Four-antigen combined toxoplasma gondii-resistant self-replicating mRNA vaccine and preparation method thereof

Abstract

The invention discloses a four-antigen combined replicative mRNA (messenger ribonucleic acid) vaccine for resisting toxoplasma gondii and a preparation method thereof. The invention provides an mNRA vaccine for preventing animal toxoplasmosis. The vaccine not only aims at a plurality of antigen epitopes of toxoplasmosis, but also aims at a plurality of antigen epitopes of toxoplasmosis, including main vaccine candidate sites of toxoplasmosis, such as coryneform protein ROP18 and micronematode protein MIC13; and two sites, namely TGME49268230 and TGME49237490, aiming at the sexual reproduction period of the toxoplasma gondii are introduced into the TGME49268230 and the TGME49237490. In addition, a sequence of RNA-dependent RNA polymerase RdRP is also added, so that the immune effect can be greatly improved; and compared with a DNA vaccine, mRNA does not need to enter a cell nucleus to play a role and is not integrated into a genome of a subject, so that the risk of causing insertion mutation is reduced. In conclusion, the vaccine can be used for preventing acute and chronic and congenital infection of toxoplasma gondii.

Description

Technical Field

[0001] The present invention relates to the field of genetic engineering technology, and in particular to a four-antigen combined anti-Toxoplasma self-replicating mRNA vaccine and a preparation method thereof. Background Art

[0002] Toxoplasma gondii is a widespread and seriously harmful parasite. It can infect almost all warm-blooded animals, including humans. Toxoplasmosis caused by its infection is a zoonosis that is prevalent worldwide. Humans are usually infected with toxoplasmosis by ingesting tissue cysts in raw or undercooked meat, oocysts in food or water, or by infecting the fetus through the placental barrier. Generally, toxoplasmosis infection in people with normal immunity generally has no obvious symptoms, but in immunocompromised people such as organ transplant patients and AIDS patients, toxoplasmosis is one of the common causes of death, causing toxoplasmic encephalitis, peritonitis, etc.; Toxoplasma can infect the fetus through the placental barrier, directly affecting the development of the fetus, and even causing miscarriage, premature birth, and stillbirth, posing a serious threat to eugenics. Children who survive the infection often suffer from intellectual disability and epilepsy due to congenital brain damage, so it is extremely important to develop prevention and treatment methods for toxoplasmosis.

[0003] Vaccination is considered an effective means of disease control and prevention. Over the past thirty years, a large number of experimental studies have been conducted at home and abroad to fight Toxoplasma infection and reduce or eliminate host tissue cyst formation, involving live attenuated vaccines, protein vaccines, DNA vaccines, mRNA vaccines, etc. The main vaccine candidate molecules for Toxoplasma gondii are concentrated on surface antigen SAG, rod protein ROP, microneme protein MIC and dense granule protein GRA. So far, the only commercial Toxoplasma vaccine known in the world is It is a live attenuated vaccine developed with attenuated strain S48. It is mainly used in goat breeding and has the risk of reversion to virulence. DNA vaccines can promote host cells to specifically express vaccine-encoded antigens and induce humoral immunity and stronger cellular immunity than protein vaccines. For example, Toxoplasma gondii subunit vaccines containing rGRA2 or rGRA5 can induce humoral immunity and cellular immunity, thereby producing partial protection for vaccinated BALB / c mice, while DNA vaccines encoding Toxoplasma gondii GRA14 antigen can induce strong specific immune responses, and the average survival time of BALB / c mice immunized with RH strains has been significantly extended.

[0004] In recent years, research on Toxoplasma mRNA vaccines has also made certain progress. Unlike DNA vaccines, mRNA vaccines only need to pass through the cell membrane and enter the cytoplasm to take effect, while plasmid DNA needs to further enter the cell nucleus to produce an effect. Therefore, RNA vaccines not only do not have the risk of foreign genetic material integrating into chromosomes, but also can produce stronger immune responses and higher safety.

