CN118389562A - A method for preparing attenuated mutant of Toxoplasma gondii and its application - Google Patents

Abstract

The present invention discloses a method for preparing an attenuated mutant of Toxoplasma gondii and its application, wherein the method uses a CRISPR/Cas9 system to knock out a gene composition in the Toxoplasma genome; the gene composition is composed of an ompdc gene, an ldh1 gene and a gra4 gene. The present invention uses gene editing technology to construct an attenuated Toxoplasma gondii strain with strong immune activation ability and high safety, which has a weak immunosuppressive effect on the host and can only survive in the host for a short period of time, while inducing a high level of immune response in the host, providing a new immunotherapy and prevention method for resisting Toxoplasma infection, and has important application value for human health and animal husbandry development.

Description

A method for preparing attenuated mutant of Toxoplasma gondii and its application

Technical Field

The present invention relates to the field of genetic engineering, and in particular to a method for preparing a toxoplasma gondii attenuated mutant and an application thereof.

Background technique

Toxoplasma gondii, also known as Toxoplasma gondii, is a protozoan that specializes in intracellular parasitism in the phylum Apicomplexa. It infects many warm-blooded animals including humans, livestock, and birds, causing zoonotic parasitic diseases. Toxoplasma can cause fatal acute infections in people with low or impaired immunity (such as AIDS, cancer, organ transplant patients, or individuals who use immunosuppressants for a long time), and can cause chronic infections in intermediate hosts with normal immune systems (mammals including humans), causing disorders of the endocrine system and nervous system, and greatly increasing the risk of depression and schizophrenia. Women infected with Toxoplasma during pregnancy (especially in the early stages) will have adverse effects on the development of the fetus, causing premature birth, malformations, abnormal development of the nervous system, and even fetal death. In animal husbandry production, Toxoplasma infection of important economic animals such as pigs and sheep can easily lead to animal miscarriage and stillbirth, and the quality of milk, meat and other products will decline, causing huge economic losses. There is also a risk of transmitting Toxoplasma to humans through food.

After being attacked by the immune system, Toxoplasma gondii will form cysts with cyst walls in muscle tissue, brain tissue and other parts, and exist independently outside the cells. It has strong immune escape ability and is difficult to remove, so there is currently no good vaccine for Toxoplasma infection. In addition, the protein vaccine and DNA vaccine in the laboratory research stage have a large gap between the infection situation of the real Toxoplasma invading cells after entering the host body, and the immune protection effect produced is low. At present, the only Toxoplasma vaccine approved for listing is a strain weakened by passage in the laboratory, which can be replicated in the host body, and there is a risk of strong virulence. Prior art CN108434447A discloses a Toxoplasma attenuated live vaccine lacking OMPDC and LDH1 genes. Although it has the safety advantage of almost no toxicity, it has the problem of suppressing the host's innate immune response and weak immunogenicity.

Summary of the invention

In order to solve the above problems existing in the prior art, the present invention provides a method for preparing a toxoplasma gondii attenuated mutant and its application.

The first object of the present invention is to provide a method for preparing attenuated mutants of Toxoplasma gondii.

The second object of the present invention is to provide application of the method in reducing the virulence of Toxoplasma gondii and/or enhancing the immunogenicity of Toxoplasma gondii.

The third object of the present invention is to provide application of the method in preparing products for preventing and/or treating Toxoplasma gondii infection.

The fourth object of the present invention is to provide a biological material for preparing attenuated mutants of Toxoplasma gondii.

The fifth object of the present invention is to provide the use of the biological material in reducing the virulence of Toxoplasma gondii and/or enhancing the immunogenicity of Toxoplasma gondii.

The sixth object of the present invention is to provide the use of the biological material in the preparation of products for preventing and/or treating Toxoplasma gondii infection.

In order to achieve the above object, the present invention is implemented by the following scheme:

Uridylic acid (UMP) is one of the nucleotides that make up RNA, and in all organisms, it is a precursor for the synthesis of other pyrimidine nucleotides. In Toxoplasma, uridine acid can be synthesized by the de novo pathway and the salvage pathway, of which the de novo pathway plays a major role, and the salvage pathway is insufficient to maintain the normal supply of uridine acid. In the de novo synthesis pathway, orotidine nucleotide-5-phosphate decarboxylase (OMPDC) is the last key enzyme, catalyzing the decarboxylation reaction of orotidine nucleotide to generate uridine acid. The loss of the ompdc gene will cause a serious deficiency in the synthesis of uridine acid in Toxoplasma, thus losing the ability to replicate. Lactate dehydrogenase (LDH) catalyzes the mutual conversion between pyruvate and lactate in the glycolysis pathway. Toxoplasma has two ldh genes: ldh1 and ldh2 . The ldh1 gene is specifically expressed in Toxoplasma tachyzoites. The loss of the ldh1 gene will cause Toxoplasma tachyzoites to fail to replicate normally in animals, but can grow normally in vitro.

Dense granule proteins (GRAs) are a series of proteins secreted into the vesicles by Toxoplasma gondii during its invasion of host cells and involved in the formation of the vesicle walls. This invention proposes for the first time that the GRA4 gene has a strong inhibitory effect on the host IFN-I pathway, thereby reducing the innate immune response of host cells.

