Use of tgme49_219828 protein in preparing toxoplasma vaccine

Abstract

This invention discloses the application of the TGME49_219828 protein in the preparation of Toxoplasma gondii vaccines, belonging to the field of biomedicine. The amino acid sequence of the TGME49_219828 protein is shown in SEQ ID NO:1, and the nucleotide sequence of the encoding gene is shown in SEQ ID NO:2. Subunit vaccines prepared using this protein can reduce the amount of Toxoplasma gondii oocysts excreted in cat feces, blocking environmental transmission at the source and reducing the risk of infection in humans and susceptible animals. This invention provides a new strategy for the prevention and control of Toxoplasma gondii zoonotic diseases.

Description

Technical Field

[0001] This invention belongs to the field of biomedicine and relates to the application of TGME49_219828 protein in the preparation of Toxoplasma gondii vaccines, especially a subunit vaccine for blocking the transmission of Toxoplasma gondii in the definitive host cat. Background Technology

[0002] Toxoplasma gondii is an obligate intracellular parasitic protozoan that can infect humans and almost all warm-blooded animals, causing toxoplasmosis. Toxoplasmosis infection is particularly dangerous for immunocompromised individuals and pregnant women, leading to serious complications such as toxoplasmic meningoencephalitis and retinochoroiditis. Vertical transmission through the placenta can also cause miscarriage, premature birth, or even stillbirth. Surviving children often suffer from congenital brain damage and intellectual disability, seriously endangering public health and livestock production.

[0003] The life cycle of Toxoplasma gondii is complex. Its sexual reproductive stage occurs only in the intestinal epithelial cells of felines. The oocysts excreted after infection by the definitive host have strong environmental resistance and infectivity, and are the main source of widespread transmission of Toxoplasma gondii. Therefore, blocking the sexual reproduction of Toxoplasma gondii in the definitive host, the cat, and reducing the excretion of oocysts are key strategies for controlling the spread of toxoplasmosis at its source.

[0004] Currently, clinical treatment for toxoplasmosis mainly relies on drugs such as pyrimethamine and sulfadiazine. However, these drugs are only effective against tachyzoites in the acute infection phase and are ineffective against tissue cysts in the chronic infection phase, and they also have significant toxic side effects. Globally, only one Toxoplasma gondii vaccine (Toxovax) has been approved for the prevention of toxoplasmosis in sheep. This vaccine is made using a passaged attenuated S48 strain, but it carries the risk of virulence reversion and restoration of oocyst formation ability, limiting its widespread application. Therefore, developing safe and effective new Toxoplasma gondii vaccines has become a crucial issue that urgently needs to be addressed in this field.

[0005] Recombinant subunit vaccines have become an important direction in Toxoplasma gondii vaccine development due to their advantages such as lack of infectious components, controllable production process, ease of standardization, and high safety. In recent years, Toxoplasma gondii vaccine research has made some progress, involving multiple technical routes such as nucleic acid vaccines, gene-deleted vaccines, and mRNA vaccines. Regarding candidate antigen screening, Toxoplasma gondii invasion-related proteins such as surface antigens (SAGs), dense particulate antigens (GRAs), rod-shaped proteins (ROPs), and microneedle proteins (MICs) have been widely used in vaccine research. However, these vaccines mainly target intermediate hosts (such as mice, humans, and livestock) and cannot block the shedding of oocysts in the definitive host (cat), thus limiting their effectiveness in controlling environmental pollution and breaking the transmission chain.

[0006] Notably, with the development of omics technologies, researchers have begun to focus on the identification of Toxoplasma gondii sexual reproduction stage-specific antigens. Studies have shown that members of the AP2 transcription factor family (AP2XI-2 and AP2XII-1) are key repressors regulating sexual differentiation in Toxoplasma gondii, and their double knockout can induce merozoite-specific gene expression, effectively mimicking the phenotypic characteristics of the sexual reproduction stage in vivo. Based on transcriptomic analysis, a number of genes specifically upregulated at the merozoite stage have been identified, including some subtelomeric region genes and hypothetical proteins with unknown functions. These sexual reproduction stage-specific antigens provide new insights for developing transmission-blocking vaccines targeting the definitive host.

