Construction of Toxoplasma gondii strain lacking progesterone response kinase and its application as attenuated vaccine

Abstract

This invention discloses the construction of a Toxoplasma gondii progesterone-responsive kinase-deficient strain and its application as a live attenuated vaccine. The Toxoplasma gondii progesterone-responsive kinase-deficient strain is derived from Toxoplasma gondii RHΔ... ku80 As a maternal strain, gene editing technology was used to knock out the enzyme encoding progesterone-responsive kinase. prk The gene-deleted strain was named RHΔ ku80 Δ prk Experiments have shown that RHΔ ku80 Δ prk The virulence of the strain was significantly reduced, and immunized mice showed good immunoprotection against acute infection with highly virulent and moderately virulent Toxoplasma gondii strains, and significantly inhibited brain cyst formation in a chronic infection model. Therefore, RHΔ ku80 Δ prk This invention has potential application value as a live attenuated vaccine against Toxoplasma gondii. It has significant application prospects.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to the construction of a Toxoplasma gondii progesterone-responsive kinase-deficient strain and its application as an attenuated vaccine. Background Technology

[0002] Toxoplasma gondii ( Toxoplasma gondii Toxoplasma gondii, also known as the parasite, is an obligate intracellular parasitic protozoan belonging to the subphylum Apicocytocium. It can infect almost all warm-blooded animals and humans, and is a widespread and serious zoonotic pathogen. Toxoplasmosis infection is prevalent in both human and animal populations, and its pathogenicity is influenced by various factors such as host species, physiological state, and health status. In immunocompetent hosts, infection often presents as a latent infection; however, in young animals, infants, immunocompromised individuals, and pregnant hosts, infection with Toxoplasma gondii can lead to severe or fatal disease.

[0003] Currently, sulfonamides are mainly used for the prevention and treatment of acute toxoplasmosis. These drugs can effectively inhibit parasite proliferation, but they are difficult to completely eliminate the parasite from the host and are almost ineffective against chronic or latent infections. To date, there are no drugs that can effectively treat chronic / late-onset toxoplasmosis infections. In the 1990s, Europe developed a live attenuated toxoplasmosis vaccine to prevent miscarriages caused by toxoplasmosis in sheep, but due to its unstable immunogenicity and potential public health safety risks, it failed to be widely adopted. Apart from this vaccine, no other commercially available toxoplasmosis vaccines have been developed. Therefore, developing a safe and effective toxoplasmosis vaccine has significant scientific importance and application prospects.

[0004] Progesterone-responsive kinase (PRK) belongs to the serine / threonine protein kinase family and is a kinase involved in the regulation of cytoskeletal protein phosphorylation, playing an important role in cell division and cell cycle regulation. However, to date, there are no reports on the function, virulence, and immunoprotective capabilities of PRK in Toxoplasma gondii. Summary of the Invention

[0005] The purpose of this invention is to prevent toxoplasmosis.

[0006] This invention first protects *Toxoplasma gondii* strains lacking the progesterone-responsive kinase. These strains are obtained by modifying the gene encoding the progesterone-responsive kinase in the maternal *Toxoplasma gondii* strain. prk It is obtained by knocking out genes.

[0007] The progesterone-responsive kinase described above can be a protein with the amino acid sequence shown in SEQ ID No. 2.

[0008] Of these, SEQ ID No.2 consists of 789 amino acid residues.

[0009] The gene encoding progesterone-responsive kinase mentioned above may be b1), b2), or b3. b1) The coding region is the DNA molecule shown in SEQ ID No. 1; b2) The nucleotide sequence is the DNA molecule shown in SEQ ID No. 1; b3) The nucleotide sequence is the DNA molecule shown in SEQ ID No. 35.

[0010] Of these, SEQ ID No. 1 consists of 2370 nucleotides. SEQ ID No. 35 consists of 2983 nucleotides. The nucleotides shown in SEQ ID No. 1 or SEQ ID No. 35 encode the amino acid sequence shown in SEQ ID No. 2.

[0011] In the Toxoplasma gondii progesterone-responsive kinase-deficient strain, the gene encoding the progesterone-responsive kinase in the Toxoplasma gondii maternal strain can be knocked out using CRISPR / Cas9 technology.

[0012] In the Toxoplasma gondii progesterone-responsive kinase-deficient strain, knocking out the gene encoding the progesterone-responsive kinase in the Toxoplasma gondii maternal strain can be achieved by replacing the gene encoding any of the aforementioned progesterone-responsive kinases with the gene encoding chloramphenicol acetyltransferase.

[0013] In the *Toxoplasma gondii* progesterone-responsive kinase-deficient strain, knocking out the gene encoding the progesterone-responsive kinase in the maternal *Toxoplasma gondii* strain can specifically involve removing the nucleotide sequence shown in SEQ ID No. 35. prk The gene was replaced with a DNA fragment whose nucleotide sequence is shown in SEQ ID No. 19. The DNA fragment whose nucleotide sequence is shown in SEQ ID No. 19 contains the gene encoding chloramphenicol acetyltransferase.

[0014] The amino acid sequence of any of the chloramphenicol acetyltransferases described above may be as shown in SEQ ID No. 34.

