Toxoplasma gondii attenuated vaccine strain RHdeltarop67 as well as construction method and application thereof

Abstract

The invention discloses a toxoplasma gondii attenuated vaccine strain RH delta rop67 as well as a construction method and application thereof, and belongs to the technical field of parasitic disease prevention and control and biological product preparation. The attenuated vaccine strain is constructed by performing targeted knockout on the ROP67 gene in a toxoplasma gondii strain RH delta ku80 through a CRISPR / Cas9 mediated gene editing technology. Compared with a wild type strain, the attenuated vaccine strain shows remarkable attenuation characteristic and good immunogenicity. A test result shows that the attenuated vaccine strain can induce a host to generate specific immune response mainly based on cellular immunity, maintains a protection effect on toxoplasma gondii infection in a relatively long immune period, and has a protection effect on tachyzoite infection and a chronic infection stage of toxoplasma gondii strains with different virulence; the survival ability of a host to tachyzoite infection can be improved, and the formation level of cysts in tissues is reduced. The invention provides a technical scheme with long-term immune potential for research and development of toxoplasma gondii attenuated vaccines.

Description

Technical Field

[0001] This invention relates to the field of parasitic disease prevention and control and biological product preparation technology, and in particular to a Toxoplasma gondii attenuated vaccine strain RHΔrop67 and its construction method and application. Background Technology

[0002] Toxoplasma gondii ( Toxoplasma gondii Toxoplasma gondii is a typical obligate intracellular parasitic protozoan that can infect a variety of warm-blooded animals, including humans and many livestock. It exhibits strong host adaptability, diverse transmission routes, and can establish long-term persistent infections within the host. Toxoplasmosis infection has significant impacts on livestock production, reproductive safety, and public health, especially in pregnant women and immunocompromised individuals, where infection is more likely to lead to serious pathological consequences. In most immunocompetent individuals, Toxoplasmosis infection is often latent; however, when the host's immune regulation capacity declines, serious clinical manifestations such as miscarriage, fetal developmental abnormalities, and central nervous system damage can occur, even endangering life. Related studies have shown that Toxoplasma gondii can invade brain tissue and remain dormant for a long time, leading to neurological symptoms such as headaches, drowsiness, and cognitive impairment, posing a persistent threat to the host's health.

[0003] Currently, the prevention and control of toxoplasmosis still relies primarily on drug treatment. However, existing chemical drugs mainly target the tachyzoite stage to inhibit infection, making it difficult to effectively eliminate tissue cysts formed during chronic infection. Furthermore, long-term or repeated use may cause toxic side effects and drug resistance, limiting their application in large-scale control. Therefore, exploring safer and more sustainable control strategies is of significant practical importance.

[0004] Vaccination is widely recognized as one of the effective means of preventing and controlling Toxoplasma gondii infection. In recent years, research on Toxoplasma gondii vaccines has deepened, resulting in the development of various vaccine forms, including nucleic acid vaccines, recombinant protein vaccines, nanodelivery vaccines, vector vaccines, and live attenuated vaccines. Among these, live attenuated vaccines have a significant advantage in inducing systemic immune responses due to their ability to replicate to a limited extent within the host and mimic the natural infection process. Studies have reported that various attenuated Toxoplasma gondii strains can produce certain protective effects in different hosts. For example, some attenuated strains can induce a specific immune response against Toxoplasma gondii, reducing post-infection clinical symptoms or pathogen burden and, to some extent, blocking transmission. However, traditional attenuated strains are mostly obtained through natural mutation or continuous passage, resulting in complex genetic backgrounds and potential risks of insufficient stability and virulence reversion, which affect their safety and widespread application.

[0005] With the analysis of Toxoplasma gondii genome information and the maturation of gene editing technologies such as CRISPR / Cas9, targeted modification of pathogenicity-related genes at the molecular level to construct attenuated strains with clear genetic backgrounds and controllable virulence has gradually become an important direction in Toxoplasma gondii vaccine research. Rod-like proteins play a crucial role in Toxoplasma gondii's invasion of host cells and the regulation of the host's immune response; their loss of function may significantly affect the parasite's pathogenicity and immunogenicity. However, the impact of deletions of different rod-like protein genes on vaccine safety and immunoprotective efficacy still requires further systematic evaluation.