Based on this, the present invention requests protection for a method for preparing an attenuated mutant of Toxoplasma gondii, using the CRISPR/Cas9 system to knock out a gene composition in the Toxoplasma gondii genome; the gene composition consists of an ompdc gene, an ldh1 gene and a gra4 gene, the accession number of the ompdc gene is TGME49_259690, the accession number of the ldh1 gene is TGME49_232350, and the accession number of the gra4 gene is TGME49_310780.

Preferably, the CRISPR/Cas9 system comprises a gene encoding sgRNA, and the sgRNA targets and knocks out the gene composition; the nucleotide sequence of the targeting site of the sgRNA is as shown in SEQ ID NOs: 1 to 3; the targeting site of the nucleotide sequence as shown in SEQ ID NO: 1 is the ompdc gene; the targeting site of the nucleotide sequence as shown in SEQ ID NO: 2 is the ldh1 gene; the targeting site of the nucleotide sequence as shown in SEQ ID NO: 3 is the gra4 gene.

More preferably, the nucleotide sequence of the gene encoding the sgRNA for targeted knockout of the gra4 gene is as shown in SEQ ID NO: 4 or as shown in the complete complementary sequence of the sequence shown in SEQ ID NO: 4.

Further preferably, the nucleotide sequence of the sgRNA for targeted knockout of the gra4 gene is shown in SEQ ID NO: 4. Even further preferably, the nucleotide sequence of the primer used to synthesize the sgRNA having a nucleotide sequence as shown in SEQ ID NO: 4 is shown in SEQ ID NOs: 5-6.

Preferably, the CRISPR/Cas9 system uses the pSAG1-Cas9-TgU6-ccdb-sgMIC3 vector as the basic skeleton.

Preferably, the CRISPR/Cas9 system is used to knock out the gra4 gene in the genome of a gene-deficient Toxoplasma gondii; the gene-deficient Toxoplasma gondii is a Toxoplasma gondii lacking the ompdc gene and the ldh1 gene.

More preferably, the method comprises the following steps: co-transfecting the homologous recombination template and the CRISPR/Cas9 system into host cells containing tachyzoites of the gene-deficient Toxoplasma gondii to obtain monoclonal cells, collecting the culture fluid of the monoclonal cells, and screening and identifying the monoclonal cells; the homologous recombination template comprises the 5' homologous arm and the 3' homologous arm of the gra4 gene.

More preferably, the nucleotide sequence of the 5' homology arm of the gra4 gene is shown in SEQ ID NO: 21. More preferably, the nucleotide sequence of the 3' homology arm of the gra4 gene is shown in SEQ ID NO: 22.

Further preferably, the host cell is an HFF cell. Further preferably, the screening is pyrimethamine screening. Further preferably, the identification is PCR amplification.

Further preferably, the PCR amplification includes three PCRs, and the nucleotide sequences of the primers used are shown in SEQ ID NOs: 16 to 21; the first PCR (i.e., PCR1) is used to detect whether the 5' homologous arm of the gra4 gene is successfully integrated into the genome of Toxoplasma gondii, and amplification to the target band indicates successful integration; the second PCR (i.e., PCR2) is used to detect whether the 3' homologous arm of the gra4 gene is successfully integrated into the genome of Toxoplasma gondii, and amplification to the target band indicates successful integration; the third PCR (i.e., PCR3) is used to detect whether the knockout gene gra4 still exists in the Toxoplasma genome, and if the target band is not amplified, it indicates that the gra4 gene has been successfully knocked out; if the results of the three PCR identifications are all consistent, it means that the ompdc gene, ldh1 gene and gra4 gene have been knocked out simultaneously in the genome of Toxoplasma gondii.

Specifically, the nucleotide sequences of the primers used in the first PCR are shown in SEQ ID NOs: 16-17; the nucleotide sequences of the primers used in the second PCR are shown in SEQ ID NOs: 18-19; and the nucleotide sequences of the primers used in the third PCR are shown in SEQ ID NOs: 20-21.

Preferably, the Toxoplasma gondii is type II Toxoplasma gondii. More preferably, the Toxoplasma gondii is strain ME49.

Further preferably, the gene-deficient Toxoplasma gondii is the ME49 strain lacking the ompdc gene and the ldh1 gene, i.e., the "△ ompdc △ ldh1 strain" in the prior art CN108434447A. The attenuated Toxoplasma gondii mutant prepared by the method is the ME49△ ompdc △ ldh1 △ gra4 strain.

The attenuated mutants of Toxoplasma gondii prepared by any of the methods described should also be within the protection scope of the present invention.

The use of any of the methods described above in reducing the virulence of Toxoplasma gondii and/or enhancing the immunogenicity of Toxoplasma gondii should also be within the scope of protection of the present invention.

Preferably, the reducing the virulence of Toxoplasma includes inhibiting the replication of Toxoplasma in the host. Preferably, the enhancing the immunogenicity of Toxoplasma includes enhancing the ability of Toxoplasma to induce an immune response in the host.

More preferably, the enhancing the immune response induced by Toxoplasma gondii in the host comprises promoting the increase of interferon content in the host. Further preferably, the interferon comprises IFN-β.

The use of any of the methods in the preparation of products for preventing and/or treating Toxoplasma gondii infection should also be within the scope of protection of the present invention.