[0015] The nucleotide sequence of the gene encoding any of the chloramphenicol acetyltransferases described above is shown in SEQ ID No. 19 from position 500 to 1159 starting from the 5' end.

[0016] In the *Toxoplasma gondii* progesterone-responsive kinase-deficient strain, the maternal strain of *Toxoplasma gondii* can be *Toxoplasma gondii* RHΔ ku80 strain.

[0017] The Toxoplasma gondii progesterone-responsive kinase-deficient strain mentioned above can specifically be the RHΔ strain prepared in the examples. ku80 Δ prk .

[0018] This invention also protects a vaccine for the prevention of Toxoplasma gondii infection. The vaccine is any of the Toxoplasma gondii progesterone-responsive kinase-deficient strains described above.

[0019] The aforementioned vaccine may be an attenuated live vaccine. The attenuated live vaccine may be prepared by culturing and amplifying any of the *Toxoplasma gondii* progesterone-responsive kinase-deficient strains described above, and then preparing the vaccine.

[0020] In any of the vaccines described above, the developmental stage of the Toxoplasma gondii progesterone-responsive kinase-deficient strain may be tachyzoite.

[0021] This invention also protects a method for increasing the level of Toxoplasma gondii-specific IgG antibodies, which is achieved by injecting any of the above-described Toxoplasma gondii progesterone-responsive kinase-deficient strains into an animal.

[0022] The present invention also protects the use of any of the above-described Toxoplasma gondii progesterone-responsive kinase-deficient strains in the preparation of vaccines for the prevention of Toxoplasma gondii infection.

[0023] In the above applications, the vaccine for preventing Toxoplasma gondii infection can be an attenuated live vaccine. The attenuated live vaccine can be prepared by culturing and amplifying any of the Toxoplasma gondii progesterone-responsive kinase-deficient strains described above, and then preparing the vaccine.

[0024] In the above applications, the developmental stage of the Toxoplasma gondii progesterone-responsive kinase-deficient strain can be tachyzoite.

[0025] The application of any of the above-described Toxoplasma gondii progesterone-responsive kinase-deficient strains in constructing a mouse model of Toxoplasma gondii infection is also within the scope of protection of this invention.

[0026] This invention addresses the technical challenge of the lack of safe and effective vaccines for toxoplasmosis by constructing a Toxoplasma gondii progesterone-responsive kinase-deficient strain—RHΔ—using gene editing technology. ku80 Δ prk The virulence of this insect strain was significantly reduced. This invention also evaluated RHΔ ku80 Δ prk The feasibility of RHΔ as a candidate strain for a live attenuated vaccine against Toxoplasma gondii was found. ku80 Δ prk Immunized mice showed good immunoprotection against acute infection with highly virulent and moderately virulent Toxoplasma gondii strains and significantly inhibited brain cyst formation in a chronic infection model. Therefore, RHΔ ku80 Δ prk This invention has potential application value as a live attenuated vaccine against Toxoplasma gondii. It has significant application prospects. Attached Figure Description

[0027] Figure 1 For example, RHΔ in Example 1 ku80 Δ prk The PCR amplification detection results.

[0028] Figure 2 For step one of Example 2, RHΔ ku80 Δ prk Plaque formation.

[0029] Figure 3 For step two RHΔ in Example 2 ku80 Δ prk Survival rate and relative weight changes in infected mice.

[0030] Figure 4 The step 2 in Example 3 involves ELISA detection of Toxoplasma gondii-specific IgG antibody levels in mouse serum.

[0031] Figure 5 For step 3 of Example 3, RHΔ ku80 Survival rate and relative weight changes in mice after tachyzoite attack.

[0032] Figure 6 For step 3 of Example 3, RHΔ ku80 Morphology of brain cysts and PCR detection results of Toxoplasma gondii gene in brain tissue of mice immunized after tachyzoite challenge.

[0033] Figure 7 The survival rate and relative weight changes of mice after being challenged with different doses of Pru tachyzoites in step 3 of Example 3.

[0034] Figure 8 This is a statistical analysis of the number of brain cysts in mice after being challenged with different doses of Pru tachyzoites in step 3 of Example 3, and the results of PCR detection of Toxoplasma gondii gene in brain tissue.

[0035] Figure 9 The results show the survival rate, relative weight change, and brain cyst statistics of mice after Pru ​​brain cyst attack in step 3 of Example 3. Detailed Implementation

[0036] The present invention will now be described in further detail with reference to specific embodiments. The given embodiments are merely illustrative of the invention and not intended to limit its scope. The embodiments provided below can serve as a guide for further improvements by those skilled in the art and do not constitute a limitation on the invention in any way.

[0037] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Unless otherwise specified, the materials and reagents used in the following examples are commercially available.

[0038] In the quantitative experiments described below, three replicate experiments were performed.

[0039] The plasmid pSAG1::Cas9-U6::sgUPRT is disclosed in the following literature: Bang Shen, Kevin Brown, Shaojun Long, and L. David Sibley. Development of CRISPR / Cas9 for Efficient Genome Editing in Toxoplasma gondii. In Vitro Mutagenesis: Methods and Protocols, Methods in Molecular Biology, vol. 1498. Its name in the literature is pSAG1::Cas9-U6::sgUPRT. This biological material is available to the public from China Agricultural University (i.e., the applicant). This biological material is intended solely for replicating experiments related to this invention and may not be used for any other purpose.