[0007] HMGB family proteins are a class of non-histone nucleoproteins with HMG (high mobility group) domains, participating in various important biological functions in eukaryotes, including DNA replication, transcription, and recombination. SRY proteins, characterized by their HMG domains, are sex-determining factors in mammals, while HMGB2 is considered a male fertility regulator and plays a crucial role in turtle spermatogenesis. However, research on the function of HMGB family proteins in the sexual reproduction of Toxoplasma gondii remains lacking. Transcriptome data from the ToxoDB database show that the TGME49_219828 protein (NCBI accession number XP_018634785.1) is specifically highly expressed during the intestinal epithelial stage in cats, suggesting a possible close association with the sexual reproduction of Toxoplasma gondii. Based on this, immunizing the definitive host cat with the TGME49_219828 protein as an antigen is expected to induce a specific immune response, block the sexual reproduction process of Toxoplasma gondii, reduce oocyst expulsion, and thus effectively block the transmission of Toxoplasma gondii. Summary of the Invention

[0008] The purpose of this invention is to provide the application of the TGME49_219828 protein in the preparation of a Toxoplasma gondii vaccine. The TGME49_219828 protein contained in this vaccine is a protein specific to the sexual reproduction stage of Toxoplasma gondii. Immunizing the definitive host cat with this vaccine can reduce the amount of Toxoplasma gondii oocysts excreted in cats, thereby reducing oocyst contamination in the environment and protecting humans and susceptible animals.

[0009] To achieve the above objectives, the present invention provides the following technical solution: The application of TGME49_219828 protein in the preparation of Toxoplasma gondii vaccine, wherein the amino acid sequence of TGME49_219828 protein is shown in SEQ ID NO:1.

[0010] Furthermore, the nucleotide sequence of the gene encoding the TGME49_219828 protein is shown in SEQ ID NO:2.

[0011] Furthermore, the Toxoplasma gondii vaccine is a transmission-blocking subunit vaccine used to induce specific antibodies in cats and inhibit the formation of Toxoplasma gondii oocysts.

[0012] Furthermore, the Toxoplasma gondii vaccine consists of the TGME49_219828 protein and a pharmaceutically acceptable carrier and adjuvant.

[0013] Furthermore, the adjuvant is a Freund's complete adjuvant or a Freund's incomplete adjuvant, and the volume ratio of antigen to adjuvant is 1:1.

[0014] Furthermore, the preparation method of the TGME49_219828 protein includes the following steps: 1) Extract total DNA from Toxoplasma gondii tachyzoites; 2) Using the extracted total DNA as a template, primers were designed for PCR amplification to obtain the target fragment with the nucleotide sequence shown in SEQ ID NO:2; 3) The target fragment was cloned into the pET28a vector to construct the recombinant expression plasmid; 4) The recombinant expression plasmid was transformed into E. coli BL21(DE3) and IPTG was used to induce expression. 5) Collect the bacterial cells, break them up, separate the supernatant, and purify them by nickel affinity chromatography to obtain the TGME49_219828 recombinant protein.

[0015] Furthermore, the sequences of the primers are shown in SEQ ID NO:3 and SEQ ID NO:4.

[0016] Further, in step 4), the IPTG induction concentration is 0.8 mmol / L, and the induction is performed at 37°C for 4 hours.

[0017] Further, the elution buffer for nickel column affinity chromatography in step 5) is a buffer solution containing 20~1000 mmol / L imidazole.

[0018] The beneficial effects of this invention are: This invention is the first to utilize the Toxoplasma gondii sexual reproduction stage-specific antigen TGME49_219828 to immunize the definitive host (cat), thereby reducing fecal oocyst excretion and blocking environmental transmission at its source, overcoming the limitation of existing vaccines that only reduce host infection rates. By reducing oocyst contamination in the environment, this invention directly reduces the infection risk to humans and susceptible animals, providing a new technical means for the comprehensive prevention and control of toxoplasmosis. This invention also boasts advantages such as outstanding safety, high protein yield, stable activity, and low cost in its prokaryotic expression system. Attached Figure Description

[0019] Figure 1PCR amplification results of the coding sequence of Toxoplasma gondii TGME49_219828. Lane M: DNA molecular weight standard; Lane 1: Amplified product of the coding sequence of TGME49_219828.

[0020] Figure 2 PCR identification results of the recombinant plasmid pET28a-219828. Lane M: DNA molecular weight standard; Lane 1: Plasmid identification product contained in the bacterial culture.

[0021] Figure 3 SDS-PAGE analysis results of pET28a-219828 expression product. Lane M: protein molecular weight standard; Lane 1: uninduced His-219828; Lane 2: His-219828 supernatant after induction; Lane 3: His-219828 precipitation after induction.

