CN118308431A - Toxoplasma gondii three-gene knockout vaccine insect strain and application thereof - Google Patents

Abstract

The invention discloses a toxoplasma gondii three-gene knockout vaccine insect strain and application thereof. The invention uses gene editing technique to knock out three genes of sedoheptulose-1, 7-diphosphatase TgSBPase, transaldolase TgTAL and ribulose-5-phosphate epimerase TgRuPE of toxoplasma, so as to successfully construct TgSBPase, tgTAL and TgRuPE three-gene knocked-out strains; after the three genes are knocked out, toxoplasma gondii can be subjected to in vitro subculture, has the characteristic of almost no toxicity in vivo, can effectively resist the infection of wild toxoplasmosis strains after mice are immunized with the three-gene knocked-out insect strains, has good immune protection effect, and provides a method and means for developing efficient, nontoxic and safe toxoplasmosis-resistant vaccines.

Description

Toxoplasma gondii three-gene knockout vaccine insect strain and application thereof

Technical Field

The invention belongs to the technical field of genetic engineering. More particularly relates to a toxoplasma tri-gene knockout vaccine insect strain and application thereof.

Background

Toxoplasmosis is a zoonosis caused by Toxoplasma gondii, and seriously endangers human health and sustainable development of animal husbandry. The healthy population or animal shows no symptoms or only slight clinical symptoms after infection of toxoplasma, but the immunocompromised or defective population and animal can have serious consequences after infection of toxoplasma, and even endanger life. Toxoplasmosis can damage eyes, brains and the like of organisms to cause toxoplasmosis and encephalopathy, and can also cause bad results of abortion, stillbirth and the like of pregnant women or pregnant animals. Toxoplasma transmission routes are complex and various, and most commonly, the toxoplasma transmission is carried out orally, and a human or animal is generally infected by drinking water containing toxoplasma egg bag pollution or by eating raw meat or uncooked meat containing tissue encapsulation by mistake; toxoplasmosis can also cause congenital toxoplasmosis in the fetus by vertical transmission. Only medicines such as pyrimethamine, sulfadiazine and the like are clinically effective on toxoplasmosis tachyzoites, can inhibit the growth and reproduction of toxoplasmosis, but have side effects and drug resistance after long-term use, and are very lack of medicines and vaccines for resisting toxoplasmosis at present; therefore, the development of safe and effective toxoplasmosis vaccine is particularly important for preventing toxoplasmosis.

Because of the close association of metabolic pathways with the growth and reproduction of the insect bodies and virulence, the compound is a potential target for drug and vaccine development. Wherein, glucose is transported into the cytoplasm of the insect body through a glucose transporter GT1, and glucose-6-phosphate (G6P) is generated under the catalysis of hexokinase HK to participate in glycolysis and pentose phosphate metabolism. And the pentose phosphate metabolic pathway (PPP) is closely linked to the glycolytic pathway, and is the second important pathway of glucose catabolism. PPP is generally divided into two stages, oxidation and non-oxidation, both of which are involved in ribose-5-phosphate (R5P) formation. R5P is a key precursor for nucleotide biosynthesis, directly affecting RNA and DNA synthesis. The prior researches show that in the PPP stage, ribulose-5-phosphate isomerase, glucose-6-phosphate dehydrogenase and transketolase are related to the growth and virulence of the toxoplasma, and the knockout of the genes can reduce the growth and reproductive capacity of toxoplasma and weaken the virulence of toxoplasma in vivo. However, the research on other related enzymes generated by R5P in toxoplasma is less, the relation between the related enzymes and the growth and the toxicity of the toxoplasma and whether the related enzymes can be used as vaccine targets is unknown, so that more effective and nontoxic attenuated toxoplasmosis-preventing and treating strains or vaccines are necessary to be developed, and more methods and technical support are provided for toxoplasmosis resistance.

Disclosure of Invention

The invention aims to overcome the defects of the existing toxoplasmosis-resistant attenuated insect strain or vaccine and provide a toxoplasmosis three-gene knockout vaccine insect strain and application thereof.

The first aim of the invention is to provide a construction method of a toxoplasma gondii three-gene knockout strain.

The second object of the invention is to provide a toxoplasma gondii three-gene knockout strain.

A third object of the present invention is to provide the use of the genes Toxotrope-1, 7-bisphosphatase TgSBPase, transaldolase TgTAL and ribulose-5-phosphate epimerase TgRuPE.

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

A fifth object of the invention is to provide a toxoplasma vaccine.

A sixth object of the present invention is to provide a medicament for controlling toxoplasmosis.

The above object of the present invention is achieved by the following technical scheme:

The invention provides a construction method of a toxoplasma gondii three-gene knockout strain, which is characterized in that a CRISPR gene editing technology is adopted to knockout sedoheptulose-1,7-bisphosphatase (sedoheptulose-1, 7-bisphosphatase) TgSBPase, transaldolase (transaldolase) TgTAL and ribulose-5-phosphate epimerase (ribulose-5-phosphate epimerase) TgRuPE genes of toxoplasma gondii to obtain the toxoplasma gondii three-gene knockout strain; wherein, the nucleotide sequence of TgSBPase gene (gene ID: TGGT1_ 235700) is shown in SEQ ID NO:1, wherein the nucleotide sequence of TgTAL gene (gene ID: TGGT1_ 229360) is shown in SEQ ID NO:2, the nucleotide sequence of TgRuPE gene (gene ID: TGGT1_ 247670) is shown as SEQ ID NO: 3.