[0040] Toxoplasma gondii RHΔ ku80 The strain was disclosed in the following literature: Barbara A. Fox, Jessica G. Ristuccia, Jason P. Gigley, and David J. Bzik. Efficient Gene Replacements in Toxoplasma gondii Strains Deficient for Nonhomologous End Joining. EUKARYOTIC CELL, Apr. 2009, pp. 520-529. Its name in the literature (specifically Table 1) is RHΔ. ku80 The biological material can be obtained from China Agricultural University (i.e., the applicant). This biological material is solely for the purpose of replicating the relevant experiments of this invention and may not be used for any other purpose. In the following text, *Toxoplasma gondii* RHΔ ku80 RHΔ (short for strain) ku80 .

[0041] The Toxoplasma gondii strain Pru is disclosed in the following literature: Chen J, Li ZY, Zhou DH, Liu GH, Zhu XQ. Genetic diversity among Toxoplasma gondii strains from different hosts and geographical regions revealed by sequence analysis of GRA5 gene. ParasitVectors. 2012 Dec 3;5:279; its name in the literature is Prugniaud (PRU). This biological material is available to the public from China Agricultural University (i.e., the applicant). This biological material is only for the purpose of replicating the relevant experiments of this invention and may not be used for any other purpose. In the following text, the Toxoplasma gondii strain Pru is abbreviated as Pru.

[0042] The original pTCR plasmid used in the following examples is disclosed in the following literature: Wang Hui, Identification and function of Toxoplasma gondii high-mobility group group B1. Doctoral dissertation, China Agricultural University, 2015; its name in the literature is "basic plasmid pTCR". The original pTCR plasmid was obtained by modifying the knockout plasmid pTCY. Specifically, the relevant elements of the knockout plasmid pTCY were replaced with a nucleotide sequence as shown in SEQ ID No. 19: tubulin 5'UTR-chloramphenicol acetyltransferase-red fluorescence-tubulin 3'UTR (tubulin 5'UTR-CAT-RFP-tubulin 3'UTR). The tubulin 5'UTR-CAT-RFP-tubulin 3'UTR fusion fragment sequentially includes the tubulin 5'UTR, the gene encoding chloramphenicol acetyltransferase, the gene encoding red fluorescent protein, and the tubulin 3'UTR. The nucleotide sequence of the gene encoding chloramphenicol acetyltransferase is shown in SEQ ID No. 19 from position 500 to 1159 starting from the 5' end. The amino acid sequence of chloramphenicol acetyltransferase is shown in SEQ ID No. 34.

[0043] Unless otherwise specified, the mice used in the following examples are all healthy 6-week-old female ICR mice.

[0044] Example 1: Toxoplasma gondii prk Gene-deleted strain—RHΔ ku80 Δ prk Construction I. Construction of pSAG1-CAS9-U6-sgRNA (prk) plasmid for targeted RNA delivery 1. Use the E-CRISP website (http: / / www.e-crisp.org / E-CRISP / ) to design the knockout process. prk Gene-specific sgRNA sequences.

[0045] prk The nucleotide sequence of the gene is shown in SEQ ID No. 1. The prk gene encodes a progesterone-responsive kinase. The amino acid sequence of the progesterone-responsive kinase is shown in SEQ ID No. 2.

[0046] Knockout prk The nucleotide sequence of the gene-specific sgRNA is 5'-CGCATGTGTGGAGCAACAGC-3' (SEQ ID No. 3).

[0047] 2. Obtaining the three segments Using plasmid pSAG1::Cas9-U6::sgUPRT as a template, PCR amplification was performed using primer pair consisting of primer F1: 5'-CAAGTAAGCAGAAGCACGCTGTATTTCCGGGAGGGTGC-3' (SEQ ID No. 4) and primer R1: 5'-GCTGTTGCTCCACACATGCGAACTTGACATCCCCATTTA-3' (SEQ ID No. 5), and a fragment of approximately 550 bp was recovered.

[0048] Using plasmid pSAG1::Cas9-U6::sgUPRT as a template, PCR amplification was performed using primer pair consisting of primer F2: 5'-CGCATGTGTGGAGCAACAGCGTTTTAGAGCTAGAAATAG-3' (SEQ ID No. 6) and primer R2: 5'-GGTACCCAATTCGCCCTATA-3' (SEQ ID No. 7), and fragment 2 of approximately 2900 bp was recovered.

[0049] Using plasmid pSAG1::Cas9-U6::sgUPRT as a template, PCR amplification was performed using primer pair consisting of primer F3: 5'-TATAGGGCGAATTGGGTACCGGGCCCCCCCTCGAGGTCG-3' (SEQ ID No. 8) and primer R3: 5'-AGCGTGCTTCTGCTTACTTG-3' (SEQ ID No. 9), and a fragment of approximately 6200 bp was recovered.

