Toxoplasma gondii attenuated strain as well as construction method and application thereof

Abstract

The invention discloses a toxoplasma gondii attenuated strain as well as a construction method and application thereof. The toxoplasma gondii attenuated strain delta MIC1 delta MIC3 disclosed by the invention is obtained by knocking out MIC1 and MIC3 genes from a TIR1 strain by utilizing a CRISPR-Cas9 (clustered regularly interspaced short palindromic repeats-associated 9) technology. The toxoplasma gondii attenuated strain delta MIC1 delta MIC3 can effectively enhance macrophage functions including phagocytic ability, proinflammatory factor secretion and the like, and provides theoretical basis and technical support for clarification of an immunoregulation network of toxoplasma gondii-host interaction and development of vaccine design based on attenuated strains.

Description

Technical Field

[0001] The present invention relates to the field of genetic engineering, and in particular to a toxoplasma gondii attenuated strain and a construction method and application thereof. Background Art

[0002] Toxoplasma gondii is a widespread intracellular parasitic protozoan that can infect all warm-blooded animals, including humans, causing toxoplasmosis. The pathogen infects the host through ingestion of oocysts in feces of cats, consumption of undercooked infected meat, or vertical transmission from mother to child. For individuals with normal immune function, the infection is mostly latent or self-limiting; however, in immunocompromised patients (such as AIDS patients and organ transplant recipients), Toxoplasma can activate latent infection and cause fatal encephalitis. In addition, infection in pregnant women can cause fetal miscarriage, stillbirth, or congenital malformations, causing serious public health problems. Currently, there is no ideal vaccine for toxoplasmosis worldwide. Traditional live attenuated vaccines are difficult to meet clinical needs due to the high risk of reversion to virulence and limited immune protection.

[0003] Macrophages, as core effector cells of the innate immune system, play a key role in resisting Toxoplasma infection. Their functions include phagocytosis of pathogens, secretion of pro-inflammatory cytokines (such as IFN-γ, IL-12, and TNF-α), activation of Th1 immune responses, and promotion of tissue repair. However, Toxoplasma gondii actively suppresses the activation and killing functions of host macrophages by secreting effector proteins (such as the MIC protein family), thereby evading immune clearance. Among them, the micronematic adhesion proteins MIC1 and MIC3 are key molecules for Toxoplasma to invade host cells. They not only mediate the adhesion of the parasite to the host cell membrane but also interfere with host immune signaling pathways, weakening the immune regulatory capacity of macrophages. Therefore, the double knockout strain ΔMIC1ΔMIC3 not only reduces the intracellular parasitic ability of Toxoplasma to weaken its virulence, but also relieves its inhibitory effect on host immune cells, thereby enhancing the anti-infection function of macrophages.

[0004] Although gene editing technologies (such as CRISPR-Cas9) have made some progress in the construction of attenuated strains of Toxoplasma gondii, existing research has mostly focused on single gene knockout or random mutations, with drawbacks such as imprecise virulence regulation and unclear immune regulation mechanisms. For example, while traditional attenuated strains (such as the Δku80 strain) can reduce pathogenicity, their immune activation effect is insufficient, making it difficult to induce long-lasting protective immunity. In addition, some gene editing strategies may destroy essential genes of Toxoplasma gondii, making it difficult to stably propagate the strain. Summary of the Invention

[0005] In view of this, the present invention provides a Toxoplasma gondii attenuated strain, its construction method and application. An attenuated strain with double gene knockout of ΔMIC1ΔMIC3 constructed based on CRISPR-Cas9 technology, by specifically targeting the MIC1 / MIC3 adhesion protein gene, while reducing the invasion ability of the parasite, significantly enhancing the phagocytic activity of macrophages, the secretion of pro-inflammatory factors and the ability to activate Th1-type immune responses, providing a new idea for the development of safe and effective prevention and treatment strategies for toxoplasmosis.

[0006] The technical solution of the present invention is implemented as follows:

[0007] In the first aspect, the present invention provides a Toxoplasma gondii attenuated strain, lacking the MIC1 gene and the MIC3 gene.

[0008] On the basis of the above technical solution, further, the nucleotide sequence of the MIC1 gene is as shown in SEQ ID NO.1, and the amino acid sequence of the MIC1 gene is as shown in SEQ ID NO.2.

[0009] On the basis of the above technical solution, further, the nucleotide sequence of the MIC3 gene is as shown in SEQ ID NO.3, and the amino acid sequence of the MIC3 gene is as shown in SEQ ID NO.4.