The invention focuses on R5P generation related enzymes, including TgRuPE, tgTAL and TgSBPase of ribose xenobiotic pathway in PPP stage, and finds that the knockout of TgSBPase, tgTAL, tgRuPE single gene has no influence on toxoplasma gondii, and the knockout of two or three genes affects the growth and reproduction of toxoplasma gondii and is related to toxoplasma virulence. Further, by constructing a genetically modified insect strain, a genetically modified insect strain (delta sbpase delta tal delta rupe) with TgSBPase, tgTAL, tgRuPE knocked out genes is obtained, after the three genes of TgSBPase, tgTAL, tgRuPE are deleted on a toxoplasma I-type wild DiCre insect strain, the insect body can grow in vitro in a passaged way, the toxicity is obviously weakened, the three-gene knocked-out insect strain is almost nontoxic, and the death rate of a mouse is still 0% under the condition that the infection dose is 10 ⁵ delta sbpase delta tal delta rupe/mouse; immunization delta sbpase delta tal delta rupe can effectively resist wild type insect re-infection, and the immune protection effect is 100%. Meanwhile, tgSBPase, tgTAL and TgRuPE genes serving as targets can be used for screening or preparing medicines for preventing and treating toxoplasmosis, have important significance for preventing and treating toxoplasmosis and researching and developing vaccines, and protect healthy development of human beings and animal husbandry.

The invention provides a specific method for constructing a toxoplasma gondii three-gene knockout strain, which comprises the following steps:

S1, directly knocking out TgSBPase genes by taking a wild toxoplasma strain as a starting insect strain to construct a DiCre delta sbpase insect strain;

S2, knocking out TgTAL genes to construct delta sbpase deltatal insect strains on the basis of DiCre delta sbpase insect strains;

S3, finally, knocking out TgRuPE genes on the delta sbpase delta tal insect strain to obtain a toxoplasma gondii three-gene knocked-out insect strain delta sbpase delta tal delta rupe.

Preferably, in the step S1, the construction method of DiCre delta sbpase insect strains is as follows: using pSAG-Cas 9-TgU6-sgTgUPRT plasmid as a template to construct TgSBPase specific CRISPR/Cas9 knockout plasmid; and amplifying the Ko-SBPase-loxp-DHFR-loxp homologous fragment by taking ploxp-DHFR-loxp plasmid as a template, and co-transfecting the plasmid and the fragment into a wild toxoplasma strain, and carrying out drug screening and PCR identification to obtain the DiCre delta SBPase insect strain.

More preferably, the primer sequence for constructing TgSBPase-specific CRISPR/Cas9 knockout plasmid is as set forth in SEQ ID NO:4 to 5; the primer sequence for amplifying the Ko-SBPase-loxp-DHFR-loxp homologous fragment is shown in SEQ ID NO:6 to 7.

Further, the construction method of the Delta sbpase Deltatal insect strain in the step S2 comprises the following steps: firstly, removing DHFR tags in DiCre Delta sbpase insect strains; using pSAG-Cas 9-TgU6-sgTgUPRT plasmid as a template, designing TgTAL gene gRNA primer, and preparing knockout plasmid pSAG-Cas 9-TgU6-sgTgTAL; and amplifying the Ko-TAL-loxp-DHFR-loxp homologous fragments by using ploxp-DHFR-loxp plasmid as a template, and co-transfecting the plasmids and the fragments into DiCre delta sbpase insect strains, and performing drug screening and PCR identification to obtain delta sbpase delta TAL insect strains.

More preferably, the primer sequence of TgTAL gene gRNA is as set forth in SEQ ID NO:12 and SEQ ID NO:5 is shown in the figure; the primer sequence for amplifying the Ko-TAL-loxp-DHFR-loxp homologous fragment is shown in SEQ ID NO:13 to 14.

Further, the construction method of the Δ sbpase ΔtalΔ rupe insect strain in the step S3 is as follows: firstly removing DHFR tags in the delta sbpase delta tal insect strain; then taking pSAG-Cas 9-TgU6-sgTgUPRT plasmid as a template, designing TgRuPE gene gRNA primer, and preparing knockout plasmid pSAG-Cas 9-TgU6-sgTgRuPE; and amplifying the Ko-RuPE-YFP-DHFR homologous fragment by taking pTub-YFP-DHFR plasmid as a template, and co-electrotransfecting the plasmid and the fragment into the delta sbpase delta tal insect strain, and carrying out drug screening and PCR identification to obtain the delta sbpase delta tal delta rupe insect strain.

More preferably, the primer sequence of TgRuPE gene gRNA is as set forth in SEQ ID NO:21 and SEQ ID NO:5 is shown in the figure; the primer sequence for amplifying the Ko-RuPE-YFP-DHFR homologous fragment is shown as SEQ ID NO:22 to 23.

Preferably, the wild toxoplasma strain in step S1 is a toxoplasma RH (dice) insect strain.

Further, pyrimethamine is used for drug screening.

Preferably, the DHFR signature in the insect strain is removed in steps S2, S3 by treatment with rapamycin (Rapa) for 2d.

The invention provides a toxoplasma gondii three-gene knockout insect strain which is constructed by the method.

The invention provides application of toxoplasmosis sedoheptulose-1, 7-bisphosphatase TgSBPase, transaldolase TgTAL and ribulose-5-phosphate epimerase TgRuPE genes serving as targets in screening or preparing medicines for preventing and treating toxoplasmosis.

