Toxoplasma gondii TgIMC1-CS 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 TgIMC1-CS nano vaccine as well as a preparation method and application thereof. The vaccine is prepared by coating pVAX1-TgIMC1 DNA (deoxyribonucleic acid) plasmid with a coating substance, the pVAX1-TgIMC1 DNA plasmid is derived from toxoplasma gondii inner membrane skeleton protein 1, and the amino acid sequence of the pVAX1-TgIMC1 DNA plasmid is shown in SEQ ID NO. 1. The vaccine provided by the invention is high in immunomodulatory factor and anti-inflammatory factor titer, can induce an organism to generate specific immune response, so that the prevention effect of animals on the disease is improved, and the vaccine is non-toxic and 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 TgIMC1-CS nano vaccine and a preparation method and application thereof. Background Art

[0002] Toxoplasma gondii (T.gondii) can infect a wide range of hosts, including a variety of mammals, birds, and cold-blooded animals including humans. The toxoplasmosis caused by it is an important zoonosis. In patients and animals, toxoplasmosis mainly manifests as headache, lethargy, and coma. Severe infection can cause death of patients and animals. In pregnant women and pregnant animals, it can cause symptoms such as miscarriage, malformed fetuses, and stillbirths. Currently, sulfadiazine and pyrimethamine are the most widely used drugs for toxoplasmosis, but they have strong toxic side effects and are prone to drug resistance after long-term use. At present, only the attenuated live vaccine based on the Toxoplasma S48 strain is available for abortion caused by toxoplasmosis in sheep, but its biosafety has been questioned and there is a possibility of toxicity reversal.

[0003] In recent years, subunit vaccines based on recombinant proteins have become a hot topic of research. They can effectively avoid the biosafety risks caused by using pathogens to produce vaccines, and at the same time have the advantages of safe use, stable properties and low production costs. Chitosan (CS) nanoadjuvants can accelerate, prolong or enhance antigen-specific immune responses and are often used together with vaccines to form vaccine preparations.

[0004] For example, in the prior art, the Chinese invention patent with application number 202010129028.0 discloses a nanomaterial subunit vaccine for Toxoplasma gondii and its preparation method and application. The vaccine is formed by coating Toxoplasma gondii recombinant protein PSMB1 with chitosan to form nanoparticles. The recombinant protein TgPSMB1 comes from the proteasome subunit 1 of Toxoplasma gondii. After prokaryotic expression, it is coated with nanomaterial chitosan to form a new vaccine form. After evaluating the immune protection efficacy of the vaccine, it was found that the vaccine can prolong the survival time of infected mice, indicating that it can provide partial immune protection, but the vaccine's immunomodulatory factors and anti-inflammatory factors are less cost-effective, and the toxicity is unknown, and there are certain biosafety risks.

[0005] Full hidden dangers. Summary of the invention

[0006] Based on this, the present application provides a Toxoplasma gondii TgIMC1-CS nanovaccine and its preparation method and application to solve the technical problems in the prior art that vaccine immunomodulatory factors and anti-inflammatory factors have low cost-effectiveness, unknown toxicity, and certain biosafety risks.

[0007] The technical solution of the present invention to solve the above technical problems is as follows:

[0008] A Toxoplasma gondii TgIMC1-CS nano vaccine is prepared by coating a pVAX1-TgIMC1 DNA plasmid with a coating material. The pVAX1-TgIMC1 DNA plasmid is derived from Toxoplasma gondii inner membrane skeleton protein 1, and its amino acid sequence is shown in SEQ ID NO.1.

[0009] Preferably, in the above-mentioned Toxoplasma gondii TgIMC1-CS nanovaccine, the coating material includes at least one of chitosan, polylactic acid, polycaprolactone, polybutylene glycol succinate, polyaniline, polycarbonate, glycolide-caprolactone copolymer, and glycolide-caprolactone copolymer.

[0010] A method for preparing a Toxoplasma gondii TgIMC1-CS nano vaccine comprises the following steps:

[0011] S10. The Toxoplasma gondii IMC1 gene sequence TGRH88_075820 was codon-optimized to obtain a nucleotide sequence shown in SEQ ID NO.2. The nucleotide sequence was fully synthesized, and the obtained gene fragment was amplified by PCR using primers shown in SEQ ID NO.3 and SEQ ID NO.4, and then ligated to the linearized pVAX1 vector using T4 DNA ligase to obtain a pVAX1-TgIMC1 DNA plasmid;

[0012] S20. After the pVAX1-TgIMC1 DNA plasmid is verified by double enzyme digestion, the plasmid with correct sequencing is transferred to E. coli DH5α, and the plasmid is extracted in large quantities using a plasmid DNA extraction kit to remove endotoxins therein to obtain an endotoxin-free pVAX1-TgIMC1 DNA plasmid;

[0013] S30. Add the endotoxin-free pVAX1-TgIMC1 DNA plasmid and sodium tripolyphosphate solution dropwise to the chitosan solution, stir magnetically and then ultrasonically disrupt to obtain TgIMC1-CS nanovaccine aqueous solution;

[0014] S40. Centrifuge the TgIMC1-CS nanovaccine aqueous solution, collect the precipitate, resuspend it in distilled water, and then filter and freeze-dry to obtain the Toxoplasma gondii TgIMC1-CS nanovaccine.

[0015] Preferably, the method for preparing the Toxoplasma gondii TgIMC1-CS nanovaccine is characterized in that step S20 is specifically:

[0016] The pVAX1-TgIMC1 DNA plasmid was double-digested and verified and sequenced using a DNA analyzer; the correctly sequenced plasmid was transferred into E. coli DH5α competent cells, the plasmid was extracted in large quantities using a plasmid DNA extraction kit, and the endotoxin was removed using an endotoxin removal kit to reduce the endotoxin content in the plasmid to less than 0.01EU / ml, and the concentration of the pVAX1-TgIMC1 DNA plasmid was adjusted to 800 to 1500ng / μl using double distilled water to obtain an endotoxin-free pVAX1-TgIMC1 DNA plasmid.