[0010] Nucleotide sequence of the MIC1 gene (5'-3') of SEQ ID NO.1:

[0011]

[0012] Amino acid sequence (5'-3') of SEQ ID NO.2 MIC1 gene:

[0013] MGQALFLTVLLPVLFGVGPEAYGEASHSHSPASGRYIQQMLDQRCQEIAAELCQSGLRKMCVPSSRIVARNAVGITHQNTLQWRCFDTASLLESNQENNGVNCVDDCGHTIPCPGGVHRQNSNHATRHEILSKLVEEGVQRFCSPYQASANKYCNDKFPGTIARRSKGFGNNVEVAWRCYEKASLLYSVYAECASNCGTTWYCPGGRRGTSTELDKRHYTEEEGIRQAIGSVDSPCSEVEVCLPKDENPPLCLDESGQISRTGGGPPSQPPEMQQPADRSDERGGGKEQSPGGEAQPDHPTKGGNIDLPEKSTSPEKTPKTEIHGDSTKATLEEGQQLTLTFISTKLDVAVGSCHSLVANFLDGFLKFQTGSNSAFDVVEVEEPAGPAVLTIGLGHKGRLAVVLDYTRLNAALGSAAYVVEDSGCSSSEEVSFQGVGSGATLVVTTLGESPTAVSA.

[0014] Nucleotide sequence (5'-3') of SEQ ID NO.3 MIC3 gene:

[0015]

[0016] Amino acid sequence (5'-3') of SEQ ID NO.4 MIC3 gene:

[0017] MRGGTSALLHALTFSGAVWMCTPAEALPIQKSVQLGSFDKVVPSREVVSESLAPSFAVTETHSSVQSPSKQETQLCAISSEGKPCRNRQLHTDNGYFIGASCPKSACCSKTMCGPGGCGEFCSSNWIFCSSSLIYHPDKSYGGDCSCEKQGHRCDKNAECVENLDAGGGVHCKCKDGFVGTGLTCSEDPCSKRGNAKCGPNGTCIVVDSVSYTCTCGDGETLVNLPEGGQGCKRTGCHAFRENCSPGRCIDDASHENGYTCECPTGYSREVTSKAEESCVEGVEVTLAEKCEKEFGISASSCKCDNGYSGSASATSHHGKGESGSEGSLSEKMNIVFKCPSGYHPRYHAHTVTCEKIKHFALDGAGNHDTTTYVARRRYPASL.

[0018] Weak strain of Toxoplasma gondii:

[0019] RHΔku80Δhxgprt; TUB1:TIR1-3FLAG, SAG1:CAT; ΔMIC3::dhfr-ts:dhfr; ΔMIC1::dhfr-ts:hxgprt (ΔMIC1ΔMIC3). To systematically evaluate its immune regulation efficacy, multi-dimensional analysis techniques such as CCK-8 method, cell scratch assay, Transwell migration model and fluorescence probes (such as DCFH-DA labeling ROS) were used to quantitatively analyze the regulatory effects of this weak strain on macrophage proliferation, migration, chemotaxis, reactive oxygen species, phagocytic function and apoptosis. Further, the mRNA expression levels of inflammatory cytokines IFN-γ, IL-6, IL-1 and INOS were measured by qPCR after the parasites infected macrophages, providing a theoretical basis and technical support for clarifying the immune regulation network of Toxoplasma gondii-host interaction and developing a vaccine design based on the weak strain.

[0020] In a second aspect, the present invention provides a method for constructing the above-mentioned weak strain of Toxoplasma gondii, by knocking out the MIC1 gene and MIC3 gene through CRISPR-Cas9 technology to obtain the weak strain of Toxoplasma gondii.

[0021] On the basis of the above technical solutions, further, it includes the following steps:

[0022] Step S1: Construct the CRISPR-Cas9 knockout plasmids for the MIC1 gene and the MIC3 gene;

[0023] Step S2: Amplify the HXGPRT homologous fragment of the MIC1 gene and the DHFR homologous fragment of the MIC3 gene;

[0024] Step S3: Co-electroporate the knockout plasmid in Step S1, the HXGPRT homologous fragment and the DHFR homologous fragment in Step S2 into the parental Toxoplasma gondii strain, and obtain the attenuated Toxoplasma gondii strain through drug screening and PCR identification.

[0025] Among them, in Step S2, using the PL-AID-6TY-HXGPRT plasmid as a template, the HXGPRT fragment for homologous replacement of the MIC1 gene is obtained by PCR amplification;

[0026] Using the PL-AID-6TY-DHFR plasmid as a template, the DHFR fragment for homologous replacement of the MIC3 gene is obtained by PCR amplification.

[0027] On the basis of the above technical solutions, further, the parental Toxoplasma gondii strain is the Toxoplasma gondii type I strain.

[0028] The Toxoplasma gondii type I strain in this application is the TIR1 strain.

[0029] In the third aspect, the present invention provides an application of the above attenuated Toxoplasma gondii strain in the preparation of a Toxoplasma gondii vaccine.

[0030] In the fourth aspect, the present invention provides a Toxoplasma gondii vaccine.

[0031] Compared with the prior art, the present invention has the following beneficial effects:

[0032] (1) The present invention uses the CRISPR-Cas9 technology, with the TIR1 Toxoplasma gondii as the parental strain, to knockout the MIC1 and MIC3 genes of Toxoplasma gondii, and constructs the ΔMIC1ΔMIC3 strain. This strain can effectively enhance the function of macrophages, including phagocytosis ability and secretion of pro-inflammatory factors such as TNF-α and IL-12 while reducing the invasion ability of the parasite.