[0006] Therefore, constructing an attenuated Toxoplasma gondii vaccine strain with both good safety and immunoprotective ability based on a specific rod-shaped protein gene deletion strategy has important research value and application prospects for the prevention and control of Toxoplasma gondii infection and the development of novel vaccines. Summary of the Invention

[0007] The purpose of this invention is to provide a Toxoplasma gondii attenuated vaccine strain RHΔrop67, its construction method, and its application, to solve the problems existing in the prior art. Using CRISPR / Cas9-mediated gene editing technology, the rod-shaped protein-related gene ROP67 is knocked out in the Toxoplasma gondii RHΔku80 strain, resulting in the construction of a corresponding gene-deleted Toxoplasma gondii attenuated vaccine strain. This attenuated vaccine strain has a clear genetic background, significantly reduced virulence, and provides immunoprotection against Toxoplasma gondii tachyzoites and cysts. It overcomes the shortcomings of existing attenuated vaccines in terms of safety, stability, and immunogenicity evaluation, providing a reliable experimental basis and technical support for the prevention of Toxoplasma gondii infection and the development of novel vaccines.

[0008] To achieve the above objectives, the present invention provides the following solution: This invention provides a Toxoplasma gondii attenuated vaccine strain RHΔrop67, which is obtained by knocking out the ROP67 gene of the Toxoplasma gondii strain RHΔku80. The accession number of the ROP67 gene in the Toxoplasma gondii genome database TOXODB is TGME49_305590.

[0009] The present invention also provides the application of the aforementioned Toxoplasma gondii attenuated vaccine strain RHΔrop67 in the preparation of a vaccine for the prevention and control of Toxoplasma gondii infection.

[0010] The present invention also provides a vaccine for preventing Toxoplasma gondii infection, comprising the aforementioned Toxoplasma gondii attenuated vaccine strain RHΔrop67.

[0011] The present invention also provides a method for constructing the Toxoplasma gondii attenuated vaccine strain RHΔrop67, which includes the step of knocking out the ROP67 gene of the Toxoplasma gondii strain RHΔku80.

[0012] Preferably, the knockout of the ROP67 gene in the Toxoplasma gondii RHΔku80 strain includes the following steps: Construct a CRISPR / Cas9 knockout plasmid targeting the ROP67 gene; Homologous arms at the 5′ and 3′ ends of the ROP67 gene, the selection marker DHFR fragment, and the pUC19 vector backbone were amplified, digested with enzymes, and ligated to obtain homologous DHFR plasmids; homologous DHFR fragments were amplified using the homologous DHFR plasmids. The CRISPR / Cas9 knockout plasmid and the homologous DHFR fragment were jointly introduced into the Toxoplasma gondii RHΔku80 tachyzoite, and after screening and monoclonalization, the Toxoplasma gondii attenuated vaccine strain RHΔrop67 was obtained.

[0013] Preferably, using pSAG1-Cas9-U6-SgUPRT as a template, PCR amplification is performed using primer pairs as shown in SEQ ID NO.1-2 to replace the SgRNA targeting UPRT in pSAG1-Cas9-U6-SgUPRT with SgRNA targeting ROP67, thereby obtaining a CRISPR / Cas9 knockout plasmid targeting the ROP67 gene.

[0014] Preferably, the nucleotide sequence of the SgRNA targeting ROP67 is shown in SEQ ID NO.3.

[0015] Preferably, the primer sequences for amplifying the 5′ and 3′ homologous arms of the ROP67 gene are shown in SEQ ID NO.4 to SEQ ID NO.7; The primer sequences for amplifying the DHFR selection marker fragment are shown in SEQ ID NO.8 to SEQ ID NO.9; The primer sequences for amplifying the pUC19 vector backbone are shown in SEQ ID NO.10 to SEQ ID NO.11; The primer sequences for amplifying the homologous DHFR fragment are shown in SEQ ID NO.12 to SEQ ID NO.13.

[0016] The present invention discloses the following technical effects: This invention uses the RHΔku80 strain as a background, knocking out the rop67 gene to obtain the Toxoplasma gondii gene-deleted strain RHΔrop67. The RHΔrop67 strain exhibits good attenuation properties and immunogenicity, effectively inducing a specific immune response in the host, primarily cellular immunity. Animal immunization experiments show that this attenuated vaccine strain provides protection against Toxoplasma gondii tachyzoites and cysts after immunization, improves the host's survival against Toxoplasma gondii infections of varying virulentity, and reduces cyst formation levels in tissues during chronic infection stages. This invention has positive significance in the construction of attenuated Toxoplasma gondii vaccines and the evaluation of their immunoprotective effects, providing a reliable experimental basis and technical support for the prevention and control of Toxoplasma gondii infection and the development of novel vaccines. Attached Figure Description