[0017] Preferably, in the method for preparing the Toxoplasma gondii TgIMC1-CS nanovaccine, in step S30, the chitosan solution is prepared as follows:

[0018] Chitosan is dissolved in an acetic acid aqueous solution as an organic phase, and the pH value is adjusted to 4 to 7 with a NaOH solution to obtain a chitosan solution with a concentration of 1 to 3 mg / ml; wherein the ratio of chitosan to the acetic acid aqueous solution is (1 to 3) mg:1 ml.

[0019] Preferably, in the above-mentioned method for preparing Toxoplasma gondii TgIMC1-CS nanovaccine, in step S30, the ratio of the chitosan solution, the endotoxin-free pVAX1-TgIMC1 plasmid and the sodium tripolyphosphate solution is (3 to 7) ml: (1 to 3) mg: 1 ml; wherein the concentration of the sodium tripolyphosphate solution is 1 to 3 mg / ml.

[0020] Preferably, in the method for preparing the above-mentioned Toxoplasma gondii TgIMC1-CS nanovaccine, in step S30, the ultrasonic fragmentation conditions are: 4° C., power 140 to 160 W, interval 1 to 2 s, ultrasonic 1 to 2 s, and ultrasonic time 4 to 5 min.

[0021] The use of the Toxoplasma gondii TgIMC1-CS 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 Inner Membrane Complex 1 (IMC1) constitutes a network under the subcellular membrane, providing the necessary support for the formation of Toxoplasma cells and ensuring that the cells can maintain a normal morphology. IMC1 is also involved in the formation and function regulation of the Toxoplasma cytoskeleton, and is an important component of the Toxoplasma membrane structure. It plays an important role in the invasion and proliferation of Toxoplasma and other life activities. In the process of Toxoplasma infection of host cells, IMC1 can participate in the formation of white actin-myosin motors. The actin-myosin motor is the main power source for the substrate-mediated gliding movement of Toxoplasma. The lack of IMC1 protein will lead to a decrease in the movement ability of Toxoplasma, making it difficult for Toxoplasma to move and spread effectively in the host, thereby affecting its survival and infection ability. In addition, the IMC1 protein is also an important component of the Toxoplasma gliding body, which is closely related to the parasite's gliding, invasion and escape from host cells. The Toxoplasma gondii TgIMC1-CS nanovaccine (hereinafter referred to as TgIMC1-CS nanovaccine) prepared in the present application using IMC1 protein as the key inner membrane antigen of Toxoplasma gondii can induce the body to produce a specific immune response, thereby improving the preventive effect of animals against the disease.

[0024] 2. When chitosan (CS) nanomaterials are used as nanomaterials, they have low irritation to animal tissues and cells, are not easy to cause adverse biological reactions such as immune rejection, can reduce immunogenicity in animals, and reduce side effects caused by the use of vaccines; at the same time, chitosan can be gradually decomposed into harmless small molecules by biological enzymes in the body, and finally absorbed or metabolized and excreted from the body, with good biocompatibility and degradability. In addition, the targeting and easy modification of CS nanomaterials can enable CS nanovaccines to efficiently penetrate into target cells and tissues, thereby improving the immune effect. CS nanomaterials can effectively load a large amount of IMC1 protein, causing a high level of antigen cross-presentation. At the same time, the sustained-release characteristics of CS nanomaterials can ensure the continuous release of antigens in the body, prolong the stimulation time of the vaccine, and thus enhance the immune effect of animals. Therefore, the nanovaccine prepared based on chitosan has good efficiency and safety, is suitable for the efficient prevention of toxoplasmosis, and can significantly reduce the risk of infection with Toxoplasma gondii, thereby protecting the health of animals.

[0025] 3. The vaccine of the present invention is based on DNA vaccine and contains only part of the nucleotide sequence of pathogenic microorganisms, so it has the advantages of safety, low toxicity and stable properties. Experiments have shown that the 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, thereby controlling the spread of Toxoplasma gondii. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 (a): Double enzyme digestion identification of recombinant plasmid pVAX1-TgIMC1; M: DL5,000 Marker; (b): Western blot analysis of the expression of pVAX1-TgIMC1; M: protein Marker (kDa); 1: HEK 293-T cells were transfected with pVAX1-TgIMC1 plasmid and recognized by rat anti-TgIMC1 serum; 2: HEK 293-T cells were transfected with pVAX1 plasmid and recognized by rat anti-TgIMC1 serum.

[0028] Figure 2 The expression of pVAX1-TgIMC1; HEK 293-T cells were transfected with pVAX1-TgIMC1 plasmid, incubated with rat anti-TgIMC1 serum, and then incubated with anti-rat CY3 secondary antibody. Before observation under laser confocal microscopy, the samples were stained with DAPI.

[0029] Figure 3 Scanning electron microscopy results of TgIMC1-CS nanovaccine; scale bar is 500 nm.

[0030] Figure 4 This is the in vitro sustained release characteristics of TgIMC1-CS nanovaccine.

[0031] Figure 5 The levels of urea nitrogen (a) and creatinine (b) in mice after immunization.

[0032] Figure 6 The changes in the levels of total IgG (a), IgG1 (b) and IgG2a (c) antibodies in the serum of mice after immunization; *P<0.05, **P<0.01, ***P<0.001.

[0033] Figure 7 Changes of IFN-γ(a), TGF-β(b), IL-4(c), IL-6(d), IL-10(e) and IL-17(f) in the serum of mice after immunization; **P<0.01, ***P<0.001.

[0034] Figure 8 The expressions of CD83 (a, b) and CD86 (a, c) molecules on splenic dendritic cells after immunization of mice; *P<0.05, ***P<0.001.

[0035] Fig. 9The expressions of MHC-I (a, b) and MHC-II molecules (a, c) on splenic dendritic cells of mice after immunization; *P<0.05, **P<0.01, ***P<0.001.