[0033] (2) The attenuated Toxoplasma gondii strain of the present invention lays an important foundation for clarifying the immune regulation network of Toxoplasma gondii-host interaction. Description of the Drawings

[0034] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required for the description of the embodiments or the prior art. Obviously, the drawings in the following description are only some embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can be obtained based on these drawings.

[0035] Figure 1 For the construction of the Toxoplasma gondii gene deletion strain ΔMIC1ΔMIC3, where (A) is the schematic diagram of the construction of the ΔMIC1ΔMIC3 strain; (B) is the PCR identification result of ΔMIC1ΔMIC3.

[0036] Figure 2 In (A), the results of CCK-8 determination after Toxoplasma gondii TIR1 and ΔMIC1ΔMIC3 infect macrophages for 0 h, 24 h, and 48 h are shown; in (B) and (C), the results of scratch experiments after Toxoplasma gondii TIR1 and ΔMIC1ΔMIC3 infect macrophages for 0 h, 12 h, and 24 h are shown; in (D) and (E), the results of chemotaxis experiments after Toxoplasma gondii TIR1 and ΔMIC1ΔMIC3 infect macrophages are shown.

[0037] Figure 3 In (A) and (B), the results of the reactive oxygen species (ROS) experiment after Toxoplasma gondii TIR1 and ΔMIC1ΔMIC3 infect macrophages are shown; in (C) and (D), the results of the phagocytosis experiment after Toxoplasma gondii TIR1 and ΔMIC1ΔMIC3 infect macrophages are shown.

[0038] Figure 4 In (A) and (B), the results of the apoptosis experiment after Toxoplasma gondii TIR1 and ΔMIC1ΔMIC3 infect macrophages are shown; in (C), the results of the qPCR experiment for detecting IFN-γ (A), IL-6 (B), IL-1 (C), and INOS (D) after Toxoplasma gondii TIR1 and ΔMIC1ΔMIC3 infect macrophages are shown. Detailed implementation manners

[0039] The following will clearly and completely describe the technical solutions in the embodiments of the present invention in combination with the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, rather than all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts belong to the scope of protection of the present invention.

[0040] Cell culture medium: Add 1% penicillin-streptomycin-amphotericin B solution and 10% fetal bovine serum to DMEM medium respectively.

[0041] Toxoplasma gondii culture medium: The same as the cell culture medium.

[0042] Parasite strain: Toxoplasma gondii type I TIR1 strain, RHΔku80Δhxgprt; TUB1: TIR1-3FLAG, SAG1: CAT.

[0043] Example 1: Construction and phenotypic analysis of Toxoplasma gondii attenuated strain TIR1ΔMIC1ΔMIC3

[0044] 1. Construction of CRISPR-Cas9 knockout plasmids for MIC1 and MIC3

[0045] (1) Design of sgRNAs for MIC1 and MIC3

[0046] Obtain the gene sequences of MIC1 (TGGT1_291890) and MIC3 (TGGT1_319560) using the Toxoplasma gondii whole genome TOXODB website (http: / / www.toxodb.org / toxo / ), and design sgRNAs for the CRISPR-Cas9 knockout plasmids.

[0047] (2) Construction of pSAG1-Cas9-U6-SgMIC1 and pSAG1-Cas9-U6-SgMIC3

[0048] Using pSAG1-Cas9-U6-SgUPRT as a template, construct the MIC1 sgRNA plasmid. Using SEQ ID NO.5 and SEQ ID NO.6 as primers, amplify plasmid backbone fragment 1. Using SEQ ID NO.7 and SEQ ID NO.8 as primers, amplify plasmid backbone fragment 2. Using SEQ ID NO.9 (universal primer), SEQ ID NO.10, and SEQ ID NO.11 as primers, amplify the specific sgRNA gene fragments of the amino terminus and carboxyl terminus of MIC1 by PCR. Use the same method to construct plasmid backbone fragment 1 and plasmid backbone fragment 2 of the MIC3 sgRNA plasmid, and respectively prepare the specific sgRNA gene fragments of the amino terminus and carboxyl terminus of MIC3 using SEQ ID NO.9 (universal primer), SEQ ID NO.12, and SEQ ID NO.13.

[0049] Among them, the primers used for constructing MIC1 and MIC3 are shown in Table 1.

[0050] Table 1

[0051]

[0052] Amplify the target specific plasmid fragments using 2×Phanta Max Master Mix, and configure the system on ice as shown in Table 2.

[0053] Table 2

[0054]

[0055] After the reaction system is configured and fully mixed, react according to the following procedure in Table 3.

[0056] Table 3

[0057]

[0058]

[0059] After the reaction ends, perform gel nucleic acid electrophoresis on the amplification product to verify whether the size of the target band is correct. If the band sizes are all as expected, they can be recovered by gel extraction.