[0017] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0018] Figure 1 This is a schematic diagram of the construction and identification results of the Toxoplasma gondii ROP67 gene deletion strain (RHΔrop67) in Example 1; where A represents a schematic diagram of the gene knockout strategy of the RHΔrop67 strain, and B represents the PCR identification results of the RHΔrop67 strain. Figure 2 The results of the evaluation of the virulence effect of the RHΔrop67 gene-deleted strain in Example 1 on mice are shown; the survival curves of mice infected with different doses of RHΔrop67 tachyzoites and RH wild-type tachyzoites are shown to compare their pathogenicity differences. Figure 3 The results of the protective effect of RHΔrop67 immunization against Toxoplasma gondii infection on day 30 post-immunization in Example 1 are presented. In this case, immunized mice and control mice were infected with different doses of PYS tachyzoites (A, B), RH tachyzoites (C, D), Pru tachyzoites (E), and different numbers of Pru cysts (F, G), respectively. The survival status and the number of cysts in the brain tissue of infected mice (H) were compared. Figure 4 The results of the evaluation of the protective effect of RHΔrop67 immunization against Toxoplasma gondii infection on day 75 after immunization in Example 1 are shown. The immunized mice and the control mice were infected with different doses of PYS tachyzoites (A, B), RH tachyzoites (C, D), Pru tachyzoites (E) and different numbers of Pru cysts (F, G), and their survival was compared. Figure 5The results of the protective effect of RHΔrop67 immunization against Toxoplasma gondii infection on day 120 post-immunization in Example 1 are shown. The immunized mice and control mice were infected with different doses of PYS tachyzoites (A, B), RH tachyzoites (C, D), Pru tachyzoites (E) and different numbers of Pru cysts (F, G), and their survival and the number of cysts in the brain tissue of the infected mice (H) were compared. Figure 6 The results show the detection of the host immune response characteristics induced by RHΔrop67 in Example 1; where A to C represent the levels of anti-Toxoplasma gondii specific IgG, IgG1, and IgG2a antibodies in mouse serum on days 15, 30, 45, 60, and 75 after immunization, respectively; and D represents the comparison results of IgG2a and IgG1 antibody levels. Figure 7 The results show the detection of cytokines such as IL-2 (A), IL-4 (B), IL-10 (C), IL-12 (D), and IFN-γ (E) in the culture supernatant of spleen cells from mice in the immunized group and the control group on days 30, 75, and 120 after immunization. Detailed Implementation

[0019] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0020] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0021] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0022] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be apparent to those skilled in the art. This specification and embodiments are merely exemplary.

[0023] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0024] Example 1 1. Construction of RHΔrop67 1.1 Source of insect strains The Toxoplasma gondii type I RHΔku80 strain was provided and preserved long-term by the Parasitic Diseases Laboratory of Shanxi Agricultural University and used in the experiment of constructing gene-deleted strains in this embodiment.

[0025] 1.2 Construction of gene knockout strains Using CRISPR / Cas9-mediated site-specific genome editing technology, the ROP67 gene was knocked out in the RHΔku80 strain to obtain a strain with the RHΔrop67 gene deletion. The specific operation process is as follows.

[0026] The culture conditions were as follows: the host cell culture medium was DMEM medium supplemented with penicillin (100 μg / mL), streptomycin (100 μg / mL), HEPES (10 mM), and fetal bovine serum (10%). The Toxoplasma gondii culture medium was identical to the host cell culture medium, except that the fetal bovine serum concentration was adjusted to 2%.

[0027] 1.2.1 Construction of CRISPR / Cas9 knockout plasmid targeting ROP67 The gene sequence of ROP67 (gene ID: TGME49_305590) was obtained from the Toxoplasma gondii genome database TOXODB (http: / / www.toxodb.org / toxo / ), and an SgRNA for gene knockout was designed based on it. Its sequence is shown in SEQ ID NO.3.

[0028] Using pSAG1-Cas9-U6-SgUPRT plasmid as a backbone template, PCR amplification was performed using the upstream primer shown in SEQ ID NO.1 and the downstream primer shown in SEQ ID NO.2. The SgRNA targeting UPRT in the original plasmid was replaced with SgRNA targeting ROP67, thereby constructing the CRISPR / Cas9 knockout plasmid pSAG1::Cas9-U6::SgROP67 targeting ROP67.

[0029] The primer sequences used for constructing the knockout plasmid are listed in Table 1.

[0030] Table 1 The PCR amplification reaction system used to construct the CRISPR / Cas9 knockout plasmid targeting ROP67 is shown in Table 2, and the PCR amplification reaction procedure is shown in Table 3.

[0031] Table 2 Table 3 After PCR amplification, the obtained PCR products were processed using the Q5 site-directed mutagenesis kit and digested with DpnI enzyme at 37℃ to remove the template plasmid. The DpnI enzyme digestion reaction system is shown in Table 4.

[0032] Table 4 Subsequently, the product treated with DpnI enzyme was subjected to KLD cyclization reaction at 25℃, and the reaction system is shown in Table 5.

