Gene-deleted recombinant toxoplasma gondii strain vaccine and construction method thereof

Abstract

The invention belongs to the field of gene engineering, and relates to a gene-deleted recombinant toxoplasma gondii strain vaccine and a construction method thereof, in particular to a gene-deleted recombinant toxoplasma gondii strain, a drug and / or a biological product containing the gene-deleted recombinant toxoplasma gondii strain, a construction method and application of the gene-deleted recombinant toxoplasma gondii strain. The toxoplasmosis vaccine with attenuated activity developed based on the recombinant toxoplasmosis strain is suitable for immunoprophylaxis of toxoplasmosis of various mammals, can block transmission of toxoplasmosis to people and other animals, and is especially suitable for immunoprophylaxis of cat toxoplasmosis infection and / or toxoplasmosis. The vaccine is easy to produce, high in safety, excellent in efficacy, long in immunization duration, capable of distinguishing vaccine immunization from natural infection, free of pollution to animals and the environment, capable of effectively reducing residues of toxoplasma oocysts in the environment and reducing the risk that people are infected with toxoplasmosis, and has huge application potential and important public health significance.

Description

Technical Field

[0001] The present disclosure belongs to the field of genetic engineering, and relates to a gene-deficient recombinant Toxoplasma gondii strain vaccine and a construction method thereof, specifically, a gene-deficient recombinant Toxoplasma gondii strain, a drug and / or biological product containing the same, a construction method and application thereof. The present disclosure provides a recombinant Toxoplasma gondii strain with genes deficient in Toxoplasma cyst, schizogony and sexual (gamete) reproductive development, a construction method and application thereof in drugs or biological products. Background Art

[0002] Companion animals are closely related to human life and have a certain impact on human happiness index. Dogs and cats are the main companion animals kept worldwide. As companion animals come into close contact with humans, the risk of zoonotic diseases caused by the zoonotic pathogens they carry increases. Among the zoonotic pathogens carried by cats, the most serious one is Toxoplasma gondii. For example, Toxoplasma infection causes miscarriage in pregnant women, blindness and intellectual disability in fetuses, and activation of Toxoplasma cysts causes disability and death in people with reduced immune capacity. Therefore, the prevention or treatment of zoonotic diseases is related to national plans such as human life and health, eugenics, and has important public health significance.

[0003] The life cycle of Toxoplasma gondii is divided into schizogony and sexual (gametic) reproduction in the definitive host cat and asexual reproduction stages in the intermediate host. The asexual reproduction stage includes the tachyzoite stage of rapid reproduction of the parasite during the acute infection period and the bradyzoite / cyst stage during the chronic infection period, while the stages in cats include the schizogony stage, the gametic reproduction stage, and the oocyst formation and sporulation development stage in the environment after the oocyst is expelled from the cat. Pseudocysts containing tachyzoites, cysts containing bradyzoites, and oocysts containing sporozoites are infective to both intermediate and definitive hosts. After tachyzoites, bradyzoites or sporozoites infect the definitive host cat, the invasion process is the same as that of the intermediate host. They asexually proliferate in the epithelial cells of the small intestinal mucosa of cats, and after several generations of fission proliferation, some merozoites develop into female gametes and some develop into male gametes. The female and male gametes combine and fertilize to become zygotes, and finally develop into oocysts that are excreted from the body with feces. Under suitable external environmental conditions, the oocysts mature after 2-5 days of development. Mature oocysts are infectious and are the main source of toxoplasmosis infection in humans and other intermediate hosts. Therefore, the key to the prevention and control of toxoplasmosis in cats lies in the prevention and control of toxoplasmosis in cats. Controlling the excretion of oocysts by cats into the external environment is a way to block the spread of toxoplasmosis and is also a source prevention and control technology.

[0004] In view of the characteristics of Toxoplasma's reproduction and development in the definitive host and the intermediate host, the development of a cat toxoplasmosis vaccine must meet two objectives at the same time: (1) provide immune protection against the sexual reproduction stage after immunization, that is, vaccinated cats cannot excrete oocysts after being infected with Toxoplasma gondii again, ensuring safety for humans and the environment; (2) provide immune protection against the development stage from tachyzoites to bradyzoites (cysts) after immunization, that is, reduce or block the formation of cysts. Summary of the invention

[0005] In view of the current state of the art and the above-mentioned technical difficulties, the inventor team of the present disclosure combines CRISPR / Cas9 technology and conditional knockout system as an effective strategy, specifically, the development of vaccine components based on live worm components, in order to seek to break through the technical limitations of the safety of live vaccines for toxoplasmosis, so as to provide a gene-deleted vaccine for toxoplasmosis, its construction method and application. Specifically, the present disclosure provides a recombinant Toxoplasma strain with defects in Toxoplasma cysts, schizogony and sexual (gamete) reproductive development, an attenuated live vaccine, a construction method of the recombinant Toxoplasma strain and its use. The vaccine is suitable for immunization against toxoplasmosis in a variety of mammals, especially toxoplasmosis in cats, and can effectively block the spread of toxoplasmosis to humans and other animals.

[0006] The first aspect of the present disclosure provides a gene-deficient recombinant Toxoplasma gondii strain, comprising:

[0007] The Cre enzyme is specifically expressed only in the cyst, schizogony and / or gametogenesis stages; and

[0008] LoxP sequences are sequentially inserted upstream and downstream of developmentally essential genes, key genes regulating cyst formation, key genes regulating late schizogony and / or gamete reproduction development, or their internal positions to knock out at least one of the developmentally essential genes, key genes regulating cyst formation, late schizogony genes, and key genes regulating gamete reproduction development, to obtain a genetically recombinant Toxoplasma gondii strain.

[0009] Furthermore, two or more genes among the developmentally essential genes, key genes regulating cyst formation, late schizogony genes and key genes regulating gamete reproductive development are knocked out.

[0010] Further, the Toxoplasma gondii strain has defects in cysts, late schizogony and / or gametogenesis. Preferably, the genetically recombinant Toxoplasma gondii strain has defects in cysts; preferably, the Toxoplasma gondii strain has defects in late schizogony; preferably, the Toxoplasma gondii strain has defects in gametogenesis; more preferably, the Toxoplasma gondii strain has defects in cysts and late schizogony; or has defects in cysts and gametogenesis, or has defects in late schizogony and gametogenesis. More preferably, the Toxoplasma gondii strain has defects in cysts, late schizogony and gametogenesis.

[0011] The target gene is any combination of essential genes that are continuously expressed in different life cycle stages of Toxoplasma and regulatory genes that are specifically expressed in stages.

[0012] Preferably, the target gene is a combination of at least two genes selected from essential genes of life history stages, AP2 transcription factor family genes, CST family genes, OWP-type genes, eIF4, AMA1, IMP1, BAG1, mNG, ENO1, LDH2, and genes specifically expressed in schizogony 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 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] A second aspect of the present disclosure provides a live attenuated vaccine for preventing Toxoplasma infection and / or toxoplasmosis, the active ingredients of which include at least the recombinant Toxoplasma strain described in any one of the above items; preferably, the vaccine is a monovalent vaccine or a multivalent vaccine; more preferably, the multivalent vaccine contains the genetically recombinant Toxoplasma strain described in any one of the above items, and also contains other isolated Toxoplasma strains that do not have cross-immune protection efficacy.

