Recombinant toxoplasma gondii strain, construction method and application thereof

Abstract

The invention belongs to the field of gene engineering, and relates to a recombinant toxoplasma gondii strain prepared through a gene editing technology, a veterinary drug and / or a biological product containing the strain and application of the recombinant toxoplasma gondii strain and the veterinary drug and / or the biological product. Preferably, the recombinant toxoplasma gondii strain comprises a Tg-SEX1 / SEX2: pBCP1 (Cas9)-CST1 strain, a Tg-SEX1 / SEX2: pP21 (Cas9)-CST2 strain, a Tg-SEX1 / SEX2 / CST2 strain and / or a Tg-SEX1 / SEX2 / CST1 strain, and the recombinant toxoplasma gondii strain can be used for preparing the recombinant toxoplasma gondii strain. Based on the recombinant toxoplasma gondii strain, an attenuated live vaccine is developed, and the attenuated live vaccine is suitable for immunoprophylaxis of toxoplasmosis of various mammals, especially felines, has extremely high safety and immune efficacy, and is an effective solution for blocking transmission of toxoplasmosis to human beings and other animals. The vaccine is free of pollution to animals and breeding and feeding environments, residues of toxoplasma gondii cysts and oocysts in animal bodies and environments are effectively reduced, and the risk of toxoplasma gondii infection is greatly reduced or eradicated. The vaccine strain can also be used as a carrier to deliver dominant antigens or therapeutic proteins of other pathogens, and has a wide application prospect.

Description

Technical Field

[0001] The present application belongs to the field of genetic engineering and relates to a recombinant Toxoplasma gondii strain, a construction method and its application. Specifically, it relates to a recombinant Toxoplasma gondii strain prepared by gene editing technology, a veterinary drug and / or biological product containing the strain, a construction method and its use. Background Art

[0002] Globally, dogs and cats are the most common companion animals. According to statistics from 2022, the total number of dogs and cats kept in China exceeded 100 million, with cats surpassing dogs to become the most important companion animal. As companion animals come into increasingly frequent contact with humans, the zoonotic pathogens they carry increase the risk of transmission of these diseases. Among the zoonotic pathogens carried by cats, Toxoplasma gondii is particularly harmful. For example, Toxoplasma infection can cause miscarriage in pregnant women, blindness in fetuses, and intellectual disability, while the activation of Toxoplasma cysts increases the risk of disability and mortality in immunocompromised individuals. In developed countries like Europe and the United States, toxoplasmosis is widespread, while in China, the prevalence of Toxoplasma antibodies in the human population is close to 20%. Therefore, the prevention and treatment of zoonotic diseases has important public health implications for protecting human life and health and promoting eugenics and reproductive health.

[0003] The life cycle of Toxoplasma gondii includes schizont and sexual reproduction stages in the definitive host, cats, and asexual reproduction stages in intermediate hosts. The asexual reproduction stages are divided into the tachyzoite stage during acute infection and the bradyzoite / cyst stage during chronic infection. In cats, the life cycle includes schizonts, gametogenesis, and the formation and excretion of oocysts. After excretion, the oocysts undergo sporulation in a suitable environment. Tachyzoites, bradyzoites, and sporozoites are all infective and can infect intermediate and definitive hosts. When tachyzoites, bradyzoites, or sporozoites infect the definitive host, cats, they asexually reproduce within the epithelial cells of the feline small intestine, forming merozoites. After several generations of fission reproduction, some merozoites develop into female gametes and some into male gametes. The male and female gametes fuse to form a zygote, which ultimately develops into an oocyst and is excreted in the feces. Under optimal conditions of temperature, humidity, and oxygen partial pressure, oocysts mature after 2-5 days of development. Mature oocysts are infective and serve as the primary source of infection for humans and other intermediate hosts. Therefore, controlling cat oocyst excretion into the external environment is key to preventing the spread of toxoplasmosis and an important technical means of achieving source control.

[0004] Considering the characteristics of Toxoplasma gondii's reproduction and development in the final host and intermediate host, as well as public health and safety considerations, cat toxoplasmosis vaccine is the best strategy for toxoplasmosis prevention and control. Its vaccine characteristics must meet two goals at the same time: (1) Safety: The complexity of Toxoplasma antigens determines that live worm immunization provides the best immune protection. The safety of live worm immunization must achieve safety for the immunized animals and the environment, that is, no cysts and oocysts can be formed after immunization; (2) Effectiveness: After immunization, it can block the formation of oocysts and reduce the risk of oocyst excretion. At the same time, it provides immune protection from the development stage of tachyzoites to bradyzoites (cysts), that is, it blocks the formation of cysts and avoids the rupture of cysts when the cat's immunity is low, which may cause the risk of acute Toxoplasma infection and continuous excretion of oocysts again. Summary of the Invention

[0005] In response to the current state of the art and the aforementioned technical difficulties, the inventors of this application combined CRISPR / Cas9 technology with a CRISPR / Cas9-based kill switch technology to develop genetically recombinant Toxoplasma gondii. They then developed a live attenuated vaccine based on live parasites, seeking to overcome the technical limitations of live toxoplasmosis vaccines. The invention also provides a toxoplasmosis vaccine, its construction method, and its application. The vaccine is suitable for immunizing a variety of mammals against toxoplasmosis, especially cats, and is an effective solution for blocking the spread of toxoplasmosis to humans and other animals.

[0006] In a first aspect, the present application provides a recombinant Toxoplasma gondii strain, comprising:

[0007] In the final host, the specific gene deletion of the gametogenesis / sexual reproduction stage of Toxoplasma gondii results in gametogenesis / sexual reproduction disorder; and / or

[0008] Knockout of essential genes, important genes or evolutionarily conserved genes for cyst development results in cyst development defects.

[0009] Preferably, the deleted gene is selected from at least one gene of the AP2 transcription factor family gene, SEX1, SEX2, SEX3, SEX4, SEX5, SEX6, SEX7, SEX8, SEX9, OWP1-OWP12, SSP, SOD3, or any combination thereof; more preferably, the deleted gene is a combination of SEX1 and SEX2

[0010] Preferably, the essential gene or important gene or evolutionary conserved gene for cyst development is selected from at least one of BAG1, BAG5, BFD1, LDH2, BCP1, P21, ENO1, MAG1, MAG2, SAG4, PMA1, CST1, CST2, BSR4, MIC12, MIC13, MIC17A, MIC17C, MIC12, MIC13, MIC17A, MIC17C, GRA4, GRA6, GRA12, ROP4, MCP4, BPK1, SRS9, or any combination thereof; more preferably, the knocked-out gene is GST1 and / or GST2.

[0011] Preferably, the cyst development defect is achieved by: regulating the expression of the Cas9 protein from at least one promoter of a cyst development-stage-specific gene, designing and expressing a guide RNA (gRNA) specific for at least one gene essential for cyst development, important for cyst development, or evolutionarily conserved; and / or directly knocking out at least one of these genes. Preferably, the Toxoplasma gondii strain exhibits cyst development defects, and / or impaired gamete / sexual reproduction, and / or defective or absent gamete, zygote, and oocyst formation.

[0012] Preferably, the gene for designing gRNA is at least one gene selected from MIC8, PKG, MYOA, CDPK1, GAP45, AKMT, DOC2.1, MAPKL1, His3, His4, His2A, His2B, CST1, CST2, GRA12, ITS1, ITS2, or any combination thereof.