[0022] Figure 4 SDS-PAGE purification results of pET28a-219828 expression product. Lane M: Protein molecular weight standard; Lane 1: Purified His-219828.

[0023] Figure 5 Results of a vaccine prepared using the Tg_219828 protein on the excretion of feline Toxoplasma gondii oocysts. In the figure, ***P<0.001.

[0024] Figure 6 Western blot analysis results of TGME49_219828 protein with different feline sera. A: Oocyst-positive feline serum (having undergone sexual reproduction); B: Oocyst-negative feline serum (not having undergone sexual reproduction); C: Toxoplasma gondii-negative feline serum; M: Protein molecular weight standard. The results showed that TGME49_219828 protein reacted only with oocyst-positive feline serum. Detailed Implementation

[0025] The present invention will be further described in detail below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Experimental methods in the following embodiments that do not specify specific conditions are generally performed according to conventional conditions or conditions described in reference books such as *Molecular Cloning: A Laboratory Guide* (New York: Cold Spring Harbor Laboratory, 1989), or according to the methods recommended in the manufacturer's operating manual.

[0026] Example 1: Preparation of recombinant TGME49_219828 protein (Tg_219828) This embodiment aims to express and purify the Toxoplasma gondii TGME49_219828 protein in prokaryotes as an antigen for subsequent vaccine preparation.

[0027] 1.1 Total DNA extraction from Toxoplasma gondii tachyzoites Total DNA was extracted from tachyzoites of Toxoplasma gondii strain ME49 using a kit from Novizan. Tachyzoites of Toxoplasma gondii strain ME49 were collected, centrifuged, and the supernatant was discarded. 200 μL of GA buffer and 20 μL of proteinase K solution were added, mixed well, and then 200 μL of GB buffer was added. The mixture was incubated at 70°C for 10 min. 200 μL of anhydrous ethanol was added, and the mixture was vortexed and transferred to an adsorption column. The column was centrifuged at 12000 rpm for 60 s. The cells were washed sequentially with 500 μL of GD buffer and 600 μL of PW buffer, and finally eluted with 50 μL of ddH2O. The total DNA concentration and purity were determined using a UV spectrophotometer.

[0028] 1.2 Amplification of the Target Fragment and Vector Primers were designed based on the TGME49_219828 gene sequence (NCBI accession number XP_018634785.1): Upstream primer (219828-F): CAAATGGGTCGCGGATCCATGGCACCGAAGAAGGTGAC (SEQ ID NO: 3) Downstream primer (219828-R): GGTGGTGGTGGTGCTCGAGTTTCTTCTTGTTGTAGAGAGACATTTCACG (SEQ ID NO: 4) Using extracted Toxoplasma gondii DNA as a template, PCR amplification was performed using Phanta Max Super-Fidelity DNA polymerase. The PCR reaction mixture (50 μL) consisted of: 2 μL cDNA template, 2 μL each of forward and reverse primers, 1 μL dNTP mix, 25 μL 2×Phanta Max Buffer, 1 μL polymerase, and ddH2O to a final volume of 50 μL. The reaction program was: 95℃ pre-denaturation for 5 min; 35 cycles (95℃ denaturation for 15 s, 57℃ annealing for 15 s, 72℃ extension for 30 s); and 72℃ final extension for 10 min.

[0029] The PCR product was detected by agarose gel electrophoresis, and a specific band was obtained at 294 bp, consistent with the expected size. Figure 1 Simultaneously, the pET28a vector was amplified, yielding a 5369 bp fragment.

[0030] 1.3 Target Fragment Recovery Using the Novizan DNA Agarose Gel Extraction Kit, the target band was excised, and an equal volume of sol was added. The gel was incubated at 55°C until completely melted. The melted liquid was added to the extraction column, centrifuged at 12,000 rpm for 1 min, and washed sequentially with 300 μL GDP and 650 μL GW. Finally, the DNA was eluted with 30 μL of preheated sterile water.

[0031] 1.4 Construction of the pET28a-219828 recombinant plasmid The TGME49_219828-CDS fragment was ligated to the pET28a vector using homologous recombination. The ligation system (10 μL) consisted of 2 μL of 5×CE MultiS buffer, appropriate amounts of linearized vector and insert fragment, 1 μL of Exnase™ Multis, and ddH2O to a final volume of 10 μL. After incubation at 37°C for 30 min, the ligation product was transformed into *E. coli* DH5α competent cells and plated onto cells containing kanamycin (Kanamycin). + LB agar plates were incubated overnight at 37°C. Single colonies were picked for PCR identification. Figure 2 After the positive clones were expanded and cultured, plasmids were extracted and sequenced for verification. The sequencing results showed a complete match with the CDS sequence of TGME49_219828, with no frameshift or mutation, indicating that the recombinant expression plasmid pET28a-219828 was successfully constructed.

