Recombinant toxoplasma gondii vaccine having undergone gene deletion and construction method therefor

Abstract

Belonging to the field of genetic engineering, a recombinant Toxoplasma gondii vaccine having undergone gene deletion and a construction method therefor are provided. Specifically, a recombinant Toxoplasma gondii strain having undergone gene deletion, a medicament and / or biological product containing same, a construction method, and a use thereof are provided. A live attenuated toxoplasmosis vaccine developed on the basis of the recombinant Toxoplasma gondii strain is suitable for immunoprophylaxis of toxoplasmosis in various mammals, can block transmission of toxoplasmosis to humans and other animals, and is particularly suitable for immunoprophylaxis of feline Toxoplasma gondii infection and / or toxoplasmosis. The provided vaccine is easy to produce, is high in safety, has excellent efficacy, has long immune duration, is capable of distinguishing vaccine immunization from natural infection, does not harm animals or the environment, is capable of effectively reducing residual Toxoplasma oocysts in the environment, and is capable of reducing the risk of human infection with toxoplasmosis.

Description

A gene-deleted recombinant Toxoplasma gondii strain vaccine and its construction method Technical Field

[0001] This disclosure belongs to the field of genetic engineering and relates to a gene-deleted recombinant Toxoplasma gondii strain vaccine and its construction method. Specifically, it discloses a gene-deleted recombinant Toxoplasma gondii strain, a drug and / or biological product containing the strain, a construction method, and its application. This disclosure provides a recombinant Toxoplasma gondii strain with gene deletions for Toxoplasma gondii cyst formation, schizogenesis, and sexual (gamete) reproduction, a construction method, and its application in drugs or biological products. Background Technology

[0002] Companion animals are closely intertwined with human life and significantly influence human happiness. Globally, dogs and cats are the most popular companion animals. Reports indicate that in 2022, the number of dogs and cats in China exceeded 100 million, with cats surpassing dogs to become the most common companion animal. With closer contact between companion animals and humans, the risk of zoonotic disease transmission from these animals is amplified. Among the zoonotic pathogens carried by cats, Toxoplasma gondii is the most dangerous. For example, Toxoplasma gondii infection can cause miscarriage in pregnant women, blindness in the fetus, and intellectual disabilities. Activation of Toxoplasma gondii cysts can lead to disability and death in individuals with weakened immune systems. In developed countries such as Europe and the United States, the disease is widespread. Reports indicate that the Toxoplasma gondii antibody positivity rate in the Chinese population is nearly 20%. Therefore, the prevention and treatment of zoonotic diseases are crucial to national health and well-being, and have significant public health implications.

[0003] The life cycle of Toxoplasma gondii consists of two stages: schizogony and sexual (gametosis) reproduction in the definitive host, the cat, and asexual reproduction in the intermediate host. The asexual reproduction stage includes the tachyzoite stage during acute infection and the bradyzoite / cyst stage during chronic infection. The cat's stages include schizogony, gametosis, oocyst formation, and sporulation in the environment after the oocyst is expelled from the cat. Pseudocysts containing tachyzoites, cysts containing bradyzoites, and oocysts containing sporozoites are infectious to both the intermediate and definitive hosts. After tachyzoites, bradyzoites, or sporozoites infect the definitive host, the cat, the invasion process is the same as that of the intermediate host. Asexual reproduction occurs within the epithelial cells of the small intestine mucosa of felines, forming merozoites. After several generations of merozoan proliferation, some merozoites develop into female gametes, and some into male gametes. The female and male gametes combine for fertilization to form a zygote, which finally develops into an oocyst and is excreted in feces. Under suitable environmental conditions, the oocyst matures in 2-5 days. Mature oocysts are infectious and are the main source of toxoplasmosis infection in humans and other intermediate hosts. Therefore, the key to controlling toxoplasmosis lies in controlling feline toxoplasmosis. Controlling the release of oocysts from cats into the external environment is the key to blocking the transmission of toxoplasmosis and is a source-based control technology.

[0004] Based on the characteristics of Toxoplasma gondii's reproduction and development in the definitive and intermediate hosts, the development of feline toxoplasmosis vaccines must simultaneously meet two objectives: (1) Provide immune protection against sexual reproduction after immunization, that is, after a cat is reinfected with Toxoplasma gondii, it will not be able to excrete oocysts, thus ensuring safety for humans and the environment; (2) Provide immune protection against the development of tachyzoites into bradyzoites (cysts) after immunization, that is, reduce or block cyst formation. Summary of the Invention

[0005] To address the current state of the technology and the aforementioned technical challenges, the inventors of this disclosure combine CRISPR / Cas9 technology with a conditional knockout system as an effective strategy. Specifically, they develop vaccine components based on live parasite parts to overcome the technical limitations in the safety of live toxoplasmosis vaccines, providing a toxoplasmosis gene-deleted vaccine, its construction method, and its applications. Specifically, this disclosure provides a recombinant Toxoplasma gondii strain with developmental defects in Toxoplasma gondii cysts, schizogony, and sexual (gamete) reproduction, an attenuated live vaccine, a method for constructing the recombinant Toxoplasma gondii strain, and its uses. The vaccine is suitable for immunization against toxoplasmosis in various mammals, especially feline toxoplasmosis, and can effectively block the transmission of toxoplasmosis to humans and other animals.

[0006] The first aspect of this disclosure provides a gene-deleted recombinant Toxoplasma gondii strain, comprising:

[0007] Cre enzymes are specifically expressed only during the cyst, schizogony, and / or gametosis stages; and

[0008] LoxP sequences are sequentially inserted upstream, downstream, or internally of developmental essential genes, key genes regulating cyst formation, and key genes regulating late schizogenes and / or gamete reproduction to knock out at least one of the aforementioned developmental essential genes, key genes regulating cyst formation, late schizogenes genes, and key genes regulating gamete reproduction, thereby obtaining a genetically recombinant Toxoplasma gondii strain.

[0009] Furthermore, knock out two or more of the aforementioned developmental essential genes, key genes regulating cyst formation, late schizogenesis genes, and key genes regulating gamete reproduction and development.

[0010] Further, the *Toxoplasma gondii* strain exhibits developmental defects in cyst formation, late schizogony, and / or gamete reproduction. Preferably, the genetically recombinant *Toxoplasma gondii* strain exhibits cyst developmental defects; preferably, the *Toxoplasma gondii* strain exhibits late schizogony developmental defects; preferably, the *Toxoplasma gondii* strain exhibits gamete reproduction developmental defects; more preferably, the *Toxoplasma gondii* strain exhibits defects in both cyst formation and late schizogony developmental defects; or it exhibits defects in both cyst formation and gamete reproduction developmental defects, or it exhibits defects in both late schizogony and gamete reproduction developmental defects. More preferably, the *Toxoplasma gondii* strain exhibits defects in cyst formation, late schizogony developmental defects, and gamete reproduction developmental defects.

[0011] The target gene is any combination of essential genes that are continuously expressed at different life stages of Toxoplasma gondii and stage-specific regulatory genes.

[0012] Preferably, the target gene is a combination of at least two genes selected from essential genes for life stage, AP2 transcription factor family genes, CST family genes, OWP-like genes, eIF4, AMA1, IMP1, BAG1, mNG, ENO1, LDH2, and genes specifically expressed during fission and sexual reproduction stages.

[0013] Preferably, the target gene is selected from AP2IX-5, AP2XII-2, AP2X-4, AP2XII-1, AP2XI-4, AP2IV-4, AP2IV-3, AP2XII-1, AP2IX-1, AP2XI-2, AP2IX-9, AP2IX-6, AP2III-4, SAG4, SAG5, AMA1, CDPK1, CDPK2, CDPK3, CDPK5, HSP40, HSP60, ROP1, ROP2, One or more of the following: ROP5, ROP7, ROP8, ROP9, ROP13, ROP16, ROP17, ROP18, ROP21, ROP35, ROP38, ROP54, MIC1, MIC2, MIC3, MIC4, MIC6, MIC8, MIC11, MIC13, HSP40, HSP60, HSP70, OWP1, OWP2, IMP1, BAG1, BFD1, eIF4A, MYR1, HDAC, and LDH2.

[0014] The second aspect of this disclosure provides a live attenuated vaccine for the prevention of Toxoplasma gondii infection and / or toxoplasmosis, the active ingredient of which includes at least the recombinant Toxoplasma gondii strain described in any one of the preceding claims; preferably, the vaccine is a monovalent vaccine or a multivalent vaccine; more preferably, the multivalent vaccine comprises the recombinant Toxoplasma gondii strain described in any one of the preceding claims, and further comprises other isolated Toxoplasma gondii isolates that do not possess cross-immunoprotective efficacy.

