AP2III-4 gene-deleted toxoplasma gondii live vaccine and construction method thereof

Abstract

The invention discloses a toxoplasma gondii live vaccine without an AP2III-4 gene and a construction method of the toxoplasma gondii live vaccine. In particular discloses an application of an AP2III-4 protein, an AP2III-4 gene or an inhibitory factor of the protein AP2III-4 in inhibiting or preventing the toxoplasma gondii from generating an oocyst. According to the invention, an AP2III-4 gene in toxoplasma gondii is knocked out through a gene editing technology, and a toxoplasma gondii strain with the deleted AP2III-4 gene is obtained. Results show that the strain maintains relatively high immune protection efficacy while losing the oocyst forming capability. When the toxoplasma gondii strain is prepared into a live vaccine immune animal, oocyst discharge can be prevented, a host is triggered to generate a lasting and effective immune reaction, and pollution of toxoplasma gondii in the environment is effectively reduced; the method is of great significance in controlling transmission of toxoplasmosis in pets and human beings, guaranteeing public health safety and promoting sustainable development of pet health industry.

Description

Technical Field

[0001] The present invention relates to the technical field of Toxoplasma vaccines, and in particular to a defective Toxoplasma strain lacking the AP2III-4 gene, a construction method thereof, and a vaccine containing the strain. Background Art

[0002] Toxoplasma gondii, also known as Toxoplasma gondii, is a zoonotic obligate cellular parasite that can grow in almost any warm-blooded cell. Nearly all warm-blooded animals can serve as intermediate hosts for Toxoplasma, where it undergoes an acute infection phase (the tachyzoite stage) and a chronic infection phase (the tissue cyst stage). Toxoplasmosis, caused by infection with Toxoplasma gondii, poses economic and health risks to the livestock industry and food safety. Currently, the mainstay of treatment for toxoplasmosis is drug therapy—a combination of pyrimethamine and sulfadiazine. However, the success rate of this drug therapy remains low and it is ineffective against Toxoplasma cysts. Cats, as the definitive hosts of Toxoplasma, excrete large numbers of oocysts in their feces after ingesting raw meat containing tissue cysts or consuming water or food contaminated with Toxoplasma oocysts. Under favorable conditions, the oocysts can sporulate and become infectious. Humans are primarily infected with Toxoplasma gondii through ingestion of undercooked meat or contact with meat products. Cats, as one of the most common companion animals, offer another potential route of infection through contact with feces from cats excreting oocysts. Although most humans are latently infected, infection in pregnant women can lead to miscarriage, premature birth, and organ abnormalities in the fetus. In immunocompromised patients (such as those with HIV and organ transplant recipients), infection can cause severe central nervous system infections.

[0003] Research has found that live Toxoplasma vaccines are the most effective way to prevent Toxoplasma infection in animals. Currently, the only commercially available toxoplasmosis vaccine is based on the S48 strain isolated from stillborn sheep and is only used in sheep. This strain has been attenuated through laboratory passage, posing a risk of reversion to virulence and limiting its use. Therefore, there is an urgent need to develop a toxoplasmosis vaccine with high safety and efficacy for immunization of pet cats. Summary of the Invention

[0004] One objective of the present invention is to provide a novel function for the AP2III-4 gene of Toxoplasma gondii. Another objective is to provide a deficient strain of Toxoplasma gondii lacking the AP2III-4 gene, as well as a vaccine containing the strain. The technical problems addressed by the present invention are not limited to the technical subject matter described herein. Other technical subjects not described herein will be readily apparent to those skilled in the art from the following description.

[0005] To achieve the above objectives, the present invention first provides the use of an AP2III-4 protein, an AP2III-4 gene, or an inhibitor of the AP2III-4 protein in any of the following:

[0006] A1) Application in inhibiting or preventing the production of oocysts by Toxoplasma gondii;

[0007] A2) use in the preparation of a product for reducing the number of oocysts excreted by animals infected with Toxoplasma gondii;

[0008] A3) Use in the preparation of a product for preventing or treating Toxoplasma gondii infection or toxoplasmosis;

[0009] The amino acid sequence of the AP2III-4 protein may be as shown in SEQ ID NO: 1, and the AP2III-4 gene encodes the AP2III-4 protein.

[0010] Furthermore, the AP2III-4 gene may be any gene capable of encoding the AP2III-4 protein.

[0011] Furthermore, the AP2III-4 gene may be a genomic gene encoding the AP2III-4 protein, or may be a coding gene encoding the AP2III-4 protein.

[0012] In the above application, the coding sequence of the AP2III-4 gene may be as shown in SEQ ID NO: 2.

[0013] Furthermore, the genomic sequence of the AP2III-4 gene may be positions 2241912-2249335 of GenBank Accession No. NC_031470.1 (Update Date 26-Oct-2024).

[0014] Furthermore, the application can be achieved by down-regulating the content and / or activity of the AP2III-4 protein.

[0015] Furthermore, the application can inhibit or prevent Toxoplasma gondii from producing oocysts and / or reduce the number of oocysts excreted by animals infected with Toxoplasma gondii by downregulating the content and / or activity of the AP2III-4 protein in Toxoplasma gondii. Downregulating the content and / or activity of the AP2III-4 protein can be achieved by reducing the expression of the AP2III-4 gene in Toxoplasma gondii (e.g., knocking out or silencing the AP2III-4 gene to reduce or inactivate the AP2III-4 gene activity).

[0016] The inhibitor of protein AP2III-4 may be any substance that reduces the activity and / or content of protein AP2III-4 by regulating expression at the gene level or protein level.

[0017] The gene-level expression regulation may include expression regulation at the chromatin level (e.g., histone modification, chromatin remodeling), transcriptional level (e.g., regulation of promoters, transcription factors, co-regulators), post-transcriptional level (e.g., RNA splicing, microRNA regulation), and post-translational level (e.g., ubiquitination, sumoylation, acetylation, glycosylation, phosphorylation, methylation, NEDD8 modification, etc.). The protein-level regulation may include regulation of protein activity and / or content by protein degradation, protein interaction, or other methods capable of regulating protein activity.