[0015] The third aspect of the present disclosure provides a method for constructing a gene-deficient recombinant Toxoplasma gondii strain, which comprises the steps of:

[0016] S1. Constructing a genetically recombinant insect strain that specifically expresses Cre enzyme in the cyst formation, schizogony and / or gametogenesis stages, comprising:

[0017] Constructing a shuttle transfection vector with dual expression cassettes or multiple expression cassettes, which includes a promoter for expressing genes using the characteristics of the cyst, schizogony and / or gamete reproduction stages, sequentially connecting the gene sequence of the Cre enzyme and the 3' non-transcribed region of Toxoplasma gondii, and introducing homology arms at both ends;

[0018] Targeting the shuttle transfection vector to the position of the first target gene or its vicinity, thereby constructing a recombinant Toxoplasma gondii strain ToxoBJ-cre strain expressing the Cre enzyme;

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

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

[0021] Furthermore, 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 cycle stages of Toxoplasma gondii and regulatory genes that are stage-specifically expressed.

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

[0023] Preferably, the second target gene is one or more genes selected from essential genes for life history stages, AP2 transcription factor family genes, CST family genes, OWP-type genes, eIF4, AMA1, IMP1, BAG1, mNG, ENO1, LDH2, and genes specifically expressed in schizogony 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, one or more genes derived from developmental essential genes, cyst formation-related genes, and fission reproduction and gamete reproduction-related genes.

[0025] Preferably, the promoters of genes specifically expressed in the cyst, schizogony and / or gamete reproduction stages include promoters of some genes of the AP2 transcription factor family, promoters of some genes of the BAG family, promoters of some genes of the OWP class, promoters of some genes of the CST family, promoters of some genes of the AMA family, 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 Tg-ΔBFD1 / OWP1 strain and Tg-ΔLDH2 / AP2XII-1 strain.

[0027] A fourth aspect of the present disclosure provides use of any of the above-mentioned recombinant Toxoplasma gondii strains in the preparation of a vaccine for preventing toxoplasmosis in mammals.

[0028] A fifth aspect of the present disclosure provides the use of the recombinant Toxoplasma gondii strain described in any one of the above items in the preparation of drugs and / or biological products for preventing, alleviating and / or treating toxoplasmosis in mammals, wherein the effective active components of the drugs and / or biological products at least include the genetically recombinant Toxoplasma gondii strain described in any one of the above items.

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

[0030] A sixth aspect of the present disclosure provides a drug and / or biological product for preventing, alleviating and / or treating toxoplasmosis in mammals, wherein the drug / biological product comprises an effective dose of the recombinant Toxoplasma gondii strain.

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

[0032] A seventh aspect of the present disclosure provides a method for preventing, alleviating and / or treating toxoplasmosis in mammals, which at least comprises administering an effective dose of a recombinant Toxoplasma gondii strain to a subject in need thereof.

[0033] Preferably, in the method for preventing toxoplasmosis in mammals, the effective dose is 10 2 ~10 8 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 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 time, each tachyzoite or bradyzoite is counted.

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

[0035] Preferably, the administration route is a vaccination route.

[0036] The method comprises a single immunization and / or a booster immunization; preferably, after the first immunization, a booster immunization is performed on the 30th to 90th day, and the booster immunization is to administer an effective dose to the subject more than once.

[0037] Beneficial Effects

[0038] The incomplete development of genetically modified Toxoplasma gondii strains in mammals, especially cats, can meet all the needs of cat Toxoplasma infection and / or toxoplasmosis vaccine research and development. The combination of CRISPR / Cas9 technology and conditional knockout system provides a feasible technical strategy for creating a new Toxoplasma vaccine.

[0039] Experimental results show that the developed cat toxoplasmosis vaccine can reduce the morbidity and mortality of animals caused by Toxoplasma infection, and can also effectively reduce the risk of toxoplasmosis spreading to humans. It is especially suitable for the immunization and prevention of toxoplasmosis in cats.

[0040] The present invention provides a novel design strategy for a gene-deleted attenuated active parasite strain vaccine for preventing toxoplasmosis in cats and a target gene combination ratio strategy. Under the guidance of the strategy, a vaccine for toxoplasmosis in cats is developed to ensure the safety and immune efficacy of the vaccine for toxoplasmosis in cats.

[0041] The cat toxoplasmosis gene-deficient vaccine provided by the present invention can prevent the formation of oocysts, or the excreted oocysts are minute in amount and have no infection function, and has high safety and immune efficacy.

[0042] The attenuated toxoplasmosis vaccine developed based on the recombinant Toxoplasma gondii strain is suitable for the immune prevention of toxoplasmosis in various mammals, can block the transmission of toxoplasmosis to humans and other animals, and is particularly suitable for the immune prevention of toxoplasmosis in cats. The vaccine and / or product of the present invention is easy to produce, highly safe, and has excellent efficacy. The vaccine has a long duration of immunity, can distinguish vaccine immunity from natural infection, is non-polluting to animals and the environment, can effectively reduce the residue of Toxoplasma oocysts in the environment, and reduce the risk of human infection with toxoplasmosis. It has huge application potential and important public health significance. BRIEF DESCRIPTION OF THE DRAWINGS

[0043] Figure 1 The shuttle transfection vector for constructing a dual expression frame according to the present invention is shown. Homology arms are introduced at both ends at the same time, and the shuttle transfection vector is targeted to the Toxoplasma UPRT gene site using CRISPR / Cas9 technology, thereby constructing a recombinant Toxoplasma strain ToxoBJ-Cre expressing Cre enzyme, in which the promoters of SRS22A and HAP2 genes are used to regulate the specific expression of Cre enzyme in the cyst and gamete reproductive stages.

[0044] Figure 2 The results of the identification of recombinant Toxoplasma gondii (ToxoBJ-Cre) expressing Cre enzyme are shown.

[0045] Figure 2A shows the identification of the transcriptional level of the ToxoBJ-Cre strain after continuous passage, with identification performed every 5 generations. The primers were used to amplify a partial fragment of the Cre enzyme gene. The results showed that the expression of the Cre enzyme was genetically stable.

[0046] Figure 2 B shows the fecundity of the parental strain (ToxoBJ) and the recombinant strain (ToxoBJ-Cre strain) 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.

[0047] Figure 2 C shows the survival curves of mice with different intraperitoneal inoculation doses (50, 500, and 5000 tachyzoites / mouse) of the parental strain (ToxoBJ strain) and the recombinant strain (ToxoBJ-Cre strain), indicating that there is no significant difference in virulence between the two strains to mice, and genetic manipulation has not increased pathogenicity to mice.

[0048] Figure 2 D shows the expression of Cre enzyme in the tachyzoites, cysts, merozoites, gametes and oocysts of the recombinant insect strain (ToxoBJ-Cre strain) by immunoblotting. The results showed that Cre enzyme was specifically expressed only in the cyst and gamete stages.