The invention provides application of a reagent for deleting or knocking out genes of Toxoplasma gondii sedoheptulose-1, 7-bisphosphatase TgSBPase, transaldolase TgTAL and ribulose-5-phosphate epimerase TgRuPE in construction of three-gene knocked-out insect strains of Toxoplasma gondii or preparation of medicines for preventing and treating toxoplasmosis.

Further, agents that delete or knock out toxoplasma TgSBPase, tgTAL and TgRuPE genes affect toxoplasma TgSBPase, tgTAL, tgRuPE gene complete expression, including but not limited to siRNA, CRISPR/Cas9, chemical agents.

The invention provides application of toxoplasma gondii three-gene knockout strain in preparing toxoplasma gondii vaccine.

The invention provides a toxoplasma vaccine which contains toxoplasma three-gene knockout insect strains.

Further, the vaccine is a attenuated vaccine.

The invention also provides a medicine for preventing and treating toxoplasmosis, which contains a reagent for deleting or knocking out genes of toxoplasmosis sedoheptulose-1, 7-bisphosphatase TgSBPase, transaldolase TgTAL and ribulose-5-phosphate epimerase TgRuPE.

The invention has the following beneficial effects:

By researching three genes of toxoplasmosis sedoheptulose-1, 7-bisphosphatase TgSBPase, transaldolase TgTAL and ribulose-5-phosphate epimerase TgRuPE, it is found that when TgSBPase, tgTAL, tgRuPE single genes are knocked out respectively, no obvious influence is caused on toxoplasmosis, two or three of the genes can influence the growth and reproduction of toxoplasmosis, and the genes are related to the virulence of the toxoplasmosis, and a toxoplasmosis TgSBPase, tgTAL, tgRuPE three-gene knocked-out strain is successfully constructed; after TgSBPase, tgTAL, tgRuPE genes are knocked out simultaneously, the vaccine strain can be subjected to in vitro subculture and has the characteristic of almost no toxicity in vivo, and after immunization, the vaccine strain can effectively resist the infection of wild insect strains, shows good immune protection effect and has good immune protection effect on mice. The three-gene knockout strain delta sbpase delta tal delta rupe provided by the invention provides a method and means for developing more effective and nontoxic toxoplasmosis-resistant medicaments and vaccines.

Drawings

Fig. 1is a schematic representation of knockdown toxoplasma DiCre delta sbpase.

FIG. 2 shows the results of PCRs identification of DiCre Δ sbpase monoclonal strain.

Fig. 3 is a schematic representation of knockout toxoplasma delta sbpase deltatal.

FIG. 4 shows the results of PCRs identification of the Δ sbpase Δtal monoclonal strain.

Fig. 5 is a schematic representation of knockout toxoplasma delta sbpase delta tal delta rupe.

FIG. 6 shows the PCRs identification results of the Δ sbpase ΔtalΔ rupe monoclonal strain.

FIG. 7 shows the results of plaque assay of Δ sbpase Δtal insect strain.

Fig. 8 shows plaque assay results for Δ sbpase ΔtalΔ rupe insect strains.

FIG. 9 shows the results of a test for virulence of the strain Delta sbpase.

FIG. 10 shows the results of a Delta sbpase Deltatal insect strain virulence experiment.

Fig. 11 shows the results of a strain virulence gradient experiment of Δ sbpase ΔtalΔ rupe.

FIG. 12 shows the results of immunoprotection experiments in mice infected with wild-type RH Δku80 by the immunized Δ sbpase ΔtalΔ rupe strain.

FIG. 13 shows the results of immunoprotection experiments in which mice of the strain Delta sbpase DeltatalDelta rupe were infected with wild-type ME 49.

Detailed Description

The invention is further illustrated in the following drawings and specific examples, which are not intended to limit the invention in any way. Unless specifically stated otherwise, the reagents, methods and apparatus employed in the present invention are those conventional in the art.

Reagents and materials used in the following examples are commercially available unless otherwise specified.

Example 1 construction of Toxoplasma gondii Single Gene knockout insect strain

In the embodiment, a wild toxoplasma RH (Dicre) insect strain I (benefit of agricultural university in China) is taken as an insect starting strain, and a ToxoDB (www.toxodb.org) toxoplasma website is searched for a gene of a native tendo heptulose-1,7-bisphosphatase TgSBPase in the toxoplasma I type insect strain, and the nucleotide sequence of the gene is shown as SEQ ID NO.1 (gene ID: TGGT1_235700, sedoheptpulose-1, 7-bisphosphatase).

1. Construction of pSAG1-Cas9-TgU6-sgTgSBPase plasmid

(1) Designing a gRNA primer in an E-CRISP DESIGN website (http:// www.e-CRISP. Org/E-CRISP/design rispr. Html), searching a targeting site from a TgSBPase genome sequence, designing a plasmid according to the gRNA sequence, and designing a target primer sequence shown in a table 1;

TABLE 1 construction of specific primers for TgSBPase CRISPR/Cas9 plasmids

(2) Construction of TgRuPE-specific CRISPR/Cas9 plasmids was performed using commercial pSAG-Cas 9-TgU6-sgTgUPRT plasmids as templates, PCR amplification reaction systems and procedures are shown in tables 2 and 3;