[0032] 1.5 Construction and protein expression of the Tg_219828 expression strain The correctly sequenced recombinant plasmid was transformed into E. coli BL21(DE3) competent cells and plated on Kana. + LB plates were incubated overnight at 37°C. Single colonies were selected for optimization of induction conditions: IPTG concentrations were set at 0.2, 0.5, 0.8, and 1.0 mmol / L, and induction was performed at 37°C for 4 h. SDS-PAGE analysis showed that under the conditions of 0.8 mmol / L IPTG and 37°C induction for 4 h, a clear protein band appeared around 15.6 kDa, and it mainly existed in the supernatant in a soluble form. Figure 3 ).

[0033] 1.6 Purification of Tg_219828 protein The bacterial cells were collected by centrifugation of 1 L of induced expression culture, resuspended, and then disrupted 5 times at 4℃ and 1000 bar. The supernatant was separated by centrifugation at 7000 r / min for 40 min. The supernatant was filtered through a 0.45 μm filter and bound to a nickel column at 4℃ for 30 min. The supernatant was washed sequentially with His Binding Buffer and eluted with a 20–1000 mmol / L imidazole gradient. The eluted product was dialyzed against PBS, and SDS-PAGE analysis showed that high-purity target protein was obtained. Figure 4 Protein concentration was determined using the BCA method and stored at -80℃ for later use.

[0034] Example 2: Immunoprotective efficacy experiment of Tg_219828 recombinant subunit vaccine 1. Preparation and Immunization Methods of Tg_219828 Recombinant Subunit Vaccine Vaccine preparation: The target protein is diluted to 400 μg / ml. For the first immunization, 0.5 mL of the target protein is mixed with an equal volume of Freund's complete adjuvant and emulsified before immunization. For the second immunization, Freund's incomplete adjuvant is used, while other components and proportions remain unchanged.

[0035] Immunization method: Subcutaneous injection at multiple sites, 1 mL / animal of antigen emulsified with Freund's complete adjuvant; 3 weeks later, subcutaneous injection at multiple sites, 1 mL / animal of antigen emulsified with Freund's incomplete adjuvant.

[0036] 2. Experimental grouping and immunization schedule Nine cats that tested negative for Toxoplasma gondii serologically and in fecal samples were randomly divided into three groups of three each: Group A was the negative control group, treated without immunization or toxoplasmosis treatment; Group B was the infected control group, treated without immunization but with toxoplasmosis treatment; and Group C was the protein immunization group, treated with immunization followed by toxoplasmosis treatment.

[0037] Table 1. Experimental Groups and Immunization Procedures

[0038] The experiment began on day 0, the first immunization on day 1, the second immunization on day 21, and the challenge experiment on day 28 (approximately 800 PRU cysts were administered via gavage). Intravenous blood was collected weekly during the experiment.

[0039] 2.3 Safety Evaluation Blood routine tests: 28 days after immunization, WBC, RBC, HGB, PLT, Neu, HCT and other indicators were measured, and there were no significant differences compared with before immunization (Table 2).

[0040] Table 2. Blood routine test results before and after immunization

[0041] Serum biochemical tests: AST, TBIL, ALT, TP, BUN, CRE and other indicators were detected, and there were no significant differences before and after immunization (Table 3).

[0042] Table 3. Serum biochemical test results before and after immunization

[0043] The above results indicate that the Tg_219828 subunit vaccine has good safety in cats.

[0044] 2.4 Measurement of oocyte expulsion volume From day 0 to day 30 post-inoculation, fecal samples were collected daily from each group. 2 g of feces was weighed, and 5-10 times its volume of tap water was added. After thorough mixing, the mixture was filtered through 160-mesh and 100-mesh sieves. The filtrate was centrifuged (3000 r / min, 10 min), and the precipitate was collected. 58 mL of saturated saline solution was added, and the mixture was centrifuged again (3000 r / min, 10 min) to allow the oocysts to float. The supernatant was collected, and the number of oocysts was counted under a microscope to calculate the number of oocysts per gram of feces (OPG).