[0033] Table 5 The obtained KLD circularized product was transformed into DH5α competent E. coli. After plating and culturing, single colonies were selected for amplification and culture, and the obtained recombinant plasmids were sequenced for identification. The correctly sequenced CRISPR / Cas9 knockout plasmids were further amplified and then extracted using an endotoxin-free plasmid extraction kit to obtain the ROP67-targeting CRISPR / Cas9 knockout plasmid pSAG1::Cas9-U6::SgROP67, which was then used for further processing.

[0034] 1.2.2 Construction of ROP67 homologous recombination electroporation fragment (DHFR homologous fragment) To obtain the homologous recombination electroporation fragment for ROP67 gene knockout, amplification primers for the 5′ and 3′ homologous arms were designed upstream of the start codon and downstream of the stop codon of the ROP67 gene, respectively. The primer sequences are shown in SEQ ID NO. 4–SEQ ID NO. 7. Simultaneously, primers were designed for amplifying the selection marker gene DHFR fragment (SEQ ID NO. 8–SEQ ID NO. 9), for amplifying the vector backbone pUC19 (SEQ ID NO. 10–SEQ ID NO. 11), and for amplifying the complete homologous DHFR fragment of ROP67 (SEQ ID NO. 12–SEQ ID NO. 13). The primer information is summarized in Table 6.

[0035] The amplification templates for the 5′ and 3′ homologous arms were Toxoplasma gondii genomic DNA, the amplification template for the DHFR fragment was the pUPRT-DHFR-D plasmid, and the amplification template for the pUC19 backbone fragment was the pUC19 plasmid.

[0036] The PCR amplification reaction system for each fragment was consistent with that shown in Table 2. The PCR reaction program used the conditions shown in Table 3. The extension time for amplifying the 5′ and 3′ homologous arms was set to 2 min, and the extension time for amplifying the DHFR fragment and the pUC19 backbone fragment was set to 3 min.

[0037] Table 6 The amplified 5′ homologous arm fragment (40 ng), 3′ homologous arm fragment (40 ng), DHFR fragment (100 ng), and pUC19 backbone fragment (100 ng) were purified by gel extraction, mixed in the above proportions, and subjected to DpnI digestion at 37 °C. The digestion reaction system is shown in Table 4.

[0038] Subsequently, the fragments digested with DpnI were ligated using the ClonExpress MultiS one-step cloning method at 37 °C. The ligation reaction system is shown in Table 7.

[0039] Table 7 The ligation reaction product was transformed into DH5α competent Escherichia coli. After plate culture, single colonies were selected for amplification culture, and the resulting recombinant plasmid was sequenced for identification. Using the homologous DHFR recombinant plasmid with correct sequencing results as a template, the homologous DHFR fragment was amplified by PCR using the primers shown in SEQ ID NO.12 and SEQ ID NO.13. The amplification product was recovered by gel extraction and used for later use.

[0040] 1.2.3 Construction, screening and identification of RHΔrop67 Human foreskin fibroblasts (HFF) were seeded in 25 T cell culture flasks, and 6 mL of DMEM medium containing 10% fetal bovine serum was added. The cells were cultured at 37 °C and 5% CO2. After a dense monolayer of cells formed, the medium was replaced with DMEM medium containing 2% fetal bovine serum, and 1 mL of freshly released RHΔku80 tachyzoites (hereinafter referred to as RH) was added. The cells were cultured for approximately 65 h. After culture, the tachyzoites were collected and purified for subsequent electroporation experiments.

[0041] The extracted CRISPR / Cas9 knockout plasmid pSAG1::Cas9-U6::SgROP67 was mixed with the gel recovery product of the homologous DHFR fragment and, after aseptic treatment, co-transfected into RH tachyzoites. Following electrotransfection, drug screening was performed using pyrimethamine, and positive monoclonal strains were obtained by 96-well plate monoclonalization. The selected monoclonal strains were then expanded and their genomic DNA was extracted. The knockout status of the ROP67 gene was identified using PCR.

[0042] PCR identification specifically includes the following steps: (1) PCR amplification (PCR1) was performed using the primers shown in SEQ ID NO.14 and SEQ ID NO.15 to detect whether the 5′ end of the targeted knockout site was successfully replaced by a homologous DHFR fragment; (2) PCR amplification (PCR2) was performed using the primers shown in SEQ ID NO.16 and SEQ ID NO.17 to detect whether the ROP67 gene was knocked out at the genomic level; (3) PCR amplification (PCR3) was performed using the primers shown in SEQ ID NO.18 and SEQ ID NO.19 to detect whether the 3′ end of the targeted knockout site was successfully replaced by the homologous DHFR fragment.

[0043] The PCR amplification reaction system is shown in Table 8, the reaction procedure is shown in Table 9, and the primer sequences are shown in Table 10.