TABLE 2PCR reaction System

TABLE 3PCR reaction procedure

After the reaction is finished, 1 mu L of Dpn I is directly added into the PCR product, and the mixture is placed in a PCR instrument for digestion for 45min at 37 ℃;

(3) Preparing a reaction system shown in the following table 4 in a new PCR tube, uniformly mixing the liquids, and placing the mixture in a PCR for reaction for 15min at 25 ℃;

TABLE 4PCR reaction System

(4) Adding all the PCR products in the step (3) into competent cells DH5 alpha of the escherichia coli, performing heat shock at 42 ℃ on bacterial liquid, and performing overnight culture by using a solid LB/Amp ⁺ culture medium for about 12 hours; and (3) placing the monoclonal colony in an LB/Amp ⁺ liquid culture medium for amplification culture and identification, further sequencing the bacterial liquid which is identified to be correct by PCR, analyzing according to a sequencing result, and if the monoclonal colony is compared to show that the target sequence is completely covered, then the construction of the pSAG-Cas 9-TgU6-sgTgSBPase plasmid is successful.

2. Amplification of Ko-SBPase-loxp-DHFR x-loxp homologous fragments

The ploxp-DHFR-loxp plasmid (benefit of agricultural university of China) was used as a template for PCR amplification using the primers shown in Table 5 and high-fidelity enzyme, and after amplification, the Ko-SBPase-loxp-DHFR-loxp homologous fragments with correct band sizes were subjected to gel recovery and stored at-20℃for a long period of time.

TABLE 5 amplification of Ko-SBPase-loxp-DHFR-loxp fragment primers

3. Construction of Toxoplasma gondii knockout Strain DiCre Δ sbpase

(1) Collecting the toxoplasma DiCre tachyzoites in the larger sodium insect bubbles, and centrifuging at 3000rpm for 8min at room temperature; the insect bodies in the sediment are resuspended by 8mL Cytomix(120mM KCl,0.15mM CaCl₂,10mM K₂HPO₄/KH₂PO₄,25mM HEPES,2mM EGTA,5mM MgCl₂,pH＝7.6), and are centrifuged for 8min at 3000rpm at room temperature; the pellet was resuspended in worms using 250 μ L cytomix;

(2) 50 μ L cytomix, 7500ng of pSAG-Cas 9-TgU-sgTgSBPase plasmid and 1500ng of kosbpase-loxp-DHFR x-loxp homology fragment were added to a 1.5mL EP tube and the resuspended bodies of step (1) above were all transferred to the EP tube;

(3) The liquid in the EP pipe is fully and uniformly mixed by adopting a pipetting gun for blowing and sucking, and all the liquid is added into a 4mm electric rotating cup, so that no bubbles are generated; electrotransfection was performed using a Bio-Rad electrotransport machine, with the metal surfaces on both sides of the electrotransport cup aligned to the sheet metal electrodes in the machine: 1600V, 25 mu F, 50Ω and 4mm electric shock are carried out once, and the electric rotating cup is slightly pulled to mix the liquid uniformly; electric shock of 1500V, 25 muF, 50Ω and 4mm is carried out once, and the liquid is evenly mixed by lightly pulling the electric rotating cup; electric shock of 1500V, 25 μF, 50Ω, 4mm was performed once, and after standing for 2min, the liquid in the electric rotating cup was added to human-derived fiber HFF cells (ATCC cell bank commercially available) containing 2% FBSDMEM medium for culture;

(4) When larger sodium worm bubbles are visible in the cells, pyrimethamine is diluted to a working concentration of 1 μm and added to about 5mL of 2% FBSDMEM medium, and the medium is used for drug screening;

(5) After each subsequent subculture, fresh human-derived fibroblasts HFF containing 1. Mu.M pyrimethamine in 2% FBSDMEM medium were used, and after 2-4 pyrimethamine selection cultures, the electrotransfer strain pool was identified by PCR using the primers shown in Table 6; the results show that the PCR3 and the PCR4 of the wild DiCre have bands at the target size, and the PCR4 of the electrotransfer strain library is amplified to obtain two target bands, which indicates that the electrotransfer strain library contains the insect strain which is successfully knocked out TgSBPase gene; the construction schematic of knockdown toxoplasma DiCre Δ sbpase is shown in figure 1;

Monoclonal strain PCRs identification primers of Table 6DiCre delta sbpase

(6) For subsequent secondary knockouts we treated Rapa with a pool of identified correct insect strains for 2d to remove DHFR panel tags and screened for monoclonal insect strains. Collecting a population pool of insect-transformed strains, diluting and counting, adding the population pool into a 96-well plate for adherent growth of HFF cells, and adding 1 toxoplasma tachyzoite into each well; after 5-7d of culture, observing whether monoclonal insect strains exist in the 96-well plate, scraping HFF cells in the wells containing the monoclonal insect strains by using a gun head, adding all HFF cells into a 24-well plate for growing HFF cells in an adherence manner, and performing expansion culture; when 50% of tachyzoites in the Nachong blebs in the 24-well plate escape, scraping off cells in the well, carrying out monoclonal strain identification by half of the cells for extracting DNA, and continuously culturing the rest of the cells in a new 24-well plate;

(7) The monoclonal insect strain was identified by PCR using the primers shown in Table 6; the results are shown in fig. 2, which shows that PCR3 and PCR4 of the wild-type DiCre and electrotransplantable strain libraries are both striped at the target size, while PCR3 of the monoclonal strain is striped at the target size, PCR4 only amplifies one stripe, indicating that the monoclonal strain successfully knocks out TgSBPase gene and DHFR-sieve gene, resulting in DiCre delta sbpase strain.

