A nano - material multi - epitope vaccine for preventing Toxoplasma gondii infection, its preparation method and application

Abstract

The present invention provides a nano - material multi - epitope peptide vaccine for preventing animal toxoplasmosis gondii. The vaccine uses PLGA to encapsulate multi - epitope peptides and profilin immune agonists to form nanoparticles. The core composition of the multi - epitope peptide is six dominant peptide segments, namely SAG1 (238‑256) , MIC3 (109‑128) , GRA7 (20‑28) , MIC3 (27‑35) , AS15 peptide, MIC3 (368‑381) . After the multi - epitope peptide is expressed prokaryotically, it is coated with PLGA nano - materials and combined with a profilin immune agonist coated with PLGA nano - materials to form a new vaccine form. Through experiments on a mouse model, it is found that the multi - epitope peptide has good immunogenicity and can effectively stimulate the body to produce humoral immunity and cellular immunity; the combined use of PLGA - rMEP and PLGA - rProfilin can significantly extend the survival time of mice infected with the Toxoplasma gondii RH strain. Therefore, the use of this vaccine can provide partial immune protection and provide an effective candidate vaccine for preventing continued Toxoplasma gondii infection.

Description

Technical Field

[0001] The present invention belongs to the technical field of biological veterinary drugs, and particularly relates to a nano-material multi-epitope vaccine for preventing Toxoplasma gondii infection, a preparation method thereof, and an application thereof. Background Art

[0002] Toxoplasma gondii, also known as Toxoplasma, belongs to the order Eucoccidia and is a common intracellular parasite. Toxoplasma gondii has a wide range of hosts and can infect almost all warm-blooded vertebrates including humans, such as pigs, cows, horses, rodents, etc., causing huge economic losses to humans and livestock. Toxoplasma gondii infection includes congenital infection and acquired infection, and its infection stages can be divided into three periods: tachyzoite stage, bradyzoite stage, and sporozoite stage. The development of its vaccine is of great significance for the prevention of congenital infection and acquired infection.

[0003] With the development of immunology, the use of Toxoplasma gondii antigen epitopes with high conservation and strong immunogenicity to develop Toxoplasma gondii vaccines has become a new idea for developing Toxoplasma gondii vaccines. At present, scholars have identified a variety of proteins that are stably expressed in different periods of the Toxoplasma gondii life cycle. Among them, TgSAG1 is stably expressed in the tachyzoite stage and is crucial for the adhesion and invasion of host cells. It is an important surface antigen of Toxoplasma gondii. In 2006, Heber et al. identified its dominant antigen epitope sequence as TgSAG1(238 - 256). TgGRA7 is expressed in both the tachyzoite and bradyzoite infection stages and can stimulate the host to produce strong cellular and humoral immunity. It is a good vaccine candidate antigen. In 2012, Cong et al. found that TgGRA7(20 - 28) as a CD8+T cell epitope can significantly stimulate mice to increase the content of TNF-γ in the body and reduce the number of cerebral cysts in mice. TgMIC3 is expressed in all stages of Toxoplasma gondii infection and can produce persistent immune protection after immunizing the host. It is the most commonly used vaccine candidate antigen in the MIC family. However, there is no report on the dominant antigen epitope of TgMIC3 at present. The AS15 peptide is a Toxoplasma gondii CD4+T cell epitope discovered by Grover et al. and can play an important immunomodulatory role to protect animals from partial Toxoplasma gondii infection. However, existing multi-epitope vaccines usually have low immunogenicity and thus have poor effects in eliciting protective immunity. Summary of the Invention

[0004] Aiming at the deficiencies of the prior art, the purpose of the present invention is to provide a nano-material multi-epitope peptide vaccine for preventing Toxoplasma gondii infection, which can safely and effectively resist the acute infection of Toxoplasma gondii, thereby controlling the spread of Toxoplasma gondii.

[0005] Another object of the present invention is to provide a co-delivery system of PLGA nanoparticles with Profilin protein as a TLR ligand, which can be used as a carrier for drugs to prevent infections in rodents, pigs, and horses, thereby achieving efficient drug delivery and enhancing the body's immune response ability.

[0006] The object of the present invention can be achieved by the following technical solutions:

[0007] On the one hand, the present application provides a vaccine for preventing Toxoplasma gondii infection, and the vaccine includes: a multi-epitope peptide coated with poly (lactic-co-glycolic acid) and a Profilin protein coated with poly (lactic-co-glycolic acid) with a mass ratio of (1:10) to (10:1);

[0008] The dominant antigenic epitopes of the recombinant multi-epitope peptide include SAG1 (238-256) , MIC3 (109-128) , GRA7 (20-28) , MIC3 (27-35) , AS15 peptide, MIC3 (368-381) Any two or more of them.

[0009] In one embodiment, the multi-epitope peptide is obtained by connecting the dominant antigenic epitopes in the order of SAG1 (238-256) , MIC3 (109-128) , GRA7 (20-28) , MIC3 (27-35) , AS15 peptide, MIC3 (368-381) through a flexible linker peptide.

[0010] In one embodiment, the flexible linker peptide is SAPGTP.

[0011] In one embodiment, the mass ratio of the poly (lactic-co-glycolic acid) coated multi-epitope peptide to the poly (lactic-co-glycolic acid) coated Profilin protein is (1:5) to (5:1), preferably (1:3) to (3:1), and more preferably 1:1.