[0049] Figure 2 E-2F shows the expression of Cre enzyme in the cysts of the recombinant strain (ToxoBJ-Cre) and in the final host at different stages of development, as identified by indirect immunofluorescence technology. The results showed that Cre enzyme was expressed in the cysts (mouse brain tissue smear staining, Figure 2 E) and gamete stage (membrane staining of cat intestinal tissue, Figure 2 F) Specific expression.

[0050] Figure 3 The schematic diagram shows the principle of constructing the Tg-ΔBFD1 / OWP1 strain based on the ToxoBJ-cre strain according to the present invention.

[0051] The ToxoBJ-cre strain was used as the parent strain. CRISPR / Cas9 technology and the strategy of homologous recombination were used to introduce LoxP sequences on both sides of the gene loci of TgBFD1 and TgOWP1. The transfected strains were subjected to dual drug screening with pyrimethamine and chloramphenicol. After continuous passaging and monoclonal isolation and identification, the Tg-ΔBFD1 / OWP1 strain was obtained.

[0052] Figure 4 The identification results of the Tg-ΔBFD1 / OWP1 strain are shown.

[0053] Figure 4A shows the results of PCR identification of the Tg-ΔBFD1 / OWP1 monoclonal strain, in which the genomic positions of the four PCR amplifications identified are Figure 3 The monoclonal strain was identified again after 40 consecutive in vitro passages, and its genomic genetic traits remained stable.

[0054] Figure 4 B shows the fecundity test of the Tg-ΔBFD1 / OWP1 monoclonal strain and the parental strain ToxoBJ-cre. Plaque forming unit analysis was performed 24 hours after inoculation, and the results showed that there was no significant difference between the two.

[0055] Figure 4 C shows the survival curves of mice with different intraperitoneal inoculation doses (50, 500, and 5000 tachyzoites / mouse) of the parental strain (ToxoBJ-cre strain) and Tg-ΔBFD1 / OWP1 tachyzoites. The results showed that there was no significant difference in the virulence of the two to mice, and genetic manipulation did not increase the pathogenicity of tachyzoites to mice.

[0056] Figure 5 The schematic diagram of the principle of constructing the Tg-ΔLDH2 / AP2XII-1 strain based on the ToxoBJ-cre strain according to the present invention is shown, wherein the ToxoBJ-cre strain is used as the parent strain, and the CRISPR / Cas9 technology and the homologous recombination strategy are used to introduce LoxP sequences on both sides of the gene sites of TgLDH2 and TgAP2XII-1, and the transfected strain is subjected to dual drug screening with pyrimethamine and chloramphenicol, and then after continuous passage and single clone separation and identification, the Tg-ΔLDH2 / AP2XII-1 strain is obtained.

[0057] Figure 6 The identification results of the Tg-ΔLDH2 / AP2XII-1 strain are shown.

[0058] Figure 6 A shows the results of PCR identification of the Tg-ΔLDH2 / AP2XII-1 monoclonal strain, in which the genomic positions of the four PCR amplifications identified were Figure 5 After 40 generations of continuous in vitro passage, the monoclonal strain was identified again, and the genetic characteristics of the genome remained stable.

[0059] Figure 6 B shows the fecundity test of the Tg-ΔLDH2 / AP2XII-1 monoclonal strain and the parental strain ToxoBJ-cre. Plaque forming unit analysis was performed 24 hours after inoculation, and the results showed that there was no significant difference between the two.

[0060] Figure 6C shows the survival curves of mice with different intraperitoneal inoculation doses (50, 500, and 5000 tachyzoites / mouse) of the parental strain (ToxoBJ-cre strain) and Tg-ΔLDH2 / AP2XII-1 tachyzoites. The results showed that there was no significant difference in the virulence of the two to mice, and genetic manipulation did not increase the pathogenicity of tachyzoites to mice. DETAILED DESCRIPTION

[0061] In order to more clearly illustrate the embodiments of the present disclosure or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only one embodiment of the present disclosure, and for ordinary technicians in this field, other embodiments can also be obtained based on these drawings.

[0062] definition

[0063] In the present disclosure, unless otherwise specified, the scientific and technical terms and abbreviations used herein all represent the meanings commonly understood by those skilled in the art. In addition, the protein and nucleic acid chemistry, molecular biology, cell and tissue culture, microbiology, immunology and laboratory operation procedures used herein are the terms and routine procedures widely used in the corresponding fields. At the same time, in order to better understand the content of the present disclosure, the definitions and explanations of the relevant terms are provided below.

[0064] As used herein, the terms "a" and "an" and "the" and similar referents refer to both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context.

[0065] 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 the manner in which the value is measured or determined or on the limitations of the measurement system.

[0066] As used herein and unless otherwise specified, the term "about" or "approximately" means within plus or minus 10% of a given value or range, e.g., plus or minus 1%, 2%, 5%, 9%, 10%, etc. Where an integer is required, the term means within plus or minus 10% of a given value or range, rounded up or down to the nearest integer.

[0067] 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.

[0068] As used herein, "and any combination thereof" refers to any one of the optional items, or a combination of any two or more items.

[0069] As used in this article, the term "CRISPR / Cas9 technology" refers to a tool that has been optimized with Toxoplasma-related regulatory elements and can be used for precise editing of Toxoplasma genes. Specifically, the promoter responsible for expressing the Cas9 endonuclease is the promoter of Toxoplasma tublin, SAG1, actin and other genes, and the promoter responsible for expressing the guide RNA is the Toxoplasma U6 promoter.

[0070] As used in this article, the term "conditional knockout system" refers to a genetic manipulation technology for knocking out Toxoplasma genes under specific conditions and life history stages. The conditional knockout technology used in this article is mainly developed based on the Cre-loxP system, and other genetic manipulation technologies that can achieve the above purpose are also applicable.

[0071] As used in this article, the term "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.

[0072] 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 cycle stage of Toxoplasma, such as tachyzoites, bradyzoites, schizonts and gametes. If this type of gene is missing, Toxoplasma cannot complete one or more of the above life cycle stages, resulting in developmental defects.

[0073] As used in this article, the term "life history stage essential genes" refers to a class of genes that are involved in each life history stage of Toxoplasma, such as the tachyzoite, bradyzoite, schizont and gamete development stages, 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.

[0074] The first aspect of the present disclosure provides a gene-deficient recombinant Toxoplasma gondii strain, comprising:

[0075] The Cre enzyme is specifically expressed only in the cyst, schizogony and / or gametogenesis stages; and

[0076] LoxP sequences are sequentially inserted upstream and downstream of developmentally essential genes, key genes regulating cyst formation, and key genes regulating schizogony and / or gametic reproduction development, or at their internal positions, to knock out at least one of the developmentally essential genes, key genes regulating cyst formation, late schizogony genes, and key genes regulating gametic reproduction development, to obtain a genetically recombinant Toxoplasma gondii strain.

[0077] In some embodiments, two or more genes selected from the group consisting of developmentally essential genes, key genes regulating cyst formation, late schizogony genes, and key genes regulating gamete reproduction development are knocked out.