[0018] In the above application, the inhibitory factor may include any one of the following:

[0019] B1) a substance that inhibits the expression of the AP2III-4 gene;

[0020] B2) A substance that inhibits the activity and / or function of the AP2III-4 protein.

[0021] Furthermore, the substances may include nucleic acid molecules, carbohydrates, lipids, small molecule compounds, antibodies, polypeptides, proteins, recombinant vectors (such as gene editing vectors), recombinant cells and viral vectors (such as lentivirus and adeno-associated virus).

[0022] Furthermore, the nucleic acid molecules may include (1) double-stranded RNA (dsRNA), small interfering RNA (siRNA), microRNA (miRNA) and short hairpin RNA (shRNA) used in RNA interference technology; (2) antisense RNA (asRNA) and antisense oligonucleotides (AON) used in antisense nucleic acid technology; (3) gRNA and sgRNA used in gene editing technology; (4) aptamers and ribozymes, etc.

[0023] In the above application, the inhibitory factor may include any one of the following:

[0024] C1) gRNA, wherein the gRNA can target the AP2III-4 gene;

[0025] C2) gRNA, the target sequence of which may be as shown in SEQ ID NO: 3;

[0026] C3) a DNA molecule encoding the gRNA described in C1) or C2);

[0027] C4) an expression cassette or recombinant vector containing the gRNA described in C1) or C2), or containing the DNA molecule described in C3);

[0028] C5) A CRISPR / Cas9 system containing the gRNA described in C1) or C2).

[0029] The present invention also provides a defective strain of Toxoplasma gondii, which can be obtained by reducing the expression level of the AP2III-4 gene in Toxoplasma gondii.

[0030] Furthermore, the defective Toxoplasma gondii strain may be a Toxoplasma gondii strain with a deleted AP2III-4 gene.

[0031] The present invention also provides a method for constructing a defective Toxoplasma gondii strain, which comprises reducing the expression level of the AP2III-4 gene in Toxoplasma gondii to obtain a Toxoplasma gondii strain with lost or reduced oocyst formation ability, namely the defective Toxoplasma gondii strain.

[0032] In the above construction method, reducing the expression level of the AP2III-4 gene in Toxoplasma gondii may be achieved by knocking out the AP2III-4 gene in Toxoplasma gondii by gene editing or homologous recombination.

[0033] It is well known to those skilled in the art to knock out a target gene using gene editing methods (including zinc finger nuclease gene editing methods, TALEN gene editing methods, and CRISPR gene editing methods, etc.) or homologous recombination methods (including complete gene knockout and conditional gene knockout).

[0034] For example, a gRNA targeting the gene can be designed according to the target gene, and then the DNA molecule encoding the gRNA and the Cas protein gene can be connected to the same vector to transform the receptor; the DNA molecule encoding the gRNA and the Cas protein gene can also be connected to different vectors to transform the receptor together; or a backbone vector already containing the Cas protein gene (such as PX459 vector, PX458 vector, PX461 vector, PX462 vector, PX551 vector, PX552 vector, pGK1.1 vector, PX330 vector, PX335 vector, PX165 vector, eSpCas9 (1.1) vector, etc.) can be selected as the expression vector of the gRNA, and the DNA molecule encoding the gRNA can be cloned into the backbone vector to construct a gene editing vector targeting the target gene. After the resulting gene editing vector is introduced into the receptor, the transcribed guide RNA (gRNA) can target the target gene through base complementary pairing. The Cas protein causes a double-strand break in the DNA at the target site of the target gene. Through the organism's own DNA damage repair response mechanism, the sheared region undergoes genetic mutation during the repair process, resulting in a frameshift mutation or premature translation termination in the coding gene, thereby achieving the knockout of the target gene. It is also possible to design two editing targets upstream and downstream of the target gene's genomic sequence. The Cas protein causes a double-strand break in the DNA upstream and downstream of the target gene. Through the organism's own DNA damage repair response mechanism, the sequences at the upstream and downstream ends of the break are connected, thereby achieving the knockout of the target gene.

[0035] Although CRISPR / Cas9 gene editing technology is used to knock out the AP2III-4 gene in Toxoplasma gondii in some embodiments of the present invention, the present invention is not limited to this specific method. Those skilled in the art may use other known gene knockout methods (e.g., homologous recombination methods) to delete or inactivate the AP2III-4 gene in Toxoplasma gondii. These methods may also be used in the present invention. These alternative methods do not depart from the scope of the present invention and are intended to be included in the present invention.

[0036] Knocking out a target gene using homologous recombination is well known to those skilled in the art. For example, a homologous recombination vector can be constructed containing an upstream homologous arm of the target gene, a donor gene (such as a reporter gene or a selectable marker gene), and a downstream homologous arm. Homologous recombination then occurs in the recipient under the action of a recombinase (such as Cre, Flp, Dre, VCre, or SCre) or a site-specific recombinase system (such as Bxb1, Cre-LoxP, FLP-Frt, Dre-Rox, Cre-ERT2, Cre-ERT, or ER-Cre-ER system). After homologous recombination occurs, the target gene is replaced by the donor gene, thereby achieving knockout of the target gene.

[0037] In the above construction method, knocking out the AP2III-4 gene in Toxoplasma gondii can be performed using the CRISPR / Cas9 system, which comprises the gRNA described herein (the gRNA described in C1) or C2) herein).

[0038] Furthermore, the CRISPR / Cas9 system also includes Cas9 protein.

[0039] Furthermore, the Cas9 protein described herein is not limited to a specific protein, as long as it can be used in conjunction with the gRNA of the present invention.

[0040] Further, the Cas9 protein described herein may include Streptococcus pyogenes Cas9 (spCas9, subtype II-A), spCas9 HF (high fidelity), notchase Cas9 (nCas9), Staphylococcus aureus Cas9 (saCas9, subtype II-A), Neisseria meningitidis Cas9 (NmCas9, subtype II-C), Francisella novicida Cas9 (FnCas9, subtype II-B), Streptococcus thermophilus Cas9 (St1Cas9, St3Cas9), Campylobacter jejuni Cas9 (CjCas9) and Treponema sp. Cas9, as well as Cas9 orthologs of other organisms but are not limited thereto. The Cas9 protein may also include high-fidelity Cas9 mutants (such as SpCas9-HF1, eSpCas9-1.1 and TrueCut TM HiFi Cas9 protein) etc.

