Toxoplasma gondii TgGAP45-PLGA nano vaccine as well as preparation method and application thereof

Abstract

The invention belongs to the technical field of biological veterinary drugs, and relates to a toxoplasma gondii TgGAP45-PLGA nano vaccine as well as a preparation method and application thereof. The vaccine is prepared by coating recombinant protein GAP45 with a coating substance, wherein the protein is derived from toxoplasma gondii slider histone; the amino acid sequence of the gene is shown in SEQ ID NO.1. After the immune protection effect of the vaccine is evaluated, it is found that the toxoplasma gondii TgGAP45-PLGA nano vaccine prepared with GAP45 as the antigen can have strong immune stimulation ability, promote activation of an immune system and remarkably reduce the infection intensity of toxoplasma gondii in a mouse body, and the toxoplasma gondii TgGAP45-PLGA nano vaccine has higher vaccine valence, is free of toxicity and is high in biological safety.

Description

Technical Field

[0001] The invention belongs to the technical field of biological veterinary drugs and relates to a Toxoplasma gondii TgGAP45-PLGA nano vaccine and a preparation method and application thereof. Background Art

[0002] Toxoplasma gondii, also known as Toxoplasma gondii, is a parasite of the genus Toxoplasma in the family Toxoplasma of the order Eucocidia and the order Toxoplasma. It is an obligate intracellular parasite. Toxoplasma gondii has a wide range of hosts and can infect almost all warm-blooded vertebrates, including humans, causing huge losses to human health and the development of animal husbandry. Commonly used drugs for the treatment of Toxoplasma gondii are sulfadiazine and pyrimethamine, but sulfonamides have many side effects and have no obvious preventive effect on host cells infected with Toxoplasma gondii again. Therefore, the development of Toxoplasma vaccines is of great significance for blocking congenital infection and preventing acquired infection. Currently, only the live attenuated S48 strain vaccine has been approved for use in sheep in Europe and New Zealand to prevent Toxoplasma gondii infection, but its biosafety has been questioned, and the vaccine may have a relapse of toxicity.

[0003] In recent years, nano-subunit vaccines have become a hot topic in the research of Toxoplasma vaccines. The vaccines do not contain the entire pathogen, but only the antigen part, so they can effectively avoid biosafety risks. In addition, the vaccines have the advantages of high safety, good immune effect and low production cost. For example, in the prior art,

[0004] The Chinese invention patent with publication number CN111407886A discloses a preparation method and application of a Toxoplasma gondii nanomaterial subunit vaccine, which mainly uses PLGA to coat the Toxoplasma gondii recombinant protein H2A1 to form nanoparticles. The H2A1 recombinant protein comes from the Toxoplasma gondii histone, and after prokaryotic expression, it is coated with the nanomaterial PLGA to form a new vaccine form. The vaccine can prolong the survival time of infected mice to a certain extent, but the vaccine price-effectiveness is not excellent enough, and the toxicity is unknown, which poses certain biosafety risks. Summary of the invention

[0005] Based on this, the present application provides a Toxoplasma gondii TgGAP45-PLGA nanovaccine and its preparation method and application, in order to solve the technical problems in the prior art that the vaccine is not sufficiently cost-effective, has unknown toxicity, and has certain biosafety risks.

[0006] The technical solution of the present invention to solve the above technical problems is as follows:

[0007] A Toxoplasma gondii TgGAP45-PLGA nano vaccine is prepared by coating a recombinant protein GAP45, the protein is derived from the glial body histone of Toxoplasma gondii, and the amino acid sequence thereof is shown in SEQ ID NO.1.

[0008] Preferably, in the above-mentioned Toxoplasma gondii TgGAP45-PLGA nanovaccine, the coating comprises at least one of polylactic acid-glycolic acid, polylactic acid, polycaprolactone, polybutylene glycol succinate, polyaniline, polycarbonate, glycolide-caprolactone copolymer or glycolide-caprolactone copolymer.

[0009] A method for preparing a Toxoplasma gondii TgGAP45-PLGA nano vaccine comprises the following steps:

[0010] S10. Prepare polyvinyl alcohol solution for standby use;

[0011] S20. The coated material is dissolved in dichloromethane as an oil phase for later use;

[0012] S30. Add the polyvinyl alcohol solution to the oil phase, shake it and then ultrasonically break it, then add the recombinant protein GAP45, shake it and then ultrasonically break it to obtain white colostrum;

[0013] S40. adding the polyvinyl alcohol solution to the white colostrum, shaking and then ultrasonically crushing to obtain a double emulsion;

[0014] S50. The organic solvent in the multiple emulsion is evaporated and removed, the precipitate is separated by centrifugation, and the lyophilization is performed after resuspending to obtain the Toxoplasma gondii TgGAP45-PLGA nano vaccine lyophilized powder.

[0015] Preferably, in the method for preparing the Toxoplasma gondii TgGAP45-PLGA nano-vaccine, in step S30, the preparation method of the recombinant protein GAP45 is as follows:

[0016] Total RNA was extracted from Toxoplasma gondii and reverse transcribed to synthesize cDNA. PCR primers were designed based on GenBank: TGME49_223940 sequence for amplification. The primer sequences are shown in SEQ ID NO.2 and SEQ ID NO.3. The amplified product was double-digested with KpnI and EcoRI. After verification by agarose gel electrophoresis, the amplified band was excised and recovered by gel recovery kit. The linearized pET-32a empty vector digested with KpnI and EcoRI was ligated with T4 DNA ligase to obtain the pET-32a-TgGAP45 recombinant plasmid, which was transformed into Escherichia coli BL21 DE3; the above-mentioned Escherichia coli was inoculated into LB liquid culture medium containing ampicillin for expansion culture, and after adding IPTG, it was continued to be placed in a shaker for shaking culture to induce large-scale expression of the recombinant protein TgGAP45, and its induced expression in Escherichia coli BL21DE3 was identified by SDS-PAGE gel electrophoresis, the bacteria were recovered by centrifugation and ultrasonically broken, and the recombinant protein was purified by Ni-NTA chromatography column to obtain the recombinant protein GAP45.