[0005] Chahal prepared a Toxoplasma RNA vaccine that carries the codes for GRA6, ROP2A, ROP18, SAG1, SAG2A, and AMA1 at the same time. Mice immunized with it stimulated complete immunity to a lethal dose of the attenuated type II strain of Toxoplasma, the PRU strain. In addition, a large number of studies have shown that vaccines targeting a single antigen or a small combination of antigens can only provide limited anti-Toxoplasma infection effects, and multi-antigen combined immunization can induce the body's immune response at a higher level. In addition, more than a dozen mRNA vaccines have entered clinical trials for the treatment of malignant tumors and various infectious diseases, such as influenza virus vaccines, Zika virus vaccines, HIV virus vaccines, rabies virus vaccines, and malarial parasite vaccines, all of which have achieved good animal experimental results. Usually, the protein required for mRNA encoding is used by the ribosomes in the cell to complete translation and protein production, and the duration in the cell is short.

[0006] In summary, there is an urgent need for self-replicating mRNA vaccines with multiple antigen targets to protect against Toxoplasma infection. Summary of the invention

[0007] In view of the deficiencies in the prior art, the present invention provides a four-antigen combined self-replicating mRNA vaccine (4X-mRNA-LNP) against Toxoplasma gondii.

[0008] The present invention is different from the mainstream mRNA vaccine in that the mRNA used in this study carries a sequence that can express RNA polymerase (RNA-dependent RNA polymerase RdRP). After this RNA polymerase is produced, it can use itself as a template to produce more cRNA, thereby greatly increasing the duration of mRNA in the cell and increasing the immune response.

[0009] Specifically, the mRNA was verified in vitro, and mice were immunized to measure the levels of IgG, IgG1, IgG2a antibodies, cytokines such as interferon-γ (IFN-γ), interleukin-2, IL-4 and IL-10, T cell levels, insect load, and lymphocytes, and pathological sections and lymphocyte proliferation experiments were carried out simultaneously. At the same time, the mRNA vaccine of two sites was simultaneously tested to verify the effectiveness of 4X-mRNA-LNP.

[0010] The invention provides a recombinant self-replicating mRNA plasmid, comprising a plasmid backbone sequence, a replicase gene, a gene encoding a rhoptry protein ROP18, a gene encoding a TGME49268230 protein, a gene encoding a TGME49237490 protein and a gene encoding a microneme protein MIC13, wherein the gene sequence encoding the rhoptry protein ROP18 is shown as SEQ ID NO.1, the gene sequence encoding the TGME49268230 protein is shown as SEQ ID NO.2, the gene sequence encoding the TGME49237490 protein is shown as SEQ ID NO.3, and the gene sequence encoding the microneme protein MIC13 is shown as SEQ ID NO.4.

[0011] Preferably, the gene sequence encoding the rhoptry protein ROP18, the gene sequence encoding the TGME49268230 protein, the gene sequence encoding the TGME49237490 protein and the gene sequence encoding the microneme protein MIC13 are the nucleotide sequences of 1-621 bp, 658-1290 bp, 1327-2154 bp and 2191-3453 bp respectively as shown in SEQ ID NO. 9. In view of the fact that the combined antigen sequence is too long and difficult to express, the main antigen expression site is truncated and optimized.

[0012] The replicase gene is a viral RNA polymerase gene, which is derived from an alpha virus, and the gene sequence is shown in SEQ ID NO.17, and the amino acid sequence is shown in SEQ ID NO.18.

[0013] After this RNA polymerase is produced, it can use itself as a template to produce more cRNA, thereby greatly increasing the duration of mRNA in the cell and increasing the immune response.

[0014] The plasmid backbone sequence includes a subgenomic promoter (SGP), a 5'UTR region, a 3'UTR region and a 3' terminal Poly A tail. The recombinant mRNA plasmid can be constructed in any cloning vector.

[0015] The present invention also provides the use of the recombinant self-replicating mRNA plasmid in preparing a vaccine for preventing Toxoplasma gondii infection.