Example 2 construction of Toxoplasma gondii double Gene knockout insect strain

The results of plaque and virulence experiment verification on the delta sbpase insect strain show that knockout TgSBPase has no obvious effect on toxoplasma in-vitro growth and in-vivo virulence (specific results are shown in example 4); meanwhile, the method has no influence on the growth rate, toxicity and other aspects of toxoplasma in vivo and in vitro after knocking out TgTAL or TgRuPE genes respectively. Based on this, in this example, an insect-developed strain was made from the toxoplasma DiCre Δ sbpase strain constructed in example 1, and the transaldolase TgTAL gene, the nucleotide sequence of which is shown in SEQ ID NO.2 (Gene ID: TGGT. DELTA.229360, transaldolase), was searched from ToxoDB (www.toxodb.org) toxoplasma website, and a double-gene deletion strain was further constructed.

1. Construction of pSAG1-Cas9-TgU6-sgTgTAL plasmid

(1) Designing gRNA in an E-CRISP DESIGN website (http:// www.e-CRISP. Org/E-CRISP/design rispr. Html), searching for a targeting site in a TgTAL genome sequence, designing a specific plasmid according to the gRNA sequence, and designing a target specific primer sequence as shown in table 7;

TABLE 7 construction of specific primers for TGTAL CRISPR/Cas9 plasmids

(2) The CRISPR/Cas9 plasmid construction and identification were performed with reference to the construction method of example 1, and sequencing results showed successful construction of pSAG-Cas 9-TgU6-sgTgTAL plasmid.

2. Amplification of Ko-TAL-loxp-DHFR x-loxp homologous fragments

The gel was recovered using ploxp-DHFR-loxp plasmid (purchased from http:// www.addgene.org) as template, and PCR amplification was performed using the primers shown in Table 8 and high fidelity enzyme, and the kol-TAL-loxp-DHFR-loxp homologous fragments with correct band sizes.

TABLE 8 amplification of Ko-TAL-loxp-DHFR-loxp fragment primers

3. Construction of toxoplasma gondii Gene knockout pest strain delta sbpase delta tal

(1) Fresh toxoplasma DiCre Δ sbpase tachyzoites were collected, resuspended in 250 μ L cytomix and electrotransfected by thoroughly mixing 7500ng of the pSAG1-Cas9-TgU6-sgTgTAL plasmid and 1500ng of the Ko-TAL-loxp-DHFR x-loxp homologous fragment with the resuspended tachyzoites as in step 3 of example 1, and after completion of electrotransfection, HFF cells containing 2% FBSDMEM medium were added for culture;

(2) When larger sodium worm bubbles are visible in the cells, pyrimethamine is diluted to a working concentration of 1 mu M and then added into about 5mL of 2% FBSDMEM culture medium, and the culture medium is used for medicine screening;

(3) Thereafter, screening culture was performed by using HFF cells containing 2% FBSDMEM medium of 1. Mu.M pyrimethamine through 2-4 subcultures, and the pool of electrotransfer strains was identified by PCR using the primers shown in Table 9; the result shows that no band exists at the corresponding size of DiCre Δ sbpase insect strains, and the target band is amplified by both PCR1 and PCR2 of the electrotransfer insect strain library, which indicates that the electrotransfer insect strain library successfully integrates DHFR drug screening labels; the construction schematic of the knockdown toxoplasma delta sbpase deltatal is shown in figure 3;

Monoclonal strain PCRs identification primers of Table 9 delta sbpase delta tal

(4) After identification proves that the DHFR drug screening tag is successfully integrated into the electrotransfer strain library, screening of monoclonal insect strains is carried out. Collecting a pool dilution count of the transgenic strains, screening by using a 96-well plate with HFF cells grown in an adherence manner, and adding 1 toxoplasma tachyzoite into each well; culturing for about 7 days, selecting insect strains with single plaques in a 96-well plate, scraping HFF cells in the wells containing the monoclonal insect strains by using a gun head, adding all the HFF cells into a 24-well plate with the HFF cells growing on the wall, and performing expansion culture; when 50% of tachyzoites in the insect strain Nachong blebs in the 24-well plate escape, scraping cells in the well, extracting half of DNA for monoclonal insect strain identification, and continuously culturing the rest part to a new 24-well plate;

(5) The monoclonal insect strain is subjected to PCR identification by using the primers shown in table 9, the result is shown in fig. 4, the corresponding size of DiCre delta sbpase insect strain is not provided with a band, and the target band is amplified by both PCR1 and PCR2 of the monoclonal insect strain, which indicates that the electrotransfer insect strain successfully integrates DHFR drug screening label; and the PCR3 of the monoclonal insect strain has no band at the corresponding size, and DiCre delta sbpase is amplified to obtain a target band, so that the monoclonal insect strain knocked out of the TgTAL gene is successfully screened out, and the delta sbpase delta tal insect strain is obtained.

Example 3 construction of Toxoplasma gondii Trigenic knockout strain Delta sbpase DeltatalDelta rupe

A strain was produced from the toxoplasma delta sbpase deltatal insect strain constructed in example 2, and the ribulose-5-phosphate epimerase TgRuPE gene of toxoplasma type I insect strain was searched from ToxoDB (www.toxodb.org) toxoplasma website, and the nucleotide sequence thereof was as shown in SEQ ID NO.3 (Gene ID: TGGT 1-247670, ribulose-5-phosphate epimerase).