[0041] The method of the present invention can be implemented using any Cas9 protein known in the art. Those skilled in the art can make appropriate selections of the coding sequence of the Cas9 protein without departing from the principles of the embodiments of the present invention.

[0042] Furthermore, the method for constructing the Toxoplasma gondii deficient strain described herein may include the following steps:

[0043] (1) constructing a DNA molecule encoding gRNA into a Cas9 expression vector to obtain a CRISPR / Cas9 gene editing vector; the gRNA is a gRNA targeting the AP2III-4 gene;

[0044] (2) introducing the CRISPR / Cas9 gene editing vector into Toxoplasma gondii;

[0045] (3) After screening and identification, a Toxoplasma gondii with AP2III-4 gene knockout was obtained, which is the Toxoplasma gondii defective strain.

[0046] Furthermore, the target sequence of the gRNA may be SEQ ID NO: 3.

[0047] Furthermore, the Cas9 expression vector in step (1) contains a Cas9 gene and is capable of expressing a Cas9 protein. Furthermore, the Cas9 expression vector may also contain one or more of the following elements: a replication origin (ori), a promoter (such as a U6 promoter), an enhancer (such as a CAG enhancer), a tag (such as a FLAG tag), a terminator (such as a bGH poly(A) terminator), a resistance gene (such as a Kana antibiotic resistance gene, an ampicillin resistance gene), a promoter of a resistance gene, a screening gene (such as a bar gene), a promoter of a screening gene, a promoter of a Cas9 gene (such as an Ubi promoter), and a framework region (scaffold) of a gRNA.

[0048] The Cas9 expression vector can be obtained commercially. After designing the gRNA targeting the target gene, the DNA molecule encoding the gRNA can be easily inserted into the commercial Cas9 expression vector, and the Cas9 protein and gRNA are expressed simultaneously, thereby editing the target gene. In addition, conventional methods in the art can also be used to construct a Cas9 expression vector. For example, the Streptococcus pyogenes genome can be used as a template to amplify the Cas9 gene, and then the Cas9 gene can be cloned into a backbone expression vector (such as pET28a, pET32a, etc.) to obtain a Cas9 expression vector.

[0049] Furthermore, the introduction method in step (2) can be electroporation.

[0050] In step (2), a homologous donor DNA containing upstream and downstream homologous arms of the AP2III-4 gene and a reporter gene can be further provided, that is, the CRISPR / Cas9 gene editing vector obtained in step (1) and the homologous donor DNA are introduced into Toxoplasma gondii together.

[0051] The purpose of introducing homologous donor DNA is to introduce a reporter gene while knocking out the AP2III-4 gene (to facilitate subsequent detection). Therefore, homologous donor DNA is usually not necessary. Suitable homologous donor DNA can be selected according to the needs of those skilled in the art, or no homologous donor DNA can be provided.

[0052] The present invention also provides a vaccine for preventing Toxoplasma infection or toxoplasmosis, wherein the vaccine comprises the defective Toxoplasma strain described herein, or the defective Toxoplasma strain obtained by any of the construction methods described herein.

[0053] The active ingredients of the vaccine include the defective Toxoplasma gondii strain described herein, or the defective Toxoplasma gondii strain obtained by any of the construction methods described herein.

[0054] When the vaccine of the present invention is used to inoculate animals, the bradyzoites or cysts of the defective Toxoplasma gondii strain can be prepared into a suspension in PBS or DMEM for oral inoculation.

[0055] The present invention also provides the use of the defective Toxoplasma gondii strain described herein, or the defective Toxoplasma gondii strain obtained by any of the construction methods described herein, in the preparation of products for preventing or treating Toxoplasma infection or toxoplasmosis.

[0056] The product described herein can be a reagent, a preparation, a product, a medicament, a pharmaceutical composition, or a vaccine (eg, a live vaccine).

[0057] The Toxoplasma gondii (or wild-type Toxoplasma gondii) described herein may be Toxoplasma gondii containing the AP2III-4 gene.

[0058] The animal mentioned herein may be a non-human mammal (eg, cat, dog, etc.).

[0059] Furthermore, the Toxoplasma gondii described herein may be a Toxoplasma gondii type II strain (eg, Toxoplasma gondii Pru strain).

[0060] The present invention discovers for the first time that the Toxoplasma gondii AP2III-4 protein and AP2III-4 gene regulate the reproductive stage of Toxoplasma gondii in the intestines of its final host, the cat. Downregulating the content and / or activity of the AP2III-4 protein in Toxoplasma can inhibit or prevent the production of oocysts and / or reduce the number of oocysts excreted by infected animals. The present invention uses gene editing technology to knock out the AP2III-4 gene in Toxoplasma gondii, generating the AP2III-4 gene-deficient Toxoplasma strain PruΔAP2III-4. Animal immunization experiments demonstrate that while the PruΔAP2III-4 strain loses the ability to form oocysts, it maintains a high level of immune protection. Upon immunization, it can stimulate the production of extremely high levels of Toxoplasma antibodies in the host, making it an ideal strain for a live vaccine.

[0061] The present invention has the following beneficial effects: Compared with wild-type strains, the AP2III-4 gene-deficient Toxoplasma gondii strain provided by the present invention can undergo schizogony in the final host, cats, but cannot form oocysts, thus being highly safe for humans, the external environment, and the host. Immunization of cats with the AP2III-4 gene-deficient strain can prevent the excretion of oocysts and induce a long-lasting and effective immune response in the host. Furthermore, the strain provides high immune protection against chronic (cyst) reinfection with high doses of Toxoplasma gondii, providing long-term and effective protection with a single immunization. Therefore, the AP2III-4 gene-deficient strain has great potential for the preparation of a genetically engineered live vaccine for the prevention of toxoplasmosis in cats.

[0062] The AP2III-4 gene-deficient Toxoplasma gondii strain of the present invention is prepared into live vaccine-vaccinated animals, which can prevent animals infected with Toxoplasma gondii from excreting oocysts and effectively reduce Toxoplasma contamination in the environment. It is of great significance for controlling the spread of toxoplasmosis in pets and humans, ensuring public health safety, and promoting the sustainable development of the pet health industry.

