with Toxoplasma oocysts, or accidentally eating raw or undercooked meat containing tissue cysts; toxoplasmosis can also be transmitted vertically to cause congenital toxoplasmosis in the fetus. Clinically, only drugs such as pyrimethamine and sulfadiazine are effective against Toxoplasma tachyzoites and can inhibit the growth and reproduction of Toxoplasma. However, long-term use can produce side effects and cause drug resistance. Currently, there is a severe lack of drugs and vaccines against toxoplasmosis. Therefore, the development of a safe and effective Toxoplasma vaccine is particularly important for the prevention of toxoplasmosis.

[0003] Due to the close relationship between metabolic pathways and the growth, reproduction and virulence of the parasite, it is a potential target for drug and vaccine development. Among them, glucose is transported into the parasite cytoplasm through the glucose transporter GT1, and glucose-6-phosphate (G6P) is generated under the catalysis of hexokinase HK, participating in glycolysis and pentose phosphate metabolism. The pentose phosphate metabolic pathway (PPP) is closely linked to the glycolysis pathway and is the second important pathway for glucose catabolism. PPP is usually divided into two stages: oxidation reaction and non-oxidation reaction, both of which participate in the generation of ribose-5-phosphate (R5P). R5P is a key precursor for nucleotide biosynthesis and directly affects the synthesis of RNA and DNA. Existing studies have shown that in the PPP stage, ribulose-5-phosphate isomerase, glucose-6-phosphate dehydrogenase, and transketolase are related to the growth and virulence of the parasite. Knocking out these genes will reduce the growth and reproduction ability of Toxoplasma and weaken the virulence of Toxoplasma. However, there are still few studies on other enzymes related to R5P production in Toxoplasma, and their relationship with parasite growth and virulence, and whether they can be used as vaccine targets are still unknown. Therefore, it is necessary to develop more effective and non-toxic attenuated strains or vaccines that can prevent and treat toxoplasmosis, and provide more methods and technical support for the fight against toxoplasmosis. Summary of the invention

[0004] The technical problem to be solved by the present invention is to overcome the deficiencies of existing attenuated strains or vaccines against toxoplasmosis, and to provide a Toxoplasma triple-gene knockout vaccine strain and its application.

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

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

[0007] The third object of the present invention is to provide the application of Toxoplasma gondii sedoheptulose-1,7-bisphosphatase TgSBPase, transaldolase TgTAL and ribulose-5-phosphate isomerase TgRuPE genes.

[0008] The fourth object of the present invention is to provide the use of a reagent for deleting or knocking out the genes of Toxoplasma sedoheptulose-1,7-bisphosphatase TgSBPase, transaldolase TgTAL and ribulose-5-phosphate epimerase TgRuPE.

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

[0010] The sixth object of the present invention is to provide a drug for preventing and treating toxoplasmosis.

[0011] The above-mentioned purpose of the present invention is achieved through the following technical solutions:

[0012] The invention provides a method for constructing a Toxoplasma gondii triple gene knockout strain, wherein sedoheptulose-1,7-bisphosphatase TgSBPase, transaldolase TgTAL and ribulose-5-phosphate epimerase TgRuPE genes of Toxoplasma gondii are knocked out by CRISPR gene editing technology to obtain a Toxoplasma triple gene knockout strain; wherein the nucleotide sequence of the TgSBPase gene (gene ID: TGGT1_235700) is shown in SEQ ID NO: 1, the nucleotide sequence of the TgTAL gene (gene ID: TGGT1_229360) is shown in SEQ ID NO: 2, and the nucleotide sequence of the TgRuPE gene (gene ID: TGGT1_247670) is shown in SEQ ID NO: 3.

[0013] The present invention focuses on enzymes related to R5P production, including TgRuPE and TgTAL in the PPP stage and TgSBPase in the ribogluconeogenesis pathway. It was found that knocking out TgSBPase, TgTAL, and TgRuPE genes separately had no effect on Toxoplasma, while knocking out two or three of them at the same time affected the growth and reproduction of Toxoplasma and was related to the virulence of the parasite. Further, through the construction of genetically modified strains, a genetically modified strain (ΔsbpaseΔtalΔrupe) with three genes of TgSBPase, TgTAL, and TgRuPE knocked out was obtained. After the three genes of TgSBPase, TgTAL, and TgRuPE were deleted from the wild-type DiCre strain of Toxoplasma type I, the parasite could be passaged and grown in vitro, and the virulence was significantly weakened. The three-gene knockout strain was almost non-toxic, and the infection dose was 10 5 Under the condition of ΔsbpaseΔtalΔrupe / mouse, the mortality rate of mice was still 0%; ΔsbpaseΔtalΔrupe vaccination can effectively resist reinfection of wild-type parasites, and the immune protection effect is 100%. At the same time, TgSBPase, TgTAL and TgRuPE genes can be used as targets to screen or prepare drugs for the prevention and treatment of toxoplasmosis, which is of great significance for the prevention and treatment of toxoplasmosis and vaccine development, and protects the healthy development of humans and animal husbandry.