1. Removal of DHFR tag

DHFR-tagged was removed after pretreatment of the Δ sbpase Δtal strain with Rapa for 2 d.

2. Construction of pSAG1-Cas9-TgU6-sgTgRuPE plasmid

(1) Designing gRNA in E-CRISP DESIGN website (http:// www.e-CRISP. Org/E-CRISP/design rispr. Html), searching for a targeting site in TgRuPE genome sequence, designing specific plasmid according to gRNA sequence, and designing the obtained specific primer sequence as shown in table 10;

Table 10 construction of TGTAL CRISPR/Cas9 plasmid specific primers

(2) The construction method of reference example 1 successfully constructed pSAG-Cas 9-TgU6-sgTgRuPE plasmids.

2. Amplification of Ko-RuPE-YFP-DHFR homologous fragment

The preparation method comprises the steps of taking pTub-YFP-DHFR (which is obtained by constructing a conventional method in the field, tub is a promoter, loxp is a characteristic sequence recognized by Cre protein, YFP is a fluorescent reporter gene, DHFR is pyrimethamine drug screening gene) as a template, carrying out PCR (polymerase chain reaction) amplification by using high-fidelity enzyme and primers shown in a table 11, recovering amplified Ko-RuPE-YFP-DHFR homologous fragment gel, and storing at the temperature of minus 20 ℃.

TABLE 11 amplification of Ko-RuPE-YFP-DHFR fragment primers

3. Construction of toxoplasma gondii Gene knockout strain delta sbpase delta taldelta rupe

(1) Fresh toxoplasma Δ sbpase Δtal tachyzoites were collected, resuspended in 250 μ L cytomix and electrotransfected with 50 μ L cytomix, 7500ng of the pSAG1-Cas9-TgU6-sgTgRuPE plasmid, 1500ng of the Ko-RuPE-YFP-DHFR homolog fragment and resuspended tachyzoites thoroughly mixed as in step 3 of example 1, following electrotransfection, HFF cells were added with 2% FBS DMEM medium after electrotransfection was completed;

(2) When larger sodium worm bubbles are visible in the cells, pyrimethamine is diluted to a working concentration of 1 mu M and then added into about 5mL of 2% FBSDMEM culture medium, and the culture medium is used for medicine screening;

(3) Thereafter, screening culture was performed by using HFF cells containing 2% FBSDMEM medium of 1. Mu.M pyrimethamine through 2-4 subcultures, and the pool of electrotransfer strains was identified by PCR using the primers shown in Table 12; the result shows that no band exists at the corresponding size of the Delta sbpase Deltatal insect strain, and the target band is amplified by both PCR1 and PCR2 of the electrotransfer insect strain library, which indicates that the electrotransfer insect strain library successfully integrates YFP-DHFR drug sieve labels; the construction schematic of the knockout toxoplasma delta sbpase delta tal delta rupe is shown in figure 5;

monoclonal strain PCRs identification primers of Table 12 delta sbpase delta tal delta rupe

(4) And (3) screening the monoclonal insect strains after the identification proves that the YFP-DHFR drug screening label is successfully integrated into the electrotransfer insect strain library. Collecting a pool dilution count of the transgenic strains, screening by using a 96-well plate with HFF cells grown in an adherence manner, and adding 1 toxoplasma tachyzoite into each well; culturing for about 7 days, selecting insect strains with single plaques in a 96-well plate, scraping HFF cells in the wells containing the monoclonal insect strains by using a gun head, adding all the HFF cells into a 24-well plate for adherent growth, and performing expansion culture; when 50% of tachyzoites in the insect strain Nachong blebs in the 24-well plate escape, scraping cells in the well, extracting half of DNA for monoclonal insect strain identification, and continuously culturing the rest part to a new 24-well plate;

(5) The monoclonal insect strain is subjected to PCR identification by using the primers shown in table 12, and the result is shown in fig. 6, wherein no band exists at the corresponding size of the delta sbpase delta tal insect strain, and the target band is amplified by both PCR1 and PCR2 of the monoclonal insect strain, so that the electrotransfer insect strain is successfully integrated with YFP-DHFR drug sieve labels; and the PCR3 of the monoclonal strain has no band at the corresponding size, and the target band is amplified by delta sbpase delta tal, so that the monoclonal strain which knocks out the TgRuPE gene in the delta sbpase delta tal strain is successfully screened out, and the delta sbpase delta tal delta rupe strain is obtained.

Example 4 in vitro plaque assay of Gene knockout strains

(1) Culturing wild-type DiCre insect strains, and obtaining DiCre delta sbpase, delta sbpase delta tal and delta sbpase delta tal delta rupe insect strains by constructing in example 1, example 2 and example 3, and collecting intracellular tachyzoites;

(2) Adding the tachyzoites collected in the previous step into a 6-well plate for adherent growth of HFF cells, adding 100 tachyzoites into each well, and culturing in a cell incubator at 37 ℃ for 7d;

(3) After washing 2 times with PBS, 1mL of 4% PFA was added to each well and the mixture was left to stand at 37℃for 20min;

(4) After washing 2 times with PBS, 1mL of 0.1% crystal violet was added to each well and the wells were stained at 37℃for 20min;

(5) After washing 1 time with PBS, air-dried, the plaques were scanned with a scanner, the pictures were saved, the plaque was counted and the area was measured, and the statistical results were analyzed using Student's t test.