[0044] Table 8 Table 9 Table 10 2. Evaluation of toxicity in mice (1) Survival assessment of mice infected with RHΔrop67 tachyzoites ① Experimental Animals and Grouping: Six- to eight-week-old SPF-grade female Kunming mice (purchased from Beijing Spefol Biotechnology Co., Ltd.) were selected. The mice were housed in an environment with a temperature of approximately 25°C and a relative humidity of 50%–60%, with free access to food and water. Bedding was changed every 2–3 days, and their health was observed daily, morning and evening. Before the experiment, all mice underwent a 7-day acclimatization period to reduce stress.

[0045] ② Preparation of tachyzoites: RHΔrop67 tachyzoites were diluted to a final concentration of 5 × 10⁻⁶. 2 5 × 10 3 5 × 10 4 5 × 10 5 5 × 10 6 Cells / mL; simultaneously, dilute RH wild-type tachyzoites to a final concentration of 5 × 10⁻⁶. 2 per mL.

[0046] ③ Inoculation of mice: Take 200 μL of the diluted tachyzoite suspension and inoculate mice by intraperitoneal injection. Six mice are inoculated for each strain in each group.

[0047] ④ Observe and record: After infection, observe once in the morning and once in the evening, and continuously record the changes in clinical symptoms and survival status of mice.

[0048] 3. Evaluation of the immunoprotective effect of RHΔrop67 immunization against acute and chronic Toxoplasma gondii infection (30 days) (1) Immunization of mice: After culturing, collecting and counting, the freshly released RHΔrop67 tachyzoites were diluted to 1×10⁻⁶. 4 Immunization of Kunming mice (2 × 10⁶ cells / mL) with more than 200 μL of diluted tachyzoites via intraperitoneal injection (i.e., 2 × 10⁶ cells / mL) 3 Each tachyzoite was counted. The blank control group mice were raised under the same conditions but were not immunized.

[0049] (2) Acute infection in mice: On day 30 post-immunization (or day 30 feeding for the control group), mice were infected with 1 × 10⁻⁶ mg / L of iodine. 2 Or 1 × 10 3 One RH or PYS tachyzoite, and 5 × 10 4 Six mice in each of the immunized and control groups were attacked by intraperitoneal injection of Pru tachyzoites. The disease incidence and survival were observed and recorded twice daily after infection.

[0050] (3) Preparation of Toxoplasma gondii Pru brain cysts: After culturing the Pru strain in HFF cells, collecting and counting the cells, take 2 × 10⁻⁶ cells. 2One Pru tachyzoite was injected intraperitoneally into blank Kunming mice. The health status of the mice was continuously monitored. On days 30, 75 and 120 after RHΔrop67 immunization, brain tissue was collected from chronically infected surviving mice, brain cysts were isolated and counted, and diluted to 10 cysts / 200 μL and 40 cysts / 200 μL, respectively, for subsequent chronic infection experiments.

[0051] (4) Chronic infection in mice: On day 30 post-immunization (or day 30 post-control group), mice in the immunized group and the control group were attacked by gavage with 10 or 40 Pru capsules, respectively, with 8 mice in each group. Clinical manifestations and survival status were observed twice daily during the infection period. On day 35 post-infection, surviving mice were sacrificed and the number of brain tissue capsules was counted.

[0052] 4. Evaluation of the medium- and long-term protective effect of RHΔrop67 immunization against Toxoplasma gondii infection (75 days and 120 days). (1) Acute infection in mice: On day 75 or day 120 post-immunization (or the corresponding time for the control group), mice were fed with 100 or 1000 RH or PYS tachyzoites, respectively, and 5 × 10⁻⁶ spores. 4 Six mice were infected by intraperitoneal injection of Pru tachyzoites. The disease status and survival status of the mice were recorded twice daily after infection.

[0053] (2) Chronic infection in mice: Mice were infected by gavage with 10 or 40 Pru cysts on day 75 or day 120 post-immunization (or the corresponding time for the control group), with 8 mice in each group. Health status was continuously monitored, and brain tissue of surviving mice was collected on day 30 post-infection and the number of cysts was counted.

[0054] 5. Detection of RHΔrop67-induced immune response (1) Serum antibody level detection (15-75 days) Blood samples were collected from mice in the immunized group and the control group on days 15, 30, 45, 60, and 75 post-immunization. After standing at 4°C for 2 hours, the samples were centrifuged at 4000 × g for 10 minutes. Serum was collected and stored at −20°C for later use. The levels of anti-Toxoplasma gondii specific IgG, IgG1, and IgG2a antibodies in the serum were detected using ELISA. The specific procedures are as follows: ① Antigen coating: Add 100 μL (approximately 1 μg / mL) of Toxoplasma gondii-specific soluble antigen to each well of an ELISA plate. Incubate at 37 °C for 1.5–2 h, then store overnight at 4 °C. Wash three times with 0.5% PBST and pat dry before use.