[0013] Preferably, the Toxoplasma gondii strains include Tg-△SEX1 / SEX2:pBCP1(Cas9)-CST1 strain, Tg-△SEX1 / SEX2:pP21(Cas9)-CST2 strain, Tg-△SEX1 / SEX2 / CST2 strain and / or Tg-△SEX1 / SEX2 / CST1 strain.

[0014] In a second aspect, the present application provides a live vaccine for preventing toxoplasmosis, the active ingredient of which comprises at least the recombinant Toxoplasma gondii strain described in any one of the first aspects above;

[0015] Preferably, the vaccine is a monovalent vaccine or a multivalent vaccine;

[0016] More preferably, the multivalent vaccine comprises the recombinant Toxoplasma gondii strain described in any one of the first aspects above, and further comprises other isolated Toxoplasma gondii isolates that do not have cross-immune protection efficacy.

[0017] In a third aspect, the present application provides a construct for encoding the recombinant Toxoplasma gondii strain described in any one of the first aspects.

[0018] In a fourth aspect, the present application provides a method for constructing a recombinant Toxoplasma gondii strain, comprising the following steps:

[0019] S1. Constructing a transfection vector that causes gamete reproduction / sexual reproduction stage-specific gene knockout and controls cyst development defects, comprising:

[0020] Introducing homologous arms at the upstream and downstream positions of the gamete reproduction / sexual reproduction stage-specific gene, and sequentially inserting the expression frame of the regulating Cas9 gene, the expression frame of the gRNA, and the expression frame of the screening gene between the two homologous arms; or

[0021] Directly knock out at least one of the key genes for gamete development and cyst development;

[0022] S2. Co-transfecting the transfection vector and the plasmid of the cyst-specific gene gRNA into Toxoplasma gondii tachyzoites,

[0023] The transfection vector is targeted to the position of a gene specific to the gamete reproduction / sexual reproduction stage, thereby preparing a genetically recombinant Toxoplasma gondii strain with gamete reproduction / sexual reproduction disorders and cyst development defects;

[0024] Preferably, the gamete reproduction / sexual reproduction stage-specific gene is at least one gene selected from the group consisting of AP2 transcription factor family genes, SEX1 (TGME49_285940), SEX2 (TGME49_255310), SEX3 (TGME49_293250), SEX4 (TGME49_295640), SEX5 (TGME49_239670), SEX6 (TGME49_291150), SEX7 (TGME49_247270), SEX8 (TGME49_234060), SEX9 (TGME49_313725), OWP1-OWP12, SSP, and SOD3; more preferably, SEX1 gene; and / or

[0025] The essential gene, important gene or evolutionary conserved gene for cyst development is selected from at least one of BAG1, BAG5, BFD1, LDH2, BCP1, P21, ENO1, MAG1, MAG2, SAG4, PMA1, CST1, CST2, BSR4, MIC12, MIC13, MIC17A, MIC17C, MIC12, MIC13, MIC17A, MIC17C, GRA4, GRA6, GRA12, ROP4, MCP4, BPK1, SRS9 or any combination thereof; and / or

[0026] The promoter gene for regulating the expression of the Cas gene is selected from at least one gene or any combination thereof selected from BAG1, BAG5, BFD1, LDH2, BCP1, P21, ENO1, MAG1, MAG2, SAG4, PMA1, CST1, CST2, BSR4, MIC12, MIC13, MIC17A, MIC17C, MIC12, MIC13, MIC17A, MIC17C, GRA4, GRA6, GRA12, ROP4, MCP4, BPK1, and SRS9, which are specifically expressed in the cyst stage of Toxoplasma gondii; and / or

[0027] The gene for designing the gRNA is at least one gene selected from MIC8, PKG, MYOA, CDPK1, GAP45, AKMT, DOC2.1, MAPKL1, His3, His4, His2A, His2B, CST1, CST2, GRA12, ITS1, ITS2, or any combination thereof.

[0028] In a fifth aspect, the present application provides the use of the recombinant Toxoplasma gondii strain according to any one of the first aspects above in the preparation of a vaccine for preventing toxoplasmosis and / or other diseases in mammals.

[0029] In a sixth aspect, the present application provides the use of the recombinant Toxoplasma gondii strain according to any one of the first aspects in the preparation of drugs and / or biological products for preventing, alleviating and / or treating toxoplasmosis and / or other diseases in mammals, wherein the effective active ingredients of the drugs and / or biological products include at least the recombinant Toxoplasma strain.

[0030] In a seventh aspect, the present application provides a drug and / or biological product for preventing, alleviating and / or treating toxoplasmosis and / or other diseases in mammals, wherein the drug / biological product comprises an effective dose of the recombinant Toxoplasma gondii strain according to any one of the first aspects;

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

[0032] In an eighth aspect, the present application provides a method for preventing, alleviating and / or treating toxoplasmosis and / or other diseases in mammals, which at least comprises 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 tachyzoites or bradyzoites of the Toxoplasma gondii strain;

[0034] Preferably, the effective dose is 10 7 ~10 8 or 105 ~10 6 or 10 4 ~10 5 or 10 3 ~10 4 or 10 2 ~10 3 tachyzoites or bradyzoites;

[0035] 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 tachyzoites or bradyzoites.

[0036] Preferably, the administration route comprises intramuscular injection, subcutaneous injection or oral administration. Preferably, the administration route is a vaccination route.

[0037] Preferably, the method comprises a single immunization and / or a booster immunization; preferably, the booster immunization is performed on day 14 to 90 after the first immunization, and the booster immunization is to administer an effective dose to the subject more than once.

[0038] Beneficial effects

[0039] The incomplete development of genetically modified Toxoplasma gondii strains in mammals, especially cats, can meet all the requirements for the development of feline toxoplasmosis vaccines. The combination of CRISPR / Cas9 technology and the CRISPR / Cas9-kill system provides a feasible solution for a toxoplasmosis vaccine. Experimental results show that the live attenuated feline toxoplasmosis vaccine developed based on the recombinant Toxoplasma gondii strain can significantly reduce the morbidity and mortality of animals caused by toxoplasmosis infection, effectively reduce the risk of toxoplasmosis transmission to humans, and is also suitable for the immunization and prevention of toxoplasmosis in a variety of mammals, especially felines.

[0040] This invention provides a live vaccine design strategy and target gene combination ratio scheme. The developed feline toxoplasmosis vaccine ensures safety and immune efficacy. Using a recombinant strain of the parasite prepared using gene editing technology as its active ingredient, the vaccine prevents oocyst formation or, if excreted, produces minimal, non-infectious oocysts, demonstrating high safety and immune efficacy.