[0012] In one embodiment, the injection dose of the vaccine is 20 μg, and the doses of the poly (lactic-co-glycolic acid) coated multi-epitope peptide and the poly (lactic-co-glycolic acid) coated Profilin protein are 10 μg:10 μg.

[0013] Optionally, the poly (lactic-co-glycolic acid) (PLGA) in the present application can be a commercially available product, such as PLGA with a mass ratio of lactic acid to glycolic acid of 50%:50%.

[0014] In one embodiment, the coating ratio of the poly (lactic-co-glycolic acid) copolymer to the multi-epitope peptide or the Profilin protein is 100:(0.1 - 1), preferably 100:0.5.

[0015] In one embodiment, the particle size of the multi-epitope peptide coated with poly (lactic-co-glycolic acid) copolymer and / or the Profilin protein coated with poly (lactic-co-glycolic acid) copolymer is 300 - 1500 nm, preferably 500 - 1000 nm.

[0016] On the other hand, the present application provides a method for preparing the above-mentioned vaccine for preventing Toxoplasma gondii infection, comprising:

[0017] Step 1), respectively prepare the lyophilized powders of the multi-epitope peptide coated with poly (lactic-co-glycolic acid) copolymer and the Profilin protein coated with poly (lactic-co-glycolic acid) copolymer;

[0018] Step 2), mix and compound according to the mass ratio to obtain the product.

[0019] In one embodiment, the preparation method of the recombinant multi-epitope peptide coated with poly (lactic-co-glycolic acid) copolymer or the Profilin protein coated with poly (lactic-co-glycolic acid) copolymer in the step 1) is as follows:

[0020] Step a), dissolve the poly (lactic-co-glycolic acid) copolymer in an oily solvent, add a surfactant to form an oil phase, and add the multi-epitope peptide or the Profilin protein to the oil phase;

[0021] Step b), prepare an aqueous solution of polyvinyl alcohol as the water phase, and mix the oil phase obtained in step a) with the water phase to form a primary emulsion;

[0022] Step c), add the primary emulsion obtained in step b) to the aqueous solution of polyvinyl alcohol to obtain a multiple emulsion;

[0023] Step d), remove the solvent in the multiple emulsion, centrifuge to obtain a precipitate, wash and dry to obtain the lyophilized powder.

[0024] In one embodiment, the mass ratio of the poly (lactic-co-glycolic acid) copolymer to the multi-epitope peptide or the Profilin protein in the step a) is 100:(0.1 - 1); and / or,

[0025] The mass concentration of the aqueous solution of polyvinyl alcohol in the step b) is 0.1% - 1%, and the mass concentration of the aqueous solution of polyvinyl alcohol in the step c) is 4% - 8%; and / or,

[0026] In the step b), the primary emulsion is prepared by the ultrasonic method, and the parameters are: 180 W, 20% power, 1 s ultrasonic wave, 1 s interval, for a total of 5 min; and / or,

[0027] In the step c), the ultrasonic method is used to prepare the emulsion, and the parameters are: 180W, 50% power, 1s ultrasound, 1s interval, and a total of 20 minutes.

[0028] Preferably, the multi-epitope peptide or Profilin protein coated with PLGA nanoparticles is prepared by the following method:

[0029] Prepare 0.5% and 2% polyvinyl alcohol solutions respectively, take 100 mg of PLGA and dissolve it in 10 mL of dichloromethane, add Span-80 to a concentration of 0.8% as the oil phase. Take 500 μL of 1 mg / mL multi-epitope peptide or Profilin protein solution and add it to the oil phase. The above oil phase and water phase are fully mixed and ultrasonically crushed to form colostrum; the colostrum is added drop by drop into 50 mL of 2% polyvinyl alcohol, and ultrasonically crushed again on ice to obtain a double emulsion. The organic solvent in the double emulsion is evaporated and removed, and the precipitate is separated by high-speed centrifugation. The finished product is washed 5 times in 30 mL of ultrapure water. After reduced pressure drying, PLGA nanoparticle freeze-dried powder can be obtained.

[0030] In one embodiment, the multi-epitope peptide or the Profilin protein is prepared by the following method:

[0031] The gene encoding the multi-epitope peptide or the Profilin protein is introduced into a vector to obtain a recombinant vector, the recombinant vector is transformed into a chassis strain to obtain a recombinant strain, the recombinant strain is cultured to induce it to express the multi-epitope peptide or the Profilin protein, and the multi-epitope peptide or the Profilin protein is collected and purified.

[0032] Preferably, the vector may be a conventional vector in the prior art, preferably a plasmid; the chassis strain may be a commonly used chassis strain in the prior art, preferably Escherichia coli.

[0033] Preferably, the multi-epitope peptide is prepared by the following method:

[0034] SAG1 (238-256) MIC3 (109-128) 、GRA7 (20-28) MIC3 (27-35) 、AS15 peptide、MIC3 (368-381)Sequentially linked, flexible linker peptides "SAPGTP" are used to connect each antigenic epitope between the multi-gene fragments. The target gene is synthesized by a biological company and ligated to the pET-28a(+) vector to obtain a recombinant plasmid capable of expressing the multi-epitope peptide. The recombinant plasmid capable of expressing the multi-epitope peptide is transformed into BL21 Escherichia coli and expanded in LB liquid medium containing ampicillin resistance. IPTG is added to a final concentration of 0.8 mmol / L to induce the expression of the multi-epitope peptide. The bacterial cells are collected by centrifugation, ultrasonically disrupted at low temperature, filtered through a 0.22 μm filter, and then passed through a Ni affinity chromatography column, and washed with different gradients of imidazole. The eluate is collected and the purification effect is detected by SDS-PAGE. The purified multi-epitope peptide is dialyzed, and the protein activity is detected by Western blot method.

