Recombinant protein Sex-Ag-1 and application thereof in preparation of cat toxoplasma gondii subunit vaccine

Abstract

The invention discloses a recombinant protein Sex-Ag-1 and an application of the recombinant protein Sex-Ag-1 in preparation of a cat toxoplasma gondii subunit vaccine. The high-purity recombinant protein is obtained by screening a coding gene of a specific antigen Sex-Ag-1 in a sexual reproduction stage of toxoplasma gondii, cloning the coding gene to a prokaryotic expression vector, and performing induced expression and purification. The protein can be used for preparing a toxoplasma gondii subunit vaccine for cats, and the formation and discharge of toxoplasma gondii oocysts are blocked by immunizing felines to excite specific immune response, so that the diffusion of toxoplasmosis is controlled from a transmission source. The vaccine has the characteristics of strong targeting property, high safety, stable immune effect and the like, and a new technical scheme is provided for solving the problems that the existing toxoplasmosis prevention and control traditional Chinese medicine treatment is limited, effective vaccines are lacked and the like.

Description

Technical Field

[0001] This invention relates to the fields of biology and genetic engineering, specifically to a recombinant protein, Sex-Ag-1, and its application in the preparation of feline Toxoplasma gondii subunit vaccines. Background Technology

[0002] Toxoplasmosis is an important zoonotic parasitic disease caused by Toxoplasma gondii. This pathogen can infect almost all warm-blooded animals, including humans, livestock, and wild animals, and is widely distributed globally. Epidemiological surveys indicate that approximately one-third of the world's population is infected with Toxoplasma gondii, with infection rates exceeding 80% in some regions.

[0003] Toxoplasma gondii has a complex life cycle and a wide range of hosts, including both asexual and sexual reproductive stages. Felines are the only definitive host for Toxoplasma gondii, completing sexual reproduction and releasing oocysts within their intestines. These oocysts can survive in the environment for months or even years, making them the primary source of Toxoplasma gondii transmission. Intermediate hosts (including humans) become infected through ingestion of food, water, or undercooked meat containing oocysts contaminated with them.

[0004] Currently, the prevention and control of toxoplasmosis mainly relies on chemical drug treatment and hygiene management measures, but the following problems exist: (1) Limitations of drug treatment: Existing anti-Toxoplasma gondii drugs (such as sulfonamides, pyrimethamine, etc.) mainly target the tachyzoite stage, and have limited effect on bradyzoites and oocysts. Furthermore, long-term use can easily lead to drug resistance. (2) Lack of effective vaccines: Although there are many candidate vaccines under research, very few vaccines have actually been commercialized. Existing vaccines have problems such as unstable immunization effects and poor safety. (3) Insufficient diagnostic techniques: The sensitivity and specificity of traditional serological diagnostic methods need to be improved. Summary of the Invention

[0005] The purpose of this invention is to overcome the shortcomings of existing technologies. This invention provides a recombinant protein, Sex-Ag-1, and its application in the preparation of a feline Toxoplasma gondii subunit vaccine. This invention involves screening to obtain the encoding gene of Sex-Ag-1, a sexual reproduction-specific antigen of Toxoplasma gondii, cloning it into a prokaryotic expression vector, and transforming it into Escherichia coli for induced expression. The expressed recombinant protein is then purified using a Ni-ANT process to obtain a high-purity target protein with natural immunogenicity.

[0006] Since the sexual reproductive stage of Toxoplasma gondii is a key link in its life cycle, a subunit vaccine was prepared by combining purified recombinant protein Sex-Ag-1 with aluminum hydroxide adjuvant. This vaccine can induce a specific immune response in the body, significantly inhibiting the sexual reproduction of Toxoplasma gondii and the formation and release of oocysts in the cat's intestines, thereby blocking the spread of toxoplasmosis at its source. This invention provides a safe, efficient, and easily scalable vaccine candidate strategy for the prevention and control of feline toxoplasmosis, and has important public health significance.