[0017] Preferably, in the method for preparing the Toxoplasma gondii TgGAP45-PLGA nano vaccine, step S30 comprises the following steps:

[0018] S31. Add the polyvinyl alcohol solution dropwise to the oil phase, shake vigorously after the addition is completed until the liquid becomes milky white, and then ultrasonically crush, and after the completion of the ultrasonication, a milky white solvent without stratification is formed;

[0019] S32. Add the recombinant protein GAP45 dropwise into the milky white solvent without stratification, vortex and shake after the dropwise addition, and then perform ultrasonic crushing to obtain white colostrum after the ultrasonication.

[0020] Preferably, in the method for preparing the above-mentioned Toxoplasma gondii TgGAP45-PLGA nanovaccine, in step S32, the concentration of the recombinant protein GAP45 is 1000 to 2000 mg / mL.

[0021] The use of the Toxoplasma gondii TgGAP45-PLGA nano vaccine in the preparation of drugs for preventing mammals from being infected with toxoplasmosis.

[0022] The above technical solution has at least the following advantages:

[0023] 1. The Toxoplasma gondii gliding body protein GAP45 protein (hereinafter referred to as GAP45) belongs to the family of microtubule-associated proteins (MAPs). It interacts with microtubules and participates in the composition and function regulation of the cytoskeleton. It is an important component of the Toxoplasma gliding body and is closely related to the parasite's gliding, invasion and escape from host cells. Toxoplasma is a parasitic protozoan that can infect a variety of animals and replicate and reproduce in them. In this process, the GAP45 protein helps Toxoplasma cells attach to and invade host cells, and promotes the growth and reproduction of parasites in host cells. GAP45, together with myosin A, myosin light chain 1 (MLC1), etc., form the actomyosin motor (AMM), which provides power for the movement of Toxoplasma. As a surface protein, GAP45 is highly conserved and highly specific, which can trigger the host's immune response and produce specific immunity against Toxoplasma. The Toxoplasma gondii TgGAP45-PLGA nanovaccine (hereinafter referred to as TgGAP45-PLGA nanovaccine) prepared in this application using GAP45 as an antigen can have strong immunostimulatory ability, promote the activation of the immune system, significantly reduce the infection intensity of Toxoplasma gondii in mice, and have a higher vaccine cost-effectiveness.

[0024] 2. PLGA nanoadjuvants have targeted and sustained-release properties, which can ensure that vaccine antigens are slowly released in the host and targeted to specific immune cells, helping to improve the immune effect and durability of the vaccine.

[0025] 3. This vaccine is prepared based on subunit vaccines and does not directly use pathogens, so it can effectively avoid biosafety risks; and experiments have shown that this vaccine has no significant effect on the kidney and liver function of mice, does not produce side effects, and is safe and non-toxic.

[0026] 4. The Toxoplasma gondii TgGAP45-PLGA nanovaccine described in this application can be used for immune protection against chronic, acute and congenital Toxoplasma gondii infections. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1(A) is the double enzyme digestion identification of the recombinant plasmid pET32a-TgGAP45, M: DL5000 Marker; (B) is the SDS-PAGE analysis of the purified GAP45 recombinant protein, M: protein Marker (kDa); 1: purified recombinant TgGAP45 protein; (C) is the Western blot analysis of natural and recombinant TgGAP45 proteins, M: protein Marker (kDa); 1: the serum of rats artificially infected with Toxoplasma gondii recognizes rTgGAP45 protein; 2: the serum of normal rats recognizes rTgGAP45 protein; 3: the serum of rats immunized with rTgGAP45 recognizes the soluble protein of whole Toxoplasma gondii tachyzoites; 4: the serum of normal rats recognizes the soluble protein of whole Toxoplasma gondii tachyzoites.

[0028] Figure 2 This is the scanning electron microscopy result of Toxoplasma gondii TgGAP45-PLGA nanovaccine, the scale bar is 500nm.

[0029] Figure 3 It is the in vitro sustained release characteristic of nanospheres.

[0030] Figure 4 The levels of urea nitrogen (A) and creatinine (B) in immunized mice.

[0031] Figure 5 Changes in total IgG (A), IgG1 (B) and IgG2a (C) antibody levels in the serum of mice after immunization with TgGAP45-PLGA nanovaccine.

[0032] Figure 6 Changes in serum IFN-γ (A), TGF-β (B), IL-4 (C), IL-6 (D), IL-10 (E) and IL-17 (F) after mice were immunized with TgGAP45-PLGA nanovaccine.

[0033] Figure 7 The expression of CD83 (A, C) and CD86 molecules (B, C) in splenic dendritic cells after mice were immunized with TgGAP45-PLGA nanovaccine.

[0034] Figure 8 The expression of MHC-I (A, C) and MHC-II molecules (B, C) in splenic dendritic cells after mice were immunized with TgGAP45-PLGA nanovaccine.

[0035] Fig. 9 This is the proliferation of spleen lymphocytes after mice were immunized with TgGAP45-PLGA nanovaccine.

[0036] Fig.10The differentiation of CD4 (A, C) and CD8 (B, C) T lymphocytes in spleen of mice immunized with TgGAP45-PLGA nanovaccine.

[0037] Fig.11 The infection status of spleen tissue after the mouse was intraperitoneally infected with 100 Toxoplasma gondii tachyzoites. The scale bar is 100 μm.

[0038] Fig.12 The infection status of myocardial tissue in mice after intraperitoneal infection with 100 Toxoplasma gondii tachyzoites. DETAILED DESCRIPTION

[0039] It should be noted that, in the absence of conflict, the embodiments and features in the embodiments of the present invention can be combined with each other. The technical solution of the present invention will be further described below in conjunction with the drawings of the embodiments of the present invention, and the present invention is not limited to the following specific implementation methods.

[0040] In a specific embodiment of the present invention, a Toxoplasma gondii TgGAP45-PLGA nano vaccine is provided, wherein the vaccine is prepared by coating a recombinant protein GAP45 with a coating material, wherein the protein is derived from the glial body histone of Toxoplasma gondii, and the amino acid sequence thereof is shown in SEQ ID NO.1.