[0016] The present invention also provides a self-replicating mRNA for combined use of four antigens against Toxoplasma gondii, comprising a 5'UTR region, a 3'UTR region and a 3' terminal PolyA tail, a replicase sequence, an mRNA encoding a rhoptry protein ROP18, an mRNA encoding a TGME49268230 protein, an mRNA encoding a TGME49237490 protein and an mRNA encoding a microneme protein MIC13; the amino acid sequence of the rhoptry protein ROP18 is shown in SEQ ID NO.5, the amino acid sequence of the TGME49268230 protein is shown in SEQ ID NO.6, the amino acid sequence of the TGME49237490 protein is shown in SEQ ID NO.7, and the amino acid sequence of the microneme protein MIC13 is shown in SEQ ID NO.8.

[0017] Preferably, the amino acid sequence of the rhoptry protein ROP18, the amino acid sequence of the TGME49268230 protein, the amino acid sequence of the TGME49237490 protein and the amino acid sequence of the microneme protein MIC13 are the amino acid sequences of positions 1-207, 220-430, 443-718 and 731-1151 as shown in SEQ ID NO. 10, respectively. Preferably, it further comprises a 5' cap structure, and the 5' cap structure is a 7-methylguanosine cap Cap1 (m7GpppXm) structure.

[0018] The present invention also provides a four-antigen combined anti-Toxoplasma self-replicating mRNA vaccine, comprising the four-antigen combined anti-Toxoplasma self-replicating mRNA.

[0019] The mRNA vaccine for preventing Toxoplasma infection provided by the present invention has been found to have a strong immune protection effect against Toxoplasma infection through cell experiments and animal experiments. 4X-mRNA-LNP is a promising vaccine against Toxoplasma infection.

[0020] The beneficial effects of the present invention are:

[0021] The present invention provides an mNRA vaccine for preventing animal toxoplasmosis. The vaccine is not only directed against multiple antigenic epitopes of Toxoplasma, including the main vaccine candidate sites of Toxoplasma, the rod protein ROP18, the microneme protein MIC13; and two sites for the sexual reproduction period of Toxoplasma are introduced: TGME49268230 and TGME49237490. In addition, a sequence capable of expressing RNA polymerase (RNA-dependent RNA polymerase RdRP) is added, which can greatly enhance the effect of immunity, and compared with DNA vaccines, mRNA does not need to enter the cell nucleus to exert its effect, and will not be integrated into the genome of the subject, thereby reducing the risk of insertion mutations. In summary, the vaccine can be used to prevent acute, chronic and congenital infections of Toxoplasma, so the mRNA vaccine has the potential to prepare anti-Toxoplasma mRNA vaccines. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 This is the basic structural model of 4X-mRNA-LNP of the present invention;

[0023] Figure 2 This is the B cell epitope prediction map of 4X protein and SAG1 protein;

[0024] Figure 3 Band diagram for WB identification of HEK-293T cells and C2C12 cells transfected with 4X-mRNA-LNP in vitro;

[0025] Figure 4 Flow chart of blood collection for immunization;

[0026] Figure 5 It is the level of IgG, IgG1, and IgG2a antibodies after immunization; A represents the level of IgG antibodies, and B represents the level of IgG1 and IgG2a antibody subtypes;

[0027] Figure 6 It represents the T cell subsets after immunization;

[0028] Figure 7 The graphs are the cytokine expression levels in the supernatant of spleen cells of each group; AE are the expression levels of IL-2, IL-4, IL-10, IL-12, and IFN-γ, respectively;

[0029] Figure 8 Figure 2 shows the number of Toxoplasma gondii in the liver, spleen, and lung of each group;

[0030] Fig. 9 HE-stained pathological sections of liver, spleen and lung in each group;

[0031] Fig.10 This is the survival curve of the immune protection experiment against RH virus after immunization of each group.