[0007] To achieve the above objectives, the technical solution designed by the present invention is as follows: The present invention provides a recombinant protein Sex-Ag-1, the amino acid sequence of which is as shown in SEQ ID NO:1.

[0008] The gene encoding the recombinant protein Sex-Ag-1 described above, the nucleotide sequence of which is shown in SEQ ID NO: 2. The present invention also provides a recombinant plasmid pET28a-Sex-Ag-1, wherein the recombinant plasmid pET28a-Sex-Ag-1 contains an expression vector of the above-mentioned gene.

[0009] Furthermore, the expression vector is pET28a.

[0010] The present invention also provides a recombinant host cell containing the recombinant plasmid pET28a-Sex-Ag-1 as described in claim 3, wherein the host cell is Escherichia coli BL21(DE3).

[0011] This invention also provides a method for purifying recombinant protein Sex-Ag-1, comprising the following steps: 1) The recombinant host cells described above were activated in LB / Kana liquid medium to obtain activated bacterial solution; 2) The activated bacterial solution was inoculated into LB / Kana liquid medium, cultured, and induced after the logarithmic phase; the bacterial solution was collected, resuspended, lysed, precipitated, and purified to obtain the recombinant protein Sex-Ag-1.

[0012] The present invention also provides the use of the above-mentioned recombinant protein Sex-Ag-1 in the preparation of a vaccine for the prevention of feline toxoplasmosis infection.

[0013] The present invention also provides a vaccine for preventing feline toxoplasmosis infection, the vaccine comprising a solution of recombinant protein Sex-Ag-1 and an aluminum hydroxide solution.

[0014] Furthermore, the concentration of the recombinant protein Sex-Ag-1 solution is 4 mg / mL, and the concentration of the aluminum hydroxide solution is 0.3 mg / mL; and the volume ratio of the recombinant protein Sex-Ag-1 solution to the aluminum hydroxide solution is 1:1.

[0015] This invention also provides a method for preparing a vaccine to prevent feline toxoplasmosis infection, comprising the following steps: A vaccine formulation is prepared by mixing a 0.3 mg / mL aluminum hydroxide solution with a 4 mg / mL recombinant protein Sex-Ag-1 solution at a volume ratio of 1:1 and then dispensing the mixture.

[0016] The beneficial effects of this invention are: 1. Significantly blocks oocyst release and reduces the risk of environmental transmission. This invention uses recombinant protein Sex-Ag-1 for immunization. The number of Toxoplasma gondii oocysts in the feces of cats in the challenged immunized group is only 0.07% of that in the control group, achieving a reduction of >99.9% in the number of oocysts. This can effectively cut off the key transmission link between cats, environment and host, and reduce the risk of zoonotic transmission from the source.

[0017] 2. It has good safety and is suitable for front-line promotion. Throughout the immunization process, monitoring showed that the immunized cats did not exhibit adverse reactions such as fever, weight loss, depression, or diarrhea. Their body temperature and weight remained stable, indicating that the vaccine is well-tolerated and has a high safety margin, making it suitable for widespread use in pet clinical and breeding settings.

[0018] 3. Reduce dependence on chemical drugs and alleviate drug resistance pressure. By establishing protection through active immunization, the long-term or repeated use of drugs such as sulfonamides and pyrimethamine in clinical practice can be reduced, thereby reducing the risk of drug resistance and the burden of drug toxicity and side effects, and improving the prevention and control system's reliance on chemical drugs alone.

[0019] 4. Mature technology, controllable costs, and easy to scale up production. This invention is based on the pET28a-Sex-Ag-1 / BL21(DE3) prokaryotic expression system. The process route is clear: the gene amplification-cloning-expression-purification link is highly standardized, with stable yield, simple purification, and low cost, which is conducive to industrial scale-up and quality consistency control.