A biological material for preparing an attenuated mutant of Toxoplasma gondii, which is any one of the following (1) to (3):

(1) A nucleic acid molecule having a sequence as shown in SEQ ID NO: 4 or a completely complementary sequence to that shown in SEQ ID NO: 4;

(2) sgRNA synthesized from the nucleic acid molecule described in (1);

(3) A gene editing vector containing the sgRNA described in (2).

Preferably, the nucleotide sequence of the sgRNA in (2) is as shown in SEQ ID NO: 4.

Preferably, the gene editing vector in (3) is a CRISPR gene editing vector containing the gene encoding the sgRNA.

More preferably, the gene editing vector described in (3) uses the pSAG1-Cas9-TgU6-ccdb-sgMIC3 vector as a backbone.

The use of the biological material in reducing the virulence of Toxoplasma gondii and/or enhancing the immunogenicity of Toxoplasma gondii should also be within the protection scope of the present invention.

Preferably, the reducing the virulence of Toxoplasma includes inhibiting the replication of Toxoplasma in the host. Preferably, the enhancing the immunogenicity of Toxoplasma includes enhancing the ability of Toxoplasma to induce an immune response in the host.

More preferably, the enhancing the immune response induced by Toxoplasma gondii in the host comprises promoting the increase of interferon content in the host. Further preferably, the interferon comprises IFN-β.

The use of the biological material in the preparation of products for preventing and/or treating Toxoplasma gondii infection should also be within the protection scope of the present invention.

Compared with the prior art, the present invention has the following beneficial effects:

The present invention adopts gene editing technology to construct an attenuated Toxoplasma gondii strain with strong immune activation ability and high safety. The strain has weak immunosuppressive effect on the host and can only survive for a short period of time in the host. At the same time, it can induce the host to produce a high level of immune response, providing a new immunotherapy and prevention method for resisting Toxoplasma infection, and has important application value for human health and animal husbandry development.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 shows the detection results of the dual luciferase reporter system in HEK293T cells with different treatments; A is the relative fluorescence intensity detection result of IFN-β in 293T (cGAS/STING) cells transfected with empty vector or overexpression vector of 13 protein molecules with GRA domain under ME49 gDNA stimulation; B is the relative fluorescence intensity detection result of IFN-β in wild-type HEK293T cells transfected with empty vector or overexpression vector of 13 protein molecules with GRA domain under ME49 gRNA stimulation; C is the relative fluorescence intensity detection result of IFN-β in 293T (cGAS/STING) cells transfected with empty vector or GRA4 overexpression vector under different doses of ME49 gDNA stimulation; D is the relative fluorescence intensity detection result of IFN-β in wild-type HEK293T cells transfected with empty vector or GRA4 overexpression vector under different doses of ME49 gRNA stimulation; ** indicates p < 0.05, *** indicates p <0.001; EV indicates empty vector (pcDNA3.1 vector).

Figure 2 is a schematic diagram of the process for constructing the pSAG1-Cas9-TgU6-sggra4 vector.

Figure 3 is a map of the pSAG1-Cas9-TgU6-sggra4 vector.

FIG4 is a schematic diagram of three PCR identifications of the gene editing of the insect strain genome DNA.

FIG. 5 shows the PCR identification results of the genomic DNA of each insect strain.

Figure 6 shows the safety evaluation results of ME49△ ompdc △ ldh1 △ gra4 strain; A shows the effect on the body weight of mice; B shows the effect on the transcription level of ITS-1 gene of the strain in the spleen; ns, no significant difference; *** p <0.001.

Figure 7 shows the evaluation results of the immune effect of the ME49△ ompdc △ ldh1 △ gra4 strain; A and B respectively show the effects on the transcription levels of the strain's Ifnb gene and Isg56 gene in the spleen of mice; C shows the effect on the IFN-β content in the serum of mice; ns, no significant difference; *** p ＜0.001.

Detailed ways

The present invention is further described in detail below in conjunction with the accompanying drawings and specific examples of the specification. The examples are only used to explain the present invention and are not used to limit the scope of the present invention. The test methods used in the following examples are conventional methods unless otherwise specified; the materials and reagents used are reagents and materials that can be obtained from commercial channels unless otherwise specified.

Example 1 Effect of protein molecules with GRA domain on the IFN-I signaling pathway activated by Toxoplasma gondii nucleic acid

1. Construction of protein overexpression vector with GRA domain

Using the pcDNA3.1 vector as the basic framework, GRA1 (gene number: TGME49_270250), GAR2 (gene number: TGME49_227620), GRA3 (gene number: TGME49_227280), GRA4 (gene number: TGME49_310780), GRA5 (gene number: TGME49_286450), GRA6 (gene number: TGME49_275440), The cDNA fragments of GRA7 (Gene ID: TGME49_203310), GRA8 (Gene ID: TGME49_254720), GRA9 (Gene ID: TGME49_251540), GRA10 (Gene ID: TGME49_268900), GRA11b (Gene ID: TGME49_237800), GRA12b (Gene ID: TGME49_275860), and GRA15 (Gene ID: TGME49_275470) were inserted into the CMV of pcDNA3.1 vector. A total of 13 overexpression vectors of protein molecules with GRA domains were obtained between the promoter site and the BGH pA site, namely, GRA1 overexpression vector, GAR2 overexpression vector, GRA3 overexpression vector, GRA4 overexpression vector, GRA5 overexpression vector, GRA6 overexpression vector, GRA7 overexpression vector, GRA8 overexpression vector, GRA9 overexpression vector, GRA10 overexpression vector, GRA11b overexpression vector, GRA12b overexpression vector and GRA15 overexpression vector.