[0032] Among them, vaccinated means vaccination with 4X-mRNA-LNP vaccine. DETAILED DESCRIPTION

[0033] Example 1 Construction of four antigen mRNA vaccines

[0034] 1.1 Antigen design

[0035] Through the ToxoDB gene search website, four targets were screened for the main targets of Toxoplasma epitopes and the specific antigen genes of the sexual reproduction period, namely: 1. TGME49268230, ORF length is 951bp (the gene sequence is shown in SEQ ID NO.2, and the encoded protein amino acid sequence is shown in SEQ ID NO.6); 2. TGME49237490, ORF length is 756bp (the gene sequence is shown in SEQ ID NO.3, and the encoded protein amino acid sequence is shown in SEQ ID NO.7); 3. MIC13, ORF length is 1407bp (the gene sequence is shown in SEQ ID NO.4, and the encoded protein amino acid sequence is shown in SEQ ID NO.8); 4. ROP18, ORF length is 1665bp (the gene sequence is shown in SEQ ID NO.1, and the encoded protein amino acid sequence is shown in SEQ ID NO.5); 4 antigen self-replicating mRNA plasmids A: ROP18-TGME49237490-

[0036] TGME49268230-MIC13, the expected expressed protein size is 130 kDa.

[0037] In view of the fact that the combined antigen sequence is too long and difficult to express, the main antigen expression site is truncated and optimized. The optimized combined antigen amino acid sequence is shown in SEQ ID NO.10, and the nucleotide sequence is shown in SEQ ID NO.9, wherein the amino acid sequences of rhoptry protein ROP18, TGME49268230 protein, TGME49237490 protein and microneme protein MIC13 are respectively the amino acid sequences of positions 1-207, 220-430, 443-718 and 731-1151 shown in SEQ ID NO.10, and the gene sequence encoding the rhoptry protein ROP18, the gene sequence encoding the TGME49268230 protein, the gene sequence encoding the TGME49237490 protein and the gene sequence encoding the microneme protein MIC13 are respectively shown in SEQ ID The nucleotide sequences of 1-621 bp, 658-1290 bp, 1327-2154 bp, and 2191-3453 bp are shown in NO. 9. During preparation, the combined antigen also has a signal peptide (amino acid sequence as shown in SEQ ID NO. 12, nucleotide sequence as shown in SEQ ID NO. 11) and a His-Tag tag (amino acid sequence as shown in SEQ ID NO. 14, nucleotide sequence as shown in SEQ ID NO. 13).

[0038] 1.2 Antigen preparation process

[0039] The specific process was carried out in cooperation with Ningbo Junjian Biotechnology Co., Ltd., including mRNA codon optimization, plasmid construction and verification, mRNA synthesis, capping Cap1 (m7GpppXm) tailing (alpha virus vector), adding RdRP sequence (alpha virus vector), linearized LNP encapsulation, and particle size, potential, and encapsulation rate detection. The basic information of LNP encapsulation is shown in Table 1 below, and the basic structural model is shown in Figure 1 As shown, the mRNA vaccine 4X-mRNA-LNP (sequence as shown in SEQ ID NO.19) was obtained. The cap structure increases its stability, the UTR structure increases its half-life, the RdRP enables the target mRNA to replicate autonomously to obtain more cRNA, and the A tail structure increases its stability and conversion efficiency. The specific capping process and encapsulation material information are shown in 1.2.1 and 1.2.2.

[0040] 1.2.1 mRNA capping

[0041] The in vitro transcribed mRNA was capped using the vaccinia virus capping system. The specific reaction system was as follows: 10 μg RNA, supplemented with RNase-free double-distilled water to 10 μl, 65°C for 10 min, immediately put on ice for 5 min to denature the RNA and remove the secondary structure; 2 μl 10× capping reaction buffer, 1 μl 10 mM GTP, 1 μl 32 mM SAM, 1 μl RNase inhibitor, 1 μl vaccinia capping enzyme, gently mix, and incubate at 37°C for 1 h.