[0041] This application develops a live attenuated vaccine based on the recombinant Toxoplasma gondii strain, which is suitable for the immune prevention of toxoplasmosis in various mammals, has extremely high safety and immune efficacy, and is particularly suitable for the immune prevention of toxoplasmosis in cats. It is an effective solution to block the spread of toxoplasmosis to humans and other animals. This type of vaccine and / or biological product is easy to produce, highly safe, and effective. The vaccine's immunity lasts for a long time and can distinguish between vaccine immunity and natural infection. It is not only pollution-free to the animals themselves and the breeding and feeding environment, but also effectively reduces the residues of Toxoplasma cysts and oocysts in the animal body and the environment, thereby greatly reducing or eliminating the risk of human Toxoplasma infection. The vaccine strain can also be used as a carrier to deliver dominant antigens or therapeutic proteins of other pathogens, and has broad application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0042] Figure 1 The present invention demonstrates the construction of a transfection vector for the recombinant Toxoplasma gondii strain Tg-ΔSEX1 / SEX2:pBCP1(Cas9)-CST1 using gene editing technology. Homology arms were introduced at both ends, and the transfection vector was targeted to the SEX1 and SEX2 gene loci using CRISPR / Cas9 technology. Furthermore, CRISPR / Cas9-kill technology was used to regulate cyst development defects.

[0043] Figure 2 The identification results of Tg-ΔSEX1 / SEX2:pBCP1(Cas9)-CST1 are shown.

[0044] Figure 2 A shows the genome identification of Tg-△SEX1 / SEX2:pBCP1(Cas9)-CST1 strain after serial passage, with identification performed every 5 generations. The primers for each PCR amplification were Figure 1 The results showed that the recombinant Toxoplasma gondii strain was genetically stable;

[0045] Figure 2 B shows the fecundity of the parental strain (ToxoBJ) and Tg-△SEX1 / SEX2:pBCP1(Cas9)-CST1 verified by plaque assay. Plaque forming unit analysis was performed 24 hours after inoculation, and the results showed that there was no statistically significant difference between the two.

[0046] Figure 3 The transfection vector for the recombinant Toxoplasma gondii strain Tg-ΔSEX1 / SEX2:pP21(Cas9)-CST2 was constructed according to the present invention. Homology arms were introduced at both ends, and the transfection vector was targeted to the SEX1 and SEX2 gene loci of Toxoplasma gondii using CRISPR / Cas9 technology. Furthermore, CRISPR / Cas9-kill technology was used to regulate cyst development defects.

[0047] Figure 4 The identification results of Tg-ΔSEX1 / SEX2:pP21(Cas9)-CST2 are shown.

[0048] Figure 4 A shows the genome identification of Tg-△SEX1 / SEX2:pP21(Cas9)-CST2 strain after serial passage, with identification performed every 5 generations. The primers for each PCR amplification were Figure 3 The results showed that the recombinant Toxoplasma gondii strain was genetically stable;

[0049] Figure 4 B shows the fecundity of the parental strain (ToxoBJ) and Tg-△SEX1 / SEX2:pP21(Cas9)-CST2 verified by plaque assay. Plaque forming unit analysis was performed 24 hours after inoculation, and the results showed that there was no significant difference between the two.

[0050] Figure 5 The transfection vector for the recombinant Toxoplasma gondii strain Tg-ΔSEX1 / SEX2 / CST2 constructed according to the present invention is shown. Homology arms are introduced at both ends, and the transfection vector is targeted to the SEX1, SEX2, and CST2 gene loci of Toxoplasma gondii using CRISPR / Cas9 technology.

[0051] Figure 6 The identification results of Tg-ΔSEX1 / SEX2 / CST2 are shown.

[0052] Figure 6 A shows the genome identification of Tg-△SEX1 / SEX2 / CST2 strain after continuous passage, with identification performed every 5 generations. The primers for each PCR amplification were Figure 5 The results showed that the recombinant Toxoplasma gondii strain was genetically stable;

[0053] Figure 6 B shows the fecundity of the parental strain (ToxoBJ) and Tg-ΔSEX1 / SEX2 / CST2 verified by plaque assay. Plaque forming unit analysis was performed 24 hours after inoculation, and the results showed that there was no significant difference between the two strains.

[0054] Figure 7 The transfection vector for the recombinant Toxoplasma gondii strain Tg-ΔSEX1 / SEX2 / CST1 is constructed according to the present invention. Homology arms are introduced at both ends, and the transfection vector is targeted to the SEX1, SEX2, and CST1 gene loci of Toxoplasma gondii using CRISPR / Cas9 technology.

[0055] Figure 8 The identification results of Tg-ΔSEX1 / SEX2 / CST1 are shown.

[0056] Figure 8 A shows the genome identification of Tg-△SEX1 / SEX2 / CST1 strain after serial passage, with identification performed every 5 generations. Figure 5 The results showed that the recombinant Toxoplasma gondii strain was genetically stable;

[0057] Figure 8 B shows the fecundity of the parental strain (ToxoBJ) and Tg-ΔSEX1 / SEX2 / CST1 verified by plaque assay. Plaque forming unit analysis was performed 24 hours after inoculation, and the results showed that there was no significant difference between the two strains. DETAILED DESCRIPTION

[0058] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments or the description of the prior art. Obviously, the drawings described below are only one embodiment of the present invention. For ordinary technicians in this field, other embodiments can also be obtained based on these drawings.

[0059] definition

[0060] Unless otherwise indicated, all scientific and technical terms and abbreviations used herein have the meanings commonly understood by those skilled in the art. Furthermore, terms and laboratory procedures related to protein and nucleic acid chemistry, molecular biology, cell and tissue culture, microbiology, and immunology used herein are those commonly used in the respective fields. To facilitate a better understanding of the present application, definitions and explanations of relevant terms are provided below.

[0061] As used herein, the terms "a," "an," "the," and similar referents refer to both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context.

[0062] As used herein, the terms "about," "substantially," and "similar to" mean within an acceptable error range for the particular value as determined by one of ordinary skill in the art, which error range may depend in part on how the value is measured or determined or on the limitations of the measurement system.

[0063] As used herein and unless otherwise specified, the term "about" or "approximately" means within plus or minus 10% of a given value or range, for example, plus or minus 1%, 2%, 5%, 9%, 10%, etc. Where a whole number is required, the term means within plus or minus 10% of a given value or range, rounded up or down to the nearest whole number.

[0064] As used herein, the term "and / or" should be understood to include a combination of preceding and following options as well as any one of the options.

[0065] As used herein, "and any combination thereof" refers to any one, any two or more combinations of the options.

[0066] As used in this article, the term "CRISPR / Cas9 technology" refers to a tool with optimized Toxoplasma-related regulatory elements that can be used for precise editing of Toxoplasma genes. Specifically, the promoter responsible for expressing the Cas9 nuclease is the promoter of Toxoplasma tublin, SAG1, actin and other genes, and the promoter responsible for expressing gRNA is the Toxoplasma U6 promoter.

[0067] As used herein, the term "CRISPR / Cas9-based kill switch" or "CRISPR / Cas9-kill system" refers to a genetic manipulation technology for knocking out Toxoplasma genes under specific conditions and life history stages. It mainly uses the promoter of stage-specifically expressed genes to control the expression of Cas9, and uses the stage-expressed Cas9 to cut and missense repair developmentally essential genes and evolutionarily conserved genes, thereby achieving gene deletion under specific conditions and life history stages. Other genetic manipulation technologies that can achieve the above-mentioned purposes are also applicable.

[0068] As used herein, the term "target gene" or "target gene" or "gene target" or "gene of interest" refers to genes, functional domains or regulatory sequences related to the growth and development of Toxoplasma gondii. This article mainly refers to the coding region or functional domain or regulatory region of essential genes or key regulatory genes in the growth and development of Toxoplasma gondii.

