Toxoplasma gondii GRA1-mRNA-LNP vaccine and application thereof

Abstract

The invention discloses a toxoplasma gondii GRA1-mRNA-LNP vaccine and application thereof, and belongs to the technical field of biological medicine. The target antigen-GRA1 protein of the toxoplasma gondii mRNA vaccine is determined through bioinformatics analysis and actual expression quantity analysis, GRA1-mRNA is obtained on the basis of the target antigen-GRA1 protein, GRA1-mRNA is modified, modified GRA1-mRNA molecules are obtained and wrapped with LNP, the GRA1-mRNA-LNP vaccine is successfully prepared, mice are immunized through intramuscular injection, and after three times of immunization, the GRA1-mRNA-LNP vaccine is obtained. The toxoplasma gondii vaccine has the advantages that the toxoplasma gondii vaccine and the toxoplasma gondii vaccine can generate immune response in a mouse body, have a higher immune protection effect, can efficiently induce IL-4 and IFN-gamma, effectively reduce the death rate of the mouse after toxoplasma gondii infection, and have potential utilization value.

Description

Technical Field

[0001] The present application relates to the field of biomedicine technology, and more specifically to a Toxoplasma gondii GRA1-mRNA-LNP vaccine and its application. Background Art

[0002] Toxoplasmosis is a global zoonosis caused by the obligate intracellular protozoan Toxoplasma gondii. Currently, approximately one-third of the global population tests positive for serum antibodies to Toxoplasma gondii. Toxoplasma gondii is an obligate intracellular parasite that infects most warm-blooded mammals, including humans. Antibody positivity rates are particularly high in cancer patients, those infected with human immunodeficiency virus (HIV), and those with AIDS, with rates reaching 63.0%, 44.8%, and 95.0%, respectively. Current anti-Toxoplasma drugs primarily include sulfonamides and antibiotics, which inhibit folate metabolism. Following treatment, the parasites differentiate into bradyzoites, entering a chronic infection phase to evade the drug's killing effects. After the drug's effects wear off, the parasites re-differentiate into tachyzoites, rapidly replicating. Consequently, current drugs are ineffective in eradicating Toxoplasma and have limited efficacy. At the same time, drugs for treating toxoplasmosis also have obvious side effects. They only have a certain killing effect on tachyzoites but almost no effect on bradyzoites. Long-term use will make Toxoplasma gondii resistant to drugs and reduce the effectiveness of drugs. Due to Toxoplasma's unique invasion and immune evasion mechanism, there is currently a lack of available drugs and vaccines for Toxoplasma.

[0003] An mRNA vaccine is a new type of vaccine developed using messenger RNA (mRNA) technology. Its core principle is to deliver mRNA sequences encoding antigenic proteins from specific pathogens (such as viruses) into human cells, allowing them to produce the antigens themselves, thereby triggering an immune response. However, currently, there is little research and development of mRNA vaccines targeting Toxoplasma gondii.

[0004] Therefore, how to provide a Toxoplasma gondii mRNA vaccine is a technical problem that needs to be urgently solved by those skilled in the art. Summary of the Invention

[0005] In view of this, the present application provides a Toxoplasma gondii GRA1-mRNA-LNP vaccine and its application. The target antigen of the Toxoplasma gondii mRNA vaccine - GRA1 protein is determined through bioinformatics analysis and analysis of actual expression levels, and on this basis, the GRA1-mRNA-LNP vaccine is successfully prepared. This vaccine can effectively activate humoral immunity and cellular immunity, and achieve the prevention and treatment effect of Toxoplasma gondii.

[0006] In order to achieve the above objectives, this application adopts the following technical solutions:

[0007] The first object of the present application is to provide: a Toxoplasma gondii GRA1-mRNA-LNP vaccine, comprising an mRNA molecule, wherein the mRNA molecule comprises a nucleotide sequence encoding the GRA1 protein; the nucleotide sequence encoding the GRA1 protein is any one of the following:

[0008] 1) as shown in SEQ ID NO.5;

[0009] 2) or a nucleotide sequence encoding a protein with the same function after one or more bases are replaced, deleted and / or added to the sequence shown in SEQ ID NO. 5.