As shown in FIGS. 7 and 8, the plaque sizes and numbers of DiCre and DiCre. DELTA. sbpase insect strains were almost unchanged, and the plaque sizes and numbers of the. DELTA. sbpase. DELTA.tal insect strains were reduced. The plaque of the delta sbpase delta tal delta rupe insect strain is obviously smaller than that of a control group delta sbpase delta tal, and the quantity is obviously reduced, so that the three-gene knockout insect strain can obviously slow down the in-vitro growth of toxoplasma gondii, the effect is better than that of single-gene and double-gene knockout insect strains, and the in-vitro growth of toxoplasma gondii can be influenced after TgSBPase, tgTAL, tgRuPE genes are knocked out.

Example 5 in vivo toxicity experiments of Gene knockout strains

(1) Wild-type RH Δku80 strain (benefit of agricultural university in China) and Δ sbpase strain were used, tachyzoites were diluted with DMEM medium containing no FBS, and 10 mice (Shangshi Biotechnology Co., ltd.) were inoculated with RH Δku80 and Δ sbpase strain at a dose of 1X 10 ², respectively. The mental state, diet and survival number of the mice were recorded, and 15d later, the mice were collected by retroorbital vein and subjected to an indirect immunofluorescence assay (IFA) to eliminate mice that were not successfully infected. The death rate of the mice is analyzed by prism software, and Kaplan-Meier survival plot is manufactured;

(2) The wild-type DiCre strain (control group genetically modified based on the wild-type RH strain) was used, and the construction of example 2 gave Δ sbpase Δtal strains, each of which was inoculated with 11/10 mice at a dose of 1X 10 ². Analyzing the death rate of the mice and preparing Kaplan-Meier survival plot;

(3) By using the construction of example 2 and example 3, Δ sbpase Δtal, Δ sbpase Δtal Δ rupe, Δ sbpase Δtal, and Δ sbpase Δtal Δ rupe, were each inoculated with 8 mice at a dose of 1×10 ², and each with 5 mice at a dose of 1×10 ⁴、1×10⁵.

The results of the measurement are shown in FIGS. 9-11, and there was little difference in virulence between the Delta sbpase and Delta sbpase Deltatal insect strains compared with the wild-type insect strain of the control. Mice vaccinated with the Δ sbpase Δtal strain had dead individuals at 8d post-infection and all mice died at 10d infection, while mice vaccinated with the Δ sbpase Δtal Δ rupe strain survived overall, indicating a significant reduction in virulence of the Δ sbpase Δtal Δ rupe strain in mice.

Example 6 immunoprotection experiments of Gene knockout strains on mice

(1) Infection is carried out by adopting wild toxoplasma RH delta ku80 and ME49 insect strains (benefit of agricultural university in China), when the RH delta ku80 and ME49 insect strains cultured in HFF cells have a plurality of large sodium insect vesicles, PBS washes out tachyzoites escaping from the cells, and the extracellular tachyzoites are collected by adding DMEM culture medium without FBS;

(2) 100. Mu.L of the tachyzoites-containing liquid was diluted 10-fold and counted under a microscope, and after completion of the counting, the tachyzoites were diluted with DMEM medium containing no FBS. Based on the virulence gradient experiments of example 5, 5 mice vaccinated with the Δ sbpase ΔtalΔ rupe strain at 1×10 ⁴、1×10⁵ doses all survived; after 21d inoculation, mice infected with 1×10 ⁴ Δ sbpase ΔtalΔ rupe insect strain and 5 unvaccinated blank mice were injected with RH Δku80 at 1×10 ⁴ tachyzoite doses, mice infected with 1×10 ⁵ Δ sbpase ΔtalΔ rupe insect strain and 5 unvaccinated blank mice were injected with ME49 at 1×10 ⁴ tachyzoite doses. The mental, appetite and mortality of the mice were observed and recorded, and the results were counted in prism5 and plotted.

The results are shown in FIG. 12, which shows that the mice in the non-immunized control group all died within 7d after infection with 1X 10 ⁴ RH Δku80 strain, and that the survival rate of mice immunized with 1X 10 ⁴ Δ sbpase ΔtalΔ rupe tachyzoites within 30d was 100%. After the non-immunized control mice were infected with 1×10 ⁴ ME49 strain, the survival rate was only 40% in 30d, as shown in fig. 13, whereas the survival rate in 30d was 100% for mice immunized with 1×10 ⁵ Δ sbpase ΔtalΔ rupe tachyzoites; the inoculation of the delta sbpase delta tal delta rupe strain is proved to have good immunity protection against infection of mice with wild toxoplasma strain, and can be used for preparing toxoplasma vaccine.

In conclusion, according to the invention, through research on three genes of sedoheptulose-1, 7-bisphosphatase TgSBPase, transaldolase TgTAL and ribulose-5-phosphate epimerase TgRuPE of toxoplasma gondii, the invention discovers that the single gene of TgSBPase, tgTAL or TgRuPE is knocked out respectively, the in-vitro growth rate and virulence of toxoplasma gondii are not influenced, and the two genes or three genes can influence the growth and propagation of toxoplasma gondii and are related to the virulence of toxoplasma gondii. The invention successfully constructs the toxoplasma TgSBPase, tgTAL, tgRuPE three-gene knockout strain, after the TgSBPase, tgTAL, tgRuPE gene is knocked out simultaneously, the strain can be subjected to in vitro subculture and has the characteristic of almost no toxicity in vivo, and the vaccine strain can effectively resist the infection of wild insect strains after immunization, shows good immune protection effect and has good immune protection effect on mice. The three-gene knockout strain delta sbpase delta tal delta rupe provided by the invention provides a method and means for developing more effective and nontoxic toxoplasmosis-resistant medicaments and vaccines.