2. Dual luciferase reporter system transfection

The pcDNA3.1-FLAG-cGAS vector and the pcDNA3.1-FLAG-STING vector were co-transfected into wild-type HEK293T cells to obtain HEK293T cells stably expressing cGAS and STING proteins, which were recorded as 293T (cGAS/STING) cells. 293T (cGAS/STING) cells (1×10 ⁶ cells/well) and wild-type HEK293T cells (1×10 ⁶ cells/well) were seeded in 24-well plates, and then the pGL3.0-IFN β-luc vector (200 ng) and pRL-TK vector (80 ng) were co-transfected with the empty vector (pcDNA3.1 vector) (500 ng) or the overexpression vectors of 13 protein molecules with GRA domains (500 ng) into the 24-well plates seeded with the two types of cells.

3. Nucleic acid stimulation

(1) DNA stimulation

The genomic DNA of the ME49 strain of Toxoplasma gondii was extracted (referred to as ME49 gDNA); 24 hours after transfection, ME49 gDNA (500 ng/well) was transfected into each 293T (cGAS/STING) cell for stimulation, and 293T (cGAS/STING) cells without DNA stimulation and transfected with an empty vector were set as negative controls. After 8 hours, samples were collected to detect the fluorescence intensity, and the relative fluorescence intensity (multiple) of IFN-β was obtained by statistics, and the formula was: .

The results are shown in A of Figure 1. Under the stimulation of ME49 gDNA, compared with the empty vector transfection, the relative fluorescence intensity of IFN-β produced by overexpressing the other 12 GRA proteins in 293T (cGAS/STING) cells had no significant difference, while the relative fluorescence intensity produced by overexpressing GRA4 protein was the lowest, indicating that GRA4 protein has a significant inhibitory effect on the IFN-β promoter activity mediated by Toxoplasma gDNA, and the inhibitory effects of the other 12 protein molecules with GRA domains on the IFN-β promoter activity mediated by Toxoplasma gDNA are significantly weaker than that of GRA4 protein.

(2) RNA stimulation

Total RNA of Toxoplasma gondii ME49 strain (denoted as ME49 RNA) was extracted; 24 h after transfection, ME49 RNA (1 μg/well) was transfected into each wild-type HEK293T cell for stimulation, and wild-type HEK293T cells without RNA stimulation and transfected with an empty vector were set as negative controls. Samples were collected 8 h later for detection, and the relative fluorescence intensity (multiple) of IFN-β was obtained by statistics.

The results are shown in Figure 1B. Under the stimulation of ME49 RNA, compared with the overexpression of the other 12 GRA proteins, the relative fluorescence intensity of IFN-β produced by overexpressing GRA4 protein in wild-type HEK293T cells relative to transfection with an empty vector was the lowest, indicating that GRA4 protein also has a significant inhibitory effect on the IFN-β promoter activity mediated by Toxoplasma RNA. The activation effects of the other 12 protein molecules with GRA domains on the IFN-I signaling pathway are significantly weaker than that of GRA4 protein.

4. Effect of GRA4 on the IFN-I signaling pathway activated by Toxoplasma nucleic acid

293T (cGAS/STING) cells (1×10 ⁶ ) and wild-type HEK293T cells (1×10 ⁶ ) were seeded in 24-well plates, and then pGL3.0-IFN β-luc vector (200 ng) and pRL-TK vector (80 ng) were co-transfected with empty vector (pcDNA3.1 vector) (500 ng) or different concentrations of GRA4 overexpression vector (400 ng or 800 ng) into the 24-well plates seeded with the two cells. 24 hours after transfection, DNA stimulation and RNA stimulation were performed according to the operation in step “3, nucleic acid stimulation” of this example, and samples were collected and detected 8 hours later to obtain the relative fluorescence intensity (multiple) of IFN-β.

The results are shown in Figure 1C. Under the stimulation of ME49 gDNA, the relative fluorescence intensity of IFN-β in 293T (cGAS/STING) cells was significantly higher than that under the unstimulated condition; after adding 400 ng of GRA4 overexpression vector, the relative fluorescence intensity was significantly reduced; after adding 800 ng of GRA4 overexpression vector, the relative fluorescence intensity of IFN-β was further significantly reduced. This indicates that the significant inhibitory effect of GRA4 on the IFN-β promoter activity mediated by Toxoplasma genomic DNA is concentration-dependent.

The results are shown in Figure 1D. Under the stimulation of ME49 RNA, the relative fluorescence intensity of IFN-β in wild-type HEK293T cells was significantly increased compared with that under unstimulated conditions; after adding 400 ng of GRA4 protein overexpression vector, the relative fluorescence intensity of IFN-β was significantly decreased, and after adding 800 ng of GRA4 protein overexpression vector, the relative fluorescence intensity of IFN-β was significantly decreased again, indicating that the significant inhibitory effect of GRA4 on Toxoplasma RNA-mediated IFN-β promoter activity is concentration-dependent.

The above results indicate that GRA4, a protein molecule with a GRA domain, can inhibit the activation of the IFN-I signaling pathway by Toxoplasma nucleic acid and is a key protein for activating the host to produce innate immune responses to resist Toxoplasma infection.