[0063] Definition of terms

[0064] In the present invention, unless otherwise specified, the scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. At the same time, in order to better understand the present invention, the definitions and explanations of relevant terms are provided below.

[0065] In the present invention, gRNA and sgRNA are used interchangeably.

[0066] The term "expression cassette" generally refers to a nucleic acid construct comprising nucleic acid elements sufficient to express a gene of interest. A typical expression cassette comprises a promoter, an MCS (multiple cloning site) and / or a terminator. An expression cassette may also include a gene of interest, a marker gene (such as a TK gene, a DHFR gene, a CAT gene, and a NEO gene), a ribosome recognition and binding site (SD), a transcription factor binding site (TFBS), an enhancer, a silencer, a repressor, an intron, a poly (A) signal sequence, and / or an mRNA splicing signal sequence. The elements in the expression cassette may be directly connected or indirectly connected via a linker.

[0067] The term "recombinant vector" generally refers to a recombinant DNA molecule constructed by connecting an exogenous target gene to a vector in vitro. It can be constructed in any suitable manner, as long as the constructed recombinant vector can carry the exogenous target gene into the recipient cell and provide the exogenous target gene with the ability to replicate, integrate, amplify and / or express in the recipient cell.

[0068] The term "inhibitor" generally refers to any substance that can inhibit or reduce (downregulate) the level and / or activity of a target protein or gene. In this context, an inhibitor of protein AP2III-4 can be a substance that inhibits the replication, transcription, translation, post-transcriptional modification, and / or post-translational modification of the AP2III-4 gene, or a substance that inhibits or reduces the content, activity, and / or function of the AP2III-4 protein.

[0069] The term "gene editing" generally refers to technologies that can alter specific gene sequences, causing base deletions, duplications, insertions, frameshift mutations, replacements, and knockouts of target genes. These technologies can achieve genomic sequence replacements, deletions, splicing, and single-base changes, effectively "editing" the genome or the sequence of a specific gene. Gene editing includes zinc finger nuclease knockout technology, TALEN gene editing technology, and CRISPR gene editing technology.

[0070] The term "reporter gene" generally refers to a group of genes encoding proteins or enzymes that are easily detected. The reporter genes may include β-glucuronidase (GUS) gene, luciferase (Luciferase) gene, chloramphenicol acetyltransferase (CAT) gene, β-galactosidase (β-gal) gene, secretory human placental alkaline phosphatase (SEAP) gene, yellow fluorescent protein (YFP) gene, green fluorescent protein (GFP) gene, enhanced green fluorescent protein (EGFP) gene, and dihydrofolate reductase (DHFR) gene.

[0071] The term "homologous recombination" generally refers to a type of genetic recombination, a molecular biology technique based on the exchange and repair of genetic material mediated by DNA sequence homology. The use of homologous recombination methods to knock out a target gene in the present invention can refer to a technique in which an exogenous gene replaces an endogenous normal homologous gene through homologous recombination, thereby inactivating the endogenous gene. This includes complete gene knockout (e.g., complete mutation of the target gene based on a replacement targeting vector or an insertion targeting vector) and conditional gene knockout (e.g., tissue-specific knockout based on the Cre-LoxP recombinase system or the FLP-FRT recombinase system).

[0072] The term "Cas9 protein" generally refers to the Cas endonuclease of the type II CRISPR system that forms a complex with crRNA and tracrRNA or with a guide RNA, which is used to specifically recognize and cut all or part of the DNA target sequence. The Cas9 protein has two different domains: the HNH domain and the RuvC domain. The HNH domain is responsible for cutting the DNA chain (target chain) that is complementary to the crRNA (or gRNA), while the RuvC domain is responsible for cutting the non-complementary chain (non-target chain). The Cas9 protein is not limited to a specific protein, as long as it can be used in conjunction with sgRNA (gRNA). The Cas9 protein may be derived from a bacterial species.

[0073] The term "gRNA" (also referred to herein as sgRNA) generally refers to a single RNA structure formed by directly (or via a linker) connecting crRNA and tracrRNA. sgRNA is a component of the CRISPR-Cas system, responsible for guiding the Cas protein to recognize and cleave the target nucleic acid molecule. In actual gene editing applications, sgRNA can be directly synthesized, expressed from a plasmid, or obtained by in vitro transcription.

[0074] The term "comprising" is not intended to be limiting, but rather inclusive and means that there may be additional elements other than the listed elements, and can be interpreted as "including, but not limited to." The term "comprising" also encompasses the terms "consisting of" and "consisting essentially of." The terms "comprising" and "including" are used interchangeably herein. BRIEF DESCRIPTION OF THE DRAWINGS

[0075] Figure 1 Schematic diagram of the knockout and identification of the AP2III-4 gene in Example 1 of the present invention.

[0076] Figure 2 This is a diagram showing the PCR identification results of the PruΔAP2III-4 insect strain in Example 1 of the present invention, wherein Pru represents the wild-type insect strain.

[0077] Figure 3 These are the intracellular proliferation rate results of the PruΔAP2III-4 strain in Example 2 of the present invention, where Pru represents the wild-type strain cultured in mouse astrocytes for 48 h, and PruΔAP2III-4 represents the knockout strain cultured in mouse astrocytes for 48 h. 1, 2, 4, 8, and ≥16 represent that the number of tachyzoites in the vacuolar membrane of the natrium is 1, 2, 4, 8, and greater than or equal to 16, respectively. The ordinate % of vacuoles represents the proportion of the vacuolar membrane of the natrium with different numbers of tachyzoites.

[0078] Figure 4These are the results of the virulence experiment on mice of the PruΔAP2III-4 strain in Example 2 of the present invention.

[0079] Figure 5 Schematic diagram of the experimental procedure of the cat immune protection experiment in Example 3 of the present invention.

[0080] Figure 6 These are the results of Toxoplasma gondii antibody detection in cat serum at different immunization times in Example 4 of the present invention. DETAILED DESCRIPTION

[0081] The present invention will be further described in detail below in conjunction with specific embodiments. The examples provided are only for illustrating the present invention and are not intended to limit the scope of the present invention. The examples provided below can serve as a guide for further improvements by those skilled in the art and are not intended to limit the present invention in any way.