[0050] 3. Using the homologous recombination seamless cloning kit (a product of Beijing TransGen Biotech Co., Ltd., catalog number CU201-02; specific product name pEASY). ®The Basic Seamless Cloning and Assembly Kit (basic version) allows for seamless ligation of fragments 1, 2, and 3 obtained in step 2 (ligation at 50°C for 15 min) to obtain the ligation product. This ligation product is then transformed into *E. coli* DH5α competent cells, gently mixed, incubated in an ice-water bath for 30 min, heat-shocked at 42°C for 60 s, and incubated in an ice-water bath for 3 min. 600 μL of LB liquid medium is then added, and the cells are incubated at 37°C and 200 rpm for 2 h. The bacterial culture is then plated onto ampicillin-resistant LB solid medium and incubated at 37°C for 12-16 h. Single colonies are picked for PCR and sequencing identification. After successful identification, the bacterial culture is expanded, and the plasmid, pSAG1-CAS9-U6-sgRNA (prk) plasmid, is extracted.

[0051] Based on the sequencing results, the structure of the pSAG1-CAS9-U6-sgRNA (prk) plasmid is described as follows: the 5'-GGCGTCTCGATTGTGAGAGC-3' (SEQ ID No. 10) in the plasmid pSAG1::Cas9-U6::sgUPRT is replaced with the DNA sequence shown in SEQ ID No. 3, and the other sequences remain unchanged.

[0052] II. Construction of pTCR-prk plasmid 1. Obtain from the ToxoDB database prk The upstream and downstream sequences of a gene.

[0053] 2. Obtaining the fragment With RHΔ ku80 Using genomic DNA as a template, PCR amplification was performed using primer pairs consisting of primer F-5': 5'-TCACTATAGGGCGAATTGGGTACCCCTTGAGCTCGCAGGACTTC-3' (SEQ ID No. 11) and primer R-5': 5'-GCGGACATGCATATCAAGCTTCATAGTCTGCTGCCTTCTTG-3' (SEQ ID No. 12), and approximately 580 bp of the 5' homologous arm was recovered.

[0054] With RHΔ ku80Using genomic DNA as a template, PCR amplification was performed using primer pairs consisting of primer F-3': 5'-GGATCCACTAGAGATATCGATGGCTAAGGCTCTGTGATG-3' (SEQ ID No. 13) and primer R-3': 5'-ACGCGGGACATGCATACTAGTCTTCGACAGTTAGTTCGTGGCG-3' (SEQ ID No. 14), and approximately 550 bp of the 3' homologous arm was recovered.

[0055] Using the original pTCR plasmid as a template, PCR amplification was performed using primer pair consisting of primer F4: 5'-AAGCTTGATATGCATGTCCGC-3' (SEQ ID No. 15) and primer R4: 5'-GATATCTCTAGTGGATCC-3' (SEQ ID No. 16), and approximately 2200 bp of the CAT-RFP drug screening frame was recovered.

[0056] Using the original pTCR plasmid as a template, PCR amplification was performed using primer pair consisting of primer F5: 5'-ACTAGTATGCATGTCCCGCGT-3' (SEQ ID No. 17) and primer R5: 5'-GGTACCCAATTCGCCCTATAGTGA-3' (SEQ ID No. 18), and approximately 5000 bp of the pTCR vector backbone was recovered.

[0057] 3. Using a homologous recombination seamless cloning kit, the 5' homologous arm, 3' homologous arm, CAT-RFP drug screening frame, and pTCR vector backbone obtained in step 2 were seamlessly ligated (ligation at 50℃ for 30 min) to obtain the ligation product. The ligation product was then transformed into *E. coli* DH5α competent cells, gently mixed, incubated in an ice-water bath for 30 min, heat-shocked at 42℃ for 60 s, and incubated in an ice-water bath for 3 min. Subsequently, 600 μL of LB liquid medium was added, and the cells were incubated at 37℃ and 200 rpm for 2 h. The bacterial culture was then plated onto ampicillin (Amp)-resistant LB solid medium and incubated at 37℃ for 12-16 h. Single colonies were picked for PCR and sequencing identification. After successful identification, the bacterial culture was expanded, and the plasmid, pTCR-prk plasmid, was extracted.

[0058] Based on the sequencing results, the structure of the pTCR-prk plasmid is described as follows: 5' homologous arms obtained in step 2 are inserted at 5'-TCACTATAGGGCGAATTGGGTACC-3' (SEQ ID No. 20) and 5'-GCGGACATGCATATCAAGCTT-3' (SEQ ID No. 21), respectively, and 3' homologous arm sequences obtained in step 2 are inserted at 5'-GGATCCACTAGAGATATC-3' (SEQ ID No. 22) and 5'-ACGCGGGACATGCATACTAGT-3' (SEQ ID No. 23), respectively.

[0059] III. Obtaining homologous template fragment solutions 1. Using the pTCR-prk plasmid constructed in step 2 as a template, PCR amplification was performed using primer pair consisting of primer F-5': 5'-CCTTGAGCTCGCAGGACTTC-3' (SEQ ID No. 24) and primer R-3': 5'-CTTCGACAGTTAGTTCGTGGCG-3' (SEQ ID No. 25), and a DNA fragment of approximately 3400 bp was recovered.