[0035] Preferably, the Profilin protein is prepared by the following method:

[0036] The target gene is synthesized by a biological company and ligated to the pET-28a(+) vector to obtain a recombinant plasmid capable of expressing the Profilin protein. The recombinant plasmid capable of expressing the Profilin protein is transformed into BL21 Escherichia coli and expanded in LB liquid medium containing ampicillin resistance. IPTG is added to a final concentration of 0.8 mmol / L to induce the expression of the recombinant Profilin protein. The bacterial cells are collected by centrifugation, ultrasonically disrupted at low temperature, filtered through a 0.22 μm filter, and then passed through a Ni affinity chromatography column, and washed with different gradients of imidazole. The eluate is collected and the purification effect is detected by SDS-PAGE. The purified multi-epitope peptide is dialyzed, and the protein activity is detected by Western blot method.

[0037] On the other hand, the present application provides the use of the vaccine in the preparation of a biological product for preventing Toxoplasma gondii infection.

[0038] In order to improve the immune protection effect of the multi-epitope peptide, the present invention encapsulates the multi-epitope peptide and a TLR11 ligand (Profilin protein) using a poly(lactic-co-glycolic acid) (PLGA) nanoadjuvant. Based on the multi-epitope peptide vaccine, since it is directly prepared using multiple antigenic dominant epitopes, it has the advantages of strong chemical stability and no carcinogenic potential, and is a safe alternative to live attenuated vaccines; it can be simulated by bioinformatics tools to achieve the high efficiency of vaccine preparation.

[0039] For the vaccine provided by this application, PLGA nanoparticles can protect antigens from enzymatic degradation, prolong their systemic circulation time, and increase the possibility of presentation to immune cells. Profilin protein is an actin modulator in Toxoplasma gondii and is involved in the gliding motility of Toxoplasma gondii. At the same time, Profilin is also a TLR11 ligand that can bind to TLR11 on macrophages and antigen-presenting cells, generating infectious cytokines IL-6 and IL-12, and then triggering the production of IFN-γ in the body. Since functional genes encoding TLR11 can be expressed in rodents, pigs, and horses, Profilin protein has broad application prospects as an immunomodulator. In addition, studies have shown that co-delivery of antigens and TLR ligands via PLGA nanoparticles can lead to selective stimulation of dendritic cells (DCs) to produce natural killer (NK) cell-activating cytokines. It can be speculated that co-delivery of multi-epitope peptides and the TLR11 ligand Profilin protein via PLGA nanoparticles can also selectively enhance the immune protection effect. Therefore, the novel multi-epitope peptide nano-vaccine constructed based on the PLGA nano-delivery system and Profilin protein can improve the immune protection of multi-epitope peptides and effectively solve the problem of low immunogenicity of multi-epitope peptides, which is of great significance for restricting the transmission of Toxoplasma gondii in animals such as rodents, pigs, and horses.

[0040] Compared with the prior art, the present invention has at least the following beneficial effects:

[0041] (1) Currently, when using PLGA nanomaterials to co-deliver Profilin protein and multi-epitope peptide vaccines, most have poor prevention and control effects on Toxoplasma gondii. The present invention fills the gap in the research of using PLGA nanomaterials for multi-epitope peptide vaccines against Toxoplasma gondii.

[0042] (2) TgSAG1, TgGRA7, and TgMIC3 are important antigenic proteins of Toxoplasma gondii, with conserved genes and stable expression in the tachyzoite stage, bradyzoite stage, and sporozoite stage. The present invention concatenates the dominant antigenic epitopes of TgSAG1, TgGRA7, and TgMIC3 to construct a novel anti-Toxoplasma multi-epitope peptide and prepares it by artificial recombination.

[0043] (3) The present invention improves the coating process of the reported PLGA nanomaterials and prepares nanoparticles with a particle size of 500 - 1000 nm.

[0044] (4) Profilin protein is an important component of Toxoplasma gondii and can play an immunomodulatory role. The present invention recombinantly prepares Profilin protein as an immunomodulator and encapsulates it with PLGA nanomaterials to improve the immune protection effect of multi-epitope peptide vaccines.

[0045] (5) The Profilin protein encapsulated in PLGA nanoparticles used in this application can be used as a carrier for preparing drugs to prevent rodents, pigs, and horses from being infected.

[0046] (6) The co-delivery of Profilin protein and multi-epitope peptide vaccine using PLGA nanoparticles described in this application can be used for immune protection against chronic, acute, and congenital Toxoplasma gondii infections. Brief Description of the Drawings

[0047] The drawings here are incorporated into the description and form a part of this description, showing embodiments consistent with the present invention, and are used together with the description to explain the principles of the present invention.