[0041] SEQ ID NO.1 is:

[0042] MSDKIIHLTDDSFDTDVLKADGAILVDFWAEWCGPCKMIAPILDEIADEYQGKLTVAKLNIDQNPGTAPKYGIRGIPTLLLLFKNGEVAATKVGALSKGQLKE FLDANLAGSGSGHMHHHHHHSSGLVPRGSGMKETAAAKFERQHMDSPDLGTDDDDKAMADIGSMGNACKKNTAKTPTRKEAEDLAEKERQEREAKEKAEAEEK ARAEAEKNAADKAEAERRAAEAREREESARKEAEAEAARKAEAEAAEAERLRKEAEKKKAEEAKRKAEEEQRAAAAEEAEQRAREEAERRKAEAAAAAERERQMQEALKQEEMSPREKYDKLASPEDSASETTMATQPQKVAEHSSAAVTDRSVVGYTVTPCDMASIDETAKYLSKRCGCDLGDQHDENECPICRHIDLSDAPLLN.

[0043] As an important protein, GAP45, a protein of the gliding body of Toxoplasma gondii, plays a key role in the life cycle of Toxoplasma gondii. GAP45 protein belongs to the family of microtubule-associated proteins (MAPs). It interacts with microtubules and participates in the regulation of the composition and function of the cytoskeleton. It is an important component of the gliding body of Toxoplasma gondii and is closely related to the parasite's gliding, invasion and escape from host cells. Toxoplasma gondii is a parasitic protozoan that can infect a variety of animals and replicate and reproduce in them. In this process, GAP45 protein helps Toxoplasma cells attach to and invade host cells, and promotes the growth and reproduction of parasites in host cells. GAP45, together with myosin A, myosin light chain 1 (MLC1), etc., form the actomyosin motor (AMM), which provides power for the movement of Toxoplasma gondii. GAP45 is a surface protein that is highly conserved and specific. It can trigger the host's immune response and produce specific immunity against Toxoplasma gondii. Therefore, it can serve as an important candidate antigen for the development of Toxoplasma vaccine.

[0044] The coating is preferably a biodegradable nano material, such as nanoparticles. In a preferred embodiment, the coating comprises at least one of polylactic acid-glycolic acid, polylactic acid, polycaprolactone, polybutylene glycol succinate, polyaniline, polycarbonate, glycolide-co-lactide copolymer or glycolide-caprolactone copolymer, preferably polylactic acid-co-glycolic acid (PLGA) in the present application.

[0045] In another specific embodiment of the present application, the method for preparing the above-mentioned Toxoplasma gondii TgGAP45-PLGA nanovaccine comprises the following steps:

[0046] S10. Prepare a polyvinyl alcohol solution for later use; specifically, weigh a certain amount of polyvinyl alcohol (PVA) and dissolve it in double distilled water. After the PVA is completely dissolved, a PVA solution is obtained. The concentration of the PVA solution is 3% to 10%, preferably 5%.

[0047] S20. Dissolve the coated material in dichloromethane as the oil phase and set aside.

[0048] S30. Add the polyvinyl alcohol solution to the oil phase, shake it and then ultrasonically break it, then add the recombinant protein GAP45, shake it and then ultrasonically break it to obtain white colostrum;

[0049] In a preferred embodiment, the above step S30 includes:

[0050] S31. Add polyvinyl alcohol solution dropwise to the oil phase, shake vigorously after adding until the liquid turns milky white, and then perform ultrasonic crushing to form a milky white solvent without stratification; specifically, add PVA solution dropwise to the oil phase, shake vigorously after adding until the liquid turns milky white, and perform ultrasonic crushing, the ultrasonic crushing power is 70 to 75W, the ultrasonication is 2 to 5s, the interval is 2 to 5s, the ultrasonication time is 3 to 7min, and a milky white solvent without stratification is formed after the ultrasonication.

[0051] S32. Add the recombinant protein GAP45 dropwise into the milky white solvent without stratification, vortex and shake after the dropwise addition, and then perform ultrasonic crushing to obtain white colostrum after the ultrasonication.

[0052] Specifically, the milky white solvent without stratification is transferred to a vortex shaker, and the recombinant protein GAP45 is added dropwise into the solvent slowly. The concentration of the recombinant protein GAP45 used should be controlled at 1000 to 2000 mg / ml. After the addition of the recombinant protein is completed, the vortex is continued for 1 to 10 minutes, and then ultrasonic crushing is performed. The settings of the ultrasonic crusher are the same as above, and white colostrum is obtained after the end.

[0053] In a preferred embodiment, the preparation method of the recombinant protein GAP45 is as follows:

[0054] Total RNA was extracted from Toxoplasma gondii and reverse transcribed to synthesize cDNA. Here, TRIZol method was used to extract Toxoplasma gondii RH tachyzoites, and then the extracted RNA was reverse transcribed into cDNA according to the instruction manual of the reverse transcription kit; PCR primers were designed according to GenBank: TGME49_223940 sequence for amplification, and the primer sequences were shown in SEQ ID NO.2 and SEQ ID NO.3; the amplified product was double-digested with KpnI and EcoRI, and after verification by agarose gel electrophoresis, the amplified band was cut and recovered by gel recovery kit; T4 DNA ligase was used to connect with the linearized pET-32a empty vector digested with KpnI and EcoRI to obtain the pET-32a-TgGAP45 recombinant plasmid, which was transformed into Escherichia coli BL21 DE3; the above-mentioned Escherichia coli was inoculated into LB liquid culture medium containing ampicillin for expansion culture, and after adding IPTG, it was continued to be placed in a shaker for shaking culture to induce large-scale expression of the recombinant protein TgGAP45; SDS-PAGE gel electrophoresis was used to identify its induced expression in Escherichia coli BL21DE3, the bacteria were recovered by centrifugation and ultrasonically broken, and the recombinant protein was purified using a Ni-NTA chromatography column to obtain the recombinant protein GAP45.