[0060] 2. Add 1 mL of pre-cooled 100% anhydrous ethanol to the DNA fragment recovered in step 1, mix well, centrifuge at 14000g for 10 min at 4°C, and collect the precipitate.

[0061] 3. Wash the precipitate collected in step 2 twice (each time by washing with 1 mL of pre-cooled 75% ethanol aqueous solution, followed by centrifugation at 4°C and 14000g for 5 min), and then dissolve it with Cytomix electroporation buffer to obtain a homologous template fragment solution.

[0062] Cytomix electroporation buffer: Dissolve 100 mL of 0.1 M KPO4 buffer (prepared from 86.6 mL of 1 M K2HPO4 and 13.4 mL of 1 M KH2PO4), 8.95 g KCl, 22.1 mg CaCl2, 1.02 g MgCl2, 5.96 g HEPES and 744 mg EDTA in an appropriate amount of ddH2O, then adjust the pH to 7.6, and finally bring the volume to 1 L with ddH2O; filter through a 0.22 μm filter, aliquot and store at 4 °C.

[0063] IV. Toxoplasma gondii prk Gene deletion strains (i.e., RHΔ) ku80 Δ prk Construction of ) 1. Collect freshly released RHΔ ku80Tachyzoites were filtered through a sterile 0.22 μm filter membrane, centrifuged at 2800 rpm for 8 min, and the supernatant was discarded to obtain purified RHΔ. ku80 Tachyzoites.

[0064] 2. Mix 750 μL Cytomix electroporation buffer, 20 μg pSAG1-CAS9-U6-sgRNA (prk) plasmid, and homologous template fragment solution (containing 100 μg homologous template fragment), then add purified RHΔ ku80 Tachyzoites were mixed and transferred to a sterile electroporation cuvette for transfection at 2050 V and ∞ resistance. HFF cells (ATCC product, catalog number Cat#CRL-1634; specific product name: Human Foreskin Fibroblasts) were then added and cultured at 37°C for 24 h. The cells were then passaged in a drug screening medium containing 40 μM chloramphenicol (solute and concentration of 12.9 mg / L chloramphenicol, solvent: anhydrous ethanol). Finally, drug-resistant tachyzoites were collected, referred to as resistant tachyzoites.

[0065] 3. Genomic DNA was extracted from the resistant tachyzoites obtained in step 2 and used as a template. PCR amplification was performed using primer pair 1, consisting of primers PCR1-F: 5'-GAGGCATTTCAGTCAGTTG-3' (SEQ ID No. 26) and primer PCR1-R: 5'-CGCCAGCGGCATCAGCACC-3' (SEQ ID No. 27), to obtain PCR amplification product 1. Genomic DNA was also extracted from the resistant tachyzoites obtained in step 2 and used as a template. PCR amplification was performed using primer pair 2, consisting of primers PCR2-F: 5'-ATGAGTGGCAGGGCGGGCT-3' (SEQ ID No. 28) and primer PCR2-R: 5'-CGGAGTGGCGGGCCTCGGC-3' (SEQ ID No. 29), to obtain PCR amplification product 2. Then the following determination is made: if the PCR amplification product 1 of a certain resistant tachyzoite contains a DNA fragment of about 500 bp and the PCR amplification product 2 contains a DNA fragment of about 700 bp, then the insect strain has the chloramphenicol acetyltransferase resistance gene and is a recombinant hybrid tachyzoite.

[0066] 4. After completing step 3, perform single-clone screening using a 96-well plate. The specific steps are as follows: (1) Collect freshly released recombinant mixed tachyzoites and dilute them at a concentration of 1-3 tachyzoites per well. Then, inoculate the tachyzoites into 96-well plates pre-coated with HFF cells and incubate at 37°C for 7 days. Select tachyzoites with only 1 plaque in each well and inoculate them into 24-well plates pre-coated with HFF cells for amplification culture to obtain monoclonal strains.

[0067] (2) After completing step (1), wait for the release of tachyzoites from the monoclonal strain, take 1 / 3 of the tachyzoites to extract genomic DNA as a template, and perform PCR amplification using primer pair 1, primer pair 2, primer pair 3 (composed of primer PCR3-F: 5'-CACCAATCCGGTTCCGTTAC-3' (SEQ ID No. 30) and primer PCR3-R: 5'-CTTGCTGGGTTCGCAGGATG-3' (SEQ ID No. 31)) and primer pair 4 (composed of primer PCR4-F: 5'-GAGACGTGGACCGCGTGATTTC-3' (SEQ ID No. 32) and primer PCR4-R: 5'-GTCAAGACCCAGAGCCTGAC-3' (SEQ ID No. 33)) to obtain PCR amplification product a, PCR amplification product b, PCR amplification product c and PCR amplification product d in sequence.

[0068] Following the steps described above, replace the monoclonal strain with RHΔ ku80 All other steps remain unchanged as a control.

[0069] Following the steps described above, replace the genomic DNA with water, keeping all other steps unchanged, as a blank control.

[0070] (3) After completing step (2), the following determination is made: if PCR amplification product a of a certain monoclonal strain contains a DNA fragment of approximately 500 bp, PCR amplification product b contains a DNA fragment of approximately 700 bp, PCR amplification product c does not contain a DNA fragment of approximately 750 bp, and PCR amplification product d does not contain a DNA fragment of approximately 850 bp, then the monoclonal strain is RHΔ ku80 prk Gene deletion strains, i.e., RHΔ ku80 Δ prk .