[0060] The specific sgRNA gene fragments of MIC1 or MIC3 are respectively homologous recombined with backbone plasmid fragment 1 and backbone plasmid fragment 2. Configure the ligation system on ice in Table 4.

[0061] Table 4

[0062]

[0063] Gently aspirate and mix the reaction system repeatedly, then centrifuge briefly to settle the liquid to the bottom of the tube. After reacting at 50 °C for 15 min, cool to 4 °C or immediately place on ice.

[0064] (3) Transform the knockout plasmids pSAG1-Cas9-U6-SgMIC1 and pSAG1-Cas9-U6-SgMIC3

[0065] Transform the above products into DH5α competent cells respectively. After spreading on plates, pick single colonies, expand the culture of the single colonies and then sequence. Re-transform the successfully sequenced CRISPR-Cas9 knockout plasmids, expand the culture, and then extract the plasmids using an endotoxin-free plasmid extraction kit for standby.

[0066] 2. Amplification of homologous templates for MIC1 and MIC3

[0067] Using the PL-AID-6TY-HXGPRT plasmid as a template and SEQ ID NO.14 and SEQ ID NO.15 as primers, obtain the HXGPRT fragment for homologous replacement of the MIC1 gene by PCR amplification, and verify it by nucleic acid electrophoresis.

[0068] Using the PL-AID-6TY-DHFR plasmid as a template and SEQ ID NO.16 and SEQ ID NO.17 as primers, obtain the DHFR fragment for homologous replacement of the MIC3 gene by PCR amplification, and verify it by nucleic acid electrophoresis.

[0069] As Figure 1 The electrophoresis results as shown in Figure 1 indicate that the size of the amplified band is consistent with the expected value, and the band is single without any background bands.

[0070] The primer sequences are shown in Table 5.

[0071] Table 5

[0072]

[0073] 3. Construction, screening and identification of ΔMIC1ΔMIC3 parasite strains

[0074] HFF cells were seeded in a T25 cell culture flask and 5 mL of DMEM medium containing 10% FBS was added. The cells were cultured in an incubator at 37°C with 5% CO2. After the cells grew to confluence, the medium was replaced with 5 mL of DMEM medium containing 2% FBS, and 1 mL of freshly released TIR1 tachyzoites was added.

[0075] When the tachyzoite release rate reached 70%-80%, the tachyzoites were collected and purified for later use.

[0076] The extracted CRISPR-Cas9 knockout plasmid, the HXGPRT homologous fragment of the MIC1 gene, and the DHFR homologous fragment of the MIC3 gene were mixed, sterilized, and co-electroporated into TIR1 tachyzoites. After screening with pyrimethamine and monoclonal screening in a 96-well plate, monoclonal parasite strains were obtained.

[0077] After the monoclonal parasite strains were amplified in culture, genomic DNA was extracted for PCR identification. The PCR reaction (PCR1) using primers SEQ ID NO.18 and SEQ ID NO.19 was used to identify whether the 5' end of the targeted knockout MIC1 gene in the monoclonal parasite strain was replaced with the homologous HXGPRT fragment.

[0078] The PCR reaction (PCR2) using primers SEQ ID NO.20 and SEQ ID NO.21 was used to identify whether the 3' end of the targeted knockout MIC1 gene in the monoclonal parasite strain was replaced with the homologous HXGPRT fragment.

[0079] The PCR reaction (PCR3) using primers SEQ ID NO.22 and SEQ ID NO.23 was used to identify whether the MIC1 gene in the monoclonal parasite strain was knocked out.

[0080] Using the same method, the PCR reaction (PCR1) using primers SEQ ID NO.24 and SEQ ID NO.25 was used to identify whether the 5' end of the targeted knockout MIC3 gene in the monoclonal parasite strain was replaced with the homologous DHFR fragment.

[0081] The PCR reaction (PCR2) using primers SEQ ID NO.26 and SEQ ID NO.27 was used to identify whether the 3'-end of the targeted knockout MIC3 gene in the monoclonal parasite strain was replaced with the homologous DHFR fragment;

[0082] The PCR reaction (PCR3) using primers SEQ ID NO.28 and SEQ ID NO.29 was used to identify whether the MIC3 gene in the monoclonal parasite strain was knocked out.

[0083] For ΔMIC1ΔMIC3, the method of direct gene knockout was adopted. Successful identification at the DNA level indicated successful acquisition of the ΔMIC1ΔMIC3-deficient parasite strain.

[0084] The primer sequence list is shown in Table 6.

[0085] Table 6

[0086] Primer Name Primer Sequence (5' to 3') SEQ ID NO.18 TCTACCTCACGTCATCC SEQ ID NO.19 AATGTCCACGTAGTTCGCGC SEQ ID NO.20 TAGGCTCCGACCACGAAG SEQ ID NO.21 TGTTGTAGAGCCGACTGAC SEQ ID NO.22 TGGACAAGGGAACGCAAG SEQ ID NO.23 CTGAGAAGGCGATGGTCT SEQ ID NO.24 AGACCTCACTGTGCACTG SEQ ID NO.25 AATGTCCACGTAGTTCGCGC SEQ ID NO.26 TAGGCTCCGACCACGAAG SEQ ID NO.27 CTTCGGCAGAGATGTCTCA SEQ ID NO.28 CGGTGACTGAGACTCACT SEQ ID NO.29 GAGGTTGCGGAAGCAGAT

[0087] Prepare the following PCR reaction system on ice, as shown in Table 7.