[0036] Fig.10 This is the proliferation of spleen lymphocytes after mice were immunized with TgIMC1-CS nanovaccine.

[0037] Fig.11 Differentiation of CD4 (a, b) and CD8 (a, c) T lymphocytes in spleen of mice immunized with TgIMC1-CS nanovaccine; *P<0.05, ***P<0.001.

[0038] Fig.12 Spleen tissue sections of mice infected with 100 Toxoplasma gondii tachyzoites in the peritoneal cavity; scale bar, 100 μm; ***P<0.001.

[0039] Fig.13 The results show the infection status of Toxoplasma gondii in myocardial tissue of mice after intraperitoneal infection with 100 Toxoplasma gondii tachyzoites; ***P<0.001. DETAILED DESCRIPTION

[0040] 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.

[0041] In a specific embodiment of the present invention, a Toxoplasma gondii TgIMC1-CS nano vaccine is provided, wherein the nano vaccine is prepared by coating a pVAX1-TgIMC1 DNA plasmid with a coating material, wherein the pVAX1-TgIMC1 DNA plasmid is derived from the inner membrane skeleton protein 1 of Toxoplasma gondii, and its amino acid sequence is shown in SEQ ID NO.1.

[0042] SEQ ID NO.1 is:

[0043] .

[0044] The Toxoplasma Inner Membrane Complex 1 (IMC1) constitutes a network under the subcellular membrane, providing the necessary support for the formation of Toxoplasma cells and ensuring that the cells can maintain a normal morphology. IMC1 is also involved in the formation and function regulation of the Toxoplasma cytoskeleton, and is an important component of the Toxoplasma membrane structure. It plays an important role in the invasion and proliferation of Toxoplasma life activities. In the process of Toxoplasma infection of host cells, IMC1 can participate in the formation of white actin-myosin motors. The actin-myosin motor is the main power source for the substrate-mediated gliding movement of Toxoplasma. The lack of IMC1 protein will lead to a decrease in the movement ability of Toxoplasma, making it difficult for Toxoplasma to move and spread effectively in the host, thereby affecting its survival and infection ability. In addition, the IMC1 protein is also an important component of the Toxoplasma gliding body, which is closely related to the parasite's gliding, invasion and escape from host cells. Therefore, IMC1 plays an important biological role in Toxoplasma cells and can be used as an important antigen for the development of Toxoplasma vaccines.

[0045] The coating is preferably a biodegradable nanomaterial, such as nanoparticles. In a preferred embodiment, the coating comprises at least one of chitosan, polylactic acid, polycaprolactone, polybutylene glycol succinate, polyaniline, polycarbonate, glycolide-caprolactone copolymer, and glycolide-caprolactone copolymer.

[0046] In another specific embodiment of the present application, the method for preparing the above-mentioned Toxoplasma gondii TgIMC1-CS nanovaccine comprises the following steps:

[0047] S10. The Toxoplasma gondii IMC1 gene sequence TGRH88_075820 was codon-optimized to obtain a nucleotide sequence shown in SEQ ID NO.2. The nucleotide sequence was fully synthesized, and the obtained gene fragment was amplified by PCR using primers shown in SEQ ID NO.3 and SEQ ID NO.4, and then ligated to the linearized pVAX1 vector using T4 DNA ligase to obtain a pVAX1-TgIMC1 DNA vaccine;

[0048] Specifically, the Toxoplasma gondii IMC1 gene sequence (TGRH88_075820) was submitted for codon optimization, and the optimized species was mouse. The obtained nucleotide sequence is shown in SEQ ID NO.2:

[0049]

[0050] The sequence was submitted to Shanghai Sangon Biotechnology for whole genome synthesis, and the obtained gene fragment was amplified by PCR with the designed primers of SEQ ID NO.3 and SEQ ID NO.4, where SEQ ID NO.3 is:

[0051] IMC1-opt-MF:5′-cgg GGTACCATGTTTAAAGATTGTGCAGACCCATGT-3′ SEQ ID NO.4 is:

[0052] IMC1-opt-MR:5′-cgc GGATCC TTAGCACTGGCAGCGACAAAC-3′.

[0053] The amplified product was verified by 1% agarose gel electrophoresis, and the correct target gene was recovered by gel excision; the amplified PCR product was ligated to the linearized pVAX1 vector using DT4 DNA ligase to obtain the pVAX1-TgIMC1 DNA vaccine.

[0054] S20. After the pVAX1-TgIMC1 DNA plasmid is verified by double enzyme digestion, the plasmid with correct sequencing is transferred to E. coli DH5α, and the plasmid is extracted in large quantities using a plasmid DNA extraction kit to remove endotoxins therein to obtain an endotoxin-free pVAX1-TgIMC1 DNA plasmid;

[0055] In a preferred embodiment, step S20 specifically comprises: performing double enzyme digestion on the pVAX1-TgIMC1 DNA plasmid and verifying and sequencing it with a DNA analyzer; transferring the correctly sequenced plasmid into E. coli DH5α competent cells, and then removing endotoxins with an endotoxin removal kit to make the endotoxin content in the plasmid lower than 0.01EU / ml, and adjusting the concentration of the pVAX1-TgIMC1 DNA plasmid to 800 to 1500ng / μl using double distilled water to obtain an endotoxin-free pVAX1-TgIMC1 DNA plasmid.

[0056] S30. Add the endotoxin-free pVAX1-TgIMC1 DNA plasmid and sodium tripolyphosphate solution dropwise to the chitosan solution, stir magnetically and then ultrasonically break to obtain TgIMC1-CS nano vaccine aqueous solution; the preparation method of the chitosan solution is as follows: dissolve chitosan in an acetic acid aqueous solution as an organic phase, and adjust the pH value to 4 to 7 with a NaOH solution to obtain a chitosan solution, wherein the concentration of the chitosan solution is 1 to 3 mg / ml; wherein the ratio of chitosan to the acetic acid aqueous solution is (1 to 3) mg: 1 ml. In a preferred embodiment, the ratio of the chitosan solution, the endotoxin-free pVAX1-TgIMC1 plasmid and the sodium tripolyphosphate solution is (3 to 7) ml: (1 to 3) mg: 1 ml; wherein the concentration of the sodium tripolyphosphate solution is 1 to 3 mg / ml.