[0055] Among them, SEQ ID NO.2: Upstream primer:

[0056] TgGAP45-F: 5'-CCG GAATTC ATGGGAAACGCGTGCAAGA-3';

[0057] SEQ ID NO.3: Downstream primer:

[0058] TgGAP45-R: 5'-CGG GGTACC TCAGTTCAACAAGGGTGCATCC-3'.

[0059] S40. adding the polyvinyl alcohol solution to the white colostrum, shaking and then ultrasonically crushing to obtain a double emulsion;

[0060] Specifically, the white colostrum is placed on a vortex shaker, and the PVA solution is continuously added drop by drop into the colostrum. After the addition is completed, the colostrum is shaken vigorously for 1 to 10 minutes, and ultrasonic crushing is performed with a power of 70 to 75 W, 2 to 5 seconds of ultrasound, 2 to 5 seconds intervals, and the time is adjusted to 5 to 15 minutes to obtain a multiple emulsion.

[0061] S50. The organic solvent in the multiple emulsion is evaporated and removed, the precipitate is separated by centrifugation, and the lyophilization is performed after resuspending to obtain the Toxoplasma gondii TgGAP45-PLGA nano vaccine lyophilized powder.

[0062] In order to completely remove the dichloromethane in the emulsion, the emulsion was placed on a magnetic stirrer in a fume hood and stirred for about 10 to 15 hours to completely evaporate the organic solvent therein, and then the liquid was subjected to ultracentrifugation to separate the supernatant and the precipitate. The precipitate was resuspended with double distilled water and transferred to a vial, and stored at -80°C for more than 2 hours to completely solidify it into a solid. The frozen sample was placed in a freeze dryer for freeze drying, and the Toxoplasma gondii TgGAP45-PLGA nano vaccine freeze-dried powder was obtained after freeze drying.

[0063] In another specific embodiment of the present invention, the above-mentioned Toxoplasma gondii TgGAP45-PLGA nano vaccine is used in the preparation of a drug for preventing mammals from being infected with toxoplasmosis.

[0064] It is worth noting that the process temperature and process time involved in the above-mentioned embodiments are the temperature or time adopted in the experimental process. Those skilled in the art can make reasonable adjustments within the error range based on the process temperature and process time provided by the present invention, which should be included in the protection scope of the present invention.

[0065] In order to make the purpose, technical solutions and advantages of the present invention more clear, the present invention is further described in detail below through experimental examples.

[0066] 1. Preparation of Toxoplasma gondii TgGAP45-PLGA Nanovaccine

[0067] 1.1 Preparation of genetically engineered bacteria

[0068] Total RNA was extracted from Toxoplasma gondii and reverse transcribed to synthesize cDNA. Two pairs of primers, TgGAP45-F (SEQ ID NO.2) and TgGAP45-R (SEQ ID NO.3), were designed based on the open reading frame of the TgGAP45 gene (GenBank: TGME49_223940), and the TgGAP45 gene fragment was amplified using PCR technology. The reaction system is as follows:

[0069] Table 1 PCR reaction system

[0070] Reagents Dosage 2 × PhantaMaxMasterMix 12.5μL TgGAP45-F 1μL TgGAP45-R 1μL T.gondiicDNA 1μL ddH2O Up to 25 μL

[0071] The PCR program is as follows:

[0072] Table 2 PCR reaction program

[0073]

[0074] The PCR product was subjected to 1% agarose gel electrophoresis at 110V for 30min, and the GAP45 gene fragment amplified by PCR was excised and recovered using a gel recovery kit. After digestion with EcoRI and HindIII restriction endonucleases, the fragment was ligated with the linearized vector pET-32a using T4 DNA ligase to obtain the pET-32a-TgGAP45 recombinant plasmid. Figure 1 A). The recombinant plasmid pET-32a-TgGAP45 was transformed into E. coli BL21 (DE3) competent cells to obtain a genetically engineered strain.

[0075] 1.2 Expression, purification and Western blot analysis of recombinant Toxoplasma gondii GAP45 protein

[0076] The recombinant expression strain obtained above was inoculated into LB liquid culture medium containing ampicillin resistance, placed in a constant temperature incubator at 37°C and 180r / min, and expanded for 4h. The bacterial solution was cultured in a shaking incubator until OD600 = 0.6, and 0.1mM IPTG was added to induce protein expression. After centrifugation of the bacterial solution, the frozen and thawed precipitate was washed with PBS and ultrasonically lysed (ultrasound twice for 20 minutes each time), centrifuged at 12000rpm for 15min, and the protein was purified by nickel column affinity chromatography. The obtained recombinant protein was repeatedly endotoxin-removed by a commercial endotoxin removal kit. After detection by an endotoxin detection kit, the endotoxin level of the purified recombinant protein was lower than 0.01EU / mL. Finally, the recombinant protein was sterilized by filtration through a 0.22μm filter membrane. Verified by SDS-PAGE, the purification result of the recombinant protein was good (attached Figure 1 B).

[0077] All experimental methods in this study complied with the animal welfare and ethics regulations of the College of Animal Science and Technology of Ningxia University and the animal welfare protection regulations of the Science and Technology Department of Ningxia Hui Autonomous Region. The license number is NXU-2023-086.

[0078] Preparation of serum from rats immunized with rTgGAP45: Select healthy rats of appropriate age and weight as immunized subjects. Rats were subcutaneously injected with 100 μg of purified rTgGAP45 recombinant protein and 100 μL of Freund's complete adjuvant mixed with emulsifier on their backs, and immunization was strengthened two weeks later. Two weeks after the second immunization, serum from rats was collected by orbital blood sampling. The collected blood was placed in a centrifuge tube at 4°C overnight, and centrifuged at 2500 r / min for 10 minutes in a 4°C centrifuge the next day to remove red blood cells and other cell debris. The rTgGAP45 antigen was fixed on the carrier using the indirect ELISA method, and serum samples containing the target antibody were added to detect the antibody titer through antigen-antibody specific binding reaction.