[0078] In some embodiments, the Toxoplasma gondii strain has defects in cyst, late schizogony and / or gametogenesis. Preferably, the genetically recombinant Toxoplasma gondii strain has defects in cyst development; preferably, the Toxoplasma gondii strain has defects in late schizogony; preferably, the Toxoplasma gondii strain has defects in gametogenesis; more preferably, the Toxoplasma gondii strain has defects in cyst and late schizogony; or has defects in cyst and gametogenesis, or has defects in late schizogony and gametogenesis. More preferably, the Toxoplasma gondii strain has defects in cyst, late schizogony and gametogenesis.

[0079] In some embodiments, the target gene is any combination selected from essential genes that are constitutively expressed in different life cycle stages of Toxoplasma gondii and regulatory genes that are specifically expressed in the stage.

[0080] In a preferred embodiment, the target gene is a combination of at least two genes selected from life history stage essential genes, AP2 transcription factor family genes, CST family genes, OWP-type genes, eIF4, AMA1, IMP1, BAG1, mNG, ENO1, LDH2, and genes specifically expressed in schizogony and sexual reproduction stages.

[0081] 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, ROP, RO One or more of ROP1, ROP2, 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.

[0082] The second aspect of the present disclosure provides a live attenuated vaccine for preventing toxoplasmosis, the active ingredients of which include at least any of the above-mentioned recombinant Toxoplasma strains. In a preferred embodiment, the vaccine is a monovalent vaccine or a multivalent vaccine; in a preferred embodiment, the multivalent vaccine includes any of the above-mentioned genetically recombinant Toxoplasma strains, and also includes antigens of other isolated Toxoplasma strains that are separated and do not have cross-immune protection efficacy.

[0083] The third aspect of the present disclosure provides a method for constructing a gene-deficient recombinant Toxoplasma gondii strain, which comprises the steps of:

[0084] S1. Constructing a genetically recombinant insect strain that specifically expresses Cre enzyme in the cyst formation, schizogony and / or gametogenesis stages, comprising:

[0085] Constructing a shuttle transfection vector with dual expression cassettes or multiple expression cassettes, which includes a promoter for expressing genes using the characteristics of the cyst, schizogony and / or gamete reproduction stages, sequentially connecting the gene sequence of the Cre enzyme and the 3' non-transcribed region of Toxoplasma gondii, and introducing homology arms at both ends;

[0086] The shuttle transfection vector is targeted to the position of the first target gene or its vicinity, thereby constructing a recombinant Toxoplasma gondii strain ToxoBJ-cre strain expressing the Cre enzyme;

[0087] S2. Using the ToxoBJ-cre strain as the operating object, two LoxP sequences in the same direction are sequentially inserted into the upstream, downstream or middle position of the second target gene or its gene locus to knock out the second target gene and construct a recombinant Toxoplasma gondii strain with gene deletion.

[0088] In some embodiments, in step S1, the first target gene is the uracil phosphoribosyltransferase homolog gene UPRT or other non-essential genes;

[0089] In some embodiments, 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 cycle stages of Toxoplasma gondii and regulatory genes that are stage-specifically expressed.

[0090] In a preferred embodiment, the second target gene is at least one of a developmentally essential gene, a key gene for cyst formation regulation, a late schizogony gene, and a key gene for gamete reproduction development regulation. In a preferred embodiment, the second target gene is two or more of a developmentally essential gene, a key gene for cyst formation regulation, a late schizogony gene, and a key gene for gamete reproduction development regulation.

[0091] In a preferred embodiment, the second target gene is one or more genes selected from life history stage essential genes, AP2 transcription factor family genes, CST family genes, OWP-type genes, eIF4, AMA1, IMP1, BAG1, mNG, ENO1, LDH2, and genes specifically expressed in schizogony and sexual reproduction stages.

[0092] 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, 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, one or more genes derived from developmental essential genes, cyst formation-related genes, and fission reproduction and gamete reproduction-related genes.

[0093] In a preferred embodiment, the promoters of genes specifically expressed in the encapsulation, schizogony and / or gametogenesis stages include promoters of partial genes of the AP2 transcription factor family, promoters of partial genes of the BAG family, promoters of partial genes of the OWP class, promoters of genes of the CST family, promoters of genes of the AMA family, 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.

[0094] In a preferred embodiment, the recombinant Toxoplasma gondii strains include Tg-ΔBFD1 / OWP1 strain and Tg-ΔLDH2 / AP2XII-1 strain.

[0095] A fourth aspect of the present disclosure provides use of any of the above-mentioned recombinant Toxoplasma gondii strains in the preparation of a vaccine for preventing toxoplasmosis in mammals.

[0096] The fifth aspect of the present disclosure provides the use of the recombinant Toxoplasma gondii strain described in any of the above items in the preparation of drugs and / or biological products for preventing, alleviating and / or treating toxoplasmosis in mammals, and the effective active components of the drugs and / or biological products at least include the genetically recombinant Toxoplasma strain described in any of the above items.

[0097] In some embodiments, the product is a vaccine, which may include a combination of multiple insect strains. Alternatively, the product is a nutritional composition suitable for mammals, such as a health food suitable for pets.

[0098] A sixth aspect of the present disclosure provides a drug and / or biological product for preventing, alleviating and / or treating toxoplasmosis in mammals, wherein the drug / biological product comprises an effective dose of a recombinant Toxoplasma gondii strain.

[0099] In a preferred embodiment, the biological product is a vaccine.

[0100] A seventh aspect of the present disclosure provides a method for preventing, alleviating and / or treating toxoplasmosis in mammals, which at least comprises administering an effective dose of a recombinant Toxoplasma gondii strain to a subject in need thereof.

[0101] In some embodiments, in the method for preventing toxoplasmosis in mammals, the effective dose is 10 2 ~10 8 Each time, each tachyzoite or bradyzoite of a genetically recombinant Toxoplasma gondii strain is administered. In a preferred embodiment, 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 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 time, each tachyzoite or bradyzoite is counted.

[0102] In some embodiments, the route of administration includes intramuscular injection, subcutaneous injection, or oral administration.

[0103] In a preferred embodiment, the route of administration is a vaccination route.

[0104] The method includes single immunization and / or booster immunization. In a preferred embodiment, booster immunization is performed on days 30 to 90 after the first immunization, and booster immunization is administering an effective dose to the subject more than once.

[0105] Example 1. Construction of a recombinant Toxoplasma gondii strain that specifically expresses Cre enzyme in the cyst and gamete reproductive stages

[0106] Taking the recombinant Toxoplasma gondii strain in which the SRS22A gene promoter (sequence as shown in SEQ ID NO.1) and the HAP2 gene promoter (sequence as shown in SEQ ID NO.2) regulate the specific expression of Cre enzyme in the cyst and gamete reproductive stages as an example, the promoter was amplified from the Toxoplasma genome and sequentially connected with the gene of Cre enzyme (SEQ ID NO.3), supplemented with the 3' non-transcribed region of the Toxoplasma gene, and a shuttle transfection vector with a double expression frame was constructed, and homology arms (such as Figure 1 shown).