[0014] The present invention provides a specific method for constructing a Toxoplasma gondii triple gene knockout strain, comprising the following steps:

[0015] S1. Using the wild-type Toxoplasma gondii strain as the starting strain, the TgSBPase gene was directly knocked out to construct the DiCreΔsbpase strain;

[0016] S2. Based on the DiCreΔsbpase strain, the TgTAL gene was knocked out to construct the ΔsbpaseΔtal strain;

[0017] S3. Finally, the TgRuPE gene was knocked out in the ΔsbpaseΔtal strain to obtain the Toxoplasma gondii triple-gene knockout strain ΔsbpaseΔtalΔrupe.

[0018] Preferably, the method for constructing the DiCreΔsbpase strain in step S1 is: using the pSAG1-Cas9-TgU6-sgTgUPRT plasmid as a template to construct a TgSBPase-specific CRISPR / Cas9 knockout plasmid; then using the ploxp-DHFR*-loxp plasmid as a template to amplify the Ko-SBPase-loxp-DHFR*-loxp homologous fragment, and co-electroporating the above plasmid and fragment into a wild-type Toxoplasma strain, performing drug screening and PCR identification to obtain the DiCreΔsbpase strain.

[0019] More preferably, the primer sequences for constructing the TgSBPase-specific CRISPR / Cas9 knockout plasmid are shown in SEQ ID NOs: 4 to 5; the primer sequences for amplifying the Ko-SBPase-loxp-DHFR*-loxp homologous fragment are shown in SEQ ID NOs: 6 to 7.

[0020] Furthermore, the construction method of the ΔsbpaseΔtal strain in step S2 is: first remove the DHFR* tag in the DiCreΔsbpase strain; use the pSAG1-Cas9-TgU6-sgTgUPRT plasmid as a template, design the TgTAL gene gRNA primer, and prepare the knockout plasmid pSAG1-Cas9-TgU6-sgTgTAL; then use the ploxp-DHFR*-loxp plasmid as a template to amplify the Ko-TAL-loxp-DHFR*-loxp homologous fragment, and co-electroporate the above plasmid and fragment into the DiCreΔsbpase strain, perform drug screening and PCR identification, and obtain the ΔsbpaseΔtal strain.

[0021] More preferably, the primer sequences of TgTAL gene gRNA are shown as SEQ ID NO: 12 and SEQ ID NO: 5; the primer sequences for amplifying Ko-TAL-loxp-DHFR*-loxp homologous fragments are shown as SEQ ID NO: 13-14.

[0022] Furthermore, the construction method of the ΔsbpaseΔtalΔrupe strain in step S3 is: first remove the DHFR* tag in the ΔsbpaseΔtal strain; then use the pSAG1-Cas9-TgU6-sgTgUPRT plasmid as a template, design the TgRuPE gene gRNA primer, and prepare the knockout plasmid pSAG1-Cas9-TgU6-sgTgRuPE; then use the pTub-YFP-DHFR* plasmid as a template to amplify the Ko-RuPE-YFP-DHFR* homologous fragment, and co-electroporate the above plasmid and fragment into the ΔsbpaseΔtal strain, perform drug screening and PCR identification, and obtain the ΔsbpaseΔtalΔrupe strain.

[0023] More preferably, the primer sequences of TgRuPE gene gRNA are shown as SEQ ID NO: 21 and SEQ ID NO: 5; the primer sequences for amplifying Ko-RuPE-YFP-DHFR* homologous fragments are shown as SEQ ID NO: 22-23.

[0024] Preferably, the wild-type Toxoplasma gondii strain in step S1 is the Toxoplasma gondii RH (DiCre) strain.

[0025] Furthermore, drug screening uses pyrimethamine.

[0026] Preferably, in steps S2 and S3, the DHFR* tag in the insect strain is removed by treating with rapamycin (Rapa) for 2 days.

[0027] The present invention provides a Toxoplasma gondii triple gene knockout strain, which is constructed by the above method.

[0028] The present invention provides the use of Toxoplasma gondii sedoheptulose-1,7-bisphosphatase TgSBPase, transaldolase TgTAL and ribulose-5-phosphate epimerase TgRuPE genes as targets in screening or preparing drugs for preventing and treating toxoplasmosis.

[0029] The present invention provides a reagent for deleting or knocking out Toxoplasma sedoheptulose-1,7-bisphosphatase TgSBPase, transaldolase TgTAL and ribulose-5-phosphate epimerase TgRuPE genes for use in constructing a Toxoplasma triple-gene knockout strain or in preparing a drug for preventing and treating toxoplasmosis.

[0030] Furthermore, agents that delete or knock out the Toxoplasma TgSBPase, TgTAL, and TgRuPE genes affect the complete expression of the Toxoplasma TgSBPase, TgTAL, and TgRuPE genes, including but not limited to siRNA, CRISPR / Cas9, and chemical reagents.

[0031] The present invention provides application of a Toxoplasma triple gene knockout strain in preparing a Toxoplasma vaccine.

[0032] The invention provides a Toxoplasma gondii vaccine, comprising a Toxoplasma gondii triple gene knockout strain.