[0079] Preparation of serum from rats artificially infected with Toxoplasma gondii: Toxoplasma tachyzoites were inoculated into ICR mice by intraperitoneal injection. The cells were inoculated three times to restore the vitality of the tachyzoites. When the parasites proliferated in large numbers, the mouse peritoneal cavity was flushed and the peritoneal lavage fluid was collected. The collected peritoneal lavage fluid was initially purified by filtration. The purified tachyzoites were ultrasonically disrupted at 4°C, with a power of 80W, 20s of ultrasound, 10s interval, and 2min of ultrasound time. The supernatant was the matrix antigen (StromalAntigen, STAg). The STAg protein content was determined by the Coomassie Brilliant Blue method and stored at -20°C. The prepared STAg was intraperitoneally injected into the rat to stimulate its immune system to produce specific antibodies. Two weeks later, serum was prepared in the same way as above.

[0080] 12% SDS-PAGE electrophoresis was used to separate parasite proteins and recombinant proteins, and the proteins were transferred to PVDF membranes by semi-dry transfer. Specific antibodies anti-Toxoplasma gondii antibodies and anti-rTgGAP45 protein serum (1:500) were used for primary antibody incubation, followed by goat anti-rabbit and goat anti-rat IgG as secondary antibodies (1:10000), and finally ECL chemiluminescence was used for color development and exposure. A specific band can be seen, and no specific band appears in negative serum (attached Figure 1 ). This indicates that the recombinant protein has good antigenicity and the rat recombinant protein antiserum can recognize specific insect proteins.

[0081] 1.3 Preparation of Toxoplasma gondii TgGAP45-PLGA nanovaccine

[0082] Weigh 5g of polyvinyl alcohol (PVA) and dissolve it in 100g of double distilled water. After PVA is completely dissolved, a 5% PVA solution is obtained. Take 100mg of PLGA and dissolve it in 2mL of dichloromethane (DCM) solution as the oil phase. Add 4mL of 5% PVA solution drop by drop to the oil phase, and shake it vigorously after the addition is completed until the liquid turns milky white. Perform ultrasonic crushing at 4°C, set the power of the ultrasonic crusher to 180W, 40% power, 3s ultrasonic, 3s interval, and ultrasonic time for 5min. After the ultrasonication is completed, a milky white and basically non-stratified solvent is formed. Transfer the solvent to a vortex shaker, take 500μL of recombinant T.gondii GAP45 protein (recombinant T.gondii GAP45, rTgGAP45) and add it drop by drop to the above solvent. The dripping speed should be as slow as possible, about 1 drop is added every 2-3s, and the concentration of the recombinant protein used should be controlled at 1500mg / ml. After the recombinant protein is added, continue to vortex and shake for 5 minutes. Ultrasonic disruption is performed at 4°C, and the settings of the ultrasonic disruptor are the same as above. After the end, white colostrum is obtained. The colostrum is placed on a vortex shaker, and 4mL of 5% PVA solution is continued to be added drop by drop to the colostrum. After the addition is completed, it is shaken vigorously for 5 minutes. Ultrasonic disruption is performed at 4°C, and the settings of the ultrasonic disruptor are the same as above. The time is adjusted to 10 minutes to obtain a double emulsion. In order to completely remove the dichloromethane in the double emulsion, the double emulsion is placed on a magnetic stirrer in a fume hood, and evaporated for about 12 hours while stirring, so that the organic solvent therein is completely evaporated. The liquid is subjected to ultracentrifugation. After centrifugation at 40000r / min for 40 minutes at 4°C, the liquid is divided into a supernatant and a precipitate. The precipitate is resuspended with double distilled water and transferred to a vial. It is stored at -80°C for at least 2 hours to completely solidify into a solid. The frozen sample is placed in a freeze dryer for freeze drying, and the duration is 24 hours. TgGAP45-PLGA vaccine dry powder was obtained after freeze-drying.

[0083] 2. Characteristics of Toxoplasma gondii TgGAP45-PLGA Nanovaccine

[0084] 2.1 Physical properties of Toxoplasma gondii TgGAP45-PLGA nanovaccine

[0085] A small amount of TgGAP45-PLGA nanovaccine freeze-dried powder was taken out and sent to the College of Life Sciences of Nanjing Agricultural University for scanning electron microscopy observation. The results are shown in the attached Figure 2As shown, the TgGAP45-PLGA nanovaccine is uniformly spherical with a relatively rough surface. The scanning electron microscope photos were analyzed using ImageJ software, and the particle size of the TgGAP-PLGA nanovaccine was 65.93±3.13nm. To determine the coating efficiency of the TgGAP45-PLGA nanovaccine, after ultracentrifugation, the supernatant after ultracentrifugation was recovered and the total volume of the supernatant was recorded. The free protein concentration in the supernatant was determined by the BCA method, and the coating efficiency of the TgGAP45-PLGA nanovaccine was calculated according to formula (1). After three independent repeated experiments, the coating efficiency of the TgGAP45-PLGA nanovaccine was 74.97±2.48%.

[0086]

[0087] 2.2 In vitro sustained release of Toxoplasma gondii TgGAP45-PLGA nanovaccine

[0088] Weigh 20 μg of TgGAP45-PLGA nanovaccine freeze-dried powder and place it in a sealed centrifuge tube. Use 5 mL of PBS solution as the sustained-release medium. Set the constant temperature water bath oscillator temperature to 37°C and the speed to 180 rpm. When sampling, centrifuge the centrifuge tube at 15,000 r / min for 5 minutes, record the volume of the supernatant, and take 20 μL of the supernatant as a sample for protein concentration determination. The sample is stored at -20°C for later use. Add 20 μL of PBS to the centrifuge tube and resuspend the precipitate, place it in the original environment, and take a sample every 12 hours. After the last sample collection, the protein concentration in the sample was determined by the BCA method, and the total protein content in the supernatant was calculated. The results are shown in the attached Figure 3 As shown, TgGAP45-PLGA nanovaccine gradually released its coated recombinant protein in the first three days, and the sustained release gradually stagnated after the fourth day, indicating that TgGAP45-PLGA nanovaccine has good sustained release properties.