[0048] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required for use in the description of the embodiments or the prior art. For those of ordinary skill in the art, without creative efforts, other drawings can also be obtained based on these drawings. In the drawings:

[0049] Figure 1 It is a schematic diagram for constructing a multi-epitope peptide;

[0050] Figure 2 It is a schematic diagram of the tertiary structure of the multi-epitope peptide;

[0051] Figure 3 It is a schematic diagram of the docking and verification of the multi-epitope peptide with the H-2-Dd molecule. In the figure, A is the schematic diagram of the docking of the MEP and the H-2-Dd molecule; B, General is the Ramachandran plot of the multi-epitope peptide, GLY is the Ramachandran plot of glycine, PRE-PRO is the Ramachandran plot of the proline pre-residue, and PRO is the Ramachandran plot of proline;

[0052] Figure 4 It is a schematic diagram of the induced expression of pET-28a(+)-MEP. In the figure, M is the protein Marker; 1-4 are the protein expression levels at 0, 2, 4, and 6 h after induction; 5 is the supernatant after ultra-cracking; 6 is the precipitate after ultra-cracking;

[0053] Figure 5 It is a schematic diagram of the purification of the pET-28a(+)-MEP recombinant protein. In the figure, M is the protein Marker; 1-2 are the pET-28a(+)-MEP proteins before and after binding to the column; 3-4 are the 60 mmol / L imidazole eluent; 5-6 are the 80 mmol / L imidazole eluent; 7-8 are the 120 mmol / L imidazole eluent; 9-14 are the 250 mmol / L imidazole eluent;

[0054] Figure 6Schematic diagram for detecting the expression of pET-28a(+)-MEP recombinant protein by Western-blot. In the figure, M is the protein Marker; MEP is pET-28a(+)-MEP;

[0055] Figure 7 Schematic diagram for the change of antibody level before and after immunizing mice with rMEP or CFA-rMEP;

[0056] Figure 8 Schematic diagram for the change of spleen lymphocyte proliferation level before and after immunizing mice with rMEP or CFA-rMEP;

[0057] Figure 9 Schematic diagram for the change of related cytokine level before and after immunizing mice with rMEP or CFA-rMEP;

[0058] Figure 10 Schematic diagram for the characterization of PLGA nanoparticles;

[0059] Figure 11 Schematic diagram for the change of antibody level before and after immunizing mice with nanoparticle vaccine;

[0060] Figure 12 Schematic diagram for the change of spleen lymphocyte proliferation level before and after immunizing mice with nanoparticle vaccine;

[0061] Figure 13 Schematic diagram for the change of related cytokine level before and after immunizing mice with nanoparticle vaccine;

[0062] Figure 14 Survival curve of mice after intraperitoneal infection with 1000 RH tachyzoites. Detailed implementation mode

[0063] To more clearly illustrate the overall concept of this application, it will be described in detail by way of examples below. In the following description, a large number of specific details are given to provide a more thorough understanding of this application. However, it is obvious to those skilled in the art that this application can be implemented without one or more of these details. In other examples, in order to avoid confusion with this application, some well-known technical features in the art are not described.

[0064] Unless otherwise specified, in the following implementation modes, if the reagent or instrument used is not indicated by the manufacturer, it is a conventional product that can be obtained through commercial purchase.

[0065] For those not specified in the examples, they are carried out according to the conventional conditions or the conditions recommended by the manufacturer.

[0066] The materials involved in the following examples are as follows:

[0067] (1) Toxoplasma gondii RH strain: Preserved by our laboratory.

[0068] (2) Experimental animals: 6 - 8-week-old SPF-grade BABL / c mice, purchased from the Experimental Animal Center of Zhejiang Academy of Medical Sciences.

[0069] (3) Plasmids and strains: The cloning vector pET-28a(+) Vector was purchased from Jierui Biotechnology Company. The MEP gene was synthesized by Jierui Biotechnology Company. Competent Escherichia coli BL21 strain was purchased from TaKaRa Company.

[0070] (4) Tool enzymes and reagents: Ni-NTA affinity chromatography was purchased from Beijing Weishi Bohui Technology Co., Ltd. Coomassie Brilliant Blue staining solution and HRP-labeled goat anti-mouse IgG antibody were purchased from Hangzhou Fude Biotechnology Co., Ltd. Anti-His tag mouse monoclonal antibody was purchased from Sigma Company. Chromogenic solution was purchased from Beijing Solarbio Science & Technology Co., Ltd. PLGA was purchased from Shandong Daigang Biotechnology Co., Ltd. Reverse transcription kit, LA Taq enzyme, and DNA marker were purchased from Takara Company. qPCR kit was purchased from Nanjing Novoprotein.

[0071] (5) Instruments: PCR instrument (Kaiyudi Biotechnology), intelligent biochemical incubator (Ningbo Jiangnan Instrument Factory), shaking incubator (Shanghai Zhichu Instrument), gel imaging system (Shanghai Peiqing Technology), nucleic acid electrophoresis instrument (Beijing Liuyi Technology), protein electrophoresis instrument (Bio-RAD, USA), ultrasonic cell disruptor (Ningbo Xinzhi Technology), microplate reader (Qingdao Shengjie Instrument System Co., Ltd.).

[0072] Example 1 Design, Preparation and Immunogenicity Evaluation of Multi-epitope Peptides of Toxoplasma gondii

[0073] I. Prediction of Dominant Antigenic Epitopes

[0074] The B-cell linear epitopes of the TgMIC3 protein were analyzed using the BCPREDS server (http: / / ailab.ist.psu.edu / bcpred / predict.html), and the sequence "SKTMCGPGGCGEFCSSNWIF" with the highest predicted score was obtained.