[0071] RHΔ ku80 Δ prk The PCR amplification detection results are shown in Figure 1 (Δ) ku80 For RHΔ ku80 Δ prk For RHΔ ku80 Δ prkH2O served as a blank control. PCR1 was performed using primer pair 1, PCR2 using primer pair 2, PCR3 using primer pair 3, and PCR4 using primer pair 4. After identification, RHΔ... ku80 Δ prk To make RHΔ ku80 In the genome prk The gene (nucleotide sequence as shown in SEQ ID No. 35) was replaced with a DNA fragment with a nucleotide sequence as shown in SEQ ID No. 19 to obtain the insect strain.

[0072] RHΔ ku80 Δ prk Continue culturing to T25 culture flasks, and after confirming that there are no errors by following the above steps again, promptly freeze and preserve the culture.

[0073] Example 2, RHΔ ku80 Δ prk In vivo morphology and in vivo toxicity experiments I. RHΔ ku80 Δ prk External appearance Plaque assays are used to comprehensively assess the invasion, proliferation, and escape capabilities of Toxoplasma gondii. The experiment was repeated three times, with the following steps for each repetition: Collection of Toxoplasma gondii strains (RHΔ) ku80 Δ prk or RHΔ ku80 Tachyzoites were counted, and 100 tachyzoites were seeded into 12-well plates covered with HFF cells. After culturing at 37°C for 7 days, the plaques were fixed with 4% paraformaldehyde at room temperature for 10 min, stained with crystal violet for 20 min, and finally washed with PBS to observe the plaque area.

[0074] Test results are shown Figure 2 (Δ) ku80 For RHΔ ku80 Δ prk For RHΔ ku80 Δ prk (1, 2, and 3 are three replicate experiments). The results showed that, compared with the maternal strain (i.e., RHΔ), ku80 Compared to RHΔ ku80 Δ prk The number and area of ​​plaques formed were significantly reduced. This indicates that... prk Gene deletion significantly inhibited the proliferation of Toxoplasma gondii.

[0075] II. RHΔ ku80 Δ prk Virulence (pathogenicity) experiments in mice The experiment was repeated three times, and the average value was taken. Five mice were infected each time. The experimental procedures for each time were as follows: The Toxoplasma gondii strain (RHΔ) was... ku80 Δ prk or RHΔ ku80 The tachyzoites of mice were intraperitoneally inoculated into mice (100 tachyzoites per mouse), and the clinical symptoms, survival rate and relative weight changes of the mice were observed and recorded continuously every day.

[0076] Relative weight change (%) = Current weight / Initial weight × 100% Survival rate (%) = (Number of surviving mice - Initial number of mice) / Initial number of mice × 100% The results showed that RHΔ ku80 The survival rate of infected mice was 0%, while RHΔ ku80 Δ prk The survival rate of infected mice was 80%. Figure 3 In A, Δ ku80 For RHΔ ku80 Δ prk For RHΔ ku80 Δ prk In addition, RHΔ ku80 Δ prk No obvious clinical symptoms were observed in the infected mice, and their body weight showed a continuous increasing trend. Figure 3 B, Δ ku80 For RHΔ ku80 Δ prk For RHΔ ku80 Δ prk ).

[0077] Therefore, RHΔ ku80 Δ prk The virulence (pathogenicity) was significantly reduced, and the survival rate of mice was increased, allowing for intraperitoneal immunization. For intraperitoneal infection, 100 RHΔ [units of something] were used. ku80 Δ prk Tachyzoescent mice died, so the dosage was reduced during subsequent intraperitoneal immunization, specifically using 80 RHΔ ku80 Δ prk Tachyzoites undergo intraperitoneal immunization.

[0078] Example 3, RHΔ ku80 Δ prk Evaluation of the protective effect of attenuated vaccines 1. Six-week-old ICR mice were randomly divided into an immunization group and a control group, and then treated as follows: Immunogroup: using RHΔ ku80 Δ prk Mice were intraperitoneally immunized with tachyzoites (80 RHΔ per mouse). ku80 Δ prkTachyzoites).

[0079] Control group: The difference from the immunization group is that RHΔ ku80 Δ prk Replace the tachyzoites with an equal volume of PBS buffer.

[0080] 2. Blood samples were collected from mice every 7 days before and after immunization via orbital blood collection. The level of Toxoplasma gondii-specific IgG antibodies in the serum of mice after immunization was detected by ELISA.

[0081] Test results are shown Figure 4 The results showed that from week 2 to week 4 post-immunization, the level of Toxoplasma gondii-specific IgG antibodies in the immunized group remained elevated and was significantly higher than that in the control group.

[0082] 3. After completing step 1, 30 days later, mice in the immunized group and the control group were challenged with different virulent strains and different infection doses. Afterwards, the clinical symptoms, relative weight changes and survival rate of the mice were monitored.