[0020] 5. The immunization procedure is clear and has good compatibility with conventional adjuvants. The procedure of subcutaneous immunization at 200 μg / kg with two booster immunizations at intervals of 21 days and 42 days, and the adjuvant is mixed with aluminum hydroxide at a 1:1 ratio, can achieve significant protective effects. This indicates that it has good compatibility with commonly used safe adjuvants, is easy to operate, and can be implemented in different institutions according to the procedure.

[0021] 6. Dual Value for Public Health and Animal Welfare It significantly reduces fecal oocyst emissions, while minimizing environmental pollution and the risk of secondary infection, thus balancing public health benefits with animal welfare; it has practical application value for farms, shelters, and urban pet management.

[0022] 7. Providing a new antigen pathway for Toxoplasma gondii vaccine development Sex-Ag-1, as a recombinant protein antigen, has obtained a clear protective signal through animal challenge verification, laying the foundation for subsequent multivalent formulations, dosage form optimization, and combined immunization strategies, and filling one of the technological gaps in the current lack of commercial vaccines and unstable immunization effects. Attached Figure Description

[0023] Figure 1 This is a graph showing the PCR identification results of the pET28a-Sex-Ag-1 monoclonal bacteria; In the figure, M represents the marker, and 1, 2, and 3 represent different monoclonal bacterial PCRs. The size of the positive monoclonal PCR identification band is 879 bp.

[0024] Figure 2 This is an SDS-PAGE analysis of Sex-Ag-1 protein eluted with 70-100mM imidazole; In the figure, M represents Protein marker, and 1, 2, 3, and 4 represent 70, 80, 90, and 100 mM imidazole eluent and the first 70 M imidazole eluent, respectively.

[0025] Figure 3 This is a graph showing the body temperature measurement results of cats during immunization with the recombinant protein Sex-Ag-1 vaccine.

[0026] Figure 4 This is a graph showing the weight measurement results of cats during the immunization period of the recombinant protein Sex-Ag-1 vaccine.

[0027] Figure 5 This is a graph showing the statistical results of oocyst release time and the number of oocysts per gram of feces (OPG) in the immune group and the control group after tissue cyst infection. Detailed Implementation

[0028] The present invention will now be described in further detail with reference to specific embodiments, so that those skilled in the art can understand it.

[0029] Example 1: Obtaining the Sex-Ag-1 gene ORF sequence of Toxoplasma gondii strain ME49 (1) Primers designed based on the genome of Toxoplasma gondii strain ME49, By consulting literature on the gamete transcriptome of coccidia and compiling and screening the potential male gamete-specific expression gene numbers reported in the literature, protein BLAST was performed on the NCBI website to identify genes with high homology to Toxoplasma gondii ME49, and the corresponding protein amino acid sequences were identified for bioinformatics analysis.

[0030] Analysis showed that the protein lacked a signal peptide but possessed a transmembrane domain, exhibited good hydrophilicity and hydrophobicity, and had a high antigenic determinant score, indicating good potential for vaccine development. Based on this, specific primers were designed for this protein: Upstream primer Sex-Ag-1-F: 5'-ATCTTCCCCATCGGTGATGT-3', Downstream primer Sex-Ag-1-R: 5'-CTCAAGACCCGTTTAGAGGC-3'; (2) Preparation of Toxoplasma gondii cDNA Toxoplasma gondii RNA was extracted from ME49 cells using a Toxoplasma gondii RNA extraction kit, and then the obtained RNA was reverse transcribed into cDNA using an RNA reverse transcription kit. The following reaction system was performed in RNase-free tubes: Heat at 65°C for 5 minutes, then quickly place on ice to cool, and let stand on ice for 2 minutes. Gently pipette to mix thoroughly, incubate at 42°C for 2 minutes. 25℃ 5 min-37℃ 45 min-85℃ 5 sec.

[0031] (3) Amplification of the target fragment Using Toxoplasma gondii ME49 strain cDNA as a template and the above primer pairs, PCR amplification was performed. PCR reaction system: PCR reaction procedure: Based on this system and procedure, target fragments and linearized vector fragments of different lengths were amplified, and then the fragments were recovered by gel extraction.