[0107] Experimental Example 1.1 Construction of shuttle vector

[0108] (1) PCR amplification of each element

[0109] Using Q5 high-fidelity DNA polymerase, the SRS22A promoter, HAP2 promoter, Cre enzyme sequence and vector backbone were amplified from the Toxoplasma genome, Lenti-sgCrebbp#1 / Cre plasmid (Addgene, sequence number: #193209) and UPRT-mCh-Nluc-P2A-neo-UPRT (Addgene, sequence number: #135015) plasmid with the primers shown in Table 1. The PCR amplification system is shown in Table 2.

[0110] Table 1. Primers

[0111]

[0112] Table 2. PCR amplification system

[0113]

[0114] The reaction conditions for PCR amplification were set according to the program shown in Table 3.

[0115] Table 3. PCR amplification reaction conditions

[0116]

[0117] The PCR products were subjected to gel electrophoresis and the target fragments were recovered by gel excision.

[0118] (2) Multi-fragment connection

[0119] The concentration of the recovered DNA fragments was determined, and multiple fragment ligation was performed using a multi-fragment ligation kit (ClonExpress MultiS One Step Cloning Kit, a product of Beijing Quanshijin Biotechnology Co., Ltd.). The optimal amount of each fragment = [0.02 × number of fragment base pairs] ng (0.03 pmol), and the general system for multi-fragment ligation is shown in Table 4 below.

[0120] Table 4. Ligation reaction system

[0121]

[0122] Ligation conditions: react at 37℃ for 30min. Transform the ligation product into competent E. coli cells and incubate on ice for 30min; heat shock in a 42℃ metal bath for 90s, then immediately cool on ice for 2-3min. Add 600μl of LB medium without resistance and shake at 37℃ for 1h. Spread the medium on an LB plate containing ampicillin resistance and culture overnight. Pick the bacteria for single cloning.

[0123] (3) Identification and sequencing

[0124] Bacterial liquid PCR identification. The bacterial liquid PCR identification reaction system is shown in Table 5 below.

[0125] Table 5. Bacterial liquid PCR reaction system

[0126]

[0127] The general conditions for bacterial solution PCR identification reaction are shown in Table 6 below.

[0128] Table 6. Bacterial liquid PCR reaction conditions

[0129]

[0130]

[0131] The PCR product was run on gel electrophoresis to identify whether it 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 instructions of Plasmid MiniPrep Kit were followed.

[0132] (3) Amplification of donor fragments

[0133] The donor fragment was amplified using the primers for amplifying the donor fragment shown in Table 7 and a mini-plasmid template.

[0134] Table 7. Primers

[0135]

[0136] CRISPR / Cas9 technology was used to target the UPRT gene site of Toxoplasma gondii (sequence information can be found on the ToxoDB website, https: / / toxodb.org / toxo / app / record / gene / TGME49-312480) and construct a recombinant Toxoplasma gondii strain expressing Cre enzyme.

[0137] 1.0×10 7 Each Toxoplasma tachyzoite was resuspended in 100μl nuclear transfer buffer, and the donor fragment and pSAG1::CAS9-U6::sgUPRT plasmid (Addgene, sequence number: #54467) were added in a ratio of 1:5, and the total amount was controlled at about 10μg. The mixture was quickly added to the electroporation cup, the nuclear transfer instrument was turned on, and the program U-033 was selected for transfection; after the nuclear transfer was completed, 1ml DMEM culture medium was quickly added to the electroporation cup, and the culture medium was aspirated into a 1.5ml centrifuge tube after blowing and aspirating evenly, and 200μl of the Toxoplasma tachyzoite solution after the transfer was added to the pre-cultured HFF cells. After 24h, the luminescence of the parasite was observed, and fluorouridine (FUDR) was added to the culture medium for screening. After 3 generations of screening, a Toxoplasma strain expressing Cre enzyme was obtained.

[0138] Experimental Example 1.2 Performance Evaluation and Analysis

[0139] Furthermore, the Toxoplasma gondii strain expressing Cre enzyme was subjected to monoclonal purification, identification, genetic stability, expression characteristics of Cre enzyme, biology and pathogenicity analysis.

[0140] (1) Purification and identification of monoclonal recombinant Toxoplasma gondii strains

[0141] The recombinant Toxoplasma gondii was counted and diluted to 2-3 tachyzoites per 10 μl, and inoculated into the pre-cultured HFF cells (96-well cell plate), with 10 μl added to each well. The number of plaques formed in each well was observed 7-10 days after inoculation, and the cell wells with single plaques were selected for digestion and amplification culture to 24-well plates. After the tachyzoites were released, 1 / 2 of the tachyzoites were taken for PCR identification, and the remaining tachyzoites were amplified and cultured.

[0142] (2) Genetic stability analysis

[0143] The correct monoclonal strain was identified and serially subcultured in HFF cells, and PCR was performed every five generations to determine the genetic stability of the Cre enzyme gene integration.

[0144] (3) Analysis of Cre enzyme expression characteristics by immunoblotting (WB) and indirect immunofluorescence assay (IFA)

[0145] The brain cysts were collected 30 days after intraperitoneal inoculation of recombinant Toxoplasma gondii tachyzoites (50 tachyzoites / mouse), and the brain cysts were orally inoculated into three 6-month-old kittens (1000 cysts / cat). One kitten was euthanized and killed 7 and 10 days after inoculation, and the intestines, merozoites and gametes were collected and divided into two parts for WB and IFA tests, respectively. The feces of the remaining kitten was collected for 7-10 days, and the oocysts were collected using the saturated saline method for WB verification.

[0146] WB operation of different life cycle stages was carried out according to the following steps: freshly collected tachyzoites, cysts, merozoites, gametes, and oocysts were resuspended in 50 μl PBS, 15 μl 5×SDS loading buffer was added, and boiled at 100°C for 10 min. An appropriate amount of sample was taken for SDS-PAGE. After electrophoresis, the protein was transferred to the NC membrane by wet transfer method and blocked with 5% skim milk at 37°C for 1 h. After blocking, the primary antibody (Anti-Cre recombinase antibody (ab216262)) and the secondary antibody ( 800CW Goat anti-Rabbit IgG Secondary Antibody), the antibody incubation condition was 37℃1h, and after each incubation, the cells were washed 3 times with PBST and imaged by an imager.

[0147] The IFA operation of insects at different life cycle stages was carried out according to the following steps: Freshly collected samples were fixed with 4% paraformaldehyde (PFA) at room temperature for 20 minutes, and the fixative was discarded. Then, permeabilization was performed with 3% BSA containing 0.25% TritonX-100 for 20 minutes at room temperature, and the permeabilization solution was discarded after the permeabilization was completed. Blocking was performed with 3% BSA for 30 minutes at room temperature. Sealing film was laid in a metal wet box, and the primary antibody (Anti-Cre recombinaseantibody (ab216262)) and secondary antibody (Alexa Fluor 594-goat anti-rabbit IgG) corresponding to the endogenous marker were incubated at 37°C for 30 minutes. After the primary and secondary antibody incubations were completed, they were rinsed 5 times with 3% BSA containing 0.05% Tween-20. After washing, the slides were sealed and dried at 37°C for 30 minutes, and then observed under a fluorescence microscope.