[0069] As used in this article, the term "developmentally essential genes" refers to a class of genes that are involved in the development of a certain life history stage of Toxoplasma, such as tachyzoites, bradyzoites, schizonts and gametes. If this type of gene is missing, Toxoplasma cannot complete any of the above life history stages, resulting in developmental defects.

[0070] As used in this article, the term "life history stage essential genes" refers to a class of genes that are involved in all life history stages of Toxoplasma, such as tachyzoites, bradyzoites, schizonts and gametes, or conserved genes in the genetic evolutionary history of Toxoplasma. If this type of gene is missing, Toxoplasma cannot complete all life history stages, resulting in developmental defects.

[0071] As used herein, the term "evolutionarily conserved genes / sequences" refers to genes / sequences that rarely change during evolution, which have high similarity and consistency among different species. The loss of such genes leads to developmental defects or stagnation of Toxoplasma gondii.

[0072] As used herein, the term "effective dose (10 2 ~10 8 The term "tachyzoites or bradyzoites)" refers to the amount administered to each cat or other animal to produce an effect, usually not less than 100 per animal.

[0073] In the first aspect, the present application provides a recombinant Toxoplasma gondii strain, which includes: in the final host, specific genes of the gamete reproduction / sexual reproduction stage of Toxoplasma are deleted, resulting in gamete reproduction / sexual reproduction disorders; and / or essential genes or important genes or evolutionarily conserved genes for cyst development are knocked out, resulting in cyst development defects.

[0074] In some embodiments, the cyst development defect is achieved by: at least one promoter of a cyst development stage-specific gene regulates the expression of a Cas9 protein, and at least one gRNA specific for a gene essential for cyst development, an important gene, or an evolutionarily conserved gene is designed and expressed; or at least one of the genes essential for cyst development, an important gene, or an evolutionarily conserved gene is directly knocked out.

[0075] In some embodiments, the Toxoplasma gondii strain has defects in cyst development, and / or gametogenesis / sexual reproduction, and / or defective or absent gamete, zygote, and oocyst formation.

[0076] In some embodiments, the specific gene of the Toxoplasma gametogenesis / sexual reproduction stage is selected from at least one gene of the AP2 transcription factor family gene, SEX1 (TGME49_285940), SEX2 (TGME49_255310), SEX3 (TGME49_293250), SEX4 (TGME49_295640), SEX5 (TGME49_239670), SEX6 (TGME49_291150), SEX7 (TGME49_247270), SEX8 (TGME49_234060), SEX9 (TGME49_313725), OWP1-OWP12, SSP, SOD3, or any combination thereof; more preferably, the deleted gene is a combination of SEX1 and SEX2; and / or

[0077] Preferably, the essential gene or important gene or evolutionary conserved gene for cyst development is selected from at least one of BAG1, BAG5, BFD1, LDH2, BCP1, P21, ENO1, MAG1, MAG2, SAG4, PMA1, CST1, CST2, BSR4, MIC12, MIC13, MIC17A, MIC17C, MIC12, MIC13, MIC17A, MIC17C, GRA4, GRA6, GRA12, ROP4, MCP4, BPK1, SRS9, or any combination thereof; more preferably, the knocked-out gene is GST1 and / or GST2.

[0078] In some embodiments, defective cyst development is achieved by:

[0079] At least one promoter of a cyst development stage-specific gene regulates the expression of the Cas9 protein, and at least one gRNA specific to a gene essential for cyst development, an important gene, or an evolutionarily conserved gene is designed and expressed; and / or

[0080] Directly knock out at least one of the essential genes, important genes or evolutionary conserved genes for cyst development.

[0081] In some embodiments, the Toxoplasma gondii strain has defects in cyst development, and / or impaired gametogenesis / sexual reproduction, and / or defective or absent gamete, zygote, and oocyst formation.

[0082] In some embodiments, the gene for designing the gRNA is at least one gene selected from MIC8, PKG, MYOA, CDPK1, GAP45, AKMT, DOC2.1, MAPKL1, His3, His4, His2A, His2B, CST1, CST2, GRA12, ITS1, ITS2, or any combination thereof.

[0083] In some embodiments, the recombinant Toxoplasma gondii strain is any one of the Tg-△SEX1 / SEX2:pBCP1(Cas9)-CST1 strain, the Tg-△SEX1 / SEX2:pP21(Cas9)-CST2 strain, the Tg-△SEX1 / SEX2 / CST2 strain and / or the Tg-△SEX1 / SEX2 / CST1 strain.

[0084] In a second aspect, the present application provides a live vaccine for preventing toxoplasmosis, the active ingredients of which include at least the recombinant Toxoplasma gondii strain described in any one of the first aspects above.

[0085] In some embodiments, the vaccine is a monovalent vaccine or a multivalent vaccine;

[0086] In some embodiments, the multivalent vaccine comprises the recombinant Toxoplasma gondii strain described in any one of the first aspects above, and also comprises other isolated Toxoplasma gondii isolates that do not have cross-immune protection efficacy.

[0087] In a third aspect, the present application provides a construct for encoding the recombinant Toxoplasma gondii strain described in any one of the first aspects.

[0088] In a fourth aspect, the present application provides a method for constructing a recombinant Toxoplasma gondii strain, comprising the following steps:

[0089] S1. Constructing a transfection vector that causes gamete reproduction / sexual reproduction stage-specific gene knockout and controls cyst development defects, comprising:

[0090] Introducing homologous arms at the upstream and downstream positions of the gamete reproduction / sexual reproduction stage-specific gene, and sequentially inserting the expression frame of the regulating Cas9 gene, the expression frame of the gRNA, and the expression frame of the screening gene between the two homologous arms; or

[0091] Directly knock out at least one of the essential genes, important genes, or evolutionarily conserved genes for gamete development and cyst development;

[0092] S2. The transfection vector and the plasmid of the cyst-specific gene gRNA are co-transfected into Toxoplasma gondii tachyzoites.

[0093] The transfection vector is targeted to the position of a gene specific to the gamete reproduction / sexual reproduction stage, thereby preparing a genetically recombinant Toxoplasma gondii strain with a deletion of the gamete reproduction / sexual reproduction specific gene and a cyst development defect;

[0094] Wherein: the gamete reproduction / sexual reproduction stage specific gene is at least one gene selected from the group consisting of AP2 transcription factor family genes, SEX1 (TGME49_285940), SEX2 (TGME49_255310), SEX3 (TGME49_293250), SEX4 (TGME49_295640), SEX5 (TGME49_239670), SEX6 (TGME49_291150), SEX7 (TGME49_247270), SEX8 (TGME49_234060), SEX9 (TGME49_313725), OWP1-OWP12, SSP, and SOD3; preferably, it is the SEX1 gene; and / or

[0095] The promoter gene regulating the expression of the Cas gene is selected from BAG1, BAG5, BFD1, LDH2, BCP1, P21, ENO1, MAG1, MAG2, SAG4, PMA1, CST1, CST2, BSR4, MIC12, MIC13, MIC17A, MIC17C, MIC12, MIC13, MIC17A, MIC17C, GRA4, GRA6, GRA12, ROP4, MCP4, BPK1, SRS9, or any combination thereof; and / or

[0096] The gene for designing gRNA is at least one gene selected from MIC8, PKG, MYOA, CDPK1, GAP45, AKMT, DOC2.1, MAPKL1, His3, His4, His2A, His2B, CST1, CST2, GRA12, ITS1, ITS2, or any combination thereof.