[0057] Furthermore, in step S30, the ultrasonic fragmentation conditions are: 4°C, power 140 to 160 W, interval 1 to 2 s, ultrasonic 1 to 2 s, and ultrasonic time 4 to 5 min.

[0058] Specifically, chitosan was weighed and dissolved in acetic acid aqueous solution as the organic phase, and the pH value was adjusted to 4 to 7 with NaOH solution; the chitosan solution was placed on a magnetic stirrer, and endotoxin-free pVAX1-TgIMC1 plasmid was added dropwise while stirring, and sodium tripolyphosphate solution was added dropwise, and stirring was continued for 20 to 40 minutes after the addition was completed, and then ultrasonic crushing was performed at 2 to 6°C, the power of the ultrasonic crusher was set to 140-160W, the interval was 1-2s, the ultrasonication was 1-2s, and the ultrasonication time was 4-5min, to obtain TgIMC1-CS nano vaccine aqueous solution.

[0059] S40. Centrifuge the TgIMC1-CS nanovaccine aqueous solution, collect the precipitate, resuspend it in distilled water, and then filter and freeze-dry to obtain the Toxoplasma gondii TgIMC1-CS nanovaccine.

[0060] Specifically, the ultrasonic aqueous solution is centrifuged at a speed of 30,000 to 50,000 r / min for 10 to 20 minutes at 2 to 6° C. After centrifugation, the aqueous solution is divided into two parts: a supernatant and a jelly-like precipitate. The precipitate is collected and resuspended in distilled water, filtered, and freeze-dried to obtain the Toxoplasma gondii TgIMC1-CS nano vaccine.

[0061] In another specific embodiment of the present invention, the above-mentioned Toxoplasma gondii TgIMC1-CS nanovaccine is used in the preparation of a drug for preventing mammals from being infected with toxoplasmosis.

[0062] 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.

[0063] 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.

[0064] Example 1 Toxoplasma gondii TgIMC1-CS nanovaccine

[0065] 1.1 Preparation of pVAX1-TgIMC1 DNA plasmid

[0066] In order to improve the expression level of Toxoplasma gondii IMC1 protein in mice, the IMC1 protein gene sequence (TGRH88_075820) was submitted to www.novopro.cn / tools / codon-optimization.html for codon optimization, and the obtained nucleotide sequence is shown in SEQ ID NO. 2. The sequence was submitted to Shanghai Sangon Biotech for whole genome synthesis.

[0067] The obtained gene fragments were amplified by PCR. The PCR amplification system was as follows: 12.5 μl of 2×T5 SuperPCR Mix polymerase, 1 μl of IMC1-opt-MF, 1 μl of IMC1-opt-MR, and 500 ng of cDNA template were added to the PCR tube, and finally supplemented to 25 μl with ddH2O. The PCR amplification program was as follows: 1 cycle of pre-denaturation at 98°C for 3 min, followed by denaturation at 98°C for 30 s, annealing at 58°C for 30 s, and extension at 72°C for 30 s, for a total of 35 cycles. Finally, the extension was completed at 72°C for 5 minutes.

[0068] The PCR product was verified by 1% agarose gel electrophoresis, and the correct target band was recovered using a gel recovery kit. The KpnI and BamHI restriction endonucleases were used as instructions for use, and the linearized pVAX1 vector was connected to obtain the pVAX1-TgIMC1 recombinant plasmid. The recombinant plasmid was verified by double enzyme digestion (see attached). Figure 1 a), transformed into E. coli DH5α competent cells, and obtained genetically engineered bacteria after single colony expansion culture. After culturing genetically engineered bacteria in large quantities, the plasmid was extracted using a commercial kit, and the endotoxin was removed by an endotoxin removal kit. The endotoxin content in pVAX1-TgIMC1 was detected by an endotoxin detection kit, and the results showed that the endotoxin content was less than 0.01EU / ml.

[0069] 1.2 Expression analysis of pVAX1-TgIMC1

[0070] All experimental protocols in this project comply with the relevant animal 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 rat experiment license number is NXU-2023-086.

[0071] Preparation of polyclonal antibody to Toxoplasma IMC1 protein: Based on the above PCR, the synthesized gene was PCR amplified, and the PCR amplification product was digested with KpnI and BamHI restriction endonucleases, and then connected to the linearized pET-32a vector to obtain the pET32a-TgIMC1 recombinant plasmid. The recombinant plasmid was transformed into E.coli BL21 (DE3) competent cells, and after induction with IPTG inducer, the recombinant Toxoplasma IMC1 protein was obtained by affinity chromatography. 200 μg of Toxoplasma IMC1 protein was mixed with complete Freund's adjuvant to immunize Wistar rats. After 2 weeks, 200 μg of Toxoplasma IMC1 protein was mixed with incomplete Freund's adjuvant, and immunization was performed 4 times at intervals of 2 weeks. All immunizations were injected subcutaneously at multiple points on the back skin of the rats. One week after the last injection, blood was collected from the eye sockets and serum was separated to obtain polyclonal antibody serum to Toxoplasma IMC1 protein.

[0072] Expression analysis of pVAX1-TgIMC1 in mammals: Human renal epithelial HEK 293-T cells were cultured in six-well plates and transfected with Lipofectamine TM The toxin-free pVAX1-TgIMC1 plasmid was transfected with 3000 reagent. The cells were fixed and cultured for 48 hours after transfection, the supernatant was discarded, and the cell samples were collected. After 12% SDS-PAGE electrophoresis, the cell samples were transferred to PVDF membranes, and the PVDF membranes were incubated with rat anti-Toxoplasma IMC1 protein serum as the primary antibody (1:500) and goat anti-rat IgG as the secondary antibody (1:10000). Untransfected cells were set as the control group, and finally ECL luminescent liquid was used for exposure and color development. The results showed that the pVAX1-TgIMC1 plasmid could be expressed by HEK 293-T cells (see attached). Figure 1 ).