[0010] As a preferred technical solution, the amino acid sequence of the GRA1 protein is any one of the following:

[0011] 1) as shown in SEQ ID NO.6;

[0012] 2) or an amino acid sequence having the same enzymatic function after substitution, deletion and / or addition of one or more amino acids in the amino acid sequence shown in SEQ ID NO. 6.

[0013] As a preferred technical solution, the mRNA molecule further includes a 5' end cap structure and / or a 3' end poly (A) tail.

[0014] As a preferred technical solution, the vaccine further comprises liposomes, lipid complexes or lipid nanoparticles, and the mRNA molecules are encapsulated in the liposomes, lipid complexes or lipid nanoparticles.

[0015] Another object of the present application is to provide: the use of the above-mentioned vaccine in the preparation of drugs for preventing and / or treating Toxoplasma gondii.

[0016] Another object of the present application is to provide a pharmaceutical composition for preventing and / or treating Toxoplasma gondii, wherein the pharmaceutical composition comprises the above-mentioned vaccine.

[0017] It can be seen from the above technical solutions that compared with the prior art, this application has the following beneficial effects:

[0018] This application determined the target antigen GRA1 protein of the Toxoplasma gondii mRNA vaccine through bioinformatics analysis and analysis of actual expression levels, and based on this, obtained GRA1-mRNA, modified it, obtained the modified GRA1-mRNA molecule, and supplemented it with LNP encapsulation to successfully prepare the GRA1-mRNA-LNP vaccine. Mice were immunized by intramuscular injection. After three immunizations, an immune response was generated in the mice, with a high immune protection effect, and the ability to efficiently induce IL-4 and IFN-γ, effectively reducing the mortality rate of mice after Toxoplasma infection, and having potential utilization value. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] In order to more clearly illustrate the embodiments of the present application or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments or the description of the prior art. Obviously, the drawings described below are merely embodiments of the present application. For ordinary technicians in this field, other drawings can be obtained based on the provided drawings without any creative work.

[0020] Figure 1 Figure 3: Bioinformatics analysis of the expression levels of the GRA1 gene in undifferentiated oocysts, sporulated oocysts, tachyzoites and bradyzoites.

[0021] Figure 2 is: the expression level of GRA1 gene in bradyzoites and tachyzoites.

[0022] Figure 3 Figure 1: Construction of the pESUMO-GRA1 prokaryotic expression plasmid, where A: GRA1 fragment amplification; B: pESUMO fragment amplification; C, D: PCR1 and PCR2 identification of DH5α-pESUMO-GRA1 bacterial solution; 1-6: DH5α-pESUMO-GRA1 No. 1-6 bacterial solution; M: DNA molecular weight standard.

[0023] Figure 4 Figure 1: SDS-PAGE detection of recombinant protein pESUMO-GRA1, where M: protein molecular weight standard; 1: induced whole bacteria; 2: uninduced whole bacteria; 3: precipitate after disruption; 4: protein after inclusion body purification.

[0024] Figure 5 For: GRA1 mouse polyclonal titer detection.

[0025] Figure 6 Figure 2: Particle size determination results of GRA1-mRNA-LNPs.

[0026] Figure 7 Figure 2: GRA1-mRNA-LNPs potential measurement results.

[0027] Figure 8 Figure 1: Western-blot analysis of GRA1 polyclonal antibody, where 1: 293T cells; 2: ME49 insect strain; 3: 293T cells transfected with GRA1-mRNA-LNP.

[0028] Figure 9 To evaluate the immune protection effect (toxicity test).

[0029] Figure 10 Figure 2: Cytokine detection results, where A: IL-4 detection; B: IFN-γ detection.

[0030] Figure 11 Figure 2: Brain cyst detection results, where A: brain cyst size statistics; B: brain cyst number statistics.

[0031] Figure 12 The following are the results of the insect load test after vaccination. DETAILED DESCRIPTION

[0032] The following will be combined with the drawings in the embodiments of this application to clearly and completely describe the technical solutions in the embodiments of this application. Obviously, the embodiments described are only part of the embodiments of this application, not all of the embodiments. Based on the embodiments in this application, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of this application.

[0033] Example 1

[0034] Screening of target antigens

[0035] (1) Screening of potential target antigens: Screen potential target antigen proteins through bioinformatics analysis. The results of bioinformatics analysis experiments are as follows: Figure 1 shown.