[0082] Unless otherwise specified, the experimental methods in the following examples are conventional methods and were performed according to the techniques or conditions described in the literature in the field or according to the product instructions. The materials and reagents used in the following examples, unless otherwise specified, were all commercially available.

[0083] The wild-type Toxoplasma gondii strain (Toxoplasma gondii strain) used in the following examples is described in the following literature: Meng Y, Zhai B, et al. (2020) Acetylome analysis of the feline small intestine following Toxoplasma gondii infection.

[0084] The pSAG1-Cas9-sgUPRT plasmid in the following examples is described in the following literature: Xia N, Yang J, et al. (2018) Functional analysis of Toxoplasmalactate dehydrogenases suggests critical roles of lactate fermentation for parasite growth in vivo. Cell Microbiol, 20(1).

[0085] The ploxP-AP2X-4-Ty-LoxP-YFP-DHFR plasmid in the following examples is described in the following literature: Zhang J, Fan F, et al. (2022) Nuclear Factor AP2X-4 Governs the Expression of Cell Cycle-and Life Stage-Regulated Genes and is Critical for Toxoplasma Growth. Microbiol Specte, 10(4).

[0086] Example 1: Construction of a Toxoplasma gondii AP2III-4 gene-deficient strain

[0087] The AP2III-4 gene-deficient strain was constructed using the wild-type Pru strain of Toxoplasma gondii as the base strain. Pru is a type II strain of Toxoplasma gondii that retains the ability to form oocysts in its definitive host, cats.

[0088] The nucleotide sequence of the coding region (CDS) of the AP2III-4 gene is shown in SEQ ID NO: 2. The amino acid sequence of the protein encoded by the AP2III-4 gene (AP2III-4 protein) is shown in SEQ ID NO: 1.

[0089] The genomic sequence of the AP2III-4 gene is at positions 2241912-2249335 of GenBank Accession No. NC_031470.1 (Update Date 26-Oct-2024).

[0090] 1. Construction of CRISPR / Cas9 system plasmid pSAG1-Cas9-sgAP2III-4

[0091] Using the pSAG1-Cas9-sgUPRT plasmid as a template, the multi-fragment seamless cloning kit ( Replace the gRNA of the UPRT gene with the gRNA of the AP2III-4 gene using the Seamless Cloning and Assembly Kit. The specific steps are as follows:

[0092] (1) gRNA design

[0093] The gRNA for the AP2III-4 gene was designed in the ToxoDB database (http: / / grna.ctegd.uga.edu / ). A gRNA with a high overall score was designed in the middle of the gene coding region (SEQ ID NO: 2). The target sequence of the designed gRNA is shown below:

[0094] Target sequence of gRNA: 5′-CGATAAAGACTGAACTGAGT-3′ (SEQ ID NO: 3).

[0095] (2) Fragment amplification

[0096] The CRISPR / Cas9 backbone is amplified in three segments, and the primer sequences are:

[0097] Clip 1:

[0098] Upstream primer: sgAP2III-4-Fw: 5′-ACTCAGTTCAGTCTTTATCGGTTTTAGAGCTAGAAATAGCA-3′;

[0099] Downstream primer: Backbone-1Rv: 5′-GCGGGACACGCCTTCCTGGC-3′;

[0100] Clip 2:

[0101] Upstream primer: Backbone-2Fw: 5′-GCGGGACACGCCTTCCTGGC-3′;

[0102] Downstream primer: Backbone-2Rv: 5′-GATCATCTTCCGCACGTCGT-3′;

[0103] Clip 3:

[0104] Upstream primer: Backbone-3Fw: 5′-ACGACGTGCGGAAGATGATC-3′;

[0105] Downstream primer: sgAP2III-4-Rv: 5′-CGATAAAGACTGAACTGAGTAACTTGACATCCCCATTTACC-3′.

[0106] All the above DNA fragments were amplified using Novozymes' high-fidelity DNA polymerase (2×Phanta Max MasterMix), and the PCR reaction system is shown in Table 1.

[0107] Table 1. PCR reaction system

[0108]

[0109] The template DNA in Table 1 is the pSAG1-Cas9-sgUPRT plasmid.

[0110] The PCR reaction conditions are shown in Table 2.

[0111] Table 2. PCR reaction conditions

[0112]

[0113] Note: High-fidelity DNA polymerases extend at least 1 kb per minute, so the extension time is determined by the specific length of the amplified fragment.

[0114] After the PCR reaction was completed, the target fragments were recovered by gel running, and the DNA fragments were purified using the AidQuick Gel Extraction Kit from Aidlab.

[0115] (3) Fragment connection

[0116] The purified DNA products were ligated using the TransGen Biotech Multi-Fragment Seamless Cloning Kit ( Seamless Cloning and Assembly Kit). The reaction system is shown in Table 3.

[0117] Table 3. Multi-fragment ligation reaction conditions

[0118]

[0119] The reaction system was gently mixed and allowed to react at 50°C for 15 minutes. After completion, the reaction was cooled on ice. The product was transferred to 50 μL of Trans1-T1 competent cells (TransGen Biotech, Trans1-T1 Phage Resistant Chemically Competent Cell), gently mixed, and placed on ice for 30 minutes. Heat shock was performed in a 42°C metal bath for 1 minute, and then immediately transferred to ice for 2 minutes. 500 μL of LB medium was added and incubated at 37°C on a shaker at 250 rpm for 1 hour. 100 μL of the solution was evenly spread on an ampicillin-resistant plate. After 24 hours, several single clones were selected for sequencing. Universal primers M13F and M13R were used for sequencing in one reaction each. Sequencing results showed that the target sequence was the target sequence of the AP2III-4 gene gRNA (SEQ ID NO: 3) and that there were no errors at the plasmid junction. The plasmid was successfully constructed and named pSAG1-Cas9-sgAP2III-4.

[0120] (4) Use PL14-large-scale plasmid extraction kit (Aidlab biotechnologies CO. Ltd) to extract plasmid pSAG1-Cas9-sgAP2III-4 for later use.