[0148] (4) Plaque assay to analyze the biological characteristics of tachyzoite proliferation: 1) Cell preparation: HFF cells were plated on a 6-well plate and cultured in a 37°C 5% CO2 cell culture incubator for 5-7 days; 2) Insect infection: Freshly released tachyzoites were purified and counted. After counting, 500 tachyzoites were inoculated onto a 6-well plate covered with HFF cells and cultured in a cell culture incubator for 7 days; 3) Staining: The cells were fixed with 75% alcohol for 10 min, stained with crystal violet, washed with PBS and double distilled water respectively, and dried for storage; 4) Statistics: Scan and take pictures, and perform statistics on plaque formation and compare and analyze.

[0149] (5) Animal experiments to analyze the pathogenicity of recombinant strains: The pathogenicity test on mice was conducted as follows: 50, 500, and 500 tachyzoites of the recombinant Toxoplasma strain and the wild-type strain were intraperitoneally inoculated into 6-week-old female BALB / c mice (n=10), respectively. Each group of mice was raised in the same environment, and the survival of the mice within 30 days of infection was counted.

[0150] The results of the above verification example are as follows Figure 2 As shown, the Cre enzyme gene was targeted to the UPRT gene site, and the recombinant Toxoplasma gondii strain ToxoBJ-Cre expressing the Cre enzyme had stable genetic traits ( Figure 2 A). Plaque assays showed that the recombinant Toxoplasma gondii strain ToxoBJ-Cre had no significant difference in reproductive capacity compared with the parent strain ( Figure 2 B); the pathogenicity to mice was not significantly different from that of the parent strain ( Figure 2 C); RT-PCR ( Figure 2 A), immunoblotting ( Figure 2 D) Indirect immunofluorescence ( Figure 2 E) and other experiments confirmed that Cre enzyme is only expressed in the cyst and gamete reproduction stages.

[0151] Example 2. Construction of Tg-ΔBFD1 / OWP1 recombinant strain

[0152] The staged deletion of BFD1 (sequence information can be found on the ToxoDB website, https: / / toxodb.org / toxo / app / record / gene / TGME49_200385; SEQ ID NO.5) and OWP1 gene (sequence information can be found on the ToxoDB website, https: / / toxodb.org / toxo / app / record / gene / TGME49_204420 ; SEQ ID NO.6) of Toxoplasma gondii, the ToxoBJ-cre strain was used as the genetic manipulation object, and the CRISPR / Cas9 technology was used to sequentially and unidirectionally insert the LoxP sequence (SEQ ID NO.4) into the upstream and downstream of the above two target genes ( Figure 2 ) and successfully constructed the insect strain Tg-ΔBFD1 / OWP1 with recombinant LoxP gene.

[0153] Experimental Example 2.1 Construction of Tg-ΔBFD1 / OWP1 recombinant strain

[0154] (1) Vector construction

[0155] According to the gRNA design program provided by the ToxoDB website, gRNA screening for BFD1 and OWP1 genes was performed respectively, and the gRNA sequence in pSAG1::CAS9-U6::sgUPRT was replaced using the Q5 site-directed mutagenesis kit to form pSAG1::CAS9-U6::sgBFD1 and pSAG1::CAS9-U6::sgOWP1 plasmids.

[0156] The left and right homologous arms of the BFD1 gene were amplified using the primers shown in Table 8, and the LoxP sequence was amplified to the upstream and downstream of the BFD1 gene by extending the primers to amplify the screening expression frame expressing the DHFR resistance gene.

[0157] Table 8. Primers

[0158]

[0159] Next, the left homology arm of BFD1, the BFD1 gene carrying the LoxP site, the 3'UTR, the DHFR resistance gene expression cassette, and the right homology arm were connected by multi-fragment connection (refer to Example 1 for the connection method) to construct a knock-in donor plasmid (Donor-BFD1) of the BFD1 gene LoxP site.

[0160] The knock-in donor plasmid (Donor-OWP1) of the Loxp site of the OWP1 gene was constructed in the same manner, and the primer sequences are shown in Table 9.

[0161] Table 9. Primers

[0162]

[0163] (2) Transfection and screening

[0164] 1.0×10 7Each Toxoplasma tachyzoite was resuspended in 100 μl nuclear transfer buffer, and the donor fragments Donor-BFD1, Donor-OWP1 and pSAG1::CAS9-U6::sgBFD1, pSAG1::CAS9-U6::sgOWP1 were added in a ratio of 1:1:5:5, and the total amount was controlled at about 10 μg. The mixed solution was quickly added to the electroporation cup, the nuclear transfer instrument was turned on, and the program U-033 was selected for transfection; after the nuclear transfer was completed, 1 ml of DMEM culture medium was quickly added to the electroporation cup, and the culture medium was aspirated into a 1.5 ml centrifuge tube after blowing and aspirating evenly, and 200 μl of the Toxoplasma tachyzoite solution after the transfer was added to the pre-cultured HFF cells. Pyrimethamine and chloramphenicol were added to the culture medium for screening. After 3 generations of screening, the recombinant Toxoplasma strain was obtained.

[0165] Verification Example 2.2 Performance Evaluation and Analysis

[0166] Furthermore, the Toxoplasma gondii strain Tg-ΔBFD1 / OWP1 with conditional knockout of the target gene BFD1 / OWP1 was subjected to monoclonal purification, identification, genetic stability, and biological and pathogenicity analysis.

[0167] For the specific verification example and its steps, please refer to Verification Example 1.2 in Example 1.

[0168] The results showed that LoxP sequences were targeted to the upstream and downstream of BFD1 and OWP1 genes ( Figure 4 A), and the recombinant Toxoplasma gondii strain with LoxP integration is genetically stable ( Figure 4 A), plaque assay showed no significant difference in reproductive capacity between it and the parent strain ( Figure 4 B); the pathogenicity to the intermediate host mice was not significantly different from that of the parent strain ( Figure 4 C) shows that a recombinant Toxoplasma gondii strain with LoxP integrated into a specific site of the genome was successfully obtained.

[0169] Example 3. Evaluation of vaccine potential

[0170] Take 10 2 ~10 8 The recombinant Toxoplasma gondii strain tachyzoites prepared in Example 2 were respectively injected intramuscularly (Table 10), subcutaneously (Table 11), and orally (Table 12) into cats for immunization. The brain, tongue, heart, chest muscle, leg muscle, lung, liver, spleen and other tissues were dissected at different time points to detect the presence of cysts. The above tissues were homogenized and inoculated into mice to detect whether symptoms and death occurred. At the same time, the fecal oocysts of the cats were detected and counted on the 3rd to 14th day after inoculation to evaluate its safety.

[0171] On the 14th day after immunization, mice were orally inoculated with Toxoplasma cysts for parasite 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 parasite infection to evaluate its effectiveness.

[0172] Table 10. Safety and efficacy evaluation of intramuscular immunization of Tg-ΔBFD1 / OWP1 strain

[0173]

[0174]

[0175] Note: - means that no cysts were detected by morphological and molecular biological methods; a: Clinical symptoms should record the number and symptoms of clinical symptoms in detail.