[0075] The CD8+ T-cell epitopes of the TgMIC3 protein were analyzed using the IEBD server (http: / / tools.iedb.org / mhci / ). The MHC I molecular types were selected as H-2-Kd, H-2-Dd, and H-2-Ld, and the sequence "LPIQKSVQL" with the highest predicted score was obtained.

[0076] Analyze the CD4+ T cell epitopes of the TgMIC3 protein using the IEBD server, select the MHC II molecular type as H-2-I, and obtain the sequence with the highest predicted score as "HDTTTYVARRRYPA".

[0077] II. Design of multi-epitope peptides

[0078] Connect the polypeptides in the following order: SAG1(238-256), MIC3(109-128), GRA7(20-28), MIC3(27-35), AS15, MIC3(368-381), as shown in Table 1. Flexible linker peptide "SAPGTP" is used to connect each antigenic epitope between the multi-gene fragments, see Figure 1 .

[0079] Table 1 Epitope sequences and types

[0080] Epitope source Epitope sequence Epitope type <![CDATA[SAG1 (238-256) > CNEKSFKDILPKLTENPWQ B cell <![CDATA[MIC3 (109-128) > SKTMCGPGGCGEFCSSNWIF B cell <![CDATA[GRA7 (20-28) > LPQFATAAT MHCⅠ <![CDATA[MIC3 (27-35) > LPIQKSVQL MHCⅠ AS15 AVEIHRPVPGTAPPS MHCⅡ <![CDATA[MIC3 (368-381) > HDTTTYVARRRYPA MHCⅡ

[0081] III. Bioinformatics analysis of chimeric multi-epitope peptides

[0082] Analyze the physicochemical properties of the multi-epitope peptide using the ProtParam server. The results show that the relative molecular mass (kDa) of the multi-epitope peptide is 12.04361, the theoretical isoelectric point (PI) is 8.83, and the average hydrophilicity (GRAVY) is -0.424. Analyze the antigenicity of the multi-epitope peptide using VaxiJen v2.0. The results show that when Escherichia coli is used as the expression system, the overall antigenicity score of the multi-epitope peptide is 0.4680 (antigen threshold is 0.4). Analyze the allergenicity of the multi-epitope peptide using the AlgPred2 service, and predict that the multi-epitope peptide is a non-allergen. Predict the secondary structure using the PSIPRED server. Its local spatial structure is as follows: 13 amino acids form an α-helix, 18 amino acids form a β-sheet, and 85 amino acids form a random coil. Predict the tertiary structure of the multi-epitope peptide using the AlphaFold2 software and perform visual analysis using the PyMOL software. The results show that the chimeric antigenic epitopes are all presented on the surface of the polypeptide, see Figure 2 .

[0083] Perform molecular docking of the multi-epitope peptide with H-2-Dd using the ZDOOCK server and perform visual analysis using the PyMOL software, see Figure 3 A. Draw a Ramachandran plot using the PyRAMA server. The results show that the amino acid conformations are almost all within a reasonable range, see Figure 3 B. Perform molecular docking and verification using the HawkDock server, and obtain a binding free energy of -34.69 kcal / mol, indicating that the multi-epitope peptide is in a low-energy stable state after binding to the MHC molecule.

[0084] IV. Expression and Purification of Multi-Epitope Recombinant Peptide MEP

[0085] The target DNA fragment was synthesized by Genomics BioSci & Tech Co., Ltd. and ligated onto the pET-28a(+) vector. The recombinant plasmid was transformed into competent Escherichia coli BL21 and cultured overnight on an LB plate containing ampicillin resistance. A single colony was picked and cultured in LB liquid medium at 37 °C and 220 r / min. When the OD450nm of the bacterial solution reached about 0.7, IPTG was added to a final concentration of 0.8 mmol / L, and 500 μL samples were taken at 0 h, 2 h, 4 h, and 6 h at 220 r / min and 37 °C. After centrifugation of the induced bacterial solution, the supernatant was discarded, and the precipitate was resuspended in pre-prepared PBS and sonicated on ice. After centrifugation, the supernatant sample and precipitate sample were separated. After boiling the samples, SDS-PAGE detection was carried out. As Figure 4 can be seen, when induced with IPTG for 6 h, the protein content in the bacterial solution reached the highest level.

[0086] After sonication lysis, the recombinant protein appeared in the supernatant sample, indicating that the recombinant multi-epitope peptide was soluble in PBS. The expanded bacterial solution was sonicated on ice, and the supernatant was passed through a Ni ion affinity chromatography column and eluted with buffers containing 60, 80, 120, 250, and 500 mmol / L imidazole. The eluted fractions were collected. The purification effect was detected by SDS-PAGE. As Figure 5 can be seen, when the concentration of the imidazole eluent was 250 mmol / L, a band with the correct molecular weight and relatively single appeared at a molecular weight of 20 kDa.

[0087] V. Western Blot Detection of the Immunological Activity of Recombinant Multi-Epitope Peptide

[0088] After dialysis, the protein was transferred to a membrane after SDS-PAGE, blocked with 5% non-fat milk for 1.5 h; mouse-derived His monoclonal antibody was used as the primary antibody and incubated overnight, washed 5 times with TBST, 5 min each time. Goat anti-mouse IgG-HRP was added as the secondary antibody and incubated at room temperature for 2 h, washed with TBST, and the chromogenic solution was added for color development and photography. As Figure 6 can be seen, the multi-epitope recombinant protein with a His tag can be recognized by the corresponding monoclonal antibody, indicating that the purified multi-epitope recombinant peptide is the desired target protein.