[0015] A third aspect of this disclosure provides a method for constructing a gene-deleted recombinant Toxoplasma gondii strain, comprising the steps of:

[0016] S1. Constructing recombinant insect strains that specifically express Cre enzymes during cyst formation, schizogony, and / or gamete reproduction stages, including:

[0017] Construct shuttle transfection vectors with dual or multiple expression frames, which include promoters that express genes using the characteristics of cyst, schizogenesis and / or gamete reproduction stages, sequentially linked to the gene sequence of Cre enzyme, the 3' non-transcribed region of Toxoplasma gondii, and homologous arms introduced at both ends.

[0018] The shuttle transfection vector was targeted to the location of or near the first target gene, thereby constructing the recombinant Toxoplasma gondii strain ToxoBJ-cre expressing Cre enzyme;

[0019] S2. Using ToxoBJ-cre strain as the target, two LoxP sequences in the same direction were sequentially inserted into the second target gene or its locus upstream, downstream or in the middle position to knock out the second target gene and construct a gene-deleted recombinant Toxoplasma gondii strain.

[0020] Further, in step S1, the first target gene is the uracil phosphoribosyltransferase homologue gene UPRT or other non-essential genes;

[0021] Further, in step S2, the second target gene to be knocked out is at least one gene selected from essential genes that are continuously expressed in different life stages of Toxoplasma gondii and regulatory genes that are expressed in stages specifically.

[0022] Preferably, the second target gene is at least one of the developmental essential genes, key genes regulating cyst formation, late schizogenes genes, and key genes regulating gamete reproduction and development; more preferably, the second target gene is two or more of the developmental essential genes, key genes regulating cyst formation, late schizogenes genes, and key genes regulating gamete reproduction and development.

[0023] Preferably, the second target gene is one or more genes selected from essential genes for life cycle stages, AP2 transcription factor family genes, CST family genes, OWP-like genes, eIF4, AMA1, IMP1, BAG1, mNG, ENO1, LDH2, and genes specifically expressed during fission and sexual reproduction stages.

[0024] Preferably, the second target gene is selected from AP2IX-5, AP2XII-2, AP2X-4, AP2XII-1, AP2XI-4, AP2IV-4, AP2IV-3, AP2XII-1, AP2IX-1, AP2XI-2, AP2IX-9, AP2IX-6, AP2III-4, SAG4, SAG5, AMA1, CDPK1, CDPK2, CDPK3, CDPK5, HSP40, HSP60, ROP1, ROP2, ROP5, ROP7, ROP8, R OP9, ROP13, ROP16, ROP17, ROP18, ROP21, ROP35, ROP38, ROP54, MIC1, MIC2, MIC3, MIC4, MIC6, MIC8, MIC11, MIC13, HSP40, HSP60, HSP70, OWP1, OWP2, IMP1, BAG1, BFD1, eIF4A, MYR1, HDAC, LDH2, derived from one or more genes among developmentally essential genes, cyst formation-related genes, and fission and gamete reproduction-related genes.

[0025] Preferably, the promoters of genes specifically expressed during the cyst, schizogenesis, and / or gamete reproduction stages include promoters of some genes from the AP2 transcription factor family, promoters of some genes from the BAG family, promoters of some genes from the OWP class, promoters of genes from the CST family, promoters of genes from the AMA family, and promoters of SRS22A, SAG4, mNG, PMA1, BSR4, CST1, ENO1, LDH2, MIC12, MIC13, MIC17A, MIC17C, MIC12, MIC13, MIC17A, MIC17C, GRA4, GRA6, GRA82, GRA11b, GRA80, ROP4, ROP7, MCP4, BPK1, SRS9, SRS22b, GRA11B, and GEX1.

[0026] Preferably, the recombinant Toxoplasma gondii strains include the Tg-ΔBFD1 / OWP1 strain and the Tg-ΔLDH2 / AP2XII-1 strain.

[0027] The fourth aspect of this disclosure provides the use of the recombinant Toxoplasma gondii strain described in any of the preceding claims in the preparation of a vaccine for the prevention of toxoplasmosis in mammals.

[0028] The fifth aspect of this disclosure provides the use of the recombinant Toxoplasma gondii strain described in any of the preceding claims in the preparation of medicaments and / or biological products for the prevention, mitigation and / or treatment of toxoplasmosis in mammals, wherein the effective active component of the medicaments and / or biological products comprises at least the recombinant Toxoplasma gondii strain described in any of the preceding claims.

[0029] The product is a vaccine and may include a combination of multiple insect strains. Alternatively, the product may be a nutritional composition suitable for mammals, such as a health food for pets.

[0030] The sixth aspect of this disclosure provides a medicament and / or biological product for the prevention, mitigation, and / or treatment of toxoplasmosis in mammals, wherein the medicament / biological product comprises an effective dose of the recombinant Toxoplasma gondii strain.

[0031] Preferably, the biological product is a vaccine.

[0032] The seventh aspect of this disclosure provides a method for preventing, mitigating, and / or treating toxoplasmosis in mammals, comprising at least administering an effective dose of a recombinant Toxoplasma gondii strain to a subject in need.

[0033] Preferably, in the method for preventing toxoplasmosis in mammals, the effective dose is 10. 2 ~10 8 Each time, one tachyzoite or bradyzoite of the aforementioned recombinant Toxoplasma gondii strain is administered; more preferably, the effective dose is 10... 7 ~10 8 Or 10 5 ~10 6 Or 10 4 ~10 5 Or 10 3 ~10 4 Or 10 2 ~10 3 One tachyzoite or bradyzoite per animal per dose; more preferably, the effective dose is 10 2 ~10 7 Or 10 3 ~10 8 Or 10 4 ~10 7 Or 10 5 ~10 8 Or 10 5 ~10 7 Each tachyzoite or bradyzoite per animal at a time.

[0034] Preferably, the route of administration includes intramuscular injection, subcutaneous injection, or oral administration.

[0035] Preferably, the route of administration is via vaccination.

[0036] The method includes a single immunization and / or a booster immunization; preferably, a booster immunization is performed 30 to 90 days after the first immunization, wherein the booster immunization is an administration of an effective dose to the subject more than once. Beneficial effects

[0037] The incomplete development of genetically modified Toxoplasma gondii strains in mammals, particularly cats, can meet all the needs for developing vaccines against feline toxoplasmosis and / or toxoplasmosis. The combination of CRISPR / Cas9 technology and conditional knockout systems provides a feasible technical strategy for creating novel Toxoplasma gondii vaccines.

[0038] Experimental results show that the developed feline toxoplasmosis vaccine reduces the morbidity and mortality rates of animals infected with toxoplasmosis, and can also effectively reduce the risk of toxoplasmosis transmission to humans, making it particularly suitable for the immunization and prevention of feline toxoplasmosis.

[0039] This invention provides a novel design strategy and target gene combination strategy for a gene-deleted attenuated live parasite vaccine for the prevention of feline toxoplasmosis. Under the guidance of this strategy, the development of a feline toxoplasmosis vaccine can ensure the safety and immunogenicity of the feline toxoplasmosis vaccine.

[0040] The feline toxoplasmosis gene-deleted vaccine provided by this invention can prevent the formation of oocysts, or the excreted oocysts are minimal and have no infectious function, thus exhibiting high safety and immunogenicity.

[0041] The attenuated toxoplasmosis vaccine developed based on the recombinant Toxoplasma gondii strain is suitable for the immunization prevention of toxoplasmosis in various mammals, and can block the transmission of toxoplasmosis to humans and other animals, especially suitable for the immunization prevention of feline toxoplasmosis. The vaccine and / or products described in this invention are easy to produce, highly safe, and have excellent efficacy. The vaccine provides a long duration of immunity, can distinguish between vaccine-induced immunity and natural infection, and does not pollute animals or the environment. It can effectively reduce the residue of Toxoplasma gondii oocysts in the environment, reduce the risk of human toxoplasmosis infection, and has great application potential and important public health significance. Attached Figure Description

[0042] Figure 1 illustrates a shuttle transfection vector with a dual expression frame constructed according to the present invention. Homologous arms are introduced at both ends, and the shuttle transfection vector is targeted to the UPRT gene site of *Toxoplasma gondii* using CRISPR / Cas9 technology. This results in the construction of the recombinant *Toxoplasma gondii* strain ToxoBJ-Cre expressing the Cre enzyme, where the promoters of the SRS22A and HAP2 genes regulate the specific expression of the Cre enzyme during the cyst and gamete reproductive stages.