[0033] Furthermore, the vaccine is a weakly attenuated vaccine.

[0034] The present invention also provides a drug for preventing and treating toxoplasmosis, comprising a reagent for lacking or knocking out the genes of Toxoplasma sedoheptulose-1,7-bisphosphatase TgSBPase, transaldolase TgTAL and ribulose-5-phosphate epimerase TgRuPE.

[0035] The present invention has the following beneficial effects:

[0036] By studying the three genes of Toxoplasma gondii, namely, sedoheptulose-1,7-bisphosphatase TgSBPase, transaldolase TgTAL, and ribulose-5-phosphate isomerase TgRuPE, it was found that knocking out the single genes of TgSBPase, TgTAL, and TgRuPE had no significant effect on Toxoplasma, while knocking out two or three of them at the same time would affect the growth and reproduction of Toxoplasma and was related to the virulence of the parasite. A Toxoplasma gondii TgSBPase, TgTAL, and TgRuPE three-gene knockout strain was successfully constructed; after knocking out the TgSBPase, TgTAL, and TgRuPE genes at the same time, it can not only be cultured in vitro, but also has almost non-toxic characteristics in vivo. After immunization, the vaccine strain can effectively resist the infection of wild toxic strains, showing good immune protection effect, and has a good immune protection effect on mice. The triple-gene knockout strain ΔsbpaseΔtalΔrupe provided by the invention provides methods and means for developing more effective and non-toxic anti-toxoplasmosis drugs and vaccines. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] Figure 1 Schematic diagram of knocking out Toxoplasma gondii DiCreΔsbpase.

[0038] Figure 2 These are the PCRs identification results of the DiCreΔsbpase monoclonal strain.

[0039] Figure 3 Schematic diagram of knocking out Toxoplasma ΔsbpaseΔtal.

[0040] Figure 4 These are the PCRs identification results of the ΔsbpaseΔtal monoclonal insect strain.

[0041] Figure 5 Schematic diagram of knocking out Toxoplasma ΔsbpaseΔtalΔrupe.

[0042] Figure 6 These are the PCRs identification results of the ΔsbpaseΔtalΔrupe monoclonal insect strain.

[0043] Figure 7 These are the results of the plaque assay of the ΔsbpaseΔtal insect strain.

[0044] Figure 8 These are the results of the plaque assay of the ΔsbpaseΔtalΔrupe insect strain.

[0045] Fig. 9 These are the results of the virulence experiment on the Δsbpase insect strain.

[0046] Fig.10These are the results of the virulence experiment on the ΔsbpaseΔtal insect strain.

[0047] Fig.11 These are the results of the virulence gradient experiment of the ΔsbpaseΔtalΔrupe insect strain.

[0048] Fig.12 These are the results of the immune protection experiment when mice immunized with the ΔsbpaseΔtalΔrupe strain were infected with the wild-type RHΔku80.

[0049] Fig.13 These are the results of the immune protection experiment when mice immunized with the ΔsbpaseΔtalΔrupe strain were infected with the wild-type ME49. DETAILED DESCRIPTION

[0050] 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.

[0051] Unless otherwise specified, the reagents and materials used in the following examples are commercially available.

[0052] Example 1 Construction of Toxoplasma gondii single gene knockout strain

[0053] In this example, the type I wild-type Toxoplasma gondii RH (DiCre) strain (kindly donated by Huazhong Agricultural University) was used as the starting strain, and the sedoheptulose-1,7-bisphosphatase TgSBPase gene in the type I Toxoplasma gondii strain was searched from the ToxoDB (www.toxodb.org) Toxoplasma website, and its nucleotide sequence is shown in SEQ ID NO.1 (gene ID: TGGT1_235700, sedoheptulose-1,7-bisphosphatase).

[0054] 1. Construction of pSAG1-Cas9-TgU6-sgTgSBPase plasmid

[0055] (1) Design gRNA primers on the E-CRISP Design website (http: / / www.e-crisp.org / E-CRISP / designcrispr.html), find the target site from the TgSBPase genome sequence, and design the plasmid according to the gRNA sequence. The designed target primer sequences are shown in Table 1;

[0056] Table 1 Specific primers for constructing TgSBPase CRISPR / Cas9 plasmid

[0057]

[0058] (2) Using the commercial pSAG1-Cas9-TgU6-sgTgUPRT plasmid as a template, the TgRuPE-specific CRISPR / Cas9 plasmid was constructed. The PCR amplification reaction system and procedure are shown in Tables 2 and 3;

[0059] Table 2 PCR reaction system

[0060]

[0061]

[0062] After the reaction, 1 μL of Dpn I was directly added to the PCR product and digested in a PCR instrument at 37°C for 45 min;

[0063] (3) Prepare the reaction system shown in Table 4 in a new PCR tube, mix the liquids, and place in a PCR tube for reaction at 25°C for 15 min;

[0064] Table 4 PCR reaction system

[0065]

[0066] (4) All PCR products of step (3) above were added to competent E. coli cells DH5α, and the bacterial solution was heat-shocked at 42°C and then lysed using solid LB / Amp + Culture the medium overnight for about 12 hours; take a single clone and place it in LB / Amp + The liquid culture medium was expanded and identified, and the bacterial liquid identified correctly by PCR was further sequenced and analyzed according to the sequencing results. If the comparison showed that the target sequence was completely covered, it meant that the pSAG1-Cas9-TgU6-sgTgSBPase plasmid was successfully constructed.