[0042] 1.2.2LNP encapsulation information

[0043] The purified mRNA stock solution is prepared by a microfluidic mixing method, and an ethanol solution containing LNP and an aqueous solution containing nucleic acid are respectively injected into two channels of the preparation system. The microscopic features designed in the channels can repeatedly control the mixing of the two phases, thereby quickly, controllably and evenly mixing to produce nanoparticles. LNP is mainly composed of ionizable cationic lipids, cholesterol, neutral auxiliary phospholipids and phospholipids modified with polyethylene glycol.

[0044] We selected a lipid mixture of DLin-MC3-DMA, CHO-HP, DSPC and DMP-PEG2000 and prepared it with anhydrous ethanol as the delivery vector. We diluted the mRNA stock solution with citrate buffer to a concentration of about 0.2 mg / ml to prepare a nucleic acid aqueous solution. We used the Nin0001 microfluidic preparation instrument from Precision NanoSystems to synthesize it at a lipid-water volume ratio of 1:3, and finally replaced the solution with 1XPBS solution to prepare the mRNA-LNP finished product.

[0045] Table 1

[0046] Particle size (nm) Point (mV) PDI Encapsulation rate (%) 75.63 0.73 0.13 95.40

[0047] 2. Comparison of surface probability, antigenic index, hydrophilicity and flexible regions of the four-antigen joint gene and SAG1

[0048] The protein B cell epitopes obtained after translation of 4X-mRNA-LNP were analyzed using the PROTEAN program in DNASTAR software. The surface probability, antigenic index, flexibility and hydrophilicity of the protein B cell epitopes obtained after translation of 4X-mRNA-LNP were more advantageous than those of SAG1 ( Figure 2 ).

[0049] Example 2 In vitro verification of mRNA expression

[0050] To determine the in vitro expression of 4X-mRNA-LNP, 1×10 6 HEK-293T cells were cultured in 6-well plates and stained with Lipofectamine according to the manufacturer's instructions. TM4X-mRNA-LNP was transfected into HEK-293T cells using 3000 reagent. After culturing for 72 h, the supernatant was discarded and 200 μl of RIPA cell lysis buffer was added to each well to collect the cell lysate and analyze it by immunoblotting using Rb pAb to 6X His tag antibody.

[0051] To further ensure that mRNA could be expressed by intramuscular injection, 4X-mRNA-LNP was transfected into skeletal myoblasts C2C12 and the above operation was repeated.

[0052] Figure 3 The results showed that the mRNA could be expressed in eukaryotic cells and even mouse muscle cells.

[0053] Example 3

[0054] 1. Immunization procedure of mRNA vaccine and detection of IgG antibody, IgG1 and IgG2a antibody levels in mice

[0055] 1.1 Immunization Procedure

[0056] Immunization was performed by intramuscular injection at days 0, 14, and 28 with 15 μg 4X-mRNA-LNP / 0.1 ml PBS, TGME49268230-mRNA-LNP (preparation method is the same as 4X-mRNA-LNP) / 0.1 ml PBS, TGME49237490-mRNA-LNP (preparation method is the same as 4X-mRNA-LNP) / 0.1 ml PBS, or an equal amount of PBS or LNP (diluted with PBS). Blood was collected from the mouse orbits at 0, 2, 4, and 6 weeks, centrifuged at 4000×g for 5 min, and stored at -20°C for later use. Figure 4 ).

[0057] 1.2. IgG antibody, IgG1, and IgG2a antibody levels in mice

[0058] The IgG antibody levels and IgG1 and IgG2a antibody subtypes in mice were detected by enzyme-linked immunosorbent assay.

[0059] The specific steps are:

[0060] 1.2.1 Dilute 4X protein (four antigen combined protein) with bicarbonate buffer (pH 9.6) to a concentration of 10 μg / ml and coat in a 96-well ELISA plate (100 μl per well) overnight;

[0061] 1.2.2 Discard the coating solution and wash the coated plate 5 times with PBST solution containing 0.05% Tween 20. Incubate with PBST containing 5% skim milk powder at 37°C for 1 hour;

[0062] 1.2.3 Wash the plate 5 times with PBST and then add 100 μl of serum diluted in PBST (1:100) containing 5% skimmed milk powder to the wells and incubate at 37°C for 1 h.