[0043] Figure 2 shows the identification results of recombinant Toxoplasma gondii (ToxoBJ-Cre) expressing Cre enzyme. Among them:

[0044] Figure 2A shows the identification of transcriptional levels in the ToxoBJ-Cre strain after continuous subculturing, with identification performed every 5 generations. Primers were used to amplify partial fragments of the Cre enzyme gene, and the results indicate that the expression of the Cre enzyme is genetically stable.

[0045] Figure 2B shows the determination of the reproductive capacity of the parent strain (ToxoBJ) and the recombinant strain (ToxoBJ-Cre strain) by plaque assay. Plaque-forming unit analysis was performed 24 hours after inoculation, and the results showed no significant difference between the two strains.

[0046] Figure 2C shows the survival curves of mice with different intraperitoneal inoculation doses (50, 500, and 5000 tachyzoites / mouse) for the parent strain (ToxoBJ strain) and the recombinant strain (ToxoBJ-Cre strain), indicating that there is no significant difference in virulence between the two strains in mice, and that genetic manipulation did not enhance pathogenicity in mice.

[0047] Figure 2D shows the expression of Cre enzyme in the tachyzoites, cysts, merozoites, gametes, and oocysts of the recombinant strain (ToxoBJ-Cre strain) using immunoblotting. The results indicate that Cre enzyme is specifically expressed only in the cyst and gamete stages.

[0048] Figures 2E-2F show the expression of Cre enzyme in different developmental stages of the recombinant strain (ToxoBJ-Cre) cysts and definitive hosts, as identified by indirect immunofluorescence. The results indicate that Cre enzyme is specifically expressed in the cyst stage (mouse brain tissue smear staining, Figure 2E) and the gamete stage (cat intestinal tissue membrane staining, Figure 2F).

[0049] Figure 3 illustrates the principle of constructing the Tg-ΔBFD1 / OWP1 strain based on the ToxoBJ-cre strain according to the present invention. Using the ToxoBJ-cre strain as the parent strain, LoxP sequences were introduced flanking the TgBFD1 and TgOWP1 gene loci using CRISPR / Cas9 technology and a homologous recombination strategy. The transfected strains underwent dual drug screening with pyrimethamine and chloramphenicol. After continuous passage and single-clone isolation and identification, the Tg-ΔBFD1 / OWP1 strain was obtained.

[0050] Figure 4 shows the identification results of the Tg-ΔBFD1 / OWP1 strain. Among them:

[0051] Figure 4A shows the results of PCR identification of the Tg-ΔBFD1 / OWP1 monoclonal strain, with the four genomic locations identified by PCR amplification marked in Figure 3. The monoclonal strain was re-identified after 40 consecutive in vitro passages, and its genomic traits remained stable.

[0052] Figure 4B shows the reproductive capacity determination of the Tg-ΔBFD1 / OWP1 monoclonal strain and the parent strain ToxoBJ-cre. Plaque-forming unit analysis was performed 24 hours after inoculation, and the results showed no significant difference between the two strains.

[0053] Figure 4C shows the survival curves of mice with different intraperitoneal inoculation doses (50, 500, and 5000 tachyzoites / mouse) of the parent strain (ToxoBJ-cre strain) and Tg-ΔBFD1 / OWP1 tachyzoites. The results indicate that there is no significant difference in virulence between the two strains in mice, and genetic manipulation did not enhance the pathogenicity of tachyzoites in mice.

[0054] Figure 5 illustrates the principle of constructing the Tg-ΔLDH2 / AP2XII-1 strain based on the ToxoBJ-cre strain according to the present invention. The ToxoBJ-cre strain is used as the parent strain. Using CRISPR / Cas9 technology and homologous recombination strategy, LoxP sequences are introduced on both sides of the gene loci of TgLDH2 and TgAP2XII-1. The transfected strains are screened with pyrimethamine and chloramphenicol. After continuous passage and single-clone isolation and identification, the Tg-ΔLDH2 / AP2XII-1 strain is obtained.

[0055] Figure 6 shows the identification results of the Tg-ΔLDH2 / AP2XII-1 strain. Among them:

[0056] Figure 6A shows the results of PCR identification of the Tg-ΔLDH2 / AP2XII-1 monoclonal strain, with the four genomic locations identified by PCR amplification marked in Figure 5. After 40 consecutive in vitro passages, the monoclonal strain was re-identified, and its genomic traits remained stable.

[0057] Figure 6B shows the reproductive capacity determination of the Tg-ΔLDH2 / AP2XII-1 monoclonal strain and the parent strain ToxoBJ-cre. Plaque-forming unit analysis was performed 24 hours after inoculation, and the results showed no significant difference between the two strains.

[0058] Figure 6C shows the survival curves of mice with different intraperitoneal inoculation doses (50, 500, and 5000 tachyzoites / mouse) of the parent strain (ToxoBJ-cre strain) and Tg-ΔLDH2 / AP2XII-1 tachyzoites. The results indicate that there is no significant difference in virulence between the two strains in mice, and genetic manipulation did not enhance the pathogenicity of tachyzoites in mice. Detailed Implementation

[0059] To more clearly illustrate the technical solutions in the embodiments of this disclosure or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only one embodiment of this disclosure. For those skilled in the art, other embodiments can be obtained based on these drawings.

[0060] definition

[0061] In this disclosure, unless otherwise stated, scientific and technical terms and abbreviations used herein have meanings commonly understood by those skilled in the art. Furthermore, the terms and laboratory procedures related to protein and nucleic acid chemistry, molecular biology, cell and tissue culture, microbiology, and immunology used herein are widely used terms and routine procedures in their respective fields. To better understand the content of this disclosure, definitions and explanations of relevant terms are provided below.

[0062] As used herein, the terms “a” and “an” as well as “the” and similar pronouns indicate singular and plural, unless otherwise specified herein or the context clearly contradicts them.

[0063] As used herein, the terms “about,” “substantially,” and “similar to” mean within an acceptable range of error for a particular value as determined by one of ordinary skill in the art, the range of error being partly dependent on how the value is measured or determined, or on the limitations of the measurement system.

[0064] As used herein and unless otherwise stated, the term “about” or “approximately” means within 10% of a given value or range, such as 1%, 2%, 5%, 9%, 10%, etc. Where an integer is required, the term means within 10% of a given value or range, rounded up or down to the nearest integer.

[0065] As used herein, the term “and / or” should be understood to include combinations of preceding and following options as well as any one option.

[0066] As used in this article, "and any combination thereof" refers to any one of the options, or any combination of two or more of them.

[0067] As used in this article, the term "CRISPR / Cas9 technology" refers to a tool optimized with Toxoplasma gondii-related regulatory elements that can be used for precise editing of Toxoplasma gondii genes. Specifically, the promoters responsible for expressing the Cas9 endonuclease are the promoters of Toxoplasma gondii genes such as tublin, SAG1, and actin, while the promoters responsible for expressing the guide RNA are the Toxoplasma gondii U6 promoter.

[0068] As used in this article, the term "conditional knockout system" refers to a genetic manipulation technique that knocks out Toxoplasma gondii genes under specific conditions and life stages. The conditional knockout technique used in this article is mainly based on the Cre-loxP system, but other genetic manipulation techniques that can achieve the above purpose are also applicable.

[0069] As used in this article, the terms “target gene” or “gene target” or “gene of interest” refer to genes, functional domains or regulatory sequences related to the growth and development of Toxoplasma gondii. In this article, it mainly refers to the coding regions or functional domains or regulatory regions of essential genes or key regulatory genes in the growth and development of Toxoplasma gondii.

[0070] As used in this article, the term "developmental essential genes" refers to a class of genes that are involved in the development of a certain life cycle stage of Toxoplasma gondii, such as the tachyzoite, bradyzoite, schizont, and gamete. The absence of these genes prevents Toxoplasma gondii from completing one or more of the above life cycle stages, resulting in developmental defects.

[0071] As used in this article, the term "life-stage essential genes" refers to a class of genes that participate in all life-stage development of Toxoplasma gondii, such as tachyzoites, bradyzoites, schizonts, and gametes, or conserved genes in the genetic evolution of Toxoplasma gondii. The absence of these genes prevents Toxoplasma gondii from completing all life-stage development, resulting in developmental defects.