[0036] Result analysis: Bioinformatics analysis showed that GRA1 was highly expressed in oocysts, tachyzoites and bradyzoites, and could be used as a potential target antigen.

[0037] (2) Confirmation of target antigen: To verify the expression of GRA1 in Toxoplasma bradyzoites and tachyzoites, bradyzoites and tachyzoites of ME49 strain were collected, RNA was extracted and reverse transcribed into cDNA, and then qPCR was performed on the cDNA. The specific experimental process is as follows:

[0038] A: Preparation of Toxoplasma gondii cDNA:

[0039] 1) Removal of genomic DNA: Extract Toxoplasma RNA, then take 2 μg of RNA extraction solution and prepare the following system on ice to remove genomic DNA. The specific system is as follows:

[0040] 5×gDNA EraserBuffer 8 μL;

[0041] gDNA Eraser 4 μL;

[0042] 2 μg total RNA;

[0043] Add RNase-free deionized water to 40 μL;

[0044] After reacting in a PCR instrument at 42°C for 5 min, cool on ice;

[0045] 2) Reverse transcription into cDNA: The specific process is as follows:

[0046] 40 μL of the product from the previous step;

[0047] Prime Script RT Enzyme Mix I 4μL;

[0048] RT Primer Mix 4 μL;

[0049] 5×Prime Script Buffer 2 16μL;

[0050] RNase-free water 16 μL;

[0051] Total volume 80 μL;

[0052] The digested RNA was reverse transcribed in a PCR instrument at 37°C for 15 min, followed by 85°C for 5 s to prepare cDNA, which was then stored at -80°C.

[0053] B: qPCR detection of GRA1 expression in Toxoplasma bradyzoites and tachyzoites

[0054] This experiment used the Novozymes fluorescence quantitative kit. According to the kit instructions, the following reaction system was prepared on ice: template 0.5 μL,

[0055] 2×AceQ Universal SYBR qPCRMasterMix 10μL

[0056] Upstream primer (10 μM) 0.8 μL,

[0057] Downstream primer (10 μM) 0.8 μL,

[0058] ddH2O 7.9 μL,

[0059] Total volume 20 μL;

[0060] The primer sequences are as follows:

[0061] F-GRA1: 5'-ACGTATCGTGTGGAGAGACC-3', SEQ ID NO.1;

[0062] R-GRA1: 5'-CGCTTCCTCTACTGTTTCGC-3', SEQ ID NO.2;

[0063] The reaction program was as follows: 95°C for 30 s; 95°C for 15 s, 58°C for 30 s (40 cycles); 72°C for 20 s;

[0064] Calculation of target gene cDNA expression: △CT = CT (target gene) - CT (internal reference GAPDH), draw a bar graph based on the results to show the relative expression level of cDNA. The experimental results are as follows Figure 2 shown.

[0065] The results analysis, by Figure 2 The results show that GRA1 is highly expressed in both bradyzoites and tachyzoites, therefore, GRA1 was selected as the target antigen.

[0066] Example 2

[0067] Construction of pESUMO-GRA1 recombinant plasmid

[0068] This application extracts RNA from the ME49 strain of Toxoplasma gondii, reverse transcribes cDNA using an RNA reverse transcription kit, amplifies the GRA1 fragment using the cDNA as a template, and amplifies the pESUMO vector using the pESUMO-BAG1 plasmid as a template. The specific process is as follows

[0069] (1) Target fragment amplification

[0070] Primers were designed using SnapGene software according to experimental needs, and the target fragments were amplified using the designed primers. The amplification reaction system was as follows: 50 ng of cDNA template, 2 μL of upstream primer (10 μM), 2 μL of downstream primer (10 μM), 25 μL of 2× PhantaMax buffer, 1 μL of dNTP Mix, 1 μL of Phanta Max Super-Fidelity DNA polymerase, and ddH2O supplemented to 50 μL.