[0089] 2.3 Toxic effects of Toxoplasma gondii TgGAP45-PLGA nanovaccine

[0090] All experimental methods in this study complied with the animal welfare and ethics regulations of the College of Animal Science and Technology of Ningxia University and the animal welfare protection regulations of the Science and Technology Department of Ningxia Hui Autonomous Region. The license numbers are NXU-2023-084 (mice) and NXU-2023-086 (rats).

[0091] To evaluate the toxicity of TgGAP45-PLGA nanovaccine, 35 healthy ICR mice were randomly divided into five experimental groups, with 7 mice in each group. The groups were as follows: Blank group: mice in this group received an equal dose of PBS as a blank group; Control group: mice in this group were injected with 300 μg of empty vector plasmid protein; PLGA group: mice in this group were injected with nanovaccine TgGAP45-PLGA containing 300 μL of empty vector plasmid protein; TgGAP45 group: mice in this group were injected with 300 μg of recombinant TgGAP45 protein; TgGAP45-PLGA group: mice in this group were injected with nanovaccine TgGAP45-PLGA containing 300 μL of rTgGAP45, and the same operation was repeated 3 days later to enhance the immune effect. During the experiment, the health status, behavioral activities and mental state of the mice were closely monitored and recorded. On the third day after the booster immunization, serum samples of mice in each group were collected. Subsequently, the levels of creatinine (Cr) and blood urea nitrogen (BUN) in the serum of mice were measured by sarcosine oxidase method and urease-indophenol method, respectively. The results showed that the levels of BUN (Appendix Figure 4 A) and Cr (attached Figure 4 B) levels were not significantly different from those in the control group (P>0.05). In addition, during the toxicity experiment, the mice behaved and were in good mental state without any abnormalities. The above results suggest that the TgGAP45-PLGA nanovaccine has no significant effect on the kidney and liver function of mice and does not produce side effects, which shows that the Toxoplasma gondii TgGAP45-PLGA nanovaccine has good safety.

[0092] 3. Toxoplasma gondii TgGAP45-PLGA nanovaccine induces humoral immunity in mice

[0093] 3.1 Experimental design

[0094] Female ICR mice weighing 18-22 g were purchased from Beijing Weitong Lihua Biological Co., Ltd. and were housed in an SPF isolation barrier system with free access to food and water. The mice were randomly divided into groups, with 26 mice in each group. The experimental groups and immunization procedures are shown in Table 3. The TgGAP45-206VG and TgGAP45-660VG emulsions were prepared using commercial MONTANIDE TM ISA 206 VG AND MONTANIDE TM It is prepared from ISA 660 VG, and the entire preparation process is strictly carried out in accordance with the instructions for use.

[0095] Table 3 Experimental groups and immunization procedures

[0096]

[0097] 3.2 Antibody levels and cytokine production

[0098] On the 10th day after the first immunization and the 10th day after the booster immunization, the blood of the mice was collected through the eye socket, and the antibody titers of total IgG, IgG1 and IgG2a in the mouse serum were determined by indirect ELISA. Briefly, rTgGAP45 was diluted with coating buffer, and 100 μl of coating buffer containing 1 μg of rTgGAP45 recombinant protein was added to each well of a 96-well ELISA plate, incubated overnight at 4°C, washed three times with PBS containing 5% skim milk, and then incubated with 20 μl of mouse serum for 2 h at room temperature. After washing three times with PBS, HRP-conjugated anti-mouse total IgG, IgG1 and IgG2a antibodies (1:5000 dilution) were used for incubation for 2 h, and then washed three times with PBS again. 100 μl of freshly prepared TMB color development solution was added to each well. After color development for 15 min at room temperature, 100 μl of freshly prepared 2 mol / L H2SO4 was added to each well to stop color development. Then place the plate on a microplate reader and read the OD450 value of each well. Figure 5 As shown, in the 2nd and 4th weeks, high levels of total IgG and IgG1 were detected in mice in the TgGAP45, TgGAP45-PLGA, TgGAP45-206VG and TgGAP45-660VG groups, which were significantly higher than the Control group (P<0.01). In the 4th week, the level of IgG2a in the TgGAP45-PLGA group was significantly higher than that in the Control group (P<0.01). By measuring the antibody level in the mouse serum, it was shown that the prepared TgGAP45-PLGA nanovaccine had similar effects as the two commercial vaccines in inducing humoral immune responses, and both were able to stimulate the body's immune system to a certain extent and produce immune protection against Toxoplasma. And compared with the vaccine disclosed in the prior art CN111407886A, it can be seen from the figure that the levels of IgG1 and IgG2a antibodies in the mouse serum of the present application are significantly higher than the levels of IgG1 and IgG2a antibodies in the prior art.

[0099] To determine the changes in cytokine levels in mouse serum, blood was collected from mice through the eye sockets on the 10th day after the first immunization and the 10th day after the booster immunization, and the levels of IFN-γ, TGF-β, IL-4, IL-6, IL-10, IL-17, and IL-10 in mouse serum were determined using commercial ELISA kits. Figure 6As shown in the results, mice in the TgGAP45-PLGA group produced high levels of IFN-γ, IL-4, and IL-17 (P<0.001), and the two commercial vaccine adjuvants could also effectively activate IL-4 and IL-17 in mice (P<0.05), indicating that both TgGAP45-PLGA nanovaccine and the two commercial vaccines could effectively activate Th1 and Th17 immune responses in mice; in addition, TgGAP45-PLGA nanovaccine could inhibit the secretion of IL-10 in mice (P<0.001), but could not inhibit the secretion of TGF-β, which may be due to the fact that TgGAP45-PLGA nanovaccine inhibited the Th2 immunity of mice. IL-10, as an anti-inflammatory cytokine, can inhibit excessive immune response. An appropriate amount of IL-10 helps prevent excessive immune damage, but too much will inhibit effective immune response.