[0072] The first aspect of this disclosure provides a gene-deleted recombinant Toxoplasma gondii strain, comprising:

[0073] Cre enzymes are specifically expressed only during the cyst, schizogony, and / or gametosis stages; and

[0074] LoxP sequences were sequentially inserted upstream, downstream, or internally of developmental essential genes, key genes regulating cyst formation, and key genes regulating schizogenes and / or gamete reproduction to knock out at least one of the following genes: developmental essential genes, key genes regulating cyst formation, late schizogenes genes, and key genes regulating gamete reproduction, thereby obtaining a genetically recombinant Toxoplasma gondii strain.

[0075] In some implementation schemes, two or more genes are knocked out, including developmental essential genes, key genes regulating cyst formation, late schizogenesis genes, and key genes regulating gamete reproduction and development.

[0076] In some embodiments, the *Toxoplasma gondii* strain exhibits defects in cyst development, late schizogony, and / or gamete development. Preferably, the recombinant *Toxoplasma gondii* strain exhibits cyst development defects; preferably, the *Toxoplasma gondii* strain exhibits late schizogony development defects; preferably, the *Toxoplasma gondii* strain exhibits gamete development defects; more preferably, the *Toxoplasma gondii* strain exhibits defects in both cyst and late schizogony development; or it exhibits defects in both cyst and gamete development, or it exhibits defects in both late schizogony and gamete development. More preferably, the *Toxoplasma gondii* strain exhibits defects in cyst, late schizogony, and gamete development.

[0077] In some embodiments, the target gene is any combination of essential genes that are continuously expressed at different life stages of Toxoplasma gondii and stage-specific regulatory genes.

[0078] In a preferred embodiment, the target gene is a combination of at least two genes selected from essential genes for life stage, AP2 transcription factor family genes, CST family genes, OWP-like genes, eIF4, AMA1, IMP1, BAG1, mNG, ENO1, LDH2, and genes specifically expressed during fission and sexual reproduction stages.

[0079] In a preferred embodiment, the target gene is selected from AP2IX-5, AP2XII-2, AP2X-4, AP2XII-1, AP2XI-4, AP2IV-4, AP2IV-3, AP2XII-1, AP2IX-1, AP2XI-2, AP2IX-9, AP2IX-6, AP2III-4, SAG4, SAG5, AMA1, CDPK1, CDPK2, CDPK3, CDPK5, HSP40, HSP60, ROP1, RO One or more of the following: P2, ROP5, ROP7, ROP8, ROP9, ROP13, ROP16, ROP17, ROP18, ROP21, ROP35, ROP38, ROP54, MIC1, MIC2, MIC3, MIC4, MIC6, MIC8, MIC11, MIC13, HSP40, HSP60, HSP70, OWP1, OWP2, IMP1, BAG1, BFD1, eIF4A, MYR1, HDAC, and LDH2.

[0080] A second aspect of this disclosure provides a live attenuated vaccine for the prevention of toxoplasmosis, the active ingredient of which includes at least the recombinant Toxoplasma gondii strain described in any one of the preceding embodiments. In a preferred embodiment, the vaccine is a monovalent or multivalent vaccine; in a preferred embodiment, the multivalent vaccine comprises the recombinant Toxoplasma gondii strain described in any one of the preceding embodiments, and further comprises antigens from other Toxoplasma gondii isolates that do not possess cross-immunoprotective efficacy.

[0081] A third aspect of this disclosure provides a method for constructing a gene-deleted recombinant Toxoplasma gondii strain, comprising the steps of:

[0082] S1. Constructing recombinant insect strains that specifically express Cre enzymes during cyst formation, schizogony, and / or gamete reproduction stages, including:

[0083] Construct shuttle transfection vectors with dual or multiple expression frames, which include promoters that express genes using the characteristics of cyst, schizogenesis and / or gamete reproduction stages, sequentially linked to the gene sequence of Cre enzyme, the 3' non-transcribed region of Toxoplasma gondii, and homologous arms introduced at both ends.

[0084] The shuttle transfection vector was targeted to the location of or near the first target gene, thereby constructing the recombinant Toxoplasma gondii strain ToxoBJ-cre expressing Cre enzyme;

[0085] S2. Using ToxoBJ-cre strain as the target, two LoxP sequences in the same direction were sequentially inserted into the second target gene or its locus upstream, downstream or in the middle position to knock out the second target gene and construct a gene-deleted recombinant Toxoplasma gondii strain.

[0086] In some implementations, in step S1, the first target gene is the uracil phosphoribosyltransferase homologue gene UPRT or other non-essential genes;

[0087] In some implementations, in step S2, the second target gene to be knocked out is at least one gene selected from essential genes that are continuously expressed at different life stages of Toxoplasma gondii and stage-specific regulatory genes.

[0088] In a preferred embodiment, the second target gene is at least one of the following: developmentally essential genes, key genes regulating cyst formation, late schizogony genes, and key genes regulating gamete reproduction and development. In a preferred embodiment, the second target gene is two or more of the following: developmentally essential genes, key genes regulating cyst formation, late schizogony genes, and key genes regulating gamete reproduction and development.

[0089] In a preferred embodiment, the second target gene is one or more genes selected from essential genes for life stage, AP2 transcription factor family genes, CST family genes, OWP-like genes, eIF4, AMA1, IMP1, BAG1, mNG, ENO1, LDH2, and genes specifically expressed during fission and sexual reproduction stages.

[0090] In a preferred embodiment, the second target gene is selected from AP2IX-5, AP2XII-2, AP2X-4, AP2XII-1, AP2XI-4, AP2IV-4, AP2IV-3, AP2XII-1, AP2IX-1, AP2XI-2, AP2IX-9, AP2IX-6, AP2III-4, SAG4, SAG5, AMA1, CDPK1, CDPK2, CDPK3, CDPK5, HSP40, HSP60, ROP1, ROP2, ROP5, ROP7, and ROP8. ROP9, ROP13, ROP16, ROP17, ROP18, ROP21, ROP35, ROP38, ROP54, MIC1, MIC2, MIC3, MIC4, MIC6, MIC8, MIC11, MIC13, HSP40, HSP60, HSP70, OWP1, OWP2, IMP1, BAG1, BFD1, eIF4A, MYR1, HDAC, LDH2, derived from one or more genes among developmentally essential genes, cyst formation-related genes, and fission and gamete reproduction-related genes.

[0091] In a preferred embodiment, the promoters of genes specifically expressed during the cyst, schizogenesis, and / or gamete reproduction stages include promoters of some genes from the AP2 transcription factor family, promoters of some genes from the BAG family, promoters of some genes from the OWP class, promoters of genes from the CST family, promoters of genes from the AMA family, and promoters of SRS22A, SAG4, mNG, PMA1, BSR4, CST1, ENO1, LDH2, MIC12, MIC13, MIC17A, MIC17C, MIC12, MIC13, MIC17A, MIC17C, GRA4, GRA6, GRA82, GRA11b, GRA80, ROP4, ROP7, MCP4, BPK1, SRS9, SRS22b, GRA11B, and GEX1.

[0092] In a preferred embodiment, the recombinant Toxoplasma gondii strains include the Tg-ΔBFD1 / OWP1 strain and the Tg-ΔLDH2 / AP2XII-1 strain.

[0093] The fourth aspect of this disclosure provides the use of the recombinant Toxoplasma gondii strain described in any of the preceding claims in the preparation of a vaccine for the prevention of toxoplasmosis in mammals.

[0094] The fifth aspect of this disclosure provides the use of the recombinant Toxoplasma gondii strain described in any of the preceding claims in the preparation of medicaments and / or biological products for the prevention, mitigation and / or treatment of toxoplasmosis in mammals, wherein the effective active component of the medicament and / or biological product comprises at least the recombinant Toxoplasma gondii strain described in any of the preceding claims.

[0095] In some embodiments, the article is a vaccine and may include a combination of multiple insect strains. Alternatively, the article is a nutritional composition suitable for mammals, such as a health food for pets.

[0096] The sixth aspect of this disclosure provides a medicament and / or biological product for the prevention, mitigation and / or treatment of toxoplasmosis in mammals, wherein the medicament / biological product comprises an effective dose of a recombinant Toxoplasma gondii strain.

[0097] In a preferred embodiment, the biological product is a vaccine.

[0098] The seventh aspect of this disclosure provides a method for preventing, mitigating, and / or treating toxoplasmosis in mammals, comprising at least administering an effective dose of a recombinant Toxoplasma gondii strain to a subject in need.