[0071] The specific primer sequences are as follows:

[0072] F-GRA1-2: 5'-ACCGCGAACAGATTGGAGGTATGGTGCGTGTGAGCGCT AT-3', SEQ ID NO.3;

[0073] R-GRA1-2: 5'-TCGAATTCGGATCCTCTAGTTTACTCTCTCTCTCCTGTTA-3', SEQ ID NO.4;

[0074] The PCR reaction procedure is as follows:

[0075] Pre-denaturation at 95°C for 5 min; denaturation at 95°C for 15 s, annealing at 58°C (depending on primer annealing temperature) for 15 s, extension at 72°C at 1 kb / min (30 cycles); extension at 72°C for 5 min; cooling at 15°C for 10 min;

[0076] Subsequently, the GRA1 target fragment was recovered using the Novozymes product recovery kit;

[0077] (2) Preparation of pESUMO vector: pESUMO vector fragment was amplified using pESUMO empty plasmid (commercial) as template;

[0078] The amplified products of step (1) and step (2) were subjected to 1% agarose gel electrophoresis. The experimental results showed that the lengths of the target fragments were the same as the expected sizes, which were 570 bp and 5736 bp respectively. Figure 3 ).

[0079] The nucleotide sequence of GRA1 is as follows:

[0080] ATGGTGCGGGTTTCTGCAATAGTGGGCGCAGCAGCCAGTGTATTTGTGTG

[0081] CCTGTCAGCTGGGGCCTATGCAGCAGAGGGCGGGGACAACCAGAGCAGT

[0082] GCTGTCAGCGACAGAGCCTCCCTTTTGGGGCTTCTCCAGGTGGCACTGG

[0083] TCAAGGCCTGGGCATCGGGGAGTCCGTTGAGCTGGAAATGATGGGCAAC

[0084] ACCTACCGCGTGGAAAGACCCACAGGAAACCCTGACCTCCTCAAGATTGC

[0085] CATCAAAACATCAGATGGCAGCTACTCTGAAGTTGGAAATGTGAACATGG

[0086] AGGAAGTGATTGACACCATGAAGTCCATGCAGAGGGATGAAGAAATCTT

[0087] CTTCCGAGCCCTGAATAAAGGAGAAACTGTGGAGGAGGCCATTGAAGAT

[0088] GTGGCCCAGGCTGAAGGTCTCAACAGCGAGCAAACCCTGCAGCTGGAGG

[0089] ACGCGGTCTCCGCTGTGGCTTCTGTGGTGCAGGATGAGATGAATGTCATT

[0090] GATGATGTCCAGCAGTTGGAGAAGGACAAGCAGCAATTGAAAGATGACA

[0091] TCGGATTTCTGACGGGAGAGAGGGAGTGA, SEQ ID NO.5;

[0092] The amino acid sequence of GRA1 is as follows:

[0093] MVRVSAIVGAAASVFVCLSAGAYAAEGGDNQSSAVSDRASLGLLSGG

[0094] TGQGLGIGESVELEMMGNTYRVERPTGNPDLLKIAIKTSDGSYSEVGNVNME

[0095] EVIDTMKSMQRDEEIFFRALNKGETVEEAIEDVAQAEGLNSEQTLQLEDAVS

[0096] AVASVVQDEMNVIDDVQQLEKDKQQLKDDIGFLTGERE, SEQ ID NO.6;

[0097] (3) Construction of pESUMO-GRA1 recombinant plasmid:

[0098] Use homologous recombination to construct the recombinant plasmid. Refer to the instructions of the Novozyme Multi-Fragment Cloning Kit and prepare the following system on ice:

[0099] 5×CE II Buffer 2 μL;

[0100] Insert fragment amplification product (0.01*number of bases) ng;

[0101] Linearized cloning vector (0.01*number of bases) ng;

[0102] II 1 μL;

[0103] Add ddH2O to 10 μL.

[0104] After the reaction system is prepared, place it in a PCR instrument at 37°C for 30 minutes. After the reaction is completed, place the reaction tube in an ice water bath to cool it down. Then, construct the pESUMO-GRA1 plasmid by ligation and transformation.

[0105] The pESUMO-GRA1 plasmid constructed above was transformed into DH5α competent cells, and DH5α-pESUMO-GRA1 was identified by PCR1 (1004 bp) and PCR2 (734 bp). After the identification was correct and the sequencing was successful, the next step of the experiment could be carried out ( Figure 3 ).