[0088] Table 7

[0089]

[0090] After the reaction system is prepared and thoroughly mixed, react according to the following procedure, as shown in Table 8.

[0091] Table 8

[0092]

[0093] Performance detection

[0094] 1. CCK8 experiment to analyze the effect of the Toxoplasma gondii weak strain ΔMIC1ΔMIC3 on the proliferation ability of macrophages. Set up a PBS group, a TIR1 group, and a ΔMIC1ΔMIC3 group, with 6 - 8 replicates in each group.

[0095] Scrape the RAW264.7 cells in the logarithmic growth phase with a cell scraper, resuspend and count them with DMEM complete medium. Inoculate 100 μL of the cell suspension containing 5000 cells into a 96-well cell plate (add PBS to the edge), shake the cell culture plate up and down and left and right by the cross method, and then place the cell culture plate in an incubator at 37 °C for incubation for 24 h.

[0096] After 24 h, discard the old medium, wash twice with PBS, add 100 μL of the reaction solution (90 μL of cell medium + 10 μL of cck8 reaction solution), place it in the cell culture incubator for culture, and measure its absorbance at 450 nm with an enzyme-linked immunosorbent assay (ELISA) reader after 2 h, recorded as 0 h.

[0097] After measuring the absorbance, wash the cells twice with PBS. Add 5×10 of the corresponding Toxoplasma gondii to each group 4 and set two time points: 24 h and 48 h. At each time point, discard the old medium and replace it with 100 μL of reaction solution. Incubate the cells in a cell culture incubator for 2 h, then measure the absorbance at 450 nm using an ELISA reader. Draw a growth curve based on the test results( Figure 2 , A).

[0098] 2. Analyze the effect of the Toxoplasma gondii weak strain ΔMIC1ΔMIC3 on the migration ability of macrophages by cell scratch assay

[0099] Set up a PBS group, a TIR1 group, and a ΔMIC1ΔMIC3 group, with 3 replicates in each group.

[0100] Scrape the Raw264.7 cells in the logarithmic growth phase with a cell scraper, resuspend them in complete DMEM medium and count them. Seed the macrophages at a density of 1×10 6 cells / mL in a 6-well plate. After shaking evenly by the cross method, incubate the cells at 37 °C and 5% CO2 until the monolayer confluence reaches over 90%. Discard the old medium and wash the cells twice with PBS.

[0101] Use a 200 μL pipette tip to draw a vertical line along the central axis of the bottom of the culture plate along a ruler to form a uniform linear injury area. Gently pipette with PBS to remove the detached cell debris, wash 3 times to thoroughly remove the residual cells, and replace with fresh medium. Take a photo under an inverted microscope and record it as the scratch width at 0 h. After taking the photo, inoculate 1×10 6 Toxoplasma gondii in each group, and take photos again to record the scratch width of the cells at 12 h and 24 h respectively. Use ImageJ software to measure the scratch width and calculate the migration rate.( Figure 2 , B, C)

[0102] 3. Analyze the effect of the Toxoplasma gondii weak strain ΔMIC1ΔMIC3 on the chemotactic ability of macrophages by Transwell assay

[0103] Set up a PBS group, a TIR1 group, and a ΔMIC1ΔMIC3 group, with 3 replicates in each group.

[0104] Scrape the Raw264.7 cells in the logarithmic growth phase with a cell scraper, resuspend them in complete DMEM medium and count them. Seed the macrophages at a density of 1×10 6 cells / mL in a 6-well plate. After shaking evenly by the cross method, incubate the cells at 37 °C and 5% CO2 until the monolayer confluence reaches over 90%. Discard the old medium and wash the cells twice with PBS. Inoculate 1×10 6 Toxoplasma gondii in the TIR1 and ΔMIC1ΔMIC3 groups and incubate for 24 h.

[0105] Place the Transwell chamber flat in a 24-well plate. Add 200 μL of cell suspension with a concentration of 1×10 5 cells / mL corresponding to each group into each Transwell chamber. Add 0.5 μL of chemokine with a concentration of 0.1 μg / μL to the lower chamber and continue culturing in a cell incubator for 48 h.