[0067] 2. Amplify Ko-SBPase-loxp-DHFR*-loxp homologous fragment

[0068] Using the ploxp-DHFR*-loxp plasmid (kindly donated by Huazhong Agricultural University) as a template, PCR amplification was performed using the primers and high-fidelity enzymes shown in Table 5. After amplification, the Ko-SBPase-loxp-DHFR*-loxp homologous fragment with the correct band size was recovered by gel and stored at -20°C for a long time.

[0069] Table 5 Primers for amplifying Ko-SBPase-loxp-DHFR*-loxp fragment

[0070]

[0071]

[0072] 3. Construction of the Toxoplasma gondii knockout strain DiCreΔsbpase

[0073] (1) Collect the Toxoplasma gondii DiCre tachyzoites in the larger vesicles and centrifuge them at 3000 rpm for 8 min at room temperature; resuspend the precipitated parasites with 8 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) and centrifuge them at 3000 rpm for 8 min at room temperature; resuspend the precipitated parasites with 250 μL cytomix;

[0074] (2) Add 50 μL cytomix, 7500 ng of pSAG1-Cas9-TgU6-sgTgSBPase plasmid and 1500 ng of Ko-SBPase-loxp-DHFR*-loxp homologous fragment to a 1.5 mL EP tube, and transfer all the resuspended worms in step (1) above to the EP tube;

[0075] (3) Use a pipette to mix the liquid in the EP tube thoroughly, and add all of it to a 4mm electrotransfer cup, taking care not to generate bubbles; use a Bio-Rad electrotransfer instrument for electrotransfection, align the metal surfaces on both sides of the electrotransfer cup with the metal sheet electrodes in the instrument: 1600V, 25μF, 50Ω, 4mm, shock once, and gently turn the electrotransfer cup to mix the liquid; 1500V, 25μF, 50Ω, 4mm, shock once, and gently turn the electrotransfer cup to mix the liquid; 1500V, 25μF, 50Ω, 4mm, shock once, let it stand for 2 minutes, and then add the liquid in the electrotransfer cup to human fibroblast HFF cells (purchased from ATCC cell bank) containing 2% FBSDMEM culture medium for culture;

[0076] (4) When larger parasite vacuoles were visible in the cells, pyrimethamine was diluted to a working concentration of 1 μM and added to approximately 5 mL of 2% FBSDMEM medium, and this medium was used for drug screening;

[0077] (5) Thereafter, each subculture was performed using fresh human fibroblast HFF cells in 2% FBSDMEM medium containing 1 μM pyrimethamine. After 2-4 rounds of pyrimethamine screening culture, the electroporated strain library was identified by PCR using the primers shown in Table 6. The results showed that both PCR3 and PCR4 of the wild-type DiCre had bands at the target size, while PCR4 of the electroporated strain library amplified two target bands, indicating that the electroporated strain library contained strains in which the TgSBPase gene was successfully knocked out. The schematic diagram of the construction of the knockout of Toxoplasma DiCreΔsbpase is shown in FIG. Figure 1 As shown;

[0078] Table 6 Primers for PCR identification of monoclonal insect strains of DiCreΔsbpase

[0079]

[0080]

[0081] (6) In order to perform secondary knockouts later, we treated the correctly identified strain library with Rapa for 2 days to remove the DHFR* drug screening label and screen for monoclonal strains. Collect the electroporated strain library, dilute and count, and add it to a 96-well plate with HFF cells growing on the wall, with one Toxoplasma tachyzoite added to each well; after 5-7 days of culture, observe whether there are monoclonal strains in the 96-well plate, use a pipette to scrape the HFF cells in the wells containing the monoclonal strains, and add all of them to a 24-well plate with HFF cells growing on the wall to expand the culture; when 50% of the tachyzoites in the vesicles in the 24-well plate escape, scrape the cells in the wells, half of which are used to extract DNA for monoclonal strain identification, and the rest are subcultured to a new 24-well plate for continued culture;

[0082] (7) Monoclonal strains were identified by PCR using the primers shown in Table 6; the results are shown in Figure 2 As shown, PCR3 and PCR4 of the wild-type DiCre and the electroporated insect strain library both have bands at the target size, while PCR3 of the monoclonal insect strain has no band at the target size, and PCR4 only amplifies one band, indicating that the monoclonal insect strain successfully knocked out the TgSBPase gene and DHFR* drug screening gene to obtain the DiCreΔsbpase insect strain.