[0106] Example 3

[0107] Preparation of GRA1 polyclonal antibody and antibody titer detection

[0108] (1) GRA1 protein purification

[0109] The correctly identified prokaryotic expression plasmid pESUMO-GRA1 was transformed into BL21 (DE3) competent cells and induced with 1mM IPTG at 37°C for 6h. 1mL of induced bacterial solution and 1mL of uninduced bacterial solution were taken, centrifuged at 10000×g for 1min, and the supernatant was discarded. 40μL of ddH2O, 10μL of DTT, and 50μL of 2×SDS loading buffer were added to the precipitate and boiled in boiling water for 10min for sample preparation. The results showed that GRA1 (39kDa) protein was induced to express, and the protein size was consistent with the predicted size. The protein was purified using the His fusion protein purification method, and the results showed that the prokaryotic expressed protein was successfully purified ( Figure 4 ), you can proceed to the next experiment.

[0110] (2) Detection of the titer of GRA1 mouse polyclonal antibody

[0111] The purified protein was injected into mice at multiple points on the back for immunization. Fourteen days after three immunizations, blood was collected from the mice's eyeballs and serum was collected. The serum from mice immunized with GRA1 protein was diluted in series and subjected to ELISA to detect the titer of GRA1 mouse polyclonal antibodies. The results showed that the serum titer reached above 1:25600 ( Figure 5 )

[0112] Example 4

[0113] Acquisition of GRA1 mRNA

[0114] (1) Synthesis of pT7-GRA1-100A plasmid

[0115] The GRA1 sequence was optimized using an online codon optimization website (https: / / www.genscript.com.cn / tools / gensmart-codon-optimization) and then synthesized by GenScript and spliced ​​into a vector, ultimately forming the pT7-GRA1-100A plasmid (which is driven by the T7 promoter and transcribes RNA with a ployA tail).

[0116] (2) Linearization of pT7-GRA1-100A plasmid

[0117] Refer to the instructions of the linearization kit of Shenji Company, place the reagents on ice, and prepare the following system:

[0118] BSPQI 0.5μL

[0119] pT7-GRA1-100A plasmid 1ng

[0120] NE Buffer 3.1 1 μL

[0121] Make up to 5 μL with DEPC H2O

[0122] Incubate at 50°C overnight and at 80°C for 20 min;

[0123] (3) In vitro transcription and co-capping

[0124] Refer to the instructions of the in vitro transcription co-capping kit of Shenji Company, place the reagents on ice, and prepare the following system:

[0125] 10×Transcription Buffer 2μL

[0126] ATP Solution 2μL

[0127] CTP Solution 2μL

[0128] GTP Solution 2μL

[0129] N1-Me-pUTP Solution 2μL

[0130] Template 1 μg

[0131] T7 RNA Polymerase Mix 2μL

[0132] Make up to 20 μL with RNase-free H2O

[0133] After mixing, centrifuge briefly and react at 37°C for 2 hours. Add 1 μL of DNase I to the reaction system and incubate at 37°C for 15 minutes to digest the transcribed DNA template. Then, purify the mRNA using the Novozymes mRNA purification kit to prepare GRA1-mRNA.

[0134] Example 5

[0135] Preparation of GRA1-mRNA-LNP vaccine

[0136] (1) Solution preparation

[0137] First, anhydrous ethanol was added to the lipid to prepare an organic phase solution with a concentration of 10 mg / mL; the encapsulated raw material (GRA1-mRNA) was diluted with acidic RNase-free sodium acetate buffer at a volume ratio of 3:1 to obtain an aqueous phase solution;

[0138] (2) Microfluidic encapsulation and initial product dilution

[0139] The microfluidic chip is loaded into the corresponding position of the nanomedicine manufacturing instrument; the organic phase solution and the aqueous phase solution are respectively loaded into disposable sterilized syringes, and after the air is exhausted, the syringes are loaded into the corresponding positions of the nanomedicine manufacturing instrument; the microfluidic parameters are set and the device is operated, and the lipids in the organic phase solution and the nucleic acids in the aqueous phase solution are self-assembled to form a nucleic acid-LNP complex preliminary product, which is diluted with PBS buffer / Tris-HCl buffer to make the LNP preliminary product more stable and facilitate subsequent processing;

[0140] (3) Buffer exchange and concentration

[0141] Depending on the volume of the diluent, use a 100kDa ultrafiltration centrifuge tube or a 100kDa TFF membrane cassette to perform buffer exchange and concentration of the LNP product to remove the remaining ethanol in the LNP product and place the LNP in a neutral buffer environment. Ultrafiltration centrifuge tubes or TFF membrane cassettes can also be used to concentrate the LNP diluent.