[0106] After 48 h, take out the chamber, discard the medium in the upper chamber, wash it 3 times with PBS, place it in a new well, add 600 μL of 4% paraformaldehyde to the lower chamber, place it in the dark at room temperature for 1 h, then take out the chamber, wash it 3 times with PBS, add 600 μL of 1% crystal violet staining solution to a new well, and stain for 1 h. Take out the chamber, dip a fluffy cotton swab in PBS, gently wipe off the cells in the upper chamber, invert the lid of the 24-well plate on the microscope stage, then place the chamber on the lid of the 24-well plate, randomly select 5 fields of view to take pictures, and count the cells stained with crystal violet. ( Figure 2 , D, E)

[0107] 4. Detection of ROS after macrophages are infected with the Toxoplasma gondii weak strain ΔMIC1ΔMIC3

[0108] Take Raw264.7 cells at 5×10 5 / mL, inoculate them into a 48-well plate, divide them into a PBS group, a TIR1 group, and a ΔMIC1ΔMIC3 group, with 3 replicates in each group and 200 μL of cell suspension in each well. The TIR1 group and the ΔMIC1ΔMIC3 group are inoculated with 1×10 5 Toxoplasma gondii for 24 h.

[0109] After 24 h, remove the medium, wash the cells twice with HBSS, add the prepared Working solution, and culture in an incubator at 37 °C and 5% CO2 for 30 min.

[0110] Remove the Working solution and wash the cells twice with HBSS.

[0111] Add HBSS again, select 3 fields of view in each well, and take pictures under an inverted microscope at Ex: 488 nm and Em: 500 - 550 nm. ( Figure 3 , A, B)

[0112] 5. Influence of the Toxoplasma gondii weak strain ΔMIC1ΔMIC3 on the phagocytic ability of macrophages after infection

[0113] Set up a PBS group, a TIR1 group, and a ΔMIC1ΔMIC3 group, with 3 replicates in each group.

[0114] Scrape the Raw264.7 cells in the logarithmic growth phase with a cell scraper, resuspend them in complete DMEM medium and count. Inoculate 2 mL of the cell suspension containing 1×10 5 cells into 6-well plates. After shaking the cell culture plates up and down and left and right by the cross method, place the cell culture plates in an incubator at 37°C and 5% CO2 for 24 h. Then, add 1×10 5 Toxoplasma gondii to each group and incubate for 24 h.

[0115] Scrape the cells with a cell scraper and place them in a 2 mL centrifuge tube, centrifuge at 1000 g for 5 min. Discard the supernatant, add 1 mL of PBS, centrifuge at 1000 g for 5 min, and discard the supernatant. Add 200 μL of FITC-dextran under light protection and incubate at 37°C for 30 minutes. Immediately after incubation, add 2 mL of pre-cooled PBS to terminate the reaction, and centrifuge at 1000×g for 5 minutes (4°C) to wash the residual fluorescent substances. Finally, detect the FITC fluorescence intensity by flow cytometry (excitation wavelength 488 nm, emission wavelength 520 nm), and evaluate the phagocytic ability of macrophages by analyzing the mean fluorescence intensity (MFI). ( Figure 3 , C, D)

[0116] 6. Effects of the attenuated Toxoplasma gondii strain ΔMIC1ΔMIC3 on the apoptosis ability of macrophages

[0117] Set up a PBS group, a TIR1 group, and a ΔMIC1ΔMIC3 group, with 3 replicates in each group.

[0118] Scrape the Raw264.7 cells in the logarithmic growth phase with a cell scraper, resuspend them in complete DMEM medium and count. Inoculate 2 mL of the cell suspension containing 1×10 6 cells into 6-well plates. After shaking the cell culture plates up and down and left and right by the cross method, place the cell culture plates in an incubator at 37°C and 5% CO2 for 24 h. Then, add 1×10 6 Toxoplasma gondii corresponding to each well and continue to culture for 24 hours to complete the infection.

[0119] After the infection is completed, collect the cells with a cell scraper and transfer them to a centrifuge tube, centrifuge at 1000×g for 5 minutes (4°C), discard the supernatant, and add 1 mL of pre-cooled PBS to wash twice (repeat the centrifugation conditions). After discarding the supernatant, add 195 μL of Annexin V-FITC binding solution to gently resuspend the cells, add 5 μL of Annexin V-FITC and 10 μL of PI staining solution in sequence, and incubate at room temperature for 15 minutes under light protection, gently resuspending the cells every 5 minutes during this period to promote uniform staining.

[0120] After the staining was completed, 1 mL of pre-cooled PBS was added to terminate the reaction. The supernatant was discarded after centrifugation at 1000×g for 5 minutes (4 °C). Finally, 200 μL of flow buffer (PBS containing calcium ions) was added to resuspend the cells, which were stored in the dark and analyzed by flow cytometry within 1 hour.

[0121] The dual-channel fluorescence signals of Annexin V-FITC (excitation wavelength 488 nm, emission wavelength 520 nm) and PI (excitation wavelength 488 nm, emission wavelength 620 nm) were analyzed by flow cytometry to calculate early apoptosis (Annexin V + / PI - ), late apoptosis (Annexin V + / PI + ), and necrosis (Annexin V - / PI + ). ( Figure 4 , A, B)

[0122] 7. Effects of IFN-γ, IL-6, IL-1, and INOS after infection of macrophages with the attenuated Toxoplasma gondii strain ΔMIC1ΔMIC3 were determined by qPCR

[0123] The PBS group, TIR1 group, and ΔMIC1ΔMIC3 group were set up, with 3 replicates in each group.