Example 2 Construction of Toxoplasma gondii ME49Δ ompdc Δ ldh1 Δ gra4 strain

1. Design and synthesis of sgRNA

The 607 bp to 626 bp region of the GRA4 gene (nucleotide sequence as shown in SEQ ID NO: 3) was used as the target site, and sgRNA (i.e., sggra4) (5’-GACGCTGGTCGTGGAGGCGT-3’ (SEQ ID NO: 4)) was designed using an online website (http://www.e-crisp.org/E-CRISP/index.html). The upstream primer used to synthesize the sgRNA was gRNA-gra4-Fw (5’-GACGCTGGTCGTGGAGGCGTGTTTTAGAGCTAGAAATAGC-3’ (SEQ ID NO: 5)), and the downstream primer was gRNA-gra4-Rv (5’-ACGCCTCCACGACCAGCGTCAACTTGACATCCCCATTTAC-3’ (SEQ ID NO: 6)).

2. Construction of gene editing vector

(1) PCR amplification

A gene editing vector for knocking out the GRA4 gene was constructed based on the CRISPR/Cas9 system. The construction process is shown in Figure 2. The main steps are: using the pSAG1-Cas9-TgU6-ccdb-sgMIC3 vector as a template, the MIC3 target-specific gRNA was replaced with the sgRNA targeting the GRA4 gene (SEQ ID NO: 4) using the NEB Q5® Site-Directed Mutagenesis Kit (Cat #E0552S). The PCR reaction system used is shown in Table 1, and the reaction procedure is shown in Table 2.

Table 1 PCR reaction system for constructing gene editing vector

Table 2 PCR reaction program for constructing gene editing vector

(2) Transformation identification

Take all the reaction products and transform them into competent cells DH5α (100 μl), apply LB/Amp plates, and invert and culture at 37℃ for 12 hours. Pick a single colony and place it in 5 ml LB/Amp liquid culture medium, and shake and culture at 37℃ and 180 rpm for 12 hours until the bacterial solution becomes turbid. Take a part of the bacterial solution to extract the vector, and use the restriction endonuclease of Thermo Company for enzyme digestion identification. The reaction system is: O Buffer, 2.0 μl; SalI endonuclease, 1.0 μl; vector, 1.0 μl; sterile deionized water (nuclease-free), 16.0 μl. After reacting at 37℃ for 4 hours, perform agarose gel electrophoresis detection. If the enzyme digestion product is two bands (size is 6 kb and 3.7 kb respectively), it is a positive clone. The positive clones were sequenced and analyzed, and the sequencing primer was M13 reverse primer. The sequencing results showed that the target sequence had been completely replaced, indicating that the gene editing vector was successfully constructed, and was recorded as pSAG1-Cas9-TgU6-sggra4 vector, and its map structure is shown in Figure 3. It was stored at -80°C with glycerol for later use.

3. Construction of homologous recombination template

(1) Obtaining homology arms

Homologous recombination templates were constructed by homologous recombination. The locus of the GRA4 gene (GeneID: TGME49_310780) was determined on ToxoDB, and the 5' homology arm (174 bp, SEQ ID NO: 21) and 3' homology arm (823 bp, SEQ ID NO: 22) were determined by the genomic sequence of the GRA4 gene. The 5' homology arm fragment (SEQ ID NO: 23) and 3' homology arm fragment (SEQ ID NO: 24) were amplified from the genomic DNA of Toxoplasma gondii ME49 using the primer pairs U5gra4-Fw (SEQ ID NO: 11) and U5gra4-Rv (SEQ ID NO: 12) and U3gra4-Fw (SEQ ID NO: 13) and U3gra4-Rv (SEQ ID NO: 14) in Table 3, and the concentration of the obtained product was determined by NanoDrop2000.

Table 3 Primers for constructing homologous recombination templates

(2) Acquisition of loxP fragments

The loxp-DHFR-loxp-Fw (SEQ ID NO: 9) and loxp-DHFR-loxp-Rv (SEQ ID NO: 10) primers in Table 3 were used to amplify the loxp-DHFR-loxp fragment from the pLinker-AID-3xHA-DHFR vector (i.e., "pLinker-AID-3xHA-DHFR" in the prior art "https://doi.org/10.1038/s41467-023-36571-4"), and the concentration of the obtained product was determined using NanoDrop2000.

(3) Connection

Using the pUC19 vector as the basic skeleton, the pUC19 vector was linearized using the primer pair pUC19 Forward (SEQ ID NO: 7) and pUC19 Reverse (SEQ ID NO: 8) in Table 3, and then connected using the Vazyme ClonExpress MultiS One Step Cloning Kit. The reaction system is shown in Table 4.