[0032] (4) Fragment recovery and sequencing Following the instructions of the Novizan DNA Agarose Gel Extraction Kit, the target fragment of a single band was excised and extracted, stored at -20 ℃, and sequenced for alignment to obtain the Sex-Ag-1 gene ORF, the nucleotide sequence of which is shown in SEQ ID NO: 2.

[0033] Example 2 Preparation of recombinant protein Sex-Ag-1 (1) Amplification of the Sex-Ag-1 gene fragment ① The ORF of the Sex-Ag-1 gene of Toxoplasma gondii strain ME49, with nucleotide sequence shown in SEQ ID NO: 2, was used to design specific primers based on the strategy of homologous recombination with the pET-28a vector: Upstream primer Sex-Ag-1-Fw: 5'-AGCAAATGGGTCGCGGATCCATGGAGTTTCGGACTCTGGAGT-3' Downstream primer Sex-Ag-1-Rw: 5'-TGGTGGTGGTGGTGCTCGAGCGGGAGGAGGAAGGGAAGAT-3' ② Prepare the following reaction system in a sterile PCR tube: the template is the cDNA of the ME49 insect strain obtained by the method in Example 1, the amplification primers are Sex-Ag-1-Fw and Sex-Ag-1-Rw, and the Sex-Ag-1 gene fragment is amplified by PCR using a high-fidelity enzyme.

[0034] PCR reaction system: The PCR reaction conditions are as follows: The target fragment was cut and recovered, and its concentration was determined for later use.

[0035] (2) Construction of pET28a-Sex-Ag-1 recombinant plasmid ① Take a new sterile PCR tube and prepare the following reaction system: After mixing thoroughly, react at 37°C for 30 minutes, then place on ice for 5 minutes; ② Transform all reaction products into DH5α competent cells, plate on LB / kana plates, and incubate overnight at 37°C with the plates inverted. Pick a single colony and place it in a sterile PV glass bottle containing 5 ml of LB / kana liquid medium, and incubate at 37°C with shaking at 180 rpm for 10 h until the bacterial culture becomes turbid. Take 500 μl of the culture and perform sequencing analysis using T7 forward and reverse primers, respectively. If the sequencing results are correct, the pET28a-Sex-Ag-1 plasmid is considered to have been successfully constructed. The correctly identified bacterial culture is then used for plasmid extraction and stored at -20°C.

[0036] (3) Expression and purification of recombinant protein Sex-Ag-1 ① Transform the correctly sequenced pET28a-Sex-Ag-1 plasmid into E. coli competent cells BL21(DE3), plate on LB / Kana plates, and incubate overnight at 37°C with the plates inverted. Pick a single colony and place it in 5 ml of LB / Kana liquid medium, incubate at 37°C with shaking at 180 rpm for 10-12 h until the bacterial culture becomes turbid. Use 2 μL of the bacterial culture as a template and perform PCR identification using the primer pairs listed below. The PCR reaction system is as follows.

[0037] Upstream primer T7-Pro-seq-Fw: 5'-ATCTTCCCCATCGGTGATGT-3', Downstream primer In-Sex-Ag-1-Rw: 5'-CTCAAGACCCGTTTAGAGGC-3'; Prepare the following reaction system in a sterile PCR tube: The PCR reaction conditions are as follows: ② PCR product identification: After amplification, take 10 μL of the PCR product, add 2 μL of 6× nucleic acid loading buffer, mix well, load the sample, and electrophoresis on a 1.0% agarose gel with 1× TAE buffer at 110-120V for 30 minutes. Observe using a gel imaging system. The PCR identification results are as follows: Figure 1 As shown, 1, 2, and 3 are different monoclonal bacterial PCRs, and the size of the positive monoclonal PCR identification band is 917 bp.