[0124] Raw264.7 cells in the logarithmic growth phase were scraped off with a cell scraper, resuspended and counted in DMEM complete medium. A cell suspension containing 2.5×10 6 cells in 2 mL was inoculated into 6-well plates. After shaking the plates up and down and left and right by the cross method, the cell culture plates were placed in an incubator at 37 °C and 5% CO2 for 24 h, and then the corresponding groups were inoculated with Toxoplasma gondii at a ratio of 1:0.5 and incubated for 24 h.

[0125] After 24 h, the old medium was discarded, and the cells were washed twice with PBS. Then, RNA was extracted according to the instructions of the RNA extraction kit. cDNA was synthesized by reverse transcription.

[0126] A standard curve was constructed by diluting the cDNA template. The primer amplification efficiency (slope method) was calculated with the logarithm of the template concentration as the abscissa and the fluorescence threshold cycle number (Ct value) as the ordinate, and the primer combination with the primer amplification efficiency meeting the requirements was screened.

[0127] The qPCR reaction was performed using the SYBR Green method, with cDNA as the template, β-actin as the internal reference gene, SEQ ID NO.30 as the primer to amplify IFN-γ, SEQ ID NO.31 as the primer to amplify IL-6, SEQ ID NO.32 as the primer to amplify IL-1β, and SEQ ID NO.33 as the primer to amplify INOS. The average Ct value and ΔCt value (ΔCt = Ct target gene - Ct internal reference gene) of the target genes (IFN-γ, IL-6, IL-1β, and INOS) were calculated.

[0128] By comparing the differences in ΔCt values among groups, the 2^(-ΔΔCt) method was used to quantitatively analyze the relative gene expression levels. ( Figure 4 , C) where the reaction program is shown in Table 9; the primer sequences are shown in Table 10.

[0129] Table 9

[0130]

[0131] Table 10

[0132]

[0133] 8. Result description

[0134] (1) Figure 2 The comparison results of the proliferation, migration, and chemotaxis abilities of macrophages after infection with Toxoplasma gondii TIR1 and ΔMIC1ΔMIC3 are shown.

[0135] 1) CCK-8 assay ( Figure 2 A): The growth curve was plotted by measuring the absorbance at 450 nm of each well at different time points. The results showed that compared with the PBS group, the proliferation abilities of macrophages in the TIR1 group and the ΔMIC1ΔMIC3 group changed at 24 h and 48 h after infection. It may be that Toxoplasma gondii infection stimulated or inhibited the proliferation of macrophages. The difference between the ΔMIC1ΔMIC3 group and the TIR1 group indicated that the double gene knockout affected the regulation of Toxoplasma gondii on macrophage proliferation. Specifically, the different trends of the two curves reflected that after knocking out the MIC1 and MIC3 genes, the effect of Toxoplasma gondii on macrophage proliferation was different from that of the wild-type TIR1 strain.

[0136] 2) Cell scratch assay ( Figure 2B, C): Using the scratch width at 0 h as a control, the migration rates at 12 h and 24 h were calculated. The results showed that macrophages in both the TIR1 group and the ΔMIC1ΔMIC3 group migrated after infection, and the change in migration ability of the ΔMIC1ΔMIC3 group was different from that of the TIR1 group. It is possible that knocking out the MIC1 and MIC3 genes altered the interaction mode between Toxoplasma gondii and macrophages, affected the signal pathways related to macrophage migration, resulting in a difference in the migration rate of macrophages between the ΔMIC1ΔMIC3 group and the TIR1 group, reflecting the unique regulatory effect of double gene knockout on macrophage migration ability.

[0137] 3) Transwell chemotaxis assay ( Figure 2 D, E): The chemotactic ability of macrophages was evaluated by counting the number of cells stained with crystal violet. The results showed that the number of macrophages migrating to the lower chamber in the ΔMIC1ΔMIC3 group and the TIR1 group was different under the action of chemokines. This indicates that knocking out the MIC1 and MIC3 genes affected the chemotactic ability of macrophages after Toxoplasma gondii infection, possibly by changing the expression of chemokine receptors on the macrophage surface or its response mechanism to chemokines, resulting in a difference in chemotactic ability between the ΔMIC1ΔMIC3 group and the TIR1 group.

[0138] (2) Figure 3 Presented the results of Toxoplasma gondii TIR1 and ΔMIC1ΔMIC3 infecting macrophages in terms of reactive oxygen species (ROS) production and phagocytic ability.

[0139] 1) ROS detection experiment ( Figure 3 A, B): Photographs were taken at specific excitation and emission wavelengths under an inverted microscope to observe the intracellular ROS level. The results showed that there were differences in the ROS levels produced by macrophages in the ΔMIC1ΔMIC3 group and the TIR1 group after infection. This indicates that knocking out the MIC1 and MIC3 genes affected the production of ROS in macrophages after Toxoplasma gondii infection, possibly by changing the intracellular redox-related signal pathways, making the ROS production mechanism of macrophages in the ΔMIC1ΔMIC3 group different from that of the TIR1 group when responding to Toxoplasma gondii infection, thus resulting in different ROS levels.