[0121] 2. Preparation of AP2III-4-5UTR-EGFP-DHFR-AP2IV-1-3UTR homologous template

[0122] (1) Using high-fidelity DNA polymerase and Pru strain genomic DNA as a template, the 5' and 3' homology arms of the AP2III-4 gene were amplified. The amplification primer sequences are as follows:

[0123] 5H-AP2III-4-Fw: 5'-CTTCCCGTTTTGGCGCCGTCCACT-3';

[0124] 5H-AP2III-4-Rv: 5'-TCGTCAAGTCGAGTGCAGGACTTACCCCGGAAATTATCACG-3';

[0125] 3H-AP2III-4-Fw: 5'-TCCTGTTGAGAAAGCGGTTGACAGCGTTTGCTCTCGGGATC-3';

[0126] 3H-AP2III-4-Rv: 5'-GCGCGCTTAGTCAAGGATTTTTTA-3'.

[0127] (2) Use high-fidelity DNA polymerase to amplify the EGFP-DHFR open reading frame from the ploxP-AP2X-4-Ty-LoxP-YFP-DHFR plasmid. The primer sequences are as follows:

[0128] EGFP-Fw: 5'-TCCTGCACTCGACTTGACGA-3';

[0129] DHFR-Rv: 5'-ACCGCTTTCTCAACAGGAAA-3'.

[0130] (3) Use high-fidelity DNA polymerase to amplify the backbone from the cloning vector T vector (the plasmid was purchased from TransGen Biotech, product number CB111). The primer sequences are as follows:

[0131] Backbone-3Fw: 5'-CCTTGACTAAGCGCGCCTTAAGTGGAGCTCCAGCTTTTGTT-3';

[0132] Backbone-3Rv: 5'-CGCCAAAACGGAGAGGTTAACGGTACCCAATTCGCCCTATA-3'.

[0133] (4) After gel purification of the amplified products of the 5' and 3' homology arms of the AP2III-4 gene, the EGFP-DHFR open reading frame, and the cloning vector backbone, the four fragment products were connected, transformed, plated, and single clones were selected using a multi-fragment kit and sent for sequencing for identification. The specific steps refer to the preparation process of the pSAG1-Cas9-sgAP2III-4 plasmid.

[0134] (5) Using the correctly sequenced bacterial solution as a template and primers 5H-AP2III-4-Fw / 3H-AP2III-4-Rv, a template for homologous recombination of the AP2III-4 gene was obtained. A 50 μL reaction system was amplified in 5 tubes. 3 μL of PCR product from each tube was run on a gel for identification. If the target band was present, the remaining PCR product was purified. The steps are as follows:

[0135] A: Add 3 volumes of isopropanol to the PCR product and place at -20°C for at least 1 hour;

[0136] B: Centrifuge at 10,000 rpm for 10 min at 4°C and discard the supernatant;

[0137] C: Add 500 μL of 75% ethanol and centrifuge at 10,000 rpm at 4°C for 5 min. Discard the supernatant to evaporate the alcohol and store at -20°C until use.

[0138] 3. Construction of the Toxoplasma gondii gene knockout strain ΔAP2III-4::EGFP-DHFR

[0139] (1) Collect 1×10 7 Freshly released Pru tachyzoites were filtered through a 5 μm filter to remove cell debris, centrifuged at 2000 rpm for 10 min, and the supernatant was discarded.

[0140] (2) Take 20 μL of buffer II (buffer II: KH2PO4 6 g, NaHCO3 0.6 g, glucose 0.2 g, add pure water to 500 mL) and add 1 mL of buffer I (buffer I: ATP-disodium salt 2 g, MgCl2-6H20 1.2 g, add water to 10 mL) and mix well to obtain a buffer mixture.

[0141] (3) Take 125 μL of the buffer mixture and add it to the reserved pSAG1-Cas9-sgAP2III-4 plasmid and the template for homologous recombination of the AP2III-4 gene. Dissolve the precipitate and mix it with the precipitate in step (1). Resuspend the worm body and take 105 μL of the suspension and add it to the nuclear transfer cup.

[0142] (4) Select the nuclear transfection instrument program: U-033. After transfection, add 500 μL of 2% culture medium preheated at 37°C, incubate for half an hour, and then inoculate into mouse astrocytes for culture. After 24 hours, replace the culture medium with a final concentration of 3 μM pyrimethamine.

[0143] (5) Identification of parasites and induction of cysts: After three generations of drug screening, PCR identification is performed. After successful identification, some of the released tachyzoites are taken and inoculated into the peritoneal cavity of mice.

[0144] (6) Screening for single clones: After 30 days, the mice inoculated intraperitoneally were killed by cervical dislocation. The brain tissue was removed and ground with a grinder. The brain tissue suspension was transferred to a 15 mL centrifuge tube and PBS was added to 7.75 mL. 2.25 mL of Percoll was added and mixed. The suspension was centrifuged at 2500 rpm for 10 min and the supernatant was discarded. The pellet was resuspended in PBS and the cysts were counted. The cysts were diluted to 100 μL / well and added to a 96-well culture plate. Single green fluorescent cysts were selected under an inverted fluorescence microscope. The selected single cysts were orally inoculated into mice.

[0145] (7) Identification of monoclonal strains: As in step (6), take an appropriate amount of cyst suspension (treated with Percoll) resuspended in PBS, centrifuge at 3000 rpm for 10 min, discard the supernatant, and extract the precipitated DNA (Tiangen Biochemical Technology (Beijing) Co., Ltd., Blood / Cell / Tissue Genomic DNA Extraction Kit) for identification. The DNA extraction method refers to the cell DNA extraction method of the kit. The identification primers are as follows:

[0146] 5'-KO-AP2III-4-Fw: 5'-AGCGTAGAAGGAATATGCGTG-3';

[0147] 5'-KO-AP2III-4-Rv: 5'-TGACTTGGACTTGGGGTTTACAG-3'(PCR1);

[0148] 3'-KO-AP2III-4-Fw:5'-TCGTTTCTGAGAGAGCACCAAAAC-3';

[0149] 3'-KO-AP2III-4-Rv: 5'-CTCTGTGGAGCGTGTCTC-3'(PCR2);

[0150] AP2III-4-CDS-Fw: 5'-TGGAAGCCTTTCCGCTCC-3';

[0151] AP2III-4-CDS-Rv: 5'-TTGCGGGTCGTACCACG-3'(PCR3).