[0176] Table 11. Safety and efficacy evaluation of subcutaneous injection of Tg-ΔBFD1 / OWP1 strain

[0177]

[0178]

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

[0180] Table 12. Safety and efficacy evaluation of oral immunization of Tg-ΔBFD1 / OWP1 strain

[0181]

[0182] Note: - means that no cysts were detected by morphological and molecular biological methods; a: Clinical symptoms should record the number and symptoms of clinical symptoms in detail.

[0183] 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 has good safety; no cysts or oocysts were detected in any cat tissues after challenge, confirming that the recombinant Toxoplasma has good immune efficacy.

[0184] Example 4. Construction of Tg-ΔLDH2 / AP2XII-1 strain

[0185] Taking the Toxoplasma gondii with staged deletion of LDH2 (sequence information available on the ToxoDB website, https: / / toxodb.org / toxo / app / record / gene / TGME49-291040; SEQ ID NO.7) and AP2XII-1 gene (sequence information available on the ToxoDB website, https: / / toxodb.org / toxo / app / record / gene / TGME49218960; SEQ ID NO.8) as an example, the ToxoBJ-cre strain was used as the genetic manipulation object, and the CRISPR / Cas9 technology was used to sequentially and unidirectionally insert the LoxP sequence (SEQ ID NO.4) into the upstream and downstream of the above target genes (the same design principle and construction steps as in Example 2, reference Figure 5 ) and constructed the LoxP-recombined Tg-ΔLDH2 / AP2XII-1 strain.

[0186] Experimental Example 4.1 Construction of Tg-ΔLDH2 / AP2XII-1 recombinant strain

[0187] (1) Vector construction

[0188] According to the gRNA design program provided by the ToxoDB website, gRNA screening for LDH2 and AP2XII-1 genes was performed respectively, and the gRNA sequence in pSAG1::CAS9-U6::sgUPRT was replaced using the Q5 site-directed mutagenesis kit to form pSAG1::CAS9-U6::sgLDH2 and pSAG1::CAS9-U6::sgAP2XII-1 plasmids.

[0189] The left and right homologous arms of the LDH2 gene were amplified using the primers shown in Table 13, and the LoxP sequence was amplified to the upstream and downstream of the LDH2 gene by extending the primers, and the screening expression frame expressing the DHFR resistance gene was amplified at the same time.

[0190] Table 13. Primers

[0191]

[0192] The left homology arm of LDH2, the LDH2 gene carrying the LoxP site, 3'UTR, the DHFR resistance gene expression cassette, and the right homology arm were connected by multi-fragment connection (refer to Example 1 for the connection method) to construct a knock-in donor plasmid (Donor-LDH2) of the LDH2 gene LoxP site.

[0193] The knock-in donor plasmid (Donor-AP2XII-1) of the AP2XII-1 gene Loxp site was constructed in the same manner, and the primers are shown in Table 14.

[0194] Table 14. Primers

[0195]

[0196] (2) Transfection and screening

[0197] 1.0×10 7 Each Toxoplasma tachyzoite was resuspended in 100 μl nuclear transfer buffer, and the donor fragments Donor-LDH2, Donor-AP2XII-1 and pSAG1::CAS9-U6::sgLDH2, pSAG1::CAS9-U6::sg AP2XII-1 were added in a ratio of 1:1:5:5, and the total amount was controlled at about 10 μg. The mixed solution was quickly added to the electroporation cup, the nuclear transfer instrument was turned on, and the program U-033 was selected for transfection; after the nuclear transfer was completed, 1 ml of DMEM culture medium was quickly added to the electroporation cup, and the culture medium was aspirated into a 1.5 ml centrifuge tube after blowing and aspirating evenly, and 200 μl of the Toxoplasma tachyzoite solution after the transfer was added to the pre-cultured HFF cells. Pyrimethamine and chloramphenicol were added to the culture medium for screening. After 3 generations of screening, the recombinant Toxoplasma strain was obtained.

[0198] Verification Example 4.2 Performance Evaluation and Analysis

[0199] The Toxoplasma gondii strain Tg-ΔΔLDH2 / AP2XII-1 with conditional knockout of the target genes LDH2 and AP2XII-1 was subjected to monoclonal purification, identification, genetic stability, and biological and pathogenicity analysis.

[0200] For the specific verification example and its steps, please refer to Verification Example 2.2 in Example 2.

[0201] The results of performance evaluation of the recombinant Tg-ΔLDH2 / AP2XII-1 strain showed that the LoxP sequence was targeted to the upstream and downstream of LDH2 and AP2XII-1 genes ( Figure 6 A), and the recombinant Toxoplasma gondii strain with LoxP integration is genetically stable ( Figure 6 A) There is no significant difference in the size of plaques formed by recombinant Toxoplasma and the parent strain after inoculation into HFF cells for 24 hours, indicating that there is no significant difference in the reproductive capacity between the recombinant Toxoplasma and the parent strain ( Figure 6 B); The survival curves and clinical symptoms of mice inoculated with different doses of the strains did not show significant effects, indicating that the pathogenicity to the intermediate host mice was not significantly different from that of the parent strain ( Figure 6 C) shows that a recombinant Toxoplasma gondii strain with LoxP integrated into a specific site of the genome was successfully obtained.

[0202] The results of the performance evaluation of the recombinant Tg-ΔLDH2 / AP2XII-1 strain showed that the LoxP sequence was targeted to the upstream and downstream of the LDH2 and AP2XII-1 genes, and the recombinant Toxoplasma strain with LoxP integration was genetically stable. There was no significant difference in the size of the plaques formed by the recombinant Toxoplasma and the parent strain after 24 hours of inoculation in HFF cells, indicating that there was no significant difference in the reproductive capacity between the recombinant Toxoplasma and the parent strain; the survival curves of mice inoculated with different doses of the strain and the clinical symptoms of the inoculated cats did not show significant effects, indicating that the pathogenicity to the intermediate host mice and the final host cats was not significantly different from that of the parent strain. This indicates that a recombinant Toxoplasma strain with LoxP integrated into a specific site of the genome was successfully obtained.

[0203] Example 5. Evaluation of vaccine potential

[0204] 10 of the Tg-ΔLDH2 / AP2XII-1 strain prepared in Example 4 2 ~10 8 After immunizing cats with tachyzoites by intramuscular injection (Table 15), subcutaneous injection (Table 16), and oral administration (Table 17), brain, tongue, heart, chest muscle, leg muscle, lung, liver, spleen and other tissues were dissected to detect the presence of cysts, and the above tissues were homogenized and inoculated into mice to detect whether symptoms and death occurred; at the same time, fecal oocysts of cats were detected and counted 3-14 days after inoculation to evaluate its safety. On the 14th day after immunization, Toxoplasma gondii cysts were orally inoculated for attack and infection, brain, tongue, heart, chest muscle, leg muscle, lung, liver, spleen and other tissues were dissected to detect the presence of cysts, and the above tissues were homogenized and inoculated into mice to detect whether symptoms and death occurred; at the same time, fecal oocysts of cats were detected and counted 3-14 days after attack and infection to evaluate its effectiveness.