[0073] To further determine the in vitro expression of pVAX1-TgIMC1, HEK 293-T cells were transfected with pVAX1-TgIMC1 for 48 h, the supernatant was discarded, the cells were rinsed three times with PBS, and then fixed with 1 ml of 4% paraformaldehyde for 12 h at 4°C. They were blocked with TBSx containing 5% bovine serum albumin (BSA), and then incubated with rat anti-Toxoplasma gondii IMC1 protein serum as the primary antibody (1:200) and CY3-coupled anti-rat IgG as the secondary antibody (1:1000), respectively. After that, they were counterstained with DAPI for 5 min, and images were taken using a laser confocal microscope (see Appendix). Figure 2 ).

[0074] 1.3 Preparation of Toxoplasma gondii TgIMC1-CS nanovaccine

[0075] Preparation of 2% sodium tripolyphosphate (TPP) solution: Place a small glass beaker on a magnetic stirrer, put in a magnetic rotor and heat, add an appropriate amount of ionized water, weigh 20 mg of TPP and add it to the small beaker, after the sodium tripolyphosphate is completely dissolved, add deionized water to make up to 10 ml, and filter with a 0.22 μm filter membrane.

[0076] Preparation of 1.0% acetic acid aqueous solution: Take 500 μl of glacial acetic acid and dissolve it in 40 ml of double distilled water. After the glacial acetic acid and water are completely mixed, the volume is adjusted to 50 ml and filtered with a 0.22 μm filter membrane to obtain a 1.0% acetic acid aqueous solution.

[0077] Accurately weigh 20 mg of chitosan and dissolve it in 10 ml of 1.0% acetic acid aqueous solution as the organic phase, and adjust the pH value to 5.0 with NaOH solution. Place 10 ml of chitosan solution on a magnetic stirrer, start stirring and ensure that the solution temperature is at 30°C, add 4 mg of endotoxin-free pVAX1-TgIMC1 plasmid drop by drop, and then add 2 ml of TPP solution drop by drop. Continue stirring for 30 minutes after the addition is completed. The mixed aqueous solution is ultrasonically crushed at 4°C, the power of the ultrasonic crusher is set to 140-160W, the interval is 1-2s, the ultrasonication is 1-2s, and the total ultrasonication time is 4-5min. At 4°C, the ultrasonicated aqueous solution is centrifuged at a speed of 40000r / min for 15min. After centrifugation, the aqueous solution was divided into two parts: supernatant and jelly-like precipitate. The precipitate was collected and resuspended in distilled water, filtered through a 0.22 μm filter and then freeze-dried to obtain the Toxoplasma gondii TgIMC1-CS nanovaccine. The nanovaccine was stored at -20°C.

[0078] Example 2 Characteristics of TgIMC1-CS Nanovaccine

[0079] 2.1 Physical properties of TgIMC1-CS nanovaccine

[0080] The prepared CS nanospheres were sent to the College of Life Sciences of Nanjing Agricultural University for scanning electron microscope photographs. Figure 3 As shown, the TgIMC1-CS nanovaccine is a uniform sphere with small protrusions on its surface. Based on the scanning electron microscope image, the average diameter of the TgIMC1-CS nanovaccine is 84.12±7.23nm using ImageJ for analysis. To determine the coating efficiency of the TgIMC1-CS 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 TgIMC1-CS nanovaccine was calculated according to formula (1). After three independent repeated experiments, the coating efficiency of the TgIMC1-CS nanovaccine was 62.40±2.02%.

[0081]

[0082] 2.2 In vitro sustained release characteristics of TgIMC1-CS nanovaccine

[0083] Weigh the nanovaccine containing 10 μg of pVAX1-TgIMC1 plasmid and place it in a closed 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, the centrifuge tube is centrifuged at 15,000 r / min for 5 minutes, and the volume of the supernatant is recorded at the same time. Take 5 μl of the supernatant as the sample for the sustained-release curve and store it at -20 ° C for standby. Add 5 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 copy number of the pVAX1-TgIMC1 plasmid in the sample was determined by absolute quantitative PCR. The primers used are:

[0084] SEQ ID NO.5: IMC1-det-F: 5′-ATGTTCAGGAGGAGGAGGGC-3′ and

[0085] SEQ ID NO.6: IMC1-det-R: 5′-TAATCCACTCGTTCTGAAGGTATG-3′, the reaction system is 1μl DNA template, 10μl 2×Universal SYBR qPCR Mix, 0.4μl upstream and downstream primers, ddH2O added to 20μl; the reaction conditions are 95℃30s; 95℃10s, 55℃15s, 72℃15s, a total of 40 cycles. At the same time, different concentration gradients of standards were set, and the standard was a pVAX1-TgIMC1 plasmid with known concentration and sequence. The cumulative release rate (%) was calculated according to formula (2). After three independent repeated experiments, the in vitro sustained release curve of TgIMC1-CS nanovaccine was drawn. The results showed that TgIMC1-CS nanovaccine had a faster release phase in the early stage, and then entered a relatively stable release period ( Figure 4 ), indicating that TgIMC1-CS nanovaccine has good sustained-release properties.

[0086]

[0087] 2.3 Toxicity of TgIMC1-CS Nanovaccine

[0088] All experimental protocols in this project comply with the relevant animal 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 mouse license number is NXU-2023-083.