[0083] Example 2 Construction of Toxoplasma gondii double gene knockout strain

[0084] After plaque and virulence experiments were performed on the Δsbpase strain, the results showed that knocking out TgSBPase had no significant effect on the in vitro growth and in vivo virulence of Toxoplasma (see Example 4 for specific results); it was also found that knocking out TgTAL or TgRuPE genes, respectively, had no effect on the in vitro and in vivo growth rate and virulence of Toxoplasma. Based on this, the DiCreΔsbpase strain of Toxoplasma gondii constructed in Example 1 was used as the starting strain in this example, and the transaldolase TgTAL gene was searched from the ToxoDB (www.toxodb.org) Toxoplasma website, and its nucleotide sequence was shown in SEQ ID NO.2 (gene ID: TGGT1_229360, transaldolase), and a double gene deletion strain was further constructed.

[0085] 1. Construction of pSAG1-Cas9-TgU6-sgTgTAL plasmid

[0086] (1) Design gRNA on the E-CRISP Design website (http: / / www.e-crisp.org / E-CRISP / designcrispr.html), find the targeting site in the TgTAL genome sequence, and design a specific plasmid based on the gRNA sequence. The designed target-specific primer sequences are shown in Table 7;

[0087] Table 7 Specific primers for constructing TgTAL CRISPR / Cas9 plasmid

[0088]

[0089] (2) CRISPR / Cas9 plasmid was constructed and identified according to the construction method of Example 1. The sequencing results showed that the pSAG1-Cas9-TgU6-sgTgTAL plasmid was successfully constructed.

[0090] 2. Amplify Ko-TAL-loxp-DHFR*-loxp homology fragment

[0091] Using ploxp-DHFR*-loxp plasmid (purchased from http: / / www.addgene.org) as a template, PCR amplification was performed using the primers and high-fidelity enzyme shown in Table 8, and the Ko-TAL-loxp-DHFR*-loxp homologous fragment with the correct band size was recovered by gel recovery.

[0092] Table 8 Primers for amplifying Ko-TAL-loxp-DHFR*-loxp fragment

[0093]

[0094] 3. Construction of the Toxoplasma gondii gene knockout strain ΔsbpaseΔtal

[0095] (1) Using the same method as in step 3 of Example 1, fresh Toxoplasma gondii DiCreΔsbpase tachyzoites were collected and resuspended in 250 μL cytomix, and 7500 ng of pSAG1-Cas9-TgU6-sgTgTAL plasmid and 1500 ng of Ko-TAL-loxp-DHFR*-loxp homology fragment were thoroughly mixed with the resuspended tachyzoites for electrotransfection using the same method as in Example 1. After electrotransfection, HFF cells containing 2% FBSDMEM medium were added for culture;

[0096] (2) When larger parasite vacuoles are visible in the cells, dilute pyrimethamine to a working concentration of 1 μM and add it to about 5 mL of 2% FBSDMEM medium, and use this medium for drug screening;

[0097] (3) After 2-4 subcultures, HFF cells were screened and cultured in 2% FBSDMEM medium containing 1 μM pyrimethamine, and the electroporated strain library was identified by PCR using the primers shown in Table 9; the results showed that there was no band at the corresponding size of the DiCreΔsbpase strain, while the target band was amplified by PCR1 and PCR2 of the electroporated strain library, indicating that the electroporated strain library had successfully integrated the DHFR* drug screening tag; the schematic diagram of the construction of the knockout of Toxoplasma ΔsbpaseΔtal is shown in Figure 3 As shown;

[0098] Table 9 PCR primers for identification of monoclonal insect strains of ΔsbpaseΔtal

[0099]

[0100] (4) After proving that the DHFR* drug screening tag has been successfully integrated into the electroporated strain library, screen for monoclonal strains. Collect the electroporated strain library, dilute and count, and use a 96-well plate with adherent HFF cells for screening. Add one Toxoplasma tachyzoite to each well; after culturing for about 7 days, select strains with only a single plaque in the 96-well plate, use a pipette to scrape the HFF cells in the wells containing the monoclonal strain, add all of them to a 24-well plate with adherent HFF cells, and expand the culture; when 50% of the tachyzoites in the vesicles of the strain in the 24-well plate have escaped, scrape the cells in the wells, extract DNA from half of them for monoclonal strain identification, and transfer the rest to a new 24-well plate for continued culture;

[0101] (5) Monoclonal strains were identified by PCR using the primers shown in Table 9. The results are as follows: Figure 4 As shown, there was no band at the corresponding size of the DiCreΔsbpase strain, while PCR1 and PCR2 of the monoclonal strain amplified the target band, indicating that the electroporated strain had successfully integrated the DHFR* drug screening tag; and there was no band at the corresponding size of PCR3 of the monoclonal strain, and DiCreΔsbpase amplified the target band, which means that the monoclonal strain with the TgTAL gene knocked out was successfully screened out, and the ΔsbpaseΔtal strain was obtained.

[0102] Example 3 Construction of Toxoplasma gondii triple gene knockout strain ΔsbpaseΔtalΔrupe

[0103] The Toxoplasma ΔsbpaseΔtal strain constructed in Example 2 was used as the starting strain, and the ribulose-5-phosphate epimerase TgRuPE gene of Toxoplasma type I strain was searched from the ToxoDB (www.toxodb.org) Toxoplasma website, and its nucleotide sequence was shown in SEQ ID NO.3 (gene ID: TGGT1_247670, ribulose-5-phosphate epimerase).