[0063] 1.2.4 Wash the plate 5 times with PBST and then add 100 μl of diluted serum and incubate at 37°C for 1 h;

[0064] 1.2.5 Repeat the above washing process and then add 1:5000 diluted goat anti-mouse IgG-HRP, goat anti-mouse IgG1-HRP or goat anti-mouse IgG2a-HRP and incubate at 37℃ for 1 hour;

[0065] 1.2.6 After washing five times, pat dry the remaining water in the wells, add colorimetric solution and incubate at 37℃ for 15 minutes;

[0066] 1.2.7 Add 50 μl 2M H2SO4 to terminate the reaction and read the absorbance at 450 nm using an ELISA reader. Figure 5 A and 5B) Compared with the blank group and the LNP group, the IgG antibody titer of the experimental group mice increased with the injection times. In addition, the IgG antibody level, as well as the IgG1 and IgG2a antibody levels were much higher than those of the control group.

[0067] 2. Flow cytometric analysis of T cells (blood)

[0068] Two weeks after the last immunization, 200 μl of whole blood was collected from the mouse through orbital blood sampling, and 5 times the volume of red blood cell lysis solution was added. The mixture was gently blown and mixed, and the red blood cells were lysed at room temperature for 2-3 minutes. The supernatant was discarded after centrifugation at 400×g for 5 minutes, and this step was repeated until the red blood cells were completely lysed. PBMC was resuspended with 100 μl FACS (PBS with 0.5% FBS), and 1 μl of fluorescently labeled FITC-CD3, PerCP-Cyanine5.5-CD4 and APC-cy7-CD8 monoclonal antibodies were added to each tube and incubated at room temperature and away from light for 15 minutes. 2 ml FACS was added to each tube for washing, and after 5 minutes at 400×g, the cells were resuspended with 500 μl FACS and detected by analytical flow cytometer. ( Figure 6 ) The results showed that the immune group CD4 + T and CD8 + The proportion of T cells was significantly increased compared with the control group, and the results are shown in Table 2.

[0069] Table 2

[0070] 4X-mRNA-LNP LNP Naive <![CDATA[CD4 + T cells]]> 36.38±2.192 36.38±2.192 12.71±2.693 <![CDATA[CD8 + T cells]]> 30.16±2.794 5.716±0.6241 5.614±0.6851

[0071] 3.4X-mRNA-LNP mice produced high levels of cytokines (IFN-γ, IL-2, IL-4, and IL-10)

[0072] Two weeks after the last immunization, the spleen cells of the mice were aseptically taken and prepared into a single cell suspension. In short: the spleen was transferred to a 70μm cell screen (the screen was placed in a 50ml centrifuge tube); the spleen was slowly ground with the inner core of a 5ml syringe, and sterile FACS (PBS containing 0.5% fetal bovine serum) was slowly added while grinding; the collected cell suspension was centrifuged at 400×g for 5min, and the supernatant was discarded; 5 times the volume of red blood cell lysis buffer was added, gently blown to mix, and lysed at room temperature for 2-3 minutes; then centrifuged at 400g for 5min, and the red supernatant was discarded; the above lysis steps were repeated until the red blood cells were completely lysed, then the supernatant was discarded and the precipitate was resuspended with FCAS. Then 1ml of 20% FBS DMEM high-glucose culture medium was added to resuspend the precipitate, and the total number of cells was counted using a modified abalone counting plate. The spleen cells (1×10 6 / 100μl) was inoculated in a sterile 96-well cell culture plate. 4X protein with a final concentration of 10μg / ml was added to the culture supernatant, and the plate was placed in a 37℃ 5% CO2 incubator. The cell culture supernatant ( Figure 7 AD). IL-2, IL-4, IL-10 and IFN-γ all showed significant increases compared with the control group, as shown in Table 3 below.