Table 4 Reaction system for constructing homologous recombination template

(2) Transformation identification

Take all the ligation products and transform them into competent cells DH5α (100 μl), apply them to LB/Amp plates, and invert and culture at 37℃ for 12 hours. Pick a single colony and place it in 5 ml LB/Amp liquid culture medium, and shake and culture at 37℃/180 rpm until the bacterial solution becomes turbid. Take the bacterial solution for PCR identification, use the pUC19 vector primer M13 (SEQ ID NO: 15; SEQ ID NO: 16) to detect whether there is a complete full-length fragment; use the 5' homology arm upstream primer U5gra4-Fw (SEQ ID NO: 11) and loxp-DHFR-loxp downstream primer loxp-DHFR-loxp-Rv (SEQ ID NO: 10) to detect whether the 5' homology arm and loxp-DHFR-loxp are completely connected; use loxp-DHFR-loxp upstream primer loxp-DHFR-loxp-Fw (SEQ ID NO: 9) and 3' homology arm downstream primer U3gra4-Rv (SEQ ID NO: 14) to detect whether loxp-DHFR-loxp and 3' homology arm are completely connected. If all three PCR results are positive, it is confirmed as a positive clone. The bacterial liquid of the positive clone was expanded and cultured (5 ml), the vector was extracted, and sequencing analysis was performed using M13 forward primer (M13-Fw) (SEQ ID NO: 15) and M13 reverse primer (M13-Rv) (SEQ ID NO: 16) for reaction. If the sequencing results were correct, it was confirmed that the vector containing the homologous recombination template was successfully constructed and recorded as pgra4-5’UTR::loxp-DHFR-loxp::gra4-3’UTR vector, which was stored at -80°C with glycerol for later use.

(3) Template fragment amplification

The pgra4-5’UTR::loxp-DHFR-loxp::gra4-3’UTR vector was amplified by PCR using U5gra4-Fw (SEQ ID NO: 11) and U3gra4-Rw (SEQ ID NO: 12) and the high-fidelity enzyme KDPlus. The recovered product was used as the homologous recombination template, recorded as gra4-5’UTR::loxp-DHFR-loxp::gra4-3’UTR. The concentration was determined and stored at -20°C for future use.

4. Gene editing of Toxoplasma gondii

(1) Insect strain

Type II Toxoplasma gondii ME49△ ompdc △ ldh1 strain (i.e., the "△ ompdc △ ldh1 strain" in the prior art CN108434447A) is a type II strain of the genus Toxoplasma, class Apicomplexa, order Coccidia, family Toxoplasma, lacking the orotidine-5-phosphate decarboxylase ( ompdc ) gene (SEQ ID NO: 1) and the lactate dehydrogenase 1 ( ldh1 ) gene (SEQ ID NO: 2), having the dense granule protein 4 (GRA4) gene (nucleotide sequence as shown in SEQ ID NO: 3), and needs to be cultured in DMEM medium supplemented with uracil (final concentration of 250 μM).

(2) Transfection

The type II Toxoplasma ME49△ ompdc △ ldh1 tachyzoites that were about to overflow from the intraparasitic bubble were repeatedly blown up three times using a 5 mL syringe equipped with a 27 g small needle. The HFF cells were lysed and filtered with a 5 μm sterile filter membrane to remove cell debris and release the parasites. The supernatant was discarded and the parasite pellet was resuspended in 8 ml of Toxoplasma gene editing-specific electroporation solution (Cytomix buffer, pH = 7.6, containing 120 mM KCl, 0.15 mM CaCl ₂ , 10 mM K ₂ HPO ₄ /KH ₂ PO ₄ , 25 mM HEPES, 2 mM EGTA and 5 mM MgCl ₂ ), and the supernatant was discarded. The pellet was centrifuged again at 1000 g for 10 min, and the supernatant was discarded.

Resuspend the insect pellet with 300 μl Cytomix, add the entire insect suspension to a 4 mm electroporation cup, add the gene editing vector pSAG1-Cas9-TgU6-sggra4 vector and the homologous recombination template gra4-5’UTR::loxp-DHFR-loxp::gra4-3’UTR to the insect suspension in the electroporation cup at a molar ratio of 5:1, and mix evenly with a pipette (avoid bubbles).

Place the electroporation cup in a Bio-Rad electroporator and set the program to 1600 V, 25 μF, 50 Ω, 4 mm. After one electroporation, change the program to 1500 V, 25 μF, 50 Ω, 4 mm and electroporate twice. Quickly aspirate the DNA-worm mixture after electroporation and add it to a T25 cell flask with HFF cells. Use growth medium (made of 89 v/v% DMEM basal medium, 10 v/v% FBS, 1 v/v% double antibody and a final concentration of 250 μM uracil (dissolved in DMSO)) and culture in a 37°C incubator.

(3) Drug screening and identification

After transfection, when 70% of the tachyzoites escaped from the HFF cells, the 5 mL syringe was replaced with a 27 g small needle and repeatedly blown three times to lyse the HFF cells. The cell debris was removed by filtering with a 5 μm sterile filter membrane to release the worms, which were added to a T25 culture flask with fresh HFF cells and cultured with drug screening medium (made of growth medium and pyrimethamine at a volume ratio of 10,000:1). After three to five generations of continuous culture with drug screening medium, the worms were diluted with drug screening medium and placed in four 96-well plates containing HFF cells for 10 to 12 days until monoclonal cells were obtained.