[0100] 4. Toxoplasma gondii TgGAP45-PLGA nanovaccine induces cellular immunity in mice

[0101] 4.1 Toxoplasma gondii TgGAP45-PLGA nanovaccine promotes the maturation of mouse splenic dendritic cells

[0102] To investigate the maturation of mouse dendritic cells induced by Toxoplasma gondii TgGAP45-PLGA nanovaccine, 5 mice were randomly selected from each group for euthanasia on the 10th day after the first immunization and the 10th day after the booster immunization. The spleen lymphocytes of mice were isolated using a spleen lymphocyte isolation kit, and then the spleen lymphocytes of mice were stained with APC-coupled anti-mouse CD11c, FITC-coupled anti-mouse CD86 and PE-coupled anti-mouse CD83 antibodies. The stained mouse spleen lymphocytes were washed three times with PBS, and the expression of CD83 and CD86 molecules in mouse spleen dendritic cells was analyzed by flow cytometry. The results are shown in the attached figure. Figure 7 As shown, all three vaccines can significantly promote the expression of CD83 and CD86 molecules in mouse splenic dendritic cells (P<0.01), indicating that all three vaccines can promote the maturation of mouse splenic dendritic cells.

[0103] 4.2 Toxoplasma gondii TgGAP45-PLGA nanovaccine promotes antigen presentation by mouse splenic dendritic cells

[0104] In order to explore the effect of Toxoplasma gondii TgGAP45-PLGA nanovaccine on antigen presentation of mouse dendritic cells, the spleen lymphocytes isolated above were stained with PE-coupled anti-mouse CD11c, FITC-coupled anti-mouse MHC-I and APC-coupled anti-mouse MHC-II antibodies. After washing with PBS three times, the expression of MHC-I and MHC-II molecules in mouse spleen dendritic cells was analyzed by flow cytometry. The results are shown in the attached figure. Figure 8 As shown in the results, TgGAP45-PLGA nanovaccine can significantly promote the expression of MHC-I molecules in mouse splenic dendritic cells (P<0.05), while TgGAP45-PLGA nanovaccine and commercial vaccine adjuvant can promote the expression of MHC-II molecules in mouse splenic dendritic cells (P<0.001). Dendritic cells are responsible for taking up the antigen components of vaccines and pathogenic microorganisms, and presenting them to T lymphocytes after processing. The splenic dendritic cells of mice immunized with TgGAP45-PLGA nanovaccine produced high levels of MHC-II molecules, indicating that the TgGAP45-PLGA nanovaccine can effectively activate mouse dendritic cells and produce corresponding functions.

[0105] 4.3 Toxoplasma gondii TgGAP45-PLGA nanovaccine promotes proliferation of mouse spleen lymphocytes

[0106] To investigate the effect of Toxoplasma gondii TgGAP45-PLGA nanovaccine on the proliferation of mouse spleen lymphocytes, 7 days after the second immunization, 3 mice in each group were taken to isolate spleen lymphocytes, and 5×105 cells / well were inoculated into 96-well cell culture plates. DMEM medium containing 10% fetal bovine serum was added to each well and cultured in an incubator at 37°C and 5% CO2 for 48 hours. After the culture was completed, 10 μL of CCK-8 reagent was added to each well. After incubation for another 2 hours, the OD450 value of each well was measured using an enzyme reader. The results are shown in the attached figure. Fig. 9 As shown in the figure, the proliferation level of spleen lymphocytes in TgGAP45 and its vaccine groups was significantly higher than that in the Control group (P<0.01). Activated dendritic cells promote the proliferation and differentiation of lymphocytes. The proliferation of spleen lymphocytes in mice was detected by CCK-8 reagent, indicating that spleen lymphocytes in mice can be activated by recombinant TgGAP45 protein and its vaccine.

[0107] 4.4 Toxoplasma gondii TgGAP45-PLGA nanovaccine promotes differentiation of mouse spleen lymphocytes

[0108] In order to evaluate the differentiation of T lymphocyte subsets in mouse spleen lymphocytes, 5 mice were randomly selected from each group for euthanasia and spleen lymphocytes were isolated on the 10th day after the first immunization and the 10th day after the booster immunization. The isolated lymphocytes were stained with FITC-coupled anti-mouse CD3e, PE-coupled anti-mouse CD8a and APC-coupled anti-mouse CD4 antibodies. After staining, the cells were washed three times with PBS, and the differentiation of CD4 and CD8 T lymphocytes in mouse spleen lymphocytes was analyzed by flow cytometry. The results are shown in the attached figure. Fig.10As shown in the figure, all three vaccines can effectively stimulate the differentiation of mouse spleen lymphocytes (P<0.05). Splenic lymphocytes activated by vaccine immunization will further differentiate into CD4 and CD8 T lymphocytes, and CD8 T lymphocytes will further differentiate into CTL cells, secrete specific antibodies, and target and kill Toxoplasma gondii.

[0109] 5. Toxoplasma gondii TgGAP45-PLGA nanovaccine protects against toxoplasmosis

[0110] 5.1 Intensity of Toxoplasma infection in mouse spleen

[0111] 14 days after the second immunization, 13 mice were selected from each group and injected intraperitoneally with 100 RH tachyzoites of Toxoplasma gondii. On the fourth day after the attack, 3 mice were randomly selected from each group for euthanasia and the spleen tissue of the mice was isolated. The spleen tissue of the mice was fixed in a 10% formalin solution for 24 hours. Subsequently, the tissue was dehydrated step by step from low to high concentration alcohol solution, and each step took 1 hour. After dehydration, the tissue was transferred to xylene for transparent treatment for 30 minutes. The tissue block was slowly immersed in melted liquid paraffin and three consecutive wax immersion operations were performed at a constant temperature of 60°C. After that, the melted paraffin was poured into a special embedding frame, and the wax-immersed tissue block was quickly embedded in it. The embedding frame was placed steadily on a condensation table to allow the paraffin to cool and solidify quickly to form a solid wax block. The embedded wax block was cut into slices with a thickness of 4-6μm. The sections were attached to glass slides and dried in an incubator for 24 hours. The sections were first dewaxed in xylene and then antigen-recovered using EDTA antigen-recovery solution with a pH of 9.0 to expose the antigenic sites. In order to eliminate endogenous peroxidase activity that may interfere with the experimental results, the sections were placed in a 3% H2O2 solution and kept in the dark for 25 minutes at room temperature. At 4°C, the sections were incubated with rabbit anti-T.gondii antibodies at a dilution ratio of 1:500 for 10 hours, the sections were fully rinsed with PBS to remove unbound antibodies, and CY3-coupled anti-rabbit IgG secondary antibodies (1:1000) were added and incubated at room temperature for 50 minutes. After being fully washed with PBS, the sections were counterstained with DAPI for 5 minutes. After being sealed with a sealing agent, the sections were placed under a fluorescence microscope and three fields of view were randomly photographed. The results are shown in the attached figure. Fig.11 As shown, the infection intensity of Toxoplasma gondii in the TgGAP45 group and its three vaccine groups was significantly reduced, among which the infection intensity of Toxoplasma gondii in the spleen tissue of mice in the TgGAP45-PLGA nanovaccine group was the lowest compared with the other two groups.