[0073] Table 3

[0074] 4X-mRNA-LNP LNP Naive IL-2 18.35±0.9547 4.224±0.1826 4.846±0.8693 IL-4 56.05±9.327 13.01±0.2463 11.14±1.686 IL-10 1269±152.5 4.122±2.643 5.002±2.867 IFN-Y 5926±2061 9.314±0.5625 8.052±0.8147

[0075] 4. Lymphocyte proliferation assay

[0076] Two weeks after the last immunization, spleen cell suspensions of 5 mice in each group were prepared as above, and spleen cells (1×10 5 Add to 96-well plate, and stimulate with 4X protein (10 μg / ml) or an equal volume of DMEM high glucose medium as negative control. In addition, no cells were added, and the medium group was added as blank control. The lymphocytes were cultured in a cell culture incubator at 37°C and 5% CO2 for 96 hours, CCK8 was added according to the instructions and incubated for 4 hours, and then the absorbance value at 450nm was read by ELISA plate reader to illustrate lymphocyte proliferation. The calculation formula is: Cell proliferation activity = (OD450 4X-OD450Blank / OD450 Control-OD450 Blank) ( Figure 7 E) The results are shown in Table 4 below.

[0077] Table 4

[0078] 4X-mRNA-LNP LNP Naive Cell proliferation activity 2.465±0.6799 1.150±0.1873 1.170±0.2403

[0079] Example 4

[0080] 1. Insect load detection

[0081] 1.1 Take 5 mice from each group and inject them with 1×10 tachyzoites of wild-type Toxoplasma gondii RH strain 5 Four days later, the mice were killed by cervical dislocation, and their liver, spleen, and lung tissues were collected.

[0082] 1.2 The collected liver, spleen and lung tissues were used to extract the genomic DNA from the precipitate obtained after centrifugation using TIANGEN's blood and tissue gene extraction kit. After the concentration was determined, it was stored at -20°C for later use.

[0083] 1.3 qPCR was performed using the obtained liver, spleen, and lung. The specific system is as follows

[0084] Upstream primer 529-F: 5'-GACTACAGACGCGATGCC-3',

[0085] Downstream primer 529-R: 5′-CTCTTCAATTCTCTCCGCCAT-3′.

[0086]

[0087] qPCR reaction conditions: 1. Pre-denaturation at 98°C for 2 minutes; 2. Denaturation at 98°C for 10 seconds; 3. Annealing at 60°C for 10 seconds; 4. Extension at 68°C for 30 seconds. Steps 2-4 were cycled 40 times. The 529bp standard curve was used to calculate the number of Toxoplasma gondii in each organ of the mouse. Figure 8 As shown, the number of Toxoplasma gondii in various organs of the immunized group was significantly lower than that of the control group, and it had a certain effect against Toxoplasma gondii.

[0088] 2. HE staining

[0089] The liver, spleen and lung tissues of each group of mice were taken and immersed in 4% paraformaldehyde (Beyotime, Shanghai, China) at room temperature for 24 hours, dehydrated with ethanol, clarified with xylene, embedded in wax blocks and sliced ​​with a microtome. The slices were then stained with hematoxylin and eosin (H&E). Fig. 9 The tissue sections of mice infected with RH strain were basically unchanged compared with normal mice, while after the LNP and negative groups were infected with RH strain, a large number of necrotic cells appeared in the spleen tissue, and lymphoid follicles were missing; liver cells were obviously separated, and the number of hemorrhagic spots increased; and the alveolar wall was significantly thickened.

[0090] 3. Experiment on the protective effect of 4X-mRNA-LNP on mice

[0091] Two weeks after the last immunization, mice were injected with 100 cells / 100 μl PBS dilution, 5 mice per group, and the death of mice was observed and recorded. Fig.10The mice in the control group died within 8 days, and the mice inoculated with TGME49268230 and TGME49237490-mRNA-LNP died within 30 days, with only one surviving. Two mice in the immunization group died on the 16th day, and the remaining three mice survived within 30 days of observation, indicating that the mice immunized with 4X-mRNA-LNP acquired the ability to resist the strong strain of wild strains, and its protective ability against Toxoplasma gondii in mice is greater than that of the vaccine immunized with a single antigen, and has the potential to become a vaccine against Toxoplasma infection.