[0038] ③ Take 400 μL of the above bacterial culture and inoculate it into 40 mL of LB / Kana liquid medium at a ratio of 1:100. Incubate the bacteria at 37℃ until the logarithmic growth phase. The OD of the bacterial culture during the growth phase is... 600The pH value was 0.5-0.6. IPTG was added at a ratio of 1:1000 and induced at 37℃ for 4 h. The cells were collected by centrifugation at 4000 rpm for 10 min, resuspended in His Binding Buffer, and then lysed. The supernatant and precipitate were separately resuspended in loading buffer. The sample was boiled in a 100°C metal bath for 10 min, then incubated on ice for 5 min before loading. SDS-PAGE analysis was performed, and Coomassie Brilliant Blue staining confirmed that the recombinant protein Sex-Ag-1 was mainly expressed in the precipitate, with a protein size of approximately 32.93 kDa. The culture volume was expanded to 1 L, the cells were collected, lysed, and the precipitate was selected for further purification.

[0039] ④ The recombinant protein Sex-Ag-1 is fused with a His tag. After denaturation and renaturation, the precipitate is inverted and bound to a nickel column at 4°C for 2 hours. Then, it is added to a pre-washed and equilibrated empty column and washed with His binding buffer until no protein flows out. The wash buffer is collected, and the target protein is eluted with 70 mM-100 mM imidazole. The eluent is collected separately and analyzed by SDS-PAGE.

[0040] The results are as follows Figure 2 As shown, 1, 2, 3, and 4 are 70, 80, 90, and 100 mM elution buffers, respectively, and 70 mM imidazole is the initial elution buffer. Then, a dialysis bag of appropriate length was boiled in a water bath for 10 minutes, and one end of the dialysis bag was sealed. An appropriate amount of protein eluted from the first elution with 70 mM imidazole was added, and the bag was sealed again and placed in 5 L of pre-cooled PBS. Dialysis was performed at 4°C, with the dialysis buffer changed every 6 hours for a total of 4 changes. The dialyzed protein was then concentrated by sucrose embedding, and the protein concentration was measured to be 4 mg / mL using a BCA assay kit.

[0041] Example 3 The vaccine for preventing feline toxoplasmosis infection consists of a 4 mg / mL solution of recombinant protein Sex-Ag-1 and a 0.3 mg / mL solution of aluminum hydroxide, wherein the volume ratio of the recombinant protein solution Sex-Ag-1 to the aluminum hydroxide solution is 1:1.

[0042] The preparation method of the above-mentioned vaccine for preventing feline toxoplasmosis infection is as follows: The original concentration of aluminum hydroxide adjuvant was 1 mg / mL. It was diluted to 0.3 mg / mL with 0.9% physiological saline and then mixed with recombinant protein Sex-Ag-1 solution (4 mg / mL) at a volume ratio of 1:1. The resulting vaccine formulation was dispensed (each vaccine had a total volume of 1 mL).

[0043] Example 4: Validation experiment of a vaccine for preventing feline toxoplasmosis infection (1) Immunization of feline toxoplasmosis and safety experiments in cats. ① Toxoplasma gondii-negative cats were selected as experimental subjects using MIC17A-iELISA and TXA-iELISA methods.

[0044] Immunization group: Three subcutaneous immunizations were administered according to the following protocol. Immunization schedule: Immunization was administered on day 0, day 21, and day 42, respectively. Immunization dose: Calculated based on the cat's weight, the dose for each immunization is 200 μg / kg; Administration method: The prepared vaccine was administered via subcutaneous injection. Control group: A control trial was conducted using the same time intervals and administration method. Control group: Injected on day 0, day 21, and day 42, synchronously with the immunization group; Control dose: The dosage for each injection is the same as that for the immunization group (calculated based on the cat's body weight); Control reagent: Use an equal volume of PBS buffer instead of the vaccine for subcutaneous injection.

[0045] ② Each cat was housed individually. After the initial immunization, the body temperature and weight of the immunized cats were measured every 3 days. Clinical symptoms during the immunization period were observed and recorded for 30 days. The measurement data were statistically analyzed after 30 days. The results of body temperature and weight measurements are as follows: Figure 3 and Figure 4 As shown.