[0104] 1. Remove DHFR* tag

[0105] The ΔsbpaseΔtal strain was pretreated with Rapa for 2 days to remove the DHFR* tag.

[0106] 2. Construction of pSAG1-Cas9-TgU6-sgTgRuPE plasmid

[0107] (1) Design gRNA on the E-CRISP Design website (http: / / www.e-crisp.org / E-CRISP / designcrispr.html), find the targeting site in the TgRuPE genome sequence, and design a specific plasmid based on the gRNA sequence. The designed specific primer sequences are shown in Table 10;

[0108] Table 10 Specific primers for constructing TgTAL CRISPR / Cas9 plasmid

[0109]

[0110] (2) The pSAG1-Cas9-TgU6-sgTgRuPE plasmid was successfully constructed according to the construction method of Example 1.

[0111] 2. Amplify the Ko-RuPE-YFP-DHFR* homologous fragment

[0112] Using pTub-YFP-DHFR* plasmid (constructed by conventional methods in the art, Tub is the promoter, Loxp is the characteristic sequence recognized by the Cre protein, YFP is the fluorescent reporter gene, and DHFR* is the pyrimethamine drug screening gene) as a template, PCR amplification was performed using a high-fidelity enzyme and the primers shown in Table 11, and the amplified Ko-RuPE-YFP-DHFR* homologous fragment was recovered from gel and stored at -20°C.

[0113] Table 11 Primers for amplifying Ko-RuPE-YFP-DHFR* fragment

[0114]

[0115]

[0116] 3. Construction of the Toxoplasma gondii knockout strain ΔsbpaseΔtalΔrupe

[0117] (1) Using the same method as in step 3 of Example 1, fresh Toxoplasma ΔsbpaseΔtal tachyzoites were collected and resuspended in 250 μL cytomix, and 50 μL cytomix, 7500 ng pSAG1-Cas9-TgU6-sgTgRuPE plasmid, 1500 ng Ko-RuPE-YFP-DHFR* homologous fragment and the resuspended tachyzoites were fully mixed and electro-transfected in the same manner as in Example 1. After electro-transfection, HFF cells containing 2% FBS DMEM medium were added for culture;

[0118] (2) When larger parasite vacuoles are visible in the cells, dilute pyrimethamine to a working concentration of 1 μM and add it to about 5 mL of 2% FBSDMEM medium, and use this medium for drug screening;

[0119] (3) After 2-4 subcultures, HFF cells were screened and cultured in 2% FBSDMEM medium containing 1 μM pyrimethamine, and the electroporated strain library was identified by PCR using the primers shown in Table 12; the results showed that there was no band at the corresponding size of the ΔsbpaseΔtal strain, while PCR1 and PCR2 of the electroporated strain library amplified the target band, indicating that the electroporated strain library had successfully integrated the YFP-DHFR* drug screening tag; the schematic diagram of the construction of the knockout of Toxoplasma ΔsbpaseΔtalΔrupe is shown in Figure 5 As shown;

[0120] Table 12 Primers for PCR identification of monoclonal insect strains of ΔsbpaseΔtalΔrupe

[0121]

[0122]

[0123] (4) After proving that the YFP-DHFR* drug screening tag has been successfully integrated into the electroporated strain library, screen for monoclonal strains. Collect the electroporated strain library, dilute and count, and use a 96-well plate with HFF cells grown on the wall for screening. Add one Toxoplasma tachyzoite to each well; after culturing for about 7 days, select strains with only a single plaque in the 96-well plate, use a pipette to scrape the HFF cells in the wells containing the monoclonal strain, add all of them to a 24-well plate with HFF cells grown on the wall, and expand the culture; when 50% of the tachyzoites in the vesicles of the strain in the 24-well plate escape, scrape the cells in the wells, extract DNA from half of them for monoclonal strain identification, and transfer the rest to a new 24-well plate for continued culture;

[0124] (5) Monoclonal strains were identified by PCR using the primers shown in Table 12. The results are as follows: Figure 6As shown, there was no band at the corresponding size of the ΔsbpaseΔtal strain, while PCR1 and PCR2 of the monoclonal strain amplified the target band, indicating that the electroporated strain had successfully integrated the YFP-DHFR* drug screening tag; and there was no band at the corresponding size of PCR3 of the monoclonal strain, and ΔsbpaseΔtal amplified the target band, which means that the monoclonal strain in which the TgRuPE gene was knocked out in the ΔsbpaseΔtal strain was successfully screened out, and the ΔsbpaseΔtalΔrupe strain was obtained.