[0205] Table 15. Safety and efficacy evaluation of intramuscular immunization of Tg-ΔLDH2 / AP2XII-1 strain

[0206]

[0207] Note: - means that no cysts were detected by morphological and molecular biological methods; a: Clinical symptoms should record the number and symptoms of clinical symptoms in detail.

[0208] Table 16. Safety and efficacy evaluation of subcutaneous injection of Tg-ΔLDH2 / AP2XII-1 strain

[0209]

[0210] Note: - means that no cysts were detected by morphological and molecular biological methods; a: Clinical symptoms should record the number and symptoms of clinical symptoms in detail.

[0211] Table 17. Safety and efficacy evaluation of oral immunization of Tg-ΔLDH2 / AP2XII-1 strain

[0212]

[0213]

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

[0215] The results in Tables 15-17 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 immune efficacy.

[0216] It can be seen from the above examples and results that the cat toxoplasmosis gene-deleted vaccine has good immune protection against cats infected with Toxoplasma cysts and oocysts after immunization via intramuscular, subcutaneous, oral and other routes. This proves that the cat toxoplasmosis gene-deleted vaccine developed by the present invention using conditional deletion and target gene selection and matching strategies can ensure high efficacy and high safety of the vaccine.

[0217] In summary, the toxoplasmosis deletion vaccine developed by the present invention using gene-deficient strains, the target genes are composed of different ratios, can effectively resist Toxoplasma infection, and has good safety and high efficacy.

[0218] The specific embodiments described above are only preferred embodiments of the present disclosure, but are not intended to limit the present disclosure. 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 disclosure.

Claims

1. A gene-deficient recombinant Toxoplasma gondii strain, characterized in that: The Cre enzyme is specifically expressed only in the cyst, schizogony, and / or gametogenesis stages; as well as LoxP sequences are sequentially inserted upstream and downstream of developmentally essential genes, key genes regulating cyst formation, key genes regulating late schizogony and / or gametic reproduction development, or their internal positions to knock out at least one of the developmentally essential genes, key genes regulating cyst formation, late schizogony genes, and key genes regulating gametic reproduction development, to obtain a recombinant Toxoplasma gondii strain.

2. The recombinant Toxoplasma gondii strain according to claim 1, characterized in that: Knocking out two or more genes among the developmentally essential genes, the key genes regulating cyst formation, the late schizogony genes and the key genes regulating gamete reproductive development; The Toxoplasma gondii strain has defects in cysts, late schizogony and / or gametogenesis.

3. The recombinant Toxoplasma gondii strain according to claim 1, characterized in that: The target gene to be knocked out is any combination of essential genes that are continuously expressed in different life history stages of Toxoplasma and regulatory genes that are specifically expressed in stages; Preferably, the target gene is a combination of at least two genes selected from the group consisting of essential genes for life history stages, AP2 transcription factor family genes, CST family genes, OWP-type genes, eIF4, AMA1, IMP1, BAG1, mNG, ENO1, LDH2, and genes specifically expressed in schizogony 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 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 Tg-ΔBFD1 / OWP1 strain and Tg-ΔLDH2 / AP2XII-1 strain.

4. A live attenuated vaccine for preventing toxoplasmosis, wherein the active ingredient comprises at least the recombinant Toxoplasma gondii strain according to any one of claims 1 to 3; Preferably, the vaccine is a monovalent vaccine or a multivalent vaccine; More preferably, the multivalent vaccine comprises the genetically recombinant Toxoplasma gondii strain according to any one of claims 1 to 3, and further comprises other isolated Toxoplasma gondii strains that are not cross-immune protective efficacy.

5. A method for constructing a gene-deficient recombinant Toxoplasma gondii strain, characterized in that: The method comprises the steps of: S1. Constructing a genetically recombinant insect strain that specifically expresses Cre enzyme in the cyst formation, schizogony and / or gametogenesis stages, comprising: Constructing a shuttle transfection vector with dual expression cassettes or multiple expression cassettes, which includes a promoter for expressing genes using the characteristics of the cyst, schizogony and / or gamete reproduction stages, sequentially connecting the gene sequence of the Cre enzyme and the 3' non-transcribed region of Toxoplasma gondii, and introducing homology arms at both ends; Targeting the shuttle transfection vector to the position of the first target gene or its vicinity, thereby constructing a recombinant Toxoplasma gondii strain ToxoBJ-cre strain expressing the Cre enzyme; S2. Using the ToxoBJ-cre strain as the operating object, two LoxP sequences in the same direction are sequentially inserted into the upstream, downstream or middle position of the second target gene or its gene locus to knock out the second target gene and construct a recombinant Toxoplasma gondii strain with gene deletion.

6. The method according to claim 5, characterized in that In step S1, the first target gene is the uracil phosphoribosyltransferase homolog 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 cycle stages of Toxoplasma gondii and regulatory genes that are specifically expressed in stages: Preferably, the second target gene is at least one of the developmentally essential genes, the key genes regulating cyst formation, the late schizogony genes and the key genes regulating gamete reproductive development; More preferably, the second target gene is two or more genes selected from the group consisting of the developmentally essential gene, the key gene regulating cyst formation, the late schizogony gene, and the key gene regulating gamete reproductive development; More preferably, the second target gene is one or more genes selected from essential genes for life history stages, AP2 transcription factor family genes, CST family genes, OWP-type genes, eIF4, AMA1, IMP1, BAG1, mNG, ENO1, LDH2, and genes specifically expressed in schizogony 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 of 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 in the cyst, schizogony and / or gamete reproduction stages include promoters of some genes of the AP2 transcription factor family, promoters of some genes of the BAG family, promoters of some genes of the OWP class, promoters of some genes of the CST family, promoters of genes of the AMA family, 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 Tg-ΔBFD1 / OWP1 strain and Tg-ΔLDH2 / AP2XII-1 strain.

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

8. Use of the recombinant Toxoplasma gondii strain according to any one of claims 1 to 3 in the preparation of a drug and / or biological product for preventing, alleviating and / or treating Toxoplasma infection and / or toxoplasmosis in mammals, characterized in that: The effective active components of the medicine and / or biological product include at least the genetically recombinant Toxoplasma gondii strain described in any one of claims 1-3.

9. A drug and / or biological product for preventing, alleviating and / or treating Toxoplasma infection and / or toxoplasmosis in mammals, characterized in that: The drug / biological product comprises an effective dose of the recombinant Toxoplasma gondii strain described in any one of claims 1 to 3; Preferably, the biological product comprises a vaccine.

10. A method for preventing, alleviating and / or treating Toxoplasma infection and / or toxoplasmosis in mammals, comprising at least administering an effective dose of a recombinant Toxoplasma strain to a subject in need thereof; Preferably, in the method for preventing mammalian toxoplasma infection and / or toxoplasmosis, the effective dose is 10 2 ~10 8 Each tachyzoite or bradyzoite of the recombinant Toxoplasma gondii strain is one; 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 per each; 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 time, each tachyzoite or bradyzoite is counted.

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

12. The method according to claim 10, characterized in that The method comprises a single immunization and / or a booster immunization; Preferably, booster immunization is performed on day 30 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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