[0142] (4) Sterile filtration

[0143] The concentrated LNP product was sterilized by filtration using a 0.22 μm syringe filter;

[0144] (5) Add cryoprotectant

[0145] Add 10% sucrose to the LNP product as a cryoprotectant to obtain a final LNP product that can be stored at low temperatures;

[0146] (6) LNP particle size distribution detection

[0147] Take 3-5 μL of LNP sample, add 1 mL of nuclease-free water to dilute, mix well and use particle size analyzer to detect. The experimental results are as follows: Figure 6 shown.

[0148] Result analysis: The average diameter was measured to be 82.14 nm (the particle size must be less than 150 nm).

[0149] (7) LNP zeta potential detection

[0150] Take 3-5 μL of LNP sample, add 1 mL of LNP storage buffer to dilute, mix and then use particle size analyzer to detect. The experimental results are as follows Figure 7 shown.

[0151] Result analysis: The zeta potential measurement value must be between -15mV and +15mV, and the zeta potential detection results of the test sample meet the requirements.

[0152] (8) LNP encapsulation efficiency detection

[0153] The encapsulation efficiency of LNP was measured using the Ribogreen method. The encapsulation efficiency indicates the proportion of mRNA encapsulated inside lipid nanoparticles (LNPs) to the total mRNA. Ribogreen is an ultra-sensitive fluorescent nucleic acid dye used to quantitatively detect the RNA content in a solution. Ribogreen cannot penetrate LNP, so the mRNA content free outside the LNP particles in the mRNA-LNP complex solution is first detected, and then TritonX-100 is used to destroy the LNP structure, so that the mRNA encapsulated inside the mRNA-LNP complex is released into the external solution, thereby detecting the total mRNA content. The encapsulation efficiency can be calculated based on the difference between the two. The encapsulation efficiency must be greater than 80%. The encapsulation efficiency of the test sample was 87.78%, which met the requirements.

[0154] Example 6

[0155] mRNA transfection in vitro

[0156] 1) Culture 293T cells in DMEM complete medium containing 10% fetal bovine serum until the cell confluence is close to 80%;

[0157] 2) Subculture into 24-well plates with a seeding density of 0.5-2×10 5 cell / well;

[0158] 3) Transfection was performed after 24 h of culture;

[0159] 4) Add 25 μL of Opti-MEM medium to a 1.5 mL sterile centrifuge tube and add 1.5 μL of Lipomaster 2000 Transfection Reagent. Mix gently with a pipette.

[0160] 5) Add 25 μL of Opti-MEM medium to a 1.5 mL sterile centrifuge tube and add 0.5 μg of mRNA. Mix gently with a pipette.

[0161] 6) Add the mRNA / opti-MEM dropwise to the Lipomaster 2000 Transfection Reagent / opti-MEM, mix gently with a pipette, and let stand at room temperature for 5 minutes before use for transfection;

[0162] 7) Add the Lipomaster 2000 Transfection Reagent / mRNA complex mixture dropwise to the culture medium and gently shake the culture dish to evenly disperse it;

[0163] 8) Incubate overnight for 24-48 hours.

[0164] In order to verify the protein expression of the above GRA1-mRNA-LNP after transfection, Western-blot test was used for detection. The experimental results are shown in Figure 2. Figure 8 As stated.

[0165] Analysis of results: The results showed that GRA1-mRNA-LNP was successfully transfected into 293T cells, GRA1 was successfully expressed, and the expression level of GRA1 protein was high.

[0166] Example 7

[0167] (1) Protective effect of GRA1-mRNA-LNP vaccine against Toxoplasma gondii infection

[0168] To investigate whether mice become immune to Toxoplasma gondii after immunization with the GRA1-mRNA-LNP vaccine, mice were challenged with the virus and the survival rates of mice in different groups were measured. The specific process is as follows:

[0169] GRA1-mRNA-LNP vaccine immunization:

[0170] Female ICR mice aged 4-6 weeks were divided into a vaccine immunization group and a PBS group. The immunization process of the vaccine immunization group was as follows: the GRA1-mRNA-LNP vaccine was diluted to 50 μg / mL with PBS, and each mouse was immunized with 10ug / 200μL of GRA1-mRNA-LNP vaccine by intramuscular injection. Each immunization was 14 days apart, for a total of 3 immunizations. Each mouse in the PBS group was injected intramuscularly with 200μL PBS, and the rest were the same as the vaccine immunization group.