[0055] ② Blocking: Add 100 μL of 5% BSA to each well, incubate at 37 °C for 1–2 h, then wash 3 times with 0.5% PBST and pat dry.

[0056] ③ Incubate with serum: Dilute serum to a ratio of 1:100 with 1% BSA, add 100 μL to each well, incubate at 37 °C for 1–2 h, and then wash 3 times with PBST.

[0057] ④ Incubation with secondary antibody: Dilute the secondary antibody with 1% BSA: Goat anti-mouse IgG1 and IgG2a at a dilution ratio of 1:5000, and IgG (HRP) at a dilution ratio of 1:3000. Add 100 μL to each well and incubate at 37 °C for 1–2 h. Wash three times with PBST.

[0058] ⑤ Termination with colorimetric reagent: Add 100 μL of TMB colorimetric solution to each well. After complete color development, add 100 μL of 2% H2SO4 to terminate the reaction.

[0059] ⑥ Determine OD 450 OD was measured at a wavelength of 450 nm. 450 The values ​​were used to calculate the antibody levels of IgG, IgG1, and IgG2a.

[0060] (2) Spleen cell culture and cytokine detection (30, 75, 120 days) Splenic cell suspensions were prepared from the spleens of mice in the immunized and control groups on days 30, 75, and 120 post-immunization. The spleen lymphocyte count was adjusted to 3 × 10⁻⁶ cells / mL. 6 Cells / mL were seeded in 96-well plates and stimulated with STAg at a final concentration of 10 μg / mL. Culture supernatants were collected at 24 h, 72 h, and 96 h after stimulation to detect the levels of cytokines such as IL-2, IL-4, IL-10, IL-12, and IFN-γ.

[0061] 6. Results and Analysis 6.1 Construction and Phenotypic Analysis of RHΔrop67 To verify the impact of ROP67 gene deletion on the biological characteristics of Toxoplasma gondii, a ROP67 gene deletion strain, RHΔrop67, was successfully constructed in the RHΔku80 background using a CRISPR / Cas9-mediated homologous recombination strategy. Figure 1 As shown in Figure A, this is a schematic diagram illustrating the construction principle of the RHΔrop67 strain; PCR identification results show that the coding sequence of ROP67 was successfully deleted, and the homologous DHFR fragment was accurately inserted into the target gene site. Figure 1 (B). The above results indicate that the ROP67 gene deletion strain RHΔrop67 was successfully constructed and can be used for subsequent functional and immunological evaluation.

[0062] 6.2 Evaluation of RHΔrop67 toxicity To assess the effect of RHΔrop67 gene deletion on Toxoplasma gondii virulence, Kunming mice were intraperitoneally infected with different doses of RHΔrop67 tachyzoites and RH wild-type tachyzoites, and the survival of the mice was continuously monitored. Results are as follows: Figure 2 As shown, mice were injected with 1 × 10 2 1 × 10 3 and 1 × 10 4 All RHΔrop67 tachyzoites maintained a 100% survival rate after injection of 1×10⁶ cells. 5 Only after 100 RHΔrop67 tachyzoescent mice began to die, and the survival rate was 83% after 35 days, with an infection rate of 1 × 10⁻⁶. 6 Even mice infected with tachyzoites showed a 50% survival rate, demonstrating a significantly better survival outcome than the wild-type RH infection group. In contrast, wild-type RH tachyzoites caused rapid death in mice at lower infection doses, specifically 1 × 10⁻⁶. 2 If one RH tachyzoite is produced, all mice will die within 13 days. These results indicate that ROP67 gene deletion significantly reduces the pathogenicity of the RH strain in mice.

[0063] 6.3 Immunization with RHΔrop67 tachyzoites provides protection against both acute and chronic Toxoplasma gondii infection (30 days after immunization). To determine the protective efficacy of the RHΔrop67 vaccine against acute toxoplasmosis infection, on day 30 of immunization, 5 × 10 4 Pru tachyzoites, 1 × 10 2 Or 1 × 10 3 One PYS tachyzoite, 1 × 10 2 Or 1 × 10 3 Mouse control and vaccinated mice were challenged with RH tachyzoites. Results showed that when challenged with different doses of PYS, RH, and Pru tachyzoites, all mice in the control group died within 15 days; mice infected with 1 × 10⁻⁶ RH tachyzoites... 3 The survival rate of mice immunized with RH tachyzoites was 33.33% (2 / 6), but the survival rate of mice infected with 1 × 10⁻⁶ RH tachyzoites was significantly higher. 2 All RH-immunized mice survived. Figure 3 Medium CD); infection 1 × 10 3 The survival rate of mice immunized with 1 × 10 PYS tachyzoites was 50% (3 / 6), while the survival rate of mice infected with 1 × 10 PYS tachyzoites was higher. 2 The survival rate of mice immunized with PYS tachyzoites was 83.33% (5 / 6). Figure 3 (Above AB); In addition, 5 × 10 4 The survival rate of mice immunized with Pru tachyzoites was also 83.33% (5 / 6). Figure 3(Middle E). Therefore, inoculation of mice with RHΔrop67 can provide effective immune protection against acute infection by highly virulent strains RH and PYS, and also provide effective protection against infection caused by the moderately virulent strain Pru tachyzoites.