[0112] 5.2 Intensity of Toxoplasma infection in mouse myocardial tissue

[0113] In order to further determine the immune protection of TgGAP45-PLGA nanovaccine on mice, 10 mice were taken from each group 7 days after the challenge, and the mice were euthanized and the myocardial tissue of the mice was isolated. The DNA in the myocardial tissue was isolated using a commercial tissue DNA extraction kit and absolute quantitative PCR amplification was performed. The primers were used to detect the 529bp repeat sequence of Toxoplasma gondii tachyzoites, and the correct 529bp sequence plasmid was used as a reference. Each sample was measured three times to quantify the infection intensity of Toxoplasma gondii in the myocardial tissue of mice. The results are shown in the attached Fig.12 As shown in the data, the infection intensity of TgGAP45, TgGAP45-PLGA, TgGAP45-206VG and TgGAP45-660VG groups was significantly lower than that of the Control group (P<0.01), and among the three vaccines we tested, the infection intensity of Toxoplasma gondii in the myocardial tissue of mice in the TgGAP45-PLGA group was the lowest, indicating that TgGAP45-PLGA nanovaccine has the best immune protection effect compared with the two commercial vaccines and can be used for acute infection of Toxoplasma gondii.

[0114] Obviously, the above embodiments of the present invention are merely examples for clearly illustrating the present invention, and are not intended to limit the embodiments of the present invention. For those skilled in the art, other different forms of changes or modifications can be made based on the above description. It is not necessary and impossible to list all the embodiments here. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention should be included in the protection scope of the present invention.

Claims

1. A Toxoplasma gondii TgGAP45-PLGA nano vaccine, characterized in that: The vaccine is prepared by coating a recombinant protein GAP45, which is derived from the glial body histone of Toxoplasma gondii, and its amino acid sequence is shown in SEQ ID NO.

1.

2. The Toxoplasma gondii TgGAP45-PLGA nano vaccine according to claim 1, characterized in that The coating material comprises at least one of polylactic acid-glycolic acid, polylactic acid, polycaprolactone, polybutylene glycol succinate, polyaniline, polycarbonate, glycolide-caprolactone copolymer, and glycolide-caprolactone copolymer.

3. A method for preparing a Toxoplasma gondii TgGAP45-PLGA nano vaccine, characterized in that: The following steps are involved: S10. Prepare polyvinyl alcohol solution for standby use; S20. The coated material is dissolved in dichloromethane as an oil phase for later use; S30. Add the polyvinyl alcohol solution to the oil phase, shake it and then ultrasonically break it, then add the recombinant protein GAP45, shake it and then ultrasonically break it to obtain white colostrum; S40. Add the polyvinyl alcohol solution to the white colostrum, shake it and then ultrasonically break it to obtain a double emulsion; S50. Volatilize and remove the organic solvent in the double emulsion, centrifuge and separate the precipitate, resuspend it and then freeze-dry it to obtain the Toxoplasma gondii TgGAP45-PLGA nano vaccine freeze-dried powder.

4. The method for preparing the Toxoplasma gondii TgGAP45-PLGA nano vaccine according to claim 3, characterized in that: In step S30, the preparation method of the recombinant protein GAP45 is as follows: Total RNA was extracted from Toxoplasma gondii and reverse transcribed to synthesize cDNA. PCR primers were designed based on GenBank: TGME49_223940 sequence for amplification. The primer sequences are shown in SEQ ID NO.2 and SEQ ID NO.

3. The amplified product was double-digested with KpnI and EcoRI. After verification by agarose gel electrophoresis, the amplified band was excised and recovered by gel recovery kit. The linearized pET-32a empty vector digested with KpnI and EcoRI was ligated with T4 DNA ligase to obtain the pET-32a-TgGAP45 recombinant plasmid, which was transformed into Escherichia coli BL21 DE3; the above-mentioned Escherichia coli was inoculated into LB liquid culture medium containing ampicillin for expansion culture, and after adding IPTG, it was continued to be placed in a shaker for shaking culture to induce large-scale expression of the recombinant protein TgGAP45, and its induced expression in Escherichia coli BL21DE3 was identified by SDS-PAGE gel electrophoresis, the bacteria were recovered by centrifugation and ultrasonically broken, and the recombinant protein was purified by Ni-NTA chromatography column to obtain the recombinant protein GAP45.

5. The method for preparing the Toxoplasma gondii TgGAP45-PLGA nano vaccine according to claim 3, characterized in that: Step S30 includes the following steps: S31. Add the polyvinyl alcohol solution dropwise to the oil phase, shake vigorously after the addition is completed until the liquid becomes milky white, and then ultrasonically crush, and after the completion of the ultrasonication, a milky white solvent without stratification is formed; S32. Add the recombinant protein GAP45 dropwise into the milky white solvent without stratification, vortex and shake after the dropwise addition, and then perform ultrasonic crushing to obtain white colostrum after the ultrasonication.

6. The method for preparing the Toxoplasma gondii TgGAP45-PLGA nano vaccine according to claim 3, characterized in that: In step S32, the concentration of the recombinant protein GAP45 is 1000 to 2000 mg / mL.

7. Use of the Toxoplasma gondii TgGAP45-PLGA nano vaccine according to claim 1 in the preparation of a drug for preventing mammals from being infected with toxoplasmosis.

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