[0097] In a fifth aspect, the present application provides the use of the recombinant Toxoplasma gondii strain according to any one of the first aspects above in the preparation of a vaccine for preventing toxoplasmosis and / or other diseases in mammals.

[0098] In a sixth aspect, the present application provides the use of the recombinant Toxoplasma gondii strain according to any one of the first aspects in the preparation of drugs and / or biological products for preventing, alleviating and / or treating toxoplasmosis and / or other diseases in mammals, wherein the effective active ingredients of the drugs and / or biological products include at least the recombinant Toxoplasma strain.

[0099] In a seventh aspect, the present application provides a drug and / or biological product for preventing, alleviating and / or treating toxoplasmosis and / or other diseases in mammals, wherein the drug / biological product comprises an effective dose of the recombinant Toxoplasma gondii strain according to any one of the first aspects.

[0100] In some embodiments, the biological product comprises a vaccine.

[0101] In an eighth aspect, the present application provides a method for preventing, alleviating and / or treating toxoplasmosis and / or other diseases in mammals, which at least comprises administering an effective dose of a recombinant Toxoplasma gondii strain to a subject in need.

[0102] In some embodiments, in the method for preventing toxoplasmosis in mammals, the effective dose is 10 2 ~10 8 tachyzoites or bradyzoites of a Toxoplasma gondii strain.

[0103] In some embodiments, the effective dose is 10 7 ~10 8 or 105 ~10 6 or 10 4 ~10 5 or 10 3 ~10 4 or 10 2 ~10 3 tachyzoites or bradyzoites.

[0104] In some embodiments, 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 tachyzoites or bradyzoites.

[0105] In some embodiments, the administration route includes intramuscular injection, subcutaneous injection or oral administration. Specifically, the administration route is a vaccination route.

[0106] In some embodiments, the method comprises a single immunization and / or a booster immunization.

[0107] In other embodiments, booster immunization is performed on days 14 to 90 after the first immunization, and the booster immunization is performed by administering an effective dose to the subject more than once.

[0108] Example 1. Construction and evaluation of the Tg-ΔSEX1 / SEX2:pBCP1(Cas9)-CST1 strain

[0109] Taking SEX1 and SEX9 as target genes respectively, CRISPR / Cas9 technology was used to achieve SEX1 and SEX2 gene deletion, and CRISPR / Cas9-kill technology was used to knock out conserved sequences during the encapsulation stage. Specifically, the left homologous arm (sequence shown in SEQ ID NO.1) and the right homologous arm (sequence shown in SEQ ID NO.2) of the SEX1 gene were amplified respectively, and the screening gene expression cassette 1 (sequence shown in SEQ ID NO.3), the Cas9 gene (sequence shown in SEQ ID NO.5) expression cassette regulated by the BCP1 gene promoter (sequence shown in SEQ ID NO.4), and the gRNA (sequence shown in SEQ ID NO.7) expression cassette for the CST1 conserved sequence regulated by the Toxoplasma U6 promoter (sequence shown in SEQ ID NO.6) were added between the two homologous arms to form three expression cassette transfection vectors; at the same time, the left homologous arm (sequence shown in SEQ ID NO.8) and the right homologous arm (sequence shown in SEQ ID NO.9) of the SEX2 gene were amplified respectively, and the screening gene expression cassette 2 (sequence shown in SEQ ID NO.10) was added between the two homologous arms to form a dual expression cassette vector. The above two transfection vectors were co-transfected with the Cas9 circular plasmids targeting the gRNA for the SEX1 gene (sequence shown in SEQ ID NO.11) and the gRNA for the SEX9 gene (sequence shown in SEQ ID NO.12) into Toxoplasma gondii tachyzoites ( Figure 1 After dual-drug pressure passage, the monoclonal strain was screened and identified, and its safety and efficacy as a toxoplasmosis vaccine was evaluated.

[0110] Experimental Example 1.1 Construction of transfection vector

[0111] (1) PCR amplification of each element

[0112] Using Q5 high-fidelity DNA polymerase, the primers listed in Table 1 were used to amplify the left and right homology arms of SEX1 and SEX9, the BCP1 promoter, the selection gene expression cassette, and the Cas9 and gRNA gene expression cassettes from the Toxoplasma genome, the pLoxP-DHFR-mCherry plasmid (Addgene, SEQ ID NO: #70147), the pCAT-YFP-TetR plasmid (Addgene, SEQ ID NO: #59018), and the pSAG1::CAS9-U6::sgUPRT plasmid (Addgene, SEQ ID NO: #54467). The PCR amplification system is shown in Table 2.

[0113] Table 1. Primers

[0114]

[0115]

[0116] Table 2. PCR amplification system

[0117]

[0118] The reaction conditions for PCR amplification were set according to the program shown in Table 3.

[0119] Table 3. PCR amplification reaction conditions

[0120]

[0121] The PCR products were subjected to gel electrophoresis and the target fragments were recovered by gel excision.

[0122] (2) Multi-fragment connection

[0123] The concentration of the recovered DNA fragments was determined, and multiple fragment ligation was performed using the ClonExpress MultiS One Step Cloning Kit (Beijing Quanshijin Biotechnology Co., Ltd.). The optimal amount of each fragment used was [0.02 × number of base pairs in the fragment] ng (0.03 pmol). The general system for multiple fragment ligation is shown in Table 4 below.

[0124] Table 4. Ligation reaction system

[0125]

[0126] Ligation conditions: React at 37°C for 30 minutes. Transform the ligation product into competent E. coli cells and incubate on ice for 30 minutes. Heat shock the cells in a 42°C metal bath for 90 seconds, then immediately cool on ice for 2-3 minutes. Add 600 μl of resistance-free LB medium and shake at 37°C for 1 hour. Spread the medium onto an LB plate containing ampicillin resistance and incubate overnight. Select the cells for single colony screening.

[0127] (3) Identification and sequencing

[0128] Bacterial liquid PCR identification. The bacterial liquid PCR identification reaction system is shown in Table 5 below.

[0129] Table 5. Bacterial liquid PCR reaction system

[0130]

[0131] The general conditions for bacterial liquid PCR identification reaction are shown in Table 6 below.

[0132] Table 6. Bacterial liquid PCR reaction conditions

[0133]

[0134] Gel electrophoresis was used to identify whether the PCR product was a positive clone. The positive clone strain was used as a template for small-scale plasmid extraction. The plasmid extraction steps were as follows: The procedure was performed according to the instructions of the Plasmid MiniPrep Kit.

[0135] (3) Amplification of donor fragments

[0136] The primers for amplifying the donor fragment were SEX1-F1 (sequence shown in SEQ ID NO.13) / SEX1-R5 (sequence shown in SEQ ID NO.22) and SEX2-F1 (sequence shown in SEQ ID NO.23) / SEX2-R2 (sequence shown in SEQ ID NO.26), respectively, and a mini-plasmid template was used to amplify the donor fragment.