[0099] In some implementations, the effective dose in methods for preventing toxoplasmosis in mammals is 10. 2 ~10 8 Each recombinant Toxoplasma gondii strain is administered with tachyzoites or bradyzoites per insect, in a preferred embodiment, the effective dose being 10 [units of something unspecified]. 7 ~10 8 Or 10 5 ~10 6 Or 10 4 ~10 5 Or 10 3 ~10 3 Or 10 2 ~10 3 One tachyzoite or bradyzoite per animal per dose. In a preferred embodiment, the effective dose is 10 2 ~10 7 Or 10 3 ~10 8 Or 10 4 ~10 7 Or 10 5 ~10 8 Or 10 5 ~10 7 Each tachyzoite or bradyzoite per animal at a time.

[0100] In some implementations, the route of administration includes intramuscular injection, subcutaneous injection, or oral administration.

[0101] In a preferred embodiment, the route of administration is vaccination.

[0102] This method includes a single immunization and / or a booster immunization. In a preferred embodiment, a booster immunization is performed 30–90 days after the first immunization, wherein the booster immunization involves administering an effective dose to the subject at least once.

[0103] Example 1. Construction of a recombinant Toxoplasma gondii strain specifically expressing Cre enzyme during cyst and gamete reproductive stages.

[0104] Taking a recombinant Toxoplasma gondii strain that uses the SRS22A gene promoter (sequence shown in SEQ ID NO.1) and the HAP2 gene promoter (sequence shown in SEQ ID NO.2) to regulate the specific expression of Cre enzyme in the cyst and gamete reproductive stages as an example, the promoter was amplified from the Toxoplasma gondii genome and sequentially linked to the Cre enzyme gene (SEQ ID NO.3), supplemented with the 3' non-transcribed region of the Toxoplasma gondii gene, to construct a shuttle transfection vector with a double expression frame, and homologous arms were introduced at both ends (as shown in Figure 1).

[0105] Experiment 1.1 Construction of the shuttle vehicle

[0106] (1) PCR amplification of each element

[0107] Using Q5 high-fidelity DNA polymerase, the SRS22A promoter, HAP2 promoter, Cre enzyme sequence, and vector backbone were amplified from the Toxoplasma gondii genome, Lenti-sgCrebbp#1 / Cre plasmid (Addgene, sequence number: #193209), and UPRT-mCh-Nluc-P2A-neo-UPRT (Addgene, sequence number: #135015) plasmids using the primers shown in Table 1. The PCR amplification systems are shown in Table 2.

[0108] Table 1. Primers

[0109] Table 2. PCR amplification system

[0110] Set the PCR amplification reaction conditions according to the procedure shown in Table 3.

[0111] Table 3. Reaction conditions for PCR amplification

[0112] PCR products were subjected to gel electrophoresis, and the target fragment was recovered by gel excision.

[0113] (2) Multi-segment connection

[0114] The concentration of recovered DNA fragments was determined, and multiple fragments were ligated using a multi-fragment ligation kit (ClonExpress MultiS One Step Cloning Kit, Beijing TransGen Biotech Co., Ltd.). The optimal amount of each fragment was [0.02 × number of fragment base pairs] ng (0.03 pmol). The general system for multi-fragment ligation is shown in Table 4 below.

[0115] Table 4. Connection Reaction System

[0116] Ligation conditions: React at 37℃ for 30 min. Transform the ligation product into competent *E. coli* cells and incubate on ice for 30 min; after heat shock in a 42℃ metal bath for 90 s, immediately cool on ice for 2-3 min. Add 600 μl of antibiotic-free LB medium and incubate at 37℃ for 1 h. Spread the medium onto LB agar plates containing ampicillin resistance and incubate overnight. Pick cells for single cloning.

[0117] (3) Identification and sequencing

[0118] Bacterial culture PCR identification. The reaction system for bacterial culture PCR identification is shown in Table 5 below.

[0119] Table 5. Bacterial PCR Reaction System

[0120] The general conditions for bacterial culture PCR identification reaction are shown in Table 6 below.

[0121] Table 6. PCR reaction conditions for bacterial culture

[0122] PCR products were subjected to gel electrophoresis to identify positive clones. Positive clones were used as templates for small-scale plasmid extraction, following the prescribed steps. Follow the instructions for the Plasmid MiniPrep Kit.

[0123] (3) Amplification of donor fragments

[0124] The donor fragments were amplified using primers shown in Table 7 and a miniature plasmid template.

[0125] Table 7. Primers

[0126] Using CRISPR / Cas9 technology, the gene was targeted to the UPRT gene locus of Toxoplasma gondii (sequence information can be found on the ToxoDB website, https: / / toxodb.org / toxo / app / record / gene / TGME49_312480) to construct a recombinant Toxoplasma gondii strain expressing Cre enzyme.

[0127] 1.0×10 7One Toxoplasma gondii tachyzoite was resuspended in 100 μl of nuclear transfer buffer. Donor fragments and pSAG1::CAS9-U6::sgUPRT plasmid (Addgene, sequence number: #54467) were added at a ratio of 1:5, with a total amount of approximately 10 μg. The mixture was quickly added to an electroporation cuvette, and the transfection instrument was turned on. Program U-033 was selected for transfection. After nuclear transfer, 1 ml of DMEM medium was quickly added to the electroporation cuvette, and after thorough mixing, the medium was transferred to a 1.5 ml centrifuge tube. 200 μl of the transfected Toxoplasma gondii tachyzoite solution was added to pre-cultured HFF cells. After 24 hours, the bioluminescence of the parasites was observed, and fluorouracil (FUDR) was added to the medium for selection. After three generations of selection, Toxoplasma gondii strains expressing Cre enzyme were obtained.

[0128] Performance Qualification and Analysis in Experiment Example 1.2

[0129] Furthermore, the Toxoplasma gondii strain expressing Cre enzyme was purified, identified, and its genetic stability, Cre enzyme expression characteristics, biological and pathogenicity were analyzed.

[0130] (1) Monoclonal purification and identification of recombinant Toxoplasma gondii strains

[0131] Recombinant Toxoplasma gondii was counted and diluted to contain 2-3 tachyzoites per 10 μl, and then inoculated into pre-cultured HFF cells (96-well plate), 10 μl per well. Plaque formation was observed in each well 7-10 days post-inoculation. Wells with single plaques were selected for digestion and amplification culture to 24-well plates. After tachyzoite release, half of the tachyzoites were used for PCR identification, and the remaining tachyzoites were amplified.

[0132] (2) Genetic stability analysis

[0133] The correctly identified monoclonal strains were continuously passaged in HFF cells, and PCR was performed every 5 generations to determine the genetic stability of Cre enzyme gene integration.

[0134] (3) Immunoblotting (WB) and indirect immunofluorescence assay (IFA) were used to analyze the expression characteristics of Cre enzyme.

[0135] Recombinant Toxoplasma gondii tachyzoites were inoculated intraperitoneally (50 tachyzoites / mouse). Brain cysts were collected 30 days later. The brain cysts were then orally inoculated into three 6-month-old kittens (1000 cysts / cat). One kitten was euthanized and one was dissected 7 and 10 days after inoculation. The kitten's intestines were collected, and merozoites and gametes were extracted and divided into two parts. Western blot (WB) and in vitro absorptive blotting (IFA) tests were performed on the merozoites and gametes, respectively. The feces of the remaining kitten were collected for 7-10 days, and oocysts were collected using the saturated saline method for WB verification.

[0136] Western blot analysis (WB) at different life stages was performed as follows: Freshly collected tachyzoites, cysts, merozoites, gametes, and oocysts were resuspended in 50 μl PBS, and 15 μl 5×SDS loading buffer was added. The samples were then boiled at 100°C for 10 min. A suitable amount of sample was subjected to SDS-PAGE. After electrophoresis, the protein was transferred to an NC membrane using a wet transfer method. The membrane was blocked with 5% skim milk at 37°C for 1 h. After blocking, the membrane was incubated with primary antibody (Anti-Cre recombinase antibody (ab216262)) and secondary antibody (ab216262) respectively. The antibody was incubated at 37°C for 1 hour with 800CW Goat anti-Rabbit IgG secondary antibody. After each incubation, the antibody was washed three times with PBST and then imaged using an imaging system.

[0137] The IFA (In vitro anatomical analysis) of worms at different life stages was performed as follows: Freshly collected samples were fixed with 4% paraformaldehyde (PFA) at room temperature for 20 min, and then the fixative was discarded. The samples were then permeabilized with 3% BSA containing 0.25% Triton X-100 at room temperature for 20 min, and the permeabilization solution was discarded after permeabilization. The samples were then blocked with 3% BSA at room temperature for 30 min. A sealing film was placed in a humidified metal chamber, and the primary antibody (Anti-Cre recombinase antibody (ab216262)) and secondary antibody (Alexa Fluor 594-goat anti-rabbit IgG) corresponding to the endogenous marker were incubated at 37°C for 30 min. After incubation with the primary and secondary antibodies, the samples were rinsed five times with 3% BSA containing 0.05% Tween-20. After washing, the samples were mounted, dried at 37°C for 30 min, and then observed under a fluorescence microscope.