[0046] The results showed that no adverse reactions such as fever, weight loss, depression, and diarrhea occurred in cats after immunization, indicating that the vaccine (containing recombinant protein Sex-Ag-1) for the prevention of feline toxoplasmosis infection has good safety for cats.

[0047] (2) Immunoprotective efficacy of vaccines used to prevent feline toxoplasmosis infection in cats: On day 30 after the last immunization, cats in the immunized group and the control group were orally administered Toxoplasma gondii tissue cysts, 800 cysts / cat, 3 cats in each group. The number of Toxoplasma gondii oocysts per gram of feces in the experimental cats was continuously measured and recorded from day 1 to day 20.

[0048] The results are as follows Figure 5As shown: In the control group, three cats shed Toxoplasma gondii oocysts from days 5 to 13 post-challenge; in the immunized group, only one cat shed Toxoplasma gondii oocysts from days 5 to 9 post-challenge. Compared to the control group, the immunized group showed a shorter oocyst shedding period and a significantly lower number of oocysts, only 0.07% of the control group's amount. This indicates that the vaccine used to prevent feline Toxoplasma gondii infection contains the recombinant protein Sex-Ag-1. Sex-Ag-1 provides good protection against wild-type strains of Toxoplasma gondii, reducing the risk of transmission due to feline oocyst shedding and demonstrating its potential as a subunit vaccine against Toxoplasma gondii.

[0049] All other parts not described in detail are existing technologies. Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, not all embodiments. People can obtain other embodiments based on these embodiments without creative effort, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A recombinant protein, Sex-Ag-1, characterized in that: The amino acid sequence of the recombinant protein Sex-Ag-1 is as described in SEQ ID NO:

1.

2. The gene encoding the recombinant protein Sex-Ag-1 as described in claim 1, characterized in that: The nucleotide sequence of the gene is shown in SEQ ID NO:

2.

3. A recombinant plasmid pET28a-Sex-Ag-1, characterized in that: The recombinant plasmid pET28a-Sex-Ag-1 contains the expression vector of the gene described in claim 2.

4. The recombinant expression vector according to claim 3, characterized in that: The expression vector is pET28a.

5. A recombinant host cell, characterized in that: The recombinant host cell contains the recombinant plasmid pET28a-Sex-Ag-1 as described in claim 3, and the host cell is Escherichia coli BL21(DE3).

6. A method for purifying recombinant protein Sex-Ag-1, characterized in that: Includes the following steps: 1) The recombinant host cells described in claim 5 are activated in LB / Kana liquid culture medium to obtain an activated bacterial solution; 2) Inoculate the activated bacterial solution into LB / Kana liquid medium, culture, and induce culture after the logarithmic phase; The bacterial culture was collected, resuspended, broken, precipitated, and purified to obtain the recombinant protein Sex-Ag-1.

7. The use of the recombinant protein Sex-Ag-1 according to claim 1 in the preparation of a vaccine for the prevention of feline toxoplasmosis infection.

8. A vaccine for preventing feline toxoplasmosis infection, characterized in that: The vaccine comprises a solution of recombinant protein Sex-Ag-1 and an aluminum hydroxide solution.

9. The vaccine according to claim 8, characterized in that: The concentration of the recombinant protein Sex-Ag-1 solution is 4 mg / mL, and the concentration of the aluminum hydroxide solution is 0.3 mg / mL; and the volume ratio of the recombinant protein Sex-Ag-1 solution to the aluminum hydroxide solution is 1:

1.

10. A method for preparing a vaccine for preventing feline toxoplasmosis infection, characterized in that: Includes the following steps: A vaccine formulation is prepared by mixing a 0.3 mg / mL aluminum hydroxide solution with a 4 mg / mL recombinant protein Sex-Ag-1 solution at a volume ratio of 1:1 and then dispensing the mixture.

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