[0064] To further evaluate the role of RHΔrop67 in preventing chronic toxoplasmosis infection, on day 30 after immunization, immunized mice and control mice were orally administered 10 or 40 cysts. Results showed that all immunized mice infected with 10 or 40 cysts survived, while only 3 mice (3 / 8) of the control group infected with 10 cysts survived (a survival rate of 37.5%), and all mice in the control group infected with 40 cysts died. Figure 3 (FG).

[0065] Cysts in the brain tissue of surviving mice were observed and counted. Two immunized mice infected with 10 cysts had cysts observed, with approximately 300 and 75 cysts respectively. Two immunized mice infected with 40 cysts had cysts observed, with approximately 150 and 100 cysts respectively. Only three control mice infected with 10 cysts survived, with approximately 2587, 1725, and 1800 cysts respectively. The number of brain cysts in the vaccinated group was significantly lower than the brain cyst burden in the unvaccinated group. Figure 3 (H). The results showed that RHΔrop67 had a protective effect against chronic infection in mice and could reduce the brain cyst burden in mice with chronic Toxoplasma gondii infection.

[0066] 6.4 Immunization with RHΔrop67 tachyzoites provides strong immune protection against long-term Toxoplasma gondii infection (75 days and 120 days after immunization). To further investigate whether the RHΔrop67 vaccine can provide long-term protection against toxoplasmosis, mice were intraperitoneally injected with three different virulences (RH, Pru, and PYS) of Toxoplasma gondii on days 75 and 120 after immunization. Simultaneously, mice were administered 10 and 40 cysts via gavage, and the survival status of the mice and the number of cysts in their brain tissue were observed.

[0067] Mice were immunized 75 days later and then treated with 5 × 10 4 One Pru tachyzoite, 1 × 10 2 Or 1 × 10 3 One PYS tachyzoite, 1 × 10 2 Or 1 × 10 3 RH tachyzoites were used to challenge control and vaccinated mice. Results showed that all control mice died within 15 days after being challenged with different doses of PYS, RH, and Pru tachyzoites. Infection rate was 1 × 10⁶. 2 The survival rate of mice immunized with PYS tachyzoites was 66.67% (4 / 6). Figure 4 A), infected 1 × 103 The survival rate of mice immunized with PYS tachyzoites was 50% (3 / 6). Figure 4 (Middle B); infection 1 × 10 2 The sum of 1 × 10 3 The survival rate of immunized mice with RH tachyzoites was 83.33% (5 / 6). Figure 4 (C and D in the middle); infection 5 × 10 4 The survival rate of mice immunized with Pru tachyzoites was 50% (3 / 6). Figure 4 (E).

[0068] To observe whether the RHΔrop67 strain could provide long-term protective immunity against chronic infection, 10 or 40 cysts were administered orally to immunized mice or control mice 75 days post-immunization. Results were as follows: Figure 4 As shown, the survival rate of control mice infected with 40 cysts was 12.5% ​​(1 / 8), and the survival rate of control mice infected with 10 cysts was 25% (2 / 8); while all immunized mice infected with either 10 or 40 cysts survived (8 / 8, 1 / 8). Figure 4 (F and G). Cysts in the brain tissue of surviving mice were observed and counted. The number of brain cysts in the two control mice infected with 10 cysts was 788 and 900, respectively, while the number of cysts in the control mice infected with 40 cysts was 1594. No cysts were observed in the brain tissue of the immunized mice infected with 10 and 40 cysts.

[0069] To observe whether the RHΔrop67 strain could provide longer-lasting protective immunity against infection, mice were challenged 120 days after immunization with different doses of PYS, RH, and Pru tachyzoites. Figure 5 As shown, all mice in the control group died within 15 days, and the infection rate was 1 × 10⁻⁶. 2 The survival rate of mice immunized with PYS tachyzoites was 100% (6 / 6). Figure 5 A), infected 1 × 10 3 One PYS tachyzoite, infected 1 × 10 2 One RH tachyzoite, infected 1 × 10 3 One RH tachyzoite and infection of 5 × 10 4 The survival rate of mice immunized with Pru tachyzoites was 33.3% (2 / 6). Figure 5 (BE).