[0089] VI. Immunogenicity Evaluation of Recombinant Multi-Epitope Peptide

[0090] 1. Experimental Design

[0091] All the protocols in this project's experimental research complied with the relevant regulations on animal welfare at Zhejiang University. BALB / c mice aged 6 - 8 weeks were randomly divided into 3 groups: the CFA-rMEP group, the PBS group, and the rMEP group. There were 10 mice in each group. The mice were subcutaneously inoculated with 100 μg of recombinant multi-epitope peptide, 100 μg of recombinant multi-epitope peptide emulsified with Freund's adjuvant at a ratio of 1:1, and 100 μL of PBS buffer at multiple points respectively. The vaccines were inoculated 4 times on day 0, day 14, day 21, and day 21. Blood samples were collected by tail amputation of the mice on day 0, day 7, day 14, day 21, and day 27 to measure the relevant antibody titers and cytokines.

[0092] 2. Antibody Titer Evaluation

[0093] 100 μl of blood samples were collected from the mice by the tail amputation method, placed in an incubator at 37°C for 1 h, left to stand overnight in a refrigerator at 4°C, and the precipitated serum was collected. After centrifuging the precipitated serum at 3000 r / min, 1 μl of the upper-layer serum was fully mixed with 150 μl of 5% skim milk and stored in a refrigerator at -20°C for later use. The Toxoplasma gondii lysate protein was diluted to 20 μg / ml with the coating buffer. 100 μl of the dilution was added to each well of a 96-well plate and incubated overnight at 4°C. After the lysate antigen was completely bound to the solid-phase carrier, the coating solution was discarded, and the wells were washed 5 times with 230 μl of PBST solution. 100 μl of 5% skim milk (prepared with PBST) was added to each well and incubated at 37°C for 2 h. The blocking solution was discarded, and the wells were washed as above with PBST solution and patted dry. 100 μl of the diluted serum was added to each well and incubated at 37°C for 1 h. The serum was discarded, and the wells were washed as above with PBST solution and patted dry. 100 μl of mouse secondary antibody diluted 1:5000 was added to each well and incubated at 37°C for 45 min. The secondary antibody was discarded, and the wells were washed again and the 96-well plate was patted dry. 100 μl of substrate chromogenic solution was added, incubated in the dark at 37°C for 15 min, and 100 μl of 2M sulfuric acid solution was added to terminate the reaction. Readings were taken 3 times at an absorbance at 450 nm using a microplate ELISA reader for the model. The final result was expressed as the D450 nm value, as shown in Figure 7 . In the third and fourth weeks, the antibody levels in the CFA-rMEP group increased significantly and were higher than those in the PBS control group.

[0094] 3. Spleen Lymphocyte Proliferation Assay

[0095] Five mice in each group were sacrificed two weeks after the last immunization. The spleens were removed and splenocyte suspensions were prepared. On the ultra-clean workbench, the mice were first soaked in 75% ethanol for 5 min, and then the spleens were taken out with sterile forceps and scissors. The spleens were placed on a 200-mesh cell strainer, cut into pieces with scissors, and pre-cooled PBS buffer was added dropwise to fully crush the spleen tissue on the strainer. After collecting the filtrate, it was centrifuged at 1500 rpm for 10 min at 4 °C to remove the supernatant, 2 - 3 ml of red blood cell lysate was added, and it was left standing on ice for 5 min. It was washed with sterile PBS solution. The cells were resuspended with cell culture medium, and after pipetting and mixing evenly, a splenocyte suspension was obtained. 10 μl of the cells were taken for cell counting, and the cell viability level was observed. The density of the above-prepared splenocyte suspension was adjusted, and 15 μg / mL of the recombinant protein was added accordingly to stimulate the splenocytes. Additionally, 15 μg / mL ConA solution, LPS solution, and MEP solution were used as positive controls, and PBS was added as a negative control. An MTT analysis kit was used to evaluate cell proliferation. As Figure 8 shown, under the stimulation of ConA, LPS solution, and MEP solution, the lymphocyte proliferation rate of the mice immunized with the CFA-rMEP group was significantly higher than that of the rMEP group and the LPS group; while there was no significant difference between the rMEP group and the LPS group.

[0096] 4. Evaluation of cytokines related to mouse spleen lymphocytes

[0097] Mouse spleen lymphocytes were collected, total RNA was extracted, reverse transcribed into cDNA, and the transcriptional levels of different cytokines were analyzed by qPCR. Relevant cytokines were retrieved from the NCBI database, specific primers were designed according to primer 5, and were synthesized by Shanghai Sangon Biotech Co., Ltd. The qPCR system is as follows:

[0098] System 20μl cDNA 1μl Primer F 0.6μl Primer R 0.6μl THUNDERBIRD SYBR qPCR Mix 12.5μl <![CDATA[ddH 2 O]]> 10.3μl

[0099] Fluorescent quantitative PCR reaction procedure: 50 °C for 3 min, 95 °C for 1 min for one cycle, 95 °C for 15 s, 60 °C for 15 s, 72 °C for 30 s, 40 cycles, and the melting curve was analyzed in the last cycle. Three qRT-PCR sample replicates were set, the CT values were taken and then analyzed. The calculation method used the CT value comparison method, and the data was analyzed by one-way ANOVA, *: P ≤ 0.05, **: P ≤ 0.01, ***: P ≤ 0.001. The final results are shown in Figure 9 , and the results showed that the levels of cytokines IL-2, IL-4, IL-8, IL-12, and IFN-γ in the CFA-rMEP group were all significantly increased, indicating that the multi-epitope peptide can stimulate the body to produce a higher level of immune protection under appropriate adjuvants.