Use a pipette tip to scrape the HFF cells and worm bodies in the well corresponding to the monoclonal cells, and combine them with the culture medium in the well to obtain the worm liquid. Then, add all the worm liquid to a 24-well plate containing fresh HFF cells and culture for about 5 days. When 70% of the HFF cells are lysed, use a sterile pipette tip to scrape 100 μl of the worm liquid again and continue to culture it in a new 24-well plate with HFF cells. Extract the genomic DNA of the remaining worm liquid and use the primers shown in Table 5 to identify the gene editing in the genomic DNA. According to the process shown in Figure 4, 3 PCR tests were performed. The ME49△ ompdc △ ldh1 strain was used as the control group. The criteria for judging the success of gene editing were as follows: PCR1 (detection primers were 5'-UpU5gra4 (SEQ ID NO: 17) and 3'-in loxp-DHFR-loxp (SEQ ID NO: 18)) was used to detect whether the 5' homologous arm of the gra4 gene was successfully integrated into the Toxoplasma genome, and amplified to 1384 bp target band (recorded as PCR1 band) indicates successful integration; PCR2 (detection primers are 5'-InDHFR (SEQ ID NO: 19) and 3'-DnU3gra4 (SEQ ID NO: 20) are used to detect whether the 3' homologous arm of the gra4 gene is successfully integrated into the Toxoplasma genome. If a target band of 1343 bp is amplified (recorded as PCR2 band), it indicates successful integration; PCR3 (detection primers are 5'-UpgRNA gra4 (SEQ ID NO: 21) and 3'-UngRNA gra4 (SEQ ID NO: 22)) is used to detect whether the knocked-out gene gra4 still exists in the genome. If the target band of 944 bp (recorded as PCR3 band) is not amplified, it indicates that the gra4 gene has been successfully knocked out, and the worm strain contained in the worm liquid is the worm strain with the ompdc gene, The PCR reaction system used in the three PCR tests is shown in Table 6, and the reaction procedures are shown in Table 7.

Table 5 PCR primers for gene editing

Table 6 PCR reaction system for gene editing

Table 7 PCR reaction procedures for gene editing

As shown in Figure 5, the PCR1 band and the PCR2 band were not amplified in the control group, but the PCR3 band was amplified; the PCR1 band and the PCR2 band were amplified in the test insect liquid, but the PCR3 band was not amplified. This indicates that the ompdc gene, the ldh1 gene and the gra4 gene have been knocked out in this insect strain, and it is recorded as the ME49△ ompdc △ ldh1 △ gra4 insect strain.

Example 3 Functional evaluation of Toxoplasma gondii ME49Δ ompdc Δ ldh1 Δ gra4 strain

1. Safety evaluation

(1) Impact on body weight

Wild-type mice (C57BL/6 inbred mice, 4-6 weeks old, female, purchased from the Experimental Animal Center of Southern Medical University, denoted as WT mice) and IFN-I receptor (IFNAR)-deficient mice (C57BL/6 inbred mice, 4-6 weeks old, female, purchased from The Jackson Laboratory, USA, denoted as Ifnar ^-/- mice, which are more susceptible to death due to Toxoplasma infection) were used as hosts.

The ME49△ ompdc △ ldh1 strain and the ME49△ ompdc △ ldh1 △ gra4 strain were respectively infected with the host at a dose of 1×10 ⁶ mice/mouse, and a total of 4 groups of mice were obtained, including Group 1 consisting of WT mice infected with the ME49△ ompdc △ ldh1 strain (denoted as WT+ME49△ ompdc △ ldh1 ), Group 2 consisting of WT mice infected with the ME49△ ompdc △ ldh1 △ gra4 strain (denoted as WT+ME49△ ompdc △ ldh1 △ gra4 ), Group 3 consisting of Ifnar ^-/- mice infected with the ME49△ ompdc △ ldh1 strain (denoted as Ifnar ^-/- +ME49△ ompdc △ ldh1 ), and Group 4 consisting of Ifnar -/- mice infected with the ME49△ ompdc △ ldh1 △ gra4 strain. ^-/- mice (denoted as Ifnar ^-/- +ME49△ ompdc △ ldh1 △ gra4 ).

The weight, physical signs and activity status of the four groups of mice were detected within 15 days after infection. As shown in A in Figure 6, the weight of WT mice and Ifnar ^-/- mice infected with WTME49△ ompdc △ ldh1 strain and ME49△ ompdc △ ldh1 △ gra4 strain continued to increase within 15 days after infection, and there was no significant difference between the groups. All mice maintained normal physical signs, no obvious abnormalities, and normal eating, drinking, sleeping and other behaviors. This shows that inoculation with ME49△ ompdc △ ldh1 △ gra4 strain does not affect the normal growth of mice.

(2) Effects on the spleen

WT mice were used as hosts, and the wild-type ME49 strain (denoted as ME49WT), ME49△ ompdc △ ldh1 strain and ME49△ ompdc △ ldh1 △ gra4 strain were infected with a dose of 1×10 ⁶ /mouse, respectively; WT mice injected with an equal volume of normal saline were used as the uninfected control group.

On the seventh day after infection, the same number of mice were randomly selected from each group, spleens were isolated, and RNA was extracted using the RNA extraction kit (TransZol Up) of Quanshijin Company. cDNA was synthesized using the reverse transcription kit (HiScript®III RT SuperMix for qPCR) of Vazyme Company. qPCR was performed using the real-time fluorescence quantitative PCR kit (ChamQUniversal SYBR qPCR Master Mix) of Vazyme Company, and the transcription level of the Toxoplasma gondii ITS-1 gene was detected using the primers TgITS-1-Fw (SEQ ID NO: 25) and TgIST-1-Rv (SEQ ID NO: 26) in Table 8 to evaluate the presence of Toxoplasma gondii in the spleen. The reaction system is shown in Table 9, and the reaction procedure is shown in Table 10.