[0089] The experimental animals were male ICR mice weighing 18-22g, and they were kept in an SPF isolation barrier system from birth to the end of the experiment. 35 mice were randomly divided into 5 groups (n=5). They were Blank group (immunized with an equal volume of PBS), Control group (immunized with 300μg pVAX1 empty plasmid), TgIMC1 group (immunized with 300μg pVAX1-TgIMC1), and TgIMC1-CS group (immunized with TgIMC1-CS containing 300μl pVAX1-TgIMC1). Each mouse was injected intramuscularly, and the injection dose was 3 times the conventional immunization. Three days after the first immunization, the mice were immunized again. Three days after the second immunization, blood was collected from the mouse orbits and serum was separated. The levels of creatinine (Cr) and blood urea nitrogen (BUN) in the mouse serum were detected by sarcosine oxidase method and urease-indophenol method, respectively. The mental and activity states of the mice were recorded during the period. The results showed that there was no significant difference between the CS group, TgIMC1 group and TgIMC1-CS group and the Control group (P>0.05, Appendix Figure 5), and the mice were in good spirits and activity during the experiment, indicating that the TgIMC1-CS nanovaccine is non-toxic to mice.

[0090] Example 3 TgIMC1-CS nanovaccine induces humoral immunity in mice

[0091] 3.1 Animal Experiment Design

[0092] Female ICR mice weighing 18-22 g were purchased from Beijing Weitong Lihua Biotechnology 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 21 mice in each group, and immunized with PBS, pVAX1 empty DNA vaccine, pVAX1 empty chitosan nanovaccine, and TgIMC1-CS nanovaccine by intramuscular injection (Table 1).

[0093] Table 1 Experimental groups and immunization procedures

[0094]

[0095] 3.2 Nanovaccine TgIMC1-CS promotes antibody secretion in mouse serum

[0096] On the 10th day after the first immunization and the 10th day after the second immunization, the serum of 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 test. Briefly, a certain amount of Toxoplasma gondii tachyzoites were dissolved in coating buffer, and after 160W total ultrasound for 5 min, the total protein concentration was determined by BCA method, and 100 μl of Toxoplasma gondii tachyzoite lysate containing 5 μg was added to each well of the 96-well ELISA plate, and incubated overnight at 4°C. After washing three times with TBS containing 5% skim milk, 20 μl of mouse serum was incubated in each well for 2 h. After washing three times with TBS, HRP-coupled anti-mouse total IgG, IgG1 and IgG2a antibodies (1:5000 dilution) were used for incubation for 2 h. After washing three times with TBS again, 100 μl of freshly prepared TMB color development solution was added to each well. After color development at room temperature for 10-15 min, 100 μl of freshly prepared 2 mol / L H2SO4 was added to each well to stop color development. The OD450 value was read by an enzyme marker for data analysis. After the first immunization and booster immunization, higher levels of total IgG were detected in the TgIMC1-CS group mice (P<0.001, Figure 6 a), and mice immunized with pVAX1-TgIMC1 and TgIMC1-CS also produced high levels of IgG1 (P<0.05, Figure 6 a) and IgG2a (P<0.05, Figure 6a). The increase in specific total IgG content plays an important role in the host's resistance to Toxoplasma gondii. IgG1 is mainly associated with Th2-type helper T cells, promoting humoral immune responses and preventing pathogens from invading cells. IgG2a is associated with Th1-type helper T cells and participates in cell-mediated immune responses. The increase in IgG1 and IgG2a indicates that the prepared TgIMC1-CS nanovaccine can induce humoral and cell-mediated immune responses in the body, thereby producing immune protection against Toxoplasma gondii.

[0097] 3.3 Nanovaccine TgIMC1-CS regulates cytokine levels in mouse serum

[0098] To determine the changes in cytokine levels in mouse serum, blood was collected from mice through the eye sockets on the 10th day after 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 the sandwich ELISA method. The results are shown in the attached Figure 7 As shown in the figure, the IFN-γ, IL-4, IL-6, IL-10, and IL-17 produced by mice in the TgIMC1-CS group were significantly higher than those in the Control group (P<0.01), while there was no significant difference in TGF-β between the two groups (P>0.05). The increase in IFN-γ and IL-4 in mouse serum indicates that the Th1 type of immunity in the immune system is activated, promoting the production of specific antibodies. IL-10 is an important anti-inflammatory cytokine. Increased IL-10 can inhibit excessive immune responses and prevent inflammatory tissue damage. There was no significant difference in TGF-β, which may be related to the fact that TGF-β is mainly involved in chronic inflammation and immune regulation, and its level will not change significantly immediately. IL-6, as an important indicator of inflammatory response, is an important reference for judging whether the vaccine can cause side effects in animals. In this study, the IL-6 level of mice did not increase significantly, indicating that the nanovaccine TgIMC1-CS cannot cause severe inflammatory response in animals. IL-17 is secreted by Th17 cells and plays an important role in stimulating the recruitment and activation of neutrophils. The increase in IL-17 indicates that the TgIMC1-CS nanovaccine activates the inflammatory pathway of the immune system and enhances the resistance of mice to Toxoplasma infection. Compared with the vaccine disclosed in the prior art 202010129028.0, it can be seen from the figure that the IFN-γ, IL-4, IL-10, and IL-17 levels in the mouse serum of the present application are significantly higher than those of the prior art.

[0099] Example 4 TgIMC1-CS nanovaccine induces cellular immunity in mice

[0100] 4.1TgIMC1-CS nanovaccine induces maturation of mouse dendritic cells

[0101] On the 10th day after the first immunization and the 10th day after the second immunization, 5 mice were randomly selected from each group for euthanasia and the mouse spleen tissue was isolated. The mouse spleen lymphocytes were isolated using a commercial spleen lymphocyte separation solution. The mouse spleen lymphocytes were cultured overnight in DMEM culture medium containing 10% FBS and 1% double antibody. The non-adherent cells were discarded the next day, and the adherent cells were collected after washing three times with PBS. The collected cells were stained with anti-mouse CD11c-APC, CD86-FITC and CD83-PE antibodies at 4°C in the dark for 40 minutes. After staining, the cells were washed three times with PBS and collected into flow tubes for analysis using a flow cytometer. The results are shown in the attached figure. Figure 8 As shown in Figure 2, the expression level of CD83 molecules was significantly higher than that of the Control group (Appendix Figure 8 a and b), the expression level of CD86 molecule was significantly higher than that of the Control group (Appendix Figure 8 a and c), CD83 is mainly involved in regulating the maturation of immune cells and the antigen presentation process, which is crucial for adaptive immune responses. CD86 is located on the surface of antigen-presenting cells and can bind to CD28 on T cells to promote T cell activation, thereby enhancing immune responses. The increase in CD83 and CD86 indicators indicates that antigen-presenting cells are in an active or mature state, and antigen presentation and T cell activation are enhanced, suggesting that the TgIMC1-CS vaccine can promote the maturation of mouse spleen dendritic cells.