[0138] (4) Plaque assay to analyze the biological characteristics of tachyzoite proliferation: 1) Cell preparation: HFF cells were seeded in 6-well plates and cultured in a 37℃ 5% CO2 cell culture incubator for 5-7 days; 2) Infection with tachyzoites: Freshly released tachyzoites were purified and counted. After counting, 500 tachyzoites were inoculated into 6-well plates filled with HFF cells and cultured statically in a cell culture incubator for 7 days; 3) Staining: Fixed with 75% alcohol for 10 min. After fixation, crystal violet was used for staining. After staining, the cells were washed with PBS and double-distilled water, and then dried and stored; 4) Statistics: Scanning and photographing were performed to statistically analyze the plaque formation.

[0139] (5) Animal experiments to analyze the pathogenicity of recombinant parasite strains: The pathogenicity test on mice was performed as follows: 50, 500, and 500 tachyzoites of recombinant Toxoplasma gondii strains and wild-type strains were intraperitoneally inoculated into 6-week-old female BALB / c mice (n=10). Each group of mice was raised in the same environment, and the survival rate of mice within 30 days of infection was recorded.

[0140] The results of the above validation examples are shown in Figure 2. The Cre enzyme gene was targeted to the UPRT gene site, and the recombinant Toxoplasma gondii strain ToxoBJ-Cre expressing Cre enzyme exhibited stable genetic traits (Figure 2A). Plaque assays showed that the reproductive capacity of this recombinant Toxoplasma gondii strain ToxoBJ-Cre was not significantly different from that of the parent strain (Figure 2B); its pathogenicity in mice was not significantly different from that of the parent strain (Figure 2C); and RT-PCR (Figure 2A), Western blotting (Figure 2D), and indirect immunofluorescence (Figure 2E) confirmed that Cre enzyme is expressed only during the cyst and gamete reproduction stages.

[0141] Example 2. Construction of the Tg-ΔBFD1 / OWP1 recombinant insect strain

[0142] Taking Toxoplasma gondii with staged deletions of BFD1 (sequence information available on ToxoDB website, https: / / toxodb.org / toxo / app / record / gene / TGME49_200385; SEQ ID NO.5) and OWP1 gene (sequence information available on ToxoDB website, https: / / toxodb.org / toxo / app / record / gene / TGME49_204420; SEQ ID NO.6) as an example, using the ToxoBJ-cre strain as the genetic manipulation object, the LoxP sequence (SEQ ID NO.4) was sequentially and in the same direction inserted upstream and downstream of the two target genes using CRISPR / Cas9 technology (Figure 2), successfully constructing the Tg-ΔBFD1 / OWP1 strain of the parasite with recombinant LoxP gene.

[0143] Experiment 2.1 Construction of Tg-ΔBFD1 / OWP1 recombinant insect strain

[0144] (1) Carrier construction

[0145] According to the gRNA design program provided on the ToxoDB website, gRNAs for the BFD1 and OWP1 genes were screened, and the gRNA sequences in pSAG1::CAS9-U6::sgUPRT were replaced using the Q5 site-directed mutagenesis kit to form plasmids pSAG1::CAS9-U6::sgBFD1 and pSAG1::CAS9-U6::sgOWP1.

[0146] The left and right homologous arms of the BFD1 gene were amplified using the primers shown in Table 8. At the same time, the LoxP sequence was amplified upstream and downstream of the BFD1 gene by lengthening primers to amplify the screening expression cassette of the DHFR resistance gene.

[0147] Table 8. Primers

[0148] Next, the left homologous arm of BFD1, the BFD1 gene carrying the LoxP site, the 3'UTR, the DHFR drug resistance gene expression cassette, and the right homologous arm were ligated using a multi-fragment ligation method (refer to Example 1 for the ligation method) to construct the knock-in donor plasmid (Donor-BFD1) for the LoxP site of the BFD1 gene.

[0149] The knock-in donor plasmid (Donor-OWP1) for the Loxp site of the OWP1 gene was constructed in the same manner, and the primer sequences are shown in Table 9.

[0150] Table 9. Primers

[0151] (2) Transfection and screening

[0152] 1.0×10 7 One Toxoplasma gondii tachyzoite was resuspended in 100 μl of nuclear transfer buffer. Donor-BFD1, Donor-OWP1 and pSAG1::CAS9-U6::sgBFD1, pSAG1::CAS9-U6::sgOWP1 were added in a ratio of 1:1:5:5, with a total amount of approximately 10 μg. The mixture was quickly added to an electroporation cuvette, and the nuclear transfer instrument was turned on. Program U-033 was selected for transfection. After nuclear transfer, 1 ml of DMEM medium was quickly added to the electroporation cuvette, and after thorough mixing, the medium was transferred to a 1.5 ml centrifuge tube. 200 μl of the transfected Toxoplasma gondii tachyzoite solution was added to pre-cultured HFF cells. Pyrimethamine and chloramphenicol were added to the medium for screening. After three generations of screening, recombinant Toxoplasma gondii strains were obtained.

[0153] Verification Example 2.2 Performance Qualification and Analysis

[0154] Furthermore, the Toxoplasma gondii strain Tg-ΔBFD1 / OWP1, which had its target gene BFD1 / OWP1 conditionally knocked out, was purified, identified, and subjected to genetic stability and biological and pathogenicity analysis.

[0155] For specific verification examples and steps, please refer to Verification Example 1.2 in Example 1.

[0156] The results showed that the LoxP sequence was precisely targeted upstream and downstream of the BFD1 and OWP1 genes (Figure 4A), and the recombinant Toxoplasma gondii strain with LoxP integration was genetically stable (Figure 4A). Plaque assays showed no significant difference in reproductive capacity compared to the parent strain (Figure 4B); the pathogenicity to the intermediate host mouse was not significantly different from that of the parent strain (Figure 4C). This indicates that a recombinant Toxoplasma gondii strain with LoxP integrated into a specific site in the genome was successfully obtained.

[0157] Example 3. Vaccine Potential Evaluation

[0158] With 10 2 ~10 8 The recombinant Toxoplasma gondii strain tachyzoites prepared in Example 2 above were used to immunize cats via intramuscular injection (Table 10), subcutaneous injection (Table 11), and oral administration (Table 12). At different time points, tissues including the brain, tongue, heart, pectoral muscles, leg muscles, lungs, liver, and spleen were dissected to detect the presence of cysts. The homogenates of these tissues were then inoculated into mice to detect the occurrence of symptoms and mortality. Simultaneously, fecal oocysts were analyzed and counted from day 3 to 14 post-inoculation in cats to assess safety.

[0159] On day 14 post-immunization, mice were orally inoculated with Toxoplasma gondii cysts for challenge infection. Tissues from the brain, tongue, heart, pectoral muscles, leg muscles, lungs, liver, and spleen were dissected and examined for the presence of cysts. The tissues were homogenized and inoculated into mice to detect the appearance of symptoms and death. Fecal oocysts were also examined and counted from day 3 to day 14 post-infection to assess the effectiveness of the treatment.

[0160] Table 10. Safety and efficacy evaluation of Tg-ΔBFD1 / OWP1 strain via intramuscular injection immunization Note: - indicates that no cysts were detected by morphological and molecular biological methods; a: Clinical symptoms should be recorded in detail, including the number of clinical symptoms and the specific symptoms.

[0161] Table 11. Safety and efficacy evaluation of subcutaneous immunization with Tg-ΔBFD1 / OWP1 strain Note: - indicates that no cysts were detected by morphological and molecular biological methods; a: Clinical symptoms should be recorded in detail, including the number of clinical symptoms and the specific symptoms.

[0162] Table 12. Safety and efficacy evaluation of oral immunization with Tg-ΔBFD1 / OWP1 strain Note: - indicates that no cysts were detected by morphological and molecular biological methods; a: Clinical symptoms should be recorded in detail, including the number of clinical symptoms and the specific symptoms.

[0163] The results in Tables 10-12 above show that no cysts or oocysts were detected in any cat tissues after immunization, confirming that the recombinant Toxoplasma gondii has good safety; no cysts or oocysts were detected in any cat tissues after challenge, confirming that the recombinant Toxoplasma gondii has good immunogenicity.