[0100] Example 2 Preparation and characterization of PLGA nanoparticles

[0101] 1. Preparation of PLGA nanoparticles by double emulsion evaporation method

[0102] Weigh 100 mg of PLGA and dissolve it in 10 mL of dichloromethane as the oil phase. Add Span-80 to a concentration of 0.8%. Use a syringe to add 500 μL of the target protein solution to the oil phase, and immediately ultrasonically disrupt it on ice (180 w, 20% power, 1 s ultrasound, 1 s interval) for 5 min until a white primary emulsion is formed. Add the primary emulsion to 50 mL of 2% polyvinyl alcohol, and ultrasonically disrupt it again on ice (180 W, 50% power, 1 s ultrasound, 1 s interval) for 20 min to obtain a double emulsion. Pour the liquid into 200 mL of 0.5% polyvinyl alcohol, stir overnight with a magnetic stirrer, and evaporate the organic solvent. Wash the finished product 5 times in 30 mL of ultrapure water. Use a vacuum drying instrument to dry it under reduced pressure to obtain PLGA nanoparticles.

[0103] 2. Characterization of rMEP-PLGA nanoparticles

[0104] Take an appropriate amount of the nanoparticle suspension before vacuum drying, incubate it on a special copper mesh for transmission electron microscopy for 3 min, and negatively stain it with phosphotungstic acid for 3 min. Observe the particle morphology through a transmission electron microscope; observe the PLGA nanoparticles after gold spraying through a scanning electron microscope. Under the electron microscope, the nanoparticles are spherical, with a smooth surface and uniform size, and no large-scale aggregation is observed. See Figure 10 A and Figure 10 B.

[0105] Precisely weigh the mass of the PLGA nanoparticles after freeze-drying. Dissolve the PLGA nanoparticles in 0.1 mol / L NaOH and 1% SDS solution, shake and then centrifuge. Take the supernatant and measure the total protein content by the BCA method; calculate the encapsulation efficiency (EE) according to the formula EE = (M_encapsulated / M_drug) × 100%. M_encapsulated is the mass of the drug encapsulated in the nanoparticles (mg); M_drug is the total mass of the drug input (mg). The average encapsulation efficiency is measured to be 54.42%. The average value of the polydispersity index (PDI) obtained by DLS is 0.203, which is lower than the threshold of 0.3; its particle size distribution is as shown in Figure 10 C.

[0106] Example 3 Evaluation of the immune effect of Toxoplasma gondii PLGA multi-epitope vaccine

[0107] 1. Design of the immune test for Toxoplasma gondii PLGA multi-epitope vaccine

[0108] All the protocols in this experimental study comply with the relevant regulations on animal welfare at Zhejiang University. Randomly divide 6-8-week-old BALB / c mice into 9 groups, with 15 mice in each group. Subcutaneously inoculate the corresponding recombinant protein (20 μg) or 100 μL of PBS buffer at multiple points in the mice. The vaccine is boosted every two weeks for a total of 3 inoculations. The grouping is as follows:

[0109]

[0110] 2. Evaluation of antibody titers after immunization with Toxoplasma gondii PLGA multi-epitope vaccine

[0111] The experimental method refers to the "Antibody titer evaluation" section in Example 1. The results are as Figure 11 shown. Both PLGA-rMEP and PLGA-rMEP + PLGA-rProfilin can stimulate the body to produce corresponding antibodies, and the IgG level shows an increasing trend. The antibody level reaches the maximum at the seventh week of detection.

[0112] 3. Splenic lymphocyte proliferation experiment after immunization with Toxoplasma gondii PLGA multi-epitope vaccine

[0113] The experimental method refers to the "Splenic lymphocyte proliferation experiment" section in Example 1. The results are as Figure 12 shown. Under the stimulation of ConA, LPS solution and MEP solution, the proliferation rate of lymphocytes in immunized mice in the PLGA-rMEP and PLGA-rMEP + PLGA-rProfilin groups is significantly increased; while there is no significant difference in the corresponding PLGA-rProfilin group and PBS group.

[0114] 4. Cytokine evaluation after immunization with Toxoplasma gondii PLGA multi-epitope vaccine

[0115] The experimental method refers to the "Evaluation of cytokines related to mouse spleen lymphocytes" section in Example 1. The results are as Figure 13 shown. The cytokines (IFN-γ) activated by Th-1 immune response and the cytokines (IL-4, IL-10) representing the activation of Th-2 immune response are significantly up-regulated at the mRNA expression level of splenic lymphocytes, indicating that the recombinant multi-epitope of PLGA material stimulates the induction of Th-1 / Th-2 mixed immune response.

[0116] 5. Acute challenge experiment

[0117] Two weeks after the third immunization, 5 BALB / c mice in each group were intraperitoneally injected with 1000 tachyzoites of Toxoplasma gondii RH strain. The mental state of the mice was observed daily, the survival time of the mice was recorded, and the survival curve was drawn. The results are as Figure 14 shown. Compared with other control groups, the PLGA-rMEP + PLGA-rProfilin group has the longest average survival time, and the mice can survive for up to 15 days at most. It shows that the vaccine can provide partial immune protection for the body.