[0137] Using CRISPR / Cas9 technology, two fragments were simultaneously targeted to the SEX1 and SEX2 gene sites of Toxoplasma gondii (sequence information can be found on the ToxoDB website, SEX1: https: / / toxodb.org / toxo / app / record / gene / TGME49_285940;SEX2: https: / / toxodb.org / toxo / app / record / gene / TGME49_255310 ) to construct a recombinant Toxoplasma gondii strain expressing Cre enzyme.

[0138] 1.0×10 7 Each Toxoplasma tachyzoite was resuspended in 100 μl of nuclear transfer buffer. The donor fragments SEX1 and SEX2 were added to the pSAG1::CAS9-U6::sgSEX1::sgSEX2 plasmid at a ratio of 1:1:5, with a total volume of approximately 10 μg. This mixture was quickly added to the electroporation cuvette. The nuclear transfer apparatus 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. After pipetting and aspiration, the medium was aspirated into a 1.5 ml centrifuge tube. 200 μl of the transfected Toxoplasma tachyzoite solution was added to pre-cultured HFF cells. After 24 hours, the parasites were observed for luminescence, and pyrimethamine and chloramphenicol were added to the culture medium for selection. After three generations of selection, a gene-edited Toxoplasma gondii strain was obtained.

[0139] Experimental Example 1.2 Performance Evaluation and Analysis

[0140] Furthermore, the gene-edited Toxoplasma gondii strain was subjected to monoclonal purification, identification, genetic stability, biology and pathogenicity analysis.

[0141] (1) Purification and identification of monoclonal gene-edited Toxoplasma gondii strains

[0142] Gene-edited Toxoplasma gondii strains were counted and diluted to contain 2-3 tachyzoites per 10 μl. The strain was then inoculated into pre-cultured HFF cells (96-well plate), with 10 μl added to each well. Seven to 10 days after inoculation, the number of plaques formed in each well was observed. Wells with single plaques were selected for digestion and amplification in 24-well plates. After tachyzoites were released, half were collected for PCR analysis, and the remaining tachyzoites were amplified.

[0143] (2) Genetic stability analysis

[0144] The correctly identified monoclonal strain was serially subcultured in HFF cells, and PCR identification was performed every five generations to determine the genetic stability of the gene editing.

[0145] (3) Plaque assay to analyze the biological characteristics of tachyzoite proliferation

[0146] Cell preparation: HFF cells were plated in 6-well plates and cultured in a 37°C 5% CO2 cell culture incubator for 5-7 days;

[0147] Insect strain infection: Freshly released tachyzoites were purified and counted. After counting, 500 tachyzoites were inoculated into a 6-well plate filled with HFF cells and cultured in a cell culture incubator for 7 days.

[0148] Staining: Fix with 75% alcohol for 10 minutes, stain with crystal violet, wash with PBS and double-distilled water, and store in an airtight container.

[0149] Statistics: Scan and take pictures, and make statistics and comparative analysis on the plaque formation.

[0150] (4) Safety analysis of recombinant Toxoplasma gondii strains

[0151] With 10 2 ~10 8 Cats were immunized with recombinant Toxoplasma gondii tachyzoites prepared in Example 2 above via intramuscular injection (Table 7), subcutaneous injection (Table 8), and oral administration (Table 9). Tissues including the brain, tongue, heart, pectoral muscle, leg muscle, lung, liver, and spleen were examined at different time points for the presence of cysts. These tissues were then homogenized and inoculated into mice to assess the development of symptoms and mortality. Fecal oocysts were also detected and counted 3-14 days after inoculation to assess safety.

[0152] (5) Analysis of the immune protection efficacy of recombinant Toxoplasma gondii strains

[0153] On the 14th day after immunization, mice were orally inoculated with Toxoplasma gondii cysts for parasitic infection. Tissues such as the brain, tongue, heart, chest muscle, leg muscle, lung, liver, and spleen were examined for the presence of cysts. The above tissues were homogenized and inoculated into mice to detect the occurrence of symptoms and death. At the same time, fecal oocysts were detected and counted 3-14 days after infection to evaluate their effectiveness.

[0154] The results of the above verification example are as follows Figure 2 As shown, SEX1 and SEX2 genes were successfully knocked out, and their genetic traits were stable ( Figure 2 A). Plaque assays showed that the gene-edited Toxoplasma gondii strain Tg-△SEX1 / SEX2:pBCP1(Cas9)-CST1 had no significant difference in reproductive capacity compared to the parent strain ( Figure 2 B).

[0155] The safety evaluation results showed that no cysts or oocysts were detected in all cat tissues after immunization, confirming that the gene-edited Toxoplasma gondii has good safety (Table 7, Table 8, Table 9).

[0156] The results of the immune protection efficacy evaluation showed that no cysts and oocysts were detected in all cat tissues after infection, confirming that the gene-edited Toxoplasma gondii had good immune efficacy (Table 7, Table 8, Table 9).

[0157] Table 7. Safety and efficacy evaluation of intramuscular immunization of Tg-△SEX1 / SEX2:pBCP1(Cas9)-CST1 strain

[0158]

[0159]

[0160] Note: - means no cysts were detected by morphological and molecular biological methods; a: Clinical symptoms: the number and symptoms of clinical symptoms should be recorded in detail.

[0161] Table 8. Safety and efficacy evaluation of subcutaneous immunization of Tg-△SEX1 / SEX2:pBCP1(Cas9)-CST1 strain

[0162]

[0163]

[0164] Note: - means no cysts were detected by morphological and molecular biological methods; a: Clinical symptoms: the number and symptoms of clinical symptoms should be recorded in detail.

[0165] Table 9. Safety and efficacy evaluation of oral immunization of Tg-△SEX1 / SEX2:pBCP1(Cas9)-CST1 strain

[0166]

[0167] Note: - means no cysts were detected by morphological and molecular biological methods; a: Clinical symptoms: the number and symptoms of clinical symptoms should be recorded in detail.

[0168] Example 2. Construction and evaluation of the Tg-ΔSEX1 / SEX2:pP21(Cas9)-CST2 strain

[0169] This embodiment has the same construction strategy as that of embodiment 1 ( Figure 3 ), mainly for the expansion of the scope of validation of specific gene promoters and knockout target genes in the cyst stage. When constructing the transfection vector, the promoter regulating Cas gene expression was replaced with the P21 (sequence shown in SEQ ID NO. 27) expression cassette and the gRNA for the CST2 gene regulated by the Toxoplasma U6 promoter (sequence shown in SEQ ID NO. 28). The remaining operations were the same as in Example 1 to evaluate the safety and efficacy of the toxoplasmosis vaccine.

[0170] The primers used to amplify the P21 gene promoter in this example are shown in Table 10, and the remaining operations are the same as those in Example 1.

[0171] Table 10. Primers

[0172]

[0173] The results of Example 2 are as follows Figure 4 As shown, SEX1 and SEX2 genes were successfully knocked out, and their genetic traits were stable ( Figure 4 A). Plaque assays showed that the gene-edited Toxoplasma gondii strain Tg-△SEX1 / SEX2:p21(Cas9)-CST2 had no significant difference in reproductive capacity compared to the parent strain ( Figure 4 B).

[0174] The safety evaluation results showed that no cysts and oocysts were detected in the tissues of all cats after immunization, confirming that the gene-edited Toxoplasma gondii has good safety (Table 11, Table 12, Table 13).