[0171] Mouse challenge test:

[0172] 1) Count freshly released Toxoplasma tachyzoites and dilute them to an appropriate concentration (toxicity test: 500 Tg / mL) using serum-free DMEM medium;

[0173] 2) Inoculate the diluted DMEM suspension into 4-6 week old female ICR mice via intraperitoneal inoculation, with 200 μL (100 Tg / mouse) inoculated per mouse;

[0174] 3) After the mice were inoculated with the parasite strain, they were raised normally, and their health status, including clinical symptoms and death time, was recorded daily.

[0175] The survival rate of mice in the GRA1 group was 100%, while that in the PBS group was only 60%. The survival rate of mice immunized with the vaccine was improved ( Figure 9 ).

[0176] (2) Effects of GRA1-mRNA-LNP vaccine on IL-4 and IFN-γ levels in mouse serum

[0177] 14 days after the three immunizations, the mouse serum was collected and cytokine detection was performed using a kit.

[0178] 1) Coating: Add 100 μL of Capture antibody diluted 250-fold in 1× Coating buffer to each well of a 96-well plate and incubate at 4°C overnight in the dark.

[0179] 2) Washing: Discard the original solution in the wells, add 300 μL of Wash Buffer to each well, and wash three times;

[0180] 3) Blocking: Add 200 μL of 1× ELISA / ELISAPOT Diluent to each well and incubate at room temperature for 1 hour;

[0181] 4) Prepare the standard during the blocking period by adding dd H2O according to the indicated dosage, incubate at room temperature for 30 minutes, and record the concentration as 500 pg / mL after vortexing.

[0182] 5) Washing: Add 300 μL of Wash Buffer to each well and wash once;

[0183] 6) Standards: In the first column, add 0, 100, 100, 100, 100, 100, 100, 100 μL of Diluent from top to bottom. Be careful not to let the tip of the pipette touch the bottom of the well. At well 0 (add 200 μL of standard stock solution), pipette 100 μL and dilute by 2-fold in sequence.

[0184] 7) Sample loading: Add 50 μL of serum sample to the remaining wells, add 50 μL of Diluent, and mix thoroughly. Leave one well blank and add only 100 μL of Diluent. Incubate at 4°C for 7-8 hours.

[0185] 8) Washing: Wash 3-5 times;

[0186] 9) Primary Antibody: Prepare the detection antibody during the incubation period, dilute it 250-fold with Diluent, add 100 μL to each well, and incubate at room temperature for 1 hour.

[0187] 10) Washing: Wash 3-5 times;

[0188] 11) Secondary antibody: Prepare Avidin-HRP (diluted 250-fold with Diluent) during incubation, add 100 μL per well, seal, and incubate at room temperature for 30 minutes.

[0189] 12) Washing: Wash 5-7 times;

[0190] 13) Color development: Add 100 μL of 1× TMB to each well and allow to develop for 15 minutes at room temperature.

[0191] 14) Stop: Add 50 μL stop solution to each well;

[0192] 15) Reading: The microplate reader was set at 450 nm to 570 nm for measurement.

[0193] In order to study the changes in cytokines in the serum of mice after immunization, the serum of mice was collected 14 days after the third immunization and cytokine detection was performed using a kit. The results showed that the serum levels of IL-4 and IFN-γ in the GRA1 group were higher than those in the control group ( Figure 10 ).

[0194] (3) Effect of GRA1-mRNA-LNP vaccine on brain cyst formation

[0195] In order to detect the brain cysts formed by Toxoplasma gondii in the brains of immunized mice, the following test was performed:

[0196] 1) Count freshly escaped ME49 tachyzoites and dilute to 500 Tg / mL using serum-free DMEM medium;

[0197] 2) 14 days after the three mRNA vaccine immunizations, each mouse was intraperitoneally injected with 200 μL of ME49 strain (100 insects);

[0198] 3) 30 days after infection with Toxoplasma gondii, the mice were euthanized and the brain tissue was removed and placed in a 15 mL centrifuge tube. The brain tissue was rinsed twice with normal saline and resuspended in 3 mL of PBS.