[0045] The results showed that no oocysts were detected in the negative control group (Group A). ​​Compared with the infected control group (Group B), the number of oocysts excreted in the feces of cats in the Tg_219828 immunized group (Group C) was significantly reduced (P<0.001), and the oocyst reduction rate was close to 100%. Figure 5 This result demonstrates that the Tg_219828 protein, as an antigen, can effectively reduce the shedding of Toxoplasma gondii oocysts in cat feces, exhibiting a good transmission blocking effect and making it an ideal candidate vaccine antigen.

[0046] Example 3: TGME49_219828 protein specifically targets the sexual reproductive stage of Toxoplasma gondii in cats. To assess whether the TGME49_219828 protein specifically targets the sexual reproduction stage of Toxoplasma gondii, we prepared infected serum from cats that had undergone sexual reproduction and those that had not, and detected the protein using Western blotting.

[0047] First, two serologically negative Toxoplasma gondii cats were selected and orally administered Toxoplasma gondii DB#3 genotype cysts. Fecal samples were collected daily after infection, and microscopic examination revealed no Toxoplasma gondii oocysts. Further tissue samples were collected from the heart, liver, spleen, lungs, kidneys, brain, and intestines. PCR amplification of the 529 bp repeat sequence of Toxoplasma gondii was performed, and specific bands were amplified in all tissue samples, confirming successful infection of both cats with the Toxoplasma gondii DB#3 strain. Since no oocysts were detected after infection, it indicates that Toxoplasma gondii only completed asexual reproduction in these cats and did not enter the sexual reproduction stage. Serum from these two cats was collected and named oocyst-negative cat serum (i.e., serum from Toxoplasma gondii-positive cats that did not undergo sexual reproduction). Serum from cats confirmed to have undergone sexual reproduction and expelled oocysts was collected as oocyst-positive cat serum, and the Toxoplasma gondii-negative cat serum served as a negative control.

[0048] Using recombinant TGME49_219828 protein as an antigen, Western blot analysis was performed with the above three types of serum. Results are as follows: Figure 6 As shown, the TGME49_219828 protein only elicits a specific immune response with oocyst-positive cat serum, but not with oocyst-negative or Toxoplasma gondii-negative cat serum. This result indicates that the TGME49_219828 protein can specifically recognize antibodies induced by the sexual reproductive stage of Toxoplasma gondii.

Claims

1. The use of TGME49_219828 protein in the preparation of a transmission-blocking subunit vaccine for inducing specific antibodies in cats and inhibiting the formation of Toxoplasma gondii oocysts, wherein the amino acid sequence of the TGME49_219828 protein is shown in SEQ ID NO:

1.

2. Use according to claim 1, wherein The nucleotide sequence of the gene encoding the TGME49_219828 protein is shown in SEQ ID NO:

2.

3. The use according to claim 1, wherein The Toxoplasma gondii vaccine consists of the TGME49_219828 protein and a pharmaceutically acceptable carrier or adjuvant.

4. Use according to claim 3, wherein the compound is ###0002### The adjuvant is either Freund's complete adjuvant or Freund's incomplete adjuvant, and the volume ratio of antigen to adjuvant is 1:

1.

5. The use according to claim 1, wherein the compound is ###0002### The preparation method of the TGME49_219828 protein includes the following steps: 1) Extract total DNA from Toxoplasma gondii tachyzoites; 2) Using the extracted total DNA as a template, primers were designed for PCR amplification to obtain the target fragment with the nucleotide sequence shown in SEQ ID NO:2; 3) The target fragment was cloned into the pET28a vector to construct the recombinant expression plasmid; 4) The recombinant expression plasmid was transformed into E. coli BL21(DE3) and IPTG was used to induce expression. 5) Collect the bacterial cells, break them up, separate the supernatant, and purify them by nickel affinity chromatography to obtain the TGME49_219828 recombinant protein.

6. Use according to claim 5, wherein The sequences of the primers are shown in SEQ ID NO:3 and SEQ ID NO:

4.

7. The use according to claim 5, wherein the compound is ###00003### or a pharmaceutically acceptable salt thereof. The IPTG induction concentration described in step 4) is 0.8 mmol / L, and induction is performed at 37°C for 4 hours.

8. The use according to claim 5, wherein the compound is ###0002### The elution buffer for nickel column affinity chromatography described in step 5) is a buffer solution containing 20-1000 mmol / L imidazole.

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