[0102] 4.2TgIMC1-CS nanovaccine promotes antigen presentation by mouse splenic dendritic cells

[0103] To analyze the effect of TgIMC1-CS nanovaccine on the MHC molecules of mouse spleen dendritic cells, mouse spleen lymphocytes were isolated on the 10th day after the first immunization and the 10th day after the second immunization using the same method as above, and mouse spleen lymphocytes were stained with anti-mouse CD11c-PE, MHC-I-FITC and MHC-II-APC, washed three times with PBS, enriched in flow tubes, and analyzed by flow cytometry. The results are shown in the attached Fig. 9 As shown in the results, TgIMC1-CS nanovaccine can significantly promote the expression of MHC-II class molecules in mouse spleen dendritic cells around the second week (P<0.001), and the expression of MHC-I and MHC-II molecules in mouse spleen dendritic cells around the fourth week can be promoted. MHC-I class molecules are mainly expressed on the surface of all nucleated cells and are responsible for presenting endogenous antigens to CD8 +T cells, thereby triggering the killing response of cytotoxic T cells. MHC-II class molecules are mainly expressed on the surface of antigen-presenting cells, presenting exogenous antigens to CD4+T cells, activating helper T cells to coordinate immune responses. Dendritic cells in the spleen of mice immunized with nanovaccines produced high levels of MHC-II class molecules, indicating that the nanovaccine can effectively activate mouse dendritic cells and produce corresponding functions.

[0104] 4.3TgIMC1-CS nanovaccine promotes the proliferation of mouse spleen dendritic cells

[0105] To analyze the effect of TgIMC1-CS nanovaccine on the proliferation of mouse spleen lymphocytes, on the 10th day after the first immunization and the 10th day after the second immunization, the mouse spleen lymphocytes were isolated using the same method as above, and 5×105 cells / well were inoculated into a 96-well plate, and DMEM medium containing 20% ​​fetal bovine serum was added to each well, and the plates were placed in an incubator at 37°C and 5% CO2 for 48 hours. After the incubation, 10 μl of CCK-8 reagent was added to each well and incubated for 4 hours, and then the OD value of each well was measured with an enzyme marker. The results are shown in the attached figure. Fig.10 As shown, the proliferation level of splenic dendritic cells produced by mice in the TgIMC1-CS group was significantly higher than that in the Control group (P<0.01). Activated dendritic cells can activate T cells by capturing and presenting antigens, promoting specific immune responses. The detection of mouse splenic lymphocytes showed that mouse splenic lymphocytes could be activated by recombinant TgTMC1 protein and its vaccine.

[0106] 4.4TgIMC1-CS nanovaccine promotes differentiation of mouse spleen lymphocytes

[0107] In order to evaluate the effect of TgIMC1-CS nanovaccine on mouse spleen T lymphocyte subsets, mouse spleen lymphocytes were isolated on the 10th day after the first and second immunizations. The isolated mouse spleen lymphocytes were stained with anti-mouse CD3e-FITC, CD8a-PE and CD4-APC. After staining, they were washed three times with PBS, and the differentiation of mouse spleen lymphocytes CD4 and CD8 T lymphocytes was analyzed by flow cytometry. The results are shown in the attached figure. Fig.11 As shown in the figure, on the 10th and 20th days after immunization, the proliferation capacity of spleen lymphocytes in the TgIMC1-CS group was significantly promoted after the first and second immunizations (P<0.001). The activated dendritic cells will further activate the proliferation of lymphocytes, leading to the differentiation of lymphocytes. By measuring the proliferation of mouse spleen lymphocytes, it is suggested that the proliferation capacity of mouse spleen lymphocytes can be activated by the recombinant TgIMC1-CS nanovaccine.

[0108] Example 5 Immune protection of TgIMC1-CS nanovaccine against toxoplasmosis

[0109] 5.1 Infection intensity of Toxoplasma gondii in mouse spleen tissue

[0110] To evaluate the immune protection effect of TgIMC1-CS nanovaccine on mice, 3 mice in each group were euthanized and the spleen tissue of mice was isolated on the 4th day after the attack. The spleen tissue of mice was placed in a commercial tissue fixative and fixed at room temperature for 24 hours. The fixed tissue was washed with physiological saline. After washing, it was placed in alcohol solutions with concentrations from low to high for step-by-step dehydration, and each concentration gradient treatment time was 1 hour. The dehydrated tissue was soaked in xylene for 40 minutes, and then the melted paraffin was poured into a special embedding frame, and the wax-impregnated tissue block was quickly embedded in it. The embedded paraffin was placed on a condensing table for cooling and solidification, and after solidification, it was cut into 4-6μm slices on a microtome. The slices were attached to a slide and dried in a constant temperature box for 24 hours. The dried slices were dewaxed in xylene. In order to eliminate the interference of endogenous peroxidase, EDTA antigen repair solution was used for antigen repair after dewaxing to fully expose the antigen sites. To eliminate endogenous peroxidase activity, tissue sections were placed in 3% H2O2 and treated for 25 minutes at room temperature in the dark. At 4°C, tissue sections were immersed in 1:500 commercial rabbit anti-T.gondii antibody and incubated at 4°C for 16 hours. After the sections were fully washed with TBS, unbound antibodies were removed, and anti-rabbit IgG-CY3 secondary antibody (1:1000) was added and incubated at room temperature in the dark for 1 hour. After being fully washed with TBS, DAPI was counterstained for 5 minutes, and the sections were sealed with sealing agent and randomly photographed under a fluorescence microscope. The results are shown in the attached figure. Fig.12 As shown, the number of Toxoplasma gondii in the spleen tissue of mice immunized with pVAX1-TgIMC1 DNA vaccine and nanovaccine TgIMC1-CS was significantly reduced, indicating that pVAX1-TgIMC1 DNA vaccine and TgIMC1-CS nanovaccine can inhibit the proliferation of parasites in the spleen.