[0199] 4) Use a 5 mL syringe to pipette until a homogenate is formed;

[0200] 5) Take 500 μL of brain tissue homogenate, add 500 μL of fixative (0.4% formaldehyde, 500 μL of 10× PBS, 400 μL of ddH2O, and 10 μL of 10% Triton-100), mix well, and incubate at 4°C for 30 min. Centrifuge at 1000 g for 10 min, and discard the supernatant.

[0201] 6) Resuspend the pellet in 1 mL of 10% FBS, centrifuge at 1000 g, 4°C for 10 min, discard the supernatant, and repeat this process three times.

[0202] 7) Add 500 μL of 10% FBS and 5 μL of DBA, resuspend the pellet, and stain at room temperature in the dark for 1 hour;

[0203] 8) Resuspend the pellet in 1 mL of 10% FBS, centrifuge at 1000 g, 4°C for 10 min, discard the supernatant, and retain the pellet. Repeat this procedure three times.

[0204] 9) Cyst counting: Add 250 μL of PBS to resuspend the pellet, add 12.5 μL / slide, cover with a coverslip, and observe and count under a fluorescence microscope. Repeat the counting for each sample three times; count and calculate the number of Toxoplasma cysts contained in each mouse brain tissue. After DBA staining of brain cysts in surviving mice, we found that the number of brain cysts in mice immunized with the mRNA-GRA1-LNP vaccine did not change significantly, but the size of the cysts was observed to be smaller ( Figure 11 ).

[0205] (4) Effect of GRA1-mRNA-LNP vaccine on the amount of worms in the ascites of mice

[0206] In order to detect the replication of Toxoplasma gondii in mice after immunization with GRA1-mRNA-LNP vaccine, freshly escaped Toxoplasma gondii ME49 were inoculated into ICR mice by intraperitoneal injection (10 5Tg / mouse), and 7 days later, the ascites of the mice were collected and the amount of Toxoplasma gondii was detected by qPCR. The results showed that compared with the PBS group, the ascites load of mice immunized with GRA1-mRNA-LNP vaccine showed a trend of decreasing ( Figure 12 ).

[0207] The various embodiments in this specification are described in a progressive manner, and each embodiment focuses on the differences from other embodiments. The same or similar parts between the various embodiments can be referenced to each other.

[0208] The above description of the disclosed embodiments is intended to enable one skilled in the art to implement or use the present application. Various modifications to these embodiments will be readily apparent to one skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the present application. Therefore, the present application is not limited to the embodiments shown herein, but is intended to conform to the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A Toxoplasma gondii GRA1-mRNA-LNP vaccine, characterized in that: The invention comprises an mRNA molecule, wherein the mRNA molecule comprises a nucleotide sequence encoding a GRA1 protein; the nucleotide sequence encoding the GRA1 protein is any one of the following: 1) as shown in SEQ ID NO.5; 2) or a nucleotide sequence encoding a protein with the same function after one or more bases are replaced, deleted and / or added to the sequence shown in SEQ ID NO.

5.

2. The vaccine according to claim 1, characterized in that The amino acid sequence of the GRA1 protein is any one of the following: 1) as shown in SEQ ID NO.6; 2) or an amino acid sequence having the same enzymatic function after substitution, deletion and / or addition of one or more amino acids in the amino acid sequence shown in SEQ ID NO.

6.

3. The vaccine according to claim 1, characterized in that The mRNA molecule further comprises a 5' end cap structure and / or a 3' end poly (A) tail.

4. The vaccine according to claim 1, characterized in that The mRNA vaccine also comprises liposomes, lipid complexes or lipid nanoparticles, in which the mRNA molecules are encapsulated.

5. Use of the vaccine according to any one of claims 1 to 4 in the preparation of a drug for preventing and / or treating Toxoplasma gondii.

6. A pharmaceutical composition for preventing and / or treating Toxoplasma gondii, characterized in that: The pharmaceutical composition comprises the vaccine according to any one of claims 1 to 4.

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