[0175] The results of the immune protection efficacy evaluation showed that no cysts and oocysts were detected in the tissues of all cats after infection, confirming that the gene-edited Toxoplasma gondii had good immune efficacy (Table 11, Table 12, Table 13).

[0176] Table 11. Safety and efficacy evaluation of intramuscular immunization of Tg-△SEX1 / SEX2:p21(Cas9)-CST2 strain

[0177]

[0178]

[0179] Note: - means no cysts were detected by morphological and molecular biological methods; a: Clinical symptoms: the number and symptoms of clinical symptoms should be recorded in detail.

[0180] Table 12. Safety and efficacy evaluation of subcutaneous immunization of Tg-△SEX1 / SEX2:p21(Cas9)-CST2 strain

[0181]

[0182]

[0183] Note: - means no cysts were detected by morphological and molecular biological methods; a: Clinical symptoms: the number and symptoms of clinical symptoms should be recorded in detail.

[0184] Table 13. Safety and efficacy evaluation of oral immunization of Tg-△SEX1 / SEX2:p21(Cas9)-CST2 strain

[0185]

[0186] Note: - means no cysts were detected by morphological and molecular biological methods; a: Clinical symptoms: the number and symptoms of clinical symptoms should be recorded in detail.

[0187] Example 3. Construction and evaluation of the Tg-ΔSEX1 / SEX2 / CST2 strain

[0188] The construction strategy of this example adopts the simultaneous knockout of genes related to gamete reproduction / sexual reproduction and cyst development ( Figure 5 ), with the target genes being SEX1, SEX2, and CST2. When constructing the transfection vector, the transfection vectors for the SEX1 and SEX2 genes were constructed based on Example 1, except that the expression cassettes regulating the Cas9 gene and gRNA were removed. The knockout vector for knocking out the CST2 gene was constructed using the same strategy as in Example 1. The left homology arm (sequence shown in SEQ ID NO. 31) and the right homology arm (sequence shown in SEQ ID NO. 32) were amplified from the Toxoplasma gondii genome using the amplification primers shown in Table 14. The remaining procedures were the same as in Example 1, and the safety and efficacy of the toxoplasmosis vaccine were evaluated.

[0189] Table 14. Primers

[0190]

[0191] The results of Example 3 are as follows Figure 6 As shown, SEX1, SEX2 and CST2 genes were successfully knocked out, and their genetic traits were stable ( Figure 6 A). Plaque assays showed that the gene-edited Toxoplasma gondii strain Tg-△SEX1 / SEX2 / CST2 had no significant difference in reproductive capacity compared to the parent strain ( Figure 6 B).

[0192] The safety evaluation results showed that no cysts or oocysts were detected in all cat tissues after immunization, confirming that the gene-edited Toxoplasma gondii has good safety (Table 15, Table 16, Table 17).

[0193] The results of the immune protection efficacy evaluation showed that no cysts and oocysts were detected in all cat tissues after infection, confirming that the gene-edited Toxoplasma gondii had good immune efficacy (Table 15, Table 16, Table 17).

[0194] Table 15. Safety and efficacy evaluation of intramuscular immunization with Tg-△SEX1 / SEX2 / CST2 strain

[0195]

[0196]

[0197] Note: - means no cysts were detected by morphological and molecular biological methods; a: Clinical symptoms: the number and symptoms of clinical symptoms should be recorded in detail.

[0198] Table 16. Safety and efficacy evaluation of subcutaneous immunization with Tg-△SEX1 / SEX2 / CST2 strain

[0199]

[0200] Note: - means no cysts were detected by morphological and molecular biological methods; a: Clinical symptoms: the number and symptoms of clinical symptoms should be recorded in detail.

[0201] Table 17. Safety and efficacy evaluation of oral immunization with Tg-△SEX1 / SEX2 / CST2 strain

[0202]

[0203] Note: - means no cysts were detected by morphological and molecular biological methods; a: Clinical symptoms: the number and symptoms of clinical symptoms should be recorded in detail.

[0204] Example 4. Construction and evaluation of the Tg-ΔSEX1 / SEX2 / CST1 strain

[0205] The construction strategy of this example adopts the simultaneous knockout of genes related to gamete reproduction / sexual reproduction and cyst development ( Figure 7 ), with the target genes being SEX1, SEX2, and CST1. When constructing the transfection vector, the transfection vectors for the SEX1 and SEX2 genes were constructed based on Example 1, except that the expression cassettes regulating the Cas9 gene and gRNA were removed. The knockout vector for knocking out the CST2 gene was constructed using the same strategy as in Example 1. The left homology arm (sequence shown in SEQ ID NO. 37) and the right homology arm (sequence shown in SEQ ID NO. 38) were amplified from the Toxoplasma gondii genome using the amplification primers shown in Table 18. The remaining procedures were the same as in Example 1, and the safety and efficacy of the toxoplasmosis vaccine were evaluated.

[0206] Table 18. Primers

[0207]

[0208] The results of Example 4 are as follows Figure 8 As shown, SEX1, SEX2 and CST1 genes were successfully knocked out, and their genetic traits were stable ( Figure 8 A). Plaque assays showed that the gene-edited Toxoplasma gondii strain Tg-△SEX1 / SEX2 / CST1 had no significant difference in reproductive capacity compared to the parent strain ( Figure 8 B).

[0209] The safety evaluation results showed that no cysts and oocysts were detected in all cat tissues after immunization, confirming that the gene-edited Toxoplasma gondii has good safety (Table 19, Table 20, Table 21).

[0210] The results of the immune protection efficacy evaluation showed that no cysts and oocysts were detected in all cat tissues after infection, confirming that the gene-edited Toxoplasma gondii had good immune efficacy (Table 19, Table 20, Table 21).

[0211] Table 19. Safety and efficacy evaluation of intramuscular immunization with Tg-△SEX1 / SEX2 / CST1 strain

[0212]

[0213]

[0214] Note: - means no cysts were detected by morphological and molecular biological methods; a: Clinical symptoms: the number and symptoms of clinical symptoms should be recorded in detail.

[0215] Table 20. Safety and efficacy evaluation of subcutaneous immunization with Tg-△SEX1 / SEX2 / CST1 strain

[0216]

[0217] Note: - means no cysts were detected by morphological and molecular biological methods; a: Clinical symptoms: the number and symptoms of clinical symptoms should be recorded in detail.

[0218] Table 21. Safety and efficacy evaluation of oral immunization with the Tg-△SEX1 / SEX2 / CST1 strain

[0219]

[0220] Note: - means no cysts were detected by morphological and molecular biological methods; a: Clinical symptoms: the number and symptoms of clinical symptoms should be recorded in detail.

[0221] As shown in the examples and results above, the recombinant Toxoplasma gondii strain demonstrated excellent immune protection against cats infected with Toxoplasma cysts and oocysts after intramuscular, subcutaneous, and oral administration. This demonstrates that the feline toxoplasmosis-specific gene-deleted vaccine developed by the present invention, utilizing a conditional deletion strategy combined with target gene selection and formulation, can ensure both high efficacy and safety.

[0222] In summary, the toxoplasmosis vaccine developed by the present invention using gene editing technology can effectively resist Toxoplasma infection through a combination of different target genes, with good safety and high efficacy.