[0125] Example 4 In vitro plaque assay of gene knockout strains

[0126] (1) Cultivating the wild-type DiCre strain and the DiCreΔsbpase, ΔsbpaseΔtal, and ΔsbpaseΔtalΔrupe strains constructed in Example 1, Example 2, and Example 3, and collecting intracellular tachyzoites;

[0127] (2) Add 100 tachyzoites collected in the previous step to each well of a 6-well plate of adherent HFF cells and culture in a 37°C cell culture incubator for 7 days;

[0128] (3) After washing twice with PBS, 1 mL of 4% PFA was added to each well and fixed at 37°C for 20 min;

[0129] (4) After washing twice with PBS, 1 mL of 0.1% crystal violet was added to each well and stained at 37°C for 20 min;

[0130] (5) Wash once with PBS and let dry. Scan the plaques with a scanner, save the image, count the plaques and measure the area. Use Student's t test to analyze the statistical results.

[0131] Plaque results Figure 7 and Figure 8 As shown, it can be seen that there is almost no difference in the size and number of plaques between DiCre and DiCreΔsbpase strains, and the size and number of plaques of ΔsbpaseΔtal strains are reduced. The plaques of ΔsbpaseΔtalΔrupe strains are significantly smaller than those of the control group ΔsbpaseΔtal, and the number is significantly reduced, indicating that the triple-gene knockout strain can significantly slow down the growth of Toxoplasma gondii in vitro, and the effect is better than that of single-gene and double-gene knockout strains. Knocking out the TgSBPase, TgTAL, and TgRuPE genes will affect the in vitro growth of Toxoplasma gondii.

[0132] Example 5 In vivo toxicity experiment of gene knockout strains

[0133] (1) The wild-type RHΔku80 strain (kindly donated by Huazhong Agricultural University) and the Δsbpase strain were used to dilute the tachyzoites using DMEM medium without FBS. The RHΔku80 and Δsbpase strains were diluted at 1×10 2 Ten mice were inoculated with each dose (Guangzhou Ruige Biotechnology Co., Ltd.). The mental state, diet and number of mice that survived were recorded. After 15 days, blood was collected from the retro-orbital vein of the mice for indirect immunofluorescence experiment (IFA), and mice that were not successfully infected were eliminated. The mortality rate of mice was analyzed using Prism software, and a Kaplan-Meier survival plot was produced;

[0134] (2) The wild-type DiCre strain (a control group after genetic modification based on the wild-type RH strain) and the ΔsbpaseΔtal strain constructed in Example 2 were used to generate 1×10 2 11 / 10 mice were inoculated with each dose. The mortality of mice was analyzed and Kaplan-Meier survival plot was prepared;

[0135] (3) The ΔsbpaseΔtal and ΔsbpaseΔtalΔrupe strains were constructed by using Example 2 and Example 3. ΔsbpaseΔtal was expressed at 1×10 2 Eight mice were inoculated with each dose of ΔsbpaseΔtalΔrupe strains at 1×10 4 , 1×10 5 Five mice were inoculated with each dose.

[0136] The results of the test are as follows Figure 9-11 As shown, there is almost no difference in virulence between the Δsbpase and ΔsbpaseΔtal strains and the wild-type strain. Mice inoculated with the ΔsbpaseΔtal strain died on the 8th day after infection, and all mice died on the 10th day after infection, while all mice inoculated with the ΔsbpaseΔtalΔrupe strain survived, indicating that the virulence of the ΔsbpaseΔtalΔrupe strain in mice was significantly reduced.

[0137] Example 6 Experiment on the immune protection of gene knockout strains on mice

[0138] (1) Wild-type Toxoplasma gondii RHΔku80 and ME49 strains (kindly donated by Huazhong Agricultural University) were used for infection. When there were many large intrazonal vacuoles of RHΔku80 and ME49 strains cultured in HFF cells, PBS was used to wash away the extracellular tachyzoites, and DMEM medium without FBS was added to collect the intracellular tachyzoites;

[0139] (2) Take 100 μL of the liquid containing tachyzoites and dilute it 10 times and count it under a microscope. After counting, calculate and dilute the tachyzoites using DMEM medium without FBS. Based on the virulence gradient experiment in Example 5, the ΔsbpaseΔtalΔrupe strain was used at 1×10 4 , 1×10 5 All five mice inoculated with each dose survived; 21 days after inoculation, 1×10 4 Mice infected with the ΔsbpaseΔtalΔrupe strain and five uninoculated blank mice were inoculated with 1×10 4 1×10 5 Mice with the ΔsbpaseΔtalΔrupe strain and five unvaccinated blank mice were inoculated with 1×10 4 The mice were injected with ME49 at a dose of 100 tachyzoites. The mental state, appetite, and mortality of the mice were observed and recorded, and the results were statistically analyzed and graphed in Prism5.

[0140] The results are as follows Fig.12 As shown, unimmunized control mice were infected with 1×10 4 After RHΔku80, all the insects died within 7 days. 4 The survival rate of mice immunized with ΔsbpaseΔtalΔrupe tachyzoites was 100% within 30 days. 4 After the ME49 strain, the survival rate was only 40% within 30 days. Fig.13 As shown, the 1×10 5 The survival rate of mice immunized with ΔsbpaseΔtalΔrupe tachyzoites within 30 days was 100%, indicating that vaccination with the ΔsbpaseΔtalΔrupe strain has good immune protection for mice against infection with wild-type Toxoplasma gondii strains and can be used to prepare Toxoplasma vaccines.