[0070] Furthermore, chronic infection experiments revealed that 120 days after immunization, the survival rate of control mice infected with 10 cysts was 25% (2 / 8, 1 / 2). Figure 5 In the control group (0 / 8 mice infected with 40 cysts), all mice died (F). Figure 5In mice infected with 10 cysts (G), the survival rate was 87.5% (7 / 8, G). Figure 5 In mice infected with 40 cysts (F), the survival rate was 50% (4 / 8). Figure 5 (G). Cysts in the brain tissue of surviving mice were observed and counted. The two control mice infected with 10 cysts had 750 and 450 brain cysts, respectively. Among the immunized mice infected with 10 cysts, two immunized mice had brain cysts, each with 37.5 cysts. Among the immunized mice infected with 40 cysts, one immunized mouse had brain cysts, with a count of 37.5 cysts. Figure 5 (H).

[0071] 6.5 RHΔrop67 can induce a strong immune response. To investigate the immunological basis of the protective effect of RHΔrop67 immunization, humoral and cellular immune responses in immunized mice were examined at different time points. Serological results showed that at multiple time points after immunization, the levels of IgG, IgG1, and IgG2a in the serum of immunized mice were significantly increased compared with those of control mice. Figure 6 (AC), and the levels of IgG2a antibodies produced by the immunized mice were significantly higher than those of IgG1 ( Figure 6 The result (D) suggests that the immune response is biased towards Th1-type immunity.

[0072] Cytokine assay results showed that at different time points after immunization, the levels of IL-2, IL-10, and IFN-γ, factors related to cellular immunity, were significantly increased in the supernatant of spleen cell culture from immunized mice, while IL-4 and IL-12 did not show significant changes at the assay time points. Figure 7 The above results indicate that RHΔrop67 immunity mainly exerts its protective effect by inducing a predominantly cell-mediated immune response.

[0073] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. A live attenuated Toxoplasma gondii vaccine strain RHΔrop67, characterized in that, The RHΔrop67 gene was obtained by knocking out the ROP67 gene in the Toxoplasma gondii strain RHΔku80. The accession number of the ROP67 gene in the Toxoplasma gondii genome database TOXODB is TGME49_305590.

2. The use of the Toxoplasma gondii attenuated vaccine strain RHΔrop67 as described in claim 1 in the preparation of a vaccine for the prevention and control of Toxoplasma gondii infection.

3. A vaccine for preventing toxoplasmosis infection, characterized in that, Includes the Toxoplasma gondii attenuated vaccine strain RHΔrop67 as described in claim 1.

4. A method for constructing the Toxoplasma gondii attenuated vaccine strain RHΔrop67 as described in claim 1, characterized in that, This includes steps to knock out the ROP67 gene in the Toxoplasma gondii RHΔku80 strain.

5. The construction method as described in claim 4, characterized in that, The knockout of the ROP67 gene in the Toxoplasma gondii RHΔku80 strain includes the following steps: A CRISPR / Cas9 knockout plasmid targeting the ROP67 gene was constructed; the 5′ and 3′ homologous arms of the ROP67 gene, the selection marker DHFR fragment, and the pUC19 vector backbone were amplified, digested with enzymes, and ligated to obtain the homologous DHFR plasmid; the homologous DHFR fragment was amplified using the homologous DHFR plasmid. The CRISPR / Cas9 knockout plasmid and the homologous DHFR fragment were jointly introduced into the Toxoplasma gondii RHΔku80 tachyzoite, and after screening and monoclonalization, the Toxoplasma gondii attenuated vaccine strain RHΔrop67 was obtained.

6. The construction method as described in claim 5, characterized in that, Using pSAG1-Cas9-U6-SgUPRT as a template, PCR amplification was performed using primer pairs as shown in SEQ ID NO.1-2 to replace the SgRNA targeting UPRT in pSAG1-Cas9-U6-SgUPRT with SgRNA targeting ROP67, thus obtaining a CRISPR / Cas9 knockout plasmid targeting the ROP67 gene.

7. The construction method as described in claim 6, characterized in that, The nucleotide sequence of the SgRNA targeting ROP67 is shown in SEQ ID NO.

3.

8. The construction method as described in claim 5, characterized in that, The primer sequences for amplifying the 5′ and 3′ homologous arms of the ROP67 gene are shown in SEQ ID NO.4 to SEQ ID NO.7; The primer sequences for amplifying the DHFR selection marker fragment are shown in SEQ ID NO.8 to SEQ ID NO.9; The primer sequences for amplifying the pUC19 vector backbone are shown in SEQ ID NO.10 to SEQ ID NO.11; The primer sequences for amplifying the homologous DHFR fragment are shown in SEQ ID NO.12 to SEQ ID NO.13.

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