[0140] 2) Phagocytic ability detection experiment ( Figure 3C, D): The phagocytic ability of macrophages was evaluated by detecting the FITC fluorescence intensity through flow cytometry. The results showed that the mean fluorescence intensity (MFI) of macrophages in the ΔMIC1ΔMIC3 group was different from that in the TIR1 group, indicating a difference in phagocytic ability between the two groups. Knocking out the MIC1 and MIC3 genes altered the invasion and immune escape modes of Toxoplasma gondii, affected the recognition and phagocytosis processes of macrophages, and made the phagocytic ability of macrophages in the ΔMIC1ΔMIC3 group different from that in the TIR1 group, reflecting the regulatory effect of double gene knockout on the phagocytic function of macrophages.

[0141] (3) Figure 4 Shows the results of Toxoplasma gondii TIR1 and ΔMIC1ΔMIC3 infecting macrophages in terms of apoptosis ability and inflammatory cytokine expression.

[0142] 1), Apoptosis detection experiment ( Figure 4 A, B): Annexin V-FITC and PI dual-channel fluorescence signals were analyzed by flow cytometry, and the proportions of early apoptosis, late apoptosis, and necrosis were calculated. The results showed that the apoptosis of macrophages in the ΔMIC1ΔMIC3 group and the TIR1 group was different after infection. Knocking out the MIC1 and MIC3 genes affected the process of Toxoplasma gondii-induced macrophage apoptosis, possibly by altering the apoptosis-related signaling pathway, resulting in a difference in the apoptosis proportion of macrophages in the ΔMIC1ΔMIC3 group compared with the TIR1 group, indicating that the regulatory effect of double gene knockout on macrophage apoptosis is different from that of the wild-type strain.

[0143] 2), qPCR detection of inflammatory cytokines ( Figure 4 C): SYBR Green method was used for qPCR reaction, and the average Ct value and ΔCt value of the target genes (IFN-γ, IL-6, IL-1β, and INOS) were calculated. The relative gene expression levels were quantitatively analyzed by the 2^(-ΔΔCt) method. The results showed that there were differences in the mRNA expression levels of IFN-γ, IL-6, IL-1β, and INOS after Toxoplasma gondii-infected macrophages in the ΔMIC1ΔMIC3 group and the TIR1 group. Knocking out the MIC1 and MIC3 genes affected the expression regulation of inflammatory cytokines after Toxoplasma gondii-infected macrophages, possibly by altering the activity of related transcription factors or signaling pathways, resulting in differences in the expression of inflammatory cytokines between the ΔMIC1ΔMIC3 group and the TIR1 group, reflecting the impact of double gene knockout on the immune regulatory function of macrophages.

[0144] The above is only the preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A Toxoplasma gondii attenuated strain, characterized in that, The MIC1 gene and the MIC3 gene are missing.

2. The attenuated strain of Toxoplasma gondii according to claim 1, characterized in that, The nucleotide sequence of the MIC1 gene is as shown in SEQ ID NO.1, and the amino acid sequence of the MIC1 gene is as shown in SEQ ID NO.

2.

3. The Toxoplasma gondii attenuated strain according to claim 1, characterized in that, The nucleotide sequence of the MIC3 gene is as shown in SEQ ID NO.3, and the amino acid sequence of the MIC3 gene is as shown in SEQ ID NO.

4.

4. The method for constructing a Toxoplasma gondii attenuated strain according to any one of claims 1 to 3, characterized in that, The MIC1 gene and the MIC3 gene are knocked out by CRISPR-Cas9 technology to obtain the attenuated Toxoplasma gondii strain.

5. The method for constructing a Toxoplasma gondii attenuated strain according to claim 4, characterized in that, It includes the following steps: Step S1: Construct a CRISPR-Cas9 knockout plasmid for the MIC1 gene and the MIC3 gene; Step S2: Amplify the HXGPRT homologous fragment of the MIC1 gene and amplify the DHFR homologous fragment of the MIC3 gene; Step S3: Co-electroporate the knockout plasmid in Step S1, the HXGPRT homologous fragment and the DHFR homologous fragment in Step S2 into the parental Toxoplasma gondii strain, and after drug screening and PCR identification, obtain the attenuated Toxoplasma gondii strain.

6. The method for constructing the attenuated strain of Toxoplasma gondii according to claim 5, wherein The parental Toxoplasma gondii strain is a Toxoplasma gondii type I strain.

7. Use of the attenuated Toxoplasma gondii strain according to any one of claims 1 to 3 in the preparation of a Toxoplasma gondii vaccine.

8. A Toxoplasma gondii vaccine, characterized in that, Containing the attenuated Toxoplasma gondii strain according to any one of claims 1 to 3.

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