[0118] The above are only embodiments of the present application and are not intended to limit the present application. For those skilled in the art, various changes and modifications can be made to the present application. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present application shall be included within the scope of the claims of the present application.

Claims

1. A vaccine for preventing Toxoplasma gondii infection, characterized in that, the vaccine comprises: a multi-epitope peptide coated with poly (lactic-co-glycolic acid) copolymer and Profilin protein coated with poly (lactic-co-glycolic acid) copolymer with a mass ratio of (1:10) to (10:1); the mass ratio of poly (lactic-co-glycolic acid) copolymer to the multi-epitope peptide or the Profilin protein is 100: (0.1 - 1); The multi-epitope peptide is obtained by connecting dominant antigen epitopes in the order of SAG1 （238-256） , MIC3 （109-128） , GRA7 （20-28） , MIC3 （27-35） , AS15 peptide, MIC3 （368-381） through a flexible linker peptide; among them, SAG1 （238-256） The epitope sequence is: CNEKSFKDILPKLTENPWQ, MIC3 （109-128） The epitope sequence is: SKTMCGPGGCGEFCSSNWIF, GRA7 （20-28） The epitope sequence is: LPQFATAAT, MIC3 （27-35） The epitope sequence is: LPIQKSVQL, the epitope sequence of AS15 peptide is: AVEIHRPVPGTAPPS, MIC3 （368-381） The epitope sequence is: HDTTTYVARRRYPA, the flexible linker peptide is SAPGTP.

2. The vaccine according to claim 1, characterized in that, the mass ratio of the poly (lactic-co-glycolic acid) copolymer-coated multi-epitope peptide to the poly (lactic-co-glycolic acid) copolymer-coated Profilin protein is (1:5) to (5:1).

3. The vaccine according to claim 2, characterized in that, the mass ratio of the poly (lactic-co-glycolic acid) copolymer-coated multi-epitope peptide to the poly (lactic-co-glycolic acid) copolymer-coated Profilin protein is (1:3) to (3:1).

4. The vaccine according to claim 3, characterized in that, the mass ratio of the poly (lactic-co-glycolic acid) copolymer-coated multi-epitope peptide to the poly (lactic-co-glycolic acid) copolymer-coated Profilin protein is 1:

1.

5. The vaccine according to claim 1, characterized in that, the particle size of the poly (lactic-co-glycolic acid) copolymer-coated multi-epitope peptide and / or the poly (lactic-co-glycolic acid) copolymer-coated Profilin protein is 300 - 1500 nm.

6. The vaccine according to claim 5, characterized in that, the particle size of the poly (lactic-co-glycolic acid) copolymer-coated multi-epitope peptide and / or the poly (lactic-co-glycolic acid) copolymer-coated Profilin protein is 500 - 1000 nm.

7. A preparation method of a vaccine for preventing Toxoplasma gondii infection as described in any one of claims 1 - 6, characterized in that, it comprises: Step 1), respectively prepare freeze-dried powders of a multi-epitope peptide coated with poly (lactic-co-glycolic acid) copolymer and a Profilin protein coated with poly (lactic-co-glycolic acid) copolymer; Step 2), mix and compound according to the mass ratio to obtain.

8. The preparation method according to claim 7, characterized in that, the preparation method of the poly (lactic-co-glycolic acid) copolymer-coated multi-epitope peptide or the poly (lactic-co-glycolic acid) copolymer-coated Profilin protein in the said Step 1) is as follows: Step a), dissolve poly (lactic-co-glycolic acid) copolymer in an oily solvent, add a surfactant, as the oil phase, add the multi-epitope peptide or Profilin protein to the oil phase; Step b), prepare an aqueous solution of polyvinyl alcohol as the water phase, mix the oil phase obtained in Step a) and the water phase to form a primary emulsion; Step c), add the primary emulsion obtained in Step b) to the aqueous solution of polyvinyl alcohol to obtain a double emulsion; Step d), remove the solvent in the double emulsion, centrifuge to obtain a precipitate, wash and dry to obtain a freeze-dried powder.

9. The preparation method according to claim 8, characterized in that, The mass concentration of the aqueous solution of polyvinyl alcohol in step b) is 0.1% - 1%, and the mass concentration of the aqueous solution of polyvinyl alcohol in step c) is 4% - 8%; and / or, In step b), the primary emulsion is prepared by the ultrasonic method, and the parameters are: 180 W, 20% power, 1 s of ultrasonic treatment, 1 s of interval, for a total of 5 min; and / or, In step c), the multiple emulsion is prepared by the ultrasonic method, and the parameters are: 180 W, 50% power, 1 s of ultrasonic treatment, 1 s of interval, for a total of 20 min.

10. According to the preparation method described in claim 7, it is characterized in that the multi-epitope peptide or the Profilin protein is obtained by the following method: The gene encoding the multi-epitope peptide or the Profilin protein is introduced into a vector to obtain a recombinant vector, the recombinant vector is transformed into a chassis strain to obtain a recombinant strain, the recombinant strain is cultured to induce the expression of the multi-epitope peptide or the Profilin protein, and the multi-epitope peptide or the Profilin protein is collected and purified.

11. Use of the vaccine according to any one of claims 1 - 6 in the preparation of a biological product for preventing Toxoplasma gondii infection.

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