[0111] 5.2 Infection intensity of Toxoplasma gondii in mouse myocardial tissue

[0112] To further evaluate the immune protection effect of TgIMC1-CS nanovaccine on mice, 5 mice in each group were euthanized and spleen tissues were isolated on the 6th day after infection, and tissue DNA was extracted using a commercial kit. The 529bp repetitive sequence of Toxoplasma gondii in mouse myocardial tissue was amplified by absolute quantitative PCR, and the plasmid containing the 529bp sequence of Toxoplasma gondii was used as a reference sequence to analyze the infection intensity of Toxoplasma gondii in mouse myocardial tissue. Three independent experiments were performed on each heart tissue DNA. The results are shown in the attached figure. Fig.13As shown, the intensity of Toxoplasma infection in the myocardial tissue of mice in the TgIMC1 group and TgIMC1-CS group was significantly lower than that in the Control group (P<0.001), indicating that the TgIMC1-CS nanovaccine can provide certain immune protection against acute toxoplasmosis and can be used to prevent acute infection of toxoplasmosis.

[0113] 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 TgIMC1-CS nano vaccine, characterized in that: The nano vaccine is prepared by coating a pVAX1-TgIMC1 DNA plasmid with a coating material. The pVAX1-TgIMC1 DNA plasmid is derived from the inner membrane skeleton protein 1 of Toxoplasma gondii, and its amino acid sequence is shown in SEQ ID NO.

1.

2. The Toxoplasma gondii TgIMC1-CS nanovaccine according to claim 1, characterized in that The coating material comprises at least one of chitosan, polylactic acid, polycaprolactone, polybutylene glycol succinate, polyaniline, polycarbonate, glycolide-lactide copolymer, and glycolide-caprolactone copolymer.

3. A method for preparing a Toxoplasma gondii TgIMC1-CS nanovaccine, characterized in that: The following steps are involved: S10. The Toxoplasma gondii IMC1 gene sequence TGRH88_075820 was codon-optimized to obtain a nucleotide sequence shown in SEQ ID NO.

2. The nucleotide sequence was fully synthesized, and the obtained gene fragment was amplified by PCR using primers shown in SEQ ID NO.3 and SEQ ID NO.4, and then ligated to the linearized pVAX1 vector using T4 DNA ligase to obtain a pVAX1-TgIMC1DNA plasmid; S20. After the pVAX1-TgIMC1 DNA plasmid is verified by double enzyme digestion, the plasmid with correct sequencing is transferred to E. coli DH5α, and the plasmid is extracted in large quantities using a plasmid DNA extraction kit to remove endotoxins therein to obtain an endotoxin-free pVAX1-TgIMC1 DNA plasmid; S30. Add the endotoxin-free pVAX1-TgIMC1 DNA plasmid and sodium tripolyphosphate solution dropwise to the chitosan solution, stir magnetically and then ultrasonically disrupt to obtain TgIMC1-CS nanovaccine aqueous solution; S40. Centrifuge the TgIMC1-CS nanovaccine aqueous solution, collect the precipitate, resuspend it in distilled water, and then filter and freeze-dry to obtain the Toxoplasma gondii TgIMC1-CS nanovaccine.

4. The method for preparing the Toxoplasma gondii TgIMC1-CS nanovaccine according to claim 3, characterized in that: Step S20 is specifically as follows: The pVAX1-TgIMC1 DNA vaccine is subjected to single enzyme digestion and verification and sequencing by a DNA analyzer; the correctly sequenced plasmid is transferred to E. coli DH5α competent cells, the plasmid is extracted in large quantities using a plasmid DNA extraction kit, and the endotoxin is removed by an endotoxin removal kit to make the endotoxin content in the plasmid lower than 0.01EU / ml, and the concentration of the pVAX1-TgIMC1 DNA plasmid is adjusted to 800 to 1500ng / μl using double distilled water to obtain an endotoxin-free pVAX1-TgIMC1 DNA plasmid.

5. The method for preparing the Toxoplasma gondii TgIMC1-CS nanovaccine according to claim 3, characterized in that: In step S30, the chitosan solution is prepared as follows: Chitosan is dissolved in an acetic acid aqueous solution as an organic phase, and the pH value is adjusted to 4 to 7 with a NaOH solution to obtain a chitosan solution with a concentration of 1 to 3 mg / ml; wherein the ratio of chitosan to the acetic acid aqueous solution is (1 to 3) mg:1 ml.

6. The method for preparing the Toxoplasma gondii TgIMC1-CS nanovaccine according to claim 3, characterized in that: In step S30, the ratio of the chitosan solution, the endotoxin-free pVAX1-TgIMC1 plasmid and the sodium tripolyphosphate solution is (3 to 7) ml: (1 to 3) mg: 1 ml; wherein the concentration of the sodium tripolyphosphate solution is 1 to 3 mg / ml.

7. The method for preparing the Toxoplasma gondii TgIMC1-CS nanovaccine according to claim 3, characterized in that: In step S30, the ultrasonic crushing conditions are: 4°C, power 140 to 160 W, interval 1 to 2 s, ultrasonic 1 to 2 s, and ultrasonic time 4 to 5 min.

8. Use of the Toxoplasma gondii TgIMC1-CS nanovaccine according to claim 1 in the preparation of a drug for preventing mammals from being infected with toxoplasmosis.

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