[0092] The present invention provides an mRNA vaccine for preventing Toxoplasma infection. Cell experiments and animal experiments show that the mRNA vaccine has a strong immune protection against Toxoplasma infection. 4X-mRNA-LNP may be a promising vaccine against Toxoplasma infection.

Claims

1. A recombinant self-replicating mRNA plasmid, characterized in that: Including plasmid backbone sequence, replicase gene, gene encoding rod protein ROP18, gene encoding TGME49_268230 protein, gene encoding TGME49_237490 protein and gene encoding microneme protein MIC13, The gene sequence encoding the rod-shaped protein ROP18 is shown in SEQ ID NO.1, the gene sequence encoding the TGME49_268230 protein is shown in SEQ ID NO.2, the gene sequence encoding the TGME49_237490 protein is shown in SEQ ID NO.3, and the gene sequence encoding the microneme protein MIC13 is shown in SEQ ID NO.

4.

2. The recombinant self-replicating mRNA plasmid according to claim 1, characterized in that The gene sequence encoding the rod-shaped protein ROP18, the gene sequence encoding the TGME49_268230 protein, the gene sequence encoding the TGME49_237490 protein and the gene sequence encoding the microneme protein MIC13 are the nucleotide sequences of 1-621bp, 658-1290bp, 1327-2154bp and 2191-3453bp shown in SEQ ID NO.9 respectively.

3. The recombinant self-replicating mRNA plasmid according to claim 1 or 2, characterized in that: The replicase gene is a viral RNA polymerase gene, and the replicase gene is derived from an alphavirus.

4. The recombinant self-replicating mRNA plasmid according to claim 1, characterized in that The plasmid backbone sequence includes a subgenomic promoter, a 5'UTR region, a 3'UTR region and a 3' terminal PolyA tail.

5. Use of the recombinant self-replicating mRNA plasmid according to any one of claims 1 to 4 in the preparation of a vaccine for preventing Toxoplasma gondii infection.

6. A self-replicating mRNA for four antigens combined with anti-Toxoplasma gondii, characterized in that: Includes 5'UTR region, 3'UTR region and 3' terminal PolyA tail, replicase sequence, mRNA encoding rhoptry protein ROP18, mRNA encoding TGME49_268230 protein, mRNA encoding TGME49_237490 protein and mRNA encoding microneme protein MIC13; The amino acid sequence of the rhoptry protein ROP18 is shown in SEQ ID NO.

5. The amino acid sequence of the TGME49_268230 protein is shown in SEQ ID NO.6, The amino acid sequence of the TGME49_237490 protein is shown in SEQ ID NO.7, The amino acid sequence of the microneme protein MIC13 is shown in SEQ ID NO.

8.

7. The self-replicating mRNA of four antigens combined with anti-Toxoplasma gondii according to claim 6, characterized in that: The amino acid sequence of the rod protein ROP18, the amino acid sequence of the TGME49_268230 protein, the amino acid sequence of the TGME49_237490 protein and the amino acid sequence of the microneme protein MIC13 are the amino acid sequences at positions 1-207, 220-430, 443-718 and 731-1151 shown in SEQ ID NO.10, respectively.

8. The self-replicating mRNA of four antigens combined with anti-Toxoplasma gondii as claimed in claim 6, characterized in that: It also comprises a 5' cap structure, wherein the 5' cap structure is a 7-methylguanosine cap structure.

9. A four-antigen combined anti-Toxoplasma self-replicating mRNA vaccine, characterized in that: A self-replicating mRNA comprising the four antigens combined against Toxoplasma gondii as described in any one of claims 6-8.

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