[0141] In summary, the present invention has studied the three genes of Toxoplasma gondii, namely, sedoheptulose-1,7-bisphosphatase TgSBPase, transaldolase TgTAL, and ribulose-5-phosphate isomerase TgRuPE, and found that knocking out the single gene of TgSBPase, TgTAL, or TgRuPE respectively has no effect on the growth rate and virulence of Toxoplasma gondii in vivo and in vitro, while knocking out two or three of the genes at the same time will affect the growth and reproduction of Toxoplasma gondii, and is related to the virulence of the parasite. The present invention successfully constructed a Toxoplasma gondii TgSBPase, TgTAL, and TgRuPE three-gene knockout strain. After knocking out the TgSBPase, TgTAL, and TgRuPE genes at the same time, it can not only be subcultured in vitro, but also has almost non-toxic characteristics in vivo. After immunization, the vaccine strain can effectively resist the infection of wild poisonous strains, showing a good immune protection effect, and has a good immune protection effect on mice. The triple-gene knockout strain ΔsbpaseΔtalΔrupe provided by the invention provides methods and means for developing more effective and non-toxic anti-toxoplasmosis drugs and vaccines.

[0142] 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. A method for constructing a Toxoplasma gondii triple gene knockout strain, characterized in that: Knockout of Toxoplasma gondii sedoheptulose-1,7-bisphosphatase by CRISPR gene editing Tg SBPase, transaldolase Tg TAL and ribulose-5-phosphate epimerase Tg RuPE gene, to obtain a Toxoplasma gondii triple gene knockout strain; Tg SBPase, Tg TAL, Tg The nucleotide sequences of the RuPE genes are shown in SEQ ID NOs: 1-3.

2. The construction method according to claim 1, characterized in that: The specific steps include: S1. Wild type DiCre To start the worm strain, directly knock out Tg SBPase gene construction DiCreΔsbpase Insect strains; S2. DiCreΔsbpase Knockout based on insect strain Tg TAL gene construction ΔsbpaseΔtal Insect strains; S3. Finally ΔsbpaseΔtal Knockout Tg RuPE gene, obtain Toxoplasma gondii triple gene knockout strain Δ sbpaseΔtalΔrupe .

3. The construction method according to claim 2, characterized in that: In step S1 DiCreΔsbpase The method for constructing the insect strain is as follows: using pSAG1-Cas9- Tg U6-sg Tg UPRT plasmid as template Tg SBPase-specific CRISPR / Cas9 knockout plasmid; then ploxp- DHFR *-loxP plasmid was used as template to amplify Ko- SBPase- loxp- DHFR* -loxP homology fragment, the above plasmid and fragment were co-electroporated into the wild-type DiCre In the process of drug screening and PCR identification, DiCreΔsbpase Insect strains; In step S2 ΔsbpaseΔtal The method of constructing the insect strain is: first remove DiCreΔsbpase Insect strains DHFR* Label; pSAG1-Cas9- Tg U6-sg Tg UPRT plasmid was used as template to design Tg TAL gene gRNA primers, preparation of knockout plasmid pSAG1-Cas9- Tg U6-sg Tg TAL; then ploxp- DHFR *-loxP plasmid was used as template to amplify Ko- TAL -loxp- DHFR* -loxP homology fragment, the above plasmid and fragment were co-transfected into DiCreΔsbpase In the insect strain, drug screening and PCR identification were performed to obtain ΔsbpaseΔtal Insect strains; In step S3 ΔsbpaseΔtalΔrupe The method of constructing the insect strain is: first remove ΔsbpaseΔtal Insect strains DHFR* Tag; then pSAG1-Cas9- Tg U6-sg Tg UPRT plasmid was used as a template to design Tg RuPE gene gRNA primers, preparation of knockout plasmid pSAG1-Cas9- Tg U6-sg Tg RuPE; then pTub-YFP- DHFR * Plasmid was used as template to amplify Ko- RuPE -YFP- DHFR* Homologous fragments were co-transfected with the above plasmid and fragments ΔsbpaseΔtal In the insect strain, drug screening and PCR identification were performed to obtain ΔsbpaseΔtalΔrupe Insect strains.

4. A Toxoplasma gondii triple gene knockout strain, characterized in that: The method is constructed by any one of claims 1 to 3.

5. Deletion or knockout of Toxoplasma gondii sedoheptulose-1,7-bisphosphatase TgSBPase , transaldolase TgTAL and ribulose-5-phosphate epimerase Tg The use of a gene reagent in constructing a Toxoplasma triple gene knockout strain or in preparing a Toxoplasma vaccine is characterized in that: The toxoplasma vaccine also knocked out Tg SBPase, Tg TAL, Tg RuPE gene, Tg SBPase, Tg TAL, Tg The nucleotide sequences of the RuPE genes are shown in SEQ ID NOs: 1-3.

6. Use of the Toxoplasma triple gene knockout strain according to claim 4 in the preparation of Toxoplasma vaccine.

7. A Toxoplasma gondii vaccine, characterized in that: Containing the Toxoplasma gondii triple gene knockout strain as described in claim 4.

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