[0152] AP2III-4 gene knockout and identification strategies Figure 1 As shown in the figure, the knockout strain has the target band in PCR1 / PCR2 but not in PCR3, while the wild strain has no target band in PCR1 / PCR2 but not in PCR3. The knockout strain is a positive monoclonal strain. Figure 2 As shown, the correctly identified AP2III-4 gene knockout strain was named PruΔAP2III-4 strain.

[0153] Example 2: Proliferation rate and virulence detection of Toxoplasma gondii AP2III-4 gene deletion strain

[0154] 1. In vitro proliferation experiment of PruΔAP2III-4 strain

[0155] The intracellular proliferation rate of the Toxoplasma gondii AP2III-4 gene deletion strain PruΔAP2III-4 constructed in Example 1 was detected by the following method:

[0156] Freshly released tachyzoites of Pru wild-type and PruΔAP2III-4 strains were collected and inoculated with 10 5 Tachyzoites were plated onto a 12-well plate filled with mouse astrocytes (isolated from suckling mice) (a sterile cell slide was placed before the cells were plated). Six hours after inoculation, uninvaded worms were washed away and the cells were cultured in an incubator. After 48 hours of culture, an IFA (indirect immunofluorescence assay) test was performed as follows:

[0157] ① Fix the cells infected with Toxoplasma gondii in 4% paraformaldehyde at 37°C for 30 minutes.

[0158] ②Permeabilize in 0.25% Triton X-100 at 37°C for 30 min.

[0159] ③ Block in 3% BSA at 37°C for 30 min.

[0160] ④ Add primary antibody raised against rabbit-derived Toxoplasma gondii GAP45 protein (Plattner, F., Yarovinsky, F., Romero, S., Didry, D., Carlier, MF, Sher, A. and Soldati-Favre, D. (2008). Toxoplasma profilin is essential for host cell invasion and TLR11-dependent induction of an interleukin-12 response. CELL HOST MICROBE 3, 77-87.), incubate at 37°C for 1 h, and wash three times with PBS.

[0161] ⑤ Add secondary antibody Cy3-labeled goat anti-mouse IgG (H+L) and nuclear dye Hoechst 33258 (both purchased from Beijing Maichen Technology Co., Ltd.) and incubate at 37°C for 1 hour. Wash three times with PBS.

[0162] ⑥ Add 10 μL of anti-fluorescence quencher to the flying slide and seal the slide, and count the number of tachyzoites in the vacuolar membrane of the parasite under a fluorescence microscope.

[0163] The results are as follows Figure 3 As shown, after 48 h of growth in mouse astrocytes, the average number of tachyzoites in the vacuolar membrane of the PruΔAP2III-4 strain was not significantly different from that of the wild-type strain.

[0164] 2. Experiments on the toxicity and brain cyst production of the PruΔAP2III-4 strain in mice

[0165] The virulence of the Toxoplasma gondii AP2III-4 gene-deficient strain PruΔAP2III-4 constructed in Example 1 was tested using the following method:

[0166] The intracellular PruΔAP2III-4 strain was collected and intraperitoneally inoculated into ICR mice (female, 7 weeks old) at a dose of 2×10 3 Tachyzoites were inoculated with 2×10 3 Pru wild-type tachyzoites served as a control group, with 6 mice in each group, and a PBS control group was also set up. The three groups of mice were housed in the same environment, and the survival of the mice was recorded daily for 25 days.

[0167] The results are as follows Figure 4 The results showed that all mice in each group survived, and there was no significant difference in the production of brain cysts between the wild-type group and the PruΔAP2III-4 group 25 days after infection.

[0168] Example 3: Cat Immunity Protection Experiment with PruΔAP2III-4 Strain

[0169] The PruΔAP2III-4 strain was used to infect cats to verify its immune protection efficacy. The experimental procedures for immunization, challenge and detection are shown in the figure below. Figure 5 The specific method is as follows:

[0170] (1) Chinese rural cats with negative Toxoplasma antibodies (purchased from Beijing Masi Biotechnology Co., Ltd.) were given 10 3 Cats in each group were housed under identical conditions. Cats were exposed to PruΔAP2III-4 brain cysts. A control group treated orally with wild-type Pru cysts at the same dose served as a control, and a blank control group treated orally with PBS served as a blank control. Fecal oocyst counts were measured and counted starting on the fourth day after challenge until no oocysts were detected. Five weeks after challenge, blood was collected from the hind limb vein of each cat in each of the three groups, and serum was stored at -20°C until further use (for Toxoplasma antibody monitoring in Example 4). As shown in Table 4, none of the three cats in the PruΔAP2III-4 group excreted oocysts, while the control group excreted a significant number of oocysts. None of the cats in the blank control group excreted oocysts.

[0171] (2) Five weeks after the first vaccination, the experimental and control cats were reinfected with 3×10 3 PruΔAP2III-4 and wild-type Pru strains produced brain cysts. A blank control group was orally administered PBS. Four days after challenge, fecal oocyst counts were again measured, as shown in Table 4. Five weeks after the second immunization, blood was collected from the hind limb vein of the three groups of cats, and serum was separated and stored at -20°C until use (for Toxoplasma antibody monitoring in Example 4). As shown in Table 4, no cats in the three groups excreted oocysts.

[0172] (3) Five weeks after the second vaccination, each of the three groups of cats received 4×10 3 Cats were challenged with brain cysts from a wild-type Pru strain. Four days after challenge, fecal oocyst counts were again measured, as shown in Table 4. Blood was collected from the hind limb vein of the three groups of cats 5 and 10 weeks after challenge, and serum was stored at -20°C until use (for Toxoplasma antibody monitoring in Example 4). As shown in Table 4, no cats in the experimental or control groups excreted oocysts after challenge, while cats in the blank group excreted a large number of oocysts.

[0173] Table 4. Inoculation of cats with AP2III-4-deficient cysts prevents subsequent oocyst excretion by wild-type cysts.