The above examples are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above examples, and any other changes, modifications, substitutions, combinations, and simplifications that do not depart from the spirit and principle of the present invention should be made in the equivalent manner, and the embodiments are included in the protection scope of the present invention.

Claims (10)

1. The construction method of the toxoplasma gondii three-gene knockout strain is characterized in that the toxoplasma gondii sedoheptulose-1, 7-bisphosphatase TgSBPase, transaldolase TgTAL and ribulose-5-phosphate epimerase TgRuPE genes are knocked out by a CRISPR gene editing technology to obtain the toxoplasma gondii three-gene knockout strain; the nucleotide sequence of TgSBPase, tgTAL, tgRuPE gene is shown in SEQ ID NO:1 to 3.

2. The construction method according to claim 1, comprising the specific steps of:

S1, directly knocking out TgSBPase genes by taking a wild toxoplasma strain as a starting insect strain to construct a DiCre delta sbpase insect strain;

S2, knocking out TgTAL genes to construct delta sbpase deltatal insect strains on the basis of DiCre delta sbpase insect strains;

S3, finally, knocking out TgRuPE genes on the delta sbpase delta tal insect strain to obtain a toxoplasma gondii three-gene knocked-out insect strain delta sbpase delta tal delta rupe.

3. The construction method according to claim 2, wherein the construction method of DiCre delta sbpase insect strain in step S1 comprises: using pSAG-Cas 9-TgU6-sgTgUPRT plasmid as a template to construct TgSBPase specific CRISPR/Cas9 knockout plasmid; amplifying a Ko-SBPase-loxp-DHFR-loxp homologous fragment by taking ploxp-DHFR-loxp plasmid as a template, and co-electrotransfecting the plasmid and the fragment into a wild toxoplasma strain, and carrying out drug screening and PCR identification to obtain a DiCre delta SBPase insect strain;

The construction method of the Delta sbpase Deltatal insect strain in the step S2 comprises the following steps: firstly, removing DHFR tags in DiCre Delta sbpase insect strains; using pSAG-Cas 9-TgU6-sgTgUPRT plasmid as a template, designing TgTAL gene gRNA primer, and preparing knockout plasmid pSAG-Cas 9-TgU6-sgTgTAL; amplifying Ko-TAL-loxp-DHFR-loxp homologous fragments by using ploxp-DHFR-loxp plasmid as a template, and co-electrotransfecting the plasmids and fragments into DiCre delta sbpase insect strains, and performing drug screening and PCR identification to obtain delta sbpase delta TAL insect strains;

The construction method of the delta sbpase delta tal delta rupe insect strain in the step S3 comprises the following steps: firstly removing DHFR tags in the delta sbpase delta tal insect strain; then taking pSAG-Cas 9-TgU6-sgTgUPRT plasmid as a template, designing TgRuPE gene gRNA primer, and preparing knockout plasmid pSAG-Cas 9-TgU6-sgTgRuPE; and amplifying the Ko-RuPE-YFP-DHFR homologous fragment by taking pTub-YFP-DHFR plasmid as a template, and co-electrotransfecting the plasmid and the fragment into the delta sbpase delta tal insect strain, and carrying out drug screening and PCR identification to obtain the delta sbpase delta tal delta rupe insect strain.

4. The method according to claim 2, wherein the wild-type toxoplasma strain in step S1 is toxoplasma RH (dice) insect strain.

5. A toxoplasma gondii three-gene knockout strain, which is characterized by being constructed by the method of any one of claims 1 to 4.

6. The application of toxoplasmosis sedoheptulose-1, 7-bisphosphatase TgSBPase, transaldolase TgTAL and ribulose-5-phosphate epimerase TgRuPE genes as targets in screening or preparing medicines for preventing and treating toxoplasmosis is characterized in that the nucleotide sequence of the TgSBPase, tgTAL, tgRuPE genes is shown as SEQ ID NO:1 to 3.

7. The application of a reagent for deleting or knocking out genes of Toxoplasma gondii sedoheptulose-1, 7-bisphosphatase TgSBPase, transaldolase TgTAL and ribulose-5-phosphate epimerase TgRuPE in constructing three-gene knocked-out strains of Toxoplasma gondii or preparing medicines for preventing and treating toxoplasmosis is characterized in that the nucleotide sequence of the TgSBPase, tgTAL, tgRuPE gene is shown as SEQ ID NO:1 to 3.

8. The use of the toxoplasma gondii tri-gene knockout strain according to claim 5 for preparing toxoplasma gondii vaccine.

9. A toxoplasma vaccine comprising the three-gene knockout strain of toxoplasma of claim 5.

10. A drug for preventing and treating toxoplasmosis, which is characterized by comprising a reagent for deleting or knocking out genes of toxoplasmosis sedoheptulose-1, 7-bisphosphatase TgSBPase, transaldolase TgTAL and ribulose-5-phosphate epimerase TgRuPE, wherein the nucleotide sequence of the TgSBPase, tgTAL, tgRuPE gene is shown as SEQ ID NO:1 to 3.