[0164] Example 4. Construction of the Tg-ΔLDH2 / AP2XII-1 insect strain

[0165] Taking Toxoplasma gondii with staged deletions of LDH2 (sequence information available on ToxoDB website, https: / / toxodb.org / toxo / app / record / gene / TGME49_291040; SEQ ID NO.7) and AP2XII-1 gene (sequence information available on ToxoDB website, https: / / toxodb.org / toxo / app / record / gene / TGME49218960; SEQ ID NO.8) as an example, using the ToxoBJ-cre strain as the genetic manipulation object, the LoxP sequence (SEQ ID NO.4) was sequentially and in the same direction inserted upstream and downstream of the above target genes using CRISPR / Cas9 technology (same design principle and construction steps as in Example 2, refer to Figure 5) to construct the LoxP recombinant Tg-ΔLDH2 / AP2XII-1 strain.

[0166] Experiment 4.1 Construction of the Tg-ΔLDH2 / AP2XII-1 recombinant insect strain

[0167] (1) Carrier construction

[0168] According to the gRNA design program provided on the ToxoDB website, gRNAs for the LDH2 and AP2XII-1 genes were screened, and the gRNA sequences in pSAG1::CAS9-U6::sgUPRT were replaced using the Q5 site-directed mutagenesis kit to form the plasmids pSAG1::CAS9-U6::sgLDH2 and pSAG1::CAS9-U6::sgAP2XII-1.

[0169] Using the primers shown in Table 13, the left and right homologous arms of the LDH2 gene were amplified respectively. The LoxP sequence was amplified upstream and downstream of the LDH2 gene by lengthening primers, and the screening expression cassette of the DHFR drug resistance gene was amplified at the same time.

[0170] Table 13. Primers

[0171] The left homologous arm of LDH2, the LDH2 gene carrying the LoxP site, the 3'UTR, the DHFR drug resistance gene expression cassette, and the right homologous arm were ligated using a multi-fragment ligation method (refer to Example 1 for the ligation method) to construct the knock-in donor plasmid (Donor-LDH2) for the LoxP site of the LDH2 gene.

[0172] The knock-in donor plasmid (Donor-AP2XII-1) for the Loxp site of the AP2XII-1 gene was constructed in the same manner, and the primers are shown in Table 14.

[0173] Table 14. Primers

[0174] (2) Transfection and screening

[0175] 1.0×10 7 One Toxoplasma gondii tachyzoite was resuspended in 100 μl of nuclear transfer buffer. Donor fragments Donor-LDH2, Donor-AP2XII-1 and pSAG1::CAS9-U6::sgLDH2, pSAG1::CAS9-U6::sgAP2XII-1 in a ratio of 1:1:5:5, with a total amount controlled at approximately 10 μg, were quickly added to an electroporation cuvette. The transfection instrument was turned on, and program U-033 was selected for transfection. After nuclear transfer, 1 ml of DMEM medium was quickly added to the electroporation cuvette, and after thorough mixing, the medium was transferred to a 1.5 ml centrifuge tube. 200 μl of the transfected Toxoplasma gondii tachyzoite solution was added to pre-cultured HFF cells. Pyrimethamine and chloramphenicol were added to the medium for screening. After three generations of screening, recombinant Toxoplasma gondii strains were obtained.

[0176] Verification Example 4.2 Performance Qualification and Analysis

[0177] Monoclonal purification, identification, genetic stability, and biological and pathogenicity analysis were performed on the Toxoplasma gondii strain Tg-ΔΔLDH2 / AP2XII-1, which had the target genes LDH2 and AP2XII-1 conditionally knocked out.

[0178] For specific verification examples and steps, please refer to Verification Example 2.2 in Example 2.

[0179] Performance evaluation of the recombinant Tg-ΔLDH2 / AP2XII-1 strain showed that the LoxP sequence was specifically targeted upstream and downstream of the LDH2 and AP2XII-1 genes (Figure 6A), and the recombinant Toxoplasma gondii strain with LoxP integration was genetically stable (Figure 6A). There was no significant difference in plaque size between the recombinant Toxoplasma gondii and the parent strain after 24 hours of culture in HFF cells, indicating no significant difference in reproductive capacity between the two strains (Figure 6B). Different doses of the strain did not significantly affect the survival curves or clinical symptoms of mice, indicating no significant difference in pathogenicity to the intermediate host mouse compared to the parent strain (Figure 6C). This demonstrates the successful acquisition of a recombinant Toxoplasma gondii strain with LoxP integrated into a specific genomic site.

[0180] Performance evaluation of the recombinant Tg-ΔLDH2 / AP2XII-1 strain revealed that the LoxP sequence was precisely targeted upstream and downstream of the LDH2 and AP2XII-1 genes. The recombinant Toxoplasma gondii strain with LoxP integration exhibited genetic stability. The plaque size formed 24 hours after inoculation into HFF cells by the recombinant Toxoplasma gondii strain was not significantly different from that of the parent strain, indicating no significant difference in reproductive capacity. Different doses of the strain did not significantly affect the survival curves of mice or the clinical symptoms of cats, indicating no significant difference in pathogenicity to the intermediate host (mice) or definitive host (cats) compared to the parent strain. This demonstrates the successful acquisition of a recombinant Toxoplasma gondii strain with LoxP integrated into a specific genomic site.

[0181] Example 5. Vaccine Potential Evaluation

[0182] 10 of the Tg-ΔLDH2 / AP2XII-1 insect strain prepared in Example 4 2 ~10 8 Cats were immunized with tachyzoites via intramuscular injection (Table 15), subcutaneous injection (Table 16), and oral administration (Table 17). After necropsy, tissues including the brain, tongue, heart, pectoral muscles, leg muscles, lungs, liver, and spleen were collected to detect the presence of cysts. These tissues were homogenized and inoculated into mice to detect symptoms and mortality. Fecal oocysts from cats were also analyzed and counted 3-14 days post-inoculation to assess safety. On day 14 post-immunization, cats were challenged with oral administration of Toxoplasma gondii cysts. After necropsy, tissues including the brain, tongue, heart, pectoral muscles, leg muscles, lungs, liver, and spleen were collected to detect the presence of cysts. These tissues were homogenized and inoculated into mice to detect symptoms and mortality. Fecal oocysts from cats were also analyzed and counted 3-14 days post-challenge to assess efficacy.

[0183] Table 15. Safety and efficacy evaluation of Tg-ΔLDH2 / AP2XII-1 strain via intramuscular injection immunization Note: - indicates that no cysts were detected by morphological and molecular biological methods; a: Clinical symptoms should be recorded in detail, including the number of clinical symptoms and the specific symptoms.

[0184] Table 16. Safety and efficacy evaluation of subcutaneous immunization with Tg-ΔLDH2 / AP2XII-1 strain Note: - indicates that no cysts were detected by morphological and molecular biological methods; a: Clinical symptoms should be recorded in detail, including the number of clinical symptoms and the specific symptoms.

[0185] Table 17. Safety and efficacy evaluation of oral immunization with Tg-ΔLDH2 / AP2XII-1 strain Note: - indicates that no cysts were detected by morphological and molecular biological methods; a: Clinical symptoms should be recorded in detail, including the number of clinical symptoms and the specific symptoms.

[0186] The results in Tables 15-17 show that no cysts or oocysts were detected in any cat tissues after immunization, confirming the good safety profile of the recombinant Toxoplasma gondii; and no cysts or oocysts were detected in any cat tissues after challenge, confirming the good immunogenicity of the recombinant Toxoplasma gondii.

[0187] The above embodiments and results demonstrate that the feline toxoplasmosis gene-deleted vaccine, administered via intramuscular, subcutaneous, or oral routes, provides good immunoprotection against Toxoplasma gondii cysts and oocysts in cats. This proves that the feline toxoplasmosis gene-deleted vaccine developed using the conditional deletion and target gene selection and matching strategy of this invention can ensure both high efficacy and high safety.

[0188] In summary, the toxoplasmosis deletion vaccine developed by this invention, using gene-deleted parasite strains, can effectively resist toxoplasmosis infection by combining target genes in different ratios, and has good safety and high efficacy.

[0189] The specific embodiments described above are merely preferred embodiments of this disclosure, but are not intended to limit this disclosure. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of this invention should be included within the scope of protection of this disclosure.