[0174]

[0175] The above results show that knockout of the AP2III-4 gene can inhibit the production of oocysts by Toxoplasma gondii. Toxoplasma gondii with the AP2III-4 gene missing loses the ability to form oocysts, effectively preventing the excretion of oocysts.

[0176] Example 4: Monitoring of Toxoplasma gondii antibodies after immunization with the PruΔAP2III-4 strain

[0177] The Toxoplasma gondii antibodies in cats immunized with the PruΔAP2III-4 strain in Example 3 were detected using the following method:

[0178] (1) Antigen coating: Prepare whole Toxoplasma gondii antigen, add 100 μL (5 μg / ml, PBS) to a 96-well plate, and incubate at 4°C overnight.

[0179] (2) Blocking: Add 300 μL PBST to each well, let stand for 5 minutes, spin dry, repeat washing 5 times, and block with 1% BSA at room temperature for 1 hour.

[0180] (3) Add primary antibody (cat serum): Wash 5 times as in step (2), add 100 μL (1:25 dilution) cat serum, and incubate at 37°C for 1 hour.

[0181] (4) Adding secondary antibody: Wash 5 times as in step (2), add HRP-labeled goat anti-cat IgG secondary antibody, and incubate at 37°C for 1 hour.

[0182] (5) Color development: Wash 5 times as in step (2), add TMB and develop color at 37°C for 15 min.

[0183] (6) Termination: Add 2 mol / L sulfuric acid solution to terminate the reaction and immediately read the value at 450 nm on a microplate reader.

[0184] The results are as follows Figure 6 As shown, cats immunized with the PruΔAP2III-4 strain initially produced antibodies to Toxoplasma gondii at levels comparable to those produced after infection with the wild-type Pru strain, and the second immunization also produced antibodies to Toxoplasma gondii at comparable levels. These results suggest that immunization with the PruΔAP2III-4 strain provides a robust humoral immune response.

[0185] In summary, the present invention provides a live vaccine for preventing Toxoplasma gondii infection in cats that lacks the AP2III-4 gene. Experiments with cats have shown that immunization with a strain of Toxoplasma gondii lacking the AP2III-4 gene not only completely prevents the excretion of oocysts, significantly reducing the risk of infection by humans and other animals from infective oocysts, but also provides long-term protection from a single immunization. Furthermore, immunization can stimulate the host to produce extremely high levels of Toxoplasma antibodies. Therefore, this strain with the AP2III-4 gene deletion has the potential to be used in the development of a genetically engineered live vaccine against Toxoplasma gondii.

[0186] The present invention has been described in detail above. For those skilled in the art, without departing from the purpose and scope of the present invention, and without the need to carry out unnecessary experimental conditions, the present invention can be implemented in a wide range under equivalent parameters, concentrations and conditions. Although the present invention provides specific embodiments, it should be understood that further improvements can be made to the present invention. In short, according to the principles of the present invention, this application is intended to include any changes, uses or improvements to the present invention, including changes that depart from the disclosed scope in this application and are made using conventional techniques known in the art.

Claims

1. Use of the AP2III-4 protein, the AP2III-4 gene or an inhibitor of the AP2III-4 protein in any of the following: A1) Application in inhibiting or preventing the production of oocysts by Toxoplasma gondii; A2) use in the preparation of a product for reducing the number of oocysts excreted by animals infected with Toxoplasma gondii; A3) Use in the preparation of a product for preventing or treating Toxoplasma gondii infection or toxoplasmosis; The amino acid sequence of the AP2III-4 protein is shown in SEQ ID NO: 1, and the AP2III-4 gene encodes the AP2III-4 protein.

2. The use according to claim 1, characterized in that The coding sequence of the AP2III-4 gene is shown in SEQ ID NO:

2.

3. The use according to claim 1 or 2, characterized in that The inhibitory factor includes any one of the following: B1) a substance that inhibits the expression of the AP2III-4 gene according to claim 1 or 2; B2) A substance that inhibits the activity and / or function of the AP2III-4 protein according to claim 1.

4. The use according to any one of claims 1 to 3, characterized in that The inhibitory factor includes any one of the following: C1) gRNA targeting the AP2III-4 gene according to claim 1 or 2; C2) gRNA, the target sequence of which is shown in SEQ ID NO: 3; C3) a DNA molecule encoding the gRNA described in C1) or C2); C4) an expression cassette or recombinant vector containing the gRNA described in C1) or C2), or containing the DNA molecule described in C3); C5) A CRISPR / Cas9 system containing the gRNA described in C1) or C2).

5. A Toxoplasma gondii-deficient strain, characterized in that: The Toxoplasma gondii defective strain is obtained by reducing the expression level of the AP2III-4 gene of claim 1 or 2 in Toxoplasma gondii.

6. A method for constructing a Toxoplasma gondii-deficient strain, characterized in that: The construction method comprises reducing the expression level of the AP2III-4 gene of claim 1 or 2 in Toxoplasma gondii, thereby obtaining a Toxoplasma gondii strain with lost or reduced oocyst formation ability, namely the Toxoplasma gondii defective strain.

7. The construction method according to claim 6, characterized in that: The reducing the expression level of the AP2III-4 gene of claim 1 or 2 in Toxoplasma gondii comprises knocking out the AP2III-4 gene of claim 1 or 2 in Toxoplasma gondii by gene editing or homologous recombination.

8. The construction method according to claim 7, characterized in that: The knockout of the AP2III-4 gene in Toxoplasma gondii according to claim 1 or 2 is performed using the CRISPR / Cas9 system, and the CRISPR / Cas9 system comprises the gRNA according to any one of claim 4.

9. A vaccine for preventing Toxoplasma gondii infection or toxoplasmosis, characterized in that: The vaccine comprises the defective Toxoplasma gondii strain according to claim 5, or the defective Toxoplasma gondii strain obtained by the construction method according to any one of claims 6-8.

10. Use of the defective Toxoplasma gondii strain according to claim 5, or the defective Toxoplasma gondii strain obtained by the construction method according to any one of claims 6 to 8, in the preparation of a product for preventing or treating Toxoplasma infection or toxoplasmosis.

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