[0083] Relative weight change (%) = Current weight / Initial weight × 100% Survival rate (%) = (Number of surviving mice - Initial number of mice) / Initial number of mice × 100% Specifically as follows: (1) RHΔ ku80 Tachyzoite attack ① After completing step 1 for 30 days, randomly group the animals (5 animals per group) and perform the following operations: Immunity + RHΔ ku80 Group: Using RHΔ ku80 The tachyzoites were used to infect the immunized mice with intraperitoneal infection (50 tachyzoites per mouse). Control +RHΔ ku80 Group: Using RHΔ ku80 The tachyzoites were used to infect the control group mice with peritoneal infection (50 tachyzoites per mouse).

[0084] With 50 RHΔ ku80 Tachyzoites were used to induce peritoneal infection in order to establish a highly virulent acute infection model.

[0085] Test results are shown Figure 5 The results showed that the control +RHΔ ku80 All mice in the group died, while the immune + RHΔ group... ku80 All mice in the group survived and continued to gain weight.

[0086] ②30 days after completing step ①, administer immunization + RHΔ ku80 The number of cysts (i.e. brain cysts) in the brain tissue of the mice was counted.

[0087] Some test results can be found Figure 6 A. The results showed that immunity + RHΔ ku80 Only one brain cyst was detected in the brain of one mouse in the group, while no brain cysts were detected in the brains of the other mice.

[0088] ③ Thirty days after completing step ①, extract immunoglobulin + RHΔ. ku80 Genomic DNA from mouse brain homogenates was used as a template for PCR amplification using primer pairs specific to Toxoplasma gondii. If a mouse's PCR amplification product contained a 529 bp target DNA fragment (positive band), it indicated that Toxoplasma gondii was detected in the mouse's brain tissue.

[0089] Following the steps above, administer immunization + RHΔ ku80 The genomic DNA of the mouse brain homogenate was replaced with the genomic DNA of the maternal parasite strain, while all other steps remained unchanged, serving as a positive control.

[0090] Some test results can be found Figure 6 B. The results showed that immunity + RHΔ ku80 Positive bands were detected in all mice in the group.

[0091] The above results indicate that using RHΔ ku80 Δ prk Tachyzoites can effectively resist RhΔ through immunization. ku80 Acute infections caused by lethal doses of highly virulent strains provide good immunoprotection.

[0092] (2) Pru tachyzoites attack insects ① After completing step 1 for 30 days, randomly group the participants and perform the following operations: Immunization + Pru 2×10 3 Group: Immunized mice were intraperitoneally infected with Pru tachyzoites (2000 Pru tachyzoites per mouse).

[0093] Control + Pru 2×10 3 Group: Control mice were intraperitoneally infected with Pru tachyzoites (2000 Pru tachyzoites per mouse).

[0094] Immunization + Pru 5×10 3 Group: Immunized mice were intraperitoneally infected with Pru tachyzoites (5000 Pru tachyzoites per mouse).

[0095] Control + Pru 5×10 3 Group: Control mice were intraperitoneally infected with Pru tachyzoites (5000 Pru tachyzoites per mouse).

[0096] 2000 Pru tachyzoites were used for intraperitoneal infection to establish a moderately virulent, low-dose acute infection model. 5000 Pru tachyzoites were used for intraperitoneal infection to establish a moderately virulent, high-dose acute infection model.

[0097] Test results are shown Figure 7 All mice survived at both infectious doses (see [link to relevant documentation]). Figure 7 (A and C in the middle).

[0098] Control + Pru 2×10 3 Mice in this group showed a continuous decrease in body weight starting from day 12, and clinical signs such as ruffled fur appeared from days 13 to 20. Immunization + Pru 2×10 3 No obvious clinical symptoms were observed in the group of mice, and their body weight continued to increase (see...). Figure 7 (Middle B). Control + Pru 5×10 3 Mice in the group showed symptoms such as weight loss and ruffled fur starting from day 8, while those receiving immunization + Pru 5×10 3 The mice in this group did not show obvious clinical symptoms and their body weight continued to increase (see...). Figure 7 (D).

[0099] ② 30 days after completing step ①, the control group + Pru 5×10 3 Group, Control + Pru 2×10 3 Group, Immunization + Pru 5×10 3 Group and Immunization + Pru 2×10 3 The number of cysts (i.e. brain cysts) in the brain tissue of the mice was counted.

[0100] Test results are shown Figure 8 A and C. The results showed that immunity + Pru 2×10 3 No brain cysts were detected in the control group mice, while the control group + Pru 2×10 3 The average number of brain cysts per mouse in the group was 224; immunization + Pru 5×10 3 No brain cysts were detected in the control group mice, and no cysts were detected in the control group mice with Pru 5×10⁻⁶ mice. 3 The average number of brain cysts per mouse in the group was 216.

[0101] ③ Thirty days after completing step ①, extract control + Pru 5×10 3 Group, Control + Pru 2×10 3 Group, Immunization + Pru 5×10 3 Group and Immunization + Pru 2×10 3Genomic DNA from mouse brain homogenates was used as a template for PCR amplification using primer pairs specific to Toxoplasma gondii. If a mouse's PCR amplification product contained a 529 bp target DNA fragment (positive band), it indicated that Toxoplasma gondii was detected in the mouse's brain tissue.