Table 8 qPCR amplification primers for gene expression levels

Table 9 qPCR reaction system for gene expression

Table 10 PCR reaction procedures for gene editing

As shown in Figure 6B, on the 7th day after infection, the mRNA expression level of ITS-1 gene in the spleen of WT mice infected with ME49△ ompdc △ ldh1 strain and ME49△ ompdc △ ldh1 △ gra4 strain was significantly reduced compared with ME49WT, and was basically the same as that of the control group, indicating that the presence of ME49△ ompdc △ ldh1 strain or ME49△ ompdc △ ldh1 △ gra4 strain could no longer be detected in the spleen of mice.

The above results indicate that the ME49△ ompdc △ ldh1 △ gra4 strain constructed by the present invention has low toxicity, can only survive in a mammalian host for a short period of time, cannot replicate effectively, and has high safety.

2. Evaluation of immune effect

WT mice were used as hosts and infected with ME49△ ompdc △ ldh1 strain and ME49△ ompdc △ ldh1 △ gra4 strain at a dose of 1×10 ⁶ per mouse, respectively; WT mice injected with normal saline were used as the control group.

On the seventh day after infection, the same number of mice were randomly selected from each group, spleens were isolated, and RNA was extracted using the RNA extraction kit (TransZol Up) of Quanshijin Company. cDNA was synthesized using the reverse transcription kit (HiScript®III RT SuperMix for qPCR) of Vazyme Company. qPCR was performed using the real-time fluorescence quantitative PCR kit (ChamQUniversal SYBR qPCR Master Mix) of Vazyme Company, and the transcription level of the Ifnb gene in the spleen of mice was detected using the primers mouse Ifnb-Fw (SEQ ID NO: 27) and mouse Ifnb-Rv (SEQ ID NO: 28) in Table 8, and the transcription level of the Isg56 gene in the spleen of mice was detected using the primers mouse Isg56-Fw (SEQ ID NO: 29) and mouse Isg56-Rv (SEQ ID NO: 30) in Table 8. The reaction system is shown in Table 9, and the reaction procedure is shown in Table 10.

In addition, the serum of each mouse was collected and the IFN-β content in the serum was detected by ELISA kit (Enzyme-linked immunosorbent assay kit) of R&D. As shown in A and B in Figure 7, compared with the ME49△ ompdc △ ldh1 strain, the mRNA expression levels of Ifnb gene and Isg56 gene in the spleen of WT mice infected with ME49△ ompdc △ ldh1 △ gra4 strain were significantly increased, by 151.2% and 82.7%, respectively. As shown in C in Figure 7, compared with the ME49△ ompdc △ ldh1 strain, the IFN-β content in the serum of WT mice infected with ME49△ ompdc △ ldh1 △ gra4 strain was significantly increased, by 62.5%.

Based on the above results, it is shown that compared with the existing ME49△ ompdc △ ldh1 strain, the ME49△ ompdc △ ldh1 △ gra4 strain constructed by the present invention not only maintains the original high safety advantage when the gra4 gene is missing, but also further significantly enhances its immunogenicity, and can activate and induce the host to produce a stronger innate immune response.

Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention rather than to limit the protection scope of the present invention. For ordinary technicians in this field, other different forms of changes or modifications can be made based on the above descriptions and ideas. It is not necessary and impossible to list all the implementation methods here. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention should be included in the protection scope of the claims of the present invention.

Claims (10)

1. A method of making an attenuated toxoplasma mutant, wherein the CRISPR/Cas9 system is used to knock out a gene composition from the toxoplasma genome; the gene composition consists of ompdc genes, ldh1 genes and gra genes.

2. The method of claim 1, wherein the CRISPR/Cas9 system comprises a gene encoding a sgRNA that targets knockout of the gene composition; the nucleotide sequence of the targeting site of the sgRNA is shown as SEQ ID NO:1 to 3.

3. The method of claim 2, wherein the nucleotide sequence of the coding gene for the sgRNA targeted to knock-out the gra gene is set forth in SEQ ID NO:4 or as shown in SEQ ID NO:4, and the complete complement of the sequence shown in (4).

4. The method of claim 1, wherein the gra gene in the gene deleted toxoplasma genome is knocked out using a CRISPR/Cas9 system; the gene-deleted toxoplasma gondii is toxoplasma gondii in which the ompdc gene and the ldh1 gene are deleted.

5. The method of claim 1, wherein the toxoplasma is toxoplasma type ii.

6. Use of the method of any one of claims 1 to 5 for reducing toxoplasma virulence and/or enhancing toxoplasma immunogenicity.

7. Use of the method according to any one of claims 1 to 5 for the preparation of a product for the prevention and/or treatment of toxoplasma infection.

8. A biomaterial for preparing an attenuated mutant of toxoplasma, characterized in that it is any one of the following (1) to (3):

(1) The sequence is shown in SEQ ID NO:4 or as shown in SEQ ID NO:4, a nucleic acid molecule represented by the complete complement of the sequence shown in (4);

(2) An sgRNA synthesized from the nucleic acid molecule of (1);

(3) A gene editing vector comprising the sgRNA described in (2).

9. Use of the biomaterial of claim 8 for reducing toxoplasma virulence and/or enhancing toxoplasma immunogenicity.

10. Use of the biomaterial according to claim 8 for the preparation of a product for the prevention and/or treatment of toxoplasma infection.