Claims

1. A gene-deleted recombinant Toxoplasma gondii strain, characterized in that, Cre enzymes are specifically expressed only during cyst formation, schizogony, and / or gametosis. as well as LoxP sequences are sequentially inserted upstream, downstream, or internally of developmental essential genes, key genes regulating cyst formation, and key genes regulating late schizogenes and / or gamete development to knock out at least one of the aforementioned developmental essential genes, key genes regulating cyst formation, late schizogenes genes, and key genes regulating gamete development, thereby obtaining recombinant Toxoplasma gondii strains.

2. The recombinant Toxoplasma gondii strain according to claim 1, characterized in that, Knock out two or more of the following genes: essential developmental genes, key genes regulating cyst formation, late schizogony genes, and key genes regulating gamete reproduction and development. The Toxoplasma gondii strain exhibited developmental defects in cyst formation, late-stage schizogenes, and / or gametogenesis.

3. The recombinant Toxoplasma gondii strain according to claim 1, characterized in that, The target genes to be knocked out are any combination of essential genes that are continuously expressed at different life stages of Toxoplasma gondii and regulatory genes that are expressed at different stages. Preferably, the target gene is a combination of at least two genes selected from essential genes for life cycle stages, AP2 transcription factor family genes, CST family genes, OWP-like genes, eIF4, AMA1, IMP1, BAG1, mNG, ENO1, LDH2, and genes specifically expressed during fission and sexual reproduction stages. Preferably, the target gene is selected from AP2IX-5, AP2XII-2, AP2X-4, AP2XII-1, AP2XI-4, AP2IV-4, AP2IV-3, AP2XII-1, AP2IX-1, AP2XI-2, AP2IX-9, AP2IX-6, AP2III-4, SAG4, SAG5, AMA1, CDPK1, CDPK2, CDPK3, CDPK5, HSP40, HSP60, ROP1, ROP2, One or more of the following: ROP5, ROP7, ROP8, ROP9, ROP13, ROP16, ROP17, ROP18, ROP21, ROP35, ROP38, ROP54, MIC1, MIC2, MIC3, MIC4, MIC6, MIC8, MIC11, MIC13, HSP40, HSP60, HSP70, OWP1, OWP2, IMP1, BAG1, BFD1, eIF4A, MYR1, HDAC, LDH2; Preferably, the recombinant Toxoplasma gondii strains include the Tg-ΔBFD1 / OWP1 strain and the Tg-ΔLDH2 / AP2XII-1 strain.

4. A live attenuated vaccine for the prevention of toxoplasmosis, wherein the active ingredient comprises at least the recombinant Toxoplasma gondii strain described in any one of claims 1-3; Preferably, the vaccine is a monovalent vaccine or a multivalent vaccine; More preferably, the multivalent vaccine comprises the recombinant Toxoplasma gondii strain according to any one of claims 1-3, and further comprises other isolated Toxoplasma gondii isolates that do not possess cross-immunoprotective efficacy.

5. A method for constructing a gene-deleted recombinant Toxoplasma gondii strain, characterized in that, The method includes the following steps: S1. Constructing recombinant insect strains that specifically express Cre enzymes during cyst formation, schizogony, and / or gamete reproduction stages, including: Construct shuttle transfection vectors with dual or multiple expression frames, which include promoters that express genes using the characteristics of cyst, schizogenesis and / or gamete reproduction stages, sequentially linked to the gene sequence of Cre enzyme, the 3' non-transcribed region of Toxoplasma gondii, and homologous arms introduced at both ends. The shuttle transfection vector was targeted to the location of or near the first target gene, thereby constructing the recombinant Toxoplasma gondii strain ToxoBJ-cre expressing Cre enzyme; S2. Using ToxoBJ-cre strain as the target, two LoxP sequences in the same direction were sequentially inserted into the second target gene or its locus upstream, downstream or in the middle position to knock out the second target gene and construct a gene-deleted recombinant Toxoplasma gondii strain.

6. The method according to claim 5, characterized in that, In step S1, the first target gene is the uracil phosphoribosyltransferase homologue gene UPRT or other non-essential genes; In step S2, the second target gene to be knocked out is at least one gene selected from essential genes that are continuously expressed in different life stages of Toxoplasma gondii and regulatory genes that are expressed in stages. Preferably, the second target gene is at least one of the following: developmental essential genes, key genes regulating cyst formation, late schizogenesis genes, and key genes regulating gamete reproduction and development. More preferably, the second target gene is two or more of the following: developmental essential genes, key genes regulating cyst formation, late schizogony genes, and key genes regulating gamete reproduction and development. More preferably, the second target gene is one or more genes selected from essential genes for life cycle stages, AP2 transcription factor family genes, CST family genes, OWP-like genes, eIF4, AMA1, IMP1, BAG1, mNG, ENO1, LDH2, and genes specifically expressed during fission and sexual reproduction stages. Preferably, the second target gene is selected from AP2IX-5, AP2XII-2, AP2X-4, AP2XII-1, AP2XI-4, AP2IV-4, AP2IV-3, AP2XII-1, AP2IX-1, AP2XI-2, AP2IX-9, AP2IX-6, AP2III-4, SAG4, SAG5, AMA1, CDPK1, CDPK2, CDPK3, CDPK5, HSP40, HSP60, ROP1, ROP2, One or more genes from the following: ROP5, ROP7, ROP8, ROP9, ROP13, ROP16, ROP17, ROP18, ROP21, ROP35, ROP38, ROP54, MIC1, MIC2, MIC3, MIC4, MIC6, MIC8, MIC11, MIC13, HSP40, HSP60, HSP70, OWP1, OWP2, IMP1, BAG1, BFD1, eIF4A, MYR1, HDAC, LDH2; Preferably, the promoters of genes specifically expressed during the cyst, schizogenesis, and / or gamete reproduction stages include promoters of some genes from the AP2 transcription factor family, promoters of some genes from the BAG family, promoters of some genes from the OWP class, promoters of genes from the CST family, promoters of genes from the AMA family, and promoters of SRS22A, SAG4, mNG, PMA1, BSR4, CST1, ENO1, LDH2, MIC12, MIC13, MIC17A, MIC17C, MIC12, MIC13, MIC17A, MIC17C, GRA4, GRA6, GRA82, GRA11b, GRA80, ROP4, ROP7, MCP4, BPK1, SRS9, SRS22b, GRA11B, and GEX1. Preferably, the recombinant Toxoplasma gondii strains include the Tg-ΔBFD1 / OWP1 strain and the Tg-ΔLDH2 / AP2XII-1 strain.

7. Use of the recombinant Toxoplasma gondii strain according to any one of claims 1-3 in the preparation of a vaccine for the prevention of Toxoplasma gondii infection and / or toxoplasmosis in mammals.

8. The use of the recombinant Toxoplasma gondii strain according to any one of claims 1-3 in the preparation of medicaments and / or biological products for the prevention, mitigation, and / or treatment of Toxoplasma gondii infection and / or toxoplasmosis in mammals, characterized in that, The effective active ingredient of the drug and / or biological product includes at least the recombinant Toxoplasma gondii strain as described in any one of claims 1-3.

9. A medicament and / or biological product for the prevention, mitigation, and / or treatment of toxoplasmosis and / or toxoplasmosis in mammals, characterized in that, The drug / biological product comprises an effective dose of any one of the recombinant Toxoplasma gondii strains according to claims 1-3 above; Preferably, the biological product includes a vaccine.

10. A method for preventing, mitigating and / or treating Toxoplasma gondii infection and / or toxoplasmosis in mammals, comprising at least administering an effective dose of a recombinant Toxoplasma gondii strain to a subject in need; Preferably, in the method for preventing mammalian toxoplasmosis infection and / or toxoplasmosis, the effective dose is 10. 2 ~10 8 Each recombinant Toxoplasma gondii strain contains either a tachyzoite or a bradyzoite per insect at a time. Preferably, the effective dose is 10 7 ~10 8 Or 10 5 ~10 6 Or 10 4 ~10 5 Or 10 3 ~10 4 Or 10 2 ~10 3 Each tachyzoite or bradyzoite per animal at a time; Preferably, the effective dose is 10 2 ~10 7 Or 10 3 ~10 8 Or 10 4 ~10 7 Or 10 5 ~10 8 Or 10 5 ~10 7 Each tachyzoite or bradyzoite per animal at a time.

11. The method according to claim 10, characterized in that, Routes of administration include intramuscular injection, subcutaneous injection, or oral administration; Preferably, the route of administration is via vaccination.

12. The method according to claim 10, characterized in that, The method includes single immunization and / or booster immunization; Preferably, a booster immunization is performed 30 to 90 days after the first immunization, wherein the booster immunization involves administering an effective dose to the subject more than once.

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