[0102] Test results are shown Figure 8 In B and D. The results showed that the control + Pru 2×10 3 Groups and controls + Pru 5×10 3 Positive bands were detected in all mice in the group, immunized with Pru 2×10 3 Group and Immunization + Pru 5×10 3 Only some mice showed weak positive bands, while no positive bands were detected in the remaining mice.

[0103] The above results indicate that using RHΔ ku80 Δ prk Immunizing mice with tachyzoites can effectively resist acute infection caused by moderately virulent strains, providing good protection.

[0104] (3) Pru brain cyst attack insect ①Preparation of Pru brain capsule A chronic infection model of Toxoplasma gondii was established in mice via intraperitoneal injection of Pru tachyzoites to prepare brain cysts. The specific steps were as follows: 5-6 week old ICR mice were selected, and each mouse was injected intraperitoneally with 2000 Pru tachyzoites. After inoculation, the mice were routinely fed for approximately 2 months to promote the formation of stable Toxoplasma gondii cysts (i.e., brain cysts) in the brain tissue. The mice were then sacrificed, and the brain tissue was completely removed. The brain tissue was placed in a sterile petri dish, and 1 ml of PBS buffer was added. The tissue was repeatedly broken up using a syringe to prepare a homogenous brain tissue homogenate. The Toxoplasma gondii brain cysts in the brain tissue homogenate were counted using an optical microscope. Finally, the Toxoplasma gondii brain cysts, i.e., Pru brain cysts, were isolated from the brain tissue homogenate.

[0105] ② After completing step 1 for 30 days, randomly group the animals (5 animals per group) and perform the following operations: Immunization + cyst group: Mice in the immunization group were infected by oral gavage with Pru brain cysts (3 Pru brain cysts per mouse).

[0106] Control + cyst group: Control mice were infected by gavage with Pru brain cysts (3 Pru brain cysts per mouse).

[0107] Three Pru brain cysts were infected via oral gavage to establish a chronic infection model.

[0108] Test results are shown Figure 9In the control + cyst group, the mice's body weight continued to decrease from day 6, their fur became ruffled on day 7, and they died on day 14, with a survival rate of 60%; all mice in the immunized + cyst group survived without obvious clinical symptoms, and their body weight continued to increase.

[0109] ③ Thirty days after completing step ②, count the brain cysts in the mouse brain tissue.

[0110] Test results are shown Figure 9 The results showed that the average number of brain cysts per mouse in the control + cyst group was 1586, while the average number of brain cysts per mouse in the immunized + cyst group was significantly reduced to 18.

[0111] The above results indicate that using RHΔ ku80 Δ prk Immunization with tachyzoites can effectively resist chronic infection of Pru brain cysts established via oral administration, and has a good protective effect.

[0112] In summary, the RHΔ constructed in Example 1 ku80 Δ prk The toxicity decreased significantly, RHΔ ku80 Δ prk Immunized mice showed good immunoprotection against acute infection with highly virulent and moderately virulent strains of Toxoplasma gondii, and significantly inhibited brain cyst formation in a chronic infection model. RHΔ ku80 Δ prk It has potential application value as a live attenuated vaccine against Toxoplasma gondii.

[0113] The present invention has been described in detail above. Those skilled in the art will recognize that the invention can be practiced in a wide range of ways with equivalent parameters, concentrations, and conditions without departing from its spirit and scope, and without requiring unnecessary experiments. While specific embodiments have been provided, it should be understood that further modifications can be made to the invention. In summary, according to the principles of the invention, this application is intended to include any changes, uses, or improvements to the invention, including changes made using conventional techniques known in the art that depart from the scope disclosed herein.

Claims

1. A Toxoplasma gondii progesterone-responsive kinase-deficient strain, which is a Toxoplasma gondii RHΔ... ku80 The nucleotide sequence of the strain's genome is shown in SEQ ID No.

35. prk The gene was replaced with a DNA fragment with a nucleotide sequence as shown in SEQ ID No. 19 to obtain the insect strain.

2. The Toxoplasma gondii progesterone-responsive kinase-deficient strain according to claim 1, characterized in that: The Toxoplasma gondii RHΔ ku80 The nucleotide sequence of the strain's genome is shown in SEQ ID No.

35. prk The gene replacement with a DNA fragment with a nucleotide sequence as shown in SEQ ID No. 19 was achieved using CRISPR / Cas9 technology.

3. A vaccine for preventing Toxoplasma gondii infection, comprising a Toxoplasma gondii progesterone-responsive kinase-deficient strain as described in claim 1 or 2.

4. The vaccine according to claim 3, characterized in that: The developmental stage of the Toxoplasma gondii progesterone-responsive kinase-deficient strain is tachyzoite.

5. The use of the Toxoplasma gondii progesterone-responsive kinase-deficient strain according to claim 1 or 2 in the preparation of a vaccine for the prevention of Toxoplasma gondii infection; The developmental stage of the Toxoplasma gondii progesterone-responsive kinase-deficient strain is tachyzoite.

6. The application of the Toxoplasma gondii progesterone-responsive kinase-deficient strain according to claim 1 or 2 in the construction of a mouse model of Toxoplasma gondii infection.

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