[0223] The specific embodiments described above are only preferred embodiments of the present invention, but are not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. A recombinant Toxoplasma gondii strain, characterized in that: In the final host, the specific gene deletion of the gametogenesis / sexual reproduction stage of Toxoplasma gondii results in gametogenesis / sexual reproduction disorder; and / or The essential genes, important genes or evolutionary conserved genes for cyst development are knocked out, resulting in cyst development defects; Wherein, preferably, the gamete reproduction / sexual reproduction stage specific gene is selected from at least one gene of the AP2 transcription factor family gene, SEX1, SEX2, SEX3, SEX4, SEX5, SEX6, SEX7, SEX8, SEX9, OWP1-OWP12, SSP, SOD3, or any combination thereof; more preferably, the deleted gene is a combination of SEX1 and SEX2; Among them, preferably, the essential gene or important gene or evolutionary conserved gene for cyst development is selected from at least one of BAG1, BAG5, BFD1, LDH2, BCP1, P21, ENO1, MAG1, MAG2, SAG4, PMA1, CST1, CST2, BSR4, MIC12, MIC13, MIC17A, MIC17C, MIC12, MIC13, MIC17A, MIC17C, GRA4, GRA6, GRA12, ROP4, MCP4, BPK1, SRS9, or any combination thereof; more preferably, the knocked-out gene is GST1 and / or GST2.

2. The recombinant Toxoplasma gondii strain according to claim 1, characterized in that The cyst developmental defect is achieved by: At least one promoter of a cyst development stage-specific gene regulates the expression of the Cas9 protein, and at least one gRNA specific to a gene essential for cyst development, an important gene, or an evolutionarily conserved gene is designed and expressed; and / or Directly knock out at least one of the essential genes, important genes or evolutionary conserved genes for cyst development.

3. The recombinant Toxoplasma gondii strain according to claim 1, characterized in that The Toxoplasma gondii strain has cyst developmental defects, and / or Gametogenesis / sexual reproduction disorders, and / or Defective or absent formation of gametes, zygotes, and oocysts.

4. The recombinant Toxoplasma gondii strain according to claim 2, characterized in that The gene for designing the gRNA is at least one gene selected from MIC8, PKG, MYOA, CDPK1, GAP45, AKMT, DOC2.1, MAPKL1, His3, His4, His2A, His2B, CST1, CST2, GRA12, ITS1, ITS2, or any combination thereof.

5. The recombinant Toxoplasma gondii strain according to claim 1, characterized in that The Toxoplasma gondii strains include Tg-△SEX1 / SEX2:pBCP1(Cas9)-CST1 strain, Tg-△SEX1 / SEX2:pP21(Cas9)-CST2 strain, Tg-△SEX1 / SEX2 / CST2 strain and / or Tg-△SEX1 / SEX2 / CST1 strain.

6. A live vaccine for preventing toxoplasmosis, the active ingredient of which comprises at least the recombinant Toxoplasma gondii strain according to any one of claims 1 to 5; 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 to 5, and further comprises other isolated Toxoplasma gondii isolates that do not have cross-immune protective efficacy.

7. A construct encoding the recombinant Toxoplasma gondii strain according to any one of claims 1 to 5.

8. A method for constructing a recombinant Toxoplasma gondii strain, characterized in that: The method comprises the following steps: S1. Constructing a transfection vector that deletes genes specific to the gamete reproduction / sexual reproduction stage and controls cyst development defects, comprising: Introducing homologous arms at the upstream and downstream positions of the gamete reproduction / sexual reproduction stage-specific gene, and sequentially inserting the expression frame of the regulating Cas9 gene, the expression frame of the gRNA, and the expression frame of the screening gene between the two homologous arms; or Directly knock out at least one of the essential genes, important genes, or evolutionarily conserved genes for gamete development and cyst development; S2. Co-transfecting the transfection vector and the plasmid of the cyst-specific gene gRNA into Toxoplasma gondii tachyzoites, The transfection vector is targeted to the position of a gene specific to the gamete reproduction / sexual reproduction stage, thereby preparing a genetically recombinant Toxoplasma gondii strain with gamete reproduction / sexual reproduction disorders and cyst development defects; Preferably, the gamete reproduction / sexual reproduction stage-specific gene is at least one gene selected from the group consisting of AP2 transcription factor family genes, SEX1, SEX2, SEX3, SEX4, SEX5, SEX6, SEX7, SEX8, SEX9, OWP1-OWP12, SSP, SOD3, or any combination thereof; preferably, SEX1 gene; and / or The essential gene, important gene or evolutionary conserved gene for cyst development is selected from at least one of BAG1, BAG5, BFD1, LDH2, BCP1, P21, ENO1, MAG1, MAG2, SAG4, PMA1, CST1, CST2, BSR4, MIC12, MIC13, MIC17A, MIC17C, MIC12, MIC13, MIC17A, MIC17C, GRA4, GRA6, GRA12, ROP4, MCP4, BPK1, SRS9 or any combination thereof; and / or The promoter gene for regulating the expression of the Cas gene is selected from at least one gene or any combination thereof selected from BAG1, BAG5, BFD1, LDH2, BCP1, P21, ENO1, MAG1, MAG2, SAG4, PMA1, CST1, CST2, BSR4, MIC12, MIC13, MIC17A, MIC17C, MIC12, MIC13, MIC17A, MIC17C, GRA4, GRA6, GRA12, ROP4, MCP4, BPK1, and SRS9, which are specifically expressed in the cyst stage of Toxoplasma gondii; and / or The gene for designing the gRNA is at least one gene selected from MIC8, PKG, MYOA, CDPK1, GAP45, AKMT, DOC2.1, MAPKL1, His3, His4, His2A, His2B, CST1, CST2, GRA12, ITS1, ITS2, or any combination thereof.

9. Use of the recombinant Toxoplasma gondii strain according to any one of claims 1 to 5 in the preparation of a vaccine for preventing toxoplasmosis and / or other diseases in mammals.

10. Use of the recombinant Toxoplasma gondii strain according to any one of claims 1 to 5 in the preparation of a drug and / or biological product for preventing, alleviating and / or treating toxoplasmosis and / or other diseases in mammals, characterized in that: The effective active ingredients of the medicine and / or biological product include at least the recombinant Toxoplasma gondii strain according to any one of claims 1 to 5.

11. A drug and / or biological product for preventing, alleviating and / or treating toxoplasmosis and / or other diseases in mammals, characterized in that: The drug / biological product comprises an effective dose of the recombinant Toxoplasma gondii strain according to any one of claims 1 to 5; Preferably, the biological product comprises a vaccine.

12. A method for preventing, alleviating and / or treating toxoplasmosis and / or other diseases in mammals, comprising at least administering an effective dose of a recombinant Toxoplasma gondii strain to a subject in need thereof; Preferably, in the method for preventing toxoplasmosis in mammals, the effective dose is 10 2 ~10 8 tachyzoites or bradyzoites of the Toxoplasma gondii strain; 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 tachyzoites or bradyzoites; 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 tachyzoites or bradyzoites.

13. The method according to claim 12, characterized in that The routes of administration include intramuscular injection, subcutaneous injection or oral administration; Preferably, the administration route is a vaccination route.

14. The method according to claim 12, characterized in that The method comprises single immunization and / or booster immunization; Preferably, booster immunization is performed on day 14 to 90 after the first immunization, wherein the booster immunization is performed by administering an effective dose to the subject more than once.

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